NO和H2O2在光／暗调控蚕豆气孔运动中的作用及其相互关系

借助表皮条分析和激光扫描共聚焦显微镜技术,对NO和H_2O_2在光/暗调控蚕豆(Vicia faba L.)气孔运动中的作用及其相互关系进行了探索。结果显示,光下外源NO供体硝普钠(SNP)和H_2O_2促进气孔关闭的效应明显大于暗中,暗中NO专一性清除剂2,4-羧基苯-4,4,5,5-四甲基咪唑-1-氧-3-氧化物(cPTIO)、一氧化氮合酶(NOS)抑制剂N~G-氮-L-精氨酸-甲酯(L-NAME)和H_2O_2清除剂抗坏血酸(Vc)、过氧化氢酶(CAT)对气孔开度的效应明显大于光下,而且光下蚕豆保卫细胞NO和H_2O_2水平比暗中明显降低。上述结果表明,光/暗通过影响保卫细胞NO和H_2O_2的水平调控气孔运动。研究还发现,光下H_2O_2既诱导NO水平增加,也诱导气孔关闭,cPTIO和L-NAME有效地逆转H_2O_2的这些效应;光下SNP既诱导H_2O_2水平增加,也诱导气孔关闭,SNP的上述效应又被Vc和CAT有效逆转。这些结果表明,NO和H_2O_2在生成及效应上均存在明显的相互作用。另外,L-NAME显著逆转暗和光下H_2O_2处理对气孔关闭和NO生成的效应表明,蚕豆保卫细胞中可能存在NOS,暗和光下H_2O_2处理可能通过提高NOS的活性促进NO水平增加,进而诱导气孔关闭。

Role and Relationship of Nitric Oxide and Hydrogen Peroxide in Light/Dark-regulated Stomatal Movement in Vicia faba

Role and relationship of NO and H_2O_2 in light/dark-regulated stomatal movement in Vicia faba L. were investigated by epidermal strip bioassay and laser-scanning confocal microscopy. Results showed that the effects of exogenous sodium nitroprusside (SNP, NO-releasing compound) and H_2O_2 on stomatal closure were more significant in light than those in the dark. Dark-induced closure of stomata was largely prevented not only by 2,4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a specific NO scavenger and NG-nitro-L-arg-methyl eater (L-NAME), an inhibitor of NO synthase (NOS) in mammalian cells that also inhibits plant NOS, but also by addition of ascorbic acid (Vc) and exogenous catalase (CAT), which are an important reducing substrate for H_2O_2 removal and an H_2O_2 scavenger, respectively. Experiments based on fluorescent probe DAF-2 DA and H_2DCF-DA showed that the level of endogenous NO and H_2O_2 in guard cells was greater in the dark than that in light. These results prove that light/dark regulates stomatal movement via influencing NO and H_2O_2 production. In addition, H_2O_2-induced NO production and stomatal closure in light were abolished partly by cPTIO and L-NAME. interestingly, SNP-induced H_2O_2 accumulation and stomatal closure were reversed by Vc and CAT in light. These show that NO and H_2O_2 cross talk in light/dark-regulated stomatal movement. Furthermore, L-NAME could reverse stomatal closure and NO generation induced by darkness and H_2O_2 in light, we presume that the NO generation in guard cells of Vicia faba is likely related to NOS-like enzyme.