[Morphological study of sexual reproductive disorders in Ligusticum chuanxiong].

Chuanxiong Rhizoma is a well-known Sichuan-specific herbal medicine. Its original plant, Ligusticum chuanxiong, has been cultivated asexually for a long time. L. chuanxiong has sexual reproductive disorders, which restricts its germplasm innovation. However, there is little research on the reproductive system of L. chuanxiong. This study is based on a comparative anatomical research approach, using morphological dissection, paraffin sectioning, staining and compression, and combined with scanning electron microscopy technology, to observe and compare the flowers, fruits, and seeds at various stages of reproductive growth of L. chuanxiong and its wild relative L. sinense. The results showed that the meiosis of pollen mother cells is abnormal in L. chuanxiong anthers, and the size and number of microspores are uneven and inconsistent in the tetrad stage. tapetum cells are not completely degenerated during anther development. During the pollen ripening stage, there are fine cracks in the anther wall, while most anthers could not release pollen normally. The surface of mature pollen grains is concave and partially deformed, and the pollens are all inactive and cannot germinate in vitro. The starch, polysaccharides, and lipids in the pollen were insufficient. The filaments of L. chuanxiong are short at the flowering stage and recurved downward. Double-hanging fruits were observed in the fruiting stage, being wrinkled; with shriveled seeds. Compared with L. sinense at the same stage, the anthers of L. sinense developed normally, and the pollen grains are vigorous and can germinate in vitro. The double-hanging fruits of L. sinense are full and normal; at the flowering period, the filaments are long and erect, significantly higher than the stigma. Mature blastocysts are visible in the ovary of both L. chuanxiong and L. sinense, and there is no significant difference in stigmas. The conclusion is that during the development of L. chuanxiong stamens, the meiosis of pollen mother cells is abnormal, and tetrad, tapetum, filament and other pollen structures develop abnormally. L. chuanxiong has the characteristic of male infertility, which is an important reason for its sexual reproductive disorders.

A Rapid Alkalinization Factor-like Peptide EaF82 Impairs Tapetum Degeneration during Pollen Development through Induced ATP Deficiency.

In plants, the timely degeneration of tapetal cells is essential for providing nutrients and other substances to support pollen development. Rapid alkalinization factors (RALFs) are small, cysteine-rich peptides known to be involved in various aspects of plant development and growth, as well as defense against biotic and abiotic stresses. However, the functions of most of them remain unknown, while no RALF has been reported to involve tapetum degeneration. In this study, we demonstrated that a novel cysteine-rich peptide, EaF82, isolated from shy-flowering 'Golden Pothos' (Epipremnum aureum) plants, is a RALF-like peptide and displays alkalinizing activity. Its heterologous expression in Arabidopsis delayed tapetum degeneration and reduced pollen production and seed yields. RNAseq, RT-qPCR, and biochemical analyses showed that overexpression of EaF82 downregulated a group of genes involved in pH changes, cell wall modifications, tapetum degeneration, and pollen maturation, as well as seven endogenous Arabidopsis RALF genes, and decreased proteasome activity and ATP levels. Yeast two-hybrid screening identified AKIN10, a subunit of energy-sensing SnRK1 kinase, as its interacting partner. Our study reveals a possible regulatory role for RALF peptide in tapetum degeneration and suggests that EaF82 action may be mediated through AKIN10 leading to the alteration of transcriptome and energy metabolism, thereby causing ATP deficiency and impairing pollen development.

Peptide signaling in anther development and pollen-stigma interactions.

Polypeptides play irreplaceable roles in cell-cell communication by binding to receptor-like kinases. Various types of peptide-receptor-like kinase-mediated signaling have been identified in anther development and male-female interactions in flowering plants. Here, we provide a comprehensive summary of the biological functions and signaling pathways of peptides and receptors involved in anther development, self-incompatibility, pollen tube growth and pollen tube guidance.

Targeted expression of bgl23-D, a dominant-negative allele of ATCSLD5, affects cytokinesis of guard mother cells and exine formation of pollen in Arabidopsis thaliana.

MAIN CONCLUSION: Targeted expression of bgl23-D, a dominant-negative allele of ATCSLD5, is a useful genetic approach for functional analysis of ATCSLDs in specific cells and tissues in plants. Stomata are key cellular structures for gas and water exchange in plants and their development is influenced by several genes. We found the A. thaliana bagel23-D (bgl23-D) mutant showing abnormal bagel-shaped single guard cells. The bgl23-D was a novel dominant mutation in the A. thaliana cellulose synthase-like D5 (ATCSLD5) gene that was reported to function in the division of guard mother cells. The dominant character of bgl23-D was used to inhibit ATCSLD5 function in specific cells and tissues. Transgenic A. thaliana expressing bgl23-D cDNA with the promoter of stomata lineage genes, SDD1, MUTE, and FAMA, showed bagel-shaped stomata as observed in the bgl23-D mutant. Especially, the FAMA promoter exhibited a higher frequency of bagel-shaped stomata with severe cytokinesis defects. Expression of bgl23-D cDNA in the tapetum with SP11 promoter or in the anther with ATSP146 promoter induced defects in exine pattern and pollen shape, novel phenotypes that were not shown in the bgl23-D mutant. These results indicated that bgl23-D inhibited unknown ATCSLD(s) that exert the function of exine formation in the tapetum. Furthermore, transgenic A. thaliana expressing bgl23-D cDNA with SDD1, MUTE, and FAMA promoters showed enhanced rosette diameter and increased leaf growth. Taken together, these findings suggest that the bgl23-D mutation could be a helpful genetic tool for functional analysis of ATCSLDs and manipulating plant growth.

Comprehensive Insight into Tapetum-Mediated Pollen Development in Arabidopsis thaliana.

In flowering plants, pollen development is a key process that is essential for sexual reproduction and seed set. Molecular and genetic studies indicate that pollen development is coordinatedly regulated by both gametophytic and sporophytic factors. Tapetum, the somatic cell layer adjacent to the developing male meiocytes, plays an essential role during pollen development. In the early anther development stage, the tapetal cells secrete nutrients, proteins, lipids, and enzymes for microsporocytes and microspore development, while initiating programmed cell death to provide critical materials for pollen wall formation in the late stage. Therefore, disrupting tapetum specification, development, or function usually leads to serious defects in pollen development. In this review, we aim to summarize the current understanding of tapetum-mediated pollen development and illuminate the underlying molecular mechanism in Arabidopsis thaliana.

The full-length Auxin Response Factor 8 isoform ARF8.1 controls pollen cell wall formation and directly regulates TDF1, AMS and MS188 expression.

Auxin Response Factor 8 plays a key role in late stamen development: its splice variants ARF8.4 and ARF8.2 control stamen elongation and anther dehiscence. Here, we characterized the role of ARF8 isoforms in pollen fertility. By phenotypic and ultrastructural analysis of arf8-7 mutant stamens, we found defects in pollen germination and viability caused by alterations in exine structure and pollen coat deposition. Furthermore, tapetum degeneration, a prerequisite for proper pollen wall formation, is delayed in arf8-7 anthers. In agreement, the genes encoding the transcription factors TDF1, AMS, MS188 and MS1, required for exine and pollen coat formation, and tapetum development, are downregulated in arf8-7 stamens. Consistently, the sporopollenin content is decreased, and the expression of sporopollenin synthesis/transport and pollen coat protein biosynthetic genes, regulated by AMS and MS188, is reduced. Inducible expression of the full-length isoform ARF8.1 in arf8-7 inflorescences complements the pollen (and tapetum) phenotype and restores the expression of the above transcription factors. Chromatin immunoprecipitation-quantitative polymerase chain reaction assay revealed that ARF8.1 directly targets the promoters of TDF1, AMS and MS188. In conclusion, the ARF8.1 isoform controls pollen and tapetum development acting directly on the expression of TDF1, AMS and MS188, which belong to the pollen/tapetum genetic pathway.

Rice anther tapetum: a vital reproductive cell layer for sporopollenin biosynthesis and pollen exine patterning.

The tapetum is the innermost layer of the four layers of the rice anther that provides protection and essential nutrients to pollen grain development and delivers precursors for pollen exine formation. The tapetum has a key role in the normal development of pollen grains and tapetal programmed cell death (PCD) that is linked with sporopollenin biosynthesis and transport. Recently, many genes have been identified that are involved in tapetum formation in rice and Arabidopsis. Genetic mutation in PCD-associated genes could affect normal tapetal PCD, which finally leads to aborted pollen grains and male sterility in rice. In this review, we discuss the most recent research on rice tapetum development, including genomic, transcriptomic and proteomic studies. Furthermore, tapetal PCD, sporopollenin biosynthesis, ROS activity for tapetum function and its role in male reproductive development are discussed in detail. This will improve our understanding of the role of the tapetum in male fertility using rice as a model system, and provide information that can be applied in rice hybridization and that of other major crops.

Tomato POLLEN DEFICIENT 2 encodes a G-type lectin receptor kinase required for viable pollen grain formation.

Pollen development is a crucial biological process indispensable for seed set in flowering plants and for successful crop breeding. However, little is known about the molecular mechanisms regulating pollen development in crop species. This study reports a novel male-sterile tomato mutant, pollen deficient 2 (pod2), characterized by the production of non-viable pollen grains and resulting in the development of small parthenocarpic fruits. A combined strategy of mapping-by-sequencing and RNA interference-mediated gene silencing was used to prove that the pod2 phenotype is caused by the loss of Solanum lycopersicum G-type lectin receptor kinase II.9 (SlG-LecRK-II.9) activity. In situ hybridization of floral buds showed that POD2/SlG-LecRK-II.9 is specifically expressed in tapetal cells and microspores at the late tetrad stage. Accordingly, abnormalities in meiosis and tapetum programmed cell death in pod2 occurred during microsporogenesis, resulting in the formation of four dysfunctional microspores leading to an aberrant microgametogenesis process. RNA-seq analyses supported the existence of alterations at the final stage of microsporogenesis, since we found tomato deregulated genes whose counterparts in Arabidopsis are essential for the normal progression of male meiosis and cytokinesis. Collectively, our results revealed the essential role of POD2/SlG-LecRK-II.9 in regulating tomato pollen development.

Anther development in Arabidopsis thaliana involves symplastic isolation and apoplastic gating of the tapetum-middle layer interface.

During flowering plant reproduction, anthers produce pollen grains, the development of which is supported by the tapetum, a nourishing maternal tissue that also contributes non-cell-autonomously to the pollen wall, the resistant external layer on the pollen surface. How the anther restricts movement of the tapetum-derived pollen wall components, while allowing metabolites such as sugars and amino acids to reach the developing pollen, remains unknown. Here, we show experimentally that in arabidopsis thaliana the tapetum and developing pollen are symplastically isolated from each other, and from other sporophytic tissues, from meiosis onwards. We show that the peritapetal strip, an apoplastic structure, separates the tapetum and the pollen grains from other anther cell layers and can prevent the apoplastic diffusion of fluorescent proteins, again from meiosis onwards. The formation and selective barrier functions of the peritapetal strip require two NADPH oxidases, RBOHE and RBOHC, which play a key role in pollen formation. Our results suggest that, together with symplastic isolation, gating of the apoplast around the tapetum may help generate metabolically distinct anther compartments.

Grass-specific ABERRANT MICROSPORE DEVELOPMENT 1 is required for maintaining pollen fertility in rice.

Pollen development includes a series of biological events that require precise gene regulation. Although several transcription factors (TFs) have been shown to play roles in maintaining pollen fertility, the major regulatory networks underlying tapetum development and pollen wall formation are largely unknown. Herein, we report that ABERRANT MICROSPORE DEVELOPMENT1 (AMD1), a protein annotated previously as unknown protein, is required for tapetum development and pollen exine patterning in rice (Oryza sativa L.). AMD1 encodes a grass-specific protein exhibiting transactivation activity in the nucleus and is spatiotemporally expressed in the tapetum and microspores during pollen development. Further biochemical assays indicate that AMD1 directly activates the transcription of DEFECTIVE POLLEN WALL (DPW) and POLYKETIDE SYNTHASE2 (OsPKS2), which are both implicated in sporopollenin biosynthesis during exine formation. Additionally, AMD1 directly interacts with TAPETUM DEGENERATION RETARDATION (TDR), a key TF involved in the regulation of tapetum degradation and exine formation. Taken together, we demonstrate that AMD1 is an important regulatory component involved in the TDR-mediated regulatory pathway to regulate sporopollenin biosynthesis, tapetum degradation, and exine formation for pollen development. Our work provides insights into the regulatory network of rice sexual reproduction and a useful target for genetic engineering of new male-sterile lines for hybrid rice breeding.

Long-Term Mild Heat Causes Post-Mitotic Pollen Abortion Through a Local Effect on Flowers.

Crop reproductive success is significantly challenged by heatwaves, which are increasing in frequency and severity globally. Heat-induced male sterility is mainly due to aborted pollen development, but it is not clear whether this is through direct or systemic effects. Here, long-term mild heat (LTMH) treatment, mimicking a heatwave, was applied locally to tomato flowers or whole plants and followed up by cytological, transcriptomic, and biochemical analyses. By analyzing pollen viability, LTMH was shown to act directly on the flowers and not via effects on other plant tissue. The meiosis to early microspore stage of pollen development was the most sensitive to LTMH and 3 days of exposure around this period was sufficient to significantly reduce pollen viability at the flower anthesis stage. Extensive cytological analysis showed that abnormalities in pollen development could first be observed after pollen mitosis I, while no deviations in tapetum development were observed. Transcriptomic and biochemical analyses suggested that pollen development suffered from tapetal ER stress and that there was a limited role for oxidative stress. Our results provide the first evidence that heat acts directly on flowers to induce pollen sterility, and that the molecular-physiological responses of developing anthers to the LTMH are different from those to severe heat shock.

A peptide-mediated, multilateral molecular dialogue for the coordination of pollen wall formation.

The surface of pollen grains is reinforced by pollen wall components produced noncell autonomously by tapetum cells that surround developing pollen within the male floral organ, the anther. Here, we show that tapetum activity is regulated by the GASSHO (GSO) receptor-like kinase pathway, controlled by two sulfated peptides, CASPARIAN STRIP INTEGRITY FACTOR 3 (CIF3) and CIF4, the precursors of which are expressed in the tapetum itself. Coordination of tapetum activity with pollen grain development depends on the action of subtilases, including AtSBT5.4, which are produced stage specifically by developing pollen grains. Tapetum-derived CIF precursors are processed by subtilases, triggering GSO-dependent tapetum activation. We show that the GSO receptors act from the middle layer, a tissue surrounding the tapetum and developing pollen. Three concentrically organized cell types, therefore, cooperate to coordinate pollen wall deposition through a multilateral molecular dialogue.

Sporophytic control of pollen meiotic progression is mediated by tapetum expression of ABORTED MICROSPORES.

Pollen development is dependent on the tapetum, a sporophytic anther cell layer surrounding the microspores that functions in pollen wall formation but is also essential for meiosis-associated development. There is clear evidence of crosstalk and co-regulation between the tapetum and microspores, but how this is achieved is currently not characterized. ABORTED MICROSPORES (AMS), a tapetum transcription factor, is important for pollen wall formation, but also has an undefined role in early pollen development. We conducted a detailed investigation of chromosome behaviour, cytokinesis, radial microtubule array (RMA) organization, and callose formation in the ams mutant. Early meiosis initiates normally in ams, shows delayed progression after the pachytene stage, and then fails during late meiosis, with disorganized RMA, defective cytokinesis, abnormal callose formation, and microspore degeneration, alongside abnormal tapetum development. Here, we show that selected meiosis-associated genes are directly repressed by AMS, and that AMS is essential for late meiosis progression. Our findings indicate that AMS has a dual function in tapetum-meiocyte crosstalk by playing an important regulatory role during late meiosis, in addition to its previously characterized role in pollen wall formation. AMS is critical for RMA organization, callose deposition, and therefore cytokinesis, and is involved in the crosstalk between the gametophyte and sporophytic tissues, which enables synchronous development of tapetum and microspores.

ADAPTOR PROTEIN-1 complex-mediated post-Golgi trafficking is critical for pollen wall development in Arabidopsis.

Primexine deposition is essential for the formation of pollen wall patterns and is precisely regulated by the tapetum and microspores. While tapetum- and/or microspore-localized proteins are required for primexine biosynthesis, how their trafficking is established and controlled is poorly understood. In Arabidopsis thaliana, AP1σ1 and AP1σ2, two genes encoding the σ subunit of the trans-Golgi network/early endosome (TGN/EE)-localized ADAPTOR PROTEIN-1 complex (AP-1), are partially redundant for plant viability, and the loss of AP1σ1 function reduces male fertility due to defective primexine formation. Here, we investigated the role of AP-1 in pollen wall formation. The deposition of Acyl-CoA SYNTHETASE5 (ACOS5) and type III LIPID TRANSFER PROTEINs (LTPs) secreted from the anther tapetum, which are involved in exine formation, were impaired in ap1σ1 mutants. In addition, the microspore plasma membrane (PM) protein RUPTURED POLLEN GRAIN1 (RPG1), which regulates primexine deposition, accumulated abnormally at the TGN/EE in ap1σ1 mutants. We show that AP-1µ recognizes the YXXΦ motif of RPG1, thereby regulating its PM abundance through endocytic trafficking, and that loss of AP1σ1 decreases the levels of other AP-1 subunits at the TGN/EE. Our observations show that AP-1-mediated post-Golgi trafficking plays a vital role in pollen wall development by regulating protein transport in tapetal cells and microspores.

Cytological Analysis and Fine Mapping of paa1 (Post-meiosis Abnormal Anther 1) Mutant with Abnormal Tapetum and Microspore Development.

To further understand the molecular mechanism for rice male reproduction, a rice male sterile mutant paa1 was screened from the rice mutant library generated by treatment with 60Coγ-rays. Genetic analysis revealed that paa1 is controlled by a single- recessive nuclear gene, and the anthers of the paa1 mutant were smaller than those of WT plants with a white color. Histological analysis demonstrated that the anthers of the paa1 mutant began to turn abnormal at the microspore stage after meiosis, with abnormal degradation of tapetum, deformed Ubisch bodies, and defective pollen exine. TUNEL assay results also confirmed the delay of tapetum PCD in paa1. Map-based cloning was performed for the PAA1 location. As a result, PAA1 was located in a 88-kb region at the end of chromosome 10, which comprises a total of seven candidate genes, and no genes related to anther development have been reported in this region. The results indicate that PAA1 is an essential gene in regulating tapetum development and pollen/microspore formation after rice meiosis.

The N-terminal acetyltransferase Naa50 regulates tapetum degradation and pollen development in Arabidopsis.

The N-terminal acetylation of proteins is a key modification in eukaryotes. However, knowledge of the biological function of N-terminal acetylation modification of proteins in plants is limited. Naa50 is the catalytic subunit of the N-terminal acetyltransferase NatE complex. We previously demonstrated that the absence of Naa50 leads to sterility in Arabidopsis thaliana. In the present study, the lack of Naa50 resulted in collapsed and sterile pollen in Arabidopsis. Further experiments showed that the mutation in Naa50 accelerated programmed cell death in the tapetum. Expression pattern analysis revealed the specific expression of Naa50 in the tapetum cells of anthers at 9-11 stages during pollen development, when tapetal programmed cell death occurred. Reciprocal cross analyses indicated that male sterility in naa50 is caused by sporophytic effects. mRNA sequencing and quantitative PCR of the closed buds showed that the deletion of Naa50 resulted in the upregulation of the cysteine protease coding gene CEP1 and impaired the expression of several genes involved in pollen wall deposition and pollen mitotic division. The collective data suggest that Naa50 balances the degradation of tapetum cells during anther development and plays an important role in pollen development by affecting several pathways.

POLLEN STERILITY, a novel suppressor of cell division, is required for timely tapetal programmed cell death in rice.

Timely programmed cell death (PCD) of the tapetum supplying nutrients to microspores is a prerequisite for normal pollen development. Here we identified a unique mutant of rice (Oryza sativa L.), pollen sterility (post), which showed aborted pollens accompanied with extra-large husks. Due to failure of timely PCD of tapetal cells, post exhibited abnormal pollen wall patterning and defective pollen grains. By map-based cloning, we identified a causal gene, POST, encoding a novel protein which is ubiquitously localized in cells. RNA in situ hybridization showed that POST is highly detected in the tapetum and microspores at stages 8 and 9. Transcriptome analysis indicated that POST could function as an important regulator of the metabolic process involved in tapetal PCD. Compared with wild-type rice, post mutant has an increased cell number resulting from elevated expression of cell cycle associated genes in grain husks. Overexpression of POST inhibits grain size in wild type, while appropriate expression of POST in post mutant can recover the seed fertility but has little effect on the large grains, illustrating that fine-tuning of POST expression could be a potential strategy for rice yield improvement. The connection between cell division and cell death conferred by POST provides novel insights into the understanding of the tapetal PCD process.

The GA-DELLA-OsMS188 module controls male reproductive development in rice.

Pollen protects male sperm and allows flowering plants to adapt to diverse terrestrial environments, thereby leading to the rapid expansion of plants into new regions. The process of anther/pollen development is coordinately regulated by internal and external factors including hormones. Currently, the molecular mechanisms underlying gibberellin (GA)-mediated male reproductive development in plants remain unknown. We show here that rice DELLA/SLR1, which encodes the central negative regulator of GA signaling, is essential for rice anther development. The slr1-5 mutant exhibits premature programmed cell death of the tapetum, lacks Ubisch bodies, and has no exine and no mature pollen. SLR1 is mainly expressed in tapetal cells and tetrads, and is required for the appropriate expression of genes encoding key factors of pollen development, which are suggested to be OsMS188-targeted genes. OsMS188 is the main component in the essential genetic program of tapetum and pollen development. Further, we demonstrate that SLR1 interacts with OsMS188 to cooperatively activate the expression of the sporopollenin biosynthesis and transport-related genes CYP703A3, DPW, ABCG15 and PKS1 for rapid formation of pollen walls. Overall, the results of this study suggest that the GA hormonal signal is integrated into the anther genetic program and regulates rice anther development through the GA-DELLA-OsMS188 module.

Microsporogénesis y micromorfología del polen de la planta Alcea rosea (Malvaceae) / Microsporogenesis and micromorphology of pollen grains of the plant Alcea rosea (Malvaceae)

Resumen Introducción: Los estudios sobre microsporogénesis, micromorfología y estructura de los granos de polen en Malvaceae son escasos. Objetivos: Describir el proceso de microsporogénesis y aspectos micromorfológicos de los granos de polen en A. rosea. Métodos: Se procesaron más de 30 andróforos de acuerdo con los protocolos estándar para incrustar y seccionar en parafina. Las secciones obtenidas se tiñeron con Azul de Safranina-Alcian, las anteras inmaduras y no fijadas se tiñeron con Azul de anilina. Se procesaron secciones de resina adicionales de los andróforos y se tiñeron con azul de toluidina. Se observaron secciones ultrafinas con microscopía electrónica de transmisión (MET). Para la observación con microscopía electrónica de barrido (MEB), el material se fijó y deshidrató en 2,2 dimetoxipropano, luego se secó hasta un punto crítico y se recubrieron con oro. Resultados: las anteras se diferencian de una masa celular en los extremos distales de los filamentos del estambre. La pared de la antera madura presenta una capa externa de células epidérmicas y una capa interna, el endotecio. Las células madre de microesporas se dividen por mitosis y luego experimentan meiosis para formar tétradas. El tapete es inicialmente celular y forma una sola capa de células y luego pierde integridad celular al invadir el lóculo de microsporangio, formando un periplasmodio. Durante la formación de la esporodermis, primero se deposita la exina y luego la intina. Para el momento de la liberación de los granos de polen, el tapete se ha degenerado por completo. Los granos de polen son pantoporados, apolares, con simetría radial, esferoidales, con espinas, báculas, gránulos y microgránulos. El téctum está perforado con fovéoleas dispuestas homogéneamente en toda la superficie y con polenkit. La exina es ancha (5-6 µm) y consta de una endexina gruesa de 3.5 a 4 µm y una ektexina fina (0.6-0.7 µm). La ultraestructura muestra columelas claramente definidas formando el infratéctum. Se aprecian tricomas nectaríferos unicelulares glandulares capitados (TG) cubriendo toda la superficie de los filamentos de los estambres. Conclusiones: La estructura y desarrollo de las anteras sigue los patrones conocidos de las angiospermas. La microsporogénesis simultánea y el depósito centrípeto de la esporodermis se han descrito previamente para Malvaceae.

Abstract Introduction: Studies on microsporogenesis, micromorphology and structure of pollen grains in Malvaceae are scarce. Objectives: To describe the process of microsporogenesis and micromorphological aspects of pollen grains in A. rosea. Methods: Androphores were processed according to standard protocols for sectioning in paraffin. The obtained sections were stained with Safranin-Alcian blue, Aniline blue was used for immature and unfixed anthers and for resin sections of the androphores, Toluidine blue. Ultrathin sections were observed with transmission electron microscopy. For observation with scanning electron microscopy the material was fixed and dehydrated in 2.2 dimethoxypropane, dried to a critical point and coated with gold. Results: Anthers differentiate from a cell mass at the distal ends of the stamen filaments. The wall of the mature anther presents an outer layer of epidermal cells and an inner layer, the endothecium. Microspore mother cells divide by mitosis and then undergo meiosis to form tetrads. The tapetum is initially cellular and forms a single layer of cells and then loses cellular integrity by invading the microsporangium locule, forming a periplasmodia, by the time the pollen grains are released it degenerated. During sporodermis formation, exine is first deposited and then intine. Pollen grains are pantoporate, apolar, with radial symmetry, spheroidal, with spines, bacula, granules and microgranules. Tectum is perforated with foveolae arranged homogeneously over the whole surface and pollenkit is present. Exine is broad and consists of a thick 3.5 to 4 µm endexine and a thin ektexine (0.6-0.7 µm). The ultrastructure shows columellae forming the infratectum. Capitate glandular unicellular nectariferous trichomes covers the whole surface of the stamen filaments. Conclusions: The structure and development of the anthers follows the known patterns for angiosperms. Simultaneous microsporogenesis and centripetal deposit of the sporodermis have been previously described for Malvaceae.