Plasticity of wheat seedling responses to K+ deficiency highlighted by integrated phenotyping of roots and root hairs over the whole root system.

The availability in the soil of potassium (K+), a poorly mobile macronutrient required in large quantities for plant growth, is generally suboptimal for crop production in the absence of fertilization, making improvement of the ability of crops to adapt to K+ deficiency stress a major issue. Increasing the uptake capacity of the root system is among the main strategies to achieve this goal. Here, we report an integrative approach to examine the effect of K+ deficiency on the development of young plant entire root system, including root hairs which are known to provide a significant contribution to the uptake of poorly mobile nutrients such as K+, in two genetically distant wheat varieties. A rhizobox-type methodology was developed to obtain highly-resolved images of root and root hairs, allowing to describe global root and root hair traits over the whole root system via image analysis procedures. The two wheat varieties responded differently to the K+ shortage: Escandia, a wheat ancestor, reduced shoot biomass in condition of K+ shortage and substantially increased the surface area of its root system, specifically by increasing the total root hair area. Oued Zenati, a landrace, conversely appeared unresponsive to the K+ shortage but was shown to constitutively express, independently of the external K+ availability, favorable traits to cope with reduced K+ availability, among which a high total root hair area. Thus, valuable information on root system adaptation to K+ deficiency was provided by global analyses including root hairs, which should also be relevant for other nutrient stresses.

Wild Wheat Rhizosphere-Associated Plant Growth-Promoting Bacteria Exudates: Effect on Root Development in Modern Wheat and Composition.

Diazotrophic bacteria isolated from the rhizosphere of a wild wheat ancestor, grown from its refuge area in the Fertile Crescent, were found to be efficient Plant Growth-Promoting Rhizobacteria (PGPR), upon interaction with an elite wheat cultivar. In nitrogen-starved plants, they increased the amount of nitrogen in the seed crop (per plant) by about twofold. A bacterial growth medium was developed to investigate the effects of bacterial exudates on root development in the elite cultivar, and to analyze the exo-metabolomes and exo-proteomes. Altered root development was observed, with distinct responses depending on the strain, for instance, with respect to root hair development. A first conclusion from these results is that the ability of wheat to establish effective beneficial interactions with PGPRs does not appear to have undergone systematic deep reprogramming during domestication. Exo-metabolome analysis revealed a complex set of secondary metabolites, including nutrient ion chelators, cyclopeptides that could act as phytohormone mimetics, and quorum sensing molecules having inter-kingdom signaling properties. The exo-proteome-comprised strain-specific enzymes, and structural proteins belonging to outer-membrane vesicles, are likely to sequester metabolites in their lumen. Thus, the methodological processes we have developed to collect and analyze bacterial exudates have revealed that PGPRs constitutively exude a highly complex set of metabolites; this is likely to allow numerous mechanisms to simultaneously contribute to plant growth promotion, and thereby to also broaden the spectra of plant genotypes (species and accessions/cultivars) with which beneficial interactions can occur.

The outward shaker channel OsK5.2 improves plant salt tolerance by contributing to control of both leaf transpiration and K+ secretion into xylem sap.

Soil salinity constitutes a major environmental constraint to crop production worldwide. Leaf K+ /Na+ homoeostasis, which involves regulation of transpiration, and thus of the xylem sap flow, and control of the ionic composition of the ascending sap, is a key determinant of plant salt tolerance. Here, we show, using a reverse genetics approach, that the outwardly rectifying K+ -selective channel OsK5.2, which is involved in both K+ release from guard cells for stomatal closure in leaves and K+ secretion into the xylem sap in roots, is a strong determinant of rice salt tolerance (plant biomass production and shoot phenotype under saline constraint). OsK5.2 expression was upregulated in shoots from the onset of the saline treatment, and OsK5.2 activity in guard cells led to a fast decrease in transpirational water flow and, therefore, reduced Na+ translocation to shoots. In roots, upon saline treatment, OsK5.2 activity in xylem sap K+ loading was maintained, and even transiently increased, outperforming the negative effect on K+ translocation to shoots resulting from the reduction in xylem sap flow. Thus, the overall activity of OsK5.2 in shoots and roots, which both reduces Na+ translocation to shoots and benefits shoot K+ nutrition, strongly contributes to leaf K+ /Na+ homoeostasis.

Non-autonomous stomatal control by pavement cell turgor via the K+ channel subunit AtKC1.

Stomata optimize land plants' photosynthetic requirements and limit water vapor loss. So far, all of the molecular and electrical components identified as regulating stomatal aperture are produced, and operate, directly within the guard cells. However, a completely autonomous function of guard cells is inconsistent with anatomical and biophysical observations hinting at mechanical contributions of epidermal origins. Here, potassium (K+) assays, membrane potential measurements, microindentation, and plasmolysis experiments provide evidence that disruption of the Arabidopsis thaliana K+ channel subunit gene AtKC1 reduces pavement cell turgor, due to decreased K+ accumulation, without affecting guard cell turgor. This results in an impaired back pressure of pavement cells onto guard cells, leading to larger stomatal apertures. Poorly rectifying membrane conductances to K+ were consistently observed in pavement cells. This plasmalemma property is likely to play an essential role in K+ shuttling within the epidermis. Functional complementation reveals that restoration of the wild-type stomatal functioning requires the expression of the transgenic AtKC1 at least in the pavement cells and trichomes. Altogether, the data suggest that AtKC1 activity contributes to the building of the back pressure that pavement cells exert onto guard cells by tuning K+ distribution throughout the leaf epidermis.

Na+ Sensitivity of the KAT2-Like Channel Is a Common Feature of Cucurbits and Depends on the S5-P-S6 Segment.

Inhibition of Shaker K+ channel activity by external Na+ was previously reported in the melon (Cucumis melo L.) inwardly rectifying K+ channel MIRK and was hypothesized to contribute to salt tolerance. In this study, two inward Shaker K+ channels, CsKAT2 from cucumber (Cucumis sativus) and ClKAT2 from watermelon (Citrullus lanatus), were identified and characterized in Xenopus oocytes. Both channels were inwardly rectifying K+ channels with higher permeability to potassium than other monovalent cations and more active when external pH was acidic. Similarly to MIRK, their activity displayed an inhibition by external Na+, thus suggesting a common feature in Cucurbitaceae (Cucumis spp., Citrullus spp.). CsKAT2 and ClKAT2 are highly expressed in guard cells. After 24 h of plant treatment with 100 mM NaCl, the three KAT2-like genes were significantly downregulated in leaves and guard cells. Reciprocal chimeras were obtained between MIRK and Na+-insensitive AtKAT2 cDNAs. The chimera where the MIRK S5-P-S6 segment was replaced by that from AtKAT2 no longer showed Na+ sensitivity, while the inverse chimera gained Na+ sensitivity. These results provide evidence that the molecular basis of the channel blockage by Na+ is located in the S5-P-S6 region. Comparison of the electrostatic property in the S5-P-S6 region in AtKAT2 and MIRK revealed four key amino acid residues potentially governing Na+ sensitivity.

Looking for Root Hairs to Overcome Poor Soils.

Breeding new cultivars allowing reduced fertilization and irrigation is a major challenge. International efforts towards this goal focus on noninvasive methodologies, platforms for high-throughput phenotyping of large plant populations, and quantitative description of root traits as predictors of crop performance in environments with limited water and nutrient availability. However, these high-throughput analyses ignore one crucial component of the root system: root hairs (RHs). Here, we review current knowledge on RH functions, mainly in the context of plant hydromineral nutrition, and take stock of quantitative genetics data pointing at correlations between RH traits and plant biomass production and yield components.

Fungal Shaker-like channels beyond cellular K+ homeostasis: A role in ectomycorrhizal symbiosis between Hebeloma cylindrosporum and Pinus pinaster.

Potassium (K+) acquisition, translocation and cellular homeostasis are mediated by various membrane transport systems in all organisms. We identified and described an ion channel in the ectomycorrhizal fungus Hebeloma cylindrosporum (HcSKC) that harbors features of animal voltage-dependent Shaker-like K+ channels, and investigated its role in both free-living hyphae and symbiotic conditions. RNAi lines affected in the expression of HcSKC were produced and used for in vitro mycorrhizal assays with the maritime pine as host plant, under standard or low K+ conditions. The adaptation of H. cylindrosporum to the downregulation of HcSKC was analyzed by qRT-PCR analyses for other K+-related transport proteins: the transporters HcTrk1, HcTrk2, and HcHAK, and the ion channels HcTOK1, HcTOK2.1, and HcTOK2.2. Downregulated HcSKC transformants displayed greater K+ contents at standard K+ only. In such conditions, plants inoculated with these transgenic lines were impaired in K+ nutrition. Taken together, these results support the hypothesis that the reduced expression of HcSKC modifies the pool of fungal K+ available for the plant and/or affects its symbiotic transfer to the roots. Our study reveals that the maintenance of K+ transport in H. cylindrosporum, through the regulation of HcSKC expression, is required for the K+ nutrition of the host plant.

Constitutive Contribution by the Rice OsHKT1;4 Na+ Transporter to Xylem Sap Desalinization and Low Na+ Accumulation in Young Leaves Under Low as High External Na+ Conditions.

HKT Na+ transporters correspond to major salt tolerance QTLs in different plant species and are targets of great interest for breeders. In rice, the HKT family is composed of seven or eight functional genes depending on cultivars. Three rice HKT genes, OsHKT1;1, OsHKT1;4 and OsHKT1;5, are known to contribute to salt tolerance by reducing Na+ accumulation in shoots upon salt stress. Here, we further investigate the mechanisms by which OsHKT1;4 contributes to this process and extend this analysis to the role of this transporter in plants in presence of low Na+ concentrations. By analyzing transgenic rice plants expressing a GUS reporter gene construct, we observed that OsHKT1;4 is mainly expressed in xylem parenchyma in both roots and leaves. Using mutant lines expressing artificial microRNA that selectively reduced OsHKT1;4 expression, the involvement of OsHKT1;4 in retrieving Na+ from the xylem sap in the roots upon salt stress was evidenced. Since OsHKT1;4 was found to be also well expressed in the roots in absence of salt stress, we extended the analysis of its role when plants were subjected to non-toxic Na+ conditions (0.5 and 5 mM). Our finding that the transporter, expressed in Xenopus oocytes, displayed a relatively high affinity for Na+, just above 1 mM, provided first support to the hypothesis that OsHKT1;4 could have a physiological role at low Na+ concentrations. We observed that progressive desalinization of the xylem sap along its ascent to the leaf blades still occurred in plants grown at submillimolar Na+ concentration, and that OsHKT1;4 was involved in reducing xylem sap Na+ concentration in roots in these conditions too. Its contribution to tissue desalinization from roots to young mature leaf blades appeared to be rather similar in the whole range of explored external Na+ concentrations, from submillimolar to salt stress conditions. Our data therefore indicate that HKT transporters can be involved in controlling Na+ translocation from roots to shoots in a much wider range of Na+ concentrations than previously thought. This asks questions about the roles of such a transporter-mediated maintaining of tissue Na+ content gradients in non-toxic conditions.

Functional Characterization of the Arabidopsis Ammonium Transporter AtAMT1;3 With the Emphasis on Structural Determinants of Substrate Binding and Permeation Properties.

AtAMT1;3 is a major contributor to high-affinity ammonium uptake in Arabidopsis roots. Using a stable electrophysiological recording strategy, we demonstrate in Xenopus laevis oocytes that AtAMT1;3 functions as a typical high-affinity NH4 + uniporter independent of protons and Ca2+. The findings that AtAMT1;3 transports methylammonium (MeA+, a chemical analog of NH4 +) with extremely low affinity (K m in the range of 2.9-6.1 mM) led to investigate the mechanisms underlying substrate binding. Homologous modeling and substrate docking analyses predicted that the deduced substrate binding motif of AtAMT1;3 facilitates the binding of NH4 + ions but loosely accommodates the binding of MeA+ to a more superficial location of the permeation pathway. Amongst point mutations tested based on this analysis, P181A resulted in both significantly increased current amplitudes and substrate binding affinity, whereas F178I led to opposite effects. Thus these 2 residues, which flank W179, a major structural component of the binding site, are also important determinants of AtAMT1;3 transport capacity by being involved in substrate binding. The Q365K mutation neighboring the histidine residue H378, which confines the substrate permeation tunnel, affected only the current amplitudes but not the binding affinities, providing evidence that Q365 mainly controls the substrate diffusion rate within the permeation pathway.

Functional characterization and physiological roles of the single Shaker outward K+ channel in Medicago truncatula.

The model legume Medicago truncatula possesses a single outward Shaker K+ channel, whereas Arabidopsis thaliana possesses two channels of this type, named AtSKOR and AtGORK, with AtSKOR having been shown to play a major role in K+ secretion into the xylem sap in the root vasculature and with AtGORK being shown to mediate the efflux of K+ across the guard cell membrane, leading to stomatal closure. Here we show that the expression pattern of the single M. truncatula outward Shaker channel, which has been named MtGORK, includes the root vasculature, guard cells and root hairs. As shown by patch-clamp experiments on root hair protoplasts, besides the Shaker-type slowly activating outwardly rectifying K+ conductance encoded by MtGORK, a second K+ -permeable conductance, displaying fast activation and weak rectification, can be expressed by M. truncatula. A knock-out (KO) mutation resulting in an absence of MtGORK activity is shown to weakly reduce K+ translocation to shoots, and only in plants engaged in rhizobial symbiosis, but to strongly affect the control of stomatal aperture and transpirational water loss. In legumes, the early electrical signaling pathway triggered by Nod-factor perception is known to comprise a short transient depolarization of the root hair plasma membrane. In the absence of the functional expression of MtGORK, the rate of the membrane repolarization is found to be decreased by a factor of approximately two. This defect was without any consequence on infection thread development and nodule production in plants grown in vitro, but a decrease in nodule production was observed in plants grown in soil.

A repertoire of cationic and anionic conductances at the plasma membrane of Medicago truncatula root hairs.

Root hairs, as lateral extensions of epidermal cells, provide large absorptive surfaces to the root and are major actors in plant hydromineral nutrition. In contact with the soil they also constitute a site of interactions between the plant and rhizospheric microorganisms. In legumes, initiation of symbiotic interactions with N2 -fixing rhizobia is often triggered at the root hair cell membrane in response to nodulation factors secreted by rhizobia, and involves early signaling events with changes in H+ , Ca2+ , K+ and Cl- fluxes inducing transient depolarization of the cell membrane. Here, we aimed to build a functional repertoire of the major root hair conductances to cations and anions in the sequenced legume model Medicago truncatula. Five root hair conductances were characterized through patch-clamp experiments on enzymatically recovered root hair protoplasts. These conductances displayed varying properties of voltage dependence, kinetics and ion selectivity. They consisted of hyperpolarization- and depolarization-activated conductances for K+ , cations or Cl- . Among these, one weakly outwardly rectifying cationic conductance and one hyperpolarization-activated slowly inactivating anionic conductance were not known as active in root hairs. All five conductances were detected in apical regions of young growing root hairs using membrane spheroplasts obtained by laser-assisted cell-wall microdissection. Combined with recent root hair transcriptomes of M. truncatula, this functional repertoire of conductances is expected to help the identification of candidate genes for reverse genetics studies to investigate the possible role of each conductance in root hair growth and interaction with the biotic and abiotic environment.

Uncovering pH at both sides of the root plasma membrane interface using noninvasive imaging.

Building a proton gradient across a biological membrane and between different tissues is a matter of great importance for plant development and nutrition. To gain a better understanding of proton distribution in the plant root apoplast as well as across the plasma membrane, we generated Arabidopsis plants expressing stable membrane-anchored ratiometric fluorescent sensors based on pHluorin. These sensors enabled noninvasive pH-specific measurements in mature root cells from the medium-epidermis interface up to the inner cell layers that lie beyond the Casparian strip. The membrane-associated apoplastic pH was much more alkaline than the overall apoplastic space pH. Proton concentration associated with the plasma membrane was very stable, even when the growth medium pH was altered. This is in apparent contradiction with the direct connection between root intercellular space and the external medium. The plasma membrane-associated pH in the stele was the most preserved and displayed the lowest apoplastic pH (6.0 to 6.1) and the highest transmembrane delta pH (1.5 to 2.2). Both pH values also correlated well with optimal activities of channels and transporters involved in ion uptake and redistribution from the root to the aerial part. In growth medium where ionic content is minimized, the root plasma membrane-associated pH was more affected by environmental proton changes, especially for the most external cell layers. Calcium concentration appears to play a major role in apoplastic pH under these restrictive conditions, supporting a role for the cell wall in pH homeostasis of the unstirred surface layer of plasma membrane in mature roots.

Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum.

Ectomycorrhizal fungi play an essential role in the ecology of boreal and temperate forests through the improvement of tree mineral nutrition. Potassium (K+ ) is an essential nutrient for plants and is needed in high amounts. We recently demonstrated that the ectomycorrhizal fungus Hebeloma cylindrosporum improves the K+ nutrition of Pinus pinaster under shortage conditions. Part of the transport systems involved in K+ uptake by the fungus has been deciphered, while the molecular players responsible for the transfer of this cation towards the plant remain totally unknown. Analysis of the genome of H. cylindrosporum revealed the presence of three putative tandem-pore outward-rectifying K+ (TOK) channels that could contribute to this transfer. Here, we report the functional characterization of these three channels through two-electrode voltage-clamp experiments in oocytes and yeast complementation assays. The expression pattern and physiological role of these channels were analysed in symbiotic interaction with P. pinaster. Pine seedlings colonized by fungal transformants overexpressing two of them displayed a larger accumulation of K+ in shoots. This study revealed that TOK channels have distinctive properties and functions in axenic and symbiotic conditions and suggested that HcTOK2.2 is implicated in the symbiotic transfer of K+ from the fungus towards the plant.

Internal Cs+ inhibits root elongation in rice.

The root system anchors the plant to the soil and contributes to plant autotrophy by taking up nutrients and water. In relation with this nutritional function, root development is largely impacted by availability of nutrients and water. Due to human activity, plants, in particular crops, can also be exposed to pollutants which can be absorbed and incorporated into the food chain. Cesium in soils is present at non-toxic concentrations for the plant (micromolar or less), even in soils highly polluted with radioactive cesium due to nuclear accidents. Here, we report on the morphological response of rice roots to Cs+ at micromolar concentrations. It is shown that Cs+ reduces root elongation without affecting root dry weight. Noteworthy, inactivation of the Cs+-permeable K+ transporter OsHAK1 prevents such effect of Cs+, suggesting that internal Cs+ triggers the modification of the root system.

Production of low-Cs+ rice plants by inactivation of the K+ transporter OsHAK1 with the CRISPR-Cas system.

The occurrence of radiocesium in food has raised sharp health concerns after nuclear accidents. Despite being present at low concentrations in contaminated soils (below µm), cesium (Cs+ ) can be taken up by crops and transported to their edible parts. This plant capacity to take up Cs+ from low concentrations has notably affected the production of rice (Oryza sativa L.) in Japan after the nuclear accident at Fukushima in 2011. Several strategies have been put into practice to reduce Cs+ content in this crop species such as contaminated soil removal or adaptation of agricultural practices, including dedicated fertilizer management, with limited impact or pernicious side-effects. Conversely, the development of biotechnological approaches aimed at reducing Cs+ accumulation in rice remain challenging. Here, we show that inactivation of the Cs+ -permeable K+ transporter OsHAK1 with the CRISPR-Cas system dramatically reduced Cs+ uptake by rice plants. Cs+ uptake in rice roots and in transformed yeast cells that expressed OsHAK1 displayed very similar kinetics parameters. In rice, Cs+ uptake is dependent on two functional properties of OsHAK1: (i) a poor capacity of this system to discriminate between Cs+ and K+ ; and (ii) a high capacity to transport Cs+ from very low external concentrations that is likely to involve an active transport mechanism. In an experiment with a Fukushima soil highly contaminated with 137 Cs+ , plants lacking OsHAK1 function displayed strikingly reduced levels of 137 Cs+ in roots and shoots. These results open stimulating perspectives to smartly produce safe food in regions contaminated by nuclear accidents.

A Dual Role for the OsK5.2 Ion Channel in Stomatal Movements and K+ Loading into Xylem Sap.

The roles of potassium channels from the Shaker family in stomatal movements have been investigated by reverse genetics analyses in Arabidopsis (Arabidopsis thaliana), but corresponding information is lacking outside this model species. Rice (Oryza sativa) and other cereals possess stomata that are more complex than those of Arabidopsis. We examined the role of the outward Shaker K+ channel gene OsK5.2. Expression of the OsK5.2 gene (GUS reporter strategy) was observed in the whole stomatal complex (guard cells and subsidiary cells), root vasculature, and root cortex. In stomata, loss of OsK5.2 functional expression resulted in lack of time-dependent outward potassium currents in guard cells, higher rates of water loss through transpiration, and severe slowdown of stomatal closure. In line with the expression of OsK5.2 in the plant vasculature, mutant plants displayed a reduced K+ translocation from the root system toward the leaves via the xylem. The comparison between rice and Arabidopsis show that despite the strong conservation of Shaker family in plants, substantial differences can exist between the physiological roles of seemingly orthologous genes, as xylem loading depends on SKOR and stomatal closure on GORK in Arabidopsis, whereas both functions are executed by the single OsK5.2 Shaker in rice.

Cloning and functional characterization of HKT1 and AKT1 genes of Fragaria spp.-Relationship to plant response to salt stress.

Commercial strawberry, Fragaria x ananassa Duch., is a species sensitive to salinity. Under saline conditions, Na+ uptake by the plant is increased, while K+ uptake is significantly reduced. Maintaining an adequate K+/Na+ cytosolic ratio determines the ability of the plant to survive in saline environments. The goal of the present work was to clone and functionally characterize the genes AKT1 and HKT1 involved in K+ and Na+ transport in strawberry and to determine the relationship of these genes with the responses of three Fragaria spp. genotypes having different ecological adaptations to salt stress. FaHKT1 and FcHKT1 proteins from F. x ananassa and F. chiloensis have 98.1% of identity, while FaAKT1 and FcAKT1 identity is 99.7%. FaHKT1 and FaAKT1 from F. x ananassa, were functionally characterized in Xenopus oocytes. FaHKT1, belongs to the group I of HKT transporters and is selective for Na+. Expression of FaAKT1 in oocytes showed that the protein is a typical inward-rectifying and highly K+-selective channel. The relative expression of Fragaria HKT1 and AKT1 genes was studied in roots of F. x ananassa cv. Camarosa and of F. chiloensis (accessions Bau and Cucao) grown under salt stress. The expression of AKT1 was transiently increased in 'Camarosa', decreased in 'Cucao' and was not affected in 'Bau' upon salt stress. HKT1 expression was significantly increased in roots of 'Cucao' and was not affected in the other two genotypes. The increased relative expression of HKT1 and decreased expression of AKT1 in 'Cucao' roots correlates with the higher tolerance to salinity of this genotype in comparison with 'Camarosa' and 'Bau'.

Nod Factor Effects on Root Hair-Specific Transcriptome of Medicago truncatula: Focus on Plasma Membrane Transport Systems and Reactive Oxygen Species Networks.

Root hairs are involved in water and nutrient uptake, and thereby in plant autotrophy. In legumes, they also play a crucial role in establishment of rhizobial symbiosis. To obtain a holistic view of Medicago truncatula genes expressed in root hairs and of their regulation during the first hours of the engagement in rhizobial symbiotic interaction, a high throughput RNA sequencing on isolated root hairs from roots challenged or not with lipochitooligosaccharides Nod factors (NF) for 4 or 20 h was carried out. This provided a repertoire of genes displaying expression in root hairs, responding or not to NF, and specific or not to legumes. In analyzing the transcriptome dataset, special attention was paid to pumps, transporters, or channels active at the plasma membrane, to other proteins likely to play a role in nutrient ion uptake, NF electrical and calcium signaling, control of the redox status or the dynamic reprogramming of root hair transcriptome induced by NF treatment, and to the identification of papilionoid legume-specific genes expressed in root hairs. About 10% of the root hair expressed genes were significantly up- or down-regulated by NF treatment, suggesting their involvement in remodeling plant functions to allow establishment of the symbiotic relationship. For instance, NF-induced changes in expression of genes encoding plasma membrane transport systems or disease response proteins indicate that root hairs reduce their involvement in nutrient ion absorption and adapt their immune system in order to engage in the symbiotic interaction. It also appears that the redox status of root hair cells is tuned in response to NF perception. In addition, 1176 genes that could be considered as "papilionoid legume-specific" were identified in the M. truncatula root hair transcriptome, from which 141 were found to possess an ortholog in every of the six legume genomes that we considered, suggesting their involvement in essential functions specific to legumes. This transcriptome provides a valuable resource to investigate root hair biology in legumes and the roles that these cells play in rhizobial symbiosis establishment. These results could also contribute to the long-term objective of transferring this symbiotic capacity to non-legume plants.

Characterization of Two HKT1;4 Transporters from Triticum monococcum to Elucidate the Determinants of the Wheat Salt Tolerance Nax1 QTL.

TmHKT1;4-A1 and TmHKT1;4-A2 are two Na+ transporter genes that have been identified as associated with the salt tolerance Nax1 locus found in a durum wheat (Triticum turgidum L. subsp. durum) line issued from a cross with T. monococcum. In the present study, we were interested in getting clues on the molecular mechanisms underpinning this salt tolerance quantitative trait locus (QTL). By analyzing the phylogenetic relationships between wheat and T. monococcum HKT1;4-type genes, we found that durum and bread wheat genomes possess a close homolog of TmHKT1;4-A1, but no functional close homolog of TmHKT1;4-A2. Furthermore, performing real-time reverse transcription-PCR experiments, we showed that TmHKT1;4-A1 and TmHKT1;4-A2 are similarly expressed in the leaves but that TmHKT1;4-A2 is more strongly expressed in the roots, which would enable it to contribute more to the prevention of Na+ transfer to the shoots upon salt stress. We also functionally characterized the TmHKT1;4-A1 and TmHKT1;4-A2 transporters by expressing them in Xenopus oocytes. The two transporters displayed close functional properties (high Na+/K+ selectivity, low affinity for Na+, stimulation by external K+ of Na+ transport), but differed in some quantitative parameters: Na+ affinity was 3-fold lower and the maximal inward conductance was 3-fold higher in TmHKT1;4-A2 than in TmHKT1;4-A1. The conductance of TmHKT1;4-A2 at high Na+ concentration (>10 mM) was also shown to be higher than that of the two durum wheat HKT1;4-type transporters so far characterized. Altogether, these data support the hypothesis that TmHKT1;4-A2 is responsible for the Nax1 trait and provide new insight into the understanding of this QTL.

The S1-S2 linker determines the distinct pH sensitivity between ZmK2.1 and KAT1.

Efficient stomatal opening requires activation of KAT-type K(+) channels, which mediate K(+) influx into guard cells. Most KAT-type channels are functionally facilitated by extracellular acidification. However, despite sequence and structural homologies, the maize counterpart of Arabidopsis KAT1 (ZmK2.1) is resistant to pH activation. To understand the structural determinant that results in the differential pH activation of these counterparts, we analysed chimeric channels and channels with point mutations for ZmK2.1 and its closest Arabidopsis homologue KAT1. Exchange of the S1-S2 linkers altered the pH sensitivity between the two channels, suggesting that the S1-S2 linker is essentially involved in the pH sensitivity. The effects of D92 mutation within the linker motif together with substitution of the first half of the linker largely resemble the effects of substitution of the complete linker. Topological modelling predicts that one of the two cysteines located on the outer face section of the S5 domain may serve as a potential titratable group that interacts with the S1-S2 linker. The difference between ZmK2.1 and KAT1 is predicted to be the result of the distance of the stabilized linkers from the titratable group. In KAT1, residue K85 within the linker forms a hydrogen bond with C211 that enables the pH activation; conversely, the linker of ZmK2.1 is distantly located and thus does not interact with the equivalent titration group (C208). Thus, in addition to the known structural contributors to the proton activation of KAT channels, we have uncovered a previously unidentified component that is strongly involved in this complex proton activation network.