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

Published: June 4th, 2021



1Department of Biochemistry and Molecular Biology, Saitama University, 2Department of Botany, University of Wisconsin

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.

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.

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....

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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 Pet.......

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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 .......

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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 Ca2+ 46,47. The protocol described above .......

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This work was supported by grants from the Japan Society for the Promotion of Science (17H05007 and 18H05491) to MT, the National Science Foundation (IOS1557899 and MCB2016177) and the National Aeronautics and Space Administration (NNX14AT25G and 80NSSC19K0126) to SG.


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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

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