Faculty Profiles - KATSUHARA Maki

Papers - KATSUHARA Maki

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Analysis of the effect of permeant solutes on the hydraulic resistance of the plasma membrane in cells of Chara corallina Reviewed

Masashi Tazawa, Randy Wayne, Maki Katsuhara

Protoplasma 2024.10

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

Abstract

In the cells of Chara corallina, permeant monohydric alcohols including methanol, ethanol and 1-propanol increased the hydraulic resistance of the membrane (Lp_m⁻¹). We found that the relative value of the hydraulic resistance (^rLp_m⁻¹) was linearly dependent on the concentration (C_s) of the alcohol. The relationship is expressed in the equation: ^rLp_m⁻¹ = ρ_mC_s + 1, where ρ_m is the hydraulic resistance modifier coefficient of the membrane. Ye et al. (2004) showed that membrane-permeant glycol ethers also increased Lp⁻¹. We used their data to estimate Lp_m⁻¹ and ^rLp_m⁻¹. The values of ^rLp_m⁻¹ fit the above relation we found for alcohols. When we plotted the ρ_m values of all the permeant alcohols and glycol ethers against their molecular weights (MW), we obtained a linear curve with a slope of 0.014 M⁻¹/MW and with a correlation coefficient of 0.99. We analyzed the influence of the permeant solutes on the relative hydraulic resistance of the membrane (^rLp_m⁻¹) as a function of the external (π₀) and internal (π_i) osmotic pressures. The analysis showed that the hydraulic resistance modifier coefficients (ρ_m) were linearly related to the MW of the permeant solutes with a slope of 0.012 M⁻¹/MW and with a correlation coefficient of 0.84. The linear relationship between the effects of permeating solutes on the hydraulic resistance modifier coefficient (ρ_m) and the MW can be explained in terms of the effect of the effective osmotic pressure on the hydraulic conductivity of water channels. The result of the analysis suggests that the osmotic pressure and not the size of the permeant solute as proposed by (Ye et al., J Exp Bot 55:449–461, 2004) is the decisive factor in a solute’s influence on hydraulic conductivity. Thus, characean water channels (aquaporins) respond to permeant solutes with essentially the same mechanism as to impermeant solutes.

DOI： 10.1007/s00709-024-02000-6

Other Link： https://link.springer.com/article/10.1007/s00709-024-02000-6/fulltext.html

In planta evidence that the HAK transporter OsHAK2 is involved in Na+ transport in rice. International journal

Shigeto Morita, Natsumi Tamba, Mineo Shibasaka, Shizuka Sasano, Taiju Kadoike, Yasuyo Urase, Masamitsu Maruyama, Aguri Fukuoka, Junta Yanai, Takehiro Masumura, Yasunari Ogihara, Shigeru Satoh, Kunisuke Tanaka, Maki Katsuhara, Hideki Nakayama

Bioscience, biotechnology, and biochemistry 87 ( 5 ) 482 - 490 2023.4

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

HAK family transporters primarily function as K+ transporters and play major roles in K+ uptake and translocation in plants, whereas several HAK transporters exhibit Na+ transport activity. OsHAK2, a rice HAK transporter, was shown to mediate Na+ transport in Escherichia coli in a previous study. In this study, we investigated whether OsHAK2 is involved in Na+ transport in the rice plant. Overexpression of OsHAK2 increased Na+ translocation from the roots to the shoots of transgenic rice. It also increased both root and whole-plant Na+ content, and enhanced shoot length under low Na+ and K+ conditions. Meanwhile, OsHAK2 overexpression increased salt sensitivity under a long-term salt stress condition, indicating that OsHAK2 is not involved in salt tolerance, unlike in the case of ZmHAK4 in maize. These results suggest that OsHAK2 is permeable to Na+ and contributes to shoot growth in rice plants under low Na+ and K+ conditions.

DOI： 10.1093/bbb/zbad020

Distinct Functions of the Atypical Terminal Hydrophilic Domain of the HKT Transporter in the Liverwort Marchantia polymorpha. Reviewed International coauthorship International journal

Shahin Imran, Masumi Oyama, Rie Horie, Natsuko I Kobayashi, Alex Costa, Ryosuke Kumano, Chiho Hirata, Sen Thi Huong Tran, Maki Katsuhara, Keitaro Tanoi, Takayuki Kohchi, Kimitsune Ishizaki, Tomoaki Horie

Plant & Cell Physiology 63 ( 6 ) 802 - 816 2022.6

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

K+/Na+ homeostasis is important for land plants, particularly under salt stress. In this study, the structure and ion transport properties of the high-affinity K+ transporter (HKT) of the liverwort Marchantia polymorpha were investigated. Only one HKT gene, MpHKT1, was identified in the genome of M. polymorpha. Phylogenetic analysis of HKT proteins revealed that non-seed plants possess HKTs grouped into a clade independent of the other two clades including HKTs of angiosperms. A distinct long hydrophilic domain was found in the C-terminus of MpHKT1. Complementary DNA (cDNA) of truncated MpHKT1 (t-MpHKT1) encoding the MpHKT_Δ596-812 protein was used to examine the functions of the C-terminal domain. Both MpHKT1 transporters fused with enhanced green fluorescent protein at the N-terminus were localized to the plasma membrane when expressed in rice protoplasts. Two-electrode voltage clamp experiments using Xenopus laevis oocytes indicated that MpHKT1 mediated the transport of monovalent alkali cations with higher selectivity for Na+ and K+, but truncation of the C-terminal domain significantly reduced the transport activity with a decrease in the Na+ permeability. Overexpression of MpHKT1 or t-MpHKT1 in M. polymorpha conferred accumulation of higher Na+ levels and showed higher Na+ uptake rates, compared to those of wild-type plants; however, phenotypes with t-MpHKT1 were consistently weaker than those with MpHKT1. Together, these findings suggest that the hydrophilic C-terminal domain plays a unique role in the regulation of transport activity and ion selectivity of MpHKT1.

DOI： 10.1093/pcp/pcac044

Calcium control of the hydraulic resistance in cells of Chara corallina Reviewed International coauthorship International journal

Masashi Tazawa, Maki Katsuhara, Randy Wayne

Protoplasma 260 ( 1 ) 299 - 306 2022.6

Identification and Characterization of Rice OsHKT1;3 Variants Reviewed International journal

Shahin Imran, Yoshiyuki Tsuchiya, Sen Thi Huong Tran, Maki Katsuhara

Plants 10 ( 10 ) 2006 - 2006 2021.9

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

In rice, the high-affinity K+ transporter, OsHKT1;3, functions as a Na+-selective transporter. mRNA variants of OsHKT1;3 have been reported previously, but their functions remain unknown. In this study, five OsHKT1;3 variants (V1-V5) were identified from japonica rice (Nipponbare) in addition to OsHKT1;3_FL. Absolute quantification qPCR analyses revealed that the transcript level of OsHKT1;3_FL was significantly higher than other variants in both the roots and shoots. Expression levels of OsHKT1;3_FL, and some variants, increased after 24 h of salt stress. Two electrode voltage clamp experiments in a heterologous expression system using Xenopus laevis oocytes revealed that oocytes expressing OsHKT1;3_FL and all of its variants exhibited smaller Na+ currents. The presented data, together with previous data, provide insights to understanding how OsHKT family members are involved in the mechanisms of ion homeostasis and salt tolerance in rice.

DOI： 10.3390/plants10102006

Physiological Role of Aerobic Fermentation Constitutively Expressed in an Aluminum-Tolerant Cell Line of Tobacco (Nicotiana tabacum) Reviewed International journal

Tsuchiya Y, Nakamura T, Izumi Y, Okazaki K, Shinano T, Kubo Y, Katsuhara M, Sasaki T, Yamamoto Y

Plant & Cell Physiology 62 ( 9 ) 1460 - 1477 2021.9

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

<title>Abstract</title>
Aluminum (Al)-tolerant tobacco cell line ALT301 derived from SL (wild-type) hardly exhibits Al-triggered reactive oxygen species (ROS) compared with SL. Molecular mechanism leading to this phenotype was investigated comparatively with SL. Under normal growth condition, metabolome data suggested the activation of glycolysis and lactate fermentation but the repression of the tricarboxylic acid (TCA) cycle in ALT301, namely aerobic fermentation, which seemed to be transcriptionally controlled partly by higher expression of genes encoding lactate dehydrogenase and pyruvate dehydrogenase kinase. Microarray and gene ontology analyses revealed the upregulation of th e gene encoding related to APETALA2.3 (RAP2.3)-like protein, one of the group VII ethylene response factors (ERFVIIs), in ALT301. ERFVII transcription factors are known to be key regulators for hypoxia response that promotes substrate-level ATP production by glycolysis and fermentation. ERFVIIs are degraded under normoxia by the N-end rule pathway of proteolysis depending on both oxygen and nitric oxide (NO), and NO is produced mainly by nitrate reductase (NR) in plants. In ALT301, levels of the NR gene expression (NIA2), NR activity and NO production were all lower compared with SL. Consistently, the known effects of NO on respiratory pathways were also repressed in ALT301. Under Al-treatment condition, NO level increased in both lines but was lower in ALT301. These results suggest that the upregulation of the RAP2.3-like gene and the downregulation of the NIA2 gene and resultant NO depletion in ALT301 coordinately enhance aerobic fermentation, which seems to be related to a higher capacity to prevent ROS production in mitochondria under Al stress.

DOI： 10.1093/pcp/pcab098

Age dependence of the hydraulic resistances of the plasma membrane and the tonoplast (vacuolar membrane) in cells of Chara corallina Reviewed International coauthorship International journal

Masashi Tazawa, Maki Katsuhara, Randy Wayne

Protoplasma 258 ( 4 ) 793 - 801 2021.7

Erratum: Physiological role of aerobic fermentation constitutively expressed in an aluminum-tolerant cell line of tobacco (Nicotiana Tabacum) (Plant and Cell Physiology DOI: 10.1093/pcp/pcab098)

Yoshiyuki Tsuchiya, Takuji Nakamura, Yohei Izumi, Keiki Okazaki, Takuro Shinano, Yasutaka Kubo, Maki Katsuhara, Takayuki Sasaki, Yoko Yamamoto

Plant and Cell Physiology 62 ( 6 ) 1058 2021.6

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

In the originally published version of this manuscript, the ethylene signaling pathway in Arabidopsis shown in the Figure 3 was incorrectly referred. The correct pathway is as follows: Ethylene -| ETR1 -&gt
CTR1 -| EIN2 -&gt
EIN3/EIL1 -&gt
ERFs. In addition, information was missing from the Figure 3 caption. An explanation of the subgroups of the ERF family shown in the figure (Group IX, VIII, VI, VII) has been added to the caption, which also necessitated two new references in the paper. The corrected caption is: Expression of the genes encoding transcription factors (EIL, ERFs) and chitinases (CHNs) associated with ethylene signaling pathway in SL and ALT301 under normal growth condition. Gene expression analysis was performed by real-time RT-PCR. Values are the mean±SE (n=3–5 from three to five independent experiments, respectively). Significant differences between SL and ALT301 are indicated with asterisks (*P &lt
0.05, **P &lt
0.01, Welch’s t-test). Ethylene signaling pathway in Arabidopsis (Kazan 2015) is shown (top). Group names of the ERF genes are indicated, which is based on Rushton et al. (2008) and Nakano et al. (2006). Note that some of the ERF genes have several synonyms (see the National Center for Biotechnology Information [NCBI] database using the gene accessions listed in Supplementary Table S6). Abbreviations: ETR1, Ethylene Response1
CTR1, Constitutive Triple Response1
EIN2, Ethylene Insensitive2
EIN3, Ethylene Insensitive3
EIL1, Ethylene Insensitive-Like Protein1
ERF, Ethylene Response Factor. The gene Tsi1 encodes Tobacco stress-induced gene1. The two new references are: Nakano, T., Suzuki, K., Fujimura, T. and Shinshi, H. (2006) Genome-wide analysis of the ERF gene family in Arabidopsis and rice. Plant Physiol. 140: 411–432. Rushton, P.J., Bokowiec, M.T., Han, S., Zhang, H., Brannock, J.F., Chen, X., et al. (2008) Tobacco transcription factors: novel insights into transcriptional regulation in the Solanaceae. Plant Physiol. 147: 280–295. These errors have been corrected.

DOI： 10.1093/pcp/pcab131

Mechanisms Activating Latent Functions of PIP Aquaporin Water Channels via the Interaction between PIP1 and PIP2 Proteins Reviewed International journal

Mineo Shibasaka, Tomoaki Horie, Maki Katsuhara

Plant & Cell Physiology 62 ( 1 ) 92 - 99 2021.3

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

<title>Abstract</title>
Plant plasma membrane-type plasma membrane intrinsic protein (PIP) aquaporins are classified into two groups, PIP1s and PIP2s. In this study, we focused on HvPIP1;2, a PIP1 in barley (Hordeum vulgare), to dissect the molecular mechanisms that evoke HvPIP1-mediated water transport. No HvPIP1;2 protein was localized to the plasma membrane when expressed alone in Xenopus laevis oocytes. By contrast, a chimeric HvPIP1;2 protein (HvPIP1;2_24NC), in which the N- and C-terminal regions were replaced with the corresponding regions from HvPIP2;4, was found to localize to the plasma membrane of oocytes. However, HvPIP1;2_24NC showed no water transport activity in swelling assays. These results suggested that the terminal regions of PIP2 proteins direct PIP proteins to the plasma membrane, but the relocalization of PIP1 proteins was not sufficient to PIP1s functionality as a water channel in a membrane. A single amino acid replacement of threonine by methionine in HvPIP2;4 (HvPIP2;4T229M) abolished water transport activity. Co-expression of HvPIP1;2_24NC either with HvPIP2;4_12NC or with HvPIP2;4TM_12NC, in which the N- and C-terminal regions were replaced with the corresponding regions of HvPIP1;2, increased the water transport activity in oocytes. These data provided evidence that the HvPIP1;2 molecule has own water transport activity and an interaction with the middle part of the HvPIP2;4 protein (except for the N- and C-termini) is required for HvPIP1;2 functionality as a water channel. This molecular mechanism could be applied to other PIP1s and PIP2s in addition to the known mechanism that the terminal regions of some PIP2s lead some PIP1s to the plasma membrane.

DOI： 10.1093/pcp/pcaa142

Other Link： http://academic.oup.com/pcp/article-pdf/62/1/92/36718180/pcaa142.pdf

Accession difference in leaf photosynthesis, root hydraulic conductance and gene expression of root aquaporins under salt stress in barley seedlings Reviewed International coauthorship International journal

Asuka Kodama, Tamaki Watanabe, Makoto Yamaguchi, Ryohei Narita, Maki Katsuhara, Kazuhiro Sato, Taiichiro Ookawa, Tadashi Hirasawa

Plant Production Science 24 ( 1 ) 73 - 82 2021.1

A Survey of Barley PIP Aquaporin Ionic Conductance Reveals Ca2+-Sensitive HvPIP2;8 Na+ and K+ Conductance Reviewed International coauthorship International journal

Sen Thi Huong Tran, Tomoaki Horie, Shahin Imran, Jiaen Qiu, Samantha McGaughey, Caitlin S. Byrt, Stephen D. Tyerman, Maki Katsuhara

International Journal of Molecular Sciences 21 ( 19 ) 7135 - 7135 2020.9

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

Some plasma membrane intrinsic protein (PIP) aquaporins can facilitate ion transport. Here we report that one of the 12 barley PIPs (PIP1 and PIP2) tested, HvPIP2;8, facilitated cation transport when expressed in Xenopus laevis oocytes. HvPIP2;8-associated ion currents were detected with Na+ and K+, but not Cs+, Rb+, or Li+, and was inhibited by Ba2+, Ca2+, and Cd2+ and to a lesser extent Mg2+, which also interacted with Ca2+. Currents were reduced in the presence of K+, Cs+, Rb+, or Li+ relative to Na+ alone. Five HvPIP1 isoforms co-expressed with HvPIP2;8 inhibited the ion conductance relative to HvPIP2;8 alone but HvPIP1;3 and HvPIP1;4 with HvPIP2;8 maintained the ion conductance at a lower level. HvPIP2;8 water permeability was similar to that of a C-terminal phosphorylation mimic mutant HvPIP2;8 S285D, but HvPIP2;8 S285D showed a negative linear correlation between water permeability and ion conductance that was modified by a kinase inhibitor treatment. HvPIP2;8 transcript abundance increased in barley shoot tissues following salt treatments in a salt-tolerant cultivar Haruna-Nijo, but not in salt-sensitive I743. There is potential for HvPIP2;8 to be involved in barley salt-stress responses, and HvPIP2;8 could facilitate both water and Na+/K+ transport activity, depending on the phosphorylation status.

DOI： 10.3390/ijms21197135

Na+ Transporter SvHKT1;1 from a Halophytic Turf Grass Is Specifically Upregulated by High Na+ Concentration and Regulates Shoot Na+ Concentration Reviewed International journal

Yuki Kawakami, Shahin Imran, Maki Katsuhara, Yuichi Tada

International Journal of Molecular Sciences 21 ( 17 ) 6100 - 6100 2020.8

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

We characterized an Na+ transporter SvHKT1;1 from a halophytic turf grass, Sporobolus virginicus. SvHKT1;1 mediated inward and outward Na+ transport in Xenopus laevis oocytes and did not complement K+ transporter-defective mutant yeast. SvHKT1;1 did not complement athkt1;1 mutant Arabidopsis, suggesting its distinguishable function from other typical HKT1 transporters. The transcript was abundant in the shoots compared with the roots in S. virginicus and was upregulated by severe salt stress (500 mM NaCl), but not by lower stress. SvHKT1;1-expressing Arabidopsis lines showed higher shoot Na+ concentrations and lower salt tolerance than wild type (WT) plants under nonstress and salt stress conditions and showed higher Na+ uptake rate in roots at the early stage of salt treatment. These results suggested that constitutive expression of SvHKT1;1 enhanced Na+ uptake in root epidermal cells, followed by increased Na+ transport to shoots, which led to reduced salt tolerance. However, Na+ concentrations in phloem sap of the SvHKT1;1 lines were higher than those in WT plants under salt stress. Based on this result, together with the induction of the SvHKT1;1 transcription under high salinity stress, it was suggested that SvHKT1;1 plays a role in preventing excess shoot Na+ accumulation in S. virginicus.

DOI： 10.3390/ijms21176100

Ectopic expression of a rice plasma membrane intrinsic protein (OsPIP1;3) promotes plant growth and water uptake Reviewed International coauthorship International journal

Siyu Liu, Tatsuya Fukumoto, Patrizia Gena, Peng Feng, Qi Sun, Qiang Li, Tadashi Matsumoto, Toshiyuki Kaneko, Hang Zhang, Yao Zhang, Shihua Zhong, Weizhong Zeng, Maki Katsuhara, Yoshichika Kitagawa, Aoxue Wang, Giuseppe Calamita, Xiaodong Ding

The Plant Journal 102 ( 4 ) 779 - 796 2020.5

Expression and Ion Transport Activity of Rice OsHKT1;1 Variants Reviewed International journal

Shahin Imran, Tomoaki Horie, Maki Katsuhara

Plants 9 ( 1 ) 16 - 16 2019.12

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

OsHKT1;1 in rice, belongs to the high-affinity K+ Transporter family, has been found to be involved in salt tolerance. OsHKT1;1 in japonica rice (Nipponbare) produces mRNA variants, but their functions remain elusive. In salt tolerant rice, Pokkali, eight OsHKT1;1 variants (V1-V8) were identified in addition to the full-length OsHKT1;1 (FL) cDNA. Absolute quantification by qPCR revealed that accumulation of OsHKT1;1-FL mRNA is minor in contrast to that of OsHKT1;1-V1, -V2, -V4, and -V7 mRNAs, all of which are predominant in shoots, while only V1 and V7 mRNAs are predominant in roots. Two electrode voltage clamp (TEVC) experiments using Xenopus laevis oocytes revealed that oocytes-expressing OsHKT1;1-FL from Pokkali exhibited inward-rectified currents in the presence of 96 mM Na+ as reported previously. Further TEVC analyses indicated that six of eight OsHKT1;1 variants elicited currents in a Na+ or a K+ bath solution. OsHKT1;1-V6 exhibited a similar inward rectification to the FL protein. Contrastingly, however, the rests mediated bidirectional currents in both Na+ and K+ bath solutions. These data suggest possibilities that novel mechanisms regulating the transport activity of OsHKT1;1 might exist, and that OsHKT1;1 variants might also carry out distinct physiological roles either independently or in combination with OsHKT1;1-FL.

DOI： 10.3390/plants9010016

Dynamics of the contents and distribution of ABA, auxins and aquaporins in developing caryopses of an ABA-deficient barley mutant and its parental cultivar Reviewed

Oksana A. Seldimirova, Guzel R. Kudoyarova, Maki Katsuhara, Ilshat R. Galin, Denis Yu. Zaitsev, Natalia N. Kruglova, Dmitry S. Veselov, Stanislav Yu. Veselov

Seed Science Research 29 ( 4 ) 261 - 269 2019.12

Language：English Publishing type：Research paper (scientific journal) Publisher：Cambridge University Press (CUP)

<title>Abstract</title>Dynamics of abscisic acid (ABA) and indole-3-acetic acid (IAA) contents were followed in developing barley caryopses of the ABA-deficient mutant AZ34 and its parental cultivar Steptoe. Distribution of these hormones and HvPIP2 aquaporins (AQPs) was studied with the help of immunohistochemical methods in the roots and coleorhiza of developing embryos. In Steptoe, maturation of the caryopsis was accompanied by vast accumulation of ABA, while this hormone accumulated more slowly in the caryopsis of AZ34 and its content was lower than in Steptoe. Accumulation of ABA was accompanied by a decline in IAA level in the developing caryopsis, the process being delayed in AZ34 in accordance with the slower accumulation of ABA. ABA accumulated to high levels in the coleorhiza cells of Steptoe, while the effect was absent in AZ34. The high level of ABA was likely to be important for maintaining the barrier function of the coleorhiza, preventing germination of seminal roots and enabling seed dormancy, while the absence of ABA accumulation in coleorhiza of AZ34 may be responsible for the initiation of root germination inside the caryopsis. The abundance of HvPIP2 AQPs in the seminal roots was higher at the beginning of maturation of Steptoe caryopsis and declined afterwards, while the levels of APQs increased later in AZ34 in accordance with the delay in ABA accumulation. These results suggest the importance of ABA accumulation in coleorhiza for preventing precocious growth of seminal roots, and suggest regulation of IAA and aquaporin levels by this hormone during maturation of embryos.

DOI： 10.1017/s0960258519000229

The Mechanism of SO₂ -Induced Stomatal Closure Differs from O₃ and CO₂ Responses and Is Mediated by Non-Apoptotic Cell Death in Guard Cells. Reviewed International journal

Ooi L, Matsuura T, Munemasa S, Murata Y, Katsuhara M, Hirayama T, Mori IC

Plant, cell & environment 42 ( 2 ) 437 - 447 2019.2

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

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

High-affinity K+ transporters from a halophyte, Sporobolus virginicus, mediate both K+ and Na+ transport in transgenic Arabidopsis, X. laevis oocytes, and yeast. Reviewed

Tada Y, Endo C, Katsuhara M, Horie T, Shibasaka M, Nakahara Y, Kurusu T

Plant & Cell Physiology 60 ( 1 ) 176 - 187 2019.1

Functional screening of salt tolerance genes from a halophyte Sporobolus virginicus and transcriptomic and metabolomic analysis of salt tolerant plants expressing glycine-rich RNA-binding protein Reviewed

Yuichi Tada, Ryuichi Kawano, Shiho Komatsubara, Hideki Nishimura, Maki Katsuhara, Soichi Ozaki, Shin Terashima, Kentaro Yano, Chisato Endo, Muneo Sato, Mami Okamoto, Yuji Sawada, Masami Yokota Hirai, Takamitsu Kurusu

Plant Science 278 54 - 63 2019.1

Energy costs of salinity tolerance in crop plants Invited Reviewed

Stephen D. Tyerman, Rana Munns, Wieland Fricke, Borjana Arsova, Bronwyn J. Barkla, Jayakumar Bose, Helen Bramley, Caitlin Byrt, Zhonghua Chen, Timothy D. Colmer, Tracey Cuin, David A. Day, Kylie J. Foster, Matthew Gilliham, Sam W. Henderson, Tomoaki Horie, Colin L. D. Jenkins, Brent N. Kaiser, Maki Katsuhara, Darren Plett, Stanley J. Miklavcic, Stuart J. Roy, Francisco Rubio, Sergey Shabala, Megan Shelden, Kathleen Soole, Nicolas L. Taylor, Mark Tester, Michelle Watt, Stefanie Wege, Lars H. Wegner, Zhengyu Wen

New Phytologist 221 ( 1 ) 25 - 29 2019.1

A Cyclic Nucleotide-Gated Channel, HvCNGC2-3, Is Activated by the Co-Presence of Na+ and K+ and Permeable to Na+ and K+ Non-Selectively Reviewed International journal

Izumi Mori, Yuichi Nobukiyo, Yoshiki Nakahara, Mineo Shibasaka, Takuya Furuichi, Maki Katsuhara

Plants 7 ( 3 ) 61 - 61 2018.7

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

Cyclic nucleotide-gated channels (CNGCs) have been postulated to contribute significantly in plant development and stress resistance. However, their electrophysiological properties remain poorly understood. Here, we characterized barley CNGC2-3 (HvCNGC2-3) by the two-electrode voltage-clamp technique in the Xenopus laevis oocyte heterologous expression system. Current was not observed in X. laevis oocytes injected with HvCNGC2-3 complementary RNA (cRNA) in a bathing solution containing either Na+ or K+ solely, even in the presence of 8-bromoadenosine 3′,5′-cyclic monophosphate (8Br-cAMP) or 8-bromoguanosine 3′,5′-cyclic monophosphate (8Br-cGMP). A weakly voltage-dependent slow hyperpolarization-activated ion current was observed in the co-presence of Na+ and K+ in the bathing solution and in the presence of 10 µM 8Br-cAMP, but not 8Br-cGMP. Permeability ratios of HvCNGC2-3 to K+, Na+ and Cl− were determined as 1:0.63:0.03 according to reversal-potential analyses. Amino-acid replacement of the unique ion-selective motif of HvCNGC2-3, AQGL, with the canonical motif, GQGL, resulted in the abolition of the current. This study reports a unique two-ion-dependent activation characteristic of the barley CNGC, HvCNGC2-3.

DOI： 10.3390/plants7030061

T-DNA Tagging-Based Gain-of-Function of OsHKT1;4 Reinforces Na Exclusion from Leaves and Stems but Triggers Na Toxicity in Roots of Rice Under Salt Stress Reviewed

Oda Y, Kobayashi NI, Tanoi K, Ma JF, Itou Y, Katsuhara M, Itou T, Horie T

International Journal of Molecular Sciences 19 ( 1 ) 2018.1

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

The high affinity K+ transporter 1;4 (HKT1;4) in rice (Oryza sativa), which shows Na+ selective transport with little K+ transport activity, has been suggested to be involved in reducing Na in leaves and stems under salt stress. However, detailed physiological roles of OsHKT1;4 remain unknown. Here, we have characterized a transfer DNA (T-DNA) insertion mutant line of rice, which overexpresses OsHKT1;4, owing to enhancer elements in the T-DNA, to gain an insight into the impact of OsHKT1;4 on salt tolerance of rice. The homozygous mutant (the O/E line) accumulated significantly lower concentrations of Na in young leaves, stems, and seeds than the sibling WT line under salt stress. Interestingly, however, the mutation rendered the O/E plants more salt sensitive than WT plants. Together with the evaluation of biomass of rice lines, rhizosphere acidification assays using a pH indicator bromocresol purple and (NaCl)-Na-22 tracer experiments have led to an assumption that roots of O/E plants suffered heavier damages from Na which excessively accumulated in the root due to increased activity of Na+ uptake and Na+ exclusion in the vasculature. Implications toward the application of the HKT1-mediated Na+ exclusion system to the breeding of salt tolerant crop cultivars will be discussed.

DOI： 10.3390/ijms19010235

Other Link： http://orcid.org/0000-0001-7545-2771

Identification of an H2O2permeable PIP aquaporin in barley and a serine residue promoting H2O2transport Reviewed

Rhee J, Horie T, Sasano S, Nakahara Y, Katsuhara M

Physiologia Plantarum 159 ( 1 ) 120 - 128 2017.1

Exogenous application of abscisic acid (ABA) increases root and cell hydraulic conductivity and abundance of some aquaporin isoforms in the ABA-deficient barley mutant Az34 Reviewed

Guzel Sharipova, Dmitriy Veselov, Guzel Kudoyarova, Wieland Fricke, Ian C. Dodd, Maki Katsuhara, Takuya Furuichi, Igor Ivanov, Stanislav Veselov

Annals of Botany 118 ( 4 ) 777 - 785 2016.10

Genome-Wide Characterization of Major Intrinsic Proteins in Four Grass Plants and Their Non-Aqua Transport Selectivity Profiles with Comparative Perspective

Abul Kalam Azad, Jahed Ahmed, Md. Asraful Alum, Md. Mahbub Hasan, Takahiro Ishikawa, Yoshihiro Sawa, Maki Katsuhara

PLOS ONE 11 ( 6 ) e0157735 2016.6

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

Major intrinsic proteins (MIPs), commonly known as aquaporins, transport not only water in plants but also other substrates of physiological significance and heavy metals. In most of the higher plants, MIPs are divided into five subfamilies (PIPs, TIPs, NIPs, SIPs and XIPs). Herein, we identified 68, 42, 38 and 28 full-length MIPs, respectively in the genomes of four monocot grass plants, specifically Panicum virgatum, Setaria italica, Sorghum bicolor and Brachypodium distachyon. Phylogenetic analysis showed that the grass plants had only four MIP subfamilies including PIPs, TIPs, NIPs and SIPs without XIPs. Based on structural analysis of the homology models and comparing the primary selectivity-related motifs [two NPA regions, aromatic/arginine (ar/R) selectivity filter and Froger's positions (FPs)] of all plant MIPs that have been experimentally proven to transport non-aqua substrates, we predicted the transport profiles of all MIPs in the four grass plants and also in eight other plants. Groups of MIP subfamilies based on ar/R selectivity filter and FPs were linked to the non-aqua transport profiles. We further deciphered the substrate selectivity profiles of the MIPs in the four grass plants and compared them with their counterparts in rice, maize, soybean, poplar, cotton, Arabidopsis thaliana, Physcomitrella patens and Selaginella moellendorffii. In addition to two NPA regions, ar/R filter and FPs, certain residues, especially in loops B and C, contribute to the functional distinctiveness of MIP groups. Expression analysis of transcripts in different organs indicated that non-aqua transport was related to expression of MIPs since most of the unexpressed MIPs were not predicted to facilitate the transport of non-aqua molecules. Among all MIPs in every plant, TIP (BdTIP1;1, SiTIP1;2, SbTIP2;1 and PvTIP1;2) had the overall highest mean expression. Our study generates significant information for understanding the diversity, evolution, non-aqua transport profiles and insight into comparative transport selectivity of plant MIPs, and provides tools for the development of transgenic plants.

DOI： 10.1371/journal.pone.0157735

Phosphorus deprivation effects on water relations of Nicotiana tabacum plant via reducing plasma membrane permeability

M. Mahdieh, A. Mostajeran, M. Katsuhara

RUSSIAN JOURNAL OF PLANT PHYSIOLOGY 63 ( 1 ) 54 - 61 2016.1

Language：English Publishing type：Research paper (scientific journal) Publisher：MAIK NAUKA/INTERPERIODICA/SPRINGER

Plants grown in phosphorus-deprived solutions often exhibit disruption of water transport due to reduction in root hydraulic conductivity (Lp (r) ). To uncover the relationship between root Lp (r) and water permeability coefficient (P (f)) of plasma membrane and the role of aquaporins, we evaluated P (f) of plasma membrane and also PIP-type aquaporin gene expression in tobacco (Nicotiana tabacum L.) plant roots after seven days P-deprivation. The results showed significant reduction in sap flow rate (J (v)) and osmotic root hydraulic conductivity (L (pr-o)) in P-deprived roots. These effects were reversed 24 h after P-resupplying. Interestingly, the P (f) of root protoplasts was 57% lower in P-deprived plants compared with P-sufficient ones. The expression of NtPIP1;1 and NtPIP2;1 aquaporins did not change significantly in P-deprived plants compared with P-sufficient ones, but the copy number of NtAQP1 increased significantly in P-deprived plants. P-deprivation did not change Lp r-o significantly in antisense NtAQP1 plants. Taken together, these findings suggest that P-deprivation may play an important role in modulation of root hydraulic conductivity by affecting P (f) in transcellular pathway of water flow across roots and aquaporins. Finally, we concluded that dominant water transport pathway under P-deprivation was transcellular one.

DOI： 10.1134/S102144371601012X

OsHKT1;4-mediated Na+ transport in stems contributes to Na+ exclusion from leaf blades of rice at the reproductive growth stage upon salt stress

Kei Suzuki, Naoki Yamaji, Alex Costa, Eiji Okuma, Natsuko I. Kobayashi, Tatsuhiko Kashiwagi, Maki Katsuhara, Cun Wang, Keitaro Tanoi, Yoshiyuki Murata, Julian I. Schroeder, Jian Feng Ma, Tomoaki Horie

BMC PLANT BIOLOGY 16 ( 1 ) 22 2016.1

Language：English Publishing type：Research paper (scientific journal) Publisher：BIOMED CENTRAL LTD

Background: Na+ exclusion from leaf blades is one of the key mechanisms for glycophytes to cope with salinity stress. Certain class I transporters of the high-affinity K+ transporter (HKT) family have been demonstrated to mediate leaf blade-Na+ exclusion upon salinity stress via Na+-selective transport. Multiple HKT1 transporters are known to function in rice (Oryza sativa). However, the ion transport function of OsHKT1;4 and its contribution to the Na+ exclusion mechanism in rice remain to be elucidated.
Results: Here, we report results of the functional characterization of the OsHKT1; 4 transporter in rice. OsHKT1; 4 mediated robust Na+ transport in Saccharomyces cerevisiae and Xenopus laevis oocytes. Electrophysiological experiments demonstrated that OsHKT1; 4 shows strong Na+ selectivity among cations tested, including Li+, Na+, K+, Rb+, Cs+, and NH4+, in oocytes. A chimeric protein, EGFP-OsHKT1;4, was found to be functional in oocytes and targeted to the plasma membrane of rice protoplasts. The level of OsHKT1; 4 transcripts was prominent in leaf sheaths throughout the growth stages. Unexpectedly however, we demonstrate here accumulation of OsHKT1;4 transcripts in the stem including internode II and peduncle in the reproductive growth stage. Moreover, phenotypic analysis of OsHKT1;4 RNAi plants in the vegetative growth stage revealed no profound influence on the growth and ion accumulation in comparison with WT plants upon salinity stress. However, imposition of salinity stress on the RNAi plants in the reproductive growth stage caused significant Na+ overaccumulation in aerial organs, in particular, leaf blades and sheaths. In addition, Na-22(+) tracer experiments using peduncles of RNAi and WT plants suggested xylem Na+ unloading by OsHKT1;4.
Conclusions: Taken together, our results indicate a newly recognized function of OsHKT1;4 in Na+ exclusion in stems together with leaf sheaths, thus excluding Na+ from leaf blades of a japonica rice cultivar in the reproductive growth stage, but the contribution is low when the plants are in the vegetative growth stage.

DOI： 10.1186/s12870-016-0709-4

OsHKT2;2/1-mediated Na+ influx over K+ uptake in roots potentially increases toxic Na+ accumulation in a salt-tolerant landrace of rice Nona Bokra upon salinity stress

Kei Suzuki, Alex Costa, Hideki Nakayama, Maki Katsuhara, Atsuhiko Shinmyo, Tomoaki Horie

JOURNAL OF PLANT RESEARCH 129 ( 1 ) 67 - 77 2016.1

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

HKT transporters are Na+-permeable membrane proteins, which mediate Na+ and K+ homeostasis in K+-depleted and saline environments in plants. Class II HKT transporters, a distinct subgroup found predominantly in monocots, are known to mediate Na+-K+ co-transport in principle. Here we report features of ion transport functions of No-OsHKT2;2/1, a class II transporter identified in a salt tolerant landrace of indica rice, Nona Bokra. We profiled No-OsHKT2;2/1 expression in organs of Nona Bokra plants with or without salinity stress. Dominant accumulation of the No-OsHKT2;2/1 transcript in K+-starved roots of Nona Bokra plants largely disappeared in response to 50 mM NaCl. We found that No-OsHKT2;2/1 expressed in the high-affinity K+ uptake deficient mutant of Saccharomyces cerevisiae and Xenopus laevis oocytes shows robust K+ selectivity even in the presence of a large amount of NaCl as reported previously. However, No-OsHKT2;2/1-expressing yeast cells exhibited Na+ hypersensitive growth under various concentrations of K+ and Na+ as the cells expressing Po-OsHKT2;2, a similar class II transporter from another salt tolerant indica rice Pokkali, when compared with the growth of cells harboring empty vector or cells expressing OsHKT2;4. The OsHKT2;4 protein expressed in Xenopus oocytes showed strong K+ selectivity in the presence of 50 mM NaCl in comparison with No-OsHKT2;2/1 and Po-OsHKT2;2. Together with apparent plasma membrane-localization of No-OsHKT2;2/1, these results point to possibilities that No-OsHKT2;2/1 could mediate destructive Na+ influx over K+ uptake in Nona Bokra plants upon salinity stress, and that a predominant physiological function of No-OsHKT2;2/1 might be the acquisition of Na+ and K+ in K+-limited environments.

DOI： 10.1007/s10265-015-0764-1

Yeast functional screen to identify genes conferring salt stress tolerance in Salicornia europaea

Yoshiki Nakahara, Shogo Sawabe, Kenta Kainuma, Maki Katsuhara, Mineo Shibasaka, Masanori Suzuki, Kosuke Yamamoto, Suguru Oguri, Hikaru Sakamoto

FRONTIERS IN PLANT SCIENCE 6 920 2015.10

Language：English Publishing type：Research paper (scientific journal) Publisher：FRONTIERS MEDIA SA

Salinity is a critical environmental factor that adversely affects crop productivity. Halophytes have evolved various mechanisms to adapt to saline environments. Salicornia europaea L. is one of the most salt tolerant plant species. It does not have special salt-secreting structures like a salt gland or salt bladder, and is therefore a good model for studying the common mechanisms underlying plant salt tolerance. To identify candidate genes encoding key proteins in the mediation of salt tolerance in S. europaea, we performed a functional screen of a cDNA library in yeast. The library was screened for genes that allowed the yeast to grow in the presence of 1.3 M NaCl. We obtained three full-length S. europaea genes that confer salt tolerance. The genes are predicted to encode (1) a novel protein highly homologous to thaumatin-like proteins, (2) a novel coiled-coil protein of unknown function, and (3) a novel short peptide of 32 residues. Exogenous application of a synthetic peptide corresponding to the 32 residues improved salt tolerance of Arabidopsis. The approach described in this report provides a rapid assay system for large-scale screening of S. europaea genes involved in salt stress tolerance and supports the identification of genes responsible for such mechanisms. These genes may be useful candidates for improving crop salt tolerance by genetic transformation.

DOI： 10.3389/fpls.2015.00920

Control of the Water Transport Activity of Barley HvTIP3;1 Specifically Expressed in Seeds

Shigeko Utsugi, Mineo Shibasaka, Masahiko Maekawa, Maki Katsuhara

PLANT AND CELL PHYSIOLOGY 56 ( 9 ) 1831 - 1840 2015.9

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

Tonoplast intrinsic proteins (TIPs) are involved in the transport and storage of water, and control intracellular osmotic pressure by transporting material related to the water potential of cells. In the present study, we focused on HvTIP3;1 during the periods of seed development and desiccation in barley. HvTIP3;1 was specifically expressed in seeds. An immunochemical analysis showed that HvTIP3;1 strongly accumulated in the aleurone layers and outer layers of barley seeds. The water transport activities of HvTIP3;1 and HvTIP1;2, which also accumulated in seeds, were measured in the heterologous expression system of Xenopus oocytes. When they were expressed individually, HvTIP1;2 transported water, whereas HvTIP3;1 did not. However, HvTIP3;1 exhibited water transport activity when co-expressed with HvTIP1;2 in oocytes, and this activity was higher than when HvTIP1;2 was expressed alone. This is the first report to demonstrate that the water permeability of a TIP aquaporin was activated when co-expressed with another TIP. The split-yellow fluorescent protein (YFP) system in onion cells revealed that HvTIP3;1 interacted with HvTIP1;2 to form a heterotetramer in plants. These results suggest that HvTIP3;1 functions as an active water channel to regulate water movement through tissues during the periods of seed development and desiccation.

DOI： 10.1093/pcp/pcv104

Dynamic Regulation of the Root Hydraulic Conductivity of Barley Plants in Response to Salinity/Osmotic Stress

Toshiyuki Kaneko, Tomoaki Horie, Yoshiki Nakahara, Nobuya Tsuji, Mineo Shibasaka, Maki Katsuhara

PLANT AND CELL PHYSIOLOGY 56 ( 5 ) 875 - 882 2015.5

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

Salinity stress significantly reduces the root hydraulic conductivity (Lp(r)) of several plant species including barley (Hordeum vulgare). Here we characterized changes in the Lp(r) of barley plants in response to salinity/osmotic stress in detail using a pressure chamber. Salt-tolerant and intermediate barley cultivars, K305 and Haruna-nijyo, but not a salt-sensitive cultivar, I743, exhibited characteristic time-dependent Lp(r) changes induced by 100 mM NaCl. An identical response was evoked by isotonic sorbitol, indicating that this phenomenon was triggered by osmotic imbalances. Further examination of this mechanism using barley cv. Haruna-nijyo plants in combination with the use of various inhibitors suggested that various cellular processes such as protein phosphorylation/dephosphorylation and membrane internalization appear to be involved. Interestingly, the three above-mentioned barley cultivars did not exhibit a remarkable difference in root cell sap osmolality under hypertonic conditions, in contrast to the case of Lp(r). The possible biological significance of the regulation of Lp(r) in barley plants upon salinity/osmotic stress is discussed.

DOI： 10.1093/pcp/pcv013

Osmotic stress decreases PIP aquaporin transcripts in barley roots but H2O2 is not involved in this process

Maki Katsuhara, Nobuya Tsuji, Mineo Shibasaka, Sanjib Kumar Panda

JOURNAL OF PLANT RESEARCH 127 ( 6 ) 787 - 792 2014.11

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

Previous reports indicate that salt stress reduces the root hydraulic conductance and the expression of plasmamembrane-type aquaporins (PIPs). As a molecular mechanism for this phenomenon, the present study found evidence that the osmotic component, but probably not an ion-specific component, decreases PIP transcripts. Eight of ten PIP transcripts were reduced to less than half by 360 mM mannitol treatment for 12 h in comparison with control samples. A large decrease of HvPIP2; 1 protein was also recorded. This reduction of both transcripts and proteins of HvPIP2s should be physiologically effective for preventing or reducing dehydration at an initial phase of severe salt/osmotic stress. Root cell sap osmolality increased from 278 to 372 mOsm 24 h after 360 mM mannitol treatment. These steps can secure survival and growth recovery with water reabsorption in barley. Our data also suggest that H2O2 seems not to be the main cause of osmotic stress-induced transcriptional down-regulation within the concentrations (20-500 mu M) and time periods (24 h) examined, although H2O2 was previously proposed to be involved in the mechanisms of salinity/osmotic tolerance.

DOI： 10.1007/s10265-014-0662-y

Expression of root HvPIPs of barley seedling under chilling stress

Rui-Jun Duan, Hui-Yan Xiong, Katsuhara Maki

Zhiwu Shengli Xuebao/Plant Physiology Journal 50 ( 8 ) 1203 - 1208 2014.8

Language：Chinese Publishing type：Research paper (scientific journal) Publisher：Science Press

The plant plasma membrane aquaporin (plasma membrane intrinsic proteins, PIPs) is a kind of membrane intrinsic proteins which has high selectivity and water transport. They play important roles in many physiological activities in plant growth and development. In this study, seedlings of barley variety 'Haruna-nijo' were treated with chilling stress. Expression of water channel protein PIPs of root in two processes which were stress period (4℃, 48 h) and recovery period (16℃, 48 h) were analyzed with quantitative real-time reverse transcriptase PCR (qRT-PCR). And root hydraulic conductivity (Lpr), root length and seedling height were analyzed at the same time. The results showed that: after 4℃ and 48 h stress, the growth of the barley seedlings was significantly inhibited, but root growth was no significant changed
after 16℃, 48 h recovery, shoot height of stressed-seedling caught up that of control seedlings and root growth did not change significantly
root hydraulic conductivity decreased in the period of chilling stress and increased rapidly in recovery period but there were no significant difference. The results of qRT-PCR showed: the highest expression were HvPIP1
2 and HvPIP1
3, the lowest expression were HvPIP1
1 and HvPIP2
3. Compared with the control group, HvPIPs expression were decreased in all after cold treatment, in which HvPIP1
2, HvPIP1
3, HvPIP1
4, HvPIP1
5, HvPIP2
1, HvPIP2
2 were down-regulated significantly. In recovery period, most of HvPIPs expression were increased, espeacally HvPIP1
1, HvPIP1
2, HvPIP1
5, HvPIP2
3 were up-regulated significantly, but HvPIP1
4, HvPIP2
5 were down-regulated. This study found that HvPIPs expressions of barley root were down-regulated overall in chilling stress and in recovery growth most HvPIPs were up-regulated. Combined with changes of root hydraulic conductivity and plant growth, the role of barley HvPIPs in chilling resistance reaction speculated that HvPIPs effected water absorption were different in different stages.

DOI： 10.13592/j.cnki.ppj.2014.0242

CO2 Transport by PIP2 Aquaporins of Barley

Izumi C. Mori, Jiye Rhee, Mineo Shibasaka, Shizuka Sasano, Toshiyuki Kaneko, Tomoaki Horie, Maki Katsuhara

PLANT AND CELL PHYSIOLOGY 55 ( 2 ) 251 - 257 2014.2

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

CO2 permeability of plasma membrane intrinsic protein 2 (PIP2) aquaporins of Hordeum vulgare L. was investigated. Five PIP2 members were heterologously expressed in Xenopus laevis oocytes. CO2 permeability was determined by decrease of cytosolic pH in CO2-enriched buffer using a hydrogen ion-selective microelectrode. HvPIP2; 1, HvPIP2; 2, HvPIP2; 3 and HvPIP2; 5 facilitated CO2 transport across the oocyte cell membrane. However, HvPIP2; 4 that is highly homologous to HvPIP2; 3 did not. The isoleucine residue at position 254 of HvPIP2; 3 was conserved in PIP2 aquaporins of barley, except HvPIP2; 4, which possesses methionine instead. CO2 permeability was lost by the substitution of the Ile254 of HvPIP2; 3 by methionine, while water permeability was not affected. These results suggest that PIP2 aquaporins are permeable to CO2. and the conserved isoleucine at the end of the E-loop is crucial for CO2 selectivity.

DOI： 10.1093/pcp/pcu003

Functional and molecular characteristics of rice and barley NIP aquaporins transporting water, hydrogen peroxide and arsenite

Maki Katsuhara, Shizuka Sasano, Tomoaki Horie, Tadashi Matsumoto, Jiye Rhee, Mineo Shibasaka

PLANT BIOTECHNOLOGY 31 ( 3 ) 213 - U173 2014

Language：English Publishing type：Research paper (scientific journal) Publisher：JAPANESE SOC PLANT CELL & MOLECULAR BIOLOGY

Mercury-sensitive water transport activities were detected in seven NIP (Nodulin 26-like intrinsic protein) type aquaporins among eleven NIPs examined. Amino acid substitutions in rice OsNIP3;3 revealed that mercury-sensitivity depended on a histidine (but not on a cysteine) in apoplastic loop C in plant NIP aquaporins, although the cysteine is involved in the mercury-sensitivity of animal aquaporins. Rice OsNIP3;3 was also first identified as a unique aquaporin facilitating all water, hydrogen peroxide and arsenite transports. In rice OsNIP3;2, hydrogen peroxide and arsenite transport activities were detected, but water transport was not. Barley HvNIP1;2- or rice OsNIP2;1-expressing yeast cells showed the arsenite transport activity but not the H2O2 transport activity. The present work revealed novel molecular mechanisms of water and other low molecular weight compounds transport/selection in barley and rice NIP aquaporins, including the histidine-related mercury-sensitivity in the water transport of aquaporins.

DOI： 10.5511/plantbiotechnology.14.0421a

The photosynthetic response of tobacco plants overexpressing ice plant aquaporin McMIPB to a soil water deficit and high vapor pressure deficit

Miki Kawase, Yuko T. Hanba, Maki Katsuhara

JOURNAL OF PLANT RESEARCH 126 ( 4 ) 517 - 527 2013.7

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

We investigated the photosynthetic capacity and plant growth of tobacco plants overexpressing ice plant (Mesembryanthemum crystallinum L.) aquaporin McMIPB under (1) a well-watered growth condition, (2) a well-watered and temporal higher vapor pressure deficit (VPD) condition, and (3) a soil water deficit growth condition to investigate the effect of McMIPB on photosynthetic responses under moderate soil and atmospheric humidity and water deficit conditions. Transgenic plants showed a significantly higher photosynthesis rate (by 48 %), higher mesophyll conductance (by 52 %), and enhanced growth under the well-watered growth condition than those of control plants. Decreases in the photosynthesis rate and stomatal conductance from ambient to higher VPD were slightly higher in transgenic plants than those in control plants. When plants were grown under the soil water deficit condition, decreases in the photosynthesis rate and stomatal conductance were less significant in transgenic plants than those in control plants. McMIPB is likely to work as a CO2 transporter, as well as control the regulation of stomata to water deficits.

DOI： 10.1007/s10265-013-0548-4

Overexpression of Alternative Oxidase Gene Confers Aluminum Tolerance by Altering the Respiratory Capacity and the Response to Oxidative Stress in Tobacco Cells

Sanjib Kumar Panda, Lingaraj Sahoo, Maki Katsuhara, Hideaki Matsumoto

MOLECULAR BIOTECHNOLOGY 54 ( 2 ) 551 - 563 2013.6

Language：English Publishing type：Research paper (scientific journal) Publisher：HUMANA PRESS INC

Aluminum (Al) stress represses mitochondrial respiration and produces reactive oxygen species (ROS) in plants. Mitochondrial alternative oxidase (AOX) uncouples respiration from mitochondrial ATP production and may improve plant performance under Al stress by preventing excess accumulation of ROS. We tested respiratory changes and ROS production in isolated mitochondria and whole cell of tobacco (SL, ALT 301) under Al stress. Higher capacities of AOX pathways relative to cytochrome pathways were observed in both isolated mitochondria and whole cells of ALT301 under Al stress. AOX1 when studied showed higher AOX1 expression in ALT 301 than SL cells under stress. In order to study the function of tobacco AOX gene under Al stress, we produced transformed tobacco cell lines by introducing NtAOX1 expressed under the control of the cauliflower mosaic virus (CaMV) 35 S promoter in sensitive (SL) Nicotiana tabacum L. cell lines. The enhancement of endogenous AOX1 expression and AOX protein with or without Al stress was in the order of transformed tobacco cell lines > ALT301 > wild type (SL). A decreased respiratory inhibition and reduced ROS production with a better growth capability were the significant features that characterized AOX1 transformed cell lines under Al stress. These results demonstrated that AOX plays a critical role in Al stress tolerance with an enhanced respiratory capacity, reducing mitochondrial oxidative stress burden and improving the growth capability in tobacco cells.

DOI： 10.1007/s12033-012-9595-7

Water and CO2 permeability of SsAqpZ, the cyanobacterium Synechococcus sp. PCC7942 aquaporin

Xiaodong Ding, Tadashi Matsumoto, Patrizia Gena, Chengwei Liu, Marialuisa Pellegrini-Calace, Shihua Zhong, Xiaoli Sun, Yanming Zhu, Maki Katsuhara, Ikuko Iwasaki, Yoshichika Kitagawa, Giuseppe Calamita

Biology of the Cell 105 ( 3 ) 118 - 128 2013.3

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

Background information: Cyanobacteria possess Aquaporin-Z (AqpZ) membrane channels which have been suggested to mediate the water efflux underlying osmostress-inducible gene expression and to be essential for glucose metabolism under photomixotrophic growth. However, preliminary observations suggest that the biophy-sical properties of transport and physiological meaning of AqpZ in such photosynthetic microorganisms are not yet completely assessed. Results: In this study, we used Xenopus laevis oocyte and proteoliposome systems to directly demonstrate the water permeability of the cyanobacterium Synechococcus sp. PCC7942 aquaporin, SsAqpZ. By an in vitro assay of intracellular acidification in yeast cells, SsAqpZ was found to transport also CO2. Consistent with this result, during the entire exponential phase of growth, Synechococcus SsAqpZ-null-mutant cells grew slower than the corresponding wild-type cells. This phenotype was stronger with higher levels of extracellular CO2. In line with the conversion of CO2 gas into HCO3- ions under alkaline conditions, the impairment in growth of the SsAqpZ-null strain was weaker in more alkaline culture medium. Conclusions: Cyanobacterial SsAqpZ may exert a pleiotropic function in addition to the already reported roles in macronutrient homeostasis and osmotic-stress response as it appears to constitute an important pathway in CO2 uptake, a fundamental step in photosynthesis. © 2013 Soçiété Française des Microscopies and Soçiété de Biologie Cellulaire de France.

DOI： 10.1111/boc.201200057

Rice sodium-insensitive potassium transporter, OsHAK5, confers increased salt tolerance in tobacco BY2 cells

NAKAYAMA Pulla K

生物工学会誌 : seibutsu-kogaku kaishi 91 ( 2 ) 85 - 85 2013.2

Aquaporin OsPIP1;1 promotes rice salt resistance and seed germination

Chengwei Liu, Tatsuya Fukumoto, Tadashi Matsumoto, Patrizia Gena, Daniele Frascaria, Tomoyuki Kaneko, Maki Katsuhara, Shihua Zhong, Xiaoli Sun, Yanming Zhu, Ikuko Iwasaki, Xiaodong Ding, Giuseppe Calamita, Yoshichika Kitagawa

PLANT PHYSIOLOGY AND BIOCHEMISTRY 63 151 - 158 2013.2

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER

OsPIP1;1 is one of the most abundant aquaporins in rice leaves and roots and is highly responsible to environmental stresses. However, its biochemical and physiological functions are still largely unknown. The oocyte assay data showed OsPIP1;1 had lower water channel activity in contrast to OsPIP2;1. EGFP and immunoelectron microscopy studies revealed OsPIP1;1 was predominantly localized in not only plasma membrane but also in some ER-like intracellular compartments in the cells. OsPIP1;1 exhibited low water channel activity in Xenopus oocytes but coexpression of OsPIP2;1 significantly enhanced its water permeability. Stop-flow assay indicated that 10His-OsPIP1;1-reconstituted proteoliposomes had significantly higher water permeability than the control liposomes. Overexpression of OsPIP1;1 greatly altered many physiological features of transgenic plants in a dosage-dependent manner. Moderate expression of OsPIP1;1 increased rice seed yield, salt resistance, root hydraulic conductivity, and seed germination rate. This work suggests OsPIP1;1 functions as an active water channel and plays important physiological roles. Published by Elsevier Masson SAS.

DOI： 10.1016/j.plaphy.2012.11.018

Salinity tolerance mechanisms in glycophytes: An overview with the central focus on rice plants. Reviewed

Horie T, Karahara I, Katsuhara M

Rice (New York, N.Y.) 5 ( 1 ) 11 - 11 2012.12

Influence of Low Air Humidity and Low Root Temperature on Water Uptake, Growth and Aquaporin Expression in Rice Plants

Tsuneo Kuwagata, Junko Ishikawa-Sakurai, Hidehiro Hayashi, Kiyoshi Nagasuga, Keiko Fukushi, Arifa Ahamed, Katsuko Takasugi, Maki Katsuhara, Mari Murai-Hatano

PLANT AND CELL PHYSIOLOGY 53 ( 8 ) 1418 - 1431 2012.8

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

The effects of low air humidity and low root temperature (LRT) on water uptake, growth and aquaporin gene expression were investigated in rice plants. The daily transpiration of the plants grown at low humidity was 1.5- to 2-fold higher than that at high humidity. LRT at 13 degrees C reduced transpiration, and the extent was larger at lower humidity. LRT also reduced total dry matter production and leaf area expansion, and the extent was again larger at lower humidity. These observations suggest that the suppression of plant growth by LRT is associated with water stress due to decreased water uptake ability of the root. On the other hand, the net assimilation rate was not affected by low humidity and LRT, and water use efficiency was larger for LRT. We found that low humidity induced coordinated up-regulation of many PIP and TIP aquaporin genes in both the leaves and the roots. Expression levels of two root-specific aquaporin genes, OsPIP2;4 and OsPIP2;5, were increased significantly after 6 and 13 d of LRT exposure. Taken together, we discuss the possibility that aquaporins are part of an integrated response of this crop to low air humidity and LRT.

DOI： 10.1093/pcp/pcs087

Hydrogen peroxide permeability of plasma membrane aquaporins of Arabidopsis thaliana

Cortwa Hooijmaijers, Ji Ye Rhee, Kyung Jin Kwak, Gap Chae Chung, Tomoaki Horie, Maki Katsuhara, Hunseung Kang

JOURNAL OF PLANT RESEARCH 125 ( 1 ) 147 - 153 2012.1

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

Although aquaporins have been known to transport hydrogen peroxide (H(2)O(2)) across cell membranes, the H(2)O(2)-regulated expression patterns and the permeability of every family member of the plasma membrane intrinsic protein (PIP) toward H(2)O(2) have not been determined. This study investigates the H(2)O(2)-regulated expression levels of all plasma membrane aquaporins of Arabidopsis thaliana (AtPIPs), and determines the permeability of every AtPIP for H(2)O(2) in yeast. Hydrogen peroxide treatment of Arabidopsis down-regulated the expression of AtPIP2 subfamily in roots but not in leaves, whereas the expression of AtPIP1 subfamily was not affected by H(2)O(2) treatment. The growth and survival of yeast cells that expressed AtPIP2;2, AtPIP2;4, AtPIP2;5, or AtPIP2;7 was reduced in the presence of H(2)O(2), while the growth of yeast cells expressing any other AtPIP family member was not affected by H(2)O(2). These results show that only certain isoforms of AtPIPs whose expression is regulated by H(2)O(2) treatment are permeable for H(2)O(2) in yeast cells, and suggest that the integrated regulation of aquaporin expression by H(2)O(2) and the capacity of individual aquaporin to transport H(2)O(2) are important for plant response to H(2)O(2).

DOI： 10.1007/s10265-011-0413-2

Functional characterization of a novel plasma membrane intrinsic protein2 in barley

Mineo Shibasaka, Sizuka Sasano, Sigeko Utsugi, Maki Katsuhara

Plant Signaling and Behavior 7 ( 12 ) 1642 - 1646 2012

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

Water homeostasis is crucial to the growth and survival of plants. Plasma membrane intrinsic proteins (PIPs) have been shown to be primary channels mediating water uptake in plant cells. We characterized a novel PIP2 gene, HvPIP2
8 in barley (Hordeum vulgare). HvPIP2
8 shared 72-76% identity with other HvPIP2s and 74% identity with rice OsPIP2
8. The gene was expressed in all organs including the shoots, roots and pistil at a similar level. When HvPIP2
8 was transiently expressed in onion epidermal cells, it was localized to the plasma membrane. HvPIP2
8 showed transport activity for water in Xenopus oocytes, however its interaction with HvPIP1
2 was not observed. These results suggest that HvPIP2
8 plays a role in water homeostasis although further functional analysis is required in future. © 2012 Landes Bioscience.

DOI： 10.4161/psb.22294

Effect of nutrient deficiencies on the water transport properties in figleaf gourd plants

Ji Ye Rhee, Gap Chae Chung, Maki Katsuhara, Sung-Ju Ahn

HORTICULTURE ENVIRONMENT AND BIOTECHNOLOGY 52 ( 6 ) 629 - 634 2011.12

Language：English Publishing type：Research paper (scientific journal) Publisher：KOREAN SOC HORTICULTURAL SCIENCE

Effects of nitrogen, phosphorus, and potassium deficiencies on water transport properties in figleaf gourd plants were studied. Plants were treated for different period of deficiency and physiological parameters such as stomatal conductance, photosynthesis and transpiration were measured. Cell and root pressure probes were utilized to measure turgor and root pressures, half-times of water exchange and hydraulic conductivities to analyze water transport properties. When plants were grown in nitrogen or phosphorus deficient nutrient solutions, they became insensitive to mercury, suggesting that aquaporin was closed resulting in reduced hydraulic conductivity. Inclusion of tungstate, however, restored the sensitivity of cells to mercury, indicating the importance of internal nutrient concentration, not the incoming nutrient supply. The hydrostatic hydraulic conductivity of roots grown in nitrogen deficient solution, representing apoplastic pathway of water transport, was reduced but this reduction was dramatically recovered by the application of tungstate, indicating the importance of nutrient availability from storage pools in relation to water status of plants.

DOI： 10.1007/s13580-011-0046-3

K+ Transport by the OsHKT2;4 Transporter from Rice with Atypical Na+ Transport Properties and Competition in Permeation of K+ over Mg2+ and Ca2+ Ions

Tomoaki Horie, Dennis E. Brodsky, Alex Costa, Toshiyuki Kaneko, Fiorella Lo Schiavo, Maki Katsuhara, Julian I. Schroeder

PLANT PHYSIOLOGY 156 ( 3 ) 1493 - 1507 2011.7

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

Members of class II of the HKT transporters, which have thus far only been isolated from grasses, were found to mediate Na+-K+ cotransport and at high Na+ concentrations preferred Na+-selective transport, depending on the ionic conditions. But the physiological functions of this K+-transporting class II of HKT transporters remain unknown in plants, with the exception of the unique class II Na+ transporter OsHKT2;1. The genetically tractable rice (Oryza sativa; background Nipponbare) possesses two predicted K+-transporting class II HKT transporter genes, OsHKT2;3 and OsHKT2;4. In this study, we have characterized the ion selectivity of the class II rice HKT transporter OsHKT2;4 in yeast and Xenopus laevis oocytes. OsHKT2;4 rescued the growth defect of a K+ uptake-deficient yeast mutant. Green fluorescent protein-OsHKT2;4 is targeted to the plasma membrane in transgenic plant cells. OsHKT2;4-expressing oocytes exhibited strong K+ permeability. Interestingly, however, K+ influx in OsHKT2;4-expressing oocytes did not require stimulation by extracellular Na+, in contrast to other class II HKT transporters. Furthermore, OsHKT2;4-mediated currents exhibited permeabilities to both Mg2+ and Ca2+ in the absence of competing K+ ions. Comparative analyses of Ca2+ and Mg2+ permeabilities in several HKT transporters, including Arabidopsis thaliana HKT1;1 (AtHKT1;1), Triticum aestivum HKT2;1 (TaHKT2;1), OsHKT2;1, OsHKT2;2, and OsHKT2;4, revealed that only OsHKT2;4 and to a lesser degree TaHKT2;1 mediate Mg2+ transport. Interestingly, cation competition analyses demonstrate that the selectivity of both of these class II HKT transporters for K+ is dominant over divalent cations, suggesting that Mg2+ and Ca2+ transport via OsHKT2;4 may be small and would depend on competing K+ concentrations in plants.

DOI： 10.1104/pp.110.168047

Mechanisms of Water Transport Mediated by PIP Aquaporins and Their Regulation Via Phosphorylation Events Under Salinity Stress in Barley Roots

Tomoaki Horie, Toshiyuki Kaneko, Genki Sugimoto, Shizuka Sasano, Sanjib Kumar Panda, Mineo Shibasaka, Maki Katsuhara

PLANT AND CELL PHYSIOLOGY 52 ( 4 ) 663 - 675 2011.4

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

Water homeostasis is crucial to the growth and survival of plants under water-related stress. Plasma membrane intrinsic proteins (PIPs) have been shown to be primary channels mediating water uptake in plant cells. Here we report the water transport activity and mechanisms for the regulation of barley (Hordeum vulgare) PIP aquaporins. HvPIP2 but not HvPIP1 channels were found to show robust water transport activity when expressed alone in Xenopus laevis oocytes. However, the co-expression of HvPIP1 with HvPIP2 in oocytes resulted in significant increases in activity compared with the expression of HvPIP2 alone, suggesting the participation of HvPIP1 in water transport together with HvPIP2 presumably through heteromerization. Severe salinity stress (200 mM NaCl) significantly reduced root hydraulic conductivity (Lp(r)) and the accumulation of six of 10 HvPIP mRNAs. However, under relatively mild stress (100 mM NaCl), only a moderate reduction in Lp(r) with no significant difference in HvPIP mRNA levels was observed. Sorbitol-mediated osmotic stress equivalent to 100 and 200 mM NaCl induced nearly identical Lp(r) reductions in barley roots. Furthermore, the water transport activity in intact barley roots was suggested to require phosphorylation that is sensitive to a kinase inhibitor, staurosporine. HvPIP2s also showed water efflux activity in Xenopus oocytes, suggesting a potential ability to mediate water loss from cells under hypertonic conditions. Water transport via HvPIP aquaporins and the significance of reductions of Lp(r) in barley plants during salinity stress are discussed.

DOI： 10.1093/pcp/pcr027

Rice sodium-insensitive potassium transporter, OsHAK5, confers increased salt tolerance in tobacco BY2 cells

Tomoaki Horie, Mitsuo Sugawara, Tomoyuki Okada, Koichiro Taira, Pulla Kaothien-Nakayama, Maki Katsuhara, Atsuhiko Shinmyo, Hideki Nakayama

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 111 ( 3 ) 346 - 356 2011.3

Language：English Publishing type：Research paper (scientific journal) Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN

Potassium ion (K(+)) plays vital roles in many aspects of cellular homeostasis including competing with sodium ion (Na(+)) during potassium starvation and salt stress. Therefore, one way to engineer plant cells with improved salt tolerance is to enhance K(+) uptake activity of the cells, while keeping Na(+) out during salt stress. Here, in search for Na(+)-insensitive transporter for this purpose, bacterial expression system was used to characterize two K(+) transporters, OsHAK2 and OsHAK5, isolated from rice (Oryza sativa cv. Nipponbare). The two OsHAK transporters are members of a KT/HAK/KUP transporter family, which is one of the major K(+) transporter families in bacteria, fungi and plants. When expressed in an Escherichia cola K(+) transport mutant strain LB2003, both OsHAK transporters rescued the growth defect in K(+)-limiting conditions by significantly increasing the K(+) content of the cells. Under the condition with a large amount of extracellular Na(+), we found that OsHAK5 functions as a Na(+)-insensitive K(+) transporter, while OsHAK2 is sensitive to extracellular Na(+) and exhibits higher Na(+) over K(+) transport activities. Moreover, constitutive expression of OsHAK5 in cultured-tobacco BY2 (Nicotiana tabacum cv. Bright Yellow 2) cells enhanced the accumulation of K(+) but not Na(+) in the cells during salt stress and conferred increased salt tolerance to the cells. Transient expression experiment indicated that OsHAK5 is localized to the plant plasma membrane. These results suggest that the plasma-membrane localized Na(+) insensitive K(+) transporters, similar to OsHAK5 identified here, could be used as a tool to enhance salt tolerance in plant cells. (C) 2010, The Society for Biotechnology, japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2010.10.014

Hormonal treatment of the bark of rubber trees (Hevea brasiliensis) increases latex yield through latex dilution in relation with the differential expression of two aquaporin genes

Kessarin Tungngoen, Unchera Viboonjun, Panida Kongsawadworakul, Maki Katsuhara, Jean-Louis Julien, Soulaiman Sakr, Herve Chrestin, Jarunya Narangajavana

JOURNAL OF PLANT PHYSIOLOGY 168 ( 3 ) 253 - 262 2011.2

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER GMBH, URBAN & FISCHER VERLAG

Natural rubber is synthesized in laticifers in the inner liber of the rubber tree (Hevea brasiliensis). Upon bark tapping, the latex is expelled due to fiber turgor pressure. The mature laticifers are devoid of plasmodesmata; therefore a corresponding decrease-in the total latex solid content is likely to occur due to water influx inside the laticifers. Auxins and ethylene used as efficient yield stimulants in mature untapped rubber trees, but, bark treatments with abscisic acid (ABA) and salicylic acid (SA) could also induce a transient increase latex yield. We recently reported that there are three aquaporin genes, HbPIP2;1, HbT1P1;1 and HbPIP1;1, that are regulated differentially after ethylene bark treatment. HbPIP2:1 was up-regulated in both the laticifers and the inner liber tissues, whereas HbT1P1:1 was up-regulated in the latex cells, but very markedly down-regulated in the inner liber tissues. Conversely, HbPIP1:1 was down-regulated in both tissues. In the present study, HbPIP2;1 and HbT1P1:1 showed a similar expression in response to auxin, ABA and SA, as seen in ethylene stimulation, while HbPIP1;1 was slightly regulated by auxin, but neither by ABA nor SA. The analysis of the HbPIP1:1 promoter region indicated the presence of only ethylene and auxin responsive elements. In addition, the poor efficiency of this HbPIP1;1 in increasing plasmalemma water conductance was confirmed in Xenopus oocytes. Thus, an increase in latex yield in response to all of these hormones was proposed to be the major function of aquaporins, HbPIP2:1 and HbTIP1;1. This study emphasized that the circulation of water between the laticifers and their surrounding tissues that result in latex dilution, as well as the probable maintenance of the liber tissues turgor pressure, favor the prolongation of latex flow. (C) 2010 Elsevier GmbH. All rights reserved.

DOI： 10.1016/j.jplph.2010.06.009

Abiotic stresses modulate expression of major intrinsic proteins in barley (Hordeum vulgare)

Ayalew Ligaba, Maki Katsuhara, Mineo Shibasaka, Gemechis Djira

COMPTES RENDUS BIOLOGIES 334 ( 2 ) 127 - 139 2011.2

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER

In one of the most important crops, barley (Hordeum vulgare L), gene expression and physiological roles of most major intrinsic proteins (MIPs) remained to be elucidated. Here we studied expression of five tonoplast intrinsic protein isoforms (HvTIP1;2, HvTIP2:1, HvTIP2;2,HvTIP2;3 and HvTIP4:1), a NOD26-like intrinsic protein (HvNIP2;1) and a plasma membrane intrinsic protein (HvPIP2;1) by using the quantitative real-time RT-PCR. Five-day-old seedlings were exposed to abiotic stresses (salt, heavy metals and nutrient deficiency), abscisic acid (ABA) and gibberellic acid (GA) for 24 h. Treatment with 100 mM NaCl, 0.1 mM ABA and 1 mM GA differentially regulated gene expression in roots and shoots. Nitrogen and prolonged P-deficiency downregulated expression of most MIP genes in roots. Intriguingly, gene expression was restored to the values in the control three days after nutrient supply was resumed. Heavy metals (0.2 mM each of Cd, Cu, Zn and Cr) downregulated the transcript levels by 60-80% in roots, whereas 0.2 mM Hg upregulated expressions of most genes in roots. This was accompanied by a 45% decrease in the rate of transpiration. In order to study the physiological role of the MIPs, cDNA of three genes (HvTIP2:1, HvTIP2:3 and HvNIP2;1) have been cloned and heterologous expression was performed in Xenopus laevis oocytes. Osmotic water permeability was determined by a swelling assay. However, no water uptake activity was observed for the three proteins. Hence, the possible physiological role of the proteins is discussed. (C) 2010 Academie des sciences. Published by Elsevier Masson SAS. All rights reserved.

DOI： 10.1016/j.crvi.2010.11.005

Early response in water relations influenced by NaCl reflects tolerance or sensitivity of barley plants to salinity stress via aquaporins

Maki Katsuhara, Ji Ye Rhee, Genki Sugimoto, Gap Chae Chung

SOIL SCIENCE AND PLANT NUTRITION 57 ( 1 ) 50 - 60 2011.2

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

Barley varieties, K305 and I743, which are sodium chloride (NaCl) tolerant and sensitive respectively, were hydroponically grown to determine the short-term effects of NaCl on the cell water relations in roots using a cell pressure probe, and on the transcript levels of 10 barley PIP aquaporin genes (HvPIPs) in roots. Stomatal conductance, as an indicator of sensitivity to NaCl, was decreased to less than half values of control upon exposure to 100 mmol L-1 NaCl for 24 h in I743 whereas tolerant variety, K305, was able to maintain original conductance. Osmotic half-times of water exchange in cortical cells allowed for a clear distinction between the two varieties up to 200 mmol L-1 NaCl. With treatment duration of up to 12 h with 100 mmol L-1 NaCl, the elastic modulus was reduced in I743 but increased in K305. Hydrostatic half-times of water exchange in K305 increased rapidly, whereas this value remained unchanged in I743. Application of abscisic acid (ABA) after 1 h NaCl treatment restored the hydraulic conductivity of cells (Lp) in K305 but not in I743 whereas the opposite results were obtained when mercury chloride (HgCl2) was applied, verifying the contrasting gating response of aquaporins in two varieties. Reduced expression of HvPIPs was consistent with the reduction of hydraulic conductivity of both varieties after 24 h NaCl, but without any significant differences between them, indicating the importance of the activities of existing aquaporins rather than de novo synthesis to cope with short-term effects of salt stress.

DOI： 10.1080/00380768.2010.541870

Water and H2O2 Permeability of Aquaporins Isolated from Cucumber and Fig leaf Gourd

Ji Ye Rhee, Maki Katsuhara, Hunseung Kang, Gap Chae Chung

HORTICULTURE ENVIRONMENT AND BIOTECHNOLOGY 51 ( 3 ) 167 - 172 2010.6

Language：English Publishing type：Research paper (scientific journal) Publisher：KOREAN SOC HORTICULTURAL SCIENCE

Cucumber aquaporin gene, CsPIP1;1 and figleaf gourd aquaporin gene CfPIP2;1 were injected to Xenopus oocytes in order to study water transport activity. While CsPIP1;1 did not transport any water and was not responsive to mercury, oocyte injected with CfPIP2;1 rapidly increased their volume without saturation indicating active water transport. Calculation of water permeability of oocytes clearly distinguished the two genes. In response to exogenous application of H2O2, only CfPIP2;1 responded, reducing osmotic water permeability. Transgenic Arabidopsis plants overexpressing aquaporin genes did not respond to applied H2O2.

Insights into the salt tolerance mechanism in barley (Hordeum vulgare) from comparisons of cultivars that differ in salt sensitivity

Ayalew Ligaba, Maki Katsuhara

JOURNAL OF PLANT RESEARCH 123 ( 1 ) 105 - 118 2010.1

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

Although barley (Hordeum vulgare L.) is a salt-tolerant crop, the underlying physiological and molecular mechanisms of salt tolerance remain to be elucidated. Therefore, we investigated the response of salt-tolerant (K305) and salt-sensitive (I743) cultivars to salt stress at both physiological and molecular levels. Salt treatment increased xylem sap osmolarity, which was attributed primarily to a rise in Na(+) and Cl(-) concentration; enhanced accumulation of the ions in shoots; and reduced plant growth more severely in I743 than K305. The concentration of K(+) in roots and shoots decreased during 8 h of salt treatment in both cultivars but with no marked difference between cultivars. Hence, the severe growth reduction in I743 is attributed to the elevated levels of (mainly) Na(+) in shoots. Analysis of gene expression using quantitative RT-PCR showed that transcripts of K(+)-transporters (HvHAK1 and HvAKT1), vacuolar H(+)-ATPase and inorganic pyrophosphatase (HvHVA/68 and HvHVP1) were more abundant in shoots of K305 than in shoots of I743. Expression of HvHAK1 and Na(+)/H(+) antiporters (HvNHX1, HvNHX3 and HvNHX4) was higher in roots of K305 than in I743 with prolonged exposure to salt. Taken together, these results suggest that the better performance of K305 compared to I743 during salt stress may be related to its greater ability to sequester Na(+) into sub-cellular compartments and/or maintain K(+) homeostasis.

DOI： 10.1007/s10265-009-0272-2

Differential Sodium and Potassium Transport Selectivities of the Rice OsHKT2;1 and OsHKT2;2 Transporters in Plant Cells

Xuan Yao, Tomoaki Horie, Shaowu Xue, Ho-Yin Leung, Maki Katsuhara, Dennis E. Brodsky, Yan Wu, Julian I. Schroeder

PLANT PHYSIOLOGY 152 ( 1 ) 341 - 355 2010.1

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

Na+ and K+ homeostasis are crucial for plant growth and development. Two HKT transporter/channel classes have been characterized that mediate either Na+ transport or Na+ and K+ transport when expressed in Xenopus laevis oocytes and yeast. However, the Na+/K+ selectivities of the K+-permeable HKT transporters have not yet been studied in plant cells. One study expressing 5' untranslated region-modified HKT constructs in yeast has questioned the relevance of cation selectivities found in heterologous systems for selectivity predictions in plant cells. Therefore, here we analyze two highly homologous rice (Oryza sativa) HKT transporters in plant cells, OsHKT2;1 and OsHKT2;2, that show differential K+ permeabilities in heterologous systems. Upon stable expression in cultured tobacco (Nicotiana tabacum) Bright-Yellow 2 cells, OsHKT2;1 mediated Na+ uptake, but little Rb+ uptake, consistent with earlier studies and new findings presented here in oocytes. In contrast, OsHKT2; 2 mediated Na+-K+ cotransport in plant cells such that extracellular K+ stimulated OsHKT2;2-mediated Na+ influx and vice versa. Furthermore, at millimolar Na+ concentrations, OsHKT2; 2 mediated Na+ influx into plant cells without adding extracellular K+. This study shows that the Na+/K+ selectivities of these HKT transporters in plant cells coincide closely with the selectivities in oocytes and yeast. In addition, the presence of external K+ and Ca2+ down-regulated OsHKT2;1-mediated Na+ influx in two plant systems, Bright-Yellow 2 cells and intact rice roots, and also in Xenopus oocytes. Moreover, OsHKT transporter selectivities in plant cells are shown to depend on the imposed cationic conditions, supporting the model that HKT transporters are multi-ion pores.

DOI： 10.1104/pp.109.145722

A bacterial biosensor for oxidative stress using the constitutively expressed redox-sensitive protein roGFP2. Reviewed International journal

Arias-Barreiro CR, Okazaki K, Koutsaftis A, Inayat-Hussain SH, Tani A, Katsuhara M, Kimbara K, Mori IC

Sensors (Basel, Switzerland) 10 ( 7 ) 6290 - 6306 2010

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

A highly specific, high throughput-amenable bacterial biosensor for chemically induced cellular oxidation was developed using constitutively expressed redox-sensitive green fluorescent protein roGFP2 in E. coli (E. coli-roGFP2). Disulfide formation between two key cysteine residues of roGFP2 was assessed using a double-wavelength ratiometric approach. This study demonstrates that only a few minutes were required to detect oxidation using E. coli-roGFP2, in contrast to conventional bacterial oxidative stress sensors. Cellular oxidation induced by hydrogen peroxide, menadione, sodium selenite, zinc pyrithione, triphenyltin and naphthalene became detectable after 10 seconds and reached the maxima between 80 to 210 seconds, contrary to Cd(2+), Cu(2+), Pb(2+), Zn(2+) and sodium arsenite, which induced the oxidation maximum immediately. The lowest observable effect concentrations (in ppm) were determined as 1.0 × 10(-7) (arsenite), 1.0 × 10(-4) (naphthalene), 1.0 × 10(-4) (Cu(2+)), 3.8 × 10(-4) (H(2)O(2)), 1.0 × 10(-3) (Cd(2+)), 1.0 × 10(-3) (Zn(2+)), 1.0 × 10(-2) (menadione), 1.0 (triphenyltin), 1.56 (zinc pyrithione), 3.1 (selenite) and 6.3 (Pb(2+)), respectively. Heavy metal-induced oxidation showed unclear response patterns, whereas concentration-dependent sigmoid curves were observed for other compounds. In vivo GSH content and in vitro roGFP2 oxidation assays together with E. coli-roGFP2 results suggest that roGFP2 is sensitive to redox potential change and thiol modification induced by environmental stressors. Based on redox-sensitive technology, E. coli-roGFP2 provides a fast comprehensive detection system for toxicants that induce cellular oxidation.

DOI： 10.3390/s100706290

Regulation mechanism of water permeability under hypertonic stress in barley root

Kaneko Toshiyuki, Horie Tomoaki, Shibasaka Mineo, Katsuhara Maki

Plant and Cell Physiology Supplement 2010 0034 - 0034 2010

Publisher：The Japanese Society of Plant Physiologists

The salt stress is one of the representative abiotic stresses that decreases growth and productivity of crops plants. The root water permeability of three barley (Hordeum Vulgare) cultivars that show a difference in the salt sensitivity was measured under salinity stress using a pressure chamber. Water permeability of salt sensitive variety did not show any difference in response to salt stress, in contrast to significant reductions of water permeability found in more salt tolerant varieties. Root water permeability of tolerant cultivars exposed to 100 mM NaCl was found to be extremely down-regulated after 1h. Interestingly, however, water permeability was partially recovered after 4 h, which was followed by a re-down-regulation afterwards. Further measurements using various inhibitors indicated that at least protein phosphorylation and recycling plays an essential role in the regulation mechanism.

DOI： 10.14841/jspp.2010.0.0034.0

Involvement of HbPIP2;1 and HbTIP1;1 Aquaporins in Ethylene Stimulation of Latex Yield through Regulation of Water Exchanges between Inner Liber and Latex Cells in Hevea brasiliensis

Kessarin Tungngoen, Panida Kongsawadworakul, Unchera Viboonjun, Maki Katsuhara, Nicole Brunel, Soulaiman Sakr, Jarunya Narangajavana, Herve Chrestin

PLANT PHYSIOLOGY 151 ( 2 ) 843 - 856 2009.10

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

Natural rubber is synthesized in specialized articulated cells (laticifers) located in the inner liber of Hevea brasiliensis. Upon bark tapping, the laticifer cytoplasm (latex) is expelled due to liber tissue turgor pressure. In mature virgin (untapped) trees, short-term kinetic studies confirmed that ethylene, the rubber yield stimulant used worldwide, increased latex yield, with a concomitant decrease in latex total solid content, probably through water influx in the laticifers. As the mature laticifers are devoid of plasmodesmata, the rapid water exchanges with surrounding liber cells probably occur via the aquaporin pathway. Two full-length aquaporin cDNAs (HbPIP2;1 and HbTIP1;1, for plasma membrane intrinsic protein and tonoplast intrinsic protein, respectively) were cloned and characterized. The higher efficiency of HbPIP2;1 than HbTIP1;1 in increasing plasmalemma water conductance was verified in Xenopus laevis oocytes. HbPIP2;1 was insensitive to HgCl(2). In situ hybridization demonstrated that HbPIP2;1 was expressed in all liber tissues in the young stem, including the laticifers. HbPIP2;1 was up-regulated in both liber tissues and laticifers, whereas HbTIP1;1 was down-regulated in liber tissues but up-regulated in laticifers in response to bark Ethrel treatment. Ethylene-induced HbPIP2;1 up-regulation was confirmed by western-blot analysis. The promoter sequences of both genes were cloned and found to harbor, among many others, ethylene-responsive and other chemical-responsive (auxin, copper, and sulfur) elements known to increase latex yield. Increase in latex yield in response to ethylene was emphasized to be linked with water circulation between the laticifers and their surrounding tissues as well as with the probable maintenance of liber tissue turgor, which together favor prolongation of latex flow.

DOI： 10.1104/pp.109.140228

New production method for greening concrete blocks.

Katsuhara M

Environment Solution Technology 8 ( 3 ) 32 - 36 2009

Biomarkers of Green Roof Vegetation: Anthocyanin and Chlorophyll as Stress Marker Pigments for Plant Stresses of Rooftop Environments.

Mori, I.C, Utsugi,S, Tanakamaru, S, Tani, A, Enomoto, T, Katsuhara, M

Journal of Environmental Engineering and Management 19 21 - 27 2009

Function and regulation of PIP-type water channels in roots of barley under salinity stress

Horie Tomoaki, Kaneko Toshiyuki, Sugimoto Genki, Shibasaka Mineo, Katsuhara Maki

Plant and Cell Physiology Supplement 2009 S0029 - S0029 2009

Publisher：The Japanese Society of Plant Physiologists

The plasma membrane intrinsic protein (PIP) forms the pore for water transport as a water channel in plant cells. We have studied the molecular mechanism of water transport in roots of barley (Hordeum Vulgare cv. Haruna-nijo). Quantitative RT-PCR analyses using roots of barley plants revealed significant reductions in the accumulation of 6 out of 10 HvPIP mRNA in response to 200 mM NaCl. Characterization of water permeability of barely roots using a pressure chamber showed that water permeability was extremely down-regulated in 200 mM NaCl condition. Furthermore, phosphorylation was found to regulate water transport activity. HvPIP1 proteins did not show apparent water transport activity when they were expressed alone in Xenopus laevis oocytes. However, co-expression of HvPIP1 proteins with HvPIP2 proteins was shown to enhance water transport activity of HvPIP2 proteins. The regulation mechanism of water transport via HvPIP proteins in barley roots under salinity stress will be discussed.

DOI： 10.14841/jspp.2009.0.s0029.0

Characterization of four plasma membrane aquaporins in tulip petals: A putative homolog is regulated by phosphorylation

Abul Kalam Azad, Maki Katsuhara, Yoshihiro Sawa, Takahiro Ishikawa, Hitoshi Shibata

PLANT AND CELL PHYSIOLOGY 49 ( 8 ) 1196 - 1208 2008.8

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

We suggested previously that temperature-dependent tulip (Tulipa gesneriana) petal movement that is concomitant with water transport is regulated by reversible phosphorylation of an unidentified plasma membrane intrinsic protein (PIP). In this study, four full-length cDNAs of PIPs from tulip petals were identified and cloned. Two PIPs, namely TgPIP1;1 and TgPIP1;2, are members of the PIP1 subfamily, and the remaining two PIPs, namely TgPIP2;1 and TgPIP2;2, belong to the PIP2 subfamily of aquaporins and were named according to the nomenclature of PIP genes in plants. Of these four homologs, only TgPIP2;2 displayed significant water channel activity in the heterologous expression assay using Xenopus laevis oocytes. The water channel activity of this functional isoform was abolished by mercury and was affected by inhibitors of protein kinase and protein phosphatase. Using a site-directed mutagenesis approach to substitute several serine residues with alanine, and assessing water channel activity using the methylotrophic yeast Pichia pastoris expression assay, we showed that Ser35, Ser116 and Ser274 are the putative phosphorylation sites of TgPIP2;2. Real-time reverse transcriptionPCR analysis revealed that the transcript levels of TgPIP1;1 and TgPIP1;2 in tulip petals, stems, leaves, bulbs and roots are very low when compared with those of TgPIP2;1 and TgPIP2;2. The transcript level of TgPIP2;1 is negligible in roots, and TgPIP2;2 is ubiquitously expressed in all organs with significant transcript levels. From the data reported herein, we suggest that TgPIP2;2 might be modulated by phosphorylation and dephosphorylation for regulating water channel activity, and may play a role in transcellular water transport in all tulip organs.

DOI： 10.1093/pcp/pcn095

Barley plasma membrane intrinsic proteins (PIP aquaporins) as water and CO(2) transporters

Maki Katsuhara, Yuko T. Hanba

PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY 456 ( 4 ) 687 - 691 2008.7

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

We identified barley aquaporins and demonstrated that one, HvPIP2;1, transports water and CO(2). Regarding water homeostasis in plants, regulations of aquaporin expression were observed in many plants under several environmental stresses. Under salt stress, a number of plasma membrane-type aquaporins were down-regulated, which can prevent continuous dehydration resulting in cell death. The leaves of transgenic rice plants that expressed the largest amount of HvPIP2;1 showed a 40% increase in internal CO(2) conductance compared with leaves of wild-type rice plants. The rate of CO(2) assimilation also increased in the transgenic plants. The goal of our plant aquaporin research is to determine the key aquaporin species responsible for water and CO(2) transport, and to improve plant water relations, stress tolerance, CO(2) uptake or assimilation, and plant productivity via molecular breeding of aquaporins.

DOI： 10.1007/s00424-007-0434-9

Drought stress alters water relations and expression of PIP-type aquaporin genes in Nicotiana tabacum plants

Majid Mahdieh, Akbar Mostajeran, Tomoaki Horie, Maki Katsuhara

PLANT AND CELL PHYSIOLOGY 49 ( 5 ) 801 - 813 2008.5

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

Plasma membrane intrinsic proteins (PIPs), a type of aquaporins, mediate water transport in many plant species. In this study, we investigated the relationship between the functions of PIP-type water channels and water relations of tobacco plants (Nicotiana tabacum cv. Samsun) under drought stress. Drought stress treatments have led to reductions in the stomatal conductance, transpiration, water potential and turgor pressure in leaves, and also the sap flow rate and osmotic hydraulic conductance in roots. In contrast, leaf osmotic pressure was increased in response to drought stress. Interestingly, the accumulation of NtPIP1;1 and NtPIP2;1 transcripts was significantly decreased, but only that of the NtAQP1 transcript was increased under drought stress. Functional analysis using Xenopus laevis oocytes revealed that NtPIP2;1 shows marked water transport activity, but the activities of NtAQP1 and NtPIP1;1 are weak or almost negligible, respectively, when expressed alone. However, co-expression of NtPIP1;1 with NtPIP2;1 significantly enhanced water transport activity compared with that of NtPIP1;1- or NtPIP2;1-expressing oocytes, suggesting that these two aquaporins may function as a water channel, forming a heterotetramer. Heteromerization of NtPIP1;1 and NtPIP2;1 was also suggested by co-expression analyses of NtPIP1;1-GFP (green fluorescent protein) and NtPIP2;1 in Xenopus oocytes. Re-watering treatments recovered water relation parameters and the accumulation of the three NtPIP transcripts to levels similar to control conditions. These results suggest that NtPIP1;1 and NtPIP2;1 play an important role in water transport in roots, and that expression of NtPIP1;1 and NtPIP2;1 is down-regulated in order to reduce osmotic hydraulic conductance in the roots of tobacco plants under drought stress.

DOI： 10.1093/pcp/pcn054

Presence of aquaporin and V-ATPase on the contractile vacuole of Amoeba proteus

Eri Nishihara, Etsuo Yokota, Akira Tazaki, Hidefurni Orii, Maki Katsuhara, Kensuke Kataoka, Hisako Igarashi, Yoshinori Moriyama, Teruo Shimmen, Seiji Sonobe

BIOLOGY OF THE CELL 100 ( 3 ) 179 - 188 2008.3

Language：English Publishing type：Research paper (scientific journal) Publisher：PORTLAND PRESS LTD

Background information. The results of water permeability measurements suggest the presence of an AQP (aquaporin) in the membrane of the CV (contractile vacuole) in Amoeba proteus [Nishihara, Shimmen and Sonobe (2004) Cell Struct. Funct. 29, 85-90].
Results. In the present study, we cloned an AQP gene from A. proteus [ApAQP (A. proteus AQP)] that encodes a 295-amino-acid protein. The protein has six putative TMs (transmembrane domains) and two NPA (Asn-Pro-Ala) motifs, which are conserved among various AQPs and are thought to be involved in the formation of water channels that span the lipid bilayer. Using Xenopus oocytes, we have demonstrated that the ApAQP protein product can function as a water channel. Immuncifluorescence microscopy with anti-ApAQP antibody revealed that ApAQP is detected on the CV membrane and on the vesicles around the CV. The presence of V-ATPase (vacuolar H+-ATPase) on the vesicle membrane around the CV was also detected.
Conclusions. Our data on ApAQP allow us to provide the first informed explanation of the high water permeability of the CV membrane in amoeba. Moreover, the results suggest that vesicles possessing V-ATPase are involved in generating an osmotic gradient. Based on our findings, we propose a new hypothesis for the mechanism of CV function.

DOI： 10.1042/BC20070091

Female mating receptivity inhibited by injection of male-derived extracts in Callosobruchus chinensis

Takashi Yamane, Yoshinobu Kimura, Maki Katsuhara, Takahisa Miyatake

JOURNAL OF INSECT PHYSIOLOGY 54 ( 2 ) 501 - 507 2008.2

Language：English Publishing type：Research paper (scientific journal) Publisher：PERGAMON-ELSEVIER SCIENCE LTD

The effects of male-derived extracts on female receptivity to remating were investigated in Callosobruchus chinensis (Coleoptera: Bruchidae). Injection of aqueous extracts of male reproductive tracts into the abdomen of females reduced receptivity. When aqueous extracts of male reproductive tracts were divided to three molecular weight (MW) fractions by ultrafiltration: < 3, 3-14, and > 14 kDa, the filtrate containing MW substances < 3 kDa reduced female receptivity 3 h and I day after injection, whereas the fraction containing MW substances > 14 kDa inhibited receptivity 2 and 4 days after injection. Finally, male reproductive tract organs were divided into accessory gland, seminal vesicle, and testis. Aqueous extracts of testis reduced receptivity of females on the second day and at 3 h, and aqueous extracts of accessory gland reduced receptivity of females on the second day after injection. On the other hand, aqueous extracts of seminal vesicle did not reduce female receptivity. The results indicate that more than one mechanism may be involved in producing the effects of male-derived substances on female receptivity; low MW male-derived substances, which possibly exist in testis, cause short-term inhibition, while high MW substances, which possibly exist in the accessory gland, inhibit female mating later than low MW substances in C chinensis. (c) 2007 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jinsphys.2007.11.009

Expanding roles of plant aquaporins in plasma membranes and cell organelles

KATSUHARA M.

Funct. Plant Biol. 35 ( 1 ) 1 - 14 2008

Barley root hydraulic conductivity and aquaporins expression in relation to salt tolerance

Maki Katsuhara, Mineo Shibasaka

SOIL SCIENCE AND PLANT NUTRITION 53 ( 4 ) 466 - 470 2007.8

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

Root hydraulic conductivity (Lp(r)) and aquaporins were investigated in barley seedlings under salt stress of 100 mmol L-1 NaCl in hydroponic culture for 24 and 48 h. No reduction in root elongation was observed in stressed seedlings, and more that 75% of root and shoot growth was maintained under salt stress of 100 mmol L-1 NaCl. A slight increase in HvPIP2;1 transcript, which was the most abundant of the three aquaporins investigated, was observed. No significant changes in LPr and HvPIP2;1 protein were detected. The possible reason for these complicated results and the function of aquaporins in LPr under salt stress are discussed.

DOI： 10.1111/j.1747-0765.2007.00154.x

An aluminum-activated citrate transporter in barley.

Jun Furukawa, Naoki Yamaji, Hua Wang, Namiki Mitani, Yoshiko Murata, Kazuhiro Sato, Maki Katsuhara, Kazuyoshi Takeda, Jian Feng Ma

Plant & cell physiology 48 ( 8 ) 1081 - 91 2007.8

Language：English

Soluble ionic aluminum (Al) inhibits root growth and reduces crop production on acid soils. Al-resistant cultivars of barley (Hordeum vulgare L.) detoxify Al by secreting citrate from the roots, but the responsible gene has not been identified yet. Here, we identified a gene (HvAACT1) responsible for the Al-activated citrate secretion by fine mapping combined with microarray analysis, using an Al-resistant cultivar, Murasakimochi, and an Al-sensitive cultivar, Morex. This gene belongs to the multidrug and toxic compound extrusion (MATE) family and was constitutively expressed mainly in the roots of the Al-resistant barley cultivar. Heterologous expression of HvAACT1 in Xenopus oocytes showed efflux activity for (14)C-labeled citrate, but not for malate. Two-electrode voltage clamp analysis also showed transport activity of citrate in the HvAACT1-expressing oocytes in the presence of Al. Overexpression of this gene in tobacco enhanced citrate secretion and Al resistance compared with the wild-type plants. Transiently expressed green fluorescent protein-tagged HvAACT1 was localized at the plasma membrane of the onion epidermal cells, and immunostaining showed that HvAACT1 was localized in the epidermal cells of the barley root tips. A good correlation was found between the expression of HvAACT1 and citrate secretion in 10 barley cultivars differing in Al resistance. Taken together, our results demonstrate that HvAACT1 is an Al-activated citrate transporter responsible for Al resistance in barley.

DOI： 10.1093/pcp/pcm091

An efflux transporter of silicon in rice

Jian Feng Ma, Naoki Yamaji, Namiki Mitani, Kazunori Tamai, Saeko Konishi, Toru Fujiwara, Maki Katsuhara, Masahiro Yano

NATURE 448 ( 7150 ) 209 - U12 2007.7

Language：English Publishing type：Research paper (scientific journal) Publisher：NATURE PUBLISHING GROUP

Silicon is an important nutrient for the optimal growth and sustainable production of rice(1-4). Rice accumulates up to 10% silicon in the shoot, and this high accumulation is required to protect the plant from multiple abiotic and biotic stresses(1-5). A gene, Lsi1, that encodes a silicon influx transporter has been identified in rice(6). Here we describe a previously uncharacterized gene, low silicon rice 2 (Lsi2), which has no similarity to Lsi1. This gene is constitutively expressed in the roots. The protein encoded by this gene is localized, like Lsi1, on the plasma membrane of cells in both the exodermis and the endodermis, but in contrast to Lsi1, which is localized on the distal side, Lsi2 is localized on the proximal side of the same cells. Expression of Lsi2 in Xenopus oocytes did not result in influx transport activity for silicon, but preloading of the oocytes with silicon resulted in a release of silicon, indicating that Lsi2 is a silicon efflux transporter. The identification of this silicon transporter revealed a unique mechanism of nutrient transport in plants: having an influx transporter on one side and an efflux transporter on the other side of the cell to permit the effective transcellular transport of the nutrients.

DOI： 10.1038/nature05964

Characterization of silicon permeability of NIP in gramineous plants Reviewed

Mitani Namiki, Yamaji Naoki, Katsuhara Maki, Ma Jian Feng

PLANT AND CELL PHYSIOLOGY 48 S30 2007

Gene expression and functional analysis of a candidate gene related to Al-activated citrate transporter in barley Reviewed

Furukawa Jun, Yamaji Naoki, Wang Hua, Katsuhara Maki, Sato Kazuhiro, Takeda Kazuyoshi, Ma Jian Feng

Plant and Cell Physiology 48 S44 2007

Molecular mechanisms of water uptake and transport in plant roots: research progress with water channel aquaporins

Katsuhara Maki

Plant Root 1 22 - 26 2007

Language：English Publisher：Japanese Society for Root Research

Aquaporins (water channels) are membrane proteins which facilitate the transport of water and low molecular weight compounds across biological membranes. The author and collaborators identified barley aquaporins and investigated the level of transcripts in roots, water transport activity, tissue localization, expression reduction by salt stress, and diurnal changes in the expression of a particular aquaporin. Over-expression of a barley aquaporin, HvPIP2;1, increased the shoot/root ratio and raised salt sensitivity in transgenic rice plants. Aquaporin research is providing significant insights into the water relations of plant roots.

DOI： 10.3117/plantroot.1.22

Low Temperature and Aquaporins, a Molecular Mechanism of Water Transport

KATSUHARA Maki, CHUNG Gap Chae, SAKURAI Junko, MURAI Mari, IZUMI Yohei, TSUMUKI Hisaaki

Cryobiology and cryotechnology 53 ( 1 ) 21 - 32 2007

Language：English Publisher：Japanese Society for Cryobiology and Cryotechnology

Aquaporins are membrane proteins facilitating the membrane transport of water and low molecular weight compounds. Recent studies have indicated that aquaporins are involved in cellular tolerance mechanisms under low temperature stress in plants and insects. Critical roles of aquaporins in cold tolerance and the influence of H_2O_2 were suggested in the cucumber and figleaf gourd. Reduction of water uptake was observed in rice under chilling stress, in which the down-regulation of aquaporin expression and probable inactivation of aquaporins were involved. Glycerol and water transport by aquaporins was investigated, and revealed to be essential for freezing tolerance in insect cells.

DOI： 10.20585/cryobolcryotechnol.53.1_21

Synchronous changes of aquaporins in rice seedlings during early phase of salt-stress.

Mineo Shibasaka, Maki Katsuhara

PLANT AND CELL PHYSIOLOGY 48 S162 - S162 2007

Comparison of transpiration rates of naturalized and domestic Sedum species

Izumi Mori, Takashi Enomoto, Maki Katsuhara

PLANT AND CELL PHYSIOLOGY 48 S227 - S227 2007

The BnALMT1 Protein That is an Aluminum-Activated Malate Transporter is Localized in the Plasma Membrane. International journal

Ayalew Ligaba, Maki Katsuhara, Wataru Sakamoto, Hideaki Matsumoto

Plant signaling & behavior 2 ( 4 ) 1 - 2 2007

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

We have previously reported that Al-induces citrate and malate efflux from P-sufficient and P-deficient plants of rape (Brassica napus L.) and that P-deficiency alone could not induce this response. Further investigation showed that the transcript of two genes designated BnALMT1 and BnALMT2 is accumulated in roots by Al-treatment. Transgenic tobacco cells (Nicotiana tabacum) and Xenopus laevis oocytes expressing the BnALMT1 and BnALMT2 proteins released more malate than control cells in the presence of Al, indicating that the BnALMT genes encode an Al-activated malate transporter. The transgenic tobacco cells exposed to toxic level of Al grew better than control cells indicating that the genes can enhance Al-resistance of plant cells. In this study we showed the subcellular localization of BnALMT1 fused to the green fluoresce protein (GFP). The BnALMT1:: GFP construct was transiently expressed in protoplasts prepared from Arabidopsis leaves using the polyethylene glycol (PEG) method. The result showed that the BnALMT1 protein is localized in the plasma membrane. This provides further evidence that the BnALMT proteins facilitate the transport of malate across the plasma membrane (PM).

The BnALMT1 and BnALMT2 genes from rape encode aluminum-activated malate transporters that enhance the aluminum resistance of plant cells

Ayalew Ligaba, Maki Katsuhara, Peter R. Ryan, Mineo Shibasaka, Hideaki Matsumoto

PLANT PHYSIOLOGY 142 ( 3 ) 1294 - 1303 2006.11

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

The release of organic anions from roots can protect plants from aluminum (Al) toxicity and help them overcome phosphorus ( P) deficiency. Our previous findings showed that Al treatment induced malate and citrate efflux from rape ( Brassica napus) roots, and that P deficiency did not induce the efflux. Since this response is similar to the malate efflux from wheat ( Triticum aestivum) that is controlled by the TaALMT1 gene, we investigated whether homologs of TaALMT1 are present in rape and whether they are involved in the release of organic anions. We isolated two TaALMT1 homologs from rape designated BnALMT1 and BnALMT2 (B. napus Al-activated malate transporter). The expression of these genes was induced in roots, but not shoots, by Al treatment but P deficiency had no effect. Several other cations ( lanthanum, ytterbium, and erbium) also increased BnALMT1 and BnALMT2 expression in the roots. The function of the BnALMT1 and BnALMT2 proteins was investigated by heterologous expression in cultured tobacco ( Nicotiana tabacum) cells and in Xenopus laevis oocytes. Both transfection systems showed an enhanced capacity for malate efflux but not citrate efflux, when exposed to Al. Smaller malate fluxes were also activated by ytterbium and erbium treatment. Transgenic tobacco cells grew significantly better than control cells following an 18 h treatment with Al, indicating that the expression of BnALMT1 and BnALMT2 increased the resistance of these plant cells to Al stress. This report demonstrates that homologs of the TaALMT1 gene from wheat perform similar functions in other species.

DOI： 10.1104/pp.106.085233

A silicon transporter in rice

JF Ma, K Tamai, N Yamaji, N Mitani, S Konishi, M Katsuhara, M Ishiguro, Y Murata, M Yano

NATURE 440 ( 7084 ) 688 - 691 2006.3

Language：English Publishing type：Research paper (scientific journal) Publisher：NATURE PUBLISHING GROUP

Silicon is beneficial to plant growth and helps plants to overcome abiotic and biotic stresses by preventing lodging ( falling over) and increasing resistance to pests and diseases, as well as other stresses(1-3). Silicon is essential for high and sustainable production of rice(4), but the molecular mechanism responsible for the uptake of silicon is unknown. Here we describe the Low silicon rice 1 (Lsi1) gene, which controls silicon accumulation in rice, a typical silicon-accumulating plant. This gene belongs to the aquaporin family(5) and is constitutively expressed in the roots. Lsi1 is localized on the plasma membrane of the distal side of both exodermis and endodermis cells, where casparian strips are located. Suppression of Lsi1 expression resulted in reduced silicon uptake. Furthermore, expression of Lsi1 in Xenopus oocytes showed transport activity for silicon only. The identification of a silicon transporter provides both an insight into the silicon uptake system in plants, and a new strategy for producing crops with high resistance to multiple stresses by genetic modification of the root's silicon uptake capacity.

DOI： 10.1038/nature04590

Hydraulic conductivity and aquaporins of cortical cells in gravitropically bending roots of Pisum sativum L.

N Miyamoto, M Katsuhara, T Ookawa, K Kasamo, T Hirasawa

PLANT PRODUCTION SCIENCE 8 ( 5 ) 515 - 524 2005.12

Language：English Publishing type：Research paper (scientific journal) Publisher：CROP SCIENCE SOC JAPAN

We examined the differential elongation of gravitropically bending roots of Pisum sativum L. in terms of cell enlargement and water uptake by cells in the growing tissue. Hydraulic conductivity between the elongating and mature tissues (Lp) was estimated from the equation G = A x Lp x AT, where G is the water-uptake rate, A is the surface area of a single cell and AP is the driving force. The rate of entry of water into a cell was estimated from the rate of increase in the volumes of cells in the outer cortex, which were calculated from longitudinal sections at given times. Gravitropic bending occurred I h after the application of gravi-stimulation and the curvature increased rapidly for the next 3 h. The biggest difference in the partial elongation rate between opposite sides of a root was found in the region 3 to 4 mm from the root tip at the start of stimulation. Cell enlargement rate was 2.8 to 3.8 times greater on the upper side of the root than on the lower side. The water potential and the osmotic potential, in both the elongating and mature tissues, were the same on both sides of the root. Therefore, there was no difference in the driving force for water flow. Hydraulic conductivity was 2.3 to 4.2 times greater on the upper side of the root than on the lower side. There was no difference between the upper and lower sides of the root in the amounts of 19-kD and 24-kD proteins in membrane fractions, which we assumed to be aquaporins (putative aquaporins), as estimated with two preparations of polyclonal antibodies. The differential elongation that Occurred during root gravitropism was caused by a difference in Lp. However, the difference in Lp did not appear to be regulated by the concentration in cell membranes of the putative aquaporins.

DOI： 10.1626/pps.8.515

Salt stress-induced lipid peroxidation is reduced by glutathione S-transferase, but this reduction of lipid peroxides is not enough for a recovery of root growth in Arabidopsis

M Katsuhara, T Otsuka, B Ezaki

PLANT SCIENCE 169 ( 2 ) 369 - 373 2005.8

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER IRELAND LTD

Reactive oxygen species (ROS)-related membrane lipid peroxidation in the root of Arabidopsis thaliana was fluorescently visualized and investigated under salt stress. In the control roots without salt stress, more fluorescence was observed in the elongating region than in the meristematic region. Salt stress of 100 mM NaCl enhanced the fluorescence in both, indicating that salt stress-induced ROS, and consequently membrane lipid peroxidation. In transgenic tobacco glutathione S-transferase over-expressing Arabidopsis (the parB plants), less fluorescence was observed than in the non-transgenic control plants. In the salt-stressed parB plant roots, the fluorescent brightness was reduced to 46% of that of the non-transgenic plant in the meristematic region. However, the inhibition of root growth was not improved in parB plants under salt stress at pH 5.7. That is, 100 mM of salt stress reduced the root growth to 40% or less both in the parent control plants and the parB plants. The root tissue osmotic pressure was almost the same between the two tested lines, which may be one of the reasons why no difference was observed in root growth between the two lines. These results suggested that salt stress-induced oxidative stress, and the introduction/overexpression of a glutathione S-transferase gene may have reduced the amount of ROS but the removal of ROS was not sufficient to effect salt tolerance because salt stress also caused an osmotic imbalance reducing the root growth. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.plantsci.2005.03.030

A novel cyanobacterial SmtB/ArsR family repressor regulates the expression of a CPx-ATPase and a metallothionein in response to both Cu(I)/Ag(I) and Zn(II)/Cd(II)

T Liu, S Nakashima, K Hirose, M Shibasaka, M Katsuhara, B Ezaki, DP Giedroc, K Kasamo

JOURNAL OF BIOLOGICAL CHEMISTRY 279 ( 17 ) 17810 - 17818 2004.4

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

A novel SmtB/ArsR family metalloregulator, denoted BxmR, has been identified and characterized from the cyanobacterium Oscillatoria brevis. Genetic and biochemical evidence reveals that BxmR represses the expression of both bxa1, encoding a CPx-ATPase metal transporter, as well as a divergently transcribed operon encoding bxmR and bmtA, a heavy metal sequestering metallothionein. Derepression of the expression of all three genes is mediated by both monovalent (Ag(I) and Cu(I)) and divalent (Zn(II) and Cd(II)) heavy metal ions, a novel property among SmtB/ArsR metal sensors. Electrophoretic gel mobility shift experiments reveal that apoBxmR forms multiple resolvable complexes with oligonucleotides containing a single 12-2-12 inverted repeat derived from one of the two operator/promoter regions with similar apparent affinities. Preincubation with either monovalent or divalent metal ions induces disassembly of both the BxmR-bxa1 and BxmR-bxmR/bmtA operator/promoter complexes. Interestingly, the temporal regulation of expression of bxa1 and bmtA mRNAs is different in O. brevis with bxa1 induced first upon heavy metal treatment, followed by bmtA/bxmR. A dynamic interplay among Bxa1, BmtA, and BxmR is proposed that maintains metal homeostasis in O. brevis by balancing the relative rates of metal storage and efflux of multiple heavy metal ions.

DOI： 10.1074/jbc.M310560200

A wheat gene encoding an aluminum-activated malate transporter

T Sasaki, Y Yamamoto, B Ezaki, M Katsuhara, SJ Ahn, PR Ryan, E Delhaize, H Matsumoto

PLANT JOURNAL 37 ( 5 ) 645 - 653 2004.3

Language：English Publishing type：Research paper (scientific journal) Publisher：BLACKWELL PUBLISHING LTD

The major constraint to plant growth in acid soils is the presence of toxic aluminum (Al) cations, which inhibit root elongation. The enhanced Al tolerance exhibited by some cultivars of wheat is associated with the Al-dependent efflux of malate from root apices. Malate forms a stable complex with Al that is harmless to plants and, therefore, this efflux of malate forms the basis of a hypothesis to explain Al tolerance in wheat. Here, we report on the cloning of a wheat gene, ALMT1 (aluminum-activated malate transporter), that co-segregates with Al tolerance in F-2 and F-3 populations derived from crosses between near-isogenic wheat lines that differ in Al tolerance. The ALMT1 gene encodes a membrane protein, which is constitutively expressed in the root apices of the Al-tolerant line at greater levels than in the near-isogenic but Al-sensitive line. Heterologous expression of ALMT1 in Xenopus oocytes, rice and cultured tobacco cells conferred an Al-activated malate efflux. Additionally, ALMT1 increased the tolerance of tobacco cells to Al treatment. These findings demonstrate that ALMT1 encodes an Al-activated malate transporter that is capable of conferring Al tolerance to plant cells.

DOI： 10.1111/j.1365-313X.2003.01991.x

Overexpression of the barley aquaporin HvPIP2;1 increases internal CO2 conductance and CO2 assimillation in the leaves of transgenic rice plants

YT Hanba, M Shibasaka, Y Hayashi, T Hayakawa, K Kasamo, Terashima, I, M Katsuhara

PLANT AND CELL PHYSIOLOGY 45 ( 5 ) 521 - 529 2004

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

The internal conductance for CO2 diffusion (g(i)) and CO2 assimilation rate were measured and the related anatomical characteristics were investigated in transgenic rice leaves that overexpressed barley aquaporin HvPIP2;1. This study was performed to test the hypothesis that aquaporin facilitates CO2 diffusion within leaves. The g(i) value was estimated for intact leaves by concurrent measurements of gas exchange and carbon isotope ratio. The leaves of the transgenic rice plants that expressed the highest levels of Aq-anti-HvPIP2;1 showed a 40% increase in g(i) as compared to g(i) in the leaves of wild-type rice plants. The increase in g(i) was accompanied by a 14% increase in CO2 assimilation rate and a 27% increase in stomatal conductance (g(s)). The transgenic plants that had low levels of Aq-anti-HvPIP2;1 showed decreases in g(i) and CO2 assimilation rate. In the plants with high levels of Aq-anti-HvPIP2;1, mesophyll cell size decreased and the cell walls of the epidermis and mesophyll cells thickened, indicating that the leaves had become xeromorphic. Although such anatomical changes could partially offset the increase in g(i) by the aquaporin, the increase in aquaporin content overcame such adverse effects.

DOI： 10.1093/pcp/pch070

Changes in roots and salt sensitivity of rice plants over-expressing aquaporin

M Katsuhara, K Koshio, M Shibasaka, Y Hayashi, T Hayakawa, K Kasamo

PLANT AND CELL PHYSIOLOGY 45 S174 - S174 2004

Expression of an aquaporin at night in relation to the growth and root water permeability in barley seedlings

M Katsuhara, K Koshio, M Shibasaka, K Kasamo

SOIL SCIENCE AND PLANT NUTRITION 49 ( 6 ) 883 - 888 2003.12

Language：English Publishing type：Research paper (scientific journal) Publisher：JAPANESE SOC SOIL SCIENCE PLANT NUTRITION

Both root and shoot of barley seedlings showed continuous growth at night as well as in the daytime. Root hydraulic conductivity (Lp(r)) was monitored, and high Lp(r) values were recorded at night. For revealing the molecular mechanism, the presence of an aquaporin HvPIP2;1 in barley roots was investigated. The accumulation of the HvPIP2;1 transcript was mostly observed from the evening to the middle of the night. HvPIP2;1 protein was most abundant around midnight. As for tissue localization, HvPIP2;1 protein was abundant in cells involved in water transport. These results suggested the possible involvement of HvPIP2;1 in the mechanism of water transport in roots.

Over-expression of a barley aquaporin increased the shoot/root ratio and raised salt sensitivity in transgenic rice plants

M Katsuhara, K Koshio, M Shibasaka, Y Hayashi, T Hayakawa, K Kasamo

PLANT AND CELL PHYSIOLOGY 44 ( 12 ) 1378 - 1383 2003.12

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

Barley HvPIP2;1 is a plasma membrane aquaporin and its expression was down-regulated after salt stress in barley [Katsuhara et al. (2002) Plant Cell Physiol. 43: 885]. We produced and analyzed transgenic rice plants over-expressing barley HvPIP2;1 in the present study. Overexpression of HvPIP2;1 increased (1) radial hydraulic conductivity of roots (Lp(r)) to 140%, and (2) the mass ratio of shoot to root up to 150%. In these transgenic rice plants under salt stress of 100 mM NaCl, growth reduction was greater than in non-transgenic plants. A decrease in shoot water content (from 79% to 61%) and reduction of root mass or shoot mass (both less than 40% of non-stressed plants) were observed in transgenic plants under salt stress for 2 weeks. These results indicated that over-expression of HvPIP2;1 makes rice plants sensitive to 100 mM NaCl. The possible involvement of aquaporins in salt tolerance is discussed.

DOI： 10.1093/pcp/pcg167

A metallothionein and CPx-ATPase handle heavy-metal tolerance in the filamentous cyanobacterium Oscillatoria brevis

T Liu, S Nakashima, K Hirose, Y Uemura, M Shibasaka, M Katsuhara, K Kasamo

FEBS LETTERS 542 ( 1-3 ) 159 - 163 2003.5

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

A metallothionein (BmtA) and a CPx-ATPase (Bxa1) have been identified and characterized from the cyanobacterium Oscillatoria brevis. Both bmtA and bxa1 expression can be markedly induced in vivo by Zn2+ or Cd2+. Over-expression of bmaA or bxa1 in Escherichia coli enhances Zn2+ and Cd2+ tolerance in the transformant. Dynamic studies on the expression of two genes showed that the maximum expression of bxa1 induced by Zn2+ and Cd2+ was much quicker than that of bmtA, suggesting distinct physiological roles of metallothionein and CPx-ATPase in the handling of surplus metal. (C) 2003 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

DOI： 10.1016/S0014-5793(03)00370-3

Increase in CO2 permeability (diffusion conductance) in leaves of transgenic rice plant over-expressing barley aquaporin

M Katsuhara, Y Hanba, M Shibasaka, Y Hayashi, T Hayakawa, K Kasamo

PLANT AND CELL PHYSIOLOGY 44 S86 - S86 2003

A novel histidine-rich CPx-ATPase from the filamentous cyanobacterium Oscillatoria brevis related to multiple-heavy-metal cotolerance

L Tong, S Nakashima, M Shibasaka, M Katsuhara, K Kasamo

JOURNAL OF BACTERIOLOGY 184 ( 18 ) 5027 - 5035 2002.9

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

A novel gene related to heavy-metal transport was cloned and identified from the filamentous cyanobacterium Oscillatoria brevis. Sequence analysis of the gene (the Bxa1 gene) showed that its product possessed high homology with heavy-metal transport CPx-ATPases. The CPC motif, which is proposed to form putative cation transduction channel, was found in the sixth transmembrane helix. However, instead of the CXXC motif that is present in the N termini of most metal transport CPx-ATPases, Bxa1 contains a unique Cys-Cys (CC) sequence element and histidine-rich motifs as a putative metal binding site. Northern blotting and real-time quantitative reverse transcription-PCR showed that expression of Bxa1 mRNA was induced in vivo by both monovalent (Cu+ and Ag+) and divalent (Zn2+ and Cd2+) heavy-metal ions at similar levels. Experiments on heavy-metal tolerance in Escherichia coli with recombinant Bxa1 demonstrated that Bxa1 conferred resistance to both monovalent and divalent heavy metals. This is the first report of a CPx-ATPase responsive to both monovalent and divalent heavy metals.

DOI： 10.1128/JB.184.18.5027-5035.2002

Functional analysis of water channels in barley roots

M Katsuhara, Y Akiyama, K Koshio, M Shibasaka, K Kasamo

PLANT AND CELL PHYSIOLOGY 43 ( 8 ) 885 - 893 2002.8

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

We identified three genes homologous to water channels in the plasma membrane type subfamily from roots of barley seedlings. These genes were designated HvPIP2;1, HvPIP1;3, and HvPIP1;5 after comparison to Arabidopsis aquaporins. Competitive reverse transcription (RT)-PCR was applied in order to distinguish and to quantify their transcripts. The HvPIP2;1 transcript was the most abundant among the three in roots. Salt stress (200 mM NaCl) down-regulated HvPIP2;1 (transcript and protein), but had almost no effect on the expressions of HvPIP1;3, or HvPIP1;5. Approximately equal amounts of the transcripts of the three were detected in shoots, and salt stress enhanced the expression of HvPIP2;1 but not of HvPIP1;3, or HvPIP1;5. HvPIP2;1 protein was confirmed to be localized in the plasma membrane. Functional expression of HvPIP2;1 in Xenopus oocytes confirmed that HvPIP2;1 encoded an aquaporin that transports water. This water permeability was reduced by HgCl2, which is a typical water channel inhibitor. This activity was not modified by some inhibitors against protein kinase and protein phosphatase.

DOI： 10.1093/pcp/pcf102

Different mechanisms of four aluminum (Al)-resistant transgenes for Al toxicity in Arabidopsis

B Ezaki, M Katsuhara, M Kawamura, H Matsumoto

PLANT PHYSIOLOGY 127 ( 3 ) 918 - 927 2001.11

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

We have characterized the mechanism of action of four transgenes (AtBCB [Arabidopsis blue copper-binding protein], parB [tobacco {Nicotiana tabacum} glutathione S-transferase], NtPox [tobacco peroxidase], and NtGDI1 [tobacco GDP dissociation inhibitor]) that independently Al resistance on transgenic Arabidopsis. All four transgenic lines showed lower deposition of callose after Al treatment than the Landsberg erecta ecotype of Arabidopsis, confirming that the four genes function to ameliorate Al toxicity. Influx and efflux experiments of Al ions suggested that the AtBCB gene may suppress Al absorption, whereas expression of the NtGDI1 gene promotes a release of Al in the root tip region of Arabidopsis. The total enzyme activities of glutathione S-transferases or peroxidases in transoenic lines carrying either the parB or NtPox genes were significantly higher than in the Landsberg erecta ecotype of Arabidopsis, and these enzyme activities were maintained at higher levels during Al stress. Furthermore, lipid peroxidation caused by Al stress was repressed in these two transgenic lines, suggesting that overexpression of these two genes diminishes oxidative damage caused by Al stress. Al-treated roots of trans-enic plants were also stained by 4',6-diamino-2-phenylindole to monitor cell death caused by Al toxicity. The result suggested that cell death is repressed in the NtPox line. Analysis of F-1 hybrids between the four transgenic lines suggests that more resistant transgenic plants can be constructed by combinations of these four genes.

DOI： 10.1104/pp.127.3.918

Isolation of barley saLT gene: Its relation to salt tolerance and to hormonal regulation by abscisic acid and jasmonic acid

M Katsuhara, M Yamada, K Kasamo

SOIL SCIENCE AND PLANT NUTRITION 47 ( 1 ) 187 - 193 2001.3

Language：English Publishing type：Research paper (scientific journal) Publisher：JAPANESE SOC SOIL SCIENCE PLANT NUTRITION

A homologous gene to rice salT was isolated from barley and designated as Hv-salT, Since the expression of rice salT was regulated by plant hormones (abscisic acid and jasmonic acid) and salt stress, the effects of these plant hormones and salt stress on the growth of barley seedlings and the expression of Hv-salT were investigated. In contrast to rice salT, almost no changes in the expression of Hv-salT by hormones and salt stress were detected.

Expression and stress-dependent induction of potassium channel transcripts in the common ice plant

H Su, D Golldack, M Katsuhara, CS Zhao, HJ Bohnert

PLANT PHYSIOLOGY 125 ( 2 ) 604 - 614 2001.2

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

We have characterized transcripts for three potassium channel homologs in the AKT/KAT subfamily (Shaker type) from the common ice plant (Mesembryanthemum crystallinum), with a focus on their expression during salt stress (up to 500 mM NaCl). Mkt1 and 2, Arabidopsis AKT homologs, and Kmt1, a KAT homolog, are members of small gene families with two to three isoforms each. Mkt1 is root specific; Mkt2 is found in leaves, flowers, and seed capsules; and Kmt1 is expressed in leaves and seed capsules. Mkt1 is present in all cells of the root, and in leaves a highly conserved isoform is detected present in all cells with highest abundance in the vasculature. MKT1 for which antibodies were made is localized to the plasma membrane. Following salt stress, MKT1 (transcripts and protein) is drastically down-regulated, Mkt2 transcripts do not change significantly, and Kmt1 is strongly and transiently (maximum at 6 h) up-regulated in leaves and stems. The detection and stress-dependent behavior of abundant transcripts representing subfamilies of potassium channels provides information about tissue specificity and the complex regulation of genes encoding potassium uptake systems in a halophytic plant.

DOI： 10.1104/pp.125.2.604

Identification and characterization of a putative heavy metal transport gene from a cyanobacterium Oscillatoria Brevis :

Liu Tong, NAKASHIMA Susumu, SHIBASAKA Mineo, KATSUHARA Maki, KASAMO Kunihiro

Plant and cell physiology 42 s208 2001

In situ RNA hybridization using Technovit resin in Arabidopsis thaliana

K Takechi, W Sakamoto, M Katsuhara, M Murata, F Motoyoshi

PLANT MOLECULAR BIOLOGY REPORTER 17 ( 1 ) 43 - 51 1999

Language：English Publishing type：Research paper (scientific journal) Publisher：TRANSACTION PERIOD CONSORTIUM

A protocol is described for RNA in situ hybridization using thin sections prepared by Technovit resin. Technovit is a widely used resin for histological examinations. Since it does not require time-consuming processes such as removal of the resin and can be performed without high temperature treatment, a high resolution of sections could be possible compared to other resins and paraffin. Thin sections (approximately 4 mu m) were made from inflorescences of Arabidopsis thaliana embedded in Technovit 8100 resin, and in situ hybridization was performed using the protocol described in this article. Hybridization signals were observed using LEAFY and other genes as probes, showing that this resin can be used for in situ analysis. In our experiments, the most important factor for a successful in situ, hybridization pattern was to optimize the RNase A concentration after hybridization. We routinely used RNase A at a concentration of 2-5 ng/ml, a concentration much lower than that used for paraffin embedding method. Thus, the use of the Technovit resin for plant tissue embedding results in a faster protocol and greater quality than allowed by paraffin sections.

Intracellular pH and proton-transport in barley root cells under salt stress: In vivo 31P-NMR study

Maki Katsuhara, Yoshiaki Yazaki, Katsuhiro Sakano, Toshio Kawasaki

Plant and Cell Physiology 38 ( 2 ) 155 - 160 1997

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

Salt stress-induced changes of intracellular pH and in levels of phosphorous compounds were monitored in intact root tips of barley seedlings (Hordeum vulgare cv. Akashinriki) by in vivo 31P-nuclear magnetic resonance (NMR) spectroscopy. Vacuolar alkalization was observed after treatment with both 300 and 500 mM NaCl. Much of the observed apparent alkalization of the cytoplasm was eliminated when the effect of Na+ ions on the titration curve was considered. Within 1 h after the initiation of salt stress, levels of glucose-6-phosphate and UDP-glucose decreased markedly, and such decreases might lead directly or indirectly to cell death. Simultaneous measurements of the external and intracellular pH revealed the promotion of external acidification and internal alkalization during salt stress. Possible mechanisms of Na+/H+ antiport at the tonoplast and the role of proton-pump in the plasma membrane are discussed.

DOI： 10.1093/oxfordjournals.pcp.a029146

Salt stress induced nuclear and DNA degradation in meristematic cells of barley roots

Maki Katsuhara, Toshio Kawasaki

Plant and Cell Physiology 37 ( 2 ) 169 - 173 1996

Language：English Publishing type：Research paper (scientific journal) Publisher：Japanese Society of Plant Physiologists

Root growth of barley (Hordeum vulgare L., ev. Akashinriki) was inhibited by 200 mM NaCl, when 1 mM CaCl2 was present in the hydroponic culture solution. Increasing the CaCl2 up to 10 mM partially prevented this inhibition. However, inhibition also occurred with 100 mM NaCl in the presence of 0.1 mM CaCl2. The nuclei of meristematic cells in roots in which growth had been inhibited by salt stress were studied after staining with DAPI (4',6-diamino-2-phenylindol). Nuclear deformation of the cells occurred with 12 h of salt stress with 500 mM NaCl, and was followed by degradation. The nuclear degradation was also observed when the roots were exposed to more than 300 mM NaCl for 24 h. Biochemical analysis revealed that nuclear degradation was accompanied by apoptosislike DNA fragmentation. The intracellular mechanisms of nuclear degradation in cells after salt stress are discussed.

DOI： 10.1093/oxfordjournals.pcp.a028928

Distribution and Production of trans-Aconitic Acid in Barnyard Grass (Echinochloa crus-galli var. oryzicola) as Putative Antifeedant against Brown Planthoppers :

KATSUHARA Maki, SAKANO Katsuhiro, SATO Mamoru, KAWAKITA Hiromu, KAWABE Susumu

Plant and cell physiology 34 ( 2 ) 251 - 254 1993

Language：English Publisher：Japanese Society of Plant Physiologists

A quantitative analysis of organic acid in leaves of barnyard grass revealed that the contents of trans-aconitic acid were high, suggesting that this compound may act as an antifeedant against brown planthoppers. However, trans-aconitic acid could not be detected in the phloem sap which was considered to be the main nutrient source for brown planthoppers. trans-Aconitic acid was formed in vitro from cis-aconitic acid through the aconitate isomerase activity which was detected only in the leaf sheath, but not in the leaf blade.

Other Link： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=181825

Detection of mycoplasmalike organisms in the phloem sap collected from mulberry trees with dwarf disease.

KAWAKITA HIROMU, KATSUHARA MAKI

Journal of Insect Biotechnology and Sericology 62 ( 2 ) 125 - 131 1993

Language：Japanese Publisher：The Japanese Society of Sericultural Science

Mycoplasmalike organisms (MLOs), the pathogens of mulberry dwarf disease, were succesufully isolated by stylectomy method (cutting of insect stylets by YAG laser) using a homopterous insect, Ricania japonica. The MLOs were detectable in the phloem sap collected from diseased mulberry plants as fluorescent particles stained with 4, 6-diamidino-2-phenylindole (DAPI) under a fluorescence microscope but were not detected in the sap of healthy mulberry plants. The presence of MLOs in the phloem sap was also confirmed by electronmicroscopy.<br>On the other hand, two kinds of leafhoppers, vectors of mulberry dwarf MLOs, and other some tested hemipterous insects could not used for this laser stylectomy. Scanning-electronmicroscopical observation showed that the stylets of these leafhoppers which were not suitable for the stylectomy method were pushed out or broken after laser cutting, whwereas stylets of Ricania japonica remained firmly in the tissues.

DOI： 10.11416/kontyushigen1930.62.125

Other Link： http://agriknowledge.affrc.go.jp/RN/2010510238

Cytoplasmic Alkalization and Cytoplasmic Streaming Induced by Light and Histidine in Leaf Cells of Egeria densa: in vivo 31P-NMR study

Yoshito Tominaga, Kazuyuki Kuchitsu, Maki Katsuhara, Masashi Tazawa, Shigetoh Miyachi

Plant and Cell Physiology 32 ( 2 ) 261 - 268 1991.3

Patch-clamp study on ion channels in the tonoplast of nitellopsis obtusa

Maki Katsuhara, Tetsuro Mimura, Masashi Tazawa

Plant and Cell Physiology 32 ( 2 ) 179 - 184 1991

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

Cytoplasmic drops covered with the tonoplast were prepared from internodal cells of Nitellopsis grown in fresh water. Applying the patch-clamp technique and the microinjection technique to such drops, we characterized the ion channels in the tonoplast. Both in cell-free patches and in the cytoplasmic-drop-attached patches, the tonoplast K+ channel was identified. The permeability ratio between Na+ and K+ was calculated to be 0.2. This channel would provide a molecular basis for the Na+/K+ exchange at the tonoplast. In cell-free patches, the K+channel was not activated by Ca2+. However, in the case of attached patches, microinjection of Ca2+ into a drop activated the K+ channel with a lag of a few seconds, suggesting that some cytoplasmic factor(s) may mediate the activation of the K+ channel by Ca2+. The conductance of this channel was not changed by cytoplasmic Ca2+, but the probability of opening increased markedly. In addition to the K+ channel, a second type of channel was also identified in cell-free patches. This channel may be the Cl- channel. © 1991 The Japanese Society of Plant Physiologists(JSSP).

DOI： 10.1093/oxfordjournals.pcp.a078062

Mechanism of calcium‐dependent salt tolerance in cells of Nitellopsis obtusa: role of intracellular adenine nucleotides

M. KATSUHARA, M. TAZAWA

Plant, Cell & Environment 13 ( 2 ) 179 - 184 1990

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

Abstract. The tonoplasts of internodal cells of Nitellopsis were removed by perfusing the vacuoles with media containing a Ca2 chelator, EGTA. Treatment of tonoplast‐free cells with 100 mol m3 NaCl induces a large membrane depolarization, a drastic decrease in the membrane resistance and an increase in Na+ influx. These events are identical to those that occur in intact cells subjected to high NaCl. These responses to NaCl are prevented if 10 mol m3 Ca2+ is supplied together with 100 mol m3 NaCl. The protective effect of Ca2+ is evident only when the intracellular ATP concentration exceeds 0.1 mol m3 and does not occur full when the intracellular ATP is removed. AMP at concentrations greater than 0.5 mol m3 or 0.25 mol m3 AMPPNP can replace ATP. It is concluded that ATP does not act as an energy source nor as a substrate for protein phosphorylation. ATP seems to exert its effects as a coeffector with Ca2+ in regulating the Na+ permeability of the plasma membrane. Copyright © 1990, Wiley Blackwell. All rights reserved

DOI： 10.1111/j.1365-3040.1990.tb01290.x

ATP-regulated ion channels in the plasma membrane of a characeae alga, Nitellopsis obtusa

Maki Katsuhara, Tetsuro Mimura, Masashi Tazawa

Plant Physiology 93 ( 1 ) 343 - 346 1990

Language：English Publishing type：Research paper (scientific journal) Publisher：American Society of Plant Biologists

Using the patch-clamp technique, we recorded single-channel currents across the excised patch of the plasma membrane of Nitellopsis. Both K+ and Na+ can pass this channel, but currents were not carried by Cl-. Upon the addition of ATP or AMP to the cytoplasmic side, the frequency of channel opening decreased. This is the first report on an ATP-regulated channel in plant cells.

DOI： 10.1104/pp.93.1.343

Patch-clamp study on a Ca2+-regulated K+ channel in the tonoplast of the brackish characeae lamprothamnium succinctum

Maki Katsuhara, Tetsuro Mimura, Masashi Tazawa

Plant and Cell Physiology 30 ( 4 ) 549 - 555 1989

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

Cytoplasmic drops were prepared from internodal cells of the brackish Characeae Lamprothamnium succinctum. Applying the patch-clamp technique to single drops covered with tonoplast, we demonstrated the presence of Ca2+-regulated K+ channels in the tonoplast. In a cell-attached mode, the selectivity of such channels for K+ was about 50 times that for Na+. This channel showed a tendency to rectify in an outward direction. In the negative region of the pipette voltage, the conductance of this channel was 50 pS, while it was 100 pS in the positive voltage region. When the pipette voltage was increased above 50 mV, two conductance levels were found in the cell-attached mode as well as in the excised patch (cytoplasmic-side-out patch), which was obtained by pulling the patch pipette from the cytoplasmic drop under conditions of low levels of Ca2+. Using the excised patch, we controlled the level of Ca2+ on the cytoplasmic side of the channels. At a low level of Ca2+ (pCa=8) on the cytoplasmic side, the open frequency was very low and the opening time was short. An increase in Ca2+ on the cytoplasmic side (pCa = 5) increased both the frequency and the duration of opening. However, the conductance of the channels did not change. This regulation by Ca2+ of the K+ channels was reversible, that is, addition of EGTA on the cytoplasmic side inactivated the channels. The present study demonstrates a direct action of Ca2+ on the K+ channels. The physiological role of the K+ channel in the regulation of turgor in Lamprothamnium is discussed. © 1989 The Japanese Society of Plant Physiologists (JSPP).

DOI： 10.1093/oxfordjournals.pcp.a077774

Rapid cytoplasmic alkalization and dynamics of intracellular compartmentation of inorganic phosphate during adaptation against salt stress in a halotolerant unicellular green alga dunaliella tertiolecta:³¹P-nuclear magnetic resonance study Reviewed

Kuchitsu, K., Katsuhara, M., Miyachi, S.

Plant and Cell Physiology 30 ( 3 ) 407 - 414 1989

ATP IS ESSENTIAL FOR CALCIUM-INDUCED SALT TOLERANCE IN NITELLOPSIS-OBTUSA

M KATSUHARA, M TAZAWA

PROTOPLASMA 138 ( 2-3 ) 190 - 192 1987

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