Tran STH , Horie T , Imran S , Qiu J , McGaughey S , Byrt CS , Tyerman SD , Katsuhara M .

20K06708 KAKENHI Grant-in-Aid for Scientific Research (C)

	Figure 1. Electrophysiological survey to test for HvPIP2 ion transport (A,B) Current–voltage relationships of X. leavis oocytes expressing each HvPIP2 in the presence of 86.4 mM NaCl and 9.6 mM KCl with 30 µM Ca2+ (A) or 1.8 mM Ca2+ (B). A total of 10 ng of each HvPIP2 cRNA or water (control) was injected into X. laevis oocytes. (C) Relationships between the external free Ca2+ concentration and HvPIP2;8-mediated Na+ conductance in the presence of 86.4 mM NaCl and 9.6 mM KCl (R2 = 0.93). The free Ca2+ concentrations are given in Methods. A step pulse protocol of −120 mV to +30 mV with a 15 mV increment was applied on every oocyte. Ionic conductance was calculated based on the data obtained from V = −75 mV to −120 mV of the membrane potential. Data are the means ± SE (n = 5 for A,C, and n = 7 for B).
	Figure 2. Monovalent alkaline cation selectivity of HvPIP2;8 and the effect of the interaction of K+ and Na+ on HvPIP2;8-mediated ion conductance activity. Current–voltage relationships obtained from oocytes either expressing HvPIP2;8 (A) or injected with water (B) HvPIP2;8 displays a different monovalent alkaline cation selectivity. Oocytes were successively immersed in bath solutions with a high calcium condition, supplemented with Na+, K+, Cs+, Rb+, and Li+ (as chloride salts) at the concentration of 96 mM. (C) Inhibition of HvPIP2;8-mediated Na+ transport by monovalent alkaline cations in the presence of 48 mM NaCl with 48 mM of each alkaline cation. (D) The effect of external Na+/K+ concentration ratios on the conductance of HvPIP2;8-expressing oocytes from V (membrane potential) = −75 mV to −120 mV. The total concentration of (Na + K) was constantly 96 mM. X. laevis oocytes were injected with 10 ng of HvPIP2;8 cRNA for the recording of the conductance in every experiment. Data are the means ± SE (n = 7 to 8 for A, n = 5 for B, n = 4 to 5 for C, and n = 5 to 6 for D).
	Figure 3. HvPIP2;8-mediated Na+ transport is Cl− independent. (A) Current–voltage relationships obtained from oocytes either expressing HvPIP2;8 or injected with water in the presence of either 96 mM NaCl or 96 mM Na-gluconate. Inset: Expanded current voltage curves around the reversal potential. (B) Current–voltage relationships obtained from oocytes either expressing HvPIP2;8 or injected with water in the presence of either 96 mM NaCl or 96 mM Choline-Cl. All solutions contained 30 µM Ca2+. X. laevis oocytes were injected with 10 ng of HvPIP2;8 cRNA. Data are the means ± SE (n = 7 to 8).
	Figure 4. Effects of divalent cations on the ion current responses in oocytes expressing HvPIP2;8. (A) Effect of divalent cations on the ion currents of the HvPIP2;8-transporter; bath solutions with a high 1.8 mM Ca2+ background calcium conditions were successively replaced with either 1.8 mM Ca2+, Ba2+, Cd2+, and Mg2+ (as chloride salts), at concentrations of 86.4 mM NaCl and 9.6 mM KCl. (B) Box plot summary of the ionic conductance presented in (A); the ionic conductances were calculated from V = −75 mV to −120 mV. (C) Relief of Ca2+ inhibition by the addition of Mg2+ on the ion current responses in oocytes expressing HvPIP2;8; note the different range of the Y-axis from the plot in (A). (D) Box plot summary of the ionic conductance presented in (C). Steady-state current–voltage curves of the X. laevis oocytes injected with 10 ng of cRNA per oocyte were recorded. Currents from the oocytes injected with water were the negative controls from the same batch. Significant differences (p < 0.05) are indicated by different letters using one-way ANOVA with Duncan’s multiple comparisons test. Data are the means ± SE of three independent experiments, (n = 6 for A,B).
	Figure 5. Co-expression of HvPIP1s and HvPIP2;8 reduces HvPIP2;8-dependent currents in X. laevis oocytes. (A) Current–voltage relationships obtained from oocytes either expressing HvPIP2;8 alone, each HvPIP1 alone, or injected with water; each HvPIP1 of the five HvPIP1s did not show ion channel activity when expressed alone. (B) Co-expression of HvPIP2;8 with each HvPIP1 largely inhibited the ion channel activity of HvPIP2;8. (C,D) Box plot summary of the ionic conductance for data shown in (A,B), respectively. Oocytes were injected with 10 ng cRNA of HvPIP2;8, 40 ng cRNA of each HvPIP1 in both the solo- and co-expression analyses. The bath solution included 86.4 mM NaCl, 9.6 mM KCl, 1.8 mM MgCl2, 1.8 mM EGTA, 1.8 mM CaCl2, 10 mM HEPES, and a pH 7.5 with Tris, and therefore the free Ca2+ concentration was 30 µM. Significant differences (p < 0.05) are indicated by different letters using one-way ANOVA with Duncan’s multiple comparisons test. Data are the means ± SE (n = 8).
	Figure 6. Phosphorylation mimic HvPIP2;8 S285D influences HvPIP2;8-facilitated cation transport. Oocytes were injected with 46 nL water (Control) or with 46 nL water (n = 11) containing 23 ng HvPIP2;8 WT (n = 30) or HvPIP2;8 S285D (n = 40) cRNA. Ionic conductance and osmotic water permeability (Pos) of the cRNA-injected oocytes were determined via the TEVC and the swelling assay, respectively. (A) Na+ conductance relative to H2O-injected control (dotted line). Currents were recorded in “Na100” (100 mM NaCl; 2 mM KCl, 1 mM MgCl2, 5 mM HEPES, 50 μM CaCl2, 100 mM NaCl or 100 mM KCl, osmolality 220 mosmol Kg−1 (adjusted with D-mannitol), and pH 8.5). (B) Pos relative to H2O-injected control (dotted line). Data in (A,B) was collected from three different frogs and is shown as the mean ± SE, where each data point represents an individual oocyte; for each oocyte, both the ionic conductance and Pos were measured; data from (C,D) is from one batch and again for each oocyte both the ionic conductance and Pos were measured. Significant differences (p < 0.05) are indicated by different letters (one-way ANOVA, Fisher’s post-test). (C) Relationships between the mean Pos and mean ionic conductance for HvPIP2;8 WT and HvPIP2;8 S285D with the mean of the controls subtracted. This data is from oocytes from the same three independent frogs as shown in (A,B). (D) Kinase inhibitor H7 influenced the relationship between the ionic conductance and water permeability in HvPIP2;8 S285D-expressing oocytes. Oocytes were either untreated or were pre-treated in a low Na+ Ringer solution that contained 10 µM H7 dihydrochloride (H7) for 2 h before TEVC and the swelling assay. An individual conductance was plotted against the corresponding Pos for each oocyte, and the mean for the water-injected controls is shown (black circle, dotted line). Linear regression of Pos versus ionic conductance was only significant for HvPIP2;8 S285D without H7 treatment (p < 0.005).
	Figure 7. The expression level of the HvPIP2;8 transcripts in a barley cultivar, Haruna-Nijo, detected by qPCR. Five-day-old barley seedlings were prepared by hydroponic culture and further grown on the culture solution with or without NaCl (100 mM or 200 mM) for 1 day or 5 days. Transcript levels of HvPIP2;8 in shoots and roots were investigated by absolute quantification. Absolute amounts of transcripts (copies/µg RNA) were displayed. Significant differences (p < 0.05) are indicated by different letters using one-way ANOVA with Duncan’s multiple comparisons test. Data are the means ± SE, and n = 3.

Amtmann, Multiple inward channels provide flexibility in Na+/K+ discrimination at the plasma membrane of barley suspension culture cells. 1997, Pubmed

Amtmann, Multiple inward channels provide flexibility in Na+/K+ discrimination at the plasma membrane of barley suspension culture cells. 1997, Pubmed
Besse, Developmental pattern of aquaporin expression in barley (Hordeum vulgare L.) leaves. 2011, Pubmed , Xenbase
Bienert, Heterotetramerization of Plant PIP1 and PIP2 Aquaporins Is an Evolutionary Ancient Feature to Guide PIP1 Plasma Membrane Localization and Function. 2018, Pubmed , Xenbase
Boursiac, Stimulus-induced downregulation of root water transport involves reactive oxygen species-activated cell signalling and plasma membrane intrinsic protein internalization. 2008, Pubmed , Xenbase
Boursiac, Early effects of salinity on water transport in Arabidopsis roots. Molecular and cellular features of aquaporin expression. 2005, Pubmed
Byrt, Non-selective cation channel activity of aquaporin AtPIP2;1 regulated by Ca2+ and pH. 2017, Pubmed , Xenbase
Chaumont, Aquaporins: highly regulated channels controlling plant water relations. 2014, Pubmed
Chen, Root plasma membrane transporters controlling K+/Na+ homeostasis in salt-stressed barley. 2007, Pubmed
Choi, Salt stress-induced Ca2+ waves are associated with rapid, long-distance root-to-shoot signaling in plants. 2014, Pubmed
Coffey, Root and cell hydraulic conductivity, apoplastic barriers and aquaporin gene expression in barley (Hordeum vulgare L.) grown with low supply of potassium. 2018, Pubmed
Davenport, A weakly voltage-dependent, nonselective cation channel mediates toxic sodium influx in wheat. 2000, Pubmed
Demidchik, Sodium fluxes through nonselective cation channels in the plasma membrane of protoplasts from Arabidopsis roots. 2002, Pubmed
Dolan, Cell expansion in roots. 2004, Pubmed
Engh, Crystal structures of catalytic subunit of cAMP-dependent protein kinase in complex with isoquinolinesulfonyl protein kinase inhibitors H7, H8, and H89. Structural implications for selectivity. 1996, Pubmed
Essah, Sodium influx and accumulation in Arabidopsis. 2003, Pubmed
Grondin, Aquaporins Contribute to ABA-Triggered Stomatal Closure through OST1-Mediated Phosphorylation. 2015, Pubmed , Xenbase
Horie, Salinity tolerance mechanisms in glycophytes: An overview with the central focus on rice plants. 2012, Pubmed
Horie, Rice OsHKT2;1 transporter mediates large Na+ influx component into K+-starved roots for growth. 2007, Pubmed
Horie, Mechanisms of water transport mediated by PIP aquaporins and their regulation via phosphorylation events under salinity stress in barley roots. 2011, Pubmed , Xenbase
Horie, HKT transporter-mediated salinity resistance mechanisms in Arabidopsis and monocot crop plants. 2009, Pubmed
Hove, Identification and Expression Analysis of the Barley (Hordeum vulgare L.) Aquaporin Gene Family. 2015, Pubmed
Ikeda, Characterization of aquaporin-6 as a nitrate channel in mammalian cells. Requirement of pore-lining residue threonine 63. 2002, Pubmed , Xenbase
Isayenkov, Plant Salinity Stress: Many Unanswered Questions Remain. 2019, Pubmed
Ismail, Genomics, Physiology, and Molecular Breeding Approaches for Improving Salt Tolerance. 2017, Pubmed
Kaneko, Dynamic regulation of the root hydraulic conductivity of barley plants in response to salinity/osmotic stress. 2015, Pubmed
Katsuhara, Over-expression of a barley aquaporin increased the shoot/root ratio and raised salt sensitivity in transgenic rice plants. 2003, Pubmed
Katsuhara, Expanding roles of plant aquaporins in plasma membranes and cell organelles. 2008, Pubmed
Katsuhara, Functional analysis of water channels in barley roots. 2002, Pubmed , Xenbase
Kim, Modulation of lysophosphatidic acid-induced Cl- currents by protein kinases A and C in the Xenopus oocyte. 2000, Pubmed , Xenbase
Knipfer, Aquaporin-facilitated water uptake in barley (Hordeum vulgare L.) roots. 2011, Pubmed
Kourghi, Divalent Cations Regulate the Ion Conductance Properties of Diverse Classes of Aquaporins. 2017, Pubmed , Xenbase
Laloux, Plant and Mammal Aquaporins: Same but Different. 2018, Pubmed
Li, Single-molecule analysis of PIP2;1 dynamics and partitioning reveals multiple modes of Arabidopsis plasma membrane aquaporin regulation. 2011, Pubmed
Liu, Ectopic expression of a rice plasma membrane intrinsic protein (OsPIP1;3) promotes plant growth and water uptake. 2020, Pubmed
Martinez-Ballesta, The Expanding Role of Vesicles Containing Aquaporins. 2018, Pubmed
Maurel, Aquaporins in Plants. 2015, Pubmed
Mori, 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. 2018, Pubmed , Xenbase
Munns, Mechanisms of salinity tolerance. 2008, Pubmed
Nyblom, Structural and functional analysis of SoPIP2;1 mutants adds insight into plant aquaporin gating. 2009, Pubmed
Otto, Aquaporin tetramer composition modifies the function of tobacco aquaporins. 2010, Pubmed
Prak, Multiple phosphorylations in the C-terminal tail of plant plasma membrane aquaporins: role in subcellular trafficking of AtPIP2;1 in response to salt stress. 2008, Pubmed
Qiu, Phosphorylation influences water and ion channel function of AtPIP2;1. 2020, Pubmed
Rodrigues, Aquaporins facilitate hydrogen peroxide entry into guard cells to mediate ABA- and pathogen-triggered stomatal closure. 2017, Pubmed
Sanders, Calcium at the crossroads of signaling. 2002, Pubmed
Shibasaka, Functional characterization of a novel plasma membrane intrinsic protein2 in barley. 2012, Pubmed , Xenbase
Tyerman, Pathways for the permeation of Na+ and Cl- into protoplasts derived from the cortex of wheat roots. 1997, Pubmed
Törnroth-Horsefield, Structural mechanism of plant aquaporin gating. 2006, Pubmed
Vandeleur, The role of plasma membrane intrinsic protein aquaporins in water transport through roots: diurnal and drought stress responses reveal different strategies between isohydric and anisohydric cultivars of grapevine. 2009, Pubmed , Xenbase
Vitali, Cooperativity in proton sensing by PIP aquaporins. 2019, Pubmed , Xenbase
Wang, Reconstitution of CO2 Regulation of SLAC1 Anion Channel and Function of CO2-Permeable PIP2;1 Aquaporin as CARBONIC ANHYDRASE4 Interactor. 2016, Pubmed , Xenbase
Yao, Differential sodium and potassium transport selectivities of the rice OsHKT2;1 and OsHKT2;2 transporters in plant cells. 2010, Pubmed , Xenbase
Yool, New roles for old holes: ion channel function in aquaporin-1. 2002, Pubmed
Zelazny, FRET imaging in living maize cells reveals that plasma membrane aquaporins interact to regulate their subcellular localization. 2007, Pubmed , Xenbase