Takamitsu Kurusu

Plant mechanosensing and Ca2+ transport

Mechanical stimuli generate Ca(2+) signals and influence growth and development in plants. Recently, candidates for Ca(2+)-permeable mechanosensitive (MS) channels have been identified. These channels are thought to be responsible for sensing osmotic shock, touch, and gravity. One candidate is the MscS-like (MSL) protein family, a homolog of the typical bacterial MS channels. Some of the MSL proteins are localized to plastids to maintain their shape and size. Another candidate is the mid1-complementing activity (MCA) protein family, which is structurally unique to the plant kingdom. MCA proteins are localized in the plasma membrane and are suggested to be involved in mechanosensing and to be functionally related to reactive oxygen species (ROS) signaling. Here, we review their structural features and role in planta.

Regulation of Microbe-Associated Molecular Pattern-Induced Hypersensitive Cell Death, Phytoalexin Production, and Defense Gene Expression by Calcineurin B-Like Protein-Interacting Protein Kinases, OsCIPK14/15, in Rice Cultured Cells

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.Cytosolic free Ca(2+) mobilization induced by microbe/pathogen-associated molecular patterns (MAMPs/PAMPs) play key roles in plant innate immunity. However, components involved in Ca(2+) 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 has yet to be clarified. Recently we identified two CIPKs, OsCIPK14 and OsCIPK15, which are rapidly induced by MAMPs, involved in various MAMP-induced 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.

Plasma membrane protein OsMCA1 is involved in regulation of hypo-osmotic shock-induced Ca2+influx and modulates generation of reactive oxygen species in cultured rice cells

BackgroundMechanosensing and its downstream responses are speculated to involve sensory complexes containing Ca2+-permeable mechanosensitive channels. On recognizing osmotic signals, plant cells initiate activation of a widespread signal transduction network that induces second messengers and triggers inducible defense responses. Characteristic early signaling events include Ca2+ influx, protein phosphorylation and generation of reactive oxygen species (ROS). Pharmacological analyses show Ca2+ influx mediated by mechanosensitive Ca2+ channels to influence induction of osmotic signals, including ROS generation. However, molecular bases and regulatory mechanisms for early osmotic signaling events remain poorly elucidated.ResultsWe here identified and investigated OsMCA1, the sole rice homolog of putative Ca2+-permeable mechanosensitive channels in Arabidopsis (MCAs). OsMCA1 was specifically localized at the plasma membrane. A promoter-reporter assay suggested that OsMCA1 mRNA is widely expressed in seed embryos, proximal and apical regions of shoots, and mesophyll cells of leaves and roots in rice. Ca2+ uptake was enhanced in OsMCA1-overexpressing suspension-cultured cells, suggesting that OsMCA1 is involved in Ca2+ influx across the plasma membrane. Hypo-osmotic shock-induced ROS generation mediated by NADPH oxidases was also enhanced in OsMCA1-overexpressing cells. We also generated and characterized OsMCA1-RNAi transgenic plants and cultured cells; OsMCA1-suppressed plants showed retarded growth and shortened rachises, while OsMCA1-suppressed cells carrying Ca2+-sensitive photoprotein aequorin showed partially impaired changes in cytosolic free Ca2+ concentration ([Ca2+]cyt) induced by hypo-osmotic shock and trinitrophenol, an activator of mechanosensitive channels.ConclusionsWe have identified a sole MCA ortholog in the rice genome and developed both overexpression and suppression lines. Analyses of cultured cells with altered levels of this putative Ca2+-permeable mechanosensitive channel indicate that OsMCA1 is involved in regulation of plasma membrane Ca2+ influx and ROS generation induced by hypo-osmotic stress in cultured rice cells. These findings shed light on our understanding of mechanical sensing pathways.

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.

Dynamic intracellular reorganization of cytoskeletons and the vacuole in defense responses and hypersensitive cell death in plants

Plants have evolved various means for controlled and organized cell destruction, known as programmed cell death (PCD). In plant immune responses against microbial infection, hypersensitive cell death as a form of PCD is a crucial event to prevent the spread of biotrophic pathogens. Recent live cell imaging techniques have revealed dynamic features and significant roles of cytoskeletons and the vacuole during defense responses and the PCD. Actin microfilaments (MFs) focus on the infection sites and function as tracks for the polar transport of antimicrobial materials. To accomplish hypersensitive cell death, further dynamic changes in cytoskeletons are induced. MFs play a role in the structural and functional regulation of the vacuole, leading to execution of the PCD. We here overview spatiotemporal dynamic changes in the cytoskeletons and the vacuoles triggered by signals from pathogens, and propose a hypothetical model for MF-regulated vacuole-mediated PCD in plant immunity.

Plant signaling networks involving Ca2+ and Rboh/Nox-mediated ROS production under salinity stress

Salinity stress, which induces both ionic and osmotic damage, impairs plant growth and causes severe reductions in crop yield. Plants are equipped with defense responses against salinity stress such as regulation of ion transport including Na+ and K+, accumulation of compatible solutes and stress-related gene expression. The initial Ca2+ influx mediated by plasma membrane ion channels has been suggested to be crucial for the adaptive signaling. NADPH oxidase (Nox)-mediated production of reactive oxygen species (ROS) has also been suggested to play crucial roles in regulating adaptation to salinity stress in several plant species including halophytes. Respiratory burst oxidase homolog (Rboh) proteins show the ROS-producing Nox activity, which are synergistically activated by the binding of Ca2+ to EF-hand motifs as well as Ca2+-dependent phosphorylation. We herein review molecular identity, structural features and roles of the Ca2+-permeable channels involved in early salinity and osmotic signaling, and comparatively discuss the interrelationships among spatiotemporal dynamic changes in cytosolic concentrations of free Ca2+, Rboh-mediated ROS production, and downstream signaling events during salinity adaptation in planta.

Establishment of Monitoring Methods for Autophagy in Rice Reveals Autophagic Recycling of Chloroplasts and Root Plastids during Energy Limitation

Autophagy is responsible for the degradation of leaf chloroplasts and root plastids in rice plants and functions during energy limitation caused by interruption of photosynthesis. Autophagy is an intracellular process leading to vacuolar or lysosomal degradation of cytoplasmic components in eukaryotes. Establishment of proper methods to monitor autophagy was a key step in uncovering its role in organisms, such as yeast (Saccharomyces cerevisiae), mammals, and Arabidopsis (Arabidopsis thaliana), in which chloroplastic proteins were found to be recycled by autophagy. Chloroplast recycling has been predicted to function in nutrient remobilization for growing organs or grain filling in cereal crops. Here, to develop our understanding of autophagy in cereals, we established monitoring methods for chloroplast autophagy in rice (Oryza sativa). We generated transgenic rice-expressing fluorescent protein (FP) OsAuTophaGy8 (OsATG8) fusions as autophagy markers. FP-ATG8 signals were delivered into the vacuolar lumen in living cells of roots and leaves mainly as vesicles corresponding to autophagic bodies. This phenomenon was not observed upon the addition of wortmannin, an inhibitor of autophagy, or in an ATG7 knockout mutant. Markers for the chloroplast stroma, stromal FP, and FP-labeled Rubisco were delivered by a type of autophagic body called the Rubisco-containing body (RCB) in the same manner. RCB production in excised leaves was suppressed by supply of external sucrose or light. The release of free FP caused by autophagy-dependent breakdown of FP-labeled Rubisco was induced during accelerated senescence in individually darkened leaves. In roots, nongreen plastids underwent both RCB-mediated and entire organelle types of autophagy. Therefore, our newly developed methods to monitor autophagy directly showed autophagic degradation of leaf chloroplasts and root plastids in rice plants and its induction during energy limitation.

Autophagy supports biomass production and nitrogen use efficiency at the vegetative stage in rice

Characterization of a rice mutant defective in autophagy highlights its importance in nitrogen remobilization from senescent leaves, biomass increase, and nitrogen use efficiency in the vegetative plant. 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. 15N 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.

Regulation of a Proteinaceous Elicitor-induced Ca2+ Influx and Production of Phytoalexins by a Putative Voltage-gated Cation Channel, OsTPC1, in Cultured Rice Cells

Background: Molecular mechanisms for elicitor-induced changes in cytosolic Ca2+ concentration and its molecular link with regulation of phytoalexin biosynthesis in plant immunity remain mostly unknown. Results: TvX-induced Ca2+ influx and the phytoalexin accumulations were suppressed in Ostpc1 knock-out cells. Conclusion: OsTPC1 plays a role in TvX-induced Ca2+ influx consequently required for the regulation of phytoalexin biosynthesis. Significance: Voltage-dependent plasma membrane Ca2+-permeable channel activity of the plant TPC1 was shown for the first time. Pathogen/microbe- or plant-derived signaling molecules (PAMPs/MAMPs/DAMPs) or elicitors induce increases in the cytosolic concentration of free Ca2+ followed by a series of defense responses including biosynthesis of antimicrobial secondary metabolites called phytoalexins; however, the molecular links and regulatory mechanisms of the phytoalexin biosynthesis remains largely unknown. A putative voltage-gated cation channel, OsTPC1 has been shown to play a critical role in hypersensitive cell death induced by a fungal xylanase protein (TvX) in suspension-cultured rice cells. Here we show that TvX induced a prolonged increase in cytosolic Ca2+, mainly due to a Ca2+ influx through the plasma membrane. Membrane fractionation by two-phase partitioning and immunoblot analyses revealed that OsTPC1 is localized predominantly at the plasma membrane. In retrotransposon-insertional Ostpc1 knock-out cell lines harboring a Ca2+-sensitive photoprotein, aequorin, TvX-induced Ca2+ elevation was significantly impaired, which was restored by expression of OsTPC1. TvX-induced production of major diterpenoid phytoalexins and the expression of a series of diterpene cyclase genes involved in phytoalexin biosynthesis were also impaired in the Ostpc1 cells. Whole cell patch clamp analyses of OsTPC1 heterologously expressed in HEK293T cells showed its voltage-dependent Ca2+-permeability. These results suggest that OsTPC1 plays a crucial role in TvX-induced Ca2+ influx as a plasma membrane Ca2+-permeable channel consequently required for the regulation of phytoalexin biosynthesis in cultured rice cells.

Comprehensive analysis of transcriptome response to salinity stress in the halophytic turf grass Sporobolus virginicus.

The turf grass Sporobolus virginicus is halophyte and has high salinity tolerance. To investigate the molecular basis of its remarkable tolerance, we performed Illumina high-throughput RNA sequencing on roots and shoots of a S. virginicus genotype under normal and saline conditions. The 130 million short reads were assembled into 444,242 unigenes. A comparative analysis of the transcriptome with rice and Arabidopsis transcriptome revealed six turf grass-specific unigenes encoding transcription factors. Interestingly, all of them showed root specific expression and five of them encode bZIP type transcription factors. Another remarkable transcriptional feature of S. virginicus was activation of specific pathways under salinity stress. Pathway enrichment analysis suggested transcriptional activation of amino acid, pyruvate, and phospholipid metabolism. Up-regulation of several unigenes, previously shown to respond to salt stress in other halophytes was also observed. Gene Ontology enrichment analysis revealed that unigenes assigned as proteins in response to water stress, such as dehydrin and aquaporin, and transporters such as cation, amino acid, and citrate transporters, and H+-ATPase, were up-regulated in both shoots and roots under salinity. A correspondence analysis of the enriched pathways in turf grass cells, but not in rice cells, revealed two groups of unigenes similarly up-regulated in the turf grass in response to salt stress; one of the groups, showing excessive up-regulation under salinity, included unigenes homologos to salinity responsive genes in other halophytes. Thus, the present study identified candidate genes involved in salt tolerance of S. virginicus. This genetic resource should be valuable for understanding the mechanisms underlying high salt tolerance in S. virginicus. This information can also provide insight into salt tolerance in other halophytes.