Simultaneous imaging of ER and cytosolic Ca2+ dynamics reveals long-distance ER Ca2+ waves in plants

Abstract

Calcium ions (Ca2+) play a key role in cell signaling across organisms. In plants, a plethora of environmental and developmental stimuli induce specific Ca2+ increases in the cytosol as well as in different cellular compartments including the endoplasmic reticulum (ER). The ER represents an intracellular Ca2+ store that actively accumulates Ca2+ taken up from the cytosol. By exploiting state-of-the-art genetically encoded Ca2+ indicators (GECIs), specifically the ER-GCaMP6-210 and R-GECO1, we report the generation and characterization of an Arabidopsis (Arabidopsis thaliana) line that allows for simultaneous imaging of Ca2+ dynamics in both the ER and cytosol at different spatial scales. By performing analyses in single cells, we precisely quantified i) the time required by the ER to import Ca2+ from the cytosol into the lumen; and ii) the time required to observe a cytosolic Ca2+ increase upon the pharmacological inhibition of the ER-localized type IIA Ca2+-ATPases. Furthermore, live imaging of mature, soil-grown plants revealed the existence of a wounding-induced, long-distance ER Ca2+ wave propagating in injured and systemic rosette leaves. This technology enhances high-resolution analyses of intracellular Ca2+ dynamics at the cellular level and in adult organisms and paves the way to develop new methodologies aimed at defining the contribution of subcellular compartments in Ca2+ homeostasis and signaling.

CamelliA-based simultaneous imaging of Ca2+ dynamics in subcellular compartments

“…The mock treatment, however, did not induce any detectable changes in the Ca 2+ levels in the above four compartments ( Supplemental Figure S18 ). Altogether, these observations are in agreement with salt-induced changes in Ca 2+ in different subcellular compartments reported in previous studies ( Knight et al, 1997 ; Bonza et al, 2013 ; Corso et al, 2018 ; Kelner et al, 2018 ), but differ in that the simultaneous imaging of Ca 2+ in multiple subcellular compartments from the same cell, enabling accurate analysis of the spatiotemporal correlation of the Ca 2+ dynamics across two ( Resentini et al, 2021 ) or four subcellular compartments with our toolset.…”

“…We demonstrated multiplexed imaging of the Ca 2+ dynamics in the cytosol, the nuclei, the sub-PM region, and the ER in several types of cells ( Figures 5–7 ). Although simultaneous imaging of nuclear and cytosolic Ca 2+ dynamics in plant cells has been reported ( Kelner et al, 2018 ; Luo et al, 2020 ; Resentini et al, 2021 ), our toolset has greatly expanded the choices of GECIs with different colors and targeted various compartments.…”

“…Importantly, the diverse spectral properties render them ideal for imaging Ca 2+ dynamics in multiple subcellular compartments ( Whitaker, 2010 ; Keinath et al, 2015 ). Single-colored GECIs have been successfully applied to imaging Ca 2+ dynamics in plant cells ( Costa and Kudla, 2015 ; Keinath et al, 2015 ; Waadt et al, 2017 ; Walia et al, 2018 ; De Vriese et al, 2019 ; Marhavy et al, 2019) , but have only been used for simultaneous Ca 2+ imaging in two subcellular compartments ( Kelner et al, 2018 ; Luo et al, 2020 ; Resentini et al, 2021 ).…”

Auxin analog-induced Ca2+ signaling is independent of inhibition of endosomal aggregation in Arabidopsis roots

“…responses in all tissues of the root meristem, possibly involving complex signal propagation and amplification mechanisms based on plasmodesmata and electrical signaling (Fichman et al, 2021;Nguyen et al, 2018;Resentini et al, 2021a). Together, these data demonstrate that 1-NAA-induced Ca 2+ signaling depends on TIR1/AFB and CNGC14 activities.…”

“…This discrepancy between the expression domain and the observed effects suggests non-cell autonomous coordination of auxin-induced Ca 2+ signaling in the root meristem. Such systemic Ca 2+ responses are emerging as a prevalent theme in plant Ca signaling, in which different Ca 2+ channel types, in conjunction with electrical signals, reactive oxygen species and plasmodesmata, are involved in initiation and propagation of intercellular Ca 2+ signaling (Choi et al, 2014;Fichman and Mittler, 2020;Fichman et al, 2021;Resentini et al, 2021a;Toyota et al, 2018). Intercellular coordination of auxin-induced Ca 2+ via similar mechanism can be expected, given the requirement of coordination of growth of different tissues during gravitropic bending.…”