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ABSTRACT: 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.
Trends in Plant Science 01/2013; · 11.05 Impact Factor
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ABSTRACT: 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.
Plant signaling & behavior 11/2012; 8(1).
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ABSTRACT: Two-pore channels (TPCs) are cation channels with a voltage-sensor domain conserved in plants and animals. Rice OsTPC1 is predominantly localized to the plasma membrane (PM), and assumed to play an important role as a Ca ( 2+) -permeable cation channel in the regulation of cytosolic Ca ( 2+) rise and innate immune responses including hypersensitive cell death and phytoalexin biosynthesis in cultured rice cells triggered by a fungal elicitor, xylanase from Trichoderma viride. In contrast, Arabidopsis AtTPC1 is localized to the vacuolar membrane (VM). To gain further insights into the intracellular localization of OsTPC1, we stably expressed OsTPC1-GFP in tobacco BY-2 cells. Confocal imaging and membrane fractionation revealed that, unlike in rice cells, the majority of OsTPC1-GFP fusion protein was targeted to the VM in tobacco BY-2 cells. Intracellular localization and functions of the plant TPC family is discussed.
Plant signaling & behavior 09/2012; 7(11).
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ABSTRACT: Mechanosensing and its downstream responses are speculated to involve sensory complexes containing Ca ( 2+) -permeable mechanosensitive channels. On recognizing hypo-osmotic stress, plant cells initiate activation of a widespread signal transduction network involving second messengers such as Ca ( 2+) to trigger inducible defense responses including the induction of transcriptional factors. ( 1) However, most of the components involved in these signaling networks still remain to be identified. Recently we identified and investigated OsMCA1, the sole homolog of the MCA family putative Ca ( 2+) -permeable mechanosensitive channels in rice. Functional characterization of the OsMCA1-suppressed cells as well as the overexpressing cells indicated that OsMCA1 is involved in the regulation of plasma membrane Ca ( 2+) influx and NADPH oxidase-mediated generation of reactive oxygen species (ROS) induced by hypo-osmotic stress. Here we will discuss possible molecular mechanisms and physiological functions of the MCA protein in hypo-osmotic signaling.
Plant signaling & behavior 07/2012; 7(7):796-8.
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Takamitsu Kurusu,
Daisuke Nishikawa,
Yukari Yamazaki,
Mariko Gotoh,
Masataka Nakano,
Haruyasu Hamada,
Takuya Yamanaka,
Kazuko Iida,
Yuko Nakagawa,
Hikaru Saji,
Kazuo Shinozaki,
Hidetoshi Iida,
Kazuyuki Kuchitsu
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ABSTRACT: Mechanosensing 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.
We 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.
We 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.
BMC Plant Biology 01/2012; 12:11. · 3.45 Impact Factor
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Haruyasu Hamada, Takamitsu Kurusu,
Eiji Okuma,
Hiroshi Nokajima,
Masahiro Kiyoduka,
Tomoko Koyano,
Yoshimi Sugiyama,
Kazunori Okada,
Jinichiro Koga,
Hikaru Saji,
Akio Miyao,
Hirohiko Hirochika,
Hisakazu Yamane,
Yoshiyuki Murata,
Kazuyuki Kuchitsu
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ABSTRACT: Pathogen/microbe- or plant-derived signaling molecules (PAMPs/MAMPs/DAMPs) or elicitors induce increases in the cytosolic concentration of free Ca(2+) 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 Ca(2+), mainly due to a Ca(2+) 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 Ca(2+)-sensitive photoprotein, aequorin, TvX-induced Ca(2+) 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 Ca(2+)-permeability. These results suggest that OsTPC1 plays a crucial role in TvX-induced Ca(2+) influx as a plasma membrane Ca(2+)-permeable channel consequently required for the regulation of phytoalexin biosynthesis in cultured rice cells.
Journal of Biological Chemistry 01/2012; 287(13):9931-9. · 4.77 Impact Factor
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Takamitsu Kurusu,
Takuya Yamanaka,
Masataka Nakano,
Akiko Takiguchi,
Yoko Ogasawara,
Teruyuki Hayashi,
Kazuko Iida,
Shigeru Hanamata,
Kazuo Shinozaki,
Hidetoshi Iida,
Kazuyuki Kuchitsu
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ABSTRACT: 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.
Journal of Plant Research 11/2011; 125(4):555-68. · 1.75 Impact Factor
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ABSTRACT: 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.
Journal of Plant Research 03/2011; 124(3):315-24. · 1.75 Impact Factor
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Takamitsu Kurusu,
Haruyasu Hamada,
Yoshimi Sugiyama,
Toshikazu Yagala,
Yasuhiro Kadota,
Takuya Furuichi,
Teruyuki Hayashi,
Kenji Umemura,
Setsuko Komatsu,
Akio Miyao,
Hirohiko Hirochika,
Kazuyuki Kuchitsu
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ABSTRACT: Microbe/pathogen-associated molecular patterns (MAMPs/PAMPs) often induce rises in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) and protein phosphorylation. Though they are postulated to play pivotal roles in plant innate immunity, their molecular links and the regulatory mechanisms remain largely unknown. To investigate the regulatory mechanisms for MAMP-induced Ca(2+) mobilization, we have established a transgenic rice (Oryza sativa) cell line stably expressing apoaequorin, and characterized the interrelationship among MAMP-induced changes in [Ca(2+)](cyt), production of reactive oxygen species (ROS) and protein phosphorylation. Oligosaccharide and sphingolipid MAMPs induced Ca(2+) transients mainly due to plasma membrane Ca(2+) influx, which were dramatically suppressed by a protein phosphatase inhibitor, calyculin A (CA). Hydrogen peroxide and hypo-osmotic shock triggered similar [Ca(2+)](cyt) elevations, which were not affected by CA. MAMP-induced protein phosphorylation, which is promoted by CA, has been shown to be required for ROS production and MAPK activation, while it negatively regulates MAMPs-induced Ca(2+) mobilization and may play a crucial role in temporal regulation of [Ca(2+)](cyt) signature.
Journal of Plant Research 11/2010; 124(3):415-24. · 1.75 Impact Factor
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ABSTRACT: 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.
Plant signaling & behavior 08/2010; 5(8):1045-7.
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Takamitsu Kurusu,
Jumpei Hamada,
Hiroshi Nokajima,
Youichiro Kitagawa,
Masahiro Kiyoduka,
Akira Takahashi,
Shigeru Hanamata,
Ryoko Ohno,
Teruyuki Hayashi,
Kazunori Okada,
Jinichiro Koga,
Hirohiko Hirochika,
Hisakazu Yamane,
Kazuyuki Kuchitsu
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ABSTRACT: Although cytosolic free Ca(2+) mobilization induced by microbe/pathogen-associated molecular patterns is postulated to play a pivotal role in innate immunity in plants, the molecular links between Ca(2+) and downstream defense responses still remain largely unknown. Calcineurin B-like proteins (CBLs) act as Ca(2+) 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 Mn(2+)-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.
Plant physiology 03/2010; 153(2):678-92. · 6.53 Impact Factor
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ABSTRACT: Elicitor-triggered transient membrane potential changes and Ca2+ influx through the plasma membrane are thought to be important during defense signaling in plants. However, the molecular bases for the Ca2+ influx and its regulation remain largely unknown. Here we tested effects of overexpression as well as retrotransposon (Tos17)-insertional mutagenesis of the rice two-pore channel 1 (OsTPC1), a putative voltage-gated Ca(2+)-permeable channel, on a proteinaceous fungal elicitor-induced defense responses in rice cells. The overexpressor showed enhanced sensitivity to the elicitor to induce oxidative burst, activation of a mitogen-activated protein kinase (MAPK), OsMPK2, as well as hypersensitive cell death. On the contrary, a series of defense responses including the cell death and activation of the MAPK were severely suppressed in the insertional mutant, which was complemented by overexpression of the wild-type gene. These results suggest that the putative Ca(2+)-permeable channel determines sensitivity to the elicitor and plays a role as a key regulator of elicitor-induced defense responses, activation of MAPK cascade and hypersensitive cell death.
The Plant Journal 07/2005; 42(6):798-809. · 6.16 Impact Factor
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ABSTRACT: Cytosolic free Ca2+ serves as an important second messenger participating in signal transduction of various environmental stresses. However, molecular bases for the plasma membrane Ca2+ influx and its regulation remain largely unknown. We here identified a gene (OsTPC1) encoding a putative voltage-gated Ca2+ channel from rice, ubiquitously expressed in mature leaves, shoots and roots as well as in cultured cells. OsTPC1 rescued the Ca2+ uptake activity and growth rate of a yeast mutant cch1. To elucidate its physiological roles, we generated transgenic rice plants and cultured cells overexpressing OsTPC1 mRNA. Furthermore, a retrotransposon (Tos17) insertional knockout mutant of OsTPC1 was isolated. OsTPC1-overexpressing cells showed hypersensitivity to excess Ca2+ but higher growth rate under Ca2+ limitation, while growth of the OsTPC1-knockout cultured cells was less sensitive to extracellular free Ca2+ concentration, suggesting that OsTPC1 has Ca2+ transport activity across the plasma membrane. OsTPC1-overexpressing plants showed reduced growth and abnormal greening of roots. Growth of Ostpc1 seedlings was comparable to the control on agar plates, while significantly reduced in adult plants. These results suggest that OsTPC1 functions as a Ca2+ -permeable channel involved in the regulation of growth and development.
Plant and Cell Physiology 07/2004; 45(6):693-702. · 4.70 Impact Factor
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ABSTRACT: Root hair formation is induced when lettuce seedlings are transferred from pH 6.0 to pH 4.0. Ethylene, auxin and light are essential to this process. To investigate the role of ethylene in root hair initiation, we isolated two 1-aminocyclopropane-1-carboxylic acid (ACC) synthase genes (Ls-ACS1 and Ls-ACS2). Seven motifs of known ACS proteins were highly conserved in Ls-ACS1 and Ls-ACS2. The Ls-ACS1 and Ls-ACS2 mRNA levels were constant at pH 6.0, which were lower than that in seedlings at pH 4.0. Ls-ACS1 and Ls-ACS2 transcripts accumulated at pH 4.0 and reached peak levels at 1 h and 30 min after acidification, respectively. Indole-3-acetic acid (IAA) induced the accumulation of both Ls-ACS1 and Ls-ACS2 transcripts, whereas ACC induced only Ls-ACS1 mRNA. These results suggest that acidification-induced auxin accumulations increase the Ls-ACS2 levels, which together with Ls-ACS2-induced ethylene raise the levels of Ls-ACS1. Furthermore, blue and white light gave the highest levels of both Ls-ACS1 mRNA and ethylene production. Darkness was less effective, and red light had an intermediate effect. The different light conditions had no effect on the levels of Ls-ACS2 mRNA. These observations support the involvement of Ls-ACS1 in the production of ethylene, which is crucial for root hair initiation.
Plant and Cell Physiology 02/2003; 44(1):62-9. · 4.70 Impact Factor
