Calcium connections: endoplasmic reticulum Ca 2+ homeostasis drives spontaneous Ca 2+ oscillations in guard cells

This study exemplifies the benefits of simultaneous subcellular Ca2+ imaging in multiple locations to further enhance our understanding of Ca2+ oscillations and Ca2+ signalling mechanisms. Cellular Ca2+ homeostasis involves interacting processes across the whole cell. The generation of Ca2+cyt oscillations often requires a source of Ca2+ from the apoplast as well as Ca2+ uptake and release from intracellular stores, notably the ER and vacuole (Brownlee Guo et al., 2022). Grenzi et al. therefore made use of Arabidopsis lines expressing two genetically encoded fluorescent Ca2+ indicators to simultaneously quantify Ca2+cyt and ER lumen Ca2+ (Ca2+ER) dynamics in the same guard cell. To achieve high sensitivity of Ca2+ imaging and differential excitation in both subcellular locations, a cytosolic localised, red-shifted R-GECO1, which is excited by green light and an ER localised, green-shifted ER-GCaMP6-210 excited by blue light, were used. This allowed the detection of spontaneous Ca2+ER oscillations alongside the spontaneous Ca2+cyt oscillations. However, the interplay between the two compartments was complex: in some cases, there was a delayed increase in Ca2+ER following a Ca2+cyt transient; sometimes, the Ca2+ increases in both the cytosol and ER were simultaneous; and in other cases, Ca2+ER decreased following a Ca2+cyt increase (Grenzi et al.). Further analysis was required to determine whether the observed Ca2+ER changes were simply a by-product of the spontaneous Ca2+cyt oscillations or an essential component. Despite the complexity of the Ca2+ER patterns, on average, it was noticeable that the ER Ca2+ indicator fluorescence decreased over time, suggesting ER Ca2+ depletion, which typically coincided with an increase in cytosolic Ca2+. This is also consistent with a Ca2+-induced Ca2+ release model of Ca2+cyt oscillation generation (Xiong et al., 2025). However, a challenge with the use of the ER-GCaMP6-210 Ca2+ reporter is that it requires blue light excitation, yet blue light perception, especially through the action of phototropin (PHOT1 and PHOT2) blue light receptors, is known to control an array of processes in guard cells, including pathways thought to trigger Ca2+ release from internal stores (Kostaki et al., 2020). Grenzi et al. therefore repeated imaging experiments under different light and dark conditions. It was confirmed that blue light induces the release of Ca2+ from the ER, while a period of darkness allows refilling of the ER Ca2+ pool. Use of the phot1/phot2 mutant line indicated that PHOT1 and/or PHOT2 are partly responsible for ER Ca2+ depletion, and seemingly in a kinase-dependent manner (Fig. 1). It is unclear whether the blue light induction of Ca2+ release is acting via inhibition of ER Ca2+ refilling, such as by inhibiting an ER Ca2+ pump, or activation of an unknown ER localised Ca2+ channel. Pathways for the transport of Ca2+ into the ER are well characterised and involve two classes of Ca2+ pump, the autoinhibited Ca2+-ATPase (ACA) and the ER-type Ca2+-ATPase (ECA), which both have isoforms present at the ER membrane in Arabidopsis (Brownlee therefore, it will be interesting to determine whether ECA mutant plants possess distinct guard cell Ca2+ phenotypes. The work by Grenzi et al. follows on from other recent studies that have further demonstrated the importance of the ER in plant cells as a source of Ca2+ for the generation of Ca2+cyt elevations (Resentini et al., 2021a; Huang et al., 2024). The ER is a key component in the formation of Ca2+ oscillations in animal cells, with tightly coupled regulation between this intracellular Ca2+ source and the extracellular Ca2+ pool, and well characterised ER Ca2+ release pathways, including inositol triphosphate (IP3) induced Ca2+ release (Xiong et al., 2025). While there are many differences in the evolution and composition of a Ca2+ signalling toolkit between plants and animals (Edel et al., 2017; Brownlee & Wheeler, 2025), the importance of ER homeostasis is conserved but with some key differences in molecular components. For example, both IP3 receptors and ryanodine receptors are absent in higher plants. A major deficiency in our understanding of plant ER Ca2+ homeostasis therefore is the lack of molecular identity of a Ca2+ release channel. Further investigation of blue light induced ER Ca2+ release is a promising route to uncover the identity of an ER Ca2+ permeable channel protein. This study exemplifies the benefits of simultaneous subcellular Ca2+ imaging in multiple locations to further enhance our understanding of Ca2+ oscillations and Ca2+ signalling mechanisms. The continued development of high sensitivity Ca2+ reporters targeted to different organelles and subcellular locations, coupled with the use of mutant lines of candidate genes, will allow many unanswered questions to be addressed, not just in model systems like the stomatal guard cells, but in other cell types and across different plant species. These include elucidating potential crosstalk in signalling and Ca2+ transfer between different subcellular compartments. There is also potential for the continued use of biosensor tools to correlate and compare the dynamics of Ca2+ with other signalling molecules, including reactive oxygen species, pH, hormones, and more, both within cells and across multiple cells at whole tissue and whole plant levels (Rowe et al., 2025). The New Phytologist Foundation remains neutral with regard to jurisdictional claims in maps and in any institutional affiliations.