Studies On The Mechanism Of Regulations Of G6PDH, CGMP, H₂O₂ And Ca²⁺ In Plant Adaptation To Salt Tolerance

In the present study, we used callus from Carex moorcroftii and Arabidopsis thaliana to study their physiological responses to salt stress. The regulative roles of glucose-6-phosphate dehydrogenase(G6PDH),3’,5’-cyclic guanosine monophosphate (cGMP), hydrogen dioxide (H2O2) and Ca2+were further investigated under salt stress. The main results were summarized as follows:1. The fruit of Carex moorcroftii were used as explants cultured in vitro to induce the callus. The experimental results showed that MS+NAA 0.5 mg·L-1+ 6-BA 1 mg·L-1+2.4-D 0.5 mg·L-1 was beneficial to the formation of callus. The induced rate of callus was about 10%. MS+NAA 0.5 mg·L-1+6-BA 0.5 mg·L-1+2.4-D 2 mg·L-1 could accelerate the growth of the callus. MS+6-BA 1 mg·L-1+NAA 1 mg·L-1 was beneficial to induce pullulation and pullulation rate reached 100%. The rooting rate almost reached 100% on 1/2MS+1AA 3 mg·L-2. In callus from Carex moorcroftii, G6PDH activity increased to its maximum in 100 mM NaCl treatment and decreased with further increased NaCl concentrations. K+/Na+ratio in 100 mM NaCl treatment did not exhibit significant difference compared with the control; however, in 300 mM NaCl treatment, it decreased. Low-concentration NaCl (100 mM) stimulated plasma membrane (PM) H+-ATPase and NADPH oxidase activities as well as Na+/H+antiporter protein expression, whereas high-concentration NaCl (300 mM) decreased their activity and expression. When G6PDH activity and expression was reduced by glycerol treatments, PM H+-ATPase and NADPH oxidase activities. Na+/H+antiporter protein level and K+/Na+ratio dramatically decreased. Simultaneously, NaCl-induced hydrogen peroxide (H2O2) accumulation was abolished. Exogenous application of H2O2increased G6PDH. PM H+-ATPase and NADPH oxidase activities, Na+/H+ antiporter protein expression and K+/Na+ratio in the control and glycerol treatments. Diphenylene iodonium (DPI). the NADPH oxidase inhibitor, which counteracted NaCl-induced H2O2 accumulation, decreased G6PDH. PM H+-ATPase and NADPH oxidase activities, Na+/H+antiporter protein level and K+/Na+ratio. Western blot result showed that G6PDH expression was stimulated by NaCl and H2O2, and blocked by DPI. Taken together, G6PDH is involved in H2O2 accumulation under salt stress. H2O2, as a signal, upregulated PM H+-ATPase activity and Na+/H+antiporter protein level, which subsequently resulted in the enhanced K+/Na+ratio. G6PDH played a central role in the process.3. Arabidopsis roots were sensitive to 100 mM NaCl treatment, which displayed a great increase in electrolyte leakage and Na+to K+ratio and a decrease in gene expression of the plasma membrane (PM) H+-ATPase. However, the application of exogenous cGMP, H2O2 or CaCl2 could alleviate the NaCl-induced injury by maintaining a lower Na+to K+ratio and increasing PM H+-ATPase gene expression. In addition, the inhibition of root elongation and seed germination in salt stress could be removed by cGMP. Further study indicated that cGMP could induce a higher NADPH contents mediated by regulating G6PDH activity to generate H2O2. The effect of cGMP and H2O2 on ionic homeostasis was abolished when Ca2+was eliminated by glycol-bis-(2-amino ethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA. Ca2+chelating) in Arabidopsis roots under salt stress. Taken together, cGMP could regulate H2O2 accumulation under salt stress, and Ca2+was necessary in the cGMP-mediated signaling pathway. H2O2, as the downstream component of cGMP signaling pathway, stimulated PM H+-ATPase gene expression. Thus, ion homeostasis was modulated for salt tolerance.