Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1038/s41598-019-54361-1

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

We have previously shown that autophagy is required for post meiotic anther development including programmed cell death-mediated degradation of the tapetum and pollen maturation in rice. However, the spatiotemporal dynamics of autophagy in the tapetum remain poorly understood. We here established an in vivo imaging technique to analyze the dynamics of autophagy in rice tapetum cells by expressing green fluorescent protein-tagged AtATG8, a marker for autophagosomes. 3D-imaging analysis revealed that the number of autophagosomes/autophagy-related structures is extremely low at the tetrad stage (stage 8), and autophagy is dramatically induced at the uninucleate stages (stage 9-10) throughout the tapetal cells during anther development. The present monitoring system for autophagy offers a powerful tool to analyze the regulation of autophagy in rice tapetal cells during pollen maturation.

DOI： 10.5511/plantbiotechnology.19.0417a

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Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.24480/bsj-review.9a5.00131

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Taylor and Francis Inc.  

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.

DOI： 10.1080/15592324.2017.1365211

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Bioimaging Society  

Autophagy contributes to various cellular processes, including innate immunity, development, and programmed cell death (PCD) in multicellular organisms. In plants, autophagy plays roles in recycling of proteins and metabolites including lipids, and is involved in many physiological processes, such as abiotic and biotic stress responses. Furthermore, rice mutants defective in autophagy showed sporophytic male sterility and reduced accumulation of lipids and starch in pollen grains at the flowering stage and exhibited reduced pollen germination activity, indicating crucial roles of autophagy during plant reproductive development. An <I>in vivo</I> imaging system has been established to analyze the dynamics of autophagy in rice as well as in cultured tobacco cells using GFP-ATG8, a marker of autophagosomes. In the present study, we review the spatiotemporal dynamics of autophagic fluxes in plant cells and discuss their possible significance in PCD and metabolic regulation, with particular focus on plant reproductive development.

DOI： 10.11169/bioimages.24.1

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PLANT BIOLOGISTS  

Much of the nitrogen in leaves is distributed to chloroplasts, mainly in photosynthetic proteins. During leaf senescence, chloroplastic proteins, including Rubisco, are rapidly degraded, and the released nitrogen is remobilized and reused in newly developing tissues. Autophagy facilitates the degradation of intracellular components for nutrient recycling in all eukaryotes, and recent studies have revealed critical roles for autophagy in Rubisco degradation and nitrogen remobilization into seeds in Arabidopsis (Arabidopsis thaliana). Here, we examined the function of autophagy in vegetative growth and nitrogen usage in a cereal plant, rice (Oryza sativa). An autophagy-disrupted rice mutant, Osatg7-1, showed reduced biomass production and nitrogen use efficiency compared with the wild type. While Osatg7-1 showed early visible leaf senescence, the nitrogen concentration remained high in the senescent leaves. N-15 pulse chase analysis revealed suppression of nitrogen remobilization during leaf senescence in Osatg7-1. Accordingly, the reduction of nitrogen available for newly developing tissues in Osatg7-1 likely led its reduced leaf area and tillers. The limited leaf growth in Osatg7-1 decreased the photosynthetic capacity of the plant. Much of the nitrogen remaining in senescent leaves of Osatg7-1 was in soluble proteins, and the Rubisco concentration in senescing leaves of Osatg7-1 was about 2.5 times higher than in the wild type. Transmission electron micrographs showed a cytosolic fraction rich with organelles in senescent leaves of Osatg7-1. Our results suggest that autophagy contributes to efficient nitrogen remobilization at the whole-plant level by facilitating protein degradation for nitrogen recycling in senescent leaves.

DOI： 10.1104/pp.15.00242

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：FRONTIERS RESEARCH FOUNDATION  

Autophagy, a major catabolic pathway in eukaryotic cells, is essential in development, maintenance of cellular homeostasis, immunity and programmed cell death (PCD) in multicellular organisms. In plant cells, autophagy plays roles in recycling of proteins and metabolites including lipids, and is involved in many physiological processes such as abiotic and biotic stress responses. However, its roles during reproductive development had remained poorly understood. Quantitative live cell imaging techniques for the autophagic flux and genetic studies in several plant species have recently revealed significant roles of autophagy in developmental processes, regulation of PCD and lipid metabolism. We here review the novel roles of autophagic fluxes in plant cells, and discuss their possible significance in PCD and metabolic regulation, with particular focus on male reproductive development during the pollen maturation.

DOI： 10.3389/fpls.2014.00457

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS INC  

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.

DOI： 10.4161/auto.28279

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Language：English   Publishing type：Research paper (international conference proceedings)  

DOI： 10.1007/978-4-431-54589-7_36

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Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bbamcr.2013.06.024

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Pharmacological evidence suggests that anion channel-mediated plasma membrane anion effluxes are crucial in early defense signaling to induce immune responses and hypersensitive cell death in plants. However, their molecular bases and regulation remain largely unknown. We overexpressed Arabidopsis SLAC1, an S-type anion channel involved in stomatal closure, in cultured tobacco BY-2 cells and analyzed the effect on cryptogein-induced defense responses including fluxes of Cl- and other ions, production of reactive oxygen species (ROS), gene expression and hypersensitive responses. The SLAC1-GFP fusion protein was localized at the plasma membrane in BY-2 cells. Overexpression of SLAC1 enhanced cryptogein-induced Cl- efflux and extracellular alkalinization as well as rapid/transient and slow/prolonged phases of NADPH oxidase-mediated ROS production, which was suppressed by an anion channel inhibitor, DIDS. The overexpressor also showed enhanced sensitivity to cryptogein to induce downstream immune responses, including the induction of defense marker genes and the hypersensitive cell death. These results suggest that SLAC1 expressed in BY-2 cells mediates cryptogein-induced plasma membrane Cl- efflux to positively modulate the elicitor-triggered activation of other ion fluxes, ROS as well as a wide range of defense signaling pathways. These findings shed light on the possible involvement of the SLAC/SLAH family anion channels in cryptogein signaling to trigger the plasma membrane ion channel cascade in the plant defense signal transduction network.

DOI： 10.1371/journal.pone.0070623

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Language：Japanese   Publishing type：Doctoral thesis  

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Autophagy has been shown to play essential roles in the growth, development and survival of eukaryotic cells. However, simple methods for quantification and visualization of autophagic flux remain to be developed in living plant cells. Here, we analyzed the autophagic flux in transgenic tobacco BY-2 cell lines expressing fluorescence-tagged NtATG8a as a marker for autophagosome formation. Under sucrose-starved conditions, the number of punctate signals of YFP-NtATG8a increased, and the fluorescence intensity of the cytoplasm and nucleoplasm decreased. Conversely, these changes were not observed in BY-2 cells expressing a C-terminal glycine deletion mutant of the NtATG8a protein (NtATG8aΔG). To monitor the autophagic flux more easily, we generated a transgenic BY-2 cell line expressing NtATG8a fused to a pH-sensitive fluorescent tag, a tandem fusion of the acid-insensitive RFP and the acid-sensitive YFP. In sucrose-rich conditions, both fluorescent signals were detected in the cytoplasm and only weakly in the vacuole. In contrast, under sucrose-starved conditions, the fluorescence intensity of the cytoplasm decreased, and the RFP signal clearly increased in the vacuole, corresponding to the fusion of the autophagosome to the vacuole and translocation of ATG8 from the cytoplasm to the vacuole. Moreover, we introduce a novel simple easy way to monitor the autophagic flux non-invasively by only measuring the ratio of fluorescence of RFP and YFP in the cell suspension using a fluorescent image analyzer without microscopy. The present in vivo quantitative monitoring system for the autophagic flux offers a powerful tool for determining the physiological functions and molecular mechanisms of plant autophagy induced by environmental stimuli. © 2013 Landes Bioscience.

DOI： 10.4161/psb.22510

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER TOKYO  

To gain insight into the cellular functions of the mid1-complementing activity (MCA) family proteins, encoding putative Ca2+-permeable mechanosensitive channels, we isolated two MCA homologs of tobacco (Nicotiana tabacum) BY-2 cells, named NtMCA1 and NtMCA2. NtMCA1 and NtMCA2 partially complemented the lethality and Ca2+ uptake defects of yeast mutants lacking mechanosensitive Ca2+ channel components. Furthermore, in yeast cells overexpressing NtMCA1 and NtMCA2, the hypo-osmotic shock-induced Ca2+ influx was enhanced. Overexpression of NtMCA1 or NtMCA2 in BY-2 cells enhanced Ca2+ uptake, and significantly alleviated growth inhibition under Ca2+ limitation. NtMCA1-overexpressing BY-2 cells showed higher sensitivity to hypo-osmotic shock than control cells, and induced the expression of the touch-inducible gene, NtERF4. We found that both NtMCA1-GFP and NtMCA2-GFP were localized at the plasma membrane and its interface with the cell wall, Hechtian strands, and at the cell plate and perinuclear vesicles of dividing cells. NtMCA2 transcript levels fluctuated during the cell cycle and were highest at the G1 phase. These results suggest that NtMCA1 and NtMCA2 play roles in Ca2+-dependent cell proliferation and mechanical stress-induced gene expression in BY-2 cells, by regulating the Ca2+ influx through the plasma membrane.

DOI： 10.1007/s10265-011-0462-6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PLANT BIOLOGISTS  

Although cytosolic free Ca2+ mobilization induced by microbe/pathogen-associated molecular patterns is postulated to play a pivotal role in innate immunity in plants, the molecular links between Ca2+ and downstream defense responses still remain largely unknown. Calcineurin B-like proteins (CBLs) act as Ca2+ sensors to activate specific protein kinases, CBL-interacting protein kinases (CIPKs). We here identified two CIPKs, OsCIPK14 and OsCIPK15, rapidly induced by microbe-associated molecular patterns, including chitooligosaccharides and xylanase (Trichoderma viride/ethylene-inducing xylanase [TvX/EIX]), in rice (Oryza sativa). Although they are located on different chromosomes, they have over 95% nucleotide sequence identity, including the surrounding genomic region, suggesting that they are duplicated genes. OsCIPK14/15 interacted with several OsCBLs through the FISL/NAF motif in yeast cells and showed the strongest interaction with OsCBL4. The recombinant OsCIPK14/15 proteins showed Mn2+-dependent protein kinase activity, which was enhanced both by deletion of their FISL/NAF motifs and by combination with OsCBL4. OsCIPK14/15-RNAi transgenic cell lines showed reduced sensitivity to TvX/EIX for the induction of a wide range of defense responses, including hypersensitive cell death, mitochondrial dysfunction, phytoalexin biosynthesis, and pathogenesis-related gene expression. On the other hand, TvX/EIX-induced cell death was enhanced in OsCIPK15-overexpressing lines. Our results suggest that OsCIPK14/15 play a crucial role in the microbe-associated molecular pattern-induced defense signaling pathway in rice cultured cells.

DOI： 10.1104/pp.109.151852

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Landes Bioscience  

Cytosolic free Ca2+ mobilization induced by microbe/pathogen-associated molecular patterns (MAMPs/PAMPs) plays key roles in plant innate immunity. However, components involved in Ca2+ signaling pathways still remain to be identified and possible involvement of the CBL (calcineurin B-like proteins)-CIPK (CBL-interacting protein kinases) system in biotic defense signaling have yet to be clarified. Recently we identified two CIPKs, OsCIPK14 and OsCIPK15, which are rapidly induced by MAMPs, involved in various MAMPinduced immune responses including defense-related gene expression, phytoalexin biosynthesis and hypersensitive cell death. MAMP-induced production of reactive oxygen species as well as cell browning were also suppressed in OsCIPK14/15-RNAi transgenic cell lines. Possible molecular mechanisms and physiological functions of the CIPKs in plant innate immunity are discussed. © 2010 Landes Bioscience.

DOI： 10.4161/psb.5.8.12407

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Language：Japanese  

J-GLOBAL

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Language：Japanese   Publisher：日本バイオイメージング学会  

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Language：Japanese   Publisher：一般社団法人日本土壌肥料学会  

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J-GLOBAL

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J-GLOBAL

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CiNii Article

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Event date： 2019.12

Language：English   Presentation type：Poster presentation  

Country：Japan  

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Event date： 2019.12

Language：English   Presentation type：Poster presentation  

Country：Japan  

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Event date： 2019.11

Language：English   Presentation type：Poster presentation  

Country：Japan  

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Event date： 2019.9

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Event date： 2019.7

Language：Japanese   Presentation type：Poster presentation  

Country：Japan  

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Event date： 2019.6

Language：English   Presentation type：Poster presentation  

Country：Spain  

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Event date： 2018.12

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2018.9

Language：English   Presentation type：Oral presentation (general)  

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Event date： 2018.7

Language：Japanese   Presentation type：Poster presentation  

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Event date： 2017.9

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Event date： 2017.9

Language：English   Presentation type：Poster presentation  

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Language：English   Presentation type：Poster presentation  

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Event date： 2017.3

Language：English   Presentation type：Poster presentation  

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Event date： 2016.9

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2016.9

Country：Japan  

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Event date： 2016.7

Language：Japanese   Presentation type：Poster presentation  

Country：Japan  

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Event date： 2016.3

Language：English   Presentation type：Poster presentation  

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Event date： 2015.12

Language：English   Presentation type：Poster presentation  

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Event date： 2015.11

Language：English   Presentation type：Poster presentation  

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Event date： 2015.9

Language：English   Presentation type：Poster presentation  

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Event date： 2015.9

Language：English   Presentation type：Poster presentation  

Country：Japan  

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Event date： 2015.7

Language：English   Presentation type：Poster presentation  

Country：Japan  

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Language：English   Presentation type：Poster presentation  

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Language：English   Presentation type：Poster presentation  

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Event date： 2014.12

Language：English   Presentation type：Poster presentation  

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Event date： 2013.12

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Country：Japan  

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Event date： 2013.11

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2013.9

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2013.9

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Event date： 2013.7

Language：Japanese   Presentation type：Poster presentation  

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Event date： 2013.6

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Event date： 2012.12

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

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Event date： 2011.1

Presentation type：Oral presentation (general)  

Venue：静岡県掛川市・ヤマハリゾートつま恋   Country：Japan  

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Event date： 2010.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：静岡県伊東市   Country：Japan  

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Event date： 2010.5

Presentation type：Poster presentation  

Venue：大阪国際会議場   Country：Japan  

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Event date： 2009.12

Language：Japanese   Presentation type：Poster presentation  

Venue：パシフィコ横浜   Country：Japan  

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Event date： 2009.9

Language：Japanese   Presentation type：Poster presentation  

Venue：就実大学（岡山）   Country：Japan  

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Event date： 2009.3

Language：Japanese   Presentation type：Poster presentation  

Venue：名古屋大学   Country：Japan  

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Event date： 2008.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：大阪大学   Country：Japan  

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Event date： 2008.6

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

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Venue：仙台  

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Presentation type：Oral presentation (general)  

Venue：京都  

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