Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Xiaohong Zhu; Aaron Taylor; Shenyu Zhang; Dayong Zhang; Ying Feng; Gaimei Liang; Jian-Kang Zhu

doi:10.3791/51945

A subscription to JoVE is required to view this content. Sign in or start your free trial.

Summary

Ca²⁺ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca²⁺ signals in Arabidopsis cells and tissues using the genetically encoded Ca²⁺ indicators Aequorin or Case12.

Abstract

Developmental and environmental cues induce Ca²⁺ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca²⁺ patterns are sensed by cellular Ca²⁺ binding proteins that initiate Ca²⁺ signaling cascades. However, we still know little about how stimulus specific Ca²⁺ signals are generated. The specificity of a Ca²⁺ signal may be attributed to the sophisticated regulation of the activities of Ca²⁺ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca²⁺ signals at both the tissue and cellular levels. Genetically encoded Ca²⁺ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca²⁺ signals. Here we describe instructions for the use of two Ca²⁺ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca²⁺ imaging and case12 based live cell confocal fluorescence Ca²⁺ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca²⁺ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca²⁺ signals at a high resolution.

Introduction

The plant cell responds to the environment via signaling that coordinates cell actions. An early cell signaling event in response to environmental stimuli is a transient Ca²⁺ increase. The pattern, or signature of a transient increase in free Ca²⁺ concentration is characterized by its amplitude, frequency, and duration. Distinct spatio-temporal Ca²⁺ signatures regulate different cellular activities¹. Specific stimuli, such as heat, cold, salt, drought, light, or plant hormones, may fine-tune the spatio-temporal activity of membrane-localized Ca²⁺ channels and/or transporters, resulting in specific Ca²⁺signatures. Although Ca²⁺ transporters have been well characterized, little is known about the molecular identities and functions of Ca²⁺ channels in plants¹. Genetic screens for mutants with altered Ca²⁺ response to stress stimuli may be an effective approach for identifying the components that compose Ca²⁺ signatures. Recently several Aequorin based Ca²⁺ detection systems have been developed that facilitate genetic screens for Ca²⁺ signaling components in response to pathogen attack and abiotic stress^2-4.

Aequorin was first used to detect Ca²⁺ signals in plants in the early 1990s⁵. Since then, Aequorin has been targeted to different cellular compartments, such as the cytoplasm⁵, nucleus⁶, chloroplasts⁷, tonoplast⁸, mitochondria⁹, and stroma¹⁰, as well as to different cell types in the root to monitor cell specific Ca²⁺ signals¹¹. Aequorin based Ca²⁺ measurements reveal the spatial and temporal Ca²⁺ response of a population of cells to stress stimuli. However, in most cases, the Ca²⁺ responses of single cells are unsynchronized in the responding tissue⁴. Therefore, Aequorin Ca²⁺ recording does not necessarily report the Ca²⁺ signal in individual cells. In recent years, genetically encoded fluorescent protein (FP)-based Ca²⁺ indicators, such as yellow cameleon (YCs)¹² and CASEs12¹³ have been used to study Ca²⁺ signaling with high subcellular resolution. YCs are fluorescence resonance energy transfer (FRET)-based Ca²⁺ indicators, containing CFP and YFP variants linked by the Ca²⁺-binding protein calmodulin and calmodulin-binding peptide M13. Calmodulin undergoes a conformational change as it binds to Ca²⁺, thereby brings CFP and YFP closer together, resulting in increased energy transfer (enhanced FRET). The FRET level over time, calculated roughly as the ratio of YFP to CFP signal intensities, reflects intracellular Ca²⁺ dynamics. Several YC versions have been used in plants. YC3.6 was targeted to the cytosol^14,15, nucleus¹⁶, mitochondria¹⁷, and plasma membrane¹⁸, and YC4.6 and D4ER were targeted to the ER^15,19, and D3cpv was targeted to the peroxisomes²⁰. Transgenic plants expressing YCs allow the live-cell imaging of Ca²⁺ dynamics within different cellular compartments of different cell types. CASEs (presumably Calcium sensor) are single circularly permuted fluorescent proteins (cpFPs) harboring a calmodulin and calmodulin-binding peptide M13. Upon binding to Ca²⁺, CASEs undergo conformational changes, leading to an increase of fluorescence intensity. The correlation between the CASE’s fluorescence response and Ca²⁺concentration allows intracellular Ca²⁺ dynamics to be measured quantitatively. The Case12 variant has 12 fold increased fluorescence in the Ca²⁺-saturated forms. N. benthiminana plants transiently expressing Case12 or Arabidopsis plants stably expressing Case 12 were used to study Ca²⁺ signaling in defense and abiotic stress^4,21. Asynchronous spatial and temporal Ca²⁺ oscillations in cells responding to pathogen attack, or to dehydration stress have been revealed with Case12 based Ca²⁺ imaging.

Here, we present detailed instructions for Aequorin based luminescence imaging of tissue- and stimuli specific Ca²⁺ dynamics in Arabidopsis seedlings, and for confocal imaging of cytosolic and nuclear Ca²⁺ dynamics in Arabidopsis root cells that express Case 12. Luminescence imaging of FAS could be adapted to analyze stress-induced Ca²⁺ dynamics in intact plants or tissues not described here, or to screen mutagenized Arabidopsis plant populations for mutants with altered stress induced Ca²⁺ signals. The live cell Ca²⁺ imaging setup could be adapted to analyze Ca²⁺ dynamics within different subcelluar compartments or in different cell types using other Ca²⁺ indicators.