Performance information

• A tonoplast-localized magnesium transporter is crucial for stomatal opening in Arabidopsis under high Mg2+ conditions.　　　　　　　　　　　　　　　
  Shin-Ichiro Inoue; Maki Hayashi; Sheng Huang; Kengo Yokosho; Eiji Gotoh; Shuka Ikematsu; Masaki Okumura; Takamasa Suzuki; Takumi Kamura; Toshinori Kinoshita; Jian Feng Ma
  The New phytologist, Number：236, First page：864, Last page：877, Jul. 2022, [Reviewed], [Lead, Corresponding], ［International magazine］
  Plant stomata play an important role in CO2 uptake for photosynthesis and transpiration, but the mechanisms underlying stomatal opening and closing under changing environmental conditions are still not completely understood. Through large-scale genetic screening, we isolated an Arabidopsis mutant (closed stomata2 (cst2)) that is defective in stomatal opening. We cloned the causal gene (MGR1/CST2) and functionally characterized this gene. The mutant phenotype was caused by a mutation in a gene encoding an unknown protein with similarities to the human magnesium (Mg2+ ) efflux transporter ACDP/CNNM. MGR1/CST2 was localized to the tonoplast and showed transport activity for Mg2+ . This protein was constitutively and highly expressed in guard cells. Knockout of this gene resulted in stomatal closing, decreased photosynthesis and growth retardation, especially under high Mg2+ conditions, while overexpression of this gene increased stomatal opening and tolerance to high Mg2+ concentrations. Furthermore, guard cell-specific expression of MGR1/CST2 in the mutant partially restored its stomatal opening. Our results indicate that MGR1/CST2 expression in the leaf guard cells plays an important role in maintaining cytosolic Mg2+ concentrations through sequestering Mg2+ into vacuoles, which is required for stomatal opening, especially under high Mg2+ conditions.
  English, Scientific journal
  DOI：https://doi.org/10.1111/nph.18410
  DOI ID：10.1111/nph.18410, PubMed ID：35976788
• Type 2C protein phosphatase clade D family members dephosphorylate guard cell plasma membrane H+-ATPase
  Mitsumasa Akiyama; Hodaka Sugimoto; Shin-ichiro Inoue; Yohei Takahashi; Maki Hayashi; Yuki Hayashi; Miya Mizutani; Takumi Ogawa; Daichi Kinoshita; Eigo Ando; Meeyeon Park; William M Gray; Toshinori Kinoshita
  Plant Physiology, Volume：188, Number：4, First page：2228, Last page：2240, Mar. 2022, [Reviewed]
  Abstract
  
  Plasma membrane (PM) H+-ATPase in guard cells is activated by phosphorylation of the penultimate residue, threonine (Thr), in response to blue and red light, promoting stomatal opening. Previous in vitro biochemical investigation suggested that Mg2+- and Mn2+-dependent membrane-localized type 2C protein phosphatase (PP2C)-like activity mediates the dephosphorylation of PM H+-ATPase in guard cells. PP2C clade D (PP2C.D) was later demonstrated to be involved in PM H+-ATPase dephosphorylation during auxin-induced cell expansion in Arabidopsis (Arabidopsis thaliana). However, it is unclear whether PP2C.D phosphatases are involved in PM H+-ATPase dephosphorylation in guard cells. Transient expression experiments using Arabidopsis mesophyll cell protoplasts revealed that all PP2C.D isoforms dephosphorylate the endogenous PM H+-ATPase. We further analyzed PP2C.D6/8/9, which display higher expression levels than other isoforms in guard cells, observing that pp2c.d6, pp2c.d8, and pp2c.d9 single mutants showed similar light-induced stomatal opening and phosphorylation status of PM H+-ATPase in guard cells as Col-0. In contrast, the pp2c.d6/9 double mutant displayed wider stomatal apertures and greater PM H+-ATPase phosphorylation in response to blue light, but delayed dephosphorylation of PM H+-ATPase in guard cells; the pp2c.d6/8/9 triple mutant showed similar phenotypes to those of the pp2c.d6/9 double mutant. Taken together, these results indicate that PP2C.D6 and PP2C.D9 redundantly mediate PM H+-ATPase dephosphorylation in guard cells. Curiously, unlike auxin-induced cell expansion in seedlings, auxin had no effect on the phosphorylation status of PM H+-ATPase in guard cells.
  Oxford University Press (OUP), Scientific journal
  DOI：https://doi.org/10.1093/plphys/kiab571
  DOI ID：10.1093/plphys/kiab571, ISSN：0032-0889, eISSN：1532-2548
• Phototropin 2 contributes to the chloroplast avoidance response at the chloroplast-plasma membrane interface.　　　　　　　　　　　　　　　
  Kazuhiro Ishishita; Takeshi Higa; Hidekazu Tanaka; Shin-Ichiro Inoue; Aeri Chung; Tomokazu Ushijima; Tomonao Matsushita; Toshinori Kinoshita; Masato Nakai; Masamitsu Wada; Noriyuki Suetsugu; Eiji Gotoh
  Plant physiology, Mar. 2020, [Reviewed], ［International magazine］
  Blue-light-induced chloroplasts movements play an important role in maximizing light utilization for photosynthesis in plants. Under a weak light condition, chloroplasts accumulate to the cell surface to capture light efficiently (chloroplast accumulation response). Conversely, chloroplasts escape from strong light and move to the side wall to reduce photodamage (chloroplast avoidance response). The blue light receptor phototropin (phot) regulates these chloroplast movements and optimizes leaf photosynthesis by controlling other responses as well as chloroplast movements. Seed plants such as Arabidopsis thaliana have the phototropins phot1 and phot2. They redundantly mediate phototropism, stomatal opening, leaf flattening, and the chloroplast accumulation response. However, the chloroplast avoidance response is induced by strong blue light and regulated primarily by phot2. Phototropins are localized mainly on the plasma membrane. However, a substantial amount of phot2 resides on the chloroplast outer envelope. Therefore, differentially localized phot2 might have different functions. To determine the functions of plasma membrane- and chloroplast envelope-localized phot2, we tethered it to these structures with their respective targeting signals. Plasma membrane-localized phot2 regulated phototropism, leaf flattening, stomatal opening, and chloroplast movements. Chloroplast envelope-localized phot2 failed to mediate phototropism, leaf flattening, and the chloroplast accumulation response but partially regulated the chloroplast avoidance response and stomatal opening. Based on the present and previous findings, we propose that phot2 localized at the interface between the plasma membrane and the chloroplasts is required for the chloroplast avoidance response and possibly for stomatal opening as well.
  English
  DOI：https://doi.org/10.1104/pp.20.00059
  DOI ID：10.1104/pp.20.00059, PubMed ID：32193212
• Raf-like kinases CBC1 and CBC2 negatively regulate stomatal opening by negatively regulating plasma membrane H+-ATPase phosphorylation in Arabidopsis.　　　　　　　　　　　　　　　
  Maki Hayashi; Hodaka Sugimoto; Hirotaka Takahashi; Motoaki Seki; Kazuo Shinozaki; Tatsuya Sawasaki; Toshinori Kinoshita; Shin-Ichiro Inoue
  Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology, Volume：19, Number：1, First page：88, Last page：98, Jan. 2020, [Reviewed], [Corresponding], ［International magazine］
  Stomatal pores, which are surrounded by pairs of guard cells in the plant epidermis, regulate gas exchange between plants and the atmosphere, thereby controlling photosynthesis and transpiration. Blue light works as a signal to guard cells, to induce intracellular signaling and open stomata. Blue light receptor phototropins (phots) are activated by blue light; phot-mediated signals promote plasma membrane (PM) H+-ATPase activity via C-terminal Thr phosphorylation, serving as the driving force for stomatal opening in guard cells. However, the details of this signaling process are not fully understood. In this study, through an in vitro screening of phot-interacting protein kinases, we obtained the CBC1 and CBC2 that had been reported as signal transducers in stomatal opening. Promoter activities of CBC1 and CBC2 indicated that both genes were expressed in guard cells. Single and double knockout mutants of CBC1 and CBC2 showed no lesions in the context of phot-mediated phototropism, chloroplast movement, or leaf flattening. In contrast, the cbc1cbc2 double mutant showed larger stomatal opening under both dark and blue light conditions. Interestingly, the level of phosphorylation of C-terminal Thr of PM H+-ATPase was higher in double mutant guard cells. The larger stomatal openings of the double mutant were effectively suppressed by the phytohormone abscisic acid (ABA). CBC1 and CBC2 interacted with BLUS1 and PM H+-ATPase in vitro. From these results, we conclude that CBC1 and CBC2 act as negative regulators of stomatal opening, probably via inhibition of PM H+-ATPase activity.
  English, Scientific journal
  DOI：https://doi.org/10.1039/c9pp00329k
  DOI ID：10.1039/c9pp00329k, PubMed ID：31904040
• Brassinosteroid Induces Phosphorylation of the Plasma Membrane H+-ATPase during Hypocotyl Elongation in Arabidopsis thaliana.　　　　　　　　　　　　　　　
  Anzu Minami; Koji Takahashi; Shin-Ichiro Inoue; Yasuomi Tada; Toshinori Kinoshita
  Plant & cell physiology, Volume：60, Number：5, First page：935, Last page：944, May 2019, [Reviewed], ［Domestic magazine］
  Brassinosteroids (BRs) are steroid phytohormones that regulate plant growth and development, and promote cell elongation at least in part via the acid-growth process. BRs have been suggested to induce cell elongation by the activating plasma membrane (PM) H+-ATPase. However, the mechanism by which BRs activate PM H+-ATPase has not been clarified. In this study, we investigated the effects of BR on hypocotyl elongation and the phosphorylation status of a penultimate residue, threonine, of PM H+-ATPase, which affects the activation, in the etiolated seedlings of Arabidopsis thaliana. Brassinolide (BL), an active endogenous BR, induced hypocotyl elongation, phosphorylation of the penultimate, threonine residue of PM H+-ATPase, and binding of the 14-3-3 protein to PM H+-ATPase in the endogenous BR-depleted seedlings. Changes in both BL-induced elongation and phosphorylation of PM H+-ATPase showed similar concentration dependency. BL did not induce phosphorylation of PM H+-ATPase in the BR receptor mutant bri1-6. In contrast, bikinin, a specific inhibitor of BIN2 that acts as a negative regulator of BR signaling, induced its phosphorylation. Furthermore, BL accumulated the transcripts of SMALL AUXIN UP RNA 9 (SAUR9) and SAUR19, which suppress dephosphorylation of the PM H+-ATPase penultimate residue by inhibiting D-clade type 2C protein phosphatase in the hypocotyls of etiolated seedlings. From these results, we conclude that BL-induced phosphorylation of PM H+-ATPase penultimate residue is mediated via the BRI1-BIN2 signaling pathway, together with the accumulation of SAURs during hypocotyl elongation.
  English, Scientific journal
  DOI：https://doi.org/10.1093/pcp/pcz005
  DOI ID：10.1093/pcp/pcz005, PubMed ID：30649552
• Excess Pyrophosphate within Guard Cells Delays Stomatal Closure　　　　　　　　　　　　　　　
  Asaoka M; Inoue S; Gunji S; Kinoshita T; Maeshima M
  Plant & Cell Physiology, Volume：60, Number：4, First page：875, Last page：887, Apr. 2019, [Reviewed], ［Domestic magazine］
  A variety of cellular metabolic reactions generate inorganic pyrophosphate (PPi) as an ATP hydrolysis byproduct. The vacuolar H+-translocating pyrophosphatase (H+-PPase) loss-of-function fugu5 mutant is susceptible to drought and displays pleotropic postgerminative growth defects due to excess PPi. It was recently reported that stomatal closure after abscisic acid (ABA) treatment is delayed in vhp1-1, a fugu5 allele. In contrast, we found that specific removal of PPi rescued all of the above fugu5 developmental and growth defects. Hence, we speculated that excess PPi itself, rather than vacuolar acidification, might delay stomatal closure. To test this hypothesis, we constructed transgenic plants expressing the yeast IPP1 gene (encoding a cytosolic pyrophosphatase) driven by a guard cell-specific promoter (pGC1::IPP1) in the fugu5 background. Our measurements confirmed stomatal closure defects in fugu5, further supporting a role for H+-PPase in stomatal functioning. Importantly, while pGC1::IPP1 transgenics morphologically mimicked fugu5, stomatal closure was restored in response to ABA and darkness. Quantification of water loss revealed that fugu5 stomata were almost completely insensitive to ABA. In addition, growth of pGC1::IPP1 plants was promoted compared to fugu5 throughout their life; however, it did not reach the wild type level. fugu5 also displayed an increased stomatal index, in violation of the one-cell-spacing rule, and phenotypes recovered upon removal of PPi by pAVP1::IPP1 (FUGU5, VHP1 and AVP1 are the same gene encoding H+-PPase), but not in the pGC1::IPP1 line. Taken together, these results clearly support our hypothesis that dysfunction in stomata is triggered by excess PPi within guard cells, probably via perturbed guard cell metabolism.
  English, Scientific journal
  DOI：https://doi.org/10.1093/pcp/pcz002
  DOI ID：10.1093/pcp/pcz002, PubMed ID：30649470
• Stomatal response to blue light in crassulacean acid metabolism plants Kalanchoe pinnata and Kalanchoe daigremontiana　　　　　　　　　　　　　　　
  Gotoh E; Oiwamoto K; Inoue S; Shimazaki K; Doi M
  Journal of Experimental Botany, Volume：70, Number：4, First page：1367, Last page：1374, Jan. 2019, [Reviewed], [Corresponding]
  English, Scientific journal
• Insights into Land Plant Evolution Garnered from the Marchantia polymorpha Genome.　　　　　　　　　　　　　　　
  John L Bowman; Takayuki Kohchi; Katsuyuki T Yamato; Jerry Jenkins; Shengqiang Shu; Kimitsune Ishizaki; Shohei Yamaoka; Ryuichi Nishihama; Yasukazu Nakamura; Frédéric Berger; Catherine Adam; Shiori Sugamata Aki; Felix Althoff; Takashi Araki; Mario A Arteaga-Vazquez; Sureshkumar Balasubrmanian; Kerrie Barry; Diane Bauer; Christian R Boehm; Liam Briginshaw; Juan Caballero-Perez; Bruno Catarino; Feng Chen; Shota Chiyoda; Mansi Chovatia; Kevin M Davies; Mihails Delmans; Taku Demura; Tom Dierschke; Liam Dolan; Ana E Dorantes-Acosta; D Magnus Eklund; Stevie N Florent; Eduardo Flores-Sandoval; Asao Fujiyama; Hideya Fukuzawa; Bence Galik; Daniel Grimanelli; Jane Grimwood; Ueli Grossniklaus; Takahiro Hamada; Jim Haseloff; Alexander J Hetherington; Asuka Higo; Yuki Hirakawa; Hope N Hundley; Yoko Ikeda; Keisuke Inoue; Shin-Ichiro Inoue; Sakiko Ishida; Qidong Jia; Mitsuru Kakita; Takehiko Kanazawa; Yosuke Kawai; Tomokazu Kawashima; Megan Kennedy; Keita Kinose; Toshinori Kinoshita; Yuji Kohara; Eri Koide; Kenji Komatsu; Sarah Kopischke; Minoru Kubo; Junko Kyozuka; Ulf Lagercrantz; Shih-Shun Lin; Erika Lindquist; Anna M Lipzen; Chia-Wei Lu; Efraín De Luna; Robert A Martienssen; Naoki Minamino; Masaharu Mizutani; Miya Mizutani; Nobuyoshi Mochizuki; Isabel Monte; Rebecca Mosher; Hideki Nagasaki; Hirofumi Nakagami; Satoshi Naramoto; Kazuhiko Nishitani; Misato Ohtani; Takashi Okamoto; Masaki Okumura; Jeremy Phillips; Bernardo Pollak; Anke Reinders; Moritz Rövekamp; Ryosuke Sano; Shinichiro Sawa; Marc W Schmid; Makoto Shirakawa; Roberto Solano; Alexander Spunde; Noriyuki Suetsugu; Sumio Sugano; Akifumi Sugiyama; Rui Sun; Yutaka Suzuki; Mizuki Takenaka; Daisuke Takezawa; Hirokazu Tomogane; Masayuki Tsuzuki; Takashi Ueda; Masaaki Umeda; John M Ward; Yuichiro Watanabe; Kazufumi Yazaki; Ryusuke Yokoyama; Yoshihiro Yoshitake; Izumi Yotsui; Sabine Zachgo; Jeremy Schmutz
  Cell, Volume：171, Number：2, First page：287, Last page：304, Oct. 2017, [Reviewed], ［International magazine］
  The evolution of land flora transformed the terrestrial environment. Land plants evolved from an ancestral charophycean alga from which they inherited developmental, biochemical, and cell biological attributes. Additional biochemical and physiological adaptations to land, and a life cycle with an alternation between multicellular haploid and diploid generations that facilitated efficient dispersal of desiccation tolerant spores, evolved in the ancestral land plant. We analyzed the genome of the liverwort Marchantia polymorpha, a member of a basal land plant lineage. Relative to charophycean algae, land plant genomes are characterized by genes encoding novel biochemical pathways, new phytohormone signaling pathways (notably auxin), expanded repertoires of signaling pathways, and increased diversity in some transcription factor families. Compared with other sequenced land plants, M. polymorpha exhibits low genetic redundancy in most regulatory pathways, with this portion of its genome resembling that predicted for the ancestral land plant. PAPERCLIP.
  English, Scientific journal
  DOI：https://doi.org/10.1016/j.cell.2017.09.030
  DOI ID：10.1016/j.cell.2017.09.030, PubMed ID：28985561
• Functional characterization of a constitutively active kinase variant of Arabidopsis phototropin 1　　　　　　　　　　　　　　　
  Jan Petersen; Shin-ichiro Inoue; Sharon M. Kelly; Stuart Sullivan; Toshinori Kinoshita; John M. Christie
  JOURNAL OF BIOLOGICAL CHEMISTRY, Volume：292, Number：33, First page：13843, Last page：13852, Aug. 2017, [Reviewed]
  Phototropins (phots) are plasma membrane-associated serine/threonine kinases that coordinate a range of processes linked to optimizing photosynthetic efficiency in plants. These photoreceptors contain two light-, oxygen-, or voltage-sensing (LOV) domains within their N terminus, with each binding one molecule of flavin mononucleotide as a UV/blue light-absorbing chromophore. Although phots contain two LOV domains, light-induced activation of the C-terminal kinase domain and subsequent receptor autophosphorylation is controlled primarily by the A'alpha-LOV2-J alpha photosensory module. Mutations that disrupt interactions between the LOV2 core and its flanking helical segments can uncouple this mode of light regulation. However, the impact of these mutations on phot function in Arabidopsis has not been explored. Here we report that histidine substitution of Arg-472 located within the A'alpha-helix of Arabidopsis phot1 constitutively activates phot1 kinase activity in vitro without affecting LOV2 photochemistry. Expression analysis of phot1 R472H in the phot-deficient mutant confirmed that it is autophosphorylated in darkness in vivo but unable to initiate phot1 signaling in the absence of light. Instead, we found that phot1 R472H is poorly functional under low-light conditions but can restore phototropism, chloroplast accumulation, stomatal opening, and leaf positioning and expansion at higher light intensities. Our findings suggest that Arabidopsis can adapt to the elevated phosphorylation status of the phot1 R472H mutant in part by reducing its stability, whereas the activity of the mutant under high-light conditions can be attributed to additional increases in LOV2-mediated photoreceptor autophosphorylation.
  AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, English, Scientific journal
  DOI：https://doi.org/10.1074/jbc.M117.799643
  DOI ID：10.1074/jbc.M117.799643, ISSN：0021-9258, eISSN：1083-351X, Web of Science ID：WOS:000407942400022
• Blue Light Regulation of Stomatal Opening and the Plasma Membrane H+-ATPase　　　　　　　　　　　　　　　
  Shin-ichiro Inoue; Toshinori Kinoshita
  PLANT PHYSIOLOGY, Volume：174, Number：2, First page：531, Last page：538, Jun. 2017, [Reviewed], [Invited], [Lead]
  AMER SOC PLANT BIOLOGISTS, English, Scientific journal
  DOI：https://doi.org/10.1104/pp.17.00166
  DOI ID：10.1104/pp.17.00166, ISSN：0032-0889, eISSN：1532-2548, Web of Science ID：WOS:000403152200007
• Brassinosteroid Involvement in Arabidopsis thaliana Stomatal Opening　　　　　　　　　　　　　　　
  Shin-ichiro Inoue; Nozomi Iwashita; Yohei Takahashi; Eiji Gotoh; Eiji Okuma; Maki Hayashi; Ryohei Tabata; Atsushi Takemiya; Yoshiyuki Murata; Michio Doi; Toshinori Kinoshita; Ken-ichiro Shimazaki
  PLANT AND CELL PHYSIOLOGY, Volume：58, Number：6, First page：1048, Last page：1058, Jun. 2017, [Reviewed], [Lead, Corresponding]
  Stomata within the plant epidermis regulate CO2 uptake for photosynthesis and water loss through transpiration. Stomatal opening in Arabidopsis thaliana is determined by various factors, including blue light as a signal and multiple phytohormones. Plasma membrane transporters, including H+-ATPase, K+ channels and anion channels in guard cells, mediate these processes, and the activities and expression levels of these components determine stomatal aperture. However, the regulatory mechanisms involved in these processes are not fully understood. In this study, we used infrared thermography to isolate a mutant defective in stomatal opening in response to light. The causative mutation was identified as an allele of the brassinosteroid (BR) biosynthetic mutant dwarf5. Guard cells from this mutant exhibited normal H+-ATPase activity in response to blue light, but showed reduced K+ accumulation and inward-rectifying K+ (K-in(+)) channel activity as a consequence of decreased expression of major K-in(+) channel genes. Consistent with these results, another BR bio-synthetic mutant, det2-1, and a BR receptor mutant, bri1-6, exhibited reduced blue light-dependent stomatal opening. Furthermore, application of BR to the hydroponic culture medium completely restored stomatal opening in dwarf5 and det2-1 but not in bri1-6. However, application of BR to the epidermis of dwarf5 did not restore stomatal response. From these results, we conclude that endogenous BR acts in a long-term manner and is required in guard cells with the ability to open stomata in response to light, probably through regulation of K-in(+) channel activity.
  OXFORD UNIV PRESS, English, Scientific journal
  DOI：https://doi.org/10.1093/pcp/pcx049
  DOI ID：10.1093/pcp/pcx049, ISSN：0032-0781, eISSN：1471-9053, Web of Science ID：WOS:000404127900007
• A Raf-like protein kinase BHP mediates blue light-dependent stomatal opening　　　　　　　　　　　　　　　
  Maki Hayashi; Shin-ichiro Inoue; Yoshihisa Ueno; Toshinori Kinoshita
  SCIENTIFIC REPORTS, Volume：7, First page：45586, Mar. 2017, [Reviewed]
  Stomata in the plant epidermis open in response to blue light and affect photosynthesis and plant growth by regulating CO2 uptake and transpiration. In stomatal guard cells under blue light, plasma membrane H+-ATPase is phosphorylated and activated via blue light-receptor phototropins and a signaling mediator BLUS1, and H+-ATPase activation drives stomatal opening. However, details of the signaling between phototropins and H+-ATPase remain largely unknown. In this study, through a screening of specific inhibitors for the blue light-dependent H+-ATPase phosphorylation in guard cells, we identified a Raf-like protein kinase, BLUE LIGHT-DEPENDENT H+-ATPASE PHOSPHORYLATION (BHP). Guard cells in the bhp mutant showed impairments of stomatal opening and H+-ATPase phosphorylation in response to blue light. BHP is abundantly expressed in the cytosol of guard cells and interacts with BLUS1 both in vitro and in vivo. Based on these results, BHP is a novel signaling mediator in blue light-dependent stomatal opening, likely downstream of BLUS1.
  NATURE PUBLISHING GROUP, English, Scientific journal
  DOI：https://doi.org/10.1038/srep45586
  DOI ID：10.1038/srep45586, ISSN：2045-2322, Web of Science ID：WOS:000398950000001
• Auxin Influx Carrier AUX1 Confers Acid Resistance for Arabidopsis Root Elongation Through the Regulation of Plasma Membrane H+-ATPase　　　　　　　　　　　　　　　
  Shin-ichiro Inoue; Koji Takahashi; Hiromi Okumura-Noda; Toshinori Kinoshita
  PLANT AND CELL PHYSIOLOGY, Volume：57, Number：10, First page：2194, Last page：2201, Oct. 2016, [Reviewed], [Lead]
  The plant plasma membrane (PM) H+-ATPase regulates pH homeostasis and cell elongation in roots through the formation of an electrochemical H+ gradient across the PM and a decrease in apoplastic pH; however, the detailed signaling for the regulation of PM H+-ATPases remains unclear. Here, we show that an auxin influx carrier, AUXIN RESISTANT1 (AUX1), is required for the maintenance of PM H+-ATPase activity and proper root elongation. We isolated a low pH-hypersensitive 1 (loph1) mutant by a genetic screen of Arabidopsis thaliana on low pH agar plates. The loph1 mutant is a loss-of-function mutant of the AUX1 gene and exhibits a root growth retardation restricted to the low pH condition. The ATP hydrolysis and H+ extrusion activities of the PM H+-ATPase were reduced in loph1 roots. Furthermore, the phosphorylation of the penultimate threonine of the PM H+-ATPase was reduced in loph1 roots under both normal and low pH conditions without reduction of the amount of PM H+-ATPase. Expression of the DR5: GUS reporter gene and auxin-responsive genes suggested that endogenous auxin levels were lower in loph1 roots than in the wild type. The aux1-7 mutant roots also exhibited root growth retardation in the low pH condition like the loph1 roots. These results indicate that AUX1 positively regulates the PM H+-ATPase activity through maintenance of the auxin accumulation in root tips, and this process may serve to maintain root elongation especially under low pH conditions.
  OXFORD UNIV PRESS, English, Scientific journal
  DOI：https://doi.org/10.1093/pcp/pcw136
  DOI ID：10.1093/pcp/pcw136, ISSN：0032-0781, eISSN：1471-9053, Web of Science ID：WOS:000388374100015
• Photosynthesis activates plasma membrane H+-ATPase via sugar accumulation　　　　　　　　　　　　　　　
  Masaki Okumura; Shin-ichiro Inoue; Keiko Kuwata; Toshinori Kinoshita
  Plant Physiology, Volume：171, Number：1, First page：580, Last page：589, May 2016, [Reviewed]
  Plant plasma membrane H+-ATPase acts as a primary transporter via proton pumping and regulates diverse physiological responses by controlling secondary solute transport, pH homeostasis, and membrane potential. Phosphorylation of the penultimate threonine and the subsequent binding of 14-3-3 proteins in the carboxyl terminus of the enzyme are required for H+-ATPase activation. We showed previously that photosynthesis induces phosphorylation of the penultimate threonine in the nonvascular bryophyte Marchantia polymorpha. However, (1) whether this response is conserved in vascular plants and (2) the process by which photosynthesis regulates H+-ATPase phosphorylation at the plasma membrane remain unresolved issues. Here, we report that photosynthesis induced the phosphorylation and activation of H+-ATPase in Arabidopsis (Arabidopsis thaliana) leaves via sugar accumulation. Light reversibly phosphorylated leaf H+-ATPase, and this process was inhibited by pharmacological and genetic suppression of photosynthesis. Immunohistochemical and biochemical analyses indicated that light-induced phosphorylation of H+-ATPase occurred autonomously in mesophyll cells. We also show that the phosphorylation status of H+-ATPase and photosynthetic sugar accumulation in leaves were positively correlated and that sugar treatment promoted phosphorylation. Furthermore, light-induced phosphorylation of H+-ATPase was strongly suppressed in a double mutant defective in ADP-glucose pyrophosphorylase and triose phosphate/phosphate translocator (adg1-1 tpt-2)
  these mutations strongly inhibited endogenous sugar accumulation. Overall, we show that photosynthesis activated H+-ATPase via sugar production in the mesophyll cells of vascular plants. Our work provides new insight into signaling from chloroplasts to the plasma membrane ion transport mechanism.
  American Society of Plant Biologists, English, Scientific journal
  DOI：https://doi.org/10.1104/pp.16.00355
  DOI ID：10.1104/pp.16.00355, ISSN：1532-2548, PubMed ID：27016447, SCOPUS ID：84964886710
• GOLDEN 2-LIKE transcription factors for chloroplast development affect ozone tolerance through the regulation of stomatal movement　　　　　　　　　　　　　　　
  Yukari Nagatoshi; Nobutaka Mitsuda; Maki Hayashi; Shin-ichiro Inoue; Eiji Okuma; Akihiro Kubo; Yoshiyuki Murata; Mitsunori Seo; Hikaru Saji; Toshinori Kinoshita; Masaru Ohme-Takagi
  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Volume：113, Number：15, First page：4218, Last page：4223, Apr. 2016, [Reviewed]
  Stomatal movements regulate gas exchange, thus directly affecting the efficiency of photosynthesis and the sensitivity of plants to air pollutants such as ozone. The GARP family transcription factors GOLDEN 2-LIKE1 (GLK1) and GLK2 have known functions in chloroplast development. Here, we show that Arabidopsis thaliana (A. thaliana) plants expressing the chimeric repressors for GLK1 and -2 (GLK1/2-SRDX) exhibited a closed-stomata phenotype and strong tolerance to ozone. By contrast, plants that overexpress GLK1/2 exhibited an open-stomata phenotype and higher sensitivity to ozone. The plants expressing GLK1-SRDX had reduced expression of the genes for inwardly rectifying K+ (K-in(+)) channels and reduced K-in(+) channel activity. Abscisic acid treatment did not affect the stomatal phenotype of 35S:GLK1/2-SRDX plants or the transcriptional activity for K-in(+) channel gene, indicating that GLK1/2 act independently of abscisic acid signaling. Our results indicate that GLK1/2 positively regulate the expression of genes for K-in(+) channels and promote stomatal opening. Because the chimeric GLK1-SRDX repressor driven by a guard cell-specific promoter induced a closed-stomata phenotype without affecting chloroplast development in mesophyll cells, modulating GLK1/2 activity may provide an effective tool to control stomatal movements and thus to confer resistance to air pollutants.
  NATL ACAD SCIENCES, English, Scientific journal
  DOI：https://doi.org/10.1073/pnas.1513093113
  DOI ID：10.1073/pnas.1513093113, ISSN：0027-8424, ORCID：45477106, Web of Science ID：WOS:000373762400077
• A Flowering Integrator, SOC1, Affects Stomatal Opening in Arabidopsis thaliana　　　　　　　　　　　　　　　
  Yuriko Kimura; Saya Aoki; Eigo Ando; Ayaka Kitatsuji; Aiko Watanabe; Masato Ohnishi; Koji Takahashi; Shin-ichiro Inoue; Norihito Nakamichi; Yosuke Tamada; Toshinori Kinoshita
  PLANT AND CELL PHYSIOLOGY, Volume：56, Number：4, First page：640, Last page：649, Apr. 2015, [Reviewed], [Invited]
  Stomatal movements are regulated by multiple environmental signals. Recent investigations indicate that photoperiodic flowering components, such as CRY, GI, CO, FT and TSF, are expressed in guard cells and positively affect stomatal opening in Arabidopsis thaliana. Here we show that SOC1, which encodes a MADS box transcription factor and integrates multiple flowering signals, also exerts a positive effect on stomatal opening. FLC encodes a potent repressor of FT and SOC1, and FRI acts as an activator of FLC. Thus, we examined stomatal phenotypes in FRI-Col, which contains an active FRI allele of accession Sf-2 by introgression. We found higher expression of FLC and lower expression of FT, SOC1 and TSF in guard cells from FRI-Col than in those from Col. Light-induced stomatal opening was significantly suppressed in FRI-Col. Interestingly, vernalization of FRI-Col partially restored light-induced stomatal opening, concomitant with a decrease of FLC and increase of FT, SOC1 and TSF. Furthermore, we observed the constitutive open-stomata phenotype in transgenic plants overexpressing SOC1-GFP (green fluorescent protein) in guard cells (SOC1-GFP overexpressor), and found that light-induced stomatal opening was significantly suppressed in a soc1 knockout mutant. RNA sequencing using epidermis from the SOC1-GFP overexpressor revealed that the expression levels of several genes involved in stomatal opening, such as BLUS1 and the plasma membrane H+-ATPases, were higher than those in background plants. From these results, we conclude that SOC1 is involved in the regulation of stomatal opening via transcriptional regulation in guard cells.
  OXFORD UNIV PRESS, English, Scientific journal
  DOI：https://doi.org/10.1093/pcp/pcu214
  DOI ID：10.1093/pcp/pcu214, ISSN：0032-0781, eISSN：1471-9053, Web of Science ID：WOS:000354730200007
• In vitro Phosphorylation Assay of Putative Blue-Light Receptor Phototropins Using Microsomal and Plasma-membrane Fractions Prepared from Vallisneria Leaves　　　　　　　　　　　　　　　
  Yuuki Sakai; Shin-ichiro Inoue; Shingo Takagi
  BIO-PROTOCOL, Volume：5, Number：21, 2015, [Invited]
  Bio-Protocol, LLC, Scientific journal
  DOI：https://doi.org/10.21769/bioprotoc.1647
  DOI ID：10.21769/bioprotoc.1647, ISSN：2331-8325
• Blue-light-induced rapid chloroplast de-anchoring in Vallisneria epidermal cells　　　　　　　　　　　　　　　
  Yuuki Sakai; Shin-Ichiro Inoue; Akiko Harada; Ken-Ichiro Shimazaki; Shingo Takagi
  JOURNAL OF INTEGRATIVE PLANT BIOLOGY, Volume：57, Number：1, First page：93, Last page：105, Jan. 2015, [Reviewed]
  In the outer periclinal cytoplasm of leaf epidermal cells of an aquatic angiosperm Vallisneria, blue light induces chloroplast de-anchoring, a rapid decline in the resistance of chloroplasts against centrifugal force. Chloroplast de-anchoring is known induced within 1min of irradiation with high-fluence-rate blue light specifically, preceding the commencement of chloroplasts migration toward the anticlinal cytoplasm. However, its regulatory mechanism has remained elusive, although pharmacological analysis suggested that a calcium release from intracellular calcium stores is necessary for the response. In search of the responsible photoreceptors, immunoblotting analysis using antibodies against phototropins demonstrated that cross-reactive polypeptides of 120-kDa exist in the plasma-membrane fraction prepared from the leaves. In vitro phosphorylation analysis revealed that 120-kDa polypeptides were phosphorylated by exposure to blue light in a fluence-dependent manner. The blue-light-induced phosphorylation activity was sensitive to a Ser/Thr kinase inhibitor, staurosporine, and unusually was retained at a high level for a long time in darkness. Furthermore, phototropin gene homologs (Vallisneria PHOTOTROPIN1 and PHOTOTROPIN2) expressed in leaves were isolated. We propose that calcium-regulated chloroplast de-anchoring, possibly mediated by phototropins, is an initial process of the blue-light-induced avoidance response of chloroplasts in Vallisneria.
  WILEY-BLACKWELL, English, Scientific journal
  DOI：https://doi.org/10.1111/jipb.12284
  DOI ID：10.1111/jipb.12284, ISSN：1672-9072, eISSN：1744-7909, Web of Science ID：WOS:000347413600010
• Mg-chelatase I subunit 1 and Mg-protoporphyrin IX methyltransferase affect the stomatal aperture in Arabidopsis thaliana　　　　　　　　　　　　　　　
  Masakazu Tomiyama; Shin-ichiro Inoue; Tomo Tsuzuki; Midori Soda; Sayuri Morimoto; Yukiko Okigaki; Takaya Ohishi; Nobuyoshi Mochizuki; Koji Takahashi; Toshinori Kinoshita
  JOURNAL OF PLANT RESEARCH, Volume：127, Number：4, First page：553, Last page：563, Jul. 2014, [Reviewed]
  To elucidate the molecular mechanisms of stomatal opening and closure, we performed a genetic screen using infrared thermography to isolate stomatal aperture mutants. We identified a mutant designated low temperature with open-stomata 1 (lost1), which exhibited reduced leaf temperature, wider stomatal aperture, and a pale green phenotype. Map-based analysis of the LOST1 locus revealed that the lost1 mutant resulted from a missense mutation in the Mg-chelatase I subunit 1 (CHLI1) gene, which encodes a subunit of the Mg-chelatase complex involved in chlorophyll synthesis. Transformation of the wild-type CHLI1 gene into lost1 complemented all lost1 phenotypes. Stomata in lost1 exhibited a partial ABA-insensitive phenotype similar to that of rtl1, a Mg-chelatase H subunit missense mutant. The Mg-protoporphyrin IX methyltransferase (CHLM) gene encodes a subsequent enzyme in the chlorophyll synthesis pathway. We examined stomatal movement in a CHLM knockdown mutant, chlm, and found that it also exhibited an ABA-insensitive phenotype. However, lost1 and chlm seedlings all showed normal expression of ABA-induced genes, such as RAB18 and RD29B, in response to ABA. These results suggest that the chlorophyll synthesis enzymes, Mg-chelatase complex and CHLM, specifically affect ABA signaling in the control of stomatal aperture and have no effect on ABA-induced gene expression.
  SPRINGER JAPAN KK, English, Scientific journal
  DOI：https://doi.org/10.1007/s10265-014-0636-0
  DOI ID：10.1007/s10265-014-0636-0, ISSN：0918-9440, eISSN：1618-0860, Web of Science ID：WOS:000338337900009
• Abscisic Acid Suppresses Hypocotyl Elongation by Dephosphorylating Plasma Membrane H+-ATPase in Arabidopsis thaliana　　　　　　　　　　　　　　　
  Yuki Hayashi; Koji Takahashi; Shin-ichiro Inoue; Toshinori Kinoshita
  PLANT AND CELL PHYSIOLOGY, Volume：55, Number：4, First page：845, Last page：853, Apr. 2014, [Reviewed]
  Plasma membrane H+-ATPase is thought to mediate hypocotyl elongation, which is induced by the phytohormone auxin through the phosphorylation of the penultimate threonine of H+-ATPase. However, regulation of the H+-ATPase during hypocotyl elongation by other signals has not been elucidated. Hypocotyl elongation in etiolated seedlings of Arabidopsis thaliana was suppressed by the H+-ATPase inhibitors vanadate and erythrosine B, and was significantly reduced in aha2-5, which is a knockout mutant of the major H+-ATPase isoform in etiolated seedlings. Application of the phytohormone ABA to etiolated seedlings suppressed hypocotyl elongation within 30 min at the half-inhibitory concentration (4.2 mu M), and induced dephosphorylation of the penultimate threonine of H+-ATPase without affecting the amount of H+-ATPase. Interestingly, an ABA-insensitive mutant, abi1-1, did not show ABA inhibition of hypocotyl elongation or ABA-induced dephosphorylation of H+-ATPase. This indicates that ABI1, which is an early ABA signaling component through the ABA receptor PYR/PYL/RCARs (pyrabactin resistance/pyrabactin resistance 1-like/regulatory component of ABA receptor), is involved in these responses. In addition, we found that the fungal toxin fusiccocin (FC), an H+-ATPase activator, induced hypocotyl elongation and phosphorylation of the penultimate threonine of H+-ATPase, and that FC-induced hypocotyl elongation and phosphorylation of H+-ATPase were significantly suppressed by ABA. Taken together, these results indicate that ABA has an antagonistic effect on hypocotyl elongation through, at least in part, dephosphorylation of H+-ATPase in etiolated seedlings.
  OXFORD UNIV PRESS, English, Scientific journal
  DOI：https://doi.org/10.1093/pcp/pcu028
  DOI ID：10.1093/pcp/pcu028, ISSN：0032-0781, eISSN：1471-9053, Web of Science ID：WOS:000334679500018
• Overexpression of plasma membrane H+-ATPase in guard cells promotes light-induced stomatal opening and enhances plant growth　　　　　　　　　　　　　　　
  Yin Wang; Ko Noguchi; Natsuko Ono; Shin-ichiro Inoue; Ichiro Terashima; Toshinori Kinoshita
  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Volume：111, Number：1, First page：533, Last page：538, Jan. 2014, [Reviewed]
  Stomatal pores surrounded by a pair of guard cells in the plant epidermis control gas exchange between plants and the atmosphere in response to light, CO2, and the plant hormone abscisic acid. Light-induced stomatal opening is mediated by at least three key components: the blue light receptor phototropin (phot1 and phot2), plasma membrane H+-ATPase, and plasma membrane inward-rectifying K+ channels. Very few attempts have been made to enhance stomatal opening with the goal of increasing photosynthesis and plant growth, even though stomatal resistance is thought to be the major limiting factor for CO2 uptake by plants. Here, we show that transgenic Arabidopsis plants overexpressing H+-ATPase using the strong guard cell promoter GC1 showed enhanced light-induced stomatal opening, photosynthesis, and plant growth. The transgenic plants produced larger and increased numbers of rosette leaves, with similar to 42-63% greater fresh and dry weights than the wild type in the first 25 d of growth. The dry weights of total flowering stems of 45-d-old transgenic plants, including seeds, siliques, and flowers, were similar to 36-41% greater than those of the wild type. In addition, stomata in the transgenic plants closed normally in response to darkness and abscisic acid. In contrast, the overexpression of phototropin or inward-rectifying K+ channels in guard cells had no effect on these phenotypes. These results demonstrate that stomatal aperture is a limiting factor in photosynthesis and plant growth, and that manipulation of stomatal opening by overexpressing H+-ATPase in guard cells is useful for the promotion of plant growth.
  NATL ACAD SCIENCES, English, Scientific journal
  DOI：https://doi.org/10.1073/pnas.1305438111
  DOI ID：10.1073/pnas.1305438111, ISSN：0027-8424, Web of Science ID：WOS:000329350700119
• Overexpression of the Mg-chelatase H subunit in guard cells confers drought tolerance via promotion of stomatal closure in Arabidopsis thaliana　　　　　　　　　　　　　　　
  Tomo Tsuzuki; Koji Takahashi; Masakazu Tomiyama; Shin-Ichiro Inoue; Toshinori Kinoshita
  Frontiers in Plant Science, Volume：4, First page：440, Oct. 2013, [Reviewed]
  The Mg-chelatase H subunit (CHLH) has been shown to mediate chlorophyll biosynthesis, as well as plastid-to-nucleus and abscisic acid (ABA)-mediated signaling. A recent study using a novel CHLH mutant, rtl1, indicated that CHLH specifically affects ABA-induced stomatal closure, but also that CHLH did not serve as an ABA receptor in Arabidopsis thaliana. However, the molecular mechanism by which CHLH engages in ABA-mediated signaling in guard cells remains largely unknown. In the present study, we examined CHLH function in guard cells and explored whether CHLH expression might influence stomatal aperture. Incubation of rtl1 guard cell protoplasts with ABA induced expression of the ABA-responsive genes RAB18 and RD29B, as also observed in wild-type (WT) cells, indicating that CHLH did not affect the expression of ABA-responsive genes. Earlier, ABA was reported to inhibit blue light (BL)-mediated stomatal opening, at least in part through dephosphorylating/inhibiting guard cell H+-ATPase (which drives opening). Therefore, we immunohistochemically examined the phosphorylation status of guard cell H+-ATPase. Notably, ABA inhibition of BL-induced phosphorylation of H+-ATPase was impaired in rtl1 cells, suggesting that CHLH influences not only ABA-induced stomatal closure but also inhibition of BL-mediated stomatal opening by ABA. Next, we generated CHLH-GFP-overexpressing plants using CER6 promoter, which induces gene expression in the epidermis including guard cells. CHLH-transgenic plants exhibited a closed stomata phenotype even when brightly illuminated. Moreover, plant growth experiments conducted under water-deficient conditions showed that CHLH transgenic plants were more tolerant of drought than WT plants. In summary, we show that CHLH is involved in the regulation of stomatal aperture in response to ABA, but not in ABA-induced gene expression, and that manipulation of stomatal aperture via overexpression of CHLH in guard cells improves plant drought tolerance. © 2013 Tsuzuki, Takahashi, Tomiyama, Inoue and Kinoshita.
  Frontiers Research Foundation, English, Scientific journal
  DOI：https://doi.org/10.3389/fpls.2013.00440
  DOI ID：10.3389/fpls.2013.00440, ISSN：1664-462X, SCOPUS ID：84898909058
• TWIN SISTER OF FT, GIGANTEA, and CONSTANS Have a Positive But Indirect Effect on Blue Light-Induced Stomatal Opening in Arabidopsis　　　　　　　　　　　　　　　
  Eigo Ando; Masato Ohnishi; Yin Wang; Tomonao Matsushita; Aiko Watanabe; Yuki Hayashi; Miho Fujii; Jian Feng Ma; Shin-ichiro Inoue; Toshinori Kinoshita
  PLANT PHYSIOLOGY, Volume：162, Number：3, First page：1529, Last page：1538, Jul. 2013, [Reviewed]
  FLOWERING LOCUS T (FT) is the major regulatory component controlling photoperiodic floral transition. It is expressed in guard cells and affects blue light-induced stomatal opening induced by the blue-light receptor phototropins phot1 and phot2. Roles for other flowering regulators in stomatal opening have yet to be determined. We show in Arabidopsis (Arabidopsis thaliana) that TWIN SISTER OF FT (TSF), CONSTANS (CO), and GIGANTEA (GI) provide a positive effect on stomatal opening. TSF, which is the closest homolog of FT, was transcribed in guard cells, and light-induced stomatal opening was repressed in tsf-1, a T-DNA insertion mutant of TSF. Overexpression of TSF in a phot1 phot2 mutant background gave a constitutive open-stomata phenotype. Then, we examined whether CO and GI, which are upstream regulators of FT and TSF in photoperiodic flowering, are involved in stomatal opening. Similar to TSF, light-induced stomatal opening was suppressed in the GI and CO mutants gi-1 and co-1. A constitutive open-stomata phenotype was observed in GI and CO overexpressors with accompanying changes in the transcription of both FT and TSF. In photoperiodic flowering, photoperiod is sensed by photoreceptors such as the cryptochromes cry1 and cry2. We examined stomatal phenotypes in a cry1 cry2 mutant and in CRY2 overexpressors. Light-induced stomatal opening was suppressed in cry1 cry2, and the transcription of FT and TSF was down-regulated. In contrast, the stomata in CRY2 overexpressors opened even in the dark, and FT and TSF transcription was up-regulated. We conclude that the photoperiodic flowering components TSF, GI, and CO positively affect stomatal opening.
  AMER SOC PLANT BIOLOGISTS, English, Scientific journal
  DOI：https://doi.org/10.1104/pp.113.217984
  DOI ID：10.1104/pp.113.217984, ISSN：0032-0889, eISSN：1532-2548, Web of Science ID：WOS:000321325700024
• Role of RPT2 in Leaf Positioning and Flattening and a Possible Inhibition of phot2 Signaling by phot1　　　　　　　　　　　　　　　
  Akiko Harada; Atsushi Takemiya; Shin-ichiro Inoue; Tatsuya Sakai; Ken-ichiro Shimazaki
  PLANT AND CELL PHYSIOLOGY, Volume：54, Number：1, First page：36, Last page：47, Jan. 2013, [Reviewed]
  We investigated the roles of the blue light receptors phototropins (phot1 and phot2) and ROOT PHOTOTROPISM 2 (RPT2) in leaf positioning and flattening, and plant growth under weak, moderate and strong white light (10, 25 and 70 mu molm(-2) s(-1)). RPT2 mediated leaf positioning and flattening, and enhanced plant growth in a phot1-dependent manner. Under weak light, phot1 alone controls these responses. Under moderate and strong light, both phot1 and phot2 affect the responses. These results indicate that plants utilize a wide range of light intensities through phot1 and phot2 to optimize plant growth. The rpt2 single mutant generally exhibited phenotypes that resembled those of the phot1 phot2 double mutant. To our surprise, when the PHOT1 gene was disrupted in the rpt2 mutant, the resulting phot1 rpt2 double mutant showed the morphology of the wild-type plant under strong light, and additional disruption of PHOT2 in the double mutant abolished this recovery. This suggested that phot2 may function in the absence of phot1 and bypass RPT2 to transmit the signal to downstream elements. Expression and light-induced autophosphorylation of phot2 were not affected in the rpt2 mutant. We conclude that RPT2 mediates leaf flattening and positioning in a phot1-dependent manner, and that phot1 may inhibit the phot2 signaling pathways. We discuss the functional role of RPT2 in phototropin signaling.
  OXFORD UNIV PRESS, English, Scientific journal
  DOI：https://doi.org/10.1093/pcp/pcs094
  DOI ID：10.1093/pcp/pcs094, ISSN：0032-0781, Web of Science ID：WOS:000315218100004
• Characterization of the Plasma Membrane H+-ATPase in the Liverwort Marchantia polymorpha　　　　　　　　　　　　　　　
  Masaki Okumura; Shin-ichiro Inoue; Koji Takahashi; Kimitsune Ishizaki; Takayuki Kohchi; Toshinori Kinoshita
  PLANT PHYSIOLOGY, Volume：159, Number：2, First page：826, Last page：+, Jun. 2012, [Reviewed]
  The plasma membrane H+-ATPase generates an electrochemical gradient of H+ across the plasma membrane that provides the driving force for solute transport and regulates pH homeostasis and membrane potential in plant cells. Recent studies have demonstrated that phosphorylation of the penultimate threonine in H+-ATPase and subsequent binding of a 14-3-3 protein is the major common activation mechanism for H+-ATPase in vascular plants. However, there is very little information on the plasma membrane H+-ATPase in nonvascular plant bryophytes. Here, we show that the liverwort Marchantia polymorpha, which is the most basal lineage of extant land plants, expresses both the penultimate threonine-containing H+-ATPase (pT H+-ATPase) and non-penultimate threonine-containing H+-ATPase (non-pT H+-ATPase) as in the green algae and that pT H+-ATPase is regulated by phosphorylation of its penultimate threonine. A search in the expressed sequence tag database of M. polymorpha revealed eight H+-ATPase genes, designated MpHA (for M. polymorpha H+-ATPase). Four isoforms are the pT H+-ATPase; the remaining isoforms are non-pT H+-ATPase. An apparent 95-kD protein was recognized by anti-H+-ATPase antibodies against an Arabidopsis (Arabidopsis thaliana) isoform and was phosphorylated on the penultimate threonine in response to the fungal toxin fusicoccin in thalli, indicating that the 95-kD protein contains pT H+-ATPase. Furthermore, we found that the pT H(+-)ATPase in thalli is phosphorylated in response to light, sucrose, and osmotic shock and that light-induced phosphorylation depends on photosynthesis. Our results define physiological signals for the regulation of pT H+-ATPase in the liverwort M. polymorpha, which is one of the earliest plants to acquire pT H+-ATPase.
  AMER SOC PLANT BIOLOGISTS, English, Scientific journal
  DOI：https://doi.org/10.1104/pp.112.195537
  DOI ID：10.1104/pp.112.195537, ISSN：0032-0889, Web of Science ID：WOS:000304834800024
• Evolutionary appearance of the plasma membrane H+-ATPase containing a penultimate threonine in the bryophyte　　　　　　　　　　　　　　　
  Masaki Okumura; Koji Takahashi; Shin-ichiro Inoue; Toshinori Kinoshita
  Plant Signaling and Behavior, Volume：7, Number：8, First page：979-982, 2012, [Reviewed], [Invited]
  The plasma membrane H+-ATPase provides the driving force for solute transport via an electrochemical gradient of H+ across the plasma membrane, and regulates ph homeostasis and membrane potential in plant cells. however, the plasma membrane H+-ATPase in non-vascular plant bryophyte is largely unknown. here, we show that the moss Physcomitrella patens, which is known as a model bryophyte, expresses both the penultimate thr-containing H±-ATPase (pt H+-ATPase) and non-pT H+-ATPase as in the green algae, and that pT H+-ATPase is regulated by phosphorylation of its penultimate Thr. A search in the P. patens genome database revealed seven H+-ATPase genes, designated PpHA (Physcomitrella patens H+-ATPase). Six isoforms are the pT H+-ATPase
  a remaining isoform is non-pT H+-ATPase. An apparent 95-kD protein was recognized by anti-H+-ATPase antibodies against an isoform of Arabidopsis thaliana and was phosphorylated on the penultimate thr in response to a fungal toxin fusicoccin and light in protonemata, indicating that the 95-kD protein contains pT H+-ATPase. Furthermore, we could not detect the pT H+-ATPase in the charophyte alga Chara braunii, which is the closest relative of the land plants, by immunological methods. these results strongly suggest the pT H+-ATPase most likely appeared for the first time in bryophyte. © 2012 Landes Bioscience.
  Landes Bioscience, English, Scientific journal
  DOI：https://doi.org/10.4161/psb.20936
  DOI ID：10.4161/psb.20936, ISSN：1559-2324, PubMed ID：22836495, SCOPUS ID：84864819460
• FLOWERING LOCUS T Regulates Stomatal Opening　　　　　　　　　　　　　　　
  Toshinori Kinoshita; Natsuko Ono; Yuki Hayashi; Sayuri Morimoto; Suguru Nakamura; Midori Soda; Yuma Kato; Masato Ohnishi; Takeshi Nakano; Shin-ichiro Inoue; Ken-ichiro Shimazaki
  CURRENT BIOLOGY, Volume：21, Number：14, First page：1232, Last page：1238, Jul. 2011, [Reviewed]
  Stomatal pores surrounded by a pair of guard cells in the plant epidermis control gas exchange for photosynthesis in response to light, CO2, and phytohormone abscisic acid [1, 2]. Phototropins (phot1 and phot2) are plant blue-light receptor kinases and mediate stomatal opening via activation of the plasma membrane H+-ATPase [3, 4]. However, the signaling mechanism from phototropins to the H+-ATPase has yet to be determined. Here, we show that FLOWERING LOCUS T(FT) is expressed in guard cells and regulates stomata! opening. We isolated an scs (suppressor of closed-stomata phenotype in phot1 phot2) 1-1 mutant of Arabidopsis thaliana and showed that scs1-1 carries a novel null early flowering 3 (elf3) allele in a phot1 phot2 background. scs1-1 (elf3 phot1 phot2 triple mutant) had an open-stomata phenotype with high H+-ATPase activity and showed increased levels of FT mRNA in guard cells. Transgenic plants overexpressing FT in guard cells showed open stomata, whereas a loss-of-function FT allele, ft-1, exhibited closed stomata and failed to activate the H+-ATPase in response to blue light. Our results define a new cell-autonomous role for FT and demonstrate that the flowering time genes ELF3 and FT are involved in the regulation of H+-ATPase by blue light in guard cells.
  CELL PRESS, English, Scientific journal
  DOI：https://doi.org/10.1016/j.cub.2011.06.025
  DOI ID：10.1016/j.cub.2011.06.025, ISSN：0960-9822, eISSN：1879-0445, Web of Science ID：WOS:000293320000026
• Immunohistochemical Detection of Blue Light-Induced Phosphorylation of the Plasma Membrane H+-ATPase in Stomatal Guard Cells　　　　　　　　　　　　　　　
  Maki Hayashi; Shin-ichiro Inoue; Koji Takahashi; Toshinori Kinoshita
  PLANT AND CELL PHYSIOLOGY, Volume：52, Number：7, First page：1238, Last page：1248, Jul. 2011, [Reviewed]
  Blue light (BL) receptor phototropins activate the plasma membrane H+-ATPase in guard cells through phosphorylation of a penultimate threonine and subsequent binding of the 14-3-3 protein to the phosphorylated C-terminus of H+-ATPase, mediating stomatal opening. To date, detection of the phosphorylation level of the guard cell H+-ATPase has been performed biochemically using guard cell protoplasts (GCPs). However, preparation of GCPs from Arabidopsis for this purpose requires > 5,000 rosette leaves and takes > 8 h. Here, we show that BL-induced phosphorylation of guard cell H+-ATPase is detected in the epidermis from a single Arabidopsis rosette leaf via an immunohistochemical method using a specific antibody against the phosphorylated penultimate threonine of H+-ATPase. BL-induced phosphorylation of the H+-ATPase was detected immunohistochemically in the wild type, but not in a phot1-5 phot2-1 double mutant. Moreover, we found that physiological concentrations of the phytohormone ABA completely inhibited BL-induced phosphorylation of guard cell H+-ATPase in the epidermis, and that inhibition by ABA in the epidermis is more sensitive than in GCPs. These results indicate that this immunohistochemical method is very useful for detecting the phosphorylation status of guard cell H+-ATPase. Thus, we applied this technique to ABA-insensitive mutants (abi1-1, abi2-1 and ost1-2) and found that ABA had no effect on BL-induced phosphorylation in these mutants. These results indicate that inhibition of BL-induced phosphorylation of guard cell H+-ATPase by ABA is regulated by ABI1, ABI2 and OST1, which are known to be early ABA signaling components for a wide range of ABA responses in plants.
  OXFORD UNIV PRESS, English, Scientific journal
  DOI：https://doi.org/10.1093/pcp/pcr072
  DOI ID：10.1093/pcp/pcr072, ISSN：0032-0781, eISSN：1471-9053, Web of Science ID：WOS:000292837500010
• Mg-chelatase H subunit affects ABA signaling in stomatal guard cells, but is not an ABA receptor in Arabidopsis thaliana　　　　　　　　　　　　　　　
  Tomo Tsuzuki; Koji Takahashi; Shin-ichiro Inoue; Yukiko Okigaki; Masakazu Tomiyama; Mohammad Anowar Hossain; Ken-ichiro Shimazaki; Yoshiyuki Murata; Toshinori Kinoshita
  JOURNAL OF PLANT RESEARCH, Volume：124, Number：4, First page：527, Last page：538, Jul. 2011, [Reviewed]
  Mg-chelatase H subunit (CHLH) is a multifunctional protein involved in chlorophyll synthesis, plastid-to-nucleus retrograde signaling, and ABA perception. However, whether CHLH acts as an actual ABA receptor remains controversial. Here we present evidence that CHLH affects ABA signaling in stomatal guard cells but is not itself an ABA receptor. We screened ethyl methanesulfonate-treated Arabidopsis thaliana plants with a focus on stomatal aperture-dependent water loss in detached leaves and isolated a rapid transpiration in detached leaves 1 (rtl1) mutant that we identified as a novel missense mutant of CHLH. The rtl1 and CHLH RNAi plants showed phenotypes in which stomatal movements were insensitive to ABA, while the rtl1 phenotype showed normal sensitivity to ABA with respect to seed germination and root growth. ABA-binding analyses using H-3-labeled ABA revealed that recombinant CHLH did not bind ABA, but recombinant pyrabactin resistance 1, a reliable ABA receptor used as a control, showed specific binding. Moreover, we found that the rtl1 mutant showed ABA-induced stomatal closure when a high concentration of extracellular Ca2+ was present and that a knockout mutant of Mg-chelatase I subunit (chli1) showed the same ABA-insensitive phenotype as rtl1. These results suggest that the Mg-chelatase complex as a whole affects the ABA-signaling pathway for stomatal movements.
  SPRINGER JAPAN KK, English, Scientific journal
  DOI：https://doi.org/10.1007/s10265-011-0426-x
  DOI ID：10.1007/s10265-011-0426-x, ISSN：0918-9440, eISSN：1618-0860, Web of Science ID：WOS:000292448700009
• Functional Analyses of the Activation Loop of Phototropin2 in Arabidopsis　　　　　　　　　　　　　　　
  Shin-ichiro Inoue; Tomonao Matsushita; Yuta Tomokiyo; Masaki Matsumoto; Keiichi I. Nakayama; Toshinori Kinoshita; Ken-ichiro Shimazaki
  PLANT PHYSIOLOGY, Volume：156, Number：1, First page：117, Last page：128, May 2011, [Reviewed], [Lead]
  Phototropins (phot1 and phot2) are autophosphorylating blue-light receptor kinases that mediate blue-light responses such as phototropism, chloroplast accumulation, and stomatal opening in Arabidopsis (Arabidopsis thaliana). Only phot2 induces the chloroplast avoidance response under strong blue light. The serine (Ser) residues of the kinase activation loop in phot1 are autophosphorylated by blue light, and autophosphorylation is essential for the phot1-mediated responses. However, the role of autophosphorylation in phot2 remains to be determined. In this study, we substituted the conserved residues of Ser-761 and Ser-763 with alanine (S761A S763A) in the phot2 activation loop and analyzed their function by investigating the phot2-mediated responses after the transformation of phot1 phot2 double mutant with this mutant phot2 gene. Transgenic plants expressing the mutant phot2 protein exhibited impaired responses in chloroplast movement, stomatal opening, phototropic bending, leaf flattening, and plant growth; and those expressing phot2 with S761D S763D mutations showed the normal responses. Substitution of both Ser-761 and Ser-763 with alanine in phot2 did not significantly affect the kinase activity in planta. From these results, we conclude that phosphorylation of Ser-761 and Ser-763 in the activation loop may be a common primary step for phot2-mediated responses.
  AMER SOC PLANT BIOLOGISTS, English, Scientific journal
  DOI：https://doi.org/10.1104/pp.111.175943
  DOI ID：10.1104/pp.111.175943, ISSN：0032-0889, Web of Science ID：WOS:000290207100010
• Phototropin signaling and stomatal opening as a model case　　　　　　　　　　　　　　　
  Shin-ichiro Inoue; Atsushi Takemiya; Ken-ichiro Shimazaki
  CURRENT OPINION IN PLANT BIOLOGY, Volume：13, Number：5, First page：587, Last page：593, Oct. 2010, [Reviewed], [Invited], [Lead]
  Phototropins are plant-specific light-activated receptor kinases that regulate diverse blue-light-induced responses, and serve to optimize plant growth under various light environments. Phototropins undergo autophosphorylation as an essential step for their signaling and induce a variety of tissue-specific or organ-specific responses, but the divergent mechanisms for these responses are unknown. It is most likely that the phototropins generate a specific output after the event of autophosphorylation. In this report, we will review the common steps of phototropin signaling and the numerous interactive proteins of phototropins, which may act as signal transducers for the diverse responses. We also describe the phototropin-mediated signaling process of stomatal guard cells and its crosstalk with abscisic acid signaling.
  CURRENT BIOLOGY LTD, English, Scientific journal
  DOI：https://doi.org/10.1016/j.pbi.2010.09.002
  DOI ID：10.1016/j.pbi.2010.09.002, ISSN：1369-5266, Web of Science ID：WOS:000284658400016
• The Arabidopsis PHYTOCHROME KINASE SUBSTRATE2 Protein Is a Phototropin Signaling Element That Regulates Leaf Flattening and Leaf Positioning　　　　　　　　　　　　　　　
  Matthieu de Carbonnel; Phillip Davis; M. Rob G. Roelfsema; Shin-ichiro Inoue; Isabelle Schepens; Patricia Lariguet; Markus Geisler; Ken-ichiro Shimazaki; Roger Hangarter; Christian Fankhauser
  PLANT PHYSIOLOGY, Volume：152, Number：3, First page：1391, Last page：1405, Mar. 2010, [Reviewed]
  In Arabidopsis (Arabidopsis thaliana), the blue light photoreceptor phototropins (phot1 and phot2) fine-tune the photosynthetic status of the plant by controlling several important adaptive processes in response to environmental light variations. These processes include stem and petiole phototropism (leaf positioning), leaf flattening, stomatal opening, and chloroplast movements. The PHYTOCHROME KINASE SUBSTRATE (PKS) protein family comprises four members in Arabidopsis (PKS1-PKS4). PKS1 is a novel phot1 signaling element during phototropism, as it interacts with phot1 and the important signaling element NONPHOTOTROPIC HYPOCOTYL3 (NPH3) and is required for normal phot1-mediated phototropism. In this study, we have analyzed more globally the role of three PKS members (PKS1, PKS2, and PKS4). Systematic analysis of mutants reveals that PKS2 (and to a lesser extent PKS1) act in the same subset of phototropin-controlled responses as NPH3, namely leaf flattening and positioning. PKS1, PKS2, and NPH3 coimmunoprecipitate with both phot1-green fluorescent protein and phot2-green fluorescent protein in leaf extracts. Genetic experiments position PKS2 within phot1 and phot2 pathways controlling leaf positioning and leaf flattening, respectively. NPH3 can act in both phot1 and phot2 pathways, and synergistic interactions observed between pks2 and nph3 mutants suggest complementary roles of PKS2 and NPH3 during phototropin signaling. Finally, several observations further suggest that PKS2 may regulate leaf flattening and positioning by controlling auxin homeostasis. Together with previous findings, our results indicate that the PKS proteins represent an important family of phototropin signaling proteins.
  AMER SOC PLANT BIOLOGISTS, English, Scientific journal
  DOI：https://doi.org/10.1104/pp.109.150441
  DOI ID：10.1104/pp.109.150441, ISSN：0032-0889, Web of Science ID：WOS:000276329500023
• Blue light-induced autophosphorylation of phototropin is a primary step for signaling　　　　　　　　　　　　　　　
  Shin-ichiro Inoue; Toshinori Kinoshita; Masaki Matsumoto; Keiichi I. Nakayama; Michio Doi; Ken-ichiro Shimazaki
  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Volume：105, Number：14, First page：5626, Last page：5631, Apr. 2008, [Reviewed], [Lead]
  Phototropins are autophosphorylating protein kinases of plant-specific blue light receptors. They regulate various blue light responses, including phototropism, chloroplast movements, hypocotyl growth inhibition, leaf flattening, and stomatal opening. However, the physiological role of autophosphorylation remains unknown. Here, we identified phosphorylation sites of Ser or Thr in the N terminus, Hinge1 region, kinase domain, and C terminus in Arabidopsis phototropin1 (phot1) by liquid chromatography-tandem mass spectrometry in vivo. We substituted these Ser or Thr residues with Ala in phot1 and analyzed their functions by inspecting the phot1-mediated responses of stomatal opening, phototropism, chloroplast accumulation, and leaf flattening after the transformation of the phot1 phot2 double mutant. Among these sites, we found that auto phosphorylation of Ser-851 in the activation loop of the kinase domain was required for the responses mentioned above, whereas the phosphorylation of the other Ser and Thr, except those in the activation loop, was not. Ser-849 in the loop may have an additional role in the responses. Immunological analysis revealed that Ser-851 was phosphorylated rapidly by blue light in a fluence-dependent manner and dephosphorylated gradually upon darkness. We conclude that autophosphorylation of Ser-851 is a primary step that mediates signaling between photochemical reaction and physiological events.
  NATL ACAD SCIENCES, English, Scientific journal
  DOI：https://doi.org/10.1073/pnas.0709189105
  DOI ID：10.1073/pnas.0709189105, ISSN：0027-8424, Web of Science ID：WOS:000254893600061
• Leaf positioning of Arabidopsis in response to blue light　　　　　　　　　　　　　　　
  Shin-ichiro Inoue; Toshinori Kinoshita; Atsushi Takemiya; Michio Doi; Ken-ichiro Shimazaki
  MOLECULAR PLANT, Volume：1, Number：1, First page：15, Last page：26, Jan. 2008, [Reviewed], [Lead]
  Appropriate leaf positioning is essential for optimizing photosynthesis and plant growth. However, it has not been elucidated how green leaves reach and maintain their position for capturing light. We show here the regulation of leaf positioning under blue light stimuli. When 1-week-old Arabidopsis seedlings grown under white light were transferred to red light (25 mu mol m(-2) s(-1)) for 5 d, new petioles that appeared were almost horizontal and their leaves were curled and slanted downward. However, when a weak blue light from above (0.1 mu mol m(-2) s(-1)) was superimposed on red light, the new petioles grew obliquely upward and the leaves were flat and horizontal. The leaf positioning required both phototropin1 (phot1) and nonphototropic hypocotyl 3 (NPH3), and resulted in enhanced plant growth. In an nph3 mutant, neither optimal leaf positioning nor leaf flattening by blue light was found, and blue light-induced growth enhancement was drastically reduced. When blue light was increased from 0.1 to 5 mu mol m(-2) s(-1), normal leaf positioning and leaf flattening were induced in both phot1 and nph3 mutants, suggesting that phot2 signaling became functional and that the signaling was independent of phot1 and NPH3 in these responses. When plants were irradiated with blue light (0.1 mu mol m(-2) s(-1)) from the side and red light from above, the new leaves became oriented toward the source of blue light. When we transferred these plants to both blue light and red light from above, the leaf surface changed its orientation to the new blue light source within a few hours, whereas the petioles initially were unchanged but then gradually rotated, suggesting the plasticity of leaf positioning in response to blue light. We showed the tissue expression of NPH3 and its plasma membrane localization via the coiled-coil domain and the C-terminal region. We conclude that NPH3-mediated phototropin signaling optimizes the efficiency of light perception by inducing both optimal leaf positioning and leaf flattening, and enhances plant growth.
  OXFORD UNIV PRESS, English, Scientific journal
  DOI：https://doi.org/10.1093/mp/ssm001
  DOI ID：10.1093/mp/ssm001, ISSN：1674-2052, Web of Science ID：WOS:000259068900004
• Possible involvement of phototropins in leaf movement of kidney bean in response to blue light　　　　　　　　　　　　　　　
  S Inoue; T Kinoshita; K Shimazaki
  PLANT PHYSIOLOGY, Volume：138, Number：4, First page：1994, Last page：2004, Aug. 2005, [Reviewed], [Lead]
  The leaf of kidney bean ( Phaseolus vulgaris) moves in response to blue light. The movement is induced by a decrease in the turgor pressure of pulvinar motor cells on the irradiated side. In this study, we investigated the initial event of the movement with respect to function of phototropin and the plasma membrane H+-ATPase in the motor cells. The results indicated that, in dark conditions, phototropin existed in a dephosphorylated state and the H+-ATPase existed in a phosphorylated state. A pulse of blue light ( 30 s) induced the phosphorylation of phototropin and the dephosphorylation of the H+-ATPase as determined by the binding behavior of 14-3-3 protein. Phototropin phosphorylation occurred rapidly, followed by the transient gradual dephosphorylation of the H+-ATPase. When the specific flavoprotein inhibitor diphenyleneiodonium and the protein kinase inhibitors K-252a and staurosporine were administered to pulvinar cells, both phototropin phosphorylation and H+-ATPase dephosphorylation were inhibited. The phosphorylation and dephosphorylation exhibited similar fluence rate dependencies to blue light. These results indicated that phototropin may function upstream of the plasma membrane H+-ATPase and decrease the activity of H+-ATPase by dephosphorylation. We provide evidence for the existence of three kinds of phototropins in pulvinar motor cells.
  AMER SOC PLANT BIOLOGISTS, English, Scientific journal
  DOI：https://doi.org/10.1104/pp.105.062026
  DOI ID：10.1104/pp.105.062026, ISSN：0032-0889, Web of Science ID：WOS:000231206600018
• Biochemical characterization of plasma membrane H+-ATPase activation in guard cell protoplasts of Arabidopsis thaliana in response to blue light　　　　　　　　　　　　　　　
  K Ueno; T Kinoshita; S Inoue; T Emi; K Shimazaki
  PLANT AND CELL PHYSIOLOGY, Volume：46, Number：6, First page：955, Last page：963, Jun. 2005, [Reviewed]
  Recent genetic analysis showed that phototropins (phot1 and phot2) function as blue light receptors in stomatal opening of Arabidopsis thaliana, but no biochemical evidence was provided for this. We prepared a large quantity of guard cell protoplasts from Arabidopsis. The immunological method indicated that phot1 was present in guard cell protoplasts from the wild-type plant and the phot2 mutant, that phot2 was present in those from the wild-type plant and the phot1 mutant, and that neither phot1 nor phot2 was present in those from the phot1 pho2 double mutant. However, the same amounts of plasma membrane H+-ATPase were found in all of these plants. H+ pumping was induced by blue light in isolated guard cell protoplasts from the wild type, from the single mutants of phototropins (phot1-5 and phot2-1), and from the zeaxanthin-less mutant (npq1-2), but not from the phot1 phot2 double mutant. Moreover, increased ATP hydrolysis and the binding of 14-3-3 protein to the H+-ATPase were found in response to blue light in guard cell protoplasts from the wild type, but not from the phot1 phot2 double mutant. These results indicate that phot1 and phot2 mediate blue light-dependent activation of the plasma membrane H+-ATPase and illustrate that Arabidopsis guard cell protoplasts can be useful for biochemical analysis of stomatal functions. We determined isogenes of the plasma membrane H+-ATPase and found the expression of all isogenes of functional plasma membrane H+-ATPases (AHA1-11) in guard cell protoplasts.
  OXFORD UNIV PRESS, English, Scientific journal
  DOI：https://doi.org/10.1093/pcp/pci104
  DOI ID：10.1093/pcp/pci104, ISSN：0032-0781, Web of Science ID：WOS:000230090400015
• Phototropins promote plant growth in response to blue light in low light environments　　　　　　　　　　　　　　　
  A Takemiya; S Inoue; M Doi; T Kinoshita; K Shimazaki
  PLANT CELL, Volume：17, Number：4, First page：1120, Last page：1127, Apr. 2005, [Reviewed], [Lead]
  Phototropins (phot1 and phot2) are plant-specific blue light receptors for phototropism, chloroplast movement, leaf expansion, and stomatal opening. All these responses are thought to optimize photosynthesis by helping to capture light energy efficiently, reduce photodamage, and acquire CO2. However, experimental evidence for the promotion of plant growth through phototropins is lacking. Here, we report dramatic phototropin-dependent effects on plant growth. When plants of Arabidopsis thaliana wild type, the phot1 and phot2 mutants, and the photl phot2 double mutant were grown under red light, no significant growth differences were observed. However, if a very low intensity of blue light (0.1 mu mol m(-2) s(-1)) was superimposed on red light, large increases in fresh weight up to threefold were found in those plants that carried functional PHOT1 genes. When the intensity of blue light was increased to 1 mu mol m(-2) s(-1), the growth enhancement was also found in the photl single mutant, but not in the double mutant, indicating that phot2 mediated similar responses as photl with a lower sensitivity. The effects occurred under low photosynthetically active radiation in particular. The well-known physiological phototropin-mediated responses, including chloroplast movement, stomatal opening, and leaf expansion, in the different lines tested indicated an involvement of these responses in the blue light-induced growth enhancement. We conclude that phototropins promote plant growth by controlling and integrating a variety of responses that optimize photosynthetic performance under low photosynthetically active radiation in the natural environment.
  AMER SOC PLANT BIOLOGISTS, English, Scientific journal
  DOI：https://doi.org/10.1105/tpc.104.030049
  DOI ID：10.1105/tpc.104.030049, ISSN：1040-4651, eISSN：1532-298X, Web of Science ID：WOS:000228375900008

• シロイヌナズナにおける液胞へのマグネシウム輸送調節機構の解明　　　　　　　　　　　　　　　
  井上晋一郎; 林真妃; 黄勝; 横正健剛; 奥村将樹; 木下俊則; 木下俊則; 馬建鋒
  Volume：69, 2023
  ISSN：2424-0575, J-Global ID：202402270862154016
• 植物のMgホメオスタシス維持を調節するイオン輸送体とシグナル伝達機構の解明　　　　　　　　　　　　　　　
  井上晋一郎; 林真妃; HUANG Sheng; 横正健剛; 後藤栄治; 池松朱夏; 奥村将樹; 鈴木孝征; 木下俊則; 木下俊則; 馬建鋒
  Volume：17th (Web), 2023
  J-Global ID：202302232810339529
• 植物の光合成と蒸散を調節するMgホメオスタシス維持機構　　　　　　　　　　　　　　　
  井上晋一郎; 林真妃; 奥村将樹; 後藤栄治; 横正健剛; 馬建鋒; 馬建鋒; 木下俊則
  Volume：66, 2020
  ISSN：2424-0575, J-Global ID：202002261207887106
• オオセキショウモにおける青色光受容体フォトトロピンの同定と解析　　　　　　　　　　　　　　　
  酒井友希; 井上晋一郎; 原田明子; 島崎研一郎; 高木慎吾
  Volume：73rd, 2009
  J-Global ID：200902253834692241
• Blue light mediates normal leaf positioning in Arabidopsis　　　　　　　　　　　　　　　
  Shin-ichiro Inoue; Toshinori Kinoshita; Atsushi Takemiya; Michio Doi; Ken-ichiro Shirnazaki
  PLANT AND CELL PHYSIOLOGY, Volume：48, First page：S233, Last page：S233, 2007
  OXFORD UNIV PRESS, English, Summary international conference
  ISSN：0032-0781, Web of Science ID：WOS:000245922701408
• Phototropins may mediate leaf movement of kidney bean in response to blue light　　　　　　　　　　　　　　　
  S Inoue; T Kinoshita; K Shimazaki
  PLANT AND CELL PHYSIOLOGY, Volume：47, First page：S207, Last page：S207, 2006
  OXFORD UNIV PRESS, English, Summary international conference
  ISSN：0032-0781, Web of Science ID：WOS:000236401401313
• Characterization of the 14-3-3 protein binding to phot1 in Arabidopsis thaliana.　　　　　　　　　　　　　　　
  S Inoue; T Kinoshita; K Shimazaki
  PLANT AND CELL PHYSIOLOGY, Volume：46, First page：S167, Last page：S167, 2005
  OXFORD UNIV PRESS, English, Summary international conference
  ISSN：0032-0781, Web of Science ID：WOS:000228104101172
• Phototropins promote plant growth in low light environment　　　　　　　　　　　　　　　
  A Takemiya; S Inoue; M Doi; T Kinoshita; K Shimazaki
  PLANT AND CELL PHYSIOLOGY, Volume：46, First page：S86, Last page：S86, 2005
  OXFORD UNIV PRESS, English, Summary international conference
  ISSN：0032-0781, Web of Science ID：WOS:000228104100342
• Blue light-dependent binding of a 14-3-3 protein to phot1 in Arabidopsis thaliana.　　　　　　　　　　　　　　　
  S Inoue; T Kinoshita; K Shimazaki
  PLANT AND CELL PHYSIOLOGY, Volume：45, First page：S84, Last page：S84, 2004
  OXFORD UNIV PRESS, English, Summary international conference
  ISSN：0032-0781, Web of Science ID：WOS:000220592700333

• 化学と生物　　　　　　　　　　　　　　　
  井上 晋一郎, [Single work]
  Oct. 2024
• アグリバイオ　　　　　　　　　　　　　　　
  井上 晋一郎, [Single work]
  2023

• Jul. 2024 - Present
  Regulatory Biology, Saitama University
• Jun. 2024 - Present
  Laboratory Course in Regulatory Biology - Basic, Saitama University
• Apr. 2024 - Present
  Seminar of Adaptational Morphology, Saitama University
• Apr. 2024 - Present
  Seminar in Plant Physiology, Saitama University
• Apr. 2024 - Present
  Laboratory Course in Regulatory Biology, Saitama University
• 2021 - Present
  Seminars in Basic Biological Science I, Nagoya University
• 2022 - 2022
  modern life science, Nagoya University

• May 2023 - Present, Japan Transporter Research Association
• Sep. 2021 - Present
• 2020 - Present
• Sep. 2005 - Present
• Dec. 2002 - Present

• マグネシウムイオンによる新奇気孔開口誘導機構の解明　　　　　　　　　　　　　　　
  Nov. 2022 - Oct. 2024
  Principal investigator
• 植物の環境応答を調節する新規マグネシウム輸送体の同定と輸送制御機構の解明　　　　　　　　　　　　　　　
  01 Apr. 2022 - 31 Mar. 2024
  Grant amount(Total)：5720000, Direct funding：4400000, Indirect funding：1320000
  Grant number：22H04805
• 気孔の開口を駆動する細胞膜プロトンポンプの活性調節機構の解明　　　　　　　　　　　　　　　
  01 Apr. 2020 - 31 Mar. 2023
  Grant amount(Total)：4290000, Direct funding：3300000, Indirect funding：990000
  Grant number：20K06703
• Identification and functional characterization of protein kinases mediating blue light-dependent stomatal opening　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (C), 01 Apr. 2015 - 31 Mar. 2018
  Inoue Shin-ichiro; HAYASHI Maki; OKUMURA Masaki, Nagoya University
  Grant amount(Total)：5200000, Direct funding：4000000, Indirect funding：1200000
  Stomata open in response to blue light, and the opening is mediated by a phototropin-activated proton pump and a consequent potassium uptake in stomatal guard cells. However, signaling for the stomatal opening is not fully understood. In this study, we performed a protein-protein interaction screening and identified CIPK23 as a phototropin interactor. As the results of functional analyses of CIPK23, we found that CIPK23 mediates stomatal opening not through the proton pump activation but through the potassium channel activation in guard cells.
  Grant number：15K07101
• Stomatal response in crassulacean acid metabolism plants　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (C), 01 Apr. 2014 - 31 Mar. 2017
  Doi Michio, Kyushu University
  Grant amount(Total)：4940000, Direct funding：3800000, Indirect funding：1140000
  We investigated stomatal blue light (BL) response in obligate crassulacean acid metabolism (CAM) plants, Kalanchoe pinnata and Kalanchoe daigremontiana. The stomatal responses to BL were determined in both intact leaves and detached epidermis using dual-beam protocol to distinguish the BL-dependent stomatal opening from the photosynthesis-dependent stomatal opening. The stomata open in response to BL superimposed on the background red light (RL). The BL-dependent stomatal opening was completely inhibited by both an inhibitor of type 1 protein phosphatase and an inhibitor of the plasma membrane H+-ATPase. A fungous toxin fusicoccin (FC), an activator of the plasma membrane H+-ATPase induced stomatal opening in the dark. BL and FC caused both activation of the plasma membrane H+-ATPase in guard cells, and stomatal opening.
  These results indicate that BL-dependent stomatal opening operates in obligate CAM plants, regardless of the difference in the modes of photosynthesis.
  Grant number：26440150
• Functional analysis of protein kinase that interacts with blue light-receptor phototropin in stomatal opening　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Young Scientists (B), 01 Apr. 2013 - 31 Mar. 2015
  INOUE Shin-ichiro, Nagoya University
  Grant amount(Total)：4550000, Direct funding：3500000, Indirect funding：1050000
  Stomata in the plant epidermis open in response to blue light and contribute to plant growth by regulating photosynthesis and transpiration. A blue light-receptor phototropin mediates the stomatal opening via promotion of an active transport of K+ in stomatal guard cells. However, signaling for the stomatal opening is not fully understood. In this study, we performed functional analysis of a protein kinase that interacts with phototropin, and characterized roles of the protein kinase in the blue light signaling for stomatal opening.
  Grant number：25840105
• Molecular mechanism of stomatal movements in response to the environmental stimuli　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area), 01 Apr. 2010 - 31 Mar. 2015
  KINOSHITA Toshinori; TAKAHASHI Koji; INOUE Shin-ichiro, Nagoya University
  Grant amount(Total)：84110000, Direct funding：64700000, Indirect funding：19410000
  Stomata in the plant epidermis regulate gas exchange between plants and atmosphere. Stomatal aperture is regulates numerous environmental signals. Opening of stomata induces uptake of CO2 for photosynthesis and transpiration. In this study, we investigated signaling pathways for stomatal opening and closing by genetic, physiological, and biochemical methods. In addition, we examined effect of stomatal aperture in the transgenic plants on plant growth and drought resistance.
  Grant number：22119005
• The transduction of light signal to ion transport in stomatal guard cells.　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (S), 01 Apr. 2009 - 31 Mar. 2015
  SHIMAZAKI Ken-ichiro; KINOSHITA Toshinori; TAKEMIYA Atsushi; INOUE Shin-ichiro; TAKAHASHI Yohei, Kyushu University
  Grant amount(Total)：205920000, Direct funding：158400000, Indirect funding：47520000
  We studied phototropin-mediated signaling components in guard cells as a model case for plant light signaling. We showed that phosphatidic acid, a phospholipid second messenger produced by ABA, inhibited protein phosphatase 1 (PP1), a positive regulator of stomatal blue light signaling. The result suggests that PP1 is a crosstalk point between blue light and ABA signaling. We identified autophosphorylation sites in phot2 by mass analysis and showed the phosphorylation of Ser-761 and Ser-763 is required for the signaling. We found a novel protein kinase named BLUS1 as a substrate for phototropins and demonstrated phosphorylation of Ser-348 in response to blue light. We demonstrated that the phosphorylation of Ser-348 is essential for the signaling and that the Ser-348 is directly phosphorylated by phototropins. We showed the expression of 11 isoforms of the H+-ATPases in guard cells and identified the isoform responsible for blue light-dependent stomatal opening as AHA1.
  Grant number：21227001
• 気孔の開口を駆動する細胞膜プロトンポンプの活性制御機構の解明　　　　　　　　　　　　　　　
  2010 - 2012
  Grant amount(Total)：5000000, Direct funding：5000000
  Grant number：10J00254
• 青色光受容体フォトトロピンのリン酸化情報伝達機構の解明　　　　　　　　　　　　　　　
  2009 - 2010
  Grant amount(Total)：4160000, Direct funding：3200000, Indirect funding：960000
  Grant number：21770052