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.