Phytohormone salicylic acid (SA) plays important roles in plant responses to environmental stress. However, knowledge about the molecular mechanisms for SA affecting the stomatal movements is limited. In this paper, we demonstrated that exogenous SA significantly induced stomatal closure and nitric oxide (NO) generation in Arabidopsis guard cells based on genetic and physiological data. These effects were significantly inhibited by the NO scavenger c-PTIO, NO synthase (NOS) inhibitor L-NAME or nitrate reductase suppressor tungstate respectively, implying that NOS and nitrate reductase (NR) participate in SA-evoked stomatal closing. Furthermore, the effects of SA promotion of stomatal closure and NO synthesis are significantly suppressed in NR single mutants of nia1, nia2 or double mutant nia1/nia2, compared with the wild type plants. This suggests that both Nia1 and Nia2 are involved in SA-stimulated stomatal closure. In addition, pharmacological experiments showed that protein kinases, cGMP and cADPR are involved in SA-mediated NO accumulation and stomatal closure induced by SA in Arabidopsis.

The effects of exogenous NO on changes in cytosolic pH of guard cells from epidermal fragments and of the pH on NO-induced stomatal closure in Vicia faba were investigated by using a pH specific fluorescence probe SNARF-1-AM and a confocal laser scanning microscopy. The results showed that treatments with 100 μmol L -1 sodium nitroprusside (SNP), a widely used NO donor, led to a significant increase in cytosolic pH (from 6.91 to 7.19, i.e. 0.28 pH unit) and stomatal closure in comparison with the control. Furthermore, SNP-induced cytosolic alkalization and stomatal closure were significantly inhibited by NO scavenger c-PTIO or weak acid butyrate, and promoted by the weak base benzylamine. Whereas two structural analogs of SNP such as Fe(II)CN and Fe(III)CN, which can not release NO, failed to induce guard cell cytoplasmic alkalization and stomatal closure. These findings suggest that cytoplasmic alkalization of guard cells mediates NO-induced stomatal closure. In addition, pH in guard cell vacuoles and cell walls was measured during the cytosolic alkalization. However, no significant changes of pH in the two regions were found, implying that the cytoplasmic protons of guard cells may not go into vacuoles or cell walls during the NO-promoted alkalization in Vicia faba.