Early Tetrad Stage Research Articles

Meiotic Defects and Premature Tapetal Degeneration Are Involved in the Low Fertility of Oncidesa Gower Ramsey, an Important Cut-flower Orchid

The dancing-lady orchid, Oncidesa Gower Ramsey, is an important cultivar for cut-flower production, but it has low pollen fertility in breeding programs. In this study, we compared the pollen germination in vitro, sporad type, and pollinia development of Oncsa. Gower Ramsey and a diploid species, Oncidium sphacelatum Lindl (one of its grandparents). In Oncsa. Gower Ramsey, the pollen germination in vitro was lower as compared with those in Onc. sphacelatum. In addition, the frequency of abnormal sporads in Oncsa. Gower Ramsey was higher than those in Onc. sphacelatum. In Oncsa. Gower Ramsey, the middle layer and the tapetum were disorganized before meiosis, and subsequently they degenerated at the early tetrad stage. In contrast, the middle layer and the tapetum of Onc. sphacelatum began to degenerate at the early tetrad stage and fully disappeared at the bicellular pollen stage. These results suggested that the abnormal meiosis caused by unbalanced genomes and the premature degeneration of the middle layer and the tapetum could probably result in the abnormal pollen development and the low fertility of Oncsa. Gower Ramsey.

Phenotype characterisation and analysis of expression patterns of genes related mainly to carbohydrate metabolism and sporopollenin in male-sterile anthers induced by high temperature in wheat (Triticum aestivum)

Male reproductive development in higher plants is highly sensitive to various stressors, including high temperature (HT). In this study, physiological male-sterile plants of wheat (Triticum aestivum L.) were established using HT induction. The physiological changes and expression levels of genes mainly related to carbohydrate metabolism and sporopollenin in male-sterile processes were studied by using biological techniques, including iodine–potassium iodide staining, paraffin sectioning, scanning electron microscopy (SEM) and fluorescent quantitative analysis. Results of paraffin sectioning and SEM revealed that parts of HT male-sterile anthers, including the epidermis and tapetum, were remarkably different from those of normal anthers. The expression levels of TaSUT1, TaSUT2, IVR1 and IVR5 were significantly lower than of normal anthers at the early microspore and trinucleate stages. The RAFTIN1 and TaMS26 genes may contribute to biosynthesis and proper ‘fixation’ of sporopollenin in the development of pollen wall; however, their expression levels were significantly higher at the early tetrad stage and early microspore stage in HT sterile anthers. The recently cloned MS1 gene was expressed at the early tetrad and early microspore stages but not at the trinucleate stage. Moreover, this gene showed extremely significant, high expression in HT sterile anthers compared with normal anthers. These results demonstrate that HT induction of wheat male sterility is probably related to the expression of genes related to carbohydrate metabolism and sporopollenin metabolism. This provides a theoretical basis and technological approach for further studies on the mechanisms of HT induction of male sterility.

Pollen wall and tapetum development in Plantago major L. (Plantaginaceae): assisting self-assembly

We have attempted to elucidate the underlying mechanisms of sporoderm development and pattern determination in Plantago major through a detailed ontogenetic study, using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). We aim to compare our observations and interpretation with those on other species. Our study of sporoderm development in Plantago from the early tetrad stage to mature pollen grains has shown that pure physical processes, including self-assembly, which are not under direct genetic control, play an important role and represent evidently one of the instruments of evolution. Our observations fit well with the sequence of self-assembling micellar mesophases and show reiteration of some of them, confirming our self-assembly hypothesis. Some attention was also paid to the possible role of rough and smooth endoplasmic reticulum in the cortical cytoplasm of the developing microspores. The tapetum and Ubisch bodies development are also traced. The importance of detailed ontogenetic studies for understanding the establishment of complex pollen walls in any species and for understanding mechanisms underlying sporoderm development was demonstrated. We also present a simulation, obtained in vitro experiments by self-assembly, mimicking pollen grain of Plantago major. It is clear that, in pollen wall development, biological processes and purely physical factors work in tandem.

Pollen Wall Substructure and Development inTanacetum vulgare(Compositae: Anthemideae): Revisiting Hypotheses on Pattern Formation in Complex Cell Walls

Premise of research. The pollen wall, arguably the most complex cell wall found in plants, is especially elaborate in the family Compositae. Through a detailed ontogenetic study of Tanacetum vulgare pollen, capturing each stage of development at a high level of resolution, we aim to understand the establishment of complex pollen walls and try to unravel the mechanisms and interactions underlying sporoderm development. Other aims are to compare our observations and interpretation with previous studies designed to elucidate the underlying mechanisms of sporoderm development and pattern determination in flowering plants and to clarify whether our earlier self-assembly hypothesis is applicable in Tanacetum.Methodology. TEM, SEM, chemical degradation, and comparative methods were used in this study.Pivotal results. The sequence of events leading to exine emergence from early tetrad stage to maturity is as follows: the appearance of spherical units, small invagination beneath spherical units, columns of spherical units, rodlike units (the future subtectal columellae), and a new layer of spacer matrix between rodlike units; growth of the inner columellae; dispersion of spacer matrix; sporopollenin accumulation on primexine; and deposition of lamellate endexine. Our observations are consistent with the sequence of self-assembling micellar mesophases.Conclusions. The microspore tetrad period is confirmed to be the most important stage, in which the complex organization of the ectexine is established through processes of self-assembly operating in a highly organized glycocalyx. Purely physical processes, including self-assembly and tensegrity, which are not under direct genetic control, play an important role. We demonstrate the complex suite of mechanisms and interactions underlying sporoderm development. It is clear that in pollen wall development, biological processes and purely physical factors are inseparably entwined.

Sporoderm and tapetum ontogeny in Juniperus communis (Cupressaceae). Connective structures between tapetum and microspores

The purpose of this work was to study in detail the successive stages of pollen wall development in Juniperus communis and intimate connection between the microspores and nutritive tapetum with TEM. Another goal was to clear up the mechanisms underlying the developmental processes. The key stages observed are: the appearance of the initial outer glycocalyx layer as a row of spherical units at the early tetrad stage, followed by the emergence of the underlying radially oriented string-like units, resulting in the formation of a reticulate layer. After sporopollenin accumulation, spherical units turn into granules. String-like units start from the microspore plasma membrane, pierce the glycocalyx layer, pass between the primexine granules and through callose jacket and come out to the anther loculus, reaching the tapetal cells. Their substructure and size are similar to viscin threads, but not their function and chemical composition. All the structures, observed in sporoderm development, correspond to subsequent mesophases of a micellar system. The latter develops by self-assembly, under genomic control, regulating increasing concentration of glycoprotein surfactants in the periplasmic space. A thick network of filaments, connecting microspores and tapetum, supplies microspores with necessary nutrients and testifies the opinion that, in the process of development, exine is a living, dynamic system, arranged from plasmodesmata-like units.

Proteome analysis of the wild and YX-1 male sterile mutant anthers of wolfberry (Lycium barbarum L.).

Pollen development is disturbed in the early tetrad stage of the YX-1 male sterile mutant of wolfberry (Lycium barbarum L.). The present study aimed to identify differentially expressed anther proteins and to reveal their possible roles in pollen development and male sterility. To address this question, the proteomes of the wild-type (WT) and YX-1 mutant were compared. Approximately 1760 protein spots on two-dimensional differential gel electrophoresis (2D-DIGE) gels were detected. A number of proteins whose accumulation levels were altered in YX-1 compared with WT were identified by mass spectrometry and the NCBInr and Viridiplantae EST databases. Proteins down-regulated in YX-1 anthers include ascorbate peroxidase (APX), putative glutamine synthetase (GS), ATP synthase subunits, chalcone synthase (CHS), CHS-like, putative callose synthase catalytic subunit, cysteine protease, 5B protein, enoyl-ACP reductase, 14-3-3 protein and basic transcription factor 3 (BTF3). Meanwhile, activities of APX and GS, RNA expression levels of apx and atp synthase beta subunit were low in YX-1 anthers which correlated with the expression of male sterility. In addition, several carbohydrate metabolism-related and photosynthesis-related enzymes were also present at lower levels in the mutant anthers. In contrast, 26S proteasome regulatory subunits, cysteine protease inhibitor, putative S-phase Kinase association Protein 1(SKP1), and aspartic protease, were expressed at higher levels in YX-1 anthers relative to WT anthers. Regulation of wolfberry pollen development involves a complex network of differentially expressed genes. The present study lays the foundation for future investigations of gene function linked with wolfberry pollen development and male sterility.

The pollen wall and tapetum are altered in the cytoplasmic male sterile line RC7 of Chinese cabbage (Brassica campestris ssp pekinensis)

Cytoplasmic male sterile line RC(7) of Chinese cabbage produces mature anthers without pollen. To understand the mechanisms involved, we examined the ultrastructural changes during development of the microspores. Development of microspores was not affected at the early tetrad stage. During the ring-vacuolated period, some large vacuoles appeared in the tapetum cells, making them larger, extending to the anther sac center during the monocyte period. At the same time, the tapetum degenerated as the microspores aborted, resulting in pollen-deficient anthers. As a result, the locules collapsed and the anthers shriveled. The callose was degraded in the pollen walls; abnormal deposits of electrodense material gave rise to irregular spike-shaped structures, rather than the characteristic rod-like shape of the B7 bacula. The internal intine wall of RC(7) was thinner than that of the B7 type. At the mitosis I microspore stage, the tapetum cells contained multiple plastids, with numerous small spherical plastoglobuli, and lipid bodies. Based on these observations, we suggest that RC(7) abortion may be due to mutated genes that normally regulate development of the pollen wall and cell walls in the RC(7) line.

Pollen wall development in Sciadopitys verticillata (Sciadopityaceae)

Pollen wall development of Sciadopitys verticillata was observed by transmission electron microscopy. The pollen of S. verticillata is non-saccate and spherical, and the exine consists of the outer thick, sculptured ectexine and the inner lamellated endexine. At the early tetrad stage, the initial ectexine and lamellae of the initial endexine begin to form on the microspore plasma membrane. The ectexine granules gradually swell. Deposition of sporopollenin materials on the ectexine granules then results it their becoming partially connected to each other. Identification of the original small ectexine granules then becomes difficult, and, finally, the ectexine appears as a homogeneous, partially discontinuous layer. The granules of the early ectexine cannot be identified. At maturity, there are four to five endexine lamellae. Recent molecular data have shown that Sciadopitys first branches off from the Cupressaceae plus Taxaceae clade, which is characterized by granular exine. Although the ectexine of Sciadopitys is similar to that of the Cupressaceae during initial development, the morphology of the ectexine is significantly different in the mature pollen. The initial stage of pollen development clearly shows the structural homology of the granular ectexine. Divergence of the exine structure occurs in the later stages.

Sporoderm ontogeny in Chamaedorea microspadix (Arecaceae): self-assembly as the underlying cause of development

The developmental events in the periplasmic space and cytoplasm of microspores of Chamaedorea microspadix during sporoderm ontogeny have been traced in detail from cytokinesis and early tetrad stage to the formation of intine in mature two-celled pollen grains. Several pre-pattern structures as seen in the periplasmic space of early microspores (dark-contrasted globules, ribbon-like striped structures, strings of beads, spherical units, radially oriented columns of spherical units, rod-like units), can easily be read as different types of micelles (spherical, cylindrical micelles, so-called segmented worms) and their intermediate forms (strings, perforated filaments, columns of spherical micelles). Funnel-like procolumellae, arranged as complexes of bent units, appear next at the middle tetrad stage. These “funnels” possibly correspond to double gyroid phase, typical for a diblock copolymer system, adapted to constraints of the narrow periplasmic space. The foot layer and few endexine lamellae with central white lines appear as laminate (“neat”) micelles, with typical gaps between the laminae; they emerge through intermediate forms of micelles as strings of spherical micelles and/or perforated laminae. Strings are probably a preferential intermediate form of micelle formation in the course of exine development in Chamaedorea: they emerge at every ontogenetic step and participate in the appearance of every exine layer. The first (channelled) layer of the two-layered intine beneath the aperture appears probably on the base of cylindrical micelles. The sequence of the sporoderm layer formation can be understood as a sequence of self-assembling mesophases (and their intermediate forms) in the colloidal system of the microspore periplasmic space, unfolding through the ontogenetic time.

A comparative light and electron microscopic analysis of microspore and tapetum development in fertile and cytoplasmic male sterile radish

To gain further insight into the abortive stages and ultrastructural changes leading to pollen degeneration of a novel cytoplasmic male sterile radish 805A, we compared differences of cellular and subcellular structure of sterile anther with fertile anther by light and electron microscopy analysis. Two types of locule degeneration in sterile anther were detected, of which the time of degeneration occurred and completed was different. In type I, abnormality of pollen mother cells (PMCs) and tapetal cells, including condensation of cytoplasm and large vacuoles within tapetal cells, was shown at PMC stage. In type II, meiosis and early tetrad stage progressed normally except for large vacuoles that appeared in tapetal cells. Ultrastructural alterations of the cellular organization were observed in the type II locules, such as chromatin condensation at the periphery of the nucleus and degeneration of the karyotheca, compared with normal pollen development. The results suggested that the cytoplasmic male sterility anther degeneration was probably caused by dysfunctions of tapetum and vacuolation of tapetum, PMCs, and microspores. Thus, the identical factors, which induced CMS in the same cytoplasmic and nuclear genetic background, might affect development of tapetum and microspore at different stages during the cytoplasmic male sterile 805A anther development.

Abnormal Vacuolization of the Tapetum During the Tetrad Stage is Associated with Male Sterility in the Recessive Genic Male Sterile Brassica napus L. Line 9012A

In the recessive genic male sterile line 9012A of Brassica napus, pollen development is affected during the tetrad stage. According to the light and electron microscopy analysis of tapetal cells and tetrads, the sterile tapetal cells swelled with expanded vacuoles at the early tetrad stage and finally filled the center of the locules where a majority of tetrads encased with the thick callose wall collapsed and degraded. We suggested that an absence of callase, which is a wall-degrading enzyme stored in the vacuoles of tapetal cells before secretion, resulted in the failure of tetrad separation. Moreover, transmission electron microscopy analysis showed that the secretory tapetal cells were not observed in sterile anthers, which indicated that the transition of the tapetum from the parietal type to the secretory type was probably aberrant. In plants, degeneration of the tapetum is thought to be the result of programmed cell death (PCD). PCD of tapetal cells was investigated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay and signals indicative of deoxyribonucleic acid fragmentation were detected much earlier in sterile anther than in fertile anther. This suggests that tapetal breakdown does not occur by the normal procession of PCD and might be following an alternative mechanism of unscheduled apoptosis in line 9012A. This research supports the hypothesis that premature PCD is associated with male sterility in B. napus.

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 development in Quercus robur (Fagaceae)

Tetrad and free microspore stages of Quercus robur L. were studied in thin sections for transmission electron microscopy, and mature pollen grains were observed with scanning electron microscopy before and following permanganate treatment. At a very early tetrad stage, there is no indication of a glycocalyx on the plasma membrane of microspores. Somewhat later, at an early tetrad stage, the first sign of the glycocalyx appears as a thin dark layer. At this stage, the glycocalyx was rather well developed, and developing columellae were apparent as part of the glycocalyx. At the early mid-tetrad stage, the exine has become better defined. Columellae consist of single tuft-units during the tetrad stages. At first, the columellae appeared to be weakly contrasted compared to the surrounding glycocalyx layer. Initially, the outer components of tufts, the binder zone, were low in contrast while the core zone was dark. The reaction of tufts to stains was reversed during the tetrad stages; the binder zone became dark, and the core, low in contrast. Our interpretation of this is that the relative distribution of spiral subunits in the core and binder subunits was changed. By the late tetrad stage, columellae appeared as distinct rodlets separated from one another, and a foot layer had been introduced. By the time of the vacuolate stage of the free microspore period, the form of the ectexine was similar to that of mature pollen. The ectexine consisted of a very thick tectum with supratectal verrucae, columellae and a foot layer. Many columellae at the vacuolate stage consisted of multiple tuft units but some remained singular. The endexine of aperture regions was lamellated. The thick tectum was penetrated by many microchannels. Tapetal cells were bordered by orbicules (Ubisch bodies) linked by a thin lamellation, the pellicle. During the vacuolate stage of free microspores, tapetal cells became invasive, surrounding the microspores in the loculus. The surface of the tectum and the prominent verrucae on it showed no indication of their rod-shaped tuft structure until exposure to acetolysis followed by potassium permanganate. Then, tuft-units were evident as part of verrucae and the rest of the tectum.

Enhancement of micronuclei frequency in the Tradescantia/micronuclei test using a long recovery time.

The Tradescantia/micronuclei test (TRAD/MCN) is a well-validated test for monitoring environmental genotoxicants. These pollutants induce at the early meiotic stage of pollen mother cells chromosome fragments which become micronuclei at the tetrad stage. The standard test protocol requires some hours of exposure of the inflorescences and a recovery time of about 24 hours to reach the early tetrad stage. Since the recovery period represents a critical step of the TRAD/MCN, experiments were performed to establish its length in plants of clone #4430 of the hybrid T. hirsutiflora x T. subacaulis which is widely used in environmental monitoring. The aim of the present research was to ascertain the exact duration of recovery time in order to improve the sensitivity of the TRAD/MCN test. First, studies were performed to select the flowers at the beginning of the meiosis, and then anthers were sampled and studied for a period of 48-86 hours. The complete meiosis in the plants examined required about 80 hours. Second, exposure to genotoxic substances followed by different recovery times was carried out to demonstrate that effectiveness of the TRAD/MCN test is closely related to the duration of the recovery time. The test was carried out by exposing inflorescences to known mutagens (sodium azide and maleic hydrazide) for six hours followed by different recovery times (24-72 hours). The results showed that the frequency of micronuclei in the pollen mother cells increased with the length of the recovery time.

Sporoderm ontogeny in Cabomba aquatica (Cabombaceae)

The study on Cabomba aquatica sporoderm ontogeny was undertaken to reveal the principles of development, their connection with the exine substructure and the resistance of the sporopollenin part of the exine to degradation. Procolumellae appear as the first elements of the exine at early tetrad stage, located in the plasma membrane glycocalyx. These procolumellae correspond to elementary exine units (tufts), by Rowley. Very soon after this, procolumellae are seen consisting of several tufts and turn into columellae. Then the grouping of the columellae occurs: sporopollenin accumulates at the proximal parts of the columellae, uniting several of them. Simultaneously a thin and interrupted tectum is introduced, and the columellae grow in height. The process of grouping of individual columellae persists at the stage of the disintegrating tetrad, so that the diameter of a new-formed complex columellae has increased 3 times. The complex columellae consist of clumps of columellae. The initial striae appear in the tectum at an early free microspore stage. Simultaneously, the foot layer as a very thin layer appears. In the course of further development some striae increase in volume (macro-striae), whereas the groups of the striae between them preserve their initial size (micro-striae). Sporopollenin Acceptor Particles (SAPs) appear on the surface of the striae. The diameter of the complex columellae is 6 times more than of procolumellae, the height of the exine increases 7–8 times. The individual columellae, which are the constructive elements of each complex columella, support adjacent supratectal striae and determine the formation of them. In each depression between the striae a row of radially oriented channels penetrates the tectum. These macro-channels are rather wide – unlike micro-channels which penetrate the columellae and the foot layer in different directions. At the stage of microspore cell vacuolisation, when the intine is introduced, the volume of the microspores increases and the exine is stretched out, the complex columellae are pulled apart and can be fragmented to pieces. The oxidative treatment with potassium permanganate brings about the etching of the superficial sporopollenin layer on the surface of the columellae and reveals a spirally arranged cluster of SAPs, the latter becoming a basis for sporopollenin accumulation in the form of a super-spiral around the complex columellae. These data give evidence of SAPs to be responsible for the growth of the elements of the exine by precisely located accumulation of receptor-dependant sporopollenin. The substructure of the exine – tuft units – becomes also evident as a result of oxidative treatment. These are revealed mainly in the tectum as the basic units of micro- and macro-stria. The binder parts of the tuft units resist degradation, but core zones do not. This gives evidence of the secondary nature of the sporopollenin deposited inside the tufts, whereas the binder zone of them seems to be the location of the primary accumulated, receptor-dependant sporopollenin.

DEX1, a Novel Plant Protein, Is Required for Exine Pattern Formation during Pollen Development in Arabidopsis

To identify factors that are required for proper pollen wall formation, we have characterized the T-DNA-tagged, dex1 mutation of Arabidopsis, which results in defective pollen wall pattern formation. This study reports the isolation and molecular characterization of DEX1 and morphological and ultrastructural analyses of dex1 plants. DEX1 encodes a novel plant protein that is predicted to be membrane associated and contains several potential calcium-binding domains. Pollen wall development in dex1 plants parallels that of wild-type plants until the early tetrad stage. In dex1 plants, primexine deposition is delayed and significantly reduced. The normal rippling of the plasma membrane and production of spacers observed in wild-type plants is also absent in the mutant. Sporopollenin is produced and randomly deposited on the plasma membrane in dex1 plants. However, it does not appear to be anchored to the microspore and forms large aggregates on the developing microspore and the locule walls. Based on the structure of DEX1 and the phenotype of dex1 plants, several potential roles for the protein are proposed.

DEX1, a Novel Plant Protein, Is Required for Exine Pattern Formation during Pollen Development in Arabidopsis

To identify factors that are required for proper pollen wall formation, we have characterized the T-DNA-tagged, dex1mutation of Arabidopsis, which results in defective pollen wall pattern formation. This study reports the isolation and molecular characterization of DEX1 and morphological and ultrastructural analyses of dex1 plants.DEX1 encodes a novel plant protein that is predicted to be membrane associated and contains several potential calcium-binding domains. Pollen wall development in dex1 plants parallels that of wild-type plants until the early tetrad stage. Index1 plants, primexine deposition is delayed and significantly reduced. The normal rippling of the plasma membrane and production of spacers observed in wild-type plants is also absent in the mutant. Sporopollenin is produced and randomly deposited on the plasma membrane in dex1 plants. However, it does not appear to be anchored to the microspore and forms large aggregates on the developing microspore and the locule walls. Based on the structure of DEX1 and the phenotype of dex1 plants, several potential roles for the protein are proposed.

Pollen development of Rondeletia odorata (Rubiaceae)

Pollen wall ontogeny of RONDELETIA: odorata was studied with transmission electron microscopy (TEM) and scanning electron microscopy (SEM) from tetrad stage until maturity. The ontogenetic sequence of wall development in RONDELETIA: follows, to some extent, the basic scheme in the angiosperms, i.e., development starts centripetally with the pro-columellae in a plasmalemma surface coating (primexine) at the early tetrad stage when the microspores are still enveloped by callose, until intine formation in young pollen grains. The main ontogenetical features of Rondeletia odorata pollen are (1) the very thin irregular foot layer, (2) development of a continuous layer of radially oriented membranous granular material under the thick endexine, (3) initiation of intine before first mitosis with characteristic radial plasmalemma invaginations, and (4) a strong stretching force upon engorgement just prior to dehiscence, which leads to reduction in thickness of all wall layers. The possible function of Golgi vesicles in the considerable increase in surface area of the plasmalemma at intine initiation is discussed. The endocingulum observed on acetolyzed and sectioned mature grains is explained ultrastructurally.

Pollen ontogeny and its wall formation in Cunninghamia lanceolata

During meiosis, microsporocyte cytoplasm shows considerable changes, expressed in greatly increased organelles and in their equatorial distribution at telophase I and II. At the early tetrad stage, the formation of microspore wall is initiated by deposition of electron dense granules against the callose wall and the appearance of vesicles outside the plasmalemma except at the distal area. After a thin layer of granules, initial ectexine, and fibrillar materials are formed, the endexine starts with the apposition of tripartite lamellae. During this time, many osmiophilic granules are present in the cytoplasm. Both ectexine and endexine continue to develop in the free microspore period. The intine is a homogeneous layer of variable thickness. Intranuclear inclusions, appearing at the early tetrad stage, coincide with exine formation. The difference between sporopollenin synthesized by the microspore and by the tapetum is discussed.

A comparative ultrastructural analysis of exine pattern development in wild-typeArabidopsis and a mutant defective in pattern formation

In order to identify factors necessary for the establishment of the reticulate pollen wall pattern, we have characterized a T-DNA tagged mutant ofArabidopsis thaliana that is defective in pattern formation. This study reports the results of an ultrastructural comparison of pollen wall formation in the mutant to wall development in wild-type plants. Pollen wall development in the mutant parallels that of wild-type until the early tetrad stage. At this point in wild-type plants, the microspore plasma membrane assumes a regular pattern of ridges and valleys. Initial sporopollenin deposition occurs on the ridges marking the beginning of probacula formation. In contrast, the plasma membrane in the mutant appears irregular with flattened protuberances and rare invaginations. As a result, the wild-type regular pattern of ridges and valleys is not formed. Sporopollenin is randomly deposited on the plasma membrane and aggregates on the locule wall; it is not anchored to the membrane. Our finding that the mutation blocks the normal invagination of the plasma membrane and disrupts the proper deposition of sporopollenin during wall formation suggests that the mutation could be in a gene responsible for pattern formation. These results also provide direct evidence that the plasma membrane plays a critical role in the establishment of the pollen wall pattern.