ACA12 IS A DEREGULATED ISOFORM OF PLASMA MEMBRANE CA2+-ATPASE OF ARABIDOPSIS THALIANA

Several environmental and endogenous stimuli activate plant signal transduction pathways through transient increases in cytosolic free calcium concentrations ([Ca2+]cyt). Strong evidence indicates that plant response specificity is triggered by the amplitude, shape, frequency and localization of these stimulus-induced Ca2+ oscillations. Due to their role in Ca2+ efflux from the cytosol, plant auto-inhibited Ca2+-ATPases (ACAs) are involved in restoring the cytosolic basal concentration of Ca2+ after its rise due to stimulus perception, therefore affecting the specificity of plant response to different stimuli. Ten ACA isoforms are present in Arabidopsis thaliana, which are divided into four clusters based on gene structure and sequence homology. Cluster I, II and IV ACAs share a common mechanism of regulation based on a N-terminal auto-inhibitory domain whose action is suppressed by the interaction with the Ca2+-sensor protein calmodulin (CaM). Cluster III isoforms (ACA12 and ACA13) compared to the other ACAs, are almost unknown and show unique features at sequence and expression levels: in particular ACA12 and ACA13 show divergences in residues involved in CaM binding and regulation while the expression levels, normally very low, are increased upon exposure to pathogens or UV stresses. By confocal microscopy, a GFP-tagged ACA12 (expressed in Arabidopsis plants) was localized at the plasma membrane (PM). ACA12 was then expressed in Saccharomyces cerevisiae strain K616, which lacks endogenous Ca2+ pumps. Our results reveal that ACA12 allows the grow of K616 in a Ca2+-depleted medium, therefore indicating that it is a functional and deregulated Ca2+ transporter. Yeast-expressed ACA12 was then purified by CaM-affinity chromatography and its activity was tested in vitro in order to study its biochemical properties. Biochemical assays results show that ACA12 is a functional Ca2+-ATPase. Moreover, as expected, its activity is not regulated by CaM. Finally, in order to try to explain ACA12 deregulated behaviour, single point mutants of ACA12 were generated and tested for K616 phenotype complementation. Taken together our results show that ACA12 has unique biochemical features suggesting for it a distinctive physiological role within the ACA family. In particular ACA12 may be involved in Ca2+-dependent signaling pathways in response to plant specific stresses such as pathogens and UV