Of No, And Cytoplasmic Ph In H <sub> 2 </ Sub> The O <sub> 2 </ Sub>-induced Stomatal Closure In The Role And Their Relationship

Plant guard cells swell to open and shrink to close stomata, thereby modulating gas exchange for photosynthesis and water status via transpiration. Opening and closing of stomata are regulated by multiple external and internal factors. The changes of nitric oxide (NO), intracellular pH and calcium in guard cells have been shown to be involved in hydrogen dioxide (H2O2)-induced stomatal closure. However.the interaction of NO with intracellular pH and the effects of calcium on NO production and intracellular pH during H2O2-induced stomatal closure remain unclear. In the present work. by means of stomatal bioassay and laser scanning confocal microscopy, we seek evidence that the interaction of NO with intracellular pH mediates Vicia faba stomatal closure caused by H2O2. Whether intracellular calcium regulates NO production and cytosol pH in guard cells is also studied. The main results are as followed:1. NO donor sodium nitroprusside (SNP) of at concentration≥100μM and 100μM H2O2 obviously induced stomatal closure (P< 0.05), and the effect of H2O2 on stomatal aperture was evidently prevented by carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-l-oxyl-3-oxide (c-PTIO). a specific NO scavenger and NG-nitro-L-argmethyl ester (L-NAME), an inhibitor of NO synthase (NOS) (P<0.05). Like SNP, the treatment of H2O2 largely increased NO fluorescence in guard cells over the control. Compared with the control. c-PTIO and L-NAME had no effect on NO levels in guard cells. However. H2O2-induced NO increase was obviously prevented by c-PTIO and L-NAME. The results show that NO mediates H2O2-induced stomatal closure.2. Weak alkalinizing agent methylamine at concentration≥2 mM and 100μM H2O2 significantly induced stomatal closure (P<0.05). and the effect of H2O2 on stomatal closure was obviously suppressed by butyric acid (P<0.05). Similar to methylamine. H2O2 evidently induced the raise of intracellular pH in guard cells over the control. Compared with the control, butyric acid, a weak acid, had no obvious effect on the intracellular pH in guard cells. However, H2O2-induced rise in intracellular pH of guard cells was largely abolished by butyric acid. The results indicate that the rise in intracellular pH in guard cells is involved in H2O2-induced stomatal closure.3. The fact that both NO production and intracellular pH rise in guard cells participate in H2O2-induced stomatal closure promotes us to explore the interrelationship between NO production and intracellular pH rise during H2O2-induced stomatal closure. Like SNP. H2O2 obviously raised intracellular pH over the control. Compared with the control c-PTIO and L-NAME had no evident effect on intracellular pH. However. H2O2-caused rise in intracellular pH were largely suppressed by c-PTIO and L-NAME. The results show that H2O2-induced NO generation raises intracellular pH in guard cells.4. Similar to methylamine. H2O2 evidently increased NO production over the control. Compared with the control, butyric acid did not cause significant change of NO level in guard cells. However, H2O2-induced NO production was obviously prevented by butyric acid. The results show that H2O2-induced rise in intracellular pH triggers NO generation in guard cells.5. Extracellular calcium chelator BAPTA and intracellular calcium chelator BAPTA-AM alone had no effect but suppressed H2O2-induced stomatal closure. NO production and the rise in intracellular pH in guard cells. The results show that intracellular calcium is required for H2O2-induced stomatal closure, NO generation and rise in intracellular pH in guard cells. In addition. the effects of BAPTA also suggest that the influx of external calcium is involved in H2O2-induced stomatal closure, NO increase and rise in intracellular pH of guard cells.In conclusion, both NO increase and intracellular pH rise in guard cells participate in stomatal closure by H2O2. NO mediates H2O2-induced rise in intracellular pH, whereas the rise of intracellular pH participates in H2O2-triggered NO production. Calcium is a prerequisite for NO production and the rise in intracellular pH in guard cells during H2O2-induced stomatal closure, besides its known action downstream of NO production. However, the mechanism by which intracellular pH alteration affects NO production or NO regulates intracellular pH still is an unresolved problem.