Microspore Stage Research Articles

Application of rice microspore-preferred promoters to manipulate early pollen development in Arabidopsis: a heterologous system.

Rice microspore-promoters. Based on microarray data analyzed for developing anthers and pollen grains, we identified nine rice microspore-preferred (RMP) genes, designated RMP1 through RMP9. To extend their biotechnological applicability, we then investigated the activity of RMP promoters originating from monocotyledonous rice in a heterologous system of dicotyledonous Arabidopsis. Expression of GUS was significantly induced in transgenic plants from the microspore to the mature pollen stages and was driven by the RMP1, RMP3, RMP4, RMP5, and RMP9 promoters. We found it interesting that, whereas RMP2 and RMP6 directed GUS expression in microspore at the early unicellular and bicellular stages, RMP7 and RMP8 seemed to be expressed at the late tricellular and mature pollen stages. Moreover, GUS was expressed in seven promoters, RMP3 through RMP9, during the seedling stage, in immature leaves, cotyledons, and roots. To confirm microspore-specific expression, we used complementation analysis with an Arabidopsis male-specific gametophytic mutant, sidecar pollen-2 (scp-2), to verify the activity of three promoters. That mutant shows defects in microspore development prior to pollen mitosis I. These results provide strong evidence that the SIDECAR POLLEN gene, driven by RMP promoters, successfully complements the scp-2 mutation, and they strongly suggest that these promoters can potentially be applied for manipulating the expression of target genes at the microspore stage in various species.

The Distribution Features of Polysaccharides and Lipids in the Development of Tomato Anthers

The regulation of nutrient transportation and transformation in developing anthers is very complex. We analyzed the distribution and features of polysaccharides and lipids in the developing anthers of tomatoes using histochemical methods. Some starches appeared in the connective somatic tissue of anthers during the sporogenous cell stage. Before meiosis of the microspore mother cell, a thick polysaccharide callose wall was formed, accompanied by a reduction in the connective tissue starches. During the tetrad stage after meiosis, the polysaccharide material in the anther did not change. At the early microspore stage, the starches in the connective cells again increased, and polysaccharide material appeared in the partial intine of pollen. At the late microspore stage, a large vacuole formed that did not contain lipids or starches, and only the pollen wall contained red polysaccharides. At this stage, the connective somatic cell starch amounts decreased, and the tapetal cells changed shape and degenerated. After microspore division, abundant lipids appeared in the bicellular pollen, and starches accumulated following pollen development. As the anthers matured, many lipids and some starches accumulated in the epidermal cells. Nutrient metabolism within the tomato pollen characteristically accumulated lipids first and then starches, while the mature pollen accumulated starches and lipids simultaneously. This characteristic pattern of nutrient metabolism in tomato pollen shows species specificity among plants.

Evaluation of haploidization efficiency in winter squash (Cucurbita maxima Duch.) and pumpkin (Cucurbita moschata Duch.) through anther culture

The production of large numbers of haploids is the crucial point of the dihaploidisation process. Although in vitro haploid plants were successfully produced by irradiated pollen technique in winter squash (Cucurbita maxima Duch.) and pumpkin (Cucurbita moschata Duch.), the frequency is still insufficient for using in a large-scale breeding programme. Thus, the present study was conducted to determine the efficacy of the anther culture technique on the production of in vitro haploids in the aforementioned species for which there have been no successful reports concerning by androgenesis. The anthers at uninucleate microspore stage were collected at different florescence times and then cultured on a solid MS medium supplemented by different combinations of 2,4-D (2,4-dichlorophenoxyacetic acid), BAP (6-benzylaminopurine), KN (kinetin) with the constant addition of NAA (naphthalene acetic acid) to induce callogenesis, embryogenesis and plantlet initiation. The combination of PGR, genotype and anther collection time played an important role in the androgenic response. The highest response was obtained from 57SI21 and G9 lines with the combination of 2.0 or 4.0 mg/l BAP + 0.05 mg/l NAA (E6 medium) at the first anther collection time. Plantlets were regenerated and rooted on MS medium supplemented by 0.01 mg/l IAA. In total, 74 plants were recovered and propagated with micro-cuttings. The ploidy analyses revealed that 35 plants (47.3 %) were haploid (n = 20), and the others (52.7 %) were diploid (2n = 40).

The Rice Eukaryotic Translation Initiation Factor 3 Subunit f (OseIF3f) Is Involved in Microgametogenesis.

Microgametogenesis is the post-meiotic pollen developmental phase when unicellular microspores develop into mature tricellular pollen. In rice, microgametogenesis can influence grain yields to a great degree because pollen abortion occurs more easily during microgametogenesis than during other stages of pollen development. However, our knowledge of the genes involved in microgametogenesis in rice remains limited. Due to the dependence of pollen development on the regulatory mechanisms of protein expression, we identified the encoding gene of the eukaryotic translation initiation factor 3, subunit f in Oryza sativa (OseIF3f). Immunoprecipitation combined with mass spectrometry confirmed that OseIF3f was a subunit of rice eIF3, which consisted of at least 12 subunits including eIF3a, eIF3b, eIF3c, eIF3d, eIF3e, eIF3f, eIF3g, eIF3h, eIF3i, eIF3k, eIF3l, and eIF3m. OseIF3f showed high mRNA levels in immature florets and is highly abundant in developing anthers. Subcellular localization analysis showed that OseIF3f was localized to the cytosol and the endoplasmic reticulum in rice root cells. We further analyzed the biological function of OseIF3f using the double-stranded RNA-mediated interference (RNAi) approach. The OseIF3f-RNAi lines grew normally at the vegetative stage but displayed a large reduction in seed production and pollen viability, which is associated with the down-regulation of OseIF3f. Further cytological observations of pollen development revealed that the OseIF3f-RNAi lines showed no obvious abnormalities at the male meiotic stage and the unicellular microspore stage. However, compared to the wild-type, OseIF3f-RNAi lines contained a higher percentage of arrested unicellular pollen at the bicellular stage and a higher percentage of arrested unicellular and bicellular pollen, and aborted pollen at the tricellular stage. These results indicate that OseIF3f plays a role in microgametogenesis.

Determination of male strobilus developmental stages by cytological and gene expression analyses in Japanese cedar (Cryptomeria japonica).

The molecular mechanisms that control male strobilus development in conifers are largely unknown because the developmental stages and related genes have not yet been characterized. The determination of male strobilus developmental stages will contribute to genetic research and reproductive biology in conifers. Our objectives in this study were to determine the developmental stages of male strobili by cytological and transcriptome analysis, and to determine the stages at which aberrant morphology is observed in a male-sterile mutant of Cryptomeria japonica D. Don to better understand the molecular mechanisms that control male strobilus and pollen development. Male strobilus development was observed for 8 months, from initiation to pollen dispersal. A set of 19,209 expressed sequence tags (ESTs) collected from a male reproductive library and a pollen library was used for microarray analysis. We divided male strobilus development into 10 stages by cytological and transcriptome analysis. Eight clusters (7324 ESTs) exhibited major changes in transcriptome profiles during male strobili and pollen development in C. japonica Two clusters showed a gradual increase and decline in transcript abundance, respectively, while the other six clusters exhibited stage-specific changes. The stages at which the male sterility trait of Sosyun was expressed were identified using information on male strobilus and pollen developmental stages and gene expression profiles. Aberrant morphology was observed cytologically at Stage 6 (microspore stage), and differences in expression patterns compared with wild type were observed at Stage 4 (tetrad stage).

Activity of selected hydrolytic enzymes in Allium sativum L. anthers

The aim of the study was to determine enzymatic activity in sterile Allium sativum anthers in the final stages of male gametophyte development (the stages of tetrads and free microspores). The analysed enzymes were shown to occur in the form of numerous isoforms. In the tetrad stage, esterase activity was predominant, which was manifested by the greater number of isoforms of the enzyme. In turn, in the microspore stage, higher numbers of isoforms of acid phosphatases and proteases were detected. The development of sterile pollen grains in garlic is associated with a high level of protease and acid phosphatase activity and lower level of esterase activities in the anther locule. Probably this is the first description of the enzymes activity (ACPH, EST, PRO) in the consecutives stages of cell wall formation which is considered to be one of the causes of male sterility in flowering plant.

Proteomic alteration of a thermo‐sensitive male sterility SP2S in rapeseed (Brassica napus) in response to mild temperature stress

AbstractWe conducted a proteomic comparison between thermo‐sensitive genic male sterility (TGMS) SP2S and its near‐isogenic line SP2F grown at 22°C. The proteomes at microsporocyte meiosis and uninucleate microspore stages were profiled using 2‐dimensional gel electrophoresis. Twenty‐five well‐reproducible spots (10 spots at microsporocyte meiosis stage and 15 spots at uninucleate microspore stage) containing 28 proteins were successfully identified by MALDI‐TOF/TOF mass spectrometry. An elongation factor EF‐2 at microsporocyte stage and 4 proteins (aconitate hydratase, triosephosphate isomerase, serine/arginine‐rich mRNA splicing factor and glutathione S‐transferase) at uninucleate microspore stage accumulated in SP2S, but more proteins were lost or reduced, including those associated with amino acid metabolism, photosynthesis, synthesis and degradation of protein, lipid metabolism, cytoskeleton, RNA modification, oxidoreductase and defence response. The dramatic decrease of tubulin, actin and Translationally controlled tumour protein (TCTP) crucial for microtubule and cell division and three enzymes for amino acid metabolite at early stage indicated a serious defect on the cytokinesis. They were important clues for us to search the TGMS genes and its interacting genes.

Premature breakdown of tapetum associated with reverse thermo-sensitive genic male-sterile line Huiyou50S in rapeseed (Brassica napus)

Thermo-sensitive genic male-sterility (TGMS) has great advantages in hybrid crop production. Different from common TGMS in Brassica napus, a reverse TGMS line named Huiyou50S is male sterile when it is cultivated in low temperature, but highly fertile when exposed to high temperature. To gain further insights into the developmental aberrations leading to this male sterility, the anther development of Huiyou50S was observed by light and electron microscopy. Firstly, it was suggested by light microscopy that the abortion of anther occurred at the tetrad to early uninucleate microspore stage. A premature breakdown of tapetum is associated with protoplast degradation in the uninucleate microspores. Under transmission electron microscope, the tapetum exhibited vacuolization from pollen mother cell (PMC) stage to tetrad microspore stage and was then disrupted before late microspore stage. Alterations in the tapetal cells included abnormal plastids and a loss of recognizable elaioplast. However, the endoplasmic reticulum seemed to be normal as in the fertile plant. The formation of primexine on the surface of tetrad microspores was postponed to the uninucleate microspore, and hence exine construction was delayed. The evidences suggested that a premature breakdown of tapetum characterized by vacuolization, less secretory activity and abnormal elaioplast, rather than programmed cell death in normal plant, was associated with microspore abortion in Huiyou50S.

Comparative analysis of microRNA profiles of rice anthers between cool-sensitive and cool-tolerant cultivars under cool-temperature stress.

Plants subjected to abiotic stress can regulate gene expression post-transcriptionally by means of small RNAs such as microRNAs. Cool-temperature stress causes abnormal tapetum hypertrophy in rice anthers, leading to pollen sterility. As a first step toward understanding the molecular mechanisms of cool tolerance in developing anthers of rice, we report here a comprehensive comparative analysis of microRNAs between cool-sensitive Sasanishiki and cool-tolerant Hitomebore cultivars. High-throughput Illumina sequencing revealed 241 known and 46 novel microRNAs. Interestingly, 15 of these microRNAs accumulated differentially in the two cultivars at the uninucleate microspore stage under cool conditions. Inverse correlations between expression patterns of microRNAs and their target genes were confirmed by quantitative RT-PCR analysis, and cleavage sites of some of the target genes were determined by 5' RNA ligase-mediated RACE experiments. Thus, our data are useful resources to elucidate microRNA-mediated mechanism(s) of cool tolerance in rice anthers at the booting stage.

Microsporogenesis and meiotic abnormalities in different ‘Oblačinska’ sour cherry (Prunus cerasus L.) clones

The Oblačinska sour cherry (Prunus cerasus) is a mixture of different clones with similar tree and fruit characteristics that is indigenous to and widely planted in commercial orchards in Serbia. Sour cherry, including Oblačinska sour cherry clones, exhibits irregular meiosis which may contribute to low fruit set in some selections. The goal of this study was to examine the process of microsporogenesis and to determine if meiosis and its anomalies effect the in vitro pollen germination and pollen tube length in four ‘Oblačinska’ sour cherry clones that differ in fruit set and yields. All clones displayed varying degrees of chromosomal abnormalities in all meiosis sub-phases. The abnormalities became evident from late pachytene with more than half of the pollen mother cells (PMC) showing abnormal conjugation of chromosomes in metaphase I. The lowest number of PMCs with laggards was in clone III/9 and the highest in clone XIII/1. In second division, the univalent and multivalent association was observed at metaphase-II, the lagging and stickiness in anaphase-II, and the phenomenon persisted up to the microspore stage. In all four ‘Oblačinska’ clones, PMCs exhibited cytomixis phenomena, however, it was only observed in the second experimental year. Cytomixis differed among the four clones but was equally frequent in all stages of meiosis. The syncytia formed, most often consisted of 2–3 PMCs that were at the same phase of meiosis, and exhibited common cytoplasm and occasionally nuclear fusion. In vitro pollen germination and pollen tube length significantly differed between the clones. Most probably abnormalities during meiosis, regardless of the good results of pollen germination, influenced the reduced potential clones XI/3 and XIII/1.

Field phenomics for response of a rice diversity panel to ten environments in Senegal and Madagascar. 2. Chilling-induced spikelet sterility

Rice spikelet sterility caused by chilling during microspore stage of panicle development is a major cause of the rareness of the indica sub-species in cool environments. A diversity panel of 200 indica accessions including traditional and improved accessions forming 4 genetic sub-groups (I1 to I4), along with 22 accessions representing other genetic groups of Oryza sativa L., was field phenotyped for spikelet sterility under flooded management. Environments were six seasonal climatic situations (sowing dates) in Senegal, ranging from cool to hot; and at two altitudes (857 and 1497m asl) and two years in Madagascar. The apex temperature during the critical phenological stage was estimated from weather records and an empirical model estimating the daily minimum water temperature. Sterility incidence was regressed for each accession against apex temperature across all environments after discarding observations potentially involving heat induced sterility. From the correlations, the slope of the temperature response and sterility at 20°C (no stress) and 15°C (chilling stress) were derived and used to evaluate the accessions and establish clusters. Indicas were on average more sensitive than temperate and tropical japonicas. Among indicas, large diversity in cold sterility was observed that formed 6 clusters, which represented the different genetic sub-groups at a distinct frequency. Sterility was greater in Senegal than in Madagascar for all clusters. Sub-group I3 indigenous to Madagascar (traditional varieties) was nearly exclusively present in the most cold tolerant cluster C1. Some improved indicas were also in C1 but belonged to sub-group I2. The phenomics resource will be used in a genome wide association (GWA) study to identify putative candidate genes.

Expression analysis of two rice pollen-specific promoters using homologous and heterologous systems

Tissue-specific promoters are a very useful tool for manipulating gene expression in a target tissue or organ; however, their range of applications in other plant species has not been determined, to date. In this study, we identified two late pollen-specific rice promoters (ProOsLPS10 and ProOsLPS11) via meta-anatomical expression analysis. We then investigated the expression of both promoters in transgenic rice (a homologous system) and Arabidopsis (a heterologous system) using ProOsLPS10 or ProOsLPS11::GFP-GUS constructs. As predicted by microarray data, both promoters triggered strong GUS expression during the late stages of pollen development in rice, with no GUS signals detected in the examined microspores and sporophytic tissues. Interestingly, these promoters exhibited different GUS expression patterns in Arabidopsis. While in Arabidopsis, the OsLPS10 promoter conferred GUS expression at the uni- and bi-cellular pollen stages, as well as at the shoot apical region during the seedling stage, the OsLPS11 promoter was not active in the pollen at any stage, or in the examined sporophytic tissues. Furthermore, by performing a complementation analysis using a sidecar pollen (scp) mutant that displays developmental defects at the microspore stage, we found evidence that OsLPS10 promoter, which can be applied in Arabidopsis, is useful for directing gene expression in the early stages of pollen development. Our results indicate that the OsLPS10 and OsLPS11 promoters can drive the expression of target genes during the late stages of pollen development in rice, but not in Arabidopsis. Our results also emphasize the necessity of confirming the applicability of an established promoter to heterologous systems.

Pollen morphology and post-tetrad wall development in the subfamily Fumarioideae (Papaveraceae)

The subfamily Fumarioideae is the most diversified group of the Papaveraceae family, a eurypalinous group whose pollen information has classically been used for studying systematic issues. In this paper we present a study of pollen morphology and development of the pollen wall in most of the Fumarioideae genera excluding Hypecoum. We describe a high variability in pollen grain ornamentation and in aperture shape and number among genera of the subfamily. Remarkable features of the pollen-wall layer development in Fumarioideae are a lamellated foot layer at the young microspore stage and a granular infratectum. In the apertures, the aperture membrane formed by ectexine is absent for the subtribe Fumariinae, and the ultrastructure of the exinous oncus shows that the endexine is arranged in three different ways among the Fumarioideae representatives. From vacuolate microspore stage onward the apertures of most Fumarioideae genera are covered by a fluffy plug, while during young bicellular stage the intine under the apertures develops from the material that accumulated in the periplasmic space. We have identified significant differences between the pollen features of the tribe Fumarieae and the tribe Hypecoeae, such as psilate exine and granular infratectum. The presence of atypical pollen features in Fumarioideae is discussed within the framework of current knowledge of pollen wall development, highlighting their implications for a better understanding of pollen wall evolution.

EMBRYOGENESIS IN ANTHER CULTURE IN DIPLOID AND TRIPLOID LOQUAT

Embryogenesis induction from anthers of diploid and triploid loquat (Eriobotrya japonica Lindl.) was evaluated. The effects of cultivar, stage of flower buds and culture media were tested. A correlation was found between diameters of the flower buds and the microspore developmental stages in both diploid and triploid loquat cultivars. Callus induction rates in the appropriate media varied according to the loquat cultivars used, ranging from about 60 (triploid) to 78% (diploid). The diploid and triploid cultivars shared the same pattern in the embryoid induction influenced by zeatin, NAA, and IBA, although the optimal media for anther embryoid induction was cultivar dependent.

Dynamics of Polysaccharides and Neutral Lipids during Anther Development in Castor (Ricinus communis)

Anthers contain starch and neutral lipids, which have key roles in microspore ontogeny and gametophyte development. In this study, we observed the dynamic changes in starch and neutral lipids in the anther developmental processes of castor (Ricinus communis) by cytochemical methods. Starch grains and neutral lipids presented a regular dynamic distribution during anther development. In young anthers, some neutral lipids accumulated in sporogenous cells, whereas neutral lipids disappeared with microspore growth. At the late microspore stage, starch grains began to accumulate in microspores, and the starch content of bicellular pollen significantly increased after microspore mitosis. At anthesis, starch grains and neutral lipids accumulated in the mature pollen grains. Visible changes occurred in anther wall cells. The epidermis, middle layer, and tapetum were degenerated, and only a single layer of endothecium remained at anthesis. The dynamic variation of starch grains and neutral lipids in tapetal cells was consistent with the changes in microspores and pollen during anther development. All these findings demonstrated that tapetal cells directly interacted with the developing gametophytes. The tapetal cells play an important role in supplying nutritional substances for microspore absorption. Moreover, the endothecium protects the pollen and contributes to anther dehiscence. The results of this study provide a foundation for the further research on sexual reproduction in angiosperms.

Pollen degeneration in three functional male-sterile lines of eggplant with the wild Solanum cytoplasms

Pollen developing process was studied to identify the stages of pollen degeneration and to elucidate the factors controlling low pollen fertility in three functional cytoplasmic male-sterile (CMS) lines of eggplant ‘Uttara’. The CMS lines of eggplant were developed by repeated backcrossing using the cytoplasms of wild Solanum species S. kurzii Brace & Prain, S. violaceum Ort., and S. virginianum L.. Anthers were squashed in 1% aceto-carmine to assess pollen staining ability and pollen degeneration at different stages of development. Unicellular microspores were released from tetrads after normal meiotic division. Pollen degeneration occurred at different stages of pollen development in CMS lines such as at unicellular microspore (29.3–36.3%), early bicellular pollen (5.5–12.2%), and late bicellular pollen (9.3–10.2%) stages. On the other hand, in eggplant, only 3.8% pollen was degenerated at unicellular microspore stage and there was negligible pollen degeneration at other stages. Among the stained pollen, abnormally stained (partly and faintly) pollen were found significantly higher in the CMS lines as compared to eggplant. Well stained pollen was varied from 23.2–31.9% in the CMS lines which was significantly lower than that of eggplant. Number of pollens per anther of CMS lines did not vary significantly from eggplant, except the CMS line with S. virginianum cytoplasm. In vitro pollen germination rate in the CMS lines was found to be significantly lower than that of eggplant. Starch accumulation and hydrolysis during pollen maturation were found incomplete in the CMS lines. Degeneration of pollen in different stages, abnormally stained pollen, incomplete starch accumulation and hydrolysis were most likely causes for low pollen fertility in these three CMS lines of eggplant.

Development and utilization of an efficient cytoplasmic male sterile system for Cai-xin (Brassica rapa L.)

Six cytoplasmic male sterile (CMS) lines of Brassica vegetable Cai-xin (Brassica rapa L. ssp. chinensis var. utilis Tsen et Lee) were newly developed by backcrossing of six local inbred lines with a new eru CMS line of the vegetable purple cai-tai (B. rapa L. var. purpurea Bailey) from South China. The sterility of these Cai-xin eru CMS lines was stable and complete. The line 4A represented typical sterility and produced six off-white and shriveled stamens without pollen grains in the anthers. Paraffin sectioning comparison of the anthers between the CMS line 4A and its maintainer line 4B showed that their developmental differentiations started at the early to middle mononucleate microspore stage. The tapetum in the CMS line 4A was vacuolized highly and expanded radically, which extruded the developing microspores and caused their adhesion with each other and failure to develope mature pollen. With six CMS lines as female parents and 6 fertile lines as pollen parents, 36 hybrids were produced and their potentials of heterosis were analyzed. In general, mid-parent heterosis was detected for most of the 14 agronomic, yield and quality traits, such as petiole length (19.01%), weight of main flower stalk (35.96%), and soluble protein content (1.20%). These results indicated that the six newly derived eru CMS lines of Cai-xin provided a novel CMS system for its breeding and heterosis utilization.

ABORTED GAMETOPHYTE 1 is required for gametogenesis in Arabidopsis.

In flowering plants, the male and female gametogenesis is a crucial step of sexual reproduction. Although many genes have been identified as being involved in the gametogenesis process, the genetic mechanisms underlying gametogenesis remains poorly understood. We reported here characterization of the gene, ABORTED GAMETOPHYTE 1 (AOG1) that is newly identified as essential for gametogenesis in Arabidopsis thaliana. AOG1 is expressed predominantly in reproductive tissues including the developing pollen grains and ovules. The AOG1 protein shares no significant amino acid sequence similarity with other documented proteins and is located mainly in nuclei of the cells. Mutation in AOG1 caused degeneration of pollen at the uninucleate microspore stage and severe defect in embryo sacs, leading to a significant reduction in male and female fertility. Furthermore, the molecular analyses showed that the aog1 mutant significantly affected the expression of several genes, which are required for gametogenesis. Our results suggest that AOG1 plays important roles in gametogenesis at the stage prior to pollen mitosis I (PMI) in Arabidopsis, possibly through collaboration with other genes.

The development of sporoderm, tapetum and Ubisch bodies in Dianthus deltoides (Caryophyllaceae): Self-assembly in action

The purpose of this work was to study in detail with TEM the successive stages of pollen wall and tapetum development in Dianthus deltoides and to clarify the mechanisms underlying the developmental processes. At the young tetrad stage a thin glycocalyx layer is poorly structured, being a mixture of glycoproteins, secreted by Golgi vesicles through the process of exocytosis. At the middle tetrad stage the plasma membrane acquires a deeply invaginated profile as a result of self-assembly cellular tensegrity, and its pinnacles determine the sites of the future spines. Then, lipoid sporopollenin (SP) precursors and monomers are added into the periplasmic space. The concentrations of all substances in the periplasmic space are under genome control. As a consequence of self-assembly hydrophilic–hydrophobic interactions, “islets” of osmiophilic lipoid substances occur in the glycocalyx mixture, first as spherical units, then as plates, with further lateral growth and the appearance of the hexagonal reticulate pattern of the future tectum. At maturity, the reticulate pattern is not evident. At the late tetrad stage spines are initiated as outgrowths of the glycocalyx in the form of cone-like liquid-crystal “skeletons”, the latter accumulate SP. Columellae are initiated very late in ontogeny, at the stage when the tetrads separate into monads, on the base of the glycocalyx rod-like units — the cylindrical micelles. The first endexine lamella with a central white line appears at the young free microspore stage on the base of laminate micelles. The second endexine layer is granulate and appears on the base of spherical micelles. Pollen grains are polyporate with aperturate membranes, consisting of lamellate endexine-1 and an underlying dilation – oncus – comprising granulate endexine-2. The pore membranes bear large cupola-like tectal spines. The tapetum secretes a peri-tapetal membrane with Ubisch bodies, first with clusters of globules, then exine-like spines on the top of clusters. These Ubisch bodies mimic the pattern of the exine. The entire sequence of developmental events can be explained as the development of a self-assembling system under initial genomic regulation.

Ultrastructure of microsporogenesis and microgametogenesis in Brachypodium distachyon.

Brachypodium distachyon has emerged as a model system for forage grass and cereal grain species. Here, we report B. distachyon pollen development at the ultrastructural level. The process of microsporogenesis and microgametogenesis in B. distachyon follows the typical angiosperm pollen development sequence. Pronounced evaginations of the nuclear envelope are observed prior to meiosis, indicating active nucleocytoplasmic exchange processes. The microspore mother cells undergo meiosis and subsequent cytokinesis, forming isobilateral tetrads. Following dissolution of the callose wall and release of free and vacuolated microspores, mitotic divisions lead to the formation of mature, three-celled pollen grains. In B. distachyon, pollen wall formation begins at the tetrad stage by the formation of the exine template (primexine). The exine is tectate-columellate, comprising a foot layer and endexine. Development of the tectum and the foot layer is complete by the free microspore stage of development, with the tectum formed discontinuously. The endexine initiates in the free microspore stage but becomes compressed in mature grains. The intine layer is deposited after mitosis and comprises three layers during the mature pollen stage of development. Pore development initiates during early free microspore development stage and Brachypodium pollen has a single germination pore consisting of a slightly raised annulus surrounding a central operculum. The tapetum is of the secretory type with loss of the tapetal cell walls beginning at about the time of microsporocyte meiosis. This is the first report on ultrastructure of microsporogenesis and microgametogenesis in B. distachyon. In general, Brachypodium microsporogenesis and microgametogenesis conform to a typical grass pollen development pattern.