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

Summary We investigated the origin and properties of spontaneous cytosolic Ca 2+ 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 Ca 2+ indicators (GECIs), we aimed to clarify how intracellular and extracellular Ca 2+ sources contribute to these dynamics. We compared GECIs with distinct biochemical properties to assess their suitability for detecting spontaneous Ca 2+ transients, testing cytosolic‐ (YC3.6, R‐GECO1, GCaMP3) and ER‐localised reporters (ER‐GCaMP6‐210). We monitored Ca 2+ dynamics in vivo under different illumination conditions and manipulated apoplastic Ca 2+ and ER Ca 2+ availability. YC3.6, R‐GECO1 and GCaMP3 reliably reported spontaneous cytosolic Ca 2+ oscillations. We further observed spontaneous Ca 2+ transients in the ER lumen. The blue excitation light required for ER‐GCaMP6‐210 induced ER Ca 2+ depletion, whereas darkness, green or red light enabled ER Ca 2+ refilling, which depended on apoplastic Ca 2+ . Our findings suggest that in guard cells, cytosolic Ca 2+ transients are primarily driven by apoplastic Ca 2+ influx, but their persistence requires proper ER Ca 2+ homeostasis. This highlights a key interplay between extracellular and intracellular Ca 2+ pools in generating and maintaining cytosolic Ca 2+ oscillations in guard cells and establishes a framework for further mechanistic dissection.