Tech Science Press Acta Medica Okayama 1851-5657 94 11 2025 Survey of Barley Sodium Transporter HvHKT1;1 Variants and Their Functional Analysis 3653 3665 EN Shahin Imran Institute of Plant Science and Resources, Okayama University Maki Katsuhara Department of Agronomy, Khulna Agricultural University Barley (Hordeum vulgare L.) employs the Na+ transporter HvHKT1;1, which is an N+-selective transporter. This study characterized the full-length HvHKT1;1 (HvHKT1;1-FL) and three mRNA variants (HvHKT1;1-V1, -V2, and -V3), which encode polypeptides of 64.7, 54.0, 40.5, and 32.9 kDa, respectively. Tissue-specific expression profiling revealed that HvHKT1;1-FL is the most abundant transcript across leaf, sheath, and root tissues under normal conditions, with the highest expression in leaves. Under 150 mM NaCl stress, HvHKT1;1-FL and its variants showed a dynamic, time-dependent expression pattern, with peak leaf expression at 2 h, sheath expression at 12 h, and root expression at 2 h, suggesting their roles in early stress response. Functional analysis using two-electrode voltage-clamp measurements demonstrated that HvHKT1;1-FL is highly selective for Na+, with minimal conductance for K+, Li+, Rb+, or Cs+. It demonstrated high Na+ transport efficiency, characterized by higher Vmax and lower Km values, while the variants showed reduced Na+ currents, lower Vmax, and higher Km values, indicating decreased Na+ transport capacity. Reversal potential analyses further confirmed Na+ selectivity, with HvHKT1;1-FL displaying the strongest preference for Na+. Notably, while all variants retained Na+ selectivity, they showed reduced efficiency, as indicated by a more negative reversal potential in low Na+ conditions. These findings highlight the functional diversity among HvHKT1;1 variants, with HvHKT1;1-FL playing a dominant role in Na+ transport. The tissue-specific regulation of these variants under salinity stress underscores their importance in barley’s adaptive responses. No potential conflict of interest relevant to this article was reported.

Oxford University Press (OUP) Acta Medica Okayama 0032-0781 66 7 2025 Oxygen supply is a prerequisite for response to aluminum in cultured cells of tobacco (Nicotiana tabacum) 1044 1060 EN Yoshiyuki Tsuchiya Institute of Plant Science and Resources, Okayama University Maki Katsuhara Institute of Plant Science and Resources, Okayama University Takayuki Sasaki Institute of Plant Science and Resources, Okayama University Yoko Yamamoto Institute of Plant Science and Resources, Okayama University Responses to aluminum (Al) were investigated in tobacco cells (cell line SL) in a calcium-sucrose solution for up to 24 h under shaking (aerobic) condition. Microarray analysis of upregulated and downregulated genes under Al exposure and following Gene Ontology (GO) enrichment analysis of biological process category revealed only one GO term to be enriched for the upregulated genes, “response to chitin,” annotated with genes encoding transcription factors (NtERF1 and NtMYB3) and MAP kinase (WIPK), and nine GO terms for the downregulated genes, including “cell wall loosening” and “lipid transport,” annotated with genes encoding expansin (NtEXPA4) and lipid transfer protein (LTP)/LTP-like (NtLTP3 and NtEIG-C29), respectively. Al triggered the production of nitric oxide (NO) then reactive oxygen species (ROS). Addition of NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide decreased the levels of NO and a part of the transcriptional changes described above, but increased the levels of ROS and a loss of growth capacity, suggesting a role of the NO to induce the transcriptional changes partly and to repress these toxic responses under Al exposure. Under non-shaking (anaerobic) condition, the cells exhibited upregulation of several hypoxia-responsive genes. The cells exposed to Al exhibited the same level of Al accumulation but much lower levels of the Al responses including NO production, ROS production, a loss of growth capacity, citrate secretion, and a part of the transcriptional changes described above, compared with the cells under shaking condition. These results suggest that coexistence of oxygen with Al is necessary to trigger the Al responses related to toxicity and tolerance. No potential conflict of interest relevant to this article was reported.

MDPI AG Acta Medica Okayama 2073-4409 14 17 2025 Discovery and Functional Characterization of Novel Aquaporins in Tomato (Solanum lycopersicum): Implications for Ion Transport and Salinity Tolerance 1305 EN Newton Chandra Paul Institute of Plant Science and Resources, Okayama University Shahin Imran Institute of Plant Science and Resources, Okayama University Anri Mitsumoto Institute of Plant Science and Resources, Okayama University Izumi C. Mori Institute of Plant Science and Resources, Okayama University Maki Katsuhara Institute of Plant Science and Resources, Okayama University Aquaporins (AQPs) are membrane proteins that facilitate the transport of water and solutes. Among AQPs, plasma membrane intrinsic proteins (PIPs) play a critical role in maintaining water balance between the internal and external cell environments. This study focuses on the tomato due to its economic importance and cultivation under moderate salinity conditions in Japan. A swelling assay using X. laevis oocyte confirmed that all five examined tomato SlPIP2 isoforms showed water transport activity. Among them, two-electrode voltage clamp (TEVC) experiments showed that only SlPIP2;1, SlPIP2;4, and SlPIP2;8 transport Na+ and K+, with no transport activity for Cs+, Rb+, Li+, or Cl−. CaCl2 (1.8 mM) reduced ionic currents by approximately 45% compared to 30 µM free-Ca2+. These isoforms function as very low-affinity Na+ and K+ transporters. Expression analysis showed that SlPIP2;4 and SlPIP2;8 had low, stable expression, while SlPIP2;1 was strongly upregulated in roots NaCl treatment (200 mM, 17days), suggesting distinct physiological roles for these ion-conducting AQPs (icAQPs). These data hypothesized that tomato icAQPs play a critical role in ion homeostasis, particularly under salinity stress. In conclusion, the first icAQPs have been identified in the dicotyledonous crop. These icAQPs are essential for plant resilience under salt stress. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 2045-2322 15 1 2025 OsPIP2;4 aquaporin water channel primarily expressed in roots of rice mediates both water and nonselective Na+ and K+ conductance 12857 EN Sen Thi Huong Tran Institute of Plant Science and Resources, Okayama University Maki Katsuhara Institute of Plant Science and Resources, Okayama University Yunosuke Mito Division of Applied Biology, Faculty of Textile Science and Technology, Shinshu University Aya Onishi Institute of Plant Science and Resources, Okayama University Ayaka Higa Division of Applied Biology, Faculty of Textile Science and Technology, Shinshu University Shuntaro Ono Institute of Plant Science and Resources, Okayama University Newton Chandra Paul Institute of Plant Science and Resources, Okayama University Rie Horie Division of Applied Biology, Faculty of Textile Science and Technology, Shinshu University Yoshihiko Harada Division of Applied Biology, Faculty of Textile Science and Technology, Shinshu University Tomoaki Horie Division of Applied Biology, Faculty of Textile Science and Technology, Shinshu University Aquaporin (AQP)-dependent water transport across membranes is indispensable in plants. Recent evidence shows that several AQPs, including plasma membrane intrinsic proteins (PIPs), facilitate the electrogenic transport of ions as well as water transport and are referred to as ion-conducting aquaporins (icAQPs). The present study attempted to identify icAQPs that exhibit cation transport activity among PIPs from rice. Electrophysiological experiments on 11 OsPIPs using Xenopus laevis oocytes revealed that OsPIP2;4 mediated the electrogenic transport of alkali monovalent cations with the selectivity sequence of Na+ ≈ K+ > Rb+ > Cs+ > Li+, suggesting non-selective cation conductance for Na+ and K+. Transcripts of OsPIP2;4 were abundant in the elongation and mature zones of roots with similar expression levels between the root stelar and remaining outer parts in the cultivar Nipponbare. Immunostaining using sections of the crown roots of Nipponbare plants revealed the expression of OsPIP2;4 in the exodermis and sclerenchyma of the surface region and in the endodermis and pericycle of the stelar region. The present results provide novel insights into OsPIP2;4-dependent non-selective Na+ and K+ transport and its physiological roles in rice. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 0140-7791 2025 Amino Acid Substitutions in Loop C of Arabidopsis PIP2 Aquaporins Alters the Permeability of CO2 EN Shaila Shermin Tania Institute of Plant Science and Resources, Okayama University Shigeko Utsugi Institute of Plant Science and Resources, Okayama University Yoshiyuki Tsuchiya Institute of Plant Science and Resources, Okayama University Shizuka Sasano Institute of Plant Science and Resources, Okayama University Maki Katsuhara Institute of Plant Science and Resources, Okayama University Izumi C. Mori Institute of Plant Science and Resources, Okayama University The transport of CO2 across biomembranes in plant cells is essential for efficient photosynthesis. Some aquaporins capable of CO2 transport, referred to as ‘COOporins’, are postulated to play a crucial role in leaf CO2 diffusion. However, the structural basis of CO2 permeation through aquaporins remains largely unknown. Here, we show that amino acids in loop C are critical for the CO2 permeability of Arabidopsis thaliana PIP2 aquaporins. We found that swapping tyrosine and serine in loop C to histidine and phenylalanine, which differ between AtPIP2;1 and AtPIP2;3, altered CO2 permeability when examined in the Xenopus laevis oocyte heterologous expression system. AlphaFold2 modelling indicated that these substitution induced a conformational shift in the sidechain of arginine in the aromatic/arginine (ar/R) selectivity filter and in lysine at the extracellular mouth of the monomeric pore in PIP2 aquaporins. Our findings demonstrate that distal amino acid substitutions can trigger conformational changes of the ar/R filter in the monomeric pore, modulating CO2 permeability. Additionally, phylogenetic analysis suggested that aquaporins capable of dual water/CO2 permeability are ancestral to those that are water-selective and CO2-impermeable, and CO2-selective and water impermeable. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 0033-183X 262 2 2024 Analysis of the effect of permeant solutes on the hydraulic resistance of the plasma membrane in cells of Chara corallina 385 395 EN Masashi Tazawa Yoshida Biological Laboratory Randy Wayne Laboratory of Natural Philosophy, Plant Biology Section, Cornell University Maki Katsuhara Institute of Plant Science and Resources (IPSR), Okayama University In the cells of Chara corallina, permeant monohydric alcohols including methanol, ethanol and 1-propanol increased the hydraulic resistance of the membrane (Lpm−1). We found that the relative value of the hydraulic resistance (rLpm−1) was linearly dependent on the concentration (Cs) of the alcohol. The relationship is expressed in the equation: rLpm−1 = ρmCs + 1, where ρm 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 Lpm−1 and rLpm−1. The values of rLpm−1 fit the above relation we found for alcohols. When we plotted the ρm 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 (rLpm−1) as a function of the external (π0) and internal (πi) osmotic pressures. The analysis showed that the hydraulic resistance modifier coefficients (ρm) 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 (ρm) 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. No potential conflict of interest relevant to this article was reported.

Elsevier BV Acta Medica Okayama 0981-9428 227 2025 The hidden cation-selective pore in ion-conducting aquaporin OsPIP2;4 from rice 110168 EN Shuntaro Ono Institute of Plant Science and Resources, Okayama University Sen Thi Huong Tran Institute of Plant Science and Resources, Okayama University Yasunori Saitoh Research Institute for Interdisciplinary Science, Okayama University Shigeko Utsugi Institute of Plant Science and Resources, Okayama University Tomoaki Horie Division of Applied Biology, Faculty of Textile Science and Technology, Shinshu University Maki Katsuhara Institute of Plant Science and Resources, Okayama University Ion-conducting aquaporins (icAQPs) transport ions as well as water. Although the molecular mechanism of how AQPs establish selective permeability for water molecules is well understood, the ion-transporting mechanism in icAQPs has not yet been fully elucidated. In this study, we investigated the molecular mechanism of cation transport in OsPIP2;4, an icAQP in rice, by homology modeling and the electrophysiological analysis using Xenopus laevis oocytes. Water and ion transport assays using OsPIP2;4 T227M and G278K mutants strongly suggested that water- and cation-transporting pathways are independent of each other. Data from amino acid substitutions V54I and A143G in OsPIP2;4 led to the identification of a novel hidden pathway for cation transport located on the side surfaces of the tetramer channel, where two protomers are in contact, which is distinct from conventional monomeric pores and the tetrameric central pore in AQPs. Moreover, the present results provide the possibility that this hypothetical hidden pore also functions in the barley icAQP HvPIP2;8. The overall structure of this novel pathway appears to differ from the structure of general cation channels. However, the arrangement of hydrophilic amino acids at the entrance of the pathway of OsPIP2;4 was found to be comparable to that of some cation channels, which implies that the molecular mechanism of dehydration of hydrated ions might resemble that of the channels. Although direct structural evidence is needed to confirm the proposed pathway, the present study can be a stepping stone toward unraveling the mechanism of dual water and ion transport through icAQPs in plants. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2223-7747 10 10 2021 Identification and Characterization of Rice OsHKT1;3 Variants 2006 EN Shahin Imran Institute of Plant Science and Resources, Okayama University Yoshiyuki Tsuchiya Institute of Plant Science and Resources, Okayama University Sen Thi Huong Tran Institute of Plant Science and Resources, Okayama University Maki Katsuhara Institute of Plant Science and Resources, Okayama University 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.</p> No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1422-0067 21 19 2020 A Survey of Barley PIP Aquaporin Ionic Conductance Reveals Ca2+-Sensitive HvPIP2;8 Na+ and K+ Conductance 7135 EN Tran Sen Thi Huong Institute of Plant Science and Resources, Okayama University Tomoaki Horie Division of Applied Biology, Faculty of Textile Science and Technology, Shinshu University Shahin Imran Institute of Plant Science and Resources, Okayama University Jiaen Qiu Australian Research Council Centre of Excellence in Plant Energy Biology, Waite Research Institute and School of Agriculture, Food and Wine, The University of Adelaide Samantha McGaughey Research School of Biology, Australian National University Caitlin S. Byrt Australian Research Council Centre of Excellence in Plant Energy Biology, Waite Research Institute and School of Agriculture, Food and Wine, The University of Adelaide Stephen D. Tyerman Australian Research Council Centre of Excellence in Plant Energy Biology, Waite Research Institute and School of Agriculture, Food and Wine, The University of Adelaide Maki Katsuhara Institute of Plant Science and Resources, Okayama University 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. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2223-7747 9 1 2019 Expression and Ion Transport Activity of Rice OsHKT1;1 Variants 16 EN Shahin Imran Institute of Plant Science and Resources, Okayama University Tomoaki Horie Division of Applied Biology, Faculty of the Textile Science and Technology, Shinshu University Maki Katsuhara Institute of Plant Science and Resources, Okayama University 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. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 0140-7791 42 2 2018 The mechanism of SO2 -induced stomatal closure differs from O3 and CO2 responses and is mediated by nonapoptotic cell death in guard cells. 437 447 EN Lia Ooi Institute of Plant Science and Resources, Okayama University Takakazu Matsuura Institute of Plant Science and Resources, Okayama University Shintaro Munemasa Graduate School of Environmental and Life Science, Okayama University Yoshiyuki Murata Graduate School of Environmental and Life Science, Okayama University Maki Katsuhara Institute of Plant Science and Resources, Okayama University Takashi Hirayama Institute of Plant Science and Resources, Okayama University Izumi C. Mori Institute of Plant Science and Resources, Okayama University 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. No potential conflict of interest relevant to this article was reported.

岡山大学環境管理センター Acta Medica Okayama 0917-1533 31 2009 岡山大学資源生物科学研究所における屋上緑化による建物冷却効果 21 25 EN Maki Katsuhara Shigemi Tanakamaru Izumi C. Mori Akio Tani Shigeko Utsugi Takashi Enomoto Toshihiko Maitani Roof greening is known to be environmentally friendly technology. Recently developed new roof greening systems, such as the thin-layer/Excel soil© system and the wetland type greening system, were tested at the roof top of buildings of Research Institute for Bioresources, Okayama University. After a multi-year test, these new systems have been established during high-temperature and less-rainfall summer seasons in the south Okayama region. Data indicated that roof greening effectively reduced the temperature of the concrete surface (more than 10°C). The room temperature under the green roof was also reduced both in a stock room (up to 6°C) and in an office room (about 2°C). We also provided the estimation indicating that this roof greening is useful for the decrease in CO(2) emission through the reduction of the electric power for air-conditioning in the summer. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 456 4 2008 Barley plasma membrane intrinsic proteins (PIP aquaporins) as water and CO2 transporters 687 691 EN Maki Katsuhara Yuko T. Hanba <p>We identified barley aquaporins and demonstrated that one, HvPIP2;1, transports water and CO2. 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 CO2 conductance compared with leaves of wild-type rice plants. The rate of CO2 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 CO2 transport, and to improve plant water relations, stress tolerance, CO2 uptake or assimilation, and plant productivity via molecular breeding of aquaporins.</p> No potential conflict of interest relevant to this article was reported.

岡山大学資源生物科学研究所 Acta Medica Okayama 0916-930X 3 2 1995 Mesembryanthemum crystallinum(ice plant)におけるカリウムチャンネルをコードするPCR産物のクローニング 145 149 EN Maki Katsuhara Hans J. Bohnert Gene fragments of potassium channels were cloned from Mesembryanthemum crystallinum by using RT-PCR (reverse transcription-polymerase chain reaction). The two fragments were isolated independently and showed high similarity with each other. About 80% identity was found between the two fragments and potassium-channel genes of Arabidopsis. Southern hybridization indicated that the potassium channel gene may be a single copy gene or that a small gene family of potassium channels exists. No potential conflict of interest relevant to this article was reported.