Proteoliposomes reconstituted with human aquaporin-1 reveal novel single-ion-channel properties.

Human aquaporin 1 (hAQP1) forms homotetrameric channels that facilitate fluxes of water and small solutes across cell membranes. In addition to water channel activity, hAQP1 displays non-selective monovalent cation-channel activity gated by intracellular cyclic GMP. Dual water and ion-channel activity of hAQP1, thought to regulate cell shape and volume, could offer a target for novel therapeutics relevant to controlling cancer cell invasiveness. This study probed properties of hAQP1 ion channels using proteoliposomes, which, unlike conventional cell-based systems such as Xenopus laevis oocytes, are relatively free of background ion channels. Histidine-tagged recombinant hAQP1 protein was synthesized and purified from the methylotrophic yeast, Pichia pastoris, and reconstituted into proteoliposomes for biophysical analyses. Osmotic water channel activity confirmed correct folding and channel assembly. Ion-channel activity of hAQP1-Myc-His6 was recorded by patch-clamp electrophysiology with excised patches. In symmetrical potassium, the hAQP1-Myc-His6 channels displayed coordinated gating, a single-channel conductance of approximately 75 pS, and multiple subconductance states. Applicability of this method for structure-function analyses was tested using hAQP1-Myc-His6 D48A/D185A channels modified by site-directed mutations of charged Asp residues estimated to be adjacent to the central ion-conducting pore of the tetramer. No differences in conductance were detected between mutant and wild-type constructs, suggesting the open-state conformation could differ substantially from expectations based on crystal structures. Nonetheless, the method pioneered here for AQP1 demonstrates feasibility for future work defining structure-function relationships, screening pharmacological inhibitors, and testing other classes in the broad family of aquaporins for previously undiscovered ion-conducting capabilities.

Root-Specific Expression of Vitis vinifera VviNPF2.2 Modulates Shoot Anion Concentration in Transgenic Arabidopsis.

Grapevines (Vitis vinifera L., Vvi) on their roots are generally sensitive to salt-forming ions, particularly chloride (Cl-) when grown in saline environments. Grafting V. vinifera scions to Cl--excluding hybrid rootstocks reduces the impact of salinity. Molecular components underlying Cl--exclusion in Vitis species remain largely unknown, however, various anion channels and transporters represent good candidates for controlling this trait. Here, two nitrate/peptide transporter family (NPF) members VviNPF2.1 and VviNPF2.2 were isolated. Both highly homologous proteins localized to the plasma membrane of Arabidopsis (Arabidopsis thaliana) protoplasts. Both were expressed primarily in grapevine roots and leaves and were more abundant in a Cl--excluding rootstock compared to a Cl--includer. Quantitative PCR of grapevine roots revealed that VviNPF2.1 and 2.2 expression was downregulated by high [NO3 -] resupply post-starvation, but not affected by 25 mM Cl-. VviNPF2.2 was functionally characterized using an Arabidopsis enhancer trap line as a heterologous host which enabled cell-type-specific expression. Constitutive expression of VviNPF2.2 exclusively in the root epidermis and cortex reduced shoot [Cl-] after a 75 mM NaCl treatment. Higher expression levels of VviNPF2.2 correlated with reduced Arabidopsis xylem sap [NO3 -] when not salt stressed. We propose that when expressed in the root epidermis and cortex, VviNPF2.2 could function in passive anion efflux from root cells, which reduces the symplasmic Cl- available for root-to-shoot translocation. VviNPF2.2, through its role in the root epidermis and cortex, could, therefore, be beneficial to plants under salt stress by reducing net shoot Cl- accumulation.

Aquaporin ion conductance properties defined by membrane environment, protein structure, and cell physiology.

Aquaporins (AQPs) are multifunctional transmembrane channel proteins permeable to water and an expanding array of solutes. AQP-mediated ion channel activity was first observed when purified AQP0 from bovine lens was incorporated into lipid bilayers. Electrophysiological properties of ion-conducting AQPs since discovered in plants, invertebrates, and mammals have been assessed using native, reconstituted, and heterologously expressed channels. Accumulating evidence is defining amino acid residues that govern differential solute permeability through intrasubunit and central pores of AQP tetramers. Rings of charged and hydrophobic residues around pores influence AQP selectivity, and are candidates for further work to define motifs that distinguish ion conduction capability, versus strict water and glycerol permeability. Similarities between AQP ion channels thus far include large single channel conductances and long open times, but differences in ionic selectivity, permeability to divalent cations, and mechanisms of gating (e.g., by voltage, pH, and cyclic nucleotides) are unique to subtypes. Effects of lipid environments in modulating parameters such as single channel amplitude could explain in part the variations in AQP ion channel properties observed across preparations. Physiological roles of the ion-conducting AQP classes span diverse processes including regulation of cell motility, organellar pH, neural development, signaling, and nutrient acquisition. Advances in computational methods can generate testable predictions of AQP structure-function relationships, which combined with innovative high-throughput assays could revolutionize the field in defining essential properties of ion-conducting AQPs, discovering new AQP ion channels, and understanding the effects of AQP interactions with proteins, signaling cascades, and membrane lipids.

Inhibition of the Aquaporin-1 Cation Conductance by Selected Furan Compounds Reduces Red Blood Cell Sickling.

In sickle cell disease (SCD), the pathological shift of red blood cells (RBCs) into distorted morphologies under hypoxic conditions follows activation of a cationic leak current (Psickle) and cell dehydration. Prior work showed sickling was reduced by 5-hydroxylmethyl-2-furfural (5-HMF), which stabilized mutant hemoglobin and also blocked the Psickle current in RBCs, though the molecular basis of this 5-HMF-sensitive cation current remained a mystery. Work here is the first to test the hypothesis that Aquaporin-1 (AQP1) cation channels contribute to the monovalent component of Psickle. Human AQP1 channels expressed in Xenopus oocytes were evaluated for sensitivity to 5-HMF and four derivatives known to have differential efficacies in preventing RBC sickling. Ion conductances were measured by two-electrode voltage clamp, and osmotic water permeability by optical swelling assays. Compounds tested were: 5-HMF; 5-PMFC (5-(phenoxymethyl)furan-2-carbaldehyde); 5-CMFC (5-(4-chlorophenoxymethyl)furan-2-carbaldehyde); 5-NMFC (5-(2-nitrophenoxymethyl)-furan-2-carbaldehyde); and VZHE006 (tert-butyl (5-formylfuran-2-yl)methyl carbonate). The most effective anti-sickling agent, 5-PMFC, was the most potent inhibitor of the AQP1 ion conductance (98% block at 100 µM). The order of sensitivity of the AQP1 conductance to inhibition was 5-PMFC > VZHE006 > 5-CMFC ≥ 5-NMFC, which corresponded with effectiveness in protecting RBCs from sickling. None of the compounds altered AQP1 water channel activity. Combined application of a selective AQP1 ion channel blocker AqB011 (80 µM) with a selective hemoglobin modifying agent 5-NMFC (2.5 mM) increased anti-sickling effectiveness in red blood cells from human SCD patients. Another non-selective cation channel known to be expressed in RBCs, Piezo1, was unaffected by 2 mM 5-HMF. Results suggest that inhibition of AQP1 ion channels and capacity to modify hemoglobin are combined features of the most effective anti-sickling agents. Future therapeutics aimed at both targets could hold promise for improved treatments for SCD.

Soybean CHX-type ion transport protein GmSALT3 confers leaf Na+ exclusion via a root derived mechanism, and Cl- exclusion via a shoot derived process.

Soybean (Glycine max) yields are threatened by multiple stresses including soil salinity. GmSALT3 (a cation-proton exchanger protein) confers net shoot exclusion for both Na+ and Cl- and improves salt tolerance of soybean; however, how the ER-localized GmSALT3 achieves this is unknown. Here, GmSALT3's function was investigated in heterologous systems and near isogenic lines that contained the full-length GmSALT3 (NIL-T; salt-tolerant) or a truncated transcript Gmsalt3 (NIL-S; salt-sensitive). GmSALT3 restored growth of K+ -uptake-defective Escherichia coli and contributed towards net influx and accumulation of Na+ , K+ and Cl- in Xenopus laevis oocytes, while Gmsalt3 was non-functional. Time-course analysis of NILs confirmed shoot Cl- exclusion occurs distinctly from Na+ exclusion. Grafting showed that shoot Na+ exclusion occurs via a root xylem-based mechanism; in contrast, NIL-T plants exhibited significantly greater Cl- content in both the stem xylem and phloem sap compared to NIL-S, indicating that shoot Cl- exclusion likely depends upon novel phloem-based Cl- recirculation. NIL-T shoots grafted on NIL-S roots contained low shoot Cl- , which confirmed that Cl- recirculation is dependent on the presence of GmSALT3 in shoots. Overall, these findings provide new insights on GmSALT3's impact on salinity tolerance and reveal a novel mechanism for shoot Cl- exclusion in plants.

Efficient CRISPR/Cas9-Mediated Knockout of an Endogenous PHYTOENE DESATURASE Gene in T1 Progeny of Apomictic Hieracium Enables New Strategies for Apomixis Gene Identification.

Most Hieracium subgenus Pilosella species are self-incompatible. Some undergo facultative apomixis where most seeds form asexually with a maternal genotype. Most embryo sacs develop by mitosis, without meiosis and seeds form without fertilization. Apomixis is controlled by dominant loci where recombination is suppressed. Loci deletion by γ-irradiation results in reversion to sexual reproduction. Targeted mutagenesis of genes at identified loci would facilitate causal gene identification. In this study, the efficacy of CRISPR/Cas9 editing was examined in apomictic Hieracium by targeting mutations in the endogenous PHYTOENE DESATURASE (PDS) gene using Agrobacterium-mediated leaf disk transformation. In three experiments, the expected albino dwarf-lethal phenotype, characteristic of PDS knockout, was evident in 11% of T0 plants, 31.4% were sectorial albino chimeras, and the remainder were green. The chimeric plants flowered. Germinated T1 seeds derived from apomictic reproduction in two chimeric plants were phenotyped and sequenced to identify PDS gene edits. Up to 86% of seeds produced albino seedlings with complete PDS knockout. This was attributed to continuing Cas9-mediated editing in chimeric plants during apomictic seed formation preventing Cas9 segregation from the PDS target. This successful demonstration of efficient CRISPR/Cas9 gene editing in apomictic Hieracium, enabled development of the discussed strategies for future identification of causal apomixis genes.

The grapevine NaE sodium exclusion locus encodes sodium transporters with diverse transport properties and localisation.

Grapevine (Vitis vinifera L.) is a valuable crop for human consumption and wine production, and is prone to suffering from salinity stress in arid regions or when exposed to low quality irrigation water. A previous study identified a quantitative trait locus (QTL) NaE, containing six High-affinity Potassium Transporter 1 genes, that was associated with shoot Na+ exclusion in grapevine. While HKT1;1 was predicted to be the most likely gene within this QTL to encode for this important salinity tolerance sub-trait, four other HKTs within the QTL remained uncharacterised; VviHKT1;2 encodes a truncated transcript unlikely to form a functional transporter. In this study, two allelic variants for each of VviHKT1;6, VviHKT1;7 and VviHKT1;8 from the heterozygous grapevine variety Cabernet Sauvignon were functionally characterised. Using the Xenopus laevis oocyte heterologous expression system, as well as transient expression in tobacco leaves, we found that the VviHKT1;6 and VviHKT1;7 alleles encoded plasma membrane localised proteins that facilitated significant non-rectifying Na+ transport. Conversely, proteins encoded by the VviHKT1;8 alleles were inwardly-rectifying, weak Na+ transporters that localised to intracellular organelles. Mining of previous RNA-seq gene expression data suggested that VviHKT1;6-8 are weakly expressed in grapevine roots, flower buds, and seeds under normal conditions and different nutrient regimes. We propose that VviHKT1;6 and VviHKT1;7 are likely to have a less significant role in grapevine leaf Na+ exclusion than VviHKT1;1, and that VviHKT1;8 is involved in endomembrane Na+ transport.

Energy costs of salt tolerance in crop plants.

Agriculture is expanding into regions that are affected by salinity. This review considers the energetic costs of salinity tolerance in crop plants and provides a framework for a quantitative assessment of costs. Different sources of energy, and modifications of root system architecture that would maximize water vs ion uptake are addressed. Energy requirements for transport of salt (NaCl) to leaf vacuoles for osmotic adjustment could be small if there are no substantial leaks back across plasma membrane and tonoplast in root and leaf. The coupling ratio of the H+ -ATPase also is a critical component. One proposed leak, that of Na+ influx across the plasma membrane through certain aquaporin channels, might be coupled to water flow, thus conserving energy. For the tonoplast, control of two types of cation channels is required for energy efficiency. Transporters controlling the Na+ and Cl- concentrations in mitochondria and chloroplasts are largely unknown and could be a major energy cost. The complexity of the system will require a sophisticated modelling approach to identify critical transporters, apoplastic barriers and root structures. This modelling approach will inform experimentation and allow a quantitative assessment of the energy costs of NaCl tolerance to guide breeding and engineering of molecular components.

Plant Cation-Chloride Cotransporters (CCC): Evolutionary Origins and Functional Insights.

Genomes of unicellular and multicellular green algae, mosses, grasses and dicots harbor genes encoding cation-chloride cotransporters (CCC). CCC proteins from the plant kingdom have been comparatively less well investigated than their animal counterparts, but proteins from both plants and animals have been shown to mediate ion fluxes, and are involved in regulation of osmotic processes. In this review, we show that CCC proteins from plants form two distinct phylogenetic clades (CCC1 and CCC2). Some lycophytes and bryophytes possess members from each clade, most land plants only have members of the CCC1 clade, and green algae possess only the CCC2 clade. It is currently unknown whether CCC1 and CCC2 proteins have similar or distinct functions, however they are both more closely related to animal KCC proteins compared to NKCCs. Existing heterologous expression systems that have been used to functionally characterize plant CCC proteins, namely yeast and Xenopus laevis oocytes, have limitations that are discussed. Studies from plants exposed to chemical inhibitors of animal CCC protein function are reviewed for their potential to discern CCC function in planta. Thus far, mutations in plant CCC genes have been evaluated only in two species of angiosperms, and such mutations cause a diverse array of phenotypes-seemingly more than could simply be explained by localized disruption of ion transport alone. We evaluate the putative roles of plant CCC proteins and suggest areas for future investigation.

Functional differences in transport properties of natural HKT1;1 variants influence shoot Na+ exclusion in grapevine rootstocks.

Under salinity, Vitis spp. rootstocks can mediate salt (NaCl) exclusion from grafted V. vinifera scions enabling higher grapevine yields and production of superior wines with lower salt content. Until now, the genetic and mechanistic elements controlling sodium (Na+ ) exclusion in grapevine were unknown. Using a cross between two Vitis interspecific hybrid rootstocks, we mapped a dominant quantitative trait locus (QTL) associated with leaf Na+ exclusion (NaE) under salinity stress. The NaE locus encodes six high-affinity potassium transporters (HKT). Transcript profiling and functional characterization in heterologous systems identified VisHKT1;1 as the best candidate gene for controlling leaf Na+ exclusion. We characterized four proteins encoded by unique VisHKT1;1 alleles from the parents, and revealed that the dominant HKT variants exhibit greater Na+ conductance with less rectification than the recessive variants. Mutagenesis of VisHKT1;1 and TaHKT1.5-D from bread wheat, demonstrated that charged amino acid residues in the eighth predicted transmembrane domain of HKT proteins reduces inward Na+ conductance, and causes inward rectification of Na+ transport. The origin of the recessive VisHKT1;1 alleles was traced to V. champinii and V. rupestris. We propose that the genetic and functional data presented here will assist with breeding Na+ -tolerant grapevine rootstocks.

Chloride: not simply a 'cheap osmoticum', but a beneficial plant macronutrient.

HIGHLIGHT: At macronutrient levels, chloride has positive effects on plant growth, which are distinct from its function in photosynthesis..

Non-selective cation channel activity of aquaporin AtPIP2;1 regulated by Ca2+ and pH.

The aquaporin AtPIP2;1 is an abundant plasma membrane intrinsic protein in Arabidopsis thaliana that is implicated in stomatal closure, and is highly expressed in plasma membranes of root epidermal cells. When expressed in Xenopus laevis oocytes, AtPIP2;1 increased water permeability and induced a non-selective cation conductance mainly associated with Na+ . A mutation in the water pore, G103W, prevented both the ionic conductance and water permeability of PIP2;1. Co-expression of AtPIP2;1 with AtPIP1;2 increased water permeability but abolished the ionic conductance. AtPIP2;2 (93% identical to AtPIP2;1) similarly increased water permeability but not ionic conductance. The ionic conductance was inhibited by the application of extracellular Ca2+ and Cd2+ , with Ca2+ giving a biphasic dose-response with a prominent IC50 of 0.32 mм comparable with a previous report of Ca2+ sensitivity of a non-selective cation channel (NSCC) in Arabidopsis root protoplasts. Low external pH also inhibited ionic conductance (IC50 pH 6.8). Xenopus oocytes and Saccharomyces cerevisiae expressing AtPIP2;1 accumulated more Na+ than controls. Establishing whether AtPIP2;1 has dual ion and water permeability in planta will be important in understanding the roles of this aquaporin and if AtPIP2;1 is a candidate for a previously reported NSCC responsible for Ca2+ and pH sensitive Na+ entry into roots.

SLAH1, a homologue of the slow type anion channel SLAC1, modulates shoot Cl- accumulation and salt tolerance in Arabidopsis thaliana.

Salinity tolerance is correlated with shoot chloride (Cl(-)) exclusion in multiple crops, but the molecular mechanisms of long-distance Cl(-) transport are poorly defined. Here, we characterize the in planta role of AtSLAH1 (a homologue of the slow type anion channel-associated 1 (SLAC1)). This protein, localized to the plasma membrane of root stelar cells, has its expression reduced by salt or ABA, which are key predictions for a protein involved with loading Cl(-) into the root xylem. Artificial microRNA knockdown mutants of AtSLAH1 had significantly reduced shoot Cl(-) accumulation when grown under low Cl(-), whereas shoot Cl(-) increased and the shoot nitrate/chloride ratio decreased following AtSLAH1 constitutive or stelar-specific overexpression when grown in high Cl(-) In both sets of overexpression lines a significant reduction in shoot biomass over the null segregants was observed under high Cl(-) supply, but not low Cl(-) supply. Further in planta data showed AtSLAH3 overexpression increased the shoot nitrate/chloride ratio, consistent with AtSLAH3 favouring nitrate transport. Heterologous expression of AtSLAH1 in Xenopus laevis oocytes led to no detectible transport, suggesting the need for post-translational modifications for AtSLAH1 to be active. Our in planta data are consistent with AtSLAH1 having a role in controlling root-to-shoot Cl(-) transport.

Identification of a Stelar-Localized Transport Protein That Facilitates Root-to-Shoot Transfer of Chloride in Arabidopsis.

Under saline conditions, higher plants restrict the accumulation of chloride ions (Cl(-)) in the shoot by regulating their transfer from the root symplast into the xylem-associated apoplast. To identify molecular mechanisms underpinning this phenomenon, we undertook a transcriptional screen of salt stressed Arabidopsis (Arabidopsis thaliana) roots. Microarrays, quantitative RT-PCR, and promoter-GUS fusions identified a candidate gene involved in Cl(-) xylem loading from the Nitrate transporter 1/Peptide Transporter family (NPF2.4). This gene was highly expressed in the root stele compared to the cortex, and its expression decreased after exposure to NaCl or abscisic acid. NPF2.4 fused to fluorescent proteins, expressed either transiently or stably, was targeted to the plasma membrane. Electrophysiological analysis of NPF2.4 in Xenopus laevis oocytes suggested that NPF2.4 catalyzed passive Cl(-) efflux out of cells and was much less permeable to NO3(-). Shoot Cl(-) accumulation was decreased following NPF2.4 artificial microRNA knockdown, whereas it was increased by overexpression of NPF2.4. Taken together, these results suggest that NPF2.4 is involved in long-distance transport of Cl(-) in plants, playing a role in the loading and the regulation of Cl(-) loading into the xylem of Arabidopsis roots during salinity stress.

Grapevine and Arabidopsis Cation-Chloride Cotransporters Localize to the Golgi and Trans-Golgi Network and Indirectly Influence Long-Distance Ion Transport and Plant Salt Tolerance.

Plant cation-chloride cotransporters (CCCs) have been implicated in conferring salt tolerance. They are predicted to improve shoot salt exclusion by directly catalyzing the retrieval of sodium (Na(+)) and chloride (Cl(-)) ions from the root xylem. We investigated whether grapevine (Vitis vinifera [Vvi]) CCC has a role in salt tolerance by cloning and functionally characterizing the gene from the cultivar Cabernet Sauvignon. Amino acid sequence analysis revealed that VviCCC shares a high degree of similarity with other plant CCCs. A VviCCC-yellow fluorescent protein translational fusion protein localized to the Golgi and the trans-Golgi network and not the plasma membrane when expressed transiently in tobacco (Nicotiana benthamiana) leaves and Arabidopsis (Arabidopsis thaliana) mesophyll protoplasts. AtCCC-green fluorescent protein from Arabidopsis also localized to the Golgi and the trans-Golgi network. In Xenopus laevis oocytes, VviCCC targeted to the plasma membrane, where it catalyzed bumetanide-sensitive (36)Cl(-), (22)Na(+), and (86)Rb(+) uptake, suggesting that VviCCC (like AtCCC) belongs to the Na(+)-K(+)-2Cl(-) cotransporter class of CCCs. Expression of VviCCC in an Arabidopsis ccc knockout mutant abolished the mutant's stunted growth phenotypes and reduced shoot Cl(-) and Na(+) content to wild-type levels after growing plants in 50 mm NaCl. In grapevine roots, VviCCC transcript abundance was not regulated by Cl(-) treatment and was present at similar levels in both the root stele and cortex of three Vitis spp. genotypes that exhibit differential shoot salt exclusion. Our findings indicate that CCC function is conserved between grapevine and Arabidopsis, but neither protein is likely to directly mediate ion transfer with the xylem or have a direct role in salt tolerance.

Shoot chloride exclusion and salt tolerance in grapevine is associated with differential ion transporter expression in roots.

BACKGROUND: Salt tolerance in grapevine is associated with chloride (Cl-) exclusion from shoots; the rate-limiting step being the passage of Cl- between the root symplast and xylem apoplast. Despite an understanding of the physiological mechanism of Cl- exclusion in grapevine, the molecular identity of membrane proteins that control this process have remained elusive. To elucidate candidate genes likely to control Cl- exclusion, we compared the root transcriptomes of three Vitis spp. with contrasting shoot Cl- exclusion capacities using a custom microarray. RESULTS: When challenged with 50 mM Cl-, transcriptional changes of genotypes 140 Ruggeri (shoot Cl- excluding rootstock), K51-40 (shoot Cl- including rootstock) and Cabernet Sauvignon (intermediate shoot Cl- excluder) differed. The magnitude of salt-induced transcriptional changes in roots correlated with the amount of Cl- accumulated in shoots. Abiotic-stress responsive transcripts (e.g. heat shock proteins) were induced in 140 Ruggeri, respiratory transcripts were repressed in Cabernet Sauvignon, and the expression of hypersensitive response and ROS scavenging transcripts was altered in K51-40. Despite these differences, no obvious Cl- transporters were identified. However, under control conditions where differences in shoot Cl- exclusion between rootstocks were still significant, genes encoding putative ion channels SLAH3, ALMT1 and putative kinases SnRK2.6 and CPKs were differentially expressed between rootstocks, as were members of the NRT1 (NAXT1 and NRT1.4), and CLC families. CONCLUSIONS: These results suggest that transcriptional events contributing to the Cl- exclusion mechanism in grapevine are not stress-inducible, but constitutively different between contrasting varieties. We have identified individual genes from large families known to have members with roles in anion transport in other plants, as likely candidates for controlling anion homeostasis and Cl- exclusion in Vitis species. We propose these genes as priority candidates for functional characterisation to determine their role in chloride transport in grapevine and other plants.