| Stoma is the main gate controlling transpiration and regulating gas exchange in leaves. Stomatal movement is regulated by multiple external and internal factors. Although guard cells abscisic acid (ABA) signaling pathway has been well studied, the mechanism of guard cells response to light/dark signal is still unclear. By means of pharmacological approach and laser scanning confocal microscope, the present work explored whether heterotrimeric GTP binding protein (G protein), protein tyrosine kinases (PTKs) and phospholipase C/phospholipase D (PLC/PLD) mediate light/dark-regulated stomatal movement, and whether the effects of G protein, PTKs and PLC/PLD on stomatal movement are related to the changes of hydrogen dioxide (H2O2), nitric oxide (NO) levels in guard cells. The main results are as follows:1. Cholera toxin (CTX), an activator of G protein, significantly induced stomatal closure in Vicia faba and Arabidopsis (WT) in light, but pertussis toxin (PTX), an inhibitor of G protein, did not. PTX obviously promoted stomatal opening in Vicia faba and Arabidopsis (WT) in the darkness, but CTX did not. Whether in light or in the darkness, stomata of Arabidopsis mutant gpal-1 and gpal-2 kept opening, and CTX and PTX did not influenced the stomatal apertures of gpal-1 and gpal-2. The results show that G protein involves in light/dark-regulated stomatal movement, light induces stomatal opening through inactivating G protein, and dark causes stomatal closure via activating G protein.H2O2 significantly induced stomatal closure in Vicia faba and Arabidopsis (WT) in light, but ascorbic acid (Vc), catalase (CAT) and diphenyleneiodonium chloride (DPI), an important reducing substrate for H2O2 removal, one.of H2O2 scavenging enzymes and an inhibitor of the H2O2-generating enzyme NADPH oxidase, did not. Vc, CAT and DPI obviously promoted stomatal opening in Vicia faba and Arabidopsis (WT) in the darkness, but H2O2 did not. The result indicates that H2O2 level within guard cells is higher in the dark than in light. The results show that H2O2 participates in light/dark-regulated stomatal movement, light induces stomatal opening through reducing H2O2, and dark causes stomatal closure via increasing H2O2.CTX increased H2O2 level in guard cells in Vicia faba and Arabidopsis (WT) in light. PTX decreased H2O2 in guard cells in Vicia faba and Arabidopsis (WT) in dark.. Whether in light or in the dark, H2O2 did not appear in guard cells of Arabidopsis mutant gpal-1 and gpal-2. The results show that inactivation of G protein decreases H2O2, and activation of G protein increases H2O2. 2. Sodium nitroprusside (SNP), a NO donor, significantly induced stomatal closure in Vicia faba and Arabidopsis (WT) in light, but c-PTIO and L-NAME, a NO scavenger and an inhibitor of nitric oxide synthase, did not. c-PTIO and L-NAME obviously promoted stomatal opening in Vicia faba and Arabidopsis (WT) in the darkness, but SNP did not. The results indicates that NO levels in guard cells are higher in the dark than in light. The results show that NO participates in light/dark-regulated stomatal movement, light induces stomatal opening through lessening NO, and dark causes stomatal closure via increasing NO.CTX increased NO in guard cells in Vicia faba and Arabidopsis (WT) in light, whereas PTX decreased NO in guard cells in Vicia faba and Arabidopsis (WT) in the dark. Whether in light or in the dark, NO did not appear in guard cells of Arabidopsis mutant gpal-1 and gpal-2. The results show that inactivation of G protein decreases NO, and activation of G protein increases NO.3. Genistein and tyrphostin A23, two specific PTKs inhibitor, significantly prevented dark-induced stomatal closure in Vicia faba and reduced H2O2 levels in guard cells in the darkness. Additionally, genistein and tyrphostin A23 not only suppressed exogenous H2O2-induced stomatal closure and reduced H2O2 levels induced by exogenous H2O2 in guard cells in light, but also reopened the closed stomata in the darkness and abolished H2O2 that had been generated by dark. The results indicate that PTKs mediates dark-induced stomatal closure via inhibiting. H2O2 scavenging, consequently increasing H2O2 levels in guard cells.4. Both genistein and tyrphostin A23 evidently inhibited dark-induced stomatal closure and reduced NO levels in guard cells in the darkness. Additionally, genistein and tyrphostin A23 not only suppressed SNP-induced stomatal closure and reduced NO levels in guard cells in light, but also reopened the closed stomata by darkness and abolished NO that had been generated by dark. The results indicate that PTKs mediates dark-induced stomatal closure via suppressing NO scavenging, consequently increasing the level of NO in guard cells.5. U73122 and n-butanol, one specific inhibitor of PLC and a selective inhibitor of phosphatidic acid production by PLD, remarkably prevented dark-induced stomatal closure in Vicia faba and reduced H2O2 levels in guard cells in the darkness. Phosphatidic acid (PA), a common product of PLC and PLD activity, evidently caused stomatal closure in Vicia faba and increased H2O2 levels in guard cells in light. The results indicate that PA generated by PLC/PLD mediated dark-induced stomatal closure via increasing H2O2 levels in guard cells of Vicia faba.6. U73122 and n-butanol not only markedly suppressed dark-induced stomatal closure in Vicia faba and reduced NO levels in guard cells of Vicia faba in the darkness, but also evidently prevented SNP-caused stomatal closure in Vicia faba in light. PA obviously induced stomatal closure, but did not increase NO levels in guard cells of Vicia faba in light. The results show that PLC/PLD not only involve in dark-induced NO production, but also mediate NO-induced stomatal closure. However, PA does not participate in PLC/PLD-mediated NO production during dark-induced stomatal closure.All together, the present results proved that G protein, PTKs and PLC/PLD mediated light/dark-regulated stomatal movement via adjusting H2O2, NO levels in guard cells.
|
PDF Full Text Request