A glycine-rich protein that facilitates exine formation during tomato pollen development

Formation of the unique and highly diverse outer cell wall, or exine, of pollen is essential for normal pollen function and survival. However, little is known about the many contributing proteins and processes involved in the formation of this wall. The tomato gene LeGRP92 encodes for a glycine-rich protein produced specifically in the tapetum. LeGRP92 is found as four major forms that accumulate differentially in protein extracts from stamens at different developmental stages. The three largest molecular weight forms accumulated during early microspore development, while the smallest molecular weight form of LeGRP92 was present in protein extracts from stamens from early microsporogenesis through anther dehiscence, and was the only form present in dehisced pollen. Light microscopy immunolocalization experiments detected LeGRP92 at only two stages, late tetrad and early free microspore. However, we observed accumulation of the LeGRP92 at the early tetrad stage of development by removing the callose wall from tetrads, which allowed LeGRP92 detection. Transmission electron microscopy confirmed the LeGRP92 accumulation from microspore mother cells, tetrads through anther dehiscence. It was observed in the callose surrounding the microspore mother cells and tetrads, the exine of microspores and mature pollen, and orbicules. Plants expressing antisense RNA had reduced levels of LeGRP92 mRNA and protein, which correlated to pollen with altered exine formation and reduced pollen viability and germination. These data suggest that the LeGRP92 has a role in facilitating sporopollenin deposition and uniform exine formation and pollen viability.     

Microspore separation in the quartet 3 mutants of Arabidopsis is impaired by a defect in a developmentally regulated polygalacturonase required for pollen mother cell wall degradation

Mutations in the QUARTET loci in Arabidopsis result in failure of microspore separation during pollen development due to a defect in degradation of the pollen mother cell wall during late stages of pollen development. Mutations in a new locus required for microspore separation, QRT3, were isolated, and the corresponding gene was cloned by T-DNA tagging. QRT3 encodes a protein that is approximately 30% similar to an endopolygalacturonase from peach (Prunus persica). The QRT3 protein was expressed in yeast (Saccharomyces cerevisiae) and found to exhibit polygalacturonase activity. In situ hybridization experiments showed that QRT3 is specifically and transiently expressed in the tapetum during the phase when microspores separate from their meiotic siblings. Immunohistochemical localization of QRT3 indicated that the protein is secreted from tapetal cells during the early microspore stage. Thus, QRT3 plays a direct role in degrading the pollen mother cell wall during microspore development.     

MS32-regulated timing of callose degradation during microsporogenesisin Arabidopsis is associated with the accumulation of stacked rough ER in tapetal cells

  In the male sterile32(ms32)mutant in Arabidopsis thaliana, pollen development is affected during meiosis of pollen mother cells (PMCs). In normal wild-type (WT) anthers, callose is deposited around PMCs before and during meiosis, and after meiosis the tetrads have a complete callose wall. In ms32, PMCs showed initial signs of some callose deposition before meiosis, but it was degraded soon after, as was part of the cellulosic wall around the PMCs. The early dissolution of callose in ms32 was associated with the occurrence of extensive stacks of rough ER (RER) in tapetal cells. The stacks of RER were also observed in the WT tapetum, but at a later stage, i.e., after the tetrads were formed and when callose is normally broken down for release of microspores. Based on these observations it is suggested that: (1) callose degradation around developing microspores is linked to the formation of RER in tapetal cells, which presumably synthesize and/or secrete callase into the anther locule, and (2) mutation in MS32 disrupts the timing of these events. Received: 27 April 1999 / Revision accepted: 21 June 1999     

Microsporogenesis and pollen development in Leymus chinensis with emphasis on dynamic changes in callose deposition

Leymus chinensis is an economically and ecologically important grass that exhibits low seed production. To better understand the causes of its low sexual reproductivity, the microsporogenesis and pollen development of this species were investigated, with emphasis on dynamic changes in callose deposition. A variety of histochemical stains were employed, including Heidenhain's hematoxylin, decolorized aniline blue, DAPI, and acetocarmine, along with a temporary mount method. Microsporogenesis and pollen development generally took place from June 12 to 26. The meiosis of microspore mother cells (MMCs) was of the successive type and the tetrad was isobilateral in shape. Mature pollen grains comprised two sperms and a vegetative nucleus. Callose initially appeared in the center of the anther locule at the premeiotic phase, and then gradually and unevenly deposited around the MMC before the commencement of meiosis. At the onset of meiosis, the accumulation of callose enclosing the MMC peaked, accompanied by the disappearance of callose in the center of the locule. At the dyad and tetrad stages, the dyads and tetrads were surrounded by callose wall and the microspores in the tetrads were isolated by a crossed cell plate composed of callose. Microspores just released from tetrads were still enclosed in callose wall, and then callose gradually disappeared in the pollen wall. Ultimately, callose almost completely disappeared from the walls of mature pollen grains. In the large numbers of sections observed, most of the cases of meiosis of the MMCs, pollen development, and callose dynamics were normal, with only a few abnormities observed. The results suggest that microsporogenesis, male gametogenesis, and callose dynamics during these processes are generally normal in this species, and that the callose wall plays an important role in the production of functional pollen grains. The small numbers of abnormities of these processes that occurred likely do not adversely affect the production of viable pollen grains. Therefore, microsporogenesis and pollen development may not be factors in the low seed production of L. chinensis.     

Two callose synthases,GSL1 and GSL5, play an essential and redundant role in plant and pollen development and in fertility

Callose, a β-1,3-glucan that is widespread in plants, is synthesized by callose synthase. Arabidopsis thaliana contains a family of 12 putative callose synthase genes (GSL1–12). The role of callose and of the individual genes in plant development is still largely uncertain. We have now used TILLING and T-DNA insertion mutants (gsl1-1, gsl5-2 and gsl5-3) to study the role of two closely related and linked genes, GSL1 and GSL5, in sporophytic development and in reproduction. Both genes are expressed in all parts of the plant. Sporophytic development was nearly normal in gsl1-1 homozygotes and only moderately defective in homozygotes for either of the two gsl5 alleles. On the other hand, plants that were gsl1-1/+ gsl5/gsl5 were severely defective, with smaller leaves, shorter roots and bolts and smaller flowers. Plants were fertile when the sporophytes had either two wild-type GSL1 alleles, or one GSL5 allele in a gsl1-1 background, but gsl1-1/+ gsl5/gsl5 plants produced an extremely reduced number of viable seeds. A chromosome with mutations in both GSL1 and GSL5 rendered pollen infertile, although such a chromosome could be transmitted via the egg. As a result, it was not possible to obtain plants that were homozygous for mutations in both the GSL genes. Pollen grain development was severely affected in double mutant plants. Many pollen grains were collapsed and inviable in the gsl1-1/gsl1-1 gsl5/+ and gsl1-1/+ gsl5/gsl5 plants. In addition, gsl1-1/+ gsl5/gsl5 plants produced abnormally large pollen with unusual pore structures, and had problems with tetrad dissociation. In this particular genotype, while the callose wall formed around the pollen mother cells, no callose wall separated the resulting tetrads. We conclude that GSL1 and GSL5 play important, but at least partially redundant roles in both sporophytic development and in the development of pollen. They are responsible for the formation of the callose wall that separates the microspores of the tetrad, and also play a gametophytic role later in pollen grain maturation. Other GSL genes may control callose formation at different steps during pollen development.     

Octad pollen formation in Cymbopetalum (Annonaceae): the binding mechanism

Our recent study of tetrad pollen formation in Annona (Annonaceae) revealed that after meiosis the callose-cellulose envelope forms a special conjugation with individual microspores and the forthcoming callose digestion is incomplete. The undigested part forms a central binder holding the four microspores of the tetrad together. This process causes the microspores to rotate 180 degrees. In this paper we describe pollen formation in another annonaceous genus, Cymbopetalum, in which the pollen is shed in octads, through use of light microscopy, epifluorescence microscopy, and TEM. In Cymbopetalum, two meiocytes, connected by abundant cytomictic channels, are produced in each sporangium. Octad pollen formation in Cymbopetalum is shown to be comparable to the synchronized formation of two connected Annona tetrads, which then integrate into a single octad. Unique features of Annona polyad formation, e.g. special binding between the callose-cellulose envelopes and microspores, incomplete callose digestion, and microspore rotation, also occur in Cymbopetalum. In addition, formation of the Cymbopetalum octad involves development of a cushion-like structure that binds the distal pronexine of all eight microspores, and there is the production of intine protrusions. The evolutionary origin of the callose-cellulose binding mechanism within the family is discussed.     

The wall ofPinus sylvestris L. pollen tubes

Summary The wall ofPinus sylvestris pollen and pollen tubes was studied by electron microscopy after both rapid-freeze fixation and freeze-substitution (RF-FS) and chemical fixation. Fluorescent probes and antibodies (JIM7 and JIM5) were used to study the distribution of esterified pectin, acidic pectin and callose. The wall texture was studied on shadow-casted whole mounts of pollen tubes after extraction of the wall matrix. The results were compared to current data of angiosperms. TheP. sylvestris pollen wall consists of a sculptured and a nonsculptured exine. The intine consists of a striated outer layer, that stretches partly over the pollen tube wall at the germination side, and a striated inner layer, which is continuous with the pollen tube wall and is likely to be partly deposited after germination. Variable amounts of callose are present in the entire intine. No esterified pectin is detected in the intine and acidic pectin is present in the outer intine layer only. The wall of the antheridial cell contains callose, but no pectin is detectable. The wall between antheridial and tube cell contains numerous plasmodesmata and is bordered by coated pits, indicating intensive communication with the tube cell. Callose and esterified pectin are present in the tip and the younger parts of the pollen tubes, but both ultimately disappear from the tube. Sometimes traces in the form of bands remain present. No acidic pectin is detected in either tip or tube. The wall of the pollen tube tip has a homogenous appearance, but gradually attains a fibrillar character at aging, perhaps because of the disappearance of callose and pectin. No secondary wall formation or callose lining can be seen wilh the electron microscope. The densily of the cellulose microfibrils (CMF) is much lower in the tip than in the tube. Both show CMF in all but axial and nontransverse orientations. In conclusion,P. sylvestris and angiosperm pollen tubes share the presence of esterified pectin in the tip, the oblique orientations of the CMF, and the gradual differentiation of the pollen tube wall, indicating a possible relation to tip growth. The presence of acidic pectin and the deposition of a secondary-wall or callose layer in angiosperms but not inP. sylvestris indicales that these characteristics are not related to tip growth, but probably represent adaptations to the fast and intrastylar growth of angiosperms.Abbreviations CMF cellulose microfibrils - II inner intine - NE nonsculptured exine - OI outer intine - RF-FS rapid-freeze fixation freeze-substitution - SE sculptured exine - SER smooth endoplasmic reliculum - SV secretory vesicles     

凤仙花花药发育中多糖和脂滴组织化学研究

以不同发育时期的凤仙花花药为实验材料,采用组织化学方法,对花药发育中的结构变化及多糖和脂滴物质分布进行观察。结果表明:(1)凤仙花的花药壁由6层细胞组成,包括1层表皮细胞,2层药室内壁细胞,2层中层细胞和1层绒毡层细胞。其中绒毡层细胞的形态不明显,很难与造孢细胞区分,且在小孢子母细胞时期退化。(2)在小孢子母细胞中出现了一些淀粉粒,但减数分裂后,早期小孢子中的淀粉粒消失,又出现了一些小的脂滴;随着花粉的发育,小孢子形成大液泡,晚期小孢子中的脂滴也消失;小孢子分裂形成二胞花粉后,营养细胞中的大液泡降解、消失,二胞花粉中又开始积累淀粉;接近开花时,成熟花粉中充满细胞质,其中包含了较多的淀粉粒和脂滴。(3)在凤仙花的花药发育中,绒毡层细胞很早退化,为小孢子母细胞和四分体小孢子提供了营养物质;其后的中层细胞退化则为后期花粉发育提供了营养物质。     

Pectic arabinan side chains are essential for pollen cell wall integrity during pollen development

Pectin is a complex polysaccharide and an integral part of the primary plant cell wall and middle lamella, contributing to cell wall mechanical strength and cell adhesion. To understand the structure–function relationships of pectin in the cell wall, a set of transgenic potato lines with altered pectin composition was analysed. The expression of genes encoding enzymes involved in pectin acetylation, degradation of the rhamnogalacturonan backbone and type and length of neutral side chains, arabinan and galactan in particular, has been altered. Upon crossing of different transgenic lines, some transgenes were not transmitted to the next generation when these lines were used as a pollen donor, suggesting male sterility. Viability of mature pollen was severely decreased in potato lines with reduced pectic arabinan, but not in lines with altered galactan side chains. Anthers and pollen of different developmental stages were microscopically examined to study the phenotype in more detail. Scanning electron microscopy of flowers showed collapsed pollen grains in mature anthers and in earlier stages cytoplasmic protrusions at the site of the of kin pore, eventually leading to bursting of the pollen grain and leaking of the cytoplasm. This phenomenon is only observed after the microspores are released and the tapetum starts to degenerate. Timing of the phenotype indicates a role for pectic arabinan side chains during remodelling of the cell wall when the pollen grain is maturing and dehydrating.     

冬季低温条件下香石竹小孢子败育的细胞学研究

利用压片法及石蜡切片法观察冬季低温下香石竹小孢子发育过程,以明确低温导致香石竹小孢子败育的因素,为杂交育种奠定基础。结果表明:(1)冬季低温下香石竹只有部分小孢子发育正常,经过小孢子母细胞、减数分裂和四分体等时期,最后发育成花粉。(2)石蜡切片法观察到冬季低温下香石竹1.5~1.6cm长花蕾中有61%的花粉母细胞发生败育,1.7~1.8cm长花蕾中有71%的花粉母细胞发生败育。(3)部分已经进入四分体时期的小孢子胼胝质未能及时溶解,妨碍了小孢子释放而导致败育。研究认为,花粉母细胞和四分体的发育异常是冬季低温下香石竹小孢子败育的主要原因。     

洋葱花药发育中钙离子分布特征

采用焦锑酸钾沉淀钙离子技术,对洋葱（Alliumcepa）花药发育中Ca^2＋分布进行了研究。在小孢子母细胞时期,小孢子母细胞中的钙沉淀颗粒很少,但绒毡层细胞的内切向壁已出现明显的钙沉淀颗粒。在四分体时期,四分体小孢子的胼胝质壁中出现较多的钙沉淀颗粒;绒毡层细胞内切向壁的钙沉淀颗粒消失,而在外切向壁和径向壁部位的钙沉淀颗粒增加。在小孢子早期,小孢子中也出现了钙沉淀颗粒,而绒毡层细胞内切向壁表面出现了很多絮状物,其上附有细小钙沉淀颗粒。到小孢子晚期,小孢子中出现一些小液泡,细胞质中的钙沉淀颗粒有所下降。此时绒毡层细胞已明显退化,但在绒毡层膜上仍有一些乌氏体和钙沉淀颗粒。在二胞花粉早期,营养细胞中的液泡收缩、消失,细胞质中又出现了较多的钙沉淀颗粒,在质体和其内部的淀粉粒表面上附有较多的钙沉淀颗粒。到二胞花粉晚期,花粉中的钙沉淀颗粒已明显下降,仅在花粉外壁中还有一地钙沉淀颗粒．     

Wall development and its autofluorescence of sterile and fertileVicia faba L. Pollen

Summary The ultrastructural changes of the pollen wall of three types of fertile and one of sterileVicia pollen were related to the autofluorescence of the pollen wall, measured by a microspectroscopic method. Till the liberation of the microspores from the tetrad, the spectrum of the ectexine shows sometimes two maxima and has a very low intensity. After this period the endexine is formed and its spectrum has one maximum with a high intensity. The differences of the pollen wall between the sterile and fertile pollen exist of the presence of one spectral maximum during the tetrad stage, a thick endexine and the absence of the intine in the sterile pollen. The different types show much differences during the tetrad stage in the callose wall as well as the ectexine. The autofluorescence illustrates the complexity and specificity of the pollen wall development.     

Release of developmental constraints on tetrad shape is confirmed in inaperturate pollen of Potamogeton

Background and Aims

Microsporogenesis in monocots is often characterized by successive cytokinesis with centrifugal cell plate formation. Pollen grains in monocots are predominantly monosulcate, but variation occurs, including the lack of apertures. The aperture pattern can be determined by microsporogenesis features such as the tetrad shape and the last sites of callose deposition among the microspores. Potamogeton belongs to the early divergent Potamogetonaceae and possesses inaperturate pollen, a type of pollen for which it has been suggested that there is a release of the constraint on tetrad shape. This study aimed to investigate the microsporogenesis and the ultrastructure of pollen wall in species of Potamogeton in order to better understand the relationship between microsporogenesis features and the inaperturate condition.

Methods

The microsporogenesis was investigated using both light and epifluorescence microscopy. The ultrastructure of the pollen grain was studied using transmission electron microscopy.

Key Results

The cytokinesis is successive and formation of the intersporal callose wall is achieved by centrifugal cell plates, as a one-step process. The microspore tetrads were tetragonal, decussate, T-shaped and linear, except in P. pusillus, which showed less variation. This species also showed a callose ring in the microsporocyte, and some rhomboidal tetrads. In the mature pollen, the thickening observed in a broad area of the intine was here interpreted as an artefact.

Conclusions

The data support the view that there is a correlation between the inaperturate pollen production and the release of constraint on tetrad shape. However, in P. pusillus the tetrad shape may be constrained by a callose ring. It is also suggested that the lack of apertures in the pollen of Potamogeton may be due to the lack of specific sites on which callose deposition is completed. Moreover, inaperturate pollen of Potamogeton would be better classified as omniaperturate.Key words: Alismatales, callose, microsporogenesis, pollen aperture, Potamogeton illinoensis, P. polygonus, P. pusillus, tetrad shape     

含笑花药发育的超微结构研究

对含笑花药发育中的超微结构变化进行观察,结果显示:(1)花粉发育中有三次液泡变化过程——第一次是小孢子母细胞在形成时内部出现了液泡,这可能与胼胝质壁的形成有关;第二次是在小孢子母细胞减数分裂之前,细胞内壁纤维素降解区域形成液泡,它的功能可能是消化原有的纤维素细胞壁;第三次是在小孢子液泡化时期,形成的大液泡将细胞核挤到边缘,产生极性。(2)含笑花粉在小孢子早期形成花粉外壁外层,花粉外壁内层在小孢子晚期形成,而花粉内壁是在二胞花粉早期形成;花粉成熟时,表面上沉积了绒毡层细胞的降解物而形成了花粉覆盖物。研究认为,含笑花粉原外壁的形成可能与母细胞胼胝质壁有关,而由绒毡层细胞提供的孢粉素物质按一定结构建成了花粉覆盖物。     

LP28, a lily pollen-specific LEA-like protein, is located in the callosic cell wall during male gametogenesis

. LP28, a pollen-specific LEA-like protein identified in Lilium longiflorum purportedly related to the desiccation tolerance of pollen, was localized during male gametogenesis using immuno-electron microscopy. At premeiotic interphase, LP28 label is absent from the microsporocyte. LP28 label was first detected in the cell wall of the microsporocyte at meiotic prophase I. LP28 gradually increased as the cell wall thickened. In the dyad, after the first meiotic division, LP28 label also appeared in the septum. In the tetrad, after the second meiotic division, LP28 was detected throughout the cell wall, including the septa. Immunolabeling of callose during meiosis indicated that the appearance and localization of LP28 was very similar to that of callose. After the microspores were released from the tetrad by digesting the callosic cell wall, LP28 was not found in the microspores. In bicellular pollen, just after microspore mitosis, LP28 appeared in the generative cell wall, which also consisted of callose. After pollen germination, LP28 also accumulated in the callosic layer of the elongated pollen tube wall and the callose plug. Thus, LP28 colocalized with the callosic cell wall during male gametogenesis. The possible role of LP28 with respect to wall formation during meiosis and pollen development is discussed.     

DEVELOPMENT OF OMNIAPERTURATE POLLEN IN TRILLIUM KAMTSCHATICUM (LILIACEAE)

Ultrastructural changes during omniaperturate pollen development in Trillium kamtschaticum Pall, was examined using transmission electron microscopy. The pollen mother cells are not enveloped within a thick callosic wall. The microspores resulting from successive meiosis are divided by scanty deposition of callosic wall in the tetrad. A primexine/exine template is not recognizable within the tetrad during formation of exinous components. Preexinous globules, originating from vesicles in the callosic wall, accumulate electron-dense materials and develop into exinous globules. The preexinous globules have ca 10 nm wide contacts with tilted and invaginated plasma membrane of the microspore within the callosic wall. After dissolution of the callosic wall, the microspores separate and mitosis subsequently leads to the formation of a generative cell and vegetative cell encased in a loose aggregation of developing exinous globules. When the generative cell is at the pollen grain surface, the channeled zone is initiated at the opposite side of the microspore on the surface of the vegetative cell. Just before pollen maturity, a new layer develops under the channeled zone. Thus, development of the omniaperturate pollen grains of T. kamtschaticum involves some processes that are distinct from those of Canna and Heliconia and some that are similar.     

Developmental and cytogenetic analyses of pollen sterility in Valeriana scandens L.

Valeriana scandens presents perfect and pistillate flowers, the latter with sterile anthers. The species is composed of two varieties with different ploidy; V. scandens var. scandens (2n = 28) and V. scandens var. candolleana (2n = 56), both of which occur in RS, Brazil. Crosses between these varieties may give rise to hybrids with pollen sterility. In this study, we analyzed the microsporogenesis and microgametogenesis of sterile and fertile anthers, and also investigate whether pollen sterility is caused by an irregular meiotic process. Developmental analysis using light microscopy and scanning electron microscopy showed that sterile anthers develop similarly to fertile anthers until the end of meiosis. After this stage, sterile tetrads do not separate as a consequence of exine fusion between adjacent microspores, which is similar to sterile pollen of Brassica ms-cdl1 mutants. In addition, vacuolated immature pollen grains degenerate after separation. The cytogenetic analysis of the microspore mother cell (MMC) showed that the diploid population of V. scandens var. scandens (2n = 28) has pollen sterility that is not caused by a cytogenetic disturbance. The MMCs analyzed from prophase I to tetrad stage showed a regular meiotic process, indicating the phenotype of V. scandens sterile pollen is a postmeiotic process formed by fusion of exine between opposite microspores.