Faculty Profiles - MUNEMASA Shintaro

Papers - MUNEMASA Shintaro

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Involvement of reactive carbonyl species in inhibition of germination and seedling growth by salt stress in rice Invited

Faruk Hossain Khan, Yoshitaka Nakashima, Toshiyuki Nakamura, Yoshimasa Nakamura, Yoshihiko Hirai, Md Anamul Hoque, Mohammad Saidur Rhaman, Shintaro Munemasa, Jun'ichi Mano, Yoshiyuki Murata

Bioscience, Biotechnology, and Biochemistry 2025.5

Dual role of cytosolic GSH in the ABA signaling pathway and plasma membrane ion channel regulation in guard cells of Vicia faba. International journal

Huifei Yin, Toshiyuki Nakamura, Yoshimasa Nakamura, Shintaro Munemasa, Yoshiyuki Murata

Journal of plant physiology 306 154447 - 154447 2025.2

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

Abscisic acid (ABA) induces stomatal closure in higher plants under drought stress. Glutathione (GSH) negatively regulates ABA-induced stomatal closure and reactive carbonyl species (RCS) play a role as signal mediators downstream of reactive oxygen species production in ABA signaling pathway in Arabidopsis thaliana. Activation of slow (S-type) anion channels and inhibition of inward-rectifying potassium ion (Kin+) channels in the plasma membrane are essential for ABA-induced stomatal closure. However, there is limited evidence regarding role of GSH in the activation of S-type anion channels and the inhibition of Kin+ channels. We used Vicia faba to clarify the regulation of these ion channels by GSH and RCS. Pretreatment of guard-cell protoplasts with the GSH-supplementing agent, glutathione monoethyl ester (GSHmee), suppressed the activation of S-type anion channels and the inactivation of Kin+ channels induced by ABA. The pretreatment with the RCS scavenger carnosine suppressed the activation of S-type anion channels and the inactivation of Kin+ channels by ABA. On patch clamping guard-cell protoplasts, the addition of GSH to the pipette (cytosolic) buffer decreased the S-type anion currents and increased the Kin+ currents. These results suggest that cytosolic GSH is involved in ABA-induced stomatal closure via negative regulation of ABA signaling and via direct regulation of ion channel activities in V. faba.

DOI： 10.1016/j.jplph.2025.154447

TGG1 and TGG2 mutations impair allyl isothiocyanate-mediated stomatal closure in Arabidopsis thaliana. International journal

Kadri Oumaima, Mohammad Shakhawat Hossain, Wenxiu Ye, Eiji Okuma, Mohammad Issak, Mohammad Mahbub Islam, Misugi Uraji, Yoshimasa Nakamura, Izumi C Mori, Shintaro Munemasa, Yoshiyuki Murata

Protoplasma 2025.2

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

Myrosinase, referred to as thioglucoside glucohydrolase (TGG), plays a crucial role in plant physiology through catalyzing the hydrolysis of glucosinolates into bioactive isothiocyanates. In Arabidopsis thaliana, the myrosinases TGG1 and TGG2 are essential for abscisic acid- and methyl jasmonate-induced stomata closure. Allyl isothiocyanate (AITC), one of myrosinase products, triggers stomatal closure in A. thaliana. We investigated stomatal responses to AITC to clarify the role of TGG1 and TGG2 in Arabidopsis guard-cell signaling. Allyl isothiocyanate at 50 μM and 100 μM induced stomatal closure in the tgg1 and tgg2 single mutants but not in the tgg1 tgg2 double mutant. Furthermore, AITC at 50 μM induced the production of reactive oxygen species and nitric oxide, cytosolic alkalization, and oscillations in cytosolic free calcium concentration in guard cells of both wild-type and mutant plants. These findings suggest that TGG1 and TGG2 are involved in AITC signaling pathway through interaction with signal component(s) downstream of these signaling events, which is not accompanied by hydrolysis of glucosinolates because of the difference in subcellular localization between enzymes (myrosinases) and substrates (glucosinolates).

DOI： 10.1007/s00709-025-02039-z

Reactive carbonyl species function downstream of reactive oxygen species in chitosan-induced stomatal closure. International journal

Israt Jahan, Md Moshiul Islam, Toshiyuki Nakamura, Yoshimasa Nakamura, Shintaro Munemasa, Jun'ichi Mano, Yoshiyuki Murata

Physiologia plantarum 177 ( 1 ) e70094 2025

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

An elicitor, chitosan (CHT), induces stomatal closure in plants, which is accompanied by salicylhydroxamic acid (SHAM)-sensitive peroxidases-mediated reactive oxygen species (ROS) production in guard cells. Reactive carbonyl species (RCS) function downstream of ROS in abscisic acid (ABA) and methyl jasmonate (MeJA) signalling in guard cells. However, the involvement of RCS in CHT-induced stomatal closure is still unknown. In this study, we used transgenic tobacco (Nicotiana tabacum) plants overexpressing Arabidopsis thaliana 2-alkenal reductase (AER-OE tobacco) and Arabidopsis wild-type (WT) plants to investigate whether RCS is involved in CHT-induced stomatal closure. Chitosan-induced stomatal closure was inhibited in the tobacco AER-OE plants. In the WT tobacco and Arabidopsis plants, CHT-induced stomatal closure was inhibited by RCS scavengers, carnosine and pyridoxamine. Chitosan significantly increased RCS production in the WT tobacco and Arabidopsis, but in the tobacco AER-OE plants, chitosan did not increase significantly RCS accumulation. Moreover, neither the application of RCS scavengers to both WT plants nor scavenging RCS by AER-OE affected the CHT-induced ROS accumulation. However, treatment with a peroxidase inhibitor, SHAM, significantly inhibited CHT-induced RCS accumulation in WT tobacco and Arabidopsis plants. Taken together, these results suggest that RCS acts downstream of ROS production in CHT signalling in guard cells of A. thaliana and N. tabacum.

DOI： 10.1111/ppl.70094

GUARD CELL HYDROGEN PEROXIDE-RESISTANT1 functions upstream of reactive carbonyl species production in Arabidopsis guard-cell abscisic acid signaling Reviewed

Oumayma Shaiek, Huifei Yin, Nodoka Uesako, Md Moshiul Islam, Mohammad Saidur Rhaman, Toshiyuki Nakamura, Yoshimasa Nakamura, Shintaro Munemasa, Jun'ichi Mano, Yoshiyuki Murata

Bioscience, Biotechnology, and Biochemistry 88 ( 12 ) 1403 - 1410 2024.9

Publishing type：Research paper (scientific journal) Publisher：Oxford University Press (OUP)

Abstract

GUARD CELL HYDROGEN PEROXIDE-RESISTANT1 (GHR1), a leucine-rich repeat receptor-like kinase, is involved in abscisic acid (ABA)-induced stomatal closure. We investigated the role of GHR1 in reactive oxygen species (ROS) signaling for ABA-induced stomatal closure. ABA induced ROS production in wild type (WT) and the ghr1 of Arabidopsis thaliana. Hydrogen peroxide induced stomatal closure, accompanying the generation of acrolein in guard cells. The reactive carbonyl species (RCS) scavengers inhibited the ABA- and H2O2-induced stomatal closure in WT. In the ghr1, H2O2 failed to induce acrolein production and stomatal closure while RCS induced stomatal closure. Thus, GHR1 functions downstream of ROS and is required for the generation of RCS in guard-cell ABA signaling. In the ghr1, Ca2+ induced stomatal closure but RCS did not activate ICa channels. The GHR1 may be also involved in a Ca2+-independent pathway for ABA-induced stomatal closure.

DOI： 10.1093/bbb/zbae135

Other Link： https://academic.oup.com/bbb/article-pdf/88/12/1403/60785758/zbae135.pdf

Quercetin Attenuates Acetaldehyde-Induced Cytotoxicity via the Heme Oxygenase-1-Dependent Antioxidant Mechanism in Hepatocytes Reviewed

Kexin Li, Minori Kidawara, Qiguang Chen, Shintaro Munemasa, Yoshiyuki Murata, Toshiyuki Nakamura, Yoshimasa Nakamura

International Journal of Molecular Sciences 25 ( 16 ) 9038 - 9038 2024.8

Publishing type：Research paper (scientific journal) Publisher：MDPI AG

It is still unclear whether or how quercetin influences the toxic events induced by acetaldehyde in hepatocytes, though quercetin has been reported to mitigate alcohol-induced mouse liver injury. In this study, we evaluated the modulating effect of quercetin on the cytotoxicity induced by acetaldehyde in mouse hepatoma Hepa1c1c7 cells, the frequently used cellular hepatocyte model. The pretreatment with quercetin significantly inhibited the cytotoxicity induced by acetaldehyde. The treatment with quercetin itself had an ability to enhance the total ALDH activity, as well as the ALDH1A1 and ALDH3A1 gene expressions. The acetaldehyde treatment significantly enhanced the intracellular reactive oxygen species (ROS) level, whereas the quercetin pretreatment dose-dependently inhibited it. Accordingly, the treatment with quercetin itself significantly up-regulated the representative intracellular antioxidant-related gene expressions, including heme oxygenase-1 (HO-1), glutamate-cysteine ligase, catalytic subunit (GCLC), and cystine/glutamate exchanger (xCT), that coincided with the enhancement of the total intracellular glutathione (GSH) level. Tin protoporphyrin IX (SNPP), a typical HO-1 inhibitor, restored the quercetin-induced reduction in the intracellular ROS level, whereas buthionine sulphoximine, a representative GSH biosynthesis inhibitor, did not. SNPP also cancelled the quercetin-induced cytoprotection against acetaldehyde. These results suggest that the low-molecular-weight antioxidants produced by the HO-1 enzymatic reaction are mainly attributable to quercetin-induced cytoprotection.

DOI： 10.3390/ijms25169038

The Metabolite of γ-Tocopherol, 2,7,8-Trimethyl-2-(2′-Carboxyethyl)-6-Hydroxychroman, Exerts Intracellular Antioxidant Activity via Up-Regulation of Heme Oxygenase-1 in Hepatocytes Invited Reviewed

Shosuke Aoyama, Tomoka Nishio, Daiki Moriya, Shintaro Munemasa, Yoshiyuki Murata, Yoshimasa Nakamura, Toshiyuki Nakamura

Nutraceuticals 4 ( 3 ) 409 - 416 2024.8

Publishing type：Research paper (scientific journal) Publisher：MDPI AG

γ-Tocopherol (γT) is the major form of vitamin E contained in plants and seed oils. Although it is readily metabolized in the liver, the function of the metabolites is not fully understood. This study investigated the antioxidant activities of the γT metabolite 2,7,8-trimethyl-2-(2′-carboxyethyl)-6-hydroxychroman (γCEHC) in comparison to its parent compound. The pretreatment of mouse hepatoma Hepa1c1c7 cells with γCEHC showed a cytoprotective effect on the hydrogen peroxide-induced cytotoxicity to a lesser extent than that of γT. A mechanistic investigation revealed that both γ-CEHC and γT significantly up-regulated the gene and protein expressions of heme oxygenase-1 (HO-1) via the promotion of the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2). Furthermore, the combination of γCEHC and γT significantly increased the gene and protein levels of HO-1 and the nuclear translocation of Nrf2, suggesting that it was an additive effect. Tin protoporphyrin IX (SnPP), a representative HO-1 inhibitor, significantly impaired the cytoprotection of γCEHC and γT against the hydrogen peroxide-induced cytotoxicity. These results suggested that not only γT but also its metabolite, γCEHC, are a promising cytoprotective factor against oxidative stress-induced cytotoxicity and that the cytoprotective effect is attributable to the cooperation of both compounds.

DOI： 10.3390/nutraceuticals4030024

Outward-rectifying potassium channels GORK and SKOR function in regulation of root growth under salt stress in Arabidopsis thaliana. Reviewed International journal

Hafsa Jahan Hiya, Yoshitaka Nakashima, Airi Takeuchi, Toshiyuki Nakamura, Yoshimasa Nakamura, Yoshiyuki Murata, Shintaro Munemasa

Journal of plant physiology 302 154322 - 154322 2024.8

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

Plants often face high salinity as a significant environmental challenge with roots being the first responders to this stress. Maintaining K+/Na+ ratio within plant cells is crucial for survival, as the intracellular K+ level decreases and the intracellular Na+ level increases under saline conditions. However, knowledge about the molecular regulatory mechanisms of K+ loss in response to salt stress through outward-rectifying K+ channels in plants is largely unknown. In this study, we found that the Arabidopsis double mutant gorkskor, in which the GORK and SKOR genes are disrupted, showed an improved primary root growth under salt stress compared to wild-type (WT) and the gork and skor single-mutant plants. No significant differences in the sensitivity to mannitol stress between the WT and gorkskor mutant were observed. Accumulation of ROS induced by salt stress was reduced in the gorkskor roots. The gorkskor mutant seedlings had significantly higher K+ content, lower Na+ content, and a greater resultant K+/Na+ ratio than the WT under salt stress. Moreover, salt-stress-induced elevation of cytosolic free Ca2+ concentration was reduced in the gorkskor roots. Taken together, these results suggest that Arabidopsis Shaker-type outward-rectifying K+ channels GORK and SKOR may redundantly function in regulation of primary root growth under salt stress and are involved in not only the late-stage response (e.g. K+ leakage) but also the early response including ROS production and [Ca2+]cyt elevation.

DOI： 10.1016/j.jplph.2024.154322

Evaluation of quercetin as a potential cytoprotector against acetaldehyde using the cultured hepatocyte model with aldehyde dehydrogenase isozyme deficiency. International journal

Yuhang Xu, Takeshi Sawamoto, Ruitong Sun, Aki Ishikura, Shintaro Munemasa, Yoshiyuki Murata, Ayano Satoh, Akiko Matsumoto, Toshiyuki Nakamura, Yoshimasa Nakamura

Bioscience, biotechnology, and biochemistry 2024.7

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

Protective effect of quercetin against acetaldehyde was evaluated using the cultured hepatocyte models with aldehyde dehydrogenase (ALDH) isozyme deficiency (aldh2-kd and aldh1a1-kd). The quercetin-induced cytoprotection against acetaldehyde in the ALDH1A1-deficient mutant (aldh1a1-kd) was weaker than that in wild type. Furthermore, quercetin did not enhance the ALDH activity in aldh1a1-kd cells, suggesting that ALDH1A1 is involved in the quercetin-induced cytoprotection.

DOI： 10.1093/bbb/zbae100

Negative regulation of chitosan-induced stomatal closure by glutathione in Arabidopsis thaliana. Reviewed International journal

Israt Jahan, Shintaro Munemasa, Toshiyuki Nakamura, Yoshimasa Nakamura, Yoshiyuki Murata

Bioscience, biotechnology, and biochemistry 2024.5

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

Chitosan (CHT) is a deacylated derivative of chitin and improves growth and yield performance, activates defensive genes, and also induces stomatal closure in plants. Glutathione (GSH) has significant functions in the growth, development, defense systems, signaling, and gene expression. Glutathione negatively regulates abscisic acid (ABA)-, methyl jasmonate (MeJA)-, and salicylic acid (SA)-induced stomatal closure. However, the negative regulation by GSH of CHT-induced stomatal closure is still unknown. Regulation of CHT-induced stomatal closure by GSH in guard cells was investigated using two GSH-deficient mutants, cad2-1 and ch1-1, and a GSH-decreasing chemical, 1-chloro-2,4-dinitrobenzene (CDNB). The cad2-1 and ch1-1 mutations and CDNB treatment enhanced CHT-induced stomatal closure. Treatment with glutathione monoethyl ester (GSHmee) restored the GSH level in the guard cells of cad2-1 and ch1-1 and complemented the stomatal phenotype of the mutants. These results indicate that GSH negatively regulates CHT-induced stomatal closure in A. thaliana.

DOI： 10.1093/bbb/zbae065

Enhancing effect of the coexisting alpha-tocopherol on quercetin absorption and metabolism. International journal

Rikito Mitsuzane, Reiko Okubo, Miyu Nishikawa, Shinichi Ikushiro, Shintaro Munemasa, Yoshiyuki Murata, Yoshimasa Nakamura, Toshiyuki Nakamura

Free radical research 1 - 16 2024.2

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

The aim of this study is to investigate the modulating effect of coexisting food components on the absorption and metabolism of quercetin and blood plasma antioxidant potentials. The combination of quercetin with α-tocopherol (αT), cellulose, or a commercially-available vegetable beverage containing αT and dietary fiber was orally administered to mice. Compared to the single administration of quercetin aglycone, the co-administration of αT with quercetin significantly increased the plasma quercetin concentration at 0.5 h, whereas the combination of quercetin and cellulose decreased it. Interestingly, the administration of quercetin mixed with the vegetable beverage showed no significant change of the quercetin concentration in the mice plasma. The treatment of the cells with the blood plasma after the co-administration of αT with quercetin significantly upregulated the gene expression of the antioxidant enzyme (heme oxygenase-1), whereas the quercetin and cellulose combination did not. In the plasma of the quercetin-administered mice, eight types of quercetin metabolites were detected and their quantities were affected by the combination with αT. The potentials of the heme oxygenase-1 gene expression by these metabolites were very limited, although several metabolites showed radical scavenging activities comparable to aglycone in the in vitro assays. These results suggested that the combination of αT potentiates the quercetin absorption and metabolism and thus the plasma antioxidant potentials, at least in part, by the quantitative changes in the quercetin metabolites.

DOI： 10.1080/10715762.2024.2317206

An outward-rectifying plant K+ channel SPORK2 exhibits temperature-sensitive ion-transport activity. Reviewed International journal

Yuki Muraoka, Gangqiang Yang, Shintaro Munemasa, Yusuke Takeuchi, Yasuhiro Ishimaru, Yoshiyuki Murata, Nobuyuki Uozumi, Minoru Ueda

Current biology 33 5488 - 5494 2023.11

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

Temperature sensing is critical for the survival of living organisms.1,2 Thermosensitive transient receptor-potential (TRP) cation channels function as thermosensors in mammals.2,3,4,5,6 In contrast to animals, land plants lack TRP genes.7,8,9 Previous patch-clamp studies in plant cells suggested the presence of ion channels whose activities are related to temperature, implying the presence of TRP-like channels.10,11,12,13,14 However, the molecular entities of such temperature-sensitive ion channels were still unknown in land plants. In this study, we observed that the unique rainfall-induced leaf-folding movement of the legume tree Samanea saman15 was temperature-sensitive by using a rainfall-mimicking assay. Chilling-induced leaf folding in S. saman was shown to be related to the swelling of the motor cells16,17 at the base of the leaflet. This swelling suggested involvement of temperature-sensitive inactivation of K+ currents, independent of fluctuations in ion channel gene expression in motor cells. These findings led us to examine the temperature sensitivity of an outward-rectifying K+ channel, SPORK2, which was reported as an ion channel responsible for the nyctinastic (circadian-rhythmic) leaf movement of S. saman.18 We also discovered that SPORK2 exhibits temperature-sensitive K+ transport activity in the Xenopus oocyte expression system. Using chimeric channels, we showed that two domains of SPORK2 regulated the temperature sensitivity. Furthermore, heterologously expressed SPORK2 in Arabidopsis guard cells induced temperature-dependent stomatal closure. Therefore, SPORK2 is an ion channel in land plants with temperature-sensitive ion-transport activity that functions similarly to mammalian TRP channels. Our current findings advance the molecular understanding of temperature-sensing mechanisms in plants.

DOI： 10.1016/j.cub.2023.10.057

The effect of exogenous dihydroxyacetone and methylglyoxal on growth, anthocyanin accumulation, and the glyoxalase system in Arabidopsis. Reviewed International coauthorship International journal

Maoxiang Zhao, Toshiyuki Nakamura, Yoshimasa Nakamura, Shintaro Munemasa, Izumi C Mori, Yoshiyuki Murata

Bioscience, biotechnology, and biochemistry 87 1323 - 1331 2023.8

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

Dihydroxyacetone (DHA) occurs in wide-ranging organisms including plants and can undergo spontaneous conversion to methylglyoxal (MG). While the toxicity of MG to plants is well-known, the toxicity of DHA to plants remains to be elucidated. We investigated the effects of DHA and MG on Arabidopsis. Exogenous DHA at up to 10 m m did not affect the radicle emergence, the expansion of green cotyledons, the seedling growth, or the activity of glyoxalase II while DHA at 10 m m inhibited the root elongation and increased the activity of glyoxalase I. Exogenous MG at 1.0 m m inhibited these physiological responses and increased both activities. DHA at 10 m m increased the MG content in the roots. These results indicate that DHA is not so toxic as MG in Arabidopsis seeds and seedlings and suggest that the toxic effect of DHA at high concentrations is attributed to MG accumulation by the conversion to MG.

DOI： 10.1093/bbb/zbad109

Cycloartenyl Ferulate Is the Predominant Compound in Brown Rice Conferring Cytoprotective Potential against Oxidative Stress-Induced Cytotoxicity Reviewed International coauthorship International journal

Hongyan Wu, Toshiyuki Nakamura, Yingnan Guo, Riho Matsumoto, Shintaro Munemasa, Yoshiyuki Murata, Yoshimasa Nakamura

International Journal of Molecular Sciences 24 ( 1 ) 822 - 822 2023.1

Publishing type：Research paper (scientific journal) Publisher：MDPI AG

Since brown rice extract is a rich source of biologically active compounds, the present study is aimed to quantify the major compounds in brown rice and to compare their cytoprotective potential against oxidative stress. The content of the main hydrophobic compounds in brown rice followed the order of cycloartenyl ferulate (CAF) (89.00 ± 8.07 nmol/g) >> α-tocopherol (αT) (19.73 ± 2.28 nmol/g) > γ-tocotrienol (γT3) (18.24 ± 1.41 nmol/g) > α-tocotrienol (αT3) (16.02 ± 1.29 nmol/g) > γ-tocopherol (γT) (3.81 ± 0.40 nmol/g). However, the percent contribution of CAF to the radical scavenging activity of one gram of whole brown rice was similar to those of αT, αT3, and γT3 because of its weaker antioxidant activity. The CAF pretreatment displayed a significant cytoprotective effect on the hydrogen peroxide-induced cytotoxicity from 10 µM, which is lower than the minimal concentrations of αT and γT required for a significant protection. CAF also enhanced the nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation coincided with the enhancement of the heme oxygenase-1 (HO-1) mRNA level. An HO-1 inhibitor, tin protoporphyrin IX (SnPP), significantly impaired the cytoprotection of CAF. The cytoprotective potential of CAF is attributable to its cycloartenyl moiety besides the ferulyl moiety. These results suggested that CAF is the predominant cytoprotector in brown rice against hydrogen peroxide-induced cytotoxicity.

DOI： 10.3390/ijms24010822

The microbiota catabolites of quercetin glycosides concertedly enhance the resistance against acetaldehyde-induced oxidative stress. Reviewed International coauthorship International journal

Kexin Li, Hongyan Wu, Minori Kidawara, Yun Lin, Ayano Satoh, Gongliang Zhang, Shintaro Munemasa, Yoshiyuki Murata, Toshiyuki Nakamura, Yoshimasa Nakamura

Free radical research 56 1 - 14 2022.12

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

3,4-Dihydroxyphenylacetic acid (DOPAC) and 3-hydroxyphenylacetic acid (OPAC) are the predominant catabolites of quercetin glycosides, such as quercetin 4'-O-β-glucoside from the onion, produced by intestinal microbiota. Although each catabolite has been reported to protect the cells from acetaldehyde-induced cytotoxicity, the effect of their combination remains to be clarified. The purpose of this study was to determine whether the combination of DOPAC and OPAC enhances the resistance against the acetaldehyde-induced oxidative stress in the cultured hepatocytes. The pretreatment of the combination of DOPAC (5 μM) and OPAC (5 μM) showed the significant protection against the acetaldehyde- and hydrogen peroxide-induced cytotoxicity, even though each compound at the same concentration did not. This combination also significantly inhibited the intracellular dichlorofluorescin diacetate-detectable reactive oxygen species (ROS) level, whereas the solo treatment did slightly, suggesting that reducing mechanisms of ROS or compounds that enhance ROS production are involved in the cytoprotective effect. The combinatory treatment significantly enhanced the gene expression of not only the aldehyde dehydrogenases (ALDHs), but also glutamate-cysteine ligase, catalytic subunit, the first rate-limiting enzyme of glutathione (GSH) synthesis. Accordingly, both the intracellular GSH level and the total ALDH activity were enhanced by DOPAC plus OPAC. Involvement of GSH in the cytoprotection as well as ALDH up-regulation by the combination were confirmed by the experiments using a GSH biosynthesis inhibitor, buthionine sulfoximine. Taken together, the present results suggested that the quercetin microbiota catabolites concertedly protect the cells from acetaldehyde through a pre-enhanced resistance against oxidative stress by the GSH-dependent up-regulation of ALDHs.

DOI： 10.1080/10715762.2022.2159820

Extracellular malate induces stomatal closure via direct activation of guard‐cell anion channel SLAC1 and stimulation of Ca²⁺ signalling Reviewed International journal

Yoshiharu Mimata, Shintaro Munemasa, Toshiyuki Nakamura, Yoshimasa Nakamura, Yoshiyuki Murata

New Phytologist 236 ( 3 ) 852 - 863 2022.11

Malate induces stomatal closure via a receptor-like kinase GHR1- and reactive oxygen species-dependent pathway in Arabidopsis thaliana Reviewed International coauthorship International journal

Yoshiharu Mimata, Shintaro Munemasa, Fahmida Akter, Israt Jahan, Toshiyuki Nakamura, Yoshimasa Nakamura, Yoshiyuki Murata

Bioscience, Biotechnology, and Biochemistry 86 ( 10 ) 1362 - 1367 2022.9

Authorship：Corresponding author Publishing type：Research paper (scientific journal) Publisher：Oxford University Press (OUP)

ABSTRACT

A primary metabolite malate is secreted from guard cells in response to the phytohormone abscisic acid (ABA) and elevated CO2. The secreted malate subsequently facilitates stomatal closure in plants. Here, we investigated the molecular mechanism of malate-induced stomatal closure using inhibitors and ABA signaling component mutants of Arabidopsis thaliana. Malate-induced stomatal closure was impaired by a protein kinase inhibitor, K252a, and also by the disruption of a receptor-like kinase GHR1, which mediates activation of calcium ion (Ca2+) channel by reactive oxygen species (ROS) in guard cells. Malate induced ROS production in guard cells while the malate-induced stomatal closure was impaired by a peroxidase inhibitor, salicylhydroxamic acid, but not by the disruption of Nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases, RBOHD and RBOHF. The malate-induced stomatal closure was impaired by Ca2+ channel blockers, verapamil, and niflumic acid. These results demonstrate that the malate signaling is mediated by GHR1 and ROS in Arabidopsis guard cells.

DOI： 10.1093/bbb/zbac122

Other Link： https://academic.oup.com/bbb/article-pdf/86/10/1362/45978292/zbac122.pdf

Negative regulation of salicylic acid-induced stomatal closure by glutathione in Arabidopsis thaliana Reviewed International coauthorship International journal

Fahmida Akter, Shintaro Munemasa, Toshiyuki Nakamura, Yoshimasa Nakamura, Yoshiyuki Murata

Bioscience, Biotechnology, and Biochemistry 86 ( 10 ) 1378 - 1382 2022.9

Publishing type：Research paper (scientific journal) Publisher：Oxford University Press (OUP)

ABSTRACT

Salicylic acid (SA) is a ubiquitous phenolic phytohormone that induces stomatal closure. Glutathione (GSH) negatively regulates stomatal closure induced by other plant hormones such as abscisic acid (ABA) and methyl jasmonate (MeJA). However, the involvement of GSH in SA-induced stomatal closure is still unknown. We investigated the regulation of SA signaling by GSH in guard cells using an Arabidopsis thaliana mutant, cad2-1, which is deficient in the first GSH biosynthesis enzyme, γ-glutamylcysteine synthetase. Application of SA decreased stomatal apertures with decreasing intracellular GSH level in guard cells. Decreasing GSH by the cad2-1 mutation and by a GSH-decreasing chemical, 1-chloro-2,4-dinitrobenzene, enhanced the SA-induced stomatal closure. Treatment with glutathione monoethyl ester restored the GSH level in the cad2-1 guard cells and complemented the stomatal phenotype of the mutant. These results indicate that GSH negatively modulates SA-induced stomatal closure in A. thaliana.

DOI： 10.1093/bbb/zbac116

Other Link： https://academic.oup.com/bbb/article-pdf/86/10/1378/45978264/zbac116.pdf

Benzyl isothiocyanate and its metabolites inhibit cell proliferation through protein modification in mouse preosteoclast RAW264.7 cells. Reviewed International coauthorship International journal

Toshiyuki Nakamura, Chiharu Tsutsui, Yu Okuda, Naomi Abe-Kanoh, Saori Okazawa, Shintaro Munemasa, Yoshiyuki Murata, Yoji Kato, Yoshimasa Nakamura

Journal of biochemical and molecular toxicology e23184 2022.8

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

Benzyl isothiocyanate (BITC), derived from cruciferous vegetables, is an organosulfur compound exerting antiproliferative effects in several human cancer cells. In this study, we assessed BITC as a potential osteoclastogenesis inhibitor and investigated its underlying mechanism. BITC at 5 μM significantly decreased the viability of the osteoclast-like differentiating RAW264.7 cells, coinciding with the downregulation of the primary biomarkers for osteoclast differentiation, such as the tartrate-resistant acid phosphatase activity and nuclear factor of activated T-cells gene expression. Not only BITC but also its metabolites, inhibited cell proliferation in the normal RAW264.7 cells, suggesting that BITC shows an anti-osteoclastogenesis effect in vivo after its ingestion and metabolism, possibly through an antiproliferative action. Both BITC and its metabolites also enhanced the DNA fragmentation and the caspase-3 activity, whereas their higher concentrations tended to suppress these effects. BITC was intracellularly accumulated when the cells were treated with its metabolites via their degradation into the free form. A quantitative experiment using the proteolysis/high performance liquid chromatography technique showed that the amount of BITC-lysine thiourea in the cells was also increased in a time-dependent manner, suggesting that lysine modification of the cellular proteins actually took place in the cells treated by BITC. Among the cellular proteins, the cleaved caspase-3 was identified as a potential target for lysine modification by BITC. Taken together, BITC dissociated from its metabolites as well as its free form might modulate osteoclastogenesis, possibly through inhibition of cell proliferation by protein modification.

DOI： 10.1002/jbt.23184

Green Tea Catechins, (-)-Catechin Gallate, and (-)-Gallocatechin Gallate are Potent Inhibitors of ABA-Induced Stomatal Closure. Reviewed International coauthorship International journal

Kanane Sato, Shunya Saito, Kohsuke Endo, Masaru Kono, Taishin Kakei, Haruka Taketa, Megumi Kato, Shin Hamamoto, Matteo Grenzi, Alex Costa, Shintaro Munemasa, Yoshiyuki Murata, Yasuhiro Ishimaru, Nobuyuki Uozumi

Advanced science 9 ( 21 ) e2201403 2022.5

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

Stomatal movement is indispensable for plant growth and survival in response to environmental stimuli. Cytosolic Ca2+ elevation plays a crucial role in ABA-induced stomatal closure during drought stress; however, to what extent the Ca2+ movement across the plasma membrane from the apoplast to the cytosol contributes to this process still needs clarification. Here the authors identify (-)-catechin gallate (CG) and (-)-gallocatechin gallate (GCG), components of green tea, as inhibitors of voltage-dependent K+ channels which regulate K+ fluxes in Arabidopsis thaliana guard cells. In Arabidopsis guard cells CG/GCG prevent ABA-induced: i) membrane depolarization; ii) activation of Ca2+ permeable cation (ICa ) channels; and iii) cytosolic Ca2+ transients. In whole Arabidopsis plants co-treatment with CG/GCG and ABA suppressed ABA-induced stomatal closure and surface temperature increase. Similar to ABA, CG/GCG inhibited stomatal closure is elicited by the elicitor peptide, flg22 but has no impact on dark-induced stomatal closure or light- and fusicoccin-induced stomatal opening, suggesting that the inhibitory effect of CG/GCG is associated with Ca2+ -related signaling pathways. This study further supports the crucial role of ICa channels of the plasma membrane in ABA-induced stomatal closure. Moreover, CG and GCG represent a new tool for the study of abiotic or biotic stress-induced signal transduction pathways.

DOI： 10.1002/advs.202201403

Plant hormone regulation of abiotic stress responses Reviewed International coauthorship International journal

Rainer Waadt, Charles A. Seller, Po-Kai Hsu, Yohei Takahashi, Shintaro Munemasa, Julian I. Schroeder

Nature Reviews Molecular Cell Biology 2022.5

Phytocytokine signalling reopens stomata in plant immunity and water loss Reviewed International coauthorship International journal

Zunyong Liu, Shuguo Hou, Olivier Rodrigues, Ping Wang, Dexian Luo, Shintaro Munemasa, Jiaxin Lei, Jun Liu, Fausto Andres Ortiz-Morea, Xin Wang, Kinya Nomura, Chuanchun Yin, Hongbo Wang, Wei Zhang, Keyan Zhu-Salzman, Sheng Yang He, Ping He, Libo Shan

Nature 2022.5

A Major Intestinal Catabolite of Quercetin Glycosides, 3-Hydroxyphenylacetic Acid, Protects the Hepatocytes from the Acetaldehyde-Induced Cytotoxicity through the Enhancement of the Total Aldehyde Dehydrogenase Activity Reviewed International journal

Yujia Liu, Takumi Myojin, Kexin Li, Ayuki Kurita, Masayuki Seto, Ayano Motoyama, Xiaoyang Liu, Ayano SATOH, Shintaro Munemasa, Yoshiyuki Murata, Toshiyuki Nakamura, Yoshimasa Nakamura

International Journal of Molecular Sciences 23 ( 3 ) 2022.2

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

Aldehyde dehydrogenases (ALDHs) are the major enzyme superfamily for the aldehyde metabolism. Since the ALDH polymorphism leads to the accumulation of acetaldehyde, we considered that the enhancement of the liver ALDH activity by certain food ingredients could help prevent alcohol-induced chronic diseases. Here, we evaluated the modulating effects of 3-hydroxyphenylacetic acid (OPAC), the major metabolite of quercetin glycosides, on the ALDH activity and acetaldehyde-induced cytotoxicity in the cultured cell models. OPAC significantly enhanced the total ALDH activity not only in mouse hepatoma Hepa1c1c7 cells, but also in human hepatoma HepG2 cells. OPAC significantly increased not only the nuclear level of aryl hydrocarbon receptor (AhR), but also the AhR-dependent reporter gene expression, though not the nuclear factor erythroid-2-related factor 2 (Nrf2)-dependent one. The pretreatment of OPAC at the concentration required for the ALDH upregulation completely inhibited the acetaldehyde-induced cytotoxicity. Silencing AhR impaired the resistant effect of OPAC against acetaldehyde. These results strongly suggested that OPAC protects the cells from the acetaldehyde-induced cytotoxicity, mainly through the AhR-dependent and Nrf2-independent enhancement of the total ALDH activity. Our findings suggest that OPAC has a protective potential in hepatocyte models and could offer a new preventive possibility of quercetin glycosides for targeting alcohol-induced chronic diseases.

DOI： 10.3390/ijms23031762

Exogenous Oxalic Acid Protects Germinating Chickpea Seeds Against Cadmium Injury Reviewed International coauthorship International journal

Lamia Sakouhi, Oussama Kharbech, Marouane Ben Massoud, Shintaro Munemasa, Yoshiyuki Murata, Abdelilah Chaoui

Journal of Soil Science and Plant Nutrition 2021.11

Neither glutamate nor alanine but arginine sensitizes BY-2 cells to arsenate Reviewed International coauthorship International journal

Nur-E-Nazmun Nahar, Md Yeasin Prodhan, Yoshiharu Mimata, Anna Yonezawa, Toshiyuki Nakamura, Yoshimasa Nakamura, Shintaro Munemasa, Yoshiyuki Murata

Bioscience, Biotechnology, and Biochemistry 2021.10

Language：English Publishing type：Research paper (scientific journal) Publisher：Oxford University Press (OUP)

<title>ABSTRACT</title>
Arsenic is toxic for plants. Our previous results showed that the application of proline enhanced the sensitivity of tobacco BY-2 cells to arsenate. In order to clarify the enhancement mechanism, we investigated the effects of other amino acids on the arsenate-stressed BY-2 cells. Glutamate at up to 10 m m did not affect the cell growth in the absence or presence of arsenate. Arginine at up to 10 m m did not affect the growth in the absence of arsenate but arginine at 10 m m enhanced the inhibition of the cell growth by arsenate. Alanine at up to 10 m m did not affect the cell growth under nonstressed condition but alanine at 10 m m significantly improved the cell growth under arsenate stress. These results suggest that alanine mitigates arsenate stress in BY-2 cells and that arginine like proline enhances the sensitivity of BY-2 cells to arsenate.

DOI： 10.1093/bbb/zbab191

White rice ethanol extract is qualitatively, but not quantitatively, equivalent to that of brown rice as an antioxidant source Reviewed International coauthorship International journal

Hongyan Wu, Toshiyuki Nakamura, Yingnan Guo, Miho Hirooka, Gongliang Zhang, Shintaro Munemasa, Yoshiyuki Murata, Akiko Fujita, Yoshimasa Nakamura

Bioscience, Biotechnology, and Biochemistry 85 ( 10 ) 2161 - 2168 2021.9

Language：English Publishing type：Research paper (scientific journal) Publisher：Oxford University Press (OUP)

<title>ABSTRACT</title>
The purpose of this study is to compare the potentials to exhibit biologically active antioxidant actions between white rice (WR) and brown rice (BR) in in vitro assays and a cellular model. The Trolox equivalent (TE) per 1 mg ethanol extract of WR for the 1,1-diphenyl-2-picrylhydrazyl assay was slightly higher than that of BR, whereas the TE per 1 g whole WR was much lower than that for BR. This tendency was very comparable to those for the oxygen radical absorbance capacity and total polyphenol content. Both of the ethanol extracts also similarly suppressed the hydrogen peroxide-induced cytotoxicity and enhanced the gene expression of drug-metabolizing enzymes. Based on the α-tocopherol quantity, its contribution to the cytoprotective effect of the rice extracts is very limited. Taken together, the ethanol extract of WR might be a qualitatively, but not quantitatively, equivalent source of antioxidative phytochemicals to that of BR.

DOI： 10.1093/bbb/zbab133

Other Link： http://academic.oup.com/bbb/article-pdf/85/10/2161/40430235/zbab133.pdf

Modulation of frequency and height of cytosolic calcium spikes by plasma membrane anion channels in guard cells Reviewed International coauthorship International journal

Md Tahjib-Ul-Arif, Shintaro Munemasa, Toshiyuki Nakamura, Yoshimasa Nakamura, Yoshiyuki Murata

Bioscience, Biotechnology, and Biochemistry 85 ( 9 ) 2003 - 2010 2021.8

Language：English Publishing type：Research paper (scientific journal) Publisher：Oxford University Press (OUP)

<title>ABSTRACT</title>
Cytosolic calcium ([Ca2+]cyt) elevation activates plasma membrane anion channels in guard cells, which is required for stomatal closure. However, involvement of the anion channels in the [Ca2+]cyt elevation remains unclear. We investigated the involvement using Arabidopsis thaliana anion channel mutants, slac1-4 slah3-3 and slac1-4 almt12-1. Extracellular calcium induced stomatal closure in the wild-type plants but not in the anion channel mutant plants whereas extracellular calcium induced [Ca2+]cyt elevation both in the wild-type guard cells and in the mutant guard cells. The peak height and the number of the [Ca2+]cyt spike were lower and larger in the slac1-4 slah3-3 than in the wild type and the height and the number in the slac1-4 almt12-1 were much lower and much larger than in the wild type. These results suggest that the anion channels are involved in the regulation of [Ca2+]cyt elevation in guard cells.

DOI： 10.1093/bbb/zbab118

Other Link： http://academic.oup.com/bbb/article-pdf/85/9/2003/39891204/zbab118.pdf

Calcium and ethylene glycol tetraacetic acid mitigate toxicity and alteration of gene expression associated with cadmium stress in chickpea (Cicer arietinum L.) shoots Reviewed International coauthorship International journal

Lamia Sakouhi, Oussama Kharbech, Marouane Ben Massoud, Charfeddine Gharsallah, Sihem Ben Hassine, Shintaro Munemasa, Yoshiyuki Murata, Abdelilah Chaoui

Protoplasma 258 ( 4 ) 849 - 861 2021.7

Language：English Publishing type：Research paper (scientific journal) Publisher：Springer Science and Business Media LLC

In the aim to estimate the protective role of calcium (Ca) and ethylene glycol tetraacetic acid (EGTA) against cadmium (Cd)-induced damage, chickpea (Cicer arietinum L.) seeds were exposed to 200 μM Cd stress for 6 days or 3 days then subjected to co-treatment of the metal with either 100 mM CaCl2 or 100 μM EGTA for 3 additional days. The addition of Ca and EGTA improved seedling growth. This protecting effect was correlated to the alleviation of the metal-induced oxidative stress, exemplified by the reduction of hydrogen peroxide (H2O2) contents. Besides, Ca and EGTA stimulated thioredoxin (Trx) and thioredoxin reductase (NTR) activities (2.75- and 1.75-fold increase when compared to Cd-stressed, respectively) protecting, thereby, protein -SH groups from the Cd-mediated oxidation, and modulated ferredoxin (Fdx) activity to a control level. Moreover, Ca and EGTA reinstated the glutathione redox steady state, mainly via preserving a high level of glutathione reduced form (GSH). This effect coincided with the maintaining of the Cd-stimulated glutathione reductase (GR) activity and the decline of glutathione peroxidase (GPX, 43% lower than Cd-stressed shoots) activity. Ca and EGTA counteracted the inhibitory effect of Cd on the activity and gene expression of Cu/Zn-superoxide dismutase (Cu/Zn-SOD) isoenzyme and modulated the activities of catalase (CAT) and ascorbate peroxidase (APX). Overall, our results provided evidence that Ca and EGTA supplement could be a promising approach in the remediation of Cd-contaminated environment.

DOI： 10.1007/s00709-020-01605-x

Other Link： https://link.springer.com/article/10.1007/s00709-020-01605-x/fulltext.html

A multidrug resistance‐associated protein inhibitor is a potential enhancer of the benzyl isothiocyanate‐induced apoptosis induction in human colorectal cancer cells Reviewed International coauthorship International journal

Qifu Yang, Toshiyuki Nakamura, Masayuki Seto, Miku Miyagawa, Wensi Xu, Beiwei Zhu, Shintaro Munemasa, Yoshiyuki Murata, Yoshimasa Nakamura

Journal of Biochemical and Molecular Toxicology 35 ( 7 ) 2021.7

A role for calcium‐dependent protein kinases in differential CO2‐ and ABA‐controlled stomatal closing and low CO 2 ‐induced stomatal opening in Arabidopsis Reviewed International coauthorship International journal

Sebastian Schulze, Guillaume Dubeaux, Paulo H. O. Ceciliato, Shintaro Munemasa, Maris Nuhkat, Dmitry Yarmolinsky, Jaimee Aguilar, Renee Diaz, Tamar Azoulay‐Shemer, Leonie Steinhorst, Jan Niklas Offenborn, Jörg Kudla, Hannes Kollist, Julian I. Schroeder

New Phytologist 229 ( 5 ) 2765 - 2779 2021.3

Oxalic Acid Mitigates Cadmium Toxicity in Cicer arietinum L. Germinating Seeds by Maintaining the Cellular Redox Homeostasis Reviewed International coauthorship International journal

Lamia Sakouhi, Oussama Kharbech, Marouane Ben Massoud, Shintaro Munemasa, Yoshiyuki Murata, Abdelilah Chaoui

Journal of Plant Growth Regulation 2021.2

INVOLVEMENT OF GLUTATHIONE IN ABSCISIC ACID SIGNALING AND METHYL JASMONATE SIGNALING IN GUARD CELLS

Akter FAHMIDA, Shintaro MUNEMASA, Yoshiyuki MURATA

Journal of Environmental Science for Sustainable Society 10 ( Supplement ) MR03_p9 - MR03_p12 2021

ELEVATION OF CYTOSOLIC CALCIUM IN GUARD CELLS

Md. TAHJIB-UL-ARIF, Shintaro MUNEMASA, Yoshiyuki MURATA

Journal of Environmental Science for Sustainable Society 10 ( Supplement ) MR02_p5 - MR02_p8 2021

Stomatal response to isothiocyanates in Arabidopsis thaliana Reviewed International coauthorship International journal

Sonya Afrin, Eiji Okuma, Md Tahjib-Ul-Arif, Md Sarwar Jahan, Toshiyuki Nakamura, Yoshimasa Nakamura, Shintaro Munemasa, Yoshiyuki Murata

Journal of Experimental Botany 71 ( 22 ) 6921 - 6931 2020.12

Language：English Publishing type：Research paper (scientific journal) Publisher：Oxford University Press (OUP)

<title>Abstract</title>Allyl isothiocyanate (AITC) induces stomatal closure accompanied by reactive oxygen species (ROS) production and glutathione (GSH) depletion in Arabidopsis thaliana. In this study, stomatal responses to three other isothiocyanates (ITCs), benzyl isothiocyanate (BITC), sulforaphane (SFN), and phenethyl isothiocyanate (PEITC), were investigated in A. thaliana. All these ITCs significantly induced stomatal closure, where PEITC and BITC were most effective. The selected ITCs also induced ROS accumulation, cytosolic alkalization, and GSH depletion in guard cells. Moreover, all ITCs increased the frequency of cytosolic free calcium ([Ca2+]cyt) spikes (transient elevation), while PEITC and BITC showed the highest frequency. There was a strong positive correlation between the number of [Ca2+]cyt spikes per guard cell and the decrease in stomatal aperture. Both cytosolic alkalization and GSH content have a positive correlation with the decrease in stomatal aperture, but ROS production did not have a significant correlation with the decrease in stomatal apertures. These results indicate that the molecules with a functional ITC group induce stomatal closure that is accompanied by GSH depletion, cytosolic alkalization, [Ca2+]cyt spikes, and ROS production, and that the former three cellular events, rather than ROS production, are highly correlated with the decrease in stomatal aperture.

DOI： 10.1093/jxb/eraa420

Other Link： http://academic.oup.com/jxb/article-pdf/71/22/6921/35332877/eraa420.pdf

MAP3Kinase-dependent SnRK2-kinase activation is required for abscisic acid signal transduction and rapid osmotic stress response Reviewed International coauthorship International journal

Yohei Takahashi, Jingbo Zhang, Po-Kai Hsu, Paulo H. O. Ceciliato, Li Zhang, Guillaume Dubeaux, Shintaro Munemasa, Chennan Ge, Yunde Zhao, Felix Hauser, Julian I. Schroeder

Nature Communications 11 ( 1 ) 12 2020.12

Language：English Publishing type：Research paper (scientific journal) Publisher：Springer Science and Business Media LLC

<title>Abstract</title>Abiotic stresses, including drought and salinity, trigger a complex osmotic-stress and abscisic acid (ABA) signal transduction network. The core ABA signalling components are snf1-related protein kinase2s (SnRK2s), which are activated by ABA-triggered inhibition of type-2C protein-phosphatases (PP2Cs). SnRK2 kinases are also activated by a rapid, largely unknown, ABA-independent osmotic-stress signalling pathway. Here, through a combination of a redundancy-circumventing genetic screen and biochemical analyses, we have identified functionally-redundant MAPKK-kinases (M3Ks) that are necessary for activation of SnRK2 kinases. These M3Ks phosphorylate a specific SnRK2/OST1 site, which is indispensable for ABA-induced reactivation of PP2C-dephosphorylated SnRK2 kinases. ABA-triggered SnRK2 activation, transcription factor phosphorylation and SLAC1 activation require these M3Ks in vitro and in plants. M3K triple knock-out plants show reduced ABA sensitivity and strongly impaired rapid osmotic-stress-induced SnRK2 activation. These findings demonstrate that this M3K clade is required for ABA- and osmotic-stress-activation of SnRK2 kinases, enabling robust ABA and osmotic stress signal transduction.

DOI： 10.1038/s41467-019-13875-y

Other Link： http://www.nature.com/articles/s41467-019-13875-y

Exogenous proline enhances antioxidant enzyme activities but does not mitigate growth inhibition by selenate stress in tobacco BY-2 cells Reviewed International coauthorship International journal

Mousumi Khatun, Daiki Matsushima, Mohammad Saidur Rhaman, Eiji Okuma, Toshiyuki Nakamura, Yoshimasa Nakamura, Shintaro Munemasa, Yoshiyuki Murata

Bioscience, Biotechnology, and Biochemistry 84 ( 11 ) 2281 - 2292 2020.11

Language：English Publishing type：Research paper (scientific journal) Publisher：Oxford University Press (OUP)

<title>Abstract</title>
Selenium (Se) causes oxidative damage to plants. Proline is accumulated as a compatible solute in plants under stress conditions and mitigates stresses. Selenate at 250 µM increased cell death and inhibited the growth of tobacco BY-2 cells while exogenous proline at 10 mM did not mitigate the inhibition by selenate. Selenate increased accumulation of Se and ROS and activities of antioxidant enzymes but not lipid peroxidation in the BY-2 cells. Proline increased Se accumulation and antioxidant enzyme activities but not either ROS accumulation or lipid peroxidation in the selenate-stressed cells. Glutathione (GSH) rather than ascorbic acid (AsA) mitigated the growth inhibition although both reduced the accumulation of ROS induced by selenate. These results indicate that proline increases both antioxidant enzyme activities and Se accumulation, which overall fails to ameliorate the growth inhibition by selenate and that the growth inhibition is not accounted for only by ROS accumulation.

Abbreviations: APX: ascorbate peroxidase; AsA: ascorbic acid; BY-2: Bright Yellow-2; CAT: catalase; DAI: days after inoculation; DW: dry weight; FW: fresh weight; GSH: glutathione; ROS: reactive oxygen species

DOI： 10.1080/09168451.2020.1799747

Reactive Carbonyl Species Mediate Methyl Jasmonate-Induced Stomatal Closure Reviewed International coauthorship International journal

Md. Moshiul Islam, Wenxiu Ye, Fahmida Akter, Mohammad Saidur Rhaman, Daiki Matsushima, Shintaro Munemasa, Eiji Okuma, Yoshimasa Nakamura, Md. Sanaullah Biswas, Jun’ichi Mano, Yoshiyuki Murata

Plant and Cell Physiology 61 ( 10 ) 1788 - 1797 2020.10

Language：English Publishing type：Research paper (scientific journal) Publisher：Oxford University Press (OUP)

<title>Abstract</title>
Production of reactive oxygen species (ROS) is a key signal event for methyl jasmonate (MeJA)- and abscisic acid (ABA)-induced stomatal closure. We recently showed that reactive carbonyl species (RCS) stimulates stomatal closure as an intermediate downstream of hydrogen peroxide (H2O2) production in the ABA signaling pathway in guard cells of Nicotiana tabacum and Arabidopsis thaliana. In this study, we examined whether RCS functions as an intermediate downstream of H2O2 production in MeJA signaling in guard cells using transgenic tobacco plants overexpressing A. thaliana 2-alkenal reductase (n-alkanal + NAD(P)+ ⇌ 2-alkenal + NAD(P)H + H+) (AER-OE tobacco) and Arabidopsis plants. The stomatal closure induced by MeJA was impaired in the AER-OE tobacco and was inhibited by RCS scavengers, carnosine and pyridoxamine, in the wild-type (WT) tobacco plants and Arabidopsis plants. Application of MeJA significantly induced the accumulation of RCS, including acrolein and 4-hydroxy-(E)-2-nonenal, in the WT tobacco but not in the AER-OE plants. Application of MeJA induced H2O2 production in the WT tobacco and the AER-OE plants and the H2O2 production was not inhibited by the RCS scavengers. These results suggest that RCS functions as an intermediate downstream of ROS production in MeJA signaling and in ABA signaling in guard cells.

DOI： 10.1093/pcp/pcaa107

Other Link： http://academic.oup.com/pcp/article-pdf/61/10/1788/34161860/pcaa107.pdf

Stomatal immunity against fungal invasion comprises not only chitin-induced stomatal closure but also chitosan-induced guard cell death Reviewed International coauthorship International journal

Wenxiu Ye, Shintaro Munemasa, Tomonori Shinya, Wei Wu, Tao Ma, Jiang Lu, Toshinori Kinoshita, Hanae Kaku, Naoto Shibuya, Yoshiyuki Murata

Proceedings of the National Academy of Sciences of the United States of America 117 ( 34 ) 20932 - 20942 2020.8

Language：English Publishing type：Research paper (scientific journal) Publisher：Proceedings of the National Academy of Sciences

Many pathogenic fungi exploit stomata as invasion routes, causing destructive diseases of major cereal crops. Intensive interaction is expected to occur between guard cells and fungi. In the present study, we took advantage of well-conserved molecules derived from the fungal cell wall, chitin oligosaccharide (CTOS), and chitosan oligosaccharide (CSOS) to study how guard cells respond to fungal invasion. In<italic>Arabidopsis</italic>, CTOS induced stomatal closure through a signaling mediated by its receptor CERK1, Ca²⁺, and a major S-type anion channel, SLAC1. CSOS, which is converted from CTOS by chitin deacetylases from invading fungi, did not induce stomatal closure, suggesting that this conversion is a fungal strategy to evade stomatal closure. At higher concentrations, CSOS but not CTOS induced guard cell death in a manner dependent on Ca²⁺but not CERK1. These results suggest that stomatal immunity against fungal invasion comprises not only CTOS-induced stomatal closure but also CSOS-induced guard cell death.

DOI： 10.1073/pnas.1922319117

Interaction of intracellular hydrogen peroxide accumulation with nitric oxide production in abscisic acid signaling in guard cells Reviewed International coauthorship International journal

Rayhanur Jannat, Takanori Senba, Daichi Muroyama, Misugi Uraji, Mohammad Anowar Hossain, Mohammad Muzahidul Islam, Yoshimasa Nakamura, Shintaro Munemasa, Izumi C Mori, Yoshiyuki Murata

Bioscience, Biotechnology, and Biochemistry 84 ( 7 ) 1418 - 1426 2020.7

Language：English Publishing type：Research paper (scientific journal) Publisher：Oxford University Press (OUP)

<title>Abstract</title>
<sec>
<title />
Reactive oxygen species and nitric oxide (NO•) concomitantly play essential roles in guard cell signaling. Studies using catalase mutants have revealed that the inducible and constitutive elevations of intracellular hydrogen peroxide (H2O2) have different roles: only the inducible H2O2 production transduces the abscisic acid (ABA) signal leading stomatal closure. However, the involvement of inducible or constitutive NO• productions, if exists, in this process remains unknown. We studied H2O2 and NO• mobilization in guard cells of catalase mutants. Constitutive H2O2 level was higher in the mutants than that in wild type, but constitutive NO• level was not different among lines. Induced NO• and H2O2 levels elicited by ABA showed a high correlation with each other in all lines. Furthermore, NO• levels increased by exogenous H2O2 also showed a high correlation with stomatal aperture size. Our results demonstrate that ABA-induced intracellular H2O2 accumulation triggers NO• production leading stomatal closure.

</sec>
<sec>
<title>Abbreviations</title>
ABA: abscisic acid; CAT: catalase; cGMP: cyclic guanosine monophosphate; DAF-2DA: 4,5-diaminofluorescein-2 diacetate; H2DCF-DA: 2ʹ,7ʹ-dichlorodihydrofluorescein diacetate; MeJA: methyljasmonate; NOS: nitric oxide synthetase; NR: nitrate reductase; POX: peroxidase; ROS: reactive oxygen species; SNAP: S-nitroso-N-acetyl-DL-penicillamine; SNP: sodium nitroprusside; NOX: NADP(H) oxidase.

</sec>

DOI： 10.1080/09168451.2020.1743168

Salicylic acid receptor NPR1 is involved in guard cell chitosan signaling Reviewed International coauthorship International journal

Yeasin Prodhan, Mohammad Issak, Shintaro Munemasa, Yoshimasa Nakamura, Yoshiyuki Murata

Bioscience, Biotechnology, and Biochemistry 84 ( 5 ) 963 - 969 2020.5

Language：English Publishing type：Research paper (scientific journal) Publisher：Oxford University Press (OUP)

<title>Abstract</title>
<sec>
<title />
Chitosan (CHT) induces stomatal closure and thus plays a crucial role in plants to adapt to the adverse environments. Our previous results of a SA-deficient mutant nahG suggest that endogenous salicylic acid (SA) is involved in the CHT signaling in guard cells. Here in order to make the involvement definite, we examined stomatal responses to CHT of another SA-deficient mutant, sid2, and an SA receptor mutant, npr1-3. The sid2 mutation impaired CHT-induced stomatal closure and reactive oxygen species production and both impairments were complemented with exogenous SA application. Moreover, the CHT-induced stomatal closure is disrupted in the npr1-3 mutant. These results suggest that endogenous SA is involved in the CHT-induced stomatal closure via the SA receptor, NPR1.

</sec>
<sec>
<title>Abbreviations</title>
SA: salicylic acid; ABA: abscisic acid; ROS: reactive oxygen species; NPR1: nonexpresser of pathogenesis-related genes1; CHT: chitosan; DAB: 3,3′-diaminobenzidine

</sec>

DOI： 10.1080/09168451.2020.1718485

The Myrosinases TGG1 and TGG2 Function Redundantly in Reactive Carbonyl Species Signaling in Arabidopsis Guard Cells Reviewed International coauthorship International journal

Mohammad Saidur Rhaman, Toshiyuki Nakamura, Yoshimasa Nakamura, Shintaro Munemasa, Yoshiyuki Murata

Plant and Cell Physiology 61 ( 5 ) 967 - 977 2020.5

Language：English Publishing type：Research paper (scientific journal) Publisher：Oxford University Press (OUP)

<title>Abstract</title>
Myrosinase (β-thioglucoside glucohydrolase, enzyme nomenclature, EC 3.2.1.147, TGG) is a highly abundant protein in Arabidopsis guard cells, of which TGG1 and TGG2 function redundantly in abscisic acid (ABA)- and methyl jasmonate-induced stomatal closure. Reactive carbonyl species (RCS) are α,β-unsaturated aldehydes and ketones, which function downstream of reactive oxygen species (ROS) production in the ABA signalling pathway in guard cells. Among the RCS, acrolein is the most highly reactive, which is significantly produced in ABA-treated guard cells. To clarify the ABA signal pathway downstream of ROS production, we investigated the responses of tgg mutants (tgg1-3, tgg2-1 and tgg1-3 tgg2-1) to acrolein. Acrolein induced stomatal closure and triggered cytosolic alkalization in wild type (WT), tgg1-3 single mutants and in tgg2-1 single mutants, but not in tgg1-3 tgg2-1 double mutants. Exogenous Ca2+ induced stomatal closure and cytosolic alkalization not only in WT but also in all of the mutants. Acrolein- and Ca2+-induced stomatal closures were inhibited by an intracellular acidifying agent, butyrate, a Ca2+ chelator, ethylene glycol tetraacetic acid (EGTA) and a Ca2+ channel blocker, LaCl3. Acrolein induced cytosolic free calcium concentration ([Ca2+]cyt) elevation in guard cells of WT plants but not in the tgg1-3 tgg2-1 double mutants. Exogenous Ca2+ elicited [Ca2+]cyt elevation in guard cells of WT and tgg1-3 tgg2-1. Our results suggest that TGG1 and TGG2 function redundantly, not between ROS production and RCS production, but downstream of RCS production in the ABA signal pathway in Arabidopsis guard cells.

DOI： 10.1093/pcp/pcaa024

Other Link： http://academic.oup.com/pcp/article-pdf/61/5/967/33207245/pcaa024.pdf

Yeast screening system reveals the inhibitory mechanism of cancer cell proliferation by benzyl isothiocyanate through down-regulation of Mis12 Reviewed International journal

Naomi Abe-Kanoh, Narumi Kunisue, Takumi Myojin, Ayako Chino, Shintaro Munemasa, Yoshiyuki Murata, Ayano Satoh, Hisao Moriya, Yoshimasa Nakamura

Scientific Reports 9 ( 1 ) 8866 - 8866 2019.12

Language：English Publishing type：Research paper (scientific journal) Publisher：Springer Science and Business Media LLC

Benzyl isothiocyanate (BITC) is a naturally-occurring isothiocyanate derived from cruciferous vegetables. BITC has been reported to inhibit the proliferation of various cancer cells, which is believed to be important for the inhibition of tumorigenesis. However, the detailed mechanisms of action remain unclear. In this study, we employed a budding yeast Saccharomyces cerevisiae as a model organism for screening. Twelve genes including MTW1 were identified as the overexpression suppressors for the antiproliferative effect of BITC using the genome-wide multi-copy plasmid collection for S. cerevisiae. Overexpression of the kinetochore protein Mtw1 counteracts the antiproliferative effect of BITC in yeast. The inhibitory effect of BITC on the proliferation of human colon cancer HCT-116 cells was consistently suppressed by the overexpression of Mis12, a human orthologue of Mtw1, and enhanced by the knockdown of Mis12. We also found that BITC increased the phosphorylated and ubiquitinated Mis12 level with consequent reduction of Mis12, suggesting that BITC degrades Mis12 through an ubiquitin-proteasome system. Furthermore, cell cycle analysis showed that the change in the Mis12 level affected the cell cycle distribution and the sensitivity to the BITC-induced apoptosis. These results provide evidence that BITC suppresses cell proliferation through the post-transcriptional regulation of the kinetochore protein Mis12.

DOI： 10.1038/s41598-019-45248-2

Other Link： http://www.nature.com/articles/s41598-019-45248-2

Ethylene Inhibits Methyl Jasmonate-Induced Stomatal Closure by Modulating Guard Cell Slow-Type Anion Channel Activity via the OPEN STOMATA 1/SnRK2.6 Kinase-Independent Pathway in Arabidopsis Reviewed International journal

Shintaro Munemasa, Yukari Hirao, Kasumi Tanami, Yoshiharu Mimata, Yoshimasa Nakamura, Yoshiyuki Murata

Plant and Cell Physiology 60 ( 10 ) 2263 - 2271 2019.10

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal) Publisher：Oxford University Press (OUP)

<title>Abstract</title>
Signal crosstalk between jasmonate and ethylene is crucial for a proper maintenance of defense responses and development. Although previous studies reported that both jasmonate and ethylene also function as modulators of stomatal movements, the signal crosstalk mechanism in stomatal guard cells remains unclear. Here, we show that the ethylene signaling inhibits jasmonate signaling as well as abscisic acid (ABA) signaling in guard cells of Arabidopsis thaliana and reveal the signaling crosstalk mechanism. Both an ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and an ethylene-releasing compound ethephon induced transient stomatal closure, and also inhibited methyl jasmonate (MeJA)-induced stomatal closure as well as ABA-induced stomatal closure. The ethylene inhibition of MeJA-induced stomatal closure was abolished in the ethylene-insensitive mutant etr1–1, whereas MeJA-induced stomatal closure was impaired in the ethylene-overproducing mutant eto1–1. Pretreatment with ACC inhibited MeJA-induced reactive oxygen species (ROS) production as well as ABA-induced ROS production in guard cells but did not suppress ABA activation of OPEN STOMATA 1 (OST1) kinase in guard cell-enriched epidermal peels. The whole-cell patch-clamp analysis revealed that ACC attenuated MeJA and ABA activation of S-type anion channels in guard cell protoplasts. However, MeJA and ABA inhibitions of Kin channels were not affected by ACC pretreatment. These results suggest that ethylene signaling inhibits MeJA signaling and ABA signaling by targeting S-type anion channels and ROS but not OST1 kinase and K+ channels in Arabidopsis guard cells.

DOI： 10.1093/pcp/pcz121

Other Link： http://academic.oup.com/pcp/article-pdf/60/10/2263/30151483/pcz121.pdf

Reactive Carbonyl Species Function as Signal Mediators Downstream of H2O2 Production and Regulate [Ca2+]cyt Elevation in ABA Signal Pathway in Arabidopsis Guard Cells Reviewed International coauthorship International journal

Md. Moshiul Islam, Wenxiu Ye, Daiki Matsushima, Mohammad Saidur Rhaman, Shintaro Munemasa, Eiji Okuma, Yoshimasa Nakamura, Md. Sanaullah Biswas, Jun’ichi Mano, Yoshiyuki Murata

Plant and Cell Physiology 60 ( 5 ) 1146 - 1159 2019.5

The mechanism of SO2-induced stomatal closure differs from O3 and CO2 responses and is mediated by nonapoptotic cell death in guard cells Reviewed International coauthorship International journal

Lia Ooi, Takakazu Matsuura, Shintaro Munemasa, Yoshiyuki Murata, Maki Katsuhara, Takashi Hirayama, Izumi C. Mori

Plant, Cell & Environment 42 ( 2 ) 437 - 447 2019.2

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

Plants closing stomata in the presence of harmful gases is believed to be a stress avoidance mechanism. SO2 , one of the major airborne pollutants, has long been reported to induce stomatal closure, yet the mechanism remains unknown. Little is known about the stomatal response to airborne pollutants besides O3 . SLOW ANION CHANNEL-ASSOCIATED 1 (SLAC1) and OPEN STOMATA 1 (OST1) were identified as genes mediating O3 -induced closure. SLAC1 and OST1 are also known to mediate stomatal closure in response to CO2 , together with RESPIRATORY BURST OXIDASE HOMOLOGs (RBOHs). The overlaying roles of these genes in response to O3 and CO2 suggested that plants share their molecular regulators for airborne stimuli. Here, we investigated and compared stomatal closure event induced by a wide concentration range of SO2 in Arabidopsis through molecular genetic approaches. O3 - and CO2 -insensitive stomata mutants did not show significant differences from the wild type in stomatal sensitivity, guard cell viability, and chlorophyll content revealing that SO2 -induced closure is not regulated by the same molecular mechanisms as for O3 and CO2 . Nonapoptotic cell death is shown as the reason for SO2 -induced closure, which proposed the closure as a physicochemical process resulted from SO2 distress, instead of a biological protection mechanism.

DOI： 10.1111/pce.13406

Methyl-β-cyclodextrin potentiates the BITC-induced anti-cancer effect through modulation of the Akt phosphorylation in human colorectal cancer cells Reviewed International coauthorship International journal

Qifu Yang, Miku Miyagawa, Xiaoyang Liu, Beiwei Zhu, Shintaro Munemasa, Toshiyuki Nakamura, Yoshiyuki Murata, Yoshimasa Nakamura

Bioscience, Biotechnology, and Biochemistry 82 ( 12 ) 2158 - 2167 2018.12

Language：English Publishing type：Research paper (scientific journal) Publisher：Oxford University Press (OUP)

<title>ABSTRACT</title>
Methyl-β-cyclodextrin (MβCD) is an effective agent for the removal of plasma membrane cholesterol. In this study, we investigated the modulating effects of MβCD on the antiproliferation induced by benzyl isothiocyanate (BITC), an ITC compound mainly derived from papaya seeds. We confirmed that MβCD dose-dependently increased the cholesterol level in the medium, possibly through its removal from the plasma membrane of human colorectal cancer cells. The pretreatment with a non-toxic concentration (2.5 mM) of MβCD significantly enhanced the BITC-induced cytotoxicity and apoptosis induction, which was counteracted by the cholesterol supplementation. Although BITC activated the phosphoinositide 3-kinase (PI3K)/Akt pathway, MβCD dose-dependently inhibited the phosphorylation level of Akt. On the contrary, the treatment of MβCD enhanced the phosphorylation of mitogen activated protein kinases, but did not potentiate their BITC-induced phosphorylation. These results suggested that MβCD might potentiate the BITC-induced anti-cancer by cholesterol depletion and thus inhibition of the PI3K/Akt-dependent survival pathway.

Abbreviations: CDs: cyclodextrins; MβCD: methyl-β-cyclodextrin; ITCs: isothiocyanates; BITC: benzyl isothiocyanate; PI3K: phosphoinositide 3-kinase; PDK1: phosphoinositide-dependent kinase-1; MAPK: mitogen activated protein kinase; ERK1/2: extracellular signal-regulated kinase1/2; JNK: c-Jun N-terminal kinase; PI: propidium iodide; FBS: fatal bovine serum; TLC: thin-layer chromatography; PBS(-): phosphate-buffered saline without calcium and magnesium; MEK: MAPK/ERK kinase; PIP2: phosphatidylinositol-4,5-bisphosphate; PIP3: phosphatidylinositol-3,4,5-trisphosphate

DOI： 10.1080/09168451.2018.1514249

Benzyl isothiocyanate ameliorates lipid accumulation in 3T3-L1 preadipocytes during adipocyte differentiation Reviewed International coauthorship International journal

Ying Liang, Ikumi Sasaki, Yuki Takeda, Beiwei Zhu, Shintaro Munemasa, Toshiyuki Nakamura, Yoshiyuki Murata, Yoshimasa Nakamura

Bioscience, Biotechnology, and Biochemistry 82 ( 12 ) 2130 - 2139 2018.12

Language：English Publishing type：Research paper (scientific journal) Publisher：Oxford University Press (OUP)

<title>ABSTRACT</title>
Benzyl isothiocyanate (BITC) is an organosulfur compound derived from cruciferous vegetables and papaya seeds. In this study, we investigated the effect of BITC on the lipid accumulation in 3T3-L1 preadipocytes during adipocyte differentiation. The treatment of BITC during the differentiation-inducing stage significantly ameliorated the lipid accumulation, whereas it had no inhibitory effect during the differentiation-maintaining stage. BITC also significantly suppressed the mRNA expression of the adipocyte-specific markers, such as CCAAT/enhancer-binding protein α (C/EBPα), C/EBPβ, C/EBPδ and peroxisome proliferator-activated receptor γ. BITC significantly inhibited the phosphorylation of extracellular signal-regulated kinase phosphorylation, whereas it enhanced that of AMP-activated protein kinase. Furthermore, BITC significantly suppressed the intracellular 2-deoxyglucose uptake as well as glucose transporter 4 expression. These results suggest that inhibition of the adipocyte differentiation and glucose uptake may mainly contribute to the inhibitory effect of BITC on the lipid accumulation in 3T3-L1 preadipocytes.

Abbreviations: PPARγ: peroxisome proliferator-activated receptor γ; CEBP: CCAAT/enhancer-binding protein; GLUT4: glucose transporter 4; AMPK: AMP-activated protein kinase; ERK1/2: extracellular signal-regulated kinase 1/2; MAPK: a mitogen-activated protein kinase; ITCs: isothiocyanates; BITC: benzyl isothiocyanate; FBS: fetal bovine serum; CS: calf serum; AITC: allyl ITC; IBMX: 3-isobutyl-1-methylxanthine; LDH: lactate dehydrogenase; KRH: Krebs-Ringer-Hepes-bicarbonate; 2-DG: 2-deoxy-d-glucose

DOI： 10.1080/09168451.2018.1514247

Abscisic acid-independent stomatal CO2 signal transduction pathway and convergence of CO2 and ABA signaling downstream of OST1 kinase Reviewed International coauthorship International journal

Po-Kai Hsu, Yohei Takahashi, Shintaro Munemasa, Ebe Merilo, Kristiina Laanemets, Rainer Waadt, Dianne Pater, Hannes Kollist, Julian I. Schroeder

Proceedings of the National Academy of Sciences of the United States of America 115 ( 42 ) E9971 - E9980 2018.10

Language：English Publishing type：Research paper (scientific journal) Publisher：Proceedings of the National Academy of Sciences

Stomatal pore apertures are narrowing globally due to the continuing rise in atmospheric [CO₂]. CO₂ elevation and the plant hormone abscisic acid (ABA) both induce rapid stomatal closure. However, the underlying signal transduction mechanisms for CO₂/ABA interaction remain unclear. Two models have been considered: (<italic>i</italic>) CO₂ elevation enhances ABA concentrations and/or early ABA signaling in guard cells to induce stomatal closure and (<italic>ii</italic>) CO₂ signaling merges with ABA at OST1/SnRK2.6 protein kinase activation. Here we use genetics, ABA-reporter imaging, stomatal conductance, patch clamp, and biochemical analyses to investigate these models. The strong ABA biosynthesis mutants <italic>nced3/nced5</italic> and <italic>aba2-1</italic> remain responsive to CO₂ elevation. Rapid CO₂-triggered stomatal closure in PYR/RCAR ABA receptor quadruple and hextuple mutants is not disrupted but delayed. Time-resolved ABA concentration monitoring in guard cells using a FRET-based ABA-reporter, ABAleon2.15, and ABA reporter gene assays suggest that CO₂ elevation does not trigger [ABA] increases in guard cells, in contrast to control ABA exposures. Moreover, CO₂ activates guard cell S-type anion channels in <italic>nced3/nced5</italic> and ABA receptor hextuple mutants. Unexpectedly, in-gel protein kinase assays show that unlike ABA, elevated CO₂ does not activate OST1/SnRK2 kinases in guard cells. The present study points to a model in which rapid CO₂ signal transduction leading to stomatal closure occurs via an ABA-independent pathway downstream of OST1/SnRK2.6. Basal ABA signaling and OST1/SnRK2 activity are required to facilitate the stomatal response to elevated CO₂. These findings provide insights into the interaction between CO₂/ABA signal transduction in light of the continuing rise in atmospheric [CO₂].

DOI： 10.1073/pnas.1809204115

Other Link： https://syndication.highwire.org/content/doi/10.1073/pnas.1809204115

Eukaryotic lipid metabolic pathway is essential for functional chloroplasts and CO2 and light responses in Arabidopsis guard cells Reviewed International coauthorship International journal

Juntaro Negi, Shintaro Munemasa, Boseok Song, Ryosuke Tadakuma, Mayumi Fujita, Tamar Azoulay-Shemer, Cawas B. Engineer, Kensuke Kusumi, Ikuo Nishida, Julian I. Schroeder, Koh Iba

Proceedings of the National Academy of Sciences of the United States of America 115 ( 36 ) 9038 - 9043 2018.9

Language：English Publishing type：Research paper (scientific journal) Publisher：Proceedings of the National Academy of Sciences

Stomatal guard cells develop unique chloroplasts in land plant species. However, the developmental mechanisms and function of chloroplasts in guard cells remain unclear. In seed plants, chloroplast membrane lipids are synthesized via two pathways: the prokaryotic and eukaryotic pathways. Here we report the central contribution of endoplasmic reticulum (ER)-derived chloroplast lipids, which are synthesized through the eukaryotic lipid metabolic pathway, in the development of functional guard cell chloroplasts. We gained insight into this pathway by isolating and examining an <italic>Arabidopsis</italic> mutant, <italic>gles1</italic> (<italic>green less stomata 1</italic>), which had achlorophyllous stomatal guard cells and impaired stomatal responses to CO₂ and light. The <italic>GLES1</italic> gene encodes a small glycine-rich protein, which is a putative regulatory component of the trigalactosyldiacylglycerol (TGD) protein complex that mediates ER-to-chloroplast lipid transport via the eukaryotic pathway. Lipidomic analysis revealed that in the wild type, the prokaryotic pathway is dysfunctional, specifically in guard cells, whereas in <italic>gles1</italic> guard cells, the eukaryotic pathway is also abrogated. CO₂-induced stomatal closing and activation of guard cell S-type anion channels that drive stomatal closure were disrupted in <italic>gles1</italic> guard cells. In conclusion, the eukaryotic lipid pathway plays an essential role in the development of a sensing/signaling machinery for CO₂ and light in guard cell chloroplasts.

DOI： 10.1073/pnas.1810458115

Guard Cell Salicylic Acid Signaling Is Integrated into Abscisic Acid Signaling via the Ca2+/CPK-Dependent Pathway Reviewed International coauthorship International journal

Md. Yeasin Prodhan, Shintaro Munemasa, Mst. Nur-E-Nazmun Nahar, Yoshimasa Nakamura, Yoshiyuki Murata

Plant Physiology 178 ( 1 ) 441 - 450 2018.9

Ion Channels Regulate Nyctinastic Leaf Opening in Samanea saman Reviewed International coauthorship International journal

Takaya Oikawa, Yasuhiro Ishimaru, Shintaro Munemasa, Yusuke Takeuchi, Kento Washiyama, Shin Hamamoto, Nobuyuki Yoshikawa, Yoshiyuki Mutara, Nobuyuki Uozumi, Minoru Ueda

Current Biology 28 ( 14 ) 2230 - 2238.e7 2018.7

Effects of calcium and EGTA on thiol homeostasis and defense-related enzymes in Cd-exposed chickpea roots Reviewed International coauthorship International journal

Lamia Sakouhi, Sondès Rahoui, Charfeddine Gharsallah, Shintaro Munemasa, Ezzeddine El Ferjani, Yoshiyuki Murata, Abdelilah Chaoui

Acta Physiologiae Plantarum 40 ( 1 ) 20 2018.1

Mechanism of Stomatal Closure in Plants Exposed to Drought and Cold Stress. International journal

Srinivas Agurla, Shashibhushan Gahir, Shintaro Munemasa, Yoshiyuki Murata, Agepati S Raghavendra

Advances in experimental medicine and biology 1081 215 - 232 2018

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

Drought is one of the abiotic stresses which impairs the plant growth/development and restricts the yield of many crops throughout the world. Stomatal closure is a common adaptation response of plants to the onset of drought condition. Stomata are microscopic pores on the leaf epidermis, which regulate the transpiration/CO2 uptake by leaves. Stomatal guard cells can sense various abiotic and biotic stress stimuli from the internal and external environment and respond quickly to initiate closure under unfavorable conditions. Stomata also limit the entry of pathogens into leaves, restricting their invasion. Drought is accompanied by the production and/or mobilization of the phytohormone, abscisic acid (ABA), which is well-known for its ability to induce stomatal closure. Apart from the ABA, various other factors that accumulate during drought and affect the stomatal function are plant hormones (auxins, MJ, ethylene, brassinosteroids, and cytokinins), microbial elicitors (salicylic acid, harpin, Flg 22, and chitosan), and polyamines . The role of various signaling components/secondary messengers during stomatal opening or closure has been a matter of intense investigation. Reactive oxygen species (ROS) , nitric oxide (NO) , cytosolic pH, and calcium are some of the well-documented signaling components during stomatal closure. The interrelationship and interactions of these signaling components such as ROS, NO, cytosolic pH, and free Ca2+ are quite complex and need further detailed examination.Low temperatures can have deleterious effects on plants. However, plants evolved protection mechanisms to overcome the impact of this stress. Cold temperature inhibits stomatal opening and causes stomatal closure. Cold-acclimated plants often exhibit marked changes in their lipid composition, particularly of the membranes. Cold stress often leads to the accumulation of ABA, besides osmolytes such as glycine betaine and proline. The role of signaling components such as ROS, NO, and Ca2+ during cold acclimation is yet to be established, though the effects of cold stress on plant growth and development are studied extensively. The information on the mitigation processes is quite limited. We have attempted to describe consequences of drought and cold stress in plants, emphasizing stomatal closure. Several of these factors trigger signaling components in roots, shoots, and atmosphere, all leading to stomatal closure. A scheme is presented to show the possible signaling events and their convergence and divergence of action during stomatal closure. The possible directions for future research are discussed.

DOI： 10.1007/978-981-13-1244-1_12

Blue light and CO2 signals converge to regulate light-induced stomatal opening Reviewed International journal

Asami Hiyama, Atsushi Takemiya, Shintaro Munemasa, Eiji Okuma, Naoyuki Sugiyama, Yasuomi Tada, Yoshiyuki Murata, Ken-ichiro Shimazaki

Nature Communications 8 1284 2017.12

Inhibition of phosphatidylinositide 3-kinase impairs the benzyl isothiocyanate-induced accumulation of autophagic molecules and Nrf2 in human colon cancer cells Reviewed International coauthorship International journal

Xiaoyang Liu, Naomi Abe-Kanoh, Yujia Liu, Beiwei Zhu, Shintaro Munemasa, Toshiyuki Nakamura, Yoshiyuki Murata, Yoshimasa Nakamura

Bioscience, Biotechnology, and Biochemistry 81 ( 11 ) 2212 - 2215 2017.11

Language：English Publishing type：Research paper (scientific journal) Publisher：Oxford University Press (OUP)

<title>Abstract</title>
The regulating role of phosphatidylinositide 3-kinase (PI3K) in benzyl isothiocyanate (BITC)-induced Nrf2 activation, contributing to the inducible expression of cytoprotective genes, was investigated. BITC significantly enhanced the accumulation of Nrf2 as well as autophagic molecules in human colorectal cancer HCT-116 cells. Experiments using a PI3K-specific inhibitor suggested that PI3K plays the key role in the non-canonical Nrf2 activation by BITC.

DOI： 10.1080/09168451.2017.1374830

3,4-Dihydroxyphenylacetic acid is a potential aldehyde dehydrogenase inducer in murine hepatoma Hepa1c1c7 cells Reviewed International coauthorship International journal

Yujia Liu, Ayuki Kurita, Sayaka Nakashima, Beiwei Zhu, Shintaro Munemasa, Toshiyuki Nakamura, Yoshiyuki Murata, Yoshimasa Nakamura

Bioscience, Biotechnology, and Biochemistry 81 ( 10 ) 1978 - 1983 2017.10

Language：English Publishing type：Research paper (scientific journal) Publisher：Oxford University Press (OUP)

<title>Abstract</title>
3,4-Dihydroxyphenylacetic acid (DOPAC) is one of the major colonic microflora-produced catabolites of quercetin glycosides, such as quercetin 4′-glucoside derived from onion. Here, we investigated whether DOPAC modulates the aldehyde dehydrogenase (ALDH) activity and protects the cells from the acetaldehyde-induced cytotoxicity in vitro. DOPAC was shown to enhance not only the total ALDH activity, but also the gene expression of ALDH1A1, ALDH2 and ALDH3A1 in a concentration-dependent manner. DOPAC simultaneously stimulated the nuclear translocation of NFE2-related factor 2 and aryl hydrocarbon receptor. The pretreatment of DOPAC completely protected the cells from the acetaldehyde-induced cytotoxicity. The present study suggested that DOPAC acts as a potential ALDH inducer to prevent the alcohol-induced abnormal reaction.

DOI： 10.1080/09168451.2017.1361809

Benzyl isothiocyanate ameliorates acetaldehyde-induced cytotoxicity by enhancing aldehyde dehydrogenase activity in murine hepatoma Hepa1c1c7 cells Reviewed International coauthorship International journal

Yujia Liu, Momoko Yamanaka, Naomi Abe-Kanoh, Xiaoyang Liu, Beiwei Zhu, Shintaro Munemasa, Toshiyuki Nakamura, Yoshiyuki Murata, Yoshimasa Nakamura

Food and Chemical Toxicology 108 305 - 313 2017.10

Exogenous proline enhances the sensitivity of Tobacco BY-2 cells to arsenate Reviewed International coauthorship International journal

Mst Nur-E-Nazmun Nahar, Mohammad Muzahidul Islam, Md Anamul Hoque, Anna Yonezawa, Md Yeasin Prodhan, Toshiyuki Nakamura, Yoshimasa Nakamura, Shintaro Munemasa, Yoshiyuki Murata

Bioscience, Biotechnology, and Biochemistry 81 ( 9 ) 1726 - 1731 2017.9

Language：English Publishing type：Research paper (scientific journal) Publisher：Oxford University Press (OUP)

<title>Abstract</title>
Arsenic causes physiological and structural disorders in plants. Proline is accumulated as a compatible solute in plants under various stress conditions and mitigates stresses. Here, we investigated the effects of exogenous proline on tobacco Bright Yellow-2 (BY-2) cultured cells under stress. Arsenate did not inhibit BY-2 cell growth at 40 and 50 μM but did it at 60 μM. Proline at 0.5 to 10 mM did not affect the cell growth but delayed it at 20 mM. At 40 μM , neither 0.5 mM nor 1 mM proline affected the cell growth but 10 mM proline inhibited it. In the presence of , 10 mM proline increased the number of Evans Blue-stained (dead) cells and decreased the number of total cells. Together, our results suggest that exogenous proline does not alleviate arsenate toxicity but enhances the sensitivity of BY-2 cells to arsenate.

DOI： 10.1080/09168451.2017.1340088

アブシジン酸誘導気孔閉口におけるカルシウムシグナル制御機構～明らかになった植物のカルシウムシグナル暗号解読システム～ Invited Reviewed

Shintaro Munemasa

Kagaku to Seibutsu 55 ( 10 ) 666 - 667 2017.9

(−)-Epigallocatechin-3-gallate inhibits human angiotensin-converting enzyme activity through an autoxidation-dependent mechanism Reviewed International coauthorship International journal

Zhe Liu, Sayaka Nakashima, Toshiyuki Nakamura, Shintaro Munemasa, Yoshiyuki Murata, Yoshimasa Nakamura

Journal of Biochemical and Molecular Toxicology 31 ( 9 ) e21932 - e21932 2017.9

Inhibition of phosphatidylinositide 3-kinase ameliorates antiproliferation by benzyl isothiocyanate in human colon cancer cells Reviewed International coauthorship International journal

Xiaoyang Liu, Chiaki Takano, Tomomi Shimizu, Shintaro Yokobe, Naomi Abe-Kanoh, Beiwei Zhu, Toshiyuki Nakamura, Shintaro Munemasa, Yoshiyuki Murata, Yoshimasa Nakamura

Biochemical and Biophysical Research Communications 491 ( 1 ) 209 - 216 2017.9

Chitosan signaling in guard cells requires endogenous salicylic acid Reviewed International coauthorship International journal

Md Yeasin Prodhan, Mohammad Issak, Toshiyuki Nakamura, Shintaro Munemasa, Yoshimasa Nakamura, Yoshiyuki Murata

Bioscience, Biotechnology, and Biochemistry 81 ( 8 ) 1536 - 1541 2017.8

Publishing type：Research paper (scientific journal) Publisher：Oxford University Press (OUP)

<title>Abstract</title>
An elicitor chitosan (CHT) induces stomatal closure but the mechanism remains to be clarified. A phytohormone salicylic acid (SA) is crucial for elicitor-induced defense signaling in plants. Here we investigated whether endogenous SA is required for CHT signaling in guard cells. In the SA-deficient nahG mutant, treatment of CHT did not induce either apoplastic reactive oxygen species (ROS) production or stomatal closure but co-treatment of CHT and SA induced both apoplastic ROS production and stomatal closure, indicating the involvement of endogenous SA in CHT-induced apoplastic ROS production and CHT-induced stomatal closure. Furthermore, CHT induced transient cytosolic free calcium concentration increments in the nahG mutant in the presence of exogenous SA but not in the absence of exogenous SA. These results provide evidence that endogenous SA is a crucial element in CHT-induced stomatal closure.

DOI： 10.1080/09168451.2017.1332979

Reactive Carbonyl Species Mediate ABA Signaling in Guard Cells Reviewed International coauthorship International journal

Md. Moshiul Islam, Wenxiu Ye, Daiki Matsushima, Shintaro Munemasa, Eiji Okuma, Yoshimasa Nakamura, Sanaullah Biswas, Jun’ichi Mano, Yoshiyuki Murata

Plant and Cell Physiology 57 ( 12 ) 2552 - 2563 2016.12

L-Met Activates Arabidopsis GLR Ca2+ Channels Upstream of ROS Production and Regulates Stomatal Movement Reviewed International coauthorship International journal

Dongdong Kong, Heng-Cheng Hu, Eiji Okuma, Yuree Lee, Hui Sun Lee, Shintaro Munemasa, Daeshik Cho, Chuanli Ju, Leah Pedoeim, Barbara Rodriguez, Juan Wang, Wonpil Im, Yoshiyuki Murata, Zhen-Ming Pei, June M. Kwak

Cell Reports 17 ( 10 ) 2553 - 2561 2016.12

Calcium and EGTA Alleviate Cadmium Toxicity in Germinating Chickpea Seeds Reviewed International coauthorship International journal

Lamia Sakouhi, Sondès Rahoui, Marouane Ben Massoud, Shintaro Munemasa, Ezzeddine EL Ferjani, Yoshiyuki Murata, Abdelilah Chaoui

Journal of Plant Growth Regulation 35 ( 4 ) 1064 - 1073 2016.12

A novel tag-free probe for targeting molecules interacting with a flavonoid catabolite Reviewed International coauthorship International journal

Sayaka Nakashima, Zhe Liu, Yuya Yamaguchi, Shunya Saiki, Shintaro Munemasa, Toshiyuki Nakamura, Yoshiyuki Murata, Yoshimasa Nakamura

Biochemistry and Biophysics Reports 7 240 - 245 2016.9

Involvement of OST1 Protein Kinase and PYR/PYL/RCAR Receptors in Methyl Jasmonate-Induced Stomatal Closure in Arabidopsis Guard Cells Reviewed International coauthorship International journal

Ye Yin, Yuji Adachi, Yoshimasa Nakamura, Shintaro Munemasa, Izumi C. Mori, Yoshiyuki Murata

Plant and Cell Physiology 57 ( 8 ) 1779 - 1790 2016.8

Galloylated Catechins as Potent Inhibitors of Angiotensin Converting Enzyme Reviewed International coauthorship International journal

Zhe Liu, Toshiyuki Nakamura, Shintaro Munemasa, Yoshiyuki Murata, Yoshimasa Nakamura

Food Science and Technology Research 22 ( 6 ) 847 - 851 2016

Mechanisms of abscisic acid-mediated control of stomatal aperture Invited Reviewed International coauthorship International journal

Shintaro Munemasa, Felix Hauser, Jiyoung Park, Rainer Waadt, Benjamin Brandt, Julian I Schroeder

Current Opinion in Plant Biology 28 154 - 162 2015.12

Identification of Open Stomata1-Interacting Proteins Reveals Interactions with Sucrose Non-fermenting1-Related Protein Kinases2 and with Type 2A Protein Phosphatases That Function in Abscisic Acid Responses Reviewed International coauthorship International journal

Rainer Waadt, Bianca Manalansan, Navin Rauniyar, Shintaro Munemasa, Matthew A. Booker, Benjamin Brandt, Christian Waadt, Dmitri A. Nusinow, Steve A. Kay, Hans-Henning Kunz, Karin Schumacher, Alison DeLong, John R. Yates, Julian I. Schroeder

Plant Physiology 169 ( 1 ) 760 - 779 2015.9

Calcium specificity signaling mechanisms in abscisic acid signal transduction in Arabidopsis guard cells Reviewed International coauthorship International journal

Benjamin Brandt, Shintaro Munemasa, Cun Wang, Desiree Nguyen, Taiming Yong, Paul G Yang, Elly Poretsky, Thomas F Belknap, Rainer Waadt, Fernando Alemán, Julian I Schroeder

eLife 4 e03599 2015.7

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal) Publisher：eLife Sciences Publications, Ltd

A central question is how specificity in cellular responses to the eukaryotic second messenger Ca2+ is achieved. Plant guard cells, that form stomatal pores for gas exchange, provide a powerful system for in depth investigation of Ca2+-signaling specificity in plants. In intact guard cells, abscisic acid (ABA) enhances (primes) the Ca2+-sensitivity of downstream signaling events that result in activation of S-type anion channels during stomatal closure, providing a specificity mechanism in Ca2+-signaling. However, the underlying genetic and biochemical mechanisms remain unknown. Here we show impairment of ABA signal transduction in stomata of calcium-dependent protein kinase quadruple mutant plants. Interestingly, protein phosphatase 2Cs prevent non-specific Ca2+-signaling. Moreover, we demonstrate an unexpected interdependence of the Ca2+-dependent and Ca2+-independent ABA-signaling branches and the in planta requirement of simultaneous phosphorylation at two key phosphorylation sites in SLAC1. We identify novel mechanisms ensuring specificity and robustness within stomatal Ca2+-signaling on a cellular, genetic, and biochemical level.

DOI： 10.7554/elife.03599

Other Link： https://cdn.elifesciences.org/articles/03599/elife-03599-v1.pdf

Open Stomata 1 Kinase is Essential for Yeast Elicitor-Induced Stomatal Closure in Arabidopsis Reviewed International coauthorship International journal

Wenxiu Ye, Yuji Adachi, Shintaro Munemasa, Yoshimasa Nakamura, Izumi C. Mori, Yoshiyuki Murata

Plant and Cell Physiology 56 ( 6 ) 1239 - 1248 2015.6

Language：English Publishing type：Research paper (scientific journal) Publisher：Oxford University Press (OUP)

We recently demonstrated that yeast elicitor (YEL)-induced stomatal closure requires a Ca2+-dependent kinase, CPK6. A Ca2+-independent kinase, Open Stomata 1 (OST1), is involved in stomatal closure induced by various stimuli including ABA. In the present study, we investigated the role of OST1 in YEL-induced stomatal closure in Arabidopsis using a knock-out mutant, ost1-3, and a kinase-deficient mutant, ost1-2. YEL did not induce stomatal closure or activation of guard cell S-type anion channels in the ost1 mutants unlike in wild-type plants. However, YEL did not increase OST1 kinase activity in wild-type guard cells. The YEL-induced stomatal closure and activation of S-type anion channels were also impaired in a gain-of-function mutant of a clade A type 2C protein phosphatase (ABA INSENSITIVE 1), abi1-1C. In the ost1 mutants like in the wild type, YEL induced H2O2 accumulation, activation of non-selective Ca2+-permeable cation (I-Ca) channels and transient elevations in cytosolic free Ca2+ concentration ([Ca2+](cyt)) in guard cells. These results suggest that OST1 kinase is essential for stomatal closure and activation of S-type anion channels induced by YEL and that OST1 is not involved in H2O2 accumulation, I-Ca channel activation or [Ca2+](cyt) elevations in guard cells induced by YEL.

DOI： 10.1093/pcp/pcv051

Diverse Stomatal Signaling and the Signal Integration Mechanism Invited Reviewed International journal

Yoshiyuki Murata, Izumi C. Mori, Shintaro Munemasa

Annual Review of Plant Biology 66 ( 1 ) 369 - 392 2015.4

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Annual Reviews

Guard cells perceive a variety of chemicals produced metabolically in response to abiotic and biotic stresses, integrate the signals into reactive oxygen species and calcium signatures, and convert these signatures into stomatal movements by regulating turgor pressure. Guard cell behaviors in response to such complex signals are critical for plant growth and sustenance in stressful, ever-changing environments. The key open question is how guard cells achieve the signal integration to optimize stomatal aperture. Abscisic acid is responsible for stomatal closure in plants in response to drought, and its signal transduction has been well studied. Other plant hormones and low-molecular-weight compounds function as inducers of stomatal closure and mediators of signaling in guard cells. In this review, we summarize recent advances in research on the diverse stomatal signaling pathways, with specific emphasis on signal integration and signal interaction in guard cell movement.

DOI： 10.1146/annurev-arplant-043014-114707

Inhibition by acrolein of light-induced stomatal opening through inhibition of inward-rectifying potassium channels in Arabidopsis thaliana Reviewed International coauthorship International journal

Md Moshiul Islam, Wenxiu Ye, Daiki Matsushima, Md Atiqur Rahman Khokon, Shintaro Munemasa, Yoshimasa Nakamura, Yoshiyuki Murata

Bioscience, Biotechnology, and Biochemistry 79 ( 1 ) 59 - 62 2015.1

Language：English Publishing type：Research paper (scientific journal) Publisher：Oxford University Press (OUP)

<title>Abstract</title>
Acrolein is a reactive α,β-unsaturated aldehyde derived from lipid peroxides, which are produced in plants under a variety of stress. We investigated effects of acrolein on light-induced stomatal opening using Arabidopsis thaliana. Acrolein inhibited light-induced stomatal opening in a dose-dependent manner. Acrolein at 100 μM inhibited plasma membrane inward-rectifying potassium (Kin) channels in guard cells. Acrolein at 100 μM inhibited Kin channel KAT1 expressed in a heterologous system using Xenopus leaves oocytes. These results suggest that acrolein inhibits light-induced stomatal opening through inhibition of Kin channels in guard cells.

DOI： 10.1080/09168451.2014.951028

Nuclear factor-kappaB sensitizes to benzyl isothiocyanate-induced antiproliferation in p53-deficient colorectal cancer cells Reviewed International coauthorship International journal

N Abe, D-X Hou, S Munemasa, Y Murata, Y Nakamura

Cell Death & Disease 5 ( 11 ) e1534 2014.11

Identification of Cyclic GMP-Activated Nonselective Ca2+-Permeable Cation Channels and Associated CNGC5 and CNGC6 Genes in Arabidopsis Guard Cells Reviewed International coauthorship International journal

Yong-Fei Wang, Shintaro Munemasa, Noriyuki Nishimura, Hui-Min Ren, Nadia Robert, Michelle Han, Irina Puzõrjova, Hannes Kollist, Stephen Lee, Izumi Mori, Julian I. Schroeder

Plant Physiology 163 ( 2 ) 578 - 590 2013.10

Language：English Publishing type：Research paper (scientific journal) Publisher：Oxford University Press (OUP)

<title>Abstract</title>
Cytosolic Ca2+ in guard cells plays an important role in stomatal movement responses to environmental stimuli. These cytosolic Ca2+ increases result from Ca2+ influx through Ca2+-permeable channels in the plasma membrane and Ca2+ release from intracellular organelles in guard cells. However, the genes encoding defined plasma membrane Ca2+-permeable channel activity remain unknown in guard cells and, with some exceptions, largely unknown in higher plant cells. Here, we report the identification of two Arabidopsis (Arabidopsis thaliana) cation channel genes, CNGC5 and CNGC6, that are highly expressed in guard cells. Cytosolic application of cyclic GMP (cGMP) and extracellularly applied membrane-permeable 8-Bromoguanosine 3′,5′-cyclic monophosphate-cGMP both activated hyperpolarization-induced inward-conducting currents in wild-type guard cells using Mg2+ as the main charge carrier. The cGMP-activated currents were strongly blocked by lanthanum and gadolinium and also conducted Ba2+, Ca2+, and Na+ ions. cngc5 cngc6 double mutant guard cells exhibited dramatically impaired cGMP-activated currents. In contrast, mutations in CNGC1, CNGC2, and CNGC20 did not disrupt these cGMP-activated currents. The yellow fluorescent protein-CNGC5 and yellow fluorescent protein-CNGC6 proteins localize in the cell periphery. Cyclic AMP activated modest inward currents in both wild-type and cngc5cngc6 mutant guard cells. Moreover, cngc5 cngc6 double mutant guard cells exhibited functional abscisic acid (ABA)-activated hyperpolarization-dependent Ca2+-permeable cation channel currents, intact ABA-induced stomatal closing responses, and whole-plant stomatal conductance responses to darkness and changes in CO2 concentration. Furthermore, cGMP-activated currents remained intact in the growth controlled by abscisic acid2 and abscisic acid insensitive1 mutants. This research demonstrates that the CNGC5 and CNGC6 genes encode unique cGMP-activated nonselective Ca2+-permeable cation channels in the plasma membrane of Arabidopsis guard cells.

DOI： 10.1104/pp.113.225045

Calcium-dependent protein Kinase CPK6 positively functions in induction by yeast elicitor of stomatal closure and inhibition by yeast elicitor of light-induced stomatal opening in Arabidopsis Reviewed International coauthorship International journal

Wenxiu Ye, Daichi Muroyama, Shintaro Munemasa, Yoshimasa Nakamura, Izumi C. Mori, Yoshiyuki Murata

Plant Physiology 163 ( 2 ) 591 - 599 2013.10

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

Yeast elicitor (YEL) induces stomatal closure that is mediated by a Ca2+-dependent signaling pathway. A Ca2+-dependent protein kinase, CPK6, positively regulates activation of ion channels in abscisic acid and methyl jasmonate signaling, leading to stomatal closure in Arabidopsis (Arabidopsis thaliana). YEL also inhibits light-induced stomatal opening. However, it remains unknown whether CPK6 is involved in induction by YEL of stomatal closure or in inhibition by YEL of light-induced stomatal opening. In this study, we investigated the roles of CPK6 in induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening in Arabidopsis. Disruption of CPK6 gene impaired induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening. Activation by YEL of nonselective Ca2+-permeable cation channels was impaired in cpk6-2 guard cells, and transient elevations elicited by YEL in cytosolic-free Ca2+ concentration were suppressed in cpk6-2 and cpk6-1 guard cells. YEL activated slow anion channels in wild-type guard cells but not in cpk6-2 or cpk6-1 and inhibited inward-rectifying K+ channels in wild-type guard cells but not in cpk6-2 or cpk6-1. The cpk6-2 and cpk6-1 mutations inhibited YEL-induced hydrogen peroxide accumulation in guard cells and apoplast of rosette leaves but did not affect YEL-induced hydrogen peroxide production in the apoplast of rosette leaves. These results suggest that CPK6 positively functions in induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening in Arabidopsis and is a convergent point of signaling pathways for stomatal closure in response to abiotic and biotic stress. © 2013 American Society of Plant Biologists. All Rights Reserved.

DOI： 10.1104/pp.113.224055

Endogenous abscisic acid is involved in methyl jasmonate-induced reactive oxygen species and nitric oxide production but not in cytosolic alkalization in Arabidopsis guard cells Reviewed International coauthorship International journal

Wenxiu Ye, Mohammad Anowar Hossain, Shintaro Munemasa, Yoshimasa Nakamura, Izumi C. Mori, Yoshiyuki Murata

Journal of Plant Physiology 170 ( 13 ) 1212 - 1215 2013.9

Neither Endogenous Abscisic Acid nor Endogenous Jasmonate Is Involved in Salicylic Acid-, Yeast Elicitor-, or Chitosan-Induced Stomatal Closure inArabidopsis thaliana Reviewed International coauthorship International journal

Mohammad Issak, Eiji Okuma, Shintaro Munemasa, Yoshimasa Nakamura, Izumi C. Mori, Yoshiyuki Murata

Bioscience, Biotechnology, and Biochemistry 77 ( 5 ) 1111 - 1113 2013.5

Language：English Publishing type：Research paper (scientific journal) Publisher：Oxford University Press (OUP)

Salicylic acid (SA), yeast elicitor (YEL), and chitosan (CHT) induced stomatal closure in Arabidopsis wild-type and aba2-2 plants, induced stomatal closure in fluridon-treated wild-type plants, and induced stomatal closure in aos mutants. These results suggest that neither endogenous abscisic acid nor endogenous jasmonic acid is involved in SA-, YEL-, or CHT-induced stomatal closure.

DOI： 10.1271/bbb.120980

Negative Regulation of Methyl Jasmonate-Induced Stomatal Closure by Glutathione in Arabidopsis Reviewed International coauthorship International journal

Nasima Akter, Eiji Okuma, Muhammad Abdus Sobahan, Misugi Uraji, Shintaro Munemasa, Yoshimasa Nakamura, Izumi C. Mori, Yoshiyuki Murata

Journal of Plant Growth Regulation 32 ( 1 ) 208 - 215 2013.3

Regulation of reactive oxygen species-mediated abscisic acid signaling in guard cells and drought tolerance by glutathione Invited Reviewed International journal

Shintaro Munemasa, Daichi Muroyama, Hiroki Nagahashi, Yoshimasa Nakamura, Izumi C. Mori, Yoshiyuki Murata

Frontiers in Plant Science 4 472 2013

FIA functions as an early signal component of abscisic acid signal cascade in Vicia faba guard cells Reviewed International journal

Yusuke Sugiyama, Misugi Uraji, Megumi Watanabe-Sugimoto, Eiji Okuma, Shintaro Munemasa, Yasuaki Shimoishi, Yoshimasa Nakamura, Izumi C. Mori, Sumio Iwai, Yoshiyuki Murata

Journal of Experimental Botany 63 ( 3 ) 1357 - 1365 2012.2

Language：English Publishing type：Research paper (scientific journal) Publisher：Oxford University Press (OUP)

An abscisic acid (ABA)-insensitive Vicia faba mutant, fia (fava bean impaired in ABA-induced stomatal closure) had previously been isolated. In this study, it was investigated how FIA functions in ABA signalling in guard cells of Vicia faba. Unlike ABA, methyl jasmonate (MeJA), H2O2, and nitric oxide (NO) induced stomatal closure in the fia mutant. ABA did not induce production of either reactive oxygen species or NO in the mutant. Moreover, ABA did not suppress inward-rectifying K+ (K-in) currents or activate ABA-activated protein kinase (AAPK) in mutant guard cells. These results suggest that FIA functions as an early signal component upstream of AAPK activation in ABA signalling but does not function in MeJA signalling in guard cells of Vicia faba.

DOI： 10.1093/jxb/err369

Negative regulation of abscisic acid-induced stomatal closure by glutathione in Arabidopsis Reviewed International coauthorship International journal

Eiji Okuma, Md. Sarwar Jahan, Shintaro Munemasa, Mohammad Anowar Hossain, Daichi Muroyama, Mohammad Mahbub Islam, Ken’ichi Ogawa, Megumi Watanabe-Sugimoto, Yoshimasa Nakamura, Yasuaki Shimoishi, Izumi C. Mori, Yoshiyuki Murata

Journal of Plant Physiology 168 ( 17 ) 2048 - 2055 2011.11

K252a-sensitive protein kinases but not okadaic acid-sensitive protein phosphatases regulate methyl jasmonate-induced cytosolic Ca2+ oscillation in guard cells of Arabidopsis thaliana Reviewed International coauthorship International journal

Mohammad Anowar Hossain, Shintaro Munemasa, Yoshimasa Nakamura, Izumi C. Mori, Yoshiyuki Murata

Journal of Plant Physiology 168 ( 16 ) 1901 - 1908 2011.11

Allyl isothiocyanate (AITC) induces stomatal closure in Arabidopsis Reviewed International coauthorship International journal

Md Atiqur Rahman Khokon, Md Sarwar Jahan, Taniya Rahman, Mohammad Anowar Hossain, Daichi Muroyama, Ikuko Minami, Shintaro Munemasa, Izumi C Mori, Yoshimasa Nakamura, Yoshiyuki Murata

Plant, Cell & Environment 34 ( 11 ) 1900 - 1906 2011.11

Methyl jasmonate signaling and signal crosstalk between methyl jasmonate and abscisic acid in guard cells Invited Reviewed International journal

Shintaro Munemasa, Izumi C. Mori, Yoshiyuki Murata

Plant Signaling & Behavior 6 ( 7 ) 939 - 941 2011.7

Chemical Genetics Reveals Negative Regulation of Abscisic Acid Signaling by a Plant Immune Response Pathway Reviewed International coauthorship International journal

Tae-Houn Kim, Felix Hauser, Tracy Ha, Shaowu Xue, Maik Böhmer, Noriyuki Nishimura, Shintaro Munemasa, Katharine Hubbard, Nora Peine, Byeong-ha Lee, Stephen Lee, Nadia Robert, Jane E. Parker, Julian I. Schroeder

Current Biology 21 ( 11 ) 990 - 997 2011.6

Involvement of Endogenous Abscisic Acid in Methyl Jasmonate-Induced Stomatal Closure in Arabidopsis Reviewed International coauthorship International journal

Mohammad Anowar Hossain, Shintaro Munemasa, Misugi Uraji, Yoshimasa Nakamura, Izumi C. Mori, Yoshiyuki Murata

Plant Physiology 156 ( 1 ) 430 - 438 2011.5

Language：English Publishing type：Research paper (scientific journal) Publisher：Oxford University Press (OUP)

<title>Abstract</title>
In this study, we examined the involvement of endogenous abscisic acid (ABA) in methyl jasmonate (MeJA)-induced stomatal closure using an inhibitor of ABA biosynthesis, fluridon (FLU), and an ABA-deficient Arabidopsis (Arabidopsis thaliana) mutant, aba2-2. We found that pretreatment with FLU inhibited MeJA-induced stomatal closure but not ABA-induced stomatal closure in wild-type plants. The aba2-2 mutation impaired MeJA-induced stomatal closure but not ABA-induced stomatal closure. We also investigated the effects of FLU and the aba2-2 mutation on cytosolic free calcium concentration ([Ca2+]cyt) in guard cells using a Ca2+-reporter fluorescent protein, Yellow Cameleon 3.6. In wild-type guard cells, FLU inhibited MeJA-induced [Ca2+]cyt elevation but not ABA-induced [Ca2+]cyt elevation. The aba2-2 mutation did not affect ABA-elicited [Ca2+]cyt elevation but suppressed MeJA-induced [Ca2+]cyt elevation. We also tested the effects of the aba2-2 mutation and FLU on the expression of MeJA-inducible VEGETATIVE STORAGE PROTEIN1 (VSP1). In the aba2-2 mutant, MeJA did not induce VSP1 expression. In wild-type leaves, FLU inhibited MeJA-induced VSP1 expression. Pretreatment with ABA at 0.1 μm, which is not enough concentration to evoke ABA responses in the wild type, rescued the observed phenotypes of the aba2-2 mutant. Finally, we found that in wild-type leaves, MeJA stimulates the expression of 9-CIS-EPOXYCAROTENOID DIOXYGENASE3, which encodes a crucial enzyme in ABA biosynthesis. These results suggest that endogenous ABA could be involved in MeJA signal transduction and lead to stomatal closure in Arabidopsis guard cells.

DOI： 10.1104/pp.111.172254

Involvement of extracellular oxidative burst in salicylic acid-induced stomatal closure in Arabidopsis Reviewed International coauthorship International journal

Md. Atiqur Rahman Khokon, Eiji Okuma, Mohammad Anowar Hossain, Shintaro Munemasa, Misugi Uraji, Yoshimasa Nakamura, Izumi C. Mori, Yoshiyuki Murata

Plant, Cell & Environment 34 ( 3 ) 434 - 443 2011.3

The Arabidopsis Calcium-Dependent Protein Kinase, CPK6, Functions as a Positive Regulator of Methyl Jasmonate Signaling in Guard Cells Reviewed International coauthorship International journal

Shintaro Munemasa, Mohammad Anowar Hossain, Yoshimasa Nakamura, Izumi C. Mori, Yoshiyuki Murata

Plant Physiology 155 ( 1 ) 553 - 561 2011.1

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal) Publisher：Oxford University Press (OUP)

<title>Abstract</title>
Previous studies have demonstrated that methyl jasmonate (MeJA) induces stomatal closure dependent on change of cytosolic free calcium concentration in guard cells. However, these molecular mechanisms of intracellular Ca2+ signal perception remain unknown. Calcium-dependent protein kinases (CDPKs) function as Ca2+ signal transducers in various plant physiological processes. It has been reported that four Arabidopsis (Arabidopsis thaliana) CDPKs, CPK3, CPK6, CPK4, and CPK11, are involved in abscisic acid signaling in guard cells. It is also known that there is an interaction between MeJA and abscisic acid signaling in guard cells. In this study, we examined the roles of these CDPKs in MeJA signaling in guard cells using Arabidopsis mutants disrupted in the CDPK genes. Disruption of the CPK6 gene impaired MeJA-induced stomatal closure, but disruption of the other CDPK genes did not. Despite the broad expression pattern of CPK6, we did not find other remarkable MeJA-insensitive phenotypes in the cpk6-1 mutant. The whole-cell patch-clamp analysis revealed that MeJA activation of nonselective Ca2+-permeable cation channels is impaired in the cpk6-1 mutant. Consistent with this result, MeJA-induced transient cytosolic free calcium concentration increments were reduced in the cpk6-1 mutant. MeJA failed to activate slow-type anion channels in the cpk6-1 guard cells. Production of early signal components, reactive oxygen species and nitric oxide, in guard cells was elicited by MeJA in the cpk6-1 mutant as in the wild type. These results provide genetic evidence that CPK6 has a different role from CPK3 and functions as a positive regulator of MeJA signaling in Arabidopsis guard cells.

DOI： 10.1104/pp.110.162750

Chitosan-Induced Stomatal Closure Accompanied by Peroxidase-Mediated Reactive Oxygen Species Production inArabidopsis Reviewed International coauthorship International journal

Md. Atiqur Rahman Khokon, Misugi Uraji, Shintaro Munemasa, Eiji Okuma, Yoshimasa Nakamura, Izumi C. Mori, Yoshiyuki Murata

Bioscience, Biotechnology, and Biochemistry 74 ( 11 ) 2313 - 2315 2010.11

Language：English Publishing type：Research paper (scientific journal) Publisher：Oxford University Press (OUP)

Chitosan induced stomatal closure in wild type-plants and NADPH oxidase knock-out mutants (atrbohD atrbohF), and reactive oxygen species (ROS) production in wild-type guard cells. Closure and production were completely abolished by catalase and a peroxidase inhibitor. These results indicate that chitosan induces ROS production mediated by peroxidase, resulting in stomatal closure.

DOI： 10.1271/bbb.100340

Yeast Elicitor-Induced Stomatal Closure and Peroxidase-Mediated ROS Production in Arabidopsis Reviewed International coauthorship International journal

Md. Atiqur Rahman Khokon, Mohammad Anowar Hossain, Shintaro Munemasa, Misugi Uraji, Yoshimasa Nakamura, Izumi C. Mori, Yoshiyuki Murata

Plant and Cell Physiology 51 ( 11 ) 1915 - 1921 2010.11

Language：English Publishing type：Research paper (scientific journal) Publisher：OXFORD UNIV PRESS

Yeast elicitor (YEL) induces stomatal closure. We investigated reactive oxygen species (ROS) production, nitric oxide (NO) production and [Ca(2+)](cyt) oscillations to clarify YEL signaling in Arabidopsis guard cells. YEL induced ROS accumulation in guard cells. A peroxidase inhibitor [salicylhydroxamic acid (SHAM)] inhibited the stomatal closure and the ROS accumulation, but neither the atrbohD atrbohF mutation nor an NADPH oxidase inhibitor [diphenylene iodonium chloride (DPI)] had any effect. An NO scavenger [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3oxide (cPTIO)] inhibited the YEL-induced stomatal closure and SHAM abolished NO production. YEL-elicited [Ca(2+)] cyt oscillations were inhibited by SHAM but not by the atrbohD atrbohF mutation. These results indicate that YEL induces stomatal closure accompanied by ROS production mediated by peroxidases and NO production.

DOI： 10.1093/pcp/pcq145

Cytosolic Alkalization and Cytosolic Calcium Oscillation in Arabidopsis Guard Cells Response to ABA and MeJA Reviewed International coauthorship International journal

Mohammad Mahbub Islam, Mohammad Anowar Hossain, Rayhanur Jannat, Shintaro Munemasa, Yoshimasa Nakamura, Izumi C. Mori, Yoshiyuki Murata

Plant and Cell Physiology 51 ( 10 ) 1721 - 1730 2010.10

Closing Plant Stomata Requires a Homolog of an Aluminum-Activated Malate Transporter Reviewed International coauthorship International journal

Takayuki Sasaki, Izumi C. Mori, Takuya Furuichi, Shintaro Munemasa, Kiminori Toyooka, Ken Matsuoka, Yoshiyuki Murata, Yoko Yamamoto

Plant and Cell Physiology 51 ( 3 ) 354 - 365 2010.3

Roles of AtTPC1, Vacuolar Two Pore Channel 1, in Arabidopsis Stomatal Closure Reviewed International coauthorship International journal

Mohammad Mahbub Islam, Shintaro Munemasa, Mohammad Anowar Hossain, Yoshimasa Nakamura, Izumi C. Mori, Yoshiyuki Murata

Plant and Cell Physiology 51 ( 2 ) 302 - 311 2010.2

Language：English Publishing type：Research paper (scientific journal) Publisher：OXFORD UNIV PRESS

Abscisic acid (ABA) induces production of reactive oxygen species (ROS) and nitric oxide (NO), elevation of the cytosolic free calcium ion concentration ([Ca(2+)](cyt)) and cytosolic pH (pH(cyt)), and activation of S-type anion channels in guard cells, causing stomatal closure. To investigate whether Arabidopsis Two pore channel 1 (AtTPC1) that encodes the slow vacuolar (SV) channel is involved in stomatal closure, we examined stomatal movements and mobilization of second messengers in the attpc1-2 loss-of-function mutant in response to ABA, methyl jasmonate (MeJA) and Ca(2+). Both ABA and MeJA elicited production of ROS and NO, [Ca(2+)] cyt oscillations, cytosolic alkalization and activation of S-type anion channel currents to lead to stomatal closure in the attpc1-2 mutant as well as the wild type. Unlike the wild type, in the attpc1-2 mutant exogenous Ca(2+) neither induced stomatal closure nor activated plasma membrane S-type anion channel currents despite [Ca(2+)] cyt elevation. These results indicate that AtTPC1 functions in response to external Ca(2+) but not to ABA and MeJA in Arabidopsis guard cells and suggest that AtTPC1 could be involved in priming of plasma membrane S-type anion channels by external Ca(2+) in Arabidopsis guard cells.

DOI： 10.1093/pcp/pcq001

MAP kinases MPK9 and MPK12 are preferentially expressed in guard cells and positively regulate ROS-mediated ABA signaling Reviewed International coauthorship International journal

Fabien Jammes, Charlotte Song, Dongjin Shin, Shintaro Munemasa, Kouji Takeda, Dan Gu, Daeshik Cho, Sangmee Lee, Roberta Giordo, Somrudee Sritubtim, Nathalie Leonhardt, Brian E. Ellis, Yoshiyuki Murata, June M. Kwak

Proceedings of the National Academy of Sciences of the United States of America 106 ( 48 ) 20520 - 20525 2009.12

Purification of the functional plant membrane channel KAT1 Reviewed International journal

Takao Hibi, Shiho Aoki, Keisuke Oda, Shintaro Munemasa, Shunsuke Ozaki, Osamu Shirai, Yoshiyuki Murata, Nobuyuki Uozumi

Biochemical and Biophysical Research Communications 374 ( 3 ) 465 - 469 2008.9

Roles of RCN1, Regulatory A Subunit of Protein Phosphatase 2A, in Methyl Jasmonate Signaling and Signal Crosstalk between Methyl Jasmonate and Abscisic Acid Reviewed International journal

Naoki Saito, Shintaro Munemasa, Yoshimasa Nakamura, Yasuaki Shimoishi, Izumi C. Mori, Yoshiyuki Murata

Plant and Cell Physiology 49 ( 9 ) 1396 - 1401 2008.8

The coronatine-insensitive 1 Mutation Reveals the Hormonal Signaling Interaction between Abscisic Acid and Methyl Jasmonate in Arabidopsis Guard Cells. Specific Impairment of Ion Channel Activation and Second Messenger Production Reviewed International journal

Shintaro Munemasa, Kenji Oda, Megumi Watanabe-Sugimoto, Yoshimasa Nakamura, Yasuaki Shimoishi, Yoshiyuki Murata

Plant Physiology 143 ( 3 ) 1398 - 1407 2007.3

CDPKs CPK6 and CPK3 Function in ABA Regulation of Guard Cell S-Type Anion- and Ca2+- Permeable Channels and Stomatal Closure Reviewed International coauthorship International journal

Izumi C Mori, Yoshiyuki Murata, Yingzhen Yang, Shintaro Munemasa, Yong-Fei Wang, Shannon Andreoli, Hervé Tiriac, Jose M Alonso, Jeffery F Harper, Joseph R Ecker, June M Kwak, Julian I Schroeder

PLoS Biology 4 ( 10 ) e327 2006.10

Language：English Publishing type：Research paper (scientific journal) Publisher：Public Library of Science (PLoS)

Abscisic acid (ABA) signal transduction has been proposed to utilize cytosolic Ca2+ in guard cell ion channel regulation. However, genetic mutants in Ca2+ sensors that impair guard cell or plant ion channel signaling responses have not been identified, and whether Ca2+-independent ABA signaling mechanisms suffice for a full response remains unclear. Calcium-dependent protein kinases (CDPKs) have been proposed to contribute to central signal transduction responses in plants. However, no Arabidopsis CDPK gene disruption mutant phenotype has been reported to date, likely due to overlapping redundancies in CDPKs. Two Arabidopsis guard cell-expressed CDPK genes, CPK3 and CPK6, showed gene disruption phenotypes. ABA and Ca2+ activation of slow-type anion channels and, interestingly, ABA activation of plasma membrane Ca2+-permeable channels were impaired in independent alleles of single and double cpk3cpk6 mutant guard cells. Furthermore, ABA- and Ca2+-induced stomatal closing were partially impaired in these cpk3cpk6 mutant alleles. However, rapid-type anion channel current activity was not affected, consistent with the partial stomatal closing response in double mutants via a proposed branched signaling network. Imposed Ca2+ oscillation experiments revealed that Ca2+-reactive stomatal closure was reduced in CDPK double mutant plants. However, long-lasting Ca2+-programmed stomatal closure was not impaired, providing genetic evidence for a functional separation of these two modes of Ca2+-induced stomatal closing. Our findings show important functions of the CPK6 and CPK3 CDPKs in guard cell ion channel regulation and provide genetic evidence for calcium sensors that transduce stomatal ABA signaling.

DOI： 10.1371/journal.pbio.0040327

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