TY - JOUR
AU - Takahashi, Hideyuki
AB - Abstract Primary roots of cucumber seedlings showed positive hydrotropism when exposed to a moisture gradient and rotated on a two-axis clinostat. To examine the role of auxin in the differential growth of the hydrotropically responding roots, we first examined the expression of auxin-inducible genes, CS-AUX/IAAs, in cucumber roots. After auxin starvation, mRNA levels of CS-IAA1 and CS-IAA3 decreased in the roots. Applying auxin to the auxin-starved roots resulted in accumulation of CS-IAA1 and CS-IAA3 mRNA. The level of expression of these genes increased when the auxin concentration was increased. CS-IAA1 mRNA accumulated in response to 10–8 M auxin, and the level increased further, depending on the dose. Auxin starvation did not result in a decrease in the level of CS-IAA2 mRNA; however, adding exogenous auxin at concentrations higher than 10–7 M increased its accumulation. In the primary roots responding hydrotropically or gravitropically, CS-IAA1 expression was greater on the concave side of the curving roots than on the convex side. The difference could be detected 30 min following stimulation by gravity or a moisture gradient, and that difference increased with time. These results support the idea that asymmetry of localization of auxin is associated with differential growth in hydrotropically responding roots. (Received December 20, 2001; Accepted May 2, 2002) Introduction Seedling roots respond positively to a moisture gradient and grow toward a wet substrate as shown in experiments using agravitropic roots of peas and clinorotation (Jaffe et al. 1985, Takahashi and Suge 1991, Takahashi et al. 1996). These findings verified early suggestions that plant roots display hydrotropism (Takahashi 1997a). When the gravitropic response is eliminated by mutation or clinorotation, the hydrotropic response can be detected, suggesting that the gravitropic response on Earth interferes with hydrotropism (Takahashi 1997a). In experiments carried out under low gravity conditions in space, we recently showed that the lateral roots of cucumber seedlings grew toward the wet substrate used as the seed-holding material and water source (Takahashi et al. 1999, Kamada et al. 2000). It was unusual to see lateral roots growing toward the shoot and deviating only 20–50° from the elongating axis of the primary roots. On earth, lateral roots grow diagravitropically or plagiogravitropically, deviating greater than 90° from the axis of the primary roots that grow downward. Because of these results, we have proposed that positive hydrotropism controls the growth direction of the lateral roots under conditions of microgravity because the same response occurs during clinorotation under conditions of normal gravity (Takahashi et al. 1999). The moisture gradient in the experimental chamber in experiments carried out in space was not measured; but, a relatively strong moisture gradient was probably maintained because the water-containing seed-holding material was placed at one end of the container in the absence of convection. Despite the obvious directional growth of the lateral roots under conditions of microgravity, there were no distinct hydrotropic responses detected in the primary roots of cucumber seedlings as shown previously (Takahashi et al. 1999). When the primary roots were placed parallel to the direction of moisture gradient, they grew down under stationary conditions and away from the wet substrate exhibiting a small curvature not larger than 40° on clinostat. The difference in the responses of the primary and the lateral roots is probably due to the direction of the moisture gradient established in the container. When seeds were placed vertically in the holder with the radicles pointing down, roots grew away from the wet holder. If a moisture gradient existed between the wet seed holder and the other end of the container, the primary roots would have elongated in an orientation parallel to the direction of the moisture gradient. In this situation, the root cap might be exposed to a symmetrical moisture gradient and might not be stimulated hydrotropically because this is where the sensory apparatus for detecting moisture gradients resides (Jaffe et al. 1985, Takahashi and Suge 1991, Takahashi and Scott 1993, Hirasawa et al. 1997). On the other hand, lateral roots grew perpendicular to the direction of the moisture gradient because they emerged transversely from the primary roots that grew away from the wet substrate. The root caps of the lateral roots would be exposed to an asymmetric moisture gradient. This might induce positive hydrotropism in the absence of gravitropic interference. Nevertheless, we cannot rule out the possibility that the primary roots of cucumber seedlings are hydrotropically insensitive to moisture gradients. Experiments in which the primary roots are placed parallel to the wet substrate so that the moisture gradient crosses the root axis transversely would provide information on whether the primary roots of cucumber seedlings are hydrotropically responsive. The seedling roots could also be rotated on a clinostat in the presence of a moisture gradient. Although the physiological status of the clinorotated plants may not be the same as that of the space-grown plants, it has been shown that clinostats serve as an alternative to experiments performed in space by mimicking certain growth responses such as agravitropism, hydrotropism and automorphogenesis that occur in microgravity (Brown et al. 1996, Hoson et al. 1997, Hoson et al. 1999, Takahashi et al. 1999, Ueda et al. 1999). The similarities were also observed in starch content and distribution of endoplasmic reticulum in columella cells of Lepidium (Hoson et al. 1997), polarity of plastid localization in Arabidopsis columella cells (Kraft et al. 2000) and peg formation in cucumber seedlings (Takahashi 1997b, Takahashi et al. 2000). We are now able to induce hydrotropic curvature in seedling roots as described above, but the mechanism of hydrotropism remains to be determined. Auxin is one factor that is thought to be important for the control of root gravitropism, and is considered to play an essential role in the regulation of differential growth (Cholodny 1927, Went 1928, Jackson and Barlow 1981, Okada and Shimura 1992, Bennett et al. 1996, Pilet 1996, Luschnig et al. 1998, Leyser 1999, Sabatini et al. 1999). In pea roots, differential hydrotropic growth occurs due to the differences in growth rate between the high- and low-water potential sides of the elongation zone; the reduction is greater on the higher water potential side than on the lower one (Takahashi and Suge 1991). The role of auxin in the hydrotropic response needs to be defined to enable comparison of the mechanisms for hydrotropism and gravitropism in developing roots. We previously reported that an inhibitor of auxin transport prevented both gravitropism and hydrotropism in pea roots (Takahashi and Suge 1991); however, we still need to identify the location of auxin in the roots responding hydrotropically. It has been suggested that auxin-inducible genes, such as SAUR (small auxin up-regulated RNAs), GH3, and AUX/IAA family, allow the localization of auxin to be determined in some plant species (McClure and Guilfoyle 1989, Li et al. 1991, Wong et al. 1996, Li et al. 1999). Likewise, we have isolated auxin-inducible genes (CS-IAA1, CS-IAA2 and CS-IAA3) from cucumber seedlings and used them as markers to identify the distribution of auxin in hypocotyl tissue (Fujii et al. 2000, Kamada et al. 2000). These auxin-inducible genes may be useful for analyzing auxin distribution in hydrotropically responsive roots of cucumber seedlings and also for determining auxin levels in various tissues. This is an important approach for comprehensive understanding of the role of auxin in tropisms and for clarifying the mechanisms by which auxin redistributes in response to different environmental cues such as gravity and moisture gradient. In the present study, we first rotated cucumber seedlings on a three-dimensional clinostat in the presence of a moisture gradient and examined whether the primary roots are hydrotropically responsive or not. Second, we tested whether expression analysis of the CS-IAA1 gene is useful for evaluating the levels of auxin in cucumber roots. Third, we determined whether auxin signals accumulate differentially in hydrotropically responding primary roots of cucumber seedlings. Results Hydrotropic response of the primary roots of cucumber seedlings on the clinostat In the present study, we attempted to induce hydrotropism in the primary roots of cucumber seedlings by placing them perpendicular to a moisture gradient (Fig. 1). To establish a moisture gradient, we placed a water-containing seedling holder on the top and filter paper soaked with a saturated solution of NaCl on the bottom of a growth container, which was then placed horizontally. The relative humidity (RH) at 2, 5, 10, 20, 30 and 40 mm from the water-containing seedling holder was approximately 97, 96, 95, 94, 93.5 and 93%, respectively. Thus, we obtained a relatively strong moisture gradient in the growth container. The primary roots of cucumber seedlings were placed vertically with the root-tip growing down and parallel to the water-containing seed holder. In this orientation, the roots of the stationary control grew down and hardly curved in the containers with either H2O or NaCl (Fig. 2A, 3). Clinorotated roots of cucumber seedlings, however, curved towards the wet seed-holding material; curvature of the roots 12 h after hydrostimulation in the growth chambers with H2O and NaCl was approximately 20° and 50°, respectively (Fig. 2B, 3). Root curvature was less when wet plastic foam was placed around the roots in the growth container. Fig. 4 shows the curvature kinetics of the primary roots of cucumber seedlings that were stimulated either hydrotropically or gravitropically. Roots of the stationary control did not curve but grew down even in the presence of a moisture gradient that crossed the path of the growing roots transversely. On the other hand, clinorotation caused the roots to grow with a distinct curvature towards the wet plastic foam, thus overcoming the gravitropic effect that normally results in downward growth. The curvature was detected within 3 h of clinorotation and by 12 h reached 50° (Fig. 4A). Gravitropic curvature commenced within 30 min, which was much earlier than the commencement of hydrotropic response (Fig. 4B). The rate of gravitropic bending of the primary roots was approximately 30° h–1, whereas the rate of hydrotropic bending was approximately 4° h–1. Accumulation of mRNA of CS-IAA1, CS-IAA2 and CS-IAA3 in cucumber roots We examined whether auxin-inducible genes isolated from cucumber seedlings could be used as markers for the localization and the level of auxin signals in cucumber roots (Fig. 5). The level of mRNA for CS-IAA1 decreased substantially after deprivation of auxin for 2 h and returned to a normal level following the addition of 10–8 M indole-3-acetic acid (IAA). Applying IAA at higher concentrations resulted in dramatic increases in the level of CS-IAA1 mRNA dependent of dose. The expression pattern of CS-IAA3 following auxin starvation and application of IAA was similar to that of CS-IAA1, but was less pronounced. An increase in the level of CS-IAA3 mRNA was detected following application of IAA at concentrations higher than 10–7 M, but the dose–response relationship was non-linear. In contrast, there was no decrease in the level of CS-IAA2 mRNA during auxin starvation although there was an increase in the level of mRNA following addition of IAA at concentrations higher than 10–7 M. Differential expression of CS-IAA1 in the roots of cucumber seedlings responding hydrotropically or gravitropically We next studied the possible involvement of auxin in inducing the hydrotropic response in the primary roots of cucumber, because during gravitropism the asymmetry of auxin distribution is known to play an important role in the differential growth of the roots. We also considered it important to compare the mechanisms underlying gravitropism and hydrotropism in developing roots. Fig. 6 shows the localization of CS-IAA1 mRNA in the hydrotropically responding primary roots of cucumber seedlings as analyzed by in situ hybridization. CS-IAA1 mRNA accumulated in the root tip including the elongation zone, which was stably detectable without exception. Accumulation of CS-IAA1 appeared to be greater in the vascular tissues of the roots under stationary conditions, whereas in the clinorotated roots it accumulated preferentially in the outer region of the root tip. Some preparations exhibited a differential accumulation of the mRNA between the convex and concave sides of the curving roots as shown in Fig. 6. However, the differential localization of the signals detected by the in situ hybridization was not stably observed, but varied depending on the preparation. It was not practical to conduct Northern blot analysis because only a small amount of the root-tip material for RNA isolation was available at once. We therefore performed quantitative RT-PCR Southern blot analysis to determine the expression level of CS-IAA1 and its localization. We examined the specificity of the probes used (Fig. 7A) and obtained titration curves of the RT-PCR products to determine the amount of template and the number of cycles required for quantitative analysis (Fig. 7B). We used 40 ng of RNA template in each reaction. Seventeen cycles were used for amplification of CS-IAA1 and CS-IAA2, while 12 cycles were used for CS-actin, which served as a control. Fig. 8 shows the levels of expression of CS-IAA1 and CS-IAA2 on the high- and low-water potential sides of the primary roots of cucumber seedlings grown on a clinostat or under stationary conditions in the presence of a moisture gradient. In clinorotated roots, expression of CS-IAA1 was much greater on the high-water potential (concave) side than on the low-water potential (convex) side, and the difference was obvious within 3 h of the start of treatment. On the other hand, there was no substantial difference in the expression of CS-IAA1 between the two sides of the stationary roots. There was no differential expression of CS-IAA2 in the hydrotropically responding roots. The expression level of CS-IAA1 in both clinorotated and stationary roots increased with time (Fig. 8). The increase was more apparent in the former than the latter, although we cannot explain this difference at present. When the primary roots of cucumber seedlings were stimulated gravitropically, increased expression of CS-IAA1 was observed on the lower (concave) side with a low water potential compared to the upper (convex) side with a high water potential (Fig. 9). The differential expression of CS-IAA1 in the gravistimulated roots was detected within 30 min of the start of treatment. There was no differential expression of CS-IAA1 in the control roots that grew straight down. It was thus found that multiple mechanisms exist for the induction of auxin redistribution, which ultimately regulates the differential growth of roots responding to hydrotropic and gravitropic stimuli. Discussion We previously showed that the lateral roots of cucumber seedlings grown under conditions of microgravity exhibited positive hydrotropism in the absence of interference from the gravitropic response (Takahashi et al. 1999). In this experiment, carried out in space, cucumber seeds were inserted into water-absorbing plastic foam so that the radicles grew away from the wet seedling holder. The primary roots initiated their elongation in an orientation parallel to the direction of the moisture gradient, whereas the lateral roots emerged from the primary roots in a direction perpendicular to the moisture gradient. From these results, we concluded that the hydrotropic response of the primary roots was obscure while the lateral roots responded hydrotropically. Thus, the moisture gradient in the growth container might induce hydrotropism in the lateral roots but not in the primary roots. The results of the present study verify our idea that the primary roots of cucumber seedlings respond to a moisture gradient and exhibit hydrotropism if gravitropic response is cancelled by clinorotation. The results are also consistent with our hypothesis that the direction of the moisture gradient established in the growth container was the cause of the poor hydrotropic response of the primary roots during a previous spaceflight experiment. Among the three auxin-inducible genes tested, CS-IAA1 was the most sensitive marker of auxin levels in cucumber roots. It is noteworthy that IAA concentration (10–8 M) that effectively elevated the expression level of CS-IAA1 in cucumber roots was in the same range of physiological concentrations as those required for root elongation (Pilet 1996). CS-IAA1 belongs to AUX/IAA family (Fujii et al. 2000). It has been shown that dominant mutations in several AUX/IAA genes in Arabidopsis confer pleiotropic auxin-related phenotypes (Rouse et al. 1998, Tian and Reed 1999, Nagpal et al. 2000, Rogg et al. 2001). Comparing the phenotypes of these mutants implied that the AUX/IAA genes have both redundant functions and different functions in auxin-related phenotypes (Nagpal et al. 2000). Although we do not know the function of the protein encoded by CS-IAA1, it might play a role in the auxin response to control root growth in cucumber seedlings. The roots respond differently to the application of IAA depending on the concentration applied and on the physiological status of the roots. For instance, IAA at concentrations lower than 10–8 M often stimulate root elongation, but higher concentrations inhibit elongation (Pilet and Saugy 1987, Evans et al. 1994). In maize, applying IAA stimulates the growth of the fast-growing roots but inhibits the growth of the slow-growing roots (Pilet 1996). Although the auxin concentration alone cannot account for the control of root elongation, a change in its concentration is a factor responsible for the growth rate in roots. In roots where the endogenous auxin concentration is lower than that optimal for growth, applying auxin may stimulate elongation. In contrast, applying IAA can inhibit elongation if roots contain a supra-optimal level of endogenous auxin. Thus, the role of auxin in root elongation remains to be clarified. It is not only the level of auxin but also the tissue sensitivity to auxin that needs to be considered to understand the relationship between auxin and root growth. Nevertheless, it is possible that tropistic stimulation induces a change in the auxin distribution resulting in differential growth of the roots; that is, a higher growth rate is maintained on the side with a low level of auxin, whereas inhibition of elongation occurs on the side with a high level of auxin. More CS-IAA1 mRNA was accumulated on the higher-water potential side than the lower-water potential side in hydrotropically responding roots of cucumber seedlings. This result supports the kinetics data that a greater inhibition of extension growth occurs on the side of higher-water potential than the side of lower-water potential during hydrotropic response in pea roots (Takahashi and Suge 1991). As discussed above, a higher level of auxin probably inhibits the extension growth for hydrotropic response in roots. The results are also consistent with the previous result that applying an inhibitor of auxin transport causes a reduction in hydrotropic response (Takahashi and Suge 1991). It has been shown that several auxin-inducible genes express differentially during gravitropic responses. In gravistimulated soybean hypocotyl and epicotyl, mRNAs of SAUR and GH3 genes are preferentially accumulated on the lower (convex) side (McClure and Guilfoyle 1989, Gee et al. 1991). In gravitropically bending hypocotyls of transgenic Arabidopsis to which LacZ reporter gene driven by promoter regions of AtAux2-22 that belongs to AUX/IAA family was introduced, lacZ staining was found primarily on the lower (convex) side (Wyatt et al. 1993). In transgenic Arabidopsis to which GUS reporter driven by IAA2 promoter that also belongs to AUX/IAA family was introduced, GUS staining was found primarily on the lower (concave) side of the gravistimulated roots (Luschnig et al. 1998). Our results in this study also showed that CS-IAA1 mRNA accumulated preferentially on the lower (convex) side in gravitropically bending roots. These available data suggest that auxin-inducible genes are preferentially expressed on the lower sides in both aerial organs and roots upon gravistimulation. The Cholodny-Went theory proposes that asymmetrically distributed auxin preferentially accumulates on the lower side in gravitropically responding organs (Cholodny 1927, Went 1928, Trewavas 1992). This theory hypothesizes that auxin stimulates elongation growth of aerial organs but inhibits elongation growth of roots, so that aerial organs bend upward whereas roots bend downward. The expression pattern of auxin-inducible genes in the gravistimulated roots of cucumber seedlings is consistent with the hypothesis of the Cholodny–Went theory. Thus, the results of the present study suggest that roots of cucumber seedlings redistribute auxin in response to both the moisture gradient and gravity. It is interesting that root-cap cells possess an apparatus for perception of both moisture gradient and gravity by which asymmetric distribution of auxin in the root-tips is brought about for hydrotropism and gravitropism in roots. However, it is possible that different mechanisms for auxin redistribution are involved in different tropisms. This is plausible because different proteins likely regulate auxin transport in different systems as has been recently revealed with PIN family of Arabidopsis genes encoding auxin-efflux carriers (Friml et al. 2002). The results of the present study show that (1) the primary roots of cucumber seedlings are hydrotropically responsive to a moisture gradient if the gravitropic response is reduced; (2) CS-IAA1 is useful for examining the localization of auxin in cucumber roots; and (3) auxin is redistributed during the hydrotropic response of seedling roots, which could counteract with auxin redistribution due to gravitropic response. The differential growth of the hydrotropically responsive roots is probably caused by the higher level of auxin on the high-water potential side, resulting in the inhibition of growth within the elongation zone on this side of the root. Materials and Methods Plant growth, tropistic stimulation and clinorotation To stimulate the primary roots of cucumber seedlings hydrotropically, seeds of cucumber (Cucumis sativus L.), cultivar Shinfushinarijibai, purchased from Watanabe Seed Co. (Kogota, Japan), were inserted into pockets made on a block (45×15×10 mm) of water-absorbing plastic foam as shown in Fig. 1. Seeds were fixed vertically on a slanted plastic block so that the primary roots grew down into the air. The plastic foam containing the fixed seeds was glued onto the inner surface of the plastic container (60×60×60 mm) and imbibed with 5 ml of distilled water. A sheet of filter paper with 1 ml of distilled water or saturated NaCl solution added was placed on the other side of the container to establish relatively weak and strong gradients of moisture between the wet plastic foam and the other end of the container. Root caps of the primary roots growing down thus cross transversely to the direction of the moisture gradient, which may stimulate the roots hydrotropically. Seeds were incubated at 24±1°C in the dark. When primary roots were 2-mm long after germination, the plastic containers were placed on a two-axes clinostat similar to that reported by Hoson et al. (1997) and were rotated at 2 rpm three-dimensionally. Root curvature was measured with a goniometer 12 h after the start of clinorotation or every 3 h during the time-course study. For comparison, the primary roots were gravistimulated by placing the plastic containers at 90° so that the seedlings were horizontal. Curvature of the gravistimulated roots was measured with a goniometer 30, 60, 120 and 180 min after the start of gravistimulation. The moisture gradient established in the container was confirmed by measuring the relative humidity (RH) at different places in the container with a HIM31 humidity and temperature indicator (Vaisala, Helsinki, Finland). The probe was placed inside the container through a small hole, and the gap was sealed. Expression analysis of auxin-inducible genes in cucumber roots To examine the dose effect of auxin on the expression of auxin-inducible genes in cucumber roots, we analyzed levels of mRNA of CS-IAA1, CS-IAA2 and CS-IAA3, under conditions of auxin starvation and auxin excess. Whole roots were excised from 36-h-old cucumber seedlings grown on wet filter paper at 25±1°C in the dark. Auxin treatment of cucumber roots was performed following the method of Theologis et al. (1985). To deplete endogenous auxin, the excised roots were kept for 1.5 h in 15 mM sucrose containing 50 µg ml–1 chloramphenicol, and for 30 min in an incubation buffer (1 mM citrate, 1 mM PIPES, 15 mM sucrose, 1 mM KCl, 50 µg ml–1 of chloramphenicol, pH 6.0). They were then kept in the incubation buffer with or without IAA for 2 h. After incubation, segments were frozen in liquid nitrogen and stored at –80°C until use. Total RNA was isolated from the roots of 36-h-old etiolated cucumber seedlings with TRI reagent (Sigma, Saint Louis, MO, U.S.A.) according to the method of Chomczynski and Mackey (1995). Total RNA (20 µg) was separated using formaldehyde agarose gel electrophoresis, transferred onto a nylon membrane and fixed by baking at 80°C for 30 min. Hybridization was carried out in 5× SSC, 50% formamide, 0.02% SDS, 0.1% lauroylsalcosine and 2% (w/v) blocking reagent (Boehringer Mannheim, Mannheim, Germany) with digoxigenin (DIG)-labeled RNA probes (CS-IAA1, CS-IAA2 and CS-IAA3) at 68°C. Non-hybridized probe was removed by washing in 0.2× SSC/0.1% SDS at 68°C and by RNaseA treatment. Detection of chemiluminescent signals was performed according to the manufacture’s instructions (Boehringer Mannheim). Expression of CS-IAA1 in roots of cucumber responding hydrotropically or gravitropically Primary roots of cucumber seedlings were stimulated hydrotropically or gravitropically as described above. The roots were then halved longitudinally into the stimulated (concave) side and the non-stimulated (convex) side. Total RNA was extracted from the root halves as described above. Quantitative RT-PCR was performed using QIAGENR OneStep RT-PCR kit and a specific primer set. The specific primers were: IAA1-F, 5′-ACCTCGAGATCACCGAGCTT-3′ and IAA1-R, 5′-CATCTTCCAAGGCACATCCC-3′ for CS-IAA1; IAA2-F, 5′-GGAGCACCATCCATGCTG-3′ and IAA2-R, 5′-GGAGCACCATCCATGCTG-3′ for CS-IAA2 and CS-actin-F, 5′-GACATTCAATGTGCCTGCTATG-3′ and CS-actin-R, 5′-CATACCGATGAGAGATGGCTG-3′ for CS-actin. cDNA was synthesized at 50°C for 30 min, and PCR was then carried out at 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min. The RT-PCR products were separated by electrophoresis on a 1.2% agarose gel and blotted onto a nylon membrane. The membrane was hybridized with oligonucletides labeled with DIG at the 5′ and 3′ ends. The probes used were: 5′-TTGCTCCGACTCTTGTCTTCTACTTCCACC-3′ for CS-IAA1; 5′-TATCTGGATCTCTCTCGGGGGATTCAGATC-3′ for CS-IAA2 and 5′-ACACCATCACCAGAATCCAGCACGATACCA-3′ for CS-actin. Hybridization was carried out at 60°C and the hybridization buffer, post-hybridization washes and DIG-detection method were the same as described above except for RNaseA treatment. Expression analysis of CS-IAA1 mRNA by in situ hybridization In situ hybridization was carried out according to Demura and Fukuda (1996) with several modifications. The primary roots of cucumber seedlings were stimulated hydrotropically for 3 h as described above. Then, seedlings were fixed with 4% paraformaldehyde and 0.25% glutaraldehyde (v/v) in 50 mM sodium phosphate buffer (pH 7.2) at 4°C overnight. The roots were excised, infiltrated with the same fixative buffer under vacuum for 30 min at room temperature, and incubated further 90 min without vacuum as a secondary fixation. The fixed samples were dehydrated through a graded series of ethanol and t-butanol, and were then embedded in Praffin (Paraplast Plus). To penetrate probes into sections, we treated sections (12-µm in thickness) fixed on slides with 16 µg ml–1 of proteinase K for 30 min. Hybridization was performed in a solution of 50% formamide, 300 mM NaCl, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA (pH 8.0), 1× Denhardt’s solution, 10% dextran sulfate, 60 mM DTT, 1 mg ml–1Escherichia coli tRNA, 500 µg ml–1 poly(A) and 1 µg ml–1 of DIG-labeled antisense or sense CS-IAA1 RNA probe at 50°C overnight. After hybridization, non-hybridized probes were washed out by electro-washing (Kobayashi et al. 1994). The hybridizing probe was detected colorimetrically using as anti-DIG Fab fragment conjugated to alkaline phosphatase. After sections were dehydrated through a graded series of ethanol and treated with xylene, DIATEX (Matsunami Glass, Osaka) and a cover slip were applied to the specimen for microscopy. Photographs were taken using an OLYMPUS BX50F microscope. Acknowledgments This work was financially supported by a Grant-in-Aid (13460113) from the Ministry of Education, Culture, Sports and Technology of Japan, the Ground Research Announcement for Space Utilization promoted by NASDA and the Japan Space Forum, and by a grant from the Institute of Space and Astronautical Science (Sagamihara, Japan). 3 Corresponding author: E-mail, hideyuki@ige.tohoku.ac.jp; Fax, +81-022-723-8218. View largeDownload slide Fig. 1 Containers used for changing the moisture gradient to induce the hydrotropic response in primary roots of cucumber seedlings. (A) normal moisture gradient; (B) moisture gradient strengthened by a saturated solution of NaCl; (C) moisture gradient weakened by wet filter paper and water-absorbing foam. View largeDownload slide Fig. 1 Containers used for changing the moisture gradient to induce the hydrotropic response in primary roots of cucumber seedlings. (A) normal moisture gradient; (B) moisture gradient strengthened by a saturated solution of NaCl; (C) moisture gradient weakened by wet filter paper and water-absorbing foam. View largeDownload slide Fig. 2 Effect of clinorotation on the hydrotropic response of primary roots of cucumber seedlings. Seeds were placed vertically in an aeroponic culture system with the radicle pointing down. Seedlings were grown under stationary conditions (A) or on a clinostat (B) for 12 h in the presence of the moisture gradient. The letter g indicates the direction of the gravitational force. View largeDownload slide Fig. 2 Effect of clinorotation on the hydrotropic response of primary roots of cucumber seedlings. Seeds were placed vertically in an aeroponic culture system with the radicle pointing down. Seedlings were grown under stationary conditions (A) or on a clinostat (B) for 12 h in the presence of the moisture gradient. The letter g indicates the direction of the gravitational force. View largeDownload slide Fig. 3 Effect of clinorotation on the hydrotropic response of primary roots of cucumber seedlings. The moisture gradient was produced using a saturated solution of NaCl. Root curvature was measured 12 h after the start of clinorotation. Data represent the mean ± SE. View largeDownload slide Fig. 3 Effect of clinorotation on the hydrotropic response of primary roots of cucumber seedlings. The moisture gradient was produced using a saturated solution of NaCl. Root curvature was measured 12 h after the start of clinorotation. Data represent the mean ± SE. View largeDownload slide Fig. 4 Curvature kinetics for hydrotropism (A) and gravitropism (B) of the primary roots of cucumber seedlings. Data represent the mean ± SE. View largeDownload slide Fig. 4 Curvature kinetics for hydrotropism (A) and gravitropism (B) of the primary roots of cucumber seedlings. Data represent the mean ± SE. View largeDownload slide Fig. 5 Northern blot analysis of the mRNA accumulation of the auxin-inducible genes, CS-IAA1, CS-IAA2 and CS-IAA3, in cucumber roots. Total RNA was isolated from the excised root sections that had not undergone any treatment (No treatment), after auxin starvation for 2 h (After auxin starvation) and 4 h (0 M IAA), and after treatment of the auxin-starved roots with exogenous IAA at different concentrations (10–12 M to 10–4 M) for a further 2 h. Total RNA (20 µg) was blotted onto a nylon membrane and hybridized with the indicated DIG-labeled probes. EtBr indicates equal loading of total RNA with ethidium bromide staining. View largeDownload slide Fig. 5 Northern blot analysis of the mRNA accumulation of the auxin-inducible genes, CS-IAA1, CS-IAA2 and CS-IAA3, in cucumber roots. Total RNA was isolated from the excised root sections that had not undergone any treatment (No treatment), after auxin starvation for 2 h (After auxin starvation) and 4 h (0 M IAA), and after treatment of the auxin-starved roots with exogenous IAA at different concentrations (10–12 M to 10–4 M) for a further 2 h. Total RNA (20 µg) was blotted onto a nylon membrane and hybridized with the indicated DIG-labeled probes. EtBr indicates equal loading of total RNA with ethidium bromide staining. View largeDownload slide Fig. 6 CS-IAA1 mRNA accumulation was analyzed by in situ hybridization in cucumber roots. Primary roots of cucumber seedlings were, in the presence of moisture gradient, grown under stationary conditions (A and B) and on clinostat (C and D). The roots grew down under stationary conditions, and the clinorotated roots curved toward the side of high water potential. Hybridization with anti-sense (A and C) or sense (B and D) probe indicates the CS-IAA1 mRNA accumulation in the root tips. View largeDownload slide Fig. 6 CS-IAA1 mRNA accumulation was analyzed by in situ hybridization in cucumber roots. Primary roots of cucumber seedlings were, in the presence of moisture gradient, grown under stationary conditions (A and B) and on clinostat (C and D). The roots grew down under stationary conditions, and the clinorotated roots curved toward the side of high water potential. Hybridization with anti-sense (A and C) or sense (B and D) probe indicates the CS-IAA1 mRNA accumulation in the root tips. View largeDownload slide Fig. 7 Quantitative RT-PCR Southern blot analysis of the response of cucumber AUX/IAA genes to auxin. Total RNA was isolated from roots after gravistimulation for 1 h. (A) Specific detection of CS-IAA1, CS-IAA2 and CS-actin mRNAs using RT-PCR and corresponding hybridization probes. Numbers on the left indicate the lengths of the RT-PCR products in base pairs. (B) Titration curves of RT-PCR products. Signal intensities of RT-PCR products are expressed as the arbitrary logarithmic values plotted against template amounts. Total RNA from roots after gravistimulation for 1 h was amplified by RT-PCR and hybridized with DIG-labeled CS-IAA1, CS-IAA2 and CS-actin oligonucleotides. PCR amplification for CS-IAA1and CS-IAA2 was performed for 17 cycles. PCR amplification for CS-actin was performed for 12 cycles. View largeDownload slide Fig. 7 Quantitative RT-PCR Southern blot analysis of the response of cucumber AUX/IAA genes to auxin. Total RNA was isolated from roots after gravistimulation for 1 h. (A) Specific detection of CS-IAA1, CS-IAA2 and CS-actin mRNAs using RT-PCR and corresponding hybridization probes. Numbers on the left indicate the lengths of the RT-PCR products in base pairs. (B) Titration curves of RT-PCR products. Signal intensities of RT-PCR products are expressed as the arbitrary logarithmic values plotted against template amounts. Total RNA from roots after gravistimulation for 1 h was amplified by RT-PCR and hybridized with DIG-labeled CS-IAA1, CS-IAA2 and CS-actin oligonucleotides. PCR amplification for CS-IAA1and CS-IAA2 was performed for 17 cycles. PCR amplification for CS-actin was performed for 12 cycles. View largeDownload slide Fig. 8 RT-PCR Southern blot analysis of the expression of CS-IAA1 and CS-IAA2 in hydrotropically stimulated roots of cucumber seedlings. Forty ng of total RNA obtained from the longitudinally halved roots following hydrotropic stimulation was amplified by RT-PCR and hybridized with DIG-labeled probes specific for CS-IAA1, CS-IAA2 and CS-actin. PCR amplification was performed for 17 cycles for CS-IAA1 and CS-IAA2 and for 12 cycles for CS-actin. HWP indicates the side of high-water potential, and LWP indicates the side of low-water potential. In the hydrotropically bending roots, the sides of HWP and LWP correspond to the concave and convex sides, respectively. The number above each band represents the relative intensity by the mean of three independent experiments. View largeDownload slide Fig. 8 RT-PCR Southern blot analysis of the expression of CS-IAA1 and CS-IAA2 in hydrotropically stimulated roots of cucumber seedlings. Forty ng of total RNA obtained from the longitudinally halved roots following hydrotropic stimulation was amplified by RT-PCR and hybridized with DIG-labeled probes specific for CS-IAA1, CS-IAA2 and CS-actin. PCR amplification was performed for 17 cycles for CS-IAA1 and CS-IAA2 and for 12 cycles for CS-actin. HWP indicates the side of high-water potential, and LWP indicates the side of low-water potential. In the hydrotropically bending roots, the sides of HWP and LWP correspond to the concave and convex sides, respectively. The number above each band represents the relative intensity by the mean of three independent experiments. View largeDownload slide Fig. 9 RT-PCR Southern blot analysis of the expression of CS-IAA1 and CS-IAA2 in gravitropically stimulated roots of cucumber seedlings. Forty ng of total RNA obtained from the longitudinally halved roots following gravitropic stimulation was amplified by RT-PCR and hybridized with DIG-labeled probes specific for CS-IAA1, CS-IAA2 and CS-actin. PCR amplification was performed for 17 cycles for CS-IAA1 and CS-IAA2 and for 12 cycles for CS-actin. HWP indicates the side of high-water potential and LWP indicates the side of low-water potential. In the gravitropically bending roots, the sides of LWP (Lower) and HWP (Upper) correspond to the concave and convex sides, respectively. The number above each band represents the relative intensity by the mean of three independent experiments for CS-IAA1 and CS-actin, and two independent experiments for CS-IAA2. View largeDownload slide Fig. 9 RT-PCR Southern blot analysis of the expression of CS-IAA1 and CS-IAA2 in gravitropically stimulated roots of cucumber seedlings. Forty ng of total RNA obtained from the longitudinally halved roots following gravitropic stimulation was amplified by RT-PCR and hybridized with DIG-labeled probes specific for CS-IAA1, CS-IAA2 and CS-actin. PCR amplification was performed for 17 cycles for CS-IAA1 and CS-IAA2 and for 12 cycles for CS-actin. HWP indicates the side of high-water potential and LWP indicates the side of low-water potential. In the gravitropically bending roots, the sides of LWP (Lower) and HWP (Upper) correspond to the concave and convex sides, respectively. The number above each band represents the relative intensity by the mean of three independent experiments for CS-IAA1 and CS-actin, and two independent experiments for CS-IAA2. References Bennett, M.J., Marchant, A., Green, H.G., May, S.T., Ward, S.P., Millner, P.A., Walker, A.R., Schulz, B. and Feldmann, K.A. ( 1996) Arabidopsis AUX1 gene: a permease-like regulator of root gravitropism. Science  273: 948–950. Google Scholar Brown, A.H., Johnsson, A., Chapman, D.K. and Heathcote, D. ( 1996) Gravitropic response of the Avena coleoptile in space and on clinostats. IV. The clinostat as a substitute for space experiments. Physiol. Plant.  98: 210–214. Google Scholar Cholodny, N. ( 1927) Wuchshormone und Tropismen bei den Pflanzen. Biol. Zentralbl.  47: 604–629. Google Scholar Chomczynski, P. and Mackey, K. ( 1995) Modification of the TRI reagent™ procedure for isolation of RNA from polysaccharide- and proteoglycan-rich sources. BioTechiques  19: 942–945. Google Scholar Demura, T. and Fukuda, H. ( 1996) In situ hybridization to cellular RNA using 35S-labelled cRNA probes. Plant Tissue Cult. Lett.  13: 343–349. Google Scholar Evans, M.L., Ishikawa, H. and Estelle, M.A. ( 1994) Responses of Arabidopsis roots to auxin studied with high temporal resolution: comparison of wild-type and auxin-response mutants. Planta  194: 215–222. Google Scholar Fujii, N., Kamada, M., Yamasaki, S. and Takahashi, H. ( 2000) Differential accumulation of Aux/IAA mRNA during seedling development and gravity response in cucumber (Cucumis sativus L.). Plant Mol. Biol.  42: 731–740. Google Scholar Friml, J., Wisniewska, J., Benková, E., Mendgen, K. and Palme, K. ( 2002) Lateral relocation of auxin efflux regulator PIN3 mediates tropism in Arabidopsis. Nature  415: 806–809. Google Scholar Gee, M.A., Hagen, G. and Guilfoyle, T.J. ( 1991) Tissue-specific and organ-specific expression of soybean auxin-responsive transcripts GH3 and SAURs. Plant Cell  3: 419–430. Google Scholar Hirasawa, T., Takahashi, H., Suge, H. and Ishihara, K. ( 1997) Water potential, turgor and cell wall properties in elongating tissues of the hydotropically bending roots of pea (Pisum sativum L.). Plant Cell Environ.  16: 99–103. Google Scholar Hoson, T., Kamisaka, S., Masuda, Y., Yamashita, M. and Buchen, B. ( 1997) Evaluation of the three-dimensional clinostat as a simulator of weightlessness. Planta  203: S 187–197. Google Scholar Hoson, T., Soga, K., Mori, Y., Saiki, M., Wakabayashi, K., Kamisaka, S., Kamigaichi, S., Aizawa, S., Yoshizaki, I., Mukai, C., Shimazu, T., Fukui, K. and Yamashita, M. ( 1999) Morphogenesis of rice and Arabidopsis seedlings in space. J. Plant Res.  112: 477–486. Google Scholar Jackson, M.B. and Barlow, P.W. ( 1981) Root geotropism and the role of growth regulators from the cap: a re-examination. Plant Cell Environ.  4: 107–123. Google Scholar Jaffe, M.J., Takahashi, H. and Biro, R.L. ( 1985) A pea mutant for the study of hydrotropism in roots. Science  230: 445–447. Google Scholar Kamada, M., Fujii, N., Aizawa, S., Kamigaichi, S., Mukai, C., Shimazu, T. and Takahashi, H. ( 2000) Control of gravimorphogenesis by auxin: accumulation pattern of CS-IAA1 mRNA in cucumber seedlings grown in space and on the ground. Planta  211: 493–501. Google Scholar Kobayashi, S., Saito, S. and Okada, M. ( 1994) A simplified and efficient method for in situ hybridization to whole Drosophila embryos, using electrophoresis for removing non-hybridized probes. Dev. Growth Differ.  36: 629–632. Google Scholar Kraft, T.F.B., van Loon, J.J.W. and Kiss, J.Z. ( 2000) Plastid position on Arabidopsis columella cells is similar in microgravity and on a random-positioning machine. Planta  211: 415–422. Google Scholar Leyser, O. ( 1999) Plant hormones: ins and outs of auxin transport. Curr. Biol.  9: R 8–R10. Google Scholar Li, Y., Hagen, G. and Guilfoyle, T.J. ( 1991) An auxin-responsive promoter is differentially induced by auxin gradients during tropisms. Plant Cell  3: 1167–1175. Google Scholar Li, Y., Wu, Y.H., Hagen, G. and Guilfoyle, T. ( 1999) Expression of the auxin-inducible GH3 Promoter/GUS fusion gene as a useful molecular marker for auxin physiology. Plant Cell Physiol.  40: 675–682. Google Scholar Luschnig, C., Gaxiola, R.A., Grisafi, P. and Fink, G.R. ( 1998) EIR1, a root-specific protein involved in auxin transport, is required for gravitropism in Arabidopsis thaliana. Genes Dev.  12: 2175–2187. Google Scholar McClure, B.A. and Guilfoyle, T.J. ( 1989) Rapid redistribution of auxin-regulated RNAs during gravitropism. Science  243: 91–93. Google Scholar Nagpal, P.L., Walker, L.M., Young, J.C., Sonawala, A., Timpte, C., Eselle, M. and Reed, J.W. ( 2000) AXR2 encodes a member of the Aux/IAA protein family. Plant Physiol.  123: 563–574. Google Scholar Okada, K. and Shimura, Y. ( 1992) Mutational analysis of root gravitropism and phototropism of Arabidopsis thaliana seedlings. Aust. J. Plant Physiol.  19: 439–448. Google Scholar Pilet, P.-E. ( 1996) Root growth and gravireaction: A reexamination of hormone and regulator implications. In Plant Roots. The Hidden Half. Edited by Waisel, Y., Eshel, A. and Kafkafi, U. pp. 285–305. Marcel Dekker, New York, Basel, Hong Kong. Google Scholar Pilet, P.-E. and Saugy, M. ( 1987) Effect on root growth of endogenous and applied IAA and ABA: A critical reexamination. Plant Physiol.  83: 33–38. Google Scholar Rogg, L.E., Lasswell, J. and Bartel, B. ( 2001) A gain-of-function mutation in IAA28 suppresses lateral root development. Plant Cell  13: 465–480. Google Scholar Rouse, D., Mackay, P., Stirnberg, P., Estelle, M. and Leyser, O. ( 1998) Changes in auxin response from mutations in an AUX/IAA gene. Science  279: 1371–1373. Google Scholar Sabatini, S., Beis, D., Wolkenfelt, H., Murfett, J., Guilfoyle, T., Malamy, J., Benfey, P., Leyser, O., Bechtold, N., Weisbeek, P. and Scheres, B. ( 1999) An auxin-depended distal organizer of pattern and polarity in the Arabidopsis root. Cell  99: 463–472. Google Scholar Takahashi, H. ( 1997) Hydrotropism: the current state of our knowledge. J. Plant Res.  110: 163–169. Google Scholar Takahashi, H. ( 1997) Gravimorphogenesis: gravity-regulated formation of the peg in cucumber seedlings. Planta  193: S 580–584. Google Scholar Takahashi, H., Kamada, M., Yamazaki, Y., Fujii, N., Higashitani, A., Aizawa, S., Yoshizaki, I., Kamigaichi, S., Mukai, C., Shimazu, T. and Fukui, K. ( 2000) Morphogenesis in cucumber seedlings is negatively controlled by gravity. Planta  210: 515–518. Google Scholar Takahashi, H., Mizuno, H., Kamada, M., Fujii, N., Higashitani, A., Kamigaichi, S., Aizawa, S., Mukai, C., Shimazu, T., Fukui, K. and Yamashita, M. ( 1999) A spaceflight experiment for the study of gravimorphogenesis and hydrotropism in cucumber seedlings. J. Plant Res.  112: 497–505. Google Scholar Takahashi, H. and Scott, T.K. ( 1993) Intensity of hydrostimulation for the induction of root hydrotropism and its sensing by the root cap. Plant Cell Environ.  16: 99–103. Google Scholar Takahashi, H. and Suge, H. ( 1991) Root hydotropism of an agravitropic pea mutant, ageotropum. Physiol. Plant.  82: 24–31. Google Scholar Takahashi, H., Takano, M., Fujii, N., Yamashita, M. and Suge, H. ( 1996) Induction of hydrotropism in clinorotated seedling roots of Alaska pea, Pisum sativum L. J. Plant Res.  110: 163–169. Google Scholar Theologis, A., Huynh, T.V. and Davis, R.W. ( 1985) Rapid induction of specific mRNAs by auxin in pea epicotyl tissue. J. Mol. Biol.  183: 53–68. Google Scholar Tian, O. and Reed, J.W. ( 1999) Control of auxin-regulated root development by the Arabidopsis thaliana SHY2/IAA3 gene . Development  126: 711–721. Google Scholar Trewavas, A.J. ( 1992) What remains of the Cholodny-Went theory? Plant Cell Environ.  15: 761–794. Google Scholar Ueda, J., Miyamoto, K., Yuda, T., Hoshino, T., Fujii, S., Mukai, C., Kamigaichi, S., Aizawa, S., Yoshizaki, I., Shimazu, T. and Fukui, K. ( 1999) Growth and development, and auxin polar transport in higher plants under microgravity conditions in space: BRIC-AUX on STS-95 space experiment. J. Plant Res.  112: 487–492. Google Scholar Went, W.F. ( 1928) Wuchsstoff und Wachstum. Recl. Trav. Bot. Neerl.  25: 1–116. Google Scholar Wong, L.M., Abel. S., Shen, N., Foata, M., Mall, Y. and Theologis, A. ( 1996) Differential activation of the primary auxin response genes, PS-IAA4/5 and PS-IAA6, during early plant development. Plant J.  9: 587–599. Google Scholar Wyatt, R.E., Ainley, W.M., Nagao, R.T., Conner, T.W. and Key, J.L. ( 1993) Expression of the Arabidopsis AtAux2-11 auxin responsive gene in transgenic plants. Plant Mol. Biol.  22: 731–749. Google Scholar
TI - Hydrotropic Response and Expression Pattern of Auxin-Inducible Gene, CS-IAA1, in the Primary Roots of Clinorotated Cucumber Seedlings
JF - Plant and Cell Physiology
DO - 10.1093/pcp/pcf093
DA - 2002-07-15
UR - https://www.deepdyve.com/lp/oxford-university-press/hydrotropic-response-and-expression-pattern-of-auxin-inducible-gene-cs-YWS2b3d0BF
SP - 793
EP - 801
VL - 43
IS - 7
DP - DeepDyve
ER -