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.