BEYOND ARABIDOPSIS: UNRAVELLING THE HIDDEN WORLD OF CA2+ SIGNALLING ACROSS SPECIES VIA MULTISCALE IMAGING TECHNOLOGIES

Confined to the site of germination, vascular plants evolved precise mechanisms to perceive and respond to environmental stimuli. Over the last decades, to in vivo investigate these processes both biological tools and imaging technologies have rapidly evolved. In particular, the use of Genetically Encoded Fluorescent Indicator (GEFI) lines has revolutionized real-time in vivo studies of second messenger dynamics in plants. This approach already revealed crucial aspects of plant biology, mainly through the use of high-end specialized commercial microscopy systems with the model species Arabidopsis thaliana (Arabidopsis). Among the various second messengers and signalling molecules, calcium ions (henceforth Ca2+) and glutamate (henceforth L-Glu) have recently reported to play a central role in long-distance signalling in Arabidopsis in response to biotic and abiotic stimuli. However, despite the power of current imaging approaches, little is known about this process in other species beyond Arabidopsis, particularly crops and crop-related species. These limitations mainly arise from the lack of GEFI lines in these species and the availability imaging platforms suitable for large-scale plant imaging. To overcome these limitations, we pursued complementary strategies: i) Generation of Solanum lycopersicum lines engineered to express different GEFIs, enabling in vivo monitoring of Ca²⁺ and other second messengers dynamics; ii) Development of the Macro Plant Projection Imaging system (MAPPI), a custom-made orthogonal, large field-of-view fluorescent imaging platform designed for shoot and root imaging in plants larger than Arabidopsis; iii) Use of a Nicotiana benthamiana (N. benthamiana) line expressing the GCaMP3 Ca2+ indicator as an alternative model to Arabidopsis for studying local and long-distance Ca2+ signalling; iv) Generation of a N. benthamiana line expressing an L-Glu indicator, to monitor in this species, both Ca2+ and L-Glu dynamics in response to external stimuli. Combining these approaches, we successfully investigated Ca²⁺ and L-Glu dynamics in N. benthamiana plants from seedling to adult developmental stages, under near-physiological conditions and in response to biotic and abiotic challenges. Moreover, the orthogonal configuration of the imaging system enabled us to examine not only leaf-to-leaf signalling, but also shoot-to-root and root-to-shoot Ca²⁺ waves triggered by stresses such as wounding, burning, flooding, and salt exposure. In conclusion, these integrated approaches establish a foundation for dissecting systemic signal propagation at the whole-plant level, across multiple species, and under a broad range of stress conditions.