Extracellular Ca²+ alleviates NaCl-induced stomatal opening through a pathway involving H₂O₂-blocked Na+ influx in Vicia guard cells.

To gain further insights into the function of extracellular Ca²+ in alleviating salt stress, Vicia faba guard cell protoplasts (GCPs) were patch-clamped in a whole-cell configuration. The results showed that 100 mM NaCl clearly induced Na+ influx across the plasma membrane in GCPs and promoted stomatal opening. Extracellular Ca²+ at 10 mM efficiently blocked Na+ influx and inhibited stomatal opening, which was partially abolished by La³+ (an inhibitor of plasma membrane Ca²+ channel) or catalase (CAT, a H₂O₂ scavenger), respectively. These results suggest that the plasma membrane Ca²+ channels and H₂O₂ possibly mediate extracellular Ca²+-blocked Na+ influx in GCPs. Furthermore, extracellular Ca²+ activated the plasma membrane Ca²+ channels under NaCl stress, which was partially abolished by CAT. These results, taken together, indicate that hydrogen peroxide (H₂O₂) likely regulates Na+ uptake by activating plasma membrane Ca²+ channels in GCPs. In accordance with this hypothesis, H₂O₂ could mimic extracellular Ca²+ to activate Ca²+ channels and block Na+ influx in guard cells. A single-cell analysis of cytosolic free Ca²+ ([Ca²+](cyt)) using Fluo 3-AM revealed that extracellular Ca²+ induced the accumulation of cytosolic Ca²+ under NaCl stress, but had few effects on the accumulation of cytosolic Ca²+ under non-NaCl conditions. All of these results, together with our previous studies showing that extracellular Ca²+ induced the generation of H₂O₂ in GCPs during NaCl stress, indicate that extracellular Ca²+ alleviates salt stress, likely by activating the H₂O₂-dependent plasma membrane Ca²+ channels, and the increase in cytosolic Ca²+ appears to block Na+ influx across the plasma membrane in Vicia guard cells, leading to stomatal closure and reduction of water loss.