PLANT PHYSIOLOGY

Aloni, Roni; Wolf, Asnat; Feigenbaum, Pua; Avni, Adi; Klee, Harry J.

doi: 10.1104/pp.117.3.841pmid: 9662526

Abstract We confirm the hypothesis thatAgrobacterium tumefaciens-induced galls produce ethylene that controls vessel differentiation in the host stem of tomato (Lycopersicon esculentum Mill.). Using an ethylene-insensitive mutant, Never ripe(Nr), and its isogenic wild-type parent we show that infection by A. tumefaciens results in high rates of ethylene evolution from the developing crown galls. Ethylene evolution from isolated internodes carrying galls was up to 50-fold greater than from isolated internodes of control plants when measured 21 and 28 d after infection. Tumor-induced ethylene substantially decreased vessel diameter in the host tissues beside the tumor in wild-type stems but had a very limited effect in the Nr stems. Ethylene promoted the typical unorganized callus shape of the gall, which maximized the tumor surface in wild-type stems, whereas the galls on the Nr stems had a smooth surface. The combination of decreased vessel diameter in the host and increased tumor surface ensured water-supply priority to the growing gall over the host shoot. These results indicate that in addition to the well-defined roles of auxin and cytokinin, there is a critical role for ethylene in determining crown-gall morphogenesis. Infection of sensitive plants by Agrobacterium tumefaciens is known to induce crown galls. Tumor growth is initiated by the integration and expression of the T-DNA of the bacterial Ti plasmid within the plant nDNA. The T-DNA encodes enzymes catalyzing the synthesis of high levels of auxin, cytokinin, and opines (Weiler and Spanier, 1981; Zambryski et al., 1989; Schell et al., 1994). Aloni et al. (1995) found that an A. tumefaciens-induced crown gall caused the development of pathologic xylem in the centripetal direction within the host stem of castor bean. This pathologic xylem was characterized by narrow vessels, giant rays, and an absence of fibers. A similar anatomy was induced experimentally in stems of elm seedlings by the ethylene-releasing agent ethrel (Yamamoto et al., 1987), indicating the possible involvement of the hormone ethylene in crown-gall development. Therefore, Aloni et al. (1995)suggested that the high auxin levels induced by the T-DNA-encoded genesiaaM and iaaH (Thomashow et al., 1984, 1986) stimulate ethylene synthesis at the base of the crown gall, where high auxin streams originating in the tumor merge. Therefore, tumor-induced ethylene might affect crown-gall morphogenesis and differentiation of host tissues adjacent to the tumor. A. tumefaciens-induced crown galls cause poor xylem development in grapevine, which impairs water flow to the young parts of the shoot above the gall (Agrios, 1988). Aloni et al. (1995)proposed the “gall-constriction hypothesis” to explain the mechanism that gives water-supply priority to the growing gall over the host shoot. The hypothesis proposes that the growing gall retards the development of its host shoot by decreasing vessel diameter in the host, substantially reducing the supply of water to the upper parts of the shoot. It was also suggested that the controlling signal that induces narrow vessels in the host is the hormone ethylene (Aloni et al., 1995), which is known to reduce vessel width (Yamamoto et al., 1987; Aloni, 1991). If this suggestion is true, one might expect that in a plant that is not sensitive to ethylene the tumor will not reduce vessel diameter, and consequently will not interrupt shoot supply and development above the gall. The working hypothesis of the present study was that we could expect normal-sized vessels in host plants that are not sensitive to ethylene. This would give water-supply priority to the host shoot over the crown gall. A limited supply of water and nutrients to the growing crown gall might therefore reduce or even inhibit tumor development. To uncover the possible role of ethylene in crown-gall morphogenesis we induced tumors with the wild-type strain C58 of A. tumefaciens in wild-type tomato (Lycopersicon esculentum Mill.) plants and the Never ripe(Nr) mutant of tomato (Lanahan et al., 1994), which is nearly insensitive to ethylene, and studied the morphogenesis and differentiation of their crown galls and host shoots. A preliminary account of some of the findings of the present study has been published previously (Aloni et al., 1997b). MATERIALS AND METHODS Plants Wild-type and Never ripe (Nr) seeds of tomato (Lycopersicon esculentum Mill.) plants were obtained from the Monsanto Company (St. Louis, MO). Nr was originally identified as a spontaneous mutation in a single fruit in a field of cv Pearson plants (Rick and Butler, 1956). We have shown that the ethylene-insensitive phenotype is regulated by a single locus (Lanahan et al., 1994). Based on triple-response assays in seedlings, we concluded that Nr is incompletely dominant and in the homozygous state confers near but not complete ethylene insensitivity (Lanahan et al., 1994; Yen et al., 1995). It is important to note that the mutant is not completely insensitive to ethylene, and this residual responsiveness likely accounts for the ethylene-associated responses observed in this study. The tomato plants were grown in standard potting soil (Shacham, Giveat Ada, Israel) in a 24°C ± 1°C growth room at a light intensity of 150 μE m−2s−1 from cool-white fluorescent lamps (Sylvania) with a 16-/8-h light/dark regime. Bacteria The wild-type strain C58 of Agrobacterium tumefaciens, obtained from the Max Planck Institut (Köln, Germany), was grown and treated as described by Aloni et al. (1995). Crown-Gall Morphogenesis To induce a tumor, a V-shaped wound was made with a razor blade in the middle of a young internode (ranging from 10 to 60 mm long) of 3-week- to 2-month-old tomato plants. The wound reached about one-half of the internode width, and was inoculated with the bacterial pellet. In each experiment we used internodes of similar length. The tumor experiment was repeated 5 times, with 5 to 20 repetitions per line. For studying vascular differentiation in the host and tumor, shoots of wild type and the Nr mutant were harvested after various periods ranging from 2 weeks to 3 months. Ethylene Measurements Ethylene evolution was studied from A. tumefaciens-induced crown galls grown on both wild-type andNr plants. For ethylene measurements whole young plants or excised internodes from older plants were kept during the experiment in either 300- or 30-mL tubes, respectively. In preliminary measurements, an increase in ethylene production occurred during the first 2 h after wounding in 3-week-old tomato plants. Therefore, to reduce the effect of wounding caused by the cuts made in the stem for preparing the excised internodes, the ethylene measurements 21 and 28 d after infection by A. tumefaciens started 2 h after the internodes were cut from the plants (i.e. the tubes containing the internodes were kept open for the first 2 h before we started to measure ethylene evolution in closed tubes). During the experiment the tubes were kept completely closed with rubber stoppers. For the experiments we used whole, small, 3- and 4-week-old plants 7 and 14 d after infection by A. tumefaciens, and excised internodes from 5- and 6-week-old plants 21 and 28 d after infection, respectively. Five-milliliter air samples from the closed tubes were analyzed for ethylene after 5 and 7 h. Ethylene was measured with a gas chromatograph (model 3350, Varian, Sugarland, TX) according to the method of Chalutz et al. (1984). The ethylene experiment (Table I) was repeated three times, with five or six repetitions per line. Table I. Evolution of ethylene from whole wild-type tomato plants and the Nr mutant 7 and 14 d after infection (DAI) by A. tumefaciens and from excised internodes 21 and 28 d after infection Examined Tissue . Whole Plants . Excised Internodes . 7 DAI . 14 DAI . 21 DAI . 28 DAI . nL g−1 fresh wt h−1 Wild type  Control 0.19  ± 0.01 0.21  ± 0.01 0.21  ± 0.01 0.23  ± 0.02  Internode – – 0.25  ± 0.02 0.38  ± 0.06  A.t.gall-a 0.58  ± 0.04* 0.53  ± 0.04* 10.87  ± 1.22* 9.01  ± 2.38* Nr  Control 0.19  ± 0.01 0.21  ± 0.02 0.29  ± 0.02 0.37  ± 0.05  Internode – – 0.28  ± 0.01 0.4  ± 0.07  A.t. gall 0.84  ± 0.04* 0.95  ± 0.08* 12.84  ± 1.88* 11.14  ± 1.92* Examined Tissue . Whole Plants . Excised Internodes . 7 DAI . 14 DAI . 21 DAI . 28 DAI . nL g−1 fresh wt h−1 Wild type  Control 0.19  ± 0.01 0.21  ± 0.01 0.21  ± 0.01 0.23  ± 0.02  Internode – – 0.25  ± 0.02 0.38  ± 0.06  A.t.gall-a 0.58  ± 0.04* 0.53  ± 0.04* 10.87  ± 1.22* 9.01  ± 2.38* Nr  Control 0.19  ± 0.01 0.21  ± 0.02 0.29  ± 0.02 0.37  ± 0.05  Internode – – 0.28  ± 0.01 0.4  ± 0.07  A.t. gall 0.84  ± 0.04* 0.95  ± 0.08* 12.84  ± 1.88* 11.14  ± 1.92* Values are means ± se (n = 5) of ethylene production. All differences between ethylene evolution from the controls and from plants and internodes carrying galls (*) were significant (P < 0.05). The controls were uninfected whole plants or internodes from uninfected plants, whereas the internodes measured 21 and 28 d after infection were internodes harvested fromA. tumefaciens-infected plants and defined as the second internode above the crown gall. F0-a A.t., A. tumefaciens. Open in new tab Table I. Evolution of ethylene from whole wild-type tomato plants and the Nr mutant 7 and 14 d after infection (DAI) by A. tumefaciens and from excised internodes 21 and 28 d after infection Examined Tissue . Whole Plants . Excised Internodes . 7 DAI . 14 DAI . 21 DAI . 28 DAI . nL g−1 fresh wt h−1 Wild type  Control 0.19  ± 0.01 0.21  ± 0.01 0.21  ± 0.01 0.23  ± 0.02  Internode – – 0.25  ± 0.02 0.38  ± 0.06  A.t.gall-a 0.58  ± 0.04* 0.53  ± 0.04* 10.87  ± 1.22* 9.01  ± 2.38* Nr  Control 0.19  ± 0.01 0.21  ± 0.02 0.29  ± 0.02 0.37  ± 0.05  Internode – – 0.28  ± 0.01 0.4  ± 0.07  A.t. gall 0.84  ± 0.04* 0.95  ± 0.08* 12.84  ± 1.88* 11.14  ± 1.92* Examined Tissue . Whole Plants . Excised Internodes . 7 DAI . 14 DAI . 21 DAI . 28 DAI . nL g−1 fresh wt h−1 Wild type  Control 0.19  ± 0.01 0.21  ± 0.01 0.21  ± 0.01 0.23  ± 0.02  Internode – – 0.25  ± 0.02 0.38  ± 0.06  A.t.gall-a 0.58  ± 0.04* 0.53  ± 0.04* 10.87  ± 1.22* 9.01  ± 2.38* Nr  Control 0.19  ± 0.01 0.21  ± 0.02 0.29  ± 0.02 0.37  ± 0.05  Internode – – 0.28  ± 0.01 0.4  ± 0.07  A.t. gall 0.84  ± 0.04* 0.95  ± 0.08* 12.84  ± 1.88* 11.14  ± 1.92* Values are means ± se (n = 5) of ethylene production. All differences between ethylene evolution from the controls and from plants and internodes carrying galls (*) were significant (P < 0.05). The controls were uninfected whole plants or internodes from uninfected plants, whereas the internodes measured 21 and 28 d after infection were internodes harvested fromA. tumefaciens-infected plants and defined as the second internode above the crown gall. F0-a A.t., A. tumefaciens. Open in new tab Ethrel Application To clarify the role of tumor-induced ethylene on vessel diameter in tomato stems, ethrel (2-chloroethyl-phosphonic acid; Sigma), which releases ethylene (Yamamoto et al., 1987), was applied in the form of lanolin paste in two concentrations, 1 and 5% (w/w), as a ring around the middle point of the fifth internode below the apical bud. To mark the border between the xylem formed before the experiment and that affected by ethrel, a very gentle longitudinal scratch was made at the site of ethrel application at the beginning of the experiment. The ethrel in lanolin paste was prepared by dissolving the ethrel in ethanol, which was then mixed with warm lanolin. To evaporate the ethanol, the mixture was kept warm and stirred with a magnetic stirrer for 15 min. The lanolin paste containing the ethrel and the control paste (lanolin with no ethrel) were reapplied weekly. Tissue was harvested after 5 weeks and the experiments were repeated 3 times, with 5 to 10 repetitions per line. Tissue Preparation and Microscopy Hand-cut sections (1–3 mm thick) of tumor and host stem tissues were cleared by boiling in 90% lactic acid for 2 to 10 min. The sections were allowed to cool for at least 1 h, stained at room temperature with a 0.4% solution of lacmoid (PolyScience, Niles, IL) in 90% lactic acid for about 60 to 75 min, and then rinsed in tap water until the red color of the tissue became blue (about 1 h). After the washing step, air bubbles within the tissue were removed by vacuum infiltration with tap water for 30 to 45 min. The sections were then transferred to 50% sodium lactate for microscopic analysis under transmitted white light (Aloni and Sachs, 1973; Aloni and Barnett, 1996). Micrographs were reproduced from color slides taken with a light microscope (model BH2, Olympus) and an OM-2 camera (Olympus) with Fujichrome 64T (64 ASA, Fuji Photo Film Co. Ltd., Tokyo, Japan) film. Statistics Statistical terminology and the test of significance for vessel diameter were with the Student's t test and according to the method of Sokal and Rohlf (1969). The Scheffe test and Tukey's honesty test for multiple comparisons were used for analyzing ethylene evolution. Data were run through SPSS statistical software (Microsoft) for these analyses. RESULTS Ethylene Evolution from A. tumefaciens-Induced Galls Ethylene production by whole 1-month-old plants, measured 7 d after infection by A. tumefaciens, was up to 4-fold greater than that in uninfected control plants (Table I). The same was true with whole plants studied 14 d after infection (Table I). TheNr plants with young growing galls produced more ethylene than wild-type plants with growing galls. The evolution of ethylene from excised internodes carrying galls was up to 50-fold greater than from excised internodes of control plants when measured 21 and 28 d after infection by A. tumefaciens(Table I). An internode carrying a gall produced up to 50-fold more ethylene than an ungalled internode from the same infected plant. However, higher levels of ethylene were also noted in some of the ungalled internodes of the infected plants. Generally, we noted a positive correlation in the levels of ethylene production between a gall-carrying internode and an ungalled internode of the same plant. The Epinastic Response of Petioles to Crown-Gall-Associated Ethylene Is Absent in the Nr Mutant Many of the shortest internodes (usually about 10 mm long) carrying crown galls in the young wild-type tomato plants showed some epinastic response (Fig. 1a) in the leaves located immediately above and below the growing crown gall, indicating ethylene production in the tumor tissues. Conversely, in all of the young Nr mutant plants the orientation of the leaves was always normal (Fig. 1b), the same as in uninfected control plants. When long internodes (longer than 40 mm) of wild-type tomato plants were infected by A. tumefaciens, their leaves did not show an epinastic response. In experiments in which the door of the growth room was left open, allowing fast air circulation, no epinastic response could be observed on any of the A. tumefaciens-infected plants. Fig. 1. Open in new tabDownload slide Comparison of A. tumefaciens-induced crown galls on wild-type tomato (a and c) and Nr mutant (b and d) stems. a, Front view of a 3-week-old tumor developed on a wild-type plant showing the typical unorganized callus shape of a young crown gall and the epinastic response of the leaves both above and below the tumor. b, Front view of a 3-week-old tumor developed on the Nr mutant, characterized by a smooth surface and leaves in the normal orientation. Note that the lower half of the gall is protected by epidermis. c, Side view of a 2-month-old tumor on a wild-type stem with numerous adventitious roots (white spots) developed both above and below the crown gall (indicated with arrowheads). d, Side view of a 2-month-old tumor on the Nr mutant showing a fibrous hard gall and a stem almost free of adventitious roots. All photographs are at the same magnification (bars = 10 mm). Fig. 1. Open in new tabDownload slide Comparison of A. tumefaciens-induced crown galls on wild-type tomato (a and c) and Nr mutant (b and d) stems. a, Front view of a 3-week-old tumor developed on a wild-type plant showing the typical unorganized callus shape of a young crown gall and the epinastic response of the leaves both above and below the tumor. b, Front view of a 3-week-old tumor developed on the Nr mutant, characterized by a smooth surface and leaves in the normal orientation. Note that the lower half of the gall is protected by epidermis. c, Side view of a 2-month-old tumor on a wild-type stem with numerous adventitious roots (white spots) developed both above and below the crown gall (indicated with arrowheads). d, Side view of a 2-month-old tumor on the Nr mutant showing a fibrous hard gall and a stem almost free of adventitious roots. All photographs are at the same magnification (bars = 10 mm). It was also noted that wild-type tomato plants were infected easily byA. tumefaciens, whereas it was necessary to make a larger wound in the Nr shoots to obtain a tumor of comparable size. Limited Development of Adventitious Roots on the Tumor-Bearing Internodes of the Nr Mutant Adventitious roots occur normally along the stems of tomato plants. Adventitious roots do not appear on the youngest internodes, and they are evident on mature internodes (usually from the fifth internode below the apical bud). Development of a crown gall substantially stimulated the development and size of numerous adventitious roots on the infected internode of the wild-type plants (Fig. 1c). Conversely, only a few small adventitious roots developed on the infected internodes of the Nr mutant plants (Fig. 1d). Smooth Surface Characterizes the Young A. tumefaciens-Induced Crown Gall of the Nr Mutant The crown galls developed on the wild-type stems had a substantially enlarged surface area (Fig. 1a) attributable to the unorganized callus shape of the tumor. During the early stages of tumor growth the epidermis was torn and the inner, fast-growing gall tissues were exposed, forming the typically unorganized callus appearance of a crown gall (Fig. 1a). The vascular elements in the tumor tissue extended up to the gall surface, with no epidermis to protect against water transpiration. Residual epidermis, characterized by nonfunctioning stomata with no starch granules, occurred at the borders of the host stem. These stomata remained continuously wide open because the epidermis was stretched by the fast-expanding tumor tissues beneath it. Conversely, 3-week-old crown galls that developed on theNr mutant (Fig. 1b) were characterized by an epidermis with active stomata, containing starch granules in a density typical of intact epidermis. Only in the area where the original wound was inflicted did a relatively smooth callus structure appear. Therefore, tumors that developed on the Nr mutant had a minimum tumor surface area and most of it was protected by epidermis. In theNr mutant the vascular tissues did not extend to the tumor surface and were protected from the atmosphere by a few cortex layers, which remained under the intact epidermis in young tumors. The gall tissues that developed on the Nr mutant had the same green color as the host stem tissues (Fig. 1b), whereas crown-gall tissues that developed on the wild-type plants had a yellowish appearance (Fig. 1a). Almost Normal Xylem Differentiation Occurs in the Host Stem Adjacent to the Tumor in the Nr Mutant In wild-type tomato stems the vessels near the tumor were very narrow (Fig. 2a), whereas the same vessels on Nr stems were almost of normal size (relatively large) (Fig. 2b). The average diameter (measured in the radial direction) of the widest vessels in the pathologic xylem of the stem adjacent to the tumor was more than 2-fold smaller (highly statistically significant at P < 0.01 by Student's ttest) in the wild-type stems than in the Nr mutant: 53 ± 4 μm versus 126 ± 5 μm, respectively (n = 25; 5 vessels from 5 plants). Beside the tumor, there was a drastic decrease in xylem production in the wild-type stems, which was characterized by wide, unlignified rays (Fig. 2a). The very large, unlignified rays in the wild-type host tissues adjacent to the tumor (Fig. 2a) made the shoot somewhat soft and breakable. Conversely, the Nr shoots had almost normal (small) lignified rays (Fig. 2b), resulting in a relatively strong stem. Fig. 2. Open in new tabDownload slide The effects of 6-week-old A. tumefaciens-induced crown galls on xylem differentiation in tomato host stems (a and b), and the effects of a 5-week application of 1% (w/w) ethrel on xylem differentiation in tomato stems (c and d), are shown in thick transverse sections cleared with lactic acid and stained with lacmoid. The crown galls were located above the micrographs (a and b), and the white region at the lower part of the photographs is the cleared pith. The border between the xylem formed after infection with A. tumefaciens or after ethrel application (upper part of each micrograph) and the intact xylem developed before the treatments (lower part) is delineated by a broken line. At the lower right of the ethrel-affected stems (c and d) there is a wound reaction resulting from a marking scratch done at the beginning of the experiment. a, Limited differentiation of pathologic xylem with very narrow vessels and wide rays (the clear regions in the upper part of the xylem) characterize the wild-type host. b, Massive xylem with wide vessels and almost normal rays occur in theNr mutant. c, Limited differentiation of xylem with narrow vessels and wide rays (clear radial regions) characterize the ethrel-affected xylem. d, Wide vessels and normal lignified rays are typical of the new xylem formed in the Nr mutant after ethrel application. Arrows, Vessels affected by A. tumefaciens; v, vessels affected by ethrel. Bars = 500 μm. Fig. 2. Open in new tabDownload slide The effects of 6-week-old A. tumefaciens-induced crown galls on xylem differentiation in tomato host stems (a and b), and the effects of a 5-week application of 1% (w/w) ethrel on xylem differentiation in tomato stems (c and d), are shown in thick transverse sections cleared with lactic acid and stained with lacmoid. The crown galls were located above the micrographs (a and b), and the white region at the lower part of the photographs is the cleared pith. The border between the xylem formed after infection with A. tumefaciens or after ethrel application (upper part of each micrograph) and the intact xylem developed before the treatments (lower part) is delineated by a broken line. At the lower right of the ethrel-affected stems (c and d) there is a wound reaction resulting from a marking scratch done at the beginning of the experiment. a, Limited differentiation of pathologic xylem with very narrow vessels and wide rays (the clear regions in the upper part of the xylem) characterize the wild-type host. b, Massive xylem with wide vessels and almost normal rays occur in theNr mutant. c, Limited differentiation of xylem with narrow vessels and wide rays (clear radial regions) characterize the ethrel-affected xylem. d, Wide vessels and normal lignified rays are typical of the new xylem formed in the Nr mutant after ethrel application. Arrows, Vessels affected by A. tumefaciens; v, vessels affected by ethrel. Bars = 500 μm. There was a negative correlation between the diameter of the host vessels beside the crown gall and tumor development. Tumor growth was faster on the wild-type plants than on the Nr mutant. On some of the Nr shoots, the development of the tumor on mature internodes was very slow and tumor growth almost stopped (Fig.1d). The Nr shoots carrying the A. tumefaciens-induced crown galls grew faster and had larger leaves and taller stems than the wild-type infected shoots (Fig. 3A). When moderate water stress was applied, the wild-type shoots above the tumors suffered from water deficiency earlier than the Nrshoots. Leaves started to turn yellow and senesce on the wild-type shoots above the tumor, whereas most of the leaves on the Nrshoots remained green and healthy (Fig. 3B). Fig. 3. Open in new tabDownload slide The effects of 4-week-old A. tumefaciens-induced crown galls located at the base of the stems on shoot development and leaf senescence in tomato plants. A, Retarded wild-type shoot (left) and a typically tallerNr shoot with large leaves (right). Note that the older leaves in the wild-type plant started to turn yellow and senesce. B, Moderate water stress caused leaf senescence in the wild-type shoot (left), whereas most of the leaves remained green and healthy on theNr shoot (right). Fig. 3. Open in new tabDownload slide The effects of 4-week-old A. tumefaciens-induced crown galls located at the base of the stems on shoot development and leaf senescence in tomato plants. A, Retarded wild-type shoot (left) and a typically tallerNr shoot with large leaves (right). Note that the older leaves in the wild-type plant started to turn yellow and senesce. B, Moderate water stress caused leaf senescence in the wild-type shoot (left), whereas most of the leaves remained green and healthy on theNr shoot (right). Ethrel Application Reduced Vessel Diameter and Xylem Production in Wild-Type Stems Application of 1% ethrel to the stem of wild-type tomato plants substantially reduced xylem formation. The xylem produced after ethrel application was characterized by very wide rays, leaving wide, unlignified radial strips in the xylem (Fig. 2c). In addition, this xylem was clearly identified by narrow vessels, which were typically narrower than the largest vessels of the xylem formed before the treatment (Fig. 2c). Application of 5% ethrel had a stronger effect, resulting in depressed xylem production characterized by a few very narrow vessels and extremely wide, unlignified rays (not shown). Conversely, application of 1% ethrel to Nr stems did not reduce xylem production. The xylem produced after ethrel application had very large vessels, some of them larger than the vessels formed before the treatment (Fig. 2d). In addition, the rays were narrow and lignified (Fig. 2d), as in the intact control plants. Application of 5% ethrel to Nr stems decreased xylem production but the vessels and rays had a normal shape (not shown). Fiber Differentiation Occurs Only in Tumor Tissues of theNr Mutant Crown galls that developed on wild-type shoots typically contained neither fibers nor sclereids and their vascular elements were surrounded by parenchyma cells only (Fig.4a); therefore, these tumors (Fig. 1c) were relatively soft. Conversely, fibrous, hard crown galls developed on the Nr mutant (Fig. 1d) as a result of fiber and sclereid differentiation within the tumor tissues (Fig. 4b). Fibers were first detected in the xylem of the 3-week-old tumors and their number increased with time. Circular vessels occurred randomly in the tumors that developed on both the wild-type and Nrshoots (Fig. 4, a and b). Fig. 4. Open in new tabDownload slide Typical patterns of vascular tissues in 6-week-old A. tumefaciens-induced crown galls on tomato stems observed in thick, longitudinal radial sections cleared with lactic acid and stained with lacmoid. a, Circular vessels surrounded by parenchyma cells in wild-type stems. b, Circular vessels surrounded by fibers in the Nr mutant. Small arrows, Circular vessels; large arrows, fibers. Both photographs are at the same magnification (bar = 100 μm). Fig. 4. Open in new tabDownload slide Typical patterns of vascular tissues in 6-week-old A. tumefaciens-induced crown galls on tomato stems observed in thick, longitudinal radial sections cleared with lactic acid and stained with lacmoid. a, Circular vessels surrounded by parenchyma cells in wild-type stems. b, Circular vessels surrounded by fibers in the Nr mutant. Small arrows, Circular vessels; large arrows, fibers. Both photographs are at the same magnification (bar = 100 μm). DISCUSSION The findings of the present study confirm that A. tumefaciens-induced crown galls produce the hormone ethylene, as proposed by Aloni et al. (1995). Similar results have been found inA. tumefaciens-induced tumors of castor bean (by using CO2-laser-activated photoacoustic spectrometry), in which up to a 70-fold higher level of ethylene production was measured in 5-week-old crown galls than in control stem tissues (C.I. Ullrich, personal communication). Our findings show that tumor-induced ethylene is a limiting and a major controlling factor of crown-gall morphogenesis. Therefore, along with auxin and cytokinin (Weiler and Spanier, 1981; Zambryski et al., 1989; Malsy et al., 1992; Schell et al., 1994), the hormone ethylene also affects tumor morphogenesis. The integration and expression of the T-DNA of the bacterial Ti plasmid within the plant nDNA (Zambryski et al., 1989) substantially elevate auxin concentrations in crown gall tissues, which may be 500 times higher in the tumor than in control tissues (Weiler and Spanier, 1981). Because high auxin levels are known to enhance ethylene synthesis (Yang and Hoffman, 1984; Abeles et al., 1992), it was suggested that tumor-induced auxin promotes the synthesis of ethylene in crown galls (Aloni et al., 1995). Our results confirm the results of Romano et al. (1993), which showed that many “auxin” effects in iaaMwere actually ethylene effects. However, we should note that auxin also has a direct effect on vascular differentiation within the tumor, in that auxin is a limiting and controlling factor in the differentiation of both vessels and sieve tubes (Roberts et al., 1988; Aloni, 1995). It is also possible that elevated cytokinin levels, which may be 1500 times higher in the tumor than in control tissues (Weiler and Spanier, 1981; Akiyoshi et al., 1983), might also promote ethylene synthesis, because cytokinin is known to stimulate ethylene production (Wright, 1980; Mattoo and White, 1991; Cary et al., 1995). In addition, it is possible that there is a synergistic effect within crown-gall tissues whereby a combination of the elevated levels of both auxin and cytokinin boosts ethylene production in the tumor. It is also possible that information in the bacterial Ti plasmid directly promotes ethylene synthesis in the tumor, although no T-DNA gene exhibits homology to any known bacterial or plant ethylene-biosynthetic gene. It should be noted that the anatomy of the vascular tissues in crown galls and host stems indicates that the gall tissues are the source of ethylene production (Aloni et al., 1995). The strongest effect of the tumor caused pathologic xylem only adjacent to the tumor center, a limited effect at its borders, and normal xylem in the stem away from the tumor, indicating regular levels of ethylene in the host stem (Aloni et al., 1995) (Fig. 4). The epinastic response observed on some of the tumor-bearing wild-type shoots but not on shoots of the Nr mutant provides developmental evidence for the production of ethylene in A. tumefaciens-induced crown galls. The tumor-induced ethylene affected the orientation of the closest leaves above and below young internodes. It is possible that some of the observed epinasty in the nearby leaves might have been cause by transport of ACC (Abeles et al., 1992; Jackson, 1994) originating in the tumor. However, no epinastic response occurred when long internodes were infected, probably because of the greater distances between the tumor and the leaves, which were somewhat older than leaves on the shortest internodes. This was also true when the door to the growth room was left open to increase air flow around the plants. Adventitious root formation is associated with increased ethylene synthesis (Liu et al., 1990; McNamara and Mitchell, 1990, 1991). Ethylene may stimulate the initiation of adventitious root primordia on tomato stems (McNamara and Mitchell, 1991). Therefore, the appearance of numerous adventitious roots both above and below the tumor on the infected internodes of the wild-type plants but not on theNr mutant is further morphogenetic evidence for the production of ethylene in crown galls. Application of ethrel (which releases ethylene) mimicked the effect of the tumor-induced ethylene on xylem differentiation in the host stem, decreasing vessel diameter and increasing ray size in the xylem of wild-type tomato plants, whereas no effect was observed in the stems of the Nr mutant. These results demonstrate that the tumor-induced signal for decreasing vessel diameter and increasing ray size in the host stem adjacent to the crown gall (Aloni et al., 1995) is the hormone ethylene. This study shows that ethylene has a major role in controlling water transport to crown galls. There are two mechanisms that influenced water transport to the tumors. The first is that ethylene substantially reduced the diameter of vessels adjacent to the tumor in wild-type tomato stems, whereas almost normal-sized vessels differentiated in theNr mutant. The rate at which water flows through a vessel is proportional to the fourth power of its radius (Poiseuille's law) (Zimmermann, 1983). Accordingly, we calculated water flow through the narrow vessels measured in the pathologic xylem beside the tumor and found a 30-fold decrease in the wild-type tomato stems compared with the Nr stems. This result confirms the gall-constriction hypothesis, which explains how a decrease in the host vessels located beside the tumor gives water-flow priority to the tumor over the host shoot (Aloni et al., 1995). The second mechanism is that ethylene substantially increased the surface of the tumor only in the wild-type plants. The epidermis was torn by the expanding inner tumor tissues, forming the typically unorganized callus appearance of a crown gall. Furthermore, the vascular elements in the tumor tissue extended up to the gall surface, which did not have an epidermis to protect against water transpiration. Residual epidermis, characterized by continuously widely open stomata, occurred only at the borders of the host stem. Schurr et al. (1996)showed that similar changes in the enlarged tumor surface substantially increased transpiration to about 15 and 7.5 times higher at the tumor surface compared with host leaves and leaves of noninfected castor bean plants, respectively. Therefore, the combination of a decrease in vessel diameter in the host and an increased surface of the tumor, both induced by ethylene, ensures water-supply priority to fast-growing crown galls. Consequently, crown galls decrease shoot development and promote leaf senescence in wild-type host plants. On the other hand, tumors on the Nr mutant were less successful in competing with the growing leaves above the tumor, and some of the crown galls on the Nr shoots even degenerated. Ethylene is known to depress polar auxin transport (Mattoo and Aharoni, 1988; Goren and Riov, 1989), and it seems likely that the circular vessels shown in this study indicate the sites of local interruptions of polar auxin movement. Circular vessels, which are induced by circular movement of auxin, were induced experimentally above sites where the polar auxin flow was interrupted (Sachs and Cohen, 1982;Aloni et al., 1997a). Fibers are usually induced by both auxin and GA (Aloni, 1979, 1987). However, high levels of auxin alone might also stimulate fiber differentiation (Aloni, 1979). Fibers differentiated in the crown galls that developed on the Nr mutant, but were completely absent in the tumors that developed on the wild-type tomato shoots. There are no reports regarding fiber differentiation in crown galls developing on other plant species (Sachs, 1991), and the limited numbers of regenerative fibers in A. tumefaciens-induced crown galls that developed on castor bean shoots were observed only in the periphery, at the base of the tumor (Aloni et al., 1995). Because ethylene is known to inhibit fiber differentiation (Yamamoto et al., 1987), we suggest that the absence of fibers in crown galls developing on wild-type plants is caused by a relatively high background level of ethylene, which prevents fiber differentiation in the tumor. Only in the Nr tumor tissues, which are nearly insensitive to ethylene, could fibers differentiate. We also suggest that background ethylene might affect other reactions and functions in tumors and should therefore be considered when crown galls are studied. Crown galls induced by A. tumefaciens produce ethylene, which is a limiting and controlling factor of tumor morphogenesis. The tumor-induced ethylene reduces vessel diameter in the host stem and enlarges the surface of the tumor, thus giving water-supply priority to the growing gall over the host shoot, resulting in fast tumor growth, retarded shoot development, and enhanced leaf senescence above the crown gall. ACKNOWLEDGMENTS We thank Prof. Cornelia I. Ullrich (Technische Hochschule, Darmstadt, Germany) for encouragement and for the gift of the wild-type strain C58 of A. tumefaciens (originally obtained from Dr. Z. Koncz, Max Planck Institut fur Zuchtungsforschung, Köln, Germany). We also thank the Monsanto Company for the gift of seeds of the wild-type tomato and the Nr mutant. LITERATURE CITED 1 Abeles FB Morgan PW Saltveit ME Jr Ethylene in Plant Biology, Ed 2. 1992 Academic Press San Diego, CA 2 Agrios GN (1988) Bacterial galls. In GN Agrios, ed, Plant Pathology. Academic Press, New York, pp 483–488 3 Akiyoshi DE Morris RO Hinz R Mischke BS Kosuge T Garfinkel DJ Gordon MP Nester EW Cytokinin/auxin balance in crown gall tumors is regulated by specific loci in the T-DNA. Proc Natl Acad Sci USA 80 1983 407 411 Google Scholar Crossref Search ADS PubMed WorldCat 4 Aloni R Role of auxin and gibberellin in differentiation of primary phloem fibers. Plant Physiol 63 1979 609 614 Google Scholar Crossref Search ADS PubMed WorldCat 5 Aloni R Differentiation of vascular tissues. Annu Rev Plant Physiol 38 1987 179 204 Google Scholar Crossref Search ADS WorldCat 6 Aloni R (1991) Wood formation in deciduous hardwood trees.In AS Raghavendra, ed, Physiology of Trees. Wiley, New York, pp 175–197 7 Aloni R The induction of vascular tissues by auxin and cytokinin. Davies PJ Plant Hormones: Physiology, Biochemistry and Molecular Biology, Ed 2. 1995 531 546 Kluwer Academic Publishers Dordrecht, The Netherlands 8 Aloni R Alexander JD Tyree MT Natural and experimentally altered hydraulic architecture of branch junctions in Acer saccharum Marsh. and Quercus velutina Lam. trees. Trees 11 1997a 255 264 Google Scholar OpenURL Placeholder Text WorldCat 9 Aloni R Barnett JR The development of phloem anastomoses between vascular bundles and their role in xylem regeneration after wounding in Cucurbita and Dahlia. Planta 198 1996 595 603 Google Scholar Crossref Search ADS PubMed WorldCat 10 Aloni R Pradel KS Ullrich CI The three-dimensional structure of vascular tissues in Agrobacterium tumefaciens-induced crown galls and in the host stems of Ricinus communis. Planta 196 1995 597 605 Google Scholar Crossref Search ADS WorldCat 11 Aloni R Sachs T The three-dimensional structure of primary phloem systems. Planta 113 1973 343 353 Google Scholar Crossref Search ADS WorldCat 12 Aloni R Wolf A Feigenbaum P Klee HJ Evidence that tumor-induced ethylene controls the morphogenesis of Agrobacterium tumefaciens-induced crown galls on tomato stems. Isr J Plant Sci 45 1997b 255 Google Scholar OpenURL Placeholder Text WorldCat 13 Cary AJ Liu W Howell SH Cytokinin action is coupled to ethylene in its effects on the inhibition of root and hypocotyl elongation in Arabidopsis thaliana seedlings. Plant Physiol 107 1995 1075 1082 Google Scholar Crossref Search ADS PubMed WorldCat 14 Chalutz E Mattoo AK Solomos T Anderson JD Enhancement by ethylene of cellulysin-induced ethylene production by tobacco leaf discs. Plant Physiol 74 1984 99 103 Google Scholar Crossref Search ADS PubMed WorldCat 15 Goren R Riov J Interference by ethylene with the abscission regarding effects of auxin in citrus leaves. Osborne DJ Jackson MB Cell Separation in Plants. 1989 191 200 Springer-Verlag Berlin 16 Jackson MB Root-to-shoot communication in flooded plants: involvement of abscisic acid, ethylene and 1-aminocyclopropane-1-carboxylic acid. Agron J 86 1994 775 782 Google Scholar Crossref Search ADS WorldCat 17 Lanahan MB Yen H-C Giovannoni JJ Klee HJ The Never ripe mutation blocks ethylene perception in tomato. Plant Cell 6 1994 521 530 Google Scholar PubMed OpenURL Placeholder Text WorldCat 18 Liu J Muckherjee I Reid DM Adventitious rooting in hypocotyls of sunflower (Helianthus annuus) seedlings. III. The role of ethylene. Physiol Plant 78 1990 268 276 Google Scholar Crossref Search ADS WorldCat 19 Malsy S van Bel AJE Kluge M Hartung W Ullrich CI Induction of crown galls by Agrobacterium tumefaciens (strain 58) reverses assimilate translocation and accumulation in Kalanchoë daigremontiana. Plant Cell Environ 15 1992 519 529 Google Scholar Crossref Search ADS WorldCat 20 Mattoo AK Aharoni N Ethylene and plant senescence. Nooden LD Leopold AD Senescence and Aging in Plants. 1988 241 280 Academic Press San Diego, CA 21 Mattoo AK White WB Regulation of ethylene biosynthesis. Mattoo AK Suttle JC The Plant Hormone Ethylene. 1991 21 42 CRC Press Boca Raton, FL 22 McNamara ST Mitchell CA Adaptive stem and adventitious root response of two tomato genotypes to flooding. Hortscience 25 1990 100 103 Google Scholar Crossref Search ADS WorldCat 23 McNamara ST Mitchell CA Roles of auxin and ethylene in adventitious root formation by flood-resistant tomato genotype. Hortscience 26 1991 57 58 Google Scholar Crossref Search ADS WorldCat 24 Rick C Butler L Phytogenetics of the tomato. Adv Genet 8 1956 267 382 Google Scholar Crossref Search ADS WorldCat 25 Roberts LW Gahan PB Aloni R Vascular Differentiation and Plant Growth Regulators. 1988 Springer-Verlag Berlin 26 Romano C Cooper M Klee H Uncoupling auxin and ethylene effects in transgenic tobacco and Arabidopsis plants. Plant Cell 5 1993 181 189 Google Scholar Crossref Search ADS PubMed WorldCat 27 Sachs T Callus and tumor development. Sachs T Pattern Formation in Plant Tissues. 1991 38 55 Cambridge University Press Cambridge, UK 28 Sachs T Cohen D Circular vessels and the control of vascular differentiation in plants. Differentiation 21 1982 22 26 Google Scholar Crossref Search ADS WorldCat 29 Schell J, Koncz C, Spena A, Palme K, Walden R (1994) The role of phytohormones in plant growth and development. In Proceeding of the XV International Botany Congress, Tokyo/Yokohama, Japan, pp 38–48 30 Schurr U Schuberth B Aloni R Pradel KS Schmundt D Jahne B Ullrich CI Structural and functional evidence for xylem-mediated water transport and high transpiration in Agrobacterium tumefaciens-induced tumors of Ricinus communis. Bot Acta 109 1996 405 411 Google Scholar Crossref Search ADS WorldCat 31 Sokal RR, Rohlf FJ (1969) Biometry, the Principals and Practice of Statistics in Biological Research. Freeman & Co, San Francisco, pp 166–172 32 Thomashow LS, Reeves S, Thomashow MF (1984) Crown gall oncogenesis: evidence that a T-DNA gene from theAgrobacterium Ti plasmid pTiA6 encodes an enzyme that catalyzes synthesis of indoleacetic acid. Proc Natl Acad Sci USA:81: 5071–5075 33 Thomashow MF Hugly S Buchholz WG Thomashow LS Molecular basis for the auxin-independent phenotype of crown gall tumor tissues. Science 231 1986 616 618 Google Scholar Crossref Search ADS PubMed WorldCat 34 Weiler EW Spanier K Phytohormones in the formation of crown gall tumors. Planta 153 1981 326 337 Google Scholar Crossref Search ADS PubMed WorldCat 35 Wright STC The effect of plant growth regulator treatments on the level of ethylene emanating from excised turgid and wilted leaves. Planta 148 1980 381 388 Google Scholar Crossref Search ADS PubMed WorldCat 36 Yamamoto F Angeles G Kozlowski TT Effect of ethrel on stem anatomy of Ulmus americana seedlings. IAWA Bull 8 1987 3 9 Google Scholar Crossref Search ADS WorldCat 37 Yang SF Hoffman NE Ethylene biosynthesis and its regulation in higher plants. Annu Rev Plant Physiol 35 1984 155 189 Google Scholar Crossref Search ADS WorldCat 38 Yen H-C Lee S Tanksley SD Lanahan MB Klee HJ Giovannoni JJ The tomato Never-ripe locus regulates ethylene-induced gene expression and is linked to a homolog of the Arabidopsis ETR1 gene. Plant Physiol 107 1995 1343 1353 Google Scholar Crossref Search ADS PubMed WorldCat 39 Zambryski P, Tempé J, Schell J (1989) Transfer and function of T-DNA genes from Agrobacterium Ti and Ri plasmids in plants. Cell 56: 193–201 40 Zimmermann MH Xylem Structure and the Ascent of Sap. 1983 Springer-Verlag Berlin Author notes 1 This study was supported by the research authority of Tel Aviv University, Israel. 2 This paper is dedicated to Prof. Judah Folkman (Harvard Medical School, Boston, MA) for his contributions on the role of angiogenesis in human and animal tumor morphogenesis. * Corresponding author; e-mail [email protected]; fax 972–3–640–9380. Copyright © 1998 American Society of Plant Physiologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Kumar Tewari, Arun; Charan Tripathy, Baishnab

doi: 10.1104/pp.117.3.851pmid: 9662527

Abstract Chlorophyll (Chl) biosynthesis in chill (7°C)- and heat (42°C)-stressed cucumber (Cucumis sativus L. cv poinsette) seedlings was affected by 90 and 60%, respectively. Inhibition of Chl biosynthesis was partly due to impairment of 5-aminolevulinic acid biosynthesis both in chill- (78%) and heat-stress (70%) conditions. Protochlorophyllide (Pchlide) synthesis in chill- and heat-stressed seedlings was inhibited by 90 and 70%, respectively. Severe inhibition of Pchlide biosynthesis in chill-stressed seedlings was caused by inactivations of all of the enzymes involved in protoporphyrin IX (Proto IX) synthesis, Mg-chelatase, and Mg-protoporphyrin IX monoester cyclase. In heat-stressed seedlings, although 5-aminolevulinic acid dehydratase and porphobilinogen deaminase were partially inhibited, one of the porphyrinogen-oxidizing enzymes, uroporphyrinogen decarboxylase, was stimulated and coproporphyrinogen oxidase and protoporphyrinogen oxidase were not substantially affected, which demonstrated that protoporphyrin IX synthesis was relatively more resistant to heat stress. Pchlide oxidoreductase, which is responsible for phototransformation of Pchlide to chlorophyllide, increased in heat-stress conditions by 46% over that of the control seedlings, whereas it was not affected in chill-stressed seedlings. In wheat (Triticum aestivum L. cv HD2329) seedlings porphobilinogen deaminase, Pchlide synthesis, and Pchlide oxidoreductase were affected in a manner similar to that of cucumber, suggesting that temperature stress has a broadly similar effect on Chl biosynthetic enzymes in both cucumber and wheat. Plants play a very important role in our lives and the environment has a significant role in plant growth and development. In different parts of the globe, especially between 20° and 30° latitudes north and south of the equator, the temperature decreases to 4°C to 8°C in the winter and increases to 40°C to 44°C in the summer. Therefore, several plant species, including annual crop plants, are exposed to both chill and heat stress during their lifetime. When plants are exposed to low- or high-temperature stress, Chl biosynthesis is affected (van Hasselt and Strikwerda, 1976; Feierabend, 1977). Biosynthesis of porphyrins and particularly that of Chl during early greening stages of seedlings has been elucidated in detail (Tripathy and Rebeiz, 1986, 1987, 1988; Hukmani and Tripathy, 1992, 1994; for recent reviews, see Leeper et al., 1991; Richards, 1992; von Wettstein et al., 1995; Porra, 1997). It is important to understand exactly how temperature stress impairs Chl biosynthesis. This will have profound importance in the generation of improved crop varieties resistant to temperature stress. There are a few reports that demonstrate that low or high temperature reduces Chl biosynthesis. Illumination of etiolated seedlings of maize at low temperature resulted in reduced Chl accumulation, and impairment of Chl biosynthesis was paralleled by an aberrant development of the thylakoid membranes (van Hasselt and Strikwerda, 1976; Hodgins and van Huystee, 1986a). A slight photoconversion of Pchlide to Chl was possible at 12°C (van Huystee and Hodgins, 1989). Chlorosis in maize was due to impaired synthesis of ALA, and incubation of leaf tissue at low temperature in ALA resulted in reduced porphyrin synthesis (Hodgins and van Huystee, 1986b). Heat-bleached rye leaves had the capacity to convert ALA to Pchlide, although they had plastidic ribosome deficiency, suggesting that the enzymes involved have to be synthesized on cytoplasmic ribosomes (Feierabend, 1977). In heat-bleached, plastidic ribosome-deficient primary leaves of rye and oat, Chl synthetase activity was partially reduced (40%), although tRNAGlu was found in substantial amounts in bleached rye plastids (Hess et al., 1992). A shift in the ratio of Chl a and Chl bwas observed in heat-bleached Euglena gracilis (Thomas and Ortiz, 1995). Although the above data are available, there has not been any systematic and comparative study on the effect of heat and chill stress on the detailed Chl biosynthetic pathway. Therefore, in the present study the effects of chill (7°C) and heat (42°C) stress on several enzymes of the Chl biosynthetic pathway were studied in the dicotyledonous horticultural plant cucumber and, when required, were compared with the monocotyledonous crop plant wheat. MATERIALS AND METHODS Plant Material Cucumber (Cucumis sativus L. cv Poinsette) and wheat (Triticum aestivum L. cv HD2329) were used as the experimental materials. The seeds were obtained from the Indian Agricultural Research Institute, New Delhi. Plant Growth Conditions The seeds were treated with 0.1% HgCl2solution for 5 min and then washed with tap water three times and spread over a single layer of moist germination paper. Cucumber and wheat seedlings were grown in the dark at 25°C for 4 and 6 d, respectively, and were transferred to low (7°C) and high (42°C) temperatures for various lengths of time. One set of seedlings remained at 25°C under identical conditions for a comparison (control). ALA Content Five pairs of cotyledons were preincubated in 60 mm LA in 50 mm phosphate buffer, pH 6.0, for 4 h in the dark at 7°C, 25°C, and 42°C. After the preincubation, cotyledons were exposed to white fluorescent light (30 μmol m−2 s−1) for 3 and 6 h while controls were left in the dark. Each time five pairs of cotyledons were weighed and hand homogenized in a prechilled mortar and pestle in 5 mL of 1 m sodium acetate buffer (pH 4.6). The homogenate was centrifuged at 10,000 rpm (12,000g) for 10 min and supernatant was taken for assay. The assay mixture consisted of 0.1 mL of supernatant, 0.4 mL of distilled water, and 25 μL of acetylacetone. The assay medium was mixed properly and heated in a boiling water bath for 10 min. Then the extract was cooled at room temperature, and an equal volume of modified Ehrlich's reagent was added and vortexed for 2 min. After 10 min of incubation, absorbance of the extract was measured at 555 nm and ALA content was determined from the standard curve of ALA (Harel and Klein, 1972). ALAD Two pairs of cotyledons, harvested from etiolated cucumber seedlings grown at different temperature regimes, were weighed and hand homogenized in 5 mL of 0.1 m Tris and 0.01 mmercaptoethanol solution, pH 7.6, in a mortar and pestle at 4°C. Homogenate was centrifuged at 10,000 rpm in a rotor (12,000g, model SM24, Sorvall) in a centrifuge (model RC5C, Sorvall) at 4°C. The supernatant was taken for ALAD assay. The crude enzyme extracts were assayed for ALAD activity according to the method of Shemin (1962) with a slight modification. The enzyme activity was determined by measuring the amount of PBG formed in 1 mL of the reaction mixture. The incubation mixture consisted of 60 mmTris, 0.2 mm ALA, 1 mm EDTA, 15 mmMgCl2, 0.5% BSA (w/v), and 0.33 mSuc, pH 7.5, and the enzyme preparation. After 10 min of preincubation, the reaction was started by adding the substrate ALA and incubation was carried out for 1 h at 28°C. The amount of PBG formed was calculated using the absorption coefficient (6.2 × 104m−1cm−1) (Hukmani and Tripathy, 1994). PBGD Two pairs of cucumber cotyledons, harvested from etiolated cucumber seedlings grown at different temperature regimes, were weighed and hand homgenized in 5 mL of 0.1 m Tris buffer (pH 7.6) at 4°C. Homogenate was centrifuged at 10,000 rpm (12,000g) for 10 min. Supernatant was taken for PBGD activity assay. The crude enzyme extracts were assayed for PBGD activity. The enzymatic activity was measured by determining the amount of uroporphyrin formed in 1 mL of reaction mixture consisting of 0.1 m Tris-HCl, 2.5 mm EDTA, 15 mm MgCl2, and 0.1% BSA (w/v), pH 7.5. The reaction was carried out for 1 h at 37°C. An aliquot of 0.85 mL of this reaction mixture was taken, to which 0.25 mL of 5 n HCl was added to stop the reaction. The porphyrinogens that were formed were oxidized in porphyrins by adding 0.1 mL of 0.1% benzoquinone to methanol. After 20 min at 4°C samples were centrifuged. A 0.1-mL aliquot was taken, to which 0.9 mL of 1 n HCl was added. Absorbance was measured at 405 nm (absorption coefficient = 5.48 × 105m−1 cm −1) (Hukmani and Tripathy, 1994). Preparation of Urogen III, Coprogen III, and Protogen IX Sodium amalgam was prepared by adding 18.31 g of mercury to 0.55 g of freshly cut and heated sodium metal under nitrogen gas (Jacobs and Jacobs, 1982). Reduction of porphyrins was carried out according to the method of Poulson and Polglosse (1974) with certain modifications. Porphyrinogens were freshly prepared by reducing porphyrins, namely uroporphyrin III, coproporphyrin III, or Proto IX. Porphyrins were dissolved in 0.1 n KOH consisting of 20% ethanol. Oxygen present in the solution was removed by flushing the solution with nitrogen gas. To this solution 5 g of 3% sodium amalgam was added and flushing the solution with the stream of nitrogen was continued. The reduction was carried out for 2 to 3 min; the solution becomes colorless within this time period and the reduction was continued for 2 to 3 min more to ensure complete reduction of the porphyrin. Immediately, the contents were filtered. The pH of the porphyrinogen solution was adjusted to 7.5 with dilute acetic acid. Proto IX Synthesis from Urogen III, Coprogen III, and Protogen IX Dark-grown cucumber seedlings were transferred to different temperature regimes, and intact plastids were isolated from cotyledons over a 50% percoll gradient. The plastids were lysed in lysis buffer consisting of 10 mm Tris-HCl and 2.5 mmNa2EDTA (pH 7.7). Lysed plastids were centrifuged at 5000 rpm for 3 min in a rotor (3000g, model SS-34, Hitachi) in a centrifuge (Hitachi) at 4°C. The supernatant was used for the enzyme assay. The reaction mixture (0.3 mL) consisted of 100 mm Tris (pH 7.5), 15 mmMgCl2, 5 mm DTT, 0.1% BSA (w/v), 0.1 mL of enzyme preparation, and 0.03 mL of urogen, coprogen, or protogen. Incubation was carried out for 1 h at 28°C (Jacobs and Jacobs, 1982). The reaction was terminated by adding 1.5 mL of 90% acetone. Pigments were quantified by spectrofluorometry (Rebeiz et al., 1975;Hukmani and Tripathy, 1992). Mg-Chelatase Etioplasts were isolated at 4°C using a modification of the previous method (Tripathy and Rebeiz, 1986). Five grams of cucumber cotyledons harvested from etiolated cucumber seedlings grown at different temperature regimes was hand homogenized in a prechilled mortar and pestle with 13 to 15 gentle strokes under a safe green light in 15 mL of chilled isolation buffer consisting of 0.5 mSuc, 20 mm Hepes, 1 mmMgCl2, 1 mmNa2EDTA, and 0.2% BSA (w/v) at a room-temperature pH of 7.7. The homogenate was passed through four layers of cheesecloth and centrifuged at 1200 rpm (200g) for 2 min. The supernatant was decanted and further centrifuged at 4000 rpm (2000g) for 7 min. All of the centrifugation was done in an SS-34 rotor at 4°C using a Hitachi centrifuge. The pelleted plastids were gently suspended with the help of a paint brush in suspension buffer containing 0.5 m Suc, 0.2 m Tris-HCl, 20 mm MgCl2, 2.5 mmNa2EDTA, 20 mm ATP, 20 mmNAD, and 8 mm Met at a room-temperature pH of 7.7. Incubation of the 0.3-mL reaction mixture consisting of 0.1 mL of chloroplast suspension, 0.1 mL of suspension buffer, 5 μL of Mg-Proto in 80% acetone, 5 μL of Proto IX, and 95 μL of distilled water was carried out at 28°C for 1 h, and 1.7 mL of ice-cold 90% ammonical acetone was added to stop the reaction. Samples were centrifuged at 5000g for 2 min to remove the pellet and an equal amount of hexane was added to the supernatant. The top hexane layer was removed and the bottom layer was again extracted with one-third of the volume of hexane. The top hexane layer was discarded and the bottom HEAR was taken for an estimation of MPE by spectrofluorometry (Hukmani and Tripathy, 1992). MPE Cyclase Plastids isolated from control, chill-, and heat-stressed cucumber seedlings were suspended in suspension buffer consisting of 0.5m Suc, 0.2 m Tris-HCl, 20 mmMgCl2, 2.5 mm EDTA, and 20 mm ATP (pH 7.7). The reaction mixture (0.3 mL) consisted of 0.1 mL of chloroplast suspension, 0.1 mL of suspension buffer, 0.5 mmS-adenosyl Met, 5 μL of Mg-Proto in 80% acetone, and 95 μL of distilled water. The pH of the reaction mixture was adjusted to 7.7 with 1 n NaOH. The incubation was carried out at room temperature (25°C) for 1 h in the dark and 1.7 mL of ice-cold 90% ammonical acetone was added to stop the reaction. The HEAR was prepared from the acetone extract and synthesis of Pchlide was estimated by spectrofluorometry. Net Synthesis of Pchlide Three-day-old etiolated cucumber seedlings grown at 25°C were transferred to 7, 42, and 25°C for 48 h in dark. To empty the endogenous Pchlide pool, seedlings were exposed to cool-white fluorescent light (30 μmol m−2s−1) for 10 min at their respective temperatures, and 10 pairs of cotyledons were excised immediately after the light treatment, weighed, and homogenized in 90% ammonical acetone. After light exposure, seedlings were kept in the dark at their respective temperatures and cotyledons were harvested after 3, 6, and 12 h. On each data point three replicates of 10 pairs of cotyledons were hand homogenized in 20 mL of ice-cold 90% ammonical acetone at 4°C, and homogenate was centrifuged at 10,000 rpm (12,000g) for 10 min. Supernatant was taken for the HEAR preparation and accumulation of Pchlide was measured spectrofluorometrically. Dry weight was measured after keeping 100 mg of cotyledon tissue for 72 h in an oven maintained at 80°C, and data points were corrected for loss of moisture both at low and high temperatures. The net synthesis of Pchlide was calculated by deducting the Pchlide value recorded after 10 min of light exposure from those recorded at 3-, 6-, and 12-h dark intervals. POR Etiolated cucumber seedlings (3 d old) grown at 25°C were transferred to 7, 42, and 25°C for 48 h in dark, and 10 pairs of cotyledons were harvested, weighed, and homogenized in 90% ammonical acetone in the dark. Subsequently, seedlings were exposed to cool-white fluorescent light (30 μmol m−2s−1) for 10 min and cotyledons were harvested, weighed, and homogenized in 90% ammonical acetone. Acetone tissue homogenates were centrifuged at 10,000 rpm in an SS-34 rotor (12,000g) for 10 min at 4°C, and from supernatant HEAR was prepared. Pchlide contents of cucumber cotyledons before and after light exposure were estimated from the HEAR fraction by spectrofluorometry (Rebeiz et al., 1975; Hukmani and Tripathy, 1992). Three replicates were taken for each data point. The percent phototransformation of Pchlide to Chlide in different temperatures was calculated as: ([(Pchlide content before phototransformation) − (Pchlide content after phototransformation)]/Pchlide content before phototransformation) × 100. Enzymatic Activities in Wheat Seedlings Etiolated wheat seedlings (6 d old) grown at 25°C were transferred to 7, 42, and 25°C for 24 h in the dark and enzymatic activities were monitored. Longer exposure (48 h) to high temperature (42°C) almost killed the wheat tissue. All of the protocols followed for wheat seedlings were identical to that of cucumber. Spectrofluorometry Fluorometric estimation of pigments was done using a photon-counting fluorometer (model 8000C, SLM, Rochester, NY). Channel A (sample) and channel C (reference) were adjusted to 20,000 counts s−1 using a tetraphenylene butadiene block as a standard and excited at 348 nm, and fluorescence emission was monitored at 422 nm. The HEAR samples were excited at 400, 420, and 440 nm and emission spectra were recorded in a ratio mode from 580 to 700 nm. Excitation and emission-slit widths were 4 nm. Emission spectra were corrected for photomultiplier response. Using appropriate equations the concentration of plant tetrapyrroles was quantified and values expressed as nanomoles per milligram of protein of per hour (Rebeiz et al., 1975; Hukmani and Tripathy, 1992). Chl and Protein Estimation Chl and protein ware estimated according to the methods ofPorra et al. (1989) and Lowry et al. (1951), respectively. Chemicals All porphyrin intermediates were purchased from Porphyrin Products (Logan, UT). DTT, ALA, ATP, NAD, Benzoquinone, LA, and Hepes were purchased from Sigma. All other chemicals were purchased from Merck (Darmstadt, Germany), Sd Fine Chemicals (Mumbai, India), BDH (Poole, UK), and Qualigens (Mumbai, India). RESULTS Chl Content Four-day-old etiolated cucumber seedlings grown at 25°C were transferred to 7°C (chill stressed), 25°C (control), and 42°C (heat stressed) and exposed to cool-white fluorescent light (30 μmol m−2 s−1) for 6, 12, 24, and 48 h. The maximum amount of Chl synthesis was observed in control seedlings at 48 h of light exposure. Chl synthesis was inhibited by 60 and 90% in heat- and chill-stressed seedlings, respectively. An initial lag period of up to 12 h was observed in chill-stressed cucumber (Fig. 1A). Fig. 1. Open in new tabDownload slide Biosynthesis of Chl (A), Pchlide (B), and ALA (C) in control (25°C, □), chill-stressed (7°C, ⋄), and heat-stressed (42°C, ○) cucumber seedlings. Experimental details are as in Methods. Each data point is the mean of three replicates, error bars represent sd, and missing error bars indicate that they are smaller than the symbols. Fig. 1. Open in new tabDownload slide Biosynthesis of Chl (A), Pchlide (B), and ALA (C) in control (25°C, □), chill-stressed (7°C, ⋄), and heat-stressed (42°C, ○) cucumber seedlings. Experimental details are as in Methods. Each data point is the mean of three replicates, error bars represent sd, and missing error bars indicate that they are smaller than the symbols. Chl Biosynthetic Intermediates To probe if inhibition of Chl biosynthesis in chill- and heat-stressed seedlings was due to reduced synthesis of tetrapyrrolic intermediates, etiolated cucumber seedlings grown at 25°C were kept for 48 h in chill- or heat-stressed conditions in the dark, and accumulation of Proto IX, MPE, and Pchlide was measured at low- or high-temperature regimes. Only the net synthesis of Pchlide could be monitored, since the amounts of MPE and Proto IX accumulated in cucumber cotyledons were negligible. In the control, the maximum amount of Pchlide was synthesized during the first 3 h (Fig. 1B). Net synthesis of Pchlide was near the maximum after 12 h of dark incubation. On the contrary, chill-stressed seedlings did not synthesize any Pchlide during the first 3 h and accumulated only small amounts of Pchlide after 12 h of dark incubation. Compared with control seedlings, Pchlide synthesis was inhibited by 90 and 70% in chill- and heat-stressed seedlings, respectively. ALA Content To study the mechanism of inhibition of Chl and Pchlide biosynthesis at different temperatures, the biosynthesis of ALA, the precursor of Chl, was monitored in chill- and heat-stressed conditions. ALA synthesis in the presence of LA was almost linear up to 6 h of illumination in control and heat-stressed seedlings. For the first 3 h ALA synthesis was completely inhibited in chill-stressed cucumber seedlings. As compared with the controls, the net synthesis of ALA was severely reduced by 78 and 70% in chill- and heat-stressed seedlings, respectively (Fig. 1C). To understand in detail the mechanism of impairment of Chl synthesis and Pchlide biosynthesis in cucumber in chill- and heat-stressed conditions, the effects of low (7°C) and high (42°C) temperatures on different enzymes involved in Chl biosynthesis were studied. ALAD The ALAD that synthesizes PBG from two molecules of ALA was determined in etiolated cucumber seedlings kept at 7, 25, and 42°C for 48 h in the dark. As compared with the control, the enzyme activity decreased by 24 and 45% in chill- and heat-stressed seedlings, respectively (Fig. 2A). Fig. 2. Open in new tabDownload slide ALAD (A) and PBGD (B) activities measured in control (25°C) and chill (7°C)- and heat (42°C)-stressed cucumber seedlings. Experimental details are as in Methods. Each data point is the mean of three replicates and error bars represent sd. Fig. 2. Open in new tabDownload slide ALAD (A) and PBGD (B) activities measured in control (25°C) and chill (7°C)- and heat (42°C)-stressed cucumber seedlings. Experimental details are as in Methods. Each data point is the mean of three replicates and error bars represent sd. PBGD The next step in the Chl biosynthetic pathway is the conversion of PBG to urogen, which is catalyzed by PBGD. The enzyme activity was estimated by measuring the amount of porphyrin synthesis from PBG in cucumber seedlings kept at 7, 25, and 42°C for the last 48 h in the dark. Compared with the control, the enzyme activity was reduced by 13 and 28% in chill- and heat-stressed seedlings, respectively, suggesting that the PBGD activity was affected more in heat than in chill stress (Fig. 2B). Proto-IX Synthesis from Urogen, Coprogen, and Protogen The subsequent steps in the Proto IX biosynthetic pathway are the conversion of urogen III to coprogen III mediated by UDC, the synthesis of protogen IX from coprogen III by coprogen oxidase, and finally the conversion of protogen to Proto IX by Protox. As compared with the controls, in chill-stressed seedlings Proto IX synthesis from urogen was severely reduced by 65%, whereas in heat-stressed seedlings the activity increased by 155% (Fig. 3A). Proto-IX synthesis from coprogen III was also reduced by 34% in chill-stressed seedlings. However, in heat-stressed seedlings, Proto IX synthesis from coprogen III was not affected (Fig. 3B). Protox activity was reduced by 60% in chill-stressed seedlings, whereas there was no significant change in enzyme activity in heat-stressed seedlings (Fig. 3C). Fig. 3. Open in new tabDownload slide UDC (A), coprogen oxidase (B), and Protox (C) activities measured in control (25°C) and chill (7°C)- and heat (42°C)-stressed cucumber seedlings. Experimental details are as inMethods. Each data point is the mean of three replicates and error bars represent sd.   Fig. 3. Open in new tabDownload slide UDC (A), coprogen oxidase (B), and Protox (C) activities measured in control (25°C) and chill (7°C)- and heat (42°C)-stressed cucumber seedlings. Experimental details are as inMethods. Each data point is the mean of three replicates and error bars represent sd.   Mg-Chelatase Mg-chelatase is the first enzyme in the Mg branch of the Chl biosynthetic pathway that inserts Mg into Proto IX to form Mg-Proto IX (Gorchein, 1972; Pardo et al., 1980; Jensen et al., 1996). In this experiment Proto IX was used as a substrate and Mg-chelatase activity was measured as the amount of Mg-Proto IX and its monoester synthesized in the plastids isolated from cucumber seedlings. As compared with the controls, the enzyme activity decreased by 60% in chill-stressed and by 65% in heat-stressed seedlings (Fig.4A). Fig. 4. Open in new tabDownload slide Mg-chelatase (A), MPE cyclase (B), and POR (C) activities measured in control (25°C) and chill (7°C)- and heat (42°C)-stressed cucumber seedlings. Experimental details are as inMethods. Each data point is the mean of three replicates and error bars represent sd. Fig. 4. Open in new tabDownload slide Mg-chelatase (A), MPE cyclase (B), and POR (C) activities measured in control (25°C) and chill (7°C)- and heat (42°C)-stressed cucumber seedlings. Experimental details are as inMethods. Each data point is the mean of three replicates and error bars represent sd. S-Adenosyl Met Methyl Transferase Plus MPE-Cyclase To analyze the effect of temperature stress on Mg-Proto:S-adenosyl Met methyl transferase and MPE cyclase (Hinchigeri et al., 1981; Bollivar and Beale, 1996), plastids isolated from cucumber seedlings grown at different temperatures were incubated with Mg-Proto IX and S-adenosyl l-Met and synthesis of Pchlide was monitored. Pchlide synthesis was reduced by 60 and 36% in chill- and heat-stressed seedlings, respectively (Fig. 4B). POR POR mediates the light-catalyzed reduction of Pchlide to Chlide, which is a key regulatory step in the biosynthesis of Chl in oxygenic photosynthetic organisms (Griffiths, 1978; von Wettstein et al., 1995). Four-day-old-etiolated cucumber seedlings grown at 25°C were transferred to 7°C or 42°C, and were kept in the dark for 48 h. They were illuminated for 10 min in light to phototransform accumulated Pchlide to Chlide. The phototransformation of Pchlide to Chlide was 75% in control and chill-stressed seedlings and 95% in heat-stressed seedlings (Fig. 4C). Effect of Temperature Stress on Certain Enzymes Involved in the Chl Biosynthetic Pathway in Wheat To compare the effects of chill and heat stress on Chl biosynthesis in dicot and monocot seedlings, wheat seedlings were used and the activities of PBGD and POR were monitored. PBG Deaminase Activity in Wheat Six-day-old-etiolated wheat seedlings grown at 25°C were transferred to 7, 25, and 42°C for 24 h in the dark. PBGD activity in chill- and heat-stressed seedlings declined by 12 and 41%, respectively (data not shown). This demonstrated that both wheat and cucumber seedlings showed a similar pattern of reduction of PBGD activity in response to temperature stress. Pchlide Synthesis in Wheat Seedlings Grown at Different Temperatures The net synthesis of Pchlide in control wheat seedlings increased after 4 to 12 h of dark incubation. However, there was a lag in Pchlide synthesis for 4 h in chill- and heat-stressed seedlings after which the rate of Pchlide synthesis increased. As compared with control seedlings, in chill-stressed seedlings Pchlide accumulation was reduced by 80%, whereas in heat-stressed seedlings reduction was 65% (Fig. 5). Chill-stressed wheat seedlings had a higher accumulation of Pchlide after 12 h of dark incubation than that of cucumber. This suggested that Chl biosynthesis in wheat as compared with cucumber was relatively more resistant to chill stress. Fig. 5. Open in new tabDownload slide Pchlide synthesis and POR activity (inset) in control (25°C, □) and chill (7°C, ⋄)- and heat (42°C, ○)-stressed wheat seedlings. Experimental details are as inMethods. Each data point is the mean of three replicates, error bars represent sd, and missing error bars indicate that they are smaller than the symbols. Fig. 5. Open in new tabDownload slide Pchlide synthesis and POR activity (inset) in control (25°C, □) and chill (7°C, ⋄)- and heat (42°C, ○)-stressed wheat seedlings. Experimental details are as inMethods. Each data point is the mean of three replicates, error bars represent sd, and missing error bars indicate that they are smaller than the symbols. POR in Wheat The percent phototransformation of Pchlide to Chlide was taken as an index of POR activity in wheat seedlings grown at different temperatures in the dark. Six-day-old etiolated wheat seedlings grown at 25°C were transferred to 7°C, 25°C, or 42°C and were kept in the dark for 24 h. They were illuminated for 10 min in light to phototransform accumulated Pchlide to Chlide. The phototransformation of Pchlide to Chlide was 70% in chill-stressed and control seedlings and 90% in heat-stressed wheat seedlings (Fig. 5, inset). POR activity had a similar response in heat-stressed cucumber seedlings. DISCUSSION Illumination of cucumber seedlings in chill- and heat-stress conditions resulted in inhibition of Chl biosynthesis by 90 and 60%, respectively (Fig. 1A), demonstrating that inhibition of Chl biosynthesis is higher in chill-stress than in heat-stress conditions. Reduced synthesis of ALA, the committed precursor of Chl, in chill- and heat-stressed seedlings (Fig. 1C) demonstrates that inhibition of Chl biosynthesis is partly due to impairment of ALA biosynthesis. ALA biosynthesis in cucumber was inhibited to a similar extent both in chill (78%)- and heat (70%)-stress conditions (Fig. 1C). However, as stated above, the inhibition of Chl biosynthesis under identical conditions in chill- and heat-stressed seedlings was 90 and 60%, respectively. To account for the discrepancy between the inhibition of ALA and Chl biosynthesis in chill- and heat-stress conditions, Pchlide synthesis was monitored. Compared with control seedlings, Pchlide synthesis was inhibited by 90 and 70% in chill- and heat-stressed seedlings, respectively (Fig. 1B), demonstrating that inhibition of Chl biosynthesis was mostly due to impairment of ALA and Pchlide biosynthesis. Enzymes involved in Proto IX and Pchlide biosynthesis were differentially affected by chill and heat stress. The ALAD was reduced by 24 and 45% in chill- and heat-stressed seedlings (Fig. 2A). Higher inhibition of ALAD activity in the heat-stress condition may be due to impairment of the enzyme. Similar to ALAD, PBGD activity was also reduced more in heat (28%)- than in chill (13%)-stress conditions (Fig. 2B). Heat stress did not impair Protox, although chill stress significantly inhibited its activity (Fig. 3). Heat stress also did not inhibit coprogen oxidase or UDC, rather, the latter was highly stimulated in high-temperature-grown seedlings. The differential inhibition pattern of Proto IX synthesis from urogen, coprogen, and protogen in chill stress suggested that both UDC and coprogen oxidase were inhibited in low-temperature-grown seedlings. These data demonstrated that Proto IX biosynthesis is relatively insensitive to heat stress as compared with chill stress. Mg-chelatase and Mg-Proto:S-adenosyl Met methyl transferase plus MPE cyclase were inhibited by both chill and heat stress (Fig. 4, A and B). POR activity increased in heat-stress conditions by 46% over that of the control (Fig. 4C). Increase in the activity of POR in heat-stressed conditions may be due to increased synthesis of the enzyme and/or conversion of nonphototransformable Pchlide to the transformable form. POR activity is not affected in chill-stressed seedlings. This is contrary to the previous report (van Huystee and Hodgins, 1989) of inhibition of phototransformation of Pchlide to Chlide in chill-stressed conditions. This discrepancy may be due to a different methodology followed by the authors. They used exogenous substrate ALA to accumulate Pchlide and then phototransform Pchlide to Chlide by exposing plants to light. However, it is known that certain amounts of Pchlide synthesized from exogenous substrate ALA are nonphototransformable (Chakraborty and Tripathy, 1992). As shown in Figure 1, the net synthesis of Pchlide was inhibited by 90% in chill stress and 70% in heat stress. Severe inhibition of Pchlide biosynthesis in chill-stressed seedlings is caused by a decline in Proto IX, Mg-Proto synthesis, and reduced conversion of Mg-Proto to Pchlide due to impairment of all of the enzymes involved in Proto IX synthesis and inhibition of Mg-chelatase and MPE cyclase activities. However, in heat-stressed seedlings, although ALAD and PBGD are partially inhibited, porphyrinogen-oxidizing enzymes were either stimulated (UDC) or unaffected (coprogen oxidase and Protox). Therefore, synthesis of Proto IX in heat-stressed seedlings is likely to be less affected than that in chill-stressed seedlings. Although heat-stressed seedlings had reduced activity of Mg-chelatase andS-adenosyl Met methyl transferase plus MPE cyclase, they accumulated higher amounts of Pchlide than chill-stressed seedlings, probably due to relatively higher synthesis of the substrate Proto IX. Taking into account the degree of inhibition of Pchlide biosynthesis in chill (90%) and heat (70%) stress, and stimulation of POR activity resulting in increased phototransformation of Pchlide to Chlide in heat stress, the calculated percent inhibition of Chl biosynthesis in chill (92%)- and heat (60%)-stressed seedlings matches well with actual inhibition of the amount of Chl measured in chill (90%)- and heat (60%)-stress conditions. To compare the effects of growth temperature on Chl biosynthesis in dicot and monocot seedlings, the activities of a few enzymes of the Chl biosynthetic pathway were monitored in wheat seedlings and compared with that of cucumber seedlings under similar temperature-stress conditions. In etiolated wheat seedlings, the PBGD activity was inhibited by 13% in chill-stress conditions and 42% in heat-stress conditions. This is similar to the pattern of inhibition of the PBGD reaction, observed in chill- and heat-stressed cucumber seedlings. Pchlide synthesis was reduced by 80% in chill-stressed seedlings, whereas in heat-stressed seedlings the reduction was only 60% (Fig. 5). A similar pattern of POR activity was observed in chill- and heat-stressed wheat seedlings as that in cucumber seedlings. These results suggest that temperature stress has a broadly similar effect on Chl biosynthetic enzymes in cucumber (dicot) and wheat (monocot). Inhibition or stimulation of different enzymes involved in porphyrin biosynthesis in chill- or heat-stressed seedlings may be due to impairment or posttranslational modifications of the enzymes in vivo. Temperature stress might have caused reduced or increased synthesis of the enzymes or corresponding mRNA. Further investigations are needed to ascertain the mechanism of inhibition or stimulation of enzymes in chill- and heat-stressed seedlings. Abbreviations: ALA 5-aminolevulinic acid ALAD 5-aminolevulinic acid dehydratase Chl chlorophyll Chlide chlorophyllide coprogen coproporphyrinogen HEAR hexane extracted acetone residue solvent mixture LA levulinic acid MPE Mg-protoporphyrin monoester PBG porphobilinogen PBGD porphobilinogen deaminase Pchlide protochlorophyllide protogen protoporphyrinogen POR Pchlide oxidoreductase Proto IX protoporphyrin IX Protox protogen oxidase UDC urogen decarboxylase urogen uroporphyrinogen LITERATURE CITED 1 Bollivar DW Beale SI The chlorophyll biosynthetic enzyme Mg-protoporphyrin IX monomethyl ester (oxidative) cyclase. Plant Physiol 112 1996 105 114 Google Scholar Crossref Search ADS PubMed WorldCat 2 Chakraborty N Tripathy BC Involvement of singlet oxygen in 5-aminolevulinic acid-induced photodynamic damage of cucumber (Cucumis sativus L.) chloroplasts. Plant Physiol 98 1992 7 11 Google Scholar Crossref Search ADS PubMed WorldCat 3 Feierabend J Capacity for chlorophyll synthesis in heat-bleached 70S ribosome-deficient rye leaves. Planta 135 1977 83 88 Google Scholar Crossref Search ADS PubMed WorldCat 4 Gorchein A Magnesium protoporphyrin chelatase activity in Rhodopseudomonas spheroides: studies with whole cells. Biochem J 127 1972 97 106 Google Scholar Crossref Search ADS PubMed WorldCat 5 Griffiths WT Reconstitution of chlorophyllide formation by isolated etioplast membranes. Biochem J 174 1978 681 692 Google Scholar Crossref Search ADS PubMed WorldCat 6 Harel E Klein S Light dependent formation of 5-aminolevulinic acid in etiolated leaves of higher plants. Biochem Biophys Res Commun 49 1972 364 370 Google Scholar Crossref Search ADS PubMed WorldCat 7 Hess WR Blank-Huber M Fieder B Borner T Rudiger W Chlorophyll synthetase and chloroplast tRNAglu are present in heat-bleached, ribosome-deficient plastids. J Plant Physiol 139 1992 427 430 Google Scholar Crossref Search ADS WorldCat 8 Hinchigeri SB Chan JCS Richards WR Purification of S-adenosyl l-methionine magnesium protoporphyrin methyltransferase by affinity chromatography. Photosynthetica 15 1981 351 359 Google Scholar OpenURL Placeholder Text WorldCat 9 Hodgins R van Huystee RB Porphyrin metabolism in chill stressed maize (Zea mays L.). J Plant Physiol 125 1986a 325 336 Google Scholar Crossref Search ADS WorldCat 10 Hodgins RR van Huystee RB Delta-aminolevulinic acid metabolism in chill-stressed maize (Zea mays L.). J Plant Physiol 126 1986b 257 268 Google Scholar Crossref Search ADS WorldCat 11 Hukmani P Tripathy BC Spectrofluorometric estimation of intermediates of chlorophyll biosynthesis: protoporphyrin IX, Mg-protoporphyrin, and protochlorophyllide. Anal Biochem 206 1992 125 130 Google Scholar Crossref Search ADS PubMed WorldCat 12 Hukmani P Tripathy BC Chlorophyll biosynthetic reactions during senescence of excised barley (Hordeum vulgare L. cv IB 65) leaves. Plant Physiol 105 1994 1295 1300 Google Scholar Crossref Search ADS PubMed WorldCat 13 Jacobs NJ Jacobs JM Assay for enzymatic protoporphyrinogen oxidation, a late step in heme synthesis. Enzyme 28 1982 862 866 Google Scholar Crossref Search ADS WorldCat 14 Jensen PE Willows RD Petersen BL Vothknecht UC Stummann BM Kannangara GC von Wettstein D Henningsen KW Structural genes for Mg-chelatase subunits in barley: xantha-F, -G and -H. Mol Genet 250 1996 383 394 Google Scholar OpenURL Placeholder Text WorldCat 15 Leeper FJ Intermediate steps in biosynthesis of chlorophylls. Scheer H Chlorophylls. 1991 407 431 CRC Press Boca Raton, FL 16 Lowry OH Rosebrough NJ Farr AL Randall RJ Protein measurement with the folin phenol reagent. J Biol Chem 193 1951 265 275 Google Scholar Crossref Search ADS PubMed WorldCat 17 Pardo AD Chereskin BM Castelfranco PA Franceschi VR Wezelman BE ATP requirement for Mg-chelatase in developing chloroplasts. Plant Physiol 65 1980 956 960 Google Scholar Crossref Search ADS PubMed WorldCat 18 Porra RJ Recent progress in porphyrin and chlorophyll biosynthesis. Photochem Photobiol 65 1997 492 516 Google Scholar Crossref Search ADS WorldCat 19 Porra RJ Thompson WA Kriedemann PE Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim Biophys Acta 975 1989 384 394 Google Scholar Crossref Search ADS WorldCat 20 Poulson R Polglasse WJ Aerobic and anaerobic coproporphyrinogen oxidase activities in extracts from Saccharomyces cerevisiae. J Biol Chem 249 1974 6367 6371 Google Scholar Crossref Search ADS PubMed WorldCat 21 Rebeiz CA Matheis JR Smith BB Rebeiz CC Dayton DF Chloroplast biogenesis: biosynthesis and accumulation of Mg-protoporphyrin IX monoester and longer wavelength metalloporphyrins by greening cotyledons. Arch Biochem Biophys 166 1975 446 465 Google Scholar Crossref Search ADS PubMed WorldCat 22 Richards WR Biosynthesis of chlorophyll chromophores of pigmented thylakoid proteins. Sundqvist C Ryberg M Pigment-Protein Complexes in Plastids: Synthesis and Assembly. 1992 91 178 Academic Press San Diego, CA 23 Shemin D 5-Aminolevulinic acid dehydratase from Rhodopseudomonas sphaeroides. Methods Enzymol 5 1962 883 884 Google Scholar Crossref Search ADS WorldCat 24 Thomas EJ Ortiz W Loss of chloroplast transcripts for proteins associated with PSII: an early event during heat-bleaching in Euglena gracilis. Plant Mol Biol 27 1995 317 325 Google Scholar Crossref Search ADS PubMed WorldCat 25 Tripathy BC Rebeiz CA Chloroplast biogenesis: demonstration of monovinyl and divinyl monocarboxylic routes of chlorophyll biosynthesis in higher plants. J Biol Chem 261 1986 13556 13564 Google Scholar Crossref Search ADS PubMed WorldCat 26 Tripathy BC, Rebeiz CA (1987) Non-equivalence of glutamic and 5-aminolevulinic acids as substrates for protochlorophyllide and chlorophyll biosynthesis in darkness. In J Biggins, ed, Progress in Photosynthesis Research, Vol 4. Nijhoff Publishers, Boston, MA, pp 439–443 27 Tripathy BC Rebeiz CA Chloroplast biogenesis 601: conversion of divinyl protochlorophyllide to monovinyl protochlorophyllide in green(ing) barley, a dark monovinyl/light divinyl plant species. Plant Physiol 87 1988 89 94 Google Scholar Crossref Search ADS PubMed WorldCat 28 van Hasselt PR Strikwerda JT Pigment degradation in discs of the thermophilic Cucumis sativus as affected by light, temperature, sugar application and inhibitors. Plant Physiol 37 1976 253 257 Google Scholar Crossref Search ADS WorldCat 29 Van Huystee RB Hodgins RR Chlorophyll synthesis from protochlorophyll (ide) in chill-stressed maize (Zea mays L.). J Exp Bot 40 1989 431 435 Google Scholar Crossref Search ADS WorldCat 30 von Wettstein D, Gough S, Kannangara CG (1995) Chlorophyll biosynthesis. Plant Cell 7: 1039–1057 Author notes 1 This work was supported by the Council of Scientific and Industrial Research (grant no. 38-922/97/EMR-II) and the Department of Science and Technology, Government of India (grant no. DST/SP/SO/A 49-95 to B.C.T.). * Corresponding author; e-mail [email protected]; fax 91–11–6165886. Copyright © 1998 American Society of Plant Physiologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Bauerle, Cynthia; Magembe, Catherine; Briskin, Donald P.

doi: 10.1104/pp.117.3.859pmid: 9742042

Abstract V-type proton-translocating ATPases (V-ATPases) (EC 3.6.1.3) are electrogenic proton pumps involved in acidification of endomembrane compartments in all eukaryotic cells. V-ATPases from various species consist of 8 to 12 polypeptide subunits arranged into an integral membrane proton pore sector (V0) and a peripherally associated catalytic sector (V1). Several V-ATPase subunits are functionally and structurally conserved among all species examined. In yeast, a 36-kD peripheral subunit encoded by the yeast (Saccharomyces cerevisiae) VMA6 gene (Vma6p) is required for stable assembly of the V0 sector as well as for V1 attachment. Vma6p has been characterized as a nonintegrally associated V0 subunit. A high degree of sequence similarity among Vma6p homologs from animal and fungal species suggests that this subunit has a conserved role in V-ATPase function. We have characterized a novel Vma6p homolog from red beet (Beta vulgaris) tonoplast membranes. A 44-kD polypeptide cofractionated with V-ATPase upon gel-filtration chromatography of detergent-solubilized tonoplast membranes and was specifically cross-reactive with anti-Vma6p polyclonal antibodies. The 44-kD polypeptide was dissociated from isolated tonoplast preparations by mild chaotropic agents and thus appeared to be nonintegrally associated with the membrane. The putative 44-kD homolog appears to be structurally similar to yeast Vma6p and occupies a similar position within the holoenzyme complex. V-ATPases are electrogenic proton pumps involved in acidification of endomembrane compartments in all eukaryotic cells (for review, seeFinbow and Harrison, 1997). V-ATPases appear to be responsible for acidification of vacuoles, lysosomes, Golgi cisternae, secretory vesicles, and clathrin-coated vesicles. In addition, V-ATPases are associated with the plasma membrane of specialized mammalian cell types. The yeast (Saccharomyces cerevisiae) V-ATPase maintains an acidic environment in the vacuolar lumen and generates a proton gradient that drives the transport of ions such as Ca2+ and basic amino acids across the vacuolar membrane (for review, see Nelson and Klionsky, 1996). In plant cells the V-ATPase operates in conjunction with a proton-translocating pyrophosphatase to maintain a proton gradient across the tonoplast that supports vacuolar uptake of K+, Ca2+, sugar, and other small metabolites by secondary transport systems (Taiz, 1992). V-ATPases from various species consist of 8 to 12 polypeptide subunits arranged in an integral membrane proton pore sector (V0) and a peripherally associated catalytic sector (V1) (Finbow and Harrison, 1997). Thus, V-ATPases display a bipartite structure similar to mitochondrial F1F0-ATPases. In yeast, combined biochemical and genetic approaches have identified 10 subunits that range from 14 to 100 kD and are required for holoenzyme assembly and ATPase activity (Table I). The V1 sector is comprised of 69-, 60-, 54-, 42-, 32-, 27-, and 14-kD polypeptides. The membrane V0sector consists of 100- and 17-kD integral membrane proteins and a tightly associated 36-kD peripheral polypeptide (Bauerle et al., 1993). Highly purified preparations of V-ATPase from red beet (Beta vulgaris) tonoplast contain at least 9 polypeptides, of which the 67-, 55-, 52-, 44-, and 32-kD subunits are reported to be peripherally associated (Parry et al., 1989). Table I. Subunit composition of V-ATPase from yeast and red beet Organism . V1Subunits . V0 Subunits . Ref. . kD Yeast 69 60 54 42 32 27 14 100 (36) 17 Graham et al. (1995) Red beet 67 55 52 (44) 42 32 29 100 16 Parry et al. (1989) Organism . V1Subunits . V0 Subunits . Ref. . kD Yeast 69 60 54 42 32 27 14 100 (36) 17 Graham et al. (1995) Red beet 67 55 52 (44) 42 32 29 100 16 Parry et al. (1989) V1 and V0 subunits of V-ATPase from yeast and red beet. Subunits are identified based on observed molecular mass. Numbers in parentheses indicate the 36-kD yeast and 44-kD beet subunits examined in this study. Open in new tab Table I. Subunit composition of V-ATPase from yeast and red beet Organism . V1Subunits . V0 Subunits . Ref. . kD Yeast 69 60 54 42 32 27 14 100 (36) 17 Graham et al. (1995) Red beet 67 55 52 (44) 42 32 29 100 16 Parry et al. (1989) Organism . V1Subunits . V0 Subunits . Ref. . kD Yeast 69 60 54 42 32 27 14 100 (36) 17 Graham et al. (1995) Red beet 67 55 52 (44) 42 32 29 100 16 Parry et al. (1989) V1 and V0 subunits of V-ATPase from yeast and red beet. Subunits are identified based on observed molecular mass. Numbers in parentheses indicate the 36-kD yeast and 44-kD beet subunits examined in this study. Open in new tab Many V-ATPase subunits are functionally and structurally conserved among all species examined (Finbow and Harrison, 1997). The V1 70-kD catalytic and 60-kD regulatory subunits share as much as 60 to 70% identity among diverse species. The 17-kD V0 proteolipid is among the most highly conserved proteins known, displaying greater than 65% sequence identity among all species examined. This high degree of sequence similarity for multiple subunits of the enzyme reflects the functional importance of this ubiquitous eukaryotic proton pump. In yeast, the 36-kD V0 subunit is encoded by theVMA6 gene (Bauerle et al., 1993). The VMA6 gene product, a 36-kD subunit, Vma6p, is required for V-ATPase activity as well as stable holoenzyme assembly. In vma6 mutants lacking the 36-kD subunit, the V0 sector does not stably assemble, and V1 particles are unable to associate with the membrane. Biochemical studies revealed that Vma6p is peripherally rather than integrally attached to the vacuolar membrane and thus represents a novel class of peripheral V0 subunits. A high degree of sequence similarity among Vma6p homologs from various species suggests that this subunit has a conserved role in V-ATPase function (Fig.1). Subunit homologs from fungal, insect, and mammalian species share 41 to 57% primary sequence identity with Vma6p. In particular, four highly conserved domains display more than 60% identity, corresponding to residue nos. 96 to 133, 172 to 193, 212 to 227, and 314 to 322 in the yeast sequence. Fig. 1. Open in new tabDownload slide Amino acid sequence alignment of Vma6p homologs. Dashes indicate spaces added to optimize alignment, periods indicate amino acid identity among all seven sequences examined, and asterisks indicate stop codons. Overall homologies with Vma6p are: S. cerevisiae (S cer), 100% (Bauerle et al., 1993); Neurospora crassa (N cras), 57% (Melnik and Bowman, 1996); Dictyostelium discoideum(D disc), 49% (Temesvari et al., 1994); Manduca sexta (M sex), 47% (Merzendorfer et al., 1997a); Bos taurus (B taur), 46% (Wang et al., 1988); Homo sapiens (H sap), 41% (van Hille et al., 1993); Mus musculus (M musc), 47% (unpublished, GenBank accession no.U21549).   Fig. 1. Open in new tabDownload slide Amino acid sequence alignment of Vma6p homologs. Dashes indicate spaces added to optimize alignment, periods indicate amino acid identity among all seven sequences examined, and asterisks indicate stop codons. Overall homologies with Vma6p are: S. cerevisiae (S cer), 100% (Bauerle et al., 1993); Neurospora crassa (N cras), 57% (Melnik and Bowman, 1996); Dictyostelium discoideum(D disc), 49% (Temesvari et al., 1994); Manduca sexta (M sex), 47% (Merzendorfer et al., 1997a); Bos taurus (B taur), 46% (Wang et al., 1988); Homo sapiens (H sap), 41% (van Hille et al., 1993); Mus musculus (M musc), 47% (unpublished, GenBank accession no.U21549).   Although gene homologs of yeast VMA6 have been isolated from various fungal and animal sources, no full-length gene homologs have been reported from any plant source. A search of the Arabidopsis gene-fragment database (using BLAST) identified sequences bearing strong homology to yeast VMA6. By aligning overlapping sequence fragments, we were able to assemble a partial sequence corresponding to a putative Arabidopsis VMA6 homolog (Fig.2). The deduced primary sequence aligned with Vma6p residue nos. 76 to 345 and displayed all four highly conserved domains (67, 86, 69, and 57% identity with the yeast Vma6p sequence, respectively). Fig. 2. Open in new tabDownload slide Amino acid sequence alignment of Vma6p and deduced partial composite sequence from Arabidopsis (A thal). Four highly conserved regions shared between yeast and Arabidopsis sequences are underlined. The sequence homology is 36% in the overlapping region (corresponding to Vma6p residue nos. 76–345).S cer, S. cerevisiae. Fig. 2. Open in new tabDownload slide Amino acid sequence alignment of Vma6p and deduced partial composite sequence from Arabidopsis (A thal). Four highly conserved regions shared between yeast and Arabidopsis sequences are underlined. The sequence homology is 36% in the overlapping region (corresponding to Vma6p residue nos. 76–345).S cer, S. cerevisiae. The existence of a putative VMA6 homolog in Arabidopsis hints that homologs of this essential yeast subunit may exist in other plant species. Thus, we sought to identify potential Vma6p subunit homologs in V-ATPase-enriched membrane preparations. Here we report the initial characterization of a novel Vma6p homolog from red beet tonoplast membranes. This 44-kD polypeptide was directly identified by immuno-cross-reactivity with antibodies raised against yeast Vma6p. Preliminary characterization suggests that the putative homolog is a peripheral subunit of the V-ATPase that is tightly associated with the membrane. MATERIALS AND METHODS Alkaline phosphatase-conjugated antibodies and Kaleidoscope protein molecular mass standards were purchased from Promega. Ready Gels for the MiniProtean II gel system were from Bio-Rad. Nitrocellulose membrane was from Schleicher & Schuell. The BCA reagent kit was from Pierce. All other reagents were from Sigma. Preparation of rabbit polyclonal antiserum against the yeast (Saccharomyces cerevisiae) 36-kD subunit has been described (Bauerle et al., 1993). Corresponding preimmune serum was collected from the same animal immediately prior to antigen exposure. Rabbit polyclonal antisera prepared against red beet (Beta vulgaris) tonoplast V-ATPase 67- and 57-kD subunits were a generous gift from Dr. Ron Poole (McGill University, Montreal, Quebec, Canada). Strains and Culture Conditions Isogenic yeast strains SEY6211a VMA6 and SEY6211avma6::LEU2 have been described (Bauerle et al., 1993). Yeast cultures were grown at 30°C with vigorous shaking in 1% yeast extract, 2% Bactopeptone, 2% dextrose buffered at pH 5.0 with 50 mm phosphate/succinate. Protein Sample Preparation, SDS-PAGE, and Immunoblot Analysis Whole yeast cell lysates were prepared in sample buffer (8m urea, 5% SDS, 1 mm EDTA, 50 mmTris-HCl, pH 6.8, and 5% β-mercaptoethanol) as described previously (Bauerle et al., 1993). Protein concentrations were determined prior to the addition of β-mercaptoethanol by the BCA assay, and 40 μg of protein was loaded per lane. Proteins were separated on 12, 15, or 10 to 20% gradient gels and electrotransferred to nitrocellulose membranes at a constant 12 V for 30 to 45 min at ambient temperature in a TransBlot semidry transfer cell (Bio-Rad). Western immunoblot analysis was performed as described before (Towbin et al., 1979). Blots were incubated with primary antibodies for 2 to 4 h in TBS containing 0.1% Tween 20 plus 2% nonfat dry milk at dilutions of 1:250, with constant rotation in a hybridization incubator (LabLine, Melrose Park, IL) at 37°C. Alkaline phosphatase-conjugated secondary antibodies were applied at a 1:5000 dilution. Immunoblot data were quantified by scanning densitometry followed by image analysis using a GS700 imaging densitometer and Molecular Analyst 2.1 software (Bio-Rad). Standard curves for molecular mass estimations were generated by regression analysis of prestained marker proteins using Molecular Analyst 2.1 software. Preparation of Tonoplast Membrane Vesicles Vacuolar membranes from red beet storage root were prepared as previously described (Poole et al., 1984), frozen in liquid N2, and stored at −80°C until use. ATPase and proton-pumping activity were assayed after thawing according to published methods (Poole et al., 1984). Membrane preparations typically displayed specific activities for ATP hydrolysis in the range of 20 μmol mg−1 h−1. Tonoplast membrane vesicles were collected by centrifugation at 100,000g, dissolved in sample buffer, and heated to 95°C for 5 min prior to SDS-PAGE. Protein loads were 20 to 40 μg per lane. Fractionation of Tonoplast Vesicles Tonoplast membranes equivalent to 400 μg of protein were thawed, diluted into 1 mL of transport buffer (250 mm sorbitol, 100 mm KCl, and 25 mm BTP-Mes, pH 7.0), and then collected by centrifugation for 30 min at 100,000g in a fixed-angle TLA 120.2 rotor (Beckman). Membrane pellets were suspended to 1 mg/mL protein in transport buffer and then incubated for 30 min on ice in the presence of 5 mm MgSO4, 0 to 200 mm KNO3, and ±5 mm Tris-ATP. An aliquot was removed for measuring proton-pumping activity, and the remaining mixture was centrifuged for 30 min to separate membrane and supernatant fractions. The membrane pellet was washed once in transport buffer, and then membranes were collected by centrifugation and resuspended in transport buffer. Wash volumes were pooled and membrane and supernatant protein was precipitated by adding TCA to 10% (v/v) and incubating for 45 min on ice. Protein was pelleted by centrifugation for 15 min at 20,000 rpm in a refrigerated microcentrifuge (Eppendorf). Protein pellets were dissolved in 50 μL of sample buffer and heated to 95°C for 5 min prior to SDS-PAGE. Partial Purification of Red Beet H+-ATPase Tonoplast membranes equivalent to 200 μg were thawed, diluted in 1 mL of transport buffer, and then collected by centrifugation as described above. Tonoplast vesicles were solubilized with Triton X-100 as previously described (Parry et al., 1989). Briefly, vesicles were resuspended in 0.70 mL of resuspension buffer (1.1 mglycerol, 5 mm Tris-Mes, pH 8.0, 1 mm EDTA, 0.5 mm BHT, and 5 mm DTT), then slowly diluted by dropwise addition of 0.75 mL of solubilization buffer (containing 8% Triton X-100 and 4 mm MgSO4), and stirred on ice. The resulting mixture was stirred gently for 20 min. The detergent-solubilized mixture was partially purified by gel filtration on Sephacryl S-400 as previously described (Parry et al., 1989). A 60- × 0.75-cm-diameter column packed with Sephacryl S-400 was preequilibrated with running buffer containing 10% glycerol, 0.3% Triton X-100, 0.05 mg/mL phospholipid (Type IV-S, Sigma), 5 mm DTT, 1 mm Tris-EDTA, 4 mmMgCl2, and 5 mm Tris-Mes, pH 8.0. The entire sample volume was loaded and the column was run at a rate of 40 mL/h at 4°C. The detergent mixture was not centrifuged to remove nonsolubilized membrane particles prior to loading. Typically, 40 1-mL fractions were collected and 50-μL aliquots were assayed for ATPase activity and protein concentration. Protein Determination The protein concentration of yeast extract was determined by BCA assay according to the supplier's instructions (Pierce). The protein concentration of tonoplast vesicle preparations and gel-filtration fractions was determined by the method of Bradford (1987). Determination of ATPase and Proton-Pumping Activity ATPase activity was determined by measuring the amount of Pi liberated from ATP at 37°C in a 20-min reaction using the Ames method as previously described (Parry et al., 1989). Gel-filtration column fractions were supplemented with sonicated 1.33-mg/mL type IV-S phospholipid to preserve V-ATPase activity. Proton-pumping activity in tonoplast vesicles was measured by the method of Giannini et al. (1995). Typically, vesicles equivalent to 10 to 20 μg of protein were suspended in transport buffer containing 250 mm sorbitol, 50 mm KCl, 5 mmMgSO4, and 5 μm acridine orange. The reaction was initiated by the addition of Tris-ATP to 5 mm final concentration, and proton pumping was monitored by observing the decrease in acridine orangeA490 with a UV/visible light spectrophotometer (model DU 640, Beckman) in “kinetics/time mode.” Rates were calculated from data collected at 10-s intervals during a 3-min reaction period. Sequence Analysis VMA6 homologous protein sequences were aligned using Align Plus 2.0 from Scientific and Educational Software (State Line, PA). Alignment parameters were determined according to the work ofMyers and Miller (1988). Arabidopsis sequence fragments homologous to yeast VMA6 were identified by a BLAST search of GenBank (Gish and States, 1993). The following Arabidopsis fragments were used to generate a derived partial amino acid sequence: T13399, Z26026, Z24482, Z30468, T20646, H36140, H36163, H37538, T13974, R30209, T44170, N97286, AA042689. The derived partial sequence reported in Figure 2 was confirmed by identifying at least two overlapping fragments along the entire length of the sequence. RESULTS A 44-kD Polypeptide from Red Beet Exhibits Immuno-Cross-Reactivity with Yeast Vma6p and Cofractionates with Tonoplast V-ATPase Immunoblot analysis with antiserum generated against the yeast 36-kD V0 subunit (α-Vma6p) consistently revealed a single band in lanes containing red beet tonoplast protein (Fig. 3, top). Immunoblot analysis using the corresponding preimmune serum did not recognize either the yeast 36-kD subunit or the cross-reactive protein in tonoplast vesicle lanes (Fig. 3, bottom). Neither α-Vma6p nor preimmune sera recognized a cross-reactive band in protein extracts from yeast vma6mutant cells lacking the 36-kD subunit. Thus, the reactivity observed in both yeast and red beet samples appears to be specifically due to α-Vma6p polyclonal antibodies present in the immune serum. The molecular mass of the cross-reacting beet protein was estimated to be 44 kD by comparison with prestained molecular mass protein standards in an adjacent lane. Fig. 3. Open in new tabDownload slide Immuno-cross-reactivity between yeast Vma6p and beet 44-kD polypeptide. Proteins were separated on a 12% gel. Top, Immunoblot with polyclonal antiserum prepared against yeast Vma6p (αVma6p). Lane 1, Forty micrograms of whole cell protein from wild-type yeast VMA6; lane 2, 40 μg of protein from yeast vma6 mutant; and lane 3, 20 μg of red beet tonoplast protein. Relative positions of molecular mass standards are indicated. Bottom, Corresponding immunoblot with related preimmune serum. Fig. 3. Open in new tabDownload slide Immuno-cross-reactivity between yeast Vma6p and beet 44-kD polypeptide. Proteins were separated on a 12% gel. Top, Immunoblot with polyclonal antiserum prepared against yeast Vma6p (αVma6p). Lane 1, Forty micrograms of whole cell protein from wild-type yeast VMA6; lane 2, 40 μg of protein from yeast vma6 mutant; and lane 3, 20 μg of red beet tonoplast protein. Relative positions of molecular mass standards are indicated. Bottom, Corresponding immunoblot with related preimmune serum. To address whether the cross-reactive protein is specifically associated with V-ATPase in tonoplast vesicles, we partially purified the enzyme and monitored cofractionation of the cross-reactive protein with peak V-ATPase fractions. Tonoplast vesicles were detergent solubilized and proteins were separated by gel-filtration chromatography. The mixture was not centrifuged prior to loading on the column, a step normally taken to remove any residual unsolubilized membranes. This allowed us to observe the distribution of the 44-kD polypeptide between fully and partially solubilized fractions. Under these conditions, solubilized V-ATPase eluted as a single broad peak separated from bulk tonoplast protein, as evidenced by protein and ATPase activity profiles (Fig. 4). ATPase specific activity (corresponding to fractions 19–21 in Fig. 4) was typically enriched greater than 10-fold in peak column fractions compared with tonoplast vesicles (TableII). A sharp early peak (fractions 13–16 in Fig. 4) with relatively lower ATPase specific activity corresponded to incompletely solubilized tonoplast vesicles eluting with the void volume. Fig. 4. Open in new tabDownload slide Gel-filtration purification of V-ATPase from detergent-solubilized red beet tonoplasts. Tonoplast membranes were detergent solubilized and proteins were separated by gel filtration as described in Methods. Equivalent aliquots of even-numbered fractions were assayed for ATPase activity (micromoles per hour of PO43− liberated) and total protein concentration (milligrams) as described in Methods. ▪, Relative protein concentration; □, relative ATPase activity. Peak ATPase specific activity (fraction no. 20) was approximately 140 μmol mg−1 h−1, representing a 7-fold enrichment. Fig. 4. Open in new tabDownload slide Gel-filtration purification of V-ATPase from detergent-solubilized red beet tonoplasts. Tonoplast membranes were detergent solubilized and proteins were separated by gel filtration as described in Methods. Equivalent aliquots of even-numbered fractions were assayed for ATPase activity (micromoles per hour of PO43− liberated) and total protein concentration (milligrams) as described in Methods. ▪, Relative protein concentration; □, relative ATPase activity. Peak ATPase specific activity (fraction no. 20) was approximately 140 μmol mg−1 h−1, representing a 7-fold enrichment. Table II. Partial purification of V-ATPase from red beet tonoplast membranes Preparation . Specific Activity . Enrichment . μmol mg−1h−1 -fold increase Tonoplast vesicles 21.7  ± 4.0 1.0 Peak column fraction 271  ± 40 12.5  ± 0.8 Preparation . Specific Activity . Enrichment . μmol mg−1h−1 -fold increase Tonoplast vesicles 21.7  ± 4.0 1.0 Peak column fraction 271  ± 40 12.5  ± 0.8 V-ATPase was partially purified from detergent-solubilized tonoplast vesicles by gel-filtration chromatography as described in Methods. Equivalent aliquots of column fractions were assayed for total protein and ATPase activity, and the fraction containing the highest ATPase specific activity was identified. The results below represent the average of three separate purifications. Open in new tab Table II. Partial purification of V-ATPase from red beet tonoplast membranes Preparation . Specific Activity . Enrichment . μmol mg−1h−1 -fold increase Tonoplast vesicles 21.7  ± 4.0 1.0 Peak column fraction 271  ± 40 12.5  ± 0.8 Preparation . Specific Activity . Enrichment . μmol mg−1h−1 -fold increase Tonoplast vesicles 21.7  ± 4.0 1.0 Peak column fraction 271  ± 40 12.5  ± 0.8 V-ATPase was partially purified from detergent-solubilized tonoplast vesicles by gel-filtration chromatography as described in Methods. Equivalent aliquots of column fractions were assayed for total protein and ATPase activity, and the fraction containing the highest ATPase specific activity was identified. The results below represent the average of three separate purifications. Open in new tab Column fractions were probed with polyclonal antiserum directed against the 67-kD subunit of the red beet V-ATPase (α-67 kD) to determine the distribution of this peripheral V1 subunit. Immunoblot results confirmed that the distribution of the 67-kD V1 subunit closely correlated with V-ATPase activity (Fig. 5, top). The 67-kD subunit distributed between both the solubilized and unsolubilized V-ATPase peaks. Very little of this V1 subunit was observed in fractions containing the bulk of soluble tonoplast protein released by detergent treatment (fraction no. 24), indicating that this subunit remained primarily associated with the enzyme complex during column purification. Fig. 5. Open in new tabDownload slide Distribution of 67-kD subunit and 44-kD polypeptide in gel-filtration fractions of detergent-solubilized beet tonoplasts. Column fractions from the experiment described in Figure 4were analyzed. Fraction numbers are indicated at the top. Arrows mark peak ATPase and protein fractions. The asterisk indicates the fraction containing the peak ATPase specific activity. Top, Equivalent volumes of each sample were separated on a 10 to 20% gradient gel and then immunoblotted with anti-67-kD antiserum. A doublet band pattern was typically observed with α67-kD antiserum when samples were separated on gradient gels. Bottom, Same samples probed with αVma6p antiserum. Fig. 5. Open in new tabDownload slide Distribution of 67-kD subunit and 44-kD polypeptide in gel-filtration fractions of detergent-solubilized beet tonoplasts. Column fractions from the experiment described in Figure 4were analyzed. Fraction numbers are indicated at the top. Arrows mark peak ATPase and protein fractions. The asterisk indicates the fraction containing the peak ATPase specific activity. Top, Equivalent volumes of each sample were separated on a 10 to 20% gradient gel and then immunoblotted with anti-67-kD antiserum. A doublet band pattern was typically observed with α67-kD antiserum when samples were separated on gradient gels. Bottom, Same samples probed with αVma6p antiserum. Distribution of the 44-kD cross-reactive protein was similar to that of the 67-kD V1 peripheral subunit and closely correlated with the observed ATPase activity peaks (Fig. 5, bottom). We were unable to detect any of the 44-kD protein in peak fractions of soluble tonoplast protein. The 44-kD protein pattern correlated closely with both the V-ATPase activity profile and the 67-kD peripheral V1 subunit pattern. This suggests that the 44-kD cross-reactive protein was tightly associated with the partially purified V-ATPase fraction from detergent-solubilized tonoplast membranes. By comparing the RF against a standard curve generated by regression analysis of prestained marker proteins, we calculated the apparent molecular mass of the cross-reacting protein to be 44.9 kD. This is within close range of 44- and 42-kD accessory subunits of beet V-ATPase previously described (Parry et al., 1989). To determine whether the cross-reactive protein comigrated with one of these previously reported accessory subunits, α-Vma6p immunoblots were compared with the protein band pattern of V-ATPase. Partially purified V-ATPase protein fractions were separated by electrophoresis and visualized directly by Coomassie staining or were transferred to nitrocellulose and then stained with amido black to reveal the subunit band pattern. Identical samples were transferred to nitrocellulose and then immunoblotted with α-Vma6p antibodies. The cross-reactive band migrated closely with the prominent 44-kD subunit observed in partially purified V-ATPase fractions (Fig. 6). Thus, the previously described 44-kD accessory subunit of beet V-ATPase appeared to be selectively immuno-cross-reactive with yeast Vma6p antibodies. Fig. 6. Open in new tabDownload slide Comigration of cross-reactive band with 44-kD accessory subunit of V-ATPase. Partially purified V-ATPase proteins were separated on a 15% polyacrylamide gel, and then duplicate lanes either were visualized by Coomassie staining (A) or were transferred to nitrocellulose and immunoblotted with α-Vma6p antiserum (B). Coomassie-stained and immunoblotted band patterns were compared by aligning band patterns of prestained marker proteins loaded in adjacent lanes. Stained gel and developed immunoblots were digitally imaged and analyzed using a scanning imaging densitometer. Fig. 6. Open in new tabDownload slide Comigration of cross-reactive band with 44-kD accessory subunit of V-ATPase. Partially purified V-ATPase proteins were separated on a 15% polyacrylamide gel, and then duplicate lanes either were visualized by Coomassie staining (A) or were transferred to nitrocellulose and immunoblotted with α-Vma6p antiserum (B). Coomassie-stained and immunoblotted band patterns were compared by aligning band patterns of prestained marker proteins loaded in adjacent lanes. Stained gel and developed immunoblots were digitally imaged and analyzed using a scanning imaging densitometer. The 44-kD Polypeptide Is Dissociated from Tonoplast Membranes by Urea Treatment Treating yeast vacuolar membranes with urea quantitatively strips Vma6p from the membrane, unlike other V0 subunits that are integrally associated with the membrane (Bauerle et al., 1993). To address whether the cross-reactive 44-kD polypeptide behaved in a similar fashion, we incubated isolated beet tonoplast vesicles in transport buffer ± 8 m urea and examined the distribution of the 44-kD polypeptide in supernatant and membrane pellet fractions (Fig. 7). Similar to yeast Vma6p, the 44-kD polypeptide was quantitatively removed from membranes treated with 8 m urea. Peripheral V1 57- and 67-kD subunits were also completely dissociated from the membrane under these conditions (C. Magembe, unpublished observations). Thus, the 44-kD polypeptide behaved like a peripherally attached subunit in membrane fractionation experiments with a strong chaotrope. Fig. 7. Open in new tabDownload slide Dissociation of 44-kD polypeptide from tonoplast membranes by urea treatment. Tonoplast membranes equivalent to 50 μg of protein were incubated in buffer alone (top) or buffer containing 8m urea (bottom) for 30 min on ice. Membranes were collected by centrifugation and protein samples from both membrane pellet and supernatant fractions were prepared as described in Methods. Proteins were separated on 12% gels and immunoblotted with α-Vma6p antiserum. Developed immunoblots were digitally imaged and analyzed using a scanning imaging densitometer. S, Supernatant; P, membrane pellet. Fig. 7. Open in new tabDownload slide Dissociation of 44-kD polypeptide from tonoplast membranes by urea treatment. Tonoplast membranes equivalent to 50 μg of protein were incubated in buffer alone (top) or buffer containing 8m urea (bottom) for 30 min on ice. Membranes were collected by centrifugation and protein samples from both membrane pellet and supernatant fractions were prepared as described in Methods. Proteins were separated on 12% gels and immunoblotted with α-Vma6p antiserum. Developed immunoblots were digitally imaged and analyzed using a scanning imaging densitometer. S, Supernatant; P, membrane pellet. KNO3 Treatment Inactivates V-ATPase and Causes Partial Dissociation of 44- and 67-kD Polypeptides Mild chaotropic agents such as KNO3 have been shown to disrupt V-ATPase activity by causing specific dissociation of V1 peripheral subunits from the V0 sector (Ward et al., 1992). It is interesting that the dissociation effect is largely dependent on the presence of MgATP and thus appears to be specifically correlated with the ATP-dependent active state of the holoenzyme. The use of such chaotropes can provide useful information about the relationship between V-ATPase activity and subunit assembly. Figure 8 illustrates the effect of KNO3 on proton pumping in tonoplast vesicles. The ATP-dependent proton gradient was rapidly dissipated by addition of gramicidin, confirming that tonoplast vesicles were tightly sealed under incubation conditions. Proton-pumping activity was vanadate insensitive (less than 15% inhibition in the presence of 200 μm vanadate), indicating that tonoplast vesicles were relatively free of contaminating plasma membrane ATPase activity (not shown). Proton-pumping activity was strongly inhibited by the presence of 200 mm KNO3 in the transport assay. Proton-pumping activity was similarly prevented by preincubating tonoplast vesicles with 200 mm KNO3plus MgATP prior to performing the assay. Following dilution of preincubated vesicles into the assay medium, the resulting KNO3 concentration during the assay was approximately 4 mm, well below the observedKi of 8 mm (refer to Fig. 10, top). Thus, inhibition in this case was due to inactivation of the V-ATPase during preincubation rather than inhibition during the assay. The inhibitory effect of KNO3 preincubation was much less pronounced in the absence of MgATP; preincubation with 200 mm KNO3 in the presence of either Mg2+ or Tris-ATP alone inhibited proton-pumping activity by less than 50% (data not shown). Fig. 8. Open in new tabDownload slide Effect of KNO3 preincubation on proton transport in beet tonoplast vesicles. Proton transport activity was monitored as a percent decrease in acridine orange fluorescence measured at 490 nm (% F). Tonoplast vesicles equivalent to 10 μg of protein were diluted in 1 mL of transport buffer containing acridine orange and equilibrated for 3 min. The reaction was started by adding 5 mm Tris-ATP, and absorbance measurements were collected every 10 s for 3 min. At the end of the reaction Gramicidin D (Gram) was added to a final concentration of 3 μm. a, Nonpreincubated vesicles assayed in the absence of KNO3 for 3 min and then for 3 min after Gramicidin D addition; b, nonpreincubated vesicles assayed in the presence of 200 mmKNO3; c, vesicles preincubated with 5 mm MgATP; and d, vesicles preincubated with 5 mm MgATP plus 200 mm KNO3. Fig. 8. Open in new tabDownload slide Effect of KNO3 preincubation on proton transport in beet tonoplast vesicles. Proton transport activity was monitored as a percent decrease in acridine orange fluorescence measured at 490 nm (% F). Tonoplast vesicles equivalent to 10 μg of protein were diluted in 1 mL of transport buffer containing acridine orange and equilibrated for 3 min. The reaction was started by adding 5 mm Tris-ATP, and absorbance measurements were collected every 10 s for 3 min. At the end of the reaction Gramicidin D (Gram) was added to a final concentration of 3 μm. a, Nonpreincubated vesicles assayed in the absence of KNO3 for 3 min and then for 3 min after Gramicidin D addition; b, nonpreincubated vesicles assayed in the presence of 200 mmKNO3; c, vesicles preincubated with 5 mm MgATP; and d, vesicles preincubated with 5 mm MgATP plus 200 mm KNO3. Fig. 10. Open in new tabDownload slide V-ATPase disruption as a function of KNO3 concentration. Tonoplast vesicles equivalent to 50 μg of protein were preincubated with 5 mm MgATP and 0 to 200 mm KNO3 for 30 min on ice. Aliquots equivalent to 10 μg of protein were removed for proton transport assays in the presence of 5 mm MgATP. The remaining sample was separated into membrane pellet and supernatant fractions and processed as described for Figure 8. Bands corresponding to 67- and 44-kD polypeptides were quantified by imaging densitometry as described in Methods. Top, Proton transport activity following preincubation; middle, distribution of 67-kD polypeptide between membrane pellet and supernatant fractions following preincubation; bottom, distribution of 44-kD polypeptide following preincubation. Fig. 10. Open in new tabDownload slide V-ATPase disruption as a function of KNO3 concentration. Tonoplast vesicles equivalent to 50 μg of protein were preincubated with 5 mm MgATP and 0 to 200 mm KNO3 for 30 min on ice. Aliquots equivalent to 10 μg of protein were removed for proton transport assays in the presence of 5 mm MgATP. The remaining sample was separated into membrane pellet and supernatant fractions and processed as described for Figure 8. Bands corresponding to 67- and 44-kD polypeptides were quantified by imaging densitometry as described in Methods. Top, Proton transport activity following preincubation; middle, distribution of 67-kD polypeptide between membrane pellet and supernatant fractions following preincubation; bottom, distribution of 44-kD polypeptide following preincubation. Dissociation of the 44-kD Polypeptide Is Dependent on ATP and KNO3 Concentration A series of membrane fractionation experiments were then conducted using KNO3 to further examine the association of the 44-kD polypeptide with tonoplast vesicles. Specifically, we sought to correlate association of the 44-kD polypeptide with proton-pumping activity. Tonoplast vesicles were preincubated at 0°C with KNO3 in the presence of MgATP, and then aliquots equivalent to 20% of the total sample were assayed for proton transport activity. The remaining membranes were pelleted, and both membrane and supernatant fractions were examined for the 67-kD V-ATPase subunit and the 44-kD polypeptide by immunoblot analysis. When compared with the control, preincubation of tonoplast vesicles with 200 mm KNO3 plus MgATP resulted in substantial dissociation of the 67-kD V1 subunit (Fig. 9, top). The 44-kD polypeptide was also partially removed from the membrane by preincubation with nitrate, although to a lesser extent than the 67-kD peripheral subunit (Fig. 9, bottom). Fig. 9. Open in new tabDownload slide Dissociation of 67-kD subunit and 44-kD polypeptide by MgATP and KNO3. Tonoplast vesicles equivalent to 50 μg of protein were preincubated with 5 mm MgATP ± 200 mm KNO3 for 30 min on ice. Membranes were collected by centrifugation and protein samples from both membrane pellet and supernatant fractions were prepared as described in Methods. Proteins were separated on 15% gels and then immunoblotted. P, Membrane pellet; S, supernatant. Top, Membrane pellet and supernatant fractions probed with α67-kD serum. Bottom, Same fractions probed with α-Vma6p serum. Fig. 9. Open in new tabDownload slide Dissociation of 67-kD subunit and 44-kD polypeptide by MgATP and KNO3. Tonoplast vesicles equivalent to 50 μg of protein were preincubated with 5 mm MgATP ± 200 mm KNO3 for 30 min on ice. Membranes were collected by centrifugation and protein samples from both membrane pellet and supernatant fractions were prepared as described in Methods. Proteins were separated on 15% gels and then immunoblotted. P, Membrane pellet; S, supernatant. Top, Membrane pellet and supernatant fractions probed with α67-kD serum. Bottom, Same fractions probed with α-Vma6p serum. Dissociation of both the 67-kD V1 subunit and the 44-kD polypeptide appeared to be a function of preincubation nitrate concentration in the presence of MgATP (Fig.10, middle and bottom). At the highest preincubation concentration of KNO3 tested (200 mm), 60% of the 67-kD subunit and 50% of the 44-kD polypeptide were dissociated from the membrane. It is interesting that complete inhibition of proton-pumping activity was achieved at much lower concentrations, with an observed preincubationKi for nitrate inhibition of approximately 8 mm. Thus, although we consistently observed concentration-dependent dissociation of V-ATPase subunits, it appeared to be only generally correlated with loss of enzyme activity. In the absence of ATP during preincubation, the observedKi for nitrate inhibition was approximately 260 mm. Some corresponding dissociation of V-ATPase subunits was observed; maximally, 25% of the 67-kD polypeptide and 10% of the 44-kD polypeptide were released during preincubation in the absence of MgATP (not shown). However, the amount of subunit dissociation observed in the absence of MgATP was not dependent on KNO3 concentration and therefore was not clearly correlated with KNO3 effects on the active V-ATPase holoenzyme. DISCUSSION In this report we present evidence supporting the identification of a red beet protein homologous to Vma6p. The putative 44-kD homolog was specifically cross-reactive with α-Vma6p polyclonal antibodies, indicating substantial sequence similarity with the yeast V0 subunit. The 44-kD polypeptide cofractionated with peak V-ATPase activity as well as the 67-kD subunit of the V1 sector, indicating that it was tightly associated with V-ATPase isolated from red beet. A 44-kD polypeptide was previously reported to be associated with highly purified V-ATPase preparations from red beet tonoplast (Parry et al., 1989). This protein was released, along with other V1 subunits, by cold inactivation conditions, leading the authors to conclude that it is a peripheral V-ATPase subunit. Our results provide additional support for the identification of this subunit and suggest further that it shares significant similarity with a homologous subunit in yeast. The existence of Vma6p homologs among animal, plant, and fungal species suggests that this conserved subunit is important in the assembly and function of V-ATPase holoenzyme. The yeast 36-kD subunit is a tightly associated peripheral component of the V0 sector of V-ATPase. Our results indicate that the 44-kD unit is also peripherally attached and thus susceptible to removal with chaotropic agents such as KNO3. KNO3 has been shown to inhibit V-ATPase activity by dissociation of V1 subunits from the membrane (Rea et al., 1987; Tu et al., 1987). At KNO3concentrations sufficient to completely inhibit V-ATPase activity in the presence of MgATP, neither the 67- nor the 44-kD subunit was quantitatively released from the membrane. Thus, the release of peripheral subunits may be a secondary consequence of a nitrate-induced conformational shift that leads to enzyme inactivation. Alternatively, some peripheral subunits may remain nonspecifically associated with the tonoplast membrane. For instance, if tonoplast membrane preparations contain a fraction of inside-out vesicles, then a portion of V-ATPase would likely be protected from dissociation by nitrate (Rea et al., 1987). Further biochemical studies are under way to determine the membrane association of this putative Vma6p homolog. Given the ubiquitous involvement of V-ATPase in acidifying internal, and in some cases, external compartments, there has been much interest in elucidating the mechanism(s) by which proton-pumping activity is regulated. V-ATPase activity appears to be regulated in part by posttranslational modifications of V1 subunits (for reviews, see Forgac, 1996; Merzendorfer et al., 1997a, 1997b). In addition, several reports have provided evidence supporting the hypothesis that V-ATPase cellular activity may also be modulated by regulated assembly-disassembly of V1 and V0 sectors. For example, selective release of peripheral V-ATPase subunits has been correlated with in vivo enzyme inactivation in response to chilling in mung bean seedlings (Matsuura-Endo et al., 1992). In Manduca sexta, regulation of V-ATPase activity during larval development appears to be correlated with a loss of V1 subunits (Sumner et al., 1995). Recently, Kane (1995) described in vivo assembly-disassembly of peripheral V-ATPase subunits in yeast cells in response to Glc deprivation. In yeast, active V-ATPase assembles by attachment of preexisting V1 particles from a cytoplasmic pool onto the V0 membrane sector. Hence, V-ATPase components may be directly triggered to mediate assembly or disassembly in response to intracellular signals. The positioning of Vma6p as a peripherally associated V0 subunit required for V1 attachment suggests a role in such a regulatory mechanism. We conclude that the 44-kD protein in red beet is a subunit of the tonoplast V-ATPase holoenzyme and is homologous to a tightly associated peripheral V0 subunit previously described in yeast. Future studies will focus on a more detailed biochemical characterization of this novel V0 subunit to understand its role in V-ATPase assembly. ACKNOWLEDGMENTS Portions of this project were completed during a sabbatical leave by C.B in the laboratory of D.P.B. The authors thank Dr. Ron Poole (McGill University, Montreal, Quebec, Canada) for providing antibodies against beet 57- and 67-kD subunits, Dr. Ben Lockhardt (University of Minnesota, Minneapolis) for use of his preparative ultracentrifuge, Lori Jahnke (Hamline University, St. Paul, MN) for photographic assistance, and Dr. Sylvia Kerr for critically reading the manuscript. The authors acknowledge helpful conversations with Dr. Lynne Gildensoph (The College of St. Catherine, St. Paul, MN). Abbreviations: BCA bicinchoninic acid BHT butylated hydroxytoluene V-ATPase V-type proton-translocating ATPase LITERATURE CITED 1 Bauerle C Ho MN Lindorfer MA Stevens TH The Saccharomyces cerevisiae VMA6 gene encodes the 36-kDa subunit of the vacuolar H+-ATPase membrane sector. J Biol Chem 268 1993 12749 12757 Google Scholar Crossref Search ADS PubMed WorldCat 2 Bradford MM A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72 1977 248 252 Google Scholar Crossref Search ADS WorldCat 3 Finbow ME Harrison MA The vacuolar H+-ATPase: a universal proton pump of eukaryotes. Biochem J 324 1997 697 712 Google Scholar Crossref Search ADS PubMed WorldCat 4 Forgac M Regulation of vacuolar acidification. Soc Gen Physiol Ser 51 1996 121 132 Google Scholar PubMed OpenURL Placeholder Text WorldCat 5 Giannini JL Nelson M Spessard GO The effect of rishitin on potato vesicle and vacuole proton transport. Phytochemistry 40 1995 1655 1658 Google Scholar Crossref Search ADS PubMed WorldCat 6 Gish W States DJ Identification of protein coding regions by database similarity search. Nat Genet 3 1993 266 272 Google Scholar Crossref Search ADS PubMed WorldCat 7 Graham LA Hill KJ Stevens TH VMA8 encodes a 32-kDa V1 subunit of the Saccharomyces cerevisiae vacuolar H+-ATPase required for function and assembly of the enzyme complex. J Biol Chem 270 1995 15037 15044 Google Scholar Crossref Search ADS PubMed WorldCat 8 Kane PM Disassembly and reassembly of the yeast vacuolar H+-ATPase in vivo. J Biol Chem 270 1995 17025 17032 Google Scholar Crossref Search ADS PubMed WorldCat 9 Matsuura-Endo C Maeshima M Yoshida S Mechanism of the decline in vacuolar H+-ATPase activity in mung bean hypocotyls during chilling. Plant Physiol 100 1992 718 722 Google Scholar Crossref Search ADS PubMed WorldCat 10 Melnik VI Bowman BJ Isolation of the vma-6 gene encoding a 41 kDa subunit of the Neurospora crassa vacuolar ATPase, and an adjoining gene encoding a ribosome-associated protein. Biochim Biophys Acta 1273 1996 77 83 Google Scholar Crossref Search ADS PubMed WorldCat 11 Merzendorfer H Graf R Huss M Harvey WR Wieczorek H Regulation of proton-translocating V-ATPases. J Exp Biol 200 1997a 225 235 Google Scholar OpenURL Placeholder Text WorldCat 12 Merzendorfer H Harvey WR Wieczorek H Sense and antisense RNA for the membrane associated 40 kDa subunit M40 of the insect V-ATPase. FEBS Lett 411 1997b 239 244 Google Scholar Crossref Search ADS WorldCat 13 Myers EW Miller W Optimal alignments in linear space. Comput Appl Biosci 4 1988 11 17 Google Scholar PubMed OpenURL Placeholder Text WorldCat 14 Nelson N Klionsky DJ Vacuolar H+-ATPase: from animals to yeast and back. Experientia 52 1996 1101 1110 Google Scholar Crossref Search ADS PubMed WorldCat 15 Parry RV Turner JC Rea PA High purity preparations of higher plant vacuolar H+-ATPase reveal additional subunits. J Biol Chem 264 1989 20025 20032 Google Scholar Crossref Search ADS PubMed WorldCat 16 Poole RJ Briskin DP Kratky Z Johnstone RM Density gradient localization of plasma membrane and tonoplast from storage tissue of growing and dormant red beet: characterization of proton-transport and ATPase in tonoplast vesicles. Plant Physiol 74 1984 549 556 Google Scholar Crossref Search ADS PubMed WorldCat 17 Rea PA Griffith CJ Manolson MF Sanders D Irreversible inhibition of H+-ATPase of higher plant tonoplast by chaotropic anions: evidence for peripheral location of nucleotide-binding subunits. Biochim Biophys Acta 904 1987 1 12 Google Scholar Crossref Search ADS WorldCat 18 Sumner J-P Dow JAT Earley FGP Klein U Jager D Wieczorek H Regulation of plasma membrane V-ATPase activity by dissociation of peripheral subunits. J Biol Chem 270 1995 5649 5653 Google Scholar Crossref Search ADS PubMed WorldCat 19 Taiz L The plant vacuole. J Exp Biol 172 1992 113 122 Google Scholar PubMed OpenURL Placeholder Text WorldCat 20 Temesvari L Rodriguez-Paris J Bush J Steck TL Cardelli J Characterization of lysosomal membrane proteins of Dictyostelium discoidium. A complex population of acidic integral membrane glycoproteins, Rab GTP-binding proteins and vacuolar ATPase subunits. J Biol Chem 269 1994 25719 25727 Google Scholar Crossref Search ADS PubMed WorldCat 21 Towbin H Staehelin T Gordon J Electrophoresis of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76 1979 4350 4354 Google Scholar Crossref Search ADS PubMed WorldCat 22 Tu S-I Nagahashi G Brouillette JN Proton pumping kinetics and origin of nitrate inhibition of tonoplast-type H+-ATPase. Arch Biochem Biophys 256 1987 625 637 Google Scholar Crossref Search ADS PubMed WorldCat 23 van Hille B Vanek M Richener H Green JR Bilbe G Cloning and tissue distribution of subunits C, D, and E of the human vacuolar H+-ATPase. Biochem Biophys Res Commun 197 1993 15 21 Google Scholar Crossref Search ADS PubMed WorldCat 24 Wang SY Moriyama Y Mandel M Hulmes JD Pan YC Danho W Nelson H Nelson N Cloning of cDNA encoding a 32-kDa protein. An accessory polypeptide of the H+-ATPase from chromaffin granules. J Biol Chem 263 1988 17638 17642 Google Scholar Crossref Search ADS PubMed WorldCat 25 Ward JM Reinders A Hsu H-T Sze H Dissociation and reassembly of the vacuolar H+-ATPase complex from oat roots. Plant Physiol 99 1992 161 169 Google Scholar Crossref Search ADS PubMed WorldCat Author notes 1 This work was supported in part by funds from the Hanna grant program (to C.B.) and a Lund Fund scholarship (to C.M.). 2 Present address: The College of St. Catherine, 601 25th Avenue S., Minneapolis, MN 55454. * Corresponding author; e-mail [email protected]; fax 1–612–523–2620. Copyright © 1998 American Society of Plant Physiologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Viksø-Nielsen, Anders; Blennow, Andreas; Hamborg Nielsen, Tom; Lindberg Møller, Birger

doi: 10.1104/pp.117.3.869pmid: 9662529

Abstract The possible involvement of potato (Solanum tuberosum L.) starch-branching enzyme I (PSBE-I) in the in vivo synthesis of phosphorylated amylopectin was investigated in in vitro experiments with isolated PSBE-I using33P-labeled phosphorylated and 3H end-labeled nonphosphorylated α(1→4)glucans as the substrates. From these radiolabeled substrates PSBE-I was shown to catalyze the formation of dual-labeled (3H/33P) phosphorylated branched polysaccharides with an average degree of polymerization of 80 to 85. The relatively high molecular mass indicated that the product was the result of multiple chain-transfer reactions. The presence of α(1→6) branch points was documented by isoamylase treatment and anion-exchange chromatography. Although the initial steps of the in vivo mechanism responsible for phosphorylation of potato starch remains elusive, the present study demonstrates that the enzyme machinery available in potato has the ability to incorporate phosphorylated α(1→4)glucans into neutral polysaccharides in an interchain catalytic reaction. Potato mini tubers synthesized phosphorylated starch from exogenously supplied 33PO43− and [U-14C]Glc at rates 4 times higher than those previously obtained using tubers from fully grown potato plants. This system was more reproducible compared with soil-grown tubers and was therefore used for preparation of 33P-labeled phosphorylated α(1→4)glucan chains. Starch is composed of the two polymers, amylose and amylopectin. The amylose molecules are essentially linear α(1→4)glucan chains, whereas the amylopectin molecules are highly branched and often contain small amounts of covalently bound phosphate (Hizukuri et al., 1970). Potato tuber starch is characterized by a high content of phosphate relative to cereal starches (Rooke et al., 1949; Hizukuri et al., 1970). The phosphate groups are located as monoesters at the C-6 (approximately 70%) and at the C-3 (approximately 30%) positions of the Glc residues (Hizukuri et al., 1970; Takeda and Hizukuri, 1982). Phosphorylation levels differ by 3-fold among potato varieties (Bay-Smidt et al., 1994) and strongly depend upon growth conditions (Nikuni et al., 1969). Small starch granules contain approximately 25% more bound phosphate per Glc residue than large granules, whereas the overall level of phosphorylation does not depend on tuber size (Nielsen et al., 1994). In a previous study it was found that phosphorylation occurs concurrently with de novo synthesis of starch in potato (Solanum tuberosum L.) tuber discs (Nielsen et al., 1994). However, the mechanism underlying phosphorylation of starch remains elusive. Neither the identity of a phosphorylated intermediate, which could be incorporated in the α(1→4)glucan chains, nor the enzyme system responsible for its incorporation are known. Since amylopectin is phosphorylated and amylose is not, it is of interest to determine whether the potato SBE (EC 2.4.1.18) can utilize phosphorylated glucans as a substrate. In the present study we have tested the possible involvement of SBE in the formation of phosphorylated starch. The normal mode of action of SBE is to catalyze the cleavage of an α(1→4)glucosidic linkage followed by a condensation of the released α(1→4)glucan to an acceptor chain thereby introducing an α(1→6)glucosidic linkage. The catalytic mechanism may involve sequential binding of the acceptor chain and then the donor chain (Borovsky et al., 1976) or, alternatively, binding of two α(1→4)glucan chains that have formed a double helix. Glucans with a DP of less than 40 do not serve as a substrate for PSBE-I at 30°C (Borovsky et al., 1976). However, at lower temperatures, where double-helix formation is facilitated, shorter chains do serve as substrates, and the presence of branch points stimulates the rate of catalysis (Borovsky et al., 1975b). This would suggest that PSBE-I acts on an α(1→4)glucan double helix rather than on two unassociated α(1→4)glucan chains. Further support for this hypothesis has been provided by monitoring the association of PSBE-I to linear malto-oligosaccharides (Blennow et al., 1998b). Maximal association took place with chains with a DP of 10 to 15, which coincides with the minimal chain length of 10 Glc residues that are required for initial double-helix formation by linear maltooligosaccharides (Gidley and Bulpin, 1987). Accordingly, an involvement of SBE in the synthesis of phosphorylated amylopectin would require that the enzyme be able to use phosphorylated α(1→4)glucans having a DP of 10 to 15 or preferably larger as a substrate. Such phosphorylated glucans can be derived from potato tuber starch by debranching with isoamylase (Blennow et al., 1998a). A radiolabeled version of the glucans can be obtained by in vivo labeling beforehand of the starch-bound phosphate, as described by Nielsen et al. (1994). Using such 33P-labeled α(1→4)glucans and nonphosphorylated α(1→4)glucans labeled with3H at the reducing end as the substrates, we demonstrate that PSBE-I catalyzes chain-transfer reactions using the phosphorylated linear glucans as donors to form branched phosphorylated polysaccharides. MATERIALS AND METHODS Chemicals and Reagents Chemicals and phosphorylase a from rabbit muscle were supplied by Sigma, isoamylase was from Megazyme (Sydney, Australia), α-amylase (Termamyl Type L) was from Novo Nordisk A/S (Bagsværd, Denmark), and radiochemicals were from Amersham. Plant Material and in Vitro Culture Potato (Solanum tuberosum L. cv Dianella) plants were grown in the greenhouse as described previously (Nielsen et al., 1994). Tubers with a diameter of approximately 5 cm were harvested from 4-month-old plants, rinsed in tap water, and used immediately for incubation experiments. Mini tubers were grown as described by Visser et al. (1994) with the following modification: Sterile, in vitro-grown plants, used as donor plants, were initially obtained by placing surface-sterilized stem sections from greenhouse-grown plants on shoot-inducing medium. The in vitro potato plants were grown at 22°C using a 14-h light period (160 μmol m−2 s−1). For tuber induction, a stem section (1 cm) with one resting auxiliary bud and one fully developed leaf was excised from a donor plant. The leaf was removed and the stem was transferred to tuber-inducing medium and placed in darkness at 14°C. After 4 weeks, the formed tubers (3 mm in diameter) were harvested and used immediately for experiments. Radiolabeling Experiments Incubation of Mini Tubers Six mini tubers (each approximately 100 mg fresh weight) were cut into halves and incubated for 4 h (total volume: 100 μL) in 300 mm Glc and 3.7 MBq33PO43−or in 300 mm sorbitol and 74 kBq [U-14C]Glc at room temperature. Three potato tuber discs (each approximately 100 mg fresh weight) were excised from soil-grown tubers (5 cm in diameter) as described previously (Nielsen et al., 1994) and incubated in the same way as the mini tubers. Isolation of Starch Granules Starch granules were isolated and washed as described previously (Nielsen et al., 1994), incorporating two additional washes of the starch in 10 mL of 100 mm phosphoric acid for 5 min at room temperature. Isolation of 33P-Labeled Phosphorylated α(1→4)Glucans Isolated 33P-labeled starch (approximately 10 mg) from the incorporation experiment was gelatinized (2 mL of water, 5 min, 100°C). After addition of sodium acetate (pH 4.0) to a final concentration of 50 mm, the starch was debranched by isoamylase (2 units, 2 h, 37°C). After the incubation period, the enzyme was inactivated by boiling (5 min) and the phosphorylated glucans produced were isolated using anion-exchange chromatography (DEAE-Sepharose, Pharmacia) as described in Blennow et al. (1998a). The33P content of each fraction was quantified by liquid-scintillation counting. Total sugar content was determined using the phenol sulfuric acid method (Dubois et al., 1956). After separation of the neutral and phosphorylated glucans, the material was lyophilized and used immediately for experiments. For enzymatic degradation, the phosphorylated glucans (0.1 mg) were dissolved in 0.5 mL of 5 mm Mes, 4 mmCaCl2, pH 6.5, and incubated with α-amylase (1 unit, 2 h, 25°C). After incubation, a 100-μL sample was immediately applied to a CarboPac PA-100 anion-exchange column (see below) and analyzed using the method described by Blennow et al. (1998a). Synthesis of α(1→4)Glucans and Reduction with NaB(3H)4 α(1→4)Glucans were synthesized by incubating (37°C, 20 h) phosphorylase a from rabbit muscle (100 units) in 20 mL of 0.35 mm maltoheptaose, 60 mm Glc-1-P, and 1 mm AMP (pH 7.0). After inactivation of the enzyme (100°C, 5 min), the resulting glucan fraction was precipitated with 80% (v/v) ethanol, lyophilized, and stored at −20°C. A modification of the method of Borovsky et al. (1976) was used to chemically synthesize a 3H-labeled nonphosphorylated α(1→4)glucan substrate. Neutral glucans (1 mg) in 100 μL of 0.1 n NaOH were reacted with 0.5 MBq NaB(3H)4 (25°C, overnight). To ensure quantitative reduction of all reducing ends, a surplus of unlabeled NaBH4 (2 mg in 200 μL of 0.1 n NaOH) was added and the reaction allowed to continue for an additional 2 h. Surplus reagent was destroyed by addition of 1n HCl until no more hydrogen evolved. Precipitated borate was removed by application of the sample to an NAP-10 column (Pharmacia) and elution of the α(1→4)glucans was performed with 1.5 mL of 50 mm phosphate buffer (pH 7.5). The3H-end-labeled α(1→4)glucan fraction was used immediately. Chain-Transfer Experiment PSBE-I was isolated to homogeneity from potato tubers by affinity chromatography. The isolated PSBE-I was free of amylases and other hydrolytic activities, as analyzed by activity measurements and zymogram analysis using the method described inViksø-Nielsen et al. (1998). For chain-transfer experiments, PSBE-I (10 ng) was incubated (25°C, 2 h) with33P-labeled phosphorylated α(1→4)glucans (1 mg, solubilized in 0.25 mL of 0.1 n NaOH and neutralized with 0.1 n HCl) and with 3H-labeled α(1→4)glucans (1 mg) in 0.5 mL of 50 mm sodium phosphate buffer, pH 7.5, 0.05% n-octylglucoside, and 0.1 mg/mL BSA. The enzyme was inactivated by boiling (5 min), and the resulting product was applied to an anion-exchange column (1 × 5 cm, DEAE-Sepharose) equilibrated in 5 mm Mes, pH 8.0. Neutral glucans were washed off the column with 15 mL of water. The phosphorylated glucans were subsequently eluted using 15 mL of 100 mm NaCl and 10 mm HCl (1.5-mL fractions). Gel-Permeation Chromatography The molecular mass distribution of the substrates and the phosphorylated product obtained from the chain-transfer reaction was analyzed using a column (830 × 26 mm) of Sephacryl S-200 (Pharmacia) as described elsewhere (Blennow et al., 1998a) and calibrated using a mixture of linear α(1→4)glucans as molecular mass markers. HPAEC A DX 500 system (Dionex Corp., Sunnyvale, CA) equipped with an S-3500 autosampler and fitted with a CarboPac PA-100 column was used to analyze the isolated, neutral, and phosphorylated α(1→4)glucan chains (Blennow et al., 1998a). Liquid-Scintillation Counting The incorporation of33PO43−and [U-14C]Glc into starch was measured using a WinSpectral 1414 liquid-scintillation counter (Wallac, Helsinki, Finland) with WinSpectral version 1.0 software and Ecoscint A scintillation liquid (National Diagnostics, Manville, NJ). Samples containing 3H and 33P were counted using a separate isotope library for each isotope and automatic correction of curve overlaps. RESULTS Incorporation of Phosphate into Starch Incorporation of33PO43−and [U-14C]Glc into starch using mini tubers was found to be 4-fold more effective than incorporation into potato tuber discs (Table I). The incorporation was linear with time for up to 4 h and continued for at least 14 h (data not shown). These results are similar to those previously reported with potato tuber discs (Nielsen et al., 1994). On this basis, the mini tuber system was chosen as the optimal experimental system for production of radiolabeled phosphorylated α-glucan chains. Table I. Incorporation of [U-14C]Glc and33PO43− into starch using potato tuber discs and potato mini tubers after 4 h of incubation Tissue . Radioactivity . [U-14C]Glc . 33PO43− . dpm mg−1α(1→4) glucan Tuber discs 250 ± 40 490 ± 70 Mini tubers 950 ± 60 2400 ± 120 Tissue . Radioactivity . [U-14C]Glc . 33PO43− . dpm mg−1α(1→4) glucan Tuber discs 250 ± 40 490 ± 70 Mini tubers 950 ± 60 2400 ± 120 Values are ± se (n = 3). Open in new tab Table I. Incorporation of [U-14C]Glc and33PO43− into starch using potato tuber discs and potato mini tubers after 4 h of incubation Tissue . Radioactivity . [U-14C]Glc . 33PO43− . dpm mg−1α(1→4) glucan Tuber discs 250 ± 40 490 ± 70 Mini tubers 950 ± 60 2400 ± 120 Tissue . Radioactivity . [U-14C]Glc . 33PO43− . dpm mg−1α(1→4) glucan Tuber discs 250 ± 40 490 ± 70 Mini tubers 950 ± 60 2400 ± 120 Values are ± se (n = 3). Open in new tab Isolation and Characterization of 33P-Labeled α(1→4)Glucan from Debranched Starch Phosphorylated α(1→4)glucan chains were isolated from isoamylase-debranched starch by anion-exchange chromatography. The neutral glucan chains were eluted from the column with 5 mmTris-HCl, pH 7.5 (Fig. 1, fractions 1–6), and the phosphorylated glucan chains were subsequently eluted with 100 mm NaCl and 10 mm HCl, pH 2.0 (fractions 13–18). Fig. 1. Open in new tabDownload slide Isolation of 33P-labeled phosphorylated α(1→4)glucans by anion-exchange chromatography of isoamylase-debranched starch isolated from potato mini tubers. The phosphorylated glucans were eluted with 100 mm NaCl and 10 mm HCl, pH 2.0. Bars represent radioactivity originating from 33P-labeled phosphorylated α(1→4)glucans. Fig. 1. Open in new tabDownload slide Isolation of 33P-labeled phosphorylated α(1→4)glucans by anion-exchange chromatography of isoamylase-debranched starch isolated from potato mini tubers. The phosphorylated glucans were eluted with 100 mm NaCl and 10 mm HCl, pH 2.0. Bars represent radioactivity originating from 33P-labeled phosphorylated α(1→4)glucans. The isolated phosphorylated glucans were lyophilized, redissolved in 0.1 n NaOH, and fractionated by HPAEC. The possible occurrence of phosphorylated glucan chains carrying more than one phosphate group and a varying internal location of the phosphate group in each glucan chain combined with the variability in chain length resulted in a broad elution profile. The radioactivity eluted in the same fractions as the phosphorylated glucan (Fig.2, A and B). To ensure that all the33P label originated from phosphate groups bound to α(1→4)glucan, a fraction of the isolated33P-labeled molecules was degraded with α-amylase and analyzed similarly. The elution profile obtained (Fig.2C) was that expected from the conversion of phosphorylated glucan into a shorter oligosaccharide. The superimposable labeling pattern (Fig.2D) documents that the 33P label detected in the nondegraded sample (Fig. 2B) is bound to the α(1→4)glucan chains. Fig. 2. Open in new tabDownload slide A, Elution profile of 33P-labeled phosphorylated α(1→4)glucan chains as determined by HPAEC/PAD. B, Distribution of 33P radioactivity in 1-mL fractions of the anion-exchange eluate of A. C, Elution of α-amylase limit phosphorylated α(1→4)glucan. D, Elution of 33P radioactivity in 1-mL fractions of the anion-exchange eluate in C. nC, Nanocoulombs.   Fig. 2. Open in new tabDownload slide A, Elution profile of 33P-labeled phosphorylated α(1→4)glucan chains as determined by HPAEC/PAD. B, Distribution of 33P radioactivity in 1-mL fractions of the anion-exchange eluate of A. C, Elution of α-amylase limit phosphorylated α(1→4)glucan. D, Elution of 33P radioactivity in 1-mL fractions of the anion-exchange eluate in C. nC, Nanocoulombs.   Synthesis of α(1→4)Glucan using Phosphorylase a To create a second, well-defined substrate for PSBE-I, nonphosphorylated α(1→4)glucan was synthesized from Glc-1-P using phosphorylase a and maltoheptaose as a primer. The chain-length distribution of the precipitated glucan fraction was determined by HPAEC and revealed an approximately binomial distribution peaking at DP 33 (Fig. 3). To produce a3H-labeled substrate distinguishable from the33P-labeled phosphorylated glucan chains, the free anomeric center was reduced with NaB(3H)4. Fig. 3. Open in new tabDownload slide Distribution of neutral α(1→4)glucan chains synthesized with phosphorylase a from maltoheptaose and Glc-1-P as determined by HPAEC/PAD using a CarboPac PA-100 column. nC, Nanocoulombs. Fig. 3. Open in new tabDownload slide Distribution of neutral α(1→4)glucan chains synthesized with phosphorylase a from maltoheptaose and Glc-1-P as determined by HPAEC/PAD using a CarboPac PA-100 column. nC, Nanocoulombs. Chain Transfer Catalyzed by PSBE-I The 33P- and3H-labeled α(1→4)glucans were tested as the substrates for PSBE-I, which was isolated to homogeneity (Fig.4) by affinity chromatography (Viksø-Nielsen et al., 1998). After incubation of the33P- and 3H-labeled glucans with PSBE-I, one-half of the reaction mixture was applied to an anion-exchange column to separate the neutral and phosphorylated products obtained from the chain-transfer process. As expected, the neutral products were exclusively 3H labeled. In contrast, the eluted phosphorylated products were co-labeled with3H and 33P (Fig.5A). This indicates that PSBE-I has catalyzed a chain-transfer reaction, resulting in the formation of a covalent linkage between the 33P- and3H-labeled α(1→4)glucans. Fig. 4. Open in new tabDownload slide SDS-PAGE and zymogram of PSBE-I isolated to homogeneity by γ-cyclodextrin-affinity chromatography. Lane A, SDS-PAGE of isolated PSBE-I. Lane B, Zymogram of isolated PSBE-I. The location of the SDS-PAGE molecular weight markers (Mr 116,000, 66,000, 45,000, and 27,000) are indicated on the left. Fig. 4. Open in new tabDownload slide SDS-PAGE and zymogram of PSBE-I isolated to homogeneity by γ-cyclodextrin-affinity chromatography. Lane A, SDS-PAGE of isolated PSBE-I. Lane B, Zymogram of isolated PSBE-I. The location of the SDS-PAGE molecular weight markers (Mr 116,000, 66,000, 45,000, and 27,000) are indicated on the left. Fig. 5. Open in new tabDownload slide Anion-exchange chromatography (DEAE-Sepharose) of products obtained from a PSBE-I-catalyzed chain-transfer reaction. A, Products from PSBE-I-catalyzed chain-transfer reactions. B, Sample as in A debranched with isoamylase. Black bars represent radioactivity originating from 33P-labeled phosphorylated α(1→4)glucans. White bars represent radioactivity originating from3H end-labeled groups. Fig. 5. Open in new tabDownload slide Anion-exchange chromatography (DEAE-Sepharose) of products obtained from a PSBE-I-catalyzed chain-transfer reaction. A, Products from PSBE-I-catalyzed chain-transfer reactions. B, Sample as in A debranched with isoamylase. Black bars represent radioactivity originating from 33P-labeled phosphorylated α(1→4)glucans. White bars represent radioactivity originating from3H end-labeled groups. To demonstrate the presence of α(1→6)linkages in the3H/33P-labeled products, the second half of the original reaction mixture was debranched with isoamylase. Separation of the debranched glucans by anion-exchange chromatography revealed a clear separation of the33P- and 3H-labeling into distinct peaks (Fig. 5B). This proves that PSBE-I has formed α(1→6)linkages between the 3H- and33P-labeled glucans. The molecular mass distribution of the 3H- and33P-labeled substrates and the products generated in the chain-transfer process (Fig. 5A, fractions 11–16) were determined using gel-permeation chromatography (Fig.6, A, B, and C, respectively). Two major groups of products were generated (Fig. 6C). The first group of products eluted around 210 mL and was labeled with3H and 33P and had a main peak around DP 80 to 85. The broad peak obtained indicates that the products synthesized in the chain-transfer process are complex oligosaccharides most likely containing more than one branch point. The second minor group of products eluted at 320 mL and was exclusively labeled with 33P. Fig. 6. Open in new tabDownload slide Gel-permeation chromatography of substrates and products in the PSBE-I catalyzed chain-transfer reactions. A, Distribution of 3H end-labeled α(1→4)glucans (Fig. 3) used as a substrate for PSBE-I. B, Distribution of33P-labeled phosphorylated α(1→4)glucans isolated from potato mini tubers (Fig. 1, fractions 13–18) used as a substrate for PSBE-I. C, Distribution of the products obtained from PSBE-I-catalyzed chain-transfer reactions (Fig. 5A, fractions 11–16) after removal of neutral chains by anion-exchange chromatography. The elution pattern of phosphorylase a-synthesized standards with a mean DP of 33, 42, and 83 is indicated. V0, Void volume. •, Radioactivity originating from 33P-labeled phosphorylated α(1→4)glucans; ○, radioactivity originating from the 3H-labeled end groups; and □, total sugar in μg mL−1. Fig. 6. Open in new tabDownload slide Gel-permeation chromatography of substrates and products in the PSBE-I catalyzed chain-transfer reactions. A, Distribution of 3H end-labeled α(1→4)glucans (Fig. 3) used as a substrate for PSBE-I. B, Distribution of33P-labeled phosphorylated α(1→4)glucans isolated from potato mini tubers (Fig. 1, fractions 13–18) used as a substrate for PSBE-I. C, Distribution of the products obtained from PSBE-I-catalyzed chain-transfer reactions (Fig. 5A, fractions 11–16) after removal of neutral chains by anion-exchange chromatography. The elution pattern of phosphorylase a-synthesized standards with a mean DP of 33, 42, and 83 is indicated. V0, Void volume. •, Radioactivity originating from 33P-labeled phosphorylated α(1→4)glucans; ○, radioactivity originating from the 3H-labeled end groups; and □, total sugar in μg mL−1. DISCUSSION In the present study we have demonstrated that potato mini tubers efficiently incorporate administered [U-14C]Glc and33PO43−into a phosphorylated starch. Earlier, Nielsen et al. (1994) used potato tuber discs as a model system for analysis of starch biosynthesis. The mini tuber system is superior to the tuber discs system because phosphorylated starch is synthesized at a rate that is 4 times higher (Table I). Furthermore, the physiological status of the mini tubers is well defined because they are synchronized with respect to age and size (Visser et al., 1994), thus providing a highly reproducible experimental system. In contrast, the physiological status of tubers harvested from normal potato plants varies, since some tubers may be actively growing while others of the same size are resting, as evidenced by a much lower activity of the starch biosynthetic machinery. Mini tubers thus constitute a suitable experimental system for the production of 33P-labeled phosphorylated amylopectin-derived glucan chains. The elution profile of the phosphorylated glucans obtained by HPAEC/PAD (Fig. 2A) was similar to the elution profiles of phosphorylated glucans isolated from fully grown potato plants (Blennow et al., 1998a). The marked preferential occurrence of phosphate in amylopectin compared with amylose suggests a functional link between the branching reactions and the mechanism of starch phosphorylation. The first studies on the substrate specificity of potato SBE using defined substrates were conducted by Borovsky and co-workers (Borovsky et al., 1975a, 1975b,1976). However, there are no reports in the literature on investigations using phosphorylated glucans as a substrate for any of the isoforms of SBE. To specifically monitor the occurrence of chain-transfer reactions, the33P-labeled phosphorylated α(1→4)glucans were used in combination with nonphosphorylated3H-end-labeled linear α(1→4)glucan chains (Fig. 6, A and B, respectively). The phosphorylated products were isolated by ion-exchange chromatography. Size fractionation by gel-filtration chromatography revealed the formation of dual-labeled (3H/33P) polysaccharides with masses in the range of 8,000 to 15,000 D, indicating that they are the products of chain-transfer reactions (Fig. 6C). The minor fraction in the mass range of 4,500 to 6,000 D (eluting around 320 mL) could be phosphorylated α(1→4)glucan chains that were not used by PSBE-I in the chain-transfer reaction or residual fragments cleaved by PSBE-I during the reaction. Two hypothetical PSBE-I catalyzed chain-transfer mechanisms are outlined in Figure 7. In reaction A, a product containing radioactivity originating from both the3H-labeled end group and the33P-labeled phosphate group is formed. Such products were indeed isolated (Fig. 5A), documenting that reaction A has taken place. Thus, we conclude that PSBE-I is capable of using a phosphorylated glucan as a donor chain. We can neither verify nor rule out that the enzyme also can use a phosphorylated glucan as an acceptor chain (reaction B). If reaction B proceeds, two mono-labeled products containing either 33P or 3H would be formed. In this case the 33P-labeled product would contain a negatively charged phosphate group, and this glucan would co-elute with the product formed in reaction A during anion-exchange chromatography. The third possibility of a chain-transfer reaction catalyzed by PSBE-I, namely an intra-chain transfer involving phosphorylated glucans, cannot be verified nor ruled out using the methods used in this study. A product originating from an intra-chain transfer process cannot be distinguished from the phosphorylated products isolated in Figure 5A, since it will co-elute with the other phosphorylated products due to the negatively charged phosphate group. Fig. 7. Open in new tabDownload slide Model of a simple inter-chain-transfer process mediated by potato SBE-I involving one phosphorylated α(1→4)glucan chain and one neutral chain. In reaction A, the phosphorylated α(1→4)glucan is used by PSBE-I as the donor chain. The phosphorylated α(1→4)glucan is cleaved and an α(1→6)linkage is formed to the 3H-labeled α(1→4)glucan. This reaction leaves an unlabeled residual fragment (RF). In reaction B, the phosphorylated chain is used as the acceptor chain by PSBE-I. In this reaction, the 3H-labeled end is cleaved off of the donor chain and subsequently an α(1→6)linkage is formed to the phosphorylated acceptor chain. This reaction leaves a residual fragment containing the 3H-labeled end group. Phosphate groups are indicated with •; ø represents the reducing end, whereas *ø represents a 3H-labeled end group. Arrows indicate the direction of formation of the α(1→6)glycosidic bond. Fig. 7. Open in new tabDownload slide Model of a simple inter-chain-transfer process mediated by potato SBE-I involving one phosphorylated α(1→4)glucan chain and one neutral chain. In reaction A, the phosphorylated α(1→4)glucan is used by PSBE-I as the donor chain. The phosphorylated α(1→4)glucan is cleaved and an α(1→6)linkage is formed to the 3H-labeled α(1→4)glucan. This reaction leaves an unlabeled residual fragment (RF). In reaction B, the phosphorylated chain is used as the acceptor chain by PSBE-I. In this reaction, the 3H-labeled end is cleaved off of the donor chain and subsequently an α(1→6)linkage is formed to the phosphorylated acceptor chain. This reaction leaves a residual fragment containing the 3H-labeled end group. Phosphate groups are indicated with •; ø represents the reducing end, whereas *ø represents a 3H-labeled end group. Arrows indicate the direction of formation of the α(1→6)glycosidic bond. SBE is localized on the starch granule surface (Kram et al., 1993) or strongly bound to starch granules (Larsson et al., 1996) and supposedly would integrate soluble-phosphorylated α(1→4)glucans into amylopectin by reaction A (Fig. 7). This would require long and unbranched glucan chains protruding from the granule surface, as proposed by Lineback (1986), and the existence of phosphorylated, soluble glucans. The possible involvement of PSBE-I in introducing soluble α(1→4)glucans into amylopectin is supported by an observed increase in the pool of soluble glucans in potato tubers in which the activity of PSBE-I has been lowered by antisense techniques (Kossmann et al., 1997). α(1→4)Glucans have been proposed to affect starch synthesis in potato tubers (Denyer et al., 1996). Potato tubers do contain small amounts of soluble, branched α(1→4)glucans, which can be detected by HPAEC analysis (data not shown). These glucans are probably synthesized in the amyloplast stroma by soluble, starch synthases or by a plastidic phosphorylase isoform with an affinity for low-molecular-mass linear α(1→4)glucans (Steup, 1988). The amount of soluble glucans that can be obtained is too low to permit an analysis of their phosphate content. Soluble α(1→4)glucans may also originate from the trimming or editing of the amylopectin molecule by debranching enzymes or amylases (Ball et al., 1996; Mouille et al., 1996). If phosphorylated α(1→4)glucans are derived from a glucan-trimming process, the chain transfer of phosphorylated α(1→4)glucans mediated by PSBE-I may not be the primary way of phosphorylating starch. In this case, it would constitute a way to reintroduce the liberated phosphorylated α(1→4)glucans into amylopectin. The current study documents that phosphorylated α(1→4)glucans participate in PSBE-I-catalyzed α-glucan-transfer reactions. It remains to be established whether this is a characteristic of potato SBEs and is thus a discriminating factor with respect to the formation of phosphorylated amylopectin, or whether SBEs from other plants likewise are able to utilize phosphorylated α(1→4)glucans for the production of phosphorylated amylopectin. The latter case would imply that the ability to form phosphorylated glucans is restricted to plants producing phosphorylated starch. ACKNOWLEDGMENTS We would like to thank Bente Wischmann and Anne Mette Bay-Smidt for their establishment and help with the mini tuber system. Abbreviations: DP degree of polymerization HPAEC high-performance anion-exchange chromatography PAD pulsed amperiometric detection PSBE-I potato starch-branching enzyme I SBE starch-branching enzyme LITERATURE CITED 1 Ball S Guan H-P James M Myers A Keeling P Mouille G Buléon A Colonna P Preiss J From glycogen to amylopectin: a model for the biogenesis of the plant starch granule. Cell 86 1996 349 352 Google Scholar Crossref Search ADS PubMed WorldCat 2 Bay-Smidt A Wischmann B Olsen CE Nielsen TH Starch bound phosphate in potato as studied by a simple method for determination of organic phosphate and 31P-NMR. Stärke 46 1994 167 172 Google Scholar OpenURL Placeholder Text WorldCat 3 Blennow A Bay-Smidt AM Olsen CE Wischmann B Møller BL The degree of starch phosphorylation is related to the chain length distribution of the neutral and the phosphorylated chains of amylopectin. Carbohydr Res 307 1998a 45 54 Google Scholar Crossref Search ADS WorldCat 4 Blennow A Viksø-Nielsen A Morell MK α-Glucan binding of potato tuber starch branching enzyme I as determined by tryptophan fluorescence quencing, affinity electrophoresis and steady state kinetics. Eur J Biochem 252 1998b 331 338 Google Scholar Crossref Search ADS WorldCat 5 Borovsky D Smith EE Whelan WJ Purification and properties of potato 1,4-α-d-glucan 6-α-(1, 4-α-glucano)-transferase. Eur J Biochem 59 1975a 615 625 Google Scholar Crossref Search ADS WorldCat 6 Borovsky D Smith EE Whelan WJ Temperature-dependence of the action of Q-enzyme and the nature of the substrate for Q-enzyme. FEBS Lett 54 1975b 201 205 Google Scholar Crossref Search ADS WorldCat 7 Borovsky D Smith EE Whelan WJ On the mechanism of amylose branching by potato Q-enzyme. Eur J Biochem 62 1976 307 312 Google Scholar Crossref Search ADS PubMed WorldCat 8 Denyer K Clarke B Hylton C Tatge H Smith AM The elongation of amylose and amylopectin chains in isolated starch granules. Plant J 10 1996 1135 1143 Google Scholar Crossref Search ADS WorldCat 9 Dubois M Gilles KA Hamilton JK Rebers PA Smith F Colorimetric method for determination of sugars and related substances. Anal Chem 28 1956 350 356 Google Scholar Crossref Search ADS WorldCat 10 Gidley MJ Bulpin PV Crystallization of malto-oligosaccharides as models of the crystalline forms of starch. Carbohydr Res 161 1987 291 300 Google Scholar Crossref Search ADS WorldCat 11 Hizukuri S Tabata S Nikuni Z Studies on starch phosphate. Part 1: estimation of glucose-6-phosphate residues in starch and the presence of other bound phosphate(s). Stärke 22 1970 338 343 Google Scholar Crossref Search ADS WorldCat 12 Kossmann J Büttcher V Abel GJW Duwenig E Emmermann M Frohberg C Lloyd JR Lorberth R Springer F Welsh T and others Starch biosynthesis and modifications of starch structure in transgenic plants. Macromol Symp 120 1997 29 38 Google Scholar Crossref Search ADS WorldCat 13 Kram AM Oostergetel GT van Bruggen EFJ Localization of starch branching enzyme in potato tuber cells with the use of immunoelectron microscopy. Plant Physiol 101 1993 237 243 Google Scholar Crossref Search ADS PubMed WorldCat 14 Larsson CT Hofvander P Khoshnoodi J Ek B Rask L Larsson H Three isoforms of starch synthase and two isoforms of starch branching enzyme are present in potato tuber starch. Plant Sci 117 1996 9 16 Google Scholar Crossref Search ADS WorldCat 15 Lineback DR Current concepts of starch structure and its impact on properties. J Jpn Soc Starch Sci 33 1986 80 88 Google Scholar Crossref Search ADS WorldCat 16 Mouille G Maddelein M-L Libessart N Talaga P Decq A Delrue B Ball S Preamylopectin processing: a mandatory step for starch biosynthesis in plants. Plant Cell 8 1996 1353 1366 Google Scholar Crossref Search ADS PubMed WorldCat 17 Nielsen TH Wischmann B Enevoldsen K Møller BL Starch phosphorylation in potato tubers proceeds concurrently with de novo biosynthesis of starch. Plant Physiol 105 1994 111 117 Google Scholar Crossref Search ADS PubMed WorldCat 18 Nikuni Z Hizukuri S Kamagi K Hasegava H Moriwaki T Fukui T Doi K Nara S Maeda I The effect of temperature during maturation period on the physico-chemical properties of potato and rice starches. Mem Sci Indstr Res Osaka Univ 26 1969 1 27 Google Scholar OpenURL Placeholder Text WorldCat 19 Rooke HS Lampitt LH Jackson EM The phosphorous compounds of wheat starch. Biochem J 45 1949 231 236 Google Scholar Crossref Search ADS PubMed WorldCat 20 Steup M Starch degradation. Preiss J The Biochemistry of Plants. 1988 255 296 Academic Press London, UK 21 Takeda Y Hizukuri S Location of phosphate groups in potato amylopectin. Carbohydr Res 102 1982 321 327 Google Scholar Crossref Search ADS WorldCat 22 Viksø-Nielsen A Blennow A Purification of starch branching enzyme from potato using γ-cyclodextrin affinity chromatography. J Chromatogr A 800 1998 382 385 Google Scholar Crossref Search ADS WorldCat 23 Visser RGF Vreugdenhil D Hendriks T Jacobsen E Gene expression and carbohydrate content during stolon to tuber transition in potatoes (Solanum tuberosum). Physiol Plant 90 1994 285 292 Google Scholar Crossref Search ADS WorldCat Author notes 1 This work was financially supported by the European Union Fishery and Agiculture Industrial Research program and by the Danish Food Technology Program (Føtek II). * Corresponding author; e-mail [email protected]; fax 45–35–28–33–33. Copyright © 1998 American Society of Plant Physiologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Gronwald, John W.; Plaisance, Kathryn L.

doi: 10.1104/pp.117.3.877pmid: 9662530

Abstract Two glutathioneS-transferase (GST) isozymes, A1/A1 and B1/B2, were purified from etiolated,O-1,3-dioxolan-2-yl-methyl-2,2,2,-trifluoro-4′-chloroacetophenone-oxime-treated sorghum (Sorghum bicolor L. Moench) shoots. GST A1/A1, a constitutively expressed homodimer, had a subunit molecular mass of 26 kD and an isoelectric point of 4.9. GST A1/A1 exhibited high activity with 1-chloro-2, 4,dinitrobenzene (CDNB) but low activity with the chloroacetanilide herbicide metolachlor. For GST A1/A1, the random, rapid-equilibrium bireactant kinetic model provided a good description of the kinetic data for the substrates CDNB and glutathione (GSH). GST B1/B2 was a heterodimer with subunit molecular masses of 26 kD (designated the B1 subunit) and 28 kD (designated the B2 subunit) and a native isoelectric point of 4.8. GST B1/B2 exhibited low activity with CDNB and high activity with metolachlor as the substrate. The kinetics of GST B1/B2 activity with GSH and metolachlor fit a model describing a multisite enzyme having two binding sites with different affinities for these substrates. Both GST A1/A1 and GST B1/B2 exhibited GSH-conjugating activity with ethacrynic acid and GSH peroxidase activity with cumene hydroperoxide, 9-hydroperoxy-trans-10,cis-12-octadecadienoic acid and 13-hydroperoxy-cis-9,trans-11-octadecadienoic acid. Both GST A1/A1 and GST B1/B2 are glycoproteins, as indicated by their binding of concanavalin A. Polyclonal antibodies raised against GST A1/A1 exhibited cross-reactivity with the B1 subunit of GST B1/B2. Comparisons of the N-terminal amino acid sequences of the GST A1, B1, and B2 subunits with other type I θ-GSTs indicated a high degree of homology with the maize GST I subunit and a sugarcane GST. GSTs (EC 2.5.1.18) are dimeric enzymes found in mammals, insects, plants, and microbes that catalyze nucleophilic attack by the thiolate anion of GSH at electrophilic centers of hydrophobic molecules (Mannervik and Danielson, 1988). In addition to catalyzing GSH conjugation, GSTs also exhibit GSH peroxidase activity and ligand-binding functions (Mannervik and Danielson, 1988; Marrs, 1996). Mammalian GSTs compose a multigene family; in rat liver at least 13 different cytosolic GST subunits are found as either heterodimers or homodimers (Ketterer and Coles, 1991). Mammalian cytosolic GSTs have been divided into four classes (α, μ, π, and θ) based on immunological, biochemical, and sequence similarities (Buetler and Eaton, 1992). It is well established that mammalian GSTs play an important role in the detoxification of electrophilic xenobiotics (Mannervik and Danielson, 1988). Although endogenous substrates for mammalian GSTs have not been clearly defined, there is evidence that α-GSTs protect against oxidative stress by detoxifying reactive products generated by lipid peroxidation (Ålin et al., 1985; Ketterer and Coles, 1991; Singhal et al., 1992). In general, plant GSTs have not been as well characterized as mammalian GSTs. Plant cytosolic GSTs belong to the archaic θ class of GSTs (Meyer et al., 1991; Marrs, 1996). This class, which is very heterogeneous in primary structure, also includes GSTs from microbes, insects, and mammals (Buetler and Eaton, 1992;Pemble and Taylor, 1992). Plant θ-GSTs have been subdivided into three types (I, II, and III) based on amino acid sequence identity and conservation of intron:exon placement (Droog et al., 1995; Marrs, 1996). The best-characterized function of plant GSTs is their role in the detoxification of certain herbicide classes such as the chloroacetanilides, thiocarbamates, and s-triazines (Lamoureux and Rusness, 1989). Plant GSTs can be induced by biotic stimuli such as pathogen invasion and abiotic stimuli, such as herbicide safeners and heavy metals (Marrs, 1996, and refs. therein). Very little is known about endogenous substrates and functions of plant GSTs. Certain plant GSTs bind auxins as nonsubstrate ligands (Bilang et al., 1993; Zettl et al., 1994). A GST encoded by the maizebronze2 gene conjugates anthocyanin prior to transport into the vacuole via a tonoplast transporter (Marrs et al., 1995). There are also increasing reports of plant GSTs exhibiting GSH peroxidase activity (Williamson and Beverley, 1987, 1988; Bartling et al., 1993; Zettl et al., 1994; Flury et al., 1996), which suggests a role in protection against oxidative stress. Our previous investigations of sorghum (Sorghum bicolor) GSTs were conducted with relatively crude enzyme fractions (Gronwald et al., 1987; Dean et al., 1990). The objectives of this study were to develop a protocol to purify GSTs from etiolated sorghum shoots and to characterize isozymes that were purified to homogeneity. The protocol we developed yielded two purified isozymes: GST A1/A1, a constitutively expressed homodimer, and GST B1/B2, a heterodimer induced by the herbicide safener fluxofenim. Kinetic analysis revealed that GST A1/A1 was best described by a random, rapid-equilibrium bireactant kinetic (Bi-Bi) mechanism, whereas GST B1/B2 was best described by a multisite model that generated kinetic constants for each subunit. We also provide the first evidence to our knowledge that plant GSTs are glycosylated. MATERIALS AND METHODS Chemicals2 [U-14C]Metolachlor, specific activity 71.5 μCi mg−1, [U-14C]alachlor, specific activity 23.6 μCi mg−1, and [U-14C]atrazine, specific activity, 19.0 μCi mg−1, were provided by Novartis (Greensboro, NC). N-(1-14C)Propyl EPTC, specific activity, 35 mCi mmol−1, was provided by ICI Americas (now Zeneca Agricultural Products, Wilmington, DE). [14C]EPTC-sulfoxide was prepared and purified as described by Casida et al. (1975). GSH and chlorotriphenyltin were obtained from Aldrich. Polybuffer 74, Sephacryl S200, and epoxy-activated Sepharose 6B were purchased from Pharmacia. Jack bean α-mannosidase was purchased from Boehringer Mannheim. All other chemicals were obtained from Sigma. S-hexyl-GSH and S-hexyl-GSH-linked Sepharose 6B were synthesized as described by Mannervik and Guthenberg (1981). 4-Hydroxynonenal-diethylacetal, provided by Dr. H. Esterbauer (Institut für Biochemie, Universitat Granz, Granz, Austria), was converted to 4HNE by acid saponification (1 mm HCl, 1 h) prior to use. 9 c,t-HPO was synthesized as described by Matthew et al. (1977) and 13 c,t-HPO was synthesized as described byHamberg and Samuelsson (1967). Linoleic hydroperoxide was separated from the free acid as described by Matthew et al. (1977) with modifications. The extracted reaction mixture was evaporated under a vacuum, reconstituted in hexane:ether (98:2, v/v), and then applied to a silica gel column (1.5 × 26 cm) equilibrated in the same solvent. The column was washed with hexane:ether (98:2, v/v), and the hydroperoxide and unreacted linoleic acid were separated using a step gradient of increasing ether concentrations (five incremental increases of 20% over 30 mL) in hexane. Fractions were monitored at 234 nm. Hydroperoxides were identified by iodometric determination (Kokatnur and Jelling, 1941) and by their unique UV spectra. Plant Material Untreated and fluxofenim-treated (0.4 g kg−1 seed) sorghum (Sorghum bicolorL. Moench var DK41Y) seeds were provided by Novartis. Forty grams of untreated or fluxofenim-treated seeds was planted 2.5 cm deep in a plastic tray (50 × 30 × 6 cm) containing 5 L of vermiculite. Each tray of seeds was watered with 4 L of deionized water and allowed to drain. Trays were covered with aluminum foil and incubated for 72 h in the dark at 30°C and 70% RH. GST Dimer and Monomer Purification Twenty-five to forty grams of 2.0-cm apical sections from 72-h-old etiolated sorghum shoots was excised and frozen in liquid N2. The tissue was ground to a fine powder using a mortar and pestle. A premixed (30 min, 4°C) extraction buffer (0.2m Tris-HCl, pH 7.8, 1.0 mm EDTA, 5.0 mm 2-mercaptoethanol, and polyvinylpolypyrrolidone [5%, w/v]) was added to the powder (5 mL g−1tissue), and the slurry was ground with mortar and pestle. All additional purification steps were conducted at 4°C. The homogenate was sequentially filtered through two layers each of cheesecloth and Miracloth (Calbiochem) and centrifuged (23,500g, 20 min). The resulting supernatant was concentrated by ultrafiltration (Amicon, Beverly, MA) to approximately 20 mL using a PM30 membrane and applied to an S200 HR gel-filtration column (2.5 × 49 cm) equilibrated with buffer A (20 mm Tris-HCl, pH 7.8, 1 mmEDTA, and 5 mm 2-mercaptoethanol). Active fractions were pooled and applied directly to a S-hexyl-GSH-linked Sepharose 6B column (1.25 × 49 cm) equilibrated in buffer A. The column was washed using a 300-mL linear gradient of 0 to 0.2m NaCl in buffer A, and GST isozymes were eluted with a 300-mL linear gradient of 0 to 5 mmS-hexyl-GSH in 0.2 m NaCl at a flow rate of 1 mL min−1. GST isozymes, which eluted as a single peak at approximately 1.5 mmS-hexyl-GSH, were quickly dialyzed and concentrated using a Centricell 20 (30,000Mr cutoff, Polysciences, Warrington, PA). The sample was applied to an FPLC anion-exchange column (Mono-Q HR 5/5, Pharmacia) equilibrated in buffer A. The isozymes were separated using a linear gradient of 0 to 0.2m NaCl in 40 mL followed by isocratic elution (10 mL). Fractions (0.5 mL) were collected at a flow rate of 0.3 mL min−1. The peak of GST(CDNB) activity that eluted at approximately 50 mm NaCl (Fig. 1, A and B, peak 2) was applied to a chromatofocusing column (Mono-P HR 5/20, Pharmacia) equilibrated in 0.025 m piperazine-iminodiacetic acid, pH 6.3, and eluted with 35 mL of 10% polybuffer 74-iminodiacetic acid, pH 4.5. GST isozymes were concentrated and dialyzed in buffer A using a Centricon 10 (Amicon). All elution profiles are representative of experiments repeated multiple times. Fig. 1. Open in new tabDownload slide FPLC anion-exchange chromatography ofS-hexyl-GSH affinity-purified GST protein from untreated (A) and fluxofenim-treated (B) sorghum shoots. Fractions were assayed for GST(CDNB) activity (○) at pH 7.5 and for GST(metolachlor) activity (•) as described in Methods. Fig. 1. Open in new tabDownload slide FPLC anion-exchange chromatography ofS-hexyl-GSH affinity-purified GST protein from untreated (A) and fluxofenim-treated (B) sorghum shoots. Fractions were assayed for GST(CDNB) activity (○) at pH 7.5 and for GST(metolachlor) activity (•) as described in Methods. The subunits of GST A1/A1 and GST B1/B2 were purified using a 25-cm, 300-Å C18 reverse-phase HPLC column (Vydac, Hesperia, CA), as described by Ostlund Farrants et al. (1987). The solvents were water (solvent A) and 0.1% (v/v) trifluoroacetic acid in acetonitrile (solvent B). Purified GST A1/A1 or GST B1/B2 was injected onto the column at 35% solvent B, and a linear gradient (35–55% solvent B over 60 min) with a flow rate of 1.5 mL min−1 was used to resolve GST proteins. Protein was detected at 220 nm. Enzyme Assays GST(CDNB) and GST(DCNB) activities were determined spectrophotometrically as described by Habig et al. (1974) with modifications. For GST(CDNB) assays, the reaction medium contained 0.1m potassium phosphate buffer pH 7.5 or 6.5, 1.0 mm GSH, 1.0 mm CDNB, 1% absolute ethanol, and protein in a total volume of 1.0 mL. For GST(DCNB) assays, the reaction medium contained 0.1 m potassium phosphate buffer, pH 7.5, 5.0 or 1.0 mm GSH, 1.0 mm DCNB, 1% absolute ethanol, and protein in a total volume of 1.0 mL. The reaction, conducted at 25°C, was initiated by the addition of CDNB or DCNB, and the change in A340 orA345, respectively, was monitored for 120 s with a spectrophotometer (model DU-65, Beckman). All initial rates were corrected for the background nonenzymatic reaction. One unit of activity is defined as the formation of 1 μmol product min−1 at 25°C (extinction coefficient at 340 nm = 9.6 mm−1cm−1 for CDNB; extinction coefficient at 345 nm = 8.5 mm−1cm−1 for DCNB). GST (metolachlor, alachlor, EPTC-sulfoxide, and atrazine) activities were determined by measuring the amount of herbicide conjugate formed as previously described (Gronwald et al., 1987; Dean et al., 1991) with modifications. All assays contained 0.1 m potassium phosphate buffer (pH 7.5 for metolachlor assays, pH 7.0 for alachlor assays, pH 6.8 for EPTC-sulfoxide assays, and pH 6.5 for atrazine assays), 1.0 mm GSH, 5.0 μm[14C]herbicide (specific activity for metolachlor and alachlor, 5 μCi μmol−1; for EPTC-sulfoxide, 2 μCi μmol−1; and for atrazine, 4.5 μCi μmol−1), 2% absolute ethanol, and protein in a final volume of 0.5 mL. Assays were initiated by the addition of radiolabeled herbicide and incubated for 1 h at 25°C. Reactions were terminated by the addition of 0.05 mL of 55% TCA and/or 0.75 mL of methylene chloride. The nonconjugated herbicide was partitioned into the organic phase by vigorous shaking for 2 min followed by centrifugation (10,000g, 5 min). A 100-μL aliquot from the aqueous phase was added to 5 mL of Ecolume (ICN) and radioactivity was counted using liquid scintillation spectroscopy. GSH conjugates of the herbicides were identified by TLC with authentic standards. The enzymatic rate of conjugation was corrected for the background nonenzymatic rate. For metolachlor, alachlor, atrazine, and EPTC-sulfoxide, 1 unit of activity is defined as the formation of 1 nmol conjugate h−1 at 25°C. Enzyme assays with ethacrynic acid were performed as described by Habig and Jakoby (1981) with the exception that the GSH concentration was increased from 0.25 to 1.0 mm in some experiments. GST assays for 4HNE were conducted as described by Ålin et al. (1985)except that the pH was increased from 6.5 to 7.5 and the GSH concentration was increased to 1.0 mm in some experiments. GST activity with cumene hydroperoxide, 9 c,t-HPO, and 13 c,t-HPO was determined using a coupled assay that measures production of GSSG (Awasthi et al., 1975). The assay medium contained 0.1 m potassium phosphate buffer, pH 7.0, 0.2 mm NADPH, 4.0 mm GSH, 1 unit of GSH reductase (type III, Sigma), 0.1 mm hydroperoxide, and 0.02 mL (1.5–3.0 μg) of purified GST isozyme in 1.0 mL. The reaction was started by the addition of hydroperoxide. All assays were conducted at 30°C (except those with ethacrynic acid, which were conducted at 25°C). Kinetic Analysis For GST A1/A1 kinetic studies, initial velocities were determined at pH 7.5 using the spectrophotometric assay described above. GSH concentrations were varied from 20 to 240 μm at fixed concentrations of CDNB that varied from 0.5 to 2.0 mm. Assay volume and ethanol concentration remained constant. Initial velocities for GST B1/B2 were determined using the standard metolachlor assay conditions described above. GSH concentrations were varied from 20 to 1280 μm at a fixed metolachlor concentration of 640 μm. Metolachlor concentrations were varied from 5 to 640 μm at a fixed GSH concentration of 1280 μm. Assay volume and ethanol concentration were kept constant. Data analysis involved determining the fit of initial-velocity data to two models: the random, rapid-equilibrium Bi-Bi model (Eq. 1) and the random, steady-state Bi-Bi model (Eq. 2). v=Vmax[A][B]αKAKB+αKA[B]+αKB[A]+[A][B]Equation 1 v=V1[A][B]+V2[A]2[B]+V3[A][B]2K1+K2[A]+K3[B]+[A][B]+K4[A]2 +K5[B]2+K6[A]2[B]+K7[A][B]2Equation 2 where v is the initial velocity; [A] is the concentration of one substrate and [B] is the concentration of the other substrate; KA andKB are dissociation constants with the free enzyme for substrates A and B, respectively; α is the parameter describing the influence of the binding of one substrate on the binding of the second; and V1,V2, and V3 andK1 through K7are combined velocity and rate constants, respectively. Nomenclature and definitions are those of Segel (1975). Kinetic constants were determined using the Grafit 3.0 computer program (Erithicus Software, Staines, UK). Goodness of fit of initial-velocity data to bireactant kinetic models was evaluated using the criteria described by Mannervik (1996). For GST B1/B2, the initial-velocity data were graphed using Eadie-Hofstee plots, and initial estimates of kinetic constants for high- and low-affinity sites were made. In initial estimates of kinetic constants, GSH and metolachlor concentrations of less than 60 and 20 μm, respectively, were used to generate constants for the high-affinity site, and concentrations greater than 320 and 80 μm, respectively, were used for determining kinetic constants for the low-affinity site. Linear regression was used to fit the data in the defined concentration ranges to a straight line and kinetic constants (Km andVmax) were determined from the axial intercepts. To correct for the contribution of the high-affinity site at high substrate concentrations and of the low-affinity site at low substrate concentrations, the method of successive corrections as described by Spears et al. (1971) was used. For the cycles of correction, GSH concentrations below 100 μm were used for the high-affinity site and those above 100 μm for the low-affinity site. For metolachlor, substrate concentrations of less than 50 μm were used for the high-affinity site and those greater than 50 μm were used for the low-affinity site. The initial estimates of Km andVmax for the high-affinity site were used to calculate the velocity of the high-affinity site at high substrate concentrations. The calculated velocities were subtracted from the observed velocities in the high substrate concentration range. From these corrected velocities in the high concentration range, a regression line of v and v/[S] was calculated and values for Vmax andKm for the low-affinity site were derived. These kinetic parameters were then used to calculate the velocity contribution by the low-affinity site in the low concentration range. New values for Vmax andKm for the high-affinity site were calculated from the corrected velocities as described above. Three cycles of successive corrections were performed for each substrate to obtain the corrected kinetic constants for the low- and high-affinity sites on GST B1/B2. Using the corrected kinetic constants, the predicted velocity of GST(metolachlor) activity for GST B1/B2 was calculated over the substrate concentration ranges examined using Equation 3: Vpredicted=V1+V2=[S]Vmax1Km1+[S]+[S]Vmax2Km2+[S]Equation 3 where V1, Vmax1, and Km1 are kinetic parameters for the corrected low-affinity site, andV2, Vmax2, and Km2 are the corrected kinetic parameters for the high-affinity site. Native Molecular Mass Native molecular mass of the GST isozymes was determined using gel-filtration chromatography. Purified GST A1/A1 or GST B1/B2 was applied to a Superose 12 column (Pharmacia) in buffer A (described above) containing 0.1 m KCl. The Superose 12 column was calibrated with BSA (66 kD), ovalbumin (45 kD), carbonic anhydrase (29 kD), and lysozyme (14 kD) in the same buffer. Apparent molecular mass of the enzyme was determined by interpolation of linear plots of logMr versus RF. Gel Electrophoresis Molecular mass of GST subunits was determined by SDS-PAGE on 8% to 25% Phast gels (Pharmacia). Native pI was determined using Phast gels (IEF 4–6.5). Gels were silver stained using the protocol of the manufacturer. Determination of I50 Values I50 values were determined for GST A1/A1 using CDNB and GSH as the substrates, and for GST B1/B2 using metolachlor and GSH as the substrates. Standard assay conditions for these substrates were as described above. I50values were obtained by nonlinear regression analysis of the appropriate data by using the Grafit 3.0 computer program. α-Mannosidase Treatment The conditions for the digestion of GST A1/A1 and GST B1/B2 with α-mannosidase were adapted from those of Haselbeck and Hösel (1988). Purified GST A1/A1 (20 μg) and GST B1/B2 (10 μg) were denatured by boiling for 2 min in buffer B (50 mm sodium citrate, pH 4.8, and 3 mm MgCl2) containing 1% (w/v) SDS and 1% (v/v) 2-mercaptoethanol. SDS was then diluted 5-fold by the addition of buffer B containing 0.5% (w/v) octylglucoside and 0.1 mm PMSF, and the protein was boiled again for 2 min. The reaction mixture was cooled to room temperature and 0.2 unit of α-mannosidase was added. After incubation overnight at 37°C, the reaction was dialyzed against buffer B and concentrated (Centricon 10, Amicon). Detection of Glycosylation HPLC-purified GST subunits A1, B1, and B2 and native GST A1/A1 and GST B1/B2 (with or without pretreatment with α-mannosidase as described above) were subjected to SDS-PAGE on 20% Phast gels. Protein was transferred to Immobilon P membranes (Millipore) as described byBraun and Abraham (1989) using 10 mm CAPS (3-cyclohexylamino-1-propanesulfonic acid), pH 11.0, and 50 mm NaCl as the transfer buffer. Blots were blocked overnight with buffer C (500 mm NaCl, 80 mmTris-HCl, pH 7.6, and 0.1% Tween 20) and then incubated for 1 h in buffer C containing 5 μg mL−1 ConA-biotin. After two 10-min washes in buffer D (0.05% Tween 20, 137 mm NaCl, 3 mm KCl, and 25 mmTris-HCl, pH 7.4), blots were incubated for 1 h with a 1:5000 dilution of avidin-alkaline phosphatase in buffer C. Blots were again washed with buffer D, and alkaline phosphatase activity was detected by the addition of 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium, as described by Blake et al. (1984). Polyclonal Antibody Production Purified GST A1/A1 protein (50 μg in 100 μL of PBS) and complete Freund's adjuvant were injected as a 1:1 emulsion into the upper wing muscles of a hen. A 25-μg booster injection using incomplete Freund's adjuvant was administered 4 weeks after the initial injection. Eggs were collected 7 to 10 d after injections and chicken IgG was purified from egg yolks as described by Jensenius et al. (1981). Titer was determined by antibody capture immunoassay (Harlow and Lane, 1988) using purified GST A1/A1. Antibody Cross-Reactivity GST A1/A1 and B1/B2 subunits purified by reverse-phase HPLC as described above were subjected to SDS-PAGE using 20% homogenous Phast gels. Protein was transferred to Immobilon P membranes as described byBraun and Abraham (1989) using 10 mm CAPS, pH 11.0, and 50 mm NaCl as the transfer buffer. Blots were blocked overnight with buffer C described above. Blots were then incubated for 1 h with 6 μg mL−1 purified GST A1/A1 antibody in buffer C. After two 10-min washes in buffer D (described above), blots were incubated with a 1:15,000 dilution of anti-chicken IgG-alkaline phosphatase in buffer C for 1 h. The blots were again washed twice for 10 min with buffer D. Alkaline phosphatase activity was detected by the addition of 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium as described by Blake et al. (1984). N-Terminal Sequencing Purified GST A1, B1, and B2 subunits were obtained by reverse-phase HPLC as described above. HPLC fractions containing the subunits from multiple runs were pooled and the volume was reduced to 50 μL using a vacuum concentrator (Savant, Farmingdale, NY). Sequence was obtained by automated Edman degradation (model 430B Sequenator, Applied Biosystems) at the Microchemical Facility of the Institute of Human Genetics (University of Minnesota, Minneapolis). Protein Determination Protein was determined using the Bio-Rad assay (Bradford, 1976) with δ-globulin as the standard. RESULTS Purification of GST A1/A1 and B1/B2 For both untreated and fluxofenim-treated etiolated sorghum shoots, gel filtration and S-hexyl-GSH affinity chromatography yielded a fraction containing purified GST proteins in the expected range of 25 to 28 kD (data not shown). The purified GST fraction from both untreated and safener-treated sorghum shoots was applied to an FPLC (Mono-Q) anion-exchange column and eluted with a salt gradient. Fractions were collected and assayed for GST(CDNB) and GST(metolachlor) activity. In untreated shoots (Fig. 1A), there were two peaks exhibiting GST activity with CDNB but little or no activity with metolachlor. The first peak (peak 1) did not adhere to the column and was collected in the flow-through. The second peak (peak 2) bound to the Mono-Q column and eluted at approximately 50 mmNaCl. Fluxofenim-treatment induced six GST peaks (peaks 3 through 8) that exhibited activity with metolachlor but little or no activity with CDNB (Fig. 1B). The constitutively expressed peaks 1 and 2 were also present in fluxofenim-treated shoots. Although there was some variability between isolations, in most cases safener treatment increased the GST(CDNB) activity of peak 2. A trend toward less induction of GST(CDNB) activity of peak 2 relative to peak 1 was observed with aging of fluxofenim-treated seeds (data not shown). The lack of binding by peak 1 (Fig. 1) was not due to column overloading, since the peak was not retained when reapplied to the column. Reapplying peak 2 to the column resulted in elution at the original salt concentration and did not lead to the appearance of the nonbinding peak 1. It was considered that the lack of binding of GSTs in peak 1 may have been due to retention ofS-hexyl-GSH, which was used to elute GST protein from theS-hexyl-GSH affinity column. However, extensive dialysis of GST protein in peak 1 to remove any bound S-hexyl-GSH did not result in binding of peak 1 protein to the column. The Mono-Q elution profiles for GST(CDNB) and GST(metolachlor) activity in untreated and fluxofenim-treated sorghum shoots (Fig. 1) were similar but not the same as those previously reported by Dean et al. (1990). The profiles were similar in that they showed that fluxofenim (formerly CGA-133205) treatment induces multiple GST isozymes that exhibit relatively high activity with a herbicidal concentration of metolachlor (5 μm). However, the Mono-Q elution profiles shown in Figure 1 differ from those of Dean et al. (1990) in the number of peaks of GST activity and the salt concentrations at which they eluted. These differences may be due to the fact that Dean et al. (1990) used a different sorghum variety and Mono-Q chromatography was performed with a crude, desalted extract instead of with purified GST protein. Although SDS-PAGE of peak 2 (Fig. 1) indicated a single band at 26 kD, native IEF gel electrophoresis indicated the presence of three isozymes in this fraction (data not shown). FPLC Mono-P chromatofocusing was used to resolve the constitutively expressed isozymes in peak 2. For both untreated and safener-treated shoots, three peaks (2a, 2b, and 2c) eluted at approximately the same pI values, 4.95 to 5.00, 4.75 to 4.90, and 4.55 to 4.60, respectively (Fig. 2). The three peaks did not elute in the same fractions because of differences in the amount of protein in the peak 2 fraction from untreated and safener-treated shoots. Of the three peaks, safener induction was evident for peak 2c, which exhibited a high level of activity with CDNB, low activity with alachlor, and little or no activity with metolachlor. Although, as mentioned above, there was variability in the relative induction of GST(CDNB) activity of peak 2 compared with peak 1 in fluxofenim-treated seeds (Fig. 1), an increase in GST(CDNB) activity of peak 2c (Fig. 2B) was always observed in safener-treated shoots. Fig. 2. Open in new tabDownload slide Chromatofocusing of peak 2 (Fig. 1) from untreated (A) and fluxofenim-treated (B) sorghum shoots. Fractions were assayed for GST(CDNB) (○), GST(metolachlor) (•), and GST(alachlor) (▴) activity as described in Methods. GST(CDNB) activity is expressed as micromoles per minute per milliliter; GST(metolachlor) and GST(alachlor) activities are expressed as nanomoles per hour per milliliter. Fig. 2. Open in new tabDownload slide Chromatofocusing of peak 2 (Fig. 1) from untreated (A) and fluxofenim-treated (B) sorghum shoots. Fractions were assayed for GST(CDNB) (○), GST(metolachlor) (•), and GST(alachlor) (▴) activity as described in Methods. GST(CDNB) activity is expressed as micromoles per minute per milliliter; GST(metolachlor) and GST(alachlor) activities are expressed as nanomoles per hour per milliliter. Preliminary experiments using SDS-PAGE and native IEF indicated that most peaks in the Mono-Q (Fig. 1B) and Mono-P (Fig. 2B) elution profile of fluxofenim-treated sorghum shoots contained multiple GST isozymes. For example, peak 5 (Fig. 1B) contained at least four native isozymes (data not shown). However, it was determined that peak 2c (Fig. 2B) and peak 6 (Fig. 1B) in safener-treated shoots contained single isozymes that were designated GST A1/A1 and GST B1/B2, respectively. As indicated by SDS-PAGE, GST A1/A1 is a homodimer with a subunit molecular mass of 26 kD, whereas GST B1/B2 is a heterodimer composed of 26-kD (B1) and 28-kD (B2) subunits in equal proportions (Fig.3). Fig. 3. Open in new tabDownload slide SDS-PAGE of GST A1/A1 (Fig. 2B, peak 2c) and GST B1/B2 (Fig. 1B, peak 6). Lanes 1 and 4, Molecular mass standards; lane 2, 100 ng of purified GST A1/A1; lane 3, 100 ng of purified GST B1/B2. Protein was visualized by silver staining. Fig. 3. Open in new tabDownload slide SDS-PAGE of GST A1/A1 (Fig. 2B, peak 2c) and GST B1/B2 (Fig. 1B, peak 6). Lanes 1 and 4, Molecular mass standards; lane 2, 100 ng of purified GST A1/A1; lane 3, 100 ng of purified GST B1/B2. Protein was visualized by silver staining. That both GST A1/A1 and GST B1/B2 represented single isozymes was confirmed by native IEF gels, which showed the presence of a single band (Fig. 4). Native IEF gels of GST A1/A1 and GST B1/B2 also indicated that both isozymes are acidic proteins with pI values of 4.9 and 4.8, respectively. NativeMr values for GST A1/A1 and GST B1/B2 determined by gel filtration were 43,000 and 50,000, respectively, indicating that both isozymes are dimers. The nativeMr for GST B1/B2 is close to the calculated value of 54,000. However, the native Mr for GST A1/A1 is significantly less than the calculated value of 52,000. These results indicate that GST A1/A1 and GST B1/B2 do not share a similar topography. Fig. 4. Open in new tabDownload slide Native IEF gels of GST A1/A1 (Fig. 2B, peak 2c) and GST B1/B2 (Fig. 1B, peak 6). A, Lane 1, IEF standards; lane 2, 300 ng of purified GST A1/A1. B, Lane 1, IEF standards; lane 2, 300 ng of purified GST B1/B2. Protein was visualized by silver staining. Fig. 4. Open in new tabDownload slide Native IEF gels of GST A1/A1 (Fig. 2B, peak 2c) and GST B1/B2 (Fig. 1B, peak 6). A, Lane 1, IEF standards; lane 2, 300 ng of purified GST A1/A1. B, Lane 1, IEF standards; lane 2, 300 ng of purified GST B1/B2. Protein was visualized by silver staining. Purification tables for GST A1/A1 and GST B1/B2 are provided in Tables I andII, respectively. S-hexyl-GSH affinity-purified GST protein represented approximately 1.3% of total soluble protein in crude extracts from safener-treated, etiolated shoots. This compares favorably with GST proteins composing 1 to 2% of the total soluble protein in etiolated maize shoots (Mozer et al., 1983). An important step in the purification was theS-hexyl-GSH affinity column. This column bound greater than 90% of the applied GST(CDNB) and GST(metolachlor) activity. Although the use of the column resulted in a large increase in specific activity, there was a significant reduction in yield. Previous investigations using S-hexyl-GSH affinity columns to purify plant GSTs have indicated that yield is sacrificed for purity (Williamson and Beverley, 1988; Irzyk and Fuerst, 1993). It should be noted that the fold purification of GST A1/A1 and GST B1/B2 indicated in Tables I and II is underestimated, since the crude fractions from sorghum shoots contained several isozymes active with the substrates (CDNB or metolachlor) assayed during purification. Table I. Purification of GST A1/A1 (Fig. 2B, peak 2c) from fluxofenim-treated sorghum shoots Fraction . Total Protein . Total Units . Specific Activity . Yield-a . Purification-a . mg μmol min−1 μmol min−1 mg−1 % -fold Crude extract 203 154 0.76 100 1 Gel filtration (S200) 99 159 1.61 103 2 Affinity (S-hexyl-GSH) 2.6 88 34 57 45 Anion-exchange (Mono-Q) 0.4 24 54 16 71 Chromatofocusing (Mono-P) 0.016 1.3 81 0.8 107 Fraction . Total Protein . Total Units . Specific Activity . Yield-a . Purification-a . mg μmol min−1 μmol min−1 mg−1 % -fold Crude extract 203 154 0.76 100 1 Gel filtration (S200) 99 159 1.61 103 2 Affinity (S-hexyl-GSH) 2.6 88 34 57 45 Anion-exchange (Mono-Q) 0.4 24 54 16 71 Chromatofocusing (Mono-P) 0.016 1.3 81 0.8 107 F0-a Yield and purification values are based on total GST(CDNB) activity in crude extracts. Open in new tab Table I. Purification of GST A1/A1 (Fig. 2B, peak 2c) from fluxofenim-treated sorghum shoots Fraction . Total Protein . Total Units . Specific Activity . Yield-a . Purification-a . mg μmol min−1 μmol min−1 mg−1 % -fold Crude extract 203 154 0.76 100 1 Gel filtration (S200) 99 159 1.61 103 2 Affinity (S-hexyl-GSH) 2.6 88 34 57 45 Anion-exchange (Mono-Q) 0.4 24 54 16 71 Chromatofocusing (Mono-P) 0.016 1.3 81 0.8 107 Fraction . Total Protein . Total Units . Specific Activity . Yield-a . Purification-a . mg μmol min−1 μmol min−1 mg−1 % -fold Crude extract 203 154 0.76 100 1 Gel filtration (S200) 99 159 1.61 103 2 Affinity (S-hexyl-GSH) 2.6 88 34 57 45 Anion-exchange (Mono-Q) 0.4 24 54 16 71 Chromatofocusing (Mono-P) 0.016 1.3 81 0.8 107 F0-a Yield and purification values are based on total GST(CDNB) activity in crude extracts. Open in new tab Table II. Purification of GST B1/B2 (Fig. 1B, peak 6) from fluxofenim-treated sorghum shoots Fraction . Total Protein . Total Units . Specific Activity . Yield1-a . Purification1-a . mg nmol h−1 nmol h−1 mg−1 % -fold Crude extract 203 278 1.37 100 1 Gel filtration (S200) 99 244 2.46 88 2 Affinity (S-hexyl-GSH) 2.6 54 21 19 15 Anion-exchange (Mono-Q) 0.0075 1.9 260 0.7 190 Fraction . Total Protein . Total Units . Specific Activity . Yield1-a . Purification1-a . mg nmol h−1 nmol h−1 mg−1 % -fold Crude extract 203 278 1.37 100 1 Gel filtration (S200) 99 244 2.46 88 2 Affinity (S-hexyl-GSH) 2.6 54 21 19 15 Anion-exchange (Mono-Q) 0.0075 1.9 260 0.7 190 F1-a Yield and purification values are based on total GST(metolachlor) activity in crude extracts. Open in new tab Table II. Purification of GST B1/B2 (Fig. 1B, peak 6) from fluxofenim-treated sorghum shoots Fraction . Total Protein . Total Units . Specific Activity . Yield1-a . Purification1-a . mg nmol h−1 nmol h−1 mg−1 % -fold Crude extract 203 278 1.37 100 1 Gel filtration (S200) 99 244 2.46 88 2 Affinity (S-hexyl-GSH) 2.6 54 21 19 15 Anion-exchange (Mono-Q) 0.0075 1.9 260 0.7 190 Fraction . Total Protein . Total Units . Specific Activity . Yield1-a . Purification1-a . mg nmol h−1 nmol h−1 mg−1 % -fold Crude extract 203 278 1.37 100 1 Gel filtration (S200) 99 244 2.46 88 2 Affinity (S-hexyl-GSH) 2.6 54 21 19 15 Anion-exchange (Mono-Q) 0.0075 1.9 260 0.7 190 F1-a Yield and purification values are based on total GST(metolachlor) activity in crude extracts. Open in new tab Characterization of GST A1/A1 and GST B1/B2 Substrate Specificity A comparison of the substrate specificities of GST A1/A1 and GST B1/B2 is presented in Table III. Two assay conditions were used for the model substrates CDNB and DCNB. The first condition was that developed by Habig et al. (1974) for assaying rat liver GSTs; the second involved a change in either pH or substrate concentration that resulted in higher activity. For GST A1/A1, GST(CDNB) activity was higher when assayed at pH 7.5 than at pH 6.5. For DCNB, activity was higher when the GSH concentration was reduced from 5 to 1 mm. These results indicate that standard assay conditions used to measure the activity of mammalian GSTs for the model substrates CDNB and DCNB may not be optimal for plant GSTs. GST A1/A1 exhibited relatively high activity with CDNB but low activity with DCNB. Compared with GST A1/A1, GST B1/B2 exhibited lower activity with CDNB and no activity with DCNB. Table III. Activity of GST A1/A1 and GST B1/B2 with various substrates Substrate . [GSH] . pH . GST A1/A1 . GST B1/B2 . mm units2-amg−1 CDNB (1.00 mm) 1.00 6.5 29.27  ± 0.92 7.32  ± 0.85 1.00 7.5 46.90  ± 0.64 10.41  ± 0.53 DCNB (1.00 mm) 5.00 7.5 0.13  ± 0.01 N.D.2-b 1.00 7.5 0.29  ± 0.06 N.D.  Metolachlor (0.005 mm) 1.00 7.5 0.77  ± 0.26 54.07  ± 1.47 Alachlor (0.005 mm) 1.00 7.0 7.00  ± 0.60 63.37  ± 1.86 Atrazine (0.005 mm) 1.00 6.5 1.11  ± 0.24 4.62  ± 0.81 EPTC-sulfoxide (0.005 mm) 1.00 6.8 N.D. 7.97  ± 0.75 Cumene hydroperoxide (0.100 mm) 4.00 7.0 0.34  ± 0.11 0.28  ± 0.12 9 c,t-Linoleic hydroperoxide (0.100 mm) 4.00 7.0 1.71  ± 0.20 1.24  ± 0.04 13 c,t-Linoleic hydroperoxide (0.100 mm) 4.00 7.0 0.38  ± 0.10 1.21  ± 0.05 4HNE (0.100 mm) 0.50 6.5 N.D. 0.07  ± 0.01 1.00 7.5 N.D. 0.13  ± 0.02 Ethacrynic acid (0.200 mm) 0.25 6.5 0.26  ± 0.04 0.32  ± 0.03 1.00 6.5 2.34  ± 0.28 0.41  ± 0.03 Substrate . [GSH] . pH . GST A1/A1 . GST B1/B2 . mm units2-amg−1 CDNB (1.00 mm) 1.00 6.5 29.27  ± 0.92 7.32  ± 0.85 1.00 7.5 46.90  ± 0.64 10.41  ± 0.53 DCNB (1.00 mm) 5.00 7.5 0.13  ± 0.01 N.D.2-b 1.00 7.5 0.29  ± 0.06 N.D.  Metolachlor (0.005 mm) 1.00 7.5 0.77  ± 0.26 54.07  ± 1.47 Alachlor (0.005 mm) 1.00 7.0 7.00  ± 0.60 63.37  ± 1.86 Atrazine (0.005 mm) 1.00 6.5 1.11  ± 0.24 4.62  ± 0.81 EPTC-sulfoxide (0.005 mm) 1.00 6.8 N.D. 7.97  ± 0.75 Cumene hydroperoxide (0.100 mm) 4.00 7.0 0.34  ± 0.11 0.28  ± 0.12 9 c,t-Linoleic hydroperoxide (0.100 mm) 4.00 7.0 1.71  ± 0.20 1.24  ± 0.04 13 c,t-Linoleic hydroperoxide (0.100 mm) 4.00 7.0 0.38  ± 0.10 1.21  ± 0.05 4HNE (0.100 mm) 0.50 6.5 N.D. 0.07  ± 0.01 1.00 7.5 N.D. 0.13  ± 0.02 Ethacrynic acid (0.200 mm) 0.25 6.5 0.26  ± 0.04 0.32  ± 0.03 1.00 6.5 2.34  ± 0.28 0.41  ± 0.03 Values are means ± se. F2-a 1 unit = 1 μmol product produced per minute except for the herbicides (metolachlor, alachlor, atrazine, and EPTC-sulfoxide) where 1 unit = 1 nmol conjugate produced per hour. F2-b N.D., No detectable activity. Open in new tab Table III. Activity of GST A1/A1 and GST B1/B2 with various substrates Substrate . [GSH] . pH . GST A1/A1 . GST B1/B2 . mm units2-amg−1 CDNB (1.00 mm) 1.00 6.5 29.27  ± 0.92 7.32  ± 0.85 1.00 7.5 46.90  ± 0.64 10.41  ± 0.53 DCNB (1.00 mm) 5.00 7.5 0.13  ± 0.01 N.D.2-b 1.00 7.5 0.29  ± 0.06 N.D.  Metolachlor (0.005 mm) 1.00 7.5 0.77  ± 0.26 54.07  ± 1.47 Alachlor (0.005 mm) 1.00 7.0 7.00  ± 0.60 63.37  ± 1.86 Atrazine (0.005 mm) 1.00 6.5 1.11  ± 0.24 4.62  ± 0.81 EPTC-sulfoxide (0.005 mm) 1.00 6.8 N.D. 7.97  ± 0.75 Cumene hydroperoxide (0.100 mm) 4.00 7.0 0.34  ± 0.11 0.28  ± 0.12 9 c,t-Linoleic hydroperoxide (0.100 mm) 4.00 7.0 1.71  ± 0.20 1.24  ± 0.04 13 c,t-Linoleic hydroperoxide (0.100 mm) 4.00 7.0 0.38  ± 0.10 1.21  ± 0.05 4HNE (0.100 mm) 0.50 6.5 N.D. 0.07  ± 0.01 1.00 7.5 N.D. 0.13  ± 0.02 Ethacrynic acid (0.200 mm) 0.25 6.5 0.26  ± 0.04 0.32  ± 0.03 1.00 6.5 2.34  ± 0.28 0.41  ± 0.03 Substrate . [GSH] . pH . GST A1/A1 . GST B1/B2 . mm units2-amg−1 CDNB (1.00 mm) 1.00 6.5 29.27  ± 0.92 7.32  ± 0.85 1.00 7.5 46.90  ± 0.64 10.41  ± 0.53 DCNB (1.00 mm) 5.00 7.5 0.13  ± 0.01 N.D.2-b 1.00 7.5 0.29  ± 0.06 N.D.  Metolachlor (0.005 mm) 1.00 7.5 0.77  ± 0.26 54.07  ± 1.47 Alachlor (0.005 mm) 1.00 7.0 7.00  ± 0.60 63.37  ± 1.86 Atrazine (0.005 mm) 1.00 6.5 1.11  ± 0.24 4.62  ± 0.81 EPTC-sulfoxide (0.005 mm) 1.00 6.8 N.D. 7.97  ± 0.75 Cumene hydroperoxide (0.100 mm) 4.00 7.0 0.34  ± 0.11 0.28  ± 0.12 9 c,t-Linoleic hydroperoxide (0.100 mm) 4.00 7.0 1.71  ± 0.20 1.24  ± 0.04 13 c,t-Linoleic hydroperoxide (0.100 mm) 4.00 7.0 0.38  ± 0.10 1.21  ± 0.05 4HNE (0.100 mm) 0.50 6.5 N.D. 0.07  ± 0.01 1.00 7.5 N.D. 0.13  ± 0.02 Ethacrynic acid (0.200 mm) 0.25 6.5 0.26  ± 0.04 0.32  ± 0.03 1.00 6.5 2.34  ± 0.28 0.41  ± 0.03 Values are means ± se. F2-a 1 unit = 1 μmol product produced per minute except for the herbicides (metolachlor, alachlor, atrazine, and EPTC-sulfoxide) where 1 unit = 1 nmol conjugate produced per hour. F2-b N.D., No detectable activity. Open in new tab GST A1/A1 exhibited relatively low activity with the chloroacetanilide herbicides metolachlor and alachlor but was selective for alachlor over metolachlor. In contrast, GST B1/B2 exhibited relatively high activity with both metolachlor and alachlor. Both GST A1/A1 and GST B1/B2 exhibited low activity with atrazine as a substrate. GST A1/A1 displayed no activity with EPTC-sulfoxide, whereas GST B1/B2 exhibited activity with this substrate. GSH peroxidase and GSH-conjugating activities of GST A1/A1 and GST B1/B2 with products of lipid peroxidation were also examined (TableIII). Cumene hydroperoxide is a model substrate for determining GSH peroxidase activity with organic hydroperoxide substrates (Mannervik and Danielson, 1988). Both GST A1/A1 and GST B1/B2 exhibited GSH peroxidase activity with this substrate. For both isozymes, GSH peroxidase activity with 9 c,t-HPO was about 5-fold greater than that with cumene hydroperoxide as a substrate. GST A1/A1 was more active with 9 c,t-HPO, whereas GST B1/B2 exhibited equivalent activity with both 9 c,t-HPO and 13c,t-HPO. 4HNE is a toxic, α,β-unsaturated aldehyde that is generated by peroxidation of arachidonic acid in rat microsomes exposed to oxidative stress (Esterbauer et al., 1986). GST B1/B2 exhibited a low level of GSH conjugation activity with 4HNE (Table III) when measured using the mammalian GST assay of Ålin et al. (1985). However, modifying the assay medium by increasing GSH concentration from 0.5 to 1.0 mm and the pH from 6.5 to 7.5 resulted in about a 2-fold increase in the activity of GST B1/B2 with 4HNE. GST A1/A1 exhibited no activity with this substrate under either set of conditions. Ethacrynic acid is a phenylacetic acid derivative used as a diuretic (Ahokas et al., 1985). It contains an electrophilic group similar to α-β-alkenals generated in mammals under oxidative stress (Danielson et al., 1987; Berhane et al., 1994). GST A1/A1 and GST B1/B2 exhibited similar activities with this substrate when assayed at a GSH concentration of 0.25 mm (Table III), the concentration typically used to assay the activity of mammalian GSTs with ethacrynic acid (Habig and Jakoby, 1981). However, when the GSH concentration was increased to 1 mm, the activity of GST A1/A1 increased 9-fold, whereas the activity of GST B1/B2 increased only slightly. Kinetics Bireactant kinetic models were used to evaluate the kinetic mechanism that best described GST A1/A1 and GST B1/B2. For GST A1/A1, we analyzed the initial-velocity data using the random, steady-state Bi-Bi equation (Eq. 2) and the random, rapid-equilibrium Bi-Bi equation (Eq. 1). Initial-velocity data exhibited a poor fit to the random, steady-state model as indicated by large standard errors and negative values for several of the kinetic parameters. The random, rapid-equilibrium model (Eq. 1) provided a better fit of the data (Fig.5). Multiple intersecting lines were generated in the reciprocal plots for both CDNB and GSH, indicating that the mechanism was sequential. The common intersection point lies below the abscissa (α = 1.8), which indicates that binding of the first substrate (GSH or CDNB) decreases the enzyme's affinity for the second substrate (Segel, 1975). This α value is similar to that of a human placental GST (α = 2.1), which was also best described by the random, rapid-equilibrium model (Ivanetich and Goold, 1989). For GST A1/A1, the Km values for GSH and CDNB were 118 μm (Ks = 65 μm) and 1913 μm(Ks = 1063 μm), respectively. Although the random, rapid-equilibrium model provided a good fit to the initial-velocity data for GST A1/A1 (Fig. 5), additional experiments involving end-product inhibition would be required to definitively demonstrate a random, rapid-equilibrium mechanism (Segel, 1975). Fig. 5. Open in new tabDownload slide Double-reciprocal plots of GST(CDNB) activity for GST A1/A1. The initial-velocity data were fitted to the random, rapid-equilibrium Bi-Bi equation (Eq. 1). A, ○, 0.02 mm; •, 0.04 mm; □, 0.08 mm; and ▪, 0.16 mm GSH concentrations;Km for CDNB = 1.91 mm. B, ○, 0.5 mm; •, 1.0 mm; and □, 2.0 mm CDNB concentrations; Km for GSH = 0.118 mm. v is expressed as micromoles per minute.   Fig. 5. Open in new tabDownload slide Double-reciprocal plots of GST(CDNB) activity for GST A1/A1. The initial-velocity data were fitted to the random, rapid-equilibrium Bi-Bi equation (Eq. 1). A, ○, 0.02 mm; •, 0.04 mm; □, 0.08 mm; and ▪, 0.16 mm GSH concentrations;Km for CDNB = 1.91 mm. B, ○, 0.5 mm; •, 1.0 mm; and □, 2.0 mm CDNB concentrations; Km for GSH = 0.118 mm. v is expressed as micromoles per minute.   Reciprocal plots of the initial-velocity data for the heterodimer GST B1/B2 using the substrates GSH and metolachlor were nonlinear (data not shown). Attempts at fitting the initial-velocity data to the random, rapid-equilibrium model (Eq. 1) did not yield a random distribution of residuals. The data were then analyzed using the random, steady-state equation (Eq. 2) because the presence of the squared terms in this model predicts nonlinear, reciprocal plots (Segel, 1975). Nonlinear, reciprocal initial-velocity plots have been observed for rat liver GST 2–2 and GST 3–3 (Jakobson et al., 1979; Ivanetich et al., 1990), and the initial-velocity data for these isozymes were fitted to the random, steady-state model. However, attempts to fit the initial-velocity data for GST B1/B2 to this model generated parameter values with high standard errors and multiple negative values. The fact that GST B1/B2 is a heterodimer suggested another possible explanation for the biphasic kinetic data. Multisite enzymes with different affinities for the same substrate will yield nonlinear, reciprocal plots (Segel, 1975). Although multisite kinetic analysis was developed to determine kinetic constants for a mixture of two enzymes acting on the same substrate, it is also applicable for one enzyme that exhibits two binding sites differing in substrate affinity (Segel, 1975). It has been established that subunits of mammalian GST heterodimers are catalytically independent and that kinetic constants are additive (Danielson and Mannervik, 1985; Tahir and Mannervik, 1986). Therefore, the possibility was considered that the biphasic pattern for the initial-velocity data for GST B1/B2 reflects the contributions of two subunits that exhibit different affinities for the substrates GSH and metolachlor. Plotting the initial-velocity data for GST B1/B2 on Eadie-Hofstee plots (Fig. 6) yielded a biphasic pattern, suggesting a multisite enzyme with two substrate-binding sites with different affinities (Segel, 1975). Initial estimates of the kinetic parameters for each site were determined by fitting a straight line to the two linear portions of the graph. Kinetic parameters for the high-affinity site were derived from the straight lines generated by linear regression below GSH and metolachlor concentrations of 60 and 20 μm, respectively. For the low-affinity site, kinetic parameters were derived from the straight lines generated by linear regression for GSH and metolachlor concentrations greater than 320 and 80 μm, respectively. This approach yielded estimated Km values of 16 and 5 μm for GSH and metolachlor, respectively, at the high-affinity site, and 370 and 286 μm, respectively, at the low-affinity site. Fig. 6. Open in new tabDownload slide Eadie-Hofstee plots for GST(metolachlor) activity for GST B1/B2. The dashed lines were generated by three rounds of successive corrections, as described by Spears et al. (1971), and used to calculate corrected Km values for the low- and high-affinity sites. A, Metolachlor was varied from 5 to 640 μm at a saturating, fixed GSH concentration of 1280 μm. The Km values for metolachlor at the low- and high-affinity sites were 421 and 3 μm, respectively. B, GSH concentration was varied from 20 to 1280 μm at a saturating, fixed metolachlor concentration of 640 μm. TheKm values for GSH at the low- and high-affinity sites were 915 and 12 μm, respectively. For both A and B, the solid, curved line is the predicted velocity calculated when the corrected kinetic constants for the low- (short dashes) and high-affinity (long dashes) sites are substituted into Equation 3. v is expressed as nanomoles per hour. Fig. 6. Open in new tabDownload slide Eadie-Hofstee plots for GST(metolachlor) activity for GST B1/B2. The dashed lines were generated by three rounds of successive corrections, as described by Spears et al. (1971), and used to calculate corrected Km values for the low- and high-affinity sites. A, Metolachlor was varied from 5 to 640 μm at a saturating, fixed GSH concentration of 1280 μm. The Km values for metolachlor at the low- and high-affinity sites were 421 and 3 μm, respectively. B, GSH concentration was varied from 20 to 1280 μm at a saturating, fixed metolachlor concentration of 640 μm. TheKm values for GSH at the low- and high-affinity sites were 915 and 12 μm, respectively. For both A and B, the solid, curved line is the predicted velocity calculated when the corrected kinetic constants for the low- (short dashes) and high-affinity (long dashes) sites are substituted into Equation 3. v is expressed as nanomoles per hour. Derivation of these parameters directly from the linear portions of the graph assumes that at low substrate concentrations the low-affinity site does not contribute significantly to the measured velocity and that at high substrate concentrations, the high-affinity site does not contribute significantly to the measured velocity. However, this assumption generally does not hold true (Spears et al., 1971), which was found to be the case for GST B1/B2. Starting with the initial estimates of kinetic parameters for the high-affinity site, three rounds of successive corrections as described by Spears et al. (1971)were performed to calculate the correctedKm values for both sites. For the high-affinity subunit of GST B1/B2, this procedure yieldedKm values for GSH and metolachlor of 12 and 3 μm, respectively. The Kmvalues for GSH and metolachlor for the low-affinity subunit of GST B1/B2 were 915 and 421 μm, respectively. The calculatedVmax values for GST(metolachlor) activity of the low- and high-affinity subunits were 0.14 and 2.19 nmol h−1, respectively. As indicated in Figure 6, the predicted velocities calculated using the corrected kinetic constants (Eq. 3) are in close agreement with the measured velocities. I50 Values The effect of inhibitors of mammalian GSTs on the activity of GST A1/A1 and GST B1/B2 was examined (TableIV). GST A1/A1 was assayed with CDNB as the substrate, whereas GST B1/B2 was assayed using metolachlor as the substrate. Under our assay conditions for GST(metolachlor) activity (5 μm metolachlor, 1 mm GSH), we were primarily measuring the inhibitor sensitivity of the high-affinity subunit of GST B1/B2, which contributed approximately 75% of the observed velocity. Of the GST inhibitors examined, Cibracon blue was the most potent and, in general, the heterodimer GST B1/B2 was less sensitive to the inhibitors. Table IV. I50 values for GST A1/A1 and GST B1/B2 measured with selected inhibitors Inhibitor . GST A1/A13-a . GST B1/B23-b . μm S-hexyl-GSH 1048  ± 42 6520  ± 339 Chlorotriphenyltin 24.9  ± 1.4 39.7  ± 2.7 Sulfobromophthalein 7.1  ± 0.5 41.7  ± 5.8 Cibacron blue 0.57  ± 0.03 5.74  ± 0.07 Inhibitor . GST A1/A13-a . GST B1/B23-b . μm S-hexyl-GSH 1048  ± 42 6520  ± 339 Chlorotriphenyltin 24.9  ± 1.4 39.7  ± 2.7 Sulfobromophthalein 7.1  ± 0.5 41.7  ± 5.8 Cibacron blue 0.57  ± 0.03 5.74  ± 0.07 Values are means ± se. F3-a Assayed using CDNB and GSH as the substrates. F3-b Assayed using metolachlor and GSH as the substrates. Open in new tab Table IV. I50 values for GST A1/A1 and GST B1/B2 measured with selected inhibitors Inhibitor . GST A1/A13-a . GST B1/B23-b . μm S-hexyl-GSH 1048  ± 42 6520  ± 339 Chlorotriphenyltin 24.9  ± 1.4 39.7  ± 2.7 Sulfobromophthalein 7.1  ± 0.5 41.7  ± 5.8 Cibacron blue 0.57  ± 0.03 5.74  ± 0.07 Inhibitor . GST A1/A13-a . GST B1/B23-b . μm S-hexyl-GSH 1048  ± 42 6520  ± 339 Chlorotriphenyltin 24.9  ± 1.4 39.7  ± 2.7 Sulfobromophthalein 7.1  ± 0.5 41.7  ± 5.8 Cibacron blue 0.57  ± 0.03 5.74  ± 0.07 Values are means ± se. F3-a Assayed using CDNB and GSH as the substrates. F3-b Assayed using metolachlor and GSH as the substrates. Open in new tab Glycosylation Initial tests to determine whether GST A1/A1 and GST B1/B2 were glycosylated were conducted using the PAS reagent, which detects the presence of unsubstituted vicinyl hydroxyls of Man, Glc, and Gal (Dyer, 1956). Purified GST A1/A1 and GST B1/B2 were electrophoresed on SDS-PAGE Phast gels, blotted onto Immobilon P membranes, and treated with the PAS reagent, as described by Strömqvist and Gruffman (1992). Glycosylation was not detected for either isozyme using this method (data not shown). However, glycosylation of GST A1/A1 (Fig.7, lane 1) and GST B1/B2 (data not shown) was demonstrated using ConA-biotin/avidin-alkaline phosphatase. ConA binds specifically to Man and Glc residues and to glucosamine with lower affinity (Poretz and Goldstein, 1970). ConA-biotin, which contains six molecules of biotin per molecule of ConA, allows for low levels of glycosylation to be visualized. Other studies have demonstrated the greater sensitivity of ConA for detecting glycoproteins compared with the PAS method (Wood and Sarinana, 1975;Bayer et al., 1987; Kuzmich et al., 1991). Fig. 7. Open in new tabDownload slide Western blot of denatured GST A1/A1 (± α-mannosidase treatment) probed with ConA-biotin/avidin-alkaline phosphatase. Lane 1, GST A1/A1 without α-mannosidase treatment; lane 2, GST A1/A1 pretreated with α-mannosidase. The amount of GST A1/A1 protein per lane was 200 ng. The position of the GST A1 subunit at 26 kD is indicated by the arrowhead on the left. The position of the heavy subunit of α-mannosidase, a mannosylated glycoprotein with aMr of 68,000, is indicated by the arrowhead on the right. Fig. 7. Open in new tabDownload slide Western blot of denatured GST A1/A1 (± α-mannosidase treatment) probed with ConA-biotin/avidin-alkaline phosphatase. Lane 1, GST A1/A1 without α-mannosidase treatment; lane 2, GST A1/A1 pretreated with α-mannosidase. The amount of GST A1/A1 protein per lane was 200 ng. The position of the GST A1 subunit at 26 kD is indicated by the arrowhead on the left. The position of the heavy subunit of α-mannosidase, a mannosylated glycoprotein with aMr of 68,000, is indicated by the arrowhead on the right. Further evidence for glycosylation of GST A1/A1 (Fig. 7, lane 2) and GST B1/B2 (data not shown) was the lack of detection by ConA-biotin/avidin-alkaline phosphatase when the enzyme was pretreated with α-mannosidase. The binding of ConA was specific for glycan moieties because the addition of methylmannoside, a competitive ligand for ConA, decreased by greater than 90% the amount of ConA-biotin that bound to either GST A1/A1 or GST B1/B2 during western analysis (data not shown). Cleavage of terminal Man residues by α-mannosidase did not alter the Mr of the GST A1, B1, and B2 subunits, as indicated by the lack of a mobility shift on SDS-PAGE after treatment (data not shown), suggesting that the extent of mannosylation of these GST subunits was minor. Reverse-phase HPLC chromatography was used to separate GST A1/A1 and GST B1/B2 subunits prior to comparing the degree of glycosylation. Purified native GST A1/A1 or GST B1/B2 was applied to a reverse-phase HPLC column and separated using a gradient of water and trifluoroacetic acid in acetonitrile (Fig. 8). The HPLC profiles verify the purity of the protein fractions used to characterize GST A1/A1 and GST B1/B2. A single protein peak was observed for the homodimeric GST A1/A1 and two peaks of equal height were observed for the heterodimeric GST B1/B2. The retention times for the 26-kD B1 subunit of GST B1/B2 and the 26-kD subunit of GST A1/A1 were similar. Western blots of the HPLC-purified subunits were probed with ConA-biotin (Fig. 9A), and on the basis of degree of ConA-biotin binding, the GST A1 subunit was shown to be the least glycosylated and the GST B2 subunit the most heavily glycosylated. Fig. 8. Open in new tabDownload slide Purification of subunits from GST A1/A1 (A) and GST B1/B2 (B) by HPLC reverse-phase chromatography. Native GST A1/A1 or B1/B2 was applied to a reverse-phase HPLC column and eluted with a gradient of water and trifluoroacetic acid in acetonitrile as described in Methods. Background absorbance due to increasing trifluoroacetic acid concentration during the gradient has been subtracted. Fig. 8. Open in new tabDownload slide Purification of subunits from GST A1/A1 (A) and GST B1/B2 (B) by HPLC reverse-phase chromatography. Native GST A1/A1 or B1/B2 was applied to a reverse-phase HPLC column and eluted with a gradient of water and trifluoroacetic acid in acetonitrile as described in Methods. Background absorbance due to increasing trifluoroacetic acid concentration during the gradient has been subtracted. Fig. 9. Open in new tabDownload slide Western blots of reverse-phase, HPLC-purified subunits of GST A1/A1 and GST B1/B2 probed with ConA-biotin/avidin-alkaline phosphatase (A) and GST A1/A1 antibody/anti-chicken IgG-alkaline phosphatase (B). Lane 1, GST B2; lane 2, GST B1; and lane 3, GST A1. The amount of GST protein per lane was 75 ng. Fig. 9. Open in new tabDownload slide Western blots of reverse-phase, HPLC-purified subunits of GST A1/A1 and GST B1/B2 probed with ConA-biotin/avidin-alkaline phosphatase (A) and GST A1/A1 antibody/anti-chicken IgG-alkaline phosphatase (B). Lane 1, GST B2; lane 2, GST B1; and lane 3, GST A1. The amount of GST protein per lane was 75 ng. Antigenic Cross-Reactivity Further evidence of the heterodimeric nature of GST B1/B2 was the difference in cross-reactivity of the B1 and B2 subunits with antibodies to GST A1/A1. GST A1/A1 antibodies cross-reacted with the 26-kD B1 subunit but not the 28-kD B2 subunit (Fig. 9B). Antibodies to the GST A1 subunit recognized other 26-kD subunits found in the multiple GST peaks in fluxofenim-treated sorghum shoots (data not shown). A difference in antigenic cross-reactivity of GST subunits of heterodimers has been observed for maize I/II (formerly GST II; seeDixon et al., 1997, for new nomenclature for maize GSTs). For maize GST I/II, antibodies generated to one subunit did not cross-react with the other subunit (Holt et al., 1995). N-Terminal Sequence Homology N-terminal sequences obtained by Edman degradation for GST A1, B1, and B2 subunits were compared with sequences for type I plant GSTs (Fig. 10), which contain the majority of published sequences for grass GSTs (Droog et al., 1995; Marrs, 1996). The N-terminal sequences for the sorghum GST A1, B1, and B2 subunits are highly homologous to each other. The N-terminal sequences of the sorghum GST subunits also exhibited a high degree of homology with maize GST I (GST I/I, new nomenclature) and a GST subunit from sugarcane. A lesser degree of homology was observed for maize GST III (GST III/III, new nomenclature) and GST IV (GST II/II, new nomenclature). Sorghum GSTs exhibited the least homology with the dehydration-induced GST from Arabidopsis (ERD11). Fig. 10. Open in new tabDownload slide Comparison of N-terminal amino acid sequences of sorghum GST A1, B1, and B2 subunits with other plant type-I GSTs. Alignments were performed using the BESTFIT program of the GCG package (Genetics Computer Group, Madison, WI). A line denotes 100% sequence identity with GST A1, two dots denote an acceptable amino acid substitution, one dot denotes a less acceptable amino acid substitution, and X denotes an unidentified amino acid residue. Acceptable and less acceptable amino acid substitutions are defined by the GCG software. Sequences from sorghum were compared with GSTs from sugarcane (Singhal et al., 1991), maize GST I, (Shah et al., 1986), maize GST III (Grove et al., 1988), maize GST IV (GST II, new nomenclature; Jepson et al., 1994; Dixon et al., 1997), wheat GST A1 (Dudler et al., 1991), Hyoscyamus muticus (Bilang et al., 1993), Arabidopsis pm239 (Bartling et al., 1993), tobacco parB (Takahashi and Nagata, 1992), Arabidopsis pm24 (Zhou and Goldsbrough, 1993), Silene cucubalus (Kutchan and Hochberger, 1992), and Arabidopsis ERD11 (Kiyosue et al., 1993). Fig. 10. Open in new tabDownload slide Comparison of N-terminal amino acid sequences of sorghum GST A1, B1, and B2 subunits with other plant type-I GSTs. Alignments were performed using the BESTFIT program of the GCG package (Genetics Computer Group, Madison, WI). A line denotes 100% sequence identity with GST A1, two dots denote an acceptable amino acid substitution, one dot denotes a less acceptable amino acid substitution, and X denotes an unidentified amino acid residue. Acceptable and less acceptable amino acid substitutions are defined by the GCG software. Sequences from sorghum were compared with GSTs from sugarcane (Singhal et al., 1991), maize GST I, (Shah et al., 1986), maize GST III (Grove et al., 1988), maize GST IV (GST II, new nomenclature; Jepson et al., 1994; Dixon et al., 1997), wheat GST A1 (Dudler et al., 1991), Hyoscyamus muticus (Bilang et al., 1993), Arabidopsis pm239 (Bartling et al., 1993), tobacco parB (Takahashi and Nagata, 1992), Arabidopsis pm24 (Zhou and Goldsbrough, 1993), Silene cucubalus (Kutchan and Hochberger, 1992), and Arabidopsis ERD11 (Kiyosue et al., 1993). DISCUSSION Two GST isozymes, GST A1/A1 (a homodimer) and GST B1/B2 (a heterodimer), were purified to homogeneity from fluxofenim-treated sorghum shoots. The two isozymes were glycosylated as indicated by their binding of ConA-biotin and exhibited GSH peroxidase activity with cumene hydroperoxide and linoleic acid hydroperoxides. Kinetic analysis indicated that GSTA1/A1 was best described by a random, rapid equilibrium Bi-Bi model for the substrates GSH and CDNB. In contrast, the best description of the kinetics of the GST B1/B2 heterodimer for the substrates GSH and metolachlor was provided by a multisite model that allowed for the determination of kinetic constants for each subunit. To our knowledge the results obtained with GST A1/A1 and B1/B2 are the first reports of glycosylation of plant GSTs. There have been few investigations of posttranslational modification of GSTs. In vitro phosphorylation and methylation of mammalian GSTs have been reported. Cytosolic rat liver GSTs were phosphorylated by a Ca2+-phospholipid-dependent protein kinase from rabbit brain (Taniguchi and Pyerin, 1989). In vitro, calmodulin-stimulated (Johnson et al., 1990) and methyltransferase-catalyzed methylation (Johnson et al., 1992) of rat liver cytosolic GSTs has been reported. There is one report of glycosylated GSTs in mammals. Human GST π and rat GST Yp were detected as glycoproteins by visualization with fluorescein isothiocyanate-ConA, a fluorescent ConA conjugate (Kuzmich et al., 1991). As with sorghum GST A1/A1 and GST B1/B2, the rat liver and human GSTs were not heavily glycosylated because they were not detected by procedures that utilized periodate oxidation for visualization (Kuzmich et al., 1991). Glycosylation of sorghum GST subunits was not limited to GST A1/A1 and GST B1/B2. Each of the eight peaks of GST activity in safener-induced sorghum shoots (Fig. 1B) contained at least one glycosylated subunit, as indicated by visualization with ConA-biotin (data not shown). The function of glycosylation of sorghum GSTs is not known. Previous research has demonstrated that glycosylation of proteins can play a role in proper protein folding, protection against proteases, solubility, and recognition phenomena (Varki, 1993). Further research is needed to determine whether glycosylation of plant GSTs is widespread and, if so, to determine its function. In all previous determinations of kinetic parameters for purified plant GSTs, initial-velocity data have been analyzed using the standard Michaelis-Menton equation for a unireaction (O'Connell et al., 1988;Williamson and Beverley, 1988; Singhal et al., 1991; Irzyk and Fuerst, 1993; Droog et al., 1995; Flury et al., 1995). However, kinetic models describing bireactant mechanisms are better suited for the determination of kinetic constants for GSTs, which utilize two substrates. For GST A1/A1, the random, rapid-equilibrium Bi-Bi model best described the kinetics with GSH and CDNB as substrates. Although the results were consistent with this model, further kinetic analysis involving end-product inhibition would be required to confirm the random, rapid equilibrium Bi-Bi model. Bireactant models have been used to characterize the kinetic mechanisms of mammalian GSTs (Jakobson et al., 1979; Schramm et al., 1984;Ivanetich and Goold, 1989; Young and Briedis, 1989; Ivanetich et al., 1990; Phillips and Mantle, 1991). For mammalian GSTs, analyses of initial-velocity data with bireactant kinetic models have indicated that the kinetic mechanism is random and sequential. However, there is a lack of agreement as to whether mammalian GSTs exhibit rapid-equilibrium or steady-state kinetics. In some cases, the random, rapid-equilibrium model provided the best fit to the data (Schramm et al., 1984; Ivanetich and Goold, 1989; Young and Briedis, 1989; Phillips and Mantle, 1991), whereas in others the kinetics were best described by the random, steady-state model (Jakobson et al., 1979; Ivanetich et al., 1990). It has been suggested that the kinetic mechanism for mammalian GSTs is isozyme specific (Ivanetich and Goold, 1989). For GST B1/B2, bireactant kinetic models did not provide a good fit to the initial-velocity data. A better description of the kinetics was obtained with a multisite enzyme analysis in which kinetic constants for each subunit could be determined by successive correction (Spears et al., 1971). This analysis provided evidence for two catalytically distinct subunits differing in substrate affinities. The results are consistent with reports of catalytic independence of the subunits of mammalian GSTs (Danielson and Mannervik, 1985; Tahir and Mannervik, 1986). In some respects, GST B1/B2 is similar to the maize heterodimer GST I/II (formerly GST II). Both heterodimers contain a herbicide safener-induced subunit that exhibits high affinity for chloroacetanilide herbicides (Irzyk and Fuerst, 1993; Holt et al., 1995; Dixon et al., 1997). The Km values for metolachlor for the high-affinity subunit of GST B1/B2 and the maize GST II subunit are 3 μm (Fig. 8) and 10.8 μm (Irzyk and Fuerst, 1993; GST IV, new nomenclature GST II/II), respectively. These subunits, by exhibiting high affinity for chloroacetanilide herbicides, would allow for detoxification of micromolar concentrations of these herbicides, which are inhibitory to plant growth (Deal and Hess, 1980; Fuerst and Gronwald, 1986; Fuerst et al., 1991). These Km values contrast sharply with the apparent Km for metolachlor of 8.9 mm for the GST III/III (formerly GST III) isozyme found in maize coleoptiles (O'Connell et al., 1988). Although the maize GST II subunit and the high-affinity subunit of sorghum GST B1/B2 both exhibit high affinity for chloroacetanilides, they differ in affinity for GSH. The high-affinity subunit of GST B1/B2 exhibited a Km of 12 μm for GSH, whereas the GST II subunit exhibited an apparentKm of 292 μm (Irzyk and Fuerst, 1993). The Km of the high-affinity subunit of B1/B2 for GSH is one of the lowest values reported in the literature for either plant or mammalian GSTs. For mammalian GSTs an apparent Km for GSH of 27 μmwas reported for rat GST 4–4 with CDNB as a substrate (Zhang et al., 1992). A heterologously expressed GST from Arabidopsis exhibited an apparent Km of 80 μm for GSH when assayed with CDNB (Bartling et al., 1993). With the exception of the pathogen-induced GSTA1 from wheat and GST ERD11 from Arabidopsis, type-I θ-GSTs have a conserved Ser in the region of residues 10 through 13 near the N terminus (domain I) (Fig.10). There is increasing evidence that an N-terminal Ser of θ-GSTs plays a critical role in catalysis and is equivalent to the domain I, N-terminal Tyr of mammalian α-, μ-, and π-GSTs that is involved in the formation of the thiolate anion of bound GSH (Blocki et al., 1993; Dirr et al., 1994). Site-directed mutagenesis studies of the θ-GST from the Australian sheep blowfly (Lucilia cuprina) and the θ-GST from the bacterium Methylophilus sp. strain DM11 indicated that the Ser-9 and Ser-12 residues, respectively, are essential for enzyme activity (Board et al., 1995; Vuilleumier and Leisinger, 1996). Furthermore, the three-dimensional structure of a θ-GST from L. cuprina showed that Ser-9 occupies a position close to that of the catalytically important Tyr of mammalian α-, μ-, and π-GSTs (Wilce et al., 1995). Recently, the first three-dimensional structure of a plant GST was reported (Reinemer et al., 1996). This enzyme, a type I GST from Arabidopsis (pm24; Fig. 10), contains no Tyr residue in the active site. However, like the GST fromL. cuprina, it contains a Ser residue that is in a location similar to that of the catalytically active Tyr in mammalian GSTs. Further research involving site-directed mutagenesis and x-ray crystallography analysis is needed to evaluate the role of the N-terminal Ser residue in the mechanism of catalysis by θ-GSTs. Both GST A1/A1 and GST B1/B2 exhibited activity with ethacrynic acid, a phenylacetic acid derivative that contains an electrophilic group similar to α-β-alkenals generated in mammals under oxidative stress (Danielson et al., 1987; Berhane et al., 1994). Ethacrynic acid can act as a substrate (Yamada and Kaplowitz, 1980), activator (Phillips and Mantle, 1991), or inhibitor (Ahokas et al., 1985; Phillips and Mantle, 1993) of mammalian GSTs, depending on the isozyme. High activity with ethacrynic acid is characteristic of mammalian π-GSTs (Mannervik and Danielson, 1988). In pea epicotyls GST activity with ethacrynic acid was inducible by treatment with GSH and a fungal elicitor (Edwards, 1996). In contrast, ethacrynic acid was a potent inhibitor (Ki = 5 μm) of auxin-inducible GSTs from tobacco (Droog et al., 1995). GST B1/B2, but not GST A1/A1, exhibited GSH-conjugating activity with 4HNE, a toxic α-β-unsaturated aldehyde that is generated by peroxidation of arachidonic acid in rat microsomes exposed to oxidative stress (Esterbauer et al., 1986). In rat liver, the α-GST 8–8 exhibits high activity with this substrate (Danielson et al., 1987). There have been no previous reports of plant GSTs exhibiting activity with 4HNE. In plants alkenals such as trans-2-hexenal are derived from 13-hydroperoxylinolenic acid after cleavage with hydroperoxide lyase (Gardner, 1991). An Arabidopsis GST active with 13-hydroperoxylinolenic acid exhibited no activity withtrans-2-hexenal (Bartling et al., 1993). Both GST A1/A1 and B1/B2 exhibited GSH peroxidase activity with cumene hydroperoxide and linoleic acid hydroperoxides. There have been an increasing number of reports of plant GSTs exhibiting GSH peroxidase activity with these substrates. In pea, GSH peroxidase activity with cumene hydroperoxide was higher in etiolated seedlings than in older green plants and was induced in roots by treatment with CuCl2 (Edwards, 1996). An auxin-binding, plasma membrane-associated GST from Arabidopsis exhibited activity with cumene hydroperoxide (Zettl et al., 1994). GSH peroxidase activity with lipid hydroperoxides as substrates has been reported for GSTs from pea seeds (Williamson and Beverley, 1987), wheat flour (Williamson and Beverley, 1988), soybean hypocotyls (Flury et al., 1996), and Arabidopsis (Bartling et al., 1993). Compared with the GSTs purified from pea seeds and wheat flour, sorghum GST A1/A1 and GST B1/B2 exhibit higher activity with both 9c,t-HPO and 13 c,t-HPO. Similar to sorghum GST B1/B2, a GST purified from pea seeds exhibited equivalent GSH peroxidase activity with 9 c,t-HPO and 13 c,t-HPO (Williamson and Beverley, 1987). In contrast, a GST isolated from wheat flour exhibited a preference for 9 c,t-HPO over 13c,t-HPO (Williamson and Beverley, 1988) similar to what was observed for sorghum GST A1/A1 (Table III). The level of GSH peroxidase activity of GST A1/A1 and GST B1/B2 with linoleic hydroperoxides is similar to that reported for linolenic and arachidonic hydroperoxides (0.4–1.2 μmol min−1mg−1) in a purified GST fraction from soybean hypocotyls (Flury et al., 1996). GSH peroxidase activity of GST A1/A1 and B1/B2 with linoleic hydroperoxides is within the range of activities reported for mammalian α-GSTs, which are distinguished by their activity with products of oxidative stress (Ketterer and Coles, 1991). Plant GSTs are members of the archaic θ-GST class, from which other GST classes evolved (Buetler and Eaton, 1992). It has been proposed that θ-GSTs originally evolved in prokaryotes to protect against oxidative stress (Pemble and Taylor, 1992). In plants, oxidative stress may signal the induction of certain GSTs (Flury et al., 1996; Marrs, 1996). Several treatments known to induce GSTs, such as wounding or exposure to ozone, ethylene, heavy metals, or pathogens, cause oxidative stress (Marrs, 1996, and refs. therein). Safener treatment of sorghum causes stress, as indicated by growth inhibition of developing seedlings (Fuerst and Gronwald, 1986), but why safeners inhibit growth is not known. Although the safener-induced GST B1/B2 exhibits activity with herbicidal concentrations of metolachlor, it is possible that the primary function of this isozyme, and perhaps others induced by the safener, is to protect against lipid peroxidation products generated by various forms of stress. At comparable substrate concentrations (4 mm GSH and 0.1 mm metolachlor or 13c,t HPO), the activity of GST B1/B2 with 13c,t-HPO is at least 2 orders of magnitude greater than that with metolachlor as a substrate. It would be of interest to determine whether treatments that cause oxidative stress, such as exposure to ozone and hydrogen peroxide, would induce GST B1/B2 in sorghum, and whether pretreating sorghum with fluxofenim would confer protection against subsequent exposure to oxidative stress. ACKNOWLEDGMENTS We express our appreciation to Novartis for providing sorghum seeds and 14C-labeled herbicides (metolachlor, alachlor, and atrazine). 4HNE was generously provided by Dr. H. Esterbauer (Institut für Biochemie, Universitat Graz, Graz, Austria). Abbreviations: alachlor 2-chloro-N-(2,6-diethylphenyl)-N-(methoxymethyl)acetamide atrazine 6-chloro-N-ethyl-N′-(1-methylethyl)-1,3,5-triazine-2,4-diamine) CDNB 1-chloro-2,4,dinitrobenzene ConA concanavalin A DCNB 1,2,-dichloro-4-nitrobenzene EPTC S-ethyldipropylcarbamothioate EPTC-sulfoxide sulfoxide derivative of EPTC fluxofenim (O-1,3-dioxolan-2-yl-methyl-2,2,2,-trifluoro-4′-chloroacetophenone-oxime) ethacrynic acid 2,3-dichloro-4(2-methylene-butyryl)phenoxyacetic acid FPLC fast- protein liquid chromatography GST glutathioneS-transferase GST(CDNB) GST activity measured with CDNB as the substrate GST(metolachlor) GST activity measured with metolachlor as the substrate 4HNE 4-hydroxynonenal9c, t-HPO 9-hydroperoxy-trans-10,cis-12-octadecadienoic acid13 c, t-HPO 13-hydroperoxy-cis-9,trans-11-octadecadienoic acid I50 inhibitor concentration producing 50% inhibition of enzyme activity metolachlor 2-chloro-N- (2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl) acetamide PAS periodic acid/Schiff LITERATURE CITED 1 Ahokas JT Nicholls FA Ravenscroft PJ Emmerson BT Inhibition of purified rat liver glutathione S-transferase isozymes by diuretic drugs. Biochem Pharmacol 34 1985 2157 2161 Google Scholar Crossref Search ADS PubMed WorldCat 2 Ålin P Danielson UH Mannervik B 4-Hydroxyalk-2-enals are substrates for glutathione transferase. FEBS Lett 179 1985 267 270 Google Scholar Crossref Search ADS PubMed WorldCat 3 Awasthi YC Beutler E Srivastava SK Purification and properties of human erythrocyte glutathione peroxidase. J Biol Chem 250 1975 5144 5149 Google Scholar Crossref Search ADS PubMed WorldCat 4 Bartling D Radzio R Steiner U Weiler EW A glutathione S-transferase with glutathione-peroxidase activity from Arabidopsis thaliana. Molecular cloning and functional characterization. Eur J Biochem 216 1993 579 586 Google Scholar Crossref Search ADS PubMed WorldCat 5 Bayer EA Ben-Hur H Wilchek M Enzyme-based detection of glycoproteins on blot transfers using avidin-biotin technology. Anal Biochem 161 1987 123 131 Google Scholar Crossref Search ADS PubMed WorldCat 6 Berhane K Widersten M Engström Å Kozarich JW Mannervik B Detoxication of base propenals and other α,β-unsaturated aldehyde products of radical reactions and lipid peroxidation by human glutathione transferases. Proc Natl Acad Sci USA 91 1994 1480 1484 Google Scholar Crossref Search ADS PubMed WorldCat 7 Bilang J MacDonald H King PJ Sturm A A soluble auxin-binding protein from Hyoscyamus muticus is a glutathione S-transferase. Plant Physiol 102 1993 29 34 Google Scholar Crossref Search ADS PubMed WorldCat 8 Blake MS Johnston KH Russell-Jones GJ Gotschlich EC A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on western blots. Anal Biochem 136 1984 175 179 Google Scholar Crossref Search ADS PubMed WorldCat 9 Blocki FA Ellis LBM Wackett LP MIF proteins are theta-class glutathione S-transferase homologs. Protein Sci 2 1993 2095 2102 Google Scholar Crossref Search ADS PubMed WorldCat 10 Board PG Coggan M Wilce MCJ Parker MW Evidence for an essential serine residue in the active site of the theta class glutathione transferases. Biochem J 311 1995 247 250 Google Scholar Crossref Search ADS PubMed WorldCat 11 Bradford MM A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72 1976 248 254 Google Scholar Crossref Search ADS PubMed WorldCat 12 Braun W Abraham R Modified diffusion blotting for rapid and efficient protein transfer with PhastSystem. Electrophoresis 10 1989 249 253 Google Scholar Crossref Search ADS PubMed WorldCat 13 Buetler TM Eaton DL Environ Carcinogen Ecotoxicol Rev C10 1992 181 203 14 Casida JE Kimmel EC Ohkawa H Ohkawa R Sulfoxidation of thiocarbamate herbicides and metabolism of thiocarbamate sulfoxides in living mice and liver enzyme systems. Pestic Biochem Physiol 5 1975 1 11 Google Scholar Crossref Search ADS WorldCat 15 Danielson UH Esterbauer H Mannervik B Structure-activity relationships of 4-hydroxyalkenals in the conjugation catalysed by mammalian glutathione transferases. Biochem J 247 1987 707 713 Google Scholar Crossref Search ADS PubMed WorldCat 16 Danielson UH Mannervik B Kinetic independence of the subunits of cytosolic glutathione transferase from the rat. Biochem J 231 1985 263 267 Google Scholar Crossref Search ADS PubMed WorldCat 17 Deal LM Hess FD An analysis of the growth inhibitory characteristics of alachlor and metolachlor. Weed Sci 28 1980 168 175 Google Scholar Crossref Search ADS WorldCat 18 Dean JV Gronwald JW Anderson MP Glutathione S-transferase activity in nontreated and CGA-154281-treated maize shoots. Z Naturforsch 46c 1991 850 855 Google Scholar OpenURL Placeholder Text WorldCat 19 Dean JV Gronwald JW Eberlein CV Induction of glutathione S-transferase isozymes in sorghum by herbicide antidotes. Plant Physiol 92 1990 467 473 Google Scholar Crossref Search ADS PubMed WorldCat 20 Dirr H Reinemer P Huber R X-ray crystal structures of cytosolic glutathione S-transferases: implications for protein architecture, substrate recognition and catalytic function. Eur J Biochem 220 1994 645 661 Google Scholar Crossref Search ADS PubMed WorldCat 21 Dixon D Cole DJ Edwards R Characterisation of multiple glutathione transferases containing the GST I subunit with activities toward herbicide substrates in maize (Zea mays). Pestic Sci 50 1997 72 82 Google Scholar Crossref Search ADS WorldCat 22 Droog FNJ Hooykaas PJJ van der Zaal BJ 2,4-Dichlorophenoxyacetic acid and related chlorinated compounds inhibit two auxin-regulated type-III tobacco glutathione S-transferases. Plant Physiol 107 1995 1139 1146 Google Scholar Crossref Search ADS PubMed WorldCat 23 Dudler R Hertig C Rebmann G Bull J Mauch F A pathogen-induced wheat gene encodes a protein homologous to glutathione-S-transferases. Mol Plant-Microbe Interact 4 1991 14 18 Google Scholar Crossref Search ADS PubMed WorldCat 24 Dyer JR Use of periodate oxidations in biochemical analysis. Methods Biochem Anal 3 1956 111 152 Google Scholar PubMed OpenURL Placeholder Text WorldCat 25 Edwards R Characterisation of glutathione transferases and glutathione peroxidases in pea (Pisum sativum). Physiol Plant 98 1996 594 604 Google Scholar Crossref Search ADS WorldCat 26 Esterbauer H Benedetti A Lang J Fulceri R Fauler G Comporti M Studies on the mechanism of formation of 4-hydroxynonenal during microsomal lipid peroxidation. Biochim Biophys Acta 876 1986 154 166 Google Scholar Crossref Search ADS PubMed WorldCat 27 Flury T Adam D Kreuz K A 2,4-D-inducible glutathione S-transferase from soybean (Glycine max). Purification, characterisation and induction. Physiol Plant 94 1995 312 318 Google Scholar Crossref Search ADS WorldCat 28 Flury T Wagner E Kreuz K An inducible glutathione S-transferase in soybean hypocotyl is localized in the apoplast. Plant Physiol 112 1996 1185 1190 Google Scholar Crossref Search ADS PubMed WorldCat 29 Fuerst EP Gronwald JW Induction of rapid metabolism of metolachlor in sorghum (Sorghum bicolor) shoots by CGA-92194 and other antidotes. Weed Sci 34 1986 354 361 Google Scholar Crossref Search ADS WorldCat 30 Fuerst EP Lamoureux GL Ahrens WH Mode of action of the dichloroacetamide antidote BAS 145–138 in corn. I. Growth responses and fate of metazachlor. Pestic Biochem Physiol 39 1991 138 148 Google Scholar Crossref Search ADS WorldCat 31 Gardner HW Recent investigations into the lipoxygenase pathway of plants. Biochim Biophys Acta 1084 1991 221 239 Google Scholar Crossref Search ADS PubMed WorldCat 32 Gronwald JW Fuerst EP Eberlein CV Egli MA Effect of herbicide antidotes on glutathione content and glutathione S-transferase activity of sorghum shoots. Pestic Biochem Physiol 29 1987 66 76 Google Scholar Crossref Search ADS WorldCat 33 Grove G Zarlengo RP Timmerman KP Li N-Q Tam MF Tu C-PD Characterization and heterospecific expression of cDNA clones of genes in the maize GSH S-transferase multigene family. Nucleic Acids Res 16 1988 425 438 Google Scholar Crossref Search ADS PubMed WorldCat 34 Habig WH Jakoby WB Assays for differentiation of glutathione S-transferases. Methods Enzymol 77 1981 398 405 Google Scholar Crossref Search ADS PubMed WorldCat 35 Habig WH Pabst MJ Jakoby WB Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J Biol Chem 249 1974 7130 7139 Google Scholar Crossref Search ADS PubMed WorldCat 36 Hamberg M Samuelsson B On the specificity of the oxygenation of unsaturated fatty acids catalyzed by soybean lipoxidase. J Biol Chem 242 1967 5329 5335 Google Scholar Crossref Search ADS PubMed WorldCat 37 Harlow E, Lane D (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 564–566 38 Haselbeck A, Hösel W (1988) Studies on the effect of the incubation conditions, various detergents and protein concentration on the enzymatic activity of N-glycosidase F (glycopeptidase F) and endoglycosidase F. In Topics in Biochemistry, no. 8. Boehringer Mannheim GmbH, Tutzing, Germany, pp 1–4 39 Holt DC Lay VJ Clarke ED Dinsmore A Jepson I Bright SWJ Greenland AJ Characterization of the safener-induced glutathione S-transferase isoform II from maize. Planta 196 1995 295 302 Google Scholar Crossref Search ADS PubMed WorldCat 40 Irzyk GP Fuerst EP Purification and characterization of a glutathione S-transferase from benoxacor-treated maize (Zea mays). Plant Physiol 102 1993 803 810 Google Scholar Crossref Search ADS PubMed WorldCat 41 Ivanetich KM Goold RD A rapid equilibrium random sequential bi-bi mechanism for human placental glutathione S-transferase. Biochim Biophys Acta 998 1989 7 13 Google Scholar Crossref Search ADS PubMed WorldCat 42 Ivanetich KM Goold RD Sikakana CNT Explanation of the non-hyperbolic kinetics of the glutathione S-transferases by the simplest steady-state random sequential bi bi mechanism. Biochem Pharmacol 39 1990 1999 2004 Google Scholar Crossref Search ADS PubMed WorldCat 43 Jakobson I Warholm M Mannervik B Multiple inhibition of glutathione S-transferase A from rat liver by glutathione derivatives: kinetic analysis supporting a steady-state random sequential mechanism. Biochem J 177 1979 861 868 Google Scholar Crossref Search ADS PubMed WorldCat 44 Jensenius J Andersen I Hau J Crone M Koch C Eggs: conveniently packaged antibodies. Methods for purification of yolk IgG. J Immunol Methods 46 1981 63 68 Google Scholar Crossref Search ADS PubMed WorldCat 45 Jepson I Lay VJ Holt DC Bright SWJ Greenland AJ Cloning and characterization of maize herbicide safener-induced cDNAs encoding subunits of glutathione S-transferase isoforms I, II and IV. Plant Mol Biol 26 1994 1855 1866 Google Scholar Crossref Search ADS PubMed WorldCat 46 Johnson JA Finn KA Siegel FL Tissue distribution of enzymic methylation of glutathione S-transferase and its effects on catalytic activity. Methylation of glutathione S-transferase 11–11 inhibits conjugating activity towards 1-chloro-2,4-dinitrobenzene. Biochem J 282 1992 279 289 Google Scholar Crossref Search ADS PubMed WorldCat 47 Johnson JA Neal TL Collins JH Siegel FL Characterization of methylation of rat liver cytosolic glutathione S-transferases by using reverse-phase h.p.l.c. and chromatofocusing. Biochem J 270 1990 483 489 Google Scholar Crossref Search ADS PubMed WorldCat 48 Ketterer B Coles B Glutathione transferases and products of reactive oxygen. Sies H Oxidative Stress: Oxidants and Antioxidants. 1991 171 194 Academic Press London 49 Kiyosue T Yamaguchi-Shinozaki K Shinozaki K Characterization of two cDNAs (ERD11 and ERD13) for dehydration-inducible genes that encode putative glutathione S-transferases in Arabidopsis thaliana L. FEBS Lett 335 1993 189 192 Google Scholar Crossref Search ADS PubMed WorldCat 50 Kokatnur VR Jelling M Iodometric determination of peroxygen in organic compounds. J Am Chem Soc 63 1941 1432 1433 Google Scholar Crossref Search ADS WorldCat 51 Kutchan TM Hochberger A Nucleotide sequence of a cDNA encoding a constitutively expressed glutathione S-transferase from cell suspension cultures of Silene cucubalus. Plant Physiol 99 1992 789 790 Google Scholar Crossref Search ADS PubMed WorldCat 52 Kuzmich S vanderVeer LA Tew KD Evidence for a glycoconjugate form of glutathione S-transferase pI. Int J Peptide Protein Res 37 1991 565 571 Google Scholar Crossref Search ADS WorldCat 53 Lamoureux GL Rusness DG The role of glutathione and glutathione S-transferases in pesticide metabolism, selectivity, and mode of action in plants and insects. Dolphin D Avramović O Poulson R Coenzymes and Cofactors, Vol III, Part B: Glutathione: Chemical, Biochemical and Medical Aspects. 1989 153 196 John Wiley & Sons New York 54 Mannervik B Regression analysis, experimental error, and statistical criteria in the design and analysis of experiments for discrimination between rival kinetic models. Purich DL Contemporary Enzyme Kinetics and Mechanism, Ed 2. 1996 111 131 Academic Press San Diego, CA 55 Mannervik B Danielson UH Glutathione transferases—structure and catalytic activity. CRC Crit Rev Biochem 23 1988 283 337 Google Scholar Crossref Search ADS PubMed WorldCat 56 Mannervik B Guthenberg C Glutathione transferase (human placenta). Methods Enzymol 77 1981 231 235 Google Scholar Crossref Search ADS PubMed WorldCat 57 Marrs KA The functions and regulation of glutathione S-transferases in plants. Annu Rev Plant Physiol Plant Mol Biol 47 1996 127 158 Google Scholar Crossref Search ADS PubMed WorldCat 58 Marrs KA Alfenito MR Lloyd AM Walbot V A glutathione S-transferase involved in vacuolar transfer encoded by the maize gene Bronze-2. Nature 375 1995 397 400 Google Scholar Crossref Search ADS PubMed WorldCat 59 Matthew JA Chan HW-S Galliard T A simple method for the preparation of pure 9-d-hydroperoxide of linoleic acid and methyl linoleate based on the positional specificity of lipoxygenase in tomato fruit. Lipids 12 1977 324 326 Google Scholar Crossref Search ADS PubMed WorldCat 60 Meyer DJ Coles B Pemble SE Gilmore KS Fraser GM Ketterer B Theta, a new class of glutathione transferases purified from rat and man. Biochem J 274 1991 409 414 Google Scholar Crossref Search ADS PubMed WorldCat 61 Mozer TJ Tiemeier DC Jaworski EG Purification and characterization of corn glutathione S-transferase. Biochemistry 22 1983 1068 1072 Google Scholar Crossref Search ADS PubMed WorldCat 62 O'Connell KM Breaux EJ Fraley RT Different rates of metabolism of two chloroacetanilide herbicides in Pioneer 3320 corn. Plant Physiol 86 1988 359 363 Google Scholar Crossref Search ADS PubMed WorldCat 63 Ostlund Farrants A-K Meyer DJ Coles B Southan C Aitken A Johnson PJ Ketterer B The separation of glutathione transferase subunits by using reverse-phase high-pressure liquid chromatography. Biochem J 245 1987 423 428 Google Scholar Crossref Search ADS PubMed WorldCat 64 Pemble SE Taylor JB An evolutionary perspective on glutathione transferases inferred from class-theta glutathione transferase cDNA sequences. Biochem J 287 1992 957 963 Google Scholar Crossref Search ADS PubMed WorldCat 65 Phillips MF Mantle TJ The initial-rate kinetics of mouse glutathione S-transferase YfYf. Evidence for an allosteric site for ethacrynic acid. Biochem J 275 1991 703 709 Google Scholar Crossref Search ADS PubMed WorldCat 66 Phillips MF Mantle TJ Inactivation of mouse liver glutathione S-transferase YfYf (Pi class) by ethacrynic acid and 5,5′-dithiobis-(2-nitrobenzoic acid). Biochem J 294 1993 57 62 Google Scholar Crossref Search ADS PubMed WorldCat 67 Poretz RD Goldstein IJ An examination of the topography of the saccharide binding sites of concanavalin A and of the forces involved in complexation. Biochemistry 9 1970 2890 2896 Google Scholar Crossref Search ADS PubMed WorldCat 68 Reinemer P Prade L Hof P Neuefeind T Huber R Zettl R Palme K Schell J Koelln I Bartunik HD and others Three-dimensional structure of glutathione S-transferase from Arabidopsis thaliana at 2.2 Å resolution: structural characterization of herbicide-conjugating plant glutathione S-transferases and a novel active site architecture. J Mol Biol 255 1996 289 309 Google Scholar Crossref Search ADS PubMed WorldCat 69 Schramm VL McCluskey R Emig FA Litwack G Kinetic studies and active site-binding properties of glutathione S-transferase using spin-labeled glutathione, a product analogue. J Biol Chem 259 1984 714 722 Google Scholar Crossref Search ADS PubMed WorldCat 70 Segel IH Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems. 1975 John Wiley & Sons New York 71 Shah DM Hironaka CM Wiegand RC Harding EI Krivi GG Tiemeier DC Structural analysis of a maize gene coding for glutathione-S-transferase involved in herbicide detoxification. Plant Mol Biol 6 1986 203 211 Google Scholar Crossref Search ADS PubMed WorldCat 72 Singhal SS Saxena M Ahmad H Awasthi S Haque AK Awasthi YC Glutathione S-transferases of human lung: characterization and evaluation of the protective role of the α-class isozymes against lipid peroxidation. Arch Biochem Biophys 299 1992 232 241 Google Scholar Crossref Search ADS PubMed WorldCat 73 Singhal SS Tiwari NK Ahmad H Srivastava SK Awasthi YC Purification and characterization of glutathione Stransferase from sugarcane leaves. Phytochemistry 30 1991 1409 1414 Google Scholar Crossref Search ADS WorldCat 74 Spears G Sneyd JGT Loten EG A method for deriving kinetic constants for two enzymes acting on the same substrate. Biochem J 125 1971 1149 1151 Google Scholar Crossref Search ADS PubMed WorldCat 75 Strömqvist M Gruffman H Periodic acid/Schiff staining of glycoproteins immobilized on a blotting matrix. Biotechniques 13 1992 744 746 Google Scholar PubMed OpenURL Placeholder Text WorldCat 76 Tahir MK Mannervik B Simple inhibition studies for distinction between homodimeric and heterodimeric isoenzymes of glutathione transferase. J Biol Chem 261 1986 1048 1051 Google Scholar Crossref Search ADS PubMed WorldCat 77 Takahashi Y Nagata T parB: an auxin-regulated gene encoding glutathione S-transferase. Proc Natl Acad Sci USA 89 1992 56 59 Google Scholar Crossref Search ADS PubMed WorldCat 78 Taniguchi H Pyerin W Glutathione S-transferase is an in vitro substrate of Ca++-phospholipid-dependent protein kinase (protein kinase C). Biochem Biophys Res Commun 162 1989 903 907 Google Scholar Crossref Search ADS PubMed WorldCat 79 Varki A Biological roles of oligosaccharides: all of the theories are correct. Glycobiology 3 1993 97 130 Google Scholar Crossref Search ADS PubMed WorldCat 80 Vuilleumier S Leisinger T Protein engineering studies of dichloromethane dehalogenase/glutathione S-transferase from Methylophilus sp. strain DM11. Ser12 but not Tyr6 is required for enzyme activity. Eur J Biochem 239 1996 410 417 Google Scholar Crossref Search ADS PubMed WorldCat 81 Wilce MCJ, Board PG, Feil SC, Parker MW (1995) Crystal structure of a theta-class glutathione transferase EMBO J 14: 2133–2143 82 Williamson G Beverley MC The purification of acidic glutathione S-transferases from pea seeds and their activity with lipid peroxidation products. Biochem Soc Trans 15 1987 1103 1104 Google Scholar Crossref Search ADS WorldCat 83 Williamson G Beverley MC Wheat glutathione S-transferase: purification and properties. J Cereal Sci 8 1988 155 163 Google Scholar Crossref Search ADS WorldCat 84 Wood JG Sarinana FO The staining of sciatic nerve glycoproteins on polyacrylamide gels with concanavalin Aperoxidase. Anal Biochem 69 1975 320 322 Google Scholar Crossref Search ADS PubMed WorldCat 85 Yamada T Kaplowitz N Binding of ethacrynic acid to hepatic glutathione S-transferases in vivo in the rat. Biochem Pharmacol 29 1980 1205 1208 Google Scholar Crossref Search ADS PubMed WorldCat 86 Young PR Briedis AV Purification and kinetic mechanism of the major glutathione S-transferase from bovine brain. Biochem J 257 1989 541 548 Google Scholar Crossref Search ADS PubMed WorldCat 87 Zettl R Schell J Palme K Photoaffinity labeling of Arabidopsis thaliana plasma membrane vesicles by 5-azido-(7-3H)indole-3-acetic acid: identification of a glutathione S-transferase. Proc Natl Acad Sci USA 91 1994 689 693 Google Scholar Crossref Search ADS PubMed WorldCat 88 Zhang P Liu S Shan S-O Ji X Gilliland GL Armstrong RN Modular mutagenesis of exons 1, 2, and 8 of a glutathione S-transferase from the Mu class. Mechanistic and structural consequences for chimeras of isoenzyme 3–3. Biochemistry 31 1992 10185 10193 Google Scholar Crossref Search ADS PubMed WorldCat 89 Zhou J Goldsbrough PB An Arabidopsis gene with homology to glutathione S-transferases is regulated by ethylene. Plant Mol Biol 22 1993 517 523 Google Scholar Crossref Search ADS PubMed WorldCat Author notes 1 This work was a cooperative investigation of the U.S. Department of Agriculture, Agricultural Research Service, and the Minnesota Agricultural Experiment Station. This is Minnesota Agricultural Experiment Station publication no. 97-1-13-0017. 2 Product names are necessary to report factually on available data; however, the U.S. Department of Agriculture neither guarantees nor warrants the standard of the product, and the use of the name by the U.S. Department of Agriculture implies no approval of the product to the exclusion of others that may also be suitable. * Corresponding author; e-mail [email protected]; fax 1–612–649–5058. Copyright © 1998 American Society of Plant Physiologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Stanković, Bratislav; Volkmann, Dieter; David Sack, Fred

doi: 10.1104/pp.117.3.893pmid: 9662531

Abstract Few studies have documented the response of gravitropically curved organs to a withdrawal of a constant gravitational stimulus. The effects of stimulus withdrawal on gravitropic curvature were studied by following individual roots of cress (Lepidium sativum L.) through reorientation and clinostat rotation. Roots turned to the horizontal curved down 62° and 88° after 1 and 5 h, respectively. Subsequent rotation on a clinostat for 6 h resulted in root straightening through a loss of gravitropic curvature in older regions and through new growth becoming aligned closer to the prestimulus vertical. However, these roots did not return completely to the prestimulus vertical, indicating the retention of some gravitropic response. Clinostat rotation shifted the mean root angle −36° closer to the prestimulus vertical, regardless of the duration of prior horizontal stimulation. Control roots (no horizontal stimulation) were slanted at various angles after clinostat rotation. These findings indicate that gravitropic curvature is not necessarily permanent, and that the root retains some commitment to its equilibrium orientation prior to gravitropic stimulation. The reorientation of most plant organs results in gravitropic curvature that normally persists for the life of the organ. This curvature is due to differential growth that at some point becomes stabilized and long-lasting. However, before stabilization, the locus of gravitropic curvature actually migrates in some organs (Firn and Digby, 1979; MacDonald et al., 1983; Tarui and Iino, 1997; for review, see Stanković et al., 1998). Thus, some curved regions later straighten, and more basal regions that were straight later become curved. Although the net result is a curved organ, loss of gravitropic curvature (axis straightening) does occur distal to the final curve. The local straightening described above occurs in organs that are kept stationary with a constant g stimulus. In contrast, straightening throughout the organ seems to occur when a gstimulus is withdrawn by placement in microgravity in spaceflight or by rotation of the plant on a clinostat on earth (for review, seeStanković et al., 1998). The loss of gravitropic curvature in space has been documented using oat coleoptiles and cress (Lepidium sativum L.) roots (Chapman et al., 1994; Volkmann and Tewinkel, 1996). Seedlings centrifuged in flight at 1gcontinued gravitropic curvature when removed from the centrifuge. Later, previously curved regions straightened in microgravity so that the organ approached the angle it was in prior to the 1gstimulus. There are also relatively few descriptions of the straightening of gravitropically curved organs resulting from the use of a clinostat in ground-based studies. In some cases, straightening apparently only occurred at very slow speeds of rotation (0.008–0.016 rpm), and not at higher speeds (roots of Artemisia absinthium and cress;Larsen, 1953, 1957). In another report, loss of gravitropic curvature took place when roots were rotated at 2 and 4 rpm (Arabidopsis; Mirza et al., 1984). But Larsen's studies did not continually follow the same roots through time and none of these reports analyzed the regions of the root responsible for straightening. Other studies on the effects of clinostat rotation on cress roots (Hensel and Iversen, 1980; Hoson et al., 1997) did not address the presence and extent of straightening. Moreover, it cannot be assumed that because curved cress roots straighten in microgravity (Volkmann and Tewinkel, 1996) they will also do so on a clinostat, since oat coleoptiles lose gravitropic curvature in space but not on a clinostat (Chapman et al., 1994). Obviously, stimulus withdrawal in space and on a clinostat are qualitatively different, since clinostat rotation results in a continuously changing stimulation that is circumlateral (one axis of clinostat rotation) or omnilateral (three-dimensional clinostat; Hoson et al., 1997), whereas in microgravity, a g stimulus is essentially eliminated. Relatively few data exist that document the response of curved organs to a withdrawal of a constant g stimulus. Further study of this response is valuable both in evaluating the stability of gravitropic curvature and in understanding how the orientation of new growth is coordinated with that of older regions. For example, for organ straightening to occur, curved regions must straighten and new growth must be coordinately aligned (Fig.1E). Such alignment is only one of several possible fates, since in theory the growth that occurs in the absence of a constant g stimulus could be random or could reference persistent or past internal signal distributions (Fig. 1, A–D). Fig. 1. Open in new tabDownload slide Theoretically possible outcomes of the direction of root growth after gravitropically curved roots are then rotated on a clinostat. In A to D, the root retains gravitropic curvature and only new growth (black segments) is affected. A, Return to original vertical. B, No change from previous direction. C, Persistence of an internal signal gradient established by a previous constantg stimulus. D, Random growth. E and F, Loss of gravitropic curvature (root straightening) on clinostat and roots either fully (E) or partially (F) return to prestimulus vertical. Fig. 1. Open in new tabDownload slide Theoretically possible outcomes of the direction of root growth after gravitropically curved roots are then rotated on a clinostat. In A to D, the root retains gravitropic curvature and only new growth (black segments) is affected. A, Return to original vertical. B, No change from previous direction. C, Persistence of an internal signal gradient established by a previous constantg stimulus. D, Random growth. E and F, Loss of gravitropic curvature (root straightening) on clinostat and roots either fully (E) or partially (F) return to prestimulus vertical. To address these issues, the behavior of individual cress roots was followed through reorientation and subsequent rotation on a clinostat. We demonstrate that clinostat rotation results both in the loss of gravitropic curvature and in the coordinated alignment of new growth to produce mostly straight roots. However, these roots do not return completely to the prestimulus vertical (Fig. 1F), and the final angle between the root and the former vertical is positively related to the length of prior stimulation. MATERIALS AND METHODS Plant Material and Experimental Procedure Seeds of garden cress (Lepidium sativum L.) were obtained from Chrysant (Bonn, Germany), allowed to imbibe in double-distilled water, and then germinated on filter paper in vertically positioned plastic Petri plates. After 24 h in darkness at 22 ± 1.5°C, roots were approximately 5 mm long. Dishes were then turned to the horizontal for 1 or 5 h in darkness and then, along with vertical controls, were placed on a clinostat (Fig.2A). The custom-made clinostat was built with a 1 rpm synchronous instrument motor (model KS, Hurst Corp., Princeton, IN). Roots were rotated for 6 h either in an “axial” configuration (the long axis of the base of the root was parallel to the axis of clinostat rotation) or in a “somersault” configuration (root axis perpendicular to the axis of rotation). Fig. 2. Open in new tabDownload slide Diagram of experiment and angle measurement. A, Vertically grown roots (24 h) were turned to the horizontal and then rotated on a clinostat in two different configurations (curved arrow, bottom center). The gravity vector is toward the bottom of the diagram. B and C, Method of angle measurement relative to the original vertical (prestimulus) reference line (0°). Tracings from gravitropically curved roots after 5 h of horizontal stimulation (B, gravity vector toward left) and then after 6 h of rotation on a clinostat (C). The boundary (arrowhead) between the hypocotyl (light shading) and the root base is distinguished by root hairs (fine lines). The seed coat (dark shading) is shown at the top. C, Decrease in the angle of the apical and middle segments indicates a loss of gravitropic curvature and root straightening. Bar in B = 0.5 cm. Fig. 2. Open in new tabDownload slide Diagram of experiment and angle measurement. A, Vertically grown roots (24 h) were turned to the horizontal and then rotated on a clinostat in two different configurations (curved arrow, bottom center). The gravity vector is toward the bottom of the diagram. B and C, Method of angle measurement relative to the original vertical (prestimulus) reference line (0°). Tracings from gravitropically curved roots after 5 h of horizontal stimulation (B, gravity vector toward left) and then after 6 h of rotation on a clinostat (C). The boundary (arrowhead) between the hypocotyl (light shading) and the root base is distinguished by root hairs (fine lines). The seed coat (dark shading) is shown at the top. C, Decrease in the angle of the apical and middle segments indicates a loss of gravitropic curvature and root straightening. Bar in B = 0.5 cm. Video Imaging and Data Analysis Roots on the clinostat were continuously illuminated with dim-green light (intensity of approximately 0.9 μmol m−2 s−1 at root level) provided by an incandescent lamp filtered through two layers of a Roscolux filter (no. 1090, Rosco Laboratories, Port Chester, NY) with a peak transmission of 526 nm and a half-bandwidth of about 58 nm. This enabled visualization of root growth and behavior using a Hi8 videocamera (model CCD-V101, Sony, Tokyo, Japan). Images were archived at 1-h intervals using a videocassette recorder (model EV-5900, Sony) and were subsequently digitized using a computer equipped with a video capture card (Snappy Play, Inc., Rancho Cordova, CA). Images were stored and processed using imaging software (Adobe Photoshop 4.0). To measure curvature, roots were divided into three segments (Fig. 2, B and C): the tipmost 2 mm, the next 4 mm (middle or subapical segment), and the entire remaining basal segment. The basal segment extended to the base of the hypocotyl, which could be distinguished from the root by the absence of root hairs on the hypocotyl and by the larger diameter and the lighter color of the root. Even though the basal segment varied in length from 2 to 7 mm (depending upon the overall length and age of the root), initial experiments indicated that it showed enough uniformity in the distribution of curvature that it could be quantified as a single segment. To test methods of delimiting root segments, root-straightening data were compared for the same sample of 10 roots divided either into three segments as above or into 2-mm segments throughout the length of each root. Since all trends were comparable using both methods for individual roots and for pooled data (data not shown), the simpler three-segment method was adopted. The angle measured was between the tangent of each of the three segments and the original vertical reference line designated as 0° (Fig. 2B). RESULTS Cress roots turned to the horizontal for 1 h curved down gravitropically at a mean of 62 ± 5° (± se). When such roots were subsequently placed on a clinostat and rotated for 6 h, some gravitropic curvature was lost and the roots mostly straightened (Fig. 3, A and C). This straightening on the clinostat occurred in part in regions of the root that previously had curved gravitropically (Fig. 3, A, C, and E). Loss of gravitropic curvature of the root was observed regardless of whether the direction of rotation of the clinostat was around the root axis (axial configuration, Fig. 3A) or perpendicular to the root axis (“somersault” configuration, Fig. 3C). Vertically grown (control) roots grew in a more or less straight direction on a clinostat, although frequently these roots slanted or curved spontaneously away from their original direction of growth (Fig. 3, B and D). Fig. 3. Open in new tabDownload slide Images of the same roots through time showing the loss of gravitropic curvature (after 1 h of horizontal stimulation) during rotation on a clinostat (A, C, and E). Control roots were kept vertical prior to placement on a clinostat (B and D). The numbers indicate the time elapsed (hours) following placement on a clinostat. Each figure (A–D) shows a different sample of six roots with each root depicted three times. The positions of the roots are different in A and B versus C and D to reflect the orientation of clinostat rotation that was either in a “somersault” configuration (A and B) or in an axial configuration (C and D). The circular arrow at the right indicates the direction of rotation of the clinostat. The original gravity vector was toward the bottom of the figure for A, B, and E. Arrowheads indicate the transition zone between the root and the hypocotyl. Bar = 1 cm for A to D. E, Tracings from a single root kept horizontal for 1 h and then rotated on a clinostat for 6 h. The white and gray images show the root at the time labeled and at the previous time point, respectively. The horizontal line indicates the transition zone between the root and the hypocotyl. The double arrow (6 h) shows the length of the root at 0 h for a comparison. Fig. 3. Open in new tabDownload slide Images of the same roots through time showing the loss of gravitropic curvature (after 1 h of horizontal stimulation) during rotation on a clinostat (A, C, and E). Control roots were kept vertical prior to placement on a clinostat (B and D). The numbers indicate the time elapsed (hours) following placement on a clinostat. Each figure (A–D) shows a different sample of six roots with each root depicted three times. The positions of the roots are different in A and B versus C and D to reflect the orientation of clinostat rotation that was either in a “somersault” configuration (A and B) or in an axial configuration (C and D). The circular arrow at the right indicates the direction of rotation of the clinostat. The original gravity vector was toward the bottom of the figure for A, B, and E. Arrowheads indicate the transition zone between the root and the hypocotyl. Bar = 1 cm for A to D. E, Tracings from a single root kept horizontal for 1 h and then rotated on a clinostat for 6 h. The white and gray images show the root at the time labeled and at the previous time point, respectively. The horizontal line indicates the transition zone between the root and the hypocotyl. The double arrow (6 h) shows the length of the root at 0 h for a comparison. The behavior of the three regions of the roots was approximated by measuring segment angles (Fig. 2, B and C) through time to determine the location, timing, and extent of straightening (Fig.4). Many roots that were kept in a horizontal orientation for 1 h contained part of the zone of gravitropic curvature in their tips (distal 2 mm). The tip angle remained unchanged during the 1st h of rotation on the clinostat (Fig.4, A and C). The tip angle subsequently decreased 25 to 30° over the next 5 h of clinostat rotation. This decrease resulted from the former zone of gravitropic curvature becoming located farther from the tip due to new root growth. Also, growth at the tip became oriented closer to the original vertical. The middle and basal segments continued curving toward the last constant gravity vector during the first 2 to 4 h of clinostat rotation, i.e. some gravitropic curvature continued to be expressed in these segments. By 5 to 6 h of rotation, the values for the angles of each of the three root segments converged, indicating a significant amount of root straightening. The same pattern and timing of root straightening occurred in both the axial and somersault configurations of root rotation (Fig. 4, A and C). In control roots the mean angles for all three segments were comparable throughout the period of clinostat rotation, indicating that on average these roots showed no preferred direction of root growth and/or that many roots were more or less straight. Thus, the straightening of gravitropically curved roots results in part from the loss of gravitropic curvature in older regions and in part from the angle of new root growth on the clinostat moving closer to the original vertical (prior to horizontal stimulation). These processes appear coordinated so that the angles of both regions become roughly aligned. Fig. 4. Open in new tabDownload slide Angles of root segments through time showing the development of gravitropic curvature and subsequent straightening on a clinostat. Negative and positive values on abscissa indicate times for both horizontal and on clinostat, respectively. A and C, Roots horizontal for 1 h. B and D, Control (vertical) roots. All roots were rotated for 6 h either in a somersault (A and B) or an axial configuration (C and D). Mean angles (± se) for each segment (tip [○], middle [□], and base [▵]) were obtained from 60 to 80 plants for each treatment. Fig. 4. Open in new tabDownload slide Angles of root segments through time showing the development of gravitropic curvature and subsequent straightening on a clinostat. Negative and positive values on abscissa indicate times for both horizontal and on clinostat, respectively. A and C, Roots horizontal for 1 h. B and D, Control (vertical) roots. All roots were rotated for 6 h either in a somersault (A and B) or an axial configuration (C and D). Mean angles (± se) for each segment (tip [○], middle [□], and base [▵]) were obtained from 60 to 80 plants for each treatment. Although these roots mostly straightened, they were still slanted 25 ± 3° away from the original vertical (Fig. 4, A and C) in a direction that indicated the retention of some gravitropic reaction. However, this angle was reduced by 37° compared with the previous angle of gravitropic curvature (62° after 1 h in a horizontal orientation). Roots rotated on the clinostat extended in length at the same rate, approximately 0.75 mm h−1, as that of horizontally and vertically grown stationary roots. Neither prior root orientation nor the configuration of rotation affected this growth rate. During the 6 h on a clinostat, the roots grew about 4.5 mm. To determine whether an extended period of horizontal stimulation would preclude root straightening after withdrawal of the directional gstimulus, roots were horizontally stimulated for 5 h. The horizontal, stationary roots reached maximal gravitropic curvature within 2 h of reorientation from the vertical, and after 5 h of horizontal stimulation the mean curvature was 88 ± 3°. After these roots were rotated on a clinostat for 6 h, the majority lost gravitropic curvature and were essentially straight regardless of the direction of clinostat rotation (Fig. 5, A and C). A small fraction of roots lost no or only some gravitropic curvature and were still curved after clinostat rotation. Fig. 5. Open in new tabDownload slide Images of roots on a clinostat as in Figure 3except that roots in A and C were horizontal for 5 h before placement on the clinostat. A and C, Despite variability most roots exhibited partial loss of gravitropic curvature (roots marked with *) and root straightening after 6 h on the clinostat. B and D, Control roots mostly resembled those in Figure 3, B and D. Bar = 1 cm. Fig. 5. Open in new tabDownload slide Images of roots on a clinostat as in Figure 3except that roots in A and C were horizontal for 5 h before placement on the clinostat. A and C, Despite variability most roots exhibited partial loss of gravitropic curvature (roots marked with *) and root straightening after 6 h on the clinostat. B and D, Control roots mostly resembled those in Figure 3, B and D. Bar = 1 cm. Measurement of the angles of subsections of roots that were horizontally stimulated for 5 h yielded results (Fig.6, A and C) comparable to roots that were horizontal for 1 h. The tip segment started to move closer to the original vertical within 1 h of clinostat rotation. By 6 h, new root growth was oriented 20 to 30° away from the angle that the tip had occupied at the start of rotation (Fig. 6, A and C). Gravitropic curvature continued to be expressed at a rate of about 2 to 5° h−1 in the middle and basal regions during the first 3 to 4 h of clinostat rotation. The reciprocal loss of tip curvature and the increase in the angles of the middle and basal segments resulted in a convergence of all three angles, indicating an alignment or straightening of much of the root axis. Roots that were fully curved gravitropically (after 5 h of horizontal stimulation) straightened on average about 35° on a clinostat, essentially the same value obtained for roots stimulated for only 1 h. But since the 5-h roots were more curved to start with, they retained more of the graviresponse at the end of clinostat rotation; i.e. the mean angle of the entire root (also equal to the convergence angle of the three segments) was 53 ± 5° for roots stimulated for 5 h compared with 25° for roots that were horizontal for 1 h. Fig. 6. Open in new tabDownload slide Angles of root segments as in Figure 4, except that roots were horizontal for 5 h before placement on the clinostat. n = 40 to 60 plants for each treatment. A and C, Note that angles converge at 50 to 60°, indicating the retention of more of a graviresponse than in roots horizontal for 1 h. Symbols are the same as those for Figure 4. Fig. 6. Open in new tabDownload slide Angles of root segments as in Figure 4, except that roots were horizontal for 5 h before placement on the clinostat. n = 40 to 60 plants for each treatment. A and C, Note that angles converge at 50 to 60°, indicating the retention of more of a graviresponse than in roots horizontal for 1 h. Symbols are the same as those for Figure 4. Control roots exhibited a range of responses to clinostat rotation, including curvature and slanting at various angles (Fig. 5, B and D). Some control roots rotated in a somersault configuration slanted slightly away from the hypocotyl hook (Figs. 5B and 6B), a direction corresponding to the loss of gravitropic curvature (Figs. 5A and 6A). But the degree of this slanting in controls was less than the loss of gravitropic curvature, and these controls exhibited much more variability (compare ses in Fig. 6, A and B). In all other controls, the final net angle of the root axis was zero. To determine whether seed position affected root straightening, seeds were positioned above or below the hypocotyl root axis in horizontal seedlings. The position of the radicle can be determined in dry cress seeds (Volkmann et al., 1986). In all experiments the seeds were planted on the substrate so that the radicle emerged to the right of the seed (Fig. 2A). For all experiments except those shown in Figure7, vertical roots were turned counterclockwise to the horizontal so that the cotyledons, the seed, and the hypocotyl hook were on the lower side of the hypocotyl-root axis (Fig. 2A). This resulted in both the hypocotyl and the gravitropically curved root forming a “C” that was a clockwise curve starting from the apex of the hook to the root tip (0 h in Figs.3A and 5A). The curvature of the hypocotyl was maintained after root straightening on a clinostat. Fig. 7. Open in new tabDownload slide Seed position does not affect root straightening. In contrast to previous figures, the seed and the hypocotyl hook were positioned on the upper side relative to the horizontal roots (A). Both root straightening and retention of some gravitropic reaction occurred after 7 h of clinostat rotation, as indicated by the slanting of most roots to the right (B), the direction of the gravity vector when roots were previously horizontal for 1 h. Bar = 1 cm. Fig. 7. Open in new tabDownload slide Seed position does not affect root straightening. In contrast to previous figures, the seed and the hypocotyl hook were positioned on the upper side relative to the horizontal roots (A). Both root straightening and retention of some gravitropic reaction occurred after 7 h of clinostat rotation, as indicated by the slanting of most roots to the right (B), the direction of the gravity vector when roots were previously horizontal for 1 h. Bar = 1 cm. In the experiments shown in Figure 7 vertical roots were turned clockwise to the horizontal so that the cotyledons, seed, and hook were on the upper side of the hypocotyl-root axis. In this case the curves in the hypocotyl (below the hook) and in the root were in opposite orientations so that the two formed an “S” (at 0 h). When these seedlings were rotated on a clinostat for 7 h, the roots straightened (Fig. 7), just as they did when the seeds were originally positioned below the root (Figs. 3 and 5). Thus, root straightening occurs regardless of the positions of the seed and the hypocotyl hook. The curve in the hypocotyl (basal to the hook) was always toward the side of the axis containing the seed regardless of orientation with respect to gravity, indicating that the hypocotyl of 1-d-old seedlings does not exhibit significant gravitropic curvature after 1 h of horizontal stimulation. Also, unlike the gravitropic curvature of the root, significant curvature of the hypocotyl was maintained throughout clinostat rotation. DISCUSSION This study documents the straightening of gravitropically curved roots following withdrawal of a constant gstimulus. Curved roots straighten on a clinostat through a combination of a loss of gravitropic curvature and the alignment of new growth closer to the prestimulus vertical. Several previous reports exist of organ straightening afterg-stimulus withdrawal (Larsen, 1953, 1957; Mirza et al., 1984; Chapman et al., 1994; Volkmann and Tewinkel, 1996; Tarui and Iino, 1997). But even in studies in which the same organs were analyzed through time, only the tip angle was measured, and the stages of straightening were not shown (Mirza et al., 1984; Chapman et al., 1994;Tarui and Iino, 1997). To our knowledge, our data provide the first visual depiction and multiregion analysis of the successive loss of gravitropic curvature in the same organs through time following stimulus withdrawal. These data also show that root straightening in cress can occur at clinostat speeds of 1 rpm, whereas Larsen (1953,1957) only found straightening at much slower speeds. This finding of root straightening shows that gravitropic curvature, at least complete curvature induced by 5 h of stimulation, can be partly or fully reversed, whereas in roots that are left stationary and that are not rotated on a clinostat, gravitropic curvature persists for the life of the organ. This reversibility of gravitropic curvature on a clinostat might result from active breakdown and wall reconditioning and/or from wall elasticity in the zone of curvature. This raises the question of when, if ever, curvature gets sufficiently plastic and rigid such that it becomes irreversible. Analyses of the responses of growing maize coleoptiles to applied tensile forces have shown that even apparent plastic deformations can actually be a type of reversible viscoelastic deformation or retarded elasticity (Hohl and Schopfer, 1992; Cosgrove, 1993). Further study of this phenomenon of loss of gravitropic curvature might prove valuable in identifying changes in the biomechanical properties of cell walls responsible for differential tropic growth. In addition to the loss of gravitropic curvature in regions of the root formed before placement on the clinostat, root straightening also involves new growth on the clinostat. Possible explanations for this outcome are that in the absence of a constant g stimulus, that new growth follows and aligns with the straightening of older regions, or, conversely, that the loss of curvature results from coordination with new growth. Root straightening cannot be due to the influence of hypocotyl position or of some automorphogenetic component (Masuda et al., 1994;Stanković et al., 1998), since it occurs regardless of whether the seed and hypocotyl were located above or below the horizontally stimulated root. Similarly, it is found regardless of whether the roots were rotated along their axis or at right angles to their axis. The phenomenon of organ straightening following tropistic curvature has been referred to as “autotropism” (Pfeffer, 1906; Firn and Digby, 1979; Hart, 1990). The various usages of these terms have recently been critically reviewed (Stanković et al., 1998). Although the migration of curvature during a constant g stimulus has been termed “autotropism” (Firn and Digby, 1979; Hart, 1990; Myers et al., 1995; Tarui and Iino, 1997), it may be more appropriate to consider these growth adjustments to be part of the overall process of gravitropism rather than a separate “tropism,” autotropism that only occurs during and in response to gravitropism (Stanković et al., 1998). In contrast, the straightening that occurs after a withdrawal of a g stimulus is clearly not part of gravitropism and for historical reasons might still be called “autotropic straightening.” But as this straightening is not a bona fide tropism in the sense of a directional response of an organ to a current environmental vector, the term autonomic straightening might be more accurate. Regardless of terminology, the findings that the root returns closer to the prestimulus vertical indicates that there is some sort of inherent, autonomic, or default tendency for disoriented organs to revert to a previous equilibrium orientation (Fig. 1, E and F). This behavior seems to reflect some persistent commitment to return root growth toward a previous alignment (before it was turned on its side). At the same time, since the final angle is still below what was the horizontal (double arrow in Fig. 1F), some gravitropic reaction seems to have been retained, even though the roots are no longer curved. This apparent persistence of some gravitropic reaction might represent the limits of autonomic straightening. This possibility is supported by the finding that roots that were horizontally stimulated for 1 and 5 h both moved about 36° closer to the prestimulus vertical after clinostat rotation. Further study is required to determine whether roots with gravitropic curvatures smaller than 36° (before clinostat rotation) return completely to the prestimulus vertical. In any case, the total amount of autonomic straightening is not simply limited by properties of the zone of gravitropic curvature (e.g. wall elasticity), but is actively and coordinately regulated in several regions of the root. ACKNOWLEDGMENTS We would like to thank K. Aram for technical assistance and Michael Evans and several anonymous reviewers for their valuable comments on the manuscript. LITERATURE CITED 1 Chapman DK Johnsson A Karlsson C Brown A Heathcote D Gravitropically-stimulated seedlings show autotropism in weightlessness. Physiol Plant 90 1994 157 162 Google Scholar Crossref Search ADS WorldCat 2 Cosgrove DJ Wall extensibility: its nature, measurement and relationship to plant cell growth. New Phytol 124 1993 1 23 Google Scholar Crossref Search ADS PubMed WorldCat 3 Firn RD Digby J A study of the autotropic straightening reaction of a shoot previously curved during autotropism. Plant Cell Environ 2 1979 149 154 Google Scholar Crossref Search ADS WorldCat 4 Hart JW (1990) Plant Tropisms and Other Growth Movements. Unwin Hyman, London, UK 5 Hensel W Iversen T-H Ethylene production during clinostat rotation and effect on root geotropism. Zeitsch für Pflanzen 97 1980 343 352 Google Scholar Crossref Search ADS WorldCat 6 Hohl M Schopfer P Physical extensibility of maize coleoptile cell walls: apparent plastic extensibility is due to elastic hysteresis. Planta 187 1992 498 504 Google Scholar PubMed OpenURL Placeholder Text WorldCat 7 Hoson T Kamisaka S Masuda Y Yamashita M Buchen B Evaluation of the three-dimensional clinostat as a simulator of weightlessness. Planta 203 1997 S187 S197 Google Scholar Crossref Search ADS PubMed WorldCat 8 Larsen P Influence of gravity on the rate of elongation and on geotropic and autotropic reactions in roots. Physiol Plant 6 1953 735 744 Google Scholar Crossref Search ADS WorldCat 9 Larsen P The development of geotropic and spontaneous curvatures in roots. Physiol Plant 10 1957 127 163 Google Scholar Crossref Search ADS WorldCat 10 MacDonald IR Hart JW Gordon DC Analysis of growth during geotropic curvature in seedling hypocotyls. Plant Cell Environ 6 1983 401 406 Google Scholar Crossref Search ADS WorldCat 11 Masuda Y Kamisaka S Yamamoto R Hoson T Nishitani K Plant responses to simulated microgravity. Adv Space Biol Med 4 1994 111 126 Google Scholar Crossref Search ADS PubMed WorldCat 12 Mirza JI Olsen GM Iversen T-H Maher EP The growth and gravitropic responses of wild-type and auxin-resistant mutants of Arabidopsis thaliana. Physiol Plant 60 1984 516 522 Google Scholar Crossref Search ADS WorldCat 13 Myers AB Glyn GH Digby J Firn RD The effect of displacement angle on the gravitropic and autotropic growth responses of sunflower hypocotyls. Ann Bot 75 1995 277 280 Google Scholar Crossref Search ADS WorldCat 14 Pfeffer W Autotropism and somatotropism. Ewart AJ Physiology of Plants. 1906 189 192 Clarendon Press Oxford, UK 15 Stanković B Volkmann D Sack FD Autotropism, automorphogenesis, and gravity. Physiol Plant 102 1998 328 335 Google Scholar Crossref Search ADS PubMed WorldCat 16 Tarui Y Iino M Gravitropism of oat and wheat coleoptiles: dependence on the stimulation angle and involvement of autotropic straightening. Plant Cell Physiol 38 1997 1346 1353 Google Scholar Crossref Search ADS PubMed WorldCat 17 Volkmann D Behrens HM Sievers A Development and gravity sensing of cress roots under microgravity. Naturwissenschaften 73 1986 438 441 Google Scholar Crossref Search ADS PubMed WorldCat 18 Volkmann D Tewinkel M Gravisensitivity of cress roots: investigations of threshold values under specific conditions of sensor physiology in microgravity. Plant Cell Environ 19 1996 1195 1202 Google Scholar Crossref Search ADS PubMed WorldCat Author notes 1 This work was supported by grants from the National Aeronautics and Space Administration (grant no. NAG2-1023) to F.S. and by Deutsche Agentur für Raumfahrtangelegenheiten (Bonn, Germany, grant no. 50 9429) and MWF (Düsseldorf) to D.V. 2 Present address: Botany Department, North Carolina State University, Raleigh, NC 27695. * Corresponding author; e-mail [email protected]; fax 1–614– 292–6345. Copyright © 1998 American Society of Plant Physiologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Bouwmeester, Harro J.; Gershenzon, Jonathan; Konings, Maurice C.J.M.; Croteau, Rodney

doi: 10.1104/pp.117.3.901pmid: 9662532

Abstract The biosynthesis of the monoterpenes limonene and carvone in the fruit of caraway (Carum carvi L.) proceeds from geranyl diphosphate via a three-step pathway. First, geranyl diphosphate is cyclized to (+)-limonene by a monoterpene synthase. Second, this intermediate is stored in the essential oil ducts without further metabolism or is converted by limonene-6-hydroxylase to (+)-trans-carveol. Third, (+)-trans-carveol is oxidized by a dehydrogenase to (+)-carvone. To investigate the regulation of monoterpene formation in caraway, we measured the time course of limonene and carvone accumulation during fruit development and compared it with monoterpene biosynthesis from [U-14C]Suc and the changes in the activities of the three enzymes. The activities of the enzymes explain the profiles of monoterpene accumulation quite well, with limonene-6-hydroxylase playing a pivotal role in controlling the nature of the end product. In the youngest stages, when limonene-6-hydroxylase is undetectable, only limonene was accumulating in appreciable levels. The appearance of limonene-6-hydroxylase correlates closely with the onset of carvone accumulation. At later stages of fruit development, the activities of all three enzymes declined to low levels. Although this correlates closely with a decrease in monoterpene accumulation, the latter may also be the result of competition with other pathways for substrate. Monoterpenes are 10-carbon members of the isoprenoid family of natural products (Gershenzon and Croteau, 1993). They are widespread in the plant kingdom (Banthorpe and Charlwood, 1980) and are often responsible for the characteristic odors of plants. These substances are believed to function principally in ecological roles, serving as herbivore-feeding deterrents, antifungal defenses, and attractants for pollinators (Langenheim, 1994). The commercial importance of monoterpenes as flavorings, fragrances, and pharmaceuticals has stimulated many efforts to increase their yield in plants. Caraway (Carum carvi L.), native to Europe, western Asia, and northern Africa, is an important monoterpene-containing herb, which contains (+)-carvone and (+)-limonene as its major monoterpene components. The caraway fruit is a schizocarp, which at harvest is separated into two halves, which are called “seeds.” In this paper we use the agricultural terms “seed” for a half-fruit and “fruit” for the entire fruit (two “seeds”). The seeds and seed oil of caraway are used traditionally in ice cream, candy, baked goods, meat, cheese, pickles, condiments, soft drinks, and alcoholic beverages (Morton, 1976). Recently, (+)-carvone extracted from caraway seeds has been introduced as an effective sprouting inhibitor of potatoes (Oosterhaven et al., 1995). The expanding commercial potential of (+)-carvone has now generated interest in maximizing the yield of this substance from caraway seeds, a goal that requires an understanding of the process of carvone biosynthesis. More than 30 years ago, Sandermann and Bruns (1965) hypothesized that in dill fruits, which also contain (+)-carvone and (+)-limonene as the main components of its essential oil, limonene is an intermediate in the biosynthesis of carvone. During fruit development the content of carvone (as a percentage of fruit weight) increases at the expense of limonene, providing support for this hypothesis. However, after measuring the changes in the absolute amounts of limonene and carvone in caraway fruits and performing in vivo-radiolabeling experiments, Von Schantz and Ek (1971) and Von Schantz and Huhtikangas (1971) showed that limonene is no longer available as a precursor for carvone biosynthesis once it has been secreted into the essential oil ducts. The pathway of (+)-limonene and (+)-carvone biosynthesis in caraway has been assumed (Bouwmeester et al., 1995b) to be analogous to the biosynthesis of (−)-limonene and (−)-carvone in spearmint (Gershenzon et al., 1989) (Fig. 1). In this process, GPP, the ubiquitous precursor of the monoterpenes, is cyclized by a monoterpene synthase to (+)-limonene. The product is either stored in the essential oil ducts or oxidized to (+)-trans-carveol by a Cyt P-450-dependent hydroxylase. Subsequently, a NAD+ or NADP+-utilizing dehydrogenase oxidizes (+)-trans-carveol to (+)-carvone, which is then stored exclusively in the essential oil ducts. Fig. 1. Open in new tabDownload slide Biosynthetic pathway of (+)-limonene and (+)-carvone in fruits of caraway. Fig. 1. Open in new tabDownload slide Biosynthetic pathway of (+)-limonene and (+)-carvone in fruits of caraway. The annual and biennial forms of caraway have been studied, and show some interesting differences in essential oil formation. Although both forms produce an essential oil that consists predominantly of carvone and limonene, the fruits of annual caraway varieties generally have a lower essential oil content than the fruits of biennial varieties (Bouwmeester and Kuijpers, 1993), probably because of a greater carbon partitioning to essential oil formation in biennial caraway (Bouwmeester et al., 1995a). In addition, biennial caraway usually has a higher carvone-to-limonene ratio. For both caraway forms, accumulation of limonene and carvone in the fruits is a developmentally regulated process. Whereas limonene accumulation predominates in the early stages of development, carvone accumulation predominates in the later stages such that, when the fruits mature, carvone and limonene contents are approximately equal (Von Schantz and Ek, 1971; Bouwmeester and Kuijpers, 1993;Bouwmeester et al., 1995a). Although it has been suggested that carvone and limonene accumulation continue until fruit maturity (Von Schantz and Ek, 1971; Von Schantz and Huhtikangas, 1971), Bouwmeester et al. (1995a) showed that carvone and limonene accumulation ceases several weeks before. The pattern of carvone and limonene accumulation in caraway fruits may be explained by the changing levels of biosynthetic enzyme activities during fruit development. In the early stages of development, when only limonene accumulates, the enzyme that converts limonene totrans-carveol may be inactive (Fig. 1). At a later stage, when both limonene and carvone are accumulating, the ratio of limonene to carvone being formed may depend on the relative activities of the enzymes in the pathway. Finally, when limonene and carvone accumulation has ceased, one or more of the enzymes in the pathway before limonene may have been inactivated. Alternatively, the formation of other components of the developing fruit, such as storage proteins, starch, or triacylglycerols, may divert the flow of carbon away from monoterpene biosynthesis. To better understand the mechanisms regulating the developmental changes of limonene and carvone accumulation in caraway fruits, we investigated the pathway of limonene and carvone biosynthesis from the ubiquitous C10 intermediate GPP. The activities of all three enzymes involved were demonstrated, and their substrate specificities determined. In addition, we studied the appearance of the relevant biosynthetic enzymes during fruit development in annual and biennial caraway in relation to the formation of monoterpenes and the accumulation of triacylglycerols, the major seed reserve. MATERIALS AND METHODS Seeds of annual caraway (Carum carvi L. var Karzo) were sown in January, 1995, in potting compost in 5-L plastic containers and after emergence thinned to one plant per pot. Taproots of biennial caraway (C. carvi L. var Bleija) were obtained from an experimental field in Wageningen, The Netherlands, in February, 1995, before regrowth had started, submerged in 2 g/L benomyl for 10 to 15 min to prevent fungal diseases, and planted individually in potting compost in 5-L plastic containers. All plants were placed in a greenhouse at 18/14°C under a cycle of 12-h light/12-h dark and 75% RH. Natural daylight was supplemented with artificial light (Philips, Eindhoven, The Netherlands) during the 12-h high-temperature period, and fertilizer was applied as required. Aphids, thrips, and spider mites were controlled by spraying with pirimicarb, imidacloprid, and methomyl. Powdery mildew was controlled by using a sulfur vaporizer. Flowering of both caraway varieties started around the beginning of May. As an umbelliferous species, caraway has several umbel orders that flower successively in time. The umbels of each umbel order are all in the same developmental stage. Dates of female flowering for 1st- to 4th-order umbels were determined for all individual plants. Because of the inherent variation in flowering time between umbel orders and the variation in flowering time between plants, a range of different developmental stages was obtained. From the beginning of flowering, houseflies were released into the greenhouse at regular intervals to ensure pollination. Because the flies are efficient pollinators of caraway, it was assumed that pollination occurred at the date of female flowering when pistils were receptive. For the developmental study, nine umbel orders were selected from both caraway varieties on May 22 and June 12 such that the samples ranged in development from 5 to 34 DAP for the biennial form and 7 to 34 DAP for the annual form (Table I). From previous experiments (Bouwmeester and Kuijpers, 1993), it was known that the formation of limonene and carvone level off at about 30 DAP. Representative umbellets of each developmental stage were used for the [14C]Suc-feeding experiment, and fruits were collected from the remaining umbels/umbellets for chemical and biochemical analyses by cutting the stalks with a small pair of scissors just below the fruits. About 0.5 g of fruits was used for the analysis of limonene, carvone, and fatty acid content, whereas about 1 g was employed in the assays of (+)-limonene synthase, (+)-limonene-6-hydroxylase, and (+)-trans-carveol dehydrogenase activities. Fruits were accurately weighed out into plastic vials, frozen in liquid N2, and stored at −80°C until further analysis. Fruit dry matter percentage and mean individual fruit weight were determined by weighing about 0.5 to 1.0 g of fruits before and after drying overnight at 105°C. Table I. Dates of pollination (female flowering) and harvest of fruits of annual and biennial caraway and their developmental stage Plant and Umbel Order . Date . Developmental Stage . Pollination . Harvest . DAP Annual  3 May 15 May 22 7  2 May 12 May 22 10  2 May 9 May 22 13  3 May 7 May 22 15  4 May 27 Jun 12 16  3 May 25 Jun 12 18  4 May 16 Jun 12 27  2 May 15 Jun 12 28  3 May 9 Jun 12 34 Biennial  3 May 17 May 22 5  4 May 15 May 22 7  3 May 12 May 22 10  3 May 8 May 22 14  4 May 23 Jun 12 20  4 May 20 Jun 12 23  4 May 16 Jun 12 27  4 May 13 Jun 12 30  3 May 9 Jun 12 34 Plant and Umbel Order . Date . Developmental Stage . Pollination . Harvest . DAP Annual  3 May 15 May 22 7  2 May 12 May 22 10  2 May 9 May 22 13  3 May 7 May 22 15  4 May 27 Jun 12 16  3 May 25 Jun 12 18  4 May 16 Jun 12 27  2 May 15 Jun 12 28  3 May 9 Jun 12 34 Biennial  3 May 17 May 22 5  4 May 15 May 22 7  3 May 12 May 22 10  3 May 8 May 22 14  4 May 23 Jun 12 20  4 May 20 Jun 12 23  4 May 16 Jun 12 27  4 May 13 Jun 12 30  3 May 9 Jun 12 34 Open in new tab Table I. Dates of pollination (female flowering) and harvest of fruits of annual and biennial caraway and their developmental stage Plant and Umbel Order . Date . Developmental Stage . Pollination . Harvest . DAP Annual  3 May 15 May 22 7  2 May 12 May 22 10  2 May 9 May 22 13  3 May 7 May 22 15  4 May 27 Jun 12 16  3 May 25 Jun 12 18  4 May 16 Jun 12 27  2 May 15 Jun 12 28  3 May 9 Jun 12 34 Biennial  3 May 17 May 22 5  4 May 15 May 22 7  3 May 12 May 22 10  3 May 8 May 22 14  4 May 23 Jun 12 20  4 May 20 Jun 12 23  4 May 16 Jun 12 27  4 May 13 Jun 12 30  3 May 9 Jun 12 34 Plant and Umbel Order . Date . Developmental Stage . Pollination . Harvest . DAP Annual  3 May 15 May 22 7  2 May 12 May 22 10  2 May 9 May 22 13  3 May 7 May 22 15  4 May 27 Jun 12 16  3 May 25 Jun 12 18  4 May 16 Jun 12 27  2 May 15 Jun 12 28  3 May 9 Jun 12 34 Biennial  3 May 17 May 22 5  4 May 15 May 22 7  3 May 12 May 22 10  3 May 8 May 22 14  4 May 23 Jun 12 20  4 May 20 Jun 12 23  4 May 16 Jun 12 27  4 May 13 Jun 12 30  3 May 9 Jun 12 34 Open in new tab The demonstration of enzyme activities, optimization of assay conditions, and investigation of substrate and product stereospecificity were carried out using bulk samples of suitable developmental stages, which were collected from plants raised in the greenhouse as described above (biennial caraway) or in the field (annual caraway). Preparation of Substrates [1-3H]GPP was synthesized as described by Croteau and Cane (1985). (+)- and (−)-Limonene were from Janssen Chimica (Geel, Belgium) and Merck-Schuchardt (Hohenbrunn, Germany), respectively. trans-Carveol was obtained by Meerwein-Ponndorf-Verley reduction of (+)-carvone using aluminum isopropoxide and dry isopropyl alcohol (Ponndorf, 1926; Johnston and Read, 1934). The resulting mixture of trans- andcis-carveol and a commercial (−)-cis/trans-carveol mixture (Janssen Chimica) were separated using a preparative gas-liquid chromatograph (model 3700, Varian, Sunnyvale, CA) equipped with a glass column (2-m × 4-mm i.d.) packed with 10% (w/w) Carbowax HP on Chromosorb 100–120 (Chrompack International, Middleburg, The Netherlands). The oven temperature was 140°C isothermal, injection port temperature was 200°C, and H2 column flow was 50 mL min−1. The desired product (as judged from the thermal conductivity detector signal) was allowed to condense in a glass capillary, and the compounds were then eluted from the capillary with pentane. Chiral analysis was performed by GC-MS using a HP 5890 series II gas chromatograph and HP 5972A mass selective detector (Hewlett-Packard) equipped with a fused silica capillary column (25-m × 0.25-mm i.d.) with octakis (6-O-methyl-2, 3-di-O-pentyl)-γ-cyclodextrin (80% [w/w] in OV1707) as the stationary phase (König et al., 1990). The oven was programmed at an initial temperature of 45°C for 1 min, with a ramp of 10°C min−1 to 200°C and a final time of 5 min. The injection port (splitless mode), interface, and MS source temperatures were 175, 290, and 180°C, respectively, and the He inlet pressure was controlled by electronic pressure to achieve a constant column flow of 1.0 mL min−1. Ionization potential was set at 70 eV, and scanning was performed both from 50 to 175 atomic mass units and in the selected ion-monitoring mode: for limonene m/z 68, 93, and 136; for camphor m/z 81, 95, and 152; for carvone m/z 82, 108, and 150; fortrans-carveol m/z 84, 109, and 152; and forcis-carveol m/z 84, 109, and 134. Analysis showed that racemization had occurred during the synthesis of the carveols. The mixtures obtained after preparative GC separation were (+)- and (−)-trans-carveol- 1.1:1 and (+)- and (−)-cis-carveol- 1.9:1. Preparative GC separation of the commercial (−)-cis/trans-carveol mixture gave purified samples of (−)-trans-carveol and (−)-cis-carveol, each with <1% of the other geometrical isomer. Enzyme Isolation During enzyme isolation and preparation of the assays, all operations were carried out on ice or at 4°C. The extraction procedure and buffer were adjusted principally to optimize hydroxylase activity (H.J. Bouwmeester, M.C.J.M. Konigs, J. Gershenzon, F. Karp, and R. Croteau, unpublished data), which also gave high levels of activity for the other two enzymes. The frozen fruit samples were ground in a prechilled mortar and pestle in prechilled buffer (10 mL/g tissue) containing 50 mm Hepes (pH 7.5), 20% (v/v) glycerol, 50 mm sodium ascorbate, 50 mmNaHSO3, 5 mmMgCl2, 2.5 mm EDTA, 2.5 mm EGTA, 5 mm DTT, 5 μm FAD, 5 μm FMN, 0.5 mm glutathione, 2 mg mL−1 BSA, 5 μg mL−1leupeptin, and 25 IU mL−1 catalase slurried with 1 g of polyvinylpolypyrrolidone and three spatula tips of purified sea sand. During grinding, additional aliquots of buffer (without polyvinylpolypyrrolidone and sea sand) were added to a total volume of 30 mL/g fruits. The homogenate was transferred to a small beaker containing polystyrene resin (0.5 g/g fruit, Amberlite XAD-4, Serva, Paramus, NJ), sonicated for 4 min in 10-s pulses (on ice), stirred carefully for 12 min, and then filtered through cheesecloth. The filtrate was centrifuged at 20,000g for 20 min (pellet discarded) and then at 150,000g for 90 min. The supernatant was used to assay limonene synthase and trans-carveol dehydrogenase activity, and the 150,000g pellet was used to assay limonene-6-hydroxylase activity. Crude fractions were diluted before assay so that the activities were linear over the time period measured for all developmental stages. Limonene Synthase For routine determination of enzyme activity, 5 μL of the 150,000g supernatant was diluted 20-fold in an Eppendorf tube with buffer A (15 mm Tris [pH 7.5], 10% [v/v] glycerol, 50 mm KCl, 1 mm sodium ascorbate, 1 mm MnCl2, and 2 mm DTT), and 35 μm [1-3H]GPP (21 Ci/mol) was added. The reaction mixture was overlaid with 1 mL of hexane to trap volatile products and the contents were mixed. After incubation for 30 min at 30°C, the vials were vigorously mixed and centrifuged briefly to separate phases. A portion of the hexane phase (750 μL) was transferred to a new Eppendorf tube containing 40 mg of silica gel (0.035–0.07 mm, pore diameter 6 nm, Janssen Chimica) to bind terpenols produced by phosphohydrolases, and after mixing and centrifugation 500 μL of the hexane layer was removed for liquid-scintillation counting in 4.5 mL of Ultima Gold cocktail (Packard, Meriden, CT). All assays were performed in duplicate or triplicate; controls that had been boiled for 5 min showed no enzymatic activity. For product identification using radio-GLC, 100 μL of the 150,000g supernatant was diluted 10-fold with buffer A in a 9-mL Teflon-lined screw-cap tube. After the addition of 3 μm [1-3H]GPP (250 Ci/mol) and a 1-mL pentane overlay to trap volatile products, the tube was carefully mixed and incubated for 1 h at 30°C. Following the assay, the tube was vigorously mixed and stored at −20°C until further analysis. After thawing, 250 μL of diethyl ether was added to the assay mixture. The organic layer was removed and passed over a short column of silica gel overlaid with anhydrous MgSO4. The assay was extracted with another 1 mL of pentane-diethyl ether (4:1, v/v), which was also passed over the silica column, and the column was washed with 1.5 mL of pentane. After addition of unlabeled α-pinene, sabinene, myrcene, limonene, γ-terpinene, and p-cymene as the carriers, monoterpenes that have all been reported to be constituents of caraway seed essential oil (Wichtmann, 1988), the mixture was slowly concentrated under a stream of N2. Radio-GLC was performed on a gas chromatograph (series 4160, Cazlo-Erba, Milano, Italy) equipped with a RAGA-90 radioactivity detector (Raytest, Straubenhardt, Germany). Sample components eluting from the column were quantitatively reduced before radioactivity measurement by passage through a conversion reactor filled with platinum chips at 800°C. Two-microliter samples were injected in the cold on-column mode. The fused silica capillary column (30-m × 0.32-mm i.d.) was coated with a film of 0.25 μm of PEG (EconoCap EC-WAX, Alltech Associates, Inc., Deerfield, IL) and operated with a He inlet pressure of 1.35 kg cm−2, giving a flow of 1 mL min−1. The oven temperature was programmed to 70°C for 5 min, followed by a ramp of 5° min−1 to 200°C and a final time of 5 min. To determine retention times and peak identities (by co-elution of radioactivity with reference standards), about 20% of the column effluent was split with an adjustable splitter to a flame-ionization detector (temperature 270°C). The remainder was directed to the conversion reactor and radiodetector. H2 was added prior to the reactor at 3 mL min−1, and CH4 as a quench gas prior to the radioactivity detector (5-mL counting tube) to give a total flow of 36 mL min−1. Limonene-6-Hydroxylase Pellets from the 150,000g centrifugation were resuspended in 3.5 mL of buffer B (50 mm Tris [pH 7.2 for the biennial form, pH 7.4 for the annual form], 20% [v/v] glycerol, 1 mm EDTA, 2 mm DTT, 1 μg mL−1 leupeptin, 5 μm FAD, and 5 μm FMN) using a glass rod and a Teflon potter. A 0.5-mL portion of the resuspended pellet was then diluted 2-fold in buffer B in a 9-mL Teflon-lined screw-cap tube, and the reaction was started by the addition of 1 mm NADPH, 5 mm Glc-6-P, 1 IU of Glc-6-P dehydrogenase, and 200 nmol (+)-limonene in 5 μL of hexane. The assays were performed in duplicate. Controls that had been boiled for 5 min showed no enzymatic activity. After incubation for 1 h at 30°C, 1 mL of diethyl ether was added, and the tubes were vigorously mixed and then stored at −20°C until further analysis. For analysis, 25 nmol camphor was added as an internal standard. The reaction mixtures were thawed, vigorously mixed, and briefly centrifugated to separate phases. The ether phase was then transferred to another vial and the water layer was re-extracted with another 1-mL portion of diethyl ether. Next, the combined diethyl ether extracts were decolorized with activated charcoal, washed with 1 mL of water and, after centrifugation, passed over a short column of silica gel overlaid with anhydrous MgSO4. The water phase was extracted with another 1 mL of diethyl ether, which was also passed over the MgSO4/silica column. After 500 μL of hexane was added, the extracts were concentrated to about 500 μL using a Gyrovap GT centrifugal evaporator (Howe, Banburry, UK) and, subsequently, a stream of N2. Samples were analyzed for camphor,cis- and trans-carveol, and carvone content using GC-MS in the selected ion-monitoring mode as described above, but with an HP-5MS column (30-m × 0.25-mm i.d., 0.25-μm film thickness, 5% [w/w] diphenyl and 95% [w/w] dimethylpolysiloxane stationary phase, Hewlett-Packard) and an oven temperature program of 45°C for 1 min, ramp of 10° min−1 to 220°C, and a final time of 5 min. To assess the levels of carveols initially present, control assays were run in which the reaction was stopped immediately after substrate addition by adding 1 mL of diethyl ether and vigorous mixing. Carveol Dehydrogenase Aliquots (50 μL) of the 150,000g supernatant were diluted 20-fold with buffer containing 50 mm Gly (pH 10.5), 10% (v/v) glycerol, and 2 mm DTT in a 9-mL Teflon-lined screw-cap tube. The reaction was started by the addition of 1 mm NAD+ and 240 nmoltrans-carveol (a mixture of 130 nmol of the [+]-enantiomer and 110 nmol of the [−]-enantiomer) in 5 μL of pentane. Assays were performed in duplicate. Controls that had been boiled for 5 min showed no enzymatic activity. After incubation for 1 h at 30°C, assays were analyzed as described for limonene-6-hydroxylase. Control assays extracted immediately after substrate addition were used to determine the levels of carvone present at the beginning of the incubation. Stereoselectivity in Substrate Utilization and Product Formation To assess the specificity of limonene-6-hydroxylase and carveol dehydrogenase, the relevant enzymes were assayed in duplicate with the following substrates: limonene-6-hydroxylase: (+)- and (−)-limonene at 200 μm; trans-carveol dehydrogenase: (+)/(−)-trans-carveol mixture (130/110 μm), (+)/(−)-cis-carveol mixture (130/70 μm), (−)-trans-carveol (200 μm), and (−)-cis-carveol (170 μm). The product specificity of limonene synthase was assayed with a preparation of annual caraway after chromatography to remove endogenous limonene. Fruits were extracted as described above in a buffer containing 25 mm Mopso (3-[N-morpholino]-2-hydroxypropanesulfonic acid), pH 6.5, 10% (v/v) glycerol, 25 mm sodium ascorbate, 25 mm NaHSO3, 5 mmMgCl2, 2.5 mm EGTA, 2 mmEDTA, 1 mm MnCl2, and 5 mm DTT. The 150,000g supernatant was loaded onto a 15- × 2.5-cm column of DEAE-cellulose (Whatman DE-52) previously equilibrated with buffer containing 15 mm Mes (pH 6.0), 10% (v/v) glycerol, 2 mm NaHSO3, 1 mm MnCl2, and 2 mm DTT. The column was washed with the equilibration buffer and the enzyme was eluted with a 0 to 600 mm KCl gradient. The combined active fractions were desalted to the limonene synthase assay buffer (buffer A), glycerol was added to 30% (v/v), and the material was stored at −80°C. After thawing, seven 200-μL aliquots of the enzyme preparation were diluted 5-fold with buffer A and the assay was started by the addition of 35 μm[1-3H]GPP (21 Ci/mol) to five of the seven vials. The other two vials were used to check endogenous limonene levels. Assays were overlaid with 1 mL of redistilled pentane and were worked up as described above, except that the pentane phases were concentrated using microdistillation to minimize losses of limonene. Enzyme products were analyzed using GC-MS on a machine equipped with a chiral column in the selected ion-monitoring mode as described above. [U-14C]Suc Feeding A set of 1.5-mL Eppendorf vials was prepared with 50 μL [U-14C]Suc (25 GBq mmol−1, 7.4 MBq mL−1 in ethanol, Amersham) in each vial. After evaporation of the solvent under a stream of N2, 100 μL of unlabeled Suc in water (30 g L−1) was added to give a final concentration of 88 mm. Three to six representative umbellets of each developmental stage (Table I) were cut under deionized water and their pedicels were placed in the Suc solution through a small hole in the cap of the vial. After incubation at 20°C in a growth cabinet with continuous, fluorescent white light at 60 to 65 μmol m−2 s−1 for 4 to 6 h, the Suc solution was taken up. A second 100-μL aliquot of unlabeled Suc was added for an additional incubation period overnight to ensure maximum uptake of the radiolabel. The second aliquot of Suc had been completely taken up by the next morning, at which time the fruits were collected, weighed, frozen in liquid N2, and stored at −80°C until further analysis. For analysis, the frozen samples were ground to a fine powder in liquid N2 with a mortar and pestle. The liquid N2 and ground fruits were transferred to a 15-mL glass vial and, after evaporation of the N2, 5 mL of pentane was added. The mixture was homogenized for 30 s with an Omni 2000 homogenizer equipped with an Omni 10010 macrogenerator and saw teeth (Omni International, Waterbury, CT). After the debris had settled, 2 mL of the supernatant was transferred to a Pyrex centrifuge tube in which the fatty acids of the triacylglycerols were transesterified to the corresponding methyl esters (Bouwmeester and Kuijpers, 1993). Before and after methylation, radioactivity was determined in aliquots of the pentane phase by liquid-scintillation spectrometry. There was no loss of radioactivity upon methylation. The total amount of pentane-soluble radioactivity per gram of fruits was calculated from the liquid-scintillation counting data. Before analysis by radio-GLC, the samples were concentrated under a stream of N2. Unlabeled limonene and carvone were added before concentration as carriers to minimize losses of radio-labeled material. However, this was unnecessary in samples from older developmental stages (after 10 DAP) because of the presence of relatively high amounts of (unlabeled) limonene and carvone in the older fruits. Before and after concentration, 1-μL aliquots were taken and diluted 1000- and 10,000-fold with hexane for analysis of the limonene-to-carvone ratio by GC-MS (see below). These ratios were used to estimate the losses of the more-volatile limonene relative to carvone during concentration. Concentrated samples were analyzed by radio-GLC as described above for the limonene synthase assay with the following modifications. A CP-Sil 8 CB capillary column (10-m × 0.53-mm i.d., coated with a 5-μm film of 5% [w/w] phenyl and 95% [w/w] dimethyl polysiloxane, Chrompack) was used with an oven temperature program of 70°C for 1 min, ramp of 25° min−1 to 260°C, and a final time of 16 min. The He inlet pressure was 0.4 kg cm−2, giving a column flow of about 1 mL min−1. Approximately 20% of the column effluent was split to the flame-ionization detector. Before the conversion reactor, H2 was added to the effluent at 2 mL min−1, and, prior to the counting tube, CH4 was added to give a total flow of 30 mL min−1. For precise measurement of radio-peak areas, the splitter was closed so that all of the column effluent was channeled solely to the radioactivity detector. GC-MS analysis to determine limonene to carvone ratios was performed as described above using an HP-5MS column with an oven temperature program as follows: 45°C for 1 min, ramp of 10° min−1 to 260°C, and a final time of 5 min. Them/z range in the scan mode was set at 50 to 300 atomic mass units. Analysis of Limonene, Carvone, and Fatty Acid Content The frozen fruit samples were homogenized with the Omni homogenizer in 10 mL of hexane containing known amounts of isobutylbenzene, camphor, and methyl decanoate as the internal standards. After the residue had settled, 5 mL of the supernatant was transferred to a Pyrex centrifuge tube, 0.2 mL of 2 n KOH in methanol was added, and the contents were vigorously mixed for 20 s to esterify the fatty acids of the triacylglycerols. After addition of 1 mL of water, the contents were mixed again for 20 s and then centrifuged for 2 min at 2000 rpm. The hexane phase was analyzed by GLC (Chrompack CP9000) using a fused-silica CP-Sil 5 CB capillary column (25-m × 0.25-mm i.d.), coated with a film of 0.25 μm of 100% (w/w) dimethyl polysiloxane (Chrompack) operated with He (50 kPa), split injection (1:60), injector temperature of 280°C, flame ionization detector at 280°C, and oven-temperature programming: 110°C for 5 min, 20°C min−1 to 220°C, and 10 min at final time. Carvone and limonene were identified by comparing retention times with authentic standards and were quantified by comparing their detector responses to that of the internal standards. RESULTS Demonstration and Characterization of Enzyme Activities When crude extracts of caraway fruits from different stages of development were assayed for monoterpene synthase activity with GPP as the substrate, limonene was the only monoterpene detected by radio-GLC, with the exception of small amounts of geraniol, a product of phosphohydrolase activity. Thus, the cyclization of GPP to limonene is the first step of carvone biosynthesis in caraway. The activity was operationally soluble (confined to the 150,000gsupernatant), displayed a pH optimum of approximately 7.5, and required a divalent metal ion (Mn2+ preferred) for catalysis. The cyclization product was almost exclusively (+)-limonene (Fig. 2). After anion-exchange chromatography to remove the endogenous limonene present in the crude extract, GPP was found to be converted to 98.4% (+)-limonene with only 1.6% of the (−)-enantiomer. Fig. 2. Open in new tabDownload slide GC-MS analysis (in selected ion-monitoring mode) of products of limonene synthase assay using octakis-(6-O-methyl-2,3-di-O-pentyl)-γ-cyclodextrin as the chiral stationary phase. Ions monitored: for limonene,m/z 68, 93, and 136; for carvone, m/z 82, 108, and 150; for trans-carveol, m/z 84, 109, and 152; and for cis-carveol, m/z84, 109, and 134. A, Reference compounds. B, Product of partially purified limonene synthase. For further details, see Methods. Fig. 2. Open in new tabDownload slide GC-MS analysis (in selected ion-monitoring mode) of products of limonene synthase assay using octakis-(6-O-methyl-2,3-di-O-pentyl)-γ-cyclodextrin as the chiral stationary phase. Ions monitored: for limonene,m/z 68, 93, and 136; for carvone, m/z 82, 108, and 150; for trans-carveol, m/z 84, 109, and 152; and for cis-carveol, m/z84, 109, and 134. A, Reference compounds. B, Product of partially purified limonene synthase. For further details, see Methods. In the second step of the carvone pathway, limonene was converted totrans-carveol by a NADPH-requiring activity from the 150,000g pellet, reminiscent of the microsomal Cyt P-450 hydroxylase from spearmint, previously implicated in the conversion of (−)-limonene to (−)-trans-carveol (Gershenzon et al., 1989; Karp et al., 1990). Hydroxylation of (+)-limonene occurred with high regio- and stereospecificity. (+)-trans-Carveol made up 97% of the total product, with (−)-trans-carveol at 2.5% and (−)-cis-carveol at 0.5% as the minor products. Assays of mixtures of (−)- and (+)-limonene showed that (−)-limonene was also used as a substrate by this enzyme but at only about 10% of the rate of (+)-limonene and with (−)-cis-carveol as the major product. (+)-trans-Carveol is converted to (+)-carvone in the third and final step of the reaction sequence. The supernatant fraction of caraway fruit extracts possessed a very active trans-carveol dehydrogenase activity. This enzyme exhibited a pH optimum of around 10 and showed an absolute requirement for NAD+. NADP+ could not substitute for NAD+, and could not act synergistically with NAD+. At a pH of 10.5, which was routinely used in experiments to characterize this activity, no general alcohol (ethanol) dehydrogenase activity was detected, as determined spectrophotometrically with ethanol as a substrate (290 mm) and a mixture of NAD+ and NADP+ (at 1 mm each) as cofactors (Sangwan et al., 1993). Substantial ethanol dehydrogenase activity was detected at pH 8.0. Carveol dehydrogenase exhibited moderate preference for its natural substrate, (+)-trans-carveol (TableII). Of the substrates tested, the mixture of (+)/(−)-trans-carveol gave the highest activity. (−)-cis-Carveol also exhibited significant activity, giving approximately 65% of the activity of the (+)/(−)-trans-carveol mixture. The very low activity with (−)-trans-carveol as a substrate (5% of the activity observed with the [+]/[−]-trans-carveol mixture) suggested that the enzyme is very sensitive to substrate chirality, a supposition confirmed by GC-MS analysis on a chiral stationary phase of the products and the unreacted substrates (Fig.3). Whereas the mixture of (+)- and (−)-trans-carveols was converted to (+)-carvone (Fig.3B), the pure (−)-enantiomer did not yield detectable products (Fig.3D). In contrast, for the cis-carveols, both the (+)/(−) mixture (Fig. 3C) and the pure (−)-enantiomer (Fig. 3E) were readily converted to (−)-carvone. The assay with (−)-cis-carveol (Fig. 3E) provides further evidence of the unreactivity of (−)-trans-carveol with this enzyme preparation, since the (−)-trans-isomer is a trace impurity (0.8%) of the (−)-cis-carveol used as a substrate. (−)-trans-Carveol made up less than 1% of the substrate before reaction, but represented nearly 25% of the unconverted carveols after reaction. Table II. Dehydrogenase activity with various carveol isomers as substrates in 50 mm Gly buffer, pH 10.5, with 10% (v/v) glycerol, 2 mm DTT, and 1 mm each of NAD+ and NADP+ Substrate . Activity . Maximum . μm nmol h−1 g−1dry wt % (+)/(−)-trans-Carveol (37/33) 45.2 100 (+)/(−)-cis-Carveol (16/8) 8.09 18 (−)-trans-Carveol (50) 2.03 5 (−)-cis-Carveol (40) 30.30 67 Substrate . Activity . Maximum . μm nmol h−1 g−1dry wt % (+)/(−)-trans-Carveol (37/33) 45.2 100 (+)/(−)-cis-Carveol (16/8) 8.09 18 (−)-trans-Carveol (50) 2.03 5 (−)-cis-Carveol (40) 30.30 67 Open in new tab Table II. Dehydrogenase activity with various carveol isomers as substrates in 50 mm Gly buffer, pH 10.5, with 10% (v/v) glycerol, 2 mm DTT, and 1 mm each of NAD+ and NADP+ Substrate . Activity . Maximum . μm nmol h−1 g−1dry wt % (+)/(−)-trans-Carveol (37/33) 45.2 100 (+)/(−)-cis-Carveol (16/8) 8.09 18 (−)-trans-Carveol (50) 2.03 5 (−)-cis-Carveol (40) 30.30 67 Substrate . Activity . Maximum . μm nmol h−1 g−1dry wt % (+)/(−)-trans-Carveol (37/33) 45.2 100 (+)/(−)-cis-Carveol (16/8) 8.09 18 (−)-trans-Carveol (50) 2.03 5 (−)-cis-Carveol (40) 30.30 67 Open in new tab Fig. 3. Open in new tabDownload slide GC-MS analyses (in selected ion-monitoring mode) of products of carveol dehydrogenase assays using octakis-(6-O-methyl-2,3-di-O-pentyl)-γ-cyclodextrin as the chiral stationary phase. Ions monitored: for limonene,m/z 68, 93, and 136; for carvone, m/z 82, 108, and 150; for trans-carveol, m/z 84, 109, and 152; for cis-carveol, m/z 84, 109, and 134. A, Reference compounds. B through E, Products of carveol dehydrogenase activity with a (+)/(−)-trans-carveol mixture (B), a (+)/(−)-cis-carveol mixture (C), (−)-trans-carveol (D), and (−)-cis-carveol (E) as the substrates. For further details, see Methods. Fig. 3. Open in new tabDownload slide GC-MS analyses (in selected ion-monitoring mode) of products of carveol dehydrogenase assays using octakis-(6-O-methyl-2,3-di-O-pentyl)-γ-cyclodextrin as the chiral stationary phase. Ions monitored: for limonene,m/z 68, 93, and 136; for carvone, m/z 82, 108, and 150; for trans-carveol, m/z 84, 109, and 152; for cis-carveol, m/z 84, 109, and 134. A, Reference compounds. B through E, Products of carveol dehydrogenase activity with a (+)/(−)-trans-carveol mixture (B), a (+)/(−)-cis-carveol mixture (C), (−)-trans-carveol (D), and (−)-cis-carveol (E) as the substrates. For further details, see Methods. Developmental Changes in Monoterpene and Fatty Acid Content and the Rate of Biosynthesis The pattern of accumulation of the monoterpenes limonene and carvone in developing caraway fruits was investigated by extracting fruits of nine different developmental stages in hexane and by analyzing the extracts with GC. The levels of fatty acids in these extracts (both free and bound as triacylglycerols) were also measured by quantifying the fatty acid methyl esters formed after hydrolysis and transesterification in methanolic KOH. The patterns of accumulation of limonene, carvone, and fatty acids and the activities of the three enzymes assayed were similar for annual and biennial caraway. Therefore, only data for annual caraway are shown. Young fruits contained very low levels of carvone and fatty acids, but high concentrations of limonene, up to 1.2 μmol fruit−1 (Fig. 4A), equivalent to approximately 130 mg g−1 fruit dry weight. From about 10 DAP, carvone content increased rapidly until about 15 DAP, at which time it became approximately the same as that of limonene. From about 15 DAP, the concentration of both monoterpenes followed a similar pattern until the end of the experiment at 35 DAP. At this stage, the monoterpene concentration of both caraway forms was similar, about 54 mg g−1 fruit dry weight, but the biennial form had a higher carvone-to-limonene ratio than the annual form (1.2 versus 0.7; data not shown). Figure 4A shows clearly that the accumulation of monoterpenes is confined to the early stages of fruit development and that limonene accumulation precedes carvone accumulation by a period of 5 to 10 d. Fig. 4. Open in new tabDownload slide Accumulation of limonene (○), carvone (▵), and fatty acids (□) (A), and changes in activities of limonene synthase (○), limonene-6-hydroxylase (▵), and trans-carveol dehydrogenase (□) (B) during development of fruits of annual caraway. Dotted lines in A indicate sigmoidal curves of the equation:y = a + b/(1 + exp[−(x − c)/d]).r2 values are 0.52 for limonene, 0.86 for carvone, and 0.99 for fatty acids. The dotted line in B indicates the carvone accumulation rate, which was calculated by taking the 1st-order derivative of the sigmoidal-curve fit to the carvone content data as shown in A. Data for A were obtained from pooled samples of 0.5 g of fruits of each developmental stage containing 40 to 130 individual fruits. Data for B were obtained from pooled samples of 1.0 g of fruits. Enzyme assays were carried out in triplicate (limonene synthase) or in duplicate (other activities) under linear conditions. Error bars indicate se. Fig. 4. Open in new tabDownload slide Accumulation of limonene (○), carvone (▵), and fatty acids (□) (A), and changes in activities of limonene synthase (○), limonene-6-hydroxylase (▵), and trans-carveol dehydrogenase (□) (B) during development of fruits of annual caraway. Dotted lines in A indicate sigmoidal curves of the equation:y = a + b/(1 + exp[−(x − c)/d]).r2 values are 0.52 for limonene, 0.86 for carvone, and 0.99 for fatty acids. The dotted line in B indicates the carvone accumulation rate, which was calculated by taking the 1st-order derivative of the sigmoidal-curve fit to the carvone content data as shown in A. Data for A were obtained from pooled samples of 0.5 g of fruits of each developmental stage containing 40 to 130 individual fruits. Data for B were obtained from pooled samples of 1.0 g of fruits. Enzyme assays were carried out in triplicate (limonene synthase) or in duplicate (other activities) under linear conditions. Error bars indicate se. The accumulation of fatty acids occurred late in fruit development, beginning at 15 to 25 DAP, and increased steadily until the end of the experiment to about 1 to 1.2 mg fruit−1 (Fig.4A). Fatty acid composition in the late stages of fruit development was 1% stearic acid, 4% palmitic acid, 35% linoleic acid, and 60% petroselinic and oleic acids (not separated in our analysis) for annual caraway and 0.5% stearic, 5% palmitic, 38% linoleic, and 57% petroselinic/oleic for the biennial form. Experiments to determine the rate of monoterpene and fatty acid biosynthesis by measuring the incorporation of [U-14C]Suc into pentane-soluble compounds showed similar trends. Overall incorporation of radioactivity was significantly higher in the younger than in the older stages, decreasing from 3 to 6% of administered Suc at 5 to 15 DAP to less than 1% after 16 to 20 DAP (Table III). In the youngest stages (5–7 DAP), the majority of label was incorporated into limonene, with less incorporation into carvone and little or no incorporation into fatty acids. No radiolabel was observed in the monoterpene intermediate trans-carveol at any developmental stage. The rate of radiolabel incorporation into limonene declined rapidly with development and was not detectable after 15 DAP. The incorporation of [U-14C]Suc into carvone increased as the rate of incorporation into limonene declined, but was also undetectable after 15 DAP. From this period onward, the only pentane-soluble compounds to incorporate [U-14C]Suc were the fatty acids. Table III. 14C-radiolabel incorporation from [U-14C]Suc into pentane-soluble compounds of caraway fruits at different developmental stages Developmental Stage . Incorporation of Label Administered . 14C Incorporation . Limonene . Carvone . Fatty acids . DAP % % of total 7 3.24 71 29 0 10 4.17 34 48 18 13 3.62 24 48 28 15 4.36 21 59 20 16 1.24 0 0 100 18 0.77 0 0 100 27 0.63 0 0 100 28 1.06 0 0 100 34 0.62 0 0 100 Developmental Stage . Incorporation of Label Administered . 14C Incorporation . Limonene . Carvone . Fatty acids . DAP % % of total 7 3.24 71 29 0 10 4.17 34 48 18 13 3.62 24 48 28 15 4.36 21 59 20 16 1.24 0 0 100 18 0.77 0 0 100 27 0.63 0 0 100 28 1.06 0 0 100 34 0.62 0 0 100 For further details, see Methods. Open in new tab Table III. 14C-radiolabel incorporation from [U-14C]Suc into pentane-soluble compounds of caraway fruits at different developmental stages Developmental Stage . Incorporation of Label Administered . 14C Incorporation . Limonene . Carvone . Fatty acids . DAP % % of total 7 3.24 71 29 0 10 4.17 34 48 18 13 3.62 24 48 28 15 4.36 21 59 20 16 1.24 0 0 100 18 0.77 0 0 100 27 0.63 0 0 100 28 1.06 0 0 100 34 0.62 0 0 100 Developmental Stage . Incorporation of Label Administered . 14C Incorporation . Limonene . Carvone . Fatty acids . DAP % % of total 7 3.24 71 29 0 10 4.17 34 48 18 13 3.62 24 48 28 15 4.36 21 59 20 16 1.24 0 0 100 18 0.77 0 0 100 27 0.63 0 0 100 28 1.06 0 0 100 34 0.62 0 0 100 For further details, see Methods. Open in new tab The level of activity of the three enzymes of monoterpene biosynthesis varied considerably over the period of fruit development (Fig. 4B). In the youngest stages, both limonene synthase andtrans-carveol dehydrogenase were active but limonene-6-hydroxylase was not; however, the maximum in all three activities occurred at about 15 DAP. From this stage, the activities of all three enzymes declined with similar kinetics until they were virtually undetectable. At all stages of development,trans-carveol dehydrogenase activity was 2 to 15 times greater than limonene synthase activity, and both were considerably higher than limonene-6-hydroxylase activity. The dotted line in Figure4B depicts the carvone accumulation rate, and represents the first derivative of the sigmoidal curve describing carvone accumulation in Figure 4A. In general, there is a good correlation between the mathematically derived rate of carvone accumulation and the limonene-6-hydroxylase activity measured in vitro, although the measured enzyme activity often underestimated the carvone accumulation rate in planta, especially during the first 15 DAP. DISCUSSION The Pathway of Monoterpene Biosynthesis in Caraway The formation of (+)-carvone in caraway fruits proceeds via a three-step pathway from GPP completely analogous to the formation of (−)-carvone in spearmint (Gershenzon et al., 1989). In the first step, GPP is cyclized to (+)-limonene by a monoterpene synthase that is very similar in its basic properties to many monoterpene synthases previously characterized from angiosperms (Alonso and Croteau, 1993;Gershenzon and Croteau, 1993). This enzyme also produces minor amounts (1.6%) of the opposite enantiomer, (−)-limonene (Fig. 2), which is also found in trace levels (0.6%) in the essential oil extracted from caraway fruits (Bouwmeester et al., 1995b). In the second step, after cyclization (+)-limonene is hydroxylated to (+)-trans-carveol by a particulate, NADPH-utilizing activity, the properties of which resemble those of other Cyt P-450 monoterpene hydroxylases (Mihaliak et al., 1993). Caraway (+)-limonene-6-hydroxylase also proved capable of oxygenating the opposite enantiomer, (−)-limonene, as has been reported for other monoterpene hydroxylases (Karp et al., 1990). Further characteristics of the caraway (+)-limonene-6-hydroxylase will be described in a subsequent paper (H.J. Bouwmeester, M.C.J.M. Konings, J. Gershenzon, F. Karp, and R. Croteau, unpublished data). The third and final step of monoterpene formation in caraway fruit is the oxidation of (+)-trans-carveol to (+)-carvone. The properties of this enzyme resemble those of other monoterpenol dehydrogenases described in the literature. As a group, the monoterpenol dehydrogenases possess a wide range of pH optima, varying from around 8.0 (Croteau and Felton, 1980; Sangwan et al., 1993) to 9.0 to 10.0 (Potty and Bruemmer, 1970; Kjonaas et al., 1985; Hallahan et al., 1995). The trans-carveol dehydrogenase studied here also has a high pH optimum, around 10.0. This finding is consistent with a trend noted earlier by Kjonaas et al. (1985) that monoterpenol dehydrogenases utilizing α,β-unsaturated alcohols have higher pH optima than monoterpenol dehydrogenases utilizing saturated alcohols. The high pH optimum for caraway (+)-trans-carveol dehydrogenase seems to have little physiological relevance because the enzyme is probably localized in the cytoplasm, where a pH of 7.0 to 7.5 can be assumed. Among other monoterpenol dehydrogenases, some require either NAD+ or NADP+, whereas some show substantial catalytic activity with both cofactors (Potty and Bruemmer, 1970; Croteau and Felton, 1980; Kjonaas et al., 1985; Sangwan et al., 1993; Hallahan et al., 1995). Carawaytrans-carveol dehydrogenase shares its requirement for NAD+ with another α,β-unsaturated monoterpenol dehydrogenase, trans-isopiperitenol dehydrogenase from peppermint (Kjonaas et al., 1985). The substrate specificity of (+)-trans-carveol dehydrogenase, like that of other monoterpenol dehydrogenases, is not very strict. Nevertheless, the oxidation of monoterpenols in other plant species has been found to be catalyzed by a dehydrogenase activity other than the general alcohol (ethanol) dehydrogenase (Croteau and Felton, 1980). This was confirmed for caraway by the absence of ethanol dehydrogenase activity at pH 10.5, near the pH optimum for trans-carveol dehydrogenase activity, and by the fact that only two of the four isomeric carveols [(+)-trans- and (−)-cis-carveol] were oxidized (Fig. 3; Table II). Caraway (+)-trans-carveol dehydrogenase exhibits only moderate substrate specificity but high enantioselectivity, in contrast to peppermint isopiperitenol dehydrogenase, which oxidizes both enantiomers oftrans-isopiperitenol but does not catalyze reaction withcis-isopiperitenol (Kjonaas et al., 1985). Further purification of trans-carveol dehydrogenase is necessary to determine whether a single activity is capable of oxidizing both (+)-trans- and (−)-cis-carveol. The low substrate specificity of both (+)-trans-carveol dehydrogenase and the second enzyme in the pathway, (+)-limonene-6-hydroxylase, stand in contrast to the high enantiomeric purity of the carvone that accumulates in caraway fruit. The enantiomeric purity of carvone thus results from the high product specificity of the first enzyme, (+)-limonene synthase, which ensures that only a single stereoisomer is made available to the later, less-specific enzymes. Regulation of Monoterpene Formation in Caraway Enzyme Control The three enzymes of monoterpene biosynthesis in caraway, limonene synthase, limonene-6-hydroxylase, and trans-carveol dehydrogenase, undergo dramatic changes in activity during fruit development (Fig. 4B), which appears to explain much of the pattern of limonene and carvone accumulation (Fig. 4, A and B). During the early stages of fruit development (5–10 DAP) there is an abundant accumulation of limonene, but very little carvone is produced. At this time, both the first and third enzymes of the pathway, limonene synthase and trans-carveol dehydrogenase, exhibit high levels of activity. However, only trace levels of the second enzyme, limonene-6-hydroxylase, were observed. The absence of significant amounts of limonene-6-hydroxylase apparently prevents the oxidation of limonene to trans-carveol. This blocks the formation of carvone, despite the consistently high levels oftrans-carveol dehydrogenase activity, leading to a buildup of limonene. From 10 DAP, there is a rise in limonene-6-hydroxylase activity that coincides with the appearance of substantial amounts of carvone. Similar changes in terpenoid accumulation patterns occur during plant development in many other plant species, including dill (Porter et al., 1983) and peppermint (Voirin and Bayet, 1996). However, to our knowledge, the enzymatic bases of such shifts in accumulation have been unexamined until now. In contrast, the rapid switches in terpenoid metabolism that occur upon pathogen infection have been extensively investigated. For example, when cell cultures of tobacco or potato are treated with fungal elicitors, there is an induction of sesquiterpenoid phytoalexin biosynthesis and a repression of sterol formation (Brindle et al., 1988; Vögeli and Chappell, 1988; Chappell, 1995). Enzymatic analyses have revealed that fungal elicitors activate farnesyl diphosphate-utilizing enzymes in phytoalexin biosynthesis while reducing the activity of squalene synthase, a branchpoint enzyme in sterol formation that also competes for farnesyl diphosphate. In caraway limonene-6-hydroxylase may serve as an important rate-controlling step in carvone formation, not only because of its limited temporal occurrence, but also as a result of its kinetics. The hydroxylation of limonene to trans-carveol appears to be a much slower transformation than the subsequent dehydrogenation to carvone, as indicated by the lack of any accumulation of the intermediate, trans-carveol. No14C-labeled trans-carveol was detected after [U-14C]Suc feeding in this study. In addition, analysis of the essential oil of caraway fruits has revealed only minute amounts of carveols: trans, 0.3 to 0.5%; andcis, 0.2% (Bouwmeester et al., 1995b). Monoterpene hydroxylation may also possess regulatory importance in the formation of monoterpenes in peppermint. In this species, (−)-limonene, an olefin precursor of oxygenated monoterpenes, makes up a much higher percentage of the total monoterpene pool in younger than in older tissue. As peppermint leaves mature, the percentage of limonene drops and the percentage of the oxygenated products menthone and menthol increases (Brun et al., 1991; Voirin and Bayet, 1995), suggesting that the 3-hydroxylation of (−)-limonene to (−)-trans-isopiperitenol, the next intermediate in menthone/menthol biosynthesis, is a regulatory step in monoterpene formation. The enzymatic changes responsible for this metabolic switch are currently under investigation (M. Rufener, J. Gershenzon, and R. Croteau, unpublished results). The correlation between limonene-6-hydroxylase activity and the profile of carvone accumulation during fruit development is not exact (Fig.4B). For example, in vitro activity is frequently insufficient to account for carvone accumulation, probably because of difficulties in quantitatively extracting and assaying this enzyme. Limonene-6-hydroxylase is a Cyt P-450-dependent oxygenase (Bolwell et al., 1994) which is found in the light membrane (microsomal) fraction of the cell (H.J. Bouwmeester, M.C.J.M. Konings, J. Gershenzon, F. Karp, and R. Croteau, unpublished data). The activities of enzymes of this class are often underestimated as a result of inefficient extraction and poor stability (Mihaliak et al., 1993; Funk et al., 1994). After extraction from caraway fruits, limonene-6-hydroxylase activity is gradually lost even at 4°C in the presence of protective agents (H.J. Bouwmeester, M.C.J.M. .Konings, J. Gershenzon, F. Karp, and R. Croteau, unpublished data). Substrate Limitation The formation of monoterpenes in caraway fruit may also be controlled by enzymes acting prior to limonene synthase, which control flux entering the monoterpene pathway (Gershenzon and Croteau, 1990). At 15 to 20 DAP, the biosynthesis of limonene and carvone in developing fruits appears to cease, based on the lack of further [14C]Suc incorporation (Table III) and the leveling off of the accumulation curves (Fig. 4A). Nevertheless, ample activities of all three enzymes of the pathway are still present, judging by the results of the in vitro assays (Fig. 4B). At this stage of development, the formation of monoterpenes could be limited by substrate partitioning to competing pathways, such as triacylglycerol synthesis. The rate of fatty acid formation in developing fruits increases dramatically at this stage (Table III; Fig. 4A). The biosynthesis of structural carbohydrates, which has been found to slightly precede triacylglycerol accumulation in caraway fruits (Luyendijk, 1956), may also compete for the supply of fixed carbon. The accumulation patterns of monoterpenes and fatty acids during caraway fruit development and the profiles of enzyme activities involved in monoterpene biosynthesis were quite similar for the two caraway forms (data not shown). However, in annual caraway, accumulation of limonene and carvone started earlier in development, reached higher levels per fruit, and ceased earlier than in biennial caraway. These differences were partially reflected in the time courses of enzyme activity for limonene synthase and limonene-6-hydroxylase. Both activities were higher in annual than in biennial caraway at early stages of development. However, the biennial form continued to accumulate limonene and carvone after the monoterpene content of annual caraway had already stabilized. The continued formation of monoterpenes in the later stages of development of biennial caraway may also explain the higher carvone-to-limonene ratio of this form. If competition for substrate is important in controlling the rate of monoterpene biosynthesis in caraway fruits, this phenomenon appears to be less important, or of later onset, in the biennial form. Other Modes of Regulation The formation of monoterpenes in developing caraway fruits may be controlled by subcellular compartmentation of the various enzymes. The first enzyme of the pathway, limonene synthase, appears to be localized in the leukoplasts, colorless plastids with few internal membranes (Gleizes et al., 1983; McCaskill and Croteau, 1995). The presence of this activity in the 150,000g supernatant is likely due to the fragility of the leukoplasts during enzyme isolation (Gleizes et al., 1983). However, limonene-6-hydroxylase, like many other Cyt P-450 oxygenases (Bolwell et al., 1994), is probably found in the ER. Hence, for the operation of the monoterpene pathway, the limonene formed in the leukoplasts must be transferred into the ER. If no limonene-6-hydroxylase activity is present in the ER, as during early fruit development, the limonene will move directly into the oil ducts, possibly via the ER network. However, when limonene-6-hydroxylase is present, limonene is hydroxylated in the ER and then oxidized to carvone. A role for the ER in transport of monoterpenes from leukoplasts to essential oil ducts is supported by the ultrastructural study of Bosabalidis (1996) with celery petioles in which the monoterpene hydrocarbon 1,3,8-menthatriene accumulates. At the stage of essential oil formation, ER elements were shown to associate with leukoplasts containing osmiophilic secretory droplets. These droplets were transferred into the ER and then transported to the essential oil ducts. The trans-carveol dehydrogenase is probably cytosolic, judging by its recovery in the 150,000gsupernatant. It is unclear whether for oxidation thetrans-carveol has to be released from the ER into the cytoplasm or whether the dehydrogenase, though it is cytoplasmic, operates in close association with the ER. The close correlation between limonene-6-hydroxylase activity and carvone accumulation (Fig.4B), and the fact that the increase in the rate of carvone accumulation during fruit development coincides with the decrease in the rate of limonene accumulation (Fig. 4A), seem to confirm that both of these monoterpenes are produced from the same pathway, and not by separate pathways localized at different cellular sites. In conclusion, the patterns of limonene and carvone accumulation during the early development of caraway fruit can be attributed largely to changes in the time course of monoterpene biosynthesis caused by alterations in the levels of limonene synthase and limonene-6-hydroxylase activities. However, at later stages of development, competition with other pathways for substrate may serve to limit monoterpene accumulation. At the enzyme level, the hydroxylation of limonene to trans-carveol seems to be a critical, rate-limiting step in carvone biosynthesis. We are currently characterizing this enzyme in more detail and plan to isolate the corresponding gene from a caraway cDNA library. Overexpression of this gene in caraway fruit should provide a rigorous evaluation of its regulatory importance in carvone biosynthesis. ACKNOWLEDGMENTS The authors wish to thank Tom Savage and Frank Karp for their helpful suggestions, Jeroen Wilmer for his help in keeping the radio-gas-liquid chromatograph operating, Jacques Davies for help with radio-GLC and GC-MS measurements, and Adrie Kooijman, Joop van Westeneng, Herman Mersbergen, and Greg Wichelns for raising the plants. Abbreviations: DAP days after pollination GPP geranyl diphosphate LITERATURE CITED 1 Alonso WR Croteau R Prenyltransferases and cyclases. Lea PJ Methods in Plant Biochemistry, Vol 9: Enzymes of Secondary Metabolism. 1993 239 260 Academic Press London 2 Banthorpe DV Charlwood BV The terpenoids. Bell EA Charlwood BV Encyclopedia of Plant Physiology, New Series, Vol 8: Secondary Plant Products. 1980 185 220 Springer-Verlag Berlin 3 Bolwell GP Bozak K Zimmerlin A Plant cytochrome P450. Phytochemistry 37 1994 1491 1506 Google Scholar Crossref Search ADS PubMed WorldCat 4 Bosabalidis AM Ontogenesis, ultrastructure and morphometry of the petiole oil ducts of celery (Apium graveolens L.). Flavour Fragrance J 11 1996 269 274 Google Scholar Crossref Search ADS WorldCat 5 Bouwmeester HJ Davies JAR Smid HG Welten RSA Physiological limitations to carvone yield in caraway (Carum carvi L.). Ind Crops Prod 4 1995a 39 51 Google Scholar Crossref Search ADS WorldCat 6 Bouwmeester HJ Davies JAR Toxopeus H Enantiomeric composition of carvone, limonene and carveols in seeds of dill, and annual and biennial caraway varieties. J Agr Food Chem 43 1995b 3057 3064 Google Scholar Crossref Search ADS WorldCat 7 Bouwmeester HJ Kuijpers A-M Relationship between assimilate supply and essential oil accumulation in annual and biennial caraway (Carum carvi L.). J Essent Oil Res 5 1993 143 152 Google Scholar Crossref Search ADS WorldCat 8 Brindle PA Kuhn PJ Threlfall DR Biosynthesis and metabolism of sesquiterpenoid phytoalexins and triterpenoids in potato cell suspension cultures. Phytochemistry 27 1988 133 150 Google Scholar Crossref Search ADS WorldCat 9 Brun N Colson M Perrin A Voirin B Chemical and morphological studies of the effects of ageing on monoterpene composition in Mentha × piperita leaves. Can J Bot 69 1991 2271 2278 Google Scholar Crossref Search ADS WorldCat 10 Chappell J Biochemistry and molecular biology of the isoprenoid biosynthetic pathway in plants. Annu Rev Plant Mol Biol 46 1995 521 547 Google Scholar Crossref Search ADS WorldCat 11 Croteau R Cane DE Monoterpene and sesquiterpene cyclases. Methods Enzymol 110 1985 383 405 Google Scholar Crossref Search ADS WorldCat 12 Croteau R Felton NM Substrate specificity of monoterpenol dehydrogenases from Foeniculum vulgare and Tanacetum vulgare. Phytochemistry 19 1980 1343 1347 Google Scholar Crossref Search ADS WorldCat 13 Funk C Lewinsohn E Vogel BS Steele CL Croteau R Regulation of oleoresinosis in grand fir (Abies grandis). Coordinate induction of monoterpene and diterpene cyclases and two cytochrome P450-dependent diterpenoid hydroxylases by stem wounding. Plant Physiol 106 1994 999 1005 Google Scholar Crossref Search ADS PubMed WorldCat 14 Gershenzon J, Croteau R (1990) Regulation of monoterpene biosynthesis in higher plants. In GHN Towers, HA Stafford, eds, Biochemistry of the Mevalonic Acid Pathway to Terpenoids, Recent Advances in Phytochemistry, Vol 24. Plenum Press, New York, pp 99–160 15 Gershenzon J, Croteau R (1993) Terpenoid biosynthesis: the basic pathway and formation of monoterpenes, sesquiterpenes and diterpenes.In TS Moore Jr, ed, Lipid Metabolism in Plants. CRC Press, Boca Raton, FL pp 339–388 16 Gershenzon J Maffei M Croteau R Biochemical and histochemical localization of monoterpene biosynthesis in the glandular trichomes of spearmint (Mentha spicata). Plant Physiol 89 1989 1351 1357 Google Scholar Crossref Search ADS PubMed WorldCat 17 Gleizes M Pauly G Carde J-P Marpeau A Bernard-Dagan C Monoterpene hydrocarbon biosynthesis by isolated leucoplasts of Citrofortunella mitis. Planta 159 1983 373 381 Google Scholar Crossref Search ADS PubMed WorldCat 18 Hallahan DL West JM Wallsgrove RM Smiley DWM Dawson GW Pickett JA Hamilton JGC Purification and characterization of an acyclic monoterpene primary alcohol:NADP+ oxidoreductase from catmint (Nepeta racemosa). Arch Biochem Biophys 318 1995 105 112 Google Scholar Crossref Search ADS PubMed WorldCat 19 Johnston RG Read J Researches in the carvone series. Part II. Some unsaturated alcohols. J Chem Soc 1934 1934 233 237 Google Scholar OpenURL Placeholder Text WorldCat 20 Karp F Mihaliak CA Harris JL Croteau R Monoterpene biosynthesis: specificity of the hydroxylation of (−)-limonene by enzyme preparations from peppermint (Mentha piperita), spearmint (Mentha spicata) and perilla (Perilla frutescens). Arch Biochem Biophys 276 1990 219 226 Google Scholar Crossref Search ADS PubMed WorldCat 21 Kjonaas RB Venkatachalam KV Croteau R Metabolism of monoterpenes: oxidation of isopiperitenol to isopiperitenone and subsequent isomerization to piperitenone by soluble enzyme preparations from peppermint (Mentha piperita) leaves. Arch Biochem Biophys 238 1985 49 60 Google Scholar Crossref Search ADS PubMed WorldCat 22 König WA Icheln D Runge T Pforr I Krebs A Cyclodextrins as chiral stationary phases in capillary gas chromatography. Part VII. Cyclodextrins with an inverse substitution pattern: synthesis and enantioselectivity. J High Res Chrom 13 1990 702 707 Google Scholar Crossref Search ADS WorldCat 23 Langenheim JH Higher plant terpenoids: a phytocentric overview of their ecological roles. J Chem Ecol 20 1994 1223 1280 Google Scholar Crossref Search ADS PubMed WorldCat 24 Luyendijk EN (1956) Over de vorming van vluchtige olie in de vruchten van enkele Umbelliferen. PhD thesis. Rijksuniversiteit Leiden, The Netherlands 25 McCaskill D Croteau R Monoterpene and sesquiterpene biosynthesis in glandular trichomes of peppermint (Mentha × piperita) rely exclusively on plastid-derived isopentenyl diphosphate. Planta 197 1995 49 56 Google Scholar Crossref Search ADS WorldCat 26 Mihaliak C Karp F Croteau R Cytochrome P-450 terpene hydroxylases. Lea PJ Methods in Plant Biochemistry, Vol 9: Enzymes of Secondary Metabolism. 1993 261 279 Academic Press London 27 Morton JF (1976) Herbs and Spices. Golden Press, New York 28 Oosterhaven K Poolman B Smid EJ S-Carvone as a natural potato sprout inhibiting, fungistatic and bacteriostatic compound. Ind Crops Prod 4 1995 23 31 Google Scholar Crossref Search ADS WorldCat 29 Ponndorf W Der reversibele Austausch der Oxydationsstufen zwischen Aldehyden oder Ketonen einerseits und primären oder sekundären Alkoholen anderseits. Z Angew Chem 39 1926 138 143 Google Scholar Crossref Search ADS WorldCat 30 Porter NG Shaw ML Shaw GJ Ellingham PJ Content and composition of dill herb oil in the whole plant and the different plant parts during crop development. New Zealand J Agr Res 26 1983 119 127 Google Scholar Crossref Search ADS WorldCat 31 Potty VH Bruemmer JH Oxidation of geraniol by an enzyme system from orange. Phytochemistry 9 1970 1003 1007 Google Scholar Crossref Search ADS WorldCat 32 Sandermann W Bruns K Biogenese von carvon in Anethum graveolens L. Planta Medica 13 1965 364 368 Google Scholar Crossref Search ADS WorldCat 33 Sangwan RS Singh-Sangwan N Luthra R Metabolism of acyclic monoterpenes: partial purification and properties of geraniol dehydrogenase from lemongrass (Cymbopogon flexuosus Stapf) leaves. J Plant Physiol 142 1993 129 134 Google Scholar Crossref Search ADS WorldCat 34 Vögeli U Chappell J Induction of sesquiterpene cyclase and suppression of squalene synthetase activities in plant cell cultures treated with fungal elicitor. Plant Physiol 88 1988 1291 1296 Google Scholar Crossref Search ADS PubMed WorldCat 35 Voirin B Bayet C Developmental changes in the monoterpene composition of Mentha × piperita leaves from individual peltate trichomes. Phytochemistry 43 1996 573 580 Google Scholar Crossref Search ADS WorldCat 36 Von Schantz M Ek BS Über die Bildung von Ätherischem Öl in Kümmel. Carum carvi L. Sci Pharm 39 1971 81 101 Google Scholar OpenURL Placeholder Text WorldCat 37 Von Schantz M Huhtikangas A Über die Bildung von limonen und carvon in Kümmel. Carum carvi. Phytochemistry 10 1971 1787 1793 Google Scholar Crossref Search ADS WorldCat 38 Wichtmann E-M (1988) Die ätherischen Öle von Kümmel (Carum carvi L.), Gemüsefenchel (Foeniculum vulgare Miller subsp. capillaceum(Gilib.) Holmboe var. azóricum (Miller) Thellung), Pastinak (Pastinaca sativa L.) und Liebstöckl (Levisticum officinale Koch) im Keimpflanzenstadium und ihre Veränderung im Verlauf der Ontogenese in Korrelation zur Anatomie der Exkretgänge. PhD thesis. University of Hamburg, Germany Author notes 1 This work was supported by the Commissie Herstructurering Akkerbouw Noorden des Lands of the Dutch Ministry of Agriculture, Nature Management, and Fisheries, the U.S. Department of Energy (grant no. DE-FG03-96ER20212), and the Washington State University Agricultural Research Center (project no. 0268). 2 Present address: Max Planck Institute of Chemical Ecology, Jena, Germany. * Corresponding author; e-mail [email protected]; fax 31–317–423110. Copyright © 1998 American Society of Plant Physiologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Su, Jin; Shen, Qingxi; David Ho, Tuan-Hua; Wu, Ray

doi: 10.1104/pp.117.3.913pmid: 9662533

Abstract To confer abscisic acid (ABA) and/or stress-inducible gene expression, an ABA-response complex (ABRC1) from the barley (Hordeum vulgare L.) HVA22gene was fused to four different lengths of the 5′ region from the rice (Oryza sativa L.) Act1 gene. Transient assay of β-glucuronidase (GUS) activity in barley aleurone cells shows that, coupled with ABRC1, the shortest minimal promoter (Act1–100P) gives both the greatest induction and the highest level of absolute activity following ABA treatment. Two plasmids with one or four copies of ABRC1 combined with the same Act1–100P and HVA22(I) of barley HVA22 were constructed and used for stable expression of uidA in transgenic rice plants. Three Southern blot-positive lines with the correct hybridization pattern for each construct were obtained. Northern analysis indicated thatuidA expression is induced by ABA, water-deficit, and NaCl treatments. GUS activity assays in the transgenic plants confirmed that the induction of GUS activity varies from 3- to 8-fold with different treatments or in different rice tissues, and that transgenic rice plants harboring four copies of ABRC1 show 50% to 200% higher absolute GUS activity both before and after treatments than those with one copy of ABRC1. Drought and high salinity are the most important environmental factors that cause osmotic stress and dramatically limit plant growth and crop productivity (Boyer, 1982). Therefore, production of drought- and NaCl-tolerant transgenic plants is very important for agriculture. In recent years different stress-tolerant transgenic plants have been obtained (Tarczynski et al., 1993; Kishor et al., 1995; Pilon-Smits et al., 1995; Holmström et al., 1996; Xu et al., 1996; Hayashi et al., 1997) by producing either a low-Mrosmoprotectant (such as Gly betaine, mannitol, inositol, Pro, fructan, or trehalose) or a late-embryogenesis-abundant protein. However, under normal environmental conditions, overproduction of these compounds or proteins need extra energy and building blocks and may hamper the normal growth of plants. Thus, it is desirable to generate transgenic plants that synthesize a high level of an osmoprotectant or a protein only under stress conditions. The phytohormone ABA is thought to mediate physiological processes in response to osmotic stress in plants (King, 1976; Jones et al., 1987). Water stress by NaCl or dehydration can cause endogenous ABA levels to increase in plant tissues (Henson, 1984; Jones et al., 1987). Mundy and Chua (1988) found that ABA controls the accumulation of specific mRNAs and proteins, both from developmental studies with seeds and physiological studies with water-stressed tissues. Specific genes are expressed under stress conditions and can also be induced in unstressed tissues by the application of exogenous ABA (Singh et al., 1987; Gomez et al., 1988; Mundy and Chua, 1988; Chandler and Robertson, 1994;Ingram and Bartels, 1996). In addition to the studies on the physiological roles of ABA, efforts are being made to investigate the molecular mechanism of ABA action, including the definition of ABREs, and the trans-acting factors that interact with ABREs. It was reported that a 75-bp fragment of the ABA-inducible wheat Em gene, when fused to a truncated CaMV 35S promoter, conferred a more than 10-fold ABA induction of GUS activity in rice (Oryza sativa L.) protoplasts (Guiltinan et al., 1990). They also found a Leu-zipper DNA-binding protein, EmBP-1, which binds the ABRE sequence (CACGTGGC) in this 75-bp region. Transient assays in rice protoplasts revealed a 40-bp ABA-responsive fragment in the rice rab 16B promoter (Ono et al., 1996). Two separate ABREs, motif I (AGTACGTGGC) and motif III (GCCGCGTGGC), are required for ABA induction; however, each can substitute for the other. The 40-bp fragment-containing motif I fused to a truncated CaMV 35S promoter showed an approximately 4- to 5-fold induction by ABA (Ono et al., 1996). The ABREs are very similar to the G-box, which, as has been pointed out by Guiltinan et al. (1990), is present in some genes that are responsive to other environmental and physiological stimuli such as light (Giuliano et al., 1988) and auxin (Liu et al., 1994). Studies on the promoter of the barley ABA-responsive HVA22gene indicate that G-box sequences are necessary but not sufficient for an ABA response (Shen and Ho, 1995). Instead, an ABA-responsive complex consisting of a G-box, namely, ABRE3 (GCC ACGTACA), and a novel coupling element, CE1 (TGCCACCGG), is sufficient for high-level ABA induction. The results of linker-scan analyses and gain-of-function studies showed that the 49-bp ABRC1 is the minimal sequence governing high-level ABA induction. In addition, the HVA22(I) of the HVA22 gene is also required for high-level ABA induction of HVA22 expression. The results of transient assay of GUS activity in barley (Hordeum vulgareL.) aleurone cells showed that four copies of this 49-bp ABRC1, linked to a truncated (−60 to +57) barley α-amylase promoter (Amy64) and coupled with the HVA22(I), can cause more than 120-fold ABA-inducibleuidA expression, whereas approximately 30-fold induction ofuidA expression can be detected with one copy of ABRC1. A similar investigation on ABA induction of a barley late-embryogenesis-abundant gene, HVA 1 (Shen et al., 1996), was conducted, and it was found that the ABRC3 of this gene consists of a 10-bp element (CCT ACGTGGC) with an ACGT core (A2) and a sequence directly upstream, named CE3 (ACGCGTGTCCTC). Only one copy of this ABRC3 is sufficient to confer ABA induction when fused to a minimal promoter (Amy64). Thus, two types of ABRCs were reported byShen and Ho (1995) and Shen et al. (1996), namely, ABRC1 (used in this study), consisting of ABRE3 and CE1 from the HVA22 gene, and ABRC3, composed of CE3 and A2 from the HVA1 gene. As mentioned above, most of the data regarding ABA-responsive expression were obtained from transient assays of GUS activity in suspension cells, protoplasts, or barley aleurone cells. In the present study we used one or four copies of ABRC1 from the barleyHVA22 gene to confer ABA and/or stress-inducibleuidA expression in transgenic rice plants. The ABA action conferred by the ABRC1-containing transgene in transgenic rice plants may be different from that of individual cells, such as barley aleurone cells, in a transient assay. Another goal for this research was to construct stress-inducible expression plasmids that can be used for subsequent production of stress-tolerant transgenic rice. MATERIALS AND METHODS Construction of Plasmids Containing (ABRC1)4 Sequences, Different Lengths of Truncated Act1 Promoters, HVA22(I), and uidA for Transient Assay of ABA-Induced GUS Activity in Barley Aleurone Cells For ABA-inducible uidA expression, a minimal promoter is required in addition to ABRC1, HVA22(I) of the barley (Hordeum vulgare L.) HVA22 gene (Shen and Ho, 1995). To elucidate the relationship between ABA-inducible uidAexpression and different lengths of minimal promoters, four fragments of the rice (Oryza sativa L.) Act1 promoter were isolated and tested as potential “minimal” promoters for transient assay of ABA-induced GUS activity in barley aleurone cells. A 789-bp Act1–229I fragment with the Act1 intron was isolated by HphI-EcoRI digestion from the plasmid pBY505 (Wang and Wu, 1995). The other three fragments (Act1–229, Act1–100I, and Act1–100) were isolated from the Act1–229I-derived intermediate plasmids (data not shown) by cutting the NruI and BstEII sites present in the Act1–229I fragment (McElroy et al., 1990) in combination with other restriction sites located in the intermediate plasmids. These four fragments of truncated Act1 promoters were used to replace the Amy64 promoter in the pQS120 plasmid (Shen and Ho, 1995), which also contained four copies of ABRC1 elements and one copy each of HVA22(I) of HVA 22, uidA, and the HVA22 3′ region, to create plasmids pJS229A, pJS229B, pJS100A, and pJS100B (Fig. 1). The four truncated Act1 promoters and all of the border regions between different functional elements were confirmed by sequence analysis. These four plasmids were used for transient assays of GUS activity in barley aleurone cells. Fig. 1. Open in new tabDownload slide Schematic diagram of plasmids used for transient assay of ABA-induced GUS activity in barley aleurone cells. For the construction of plasmid pJS100A, Act1–100I(P) was inserted into pQS120 by replacing the Amy64 promoter. Act1–100I(P) contains a truncatedAct1 promoter (−100 to +560 includingAct1 intron). Similarly, the construction of pJS100B started with Act1–100(P), which includes a truncatedAct1 promoter (−100 to +80 without Act1intron). The construction of pJS229A started with Act1–229I(P), which includes a truncated Act1 promoter (−229 to +560 including Act1 intron). The construction of pJS229B started with Act1–229(P), which includes a truncatedAct1 promoter (−229 to +80 without Act1intron). Fig. 1. Open in new tabDownload slide Schematic diagram of plasmids used for transient assay of ABA-induced GUS activity in barley aleurone cells. For the construction of plasmid pJS100A, Act1–100I(P) was inserted into pQS120 by replacing the Amy64 promoter. Act1–100I(P) contains a truncatedAct1 promoter (−100 to +560 includingAct1 intron). Similarly, the construction of pJS100B started with Act1–100(P), which includes a truncatedAct1 promoter (−100 to +80 without Act1intron). The construction of pJS229A started with Act1–229I(P), which includes a truncated Act1 promoter (−229 to +560 including Act1 intron). The construction of pJS229B started with Act1–229(P), which includes a truncatedAct1 promoter (−229 to +80 without Act1intron). Transient Assay of GUS Activity in Barley Aleurone Cells Seeds of barley cv Himalaya (1988 harvest; Department of Agronomy and Soils, Washington State University, Pullman) were used. Preparations of embryoless half-seeds and aleurone cells, particle bombardment, homogenization of the bombarded seed, and GUS and luciferase assays were conducted essentially as described previously (Lanahan et al., 1992). Test for Tissue Specificity and Histochemical Analysis Leaves and roots from 10-d-old rice (cv Kenfong) seedlings grown in solid MS (Murashige and Skoog, 1962) medium were used as transformation materials and bombarded with tungsten particles coated with the pJS100B plasmid, essentially as described by Cao et al. (1992). The bombarded leaves and roots were transferred to fresh solid MS medium and cultured in a growth room (27°C with photoperiod of 12 h) for 2 d. Then the transformed leaves and roots were induced in liquid MS medium in the presence of 20 μm ABA for 20 h and subjected to histochemical staining with a solution containing 1 mm X-gluc and 50 mm sodium phosphate buffer (pH 7.0) as described by Jefferson et al. (1987). Construction of Plasmids for Analyzing ABA- and/or Stress-InducibleuidA Expression in Transgenic Rice Plants A previous report (Shen and Ho, 1995) indicated that four copies of ABRC1 confer ABA-responsive induction of uidA expression in barley aleurone cells four times higher than that with one copy. To compare the functional difference between one and four copies of ABRC1 in transgenic rice plants, we constructed two plasmids harboring either one or four copies of ABRC1. For construction of a plasmid containing one copy of ABRC1, the ABRC1 fragment from plasmid pJS115 (Shen and Ho, 1995) was isolated by EcoRI-XbaI digestion and subcloned into EcoRI-XbaI-digested pBluescript-KS(±). An Act1–100 promoter joined to the HVA22(I), which is abbreviated as Act1–100P-HVA22(I), was excised from pJS100B (see Table I) by BamHI digestion and subcloned at the BamHI site downstream of ABRC1 in pBluescript-KS(±) to produce the ABRC1-Act1–100P-HVA22(I) fragment. Table I. ABA-induced GUS activity in barley aleurone cells Constructs . Normalized Relative GUS Activity . Induction . −ABA . +ABA . -fold pJS100A 3101  ± 452 14829  ± 3229 5 pJS100B  3677 ± 1012 77685  ± 3320 21 pJS229A 24571  ± 1963 45023  ± 4680 2 pJS229B 5627  ± 423 37454  ± 3465 7 Constructs . Normalized Relative GUS Activity . Induction . −ABA . +ABA . -fold pJS100A 3101  ± 452 14829  ± 3229 5 pJS100B  3677 ± 1012 77685  ± 3320 21 pJS229A 24571  ± 1963 45023  ± 4680 2 pJS229B 5627  ± 423 37454  ± 3465 7 Normalized relative GUS activity was calculated based on luciferase activity (Lanahan et al., 1992). Each value represents the average of four independent analyses ± se. The maximum induction value is underlined. Open in new tab Table I. ABA-induced GUS activity in barley aleurone cells Constructs . Normalized Relative GUS Activity . Induction . −ABA . +ABA . -fold pJS100A 3101  ± 452 14829  ± 3229 5 pJS100B  3677 ± 1012 77685  ± 3320 21 pJS229A 24571  ± 1963 45023  ± 4680 2 pJS229B 5627  ± 423 37454  ± 3465 7 Constructs . Normalized Relative GUS Activity . Induction . −ABA . +ABA . -fold pJS100A 3101  ± 452 14829  ± 3229 5 pJS100B  3677 ± 1012 77685  ± 3320 21 pJS229A 24571  ± 1963 45023  ± 4680 2 pJS229B 5627  ± 423 37454  ± 3465 7 Normalized relative GUS activity was calculated based on luciferase activity (Lanahan et al., 1992). Each value represents the average of four independent analyses ± se. The maximum induction value is underlined. Open in new tab We chose Act1–100 as the minimal promoter (Act1–100P) because it was the best among the four fragments listed in Figure 1, as determined by transient assay of GUS activity in barley aleurone cells (seeResults). The fragment ABRC1-Act1–100P-HVA22(I) was further cloned into the Act1 5′ region-deleted pBY505 to create the pJS104 plasmid, which contains ABRC1-Act1–100P-HVA22(I)/polylinker/Pin2 3′//CaMV 35S(P)/bar/Nos. Thebar cassette, 35S(P)/bar/Nos, was used for the selection of rice transformants. By using the same procedure (except that four tandem copies of ABRC1 were isolated from pQS120 [Shen and Ho, 1995]), pJS109, which contained 4ABRC1-Act1–100P-HVA22(I)/polylinker/Pin2 3′//35S(P)/bar/Nos, was also constructed. Both plasmids pJS104 and pJS109 may serve as expression vectors for construction of the plasmids containing stress-tolerant genes. The GUS coding sequence (uidA) was cloned into theSmaI site of pJS104 and pJS109 to create pJS105 and pJS110, respectively (the components of the latter two plasmids are shown in Fig. 2). The plasmids pJS105 and pJS110 were used for transformation of rice and for testing ABA and/or stress-inducibleuidA expression in the transgenic rice plants. Fig. 2. Open in new tabDownload slide Schematic diagram of plasmids pJS105 and pJS110. Each plasmid consists of two gene expression cassettes, theuidA cassette, in which uidA expression is regulated by the ABRC1-Act1–100P-HVA22(I) promoter complex and the potato Pin 2 3′ region, and the bar cassette, in which the bar gene is controlled by the CaMV 35S promoter and the Nos 3′ region, and serves as the selectable marker for transformation of rice. Only those restriction sites used for DNA digestion in DNA-blot hybridization are indicated.HindIII is a unique site in these two plasmids. Fig. 2. Open in new tabDownload slide Schematic diagram of plasmids pJS105 and pJS110. Each plasmid consists of two gene expression cassettes, theuidA cassette, in which uidA expression is regulated by the ABRC1-Act1–100P-HVA22(I) promoter complex and the potato Pin 2 3′ region, and the bar cassette, in which the bar gene is controlled by the CaMV 35S promoter and the Nos 3′ region, and serves as the selectable marker for transformation of rice. Only those restriction sites used for DNA digestion in DNA-blot hybridization are indicated.HindIII is a unique site in these two plasmids. Production of Transgenic Rice Plants Calli were induced in Linsmaier and Skoog medium (Linsmaier and Skoog, 1965) from mature rice cv Kenfong embryos. Suspension cultures were initiated from embryogenic calli in liquid AA medium (Cao et al., 1991). Fine suspension cells (subcultured for 3 d prior to bombardment) were bombarded with tungsten particles coated with either the pJS105 or the pJS110 plasmid, according to the procedure described by Cao et al. (1992). Resistant calli were selected in KPR medium (Zhang and Wu, 1988), supplemented with 8 mg L−1Bialaphos as a selective agent, for 6 weeks (subcultured every 2 weeks). The resistant calli were transferred to MS regeneration medium containing 3 mg L−1 Bialaphos to regenerate into plants. Regenerated plants were transplanted into sterilized soil and grown in the greenhouse (32°C day/22°C night with a supplemental photoperiod of 10 h). The presence of the transgenes in regenerated rice plants was first indicated by the herbicide resistance of the plants. To test herbicide resistance, leaves on 3-month-old transgenic rice plants were painted on both sides with 0.25% (v/v) of the herbicide Basta (containing 162 g L−1 glufosinate ammonium; Hoechst-Roussel Agri-Vet Co., Somerville, NJ) and 0.05% (v/v) Tween 20. One week later, the resistant or sensitive phenotypes were scored. DNA-Blot Hybridization Analysis of Transgenic Rice Plants Genomic DNA from transgenic rice plants was prepared as described by Zhao et al. (1989). Eight micrograms of genomic DNA was digested with restriction enzymes, electrophoresed through 0.8% (foruidA probe) and 1.2% (for probe 2 shown in Fig. 2) agarose gels, and transferred to nylon membranes (Nytran, Schleicher & Schuell). Probe preparation and hybridization were performed by following the manufacturer's instructions for nonradioactive DIG labeling and for the detection kit (Boehringer Mannheim). RNA-Blot-Hybridization Analysis of Transgenic Rice Plants Total RNA from leaves of R1 transgenic rice plants was isolated as described by Hihara et al. (1996). Five micrograms of total RNA from the transgenic rice was subjected to electrophoresis in a 1.0% formaldehyde agarose gel. After electrophoresis, RNA was transferred to a nylon membrane (Boehringer Mannheim). The 1.8-kb GUS coding region was used as a probe and labeled with [α-32P]dCTP using a random primer DNA-labeling kit (GIBCO-BRL). Gel preparation, hybridization, and washing were carried out as described by Sambrook et al. (1989). ABA, Water Deficit, and NaCl Treatments of Transgenic Rice For ABA treatment, seedlings of R1 plants were used. Rice embryos of R1 mature seeds both from transgenic and nontransgenic plants were germinated in solid one-half-strength MS medium and cultured in a growth room for 5 weeks for RNA-blot hybridization or for 2 weeks for assaying ABA-induced GUS activity. Then the 5- or 2-week-old R1 plants were transferred to liquid one-half-strength MS medium containing 50 μm ABA for 20 h in the growth room. For stress treatments (water deficit and NaCl), R1 plants grown in soil were used. R1 seeds were first germinated in one-half-strength MS medium for 7 d, and were then transplanted into soil in pots (8 × 8 in) with holes in the bottom. The pots were kept in flat-bottomed trays containing water. The seedlings were grown for an additional 7 weeks before they were exposed to stress conditions. To induce water deficit, water was withheld from the trays for up to 8 d. The absolute water content of the soil during the stress period and before treatment was determined. Nonstressed plants were supplied with water continuously from the trays. For NaCl treatment, water containing 150 mm NaCl solution was used to water 8-week-old plants, including nontransgenic plants. Leaves and roots were collected from the same plant after different periods of stress treatments and used for assaying stress-induced GUS activity. Quantitative Assay of GUS Activity in Transgenic Rice Plants To detect the GUS activity in R0 plants before treatment and in R1 transgenic rice plants after treatment with ABA, water deficit, and NaCl, a quantitative assay of GUS activity was carried out as described by Jefferson et al. (1987). Different leaves (adjacent) or roots from the same R1 plant of each line were collected before treatment or at the different stages of treatments: 20 h for ABA treatment; 4, 6, and 8 d for water stress; and 48, 72, and 96 h for NaCl treatment. Control experiments in parallel to ABA and NaCl treatments in the absence of ABA or NaCl were also performed to test for possible injury effect on GUS activity. Collected leaves or roots were frozen immediately in liquid N2 and homogenized in extraction buffer (50 mm phosphate buffer, pH 7.0, 10 mm EDTA, 0.1% Triton X-100, 0.1% Sarkosyl, 10 mm β-mercaptoethanol, and 25 μg mL−1 PMSF). After centrifugation (12,000 rpm for 15 min at 4°C) the crude extract, containing 20 μg of protein from leaves or roots, was directly used for spectrofluorometric assay. Protein concentration of the crude extract was determined by the dye-binding method of Bradford (1976) with a protein assay reagent (Bio-Rad). RESULTS The Shortest Truncated Act1 Promoter (Act1–100P) Confers the Highest ABA Induction in Barley Aleurone Cells To get ABA- and stress-inducible gene expression in transgenic rice plants, a truncated promoter (termed the “minimal promoter”) is required in addition to ABRC1 and HVA22(I) of the HVA 22gene. Before stable transformation of rice, transient expression assay of ABA-induced GUS activity was first performed in barley aleurone cells by using four different lengths of truncated Act1promoters as the minimal promoters. The results (Table I) indicated that the plasmid with the shortest promoter (Act1–100P) shows not only the highest induction (21-fold), but also the highest GUS activity after exogenous ABA application. The Act1 intron is not necessary for ABA-inducible uidA expression. In fact, it inhibits uidA expression when the HVA22 intron is also present in the plasmid (see Table I). Tissue specificity of uidA expression driven by the ABA-responsive promoter complex (4ABRC1-Act1–100P-HVA22(I)) was also tested in this study. After histochemical analysis following ABA induction, blue spots were observed in the detached leaves and roots bombarded with plasmid pJS100B (data not shown). This result indicated a lack of tissue specificity for ABA-inducibleuidA expression driven by the ABA-responsive promoter complex. According to the results mentioned above, Act1–100P was used as a minimal promoter for plasmid constructs suitable for stable transformation of rice plants. Production of Transgenic Rice Plants and Southern-Blot-Hybridization Analyses Two plasmids, pJS105 (containing one copy of ABRC1) and pJS110 (containing four copies of ABRC1), were constructed for expression ofuidA in transgenic rice plants. The structures of these two plasmids are shown in Figure 2. After particle bombardment of suspension cells by using the two plasmids, eight Basta-resistant and Southern-blot-positive lines were regenerated, of which six (three lines for each plasmid) showed the correct hybridization pattern. The other two lines had rearranged bands and, therefore, were not further studied. The six desired transgenic lines were all fertile and their R1 generations were used for further analyses. The results of Southern-blot hybridization with the 1.8-kbuidA coding region as the probe (Fig. 2, probe 1) are shown in Figure 3. Both rice genomic and plasmid DNA were digested by BamHI or HindIII.BamHI digestion released a 1.8-kb hybridizing band corresponding to the size of uidA.HindIII is a unique site in the plasmids pJS105 and pJS110, thus each hybridization band created by HindIII digestion represents one copy of the transgene uidA, except in cases when HindIII fragments cannot be resolved. Each line has its own specific hybridization pattern except the expected 1.8-kb band, indicating that these six transgenic lines were derived from independent transformation events. Fig. 3. Open in new tabDownload slide Southern-hybridization analysis ofgusA-transgenic rice plants. Eight micrograms of rice genomic DNA was digested by BamHI (two sites in the plasmids) or HindIII (a unique site in the plasmids) and separated in a 0.8% agarose gel. A DIG-labeled, 1.8-kb GUS coding region (probe 1; see Fig. 2) was used as the probe. Molecular sizes (kb) of the 1-kb DNA ladder are indicated on the left side. B,BamHI; H, HindIII; U, undigested; and NT, DNA from nontransgenic plants. Fig. 3. Open in new tabDownload slide Southern-hybridization analysis ofgusA-transgenic rice plants. Eight micrograms of rice genomic DNA was digested by BamHI (two sites in the plasmids) or HindIII (a unique site in the plasmids) and separated in a 0.8% agarose gel. A DIG-labeled, 1.8-kb GUS coding region (probe 1; see Fig. 2) was used as the probe. Molecular sizes (kb) of the 1-kb DNA ladder are indicated on the left side. B,BamHI; H, HindIII; U, undigested; and NT, DNA from nontransgenic plants. To verify that one copy of ABRC1 and four copies of ABRC1 were also integrated into the genome of transgenic rice plants, an additional Southern-blot hybridization was conducted by using the 330-bp probe 2 (see Fig. 2). The results (Fig. 4) indicated that transgenic lines 1, 2, and 5 contained one copy of ABRC1 corresponding to the size (330 bp) of the expected band of pJS105, whereas lines 3, 7, and 11 contained four copies of ABRC1 corresponding to the size (470 bp) of the expected band of pJS110. This result also showed that the one copy of ABRC1 or four copies of ABRC1, fused to the Act1–100P with HVA22(I), were integrated into the rice genome. The copy number of the transgenes was estimated both by HindIII digestion, which has only one restriction site in the plasmids (Fig.3), and by using the Act1–100P-containing band as an internal standard. Previous work (McElroy et al., 1990) indicated the presence of only one copy of the Act1 gene in the rice genome. Since there is also one copy of the Act1–100P in plasmids pJS105 and pJS110, the ratio of the intensity of hybridization bands (the 330-bp band for pJS105 transgenic lines, and 470-bp band for pJS110 transgenic lines) to the band (2.2 kb) corresponding to that of nontransgenic plants should give the copy number of the transgene in a given transgenic plant (Table II). Fig. 4. Open in new tabDownload slide Southern-hybridization analysis ofgusA-transgenic rice plants. Eight micrograms of genomic DNA was digested by EcoRV (see EcoRV sites in Fig. 2) and the digested DNA was separated in a 1.2% agarose gel. A DIG-labeled, 330-bp of probe 2 (indicated in Fig. 2) was used. Molecular sizes of the 1-kb DNA ladder are indicated on the left side. 3x and 5x plasmid DNA represent 3 and 5 genome equivalents of DNA relative to 8 μg of rice genomic DNA, respectively. NT, DNA from nontransgenic plants. Fig. 4. Open in new tabDownload slide Southern-hybridization analysis ofgusA-transgenic rice plants. Eight micrograms of genomic DNA was digested by EcoRV (see EcoRV sites in Fig. 2) and the digested DNA was separated in a 1.2% agarose gel. A DIG-labeled, 330-bp of probe 2 (indicated in Fig. 2) was used. Molecular sizes of the 1-kb DNA ladder are indicated on the left side. 3x and 5x plasmid DNA represent 3 and 5 genome equivalents of DNA relative to 8 μg of rice genomic DNA, respectively. NT, DNA from nontransgenic plants. Table II. Approximate copy number of transgenes in pJS105- and pJS110-transgenic lines . pJS105 Transgenic . pJS110 Transgenic . Lines 1 2 5 3 7 11 Transgene copy no. 9 3 1 7 1 5 . pJS105 Transgenic . pJS110 Transgenic . Lines 1 2 5 3 7 11 Transgene copy no. 9 3 1 7 1 5 Open in new tab Table II. Approximate copy number of transgenes in pJS105- and pJS110-transgenic lines . pJS105 Transgenic . pJS110 Transgenic . Lines 1 2 5 3 7 11 Transgene copy no. 9 3 1 7 1 5 . pJS105 Transgenic . pJS110 Transgenic . Lines 1 2 5 3 7 11 Transgene copy no. 9 3 1 7 1 5 Open in new tab GUS Activity in R0 Transgenic Rice Plants The promoter complex in plasmids pJS105 and pJS110 is composed of ABRC1, the Act1–100P minimal promoter, and HVA22(I), in which the Act1–100P promoter plays an important role in conferring the basal level of uidA expression. Before starting to test for ABA- and stress-induced GUS activity, we first examined the basal level of GUS activity in 4-month-old R0 transgenic plants. The results are shown in Figure5. Of the six transgenic lines, L5, L7, and L11 showed high levels of GUS activity and L2 showed low activity (≤ 1 nmol h−1 mg−1 protein). No GUS activity was detected in either leaves or roots of L1 and L3. L2 and L5 (pJS105 transformants) and L7 and L11 (pJS110 transformants) were used for assaying ABA- and stress-inducible uidAexpression. Fig. 5. Open in new tabDownload slide GUS activity in the R0 transgenic plants without any treatment. L1, L2, and L5 represent pJS105-transgenic lines 1, 2, 5; and L3, L7, and L11 represent pJS110-transgenic lines 3, 7, and 11. Mean ± sevalues of GUS activity (4-methylumbelliferone, nmol h−1mg−1 protein) are: Leaves: L1, 0.02 ± 0.01; L2, 1 ± 0.3; L5, 7 ± 2; L3, 0.02 ± 0.01; L7, 14 ± 4; L11, 12 ± 3; and NT, 0.02 ± 0.01. NT, DNA from nontransgenic plants. Roots: L1, 0.01 ± 0.01; L2, 0.8 ± 0.2; L5, 6 ± 1; L3, 0.01 ± 0.01; L7, 9 ± 2; L11, 7 ± 2; and NT, 0.01 ± 0.01. Data represent the average results of four experiments by using different tillers of the same R0 line. Bar represents the se. Fig. 5. Open in new tabDownload slide GUS activity in the R0 transgenic plants without any treatment. L1, L2, and L5 represent pJS105-transgenic lines 1, 2, 5; and L3, L7, and L11 represent pJS110-transgenic lines 3, 7, and 11. Mean ± sevalues of GUS activity (4-methylumbelliferone, nmol h−1mg−1 protein) are: Leaves: L1, 0.02 ± 0.01; L2, 1 ± 0.3; L5, 7 ± 2; L3, 0.02 ± 0.01; L7, 14 ± 4; L11, 12 ± 3; and NT, 0.02 ± 0.01. NT, DNA from nontransgenic plants. Roots: L1, 0.01 ± 0.01; L2, 0.8 ± 0.2; L5, 6 ± 1; L3, 0.01 ± 0.01; L7, 9 ± 2; L11, 7 ± 2; and NT, 0.01 ± 0.01. Data represent the average results of four experiments by using different tillers of the same R0 line. Bar represents the se. ABA-, Water Deficit-, and NaCl-Induced uidA mRNA Level in Transgenic Rice Plants To test the ABA- or stress-inducible uidA expression, we first examined the transcript level of the uidA transgene in R1 leaves before and after water-deficit treatment for 6 d in the greenhouse (see Methods). Three transgenic lines (L5, L7, and L11) were found to express uidA (data not shown). L5 from the pJS105 construct and L7 from the pJS110 construct were selected for further treatments and analyses. ABA and NaCl were also found to induce uidAexpression (Fig. 6). By densitometry tracing, the induction level varied from 6- to 8-fold. NouidA transcripts were detected in R1leaf RNA from the other two Southern-blot-positive lines (L1 and L3) or from nontransgenic plants even after water-deficit treatment. Fig. 6. Open in new tabDownload slide ABA-, water-deficit-, and NaCl-inducedgusA expression confirmed by northern-hybridization analysis. Five micrograms of total RNA was fractionated in a 1% formaldehyde agarose gel and blotted onto a nylon membrane hybridized with [α-32P]dCTP-labeled gusA coding sequence. Equal loading of the RNA samples was confirmed by ethidium bromide staining of rRNA in a parallel-running gel. Molecular sizes (kb) of two fragments from the RNA ladder are indicated on the right side. A, ABA: 50 μm for 20 h; B, basal level without any treatment; W, water deficit: water withheld for 6 d; and N, NaCl: 150 mm NaCl, for 72 h. (For detailed procedure, see Methods.) Fig. 6. Open in new tabDownload slide ABA-, water-deficit-, and NaCl-inducedgusA expression confirmed by northern-hybridization analysis. Five micrograms of total RNA was fractionated in a 1% formaldehyde agarose gel and blotted onto a nylon membrane hybridized with [α-32P]dCTP-labeled gusA coding sequence. Equal loading of the RNA samples was confirmed by ethidium bromide staining of rRNA in a parallel-running gel. Molecular sizes (kb) of two fragments from the RNA ladder are indicated on the right side. A, ABA: 50 μm for 20 h; B, basal level without any treatment; W, water deficit: water withheld for 6 d; and N, NaCl: 150 mm NaCl, for 72 h. (For detailed procedure, see Methods.) ABA-Induced GUS Activity in Transgenic Rice Plants A previous report (Shen and Ho, 1995) indicated that ABRC1 confers a high degree of ABA induction for gene expression by a transient assay in barley aleurone cells. To examine the ABA-induction level ofuidA expression conferred by the ABA-responsive promoter complex, ABRC1-Act1–100P-HVA22(I), in transgenic rice leaves and roots, a quantitative assay of GUS activity before and after ABA treatment of 2-week-old seedlings was carried out. At the 2-week stage, most R1 seedlings had two normal-sized leaves. Of 10 plants tested, 8 showed GUS activity and ABA inducibility. A lower leaf of an R1 seedling was cut off and used for GUS activity assay before applying exogenous ABA. An upper leaf of the same seedling was collected for assaying ABA-inducibleuidA expression after supplying 50 μm ABA for 20 h. The results of this analysis are given in Figure7. It is shown that the absolute level of GUS activity in pJS110-transformed plants is higher than that of pJS105-transformed plants both before and after ABA induction. A control experiment using upper leaves of L7 (with the highest GUS activity) after collection of the lower leaf was carried out in the absence of ABA and no increase of GUS activity was found. Thus, removing leaf tissues from plants did not show any adverse effect on GUS activity. Fig. 7. Open in new tabDownload slide ABA-induced GUS activity (4-methylumbelliferone, nmol h−1 mg−1 protein) in 2-week-old R1 seedlings of transgenic plants. All data were derived from the results of eight seedlings. pJS105 (one copy of ABRC1), L2 and L5; pJS110 (four copies of ABRC1), L7 and L11. NT, Nontransgenic plants. x indicates the -fold induction. Bars represents these. Left panel, Leaves. Mean ± se values of ABA-induced GUS activity are: L2, 1 ± 0.2 (−ABA), 1.2 ± 0.3 (+ABA), 1.2x; L5, 6 ± 2 (−ABA), 22 ± 5 (+ABA), 4x; L7, 15 ± 4 (−ABA), 73 ± 8 (+ABA), 5x; L11, 11 ± 3 (−ABA), 41 ± 6 (+ABA), 4x; and NT, 0.02 ± 0.01 (−ABA), 0.02 ± 0.01 (+ABA), 1x. Right panel, Roots. Mean ±se values of ABA-induced GUS activity are: L2, 0.8 ± 0.2 (−ABA), 0.9 ± 0.2 (+ABA), 1x; L5, 4 ± 2 (−ABA), 26 ± 5 (+ABA), 7x; L7, 6 ± 2 (−ABA), 48 ± 10 (+ABA), 8x; L11, 5 ± 1 (−ABA), 33 ± 5 (+ABA), 7x; and NT, 0.01 ± 0.01 (−ABA), 0.01 ± 0.01 (+ABA), 1x.   Fig. 7. Open in new tabDownload slide ABA-induced GUS activity (4-methylumbelliferone, nmol h−1 mg−1 protein) in 2-week-old R1 seedlings of transgenic plants. All data were derived from the results of eight seedlings. pJS105 (one copy of ABRC1), L2 and L5; pJS110 (four copies of ABRC1), L7 and L11. NT, Nontransgenic plants. x indicates the -fold induction. Bars represents these. Left panel, Leaves. Mean ± se values of ABA-induced GUS activity are: L2, 1 ± 0.2 (−ABA), 1.2 ± 0.3 (+ABA), 1.2x; L5, 6 ± 2 (−ABA), 22 ± 5 (+ABA), 4x; L7, 15 ± 4 (−ABA), 73 ± 8 (+ABA), 5x; L11, 11 ± 3 (−ABA), 41 ± 6 (+ABA), 4x; and NT, 0.02 ± 0.01 (−ABA), 0.02 ± 0.01 (+ABA), 1x. Right panel, Roots. Mean ±se values of ABA-induced GUS activity are: L2, 0.8 ± 0.2 (−ABA), 0.9 ± 0.2 (+ABA), 1x; L5, 4 ± 2 (−ABA), 26 ± 5 (+ABA), 7x; L7, 6 ± 2 (−ABA), 48 ± 10 (+ABA), 8x; L11, 5 ± 1 (−ABA), 33 ± 5 (+ABA), 7x; and NT, 0.01 ± 0.01 (−ABA), 0.01 ± 0.01 (+ABA), 1x.   Water-Deficit-Induced GUS Activity in Transgenic Rice Plants As mentioned previously, ABA mediates gene expression involved in plant physiological responses to stress such as drought and salinity. The ABA-induced uidA expression in this report encouraged us to explore the water-deficit-induced GUS activity in the transgenic rice. Before water-deficit treatment, the third leaf from the bottom and about one-tenth the amount of roots of 8-week-old R1 plants with four to five leaves were collected, frozen in liquid N2, and used for assaying the basal level of GUS activity. These plants were subjected to water-deficit treatment for 4, 6, and 8 d. The other three leaves from the same plant used for assaying basal level were collected at 4, 6, and 8 d, respectively, after the treatment and used for testing the induced activity. At the same time, one-tenth the amount of roots was also collected at each time point following the leaf collection. Three plants for each line were used for each experiment to calculate the degree of induction of uidA expression by water-deficit treatment. The results from three independent experiments are listed in Table III. After 4 d of treatment GUS activity in rice leaves increased only slightly. With an increase of treatment days, GUS activity increased rapidly and reached a peak at 8 d, resulting in 5- to 6-fold induction. Beyond 8 d (data not shown), the treated leaves started to wilt. In rice roots the GUS activity reached a peak after 6 d. A longer treatment (e.g. 8 d) gave a slightly reduced level ofuidA expression in the roots of transgenic rice by withholding water. Table III. Water-deficit-induced GUS activity in R1 leaves and roots of transgenic rice plants Days of Treatment . Water Content of Soil . GUS Activity . pJS105 Transgenic . pJS110 Transgenic .   . L2 . L5 . L7 . L11 . NT . Leaves . Roots . Leaves . Roots . Leaves . Roots . Leaves . Roots . Leaves . Roots . % 4-MU nmol h−1 mg−1protein 0 37 1  ± 0.2 0.9  ± 0.2 7  ± 3 6  ± 2 14  ± 4 13  ± 3 10  ± 3 7  ± 2 0.02  ± 0.01 0.01  ± 0.01 4 24 1  ± 0.2 0.9  ± 0.2 10  ± 3 11  ± 3 18  ± 4 28  ± 3 14  ± 4 16  ± 3 0.02  ± 0.01 0.01  ± 0.01 6 14 1  ± 0.2 0.9  ± 0.2 18  ± 4 34  ± 5 35  ± 6 88  ± 6 27  ± 6 41  ± 5 0.02  ± 0.01 0.01  ± 0.01 8 9 1  ± 0.2 0.9  ± 0.2 40  ± 6 31  ± 5 81  ± 7 80  ± 6 47  ± 7 38  ± 5 0.02  ± 0.01 0.01  ± 0.01 8 d/0 d 1 6 6 5 1 6 d/0 d 1 6 7 6 1 Days of Treatment . Water Content of Soil . GUS Activity . pJS105 Transgenic . pJS110 Transgenic .   . L2 . L5 . L7 . L11 . NT . Leaves . Roots . Leaves . Roots . Leaves . Roots . Leaves . Roots . Leaves . Roots . % 4-MU nmol h−1 mg−1protein 0 37 1  ± 0.2 0.9  ± 0.2 7  ± 3 6  ± 2 14  ± 4 13  ± 3 10  ± 3 7  ± 2 0.02  ± 0.01 0.01  ± 0.01 4 24 1  ± 0.2 0.9  ± 0.2 10  ± 3 11  ± 3 18  ± 4 28  ± 3 14  ± 4 16  ± 3 0.02  ± 0.01 0.01  ± 0.01 6 14 1  ± 0.2 0.9  ± 0.2 18  ± 4 34  ± 5 35  ± 6 88  ± 6 27  ± 6 41  ± 5 0.02  ± 0.01 0.01  ± 0.01 8 9 1  ± 0.2 0.9  ± 0.2 40  ± 6 31  ± 5 81  ± 7 80  ± 6 47  ± 7 38  ± 5 0.02  ± 0.01 0.01  ± 0.01 8 d/0 d 1 6 6 5 1 6 d/0 d 1 6 7 6 1 Mean ± se values of the GUS activity were calculated from the results of three independent experiments and three plants were used for each experiment. R1 plants were grown in a greenhouse and treated without water for 4, 6, and 8 d. 0 d represents the basal level before water-deficit treatment. 8 d/0 d indicates the induction of GUS activity in rice leaves by withholding water for 8 d, and 6 d/0 d indicates the induction in rice roots by withholding water for 6 d. NT, Nontransgenic; 4-MU, 4-methylumbelliferone. Maximum induction values are underlined. Open in new tab Table III. Water-deficit-induced GUS activity in R1 leaves and roots of transgenic rice plants Days of Treatment . Water Content of Soil . GUS Activity . pJS105 Transgenic . pJS110 Transgenic .   . L2 . L5 . L7 . L11 . NT . Leaves . Roots . Leaves . Roots . Leaves . Roots . Leaves . Roots . Leaves . Roots . % 4-MU nmol h−1 mg−1protein 0 37 1  ± 0.2 0.9  ± 0.2 7  ± 3 6  ± 2 14  ± 4 13  ± 3 10  ± 3 7  ± 2 0.02  ± 0.01 0.01  ± 0.01 4 24 1  ± 0.2 0.9  ± 0.2 10  ± 3 11  ± 3 18  ± 4 28  ± 3 14  ± 4 16  ± 3 0.02  ± 0.01 0.01  ± 0.01 6 14 1  ± 0.2 0.9  ± 0.2 18  ± 4 34  ± 5 35  ± 6 88  ± 6 27  ± 6 41  ± 5 0.02  ± 0.01 0.01  ± 0.01 8 9 1  ± 0.2 0.9  ± 0.2 40  ± 6 31  ± 5 81  ± 7 80  ± 6 47  ± 7 38  ± 5 0.02  ± 0.01 0.01  ± 0.01 8 d/0 d 1 6 6 5 1 6 d/0 d 1 6 7 6 1 Days of Treatment . Water Content of Soil . GUS Activity . pJS105 Transgenic . pJS110 Transgenic .   . L2 . L5 . L7 . L11 . NT . Leaves . Roots . Leaves . Roots . Leaves . Roots . Leaves . Roots . Leaves . Roots . % 4-MU nmol h−1 mg−1protein 0 37 1  ± 0.2 0.9  ± 0.2 7  ± 3 6  ± 2 14  ± 4 13  ± 3 10  ± 3 7  ± 2 0.02  ± 0.01 0.01  ± 0.01 4 24 1  ± 0.2 0.9  ± 0.2 10  ± 3 11  ± 3 18  ± 4 28  ± 3 14  ± 4 16  ± 3 0.02  ± 0.01 0.01  ± 0.01 6 14 1  ± 0.2 0.9  ± 0.2 18  ± 4 34  ± 5 35  ± 6 88  ± 6 27  ± 6 41  ± 5 0.02  ± 0.01 0.01  ± 0.01 8 9 1  ± 0.2 0.9  ± 0.2 40  ± 6 31  ± 5 81  ± 7 80  ± 6 47  ± 7 38  ± 5 0.02  ± 0.01 0.01  ± 0.01 8 d/0 d 1 6 6 5 1 6 d/0 d 1 6 7 6 1 Mean ± se values of the GUS activity were calculated from the results of three independent experiments and three plants were used for each experiment. R1 plants were grown in a greenhouse and treated without water for 4, 6, and 8 d. 0 d represents the basal level before water-deficit treatment. 8 d/0 d indicates the induction of GUS activity in rice leaves by withholding water for 8 d, and 6 d/0 d indicates the induction in rice roots by withholding water for 6 d. NT, Nontransgenic; 4-MU, 4-methylumbelliferone. Maximum induction values are underlined. Open in new tab NaCl-Induced GUS Activity in Transgenic Rice Plants To test the extent of induction of uidA expression by NaCl treatment, a 150 mm NaCl solution was used to create a salinity-stress condition. Water was withheld for 24 h from 8-week-old plants with four to five leaves grown in the greenhouse, and then a 150 mm NaCl solution was added to the plant-containing pots and the tray. The NaCl solution was changed every 24 h. Samples were collected in the same way as in the water-deficit treatment except that the third leaf of each plant used for assaying the basal level of GUS activity was collected after 24 h of withholding water (0 h treatment by NaCl). TableIV indicates the results of this analysis in the leaves and roots of transgenic rice. As compared with the results of ABA and water-deficit treatments, the GUS activity and induction levels were both lower. Similar to the water-deficit treatment, the NaCl-induced GUS activity in the roots of transgenic rice plants reached its peak at 72 h of treatment. A longer treatment (such as 96 h) showed a slightly reduced level ofuidA expression. A control experiment using leaves collected at 48, 72, and 96 h after cutting the first leaves was also performed in the absence of NaCl and no increase of GUS activity was observed. Thus, removing leaf tissues from plants did not show any adverse effect on GUS activity. Table IV. NaCl-induced GUS activity in R1 leaves and roots of transgenic rice plants NaCl Treatment . GUS Activity . pJS105 Transgenic . pJS110 Transgenic .   . L2 . L5 . L7 . L11 . NT . Leaves . Roots . Leaves . Roots . Leaves . Roots . Leaves . Roots . Leaves . Roots . h 4-MU nmol h−1 mg−1protein 0 0.9  ± 0.2 0.8  ± 0.2 6  ± 2 5  ± 1 13  ± 3 12  ± 2 10  ± 2 9  ± 2 0.02  ± 0.01 0.01  ± 0.01 48 0.9  ± 0.2 0.8  ± 0.2 10  ± 3 11  ± 3 20  ± 4 25  ± 4 16  ± 4 20  ± 4 0.02  ± 0.01 0.01  ± 0.01 72 0.9  ± 0.2 0.8  ± 0.2 14  ± 3 20  ± 5 28  ± 6 46  ± 6 21  ± 4 25  ± 5 0.02  ± 0.01 0.01  ± 0.01 96 0.9  ± 0.2 0.8  ± 0.2 17  ± 4 16  ± 3 59  ± 7 40  ± 4 28  ± 5 22  ± 3 0.02  ± 0.01 0.01  ± 0.01 96 h/0 h 1 3 4 3 1 72 h/0 h 1 4 4 3 1 NaCl Treatment . GUS Activity . pJS105 Transgenic . pJS110 Transgenic .   . L2 . L5 . L7 . L11 . NT . Leaves . Roots . Leaves . Roots . Leaves . Roots . Leaves . Roots . Leaves . Roots . h 4-MU nmol h−1 mg−1protein 0 0.9  ± 0.2 0.8  ± 0.2 6  ± 2 5  ± 1 13  ± 3 12  ± 2 10  ± 2 9  ± 2 0.02  ± 0.01 0.01  ± 0.01 48 0.9  ± 0.2 0.8  ± 0.2 10  ± 3 11  ± 3 20  ± 4 25  ± 4 16  ± 4 20  ± 4 0.02  ± 0.01 0.01  ± 0.01 72 0.9  ± 0.2 0.8  ± 0.2 14  ± 3 20  ± 5 28  ± 6 46  ± 6 21  ± 4 25  ± 5 0.02  ± 0.01 0.01  ± 0.01 96 0.9  ± 0.2 0.8  ± 0.2 17  ± 4 16  ± 3 59  ± 7 40  ± 4 28  ± 5 22  ± 3 0.02  ± 0.01 0.01  ± 0.01 96 h/0 h 1 3 4 3 1 72 h/0 h 1 4 4 3 1 Mean ± se values of NaCl-induced GUS activity were calculated from the results of three independent experiments and three plants were used for each experiment. Eight-week-old R1plants were grown in the greenhouse. After withholding water for 24 h, the third leaf or one-tenth the amount of roots was collected and used for a basal level test of GUS activity (0 h). Then, the plants were supplied with 150 mm NaCl solution. At 48, 72, and 96 h, the other three leaves or one-tenth the amount of roots were collected, respectively, and used for assaying NaCl-induced GUS activity. Maximum induction values are underlined. 96 h/0 h indicates the -fold induction of GUS activity in rice tissues after 96 h of treatment with 150 mm NaCl. 72 h/0 h indicates the induction. NT, Nontransgenic; 4-MU, 4-methylumbelliferone. Open in new tab Table IV. NaCl-induced GUS activity in R1 leaves and roots of transgenic rice plants NaCl Treatment . GUS Activity . pJS105 Transgenic . pJS110 Transgenic .   . L2 . L5 . L7 . L11 . NT . Leaves . Roots . Leaves . Roots . Leaves . Roots . Leaves . Roots . Leaves . Roots . h 4-MU nmol h−1 mg−1protein 0 0.9  ± 0.2 0.8  ± 0.2 6  ± 2 5  ± 1 13  ± 3 12  ± 2 10  ± 2 9  ± 2 0.02  ± 0.01 0.01  ± 0.01 48 0.9  ± 0.2 0.8  ± 0.2 10  ± 3 11  ± 3 20  ± 4 25  ± 4 16  ± 4 20  ± 4 0.02  ± 0.01 0.01  ± 0.01 72 0.9  ± 0.2 0.8  ± 0.2 14  ± 3 20  ± 5 28  ± 6 46  ± 6 21  ± 4 25  ± 5 0.02  ± 0.01 0.01  ± 0.01 96 0.9  ± 0.2 0.8  ± 0.2 17  ± 4 16  ± 3 59  ± 7 40  ± 4 28  ± 5 22  ± 3 0.02  ± 0.01 0.01  ± 0.01 96 h/0 h 1 3 4 3 1 72 h/0 h 1 4 4 3 1 NaCl Treatment . GUS Activity . pJS105 Transgenic . pJS110 Transgenic .   . L2 . L5 . L7 . L11 . NT . Leaves . Roots . Leaves . Roots . Leaves . Roots . Leaves . Roots . Leaves . Roots . h 4-MU nmol h−1 mg−1protein 0 0.9  ± 0.2 0.8  ± 0.2 6  ± 2 5  ± 1 13  ± 3 12  ± 2 10  ± 2 9  ± 2 0.02  ± 0.01 0.01  ± 0.01 48 0.9  ± 0.2 0.8  ± 0.2 10  ± 3 11  ± 3 20  ± 4 25  ± 4 16  ± 4 20  ± 4 0.02  ± 0.01 0.01  ± 0.01 72 0.9  ± 0.2 0.8  ± 0.2 14  ± 3 20  ± 5 28  ± 6 46  ± 6 21  ± 4 25  ± 5 0.02  ± 0.01 0.01  ± 0.01 96 0.9  ± 0.2 0.8  ± 0.2 17  ± 4 16  ± 3 59  ± 7 40  ± 4 28  ± 5 22  ± 3 0.02  ± 0.01 0.01  ± 0.01 96 h/0 h 1 3 4 3 1 72 h/0 h 1 4 4 3 1 Mean ± se values of NaCl-induced GUS activity were calculated from the results of three independent experiments and three plants were used for each experiment. Eight-week-old R1plants were grown in the greenhouse. After withholding water for 24 h, the third leaf or one-tenth the amount of roots was collected and used for a basal level test of GUS activity (0 h). Then, the plants were supplied with 150 mm NaCl solution. At 48, 72, and 96 h, the other three leaves or one-tenth the amount of roots were collected, respectively, and used for assaying NaCl-induced GUS activity. Maximum induction values are underlined. 96 h/0 h indicates the -fold induction of GUS activity in rice tissues after 96 h of treatment with 150 mm NaCl. 72 h/0 h indicates the induction. NT, Nontransgenic; 4-MU, 4-methylumbelliferone. Open in new tab In conclusion, ABA, water deficit, and 150 mm NaCl induceduidA expression both at the RNA and protein levels (GUS activity) conferred by the ABA-induced promoter in transgenic rice plants. Transgenic rice plants harboring the plasmid with four copies of ABRC1 exhibited 50% to 200% higher GUS activity than those with one copy of ABRC1 among the tested transgenic rice lines. The Act1–100P minimal promoter coupled with ABRC1 and HVA22(I) of the barley HVA22 gene conferred ABA- and stress-inducibleuidA expression in transgenic rice. These results suggest that the expression vectors pJS104 (containing one copy of ABRC1) and pJS109 (four copies of ABRC1) can be used for other plasmid constructions to produce stress-induced osmotolerant transgenic rice plants. DISCUSSION Different stress treatments and exogenous ABA application caused different extents of induction of uidA expression in both transgenic rice leaves and roots. In this study water-deficit treatment caused the highest induction of GUS activity, about 5- to 6-fold in rice leaves, followed by ABA application with a 4- to 5-fold increase, and NaCl treatment with 3- to 4-fold increase of GUS activity. In roots ABA treatment resulted in the highest induction of GUS activity, with a 7- to 8-fold increase, followed by water-deficit treatment with a 6- to 7-fold induction, and NaCl treatment with a 3- to 4-fold increase. Strong and constitutive promoters are beneficial for high-level expression of selectable marker genes, which is necessary for efficient selection and generation of transgenic plants. However, constitutively active promoters are not always desirable for plant genetic engineering because constitutive overexpression of a transgene may compete for energy and building blocks for synthesis of proteins, RNA, etc., which are also required for plant growth under normal conditions. Either one copy of ABRC1 or four tandem copies of ABRC1 coupled with Act1–100P and HVA22(I) of the HVA22 gene confer ABA- and stress-induced uidA expression in transgenic rice. Transgene expression in transgenic plants is often correlated with copy number (Hobbs et al., 1993; Matzke et al., 1994) and integration position of transgenes (position effect) in the genome (Peach and Velten, 1991; Bhattacharyya et al., 1994). Thus, it is difficult to conclude which type of promoter complex (either one copy of ABRC1 or four copies of ABRC1) would be better for generation of stress-tolerant transgenic rice plants. According to the results in this study, we prefer to use four copies of the ABRC1-containing promoter complex because it can give approximately 50% to 200% higher GUS activity (e.g. plant L7) than one copy of the ABRC1-containing promoter complex (plant L5). We are currently using the stress-induced expression vectors to construct plasmids containing other potentially useful genes for transformation of rice. We believe that transgenic rice plants with foreign genes driven by a stress-induced promoter are expected to develop and grow better than those with genes driven by a constitutive promoter because the transgenes would be highly expressed only under stress conditions. The primary goal of this study was to test recombinant gene constructs, the expression of which is induced by ABA and stress conditions in transgenic rice plants. The information obtained in this study will be valuable in future work attempting to express useful genes in transgenic plants under stress. Since it is well established that environmental stresses such as water deficit and salinity usually lead to enhanced levels of endogenous ABA (Zeevaart and Creelman, 1988), we reason that an ABA-responsive promoter could also be induced by stress conditions. Indeed, the ABA-responsive gene constructs tested in this study are all induced by water-deficit and NaCl treatment. For an ABA-/stress-responsive promoter to be useful in driving the expression of useful genes, it is better to be highly sensitive and respond quickly to ABA/stress. Indeed, in this work we have shown that the ABRC1/actin minimal promoter responds to mild water stress and salinity within a couple of days (Tables III and IV). Although not determined in this work, we believe that this construct is even more sensitive, because Shen et al. (1993) have shown that HVA22, the promoter of which contains ABRC1, is responsive to ABA concentrations as low as 10−8m, and that this gene is induced by 10−6m ABA within 40 min. Therefore, ABRC1 appears to have the desirable features in regulating transgenes encoding useful traits for protecting plants against stress conditions. Among the ABA-responsive promoter sequences, ABRC, as defined by Shen and Ho (1995) and Shen et al. (1996), appears to be necessary and sufficient for a high level of ABA induction. However, their work was essentially carried out in a highly specialized tissue, the aleurone layers of germination barley seeds. By linking ABRC to a minimal promoter derived from the actin gene, which is constitutively expressed in many cell types, we have shown that our gene constructs can be expressed in at least two major vegetative tissues, leaves and roots, in addition to the aleurone layers. Although the transgenic approach as described in this work has proven to be an efficient means to analyze promoters, ectopic functions of promoters in transgenic plants have also been observed. For example, Sieburth and Meyerowitz (1997) have recently reported that the cis elements for spatial regulation of the Arabidopsis AGAMOUS gene are located intragenically. Thus, it is conceivable that the promoter of a gene does not always contain all of the elements regulating its expression. However, it is clear from our work and from the work of Shen and Ho (1996) that ABRC1 alone is sufficient to confer a high level of ABA inducibility. It is equally significant that the gene constructs tested in this study function well in both rice and barley. Since the ABRC we used was derived from a barley gene with homologs present in many cereal grains (Q. Shen and D. Ho, unpublished data), it is conceivable that our gene constructs could work in other cereals as well. ACKNOWLEDGMENTS We thank Dr. Xiongfong Chen for help with preparation of the photos, Cathy Herlache for reading the manuscript, and Miguel Munoz for help with drawing figures in this manuscript. Abbreviations: ABRC ABA-response complex ABREs ABA-response elements CaMV cauliflower mosaic virus DIG digoxigenin HVA22(I) intron1-exon2-intron2 of barley HVA22 gene MS Murashige and Skoog Nos nopaline synthetase LITERATURE CITED 1 Bhattacharyya BA Stermer BA Dixon RA Reduced variation in transgene expression from a binary vector with selectable markers at the right and left T-DNA borders. Plant J 6 1994 957 968 Google Scholar Crossref Search ADS WorldCat 2 Boyer JS Plant productivity and environment. Science 218 1982 443 Google Scholar Crossref Search ADS PubMed WorldCat 3 Bradford M A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72 1976 248 254 Google Scholar Crossref Search ADS PubMed WorldCat 4 Bruce WB Quail PH cis-Acting elements involved in photoregulation of an oat phytochrome promoter in rice. Plant Cell 2 1990 1081 1089 Google Scholar PubMed OpenURL Placeholder Text WorldCat 5 Cao J Duan X McElory D Wu R Regeneration of herbicide resistant transgenic rice plants following microprojectile-mediated transformation of suspension culture cells. Plant Cell Rep 11 1992 586 591 Google Scholar Crossref Search ADS PubMed WorldCat 6 Cao J, Zhang W, McElory D, Wu R (1991) Assessment of rice genetic transformation techniques. In GH Toenniessen, GS Khush, eds, Rice Biotechnology. C.A.B. International, Oxon, UK, pp 175–198 7 Chandler PM Robertson M Gene expression regulated by abscisic acid and its regulation to stress tolerance. Annu Rev Plant Physiol Mol Biol 45 1994 113 114 Google Scholar Crossref Search ADS WorldCat 8 Giuliano G Pichersky E Malik VS Timko MP Scolnik PA Cashmore AR An evolutionarily conserved protein binding sequence upstream of a plant light-regulated gene. Proc Natl Acad Sci USA 85 1988 7089 7093 Google Scholar Crossref Search ADS PubMed WorldCat 9 Gomez J Sanchez-Martinez D Stiefel V Rigau J Puigdomenech P Pages M A gene induced by the plant hormone abscisic acid in response to water stress encodes a glycine-rich protein. Nature 334 1988 262 264 Google Scholar Crossref Search ADS PubMed WorldCat 10 Guiltinan MJ Marcotte WR Jr Quatrano RS A plant leucine zipper protein that recognizes an abscisic acid response element. Science 250 1990 267 271 Google Scholar Crossref Search ADS PubMed WorldCat 11 Hayashi H Transformation of Arabidopsis thaliana with codA gene for choline oxidase: accumulation of glycinebetaine and enhanced tolerance to salt and cold stress. Plant J 12 1997 133 142 Google Scholar Crossref Search ADS PubMed WorldCat 12 Henson IE Effects of atmospheric humidity on abscisic acid accumulation and water in leaves of rice (Oryza sativa L.). Ann Bot 54 1984 569 582 Google Scholar Crossref Search ADS WorldCat 13 Hihara Y Hara C Uchimiya H Isolation and characterization of two cDNA clones for mRNA that are abundantly expressed in immature anthers of rice (Oryza sativa L.). Plant Mol Biol 30 1996 1181 1193 Google Scholar Crossref Search ADS PubMed WorldCat 14 Hobbs SLA Warkentin TD Delong CMO Transgene copy number can be positively or negatively associated with transgene expression. Plant Mol Biol 21 1993 17 26 Google Scholar Crossref Search ADS PubMed WorldCat 15 Holmström KO Mäntyla E Welin B Mandal A Palva ET Tunnela OE Londesborough J Drought tolerance in tobacco. Nature 379 1996 683 684 Google Scholar Crossref Search ADS WorldCat 16 Ingram J Bartels D The molecular basis of dehydration tolerance in plants. Annu Rev Plant Physiol Mol Biol 47 1996 377 404 Google Scholar Crossref Search ADS WorldCat 17 Jefferson RA Kavanagh TA Bevan MW GUS fusion: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6 1987 3901 3907 Google Scholar Crossref Search ADS PubMed WorldCat 18 Jones H Leigh RA Tomos AD Jones RGW The effect of abscisic acid on cell turgor pressures, solute content and growth of wheat roots. Planta 170 1987 257 262 Google Scholar Crossref Search ADS PubMed WorldCat 19 King RW Abscisic acid in developing wheat grains and its relationship to grain growth and maturation. Planta 132 1976 43 51 Google Scholar Crossref Search ADS PubMed WorldCat 20 Kishor PBK Hong Z Miao G-H Hu C-AA Verma DPS Overexpression of Δ1-pyrroline-5-carboxylate synthetase increases proline production and confers osmotolerance in transgenic plants. Plant Physiol 108 1995 1387 1394 Google Scholar Crossref Search ADS PubMed WorldCat 21 Lanahan MB Ho T-HD Rogers SW Rogers JC A gibberellin response complex in cereal α-amylase gene promoters. Plant Cell 4 1992 203 211 Google Scholar PubMed OpenURL Placeholder Text WorldCat 22 Linsmaier EM Skoog F Organic growth factor requirements of tobacco tissue cultures. Physiol Plant 18 1965 100 127 Google Scholar Crossref Search ADS WorldCat 23 Liu Z-B Ulmasov T Shi X Hagen G Guilfoyle TJ Soybean GH3 promoter contains multiple auxin-inducible elements. Plant Cell 6 1994 645 657 Google Scholar PubMed OpenURL Placeholder Text WorldCat 24 Matzke AJM Neuhuber F Park YD Ambros PF Matzke MA Homology-dependent gene silencing in transgenic plants: epistatic silencing loci contain multiple copies of methylated transgenes. Mol Gen Genet 244 1994 219 229 Google Scholar Crossref Search ADS PubMed WorldCat 25 McElroy D Zhang W Cao J Wu R Isolation of an efficient actin promoter for use in rice transformation. Plant Cell 2 1990 163 171 Google Scholar PubMed OpenURL Placeholder Text WorldCat 26 Mundy J Chua N-H Abscisic acid and water-stress induce the expression of a novel rice gene. EMBO J 7 1988 2279 2286 Google Scholar Crossref Search ADS PubMed WorldCat 27 Murashige T Skoog F A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15 1962 473 497 Google Scholar Crossref Search ADS WorldCat 28 Ono A Izawa T Chua N-H Shimamoto K The rab 16B promoter of rice contains two distinct abscisic acid-responsive elements. Plant Physiol 112 1996 483 491 Google Scholar Crossref Search ADS PubMed WorldCat 29 Peach C Velten J Transgene expression variability (position effect) of CAT and GUS reporter genes driven by linked divergent T-DNA promoters. Plant Mol Biol 17 1991 49 60 Google Scholar Crossref Search ADS PubMed WorldCat 30 Pilon-Smits EAH Ebskamp MJM Improved performance of transgenic fructan-accumulating tobacco under drought stress. Plant Physiol 107 1995 125 130 Google Scholar Crossref Search ADS PubMed WorldCat 31 Sambrook J Fritsch EF Maniatis T Molecular Cloning: A Laboratory Manual, Ed 2. 1989 Cold Spring Harbor Laboratory Press Cold Spring Harbor, NY 32 Shen Q Ho T-HD Functional dissection of an abscisic acid (ABA)-inducible gene reveals two independent ABA-responsive complexes each containing a G-box and a novel cis-acting element. The Plant Cell 7 1995 295 307 Google Scholar PubMed OpenURL Placeholder Text WorldCat 33 Shen Q Uknes SJ Ho T-HD Hormone response complex of a novel abscisic acid and cycloheximide inducible barley gene. J Biol Chem 268 1993 23652 23660 Google Scholar Crossref Search ADS PubMed WorldCat 34 Shen Q Zhang P Ho T-HD Modular nature of abscisic acid (ABA) response complexes: composite promoter units that are necessary and sufficient for ABA induction of gene expression in barley. The Plant Cell 8 1996 1107 1119 Google Scholar PubMed OpenURL Placeholder Text WorldCat 35 Sieburth LE Meyerowitz EM Molecular dissection of the AGAMOUS control region show that cis elements for spatial regulation are located intragenically. Plant Cell 9 1997 355 365 Google Scholar PubMed OpenURL Placeholder Text WorldCat 36 Singh NK LaRosa PC Handa AD Hasegawa PM Bressan RA Hormonal regulation of protein synthesis associated with salt tolerance in plant cell. Proc Natl Acad Sci USA 84 1987 739 743 Google Scholar Crossref Search ADS PubMed WorldCat 37 Tarczynski MC Jensen RG Bohnert HJ Stress-protection of transgenic tobacco by production of the osmolyte mannitol. Science 259 1993 508 510 Google Scholar Crossref Search ADS PubMed WorldCat 38 Wang B Wu R A vector for inserting foreign genes and selection of transformed rice plants. Rice Biotech Quart 22 1995 8 Google Scholar OpenURL Placeholder Text WorldCat 39 Xu D Duan X Wang B Hong B Ho T-HD Wu R Expression of a late embryogenesis abundant protein gene, HVA1, from barley confers tolerance to water deficit and salt stress in transgenic rice. Plant Physiol 110 1996 249 257 Google Scholar Crossref Search ADS PubMed WorldCat 40 Zeevaart JAD Creelmann RA Metabolism and physiology of abscisic acid. Annu Rev Plant Physiol Plant Mol Biol 39 1988 439 473 Google Scholar Crossref Search ADS WorldCat 41 Zhang W Wu R Efficient regeneration of transgenic plants from rice protoplasts and correctly regulated expression of the foreign gene in the plants. Theor Appl Genet 76 1988 835 840 Google Scholar Crossref Search ADS PubMed WorldCat 42 Zhao X Wu T Xie Y Wu R Genome-specific repetitive sequences in the genus Oryza. Theor Appl Genet 78 1989 201 209 Google Scholar Crossref Search ADS PubMed WorldCat Author notes 1 This work was supported by the Rockefeller Foundation (research grant no. RF93001, allocation no. 194) to R.W. J.S. was supported by a postdoctoral fellowship of the Rockefeller Foundation. * Corresponding author; e-mail [email protected]; fax 1–607–255–2428. Copyright © 1998 American Society of Plant Physiologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Miquel, Martine; Cassagne, Claude; Browse, John

doi: 10.1104/pp.117.3.923pmid: 9662534

Abstract Chloroplast glycerolipids in a number of higher-plant species, including Arabidopsis thaliana, are synthesized by two distinct pathways termed the prokaryotic and eukaryotic pathways. The molecules of galactolipids produced by the prokaryotic pathway contain substantial amounts of hexadecatrienoic acid fatty acid. Here we describe a new class of mutants, designatedgly1, with reduced levels of hexadecatrienoic acid. Lipid fatty acid profiles indicated that gly1 mutants exhibited a reduced carbon flux through the prokaryotic pathway that was compensated for by an increased carbon flux through the eukaryotic pathway. Genetic and biochemical approaches revealed that thegly1 phenotype could not be explained by a deficiency in the enzymes of the prokaryotic pathway. The flux of fatty acids into the prokaryotic pathway is sensitive to changes in glycerol-3-phosphate (G3P) availability, and the chloroplast G3P pool can be increased by exogenous application of glycerol to leaves. Exogenous glycerol treatment of gly1 plants allowed chemical complementation of the mutant phenotype. These results are consistent with a mutant lesion affecting the G3P supply within the chloroplast. The gly1 mutants may therefore help in determining the pathway for synthesis of chloroplast G3P. Higher plants possess two distinct pathways for the synthesis of chloroplast glycerolipids in leaf cells (Roughan et al., 1980; Browse and Somerville, 1991). The chloroplasts or the plastids of the cell are the sole site of de novo fatty acid synthesis (Ohlrogge et al., 1991). The final products of fatty acid synthesis and of the soluble stearoyl ACP desaturase are 16:0-ACP and 18:1-ACP (McKeon and Stumpf, 1982;Shanklin and Somerville, 1991). These either enter the prokaryotic pathway of the chloroplast inner envelope to produce chloroplastic lipids or they are hydrolyzed to free fatty acids that are exported through the plastid envelope to the cytoplasm as CoA thioesters, thus initiating the eukaryotic pathway. Because of the specificities of the plastid acyltransferases for certain acyl-ACP substrates (Frentzen, 1993), the PA made by the prokaryotic pathway has 16:0 at thesn-2 position and, in most cases, 18:1 at thesn-1 position. This PA is used for the synthesis of PG or is converted to DAG by a PAPase (Joyard and Douce, 1977). This DAG pool is the precursor for the synthesis of MGD, DGD, and SL, the major plastid membrane lipids (Joyard et al., 1993). The PA synthesized in the ER by a different set of acyltransferases than the plastid isozymes is characteristically enriched in 18-carbon fatty acids at thesn-2 position; 16:0, when present, is confined to thesn-1 position. This PA is used to produce phospholipids such as PC, PE, and PI, which are characteristic of the various extrachloroplastic membranes of the cell. In addition, a portion of PC produced by the eukaryotic pathway is returned to the chloroplast and used in the production of chloroplast lipids (Browse and Somerville, 1991). In the majority of higher plants, PG is the only product of the prokaryotic pathway, and the remaining chloroplast lipids are synthesized entirely by the eukaryotic pathway (Browse et al., 1986b). However, in a number of species, including Arabidopsis, both pathways contribute about equally to the synthesis of MGD, DGD, and SL (Browse and Somerville, 1991), and the leaf lipids of such plants characteristically contain substantial amounts of 16:3, which is found only at the sn-2 position of galactolipid molecules produced by the prokaryotic pathway. These species have been termed 16:3 plants to distinguish them from 18:3 plants, the galactolipids of which contain predominantly α-linolenate (Jamieson and Reed, 1971; Browse and Somerville, 1991). Mutants of Arabidopsis with altered fatty acid composition have been isolated (Browse and Somerville, 1994). These mutants were identified by direct analysis of leaf or seed fatty acid composition of individual mutagenized plants by GC (Browse et al., 1986a). One of them,act1, is deficient in the activity of chloroplast GPAT, the first enzyme of the prokaryotic pathway, and its leaf fatty acid composition is characterized by greatly reduced amounts of 16:3 because the act1 mutation substantially blocks the flux of carbon into the prokaryotic pathway (Kunst et al., 1988). In this paper we report the isolation and characterization of a second class of mutants with reduced levels of 16:3. These mutants also exhibit reduced carbon flux into the prokaryotic pathway. However, the mutation does not appear to affect a step in lipid synthesis but instead may limit the supply of G3P within the chloroplast. MATERIALS AND METHODS Plant Material and Growth Conditions The lines of Arabidopsis thaliana (L.) Heynh. described here were descended from the Columbia wild type. Mutants were isolated from M2 populations obtained after mutagenesis with ethyl methanesulfonate (Haughn and Somerville, 1986) by directly analyzing the fatty acid compositions of small tissue samples by GC (Browse et al., 1986a). The mutant lines were backcrossed to the wild type three times before being used in any of the experiments reported here. Plants were grown on soil in controlled-environment chambers at 22°C under continuous fluorescent illumination (150 μmol quanta m−2 s−1). Plants used to isolate chloroplasts were grown for 11 d under continuous illumination before being transferred to a growth-type chamber in a day/night cycle at 22°C for another 12 d. The lighting regime in the growth chamber was 150 μmol quanta m−2s−1 for an 8-h day. Fatty Acid and Lipid Analysis The overall fatty acid compositions of leaves and other tissues were determined by GC after derivatization with 2.5% (v/v) H2SO4 in methanol (Miquel and Browse, 1992). When small tissue samples were analyzed, the same procedure was used except that fatty acid methyl esters were extracted into 150 μL (rather than 1 mL) of hexane. Typically, 75 to 100 μL of this extract could be recovered and transferred to a microvial for injection onto the gas chromatograph. Usually, a 1-μL aliquot taken directly from the sample was sufficient for analysis. Samples of leaf tissue were killed rapidly by immersion in liquid N2 and ground under liquid N2 before being extracted and analyzed as described previously (Miquel and Browse, 1992). Lipase Positional Analysis The fatty acid compositions at the sn-1 andsn-2 positions of individual lipids were determined by lipase digestion. After TLC lipids were extracted from the silica gel by the method of Bligh and Dyer (1959). The protocol for digestion withRhizopus sp. lipase, including purification of thelyso-derivatives and fatty acids, was that described bySiebertz and Heinz (1977), except that 50 mmH3BO4 was added to the buffer used for lipase digestion to minimize intramolecular acyl transfer on the lyso-lipids produced. Fatty methyl esters were formed from untreated lipids, lyso-lipids, and fatty acids as described above, and the fatty acid composition of each compound was determined by GC. Chloroplast Isolation Leaves of the wild type and mutant were harvested at the end of the night period. Ten grams fresh weight of leaves was homogenized using a Polytron (Kinematica, Lucern, Switzerland; two to three 5-s bursts) in 125 mL of semifrozen buffer containing 0.35 msorbitol, 25 mm Hepes-KOH, pH 7.8, and 2 mg mL−1 defatted BSA. The homogenates were filtered through two layers of prewetted Miracloth (Calbiochem) and centrifuged at 2,600g for 1 min in an HB-4 swinging-bucket rotor (Beckman). Pellets were gently resuspended with a small volume of cold wash buffer that contained 0.33 m sorbitol, 10 mm Hepes-KOH, pH 7.8, and 2.5 mm EDTA. This plastid suspension was layered on top of a preformed Percoll (Pharmacia) gradient. The gradient was self-generated by mixing 16 mL of 100% Percoll in 0.33 m sorbitol and 16 mL of wash buffer, and centrifuging for 20 min at 27,000g (SS34 rotor, Beckman). The chloroplasts were purified by centrifuging the gradient at 14,000g (HB-4 rotor) for 1 min. The intact chloroplasts (the green band deeper in the gradient) were collected, diluted at least three times with wash buffer, and then centrifuged at 2,600g for 1 min as described previously. Finally, the intact chloroplasts were suspended in wash buffer and used immediately. All operations were carried out at 4°C. Enzyme Assay The PAPase was assayed within isolated envelope membranes, according to assay 2 of Malherbe et al. (1992). Radioactive PA was synthesized in situ as purified envelope membranes were loaded with PA synthesized by acylation ofsn-[14C]G3P (specific radioactivity, 5.7 × 1012 Bq/mol; NEN Life Sciences Products). PAPase activity was then measured by following PA conversion into DAG. Typically, the reaction mixture contained 100 to 150 μg of fatty acids from envelope membranes and 2 to 3.5 mg from stromal proteins. Exogenous Glycerol Treatment Wild-type and mutant plants grown under continuous illumination as described above were 14 d old at the start of the treatment. Glycerol solutions in double-distilled water (10 or 50 mm) were sprayed on plants at different times during a period of 80 h using a perfume atomizer. Control plants were sprayed with water. The volumes used were 500 μL for each treatment of 25 plants during the first 12 h, 650 μL for treatments from 24 to 36 h, and 800 μL after 48 h until the end of the experiment. The surface of the leaves was dry within 30 min after spraying. Thirty minutes after the 36- and 80-h treatments, 6 to 15 plants from each treatment and from each genotype were harvested and weighed individually before being frozen in liquid N2. Total lipids were extracted from each sample and analyzed as described above. Other Assays Chloroplast integrity was determined as described by Heber and Santarius (1970). Protein concentration was determined by the method ofBradford (1976) using BSA as a standard. RESULTS Genetic Analysis The mutant lines JB19 and EMS 5 no. 1 were isolated without selection by screening M2 progeny of ethyl methanesulfonate-mutagenized seeds for altered leaf fatty acid composition. The mutant lines were identified as being deficient in 16:3. Genetic complementation tests indicated that the two lines have a lesion at the same locus (data not shown). Therefore, we characterized only one of the lines in detail. The representative mutant line JB19 was normal in appearance and growth characteristics but could be readily distinguished from the wild type by the reduced amount of 16:3 in its leaf lipids (Table I). The mode of inheritance of this altered fatty acid composition was determined by reciprocal crosses between line JB19 plants and wild-type Arabidopsis. Leaves of the F1 progeny showed a slightly decreased amount of 16:3 compared with the wild type (Table I), suggesting that the wild-type allele is incompletely dominant. The frequency of individuals with the mutant phenotype in the F2 population resulting from self-fertilization of F1 plants was also measured by GC of leaf samples. Of 96 F2 plants analyzed, 21 had fatty acid compositions similar to those of plants of the original JB19 line, whereas the remaining individuals had leaf fatty acid compositions similar to those of plants of the wild type or the F1 hybrid. This pattern of segregation is a good fit (χ2 = 0.374, P > 0.6) to the 3:1 hypothesis and indicates that the altered fatty acid composition is caused by a single nuclear mutation at a locus we have designatedgly1. Consequently, the two lines JB19 and EMS 5 no. 1 were designated gly1-1 and gly1-2, respectively. Table I. Fatty acid composition of total leaf lipids from wild-type (WT) and gly1-1 mutant Arabidopsis Fatty Acid . WT . (WT ×gly1-1) F1 . gly1-1 . mol % 16:0 13.9  ± 0.1 13.6  ± 0.1 13.5  ± 0.1 16:1 cis 0.7  ± 0.1 0.6  ± 0.1 0.6  ± 0.1 16:1trans 2.8  ± 0.1 2.7  ± 0.1 2.2  ± 0.1 16:2 0.9  ± 0.1 0.8  ± 0.1 0.5  ± 0.1 16:3 15.5  ± 0.1 14.3  ± 0.1 5.4  ± 0.1 18:0 0.8  ± 0.1 0.8  ± 0.1 0.8  ± 0.1 18:1 3.0  ± 0.1 3.4  ± 0.1 8.0  ± 0.2 18:2 13.8  ± 0.1 14.5  ± 0.1 18.2  ± 0.1 18:3 48.5  ± 0.1 49.2  ± 0.1 50.8  ± 0.2 Fatty Acid . WT . (WT ×gly1-1) F1 . gly1-1 . mol % 16:0 13.9  ± 0.1 13.6  ± 0.1 13.5  ± 0.1 16:1 cis 0.7  ± 0.1 0.6  ± 0.1 0.6  ± 0.1 16:1trans 2.8  ± 0.1 2.7  ± 0.1 2.2  ± 0.1 16:2 0.9  ± 0.1 0.8  ± 0.1 0.5  ± 0.1 16:3 15.5  ± 0.1 14.3  ± 0.1 5.4  ± 0.1 18:0 0.8  ± 0.1 0.8  ± 0.1 0.8  ± 0.1 18:1 3.0  ± 0.1 3.4  ± 0.1 8.0  ± 0.2 18:2 13.8  ± 0.1 14.5  ± 0.1 18.2  ± 0.1 18:3 48.5  ± 0.1 49.2  ± 0.1 50.8  ± 0.2 Homozygous (gly1-1) and heterozygous (F1 of the cross WT × gly1-1) mutant plants were grown together with the wild type. Results are means ± se,n = 24. Open in new tab Table I. Fatty acid composition of total leaf lipids from wild-type (WT) and gly1-1 mutant Arabidopsis Fatty Acid . WT . (WT ×gly1-1) F1 . gly1-1 . mol % 16:0 13.9  ± 0.1 13.6  ± 0.1 13.5  ± 0.1 16:1 cis 0.7  ± 0.1 0.6  ± 0.1 0.6  ± 0.1 16:1trans 2.8  ± 0.1 2.7  ± 0.1 2.2  ± 0.1 16:2 0.9  ± 0.1 0.8  ± 0.1 0.5  ± 0.1 16:3 15.5  ± 0.1 14.3  ± 0.1 5.4  ± 0.1 18:0 0.8  ± 0.1 0.8  ± 0.1 0.8  ± 0.1 18:1 3.0  ± 0.1 3.4  ± 0.1 8.0  ± 0.2 18:2 13.8  ± 0.1 14.5  ± 0.1 18.2  ± 0.1 18:3 48.5  ± 0.1 49.2  ± 0.1 50.8  ± 0.2 Fatty Acid . WT . (WT ×gly1-1) F1 . gly1-1 . mol % 16:0 13.9  ± 0.1 13.6  ± 0.1 13.5  ± 0.1 16:1 cis 0.7  ± 0.1 0.6  ± 0.1 0.6  ± 0.1 16:1trans 2.8  ± 0.1 2.7  ± 0.1 2.2  ± 0.1 16:2 0.9  ± 0.1 0.8  ± 0.1 0.5  ± 0.1 16:3 15.5  ± 0.1 14.3  ± 0.1 5.4  ± 0.1 18:0 0.8  ± 0.1 0.8  ± 0.1 0.8  ± 0.1 18:1 3.0  ± 0.1 3.4  ± 0.1 8.0  ± 0.2 18:2 13.8  ± 0.1 14.5  ± 0.1 18.2  ± 0.1 18:3 48.5  ± 0.1 49.2  ± 0.1 50.8  ± 0.2 Homozygous (gly1-1) and heterozygous (F1 of the cross WT × gly1-1) mutant plants were grown together with the wild type. Results are means ± se,n = 24. Open in new tab An Arabidopsis mutant, act1, with greatly reduced 16:3 amounts in its leaf lipids (1–2% of total fatty acids) has previously been characterized as being deficient in chloroplast GPAT (Kunst et al., 1988). Reciprocal crosses between gly1-1 andact1 produced F1 progeny, the fatty acid composition of which was similar to that of the wild type. This genetic complementation indicates that gly1-1 andact1 are not allelic and that gly1-1 is not deficient in chloroplast GPAT activity. Biochemical Characterization In mutant leaves the nearly 3-fold reduction in 16:3 was not accompanied by any increase in the precursors 16:0, 16:1, or 16:2, but was compensated for by increased 18:1, 18:2, and 18:3 amounts (TableI). This lack of precursor accumulation indicates that the mutation ingly1-1 is not attributable to a reduction in desaturation of 16-carbon fatty acids. In plant roots, which contain a predominance of extrachloroplastic membranes, and in seeds, which contain large amounts of triglycerides, the prokaryotic pathway does not contribute significantly to lipid synthesis. Comparison of the overall fatty acid composition of the roots and mature seeds from the mutant and the wild type showed no detectable difference. Although we have shown that the two mutations act1 and gly1-1 are not allelic (see above), the comparison between their respective overall leaf fatty acid composition suggests that the mutation in gly1-1 likely affects the flux of fatty acids into the prokaryotic pathway. Finally, because 16:3 is synthesized exclusively in chloroplasts by sequential desaturation of 16:0 acyl groups of galactolipids (Roughan et al., 1979; Roughan and Slack, 1982), the remaining amount of 16:3 ingly1-1 leaf lipids suggests that the mutation affects a biochemical step that is partially redundant, or that the mutation incompletely blocks a step in the prokaryotic pathway. The biochemical consequences of the gly1-1 mutation are shown more clearly by an analysis of individual lipids extracted from leaf tissue of wild-type and mutant plants (TableII). The data indicated that the changes in the proportions of the various polar lipids in the mutant affected only the chloroplast lipids MGD, DGD, SL, and PG. In gly1-1the mole fraction of MGD decreased by 10% compared with that in the wild type, whereas DGD increased by 19%. However, when chloroplast lipids were considered as a whole, there was no difference betweengly1-1 and the wild type. The differences in the fatty acid compositions of individual lipids were more informative regarding the nature of the mutation. In MGD, DGD, and SL the reduction in 16-carbon fatty acid amounts was compensated for by higher amounts of 18-carbon fatty acids, as indicated by the ratio C-18/C-16 fatty acids (TableII). This ratio increased by 1.5-, 1.8-, and 2.5-fold for SL, DGD, and MGD, respectively, indicating a reduced synthesis of prokaryotic-type molecules for these lipids. The data on the lipid composition and on the fatty acid compositions of individual lipids showed that the reduced synthesis of galactolipids and SL by the prokaryotic pathway in the mutant was entirely compensated for by increased production of these lipids via the eukaryotic pathway. Table II. Fatty acid composition of leaf lipids from wild-type (WT) and gly1-1 mutant Arabidopsis Glycerolipid . Total Polar Lipids . Fatty Acid Composition . C-18/C-16 . 16:0 . 16:11-a . 16:2 . 16:3 . 18:0 . 18:1 . 18:2 . 18:3 . % mol % MGD  WT 40.0 1.2 1.2 2.0 33.0 0.2 1.1 2.7 58.7 1.7  gly1-1 36.1 1.5 0.8 1.3 15.1 0.3 1.2 3.4 76.3 4.4 DGD  WT 15.6 11.6 –1-b 0.7 3.0 1.4 1.7 4.3 77.4 5.6  gly1-1 18.6 7.4 – 0.5 0.9 0.9 1.4 3.2 85.6 10.3 PG  WT 7.5 30.0 21.4 – – 2.4 7.1 8.2 30.9 1.0  gly1-1 8.2 32.0 21.7 – – 2.2 5.3 9.8 29.0 0.9 SL  WT 2.0 45.5 – – – 2.5 3.8 5.4 42.8 1.2  gly1-1 2.3 33.0 – – – 5.0 8.2 6.7 47.1 2.0 PC  WT 19.7 19.7 – – – 2.4 7.3 37.9 32.7 4.1  gly1-1 20.6 17.7 – – – 1.8 15.3 41.6 23.6 4.6 PE  WT 12.2 28.1 – – – 2.6 4.5 37.9 27.0 2.6  gly1-1 11.0 27.2 – – – 2.5 8.7 43.3 18.3 2.7 PI  WT 3.0 42.3 – – – 5.7 5.2 24.0 22.8 1.4  gly1-1 3.2 43.1 – – – 5.5 7.4 26.3 17.7 1.3 Glycerolipid . Total Polar Lipids . Fatty Acid Composition . C-18/C-16 . 16:0 . 16:11-a . 16:2 . 16:3 . 18:0 . 18:1 . 18:2 . 18:3 . % mol % MGD  WT 40.0 1.2 1.2 2.0 33.0 0.2 1.1 2.7 58.7 1.7  gly1-1 36.1 1.5 0.8 1.3 15.1 0.3 1.2 3.4 76.3 4.4 DGD  WT 15.6 11.6 –1-b 0.7 3.0 1.4 1.7 4.3 77.4 5.6  gly1-1 18.6 7.4 – 0.5 0.9 0.9 1.4 3.2 85.6 10.3 PG  WT 7.5 30.0 21.4 – – 2.4 7.1 8.2 30.9 1.0  gly1-1 8.2 32.0 21.7 – – 2.2 5.3 9.8 29.0 0.9 SL  WT 2.0 45.5 – – – 2.5 3.8 5.4 42.8 1.2  gly1-1 2.3 33.0 – – – 5.0 8.2 6.7 47.1 2.0 PC  WT 19.7 19.7 – – – 2.4 7.3 37.9 32.7 4.1  gly1-1 20.6 17.7 – – – 1.8 15.3 41.6 23.6 4.6 PE  WT 12.2 28.1 – – – 2.6 4.5 37.9 27.0 2.6  gly1-1 11.0 27.2 – – – 2.5 8.7 43.3 18.3 2.7 PI  WT 3.0 42.3 – – – 5.7 5.2 24.0 22.8 1.4  gly1-1 3.2 43.1 – – – 5.5 7.4 26.3 17.7 1.3 Values represent the averages of three samples. F1-a Sum of 16:1 cis and 16:1trans fatty acids. F1-b Amounts were <0.1 mol%. Open in new tab Table II. Fatty acid composition of leaf lipids from wild-type (WT) and gly1-1 mutant Arabidopsis Glycerolipid . Total Polar Lipids . Fatty Acid Composition . C-18/C-16 . 16:0 . 16:11-a . 16:2 . 16:3 . 18:0 . 18:1 . 18:2 . 18:3 . % mol % MGD  WT 40.0 1.2 1.2 2.0 33.0 0.2 1.1 2.7 58.7 1.7  gly1-1 36.1 1.5 0.8 1.3 15.1 0.3 1.2 3.4 76.3 4.4 DGD  WT 15.6 11.6 –1-b 0.7 3.0 1.4 1.7 4.3 77.4 5.6  gly1-1 18.6 7.4 – 0.5 0.9 0.9 1.4 3.2 85.6 10.3 PG  WT 7.5 30.0 21.4 – – 2.4 7.1 8.2 30.9 1.0  gly1-1 8.2 32.0 21.7 – – 2.2 5.3 9.8 29.0 0.9 SL  WT 2.0 45.5 – – – 2.5 3.8 5.4 42.8 1.2  gly1-1 2.3 33.0 – – – 5.0 8.2 6.7 47.1 2.0 PC  WT 19.7 19.7 – – – 2.4 7.3 37.9 32.7 4.1  gly1-1 20.6 17.7 – – – 1.8 15.3 41.6 23.6 4.6 PE  WT 12.2 28.1 – – – 2.6 4.5 37.9 27.0 2.6  gly1-1 11.0 27.2 – – – 2.5 8.7 43.3 18.3 2.7 PI  WT 3.0 42.3 – – – 5.7 5.2 24.0 22.8 1.4  gly1-1 3.2 43.1 – – – 5.5 7.4 26.3 17.7 1.3 Glycerolipid . Total Polar Lipids . Fatty Acid Composition . C-18/C-16 . 16:0 . 16:11-a . 16:2 . 16:3 . 18:0 . 18:1 . 18:2 . 18:3 . % mol % MGD  WT 40.0 1.2 1.2 2.0 33.0 0.2 1.1 2.7 58.7 1.7  gly1-1 36.1 1.5 0.8 1.3 15.1 0.3 1.2 3.4 76.3 4.4 DGD  WT 15.6 11.6 –1-b 0.7 3.0 1.4 1.7 4.3 77.4 5.6  gly1-1 18.6 7.4 – 0.5 0.9 0.9 1.4 3.2 85.6 10.3 PG  WT 7.5 30.0 21.4 – – 2.4 7.1 8.2 30.9 1.0  gly1-1 8.2 32.0 21.7 – – 2.2 5.3 9.8 29.0 0.9 SL  WT 2.0 45.5 – – – 2.5 3.8 5.4 42.8 1.2  gly1-1 2.3 33.0 – – – 5.0 8.2 6.7 47.1 2.0 PC  WT 19.7 19.7 – – – 2.4 7.3 37.9 32.7 4.1  gly1-1 20.6 17.7 – – – 1.8 15.3 41.6 23.6 4.6 PE  WT 12.2 28.1 – – – 2.6 4.5 37.9 27.0 2.6  gly1-1 11.0 27.2 – – – 2.5 8.7 43.3 18.3 2.7 PI  WT 3.0 42.3 – – – 5.7 5.2 24.0 22.8 1.4  gly1-1 3.2 43.1 – – – 5.5 7.4 26.3 17.7 1.3 Values represent the averages of three samples. F1-a Sum of 16:1 cis and 16:1trans fatty acids. F1-b Amounts were <0.1 mol%. Open in new tab In wild-type Arabidopsis the prokaryotic pathway is responsible for producing approximately 70% of the total leaf MGD, 12% of the DGD, 63% of the SL, and 85% of the PG, as indicated by the amounts of 16-carbon fatty acids at the sn-2 position of the glycerol (Browse et al., 1986b). To quantitate the effect of the mutation on the flux of fatty acids through the prokaryotic pathway, purified lipids were digested with Rhizopussp. lipase and the fatty acid compositions of the lyso-derivatives and released fatty acids were determined (TableIII). This analysis indicated that in the mutant, only 34% of the MGD, 24% of the DGD, and 39% of the SL were synthesized through the prokaryotic pathway. By contrast, the synthesis of PG was not affected. The other polar lipids contained >90% 18-carbon fatty acids at the sn-2 position of the glycerol, indicating that they were produced by the eukaryotic pathway. Table III. Mass and fatty acid compositions of wild-type (WT) and gly1-1 mutant Arabidopsis leaf lipids and their lyso-derivatives Lipid lyso-Derivative . Mass of Fatty Acids . Fatty Acid Composition . 16-Carbon . 18-Carbon . WT . gly1-1 . WT . gly1-1 . WT . gly1-1 . mol/1000 mol mol % MGD 390 348 37 20 63 80 sn-2 70 34 30 66 DGD 152 184 15 8 95 92 sn-2 12 24 88 96 PG 81 83 52 51 48 49 sn-2 83 86 17 14 SL 28 23 43 30 57 70 sn-2 63 39 37 61 PC 218 219 22 22 78 78 sn-2 1 2 99 98 PE 100 113 29 28 71 72 sn-2 1 1 99 99 Lipid lyso-Derivative . Mass of Fatty Acids . Fatty Acid Composition . 16-Carbon . 18-Carbon . WT . gly1-1 . WT . gly1-1 . WT . gly1-1 . mol/1000 mol mol % MGD 390 348 37 20 63 80 sn-2 70 34 30 66 DGD 152 184 15 8 95 92 sn-2 12 24 88 96 PG 81 83 52 51 48 49 sn-2 83 86 17 14 SL 28 23 43 30 57 70 sn-2 63 39 37 61 PC 218 219 22 22 78 78 sn-2 1 2 99 98 PE 100 113 29 28 71 72 sn-2 1 1 99 99 Polar lipids were separated by two-dimensional TLC. The mass and fatty acid compositions of the lipids and their lyso-derivatives, resulting from digestion with Rhizopussp. lipase, were determined by GC analysis as outlined in Methods. Open in new tab Table III. Mass and fatty acid compositions of wild-type (WT) and gly1-1 mutant Arabidopsis leaf lipids and their lyso-derivatives Lipid lyso-Derivative . Mass of Fatty Acids . Fatty Acid Composition . 16-Carbon . 18-Carbon . WT . gly1-1 . WT . gly1-1 . WT . gly1-1 . mol/1000 mol mol % MGD 390 348 37 20 63 80 sn-2 70 34 30 66 DGD 152 184 15 8 95 92 sn-2 12 24 88 96 PG 81 83 52 51 48 49 sn-2 83 86 17 14 SL 28 23 43 30 57 70 sn-2 63 39 37 61 PC 218 219 22 22 78 78 sn-2 1 2 99 98 PE 100 113 29 28 71 72 sn-2 1 1 99 99 Lipid lyso-Derivative . Mass of Fatty Acids . Fatty Acid Composition . 16-Carbon . 18-Carbon . WT . gly1-1 . WT . gly1-1 . WT . gly1-1 . mol/1000 mol mol % MGD 390 348 37 20 63 80 sn-2 70 34 30 66 DGD 152 184 15 8 95 92 sn-2 12 24 88 96 PG 81 83 52 51 48 49 sn-2 83 86 17 14 SL 28 23 43 30 57 70 sn-2 63 39 37 61 PC 218 219 22 22 78 78 sn-2 1 2 99 98 PE 100 113 29 28 71 72 sn-2 1 1 99 99 Polar lipids were separated by two-dimensional TLC. The mass and fatty acid compositions of the lipids and their lyso-derivatives, resulting from digestion with Rhizopussp. lipase, were determined by GC analysis as outlined in Methods. Open in new tab A previous detailed analysis of wild-type Arabidopsis showed that for every 1000 fatty acid molecules made in the chloroplast, 615 enter the eukaryotic pathway (117 as 16-carbon fatty acids and 498 as 18-carbon fatty acids). A similar analysis of gly1-1 indicated a 29% increase in flux through the eukaryotic pathway, which was made up of 85% 18-carbon fatty acid chains (Fig.1). However, the C-18/C-16 ratio in PC, PE, and PI is the same as in the corresponding lipids of the wild type (Table II). In contrast, the C-18/C-16 ratio in the galactolipids and SL of the mutant in each case is more than the ratio calculated for these lipids synthesized by the eukaryotic pathway in the wild type (table IV of Browse et al., 1986b). Thus, the additional 18-carbon fatty acids entering the eukaryotic pathway in the mutant are found specifically in the additional quantities of chloroplast lipids (galactolipids and SL) that are produced by the eukaryotic pathway in response to the loss of the prokaryotic pathway. This situation is also characteristic of the act1 mutant, in which the loss of the prokaryotic pathway is almost complete except for the synthesis of PG. In act1 there is a 50% increase of the flux of fatty acids through the eukaryotic pathway that is made up of 86% 18-carbon fatty acids (Kunst et al., 1988). Fig. 1. Open in new tabDownload slide Flow diagram of fatty acid fluxes (mol/1000 mol) during lipid synthesis by wild-type (WT) and gly1-1mutant Arabidopsis leaves. NFA, Nonesterified fatty acids. Fig. 1. Open in new tabDownload slide Flow diagram of fatty acid fluxes (mol/1000 mol) during lipid synthesis by wild-type (WT) and gly1-1mutant Arabidopsis leaves. NFA, Nonesterified fatty acids. Another analogy with the act1 mutant is that the mutationgly1-1 causes a significant increase in the amount of 18:1 and a decrease in the amount of 18:3 in all of the extrachloroplastic lipids. In these lipids there is only a slight effect on the amount of 18:2 (Table II). The increase in 18:1 amounts in these lipids in the mutant relative to the wild type reflects a 5 to 7% reduction in the extent of 18:1 desaturation, suggesting that the ER 18:1 desaturase may be unable to completely metabolize the increased flux of lipid through the eukaryotic pathway in the mutant. PAPase Activity Because the synthesis of PG by the prokaryotic pathway was not affected in the mutant, we first considered the possibility thatgly1-1 plants were deficient in the enzyme PAPase. The chloroplast PAPase provides the DAG moieties (Joyard and Douce, 1977) used for the synthesis of the prokaryotic molecular species of MGD, DGD, and SL that characterize 16:3 plants (Browse and Somerville, 1991). In the case of 18:3 plants, the chloroplast PAPase is not functional and the chloroplast lipids are synthesized from eukaryotic DAG molecular species (Browse and Somerville, 1991). Therefore, thegly1-1 phenotype could be explained by a mutation that reduced (but did not eliminate) PAPase activity. We assayed the activity of the chloroplast PAPase (Malherbe et al., 1992) and found that the activity is increased in the mutant compared with wild-type controls (Fig. 2). These results indicate that PAPase is not decreased in the mutant. Fig. 2. Open in new tabDownload slide PAPase activity from wild-type andgly1-1 mutant Arabidopsis. PAPase was assayed as described in Methods and data represent a typical experiment. ▪, Wild type; •, gly1-1. Fig. 2. Open in new tabDownload slide PAPase activity from wild-type andgly1-1 mutant Arabidopsis. PAPase was assayed as described in Methods and data represent a typical experiment. ▪, Wild type; •, gly1-1. G3P Supply for Lipid Biosynthesis in Chloroplasts Another hypothesis that could account for the phenotype ofgly1-1 plants is a defect in the availability of chloroplast G3P, which provides the glycerol backbone of the lipids synthesized by the prokaryotic pathway (Frentzen, 1993). Experiments with isolated spinach chloroplasts and intact leaf tissues indicate that flux of fatty acids into the prokaryotic pathway is sensitive to changes in G3P availability. Roughan et al. (1980) showed that chloroplasts incubated in a basal medium contained 23% of the incorporated label in glycerolipids (representing the prokaryotic pathway), whereas more than 70% was incorporated into unesterified fatty acids and acyl-CoAs, which are precursors for the eukaryotic pathway. Addition of 0.48 mm G3P to the basal medium increased glycerolipid products to 49% of the total radioactivity and decreased unesterified fatty acids plus acyl-CoAs to 40%. When glycerol was supplied to leaves of spinach plants, it increased the size of the G3P pool and increased the flux of [14C]acetate label into prokaryotic lipids (Gardiner et al., 1982). These results suggest that if thegly1-1 mutation is a lesion reducing the availability of G3P in the chloroplast, then exogenous application of glycerol should result in chemical complementation of the mutant phenotype. Because 16:3 is produced only by the prokaryotic pathway, it was possible to use this fatty acid to monitor the effects of glycerol treatments. A typical experiment in which glycerol was applied to wild-type and gly1-1 plants is described in Figure3. The control plants in these experiments received an application of water instead of glycerol solutions. In wild-type plants glycerol caused a small but consistent increase in 16:3 in the total leaf lipids. By contrast, after 80 h of treatment with 50 mm glycerol, gly1-1 plants exhibited a 2-fold increase in the proportion of 16:3 in total lipids to levels that were similar to those of untreated wild-type plants. When the individual lipids were purified and their fatty acid composition analyzed, we determined that the level of 16:3 in MGD ofgly1-1 plants after 80 h of 50 mm glycerol application had doubled to 29% compared with that of the controlgly1-1 plants. By contrast, in wild-type plants the increase was only to 3%. Therefore, raising the G3P amounts ingly1-1 plants led to a notable alleviation of the altered fatty acid composition. This suggests that the concentration of G3P ingly1-1 chloroplasts is not sufficient to meet the requirement for chloroplast lipid synthesis through the prokaryotic pathway. Application of glycerol is known to inhibit photosynthesis (Leegood et al., 1988), but both the wild-type and mutant plants remained healthy throughout the duration of our experiments. Fig. 3. Open in new tabDownload slide Effect of exogenous glycerol treatment of wild-type and gly1-1 mutant Arabidopsis on their leaf fatty acid composition. Wild-type and mutant plants were supplied with exogenous glycerol by repeatedly spraying rosette leaves with water containing 0, 10, or 50 mm glycerol at the times indicated. Thirty minutes after the 36- and 80-h treatments, 6 to 15 plants for each glycerol treatment and from each genotype were harvested and total leaf lipids were extracted from each sample. The bar graphs show 16:3 as a percentage of total fatty acids. Striped bars, Wild type; shaded bars, gly1-1; G, glycerol treatment during the 1st 12 h. Repeat treatments on subsequent days are indicated by tick marks. H, Harvest. Fig. 3. Open in new tabDownload slide Effect of exogenous glycerol treatment of wild-type and gly1-1 mutant Arabidopsis on their leaf fatty acid composition. Wild-type and mutant plants were supplied with exogenous glycerol by repeatedly spraying rosette leaves with water containing 0, 10, or 50 mm glycerol at the times indicated. Thirty minutes after the 36- and 80-h treatments, 6 to 15 plants for each glycerol treatment and from each genotype were harvested and total leaf lipids were extracted from each sample. The bar graphs show 16:3 as a percentage of total fatty acids. Striped bars, Wild type; shaded bars, gly1-1; G, glycerol treatment during the 1st 12 h. Repeat treatments on subsequent days are indicated by tick marks. H, Harvest. DISCUSSION Studies of lipid metabolism pathways using a genetic approach have revealed the existence of regulatory mechanisms that coordinate the activity of the two pathways for glycerolipid biosynthesis in higher plants. For example, the deficiency in activity of chloroplast GPAT inact1 mutants is compensated for by increased synthesis of chloroplast glycerolipids via the eukaryotic pathway (Kunst et al., 1988). This and studies of other Arabidopsis mutants (Browse et al., 1989; Kunst et al., 1989; Miquel and Browse, 1992) indicate that lipid metabolism is regulated to ameliorate the consequences of the lesion in each mutant by altering the flux through the two pathways of glycerolipid biosynthesis. Mutant gly1-1 plants are no exception, and the decreased synthesis of prokaryotic species of chloroplast lipids was entirely compensated for by increased production of these lipids via the eukaryotic pathway. This reduced synthesis through the prokaryotic pathway did not result in an increase of 16-carbon chains in the eukaryotic pathway. Instead, the 16:0-ACP was apparently elongated and desaturated to 18:1-ACP before export from the chloroplast. The overall C-18/C-16 ratio was then increased from 1.96 in the wild type to 3.5 in the mutant. The C-18/C-16 ratios of extrachloroplastic lipids were unchanged, suggesting that the amount of 16:0 export was not regulated by the availability of 16:0. Rather, the increase in the overall ratio indicated that elongation is regulated by availability of the substrate (16:0) and that this is determined by competition between alternative pathways of 16-carbon fatty acid metabolism. The mutation in gly1-1 is not an allele ofact1, the structural gene for GPAT. Direct assays of PAPase activity in wild-type and mutant plants allowed us to indirectly test for a lesion in a third enzyme of the prokaryotic pathway, LPAAT. Assays of the PAPase activity necessitated a prior loading of chloroplast envelopes with radiolabeled PA and, to this effect, purified chloroplast envelopes were incubated with stromal proteins and the appropriate cofactors (Malherbe et al., 1992). The stroma is the source of the GPAT, which forms lyso-PA, and the envelope contains the LPAAT, which forms PA (Joyard and Douce, 1977). It has been shown that the GPAT possesses a specificity for G3P and exclusively catalyzes the acylation of the sn-1 position of the acyl acceptor (Frentzen, 1986). From this we inferred that the presence of radiolabeled PA in the envelopes resulted from the sole action of the LPAAT on lyso-PA. Thus, the gly1-1mutation is not a deficiency in LPAAT activity. The results of the exogenous glycerol treatment and the fact that thegly1-1 phenotype could not be explained by a deficiency in the enzymes of the prokaryotic pathway pointed to a possible defect in the G3P supply for lipid biosynthesis in chloroplasts. The experiments in which exogenous glycerol provides for chemical complementation of the gly1-1 phenotype are consistent with a mutant lesion affecting the supply of G3P within the chloroplast. Because G3P is present in (at least) the chloroplast and cytoplasm of leaf cells it is not possible to measure the chloroplast G3P pool in vivo. In plants G3P can be synthesized via essentially three different reaction sequences (Frentzen, 1993). It can be formed from DHAP by the action of an NAD+-G3P oxidoreductase (EC 1.1.1.8 or DHAP reductase) in both the chloroplast and cytoplasm of leaves from higher plants (Santora et al., 1979; Gee et al., 1988a, 1988b, 1988c, 1989;Kirsch et al., 1992). G3P can also be formed by the pathway DHAP → glyceraldehyde 3-phosphate → glyceraldehyde → glycerol → G3P in the cytoplasm (Ghosh and Sastry, 1988). The last enzyme of the pathway, glycerol kinase, has been detected in all plant organs (Hippman and Heinz, 1976; Sadava and Moore, 1987) and especially in germinating seeds (Huang and Beevers, 1975; Hippman and Heinz, 1976;Finlayson and Dennis, 1980) and appears to be the rate-limiting enzyme of this pathway. Except in developing groundnut seeds (Ghosh and Sastry, 1988), both G3P synthesis pathways are found, therefore raising the question of the respective roles and importance of the different pathways and their regulation. Given the uncertainties surrounding the source of G3P in the chloroplast it is likely that further studies on the gly1-1 mutant will help to resolve the questions in this area of biochemistry. ACKNOWLEDGMENTS We thank Dr. Jim Tokuhisa for the gift of EMS 5 no. 1. Abbreviations: ACP acyl carrier protein DAG diacylglycerol DGD digalactosyldiacylglycerol DHAP dihydroxyacetone phosphate G3P glycerol-3-phosphate GPAT acyl-ACP:sn-G3P acyltransferase LPAAT acyl-ACP:sn-1-acylglycerol-3-phosphate acyltransferase MGD monogalactosyldiacylglycerol PA phosphatidic acid PAPase phosphatidate phosphatase PC phosphatidylcholine, PE, phosphatidylethanolamine PG phosphatidylglycerol PI phosphatidylinositol SL sulfoquinovosyldiacylglycerol (sulfolipid) X:Y a fatty acyl group containing X carbon atoms and Y double bonds (cis unless specified) 16:0 palmitate 16:1 palmitoleate 16:2 hexadecadienoic acid 16:3 hexadecatrienoic acid 18:0 stearate 18:1 oleate 18:2 linoleate 18:3 linolenate LITERATURE CITED 1 Bligh EG Dyer WJ A rapid method of total extraction and purification. Can J Biochem Physiol 37 1959 911 917 Google Scholar Crossref Search ADS PubMed WorldCat 2 Bradford MM A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72 1976 248 254 Google Scholar Crossref Search ADS PubMed WorldCat 3 Browse J Kunst L Anderson S Hugly S Somerville CR A mutant of Arabidopsis deficient in the chloroplast 16:1/18:1 desaturase. Plant Physiol 90 1989 522 529 Google Scholar Crossref Search ADS PubMed WorldCat 4 Browse J McCourt P Somerville CR Fatty acid composition of leaf lipids determined after combined digestion and fatty acid methyl ester formation from fresh tissue. Anal Biochem 152 1986a 141 145 Google Scholar Crossref Search ADS WorldCat 5 Browse J Somerville C Glycerolipids synthesis: biochemistry and regulation. Annu Rev Plant Physiol Plant Mol Biol 42 1991 467 506 Google Scholar Crossref Search ADS WorldCat 6 Browse J Somerville CR Lipids. Meyerowitz EM Somerville CR Arabidopsis. 1994 881 912 Cold Spring Harbor Laboratory Press Cold Spring Harbor, NY 7 Browse J Warwick N Somerville CR Slack CR Fluxes through the prokaryotic and the eukaryotic pathways of lipid synthesis in the “16:3” plant Arabidopsis thaliana. Biochem J 235 1986b 25 31 Google Scholar Crossref Search ADS WorldCat 8 Finlayson SA Dennis DT NAD+-specific glycerol 3-phosphate dehydrogenase from developing castor bean endosperm. Arch Biochem Biophys 199 1980 179 185 Google Scholar Crossref Search ADS PubMed WorldCat 9 Frentzen M Biosynthesis and desaturation of the different diacylglycerol moieties in higher plants. J Plant Physiol 124 1986 193 209 Google Scholar Crossref Search ADS WorldCat 10 Frentzen M Acyltransferases and triacylglycerols. Moore TS Lipid Metabolism in Plants. 1993 195 231 CRC Press Boca Raton, FL 11 Gardiner SE Roughan PG Slack CR Manipulating the incorporation of [1-14C]acetate into different leaf glycerolipids in several plant species. Plant Physiol 70 1982 1316 1320 Google Scholar Crossref Search ADS PubMed WorldCat 12 Gee R Byerrum RU Gerber D Tolbert NE Changes in the activity of the chloroplastic and cytosolic forms of dihydroxyacetone phosphate reductase during maturation of leaves. Plant Physiol 89 1989 305 308 Google Scholar Crossref Search ADS PubMed WorldCat 13 Gee RW Byerrum RU Gerber DW Tolbert NE Dihydroxyacetone phosphate reductase in plants. Plant Physiol 86 1988a 98 103 Google Scholar Crossref Search ADS WorldCat 14 Gee RW Byerrum RU Gerber DW Tolbert NE Differential inhibition and activation of two leaf dihydroxyacetone phosphate reductases. Plant Physiol 87 1988b 379 383 Google Scholar Crossref Search ADS WorldCat 15 Gee R Goyal A Gerber DW Byerrum RU Tolbert NE Isolation of dihydroxyacetone phosphate reductase from Dunaliella chloroplasts and comparison with isozymes from spinach leaves. Plant Physiol 88 1988c 896 903 Google Scholar Crossref Search ADS WorldCat 16 Ghosh S Sastry PS Triacylglycerol synthesis in developing seeds of groundnut (Arachis hypogaea): pathway and properties of enzymes of sn-glycerol 3-phosphate formation. Arch Biochem Biophys 262 1988 508 516 Google Scholar Crossref Search ADS PubMed WorldCat 17 Haughn GW Somerville CR Sulfonylurea-resistant mutants of Arabidopsis thaliana. Mol Gen Genet 204 1986 430 434 Google Scholar Crossref Search ADS WorldCat 18 Heber U, Santarius KA (1970) Direct and indirect transfer of ATP and ADP across the chloroplast envelope. Z Naturforsch 25b:718–728 19 Hippman H Heinz E Glycerol kinases in leaves. Z Pflanzenphysiol 79 1976 408 418 Google Scholar Crossref Search ADS WorldCat 20 Huang A Beevers H Enzymes of glycerol metabolism in the storage tissues of fatty acid seedlings. Plant Physiol 55 1975 555 558 Google Scholar Crossref Search ADS PubMed WorldCat 21 Jamieson GR Reed EH The occurrence of hexadeca-7,10,13-trienoic acid in the leaf lipids of angiosperms. Phytochemistry 10 1971 1837 1843 Google Scholar Crossref Search ADS WorldCat 22 Joyard J Block MA Malherbe A Marechal E Douce R Origin and synthesis of galactolipid and sulfolipid head groups. Moore TS Lipid Metabolism in Plants. 1993 231 258 CRC Press Boca Raton, FL 23 Joyard J Douce R Site of synthesis of phosphatidic acid and diacylglycerol in spinach chloroplasts. Biochim Biophys Acta 486 1977 273 285 Google Scholar Crossref Search ADS PubMed WorldCat 24 Kirsch T Gerber DW Byerrum RU Tolbert NE Plant dihydroxyacetone phosphate reductases. Plant Physiol 100 1992 352 359 Google Scholar Crossref Search ADS PubMed WorldCat 25 Kunst L Browse J Somerville C Altered regulation of lipid biosynthesis in a mutant of Arabidopsis deficient in chloroplast glycerol-3-phosphate acyltransferase activity. Proc Natl Acad Sci USA 85 1988 4143 4147 Google Scholar Crossref Search ADS PubMed WorldCat 26 Kunst L Browse J Somerville C A mutant of Arabidopsis deficient in desaturation of palmitic acid in leaf lipids. Plant Physiol 90 1989 943 947 Google Scholar Crossref Search ADS PubMed WorldCat 27 Leegood RC Labate CA Huber SC Neuhaus HE Stitt M Phosphate sequestration by glycerol and its effects on photosynthetic carbon assimilation by leaves. Planta 176 1988 117 126 Google Scholar Crossref Search ADS PubMed WorldCat 28 Malherbe A Block MA Joyard J Douce R Feedback inhibition of phosphatidate phosphatase from spinach chloroplast envelope membranes by diacylglycerol. J Biol Chem 267 1992 23546 23553 Google Scholar Crossref Search ADS PubMed WorldCat 29 McKeon TA Stumpf PK Purification and characterization of the stearoyl-acyl carrier protein desaturase and the acyl-acyl carrier protein thioesterase from maturing seeds of safflower. J Biol Chem 257 1982 12141 12147 Google Scholar Crossref Search ADS PubMed WorldCat 30 Miquel M Browse J Arabidopsis mutants deficient in polyunsaturated fatty acid synthesis: biochemical and genetic characterization of a plant oleoyl-phosphatidylcholine desaturase. J Biol Chem 267 1992 1502 1509 Google Scholar Crossref Search ADS PubMed WorldCat 31 Ohlrogge J Browse J Somerville CR The genetics of plant lipids. Biochim Biophys Acta 1082 1991 1 26 Google Scholar Crossref Search ADS PubMed WorldCat 32 Roughan PG Holland R Slack CR On the control of long-chain fatty acid synthesis in isolated intact spinach (Spinacia oleracea) chloroplasts. Biochem J 184 1979 571 574 Google Scholar Crossref Search ADS PubMed WorldCat 33 Roughan PG Holland R Slack CR The role of chloroplasts and microsomal fractions in polar lipid synthesis from [1-14C]acetate by cell-free preparations from spinach (Spinacia oleracea) leaves. Biochem J 188 1980 17 24 Google Scholar Crossref Search ADS PubMed WorldCat 34 Roughan PG Slack CR Cellular organization of glycerolipid metabolism. Annu Rev Plant Physiol 33 1982 97 132 Google Scholar Crossref Search ADS WorldCat 35 Sadava D Moore K Glycerol metabolism in higher plants: glycerol kinase. Biochem Biophys Res Commun 143 1987 977 983 Google Scholar Crossref Search ADS PubMed WorldCat 36 Santora G Gee R Tolbert NE Isolation of sn-glycerol 3-phosphate:NAD oxidoreductase from spinach leaves. Arch Biochem Biophys 196 1979 403 411 Google Scholar Crossref Search ADS PubMed WorldCat 37 Shanklin J Somerville CR The cDNA clones for stearoyl-ACP desaturase from higher plants are not homologous to yeast or mammalian genes encoding stearoyl-CoA desaturase. Proc Natl Acad Sci USA 88 1991 2510 2514 Google Scholar Crossref Search ADS PubMed WorldCat 38 Siebertz HP Heinz E Labeling experiments on the origin of hexa- and octa-decatrienoic acids in galactolipids of leaves. Z Naturforsch 32c 1977 193 205 Google Scholar OpenURL Placeholder Text WorldCat Author notes 1 This work was supported by the Centre National de la Recherche Scientifique and the Université Victor Segalen (Bordeaux, France), the National Science Foundation (grant no. IBN-9407902), and the Agricultural Research Center, Washington State University. * Corresponding author; e-mail [email protected]; fax 33–5–5651–8361. Copyright © 1998 American Society of Plant Physiologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Moreau, Patrick; Hartmann, Marie-Andrée; Perret, Anne-Marie; Sturbois-Balcerzak, Bénédicte; Cassagne, Claude

doi: 10.1104/pp.117.3.931pmid: 9662535

Abstract To investigate the intracellular transport of sterols in etiolated leek (Allium porrumL.) seedlings, in vivo pulse-chase experiments with [1-14C]acetate were performed. Then, endoplasmic reticulum-, Golgi-, and plasma membrane (PM)-enriched fractions were prepared and analyzed for the radioactivity incorporated into free sterols. In leek seedlings sterols are present as a mixture in which (24R)-24-ethylcholest-5-en-3β-ol is by far the major compound (around 60%). The other sterols are represented by cholest-5-en-3β-ol, 24-methyl-cholest-5-en-3β-ol, (24S)-24-ethylcholesta-5,22E-dien-3β-ol, and stigmasta-5,24(241)Z-dien-3β-ol. These compounds are shown to reside mainly in the PM. Our results clearly indicate that free sterols are actively transported from the endoplasmic reticulum to the PM during the first 60 min of chase, with kinetics very similar to that of phosphatidylserine. Such a transport was found to be decreased at low temperature (12°C) and following treatment with monensin and brefeldin A. These data are consistent with a membrane-mediated process for the intracellular transport of sterols to the PM, which likely involves the Golgi apparatus. Whereas mammalian and fungal cells mainly contain one major sterol, cholesterol and ergosterol, respectively, higher plant cells are characterized by a mixture of sterols in which sitosterol, stigmasterol, and 24-methylcholesterol often predominate. Sterol biosynthesis in plants has been extensively studied (Benveniste, 1986). From the conversion of farnesyl diphosphate into squalene and end products, this pathway represents a sequence of more than 30 enzyme-catalyzed reactions, all associated with membranes. It is now well established that sterols are synthesized at the level of the ER, but mainly accumulate in the PM (Hartmann and Benveniste, 1987). Thus, the neosynthesized sterols must be transferred from the ER to the PM. In contrast to recent advances in the understanding of intracellular movement of membrane proteins, relatively little attention has been paid to the intracellular transport of membrane lipids. Recent studies have been devoted to the transport of phospholipids in etiolated leek seedlings (Moreau et al., 1988; Bertho et al., 1991; Sturbois et al., 1994). It has been shown that the different phospholipid classes do not follow the same route from the ER to the PM. Moreover, low temperatures and treatment with monensin have been shown to block the transfer of only some molecular species (Bertho et al., 1991; Moreau and Cassagne, 1994; Sturbois-Balcerzak et al., 1995). As no information on the mechanisms involved in the delivery of sterol molecules to the PM is so far available, we have taken the advantage of this plant system to investigate the intracellular transport of sterols in higher plant cells. In vivo pulse-chase experiments with [1-14C]acetate clearly indicate that the sterols synthesized in the ER membranes are transferred to the PM with kinetics similar to that of PS. The effects of low temperature and treatment with monensin and brefeldin A on the transport of sterols were also investigated. MATERIALS AND METHODS Leek (Allium porrum L.) seeds were purchased from Vilmorin (La Ménitré, France) and stored overnight at 4°C before being hydrated with distillated water for 2 h. The seeds were allowed to germinate in the dark for 7 d at 22 to 24°C, as described previously (Moreau et al., 1988). Chemicals All chemicals were purchased from Sigma. [1-14C]Acetate was obtained from CEA (Saclay, France). Pulse-Chase Experiments For each experimental value, 10 batches of 20 seedlings (cut into 5- to 10-mm segments, including roots) were first incubated in 0.2 mL of 3.5 × 105 Bq of [1-14C]acetate (2 × 1012 Bq mol−1) for 120 min at 24°C or 12°C, in the presence or absence of monensin (5 μm) or brefeldin A (100 or 500 μm). Chase was made with 0.5 mL of 0.2 m unlabeled acetate for periods of time ranging from 30 to 120 min. Isolation of ER, Golgi, and PM Fractions Leek seedlings were homogenized in a buffer consisting of 10 mm KH2PO4, pH 8.2, with 0.5 m sorbitol, 5% (w/v) PVP 40, 0.5% (w/v) BSA, 2 mm salicylhydroxamic acid, and 1 mmPMSF. The homogenate was submitted to differential centrifugations at 1,000g for 10 min, 10,000g for 10 min, and 150,000g for 60 min. The resulting microsomal pellet was resuspended in 10 mmKH2PO4 and 0.5m sorbitol. One-half of the suspension was loaded onto a discontinuous Suc-density gradient consisting of 2.5 mL of 37% (w/v) Suc, 3.5 mL of 25% (w/v) Suc, and 3.5 mL of 18% (w/v) Suc. After centrifugation at 80,000g for 150 min, membranes at the 18/25% (ER fraction) and 25/37% (Golgi fraction) Suc interface were collected, diluted with 30 mm Hepes-KCl, pH 6.8, and centrifuged at 100,000g for 60 min. PMs were isolated by phase partitioning using PEG 4000 and dextran T500. The other half of the microsomal suspension was mixed with a polymer (PEG/dextran mixture) in 0.5 m sorbitol containing 10 mm KH2PO4and 40 mm NaCl, pH 7.8, to obtain final PEG 4000 and dextran T500 concentrations of 6.0% (w/w). The solution (final volume, 28 mL) was centrifuged for 15 min at 1000g and the PEG-enriched upper phase (12 mL) was recovered without disturbing the interface. Membranes were then recovered after centrifugation at 150,000g for 60 min and resuspended in 30 mmHepes-KCl, pH 6.8. Specific membrane compartments were identified by assays for the following markers: ER, NADPH-Cyt c reductase and CDP-choline phosphotransferase; Golgi apparatus, IDPase; and PM, glucan synthetase II (Moreau et al., 1988; Bertho et al., 1991). Setting the specific activities of the marker enzymes at 1 in the homogenate, we obtained the following relative enrichments in the membrane fractions: ER fraction, 8.5 for NADPH-Cyt c reductase, 3.5 for CDP-choline phosphotransferase, 2.5 for IDPase, and 0.1 for glucan synthetase II; Golgi fraction, 2.4 for NADPH-Cyt c reductase, 0.5 for CDP-choline phosphotransferase, 9.5 for IDPase, and 0.6 for glucan synthetase II; PM fraction, 0.1 for NADPH-Cyt c reductase, 0.15 for CDP-choline phosphotransferase, 0.35 for IDPase, and 4.4 for glucan synthetase II. The specific activity of succinodeshydrogenase (a mitochondrial marker) was < 0.1 in the microsomes compared with the homogenate. A low contamination of ER and Golgi fractions by plastid envelope membranes was determined by the presence of small amounts of galactolipids (< 10% of the total glycerolipids). Protein concentrations were determined by the method of Bradford (1976) using BSA as standard. Lipid Analyses Lipids were extracted by chloroform:methanol (1:1, v/v) for 30 min at room temperature, and then washed three times with distilled water. The solvent was evaporated and lipids were resuspended in an appropriate volume of chloroform:methanol (1:1, v/v) according to procedures already described (Moreau et al., 1988; Bertho et al., 1991). PS isolation was carried out on HPTLC plates (60F254, Merck, Darmstadt, Germany) developed with methylacetate:n-propanol:chloroform:methanol:aqueous 0.25% (w/v) KCl (25:25:25:10:9, v/v) according to the method of Heape et al. (1985). Neutral lipids were isolated onto HPTLC plates developed with hexane:chloroform:methanol (100:60:10, v/v) to separate into sterols (RF 0.54), fatty alcohols (RF 0.64), and free fatty acids (RF 1); with hexane:ethylether:acetic acid (90:15:2, v/v) to give diacylglycerols (RF 0.08), 4-demethylsterols (RF 0.17), fatty alcohols (RF 0.22), and free fatty acids (RF 0.29). After identification by comparison with standards, the different lipids were scraped off directly into vials and their radioactivity was determined by liquid scintillation counting (model 2000CA counter, Packard Instruments, Meriden, CT). Radioactivity of the lipids was also determined after autoradiography of the HPTLC plates (Hyperfilm MP-RPN 1675, Amersham) and scanning with a densitometer (model 76510, Camag, Muttenz, Switzerland). Both methods gave similar results and were alternately used. Sterols were identified and quantified as previously reported (Hartmann and Benveniste, 1987). After extraction from membrane fractions with hexane, sterols were subjected to TLC with dichloromethane as the developing solvent for two runs. The 4-demethylsterols (end products) were eluted and acetylated before being analyzed by GC on a glass capillary column (30 m long, 0.25-mm i.d., coated with DB-1). The temperature program used includes a fast rise from 60°C to 230°C (30°C/min), then a slow rise from 230°C to 280°C (2°C/min). A cholesterol standard (not acetylated) was added to the samples prior to analysis. Sterol identification was made by GC-MS (Rahier and Benveniste, 1989). RESULTS Free Sterol Composition of Membrane Fractions ER, Golgi, and PM fractions were prepared from 7-d-old etiolated leek seedlings and characterized by enzymatic markers (Moreau et al., 1988; Bertho et al., 1991) and labeling by anti-HDEL (Napier et al., 1992) and JIM 84 (Horsley et al., 1993) antibodies for ER and Golgi fractions, respectively (B. Sturbois-Balcerzak, L. Maneta-Peyret, M. Duvert, B. Satiat-Jeunemaitre, P. Vincent, C. Cassagne, and P. Moreau, unpublished data). These fractions were analyzed for their free 4-demethylsterol content. Data are shown in TableI. In leek seedlings, like in other plant tissue, the PM was found to be the richest membrane in free sterols (expressed in micrograms per milligram of protein). A few sterol molecules were present in the ER. The Golgi fraction contained an intermediate concentration of free sterols. PM was also characterized by the highest sterol-to-phospholipid molar ratio. In all of the fractions, sterols are present as a mixture in which sitosterol is largely predominent (62%–70%). The other sterols are represented by 24-methylcholesterol, stigmasterol, cholesterol, and isofucosterol. Such a sterol composition and the relatively high content of cholesterol (10%) are in agreement with published data concerning other plants belonging to Liliaceae (Itoh et al., 1977). Table I. Sterol composition of membrane fractions isolated from 7-d-old etiolated leek seedlings Membrane Fraction . Phospholipids . Sterols . Molar Ratio of Sterols to Phospholipids . Relative Sterol Compositions-a . Ch . 24-m . St . Si . Is . μg · mg−1 protein % ER 580 8.8 0.025 10.5 8.5 5.0 69.5 6.5 Golgi 500 18.9 0.065 8.5 8.0 3.0 66.5 14.0 PM 420 45.5 0.18 5.0 8.0 3.0 61.5 17.0 Membrane Fraction . Phospholipids . Sterols . Molar Ratio of Sterols to Phospholipids . Relative Sterol Compositions-a . Ch . 24-m . St . Si . Is . μg · mg−1 protein % ER 580 8.8 0.025 10.5 8.5 5.0 69.5 6.5 Golgi 500 18.9 0.065 8.5 8.0 3.0 66.5 14.0 PM 420 45.5 0.18 5.0 8.0 3.0 61.5 17.0 The values are from two independent lipid analyses. F0-a Ch, cholesterol; 24-m, 24-methylcholesterol; St, stigmasterol; Si, sitosterol; Is, isofucosterol. Open in new tab Table I. Sterol composition of membrane fractions isolated from 7-d-old etiolated leek seedlings Membrane Fraction . Phospholipids . Sterols . Molar Ratio of Sterols to Phospholipids . Relative Sterol Compositions-a . Ch . 24-m . St . Si . Is . μg · mg−1 protein % ER 580 8.8 0.025 10.5 8.5 5.0 69.5 6.5 Golgi 500 18.9 0.065 8.5 8.0 3.0 66.5 14.0 PM 420 45.5 0.18 5.0 8.0 3.0 61.5 17.0 Membrane Fraction . Phospholipids . Sterols . Molar Ratio of Sterols to Phospholipids . Relative Sterol Compositions-a . Ch . 24-m . St . Si . Is . μg · mg−1 protein % ER 580 8.8 0.025 10.5 8.5 5.0 69.5 6.5 Golgi 500 18.9 0.065 8.5 8.0 3.0 66.5 14.0 PM 420 45.5 0.18 5.0 8.0 3.0 61.5 17.0 The values are from two independent lipid analyses. F0-a Ch, cholesterol; 24-m, 24-methylcholesterol; St, stigmasterol; Si, sitosterol; Is, isofucosterol. Open in new tab Transport Kinetics of Sterols to the PM Leek seedlings were first incubated with [1-14C]acetate for 120 min, then with unlabeled acetate for 30 to 120 min (Figs. 1–3). Membrane fractions were prepared and analyzed for their radioactivity incorporated into free sterols. Figure 1 shows the comparative evolution of the radioactivity incorporated into sterols as a function of chase time in microsomes and purified PM. A significant increase in the sterol labeling of PM was observed whatever the chase time, whereas the amount of radioactivity associated with sterols of microsomes remained stable or decreased (after 120 min), indicating that the increase in the sterol labeling of PM was likely due to a delivery of newly synthesized sterols (made during the pulse period). To determine the origin of labeled sterols in the PM, we analyzed the sterol labeling of ER, Golgi, and PM fractions from leek seedlings after similar pulse-chase experiments. Results are presented in Figure2. At time 0, i.e. at the end of the pulse, the radioactivity associated with sterols of ER was about 2-fold higher than that present in the Golgi and PM fractions, as would be expected for the involvement of ER in the synthesis of free sterols (Hartmann and Benveniste, 1987). During the chase, an increase in the sterol labeling of PM was correlated with a decrease in the radioactivity associated with sterols of ER. The Golgi fraction appeared to have an intermediate behavior, as the sterol labeling increased after 30 and 60 min of chase and decreased after 120 min. Fig. 1. Open in new tabDownload slide Sterol labeling in the microsomes (□) and the PM (•) as a function of chase time. Leek seedlings were first incubated with [14C]- acetate for 120 min, then with unlabeled acetate for the indicated periods of time. Microsomes and PM were prepared and the radioactivity associated with sterols determined as explained in Methods. Values are expressed as percentages of the radioactivity (dpm mg−1protein) incorporated during the 120-min labeling period (n = 7), and correspond to an average of 4 (30- and 60-min chase) and 7 (120-min chase) determinations (±sd). Sterol labeling in the microsomes and PM fraction at the end of the pulse (i.e. 0-min chase) was 65,000 and 25,000 dpm mg−1protein, respectively. Fig. 1. Open in new tabDownload slide Sterol labeling in the microsomes (□) and the PM (•) as a function of chase time. Leek seedlings were first incubated with [14C]- acetate for 120 min, then with unlabeled acetate for the indicated periods of time. Microsomes and PM were prepared and the radioactivity associated with sterols determined as explained in Methods. Values are expressed as percentages of the radioactivity (dpm mg−1protein) incorporated during the 120-min labeling period (n = 7), and correspond to an average of 4 (30- and 60-min chase) and 7 (120-min chase) determinations (±sd). Sterol labeling in the microsomes and PM fraction at the end of the pulse (i.e. 0-min chase) was 65,000 and 25,000 dpm mg−1protein, respectively. Fig. 2. Open in new tabDownload slide Sterol labeling in the ER (□), Golgi (⬗), and PM (•) as a function of chase time. Pulse-chase procedures and measurements of sterol labeling were as in Figure 1. PM/ER ratios increase from 0.54 ± 0.06 (120-min pulse) to 5.07 ± 0.45 (120-min chase). The values are from three independent experiments (±sd). Fig. 2. Open in new tabDownload slide Sterol labeling in the ER (□), Golgi (⬗), and PM (•) as a function of chase time. Pulse-chase procedures and measurements of sterol labeling were as in Figure 1. PM/ER ratios increase from 0.54 ± 0.06 (120-min pulse) to 5.07 ± 0.45 (120-min chase). The values are from three independent experiments (±sd). The kinetics of transfer of sterols from the ER to the PM were compared with that of PS, a phospholipid previously shown to be transported to the PM in a vesicular pathway (Sturbois-Balcerzak et al., 1995). As shown in Figure 3, the kinetics of labeling of sterols and PS, as expressed as the ratio of radioactivities associated with the PM to those associated with the ER, were quite similar, suggesting closely related mechanisms of delivery to the cell surface for these two classes of molecules. As a control, the evolution of the labeling of other lipid classes such as diacylglycerols and free fatty acids was checked. No variation in their radioactivities was observed during the chase, indicating that these lipids were not transported. Fig. 3. Open in new tabDownload slide PM/ER ratios of sterol and PS labeling (dpm mg−1 protein) as a function of chase time. Pulse-chase procedures and measurements of lipid labeling were as in Figure 1. The data concerning sterols were taken from Figure 2 and compared with those obtained for PS, which is considered as a marker for membrane-mediated processes (Sturbois-Balcerzak et al., 1995), with free fatty acids and diacylglycerol as negative controls. For the sake of clarity, the sds from three experiments with values between 7% and 13% were omitted. Sterol labeling in the ER and PM fractions at the end of the pulse (i.e. 0-min chase) was 50,000 and 27,000 dpm mg−1 protein, respectively. •, Sterol; ▵, PS; □, diacylglycerol plus free fatty acids. Fig. 3. Open in new tabDownload slide PM/ER ratios of sterol and PS labeling (dpm mg−1 protein) as a function of chase time. Pulse-chase procedures and measurements of lipid labeling were as in Figure 1. The data concerning sterols were taken from Figure 2 and compared with those obtained for PS, which is considered as a marker for membrane-mediated processes (Sturbois-Balcerzak et al., 1995), with free fatty acids and diacylglycerol as negative controls. For the sake of clarity, the sds from three experiments with values between 7% and 13% were omitted. Sterol labeling in the ER and PM fractions at the end of the pulse (i.e. 0-min chase) was 50,000 and 27,000 dpm mg−1 protein, respectively. •, Sterol; ▵, PS; □, diacylglycerol plus free fatty acids. Effect of Low Temperature on Sterol Transport Leek seedlings were incubated with [14C]acetate for 120 min at 24°C or at 12°C, a temperature that was found to block the transport of some phospholipids (particularly PS and PE) to the PM (Sturbois-Balcerzak et al., 1995). ER, Golgi, and PM fractions were then prepared and their sterol and PS (used as a reporter for the vesicular transport) labeling was determined. To focus only on the effect of low temperature on the distribution of labeled lipids between the various cellular membranes independently of the effect of low temperature on their synthesis, we have calculated for each lipid L (PS or sterols) of each membrane fraction X (ER, Golgi, or PM) the following ratio: [L (X) 12°C/L (X) 24°C] × [L (μ) 24°C/L (μ) 12°C] (see legend of Table II). Two situations can be observed: (a) a value close to 1 for the lipid of the PM means that there is no apparent temperature block, and therefore no great change in the ratio of this lipid in the intracellular membrane fractions is expected; and (b) a value less than 1 for the lipid of the PM indicates an effect of low temperature on the transport of this lipid to the PM. The lipid not transferred would be expected to accumulate intracellulary, and thus the ratio of this lipid will be greater than 1 in one or both intracellular membrane fractions (TableII). Table II. Effect of low temperature (12°C) on the delivery of free sterols and PS to the PM Membrane Fraction . Radioactivity1-a(12°C/24°C Ratios) . Free sterols . PS . ER 1.88  ± 0.12 1.42  ± 0.18 Golgi 2.70  ± 0.23 1.85  ± 0.24 PM 0.41  ± 0.07 0.34  ± 0.08 Membrane Fraction . Radioactivity1-a(12°C/24°C Ratios) . Free sterols . PS . ER 1.88  ± 0.12 1.42  ± 0.18 Golgi 2.70  ± 0.23 1.85  ± 0.24 PM 0.41  ± 0.07 0.34  ± 0.08 Leek seedlings were labeled at 24°C or 12°C with [14C]acetate for 120 min. ER, Golgi, and PM were isolated and the radioactivity of free sterols and PS was determined. F1-a We have calculated for each lipid L of each membrane fraction X the following expression: [L (X) 12°C/L (X) 24°C] X [L (μ) 24°C/L (μ) 12°C], where L(X) 12°C andL(X) 24°C are the radioactivities (dpm mg−1 protein) of the lipid L of the membrane fraction X at 12°C and 24°C, respectively, andL (μ) 12°C and L (μ) 24°C are the radioactivities (dpm mg−1 protein) of the lipidL of microsomes at 12°C and 24°C, respectively. These calculations have been made from five experiments (±sd). Open in new tab Table II. Effect of low temperature (12°C) on the delivery of free sterols and PS to the PM Membrane Fraction . Radioactivity1-a(12°C/24°C Ratios) . Free sterols . PS . ER 1.88  ± 0.12 1.42  ± 0.18 Golgi 2.70  ± 0.23 1.85  ± 0.24 PM 0.41  ± 0.07 0.34  ± 0.08 Membrane Fraction . Radioactivity1-a(12°C/24°C Ratios) . Free sterols . PS . ER 1.88  ± 0.12 1.42  ± 0.18 Golgi 2.70  ± 0.23 1.85  ± 0.24 PM 0.41  ± 0.07 0.34  ± 0.08 Leek seedlings were labeled at 24°C or 12°C with [14C]acetate for 120 min. ER, Golgi, and PM were isolated and the radioactivity of free sterols and PS was determined. F1-a We have calculated for each lipid L of each membrane fraction X the following expression: [L (X) 12°C/L (X) 24°C] X [L (μ) 24°C/L (μ) 12°C], where L(X) 12°C andL(X) 24°C are the radioactivities (dpm mg−1 protein) of the lipid L of the membrane fraction X at 12°C and 24°C, respectively, andL (μ) 12°C and L (μ) 24°C are the radioactivities (dpm mg−1 protein) of the lipidL of microsomes at 12°C and 24°C, respectively. These calculations have been made from five experiments (±sd). Open in new tab The value obtained for free sterols was ≪ 1 for the PM, suggesting that sterols were less efficiently delivered to the PM at 12°C. Moreover, a sterol accumulation was observed in the ER and Golgi fractions (ratios > 1). In agreement with previous results (Sturbois-Balcerzak et al., 1995), the value for PS in the PM was also ≪ 1 and confirmed the arrest of the transfer of this phospholipid to the PM at 12°C. PS was shown to accumulate in the ER and Golgi fractions, since the values of ratios were found to be > 1 in both fractions. These results strongly suggest that the transport of free sterols to the PM was either slowed down or partly blocked at 12°C in a way similar to that observed for PS transfer. Their accumulation in the intracellular membranes could be explained by the decrease in the number of secretory vesicles and the increase in the surface area of the trans-Golgi/trans-Golgi network that was morphologically observed at 12°C (Sturbois-Balcerzak et al., 1995). Effect of Monensin and Brefeldin A on Sterol Transport Monensin has been shown to disturb the secretory function of the Golgi apparatus (Mollenhauer et al., 1990) and was previously used to show the intermediate position of this organelle in the delivery of some phospholipids (including PS) to the PM (Bertho et al., 1991;Sturbois-Balcerzak et al., 1995). The monensin concentration (5 μm) used led to a 25% inhibition of sterol synthesis in the microsomes (Fig. 4). Under these conditions, the amount of the radioactivity associated with sterols of the PM was decreased by 75% (Fig. 4), suggesting that the delivery of these molecules to the PM was affected. Fig. 4. Open in new tabDownload slide Effect of monensin and brefeldin A on sterol delivery to the PM. Leek seedlings were incubated with [14C]acetate for 120 min in the absence or presence of 5 μm monensin or 100 μm or 500 μm brefeldin A. Microsomes and PM were then prepared and the radioactivity (dpm mg−1 protein) incorporated into sterols was determined. The ratios of treated to untreated were then calculated for each membrane fraction. The data are from three independent experiments and the results are expressed as arbitrary units, with the control values equal to 1. M, Microsomes. Sterol labeling in the crude microsomes and PM in the absence of drug were 56,000 and 31,000 dpm mg−1 protein, respectively. Fig. 4. Open in new tabDownload slide Effect of monensin and brefeldin A on sterol delivery to the PM. Leek seedlings were incubated with [14C]acetate for 120 min in the absence or presence of 5 μm monensin or 100 μm or 500 μm brefeldin A. Microsomes and PM were then prepared and the radioactivity (dpm mg−1 protein) incorporated into sterols was determined. The ratios of treated to untreated were then calculated for each membrane fraction. The data are from three independent experiments and the results are expressed as arbitrary units, with the control values equal to 1. M, Microsomes. Sterol labeling in the crude microsomes and PM in the absence of drug were 56,000 and 31,000 dpm mg−1 protein, respectively. Brefeldin A, a fungus-derived cyclic lactone, has been largely used to dissect membrane-trafficking events in mammalian cells (Klausner et al., 1992) and has only recently been implemented in plant cells (Satiat-Jeunemaı̂tre et al., 1996). Following treatment of leek seedlings with 100 and 500 μm of brefeldin A, the sterol synthesis in the microsomes was found to be inhibited by 37% and 62%, respectively. Under these conditions, the decrease in the sterol labeling of the PM reached 73% and 88%, respectively (Fig. 4), indicating that in addition to an effect on sterol synthesis, brefeldin A also inhibited the delivery of sterols to the PM. DISCUSSION In etiolated leek seedlings, free sterols are mainly concentrated in the PM (Table I), as they are in other plant tissues (Hartmann and Benveniste, 1987). A few sterol molecules are present in the ER membranes and an intermediate amount of sterol was found in the Golgi fraction. In all of these membrane fractions, sterols are present as a mixture, with sitosterol as the major compound, suggesting that free sterols are transported together to the PM. In vivo pulse-chase experiments with [1-14C]acetate clearly indicated that sterols are actively transferred from the ER to the PM during the first 60 min of chase. These results are totally in agreement with previous data obtained with maize coleoptiles after in vivo labeling with [5-14C]mevalonic acid (Hartmann, 1980). Such a study indicated that a few molecules of biosynthetic precursors were also transported to the PM. In contrast, steryl esters were found not to be transferred. In leek seedlings kinetics of intracellular transport of free sterols from the ER to the PM were shown to be similar to that of PS. The transport of these two classes of lipids was found to be decreased by low temperature and treatment with monensin and brefeldin A (Table II; Fig. 4), suggesting similar mechanisms of delivery to the PM. Which mechanism(s) might be responsible for the movement of free sterols to the PM: a simple diffusion through the cytoplasm, a protein-mediated transport, or a vesicular transfer? It is generally accepted that a spontaneous diffusion through the aqueous medium is a slow phenomenon and therefore not a significant route for sterol transport, so an activation-collision mechanism was suggested (Steck et al., 1988). However, a significant sterol exchange through the aqueous phase was recently observed in fibroblasts (Frolov et al., 1996). This spontaneous exchange was faster from PM to intracellular membranes than in the reverse direction (Frolov et al., 1996). These results are therefore not in favor of a quantitative transport of sterols to the PM by simple diffusion. Specific carrier proteins could enhance the diffusion through the aqueous phase. Frolov et al. (1996) have observed that sterol exchange between biological membranes is highly enhanced by the sterol carrier protein SCP2. In good agreement with these in vitro studies, Puglielli et al. (1995) have shown a requirement for SCP2 in sterol transport from the ER to the PM of cultured fibroblasts. Their results pointed to a predominant SCP2-mediated transport of cholesterol in normal fibroblasts but also to the occurrence of a membrane-mediated transport revealed in the SCP2-deficient fibroblasts. Thus, a protein-stimulated as well as a membrane-mediated transport of sterols has to be considered. There is no indication in the literature of the existence of specialized proteins in plant cells that are able to transfer sterols (Kader, 1996). Although the occurrence of specific sterol-carrier proteins in plants cannot be ruled out, a much more likely way to transfer sterols is via a membrane-mediated process. Our data are compatible with such a process for several reasons. First, we estimated the t1/2 of sterol transfer to the PM to be about 30 min (Fig. 2), which is of the same order of magnitude as those obtained for phospholipids, which are known to follow a vesicular pathway (Bertho et al., 1991; Sturbois et al., 1994), and for proteins (Mitsui et al., 1985; Kappler et al., 1986). Moreover, the transport of sterols was similar to that of PS (Fig. 3), which has been shown to be membrane mediated (Sturbois et al., 1994;Sturbois-Balcerzak et al., 1995). In addition, we have observed that low temperature (12°C) partly blocked the delivery of sterols to the PM and resulted in their intracellular accumulation (as was also the case with PS; Table II). Similar results have been obtained in animal cells treated at 15°C (Kaplan and Simoni, 1985), and the existence of cholesterol-rich intracellular membranes potentially involved in cholesterol transport has been demonstrated (Kaplan and Simoni, 1985; Lange and Steck, 1985). Sterol-rich lipid particles have also been suggested as possible structures for the transport of sterols from internal membranes to the PM of yeast (Zinser et al., 1993). Other arguments favoring a membrane-mediated process come from experiments carried out with monensin and brefeldin A (Fig. 4). Although the effects of monensin on the plant secretory system are controversial (Sticher and Jones, 1988; Zhang et al., 1993;Satiat-Jeunemaı̂tre et al., 1994), it has been shown that in our system monensin led to a transport block of some phospholipid species and to their accumulation in internal membranes, including a Golgi-enriched fraction (Bertho et al., 1991; Sturbois-Balcerzak et al., 1995). We found that at a monensin concentration that only slightly affected the de novo synthesis of sterols, their delivery to the PM was dramatically inhibited (Fig. 4). Therefore, it is possible that monensin induces an accumulation of sterol molecules in an intracellular compartment resembling the Golgi-derived swollen vesicles described by Zhang et al. (1996). The other drug used in these experiments is brefeldin A, and its effect on the Golgi apparatus in plant cells has been extensively described (Satiat-Jeunemaı̂tre et al., 1996). Contrary to monensin, the effect of brefeldin A on plant cells has been widely reproduced (Satiat-Jeunemaı̂tre et al., 1996) with only few exceptions (Robinson, 1993). The results with brefeldin A are similar to those obtained with monensin (compare 5 μm monensin and 100 μm brefeldin A, Fig. 4). As a consequence, it is likely that a disturbance of the Golgi apparatus was at least to some extent at the origin of the inhibition of sterol transport to the PM. These data clearly differ from those obtained with Chinese hamster ovary cells, in which none of these drugs affected the intracellular transport of cholesterol (Kaplan and Simoni, 1985; Urbani and Simoni, 1990). In this case, the delivery of newly synthesized cholesterol to the PM would be mediated by lipid-rich vesicles not related to the Golgi apparatus (Urbani and Simoni, 1990; Liscum and Underwood, 1995). The potential occurrence of a direct ER-to-PM pathway for intracellular transport of sterols in leek seedlings also has to be considered (Kristen et al., 1987; Sturbois-Balcerzak et al., 1995). In conclusion, the present data clearly favor a membrane-mediated process for the transport of free sterols from the ER to the PM in leek seedlings. Free sterols are known to play a key role in regulating the physical properties of the PM as well as the activity of some membrane-bound enzymes such as H+-ATPase. Consequently, levels of these molecules within the PM have to be tightly regulated. Mechanisms contributing to homeostasis of sterols in higher plant cells remain to be elucidated, but certainly differ fundamentally from those operating in mammalian cells. One argument is the absence of the classic low-density lipoprotein pathway in plants. It now appears crucial to isolate membrane vesicles participating in lipid transport from plant tissues. Such experiments are currently being performed in our laboratory and are expected to determine whether free sterols and PS molecules are transported together in the same vesicles, and to shed more light on sterol trafficking in plant cells. ACKNOWLEDGMENTS We thank Mr. John F. Ackerson for critically reading the English text. Abbreviations: cholesterol cholest-5-en-3β-ol HPTLC high-performance TLC isofucosterol stigmasta-5,24(241)Z-dien-3β-ol PM plasma membrane PS phosphatidylserine sitosterol (24R)-24-ethylcholest-5-en-3β-ol stigmasterol (24S)-24-ethylcholesta-5,22E-dien-3β-ol LITERATURE CITED 1 Benveniste P Sterol biosynthesis. Annu Rev Plant Physiol 37 1986 275 308 Google Scholar Crossref Search ADS WorldCat 2 Bertho P Moreau P Morré DJ Cassagne C Monensin blocks the transfer of very long chain fatty acid containing lipids to the plasma membrane of leek seedlings: evidence for lipid sorting based on fatty acyl chain length. Biochim Biophys Acta 1070 1991 127 134 Google Scholar Crossref Search ADS PubMed WorldCat 3 Bradford MM A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72 1976 248 254 Google Scholar Crossref Search ADS PubMed WorldCat 4 Frolov A Woodford JK Murphy EJ Billheimer JT Schroeder F Spontaneous and protein-mediated sterol transfer between intracellular membranes. J Biol Chem 271 1996 16075 16083 Google Scholar Crossref Search ADS PubMed WorldCat 5 Hartmann MA (1980) These de Doctorat d'Etat, UniversitéLouis Pasteur, Strasbourg, France 6 Hartmann MA Benveniste P Plant membrane sterols: isolation, identification and biosynthesis. Methods Enzymol 148 1987 632 650 Google Scholar Crossref Search ADS WorldCat 7 Heape AM Juguelin H Boiron F Cassagne C Improved one-dimensional thin-layer chromatographic technique for polar lipids. J Chromatogr 322 1985 391 395 Google Scholar Crossref Search ADS PubMed WorldCat 8 Horsley D Coleman J Evans D Crooks K Peart J Satiat-Jeunemaitre B Hawes C A monoclonal antibody, JIM 84, recognizes the Golgi apparatus and plasma membrane in plant cells. J Exp Bot 44 1993 223 229 Google Scholar OpenURL Placeholder Text WorldCat 9 Itoh T Tamura T Mitsuhashi T Matsumoto T Sterols of Liliaceae. Phytochem 16 1977 140 141 Google Scholar Crossref Search ADS WorldCat 10 Kader JC Lipid-transfer proteins in plants. Annu Rev Plant Physiol Plant Mol Biol 47 1996 627 654 Google Scholar Crossref Search ADS PubMed WorldCat 11 Kaplan MR Simoni RD Transport of cholesterol from the endoplasmic reticulum to the plasma membrane. J Cell Biol 101 1985 446 453 Google Scholar Crossref Search ADS PubMed WorldCat 12 Kappler R Kristen U Morré DJ Membrane flow in plants: fractionation of growing pollen tubes of tobacco by preparative free-flow electrophoresis and kinetics of labeling of endoplasmic reticulum and Golgi apparatus with [3H] leucine. Protoplasma 132 1986 38 50 Google Scholar Crossref Search ADS WorldCat 13 Klausner RD Donaldson JG Lippincott-Schwartz J Brefeldin A: insights into the control of membrane traffic and organelle structure. J Cell Biol 116 1992 1071 1080 Google Scholar Crossref Search ADS PubMed WorldCat 14 Kristen U Lockhausen J Kandasamy MK Structural aspects of protein secretion in higher plant cells. Phyton 28 1987 183 191 Google Scholar OpenURL Placeholder Text WorldCat 15 Lange Y Steck TL Cholesterol-rich intracellular membranes: a precursor to the plasma membrane. J Biol Chem 260 1985 15592 15597 Google Scholar Crossref Search ADS PubMed WorldCat 16 Liscum L Underwood KW Intracellular cholesterol transport and compartmentation. J Biol Chem 270 1995 15443 15446 Google Scholar Crossref Search ADS PubMed WorldCat 17 Mitsui T Akazawa T Christeller JT Tartakoff AM Biosynthesis of rice seed α-amylase: two pathways of amylase secretion by the scutellum. Arch Biochem Biophys 241 1985 315 328 Google Scholar Crossref Search ADS PubMed WorldCat 18 Mollenhauer HH Morré DJ Rowe LD Alteration of intracellular traffic by monensin: mechanism, specificity and relationship to toxicity. Biochim Biophys Acta 1031 1990 225 246 Google Scholar Crossref Search ADS PubMed WorldCat 19 Moreau P Cassagne C Phospholipid trafficking and membrane biogenesis. Biochim Biophys Acta 1197 1994 257 290 Google Scholar Crossref Search ADS PubMed WorldCat 20 Moreau P Juguelin H Lessire R Cassagne C Plasma membrane biogenesis in higher plants: in vivo transfer of lipids to the plasma membrane. Phytochemistry 27 1988 1631 1638 Google Scholar Crossref Search ADS WorldCat 21 Napier RM Fowke L Hawes C Lewis M Pelham HRB Immunological evidence that plants use both HDEL and KDEL for targeting proteins to the endoplasmic reticulum. J Cell Sci 102 1992 261 271 Google Scholar PubMed OpenURL Placeholder Text WorldCat 22 Puglielli L Rigotti A Greco AV Santos MJ Nervi F Sterol carrier protein-2 is involved in cholesterol transfer from the endoplasmic reticulum to the plasma membrane in human fibroblasts. J Biol Chem 270 1995 18723 18726 Google Scholar Crossref Search ADS PubMed WorldCat 23 Rahier A, Benveniste P (1989) Mass spectral identification of phytosterols. In WD Nes, E Parish, eds, Analysis of Sterols and Other Significant Steroids. Academic Press, New York, pp 223–250 24 Robinson D Brefeldin A: a tool for plant cell biologists? Bot Acta 106 1993 107 109 Google Scholar Crossref Search ADS WorldCat 25 Satiat-Jeunemaı̂tre B Cole L Bourett T Howard R Hawes C Brefeldin A effects in plant and fungal cells: something new about vesicle trafficking? J Microscopy 181 1996 162 177 Google Scholar Crossref Search ADS WorldCat 26 Satiat-Jeunemaı̂tre B Fitchette-Lainé AC Alabouvette J Marty-Mazars D Hawes C Faye L Marty F Differential effects of monensin on the plant secretory pathway. J Exp Bot 45 1994 685 698 Google Scholar Crossref Search ADS WorldCat 27 Steck TL Ferenc JK Lange Y An activation-collision mechanism for cholesterol transfer between membranes. J Biol Chem 263 1988 13023 13031 Google Scholar Crossref Search ADS PubMed WorldCat 28 Sticher L Jones RL Monensin inhibits the secretion of α-amylase but not polysaccharide slime from seedling tissues of Zea mays. Protoplasma 142 1988 36 45 Google Scholar Crossref Search ADS WorldCat 29 Sturbois B Moreau P Maneta-Peyret L Morré DJ Cassagne C Cell-free transfer of phospholipids between the endoplasmic reticulum and the Golgi apparatus of leek seedling. Biochim Biophys Acta 1189 1994 31 37 Google Scholar Crossref Search ADS PubMed WorldCat 30 Sturbois-Balcerzak B Morré DJ Loreau O Noël JP Moreau P Cassagne C Effects of low temperatures on the transfer of phospholipids to the plasma membrane and on the morphology of the ER-Golgi apparatus-plasma membrane pathway of leek cells. Plant Physiol Biochem 33 1995 625 637 Google Scholar OpenURL Placeholder Text WorldCat 31 Urbani L Simoni RD Cholesterol and vesicular stomatitis virus G protein take separate routes from the endoplasmic reticulum to the plasma membrane. J Biol Chem 265 1990 1919 1923 Google Scholar Crossref Search ADS PubMed WorldCat 32 Zhang GF Driouich A Staehelin LA Effect of monensin on plant Golgi: re-examination of the monensin-induced changes in cisternal architecture and functional activities of the Golgi apparatus of sycamore suspension-cultured cells. J Cell Sci 104 1993 819 831 Google Scholar PubMed OpenURL Placeholder Text WorldCat 33 Zhang GF Driouich A Staehelin LA Monensin-induced redistribution of enzymes and products from Golgi stacks to swollen vesicles in plant cells. Eur J Cell Biol 71 1996 332 340 Google Scholar PubMed OpenURL Placeholder Text WorldCat 34 Zinser E Paltauf F Daum G Sterol composition of yeast organelle membranes and subcellular distribution of enzymes involved in sterol metabolism. J Bacteriol 175 1993 2853 2858 Google Scholar Crossref Search ADS PubMed WorldCat Author notes 1 This work was supported by the Centre National de la Recherche Scientifique and the University Victor Segalen Bordeaux 2. * Corresponding author; e-mail [email protected]; fax 33–05–56–51–83–61. Copyright © 1998 American Society of Plant Physiologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)