Integration of plastids in the plant calcium signalling network in response to environmental stimuli.

Transient elevations in intracellular free Ca2+ concentration are known to mediate plant responses to a wide variety of abiotic and biotic stimuli. To elucidate the contribution of plastids to Ca2+ homeostasis and signalling in the plant cell, we generated Arabidopsis thaliana lines stably expressing the bioluminescent Ca2+ reporter aequorin targeted to different chloroplast subcompartments, i.e. the outer envelope, the stroma, the thylakoid membrane and the thylakoid lumen. Ca2+ measurements in response to environmental stimuli were carried out in entire seedlings, as well as in cell suspension cultures, obtained from in vitro dedifferentiation of Arabidopsis explants. Monitoring of Ca2+ dynamics allowed to dissect stimulus-specific Ca2+ signals in amyloplasts and chloroplasts, as well as the occurrence of dark-induced intraplastidial Ca2+ fluxes, characterized by unique kinetic parameters. To get insights into the homeostatic mechanisms allowing for chloroplast Ca2+ fluxes, a mutant line lacking a putative plastidial Ca2+-permeable transporter was transformed with the construct encoding the stroma-targeted aequorin chimera. The obtained results shed new light on the complex network underlying the Ca2+-mediated transduction of environmental stimuli in plants at an intracellular level. This complex kit of chloroplast-targeted aequorin probes turned out to be a valuable tool for the monitoring of organellar Ca2+ dynamics during signal transduction. In the last part of the work the ability of Chlamydomonas reinhardtii, a unicellular freshwater/soil green alga, to evoke intracellular Ca2+ changes ([Ca2+]) in response to different environmental stimuli was considered. As there is no evidence in the literature about environmental stimuli that trigger cytosolic [Ca2+] variations in Chlamydomonas, the first step had necessarily to deal with the identification of environmental cues evoking cytosolic [Ca2+] elevations in the microalga. Chlamydomonas cells, loaded with the Ca2+-responsive fluorescent dye Oregon Green-BAPTA, were challenged with stimuli previously used in this work on Arabidopsis. Moreover, the alga was also challenged with a hypoosmotic shock, that resulted to trigger cytosolic [Ca2+] elevations, and the nature of the observed dynamics was investigated. Taken together, the obtained results indicate that some aspects of Ca2+ signalling do not appear to be conserved between the green alga Chlamydomonas and the higher plant Arabidopsis, but other elements in Ca2+-mediated responses to osmotic shocks are similar. These differences may be due to the different Ca2+ signalling toolkits found in plants and algae and/or to the different physiology of these model organisms, both belonging to the Viridiplantae clade. Investigation on the Ca2+ handling mechanisms operating in different photosynthetic organisms of phylogenetic interest may shed new light on the evolutionary history of the Ca2+ signalling toolkit