Title: Rapid hyperosmotic-induced Ca responses in Arabidopsis thaliana exhibit sensory potentiation and establish involvement of plastidial KEA transporters

Plants experience hyperosmotic stress when faced with saline soils and possibly drought stress, but it is currently unclear how plants perceive this stress in an environment of dynamic water availabilities. Hyperosmotic stress induces a rapid rise in intracellular Ca concentrations ([Ca]i) in plants, and this Ca response may reflect the activities of osmosensory components. Here, we find in the reference plant Arabidopsis thaliana that the rapid hyperosmotic-induced Ca response exhibited enhanced response magnitudes after pre-exposure to an intermediate hyperosmotic stress. We term this phenomenon “osmo-sensory potentiation”. The initial sensing and potentiation occurred in intact plants as well as in roots. Having established a quantitative understanding of WT responses, we investigated effects of pharmacological inhibitors and candidate channel/transporter mutants. Quintuple MSL channel mutants as well as double MCA channel mutants did not affect the response. However interestingly, double mutations in the plastid KEA transporters, kea1kea2, and a single mutation that does not visibly affect chloroplast structure, kea3, impaired the rapid hyperosmotic-induced Ca responses. These mutations did not significantly affect sensory potentiation of the response. These findings suggest that plastids may play an important role in the early steps mediating the response to hyperosmotic stimuli. Together, these findings demonstrate that the plant osmosensory components necessary to generate rapid osmotic-induced Ca responses remains responsive under varying osmolarities, endowing plants with the ability to perceive the dynamic intensities of water limitation imposed by osmotic stress. Significance Statement: The sensitivity ranges of biological sensors determine whenand to what extent responses to environmental stimuli are activated. Plants may perceive water limitation imposed by soil salinity or drought in the form of osmotic stress, among other mechanisms. Rapid osmotic stress-induced Ca responses provide the opportunity to quantitatively characterize the responses to osmotic stress under environmental and genetic perturbations. This report describes a phenomenon whereby prior exposure to osmotic stress increases the sensitivity of the rapid responses to subsequent stress. Further, mutations in specific plastidial transporters were found to reduce the stress response. These findings inform the reader of new avenues for understanding osmotic stress responses in plants. Page 2 peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/048330 doi: bioRxiv preprint first posted online Apr. 13, 2016;