Secretory tapetum of Brassica oleracea L.: polarity and ultrastructural features

Summary The ultrastructure of the secretory, binucleate tapetum of Brassica oleracea in the micro spore mother cell (MMC) stage through to the mature pollen stage is reported. The tapetal cells differentiate as highly specialized cells whose development is involved in lipid accumulation in their final stage. They start breaking down just before anther dehiscence. Nuclei with dispersed chromatin, large nucleoli and many ribosomes in the cytoplasm characterize the tapetal cells. The wall-bearing tapetum phase ends at the tetrade stage. The dissolution of tapetal walls begins from the inner tangential wall oriented towards the loculus and proceeds gradually along the radial walls to the outer tangential one. The plasmodesmata transversing the radial walls between tapetal cells persist until the mature microspore, long after loss of the inner tangential wall. After wall dissolution, the tapetal protoplasts retain their integrity and position within the anther locule. The tapetal cell membrane is in direct contact with the exine of the microspores/pollen grains and forms tubular evaginations that increase its surface area and appear to be involved in the translocation of solutes from the tapetal cells to the microspores/ pollen grains. The tapetal cells exhibit a polarity expressed by spatial differentiation in the radial direction.     

ABCG26-Mediated Polyketide Trafficking and Hydroxycinnamoyl Spermidines Contribute to Pollen Wall Exine Formation in Arabidopsis

Pollen grains are encased by a multilayered, multifunctional wall. The sporopollenin and pollen coat constituents of the outer pollen wall (exine) are contributed by surrounding sporophytic tapetal cells. Because the biosynthesis and development of the exine occurs in the innermost cell layers of the anther, direct observations of this process are difficult. The objective of this study was to investigate the transport and assembly of exine components from tapetal cells to microspores in the intact anthers of Arabidopsis thaliana. Intrinsically fluorescent components of developing tapetum and microspores were imaged in intact, live anthers using two-photon microscopy. Mutants of ABCG26, which encodes an ATP binding cassette transporter required for exine formation, accumulated large fluorescent vacuoles in tapetal cells, with corresponding loss of fluorescence on microspores. These vacuolar inclusions were not observed in tapetal cells of double mutants of abcg26 and genes encoding the proposed sporopollenin polyketide biosynthetic metabolon (ACYL COENZYME A SYNTHETASE5, POLYKETIDE SYNTHASE A [PKSA], PKSB, and TETRAKETIDE α-PYRONE REDUCTASE1), providing a genetic link between transport by ABCG26 and polyketide biosynthesis. Genetic analysis also showed that hydroxycinnamoyl spermidines, known components of the pollen coat, were exported from tapeta prior to programmed cell death in the absence of polyketides, raising the possibility that they are incorporated into the exine prior to pollen coat deposition. We propose a model where ABCG26-exported polyketides traffic from tapetal cells to form the sporopollenin backbone, in coordination with the trafficking of additional constituents, prior to tapetum programmed cell death.     

Sporoderm in Populus and Salix

Four phenomena were observed in a study of Populus tremula and P. tremula f. gigas microspores from before microspore mitosis through mature pollen which may have general significance in the ontogeny of pollen grains: 1) The exine and orbicules (Ubisch bodies) were covered by membranes. 2) The exine and the tapetal surfaces where orbicules form were covered by a polysaccharide (PAS positive) coat until after microspore mitosis; subsequently the tapetum became plasmodial. 3) Material having the staining characteristics of the nexine 2 (endexine in the sense of Fægri) accumulated on membranes in microspores in the space between the exine and the plasma membrane. That material was almost completely gone from the wall in mature pollen. The membranes on which material had accumulated migrated through the exine. Following passage through the exine these membranes were seen as empty fusiform vesicles in micrographs of anthers prepared by commonly used methods. 4) At about microspore mitosis when the cellulosic intine begins to form, microtubules about 240 A in diameter occurred near the plasma membrane and generally parallel with it. Positive acid phosphatase reactions in tapetal cells together with the morphology of orbicules and other tapetal organelles suggest that the wall of orbicules, which is like the pollen exine, may form as a residual product of a lysosome system.

Sections of mature Salix humilis pollen were compared with Populus.     

Anther, pollen and tapetum development in safflower, Carthamus tinctorius L

In safflower, the anther wall at maturity consists of a single epidermis, an endothecium, a middle layer and the tapetum. The tapetum consists mainly of a single layer of cells. However, this single-layer appearance is punctuated by loci having ‘two-celled’ groupings due to additional periclinal divisions in some tapetal cells. Meiotic division in microsporocytes gives rise to tetrads of microspores. The primexine is formed around the protoplasts of microspores while they are still enveloped within the callose wall. Just prior to microgametogenesis, the microspores enlarge through the process of vacuolation, and the exine wall pattern becomes established. Microgametogenesis results in the formation of 3-celled pollen grains. The two elongated sperm cells appear to be connected. The exine wall is highly sculptured with a distinct tectum, columellae, a foot layer, an endexine and a thin intine. Similar to other members of the Asteraceae family, the tapetum is of the invasive type. The most novel finding of this study is that in addition to the presence of invasive tapetal cells, a small population of ‘non-invasive’ tapetal cells is also present. The tapetal cells next to the anther locules in direct contact with the microspores become invasive and start to grow into the space between developing microspores. These tapetal cells synthesize tryphine and eventually degenerate at the time of gametogenesis releasing their content into the anther locules. A smaller population of non-invasive tapetal cells is formed as a result of periclinal divisions at the time of tapetum differentiation. These cells are not exposed to the anther locules until the degeneration of the invasive tapetal cells. The non-invasive tapetal cells have a different cell fate as they synthesize pollenkitt. This material is responsible for allowing some pollen grains to adhere to each other and to the anther wall after anther dehiscence. This observation explains the out-crossing ability of Carthamus species and varieties in nature.     

Microspore and tapetal development in Echinodorus cordifolìus (Alismaceae)

In the microspore tetrad period the exine begins as rods that originate from the plasma membrane. These rods are exine units that on further development become columellae as well as part of the tectum, foot layer and “transitory endexine”. The primexine matrix is very thin in the future sites of the pores. At these sites the plasma membrane and its surface coating (glycocalyx) are without exine units and adjacent to the callose envelope. The exine around the aperture margin is characterized by units of reduced height. After the exine units and primexine matrix have become ca 0.2 μm in height a fibrillar zone forms under the aperture margin. It is the exine units around the aperture that are templates for exine processes on apertures of mature pollen. Oblique sections of the early exine show that the tectum consists of the distal portions of close-packed exine units. The exine enlarges in the free microspore period but initially its substructure (tectum, columellae, foot layer and transitory endexine) is not homogeneous and unit structures are visible until after the vacuolate microspore period. There are indications of a commissural line/plane (junction plane) which separates the foot layer from the endexine during early development. Our observations of development in Echinodorus pollen extend a growing number of reports of “transitory endexines” in monocot pollen. The exine unit-structures become 0.2 μm or more in diameter and many columellae are composed of only one exine unit. Spinules become exceptionally tall, many protruding ca 0.7 μm above the level of the tectum as units only ca 0.1 μm in diameter. The outer portion of the tectum fills in around spinules and by maturity they are microechinate with their bases spread out to ca 1 μm or more. Unit structures can be seen with SEM in mature pollen following oxidation by plasma ashing and in the tapetum these units are arranged both radially, as in spinules, and parallel with the tapetal surfaces. There are clear indications of such an arrangement of units in untreated fresh pollen. Units comprising the basal part of the exine are not completely fused by sporopollenin accumulated during development. This would seem to be a characteristic feature, based on published work, of the alismacean pollen. Our use of a tracer shows, however, that there is considerable space within or between exine structure of mature Echinodorus pollen. Based upon the ca 0.1 μm size of exine-units formed early in development and exine components seen after oxidative treatment it seems that the early (primary) accumulated sporopollenin has greater resistance to oxidation than sporopollenin added, secondarily, around and between units later in development. Both primarily and secondarily accumulated sporopollenin are resistant to acetolysis but published work indicates that acetolysis alters exine material. At the microspore tetrad time and until the vacuolate stages tapetal cells are arranged as in secretory tapetums. During early microspore stages there are orbicules at the inner surface of tapetal cells. At free microspore period tapetal cells greatly elongate into the loculus and surround the microspores. By the end of the microspore vacuolate period tapetal cells release their cellular contents and microspores are for a time enveloped by tapetal organelles and translocation material.     

莴苣花药发育过程中钙的分布特征

减数分裂前,莴苣花药中的钙颗粒很少。减数分裂后,花药绒毡层细胞中的钙颗粒明显增加。同时在花药药室基质中也出现许多细小的钙颗粒。刚从四分体中释放出的小孢子内钙颗粒很少。伴随着花粉外壁物质在小孢子表面的沉积,钙颗粒开始积累在花粉壁部位。随后。小孢子中开始出现钙颗粒。当小孢子开始形成液泡后,钙颗粒向其中聚集,伴随着小液泡融合成大液泡。体积较大的钙颗粒主要集中在液泡中,而细胞质基质中的钙颗粒很少。随着二胞花粉中的大液泡消失,花粉细胞质中的钙颗粒变得很少。在以后的发育中,只有花粉壁中积累较多的钙颗粒。在莴苣花药发育过程中,钙与绒毡层细胞的退化和小孢子液泡形成以及二胞花粉中大液泡的消失有关。而花粉外壁表面积累丰富的钙与以后花粉的萌发有关。     

Chemical agents that inhibit pollen development: effects of the phenyl-cinnoline carboxylates SC-1058 and SC-1271 on the ultrastructure of developing wheat anthers (Triticum aestivum L. var Yecora rojò)

Summary Phenylcinnoline carboxylate compounds SC-1058 and SC-1271 cause complete male sterility in wheat when applied at suitable dosages at the pre-meiotic stage of anther development. Anthers from treated and untreated plants were compared using light and electron microscopy from the pre-meiotic stage through the formation of nearly mature pollen. Overall anther development is gradually slowed in treated plants and pollen development is generally arrested in the late prevacuolate or early vacuolate microspore stage, although the first pollen mitosis does sometimes occur. The sporopollenin-containing exine walls are thinner, and show abnormally developed foot and tectum layers with sparse connecting baculi. Microspore cytoplasm degenerates and the cells eventually collapse. At the early, prevacuolate, free microspore stage treated tapetal cells hypertrophy, expanding into the locule. They contain abnormally large vacuoles that appear to form from the fusion of secretory vesicles, and some vacuoles contain electrondense deposits. The sporopollenin-containing orbicular wall and Ubisch bodies are retarded in their development and are structurally deformed. Acetolysis of whole anthers and of thick sections shows that the sporopollen-in-containing structures of treated materials are greatly reduced in thickness and are less rigid than in the control. We conclude that application of these compounds causes interference with the secretory function of tapetal cells which supplies sporopollenin cell-wall polymers to the exine of the microspores and to the tapetal orbicular wall and associated Ubisch bodies. Interference with the tapetal secretion of other nutrients required for microspore development is strongly suggested.     

Localization and release of allergens from tapetum and pollen grains ofBetula pendula

Summary Although intact pollen grains are assumed to be the primary carrier of pollen allergens, specific immunoreactive components have been found in other aerosol fractions, e.g., starch grains and remains of tapetal cells Cryo-scanning-electron-microscopy results demonstrate the presence of a clear network of strands connecting the tapetum with the microspores. The distribution of protein in tapetal orbicules, pollen wall, and pollen cytoplasm was tested by histochemical stains for light microscopy and transmission electron microscopy. The protein is mainly localized at the apertures and starch grains in the cytoplasm of pollen and in the core and on the surface of tapetal orbicules. Monoclonal antibodies Bv-10, BIP3, and BIP4 have been used to locate the cellular sites of pollen and tapetal allergens inBetula pendula (syn.B. verrucosa). The application of rapid-freeze fixation prevented relocation of allergens from their native sites. The allergens are predominantly found in the starch grains and to lesser extent in the exine. We also tested interactions between mature birch pollen and human fluids: saliva, nostrils fluid, and eyes solution. The aim was to mimic more closely the in vivo situation during allergenic response. In all cases we observed several pollen grains that were burst and had released their cytoplasmic contents. In the nose the allergens are released from the pollen within minutes. In rhinitis, nasal pH is increased from the normal pH 6.0 to 8.0. When we used nasal fluid at pH 8.0, the number of ruptured pollen grains increased. The mechanism that might induce formation of small allergen-bearing particles from living plant cells is discussed.     

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。     

莴苣花药发育过程中钙的分布特征

减数分裂前,莴苣花药中的钙颗粒很少。减数分裂后,花药绒毡层细胞中的钙颗粒明显增加, 同时在花药药室基质中也出现许多细小的钙颗粒。刚从四分体中释放出的小孢子内钙颗粒很少,伴随着花粉外壁物质在小孢子表面的沉积,钙颗粒开始积累在花粉壁部位。随后,小孢子中开始出现钙颗粒。当小孢子开始形成液泡后,钙颗粒向其中聚集,伴随着小液泡融合成大液泡,体积较大的钙颗粒主要集中在液泡中,而细胞质基质中的钙颗粒很少。随着二胞花粉中的大液泡消失,花粉细胞质中的钙颗粒变得很少。在以后的发育中,只有花粉壁中积累较多的钙颗粒。在莴苣花药发育过程中,钙与绒毡层细胞的退化和小孢子液泡形成以及二胞花粉中大波泡的消失有关。而花粉外壁表面积累丰富的钙与以后花粉的萌发有关。     

An ABCG/WBC-type ABC transporter is essential for transport of sporopollenin precursors for exine formation in developing pollen

The exine of the pollen wall shows an intricate pattern, primarily comprising sporopollenin, a polymer of fatty acids and phenolic compounds. A series of enzymes synthesize sporopollenin precursors in tapetal cells, and the precursors are transported from the tapetum to the pollen surface. However, the mechanisms underlying the transport of sporopollenin precursors remain elusive. Here, we provide evidence that strongly suggests that the Arabidopsis ABC transporter ABCG26/WBC27 is involved in the transport of sporopollenin precursors. Two independent mutations at ABCG26 coding region caused drastic decrease in seed production. This defect was complemented by expression of ABCG26 driven by its native promoter. The severely reduced fertility of the abcg26 mutants was caused by a failure to produce mature pollen, observed initially as a defect in pollen-wall development. The reticulate pattern of the exine of wild-type microspores was absent in abcg26 microspores at the vacuolate stage, and the vast majority of the mutant pollen degenerated thereafter. ABCG26 was expressed specifically in tapetal cells at the early vacuolate stage of pollen development. It showed high co-expression with genes encoding enzymes required for sporopollenin precursor synthesis, i.e. CYP704B1, ACOS5, MS2 and CYP703A2. Similar to two other mutants with defects in pollen-wall deposition, abcg26 tapetal cells accumulated numerous vesicles and granules. Taken together, these results suggest that ABCG26 plays a crucial role in the transfer of sporopollenin lipid precursors from tapetal cells to anther locules, facilitating exine formation on the pollen surface.     

芝麻核雄性不育系ms86-1微粉发生的细胞学观察

芝麻(Sesamum indicum)核雄性不育系ms86-1姊妹交后代表现为可育、部分不育(即微粉)及完全不育(简称不育)3种类型。不同育性类型的花药及花粉粒形态差异明显。Alexander染色实验显示微粉植株花粉粒外壁为蓝绿色, 内部为不均一洋红色, 与可育株及不育株花粉粒的染色特征均不相同。为探明芝麻微粉发生机理, 在电子显微镜下比较观察了可育、微粉、不育类型的小孢子发育过程。结果表明, 可育株小孢子母细胞减数分裂时期代谢旺盛, 胞质中出现大量脂质小球; 四分体时期绒毡层细胞开始降解, 单核小孢子时期开始出现乌氏体, 成熟花粉时期花粉囊腔内及花粉粒周围分布着大量乌氏体, 花粉粒外壁有11–13个棱状凸起, 表面存在大量基粒棒, 形成紧密的覆盖层。不育株小孢子发育异常显现于减数分裂时期, 此时胞质中无脂质小球出现, 细胞壁开始积累胼胝质; 四分体时期绒毡层细胞未见降解; 单核小孢子时期无乌氏体出现; 成熟花粉时期花粉囊腔中未发现正常的乌氏体, 存在大量空瘪的败育小孢子, 外壁积累胼胝质, 缺乏基粒棒。微粉株小孢子在减数分裂时期可见胞质内有大量脂质小球, 四分体时期部分绒毡层发生变形, 单核小孢子时期有部分绒毡层开始降解; 绒毡层细胞降解滞后为少量发育进程迟缓的小孢子提供了营养物质, 部分小孢子发育为正常花粉粒; 这些花粉粒比较饱满, 表面有少量颗粒状突起, 但未能形成覆盖层, 花粉囊腔中及小孢子周围存在少量的乌氏体。小孢子形成的育性类型与绒毡层降解是否正常有关。     

Genetic control of male fertility in Arabidopsis thaliana: structural analyses of postmeiotic developmental mutants

Seven new male-sterile mutants (ms7–ms13) of Arabidopsis thaliana (L.) Heynh. (ecotype columbia) are described that show a postmeiotic defect of microspore development. In ms9 mutants, microspores recently released from the tetrad appear irregular in shape and are often without exines. The earliest evidence of abnormality in ms12 mutants is degeneration of microspores that lack normal exine sculpturing, suggesting that the MS12 product is important in the formation of pollen exine. Teratomes (abnormally enlarged microsporocytes) are also occasionally present and each has a poorly developed exine. In ms7 mutant plants, the tapetal cytoplasm disintegrates at the late vacuolate microspore stage, apparently causing the degeneration of microspores and pollen grains. With ms8 mutants, the exine of the microspores appears similar to that of the wild type. However, intine development appears impaired and pollen grains rupture prior to maturity. In ms11 mutants, the first detectable abnormality appears at the mid to late vacuolate stage. The absence of fluorescence in the microspores and tapetal cells after staining with 4′,6-diamidino-2-phenylindole (DAPI) and the occasional presence of teratomes indicate degradation of DNA. Viable pollen from ms10 mutant plants is dehisced from anthers but appears to have surface abnormalities affecting interaction with the stigma. Pollen only germinates in high-humidity conditions or during in-vitro germination experiments. Mutant plants also have bright-green stems, suggesting that ms10 belongs to the eceriferum (cer) class of mutants. However, ms10 and cer6 are non-allelic. The ms13 mutant has a similar phenotype to ms10, suggesting is also an eceriferum mutation. Each of these seven mutants had a greater number of flowers than congenic male-fertile plants. The non-allelic nature of these mutants and their different developmental end-points indicate that seven different genes important for the later stages of pollen development have been identified. Received: 14 August 1997 / Accepted: 7 October 1997     

Two new male-sterile mutants of Zea mays (Poaceae) with abnormal tapetal cell morphology

Two new recessive male-sterile mutants of Zea mays (Poaceae), or maize, were studied to identify the timing of pollen abortion and to examine the involvement of anther wall cell layers. The results of test crosses indicated that these mutants were not allelic with any known male-sterile mutants of maize. Light and transmission electron microscopy were used to compare pollen development in homozygous male-sterile mutants to that in fertile heterozygous siblings. In both mutants, microspores abort soon after release from the meiotic tetrad. However, the two mutations have strikingly different phenotypes. Large lipid bodies accumulate in the tapetal cells as the microspores vacuolate and die in the mutant ms25. Large vacuoles appear in both the tapetal cells and the young microspores as they begin to disintegrate in the mutant ms26. Because abnormal tapetal cell morphology is detected in both mutants, it is possible that both of these mutations affect the expression of genes in tapetal cells.     

Development of the pollen grain and tapetum of wheat (Triticum aestivum) in untreated plants and plants treated with chemical hybridizing agent RH0007

Summary A study of pollen development in wheat was made using transmission electron microscopy (TEM). Microspores contain undifferentiated plastids and mitochondria that are dividing. Vacuolation occurs, probably due to the coalescence of small vacuoles budded off the endoplasmic reticulum (ER). As the pollen grain is formed and matures, the ER becomes distended with deposits of granular storage material. Mitochondria proliferate and become filled with cristae. Similarly, plastids divide and accumulate starch. The exine wall is deposited at a rapid rate throughout development, and the precursors appear to be synthesized in the tapetum. Tapetal cells become binucleate during the meiosis stage, and Ubisch bodies form on the plasma membrane surface that faces the locule. Tapetal plastids become surrounded by an electron-translucent halo. Rough ER is associated with the halo around the plastids and with the plasma membrane. We hypothesize that the sporopollenin precursors for both the Ubisch bodies and exine pollen wall are synthesized in the tapetal plastids and are transported to the tapetal cell surface via the ER. The microspore plastids appear to be involved in activities other than precursor synthesis: plastid proliferation in young microspores, and starch synthesis later in development. Plants treated with the chemical hybridizing agent RH0007 show a pattern of development similar to that shown by untreated control plants through the meiosis stage. In the young microspore stage the exine wall is deposited irregularly and is thinner than that of control plants. In many cases the microspores are seen to have wavy contours. With the onset of vacuolation, microspores become plasmolyzed and abort. The tapetal cells in RH0007-treated locules divide normally through the meiosis stage. Less sporopollenin is deposited in the Ubisch bodies, and the pattern is less regular than that of the control. In many cases, the tapetal cells expand into the locule. At the base of one of the locules treated with a dosage of RH0007 that causes 95% male sterility, several microspores survived and developed into pollen grains that were sterile. The conditions at the base of the locule may have reduced the osmotic stress on the microspores, allowing them to survive. Preliminary work showed that the extractable quantity of carotenoids in RHOOO7-treated anthers was slightly greater than in controls. We concluded that RH0007 appears to interfere with the polymerization of carotenoid precursors into the exine wall and Ubisch bodies, rather than interfering with the synthesis of the precursors.     

Sporoderm development inNymphaea mexicana (Nymphaeaceae)

Mature pollen grains ofNymphaea mexicana have a verrucate proximal surface, a psilate distal surface and an anazonasulculus (encircling-sulcate aperture). The developmental events of microspores and tapetal cells were observed with TEM and SEM. Radially oriented substructural elements are seen in the microspore surface coating ofNymphaea mexicana from the early tetrad stage through the whole exine development. These elements, being the structural units of the microspore surface matrix (glycocalyx), are associated with sporopollenin precursor accumulation. In young free microspores, radially oriented elements are observed at both proximal and distal poles as a palisade between the endexine and plasmalemma.—Several points are discussed: (1) the initial and mature forms of exine substructure elements; (2) the significance of exine substructure for realisation of morphogenetic processes; (3) the ways by which verrucate and psilate sculpture patterns are developed.     

POLLEN PORE DEVELOPMENT AND ITS SPATIAL ORIENTATION DURING MICROSPOROGENESIS IN THE GRASS SORGHUM BICOLOR

The spatial relationships observed during microsporogenesis and pollen development in Sorghum bicolor indicate that a strong polarization exists in the anther locule and within individual microspores and pollen grains. During all developmental stages, each sporogenous cell and its derivatives lie continuously adjacent to the tapetum. The microspores and pollen grains form depressions in the tapetal orbicular wall. When the single pore of each microspore is initiated, as a gap in the primexine, it too lies adjacent to the tapetum and remains tightly appressed there until pollen maturity. A sequence of polar phenomena in microspores and pollen grains centers on an axis through the pore and perpendicular to the tapetal surface. These events include migrations of the microspore and vegetative nuclei, initial placement of the generative cell opposite the pore and its later migration, and a polar engorgement process whereby the pore end of the pollen grain (adjacent to the tapetum) fills with starch grains first. The tapetal cytoplasm completely degenerates at precisely the time of pollen engorgement, and its degradation products are believed to be available for pollen uptake at this time. The continuous association of the sporogenous cells or their cellular derivatives and their pores with the tapetum is thought to play an indispensible role in pollen development in sorghum and probably in all other grasses as well. The consistent position of the pore adjacent to the tapetum should be considered another common feature of microsporogenesis in the Gramineae. The characteristic exine pattern forms over the operculum and annulus of the pore, but the lamellae, which underlie the annulus, form a highly modified multilayered nexine. Membrane-like cores are observed in these lamellae and are believed to be involved in the initiation of sporopollenin deposition, but they are obliterated by pollen maturity. Neither the cores nor the lamellae are found in other parts of the pore or in the nonapertured wall.     

ESCRT components ISTL1 andLIP5 are required for tapetal function and pollen viability

Pollen wall assembly is crucial for pollen development and plant fertility. The durable biopolymer sporopollenin and the constituents of the tryphine coat are delivered to developing pollen grains by the highly coordinated secretory activity of the surrounding tapetal cells. The role of membrane trafficking in this process, however, is largely unknown. In this study, we used Arabidopsis thaliana to characterize the role of two late-acting endosomal sorting complex required for transport (ESCRT) components, ISTL1 and LIP5, in tapetal function. Plants lacking ISTL1 and LIP5 form pollen with aberrant exine patterns, leading to partial pollen lethality. We found that ISTL1 and LIP5 are required for exocytosis of plasma membrane and secreted proteins in the tapetal cells at the free microspore stage, contributing to pollen wall development and tryphine deposition. Whereas the ESCRT machinery is well known for its role in endosomal trafficking, the function of ISTL1 and LIP5 in exocytosis is not a typical ESCRT function. The istl1 lip5 double mutants also show reduced intralumenal vesicle concatenation in multivesicular endosomes in both tapetal cells and developing pollen grains as well as morphological defects in early endosomes/trans-Golgi networks, suggesting that late ESCRT components function in the early endosomal pathway and exocytosis.

Endosomal sorting complex required for transport proteins ISTL1 and LIP5 are required for exocytosis of both plasma membrane and secreted proteins in tapetal cells during microspore formation.