Eduardo O. Leidi

Ion Exchangers NHX1 and NHX2 Mediate Active Potassium Uptake into Vacuoles to Regulate Cell Turgor and Stomatal Function in Arabidopsis

Intracellular Na+,K+/H+ antiporters (NHXs) play central roles in maintaining ion homeostasis and pH control. Tonoplast-localized proteins NHX1 and NHX2 are critical for active K+ uptake into the vacuole, a process that is required to create osmotic potential for cell expansion and turgor regulation. These proteins are abundantly expressed in guard cells, where they contribute to stomata function. Intracellular NHX proteins are Na+,K+/H+ antiporters involved in K+ homeostasis, endosomal pH regulation, and salt tolerance. Proteins NHX1 and NHX2 are the two major tonoplast-localized NHX isoforms. Here, we show that NHX1 and NHX2 have similar expression patterns and identical biochemical activity, and together they account for a significant amount of the Na+,K+/H+ antiport activity in tonoplast vesicles. Reverse genetics showed functional redundancy of NHX1 and NHX2 genes. Growth of the double mutant nhx1 nhx2 was severely impaired, and plants were extremely sensitive to external K+. By contrast, nhx1 nhx2 mutants showed similar sensitivity to salinity stress and even greater rates of Na+ sequestration than the wild type. Double mutants had reduced ability to create the vacuolar K+ pool, which in turn provoked greater K+ retention in the cytosol, impaired osmoregulation, and compromised turgor generation for cell expansion. Genes NHX1 and NHX2 were highly expressed in guard cells, and stomatal function was defective in mutant plants, further compromising their ability to regulate water relations. Together, these results show that tonoplast-localized NHX proteins are essential for active K+ uptake at the tonoplast, for turgor regulation, and for stomatal function.

The AtNHX1 exchanger mediates potassium compartmentation in vacuoles of transgenic tomato

NHX-type antiporters in the tonoplast have been reported to increase the salt tolerance of various plants species, and are thought to mediate the compartmentation of Na(+) in vacuoles. However, all isoforms characterized so far catalyze both Na(+)/H(+) and K(+)/H(+) exchange. Here, we show that AtNHX1 has a critical involvement in the subcellular partitioning of K(+), which in turn affects plant K(+) nutrition and Na(+) tolerance. Transgenic tomato plants overexpressing AtNHX1 had larger K(+) vacuolar pools in all growth conditions tested, but no consistent enhancement of Na(+) accumulation was observed under salt stress. Plants overexpressing AtNHX1 have a greater capacity to retain intracellular K(+) and to withstand salt-shock. Under K(+)-limiting conditions, greater K(+) compartmentation in the vacuole occurred at the expense of the cytosolic K(+) pool, which was lower in transgenic plants. This caused the early activation of the high-affinity K(+) uptake system, enhanced K(+) uptake by roots, and increased the K(+) content in plant tissues and the xylem sap of transformed plants. Our results strongly suggest that NHX proteins are likely candidates for the H(+)-linked K(+) transport that is thought to facilitate active K(+) uptake at the tonoplast, and the partitioning of K(+) between vacuole and cytosol.

Loss of Halophytism by Interference with SOS1 Expression

The contribution of SOS1 (for Salt Overly Sensitive 1), encoding a sodium/proton antiporter, to plant salinity tolerance was analyzed in wild-type and RNA interference (RNAi) lines of the halophytic Arabidopsis (Arabidopsis thaliana)-relative Thellungiella salsuginea. Under all conditions, SOS1 mRNA abundance was higher in Thellungiella than in Arabidopsis. Ectopic expression of the Thellungiella homolog ThSOS1 suppressed the salt-sensitive phenotype of a Saccharomyces cerevisiae strain lacking sodium ion (Na+) efflux transporters and increased salt tolerance of wild-type Arabidopsis. thsos1-RNAi lines of Thellungiella were highly salt sensitive. A representative line, thsos1-4, showed faster Na+ accumulation, more severe water loss in shoots under salt stress, and slower removal of Na+ from the root after removal of stress compared with the wild type. thsos1-4 showed drastically higher sodium-specific fluorescence visualized by CoroNa-Green, a sodium-specific fluorophore, than the wild type, inhibition of endocytosis in root tip cells, and cell death in the adjacent elongation zone. After prolonged stress, Na+ accumulated inside the pericycle in thsos1-4, while sodium was confined in vacuoles of epidermis and cortex cells in the wild type. RNAi-based interference of SOS1 caused cell death in the root elongation zone, accompanied by fragmentation of vacuoles, inhibition of endocytosis, and apoplastic sodium influx into the stele and hence the shoot. Reduction in SOS1 expression changed Thellungiella that normally can grow in seawater-strength sodium chloride solutions into a plant as sensitive to Na+ as Arabidopsis.

How do vacuolar NHX exchangers function in plant salt tolerance

Potassium (K+) is a major osmoticum of plant cells, and the vacuolar accumulation of this element is a especially crucial feature for plants under high-salt conditions. Emerging evidence indicates that cation/proton transporters of the NHX family are instrumental in the H+-linked K+ transport that mediate active K+ uptake at the tonoplast for the unequal partitioning of K+ between vacuole and cytosol. However, and in spite of tenuous supporting evidence, NHX proteins are widely regarded as key players in the sequestration of sodium (Na+) into vacuoles to avert ion toxicity in the cytosol of plants under salinity stress. Here, we propose an updated model positing that NHX proteins fulfill a protective function to minimize salt-related stress mainly through the vacuolar compartmentalization of K+ and, in some cases, of Na+ as well thereby preventing toxic Na+-K+ ratios in the cytosol while accruing solutes for osmotic balance.

Control of vacuolar dynamics and regulation of stomatal aperture by tonoplast potassium uptake

Significance Rapid fluxes of K+ and other osmolytes in guard cells control the opening and closing of stomata and thereby gas exchange and transpiration of plants. Despite the well-established role of the plasma membrane of guard cells in stomatal function, osmolyte uptake into the cytosol represents only a transient step to the vacuole, as more than 90% of the solutes accumulate in these organelles. We show that the tonoplast-localized K+/H+ exchangers mediate the vacuolar accumulation of K+ in guard cells, and that activity of these transporters controls not only stomatal opening but also stomatal closure. We also establish vacuolar K+/H+ exchange as a critical component involved in vacuolar remodeling and the regulation of vacuolar pH during stomatal movements. Stomatal movements rely on alterations in guard cell turgor. This requires massive K+ bidirectional fluxes across the plasma and tonoplast membranes. Surprisingly, given their physiological importance, the transporters mediating the energetically uphill transport of K+ into the vacuole remain to be identified. Here, we report that, in Arabidopsis guard cells, the tonoplast-localized K+/H+ exchangers NHX1 and NHX2 are pivotal in the vacuolar accumulation of K+ and that nhx1 nhx2 mutant lines are dysfunctional in stomatal regulation. Hypomorphic and complete-loss-of-function double mutants exhibited significantly impaired stomatal opening and closure responses. Disruption of K+ accumulation in guard cells correlated with more acidic vacuoles and the disappearance of the highly dynamic remodelling of vacuolar structure associated with stomatal movements. Our results show that guard cell vacuolar accumulation of K+ is a requirement for stomatal opening and a critical component in the overall K+ homeostasis essential for stomatal closure, and suggest that vacuolar K+ fluxes are also of decisive importance in the regulation of vacuolar dynamics and luminal pH that underlie stomatal movements.

VARIATION IN CARBON ISOTOPE DISCRIMINATION AND OTHER TRAITS RELATED TO DROUGHT TOLERANCE IN UPLAND COTTON CULTIVARS UNDER DRYLAND CONDITIONS

Abstract The search for traits related to drought resistance is a main step in the selection of cotton with improved performance under limited water supply. The effect of cultivar and drought conditions on the physiological traits, such as carbon isotope discrimination (Δ), photosynthesis (A), transpiration (E), stomatal conductance (gs), leaf osmotic potential (ψo) and leaf-water content (LWC), were studied in Andalucia, Spain. The morphological traits, such as leaf area and specific leaf weight (SLW), were also evaluated. In the initial study performed with three cotton cultivars, positive associations between Δ and A, E and gs were observed under increasing water stress. The A/gs ratio was negatively associated with Δ, and a strong negative correlation was observed between SLW and Δ. In a further experiment, using a wider group of cultivars, an apparent genotypic variation in Δ was observed in plants after withholding irrigation. Genotypic variation was found for gas exchange (A, E, gs) and leaf variables (LWC, SLW, ψo). No relationship was found between Δ and gas exchange or leaf-related traits. Considering both samplings as situations of different water availability, and plotting the data together, a low but positive correlation was observed between Δ and gs or LWC, and negative between Δ and A/gs. A remarkable correlation between LWC and gas exchange traits was found, whereas SLW was negatively correlated with A, A/E, and LWC. In dryland trials (1996 and 1997), the genotypic variation in LWC was positively associated with Δ. Under these conditions, an association between genotypic variation in Δ and A or A/E was observed. A positive correlation between yield and Δ was detected in 1996. Carbon isotope discrimination might be a useful tool for selecting drought-tolerant cotton genotypes but more studies are required to define more precisely the sampling conditions and the influence of factors affecting Δ and its relationship to crop yield.

Expression of wheat Na+/H+ antiporter TNHXS1 and H+- pyrophosphatase TVP1 genes in tobacco from a bicistronic transcriptional unit improves salt tolerance

Abiotic stress tolerance of plants is a very complex trait and involves multiple physiological and biochemical processes. Thus, the improvement of plant stress tolerance should involve pyramiding of multiple genes. In the present study, we report the construction and application of a bicistronic system, involving the internal ribosome entry site (IRES) sequence from the 5′UTR of the heat-shock protein of tobacco gene NtHSF-1, to the improvement of salt tolerance in transgenic tobacco plants. Two genes from wheat encoding two important vacuolar ion transporters, Na+/H+ antiporter (TNHXS1) and H+-pyrophosphatase (TVP1), were linked via IRES to generate the bicistronic construct TNHXS1-IRES-TVP1. Molecular analysis of transgenic tobacco plants revealed the correct integration of the TNHXS1-IRES-TVP1construct into tobacco genome and the production of the full-length bicistronic mRNA from the 35S promoter. Ion transport analyses with tonoplast vesicles isolated from transgenic lines confirmed that single-transgenic lines TVP1cl19 and TNHXS1cl7 had greater H+-PPiase and Na+/H+ antiport activity, respectively, than the WT. Interestingly, the co-expression of TVP1 and TNHXS1 increased both Na+/H+ antiport and H+-PPiase activities and induced the H+ pumping activity of the endogenous V-ATPase. Transgenic tobacco plants expressing TNHXS1-IRES-TVP1 showed a better performance than either of the single gene-transformed lines and the wild type plants when subjected to salt treatment. In addition, the TNHXS1-IRES-TVP1 transgenic plants accumulated less Na+ and more K+ in their leaf tissue than did the wild type and the single gene-transformed lines. These results demonstrate that IRES system, described herein, can co-ordinate the expression of two important abiotic stress-tolerance genes and that this expression system is a valuable tool for obtaining transgenic plants with improved salt tolerance.

A constitutively active form of a durum wheat Na+/H+ antiporter SOS1 confers high salt tolerance to transgenic Arabidopsis

AbstractKey messageExpression of a truncated form of wheat TdSOS1 in Arabidopsis exhibited an improved salt tolerance. This finding provides new hints about this protein that can be considered as a salt tolerance determinant.Abstract The SOS signaling pathway has emerged as a key mechanism in preserving the homeostasis of Na+ and K+ under saline conditions. We have recently identified and functionally characterized, by complementation studies in yeast, the gene encoding the durum wheat plasma membrane Na+/H+ antiporter (TdSOS1). To extend these functional studies to the whole plant level, we complemented Arabidopsis sos1-1 mutant with wild-type TdSOS1 or with the hyperactive form TdSOS1∆972 and compared them to the Arabidopsis AtSOS1 protein. The Arabidopsis sos1-1 mutant is hypersensitive to both Na+ and Li+ ions. Compared with sos1-1 mutant transformed with the empty binary vector, seeds from TdSOS1 or TdSOS1∆972 transgenic plants had better germination under salt stress and more robust seedling growth in agar plates as well as in nutritive solution containing Na+ or Li+ salts. The root elongation of TdSOS1∆972 transgenic lines was higher than that of Arabidopsis sos1-1 mutant transformed with TdSOS1 or with the endogenous AtSOS1 gene. Under salt stress, TdSOS1∆972 transgenic lines showed greater water retention capacity and retained low Na+ and high K+ in their shoots and roots. Our data showed that the hyperactive form TdSOS1∆972 conferred a significant ionic stress tolerance to Arabidopsis plants and suggest that selection of hyperactive alleles of the SOS1 transport protein may pave the way for obtaining salt-tolerant crops.

Plant Responses to Salinity

Cotton is one of the most salt-tolerant of the major annual crops, but the variable nature of both salinity and the plant’s response to it make salinity a far from simple problem. Here we consider the response of cotton to salinity, and possible means of improving cotton yields on saline land or with saline water. This review will cover most of the research published in the last 30 years, but space does not permit a more extensive review of the older literature.

Phenotypic and genotypic characterization of rhizobia from diverse geographical origin that nodulate Pachyrhizus species.

Legumes from the genus Pachyrhizus, commonly known as yam bean, are cultivated in several countries from the American continent and constitute an alternative source for sustainable starch, oil and protein production. The endosymbionts of these legumes have been poorly studied although it is known that this legume is nodulated by fast and slow growing rhizobia. In this study we have analyzed a collection of strains isolated in several countries using different phenotypic and molecular methods. The results obtained by SDS-PAGE analysis, LPS profiling and TP-RAPD fingerprinting showed the high diversity of the strains analyzed, although all of them presented slow growth in yeast mannitol agar (YMA) medium. These results were confirmed using 16S-23S internal transcribed spacer (ITS) region and complete sequencing of the 16S rRNA gene, showing that most strains analyzed belong to different species of genus Bradyrhizobium. Three strains were closely related to B. elkanii and the rest of the strains were related to the phylogenetic group constituted by B. japonicum, B. liaoningense, B. yuanmingense and B. betae. These results support that the study of rhizobia nodulating unexplored legumes in different geographical locations will allow the discovery of new species able to establish legume symbioses.