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

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.

Calcium control of the hydraulic resistance in cells of Chara corallina.

The hydraulic resistance (the reciprocal of the hydraulic conductivity Lp) Lp-1 was measured in cells of Chara corallina by the method of transcellular osmosis. Treatment of cells with 100 mM KCl decreased Lp-1 significantly. Subsequent treatment of the cells with 70 mM CaCl2 recovered the decreased Lp-1 to the original value. To know whether K+ or Ca2+/Mg2+ acts on the cell wall and/or the membrane, the hydraulic resistances of the cell wall (Lpw-1) and that of the membrane (Lpm-1) were determined in one and the same cell. For this, a pair of cells (twin cells) were made from an internodal cell, one used for measurement of Lp-1 and the other used for the measurement of Lpw-1. From Lp-1 and Lpw-1, Lpm-1 was calculated. Both Lp-1 and Lpw-1 were decreased by K+, while Lpm-1 was not affected by K+. The same result was obtained with 5 mM EGTA. Lpw-1 was decreased more than it was by KCl but Lpm-1 remained constant after EGTA treatment. The recovery of the K+-decreased Lp-1 with Ca2+ can be explained exclusively by the recovery of Lpw-1 with Ca2+. The Ca2+ recovery of Lpw-1 was observed in the intact cell wall but not in the cell wall tube isolated from an internodal cell. The different response to Ca2+ between the intact cell wall and the isolated cell wall was discussed in relation to the tension in the cell wall which may be an important factor for the ionic regulation of hydraulic conductivity.

Distinct Functions of the Atypical Terminal Hydrophilic Domain of the HKT Transporter in the Liverwort Marchantia polymorpha.

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.

Identification and Characterization of Rice OsHKT1;3 Variants.

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.

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

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 the 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.

Greetings from the new Editor-in-Chief of the Journal of Plant Research 2021.

It is my pleasure to be writing this editorial as Editor-in-Chief of the Journal of Plant Research (JPR) for the next 4 years, following the former Editor-in-Chief, Prof. Kouki Hikosaka of Tohoku University. I will do my best to improve JPR along with all editors, board members, and readers.

Age dependence of the hydraulic resistances of the plasma membrane and the tonoplast (vacuolar membrane) in cells of Chara corallina.

Hydraulic resistances (reciprocals of hydraulic conductivities) of the cell (Lp-1), the cell wall (Lpw-1), the membrane (Lpm-1), the plasma membrane (Lppm-1), and the tonoplast (Lptp-1) were determined in individual internodal cells of Chara corallina and their dependence on the cell age was studied. The thickness of the cell wall (d) was adopted as an index of the cell age, since the cell wall of spring-grown young cells (sg-cells) was found to be significantly thinner than that of winter-spent old cells (ws-cells). Both Lpw-1 and Lpm-1 were found to increase with cell age. Since Lpm-1 is the sum of Lppm-1 and Lptp-1, their dependence on the wall thickness was studied. It was found that both Lppm-1 and Lptp-1 increase with cell age using d as a proxy and that the former is distinctly higher than the latter. The ratio Lppm-1/Lptp-1 amounts to 30 for 5 µm of d, indicating that the tonoplast is a negligible barrier to osmotic water flow. The ratio decreases with the increase in d and amounts to 5.0 for 11 µm of d, showing that the tonoplast ages faster than the plasma membrane. The physiological meaning of the age dependence of hydraulic resistance of the tonoplast was discussed in terms of the role of the vacuole in the osmoregulation of the cytoplasm.

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

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.

A Survey of Barley PIP Aquaporin Ionic Conductance Reveals Ca2+-Sensitive HvPIP2;8 Na+ and K+ Conductance.

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.

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

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.

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

Plasma membrane intrinsic proteins (PIPs) are known to be major facilitators of the movement of a number of substrates across cell membranes. From a drought-resistant cultivar of Oryza sativa (rice), we isolated an OsPIP1;3 gene single-nucleotide polymorphism (SNP) that is mostly expressed in rice roots and is strongly responsive to drought stress. Immunocytochemistry showed that OsPIP1;3 majorly accumulated on the proximal end of the endodermis and the cell surface around the xylem. Expression of GFP-OsPIP1;3 alone in Xenopus oocytes or rice protoplasts showed OsPIP1;3 mislocalization in the endoplasmic reticulum (ER)-like neighborhood, whereas co-expression of OsPIP2;2 recruited OsPIP1;3 to the plasma membrane and led to a significant enhancement of water permeability in oocytes. Moreover, reconstitution of 10×His-OsPIP1;3 in liposomes demonstrated water channel activity, as revealed by stopped-flow light scattering. Intriguingly, by patch-clamp technique, we detected significant NO3- conductance of OsPIP1;3 in mammalian cells. To investigate the physiological functions of OsPIP1;3, we ectopically expressed the OsPIP1;3 gene in Nicotiana benthamiana (tobacco). The transgenic tobacco plants exhibited higher photosynthesis rates, root hydraulic conductivity (Lpr ) and water-use efficiency, resulting in a greater biomass and a higher resistance to water deficit than the wild-type did. Further experiments suggested that heterologous expression of OsPIP1;3 in cyanobacterium altered bacterial growth under different conditions of CO2 gas supply. Overall, besides shedding light on the multiple functions played by OsPIP1;3, this work provides insights into the translational value of plant AQPs.

Expression and Ion Transport Activity of Rice OsHKT1;1 Variants.

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.

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.

Sporobolus virginicus is a halophytic C4 grass found worldwide, from tropical to warm temperate regions. One Japanese genotype showed a salinity tolerance up to 1.5 M NaCl, a three-fold higher concentration than the salinity of sea water. To identify the key genes involved in the regulation of salt tolerance in S. virginicus, we produced 3500 independent transgenic Arabidopsis lines expressing random cDNA from S. virginicus and screened 10 lines which showed enhanced salt tolerance compared with the wild type in a medium containing 150 mM NaCl. Among the selected lines, two contained cDNA coding glycine-rich RNA-binding proteins (SvGRP1 and SvGRP2). This is the first reports on the function of GRPs from halophytes in salt tolerance though reports have shown GRPs are involved in diverse biological and biochemical processes including salt tolerance in Arabidopsis and some other glycophytes. Transcriptomic analysis and GO enrichment analysis of SvGRP1-expressing Arabidopsis under salt stress revealed upregulation of polyol and downregulation of glucosinolate and indole acetic acid biosynthesis/metabolic pathways. Metabolomic analysis of the SvGRP1-transformant suggested that the increase in 3-aminoppropanoic acid, citramalic acid, and isocitric acid content was associated with enhanced salt tolerance. These findings could provide novel insight into the roles of GRPs in plant salt tolerance.

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

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.

High-Affinity K+ Transporters from a Halophyte, Sporobolus virginicus, Mediate Both K+ and Na+ Transport in Transgenic Arabidopsis, X. laevis Oocytes and Yeast.

Class II high-affinity potassium transporters (HKTs) have been proposed to mediate Na+-K+ co-transport in plants, as well as Na+ and K+ homeostasis under K+-starved and saline environments. We identified class II HKTs, namely SvHKT2;1 and SvHKT2;2 (SvHKTs), from the halophytic turf grass, Sporobolus virginicus. SvHKT2;2 expression in S. virginicus was up-regulated by NaCl treatment, while SvHKT2;1 expression was assumed to be up-regulated by K+ starvation and down-regulated by NaCl treatment. Localization analysis revealed SvHKTs predominantly targeted the plasma membrane. SvHKTs complemented K+ uptake deficiency in mutant yeast, and showed both inward and outward K+ and Na+ transport activity in Xenopus laevis oocytes. When constitutively expressed in Arabidopsis, SvHKTs mediated K+ and Na+ accumulation in shoots under K+-starved conditions, and the K+ concentration in xylem saps of transformants was also higher than in those of wild-type plants. These results suggest transporter-enhanced K+ and Na+ uploading to the xylem from xylem parenchyma cells. Together, our data demonstrate that SvHKTs mediate both outward and inward K+ and Na+ transport in X. laevis oocytes, and possibly in plant and yeast cells, depending on the ionic conditions.