Abstract
Diatoms are a group of silicified algae that play a major role in marine and freshwater ecosystems. Diatom chloroplasts were acquired by secondary endosymbiosis and exhibit important structural and functional differences from the primary plastids of land plants and green algae. Many functions of primary plastids, including photoacclimation and inorganic carbon acquisition, are regulated by calcium-dependent signaling processes. Calcium signaling has also been implicated in the photoprotective responses of diatoms; however, the nature of calcium elevations in diatom chloroplasts and their wider role in cell signaling remains unknown. Using genetically encoded calcium indicators, we find that the diatom Phaeodactylum tricornutum exhibits dynamic calcium elevations within the chloroplast stroma. Stromal calcium ([Ca2+]str) acts independently from the cytosol and is not elevated by stimuli that induce large cytosolic calcium ([Ca2+]cyt) elevations. In contrast, high light and exogenous hydrogen peroxide (H2O2) induce large, sustained [Ca2+]str elevations that are not replicated in the cytosol. Measurements using the fluorescent H2O2 sensor roGFP2-Oxidant Receptor Peroxidase 1 (Orp1) indicate that [Ca2+]str elevations induced by these stimuli correspond to the accumulation of H2O2 in the chloroplast. [Ca2+]str elevations were also induced by adding methyl viologen, which generates superoxide within the chloroplast, and by treatments that disrupt nonphotochemical quenching (NPQ). The findings indicate that diatoms generate specific [Ca2+]str elevations in response to high light and oxidative stress that likely modulate the activity of calcium-sensitive components in photoprotection and other regulatory pathways.