Intraorganellar calcium imaging in Arabidopsis seedling roots using the GCaMP variants GCaMP6m and R-CEPIA1er.

Ca acts as a universal second messenger in eukaryotes. In animals, a wide variety of environmental and developmental stimuli trigger Ca dynamics in organelles, such as the cytoplasm, nucleus, and endoplasmic reticulum (ER). However, ER Ca ([Ca]) homeostasis and its contributions in cytosolic and/or nucleosolic Ca dynamics in plants remain elusive. GCaMPs are comprised of a circularly permutated form of enhanced green fluorescent protein fused to calmodulin and myosin light-chain kinase M13 and used for monitoring Ca dynamics in mammalian cells. Here, we targeted a high-affinity variant of GCaMP with nuclear export signal in the cytoplasm (NES-GCaMP6m), with a nuclear-localised signal in the nucleus (NLS-GCaMP6m), and a low-affinity variant of GCaMP, also known as calcium-measuring organelle-entrapped protein indicators (CEPIA), with a signal peptide sequence of the ER-localised protein Calreticulin 1a in the ER lumen (CRT1a-R-CEPIA1er) for intraorganellar Ca imaging in Arabidopsis. We found that cytosolic Ca ([Ca]) increases induced by 250 mM sorbitol as an osmotic stress stimulus, 50 μM abscisic acid (ABA), or 1 mM carbachol (CCh) were mainly due to extracellular Ca influx, whereas nucleosolic Ca ([Ca]) increases triggered by osmotic stress, ABA, or CCh were contributed by [Ca] release. In addition, [Ca] dynamics presented specific patterns in response to different stimuli such as osmotic stress, ABA, or CCh, indicating that Ca signalling occurs in the ER in plants. These results provide valuable insights into subcellular Ca dynamics in response to different stresses in Arabidopsis root cells and prove that GCaMP imaging is a useful tool for furthering our understanding of plant organelle functions.