Cloning And Expression Analysis Of The H Subunit Of V-H～+-ATPase In The Leaves Of Halophyte Suaeda Salsa Under Salt Stress

Salt stress disrupts ionic and osmotic homeostasis of plant cells. The disorder ofhomeostasis occurs at both cellular and whole plant levels which lead to moleculardamage, growth inhibition and even death. For plants, one of the most importantstrategies to survive in salt environment is to re-establish the ionic and osmotichomeostasis. In the present study, effect of NaCl stress on the subunits expressions andactivities of V-H+-ATPase was analyzed, and the role of V-H+-ATPase in the Na+sequestration into the vacuole of halophyte Suaeda salsa was examined. The transgenicArabidopsis plants overexpressing SsVHA-H were also analyzed to examine salttolerance of the transgenic plants. The results were shown as follows:1. Effects of NaCl treatment on the subunit expressions and activitiy of V-H+-ATPaseinthe leaves of Suaeda salsaNorthern blot analyses indicated that the transcripts of V-H+-ATPase subunits B, Hand c were obviously up-regulated by NaCl stress. The transcript level of H subunit in400 mmol/L NaCl-treated plants was significantly higher than that in controls during0-120h and the mRNA levels of B, H and c subunits were also increased under 100 and400 mmol/L NaCl stress. The similar up-regulation of subunit B, H (located in the V1part) and subunit c (located in the V0 part) indicated that a coordinate expression ofV-H+-ATPase subunits ocurred under salt stress.Polyclonal antiserum against subunit H of V-H+-ATPase from the leaves of S. slasawas prepared using GST-SsVHA-H?4-206 immuning rabbits. Western blotting analyseswith the antiserum ATP95 and GST-SsVHA-H?4-206 revealed a significant increase ofprotein amounts of the V-H+-ATPase subunits B, H and c of S. slasa under NaCltreatment, which gave another evidence for a salt-induced coordinate up-regulation ofV-H+-ATPase subunits at translation levels. The coordinate salt-induced increase ofsubunit B,H and c of V-H+-ATPase from S. salsa at transcription and translation levelsindicated an increase of V-H+-ATPase holoenzyme amounts, which might be thereason for the increase of V-H+-ATPase activity of S. salsa under salt stress.2. Effect of overexpressing SsVHA-H on the salt tolerance of Arabidopsis plantsThe transgenic Arabidopsis plants which overexpressed SsVHA-H were moresalt-tolerant than wild type plants. No difference was observed in growth betweentransgenic plants and wild type plants in the absence of NaCl stress, which indicatedthat the insertion and overexpression of SsVHA-H had no effect on the normal growth ofArabidopsis plants under normal growth condition. However, the colour and growth ofrosette leaves of transgenic plants grown in MS medium containing 10 mmol/L LiClwere better than those of wild type plants.In MS medium containing 15 mmol/L LiClthe leaves of wild type plants became colorless and the plants were nearly dead;whilethe transgenic plants were still green and survival.In contrast to the plants grown in MS medium containing LiCl, the growth oftransgenic plants grown in MS medium containing NaCl were much worse than that ofwild type plants. The rosette leaves' colour of transgenic plants was obviously worsethan that of wild type plants. The growth of both transgenic and wild type Arabidopsisplants were suppressed by 150 mmol/L NaCl treatment in MS medium, but the growthof wild type plants were much better than that of transgenic plants. If the plants weretransplanted from normal MS medium to MS medium containing 150 mmol/L NaCl,after four days the wild type plants still survive but the transgenic plants were almostdead.Summarily, the data in this study indicated that the ability of Na+ sequestration intovacuole of S. salsa is positively related to the salt tolerance. The expressions ofV-H+-ATPase subunits B, H and c of S. salsa were significantly up-regulated by salinityat both transcription and translation levels, which led to an increase of V-H+-ATPaseholoenzyme amounts. The increase of enzyme amounts of V-H+-ATPase might be thereason for the increase of V-H+-ATPase activity of S. salsa under salt stress. Thesalt-induced increase of V-H+-ATPase activity energized the tonoplast for the Na+/H+antiporter that finally caused the Na+ compartmentalization into vacuoles. The Na+compartmentalization of S. salsa in leaf vacuoles reduced the toxicity of Na+ tocytosolic enzymes and therefore kept S. salsa plants survive in saline soils. TransgenicArabidopsis plants overexpressing SsVHA-H had greater LiCl tolerance than wild typeplants, which proved that H subunit had some kind of role in V-H+-ATPase and furtherindicated the importance of ion compartmentation in plant salt tolerance and can be usedin gene engineering for improving salt tolerance of plants. But in MS mediumcontaining NaCl wild type plants grew better than transgenic plants. These resultssuggested that overexpressing SsVHA-H in Arabidopsis disturbed osmotic tolerance butenhanced ionic tolerance. However, the detail reason for that need be further proved.The conclusive points in this study were shown as follows:1. Polyclonal antiserum against H subunit of the V-H+-ATPase of Suaeda salsa wassuccessfully prepared.2. Expression analyses of subunits B, H and c of tonoplast H+-ATPase of Suaeda salsaindicated that NaCl stress led to up-regulation and coordination of tonoplastH+-ATPase subunits at transcription and translation levels.3. Salt stress induced an increase of Na+ contents, expressions and activities oftonoplast H+-ATPase of Suaeda salsa plants, which grew very well under salt tress.These results suggested that tonoplast H+-ATPase plays an important role in Na+sequestration into vacuole.4. Ti vector containing SsVHA-H was constructed and Arabidopsis plants weretransformed with the Ti vector. RT-PCR and western blotting analyses showed thatSsVHA-H was expressed in the transgenic Arabidopsis plants. Stress tolerance of thetransgenic Arabidopsis plants was examined. The results showed that the transgenicArabidopsis plants grew much better than wild type plants in the medium containing10, 15 mmol/L LiCl, which further indicated the important role of ioncompartmentalization into vacuoles in plant salt tolerance. Surprisingly, in MSmedium containing NaCl wild type plants grew better than transgenic plants. Theseresults suggested that overexpressing SsVHA-H in Arabidopsis disturbed osmotictolerance but enhanced ionic tolerance.