The T-REX eyes on the vacuolar Ca2+ fluxes in Arabidopsis

DOCCULA, FABRIZIO GANDOLFO; LUONI, LAURA; COSTA, ALEX; XU, ZHAO LONG; BONZA, CRISTINA; ROMANO ARMADA, NELI; BASSI, ANDREA

Florencia

Congreso; Society for Experimental Biology Annual Metting, Masters of Biology; 2018

Society for Experimental Biology

In plant cells, both biotic and abiotic stresses trigger cytosolic Ca2+ concentration increases. Based on the type of stimulus, the Ca2+ elevation can display the form of single transients or repetitive oscillations. The generation of the cytosolic Ca2+ increases is mainly dependent on the entry of Ca2+ from the apoplast. Pharmacological and genetic evidence support also a role played by the internal stores. The fact that the vacuole is the main intracellular Ca2+ store makes it the principal suspect to contribute to the generation of the cytosolic Ca2+ elevations. Ca2+ dyes and genetically encoded Ca2+ sensors allowed the study of Ca2+ dynamics in the cytosol and several sub-cellular compartments, but unfortunately, the measurements of luminal vacuolar Ca2+ dynamics are missing due to its very acidic luminal pH which limits the use of GFP-based Ca2+ sensors. To overcome this limitation, we here report the generation of a Cameleon YC3.6 Ca2+ sensor targeted to the cytosolic face of the tonoplast membrane in the model species Arabidopsis thaliana. The new sensor was called T-REX which stands for Tonoplast-localized Ratiometric sEnsor for calcium fluXes detection. T-REX thus reports the Ca2+ levels at tonoplast microdomains, allowing to appreciate the contribution of luminal Ca2+ in the generation of cytosolic Ca2+ increases. Comparison of tonoplast and cytosolic Ca2+ dynamics reveals clear differences in response to different stimuli in root tip cells. Besides, such differences were particularly evident either in guard cells subjected to external Ca2+, a stimulus predicted to induce a Ca2+-induced Ca2+ release process.