Developing Novel Florescence Ratiometric Genetically Encoded Calcium Indicators In Arabidopsis

Calcium signals are core transducers and regulators in many response and developmental processes of plants.Our understanding of the underlying mechanisms of calcium signaling and its involvement in numerous cellular processes is largely dependent on the development of calcium indicators.Therefore,high spatial and temporal resolution of calcium indicator is of great significance for monitoring the dynamic changes of calcium.In this study,we further optimized the ultra-sensitive calcium indicator GCaMP6S,which has been applied in plant calcium signal research.By using UBIQUITIN10((UBQ10)as a promoter to drive expression that can be stably and highly expressed in offspring and is not susceptible to gene silencing,We constructed a series of protoplast transient expression constructs fused GCaMP6S with different subcellular localization signal peptides and constructs for generating stable transgenic lines.Using a calcium-insensitive protein,dTomato,we have developed the GCaMP6S-dTomato,a fluorescent ratiometric indicator that can quantitatively image calcium concentration change.In order to study calcium signal-in whole plant or tissues or organs,we have generated a series of ultra-sensitive calcium indicator transgenic plants.In the previous study,it is known that nitrate can rapidly induce a calcium increase in the cytoplasm of Arabidopsis cell.We have demonstrated that this ratiometric indicator GCaMP6S-dTomato has the same rapid calcium ion kinetics and sensitivity as GCaMP6S when it responds to nitrate.It can be used to mornitor calcium concentration change in a single cell or plant tissue level.More importantly,GCaMP6S-dTomato can be used for quantification calcium concentration in cells,suggesting that this optimized calcium indicator has great potential for studying plant calcium signaling.In this study,we studied the interaction between ABA and Calcium signaling using the GCaMP6S transgenic plant.ABA can cause calcium oscillations in Arabidopsis guard cells.Using optimized GCaMP6S ultra-sensitive calcium indicator,we demonstrated that ABA can cause calcium oscillations in Arabidopsis guard cells,leaf epidermal cells and mesophyll cells.Moreover,ABA induced a rapid and transient calcium concentration elevation in root cell.The discovery of these phenomena will help further reveal the deep relationship between calcium and ABA signal.In this study,we have generated a series of GCaMP6S-based quantitative and qualitative transient expression vectors and transgenic plant.This will provide a powerful tool for studying calcium signaling in plants and lay a foundation for basic research in the life sciences.