Tetrad pollen formation in quartet mutants of Arabidopsis thaliana is associated with persistence of pectic polysaccharides of the pollen mother cell wall

The quartet (qrt) mutants of Arabidopsis thaliana produce tetrad pollen in which microspores fail to separate during pollen development. Because the amount of callose deposition between microspores is correlated with tetrad pollen formation in other species, and because pectin is implicated as playing a role in cell adhesion, these cell-wall components in wild-type and mutant anthers were visualized by immunofluorescence microscopy at different stages of microsporogenesis. In wild-type, callose was detected around the pollen mother cell at the onset of meiosis and around the microspores during the tetrad stage. Microspores were released into the anther locule at the stage where callose was no longer detected. Deposition and degradation of callose during tetrad pollen formation in qrt1 and qrt2 mutants were indistinguishable from those in wild-type. Enzymatic removal of callose from wild-type microspores at the tetrad stage did not release the microspores, suggesting that callose removal is not sufficient to disperse the microspores in wild-type. Pectic components were detected in the primary wall of the pollen mother cell. This wall surrounded the callosic wall around the pollen mother cell and the microspores during the tetrad stage. In wild-type, pectic components of this wall were no longer detectable at the time of microspore release. However, in qrt1 and qrt2 mutants, pectic components of this wall persisted after callose degradation. This result suggests that failure of pectin degradation in the pollen mother cell wall is associated with tetrad pollen formation in qrt mutants, and indicates that QRT1 and QRT2 may be required for cell type-specific pectin degradation to separate microspores.     

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.     

Callose synthase (CalS5) is required for exine formation during microgametogenesis and for pollen viability in Arabidopsis

Callose (beta-1,3-glucan) is produced at different locations in response to biotic and abiotic cues. Arabidopsis contains 12 genes encoding callose synthase (CalS). We demonstrate that one of these genes, CalS5, encodes a callose synthase which is responsible for the synthesis of callose deposited at the primary cell wall of meiocytes, tetrads and microspores, and the expression of this gene is essential for exine formation in pollen wall. CalS5 encodes a transmembrane protein of 1923 amino acid residues with a molecular mass of 220 kDa. Knockout mutations of the CalS5 gene by T-DNA insertion resulted in a severe reduction in fertility. The reduced fertility in the cals5 mutants is attributed to the degeneration of microspores. However, megagametogenesis is not affected and the female gametes are completely fertile in cals5 mutants. The CalS5 gene is also expressed in other organs with the highest expression in meiocytes, tetrads, microspores and mature pollen. Callose deposition in the cals5 mutant was nearly completely lacking, suggesting that this gene is essential for the synthesis of callose in these tissues. As a result, the pollen exine wall was not formed properly, affecting the baculae and tectum structure and tryphine was deposited randomly as globular structures. These data suggest that callose synthesis has a vital function in building a properly sculpted exine, the integrity of which is essential for pollen viability.     

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.     

洋葱花药发育过程中的细胞化学研究(简报)

高等植物花药结构复杂,其发育更是一个迅速、多变的过程,如小孢子母细胞减数分裂期间的细胞质改组、胼胝质壁的形成与降解、大液泡的形成与消失、花粉内外壁的形成、绒毡层细胞的降解、营养物质的积累与转化等。除了上述花药组成细胞的形态和结构发生明显变化外。花药发育的另一个显著特点是以花粉为中心的营养物质单向运输和转化,尤其是小孢子有丝分裂形成二胞花粉后开始积累大量的营养储存物以供成熟花粉萌发时利用。     

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     

More than a leak sealant. The mechanical properties of callose in pollen tubes

While callose is a well-known permeability barrier and leak sealant in plant cells, it is largely unknown whether this cell wall polymer can also serve as a load-bearing structure. Since callose occurs in exceptionally large amounts in pollen, we assessed its role for resisting tension and compression stress in this cell. The effect of callose digestion in Solanum chacoense and Lilium orientalis pollen grains demonstrated that, depending on the species, this cell wall polymer represents a major stress-bearing structure at the aperture area of germinating grains. In the pollen tube, it is involved in cell wall resistance to circumferential tension stress, and despite its absence at the growing apex, callose is indirectly involved in the establishment of tension stress resistance in this area. To investigate whether or not callose is able to provide mechanical resistance against compression stress, we subjected pollen tubes to local deformation by microindentation. The data revealed that lowering the amount of callose resulted in reduced cellular stiffness and increased viscoelasticity, thus indicating clearly that callose is able to resist compression stress. Whether this function is relevant for pollen tube mechanics, however, is unclear, as stiffened growth medium caused a decrease in callose deposition. Together, our data provide clear evidence for the capacity of cell wall callose to resist tension and compression stress, thus demonstrating that this amorphous cell wall substance can have a mechanical role in growing plant cells.     

Analysis of sticky generative cell mutants reveals that suppression of callose deposition in the generative cell is necessary for generative cell internalization and differentiation in Arabidopsis

In flowering plants, double fertilization between male and female gametophytes, which are separated by distance, largely depends on the unique pattern of the male gametophyte (pollen): two non-motile sperm cells suspended within a tube-producing vegetative cell. A morphological screen to elucidate the genetic control governing the strategic patterning of pollen has led to the isolation of a sticky generative cell (sgc) mutant that dehisces abnormal pollen with the generative cell immobilized at the pollen wall. Analyses revealed that the sgc mutation is specifically detrimental to pollen development, causing ectopic callose deposition that impedes the timely internalization and differentiation of the generative cell. We found that the SGC gene encodes the highly conserved domain of unknown function 707 (DUF707) gene that is broadly expressed but is germline specific during pollen development. Additionally, transgenic plants co-expressing fluorescently fused SGC protein and known organelle markers showed that SGC localizes in the endoplasmic reticulum, Golgi apparatus and vacuoles in pollen. A yeast two-hybrid screen with an SGC bait identified a thaumatin-like protein that we named GCTLP1, some homologs of which bind and/or digest β-1,3-glucans, the main constituent of callose. GCTLP1 is expressed in a germline-specific manner and colocalizes with SGC during pollen development, indicating that GCTLP1 is a putative SGC interactor. Collectively, our results show that SGC suppresses callose deposition in the nascent generative cell, thereby allowing the generative cell to fully internalize into the vegetative cell and correctly differentiate as the germline progenitor, with the potential involvement of the GCTLP1 protein, during pollen development in Arabidopsis.     

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.     

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.     

Overexpression of AtTTP Affects ARF17 Expression and Leads to Male Sterility in Arabidopsis

Callose synthesis is critical for the formation of the pollen wall pattern. CalS5 is thought to be the major synthethase for the callose wall. In the Arabidopsis anther, ARF17 regulates the expression of CalS5 and is the target of miR160. Plants expressing miR160-resistant ARF17 (35S:5mARF17 lines) with increased ARF17 mRNA levels display male sterility. Here we report a zinc finger family gene, AtTTP, which is involved in miR160 maturation and callose synthesis in Arabidopsis. AtTTP is expressed in microsporocytes, tetrads and tapetal cells in the anther. Over-expression lines of AtTTP (AtTTP-OE line) exhibited reduced male fertility. CalS5 expression was tremendously reduced and the tetrad callose wall became much thinner in the AtTTP-OE line. Northern blotting hybridization and quantitative RT-PCR analysis revealed that miR160 was decreased, while the expression of ARF17 was increased in the AtTTP-OE line. Based on these results, we propose that AtTTP associates with miR160 in order to regulate the ARF17 expression needed for callose synthesis and pollen wall formation.     

The exogenous application of AtPGLR,an endo‐polygalacturonase,triggers pollen tube burst and repair

Plant cell wall remodeling plays a key role in the control of cell elongation and differentiation. In particular, fine‐tuning of the degree of methylesterification of pectins was previously reported to control developmental processes as diverse as pollen germination, pollen tube elongation, emergence of primordia or elongation of dark‐grown hypocotyls. However, how pectin degradation can modulate plant development has remained elusive. Here we report the characterization of a polygalacturonase (PG), AtPGLR, the gene for which is highly expressed at the onset of lateral root emergence in Arabidopsis. Due to gene compensation mechanisms, mutant approaches failed to determine the involvement of AtPGLR in plant growth. To overcome this issue, AtPGLR has been expressed heterologously in the yeast Pichia pastoris and biochemically characterized. We showed that AtPGLR is an endo‐PG that preferentially releases non‐methylesterified oligogalacturonides with a short degree of polymerization (< 8) at acidic pH. The application of the purified recombinant protein on Amaryllis pollen tubes, an excellent model for studying cell wall remodeling at acidic pH, induced abnormal pollen tubes or cytoplasmic leakage in the subapical dome of the pollen tube tip, where non‐methylesterified pectin epitopes are detected. Those leaks could either be repaired by new β‐glucan deposits (mostly callose) in the cell wall or promoted dramatic burst of the pollen tube. Our work presents the full biochemical characterization of an Arabidopsis PG and highlights the importance of pectin integrity in pollen tube elongation.     

Cytokinesis in plant male meiosis

In somatic cell division, cytokinesis is the final step of the cell cycle and physically divides the mother cytoplasm into two daughter cells. In the meiotic cell division, however, pollen mother cells (PMCs) undergo two successive nuclear divisions without an intervening S-phase and consequently generate four haploid daughter nuclei out of one parental cell. In line with this, the physical separation of meiotic nuclei does not follow the conventional cytokinesis pathway, but instead is mediated by alternative processes, including polar-based phragmoplast outgrowth and RMA-mediated cell wall positioning. In this review, we outline the different cytological mechanisms of cell plate formation operating in different types of PMCs and additionally focus on some important features associated with male meiotic cytokinesis, including cytoskeletal dynamics and callose deposition. We also provide an up-to-date overview of the main molecular actors involved in PMC wall formation and additionally highlight some recent advances on the effect of cold stress on meiotic cytokinesis in plants.     

山麦冬大小孢子发生及雌雄配子体发育研究

采用石蜡切片技术对百合科植物山麦冬大小孢子发生及雌雄配子体发育进行了观察研究。结果表明:(1)山麦冬花药具有4个花粉囊,花药壁的发育方式为基本型,花药壁完全分化时由表皮、药室内壁、中层及绒毡层组成。(2)绒毡层发育类型为分泌型,到四分体孢子彼此分离形成单细胞花粉阶段,绒毡层细胞开始解体退化,花粉成熟时绒毡层细胞完全消失;花粉母细胞减数分裂为连续型,四分体为左右对称形排列,成熟花粉为3-细胞花粉,单萌发沟。(3)子房3室,每室2枚胚珠,胚珠倒生型,双珠被,薄珠心,雌性孢原细胞不经过平周分裂而直接发育而成大孢子母细胞。(4)减数分裂后四分体大孢子呈线型或T型排列,合点端大孢子分化为功能大孢子,胚囊发育为蓼型;花粉母细胞减数分裂过程中,二分体、四分体细胞外方被胼胝质壁所包被,小孢子形成后胼胝质壁逐渐消失。该研究结果丰富了百合科植物生殖生物学研究的内容,也为探讨百合科植物的系统学研究提供了参考。     

A novel UDP-glucose transferase is part of the callose synthase complex and interacts with phragmoplastin at the forming cell plate

Using phragmoplastin as a bait, we isolated an Arabidopsis cDNA encoding a novel UDP-glucose transferase (UGT1). This interaction was confirmed by an in vitro protein--protein interaction assay using purified UGT1 and radiolabeled phragmoplastin. Protein gel blot results revealed that UGT1 is associated with the membrane fraction and copurified with the product-entrapped callose synthase complex. These data suggest that UGT1 may act as a subunit of callose synthase that uses UDP-glucose to synthesize callose, a 1,3-beta-glucan. UGT1 also interacted with Rop1, a Rho-like protein, and this interaction occurred only in its GTP-bound configuration, suggesting that the plant callose synthase may be regulated by Rop1 through the interaction with UGT1. The green fluorescent protein--UGT1 fusion protein was located on the forming cell plate during cytokinesis. We propose that UGT1 may transfer UDP-glucose from sucrose synthase to the callose synthase and thus help form a substrate channel for the synthesis of callose at the forming cell plate.     

Electron tomographic analysis of post-meiotic cytokinesis during pollen development in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

The mechanism of cell wall formation after male meiosis was studied in microsporocytes of Arabidopsis thaliana (L.) Heynh. by means of thin-section and immuno-electron microscopy and dual-axis electron tomography of high-pressure-frozen/freeze-substituted cells. The cellularization of four-nucleate microsporocytes involves a novel type of cell plate, called a post-meiotic-type cell plate. As in the syncytial endosperm, the microsporocyte cell plates assemble in association with mini-phragmoplasts. However, in contrast to the endosperm cell plates, post-meiotic type cell plates arise simultaneously across the entire division plane. Vesicles are transported along mini-phragmoplast microtubules by putative kinesin proteins and, prior to fusion, they become connected together by 24-nm-long linkers that resemble exocyst complexes. These vesicles fuse with each other to form wide tubules and wide tubular networks. In contrast to endosperm cell plates, the wide tubular networks in microsporocytes completely lack callose and do not appear to be constricted by dynamin rings. The most peripheral wide tubular networks begin to fuse with the plasma membrane before the more central cell plate assembly sites become integrated into a coherent cell plate. Fusion with the parental plasma membrane triggers callose synthesis and the wide tubular domains are converted into convoluted sheets. As the peripheral convoluted sheets accumulate callose and arabinogalactan proteins, they are converted into stub-like projections, which grow centripetally, i.e. toward the interior of the syncytium, fusing with the wide tubular networks already assembled in the division plane. We also demonstrate that the ribosome-excluding cell plate assembly matrix is delivered to the mini-phragmoplast with the first vesicles, and encompasses all the linked vesicles and intermediate stages in cell plate formation.Abbreviations AGP Arabinogalactan protein - MT Microtubule     

A Comparative Study of Anther and Pollen Development in Male Fertile and Male. Sterile Green Onion (Allium fistulosum L.)

Anther and pollen development in male-fertile and male-sterile green onions was studied. In the male-fertile line, both meiotic microspore mother ceils and tetrads have a callose wall. Mature pollen grains are 2-celled. The elongated generative cell with two bended ends displays a PAS positive cell wall. The tapetum has the character of both secretory and invasive types. From microspore stage onwards, many oil bodies or masses accumulate in the cytoplasm of the tapetal cells. The tapetum degenerates at middle 2-celled pollen stage. In male-sterile line, meiosis in microspore mother cells proceeds normally to form the tetrads. Pollen abortion occurs at microspore with vacuole stage. Two types of pollen abortion were observed. In type I, the protoplasts of the microspores contract and gradually disintegrate. At the same time the cytoplasm of microspores accumulates oil bodies which remain in the empty pollen. The tapetal cells behave normally up to the microspore stage and early stage of microspore abortion, but contain fewer oil bodies or masses than those in the male-fertilt line. At late stage of microspore abortion, three forms of the tapetal ceils can be observed: (1) the tapetal cells with degenerating protoplasts become flattened, (2) the tapetal cells enlarge but protoplasts retractor, (3) the cells break down and tile middle layer enlarges. In type Ⅱ, the cytoplasm degenerates earlier than the nucleus of the microspores and no protoplast is found in the anther locule. There are fibrous thickenings iii the endothecium of both types. It is difficult to verify whether the tapetum behavior and pollen abortion is the cause or the effect.     

Regulation of cell length in the Arabidopsis thaliana root by the ethylene precursor 1-aminocyclopropane- 1-carboxylic acid: a matter of apoplastic reactions

Treatment of the Arabidopsis thaliana root with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) immediately imposes a reduced maximal cell length beyond which further elongation is blocked. Here, we investigated possible apoplastic reactions involved in the inhibition of cell elongation. Five-day-old Arabidopsis seedlings were transferred to a growth medium supplemented with ACC and the effect on root cell length was recorded after 3 h of treatment. Altered characteristics in the apoplast of the nonelongating cells in the ACC-treated root, such as 'reactive oxygen species' (ROS) production and callose deposition, were detected using specific fluorochromes. The presence of functional hydroxyproline-rich glycoproteins (HRGPs) and the crosslinking of these cell-wall proteins are essential in limiting cell elongation. The ROS that drive the oxidative crosslinking of HRGPs, accumulate in the apoplast of cells in the zone where cell elongation stops. In the same cells, callose is deposited in the cell wall. The final cell length in the Arabidopsis root treated for a short period with ACC is determined in the zone of fast elongation. Both HRGPs crosslinking by ROS and callose deposition in the cell wall of this zone are suggested as causes for the reduced cell elongation.     

Callose synthesis in higher plants

Callose is a polysaccharide in the form of β-1,3-glucan with some β-1,6-branches and it exists in the cell walls of a wide variety of higher plants. Callose plays important roles during a variety of processes in plant development and/or in response to multiple biotic and abiotic stresses. It is now generally believed that callose is produced by callose synthases and that it is degraded by β-1,3-glucanases. Despite the importance of callose in plants, we have only recently begun to elucidate the molecular mechanism of its synthesis. Molecular and genetic studies in Arabidopsis have identified a set of genes that are involved in the biosynthesis and degradation of callose. In this mini-review, we highlight recent progress in understanding callose biosynthesis and degradation and discuss the future challenges of unraveling the mechanism(s) by which callose synthase operate.Key words: Arabidopsis thaliana, callose, callose synthase, glucan synthase-like, pollen, plasmodesmata, cell plate, stress