Andrés Belver

The plasma membrane Na+/H+ antiporter SOS1 is essential for salt tolerance in tomato and affects the partitioning of Na+ between plant organs

We have identified a plasma membrane Na(+)/H(+) antiporter gene from tomato (Solanum lycopersicum), SlSOS1, and used heterologous expression in yeast to confirm that SlSOS1 was the functional homolog of AtSOS1. Using post-transcriptional gene silencing, we evaluated the role played by SlSOS1 in long-distance Na(+) transport and salt tolerance of tomato. Tomato was used because of its anatomical structure, more complex than that of Arabidopsis, and its agricultural significance. Transgenic tomato plants with reduced expression of SlSOS1 exhibited reduced growth rate compared to wild-type (WT) plants in saline conditions. This sensitivity correlated with higher accumulation of Na(+) in leaves and roots, but lower contents in stems of silenced plants under salt stress. Differential distribution of Na(+) and lower net Na(+) flux were observed in the xylem sap in the suppressed plants. In addition, K(+) concentration was lower in roots of silenced plants than in WT. Our results demonstrate that SlSOS1 antiporter is not only essential in maintaining ion homeostasis under salinity, but also critical for the partitioning of Na(+) between plant organs. The ability of tomato plants to retain Na(+) in the stems, thus preventing Na(+) from reaching the photosynthetic tissues, is largely dependent on the function of SlSOS1.

Two closely linked tomato HKT coding genes are positional candidates for the major tomato QTL involved in Na+/K+ homeostasis

The location of major quantitative trait loci (QTL) contributing to stem and leaf [Na(+) ] and [K(+) ] was previously reported in chromosome 7 using two connected populations of recombinant inbred lines (RILs) of tomato. HKT1;1 and HKT1;2, two tomato Na(+) -selective class I-HKT transporters, were found to be closely linked, where the maximum logarithm of odds (LOD) score for these QTLs located. When a chromosome 7 linkage map based on 278 single-nucleotide polymorphisms (SNPs) was used, the maximum LOD score position was only 35 kb from HKT1;1 and HKT1;2. Their expression patterns and phenotypic effects were further investigated in two near-isogenic lines (NILs): 157-14 (double homozygote for the cheesmaniae alleles) and 157-17 (double homozygote for the lycopersicum alleles). The expression pattern for the HKT1;1 and HKT1;2 alleles was complex, possibly because of differences in their promoter sequences. High salinity had very little effect on root dry and fresh weight and consequently on the plant dry weight of NIL 157-14 in comparison with 157-17. A significant difference between NILs was also found for [K(+) ] and the [Na(+) ]/[K(+) ] ratio in leaf and stem but not for [Na(+) ] arising a disagreement with the corresponding RIL population. Their association with leaf [Na(+) ] and salt tolerance in tomato is also discussed.

Genetic analysis of Na + and K + concentrations in leaf and stem as physiological components of salt tolerance in Tomato

The sodium and potassium concentrations in leaf and stem have been genetically studied as physiological components of the vegetative and reproductive development in two populations of F8 lines, derived from a salt sensitive genotype of Solanum lycopersicum cv. Cerasiforme, as female parent, and two salt tolerant lines, as male parents, from S. pimpinellifolium, the P population (142 lines), and S. cheesmaniae, the C population (116 lines). Genetic parameters of ten traits under salinity and five of them under control conditions were studied by ANOVA, correlation, principal component and QTL analysis to understand the global response of the plant. Two linkage maps including some tomato flowering time and salt tolerance candidate genes encoding for SlSOS1, SlSOS2, SlSOS3, LeNHX1, LeNHX3, were used for the QTL detection. Thirteen and 20 QTLs were detected under salinity in the P and C populations, respectively, and four under control conditions. Highly significant and contributing QTLs (over 40%) for the concentrations of Na+ and K+ in stems and leaves have been detected on chromosome 7 in both the populations. This is the only genomic position where the concentration QTLs for both the cations locate together. The proportion of QTLs significantly affected by salinity was larger in the P population (64.3%, including all QTLs detected under control) than in the C population (21.4%), where the estimated genetic component of variance was larger for most traits. A highly significant association between the leaf area and fruit yield under salinity was found only in the C population, which is supported by the location of QTLs for these traits in a common region of chromososome C1. As far as breeding for salt tolerance is concerned, only two sodium QTLs (lnc1.1 and lnc8.1) map in genomic regions of C1 and C8 where fruit yield QTLs are also located but in both the cases the profitable allele corresponds to the salt sensitive, cultivated species. One of those QTLs, lnc1.1 might involve LeNHX3.

Overexpression of SlSOS2 (SlCIPK24) confers salt tolerance to transgenic tomato

The Ca(2+)-dependent SOS pathway has emerged as a key mechanism in the homeostasis of Na(+) and K(+) under saline conditions. We have identified and functionally characterized the gene encoding the calcineurin-interacting protein kinase of the SOS pathway in tomato, SlSOS2. On the basis of protein sequence similarity and complementation studies in yeast and Arabidopsis, it can be concluded that SlSOS2 is the functional tomato homolog of Arabidopsis AtSOS2 and that SlSOS2 operates in a tomato SOS signal transduction pathway. The biotechnological potential of SlSOS2 to provide salt tolerance was evaluated by gene overexpression in tomato (Solanum lycopersicum L. cv. MicroTom). The better salt tolerance of transgenic plants relative to non-transformed tomato was shown by their faster relative growth rate, earlier flowering and higher fruit production when grown with NaCl. The increased salinity tolerance of SlSOS2-overexpressing plants was associated with higher sodium content in stems and leaves and with the induction and up-regulation of the plasma membrane Na(+)/H(+) (SlSOS1) and endosomal-vacuolar K(+), Na(+)/H(+) (LeNHX2 and LeNHX4) antiporters, responsible for Na(+) extrusion out of the root, active loading of Na(+) into the xylem, and Na(+) and K(+) compartmentalization.

Effects of salt stress on H+-ATPase activity of plasma membrane-enriched vesicles isolated from sunflower roots

It has previously been shown that mild and severe salt-stress increased both ATP- and PPi-dependent H+ transport and induced a Na+/H+ exchange activity in tonoplast vesicles from sunflower seedling roots. In this study, the vanadate-sensitive H+-ATPase activity was characterized in plasma membrane-enriched vesicles isolated by discontinuous sucrose gradient centrifugation from sunflower (Helianthus annuus L.) roots exposed for 3 days to 0, 75 or 150 mM NaCl. Blots immunoassayed with an Arabidopsis thaliana plasma membrane ATPase polyclonal antibody revealed the existence of a band of about 100 kDa, which was highly enriched in the 32/43% sucrose interface. ATP hydrolyzing activity in this fraction was mostly inhibited by vanadate, and scarcely by azide, oligomycin, nitrate and molybdate, indicating that it was essentially enriched in plasma membrane vesicles. Properties of vanadate-sensitive ATPase activity, such as inhibitor sensitivity, pH optimum, substrate specificity, ion effects and kinetic data were consistent with those of other plasma membrane ATPases. Although the pH profile, ion dependence, apparent Km and the amount of antigenic protein were unaffected by salt-stress, the Vmax of the vanadate-sensitive ATPase activity (measured in the presence of detergent) was reduced as a function of salt treatments. Likewise, while mild and severe salt-stress reduced the basal ATP hydrolyzing activity (measured in the absence of detergent) about a 35 and 55%, respectively, the ATP-dependent H+ transport activity sensitive to vanadate remained unchanged. As a consequence, the ratio of H+ pumping to the basal phosphohydrolase activity was proportionally increased. These results and those previously reported at the sunflower tonoplast suggest that the activity of H+ pumps under salt stress can form part of a tolerance mechanism in sunflower roots, which could regulate the ion fluxes (Na+ and Cl−) across the tonoplast and plasma membrane.

The Na+/H+ exchanger SOS1 controls extrusion and distribution of Na+ in tomato plants under salinity conditions

Maintaining a high K+/Na+ ratio in the cell cytosol, along with the transport processes implicated in the xylem and phloem loading/unloading of Na+ in plants (long-distance transport) are key aspects in plant salt tolerance. The Ca2+-dependent SOS pathway regulating Na+ and K+ homeostasis and long-distance Na+ transport has been reported in Arabidopsis. However, Arabidopsis might not be the best model to analyze the involvement of the SOS pathway in long-distance Na+ transport due to the very short stem of these plants which do not allow a precise dissection of the relative content of Na+ in stem versus leaf. This separation would be critical to assess the role of SOS1 in xylem loading/unloading, Na+ export by roots, retention in stems and the differential distribution/accumulation in old leaves. Towards this goal, tomato might represent a superior model due to its anatomical structure and agricultural significance. We recently demonstrated the key role played by the plasma membrane Na+/H+ antiporter SlSOS1 in salt tolerance in tomato by maintaining ion homeostasis under salinity stress and in the partitioning of Na+ in plant organs.

Heterologous expression of the yeast HAL5 gene in tomato enhances salt tolerance by reducing shoot Na+ accumulation in the long term.

For salt tolerance to be achieved in the long-term plants must regulate Na(+)/K(+) homeostasis over time. In this study, we show that the salt tolerance induced by overexpression of the yeast HAL5 gene in tomato (Solanum lycopersicum) was related to a lower leaf Na(+) accumulation in the long term, by reducing Na(+) transport from root to shoot over time regardless of the severity of salt stress. Furthermore, maintaining Na(+)/K(+) homeostasis over time was associated with changes in the transcript levels of the Na(+) and K(+) transporters such as SlHKT1;2 and SlHAK5. The expression of SlHKT1;2 was upregulated in response to salinity in roots of transgenic plants but downregulated in the roots of wild-type (WT) plants, which seems to be related to the lower Na(+) transport rate from root to shoot in transgenic plants. The expression of the SlHAK5 increased in roots and leaves of both WT and transgenic plants under salinity. However, this increase was much higher in the leaves of transgenic plants than in those of WT plants, which may be associated with the ability of transgenic leaves to maintain Na(+)/K(+) homeostasis over time. Taken together, the results show that the salt tolerance mechanism induced by HAL5 overexpression in tomato is related to the appropriate regulation of ion transport from root to shoot and maintenance of the leaf Na(+)/K(+) homeostasis through modulation of SlHKT1 and SlHAK5 over time.

Effects of salt-adaptation and salt-stress on extracellular acidification and microsome phosphohydrolase activities in tomato cell suspensions

The effects of NaCl-adaptation and NaCl-stress on in vivo H+ extrusion and microsomal vanadate- and bafilomycin-sensitive ATPase and PPase activities were studied in tomato cell suspensions. Acidification of the external medium by 50 mM NaCl-adapted and non-adapted (control) tomato cells was similar. Extracellular acidification by both types of cells during the first hour of incubation with 2 μM fusicoccin (FC) in the presence of 100 mM NaCl was lightly increased while in the presence of 100 mM KCl it was increased by 3 (control)- and 6.5 (adapted)-fold. Extracellular alkalinization after 2 h of cell incubation in 100 mM NaCl indicated the possibility that a Na+/H+ exchange activity could be operating in both types of cells. Moreover, acidification induced by adding 100 mM NaCl + FC to non-adapted cells was relatively less affected by vanadate than that induced by 5 mM KCl + FC, which suggested that salt stress could induce some component other than H+ extrusion by H+-ATPase. In addition, no differences were observed in microsomal vanadate-sensitive ATPase activity among control, NaCl-adapted and NaCl-stressed cells, while K+-stimulated H+-PPase and bafilomycin-sensitive H+-ATPase activities were higher in microsomes from NaCl-adapted than in those from control cells. Likewise, the stimulation of in vivo H+ extrusion in NaCl adapted cells under NaCl or KCl stress in the presence of FC occurred with an inhibition of H+-PPase and bafilomycin-sensitive H+-ATPase activities and without changes in the vanadate-sensitive H+-ATPase activity. These results suggest that the stimulation of tonoplast proton pumps in NaCl-adapted cells, without changes in plasmalemma H+-ATPase, could serve to energize Na+ efflux across the plasmalemma and Na+ fluxes into vacuoles catalyzed by the Na+/H+ antiports.

Involvement of SlSOS2 in tomato salt tolerance.

The Ca2+-dependent SOS pathway has emerged as a key mechanism in the homeostasis of Na+ and K+ under saline conditions. We recently identified and functionally characterized by complementation studies in yeast and Arabidopsis the gene encoding the calcineurin-interacting protein kinase of the SOS pathway in tomato, SlSOS2.1 We also show evidences on the biotechnological potential of SlSOS2 conferring salt tolerance to transgenic tomato. The increased salinity tolerance of SlSOS2 overexpressing plants is associated with higher sodium content in stems and leaves. SlSOS2 overexpression upregulates the Na+/H+ exchange at the plasma membrane (SlSOS1) and K+,Na+/H+ antiport at the endosomal and vacuolar compartments (LeNHX2 and LeNHX4). Therefore, SlSOS2 seems to be involved in tomato salinity tolerance through regulation of Na+ extrusion from the root, active loading of Na+ into the xylem and Na+ and K+ compartmentalization.

The sodium transporter encoded by the HKT1;2 gene modulates sodium/potassium homeostasis in tomato shoots under salinity

Excessive soil salinity diminishes crop yield and quality. In a previous study in tomato, we identified two closely linked genes encoding HKT1-like transporters, HKT1;1 and HKT1;2, as candidate genes for a major quantitative trait locus (kc7.1) related to shoot Na+ /K+ homeostasis - a major salt tolerance trait - using two populations of recombinant inbred lines (RILs). Here, we determine the effectiveness of these genes in conferring improved salt tolerance by using two near-isogenic lines (NILs) that were homozygous for either the Solanum lycopersicum allele (NIL17) or for the Solanum cheesmaniae allele (NIL14) at both HKT1 loci; transgenic lines derived from these NILs in which each HKT1;1 and HKT1;2 had been silenced by stable transformation were also used. Silencing of ScHKT1;2 and SlHKT1;2 altered the leaf Na+ /K+ ratio and caused hypersensitivity to salinity in plants cultivated under transpiring conditions, whereas silencing SlHKT1;1/ScHKT1;1 had a lesser effect. These results indicate that HKT1;2 has the more significant role in Na+ homeostasis and salinity tolerance in tomato.