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.     

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.     

Programmed cell death progressively models the development of anther sporophytic tissues from the tapetum and is triggered in pollen grains during maturation

To characterize the spatial and temporal occurrence of programmed cell death (PCD) in Lilium anther tissues, we used both microscopical and molecular markers of apoptosis for developmental stages from meiosis to pollen release. The first hallmarks of PCD include cell condensation and shrinkage of the cytoplasm, separation of chromatin into delineated masses, and DNA fragmentation in the tapetum as early as the premeiosis stage. PCD then extended to other anther sporophytic tissues, leading to anther dehiscence. Although the PCD clearly affected the endothecium and the epidermis, these two cell layers remained alive until anther dehiscence. In pollen, no sign of PCD was found until pollen mitosis I, after what apoptotic features developed progressively in the vegetative cell. In addition, DNA ladders were detected in all sporophytic tissues and cell types throughout pollen development, whereas in the male gametophyte DNA ladders were only detected during pollen maturation. Our data suggest that PCD is a progressive and active process affecting all the anther tissues, first being triggered in the tapetum.     

Sporophytic control of anther development and male fertility by glucose-6-phosphate/phosphate translocator 1(OsGPT1) in rice

Coordination between the sporophytic tissue and the gametic pollen within anthers is tightly controlled to achieve the optimal pollen fitness. Glucose-6-phosphate/phosphate translocator(GPT) transports glucose-6-phosphate, a key precursor of starch and/or fatty acid biosynthesis, into plastids. Here, we report the functional characterization of Os GPT1 in the rice anther development and pollen fertility. Pollen grains from homozygous osgpt1 mutant plants fail to accumulate starch granules, resulting in pollen sterility. Genetic analyses reveal a sporophytic effect for this mutation. Os GPT1 is highly expressed in the tapetal layer of rice anther. Degeneration of the tapetum, an important process to provide cellular contents to support pollen development, is impeded in osgpt1 plants. In addition, defective intine and exine are observed in the pollen from osgpt1 plants. Expression levels of multiple genes that are important to tapetum degeneration or pollen wall formation are significantly decreased in osgpt1 anthers. Previously, we reported that At GPT1 plays a gametic function in the accumulation of lipid bodies in Arabidopsis pollen. This report highlights a sporophytic role of Os GPT1 in the tapetum degeneration and pollen development. The divergent functions of Os GPT1 and At GPT1 in pollen development might be a result of their independent evolution after monocots and dicots diverged.     

Wax-deficient anther1 is involved in cuticle and wax production in rice anther walls and is required for pollen development

In vegetative leaf tissues, cuticles including cuticular waxes are important for protection against nonstomatal water loss and pathogen infection as well as for adaptations to environmental stress. However, their roles in the anther wall are rarely studied. The innermost layer of the anther wall (the tapetum) is essential for generating male gametes. Here, we report the characterization of a T-DNA insertional mutant in the Wax-deficient anther1 (Wda1) gene of rice (Oryza sativa), which shows significant defects in the biosynthesis of very-long-chain fatty acids in both layers. This gene is strongly expressed in the epidermal cells of anthers. Scanning electron microscopy analyses showed that epicuticular wax crystals were absent in the outer layer of the anther and that microspore development was severely retarded and finally disrupted as a result of defective pollen exine formation in the mutant anthers. These biochemical and developmental defects in tapetum found in wda1 mutants are earlier events than those in other male-sterile mutants, which showed defects of lipidic molecules in exine. Our findings provide new insights into the biochemical and developmental aspects of the role of waxes in microspore exine development in the tapetum as well as the role of epicuticular waxes in anther expansion.     

Sporophyte-gametophyte interactions between anther and male gametophyte in petunia

Sporophyte-gametophyte interactions between anther and male gametophyte were investigated in two (fertile and sterile) clones of petunia (Petunia hybrida L.) with different reproductive strategies. Structural and functional reorganization of sporophyte tissues in the developing anther of fertile clone is closely coordinated with each of the successive stages of male gametophyte development (from meiosis to the formation of binuclear pollen) and comprises not only destruction of tapetum and three middle layers of the wall but also an activation of gas exchange and a rise in the content of sugars (sucrose, fructose, and glucose). In sterile clone, degradation of tapetum and anomalies in the development of sporogenic tissue were simultaneously observed in the prophase of meiosis. The death of microsporocytes and degeneration of tapetum were accompanied by a decrease in the level of sucrose delivered to the anther tissues and changes in the ratio between sucrose and hexoses in favor of glucose.     

New views of tapetum ultrastructure and pollen exine development in Arabidopsis thaliana

Background and Aims

The Arabidopsis thaliana pollen cell wall is a complex structure consisting of an outer sporopollenin framework and lipid-rich coat, as well as an inner cellulosic wall. Although mutant analysis has been a useful tool to study pollen cell walls, the ultrastructure of the arabidopsis anther has proved to be challenging to preserve for electron microscopy.

Methods

In this work, high-pressure freezing/freeze substitution and transmission electron microscopy were used to examine the sequence of developmental events in the anther that lead to sporopollenin deposition to form the exine and the dramatic differentiation and death of the tapetum, which produces the pollen coat.

Key Results

Cryo-fixation revealed a new view of the interplay between sporophytic anther tissues and gametophytic microspores over the course of pollen development, especially with respect to the intact microspore/pollen wall and the continuous tapetum epithelium. These data reveal the ultrastructure of tapetosomes and elaioplasts, highly specialized tapetum organelles that accumulate pollen coat components. The tapetum and middle layer of the anther also remain intact into the tricellular pollen and late uninucleate microspore stages, respectively.

Conclusions

This high-quality structural information, interpreted in the context of recent functional studies, provides the groundwork for future mutant studies where tapetum and microspore ultrastructure is assessed.     

A Brassica S locus gene promoter directs sporophytic expression in the anther tapetum of transgenic Arabidopsis

The S locus glycoprotein (SLG) gene of Brassica encodes stigmatic glycoproteins that are implicated in the pollen-stigma interaction of self-incompatibility. We have transformed the related plant Arabidopsis thaliana with a chimaeric gene consisting of the promoter region of an SLG gene fused to the reporter gene beta-glucuronidase (GUS). In transgenic plants the gene was expressed in two cell types of the flower. In stigmas, the timing and distribution of GUS activity was similar to that previously described for SLG expression in Brassica. In anthers, expression was detected at an earlier stage of flower development with GUS activity restricted to the tapetal cell layer. The novel finding of SLG-promoter activity in the anther supports the hypothesis that sporophytic control of self-incompatibility is a result of SLG-gene expression in the tapetum.     

Anther wall layers control pollen sugar nutrition inLilium

Summary Anthers ofLilium were for the first time investigated at the ultrastructural level in order to appreciate the possible ways of sugar transport in the microsporangium. Our results have shown that the cells of the outer anther wall layers and the cell of the connective were interconnected by plasmodesmata, thus allowing assimilates to travel through the symplasmic pathway from the vascular bundle to the most internal middle layer (ML 1). ML 1 was devoid of cell communication throughout pollen development. Tapetal cells were also lacking plasmodesmata on their external face towards ML 1, but adjacent tapetal cells developed lateral junctions: the tapetum could represent a syncytium. Sugars destinated to pollen in the loculus have then to cross the ML 1 and the tapetal layers by the apoplasmic pathway; it is suggested that these two envelopes could be involved in the control of sugar transport from the outer anther wall layers to the locular fluid. Before microspore mitosis, the tapetum degenerated but ML 1 remained structurally unchanged. During pollen development, the guard cells of stomata were lacking cell communication, and preserved their starch content, which could be the sign of photosynthesis within the anther wall. In order to check whether these structural disconnections in anther tissues corresponded to physiological barriers, isolated pollen and stamens were cultivated during the anther maturation phase, on a medium containing increasing concentrations of sucrose (0 M, 1/6 M, 1/2 M, 1 M). After 7 days of culture, isolated pollen was engorged with starch grains and was unable to germinate, whereas in cultivated stamens, pollen did not contain any starch grain: sporophytic tissues, however, accumulated abnormal amylaceous reserves. These results strongly suggest that the anther wall layers, in particular ML 1, starve pollen with sugars during its maturation. They are acting as a physiological buffer storing nutriment surplus in starch grains.Abbreviations ML 1 middle layer 1 - ML 2 middle layer 2 - PAS periodic acid Schiff - PATAg periodic acid thiosemicarbazide silver nitrate