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.

Enhancing crop yields through improvements in the efficiency of photosynthesis and respiration.

The rate with which crop yields per hectare increase each year is plateauing at the same time that human population growth and other factors increase food demand. Increasing yield potential ( Y p ) of crops is vital to address these challenges. In this review, we explore a component of Y p that has yet to be optimised - that being improvements in the efficiency with which light energy is converted into biomass ( ε c ) via modifications to CO2 fixed per unit quantum of light (α), efficiency of respiratory ATP production ( ε prod ) and efficiency of ATP use ( ε use ). For α, targets include changes in photoprotective machinery, ribulose bisphosphate carboxylase/oxygenase kinetics and photorespiratory pathways. There is also potential for ε prod to be increased via targeted changes to the expression of the alternative oxidase and mitochondrial uncoupling pathways. Similarly, there are possibilities to improve ε use via changes to the ATP costs of phloem loading, nutrient uptake, futile cycles and/or protein/membrane turnover. Recently developed high-throughput measurements of respiration can serve as a proxy for the cumulative energy cost of these processes. There are thus exciting opportunities to use our growing knowledge of factors influencing the efficiency of photosynthesis and respiration to create a step-change in yield potential of globally important crops.

Picrotoxin Delineates Different Transport Configurations for Malate and γ Aminobutyric Acid through TaALMT1.

Plant-derived pharmacological agents have been used extensively to dissect the structure-function relationships of mammalian GABA receptors and ion channels. Picrotoxin is a non-competitive antagonist of mammalian GABAA receptors. Here, we report that picrotoxin inhibits the anion (malate) efflux mediated by wheat (Triticum aestivum) ALMT1 but has no effect on GABA transport. The EC50 for inhibition was 0.14 nM and 0.18 nM when the ALMTs were expressed in tobacco BY2 cells and in Xenopus oocytes, respectively. Patch clamping of the oocyte plasma membrane expressing wheat ALMT1 showed that picrotoxin inhibited malate currents from both sides of the membrane. These results demonstrate that picrotoxin inhibits anion efflux effectively and can be used as a new inhibitor to study the ion fluxes mediated by ALMT proteins that allow either GABA or anion transport.

Role of TaALMT1 malate-GABA transporter in alkaline pH tolerance of wheat.

Malate exudation through wheat (Triticum aestivum L) aluminium-activated malate transporter 1 (TaALMT1) confers Al3+ tolerance at low pH, but is also activated by alkaline pH, and is regulated by and facilitates significant transport of gamma-aminobutyric acid (GABA, a zwitterionic buffer). Therefore, TaALMT1 may facilitate acidification of an alkaline rhizosphere by promoting exudation of both malate and GABA. Here, the performance of wheat near isogenic lines ET8 (Al+3 -tolerant, high TaALMT1 expression) and ES8 (Al+3 -sensitive, low TaALMT1 expression) are compared. Root growth (at 5 weeks) was higher for ET8 than ES8 at pH 9. ET8 roots exuded more malate and GABA at high pH and acidified the rhizosphere more rapidly. GABA and malate exudation was enhanced at high pH by the addition of aluminate in both ET8 and transgenic barley expressing TaALMT1. Xenopus laevis oocytes expressing TaALMT1 acidified an alkaline media more rapidly than controls corresponding to higher GABA efflux. TaALMT1 expression did not change under alkaline conditions but key genes involved in GABA turnover changed in accordance with a high rate of GABA synthesis. We propose that TaALMT1 plays a role in alkaline tolerance by exuding malate and GABA, possibly coupled to proton efflux.

A laser ablation technique maps differences in elemental composition in roots of two barley cultivars subjected to salinity stress.

In saline soils, high levels of sodium (Na+ ) and chloride (Cl- ) ions reduce root growth by inhibiting cell division and elongation, thereby impacting on crop yield. Soil salinity can lead to Na+ toxicity of plant cells, influencing the uptake and retention of other important ions [i.e. potassium (K+ )] required for growth. However, measuring and quantifying soluble ions in their native, cellular environment is inherently difficult. Technologies that allow in situ profiling of plant tissues are fundamental for our understanding of abiotic stress responses and the development of tolerant crops. Here, we employ laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) to quantify Na, K and other elements [calcium (Ca), magnesium (Mg), sulphur (S), phosphorus (P), iron (Fe)] at high spatial resolution in the root growth zone of two genotypes of barley (Hordeum vulgare) that differ in salt-tolerance, cv. Clipper (tolerant) and Sahara (sensitive). The data show that Na+ was excluded from the meristem and cell division zone, indicating that Na+ toxicity is not directly reducing cell division in the salt-sensitive genotype, Sahara. Interestingly, in both genotypes, K+ was strongly correlated with Na+ concentration, in response to salt stress. In addition, we also show important genetic differences and salt-specific changes in elemental composition in the root growth zone. These results show that LA-ICP-MS can be used for fine mapping of soluble ions (i.e. Na+ and K+ ) in plant tissues, providing insight into the link between Na+ toxicity and root growth responses to salt stress.

The VvBAP1 gene is identified as a potential inhibitor of cell death in grape berries.

Cell death (CD) in Vitis vinifera L grape berries, exemplified in Shiraz, occurs late in ripening influencing yield, berry and wine quality. Here we isolated and functionally characterised a BON1-associated gene, VvBAP1 from Shiraz berries, encoding a small protein with a C2 domain. VvBAP1 transcript increased during fruit development from veraison to harvest, and was significantly inhibited by drought stress 92 days after flowering when CD normally begins. This was correlated with high CD in Shiraz berries. The agrobacterium-mediated transient expression of VvBAP1 in tobacco leaves led to a decrease in electrolyte leakage and downregulated a marker gene (Hsr203J) for cell death. Expressing VvBAP1 in yeast (Saccharomyces cerevisiae) also alleviated cell death induced by hydrogen peroxide (H2O2). Overexpression of VvBAP1 in Arabidopsis increased resistance to H2O2 and reduced CD due to higher expression of genes involved in anti-oxidative responses. Arabidopsis overexpressing VvBAP1 displayed higher tolerance to drought accompanied by upregulation of antioxidant-related gene expression. VvBAP1 complemented an Arabidopsis bap1 knockout by abolishing its CD phenotypes. These results indicate that VvBAP1 may play a role in alleviating CD in grape berries and its downregulation under drought stress may be responsible for the generally observed increase in CD within the berry.

Aluminum-Activated Malate Transporters Can Facilitate GABA Transport.

Plant aluminum-activated malate transporters (ALMTs) are currently classified as anion channels; they are also known to be regulated by diverse signals, leading to a range of physiological responses. Gamma-aminobutyric acid (GABA) regulation of anion flux through ALMT proteins requires a specific amino acid motif in ALMTs that shares similarity with a GABA binding site in mammalian GABAA receptors. Here, we explore why TaALMT1 activation leads to a negative correlation between malate efflux and endogenous GABA concentrations ([GABA]i) in both wheat (Triticum aestivum) root tips and in heterologous expression systems. We show that TaALMT1 activation reduces [GABA]i because TaALMT1 facilitates GABA efflux but GABA does not complex Al3+ TaALMT1 also leads to GABA transport into cells, demonstrated by a yeast complementation assay and via 14C-GABA uptake into TaALMT1-expressing Xenopus laevis oocytes; this was found to be a general feature of all ALMTs we examined. Mutation of the GABA motif (TaALMT1F213C) prevented both GABA influx and efflux, and resulted in no correlation between malate efflux and [GABA]i We conclude that ALMTs are likely to act as both GABA and anion transporters in planta. GABA and malate appear to interact with ALMTs in a complex manner to regulate each other's transport, suggestive of a role for ALMTs in communicating metabolic status.

Divalent Cations Regulate the Ion Conductance Properties of Diverse Classes of Aquaporins.

Aquaporins (AQPs) are known to facilitate water and solute fluxes across barrier membranes. An increasing number of AQPs are being found to serve as ion channels. Ion and water permeability of selected plant and animal AQPs (plant Arabidopsis thaliana AtPIP2;1, AtPIP2;2, AtPIP2;7, human Homo sapiens HsAQP1, rat Rattus norvegicus RnAQP4, RnAQP5, and fly Drosophilamelanogaster DmBIB) were expressed in Xenopus oocytes and examined in chelator-buffered salines to evaluate the effects of divalent cations (Ca2+, Mg2+, Ba2+ and Cd2+) on ionic conductances. AtPIP2;1, AtPIP2;2, HsAQP1 and DmBIB expressing oocytes had ionic conductances, and showed differential sensitivity to block by external Ca2+. The order of potency of inhibition by Ca2+ was AtPIP2;2 > AtPIP2;1 > DmBIB > HsAQP1. Blockage of the AQP cation channels by Ba2+ and Cd2+ caused voltage-sensitive outward rectification. The channels with the highest sensitivity to Ca2+ (AtPIP2;1 and AtPIP2;2) showed a distinctive relief of the Ca2+ block by co-application of excess Ba2+, suggesting that divalent ions act at the same site. Recognizing the regulatory role of divalent cations may enable the discovery of other classes of AQP ion channels, and facilitate the development of tools for modulating AQP ion channels. Modulators of AQPs have potential value for diverse applications including improving salinity tolerance in plants, controlling vector-borne diseases, and intervening in serious clinical conditions involving AQPs, such as cancer metastasis, cardiovascular or renal dysfunction.

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.

GABA signalling modulates plant growth by directly regulating the activity of plant-specific anion transporters.

The non-protein amino acid, gamma-aminobutyric acid (GABA) rapidly accumulates in plant tissues in response to biotic and abiotic stress, and regulates plant growth. Until now it was not known whether GABA exerts its effects in plants through the regulation of carbon metabolism or via an unidentified signalling pathway. Here, we demonstrate that anion flux through plant aluminium-activated malate transporter (ALMT) proteins is activated by anions and negatively regulated by GABA. Site-directed mutagenesis of selected amino acids within ALMT proteins abolishes GABA efficacy but does not alter other transport properties. GABA modulation of ALMT activity results in altered root growth and altered root tolerance to alkaline pH, acid pH and aluminium ions. We propose that GABA exerts its multiple physiological effects in plants via ALMT, including the regulation of pollen tube and root growth, and that GABA can finally be considered a legitimate signalling molecule in both the plant and animal kingdoms.

Ethylene negatively regulates aluminium-induced malate efflux from wheat roots and tobacco cells transformed with TaALMT1.

An important mechanism for Al(3+) tolerance in wheat is exudation of malate anions from the root apex through activation of malate-permeable TaALMT1 channels. Here, the effect of ethylene on Al(3+)-activated efflux of malate was investigated using Al(3+)-tolerant wheat genotype ET8, which has high expression of TaALMT1. Exposure of ET8 plants to Al(3+) enhanced ethylene evolution in root apices. Treatment with the ethylene synthesis precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and ethylene gas suppressed Al(3+)-induced malate efflux from root apices, whereas the intracellular malate concentrations in roots were not affected. Malate efflux from root apices was enhanced in the presence of Al(3+) by two antagonists of ethylene biosynthesis, aminoethoxyvinylglycine (AVG) and 2-aminoisobutyric acid (AIB). An increase in Al accumulation in root apices was observed when treated with ACC, whereas AVG and AIB suppressed Al accumulation in root apices. Al(3+)-induced inhibition of root elongation was ameliorated by pretreatment with AIB. In addition, ethylene donor (Ethrel) also inhibited Al(3+)-induced malate efflux from tobacco cells transformed with TaALMT1. ACC and the anion-channel blocker niflumate had a similar and non-additive effect on Al-induced malate efflux from root apices. Treatment of ET8 plants with ACC enhanced expression of TaALMT1, suggesting that the inhibitory effect of ethylene on Al-induced malate efflux is unlikely to occur at the transcriptional level. These findings indicate that ethylene may behave as a negative regulator of Al(3+)-induced malate efflux by targeting TaALMT1-mediated malate efflux by an unknown mechanism.

Cell-specific vacuolar calcium storage mediated by CAX1 regulates apoplastic calcium concentration, gas exchange, and plant productivity in Arabidopsis.

The physiological role and mechanism of nutrient storage within vacuoles of specific cell types is poorly understood. Transcript profiles from Arabidopsis thaliana leaf cells differing in calcium concentration ([Ca], epidermis <10 mM versus mesophyll >60 mM) were compared using a microarray screen and single-cell quantitative PCR. Three tonoplast-localized Ca(2+) transporters, CAX1 (Ca(2+)/H(+)-antiporter), ACA4, and ACA11 (Ca(2+)-ATPases), were identified as preferentially expressed in Ca-rich mesophyll. Analysis of respective loss-of-function mutants demonstrated that only a mutant that lacked expression of both CAX1 and CAX3, a gene ectopically expressed in leaves upon knockout of CAX1, had reduced mesophyll [Ca]. Reduced capacity for mesophyll Ca accumulation resulted in reduced cell wall extensibility, stomatal aperture, transpiration, CO(2) assimilation, and leaf growth rate; increased transcript abundance of other Ca(2+) transporter genes; altered expression of cell wall-modifying proteins, including members of the pectinmethylesterase, expansin, cellulose synthase, and polygalacturonase families; and higher pectin concentrations and thicker cell walls. We demonstrate that these phenotypes result from altered apoplastic free [Ca(2+)], which is threefold greater in cax1/cax3 than in wild-type plants. We establish CAX1 as a key regulator of apoplastic [Ca(2+)] through compartmentation into mesophyll vacuoles, a mechanism essential for optimal plant function and productivity.