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.

1. Plant material preparation
<ol>
	<li>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.</li>
	<li>In a sterile hood, sow 13 surface-sterilized seeds on a 10 cm square plastic Pet...

The authors do not have any conflicts of interest.

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+&#160;46,47. The protocol described above ...

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 plants4,5.
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 leaves6,7. This systemic Ca2+ signal is received by intracellular Ca2+-sensing proteins, which lead&#160;to the activation of downstream defense signaling pathways, including JA biosynthesis8,9. 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 plants12,13,14,15,16,17,18, it is not well-suited for real-time imaging due to the extremely low luminescent signal it produces. F&#246;rster 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 plants19,20,21,22,23,24. 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 use25,26,27,28,29,30. 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 cells40,41. iGluSnFR is a GFP-based single-FP glutamate biosensor composed of cpGFP and a glutamate binding protein (GltI) from Escherichia coli42,43. 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Arabidopsis expressing GCaMP3</td>
			<td>Saitama University</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Arabidopsis expressing CHIB-iGluSnFR</td>
			<td>Saitama University</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism 7</td>
			<td>GraphPad Software</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamate</td>
			<td>FUJIFILM Wako</td>
			<td>072-00501</td>
			<td>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].</td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Murashige and Skoog (MS) medium</td>
			<td>FUJIFILM Wako</td>
			<td>392-00591</td>
			<td>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.</td>
		</tr>
		<tr>
			<td>Nikon SMZ25 stereomicroscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-Elements AR analysis</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1x objective lens (P2-SHR PLAN APO)</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sCMOS camera (ORCA-Flash4.0 V2)</td>
			<td>Hamamatsu Photonics</td>
			<td>C11440-22CU</td>
			<td></td>
		</tr>
		<tr>
			<td>Square plastic Petri dish</td>
			<td>Simport</td>
			<td>D210-16</td>
			<td></td>
		</tr>
	</tbody>
</table>

wide-field, real-time imaging of local and systemic wound signals in arabidopsis

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.

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&#160;(at 0 s) led to a significant increase in [Ca2+]cyt that was rapidly induced locally through the vasculature (at 40 s) (Figure ...

Watch this Scientific Journal Video about Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis at JoVE.com

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

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.

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

Plants respond to mechanical stresses such as wounding and herbivory by inducing defense responses both in the damaged and in the distal undamaged parts. ...

This protocol allows plant-wide real time imaging of the activity of the plant systemic signaling system through monitoring the dynamics of the calcium and apoplastic glutamate in response to wounding. This plant-wide real-time imaging method provides a robust tool to understand the dynamics of the rapid and long distance signals in plants, combining a high spatial temporal resolution and ease of use. This protocol offers the potential to provide a new insight into the spatial and temporal characteristics of the system calcium signaling in both biotic and abiotic stress responses in other plants species.Demonstrating the procedure will be Takuya Uemura, a post doctoral fellow from my laboratory. Begin by turning on the motorized fluorescent stereo microscope, equipped with a 1X objective lens and a sCMOS camera. Configure the device settings to irradiate with excitation light centered on 470 nanometers, selected using a filter that transmits light between 450 and 490 nanometers.Acquire emission light using a filter that transmits between 510 and 560 nanometers. Remove the lid from the dish containing the plant and place it under the objective lens. Check the fluorescent signal from the plant.Then wait for approximately 30 minutes in the dark until the plants are adapted to the new environmental conditions. Adjust the focus and magnification to see the whole plant in the field of view. Then set the acquisition parameters to detect the fluorescent signals using the microscopes imaging software.Set the recording time to 11 minutes. Image for five minutes prior to starting the experiment to acclimate the plant to the blue light irradiation from the microscope, then start recording. To determine the average baseline fluorescence record at least 10 frames before wounding or glutamate application.For real-time imaging of wound induced cytosolic calcium ion and apoplastic glutamate concentration changes, cut the petiole or the middle region of leaf L1 with scissors. For real-time imaging of glutamate triggered cytosolic calcium changes, cut approximately one millimeter from the tip of leaf L1 across the main vein with scissors. After at least 20 minutes apply 10 microliters of 100 millimolar glutamate to the leaf's cut surface.For fluorescence intensity analysis over time, define a region of interest at the place where fluorescence intensity is to be analyzed. Define two ROIs for the velocity calculation of the calcium wave. In the imaging software click on time measurement, define and circle.Measure the distance between the two regions by clicking on annotations and measurement, length and simple line. Measure the raw florescence values in each ROI over time by clicking on measure, then export the raw data to spreadsheet software, to convert the fluorescent signal into numbers at each time point. Determine the baseline fluorescence value, which is divined as F zero, by calculating the average F over the first 10 frames in the recorded data, then normalize the F data as described in the text manuscript.For calcium velocity wave analysis, define a significant signal rise point above the pre-stimulated values as representing detection of a calcium increase in each ROI. Calculate the time difference in the calcium increase between the two ROIs and the distance between them to determine the velocities of any calcium wave. Propagation of a wound-triggered change in the concentrations of the cytosolic calcium ion and apoplastic glutamate is shown here.Cutting the petiole of a leaf in plants expressing GCaMP-3 led to a significant increase in calcium. It was induced locally and then spread throughout the vasculature. The signal was rapidly propagated to neighboring leaves within a few minutes.Upon cutting a leaf and plants expressing basic chitinase I glue sniffer, a rapid apoplastic glutamate increase was observed around the cut region. Within a few minutes, the signal also propagated through the vasculature. For real-time imaging of calcium signal propagation triggered by the application of glutamate, the edge of a leaf in plants expressing CCaMP-3 was cut.This caused a local cytosolic calcium ion concentration increase, but the signal disappeared within a few minutes. After approximately 10 minutes, glutamate was applied to the cut surface, causing a rapid local increase in the concentration of cytosolic calcium and then propagation of this signal to distal leaves. To measure the changes inside a solid calcium concentration induced by wounding in the systemic leaf, the time course change of GCaMP-3 signal intensity was measured in two regions of interest.Apoplastic glutamate concentration changes in response to mechanical damage were measured as well. The glutamate signature exhibited a single peak at approximately 100 seconds after wounding. This experiment should be conducted under temperature and humidity controlled conditions because are elevated by changes in this environmental conditions.This protocol offers a potential to provide insights into the molecular mechanisms underlying long distance signaling through using mutants that the defective and putative elements of the lab signaling system.

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4.6K visualizações.  Saitama University. Este protocolo permite imagens em tempo real da atividade do sistema de sinalização sistêmica da planta através do monitoramento da dinâmica do cálcio e glutamato apoplástico em resposta ao ferimento. Este método de imagem em tempo real de toda a planta fornece uma ferramenta robusta para entender a dinâmica dos sinais de rápida e longa distância nas plantas, combinando uma alta resolução temporal espacial e facilidade de uso. Este protocolo oferece o potencial de fornecer uma no...