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 (Ca) signaling networks are implicated in most abiotic stress responses, heat-triggered changes in cytosolic Ca were investigated in leaves and pollen. Plants were engineered with a reporter called CGf, a ratiometric, genetically encoded Ca reporter with an mherry reference domain fused to an intensiometric Ca reporter CaMP6. Relative changes in [Ca] were estimated based on CGf's apparent around 220 nM. The ratiometric output provided an opportunity to compare Ca dynamics between different tissues, cell types, or subcellular locations. In leaves, CGf detected heat-triggered cytosolic Ca signals, comprised of three different signatures showing similarly rapid rates of Ca influx followed by differing rates of efflux (50% durations ranging from 5 to 19 min). These heat-triggered Ca 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 [Ca] oscillations to shift to a pattern characteristic of a growth arrest (22%), or an almost undetectable [Ca] (78%). Together, these contrasting examples of heat-triggered Ca responses in leaves and pollen highlight the diversity of Ca signals in plants, inviting speculations about their differing kinetic features and biological functions.