Dual Color Sensors for Simultaneous Analysis of Calcium Signal Dynamics in the Nuclear and Cytoplasmic Compartments of Plant Cells

Spatiotemporal changes in cellular calcium (Ca²⁺) concentrations are essential for signal transduction in a wide range of plant cellular processes. In legumes, nuclear and perinuclear-localized Ca²⁺ oscillations have emerged as key signatures preceding downstream symbiotic signaling responses. Förster resonance energy transfer (FRET) yellow-based Ca²⁺ cameleon probes have been successfully exploited to measure the spatiotemporal dynamics of symbiotic Ca²⁺ signaling in legumes. Although providing cellular resolution, these sensors were restricted to measuring Ca²⁺ changes in single subcellular compartments. In this study, we have explored the potential of single fluorescent protein-based Ca²⁺ sensors, the GECOs, for multicolor and simultaneous imaging of the spatiotemporal dynamics of cytoplasmic and nuclear Ca²⁺ signaling in root cells. Single and dual fluorescence nuclear and cytoplasmic-localized GECOs expressed in transgenic Medicago truncatula roots and Arabidopsis thaliana were used to successfully monitor Ca²⁺ responses to microbial biotic and abiotic elicitors. In M. truncatula, we demonstrate that GECOs detect symbiosis-related Ca²⁺ spiking variations with higher sensitivity than the yellow FRET-based sensors previously used. Additionally, in both M. truncatula and A. thaliana, the dual sensor is now able to resolve in a single root cell the coordinated spatiotemporal dynamics of nuclear and cytoplasmic Ca²⁺ signaling in vivo. The GECO-based sensors presented here therefore represent powerful tools to monitor Ca²⁺ signaling dynamics in vivo in response to different stimuli in multi-subcellular compartments of plant cells.