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

Abstract

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 KD 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.

Cytoplasmic Ca2+ influx mediates iron- and reactive oxygen species-dependent ferroptotic cell death in rice immunity

“…The Ca 2+ sensor was designed as the mCherry fused to the N-terminus of GCaMP6f and consisted of two tandem fluorescent proteins ( Weigand et al., 2021 ). The protein-based Ca 2+ sensor (GCaMP6fmC) was used as an alternative tool to compare two separate cytoplasmic Ca 2+ influxes in the wild type and Ca 2+ sensor transgenic rice lines.…”

“…GCaMP6f was cloned into the plant expression vector pGWB554 using standard molecular techniques, as described previously ( Choi et al., 2014 ; Weigand et al., 2021 ). The resultant construct was transformed into rice cultivar Kitaake using Agrobacterium tumefaciens strain LBA4404.…”

“…At least three regions of interest (ROIs) were selected to quantify changes in Ca 2+ levels by calculating the GCaMP6f/mCherry ratio at different time points or at 36 hpi using the ImageJ software. Green and red fluorescence signal intensities were measured using ImageJ installed with the Macro ( Weigand et al., 2021 ). Data were exported into an Excel file, and the ratio of green to red fluorescence signal intensities was calculated.…”

Membrane Fluidization Governs the Coordinated Heat-Inducible Expression of Nucleus- and Plastid Genome-Encoded Heat Shock Protein 70 Genes in the Marine Red Alga Neopyropia yezoensis

“…It has been proposed that membranelocalized calcium ion (Ca 2+ ) channels are heat sensors in terrestrial plants [36][37][38]68]. Indeed, many lines of evidence indicate the contribution of Ca 2+ influxes in the early phase of the heat stress response in terrestrial plants [36][37][38][68][69][70]; in moss, the activation of plasma membrane-embedded cyclic nucleotide-gated Ca 2+ channels depends on membrane fluidization [36][37][38]68]. Thus, it is plausible that Ca 2+ channels might be heat sensors that become activated by the heat-induced membrane fluidization in N. yezoensis; however, little is currently known about the heat sensor and heat stress signal transduction pathways.…”

Cold-induced [Ca2+]cyt elevations function to support osmoregulation in marine diatoms

“…In other plants, such as Arabidopsis ( Arabidopsis thaliana ), support for the role of high temperatures in inducing [Ca 2+ ] cyt elevations is mixed, although Ca 2+ elevations are observed within the chloroplast ( Lenzoni and Knight, 2019 ). However, a recent study demonstrated that elevated temperature-induced [Ca 2+ ] cyt elevations in Arabidopsis leaves, but not in pollen tubes ( Weigand et al, 2021 ). Potential temperature sensors in plants include the cold-sensitive regulator of G-protein signaling (COLD1/RGA1) complex in rice ( Oryza sativa ), which is proposed to either function as a Ca 2+ channel or to activate other Ca 2+ channels ( Ma et al, 2015 ).…”