Isolation, Purification And Subunit Analysis Of Tonoplast H～+-ATPase From Suaeda Salsa L.

Vacuolar H~+-ATPase is a kind of membrane-bound proton transporter, whichuses the energy released from ATP and builds up a proton gradient across thetonoplast to drive translocation of ions and metabolites. Vacuolar H~+-ATPase in thetonoplast plays crucial roles in ion balance, metabolite translocation, plant growth andso on. In a saline environment, salt-dilution halophytes alleviate salt toxicity mainlythrough the capacity of sequestrating Na~+ into the vacuole. The transportation of Na~+against the concentration gradient from the cytoplasm into the vacuole via thetonoplast Na~+/H~+ antiporter is dependent on the activities of tonoplast proton pumps(V-H~+-ATPase and V-H~+-PPase), which establish an electrochemical H~+-gradientacross the tonoplast that energizes the transporters. Vacuolar H~+-ATPase not only isextraordinarily important as a house-keeping enzyme, but also functions as a stressresponse enzyme under environmental stresses. In the present study, salt-dilutionhalophyte S. salsa cultured under different salt concentrations was used to investigatethe activities, subunit composition and subunits expression of V-H~+-ATPase byWestern, Northern blot analysis and immunoprecipitation in addition to thedetermination of some physiological parameters. The main results were shown asfollows.1. Effects of NaCl treatment on the growth of S. salsaThe growth of S. salsa was enhanced by treatment of 200 mmol/L NaCl, and thefresh weight of leaves and whole plants was increased significantly compared withthe control. The number of shoot branching was increased. Furthermore, theNaCl-treated seedlings and leaves were bigger than the controls.S. salsa plants have high capacity of accumulating Na~+ in low Na~+ concentrationenvironment. The Na~+ concentrations of cell sap from leaves were greatly increasedunder 200 mmol/L NaCl treatment. As a typical salt-accumulating halophyte, Na~+ wasmainly accumulated in leaves, and then the osmotic potential of the cell sap was muchlower in leaves than that in the soil. So S. salsa plants can modulate the osmolarity tomaintain the normal growing state under salt stress. Furthermore, low Na~+concentration environment in cytoplast can protect many enzymes from Na~+ toxicityto maintain the normal growing state under salt stress. In saline condition, K+concentrations in the cell sap from leaves were decreased significantly compared withthe controls. Since the growth of S. salsa seedlings was promoted by salt stress, thedecrease of K+ concentration in the cell sap to a certain extent wouldn't inhibit theplants growth.2. Effects of NaCl treatment on the activities of H+-ATPase in tonoplast vesiclesprepared from leaves of S. salsa.The V-H+-ATPase activities in leaves were significantly increased by 200mmol/L NaCl treatment. The increased proton transport activity of V-ATPase mayprovide stronger driving force for Na+/H+ antiporter and a wide range of secondaryactive transport processes, leading to osmotic adjustment and elimination of iontoxicity. With lower osmotic potential of the cell sap, S. salsa plants can maintain thenormal growing state under salt stress.3. Subunit composition of tonoplast H+-ATPase from Suaeda salsa L.The tonoplast vesicles were obtained from leaves of Suaeda salsa plants by thediscontinuous sucrose gradient centrifugation.By solubilizing tonoplast vesicles withTriton X-100 (2%, v/v) and purifying ATPase complex by immunoprecipitation, thepartial pure enzyme was obtained. Western blot analysis with the antisera ATP95 andATP88 directed against the V-ATPase holoenzyme leads to the similar results thatV-ATPase consists of about 12 subunits. The V1 subcomplex is probably formed bythe subunits A, B, C, D, E, F, G and H, and the V0 subcomplex by a,d,e and c(or c′).The subunit a, G and e were firstly identified in the present study in Suaeda salsa L.4. Effects of NaCl treatment on subunit expression of H+-ATPase in tonoplastvesicles prepared from leaves of S. salsa.Western blot analysis indicated that the expression of a,A,B and c subunits wassignificantly up-regulated by 200 mmol/L NaCl treatment. Moreover, the expressionof B subunit was coordinated with c subunit of V-H+-ATPase at transcript andtranslation levels under NaCl stress. Then the number of V-ATPase holoenzymewould be increased in membrane as well. There was a basically consistentrelationship between the western blot analysis and the hydrolysis activity.In conclusion, significant difference was observed in the responses of growth,ion accumulation, activities of tonoplast proton pumps and expression at the levels oftranscription and translation of tonoplast H+-ATPase of S. salsa to NaCl stress. Thegreat difference suggested that the salt tolerance of S. salsa was related with manyfactors. Investigation of expressions and activities of V-H+-ATPase of S. salsaindicated that the changes of hydrolytic activity of V-H+-ATPase were consistent withthe gene expressions and translations and Na+ accumulation in cell saps. TheV-H+-ATPase might play a very important role in Na+ sequestration into vacuoleunder salt environments. Tonoplast H+-ATPase from S. salsa consists of about 12subunits by immunoprecipitation, SDS-PAGE and Western analysis. The V1subcomplex is probably formed by the subunits A, B, C, D, E, F, G and H, and the V0subcomplex by a,d,e and c(or c′). The subunits a, G and e were tested firstly in S.salsa. The expression of some subunits of S. salsa, and the expression of somesubunits at transcript and translation levels were significantly up-regulated underNaCl stress.