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 JA induced by wounding was detected in distal, intact leaves within just a few minutes of damage elsewhere in the plant suggesting that a rapid and long-distance signal is being transmitted from the wounded leaf³. Several candidates, such as Ca²⁺, reactive oxygen species (ROS), and electrical signals, have been proposed to serve as these long-distance wound signals in plants^4,5.

Ca²⁺ is one of the most versatile and ubiquitous second messenger elements in eukaryotic organisms. In plants, caterpillar chewing and mechanical wounding cause drastic increases in the cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) both in the wounded leaf and in unwounded distant leaves^6,7. This systemic Ca²⁺ signal is received by intracellular Ca²⁺-sensing proteins, which lead to the activation of downstream defense signaling pathways, including JA biosynthesis^8,9. Despite numerous such reports supporting the importance of Ca²⁺ signals in plant wound responses, information on the spatial and temporal characteristics of Ca²⁺ signals induced by wounding is limited.

Real-time imaging using genetically encoded Ca²⁺ indicators is a powerful tool to monitor and quantify the spatial and temporal dynamics of Ca²⁺ signals. To date, versions of such sensors have been developed that enable the visualization of Ca²⁺ signals at the level of a single cell, to tissues, organs and even whole plants¹⁰. The first genetically encoded biosensor for Ca²⁺ used in plants was the bioluminescent protein aequorin derived from the jellyfish Aequorea victoria¹¹. Although this chemiluminescent protein has been used to detect Ca²⁺ changes in response to various stresses in plants^{12,13,14,15,16,17,18}, it is not well-suited for real-time imaging due to the extremely low luminescent signal it produces. Förster Resonance Energy Transfer (FRET)-based Ca²⁺ indicators, such as the Yellow cameleons, have also been successfully used to investigate the dynamics of a range of Ca²⁺ signaling events in plants^{19,20,21,22,23,24}. These sensors are compatible with imaging approaches and most commonly are composed of the Ca²⁺ binding protein calmodulin (CaM) and a CaM-binding peptide (M13) from a myosin light chain kinase, all fused between two fluorophore proteins, generally a Cyan Fluorescent Protein (CFP) and a Yellow Fluorescent Protein variant (YFP)¹⁰. Ca²⁺ binding to CaM promotes the interaction between CaM and M13 leading to a conformational change of the sensor. This change promotes energy transfer between the CFP and YFP, which increases the fluorescence intensity of the YFP while decreasing the fluorescence emission from the CFP. Monitoring this shift from CFP to YFP fluorescence then provides a measure of the increase in Ca²⁺ level. In addition to these FRET sensors, single fluorescent protein (FP)-based Ca²⁺ biosensors, such as GCaMP and R-GECO, are also compatible with plant imaging approaches and are widely used to study [Ca²⁺]_cyt changes due to their high sensitivity and ease of use^{25,26,27,28,29,30}. GCaMPs contain a single circularly permutated (cp) GFP, again fused to CaM and the M13 peptide. The Ca²⁺-dependent interaction between CaM and M13 causes a conformational change in the sensor that promotes a shift in the protonation state of the cpGFP, enhancing its fluorescent signal. Thus, as Ca²⁺ levels rise, the cpGFP signal increases.

To investigate the dynamics of Ca²⁺ signals generated in response to mechanical wounding or herbivore feeding, we have used transgenic Arabidopsis thaliana plants expressing a GCaMP variant, GCaMP3, and a wide-field fluorescence microscope⁶. This approach has succeeded in visualizing rapid transmission of a long-distance Ca²⁺ signal from the wound site on a leaf to the whole plant. Thus, an increase in [Ca²⁺]_cyt was immediately detected at the wound site but this Ca²⁺ signal was then propagated to the neighboring leaves through the vasculature within a few minutes of wounding. Furthermore, we found that the transmission of this rapid systemic wound signal is abolished in Arabidopsis plants with mutations in two glutamate receptor-like genes, Glutamate Receptor Like (GLR), GLR3.3, and GLR3.6⁶. The GLRs appear to function as amino-acid gated Ca²⁺ channels involved in diverse physiological processes, including wound response³, pollen tube growth³¹, root development³², cold response³³, and innate immunity³⁴. Despite this well-understood, broad physiological function of the GLRs, information on their functional properties, such as their ligand specificity, ion selectivity, and subcellular localization, are limited³⁵. However, recent studies reported that GLR3.3 and GLR3.6 are localized in the phloem and xylem, respectively. Plant GLRs have similarities to ionotropic glutamate receptors (iGluRs)³⁶ in mammals, which are activated by amino acids, such as glutamate, glycine, and D-serine in the mammalian nervous system³⁷. Indeed, we demonstrated that the application of 100 mM glutamate, but not other amino acids, at the wound site induces a rapid, long-distance Ca²⁺ signal in Arabidopsis, indicating that extracellular glutamate likely acts as a wound signal in plants⁶. This response is abolished in the glr3.3/glr3.6 mutant suggesting that glutamate may be acting through one or both of these receptor-like channels and indeed, AtGLR3.6 was recently shown to be gated by these levels of glutamate³⁸.

In plants, in addition to its role as a structural amino acid, glutamate has also been proposed as a key developmental regulator³⁹; however, its spatial and temporal dynamics are poorly understood. Just as for Ca²⁺, several genetically encoded indicators for glutamate have been developed to monitor the dynamics of this amino acid in living cells^40,41. iGluSnFR is a GFP-based single-FP glutamate biosensor composed of cpGFP and a glutamate binding protein (GltI) from Escherichia coli^42,43. The conformational change of iGluSnFR, that is induced by glutamate binding to GltI, results in an enhanced GFP fluorescence emission. To investigate whether extracellular glutamate acts as a signaling molecule in plant wound response, we connected the iGluSnFR sequence with the basic chitinase signal peptide secretion sequence (CHIB-iGluSnFR) to localize this biosensor in the apoplastic space⁶. This approach enabled imaging of any changes in the apoplastic glutamate concentration ([Glu]_apo) using transgenic Arabidopsis plants expressing this sensor. We detected rapid increases in the iGluSnFR signal at the wounding site. This data supports the idea that glutamate leaks out of the damaged cells/tissues to the apoplast upon wounding and acts as a damage signal activating the GLRs and leading to the long-distance Ca²⁺ signal in plants⁶.

Here, we describe a plant-wide real-time imaging method using genetically encoded biosensors to monitor and analyze the dynamics of long-distance Ca²⁺ and extracellular glutamate signals in response to wounding⁶. The availability of wide-field fluorescence microscopy and transgenic plants expressing genetically encoded biosensors provides a powerful, yet easily implemented approach to detect rapidly transmitted long-distance signals, such as Ca²⁺ waves.

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) MES and 0.5% (w/v) gellan gum; pH 5.7 adjusted with 1N KOH]. Replace the lid and wrap with surgical tape.
3. After incubation in dark at 4 °C for 2 days, place the plates horizontally at 22 °C in a growth chamber under continuous light (90-100 µmol m^-2 s^-1) for approximately 2 weeks before use. After 2 weeks, count the number of Arabidopsis leaves from oldest to youngest⁴⁴ (Figure 1). Wound responses preferentially move from the damaged leaf (n) to leaves numbered n ± 3 and n ± 5⁶.
   NOTE: In this protocol, the Petri dish will be opened for imaging the wound and glutamate effects under a fluorescence microscope. Therefore, subsequent steps in this experiment should be conducted under temperature- and humidity-controlled room conditions. This is because Ca²⁺ signals are also elicited by changes in these environmental conditions. It is also known that the blue light, emitted from the microscope during recording for excitation of the biosensor protein's fluorescence, may elicit an increase of cytosolic Ca²⁺ concentration⁴⁵ and so the plant should be acclimated to the blue light irradiation for several minutes before beginning the experiment.

2. Chemical preparation

1. Dissolve L-Glutamate 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] to make a 100 mM working solution.
   NOTE: Avoid use of salts of glutamate such as sodium glutamate to prevent potential cation-related effects on Ca²⁺ dynamics.

3. Microscope setting and conducting real-time imaging

1. Turn on the motorized fluorescence stereomicroscope equipped with a 1x objective lens (NA = 0.156) and a sCMOS camera (Figure 2) and configure the device settings to irradiate with a 470/40 nm excitation light and acquire an emission light passing through a 535/50 nm filter.
   NOTE: Any GFP-sensitive fluorescence microscope can be used to detect GCaMP3 and iGluSnFR signals in real-time, but a low power objective lens and highly sensitive camera with a wide sCMOS chip are recommended to acquire signals from the entire plant. The low power objective allows for imaging of an entire Arabidopsis plant's response and use of a highly sensitive camera permits the fast data acquisition needed to capture the rapid time course of the wound-triggered Ca²⁺ wave. For the fluorescence microscope used in this study, the maximum values of the field of view and temporal resolution are 3 cm x 3 cm and 30 frames per second (fps), respectively.
2. Remove the lid and place the dish under the objective lens.
3. Check the fluorescence signal from the plant and then wait for approximately 30 min in the dark until plants are adapted to the new environmental conditions. This adaptation step is required because changes in humidity elicit [Ca²⁺]_cyt elevation in plants that can interfere with any wound-related events.
4. Adjust the focus and magnification to see the whole plant in the field of view. In the current protocol, a 0.63x magnification was used.
5. Before starting real-time imaging, set up the acquisition parameters to detect the fluorescence signals using microscope imaging software. The settings for imaging in the current protocol are: exposure and interval times set to 1.8 s and 2 s (i.e., 0.5 fps), respectively. Set recording time to 11 min.
6. Image for 5 min prior to starting the experiment to acclimate the plant to the blue light irradiation from the microscope, then start recording by clicking on Run Now, or the equivalent command in the microscope software being used. To determine the average baseline fluorescence, record at least 10 frames (i.e., at least 20 s in the current protocol) before wounding or glutamate application (see Section 4).
   1. For real-time imaging of wound-induced [Ca²⁺]_cyt and [Glu]_apo changes, cut the petiole or the middle region of leaf L1 with scissors (Figure 3 and Figure 4).
   2. For real-time imaging of glutamate-triggered [Ca²⁺]_cyt changes, cut the edge (approximately 1 mm from the tip) of leaf 1 across the main vein with scissors. After at least 20 min recovery period, apply 10 µL of 100 mM glutamate to the leaf's cut surface (Figure 5).
      NOTE: This pre-cutting was necessary to allow glutamate access to the leaf apoplast in order to trigger responses. In addition, applying a drop of distilled water to the cut surface of leaf L1 was found to be critical to prevent the samples from desiccating during the recovery before applying glutamate.
7. After finishing the 11 min recording, save the data.

4. Data analysis

1. For fluorescence intensity analysis over time, define a region of interest (ROI) at the place where fluorescence intensity is to be analyzed (Figure 6 and Figure 7). For the velocity calculation of the Ca²⁺ wave, define 2 ROIs (ROI1 and ROI2) for analysis. In the imaging software, click on Time Measurement | Define | Circle. Measure the distance between ROI1 and ROI2 by clicking on Annotations and Measurement | Length | Simple Line (Figure 6).
2. Measure the raw fluorescence values (F) in each ROI over time by clicking on Measure. Export raw data to a spreadsheet software to convert the fluorescence signal into numbers at each time point by clicking on All to Excel | Export.
3. Determine the baseline fluorescence value, which is defined as F₀, by calculating the average of F over the first 10 frames (i.e., prior to treatment) in the recorded data.
4. Normalize the F data (by calculating ΔF/F) using the equation ΔF/F = (F−F₀)/F₀, where ΔF is the time-dependent change in fluorescence.
5. For Ca²⁺  wave velocity wave analysis, define a significant signal rise point above the pre-stimulated values as representing detection of a Ca²⁺ increase in each ROI (t₁ and t₂) using the criterion of a rise to 2× the standard deviation (2x SD) that is calculated from the F₀ data using statistical software. 95% of the F₀ data falls within 2x SD from the mean, indicating that a rise in signal above this level by chance is ≤5%. Calculate the time difference of the Ca²⁺ increase between ROI1 and ROI2 [t₂- t₁ time-lag (Δt)] and measure the distance between ROI1 and ROI2, then determine the velocities of any Ca²⁺ wave.

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