Guard cells count the number of unitary cytosolic Ca²⁺ signals to regulate stomatal dynamics

Transient stimulus-specific increases in the cytosolic Ca²⁺ concentration (“calcium signatures”) of guard cells have been proposed to regulate the opening and closure of stomatal pores on plant leaves. However, the mechanism by which these Ca²⁺ signatures are generated and translated into stomatal movement is still largely unresolved. We used a light-gated, Ca²⁺-permeable variant of ChannelRhodopsin 2 (ChR2-XXM2.0) that was stimulated by tailored light pulses to investigate this phenomenon. We found that activation of the ChR2-XXM2.0 channel provoked characteristic increases in the cytosolic concentration of Ca²⁺. We also demonstrated that the endoplasmic reticulum (ER) was involved in the generation of these calcium signatures. Using ChR2-XXM2.0 technology, we showed that transient increases in Ca²⁺ activated S-type anion channels and determined the extent and speed of stomatal closure with their number and frequency. Our data reveal that guard cells are capable of counting Ca²⁺ transients in order to optimize stomatal aperture in the prevailing environmental conditions.