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.

Background: Molecular mechanisms for elicitor-induced changes in cytosolic Ca 2ϩ concentration and its molecular link with regulation of phytoalexin biosynthesis in plant immunity remain mostly unknown. Results: TvX-induced Ca 2ϩ influx and the phytoalexin accumulations were suppressed in Ostpc1 knock-out cells. Conclusion: OsTPC1 plays a role in TvX-induced Ca 2ϩ influx consequently required for the regulation of phytoalexin biosynthesis. Significance: Voltage-dependent plasma membrane Ca 2ϩ -permeable channel activity of the plant TPC1 was shown for the first time.

Autophagy 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.

Autophagy plays crucial roles in the recycling of metabolites, and is involved in many developmental processes. Rice mutants defective in autophagy are male sterile due to immature pollens, indicating its critical role in pollen development. However, physiological roles of autophagy during seed maturation had remained unknown. We here found that seeds of the rice autophagy-deficient mutant Osatg7-1, that produces seeds at a very low frequency in paddy fields, are smaller and show chalky appearance and lower starch content in the endosperm at the mature stage under normal growth condition. We comprehensively analyzed the effects of disruption of autophagy on biochemical properties, proteome and seed quality, and found an abnormal activation of starch degradation pathways including accumulation of α-amylases in the endosperm during seed maturation in Osatg7-1. These results indicate critical involvement of autophagy in metabolic regulation in the endosperm of rice, and provide insights into novel autophagy-mediated regulation of starch metabolism during seed maturation.

Programmed cell death (PCD), organized destruction of cells, is essential in development, maintenance of cellular homeostasis, and innate immunity in multicellular organisms. In most angiosperms, development of male and female organs involves spatial and temporal regulation of PCD. The tapetum, the innermost layer of the anther, provides both nutrient and lipid components to developing microspores, and has been proposed to be degraded by PCD during the later stages of pollen maturation. Plants lack homologues of most apoptosis-related genes in animals and have evolved specifi c mechanisms for PCD. PCD is also a crucial event in plant immune responses against microbial infection that prevents the spread of pathogens. Recent live cell imaging techniques have revealed the dynamic features and signifi cant roles of the vacuole during defense responses and PCD. Disintegration or collapse of the vacuolar membrane has been suggested to trigger the fi nal step of

Despite the molecular diversity of signaling components among plants and animals, Ca 2+ has been established as a common second messenger in most eukaryotic cells. 1,2 Though electrophysiological studies have revealed several types of Ca 2+ -permeable channels localized at the plasma membrane (PM) and vacuolar membrane (VM) in many plant species, 3 their molecular identity are still largely unknown. Homologs of the major voltage-dependent Ca 2+ channels (VDCCs) are not found in plants. 3 In turn, the two-pore channel (TPC) family, originally isolated from rat, 4 is homologous to a half structure of the α1-subunit of vertebrate VDCCs. 5 Human TPCs mediate nicotinic acid adenine dinucleotide phosphate-induced Ca 2+ release from acidic organelles in HEK293 cells. 6 Arabidopsis AtTPC1 shows a slow-activating vacuolar cation channel activity 7,8 and be involved in the sucrose-induced Ca 2+ rise, 9 abscisic acid-induced repression of germination 10 and the stomatal response to extracellular Ca 2+ changes. 7,10 Tobacco NtTPC1s have roles in increasing Ca 2+ concentrations, defense-related gene expression, and regulation of hypersensitive cell death triggered by cryptogein, an elicitor from an oomycete, in tobacco BY-2 cells. 11 Rice and wheat TPC1s appear to function in responses to abiotic stresses. 12,13 Role of OsTPC1 in Xylanase Elicitor-Triggered Defense Responses in RiceCharacterization of the retrotransposon-insertional knockout mutant of rice Ostpc1 revealed that OsTPC1 affected the

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