Studies On The Mechanism Of Regulations Of Glucose-6-phosphate Dehydrogenase In The Adaptation Of Reed Callus To Salt Stress

In the present study, we used the calli from two ecotype reed (Phragmites communis Trin.; dune reed, DR and swamp reed, SR) to study their physiological responses in a long-term salt stress, and further investigated the regulative roles of glucose-6-phosphate dehydrogenase (G6PDH) and the signal-transduction mechanism.The main results are:1. Under 50 mM NaCl treatment, the growth rate of DR callus increased, but it decreased in SR callus. Further studies found: under 50 mM NaCl treatment, the levels of ion leakage, lipid peroxide, relative cell viability and reactive oxygen species (ROS; H₂O₂ and O₂^.-) significantly increased in SR callus; however, in DR callus, the levels of lipid peroxide, relative cell viability and O₂^.- slightly decreased. Ion leakage as well as H₂O₂ contents increased in DR callus in 50 mM NaCl treatment, however, they were higher in SR callus. Under higher NaCl treatment (300 mM), the levels of ion leakage, lipid peroxide and ROS contents markedly increased in both calli; however, SR callus had more significant increases than DR callus. These results suggested that DR callus had higher salt tolerance.2. Proline, glycine betaine and total soluble sugar contents decreased in DR callus under 50 mM NaCl treatment; in SR callus, proline and glycine betaine contents significantly increased, but total soluble sugar contents decreased, suggesting that proline and glycine betaine acted the main roles in relieving osmotic stress under low-concentration salt treatment in SR callus. Under 300 mM NaCl treatment, proline, glycine betaine and total soluble sugar contents in both calli markedly increased, and the proline as well as total soluble sugar contents in DR callus were higher than those in SR callus.3. Under 50 mM NaCl treatment, the activities of superoxde dismutase (SOD), catalase (CAT) in DR callus increased but the activities of peroxidase (POD), glutathione peroxidase (GPX) and the key enzymes of ascorbate-glutathione cycle (ASA-Glu; ascorbate peroxidase [APX] and glutathione reducase [GR]) decreased; in SR callus, the activities of POD, CAT, APX, GR, GPX markedly increased. These results suggested that SOD and CAT were the main scavengers of ROS in DR callus; in contrast, they were POD, CAT and ASA-Glu cycle in SR callus under 50 mM NaCl treatment. Under higher NaCl concentration (300 mM), SOD, POD, GPX, APX and GPX activities significantly increased in DR callus; however, in SR callus, the activities of CAT, GPX, APX and GR were low compared with those in 50 mM NaCl treatment. It suggested that both SOD and ASA-Glu cycle were the main scavengers of ROS under 300 mM NaCl treatment in DR callus.4. G6PDH activity was higher in SR callus than that of DR callus under 50-150 mM NaCl treatments. In contrast, at higher NaCl concentrations (300-600 mM), G6PDH activity was lower in SR callus. A similar profile was observed in GSH content in both salt-stressed calli. After G6PDH activity and its protein expression decreased under glycerol treatment, GSH content dramatically reduced (only to 50% of the control) in both calli. Under glycerol+NaCl treatment, GSH content slightly increased, but it was markedly low compared with that in control. These results suggested that G6PDH played a pivotal role in intracellular GSH maintenance not only in salt stress but in control conditions.5. In glycerol treatment alone and glycerol+NaCl treatment, the decrease in G6PDH activity and its protein expression also resulted in the dramatic decrease in GR and GPX activities in both calli. Our present results further showed that glycerol treatment could abolish NaCl-induced H₂O₂ accumulation in both calli; however, the extent of ion leakage didn't decrease, on the contrary, it markedly increased compared with that in 300 mM NaCl treatment alone. Exogenous H₂O₂ could abolish the glycerol-induced decreases in G6PDH, GR and GPX activities in both calli. Moreover, exogenous H₂O₂ increased the activities of G6PDH, GR and GPX as well as GSH content in a concentration-dependent manner in control conditions. Diphenylene iodonium (DPI), a plasma membrane (PM) NADPH oxidase inhibitor, which counteracted NaCl-induced H₂O₂ accumulation, dramatically decreased these enzymes activities and GSH content in both calli. Western-blot result further showed that G6PDH expression was stimulated by NaCl and H₂O₂, and blocked by DPI in DR callus. These results strongly suggested that G6PDH activity was involved in NADPH oxidase-dependent H₂O₂ accumulation; H₂O₂, as a signal, regulated the activities of G6PDH, GR and GPX in reed calli under the long-term salt stress.6. Exogenous N-acetyl-L-cysteine (NAC, the GSH precursor) and buthionine sulfoximine (BSO, an effective inhibitor ofγ-glutamylcysteine synthetase that catalyzes the first step in glutathione biosynthesis) treatment decreased and increased G6PDH activity in both calli, respectively. Similarly, either the decrease or increase of H₂O₂ contents in both calli also followed the NAC and BSO treatment, respectively. The addition of exogenous H₂O₂ effectively inhibited the NAC treatment-mediated decrease in G6PDH activity in both calli; DPI inhibited the BSO-induced H₂O₂ accumulation and also decreased G6PDH activity in both calli. These results suggested that the decrease induced by NAC or the increase induced by BSO in G6PDH activity might also involve H₂O₂ signaling.Taken together, G6PDH modulated GSH levels and H₂O₂ accumulation in salt stress; H₂O₂, as a signal, regulated G6PDH, GR and GPX activities, which finally resulted in the enhanced glutathione cycling rate and thus the increased GSH levels under salt stress. Thus, the enhanced anti-oxidant ability can facilitate to maintain a steady-state level of H₂O₂ in cells, and allows beneficial functions of H₂O₂ for signaling as well as a defensive response to salt stress. During this process, G6PDH plays the important regulative roles.