Vacuolar Ca～(2+) Transporters And Salt Tolerance In C₃ Halophyte Suaeda Salsa L

Salt stress disrupts homeostasis in water potential and ion distribution. This disorder of homeostasis occurs at both the cellular and the whole plant levels. Drastic changes in ion and water homeostasis lead to molecular damage, growth inhibition and even death. There exist complicated signal systems in plant salt tolerance. Cytosol Ca²⁺ as a second messenger can transfer environment signals and magnify them to make plant responses. So it is important that the Ca²⁺ transporters in the tonoplast establish Ca²⁺ homeostasis during and after signal transduction events and stress responses. The present article was divided into two parts to analyze the role of the tonoplast H⁺-ATPase, Ca²⁺-ATPase and Ca²⁺/H⁺ antiporter in salt tolerance of halophyte Suaeda salsa. The first part was mainly that Suaeda salsa L. seedlings grown in Hoagland nutrient solution were treated with different concentrations of NaCl combined with two levels of Ca²⁺ (0 mmol/L and 20 mmol/L) to study the effect of Ca²⁺ nutrition on the growth and activity of leaf tonoplast V-H⁺-ATPase and Ca²⁺-ATPase. And the second part was that SsCAX1encoding a vacuolar membrane Ca²⁺/H⁺ antiporter of a C3 halophyte Suaeda salsa L. was cloned and its function was analyzed. The results were shown as follows: 1. Effect of Ca²⁺ on growth of Suaeda salsa under NaCl treatment. The reasponses of S. salsa growth to NaCl stress depends on Ca²⁺ supply. When 20 mmo/L Ca²⁺ was present in the root medium, RGQ of NaCl-treated plants was significantly higher than that of controls. No significant difference in the RGQ was observed between control and NaCl-treated seedlings (P>0.1), when Ca²⁺ level in the nutrient solution was 0 mmol/L. 2. Effects of Ca²⁺ on Na⁺, K⁺, and Ca²⁺ concentrations in leaf cell sap of Suaeda salsa seedlings under NaCl treatment K⁺ and Ca²⁺ concentrations in leaf cell sap were significantly decreased with the increase of NaCl concentration (P<0.05) irrespective of whether plants grown in 0 mmol/L or in 20 mmol/L Ca²⁺ medium. Conversely, Na⁺ concentration was significantly increased (P<0.001). Nevertheless, under condition of both 100 mmol/L and 400 mmol/L NaCl treatment, K⁺ and Ca²⁺ concentrations were significantly increased and Na⁺ concentration was reduced (P<0.001) when the plants were grown in 20 mmol/L Ca²⁺ medium comparing with those cultured in 0 mmol/L Ca²⁺ medium. 3. Effects of Ca²⁺ on leaf plasmalemma permeability of Suaeda salsa seedling under NaCl treatment Leaf plasmalemma permeability of plants grown in 0 mmol/L Ca²⁺ medium was significantly increased by increasing the NaCl concentration. However, when plants were grown in 20 mmol/L Ca2+ medium, only 400 mmol/L NaCl treatment significantly increased leaf plasmalemma permeability. 4. Effects of Ca2+ on the activity and protein amount and subunit composition of V-H+-ATPase of Suaeda salsa leaves The leaf V-H+-ATPase activity was higher in 100 mmol/L NaCl-treated plants than that in controls when the plants were grown in Ca2+-deficient condition (0 mmol/L), but there was no significant difference between 400 mmol/L NaCl-treated plants and controls. However, leaf V-H+-ATPase activity was markedly increased with increasing NaCl concentration when high Ca2+ concentration (20 mmol/ L) was applied to the root medium. Although there was no significant difference between 20 mmol/L and 0 mmol/L Ca2+ medium without NaCl treatment, the activity of V-H+-ATPase in the NaCl-treated plants grown in 20 mmol/L Ca2+ medium was much higher than that in 0 mmol/L Ca2+ medium. In addition, there was no significant difference in the the activity of Ca2+-ATPase between NaCl-treated plants and controls. Only the activity of Ca2+-ATPase in 20 mmol/L Ca2+ medium was little higher than in 0 mmol/L Ca2+ medium. Western blot immunostaining was performed using antiserum ATP95. The antiserum ATP95 cross-reacted with four polypeptides exhibiting molecular masses of 66 kDa, 55 kDa, 36 kDa, 18 kDa and these polypeptides seemed to be subunits A, B, D and c of V-H+-ATPase. The results showed that when the plants were grown in Ca2+ -deficient (0 mmol/L) medium, there was no significant difference in subunit pattern and subunit amounts between 400 mmol/L and controls except for a little increase in subunit c amount of NaCl-treated plants. However, amounts of subunit B and c of leaf V-H+-ATPase were markedly increased by increasing NaCl concentration under high Ca2+ concentration (20 mmol/L) supplication in the root medium. Moreover, amounts of A, B, D and c subunits of V-H+-ATPase in the plants grown in 20 mmol/L Ca2+ medium were higher than those in 0 mmol/L Ca2+ medium. In conclusion, Suaeda salsa was a typical halophyte and Ca2+ was involved in salt tolerance of Suaeda salsa. Enhancement of NaCl stress to leaf V-H+-ATPase activity of halophyte Suaeda salsa was Ca2+-dependent and the changes in V-H+-ATPase activity was due to the up-regulation in amounts of the enzymes. Ca2+ nutrition was partly involved in salt tolerance of halophyte Suaeda salsa via up-regulation of subunit amounts of V-H+-ATPase. 5. cDNA cloning and sequence analyzing of tonoplast Ca2+/H+ antiporter in halophyte Suaeda salsa Based on sequence homologies to the CAX-like genes of putative Ca2+/H+ antiporter from Arabidopsis and Mung bean et al., we used reverse transcriptase (RT)-PCR amplification and 5'-and 3'-RACE amplification to obtain the full-length transcript sequences of SsCAX1 (AY518204). The cDNA of SsCAX1 was 1596 nucleotides long coding for 456 amino acids. And the protein, which a predicted molecular mass was 48.8 kDa, was encoded by a cDNA with 1371 nucleotides in ORF. Hydrophobicity analysis suggested that the SsCAX1 from S. salsa was a transmembrane protein with eleven transmembrane domains. There was a hydrophileloop between the sixth and seventh transmembrane domains. 6. Expression analysis of tonoplast Ca2+/H+ antiporter in halophyte Suaeda salsa Northern blot analysis indicated that the transcription of Ca2+/H+ antiporter was up-regulated by CaCl2 treatment. Following CaCl2 addition, amounts of SsCAX1 transcripts in leaves increased for up to 24 h and then decreased. From 48 h-72 h, the mRNA levels of SsCAX1 in CaCl2 treatment were still higher than those of controls. By RNA-blot hybridizations the tissue specificity of expression of the SsCAX1 was that SsCAX was expressed primarily in leaves and hardly expressed in stems and roots. To test whether SsCAX1 transcript could be induced by salt stress or by osmotic stress, Northern blot hybridization was carried out with total RNA isolated from plant leaves stressed by the addition of 100 mmol/L CaCl2, 100 mmol/L NaCl and 400 mmol/L mannitol for 48 h.The results suggested that SsCAX1 RNA levels increased in response to NaCl, but markedly was lower than those to CaCl2. However, SsCAX1 RNA level was not significantly changed under mannitol treatment. The results suggested that expression of the SsCAX1 was ionic stress-dependent not osmotic-denpendent. 7. An analysis of Ca2+/H+ antiporter function and intracellular location of SsCAX1 in S. salsa.leaves To determine the SsCAX1 function, we expressed the gene SsCAX1 in Saccharomyces cerevisiae strain K667, which had defect in vacuolar Ca2+ transport. The result showed that SsCAX1 could suppress the Ca2+ sensitivity of yeast strain K667. We prepared the antibody SsCAX1?1-70 of SsCAX1, and using Western-blotting we found the SsCAX1 mainly located the vacuolar membrane, not plasma membrane of leaves. When the gene of SsCAX1 was over-expressed in Arabidopsis, it is found that the transgenic plants was more salt-sensitive than the wild types. No difference was observed in growth between transgenic plants and wild type plants grown in the normal MS medium, which indicated that the insertion and overexpression of SsCAX1 had no effect on the normal growth of Arabidopsis plants. However, if the plants were grown in MS medium, which was deficit of Ca2+, the colour of leaves, the development of lateral roots and fresh and dry weight of rosette leaves of transgenic plants were all obviously worse than those of wild type plants. The growth of both transgenic and wild type Arabidopsis plants were suppressed by 75 mmol/L NaCl treatment in MS medium, but the growth of transgenic plants were much worse than that of wild type plants. The transgenic plants had worse lateral roots, rosette leaves than those of wild type plants when grown for one week in MS medium containing 15 mmol/L LiCl and were dead for two weeks in MS medium contaning 10 mmol/L LiCl. Summarily, the data in this article indicated that the ability of Na+ sequestration into vacuole of S. salsa was close correlative to its salt tolerance. The expressions of V-H+-ATPase of S. salsa were significantly up-regulated by salinity at transcription and translation levels, which indicated an increase of V-H+-ATPase holoenzymeamounts. The increase of enzyme amounts of V-H+-ATPase might be the reason for the increase of V-H+-ATPase activities of S. salsa under salt stress. The salt-induced increase of V-H+-ATPase activities energized the tonoplast for the Na+/H+ antiporter and the Ca2+/H+antiporter that finally caused the Na+ compartmentalization in vacuoles and cytosol Ca2+ regain vacuoles. One hand, the Na+ compartmentalization of S. salsa in leaf vacuoles reduced the toxicity of Na+ to cytosolic enzymes. On the other hand, cytosol Ca2+ regain vacuoles can maintain the normal cytosol Ca2+to make signal road smooth. Therefore, S. salsa plants can survive in saline soil. Transgenic Arabidopsis plants overexpressed of SsCAX1 were less salt tolerance than wild type plants, which further indicated the importance of maintenance Ca2+ homeostasis in plant salt tolerance.