Post-meiotic Anther Development Research Articles

OsMS1 functions as a transcriptional activator to regulate programmed tapetum development and pollen exine formation in rice.

OsMS1 functions as a transcriptional activator and interacts with known tapetal regulatory factors through its plant homeodomain (PHD) regulating tapetal programmed cell death (PCD) and pollen exine formation in rice. The tapetum, a hallmark tissue in the stamen, undergoes degradation triggered by PCD during post-meiotic anther development. This degradation process is indispensable for anther cuticle and pollen exine formation. Previous study has shown that PTC1 plays a critical role in the regulation of tapetal PCD. However, it remained unclear how this occurs. To further investigate the role of this gene in rice, we used CRISPR/Cas9 system to generate the homozygous mutant named as osms1, which showed complete male sterility with slightly yellow and small anthers, as well as invisible pollen grains. In addition, cytological observation revealed delayed tapetal PCD, defective pollen exine formation and a lack of DNA fragmentation according to a TUNEL analysis in the anthers of osms1 mutant. OsMS1, which encodes a PHD finger protein, was located in the nucleus of rice protoplasts and functioned as a transcription factor with transcriptional activation activity. Y2H and BiFC assays demonstrated that OsMS1 can interact with OsMADS15 and TDR INTERACTING PROTEIN2 (TIP2). It has been reported that TIP2 coordinated with TDR to modulate the expression of EAT1 and further regulated tapetal PCD in rice. Results of qPCR suggested that the expression of the genes associated with tapetal PCD and pollen wall biosynthesis, such as EAT1, AP37, AP25, OsC6 and OsC4, were significantly reduced in osms1 mutant. Taken together, our results demonstrate that the interaction of OsMS1 with known tapetal regulatory factors through its PHD finger regulates tapetal PCD and pollen exine formation in rice.

Autophagy-mediated regulation of phytohormone metabolism during rice anther development

ABSTRACTAutophagy has recently been shown to be required for postmeiotic anther development including anther dehiscence, programmed cell death-mediated degradation of the tapetum and pollen maturation in rice. Several phytohormones are known to play essential roles during male reproductive development including pollen maturation. However, the relationship between phytohormone metabolism and autophagy in plant reproductive development is unknown. We here comprehensively analyzed the effect of autophagy disruption on phytohormone contents in rice anthers at the flowering stage, and found that endogenous levels of active-forms of gibberellins (GAs) and cytokinin, trans-zeatin, were significantly lower in the autophagy-defective mutant, Osatg7–1, than in the wild type. Treatment with GA4 partially recovered maturation of the mutant pollens, but did not recover the limited anther dehiscence as well as sterility phenotype. These results suggest that autophagy affects metabolism and endogenous levels of GAs and cytokinin in rice anthers. Reduction in bioactive GAs in the autophagy-deficient mutant may partially explain the defects in pollen maturation of the autophagy-deficient mutant, but tapetal autophagy also plays other specific roles in fertilization.

OsATG7 is required for autophagy-dependent lipid metabolism in rice postmeiotic anther development

In flowering plants, the tapetum, the innermost layer of the anther, provides both nutrient and lipid components to developing microspores, pollen grains, and the pollen coat. Though the programmed cell death of the tapetum is one of the most critical and sensitive steps for fertility and is affected by various environmental stresses, its regulatory mechanisms remain mostly unknown. Here we show that autophagy is required for the metabolic regulation and nutrient supply in anthers and that autophagic degradation within tapetum cells is essential for postmeiotic anther development in rice. Autophagosome-like structures and several vacuole-enclosed lipid bodies were observed in postmeiotic tapetum cells specifically at the uninucleate stage during pollen development, which were completely abolished in a retrotransposon-insertional OsATG7 (autophagy-related 7)-knockout mutant defective in autophagy, suggesting that autophagy is induced in tapetum cells. Surprisingly, the mutant showed complete sporophytic male sterility, failed to accumulate lipidic and starch components in pollen grains at the flowering stage, showed reduced pollen germination activity, and had limited anther dehiscence. Lipidomic analyses suggested impairment of editing of phosphatidylcholines and lipid desaturation in the mutant during pollen maturation. These results indicate a critical involvement of autophagy in a reproductive developmental process of rice, and shed light on the novel autophagy-mediated regulation of lipid metabolism in eukaryotic cells.

OsC6, Encoding a Lipid Transfer Protein, Is Required for Postmeiotic Anther Development In Rice

Synthesis of lipidic components in anthers, including of the pollen exine, is essential for plant male reproductive development. Plant lipid transfer proteins (LTPs) are small, abundant lipid-binding proteins that have the ability to exchange lipids between membranes in vitro. However, their biological role in male reproductive development remains less understood. Here, we report the crucial role of OsC6 in regulating postmeiotic anther development in rice (Oryza sativa). Found in monocots, OsC6 belongs to a distinct clade from previously identified LTP1 and LTP2 family members found in both dicots and monocots. OsC6 expression is mainly detectable in tapetal cells and weakly in microspores from stage 9 to stage 11 of anther development. Immunological assays indicated that OsC6 is widely distributed in anther tissues such as the tapetal cytoplasm, the extracellular space between the tapetum and middle layer, and the anther locule and anther cuticle. Biochemical assays indicated that recombinant OsC6 has lipid binding activity. Moreover, plants in which OsC6 was silenced had defective development of orbicules (i.e. Ubisch bodies) and pollen exine and had reduced pollen fertility. Furthermore, additional evidence is provided that the expression of OsC6 is positively regulated by a basic helix-loop-helix transcription factor, Tapetum Degeneration Retardation (TDR). Extra granule-like structures were observed on the inner surface of the tdr tapetal layer when the expression of OsC6 was driven by the TDR promoter compared with the tdr mutant. These data suggest that OsC6 plays a crucial role in the development of lipidic orbicules and pollen exine during anther development in rice.

The Post-meiotic Deficicent Anther1 (PDA1) gene is required for post-meiotic anther development in rice

To understand the molecular mechanism of male reproductive development in the model crop rice, we isolated a complete male sterile mutant post-meiotic deficient anther1 (pda1) from a gamma-ray-treated rice mutant library. Genetic analysis revealed that the pda1 mutant was controlled by a recessive nucleus gene. The pda1 mutant anther seemed smaller with white appearance. Histological analysis demonstrated that the pda1 mutant anther undergoes normal early tapetum development without obvious altered meiosis. However, the pda1 mutant displayed obvious defects in postmeiotic tapetal development, abnormal degeneration occurred in the tapetal cells at stage 9 of anther development. Also we observed abnormal lipidic Ubisch bodies from the tapetal layer of the pda1 mutant, causing no obvious pollen exine formation. RT-PCR analysis indicated that the expression of genes involved in anther development including GAMYB, OsC4 and Wax-deficient anther1 (WDA1) was greatly reduced in the pda1 mutant anther. Using map-based cloning approach, the PDA1 gene was finely mapped between two markers HLF610 and HLF627 on chromosome 6 using 3,883 individuals of F(2) population. The physical distance between HLF610 and HLF627 was about 194 kb. This work suggests that PDA1 is required for post-meiotic tapetal development and pollen/microspore formation in rice.

Male reproductive development: gene expression profiling of maize anther and pollen ontogeny

BackgroundDuring flowering, central anther cells switch from mitosis to meiosis, ultimately forming pollen containing haploid sperm. Four rings of surrounding somatic cells differentiate to support first meiosis and later pollen dispersal. Synchronous development of many anthers per tassel and within each anther facilitates dissection of carefully staged maize anthers for transcriptome profiling.ResultsGlobal gene expression profiles of 7 stages representing 29 days of anther development are analyzed using a 44 K oligonucleotide array querying approximately 80% of maize protein-coding genes. Mature haploid pollen containing just two cell types expresses 10,000 transcripts. Anthers contain 5 major cell types and express >24,000 transcript types: each anther stage expresses approximately 10,000 constitutive and approximately 10,000 or more transcripts restricted to one or a few stages. The lowest complexity is present during meiosis. Large suites of stage-specific and co-expressed genes are identified through Gene Ontology and clustering analyses as functional classes for pre-meiotic, meiotic, and post-meiotic anther development. MADS box and zinc finger transcription factors with constitutive and stage-limited expression are identified.ConclusionsWe propose that the extensive gene expression of anther cells and pollen represents the key test of maize genome fitness, permitting strong selection against deleterious alleles in diploid anthers and haploid pollen. Because flowering plants show a substantial bias for male-sterile compared to female-sterile mutations, we propose that this fitness test is general. Because both somatic and germinal cells are transcriptionally quiescent during meiosis, we hypothesize that successful completion of meiosis is required to trigger maturation of anther somatic cells.

A floral third whorl-specific marker gene in the dioecious species white campion is differentially expressed in mutants defective in stamen development.

The CCLS4 gene of white campion is specifically expressed in anther epidermis and endothecium from pre- to post-meiotic stages. We report on a detailed in situ analysis of the gene's expression and show that it is a marker of the floral third whorl. The gene is expressed (1) in the anther (epidermis, parietal cells and the derived endothecium) in normally developing stamens and (2) in distinct sub-domains of third-whorl epidermis in mutants exhibiting aberrant states of parietal differentiation. Our results suggest that CCLS4 may fulfil different functions during pre- and post-meiotic anther development and reveal the complex role parietal cells may play during early stamen formation.

Enzymatic Changes in Post-meiotic Anther Development in Petunia hybrida. II. Histochemical Localization of Esterase, Peroxidase, Malate- and Alcohol dehydrogenase

Summary The histochemical localization of esterase, peroxidase, malate (MDH), and alcohol dehydrogenase (ADH) was examined in nine stages of post-meiotic anther development in Petunia hybrida . Esterase was found mostly in the tapetum at early stages and later only in the pollen grains. Peroxidase was also found in the early stages, but in the cell walls of inner wall layers and in the endothecium. MDH was observed in most tissues of the anther including stomium, inner wall layers, tapetum and endothecium, but not in pollen grains. In contrast, ADH was observed only in the pollen grains. The role of these enzymes in the differentiation of the various tissues of the anther is discussed.

Enzymatic Changes in Post-meiotic Anther Development in Petunia hybrida. I. Anther Ontogeny and Isozyme Analyses

Summary The relationship of the occurrence of isozymes of esterase, peroxidase, alcohol dehydrogenase (ADH) and malate dehydrogenase (MDH) with nine stages of the post-meiotic anther development of Petunia hybrida was examined. The esterases were primarily found in stages i to iii when tapetum degeneration was taking place, and later a single band was observed during pollen maturation. Peroxidases were also observed during the earlier stages and their occurrence was related to the differentiation of the endothecium. The activity of ADH was not observed until stage iv and it increased through to anther maturation. The occurrence of ADH was considered to be due to the anaerobic conditions of the maturing pollen and the dessication of anther tissue at later stages of development. Different forms of MDH were present at all nine stages of development and were suggested to be involved with the differentiation of various tissues.