Cytological analysis and genetic control of rice anther development

Microsporogenesis and male gametogenesis are essential for the alternating life cycle of flowering plants between diploid sporophyte and haploid gametophyte generations.Rice (Oryza sativa) is the world's major staple food,and manipulation of pollen fertility is particularly important for the demands to increase rice grain yield.Towards a better understanding of the mechanisms controlling rice male reproductive development,we describe here the cytological changes of anther development through 14 stages,including cell division,differentiation and degeneration of somatic tissues consisting of four concentric cell layers surrounding and supporting reproductive cells as they form mature pollen grains through meiosis and mitosis.Furthermore,we compare the morphological difference of anthers and pollen grains in both monocot rice and eudicot Arabidopsis thaliana.Additionally,we describe the key genes identified to date critical for rice anther development and pollen formation.     

Pollen development at high temperature and role of carbon and nitrogen metabolites

Fruit and seed crop production heavily relies on successful stigma pollination, pollen tube growth, and fertilization of female gametes. These processes depend on production of viable pollen grains, a process sensitive to high‐temperature stress. Therefore, rising global temperatures threaten worldwide crop production. Close observation of plant development shows that high‐temperature stress causes morpho‐anatomical changes in male reproductive tissues that contribute to reproductive failure. These changes include early tapetum degradation, anther indehiscence, and deformity of pollen grains, all of which are contributing factors to pollen fertility. At the molecular level, reactive oxygen species (ROS) accumulate when plants are subjected to high temperatures. ROS is a signalling molecule that can be beneficial or detrimental for plant cells depending on its balance with the endogenous cellular antioxidant system. Many metabolites have been linked with ROS over the years acting as direct scavengers or molecular stabilizers that promote antioxidant enzyme activity. This review highlights recent advances in research on anther and pollen development and how these might explain the aberrations seen during high‐temperature stress; recent work on the role of nitrogen and carbon metabolites in anther and pollen development is discussed including their potential role at high temperature.     

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.     

PERSISTENT TAPETAL CELL1 encodes a PHD-finger protein that is required for tapetal cell death and pollen development in rice

In higher plants, timely degradation of tapetal cells, the innermost sporophytic cells of the anther wall layer, is a prerequisite for the development of viable pollen grains. However, relatively little is known about the mechanism underlying programmed tapetal cell development and degradation. Here, we report a key regulator in monocot rice (Oryza sativa), PERSISTANT TAPETAL CELL1 (PTC1), which controls programmed tapetal development and functional pollen formation. The evolutionary significance of PTC1 was revealed by partial genetic complementation of the homologous mutation MALE STERILITY1 (MS1) in the dicot Arabidopsis (Arabidopsis thaliana). PTC1 encodes a PHD-finger (for plant homeodomain) protein, which is expressed specifically in tapetal cells and microspores during anther development in stages 8 and 9, when the wild-type tapetal cells initiate a typical apoptosis-like cell death. Even though ptc1 mutants show phenotypic similarity to ms1 in a lack of tapetal DNA fragmentation, delayed tapetal degeneration, as well as abnormal pollen wall formation and aborted microspore development, the ptc1 mutant displays a previously unreported phenotype of uncontrolled tapetal proliferation and subsequent commencement of necrosis-like tapetal death. Microarray analysis indicated that 2,417 tapetum- and microspore-expressed genes, which are principally associated with tapetal development, degeneration, and pollen wall formation, had changed expression in ptc1 anthers. Moreover, the regulatory role of PTC1 in anther development was revealed by comparison with MS1 and other rice anther developmental regulators. These findings suggest a diversified and conserved switch of PTC1/MS1 in regulating programmed male reproductive development in both dicots and monocots, which provides new insights in plant anther development.     

Emergence and patterning of the five cell types of the Zea mays anther locule

One fundamental difference between plants and animals is the existence of a germ-line in animals and its absence in plants. In flowering plants, the sexual organs (stamens and carpels) are composed almost entirely of somatic cells, a small subset of which switch to meiosis; however, the mechanism of meiotic cell fate acquisition is a long-standing botanical mystery. In the maize (Zea mays) anther microsporangium, the somatic tissues consist of four concentric cell layers that surround and support reproductive cells as they progress through meiosis and pollen maturation. Male sterility, defined as the absence of viable pollen, is a common phenotype in flowering plants, and many male sterile mutants have defects in somatic and reproductive cell fate acquisition. However, without a robust model of anther cell fate acquisition based on careful observation of wild-type anther ontogeny, interpretation of cell fate mutants is limited. To address this, the pattern of cell proliferation, expansion, and differentiation was tracked in three dimensions over 30 days of wild-type (W23) anther development, using anthers stained with propidium iodide (PI) and/or 5-ethynyl-2′-deoxyuridine (EdU) (S-phase label) and imaged by confocal microscopy. The pervading lineage model of anther development claims that new cell layers are generated by coordinated, oriented cell divisions in transient precursor cell types. In reconstructing anther cell division patterns, however, we can only confirm this for the origin of the middle layer (ml) and tapetum, while young anther development appears more complex. We find that each anther cell type undergoes a burst of cell division after specification with a characteristic pattern of both cell expansion and division. Comparisons between two inbreds lines and between ab- and adaxial anther florets indicated near identity: anther development is highly canalized and synchronized. Three classical models of plant organ development are tested and ruled out; however, local clustering of developmental events was identified for several processes, including the first evidence for a direct relationship between the development of ml and tapetal cells. We speculate that small groups of ml and tapetum cells function as a developmental unit dedicated to the development of a single pollen grain.     

iaaM基因在烟草花粉中的表达及其在花粉发育中的作用

试验利用花粉特异表达的启动子（Lat52)和绒毡层特异表达的启动子（TA29）引导外源生长素合成代谢基因（iaaM)在烟草花粉中表达以研究生长素在花粉发育中的作用。转Lat52-iaaM基因或转TA29－iaaM基因烟草在形态上表现出变异,如从茎上形成不定根,叶呈卷曲状等典型的生长素表达的性状。另外,与对照相比,转基因烟草花药中IAA水平显著增加,且植株矮化,开花期推迟,有的转基因烟草未能开花。上述现象表明：Lat52和TA29启动子的表达并不仅限于花粉或绒毡层,或者说这两个启动子的表达有些泄漏。转基因烟草的花药形状有较大的变异,早期的每个花药中花粉数明显减少,但这些花粉可被醋酸－洋红染色。所有能开花的转基因烟草均可收到种子,但收自某些转基因株系的种子不能萌发。所有这些结果表明生长素在花粉发育过程中起重要作用,过量的生长素会导致花粉发育的异常。     

iaaM基因在烟草花粉中的表达及其在花粉发育中的作用

试验利用花粉特异表达的启动子(Lat52)和绒毡层特异表达的启动子(TA29)引导外源生长素合成代谢基因(iaaM)在烟草花粉中表达以研究生长素在花粉发育中的作用。转Lat52-iaaM基因或转TA29-iaaM基因烟草在形态上表现出变异,如从茎上形成不定根,叶呈卷曲状等典型的生长素过量表达的性状。另外,与对照相比,转基因烟草花药中IAA水平显著增加,且植株矮化,开花期推迟,有的转基因烟草未能开花。上述现象表明:Lat52和TA29启动子的表达并不仅限于花粉或绒毡层,或者说这两个启动子的表达有些泄漏。转基因烟草的花药形状有较大的变异,早期的每个花药中花粉数明显减少,但这些花粉可被醋酸一洋红染色。所有能开花的转基因烟草均可收到种子,但收自某些转基因株系的种子不能萌发。所有这些结果表明生长素在花粉发育过程中起重要作用,过量的生长素会导致花粉发育的异常。     

Arabinogalactan proteins 6 and 11 are required for stamen and pollen function in Arabidopsis

Successful male reproductive function in plants is dependent on the correct development and functioning of stamens and pollen. AGP6 and AGP11 are two homologous Arabidopsis genes encoding cell wall-associated arabinogalactan glycoproteins (AGPs). Both genes were found to be specifically expressed in stamens, pollen grains and pollen tubes, suggesting that these genes may play a role in male organ development and function. RNAi lines with reduced AGP6 and AGP11 expression were generated. These, together with lines harboring point mutations in the coding region of AGP6, were used to show that loss of function in AGP6 and AGP11 led to reduced fertility, at least partly as a result of inhibition of pollen tube growth. Our results also suggest that AGP6 and AGP11 play an additional role in the release of pollen grains from the mature anther. Thus, our study demonstrates the involvement of specific AGPs in pollen tube growth and stamen function.     

Influence of UV-B radiation on the reproductive function of Hordeum vulgare L. plants

Irradiation of barley seedlings by ultraviolet-B radiation influences the growth and development of the sexual elements of the spike, accelerating differentiation of the sporogenous tissue of the anther and the male gametophyte, a phenomenon which is accompanied by an increase in the asynchronicity of microgametogenesis and in heterogeny of the pollen grains, and in an increase in the sterility of the pollen grains. High ultraviolet doses tend to reduce the level of sterility of the anther due to intensification of haplontic cell selection. The influence of ultraviolet irradiation on the reproductive system of the plant is expressed in genotoxic (through damage to the DNA of meristemic cells) and photo-inductive (through acceleration of efflorescence and differentiation of gametophytes) effects.     

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     

低温预处理影响水稻花药培养效率的机理初探

低温预处理延缓药壁中层和绒毡层的降解,促进表皮层和药室内壁层的发育,延缓花药过氧化物酶同工酶活性的增强。处理期间花药可溶性蛋白质、淀粉酶同工酶潜带发生明显变化。处理期间花药的~3H-TdR渗入和花粉的发育、分裂,表明花粉存在合成和充实活动。绒毡层和花粉间存在囊泡,表皮层和药室内壁层之间存在多泡体的穿壁运动,说明低温处理中药壁向花粉输送雄核发育所需的物质。在进入正常培养初期,经过低温处理的花药药壁仍有表皮层和药室内壁层的发育,多细胞花粉出现提早、数量增加,花粉退化延缓。而未经处理的花药药壁各层均迅速降解,花粉大量退化。     

Tapetum determinant1 is required for cell specialization in the Arabidopsis anther

In flowering plants, pollen formation depends on the differentiation and interaction of two cell types in the anther: the reproductive cells, called microsporocytes, and somatic cells that form the tapetum. The microsporocytes generate microspores, whereas the tapetal cells support the development of microspores into mature pollen grains. Despite their importance to plant reproduction, little is known about the underlying genetic mechanisms that regulate the differentiation and interaction of these highly specialized cells in the anther. Here, we report the identification and characterization of a novel tapetum determinant1 (TPD1) gene that is required for the specialization of tapetal cells in the Arabidopsis anther. Analysis of the male-sterile mutant, tpd1, showed that functional interruption of TPD1 caused the precursors of tapetal cells to differentiate and develop into microsporocytes instead of tapetum. As a results, extra microsporocytes were formed and tapetum was absent in developing tpd1 anthers. Molecular cloning of TPD1 revealed that it encodes a small protein of 176 amino acids. In addition, tpd1 was phenotypically similar to excess microsporocytes1/extra sporogenous cells (ems1/exs) single and tpd1 ems1/exs double mutants. These data suggest that the TPD1 product plays an important role in the differentiation of tapetal cells, possibly in coordination with the EMS1/EXS gene product, a Leu-rich repeat receptor protein kinase.     

Fused lobed anther and hooked stigma affect pollination,fertilization and fruit set in mango: A scanning electron microscopy study

Mango malformation is the most threaten disease that limits mango production, worldwide. For a long time, due to its complex nature, the cause and causal agents were strongly disputed. Diverse Fusaria, including Fusarium mangiferae, are known to be associated with the disease. There are indications that augmented level of endogenous ethylene in response to various abiotic and biotic stresses alters the morphology of reproductive organs. Here, scanning electron microscopy (SEM) of healthy and malformed reproductive organs of mango cv. Baramasi was performed to compare the functional morphology. The SEM study revealed that anthers of hermaphrodite healthy flowers were bilobed with large number of turgid pollen grains whereas malformed flowers showed fused lobed anthers with scanty deformed pollen grains. Furthermore, the stigma of healthy flowers exhibited a broad landing pad as compared to malformed stigma which showed hooked and pointed tip. All these impaired morphology of male and female reproductive organs lead to failure of sexual reproduction. This is the first evidence to show fused lobed anther with impaired pollen grains and hooked stigma with poor stigmatic receptivity are mainly responsible for restricting the pollen germination and pollen tube growth. Here we suggest that abnormal development of anthers and pistils is due to endogenously produced stress ethylene. Further, added load of cyanide, a byproduct of ethylene biosynthesis, may also contribute to the development of necrosis which lead to desiccation of anther and pistil during hypersensitive response of plants.