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

Abstract

Ca2+ acts as a universal second messenger in eukaryotes. In animals, a wide variety of environmental and developmental stimuli trigger Ca2+ dynamics in organelles, such as the cytoplasm, nucleus, and endoplasmic reticulum (ER). However, ER Ca2+ ([Ca2+]er) homeostasis and its contributions in cytosolic and/or nucleosolic Ca2+ 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 Ca2+ 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 Ca2+ imaging in Arabidopsis. We found that cytosolic Ca2+ ([Ca2+]cyt) 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 Ca2+ influx, whereas nucleosolic Ca2+ ([Ca2+]nuc) increases triggered by osmotic stress, ABA, or CCh were contributed by [Ca2+]er release. In addition, [Ca2+]er dynamics presented specific patterns in response to different stimuli such as osmotic stress, ABA, or CCh, indicating that Ca2+ signalling occurs in the ER in plants. These results provide valuable insights into subcellular Ca2+ 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.