摘要
以蚕豆(Viciafaba 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,10mmolL-1Ca2+和100μmolL-1NO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0.1mmolL-1Ca2+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10μmolL-1或100μmolL-1SNP均可选择性抑制蚕豆保卫细胞质膜内向K+通道,追加0.1mmolL-1Ca2+可显著激活质膜外向K+通道,且可被La3+所缓解,然而0.1mmolL-1Ca2+单独作用并不影响质膜外向K+通道活性。10mmolL-1Ca2+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,100μmolL-1SNP明显诱导胞内Ca2+积累,但10mmolL-1Ca2+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10mmolL-1Ca2+对气孔和质膜K+通道活性的调节并不依赖于NO。
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 Ca2+ 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-1Ca2+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 Ca2+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.
出处
《作物学报》
CAS
CSCD
北大核心
2009年第8期1491-1499,共9页
Acta Agronomica Sinica
基金
国家自然科学基金项目(30871300)资助
关键词
钙离子
一氧化氮
保卫细胞
质膜K+通道
信号转导
Calcium Nitric oxide Guard cell Plasma membrane K+ channels Signal transduction