A Ratiometric Calcium Reporter CGf Reveals Calcium Dynamics Both in the Single Cell and Whole Plant Levels Under Heat Stress

Land plants evolved to quickly sense and adapt to temperature changes, such as hot days and cold nights. Given that calcium (Ca²⁺) signaling networks are implicated in most abiotic stress responses, heat-triggered changes in cytosolic Ca²⁺ were investigated in Arabidopsis leaves and pollen. Plants were engineered with a reporter called CGf, a ratiometric, genetically encoded Ca²⁺ reporter with an mCherry reference domain fused to an intensiometric Ca²⁺ reporter GCaMP6f. Relative changes in [Ca²⁺]_cyt were estimated based on CGf’s apparent K_D around 220 nM. The ratiometric output provided an opportunity to compare Ca²⁺ dynamics between different tissues, cell types, or subcellular locations. In leaves, CGf detected heat-triggered cytosolic Ca²⁺ signals, comprised of three different signatures showing similarly rapid rates of Ca²⁺ influx followed by differing rates of efflux (50% durations ranging from 5 to 19 min). These heat-triggered Ca²⁺ signals were approximately 1.5-fold greater in magnitude than blue light-triggered signals in the same leaves. In contrast, growing pollen tubes showed two different heat-triggered responses. Exposure to heat caused tip-focused steady growth [Ca²⁺]_cyt oscillations to shift to a pattern characteristic of a growth arrest (22%), or an almost undetectable [Ca²⁺]_cyt (78%). Together, these contrasting examples of heat-triggered Ca²⁺ responses in leaves and pollen highlight the diversity of Ca²⁺ signals in plants, inviting speculations about their differing kinetic features and biological functions.