Chloroplasts, calcium and abiotic stress response: characterization of the role of cMCU and CAS in stress signaling networks

Chloroplasts have a key role in plant stress response, taking part in stress perception, signal integration and regulation of calcium, retrograde and hormonal signaling. Although many instances of chloroplast-regulated calcium signals have been described as parts of several cellular processes, the calcium-permeable channels or transporters and the calcium sensors involved are currently only partially characterized. A challenge of great importance is thus to understand how the proteins mediating the calcium signal are regulated under specific stress conditions, resulting in the transduction of specific information through calcium dynamics into adaptive effects on cellular physiology. One of the six homologs of the mitochondrial calcium uniporter (MCU) in Arabidopsis thaliana was found to be localized in the chloroplast envelope in the leaves. This chloroplast MCU (cMCU) mediates stromal calcium fluxes upon osmotic and oxidative stress response and regulates retrograde signaling. Moreover, cMCU knock-out plants have a constitutively increased stomatal closure and an enhanced drought tolerance. Here, we characterize both the impact of cMCU knock-out on the homeostasis of unstressed plants and the alterations occurring in signaling in cmcu upon drought stress. To do so, we integrated comparative proteomics with transcript analysis and quantifications of ion abundance and hormonal levels in cmcu plants. Our results on cMCU show for the first time a connection between a chloroplast calcium channel and hormone levels, since cmcu plants show a higher ABA level already in non-stressed condition. We also investigated the reasons behind this elevated ABA accumulation, discovering putatively enhanced ABA de-conjugation dynamics and a lower ABA turnover rate. Additionally, we found indications for a different regulation of chlorophyll biosynthetic enzymes and retrograde signaling mediators in cmcu plants, together with an impaired activation of osmotic stress effectors upon drought. Furthermore, we studied the interplay between cMCU and the chloroplast calcium sensor CAS. We observed at first that the lack of cMCU resulted in CAS upregulation in drought conditions, and we decided thus to employ a genetic approach and generate a cmcu cas double knock-out line. The ensuing characterization highlighted a smaller rosette area of cmcu cas plants and a reduced tolerance to hyperosmotic stress in germination assays. Moreover, stromal Ca2+ signals under osmotic stress had an increased amplitude in cas seedlings, while this phenotype was rescued in cmcu cas plants. These new data are discussed considering the proposed roles for cMCU and CAS in chloroplast calcium and retrograde signaling. Moreover, we address the hypothesis of interorganellar crosstalk between mitochondria and chloroplasts, which would play a role in stress responses and could be mediated by calcium and reactive oxygen species. To sum up, our aim in this research is to contribute to the decoding of the complex signaling network centered on chloroplasts under osmotic stress. This Ph.D. work should help better delineate the role of cMCU and CAS as players in chloroplast calcium signaling, together with their role in the complex picture of osmotic stress-responsive signaling.