Crosstalk of Nitric Oxide with Ca2+ in Stomatal Movement in Vicia faba Guard Cells

For gaining further insights into Ca2+ function in nitric oxide (NO)-regulated stomatal movement, Vicia faba guard cell protoplasts were patch-clamped in a whole-cell configuration. In the absence of extracellular Ca2+, sodium nitroprusside (SNP, the NO donor) inhibited inward rectifying K+ current at a concentration of 10 or 100 μmol L−1, but had little effect on outward rectifying K+ current. SNP significantly activated outward rectifying K+ current when CaCl2 (0.1 mmol L−1) was added to the bath solution, and LaCl3 could alleviate this effect of NO. In contrast, 0.1 mmol L−1 CaCl2 alone had little effect on inward or outward rectifying K+ current. Extracellular Ca2+ significantly inhibited inward rectifying K+ current and activated outward rectifying K+ current at the concentration of 10 mmol L−1, and this effect was not alleviated by 2-(4-carboxyphenyl)-4,4,5,5 tetramethylimidazoline (c-PTIO). When monitoring Ca2+ and NO levels in single cells by Fluo 3-AM and 4,5-diaminofluorescein diacetate (DAF-2DA), the specific fluorescence probes for Ca2+ and NO, respectively, SNP at 100 μmol L−1 evidently induced the accumulation of Ca2+ in guard cells, and the induction could be partially alleviated by LaCl3. However, CaCl2 at 0.1 mmol L−1 or 10 mmol L−1 had small effect on the accumulation of NO in the guard cells. These results indicated that NO promoted influx of Ca2+ into cytoplasm through Ca2+ channels to activate outward rectifying K+ channels and promotes K+ efflux. Alternatively, NO inhibited inward rectifying K+ channels and blocks K+ influx, thus inhibiting stomatal opening and preventing excessive loss of water in plants. In addition, extracellular Ca2+ at a concentration of 10 mmol L−1 modulated stomatal movement and plasma membrane K+ channels of V. faba guard cells in a NO-independent signaling pathway.