Investigating the origin of spontaneous cytosolic calcium oscillations in stomatal guard cells: a dialogue between apoplastic and endoplasmic reticulum stores

We investigated the origin and properties of spontaneous cytosolic Ca²⁺ oscillations in Arabidopsis thaliana and Solanum lycopersicum guard cells. Although these oscillations have been described since the 1990s, their underlying mechanisms remain unresolved. Using genetically encoded Ca²⁺ indicators (GECIs), we aimed to clarify how intracellular and extracellular Ca²⁺ sources contribute to these dynamics. We compared GECIs with distinct biochemical properties to assess their suitability for detecting spontaneous Ca²⁺ transients, testing cytosolic- (YC3.6, R-GECO1, GCaMP3) and ER-localised reporters (ER-GCaMP6-210). We monitored Ca²⁺ dynamics in vivo under different illumination conditions and manipulated apoplastic Ca²⁺ and ER Ca²⁺ availability. YC3.6, R-GECO1 and GCaMP3 reliably reported spontaneous cytosolic Ca²⁺ oscillations. We further observed spontaneous Ca²⁺ transients in the ER lumen. The blue excitation light required for ER-GCaMP6-210 induced ER Ca²⁺ depletion, whereas darkness, green or red light enabled ER Ca²⁺ refilling, which depended on apoplastic Ca²⁺. Our findings suggest that in guard cells, cytosolic Ca²⁺ transients are primarily driven by apoplastic Ca²⁺ influx, but their persistence requires proper ER Ca²⁺ homeostasis. This highlights a key interplay between extracellular and intracellular Ca²⁺ pools in generating and maintaining cytosolic Ca²⁺ oscillations in guard cells and establishes a framework for further mechanistic dissection.