Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

Summary

Extracellular glutamate-triggered systemic calcium signaling is critical for the induction of plant defense responses to mechanical wounding and herbivore attack in plants. This article describes a method to visualize the spatial and temporal dynamics of both these factors using Arabidopsis thaliana plants expressing calcium- and glutamate-sensitive fluorescent biosensors.

Abstract

Plants respond to mechanical stresses such as wounding and herbivory by inducing defense responses both in the damaged and in the distal undamaged parts. Upon wounding of a leaf, an increase in cytosolic calcium ion concentration (Ca²⁺ signal) occurs at the wound site. This signal is rapidly transmitted to undamaged leaves, where defense responses are activated. Our recent research revealed that glutamate leaking from the wounded cells of the leaf into the apoplast around them serves as a wound signal. This glutamate activates glutamate receptor-like Ca²⁺ permeable channels, which then leads to long-distance Ca²⁺ signal propagation throughout the plant. The spatial and temporal characteristics of these events can be captured with real-time imaging of living plants expressing genetically encoded fluorescent biosensors. Here we introduce a plant-wide, real-time imaging method to monitor the dynamics of both the Ca²⁺ signals and changes in apoplastic glutamate that occur in response to wounding. This approach uses a wide-field fluorescence microscope and transgenic Arabidopsis plants expressing Green Fluorescent Protein (GFP)-based Ca²⁺ and glutamate biosensors. In addition, we present methodology to easily elicit wound-induced, glutamate-triggered rapid and long-distance Ca²⁺ signal propagation. This protocol can also be applied to studies on other plant stresses to help investigate how plant systemic signaling might be involved in their signaling and response networks.

Introduction

Plants cannot escape from biotic stresses, e.g., insects feeding on them, so they have evolved sophisticated stress sensing and signal transduction systems to detect and then protect themselves from challenges such as herbivory¹. Upon wounding or herbivore attack, plants initiate rapid defense responses including accumulation of the phytohormone jasmonic acid (JA) not only at the wounded site but also in undamaged distal organs². This JA then both triggers defense responses in the directly damaged tissues and preemptively induces defenses in the undamaged parts of the plant. In Arabidopsis, the accumulation of J....

Protocol

1. Plant material preparation

1. In a 1.5 mL microtube, surface sterilize the seeds of Arabidopsis thaliana (Col-0 accession) plant expressing either GCaMP3 or CHIB-iGluSnFR by shaking with 20% (v/v) NaClO for 3 min and then wash 5 times with sterile distilled water.
   NOTE: The transgenic lines of Arabidopsis expressing GCaMP3 or CHIB-iGluSnFR have been described previously⁶.
2. In a sterile hood, sow 13 surface-sterilized seeds on a 10 cm square plastic Petri dish filled with 30 mL sterile (autoclaved) Murashige and Skoog (MS) medium [1x MS salts, 1% (w/v) sucrose, 0.01% (w/v) myoinositol, 0.05% (w/v) ME....

Results

Signal propagation of [Ca²⁺]_cyt and [Glu]_apo in response to wounding is presented in Figure 3, Figure 4, Movie S1, and Movie S2. Cutting the petiole of the leaf 1 in plants expressing GCaMP3 (at 0 s) led to a significant increase in [Ca²⁺]_cyt that was rapidly induced locally through the vasculature (at 40 s) (Figure 3

Discussion

Systemic signaling is important for plants to respond to localized external environmental stimuli and then to maintain their homeostasis at a whole plant level. Although they are not equipped with an advanced nervous system like animals, they employ rapid communication both within and between organs based on factors such as mobile electrical (and possibly hydraulic) signals and propagating waves of ROS and Ca^2+ 46,47. The protocol described above .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Arabidopsis expressing GCaMP3	Saitama University
Arabidopsis expressing CHIB-iGluSnFR	Saitama University
GraphPad Prism 7	GraphPad Software
L-Glutamate	FUJIFILM Wako	072-00501	Dissolved in a liquid growth medium [1/2x MS salts, 1% (w/v) sucrose, and 0.05% (w/v) MES; pH 5.1 adjusted with 1N KOH].
Microsoft Excel	Microsoft Corporation
Murashige and Skoog (MS) medium	FUJIFILM Wako	392-00591	composition: 1x MS salts, 1% (w/v) sucrose, 0.01% (w/v) myoinositol, 0.05% (w/v) MES, and 0.5% (w/v) gellan gum; pH 5.7 adjusted with 1N KOH.
Nikon SMZ25 stereomicroscope	Nikon
NIS-Elements AR analysis	Nikon
1x objective lens (P2-SHR PLAN APO)	Nikon
sCMOS camera (ORCA-Flash4.0 V2)	Hamamatsu Photonics	C11440-22CU
Square plastic Petri dish	Simport	D210-16