NO与Ca2+对蚕豆保卫细胞气孔运动的互作调控

以蚕豆(Vicia faba L.)为材料研究NO和Ca²⁺对蚕豆气孔运动及质膜K⁺通道的影响。结果表明，10 mmol L^-1 Ca²⁺和100 µmol L^-1 NO供体SNP均有效抑制气孔开放，NO清除剂c-PTIO不能缓解Ca²⁺抑制气孔开放，相反胞外加入0.1 mmol L^-1 Ca²⁺可以明显加强NO对气孔开放的抑制程度，该现象可被La³⁺(Ca²⁺通道抑制剂)缓解。以膜片钳技术记录全细胞K⁺电流发现，胞外10 µmol L^-1或100 µmol L^-1 SNP均可选择性抑制蚕豆保卫细胞质膜内向K⁺通道，追加0.1 mmol L^-1 Ca²⁺可显著激活质膜外向K⁺通道，且可被La³⁺所缓解，然而0.1 mmol L^-1 Ca²⁺单独作用并不影响质膜外向K⁺通道活性。10 mmol L^-1 Ca²⁺单独处理可激活质膜外向K⁺通道，但不能被c-PTIO缓解。分别用Ca²⁺和NO专一的荧光探针Fluo-3-AM和DAF-2DA标记蚕豆保卫细胞原生质体，检测胞内Ca²⁺和NO的水平变化发现，100 µmol L^-1 SNP明显诱导胞内Ca²⁺积累，但10 mmol L^-1 Ca²⁺并不能诱导NO在细胞内积累。记录保卫细胞质膜Ca²⁺通道电流发现，NO可明显激活质膜Ca²⁺通道。表明NO有效抑制气孔开放，可能主要通过激活质膜Ca²⁺通道，提高胞内Ca²⁺，激活质膜外向K⁺通道促进K⁺外流，同时，可选择性抑制内向K⁺通道阻止K⁺内流，两种途径共同作用抑制气孔开放。然而，胞外10 mmol L^-1 Ca²⁺对气孔和质膜K⁺通道活性的调节并不依赖于NO。

Crosstalk of NO with Ca2+in Stomatal Movement in Vicia faba Guard Cells

Previous studies suggested that both NO and Ca²⁺ can serve as a signalling intermediate in ABA, H₂O₂-induced stomatal movement. However, Its mechanism(s) of action is not well defined in guard cells and, generally, in higher plants. In this study, extracellular 10 mmol L^-1 Ca^{2+ significantly inhibited stomatal opening, which was not alleviated by carboxy PTIO (c-PTIO, a NO scavenger). Sodium nitroprusside (SNP, a NO donor) showed effects of inhibition on stomatal opening at concentration of 10 or 100} µmol L^-1. However, 0.1 mmol L^-1Ca^{2+facilitated NO-inhibited stomatal opening, which was alleviated by LaCl₃ (a Ca2+channel inhibitor) at concentration of 1 mmol L-1. To gain further insights into Ca2+ function in NO-regulated stomatal movement, we patch-clamped Vicia faba guard cell protoplasts in a whole-cell configuration. In the absence of extracellular Ca2+NO inhibited inward rectifying K+ current at concentration of 10 or 100 ,} µmol L^-1, but have little effects on outward rectifying K⁺ current. NO significantly activated outward rectifying K⁺ current, when CaCl₂ was added to the bath solution, at concentration of 0.1 mmol L^-1, which was alleviated by LaCl₃. In contrast, 0.1 mmol L^-1 CaCl₂ alone had little effects on inward or outward rectifying K⁺ current. Extracellular Ca^{2+significantly inhibited inward rectifying K+ current and activated outward rectifying K+ current at concentration of 10 mmol L-1, which was not alleviated by c-PTIO. A single-cell analysis of cytosolic Ca2+ and NO using Ca2+specific fluorescence probe Fluo-3-AM and DAF-2DA revealed that 100 or NO} µmol L^-1 SNP evidently induced accumulation of Ca²⁺ in the guard cells，which was partially alleviated by LaCl₃, but 0.1 or 10 mmol L^-1 CaCl₂ had few effects on the accumulation of NO in the guard cells. These results indicated that NO promotes influx of Ca²⁺ into cytoplasm through Ca²⁺ channels to activate outward rectifying K⁺ channels and promotes K⁺ eflux, alternatively, NO inhibits inward rectifying K⁺ channels and blocks K⁺ influx, thus inhibiting stomatal opening and preventing the excessive loss of water in plants. In addition, extracellular Ca²⁺ at concentration of 10 mmol L^-1 modulatesstomatal movement and plasma membrane K⁺ channels of Vicia guard cells in a NO-independent signaling pathway.