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

Chrystle Weigand,Elizabeth Brown,Jeffrey F. Harper,Emily Medina,Gad Miller,Su-Hwa Kim,Won-Gyu Choi,Moises Mares

doi:10.3389/fpls.2021.777975

Chrystle Weigand, Elizabeth Brown + Show 6 more

Open Access

https://doi.org/10.3389/fpls.2021.777975

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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.

Highlights

An important adaptive trait for many land plants is an amazing ability to sense and adapt to changing temperatures (Lamers et al, 2020; Hayes et al, 2021; Mareri et al, 2021; Heat-Triggered Ca2+ SignalsNishad and Nandi, 2021)
To determine whether the Ca2+ affinity of GCaMP6f was altered by a fusion with mCherry, both CGf and an unfused GCaMP6f reporter were separately expressed in E. coli and purified
The Hill coefficients for both CGf and GCaMP6f were in the range of 1.8–2.1 (Figure 2C), which indicates that the designed fusion did not significantly alter the expected cooperative binding of Ca2+ to the calmodulin domain

Summary

Introduction

An important adaptive trait for many land plants is an amazing ability to sense and adapt to changing temperatures (Lamers et al, 2020; Hayes et al, 2021; Mareri et al, 2021; Heat-Triggered Ca2+ SignalsNishad and Nandi, 2021). Global climate change is predicted to make periods of heat stress increasingly detrimental to plant growth and reproduction (Challinor et al, 2014; Zhao et al, 2017; Cohen et al, 2021; Zandalinas et al, 2021). The threshold at which different plants succumb to heat stress varies and can be influenced by combinatorial stresses, such as drought, light intensity, and nutrition (Lamers et al, 2020; Hayes et al, 2021; Zandalinas et al, 2021). As temperatures rise to 30–37◦C, Arabidopsis plants activate heat stress response pathways (Hayes et al, 2021). While long term exposure to temperatures around 40◦C will cause cell death, Arabidopsis can still complete its life cycle with a diurnal stress regime that includes a 1-h mid-day 40◦C heat stress, albeit with a major reduction in seed set (Tunc-Ozdemir et al, 2013)

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