In plants, increases in cytosolic Ca2+ concentration ([Ca2+]cyt), occurring in response to both biotic and abiotic stimuli, work as a key component of different signal transduction pathways. Depending on the stimulus, Ca2+ rises can display the form of a single transient, repetitive Ca2+ oscillations or sustained increase and are commonly designated as “Ca2+ signatures”. Generation and shaping of Ca2+ signatures depends on the fine-tuning of Ca2+ influxes and effluxes occurring at both the plasma membrane (PM) and membranes of the different subcellular compartments. In response to different stimuli, the opening of PM Ca2+-permeable influx channels (e.g. GLRs, CNGCs, OSCAs….) will release Ca2+ into the cytosol generating a [Ca2+]cyt increase, while the activity of the Ca2+ efflux transporters (e.g. H+/Ca2+ antiporters, Ca2+ ATPases…) will return the [Ca2+]cyt to the resting concentrations. Not only cytosol but also organelles and other subcellular compartments (e.g. chloroplasts, mitochondria, endoplasmic reticulum…) experience Ca2+ transients, hence putatively participating in the cellular Ca2+ homeostasis and potentially in the Ca2+ signature shaping process. To study these processes in planta the use of in vivo non-invasive state of the art imaging tools is required. In such a context the seminar will provide an overview of:
I. use of the genetically encoded ratiometric Förster Resonance Energy Transfer (FRET)-based Ca2+ sensor Cameleon in plant. Attention will be paid to present the developed Cameleon sensors for the analyses of Ca2+ dynamics in different subcellular compartments;
II. use of the presented technologies for the study of Ca2+ transporters involved in the regulation of cytosolic and organellar Ca2+ dynamics;
III. use of Light Sheet Fluorescence Microscopy (LSFM) for FRET-based Ca2+ imaging analyses in Arabidopsis root cells.