TY - JOUR
AU - Isogai, Akira
AB - Abstract Using an X-ray microanalysis system fitted with variable-pressure scanning electron microscopy, we noted that many calcium crystals accumulated under the stomium in the anther of Petunia. When the anther was dehisced and pollen grains were released from the stomata, the calcium crystals adhered to pollen grains and moved to the stigma together with pollen grains. In contrast, an X-ray microanalysis of the stigma surface before pollination detected no calcium emission on the stigma surface. Furthermore, pollen germination and pollen tube growth in medium without Ca occurred as in complete medium. However, after the pollen grains had been washed with abundant germination medium without calcium, pollen germination in the medium without Ca was inhibited. These results show that the calcium crystals dissolved in the aqueous drop under the exudate on the stigma and supplied calcium ions for pollen germination. In addition, calcium crystals were produced not only in the anther of Petunia but also in Nicotiana, suggesting that calcium crystals supply pollen grains with the calcium ions required for pollen germination and serve to improve reproduction efficiency in Solanaceae. (Received September 1, 2003; Accepted October 21, 2003) Introduction Several sequential steps occur from the anthesis to the fertilization of flowering plants. First, many flowering plants release pollen grains from the anther face. Next, the pollen grains adhere to the stigma surface aided by wind and insect species. On the stigma face, a pollen grain hydrates and germinates to produce a pollen tube, which enters the style. Finally, the sperm in the pollen tube encounters the egg cells and fertilization is achieved. Through the recognition system between pollen grains and stigma, or between pollen tubes and the style, only fertilization with a compatible pollen grain is achieved. However, the mechanism by which stigma recognizes a pollen grain as compatible is not clear. Pollen grains of most species normally germinate and grow a tube in a solution containing calcium, boron, and an osmoticant such as sucrose (Taylor and Hepler 1997). The absence of calcium in the growth medium results in morphological abnormalities such as coiling and swelling of the tip (Shivanna and Rangaswamy 1992). The tip region of the pollen tube exhibits pulsating growth, and changes in calcium concentration in the tip region are correlated with the growth of the pollen tube in an activating or an inhibiting form (Franklin-Tong et al. 1993, Malho’ et al. 1994, Pierson et al. 1994, Geitmann and Cresti 1998, Franklin-Tong 1999). In a growing pollen tube, the tip has a high calcium ion gradient, while calcium ions distribute uniformly in a non-growing tube (Pierson et al. 1996). The calcium gradient in the tip is formed by the influx of calcium ions from the external medium. These results show that calcium ions in the external medium play an important role in pollen germination and tube growth. Furthermore, calcium ions should function not only in in vitro germination and growth but also in in vivo germination during pollination process on the stigma. Namely, calcium ions required for pollen germination and pollen tube growth should be incorporated into the pollen tube from the stigma and the transmitting duct of the style (Shivanna and Rangaswamy 1992). However, how calcium is supplied for pollen germination on the stigma has remained unclear. The stigma surface is as variable as other morphological features of flowering plants. The stigma surface can be classified into dry stigma and wet stigma. The dry stigma has little or no surface secretion at maturity, whereas the wet stigma has a distinct surface secretion. In general, pollen from plants with dry stigmas tends to not readily germinate, while pollen from plants with wet stigmas tends to easily germinate in liquid or semi-solid media (Heslop-Harrison and Shivanna 1977). In addition, the stigma characteristics are related to the physiology of self-incompatibility (SI) systems in flowering plants. Thus, families known to have sporophytic self-incompatibility systems belong to the dry stigma group. In this system, the behavior of pollen is determined by the genotype of the pollen parent, and the inhibition of pollen germination and tube growth occurs at the stigma surface. Conversely, families with gametophytic self-incompatibility systems, in general, have wet stigmas. In this SI system, pollen behavior is determined by the gametophytic (haploid) genotype. Pollen germination occurs at the stigma surface, and pollen tube growth is inhibited in the style. Thus, physiological differences in stigma surface and pollen type affect pollen germination and pollen tube growth. However, the physiological difference between wet-stigma and dry-stigma plants has not been elucidated. In Brassica, which has a dry-type stigma and a sporophytic self-incompatibility system, we previously demonstrated, using an X-ray microanalysis (EDX) system, that calcium concentration was increased near the surface of the papillar cell after cross-pollination (Iwano et al. 1999). Then, it was suggested that calcium required for germination of the pollen tube was supplied from the papillar cell. In contrast, Petunia, one of the Solanaceae species, has a wet-type stigma and a gametophytic self-incompatibility system. In Petunia, however, the means by which calcium is supplied for pollen germination has remained unknown. In this study, in order to clarify how calcium is supplied during the pollination process in Petunia, we examined the distribution of calcium during pollination using an EDX system fitted with the variable-pressure scanning electron microscopy (VP-SEM). First, stigma and pollen grains before pollination were X-ray microanalyzed. Second, a pollinated stigma was microanalyzed, and then we discovered that calcium crystals from pollen grains exist on the pollinated-stigma surface. Third, the anther was microanalyzed and calcium crystals were shown to come from the anther stomium. Furthermore, the role of calcium crystals for pollen germination and pollen tube growth was examined by an in vitro germination assay. Results X-ray microanalysis in VP-SEM In order to examine the amount of Ca contained in a pollen grain, X-ray microanalysis of pollen grains was performed. Pollen grains were spherical and had a fine reticulated pattern formed by the baculae on the surface (Fig. 1a). A portion of a pollen grain, a square of about 7 µm, was analyzed using the EDX system. Emission of Mg-Kα, P-Kα, S-Kα, K-Kα, and Ca-Kα was detected in addition to the Kα emissions of C and O, which are constituent elements of biological materials, as shown in Fig. 1b. The intensity of the emission of P-Kα was greater than that of S-Kα, K-Kα, Ca-Kα, and Ca-Kβ. These spectrum patterns were reproducible. Moreover, there were many crystal-like grains around the pollen (Fig. 1a). Interestingly, X-ray microanalysis of the grains detected intense emission of C-Kα, O-Kα, and Ca-Kα and weak emission of K-Kα (Fig. 1c). In order to examine the distribution of crystal-like grains in the anther, the dehisced anther was observed by SEM (Fig. 2a). As shown in Fig. 2b, many crystal-like grains accumulated at the dissilient part of the anther. To examine where the crystal-like grains were produced, anthers from young buds and a bud just before dehiscence were observed. At first, the anther from a 7 mm long bud, which belongs to the microspore stage, was fixed with glutaraldehyde and osmium and embedded in Spurr’s resin. Semi-thin sections were cut and observed with light microscopy (LM) and SEM. In LM, the tapetum, connective tissue, and immature pollen grains in the locule were observed (Fig. 3a). In SEM, crystal-like grains were observed as white and high-contrast material in some connective tissue cells (an arrowhead in Fig. 3b) and in cells beneath the stomium. In higher magnification of the cells beneath the stomium, crystal-like grains existed in the vacuole (a white arrow in Fig. 3c). Furthermore, in the cross section of the anther without fixation, crystal-like grains existed in connective tissue (arrowhead in Fig. 3d) and in cells beneath the stomium (white arrow in Fig. 3d). X-ray microanalysis of the crystal-like grains showed that the X-ray spectrum was the same as that observed around pollen grains (data not shown). In the anther from a bud just before dehiscence, many crystal-like grains accumulated beneath the stomium (Fig. 3e, f), whereas few crystal-like grains were observed around the pollen. Therefore, it was shown that the crystal-like grains come from cells beneath the stomium, but not from the tapetum. The crystal-like grains in the anther and pollen grains were observed not only in Petunia, but also in Nicotianatabacum (data not shown). The stigma was X-ray microanalyzed before and after pollination to examine how Ca was supplied during the pollination. In the stigma before pollination and just after flowering, many papilla cells existed and the oily exudate surrounded the base of all papilla cells (Fig. 4a, b). The oily area of the stigma surface, which was a square of about 7 µm, was analyzed. Fig. 5a shows the X-ray spectrum of the stigma surface. In addition to the emission of Al-Kα coming from the stub, only emissions of C-Kα and O-Kα were detected. Next, after the pollen grains from SD1-homozygotes were attached to the SB1 stigma, the cross-pollinated stigma was microanalyzed. On the surface of the stigma right after pollination, many pollen grains were observed in the oily exudates and white crystal-like grains around pollen were visualized (Fig. 4c). In an X-ray microanalysis of the area, the emission of P-Kα, S-Kα, K-Kα, and Ca-Kα was detected (Fig. 5b). Although the pattern of P-Kα, S-Kα, and K-Kα emission was similar to that of the pollen in Fig. 2a, Ca-Kα emission was higher than that in pollen. The emission of Ca would come from both pollen grains and crystal-like grains. At 6 h after pollination, many pollen grains were embedded in secretion products, and crystal-like grains were not observed on the stigma surface. However, the intensity of P-Kα, S-Kα, K-Kα, and Ca-Kα emission was not so different from that just after pollination (data not shown). As a Ca increase from stigma cells was not detected during pollination in Petunia, Ca required for germination of Petunia pollen would come from crystal-like grains around pollen grains. Pollen germination assay Almost all pollen grains germinated on the complete culture medium, and the pollen tube grew normally (Fig. 6a). When Ca or K was omitted from the complete culture medium, pollen germination and pollen tube growth still occurred normally, as in the complete medium (Fig. 6b, c). Conversely, in medium without Mg, pollen tube growth was abnormal, although many pollen grains germinated (data not shown). In medium without B, pollen germination and pollen tube growth were abnormal in both conditions, with or without Ca, K, and Mg (data not shown). Therefore, Ca and K in the culture medium are not indispensable for in vitro germination of pollen grains in Petunia, while B is indispensable. However, when pollen grains were mounted on the medium without Ca, after the pollen grains had been washed with abundant germination medium without Ca, pollen germination and pollen tube growth were inhibited (Fig. 6d). Thus, Ca required for pollen germination was not supplied from the pollen cytoplasm, but rather from the calcium-crystal around the pollen. Discussion Using an X-ray microanalysis system fitted with VP-SEM, we observed that many calcium crystals accumulated under the stomium in the anther of Petunia. When the anther was dehisced and pollen grains were released from the stomata, the calcium crystals adhered to pollen and moved with pollen grains to the stigma. Finally, the calcium crystals translocated to the stigma together with pollen grains. For in vitro pollen germination and pollen tube growth, an optimal concentration of calcium ions in the extracellular germination medium is required (Steer and Steer 1989). Calcium ions in the extracellular medium are incorporated by a calcium channel to the pollen tube and form a tip-to-base calcium gradient, which regulates the direction of pollen growth. Calcium ions are also used for the synthesis of pectin below the pollen tube tip. In this study, the pollen with the crystals germinated, and the pollen tube grew normally in a pollen germination medium without calcium ions, while pollen germination was inhibited after the crystals were washed away. These findings indicate that calcium crystals in the anther play an important role in pollen germination and pollen tube growth in Petunia. The stigma of Petunia is of the wet-type and is covered with the continuous oil phase of hydrophobic exudates from secretion cells. The major component of the exudates is triglycerides (Konar and Linskens 1966, Cresti et al. 1986). X-ray microanalysis of the stigma surface before pollination revealed little calcium in the hydrophobic exudates. Just after pollination, calcium crystals were observed with pollen grains on the stigma surface, and Ca-Kα emission was detected. At 6 h after pollination, the intensity of Ca-Kα emission at the stigma surface was the same as that just after pollination. In Solanaceae, aqueous drops on the cell surface beneath the oily exudate supply the water for pollen germination to pollen (Lush et al. 1998, Lush et al. 2000). The calcium crystals may dissolve into aqueous drops and supply the pollen with the calcium ion needed for germination. The relative intensity of P-Kα, S-Kα, K-Kα, and Ca-Kα in Petunia pollen was similar to that in Brassica. In Brassica, it is suggested that the calcium required for pollen germination and pollen tube growth comes from the papilla cells of the stigma (Elleman and Dickinson 1999, Iwano et al. 1999). In Petunia, the increase in calcium concentration on the stigma, did not come from the stigma, but instead from calcium crystals. Although Bednarska and Butowt reported that calcium exists between the exine on the pollen surface of Petunia using pyroantimonate and autoradiographic methods, and speculated that calcium for germination comes from pollen (Bednarska and Butowt 1994), our data suggests that Ca for germination comes from crystals around the pollen. On the other hand, calcium was localized in the intercellular matrix of the transmitting tract (Bednarska and Butowt 1995a, Bednarska and Butowt 1995b). The calcium ions required for pollen tube growth after germination may be supplied from the transmitting tract and not from calcium crystals. In many plants, calcium crystals such as calcium oxalate and calcium carbonate occur in most organs and tissues (Franceschi and Horner 1980, Heslop-Harrison and Heslop-Harrison 1996). Although the metabolic pathway of oxalic acid production has long been studied (Hodgkinson 1977), the physiological function of calcium crystals in the plant has not been fully characterized. Oxalate is generally considered to be an endproduct of metabolism, and most oxalate was combined with Ca as an insoluble Ca salt. In the root of the quatic plant Lemna, an induced Ca deficiency caused a decrease in insoluble oxalate, while soluble oxalate increased and total oxalate only slowly declined over time (Franceschi and Horner 1980). Furthermore, Franceschi reported in Lemna that the increase in ambinent calcium rapidly induced vacuolar Ca crystals, whereas many crystals were solubilized in 3–5 h in a calcium-free medium (Franceschi 1989). This suggested that ionic Ca was in equilibrium with less soluble forms of Ca, such as calcium oxalate, and that Ca accumulated in conditions of excess Ca influx, while Ca crystal is mobilized for the Ca demand. Calcium oxalate crystal formation in plant cells would not be a simple precipitation process, but would instead involve cellular specialization. Crystals also occur in the anther of Helianthus annuus (Horner 1977) and Lycopersicon esculentum (Bonner and Dickinson 1989). During the developmental process of the anther, Ca crystals may participate in an intracellular regulatory system, as suggested in Lemna. In aged anther, Ca crystals may function as a means of removing excess oxalic acid from the plant system. In this study, however, data indicate that calcium crystals function to supply calcium ions in pollen germination and are directly related to the mechanism of pollination and fertilization in Petunia. Interestingly, the presence of calcium crystals was also observed in the anther of Nicotiana tabacum, another Solanaceae species. Therefore, calcium crystals in the anther may play a specific role in the pollination and germination of Solanaceae. Materials and Methods Petunia hybrida Vilm. PB line and Nicotianatabacum were grown in a greenhouse at 25°C. The Petuniahybrida PB is a self-incompatible diploid, and SB1- or SD2-homozygotes were used (Entani et al. 1999). The freshly opened flowers of the SB1-homozygote, whose anthers were not dehisced, were excised and stuck to an agar plate, and the anthers were then removed from the flowers. In cross-pollination, pollen grains from freshly dehisced anthers of the SD2-homozygote were attached to the SB1 stigma. For self-pollination, pollen grains from the SB1-homozygote were attached. The pollinated stigma was subsequently micro-analyzed after either 30 min or 3 h. For the observation and X-ray analysis in a VP-SEM (Hitachi, S-3500N, Tokyo, Japan), the pistil was excised from the flower and glued to the aluminum stub. Pollen grains were attached to the aluminum stub with double-sided sticky carbon tape. The aluminum stub was mounted on the cooling stage, which was set to a temperature of –10°C. The specimen was observed and analyzed fitted to a VP-SEM with an EDX (Horiba, EMAX-7000, Kyoto, Japan). The chamber pressure was 30 Pa. The accelerating voltage was 15 kV, and data was collected for 50 live seconds. Regions of interest were defined for P, S, K, and Ca as follows: P, 1.94–2.07 kev; S, 2.24–2.37 kev; K, 3.24–3.38 kev; and Ca, 3.61–3.76 kev. For morphological observation, the anthers of 20–40 mm long buds were fixed in 2.5% glutaraldehyde in a cacodylate buffer, pH 7.2, at 4°C for 2 h. These were washed in the same buffer, post-fixed in 1% osmium tetroxide at 25°C for 2 h, dehydrated with ethanol, and embedded in Spurr’s resin. Semi-thin sections (500 nm thick) were mounted on a cover glass, stained with toluidine blue, and observed by a light microscopy (Zeiss, Axiophoto 2). Furthermore, some sections on a cover glass were microanalyzed without staining using the EDX system. The condition for observation and analysis was the same as that in pistils. For in vitro germination of pollen, the pollen culture medium containing 0.07% Ca(NO3)2, 0.01% KNO3, 0.02% MgSO4, 0.01% H3BO3, 2% sucrose, and 15% PEG4000 in 20 mM MES (pH 6.0) was used (Jahnen et al. 1989). First, a freshly dehisced anther was suspended in 200 µl of pollen culture medium (ca. 5×104 grains ml–1). Aliquots of 100 µl of suspended culture medium were mounted on glass slides in a moist chamber at 25°C. In order to examine the effect of the element in the culture medium on pollen germination and pollen tube growth, culture media from which Ca, K, Mg, or B was omitted were prepared. After one hour, germinated pollen was observed and photographed using a light microscopy (Zeiss, Axiophoto 2). In order to abrogate the effect of substances existing around pollen grains, a freshly dehisced anther was suspended in 200 µl of pollen culture medium without Ca (ca. 5×104 grains ml–1). Culture medium (100 µl) was immediately suspended in 50 ml of the culture medium without Ca, centrifuged (100×g, 25°C, 3 min), and then precipitated pollen grains were resuspended in 1 ml of the culture medium without Ca. These experiments were independently repeated three times. Acknowledgments We thank Mr. Masao Wada from Hitachi Science Systems and Mr. Youji Morita from Horiba for their technical advice. This work was supported in part by Grants-in-Aid for Special Research on Priority Areas (No. 11238205 and 15208013) and Grants-in-Aid for Special Research (C) (No. 13640649) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. 1 Corresponding author: E-mail, m-iwano@bs.aist-nara.ac.jp; Fax, +81-743-72-5459. View largeDownload slide Fig. 1 Electron micrographs of P. hybrida taken by VP-SEM and X-ray spectrum from pollen grains and crystal grains. (a) Pollen grains. The shape was spheroid and had a fine reticulated pattern formed by the baculae on the surface. Many calcium crystals were observed around pollen grains. The square areas were X-ray microanalyzed. Bar = 10 µm. (b) Pollen grains. The emission of P-Kα, S-Kα, K-Kα and Ca-Kα was detected. (c) Crystal grains. Intense emission of Ca-Kα (left) and weak emission of Ca-Kβ (right) were detected. View largeDownload slide Fig. 1 Electron micrographs of P. hybrida taken by VP-SEM and X-ray spectrum from pollen grains and crystal grains. (a) Pollen grains. The shape was spheroid and had a fine reticulated pattern formed by the baculae on the surface. Many calcium crystals were observed around pollen grains. The square areas were X-ray microanalyzed. Bar = 10 µm. (b) Pollen grains. The emission of P-Kα, S-Kα, K-Kα and Ca-Kα was detected. (c) Crystal grains. Intense emission of Ca-Kα (left) and weak emission of Ca-Kβ (right) were detected. View largeDownload slide Fig. 2 Electron micrographs of a dehisced anther. (a) A dehisced anther. At the edge of the dehisced anther, calcium crystals accumulated. Bar = 500 µm. (b) Higher magnification of the edge of a dehisced anther. Calcium crystals were observed around pollen grains. Bar = 10 µm. View largeDownload slide Fig. 2 Electron micrographs of a dehisced anther. (a) A dehisced anther. At the edge of the dehisced anther, calcium crystals accumulated. Bar = 500 µm. (b) Higher magnification of the edge of a dehisced anther. Calcium crystals were observed around pollen grains. Bar = 10 µm. View largeDownload slide Fig. 3 (a) A light micrograph of a cross-section of the anther from a 7 mm long bud, which was fixed with glutaraldehyde and embedded in Spurr’s resin. The tapetum (T), connective tissue (C), and immature pollen grains (P) in the locule were observed. S, stomium. Bar = 50 µm. (b–f) Electron micrographs by VP-SEM. (b) A cross-section of the anther from a 7 mm long bud which was fixed with glutaraldehyde and embedded in Spurr’s resin. An arrowhead shows a calcium crystal in the anther connective tissue. Bar = 50 µm. (c) A higher magnification of the cells beneath the stomium. A calcium grain (arrow) was observed in the vacuole. Bar = 10 µm. (d) A cross-section of the anther from a 7mm long bud without fixation. In the connective tissue and the cells beneath the stomium, calcium crystals (arrow) were observed. Bar = 50 µm. (e) An anther just after dehiscence. Bar = 100 µm. (f) A higher magnification of the area shown by an arrow in Fig. 3e. Bar = 10 µm. View largeDownload slide Fig. 3 (a) A light micrograph of a cross-section of the anther from a 7 mm long bud, which was fixed with glutaraldehyde and embedded in Spurr’s resin. The tapetum (T), connective tissue (C), and immature pollen grains (P) in the locule were observed. S, stomium. Bar = 50 µm. (b–f) Electron micrographs by VP-SEM. (b) A cross-section of the anther from a 7 mm long bud which was fixed with glutaraldehyde and embedded in Spurr’s resin. An arrowhead shows a calcium crystal in the anther connective tissue. Bar = 50 µm. (c) A higher magnification of the cells beneath the stomium. A calcium grain (arrow) was observed in the vacuole. Bar = 10 µm. (d) A cross-section of the anther from a 7mm long bud without fixation. In the connective tissue and the cells beneath the stomium, calcium crystals (arrow) were observed. Bar = 50 µm. (e) An anther just after dehiscence. Bar = 100 µm. (f) A higher magnification of the area shown by an arrow in Fig. 3e. Bar = 10 µm. View largeDownload slide Fig. 4 Electron micrographs of stigma by VP-SEM. (a) Unpollinated stigma. Bar = 250 nm. (b) A higher magnification of an unpollinated stigma. The square area was X-ray microanalyzed. Bar = 10 µm. (c) A part of a pollinated stigma just after pollination. Bar = 10 µm. View largeDownload slide Fig. 4 Electron micrographs of stigma by VP-SEM. (a) Unpollinated stigma. Bar = 250 nm. (b) A higher magnification of an unpollinated stigma. The square area was X-ray microanalyzed. Bar = 10 µm. (c) A part of a pollinated stigma just after pollination. Bar = 10 µm. View largeDownload slide Fig. 5 X-ray spectrum from a stigma in Fig. 4. (a) Unpollinated stigma. The emission of C-Kα, O-Kα, and Al-Kα was detected. Al-Kα would be detected by the spatial relationship between the sample and X-ray detector. (b) Pollinated stigma. The emission of P-Kα, S-Kα, K-Kα, and Ca-Kα was detected. The emission of Ca-Kα was higher than that of a pollen grain. View largeDownload slide Fig. 5 X-ray spectrum from a stigma in Fig. 4. (a) Unpollinated stigma. The emission of C-Kα, O-Kα, and Al-Kα was detected. Al-Kα would be detected by the spatial relationship between the sample and X-ray detector. (b) Pollinated stigma. The emission of P-Kα, S-Kα, K-Kα, and Ca-Kα was detected. The emission of Ca-Kα was higher than that of a pollen grain. View largeDownload slide Fig. 6 In vitro germination assay. (a) Pollen tubes germinated in the complete medium containing Ca, K, Mg, and B. Ca (b) or K (c) was omitted from the complete culture medium, pollen germination and pollen tube growth occurred normally as in the complete medium. (d) Pollen grains after washing with abundant germination medium without Ca. Pollen germination and pollen tube growth were inhibited. Bar = 10 µm. View largeDownload slide Fig. 6 In vitro germination assay. (a) Pollen tubes germinated in the complete medium containing Ca, K, Mg, and B. Ca (b) or K (c) was omitted from the complete culture medium, pollen germination and pollen tube growth occurred normally as in the complete medium. (d) Pollen grains after washing with abundant germination medium without Ca. Pollen germination and pollen tube growth were inhibited. Bar = 10 µm. 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TI - Calcium Crystals in the Anther of Petunia: the Existence and Biological Significance in the Pollination Process
JF - Plant and Cell Physiology
DO - 10.1093/pcp/pch004
DA - 2004-01-15
UR - https://www.deepdyve.com/lp/oxford-university-press/calcium-crystals-in-the-anther-of-petunia-the-existence-and-biological-BT9MZdUj4c
SP - 40
EP - 47
VL - 45
IS - 1
DP - DeepDyve
ER -