Roles Of Membrane-related Translocating Proteins In Salt Tolerance Of Suaeda Salsa

High salinity disrupts ionic and osmotic homeostasis of plants. 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 the cell and plant death. Therefore, one important strategy for plants to survive under salt stress is to re-establish the homeostasis in high salt environment. Both ionic and osmotic homeostasis must be restored. The Chenopodiaceae Suaeda salsa L. is a C3 succulent euhalophyte which is native to saline soil and adapted to the high salinity region in the north of China. Previous studies showed that the fresh weight, dry weight, and leaf succulence of S. salsa were not inhibited by treatment of 100-400 mmol/L NaCl, rather than stimulated.It is well known that enzymes and many important metabolisms have generally been found to be sensitive to Na⁺ for both halophytes and non-halophytes. Surprisingly, NaCl (100-400 mmol/L) treatment can markedly increase the ion content (mainly Na⁺) in leaves of S. salsa and stimulated its growth, which suggests that there is no excessive Na⁺ in cytoplasm. High concentration of Na⁺ outside the cell will enter the cell down the concentration gradient of Na⁺. Na⁺ concentration in cytoplasm can be decreased through two ways: 1) Na⁺ can be compartmentalized in the vacuole which is the main way because the volume of vacuole is about 90% of the volume of the matured cells; 2) Na⁺ can also be transported out of the cell.Na⁺ entering the cell reduces cellular osmotic potential and ensures water uptake, which is very important to the salt tolerance of cells, because water uptake of cells ensures the cell turgor and expansion and dilutes the Na⁺ entered the cell. Therefore, high tissue water content (succulence) is very important strategy for salt tolerance of halophytes under saline environment.In the present study, a typical salt-accumulator euhalophyte S. salsa was used to investigate the roles of the vacuolar proteins in Na⁺ compartmentalization, the protein of plasma membrane in Na⁺ transport out of the cell, and the plasma membrane aquaporins in leaf succulence. The main results were as follows:1. Coordinate up-regulation of V-H⁺-ATPase and vacuolar Na⁺/H⁺ antiporter plays an important role in Na⁺ compartmentalization into vacuole. S. salsa seedlings were used to examine the adaptation strategy to high salinity at whole plant level. Our results firstly demonstrated that Na⁺ was a beneficial element rather than toxic ion for S. salsa plants. The biomass of S. salsa seedlings increased significantly after salt-treatment (100-400 mmol/L NaCl). Secondly, S. salsa had strong ability to enrich Na⁺ under saline condition. For example, 94% of Na⁺ absorbed by salt-treated S. salsa plant accumulated in the shoot, especially in the leaves, and 98% of Na⁺ in the leaves accumulated in leaf symplast. Thirdly, measurement on isolated tonoplast-enriched membrane vesicles derived from the leaves of S. salsa showed that increased V-H⁺-ATPase hydrolytic activity, V-H⁺-ATPase proton pump activity and the tonoplast Na⁺/H⁺ antiporter activity were found in salt-treated leaves compared with the control plants. Up-regulation of the tonoplast Na⁺/H⁺ antiporter activity was due to the increase of both transcription and translation. These results suggested that coordinate up-regulation of V-H⁺-ATPase activity and Na⁺/H⁺ antiporter activity play a pivotal role in Na⁺ sequestering into vacuoles of S. salsa. 2. S. salsa callus adapted to sanlinity partly via regulation of the plasma membrane H⁺-ATPase.The electrochemical potential grandient across the plasma memebrane established by plasma membrane H⁺-ATPase is the main driving force for substance exchange between the cell and environment. S. salsa calli treated with different concentrations of NaCl were used to examine the roles of plasma membrane H⁺-ATPase in salt tolerance at cellular level. The results showed that, after S. salsa calli were treated with 0, 50, 150, and 250 mmol/L NaCl for 7 days, 50 mmol/L NaCl was the optimum concentration of NaCl for growth of the calli, i.e. at 50 mmol/L NaCl, the RGR of S. salsa calli reached the highest point, the organic dry weight was significantly higher than that of the controls (P < 0.05), and the water content showed no significant difference when compared with that of the controls.The concentrations of inorganic ions (except Ca²⁺) and organic solutes of the calli increased significantly, especially for Na⁺, with the increase of NaCl.When calli were treated with 0-150 mmol/L NaCl, the activity of the PM H⁺-ATPase significantly increased. In contrast, the activity of the PM H⁺-ATPase dramatically decreased by 250 mmol/L NaCl. The proton extrusion rate markedly increased in calli treated with 50 mmol/L NaCl, while no significant difference was detected in the proton extrusion rate between 150 mmol/L NaCl-treated calli and the control. However, 250 mmol/L NaCl led to a significant decrease of the parameter.Northern blotting was performed to analyze the expression of the PM H⁺-ATPase of NaCl-treated S. salsa calli. An autoradiogram signal was markedly increased in the calli treated with 50 mmol/L NaCl from 2th to 7th day and reached the maximum value in the second day in comparison with the control. A single polypeptide was identified with the immunoblotting analysis of the PM H⁺-ATPase from calli of S. salsa, when probed with anti-Zea mays H⁺-ATPase serum (monoclonal 46E5B11D5). The protein amount varied with the different concentrations of NaCl treatments. The highest amount was observed at 150 mmol/L NaCl.These results suggest that S. salsa callus adapts to sanlinity partly via regulation of the plasma membrane H⁺-ATPase at the levels of transcript, protein amount and activity.3. Aquaporin plays an important role in leaf succulence of S. salsa under salinity.Plasma membrane aquaporins (AQPs) determine, likely among other functions, the water relations of plant cells. Seedlings were treated with 100 mmol/L NaCl for 10 days, then the effects of NaCl on leaf succulence degree, water permeability of protoplasts, expression of SsPIP, and the amount of SsPIP in leaves of S. salsa were determined. The results showed a correlation between leaf succulence and the water permeability of the protoplasts of leaves of S. salsa, under salinity. Also, the abundance of transcripts for a plasma-membrane AQP increased in the presence of NaCl. Immunoblot analyses of PIP AQPs located in plasma membrane-enriched fractions of S. salsa seedlings showed a significant increase under salinity. In conclusion, the increase in salinity-induced AQP activity is by way of an increase in both the transcripts and protein amount, which correlate with the increase in leaf succulence of S. salsa under salinity.Summarily, salt tolerance of the euhalophyte S. salsa was closely related to Na⁺ sequestration into vacuole, Na⁺ transport out of the cell, and high leaf succulence under salinity. The expression of Na⁺/H⁺ antiporter of tonoplast of S. salsa were significantly up-regulated by salinity at transcription and translation levels, which indicated an increase of Na⁺/H⁺ antiporter amounts. The increase of enzyme amounts of Na⁺/H⁺ antiporter may lead to the increase of Na⁺/H⁺ antiporter activities of S. salsa under salt stress. The salt-induced coordinate increase of Na⁺/H⁺ antiporter and V-H⁺-PPase activities caused the Na⁺ compartmentalization in vacuoles. The expression of PM H⁺-ATPase of S. salsa calli was significantly up-regulated by salinity at transcription and translation levels, which may lead to an increase of PM H⁺-ATPase enzyme amounts. Subsequently, the increase of enzyme amounts of PM H⁺-ATPase may cause the increase of PM H⁺-ATPase activities of S. salsa under salt stress. The salt-induced increase of PM H⁺-ATPase activities may energize the plasma membrane for the Na⁺/H⁺ antiporter (SOS1) to transport Na⁺ out of the cell, which reduces the toxicity of Na⁺ to cytosolic enzymes. The expressions of plasma membrane aquaporins of S. salsa were significantly up-regulated by salinity at transcription and translation levels, which may induce to an increase of plasma membrane aquaporins activities. The leaf succulence S. salsa was induced by the increase of plasma membrane aquaporins activities of S. salsa under salt stress.