Name: Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging (Video) | JoVE Journal
Uploaded: 2010-10-02T17:00:00.000+00:00
Duration: 7 min 3 s
Description: Learn whole leaf imaging to assess stomatal responses to live bacterial cells using fluorescence microscopy. This intact leaf method minimizes tissue damage and enables real-time observation of guard cell movement in plant-pathogen interaction studies.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This research was supported by a grant from the National Institute of Health and The University of Texas at Arlington set up funds to MM.

1. Growing Plants and Preparing Bacteria
<ol>
 <li>To begin this procedure sow Arabidopsis seeds on a 1:1:1 v:v:v mixture of growing medium (Redi-earth plug and seedling mix, Sun Gro), fine vermiculite, and perlite.</li>
 <li>Grow the plants in a growth chamber (22°C, 12 hours of 100 μmol/mm2/sec daily light and 65±5% humidity) and water as needed. The plants are ready to use in 4-6 weeks when they have young fully expanded leaves prior to bolting.</li>
 <li>Two days before starting the experiment prepare the bacterium culture. Streak Pseudomonas syringae from glycerol stock on modified LB medium (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl pH 7.0, and 1.5% agar) and incubate for 24 hours at 28°C. Use fresh culture to prepare the inoculum and always start culture plates from glycerol stocks as bacteria becomes less virulent after sub-culturing.</li>
 <li>Use bacteria that grew on the plate to start a 10 mL liquid culture of P. syringae in a 50 mL Erlenmeyer flask. Incubate the culture overnight at 28°C with vigorous shaking until it reaches optical density or OD at 600 nm between 0.8 and 1.</li>
 <li>Measure the OD at 600 and harvest the cells by centrifugation at 1360 xg for 10 minutes. Resuspend the cells in distilled water so that the final O.D is 0.2. This O.D corresponds to 108 colony forming units (CFU)/mL. With the bacteria ready, proceed to prepare the leaves and perform the assay.</li>
</ol>
2. Leaf Staining and Incubation with Bacteria
<ol>
 <li>In order to stain the leaves first prepare 20 μM propidium iodide or PI solution in water. 10 mL of solution is sufficient to stain 3 small leaves at a time.</li>
 <li>Retrieve the plants from the growth chamber and with forceps detach 3 young, fully-expanded leaves. Immerse the whole leaves in the PI solution for 5 minutes. Then remove the leaves and rinse them briefly with distilled water. </li>
 <li>Next place a leaf with the lower surface facing down on a microscope slide. Cut the leaf with a sharp razor blade into four pieces excluding the mid vein so that the leaf lays flat on the slide.</li>
 <li>To incubate the bacteria with the leaves, add 300 μL of bacterial suspension under the leaf pieces on the slide. Make sure that the lower surface of the leaf is in contact with the inoculum.</li>
 <li>Incubate the samples under the same environmental conditions that were used to grow the plants. At the time to observe leaves under the microscope, transfer the leaf pieces to a new slide with lower surface facing up. Note that cover slip is not used when mounting the slides. The same leaf sample can be imaged multiple times during incubation and a time course may be set up according to the research objectives.</li>
</ol>
3. Microscopy, Measurement and Data Analysis
<ol>
 <li>Here a laser scanning confocal microscope (LSM 510 Meta, Carl Zeiss Inc.) is used to examine the leaves. Observe the lower surface of the leaves to detect the fluorescence of PI (excitation 453 nm, emission 543 and 620 nm). </li>
 <li>Using the same leaf samples capture images of stomata over time. Save the images for measuring the width of the stomatal aperture.</li>
 <li>Measure stomatal aperture width of at least 60 stomata for each treatment at each time point using LSM image browser.</li>
 <li>Calculate the average and standard error for the stomatal aperture width. Statistical significance of the results can be calculated using 2-tailed, paired wise Student's t-test.</li>
</ol>
4. Representative Images of Stomatal Response to Incubation with Bacteria
<ol>
 <li>Here is a typical microscopic image (using a 20x objective) of an Arabidopsis leaf surface under laser scanning confocal microscope in bright field view (Figure 1).</li>
 <li>Propidium iodide stains the cell wall of viable cells increasing the visibility of guard cells in addition to allowing for the identification of undamaged cells for data collection (Figure 2). A range of stomatal pore openings can be seen in these micrographs, from completely closed stomata to wide open stomata.</li>
 <li>Completely closed stomata are identified by the shape of the guard cells (Figure 3). The aperture width is considered to be 0 μm. </li>
 <li>For open stomata (Figure 4) the aperture width shown is measured by using a straight line drawn across the widest area of the stomatal pore.</li>
 <li>These are typical experimental results of this stomatal assay. Arabidopis leaves were incubated in the dark with three different strains of Pseudomonas syringae. Only the bacterium Pst DC3000 was able to open dark-closed stomata, as measured by the aperture width of randomly selected 60 stomata per bacterial strain (Figure 5).</li>
</ol>
<img src="/files/ftp_upload/2185/2185fig1.jpg" alt="Cellular microstructure, microscope image, cell morphology, biological research, structural analysis." data-altupdatedat="1788235205000" data-alt="Figure 1"> Figure 1. Micrograph of the surface of an Arabidopsis leaf. One field of view of the leaf surface under laser scanning confocal microscope (LSCM) using the 20x objective. Note that stomatal aperture is not as evident when compared to the fluorescent view aided by propidium iodide staining (Figure 2).
<img src="/files/ftp_upload/2185/2185fig2.jpg" alt="Fluorescence microscopy image of plant cells; red chloroplasts, highlighted guard cells visible." data-altupdatedat="1788235205000" data-alt="Figure 2"> Figure 2. Micrograph of the surface of a propidium iodide-stained Arabidopsis leaf. One field of view of the leaf surface under laser scanning confocal microscope (LSCM) using the 20x objective. Note a range of stomatal pore opening. Yellow arrows are pointed to completely close stomata and green arrows are pointed to wide open stomata.
<img src="/files/ftp_upload/2185/2185fig3.jpg" alt="Fluorescence microscopy image, cell structure, highlighted areas, optical analysis." data-altupdatedat="1788235205000" data-alt="Figure 3"> Figure 3. Completely close stomata identified by the shape of and the opening between the guard cells. The aperture width is considered to be 0 μm.
<img src="/files/ftp_upload/2185/2185fig4.jpg" alt="Polarized light microscopy; birefringence analysis; microscale measurements; optical properties." data-altupdatedat="1788235205000" data-alt="Figure 4"> Figure 4. Open stomata showing the aperture width in μm. Measurements were taken by using the LSCM browser based on a straight line drawn across the widest area of the stomatal pore.
<img src="/files/ftp_upload/2185/2185fig5.jpg" alt="Bar graph of stomatal aperture (µm) at different time points, with DC3000 showing highest increase." data-altupdatedat="1788235205000" data-alt="Figure 5"> Figure 5. Stomatal aperture of Arabidopsis leaves incubated with three strains of Pseudomonas syringae pv. tomato: DC3118, DB29, and DC3000. Plants were kept in the dark during the experimentation time. Results are shown as the mean (n=50-70) ± standard error. Statistical significance was detected with two-tailed Student's t-test. The experiment was repeated three times with similar results.

<ol><li>Brooks, D. M., Hernandez-Guzman, G., Kloek, A. P., Alarcon-Chaidez, F., Sreedharan, A., Rangaswamy, V., Penaloza-Vazquez, A., Bender, C. L., Kunkel, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+and+characterization+of+a+well-defined+series+of+coronatine+biosynthetic+mutants+of+Pseudomonas+syringae+pv.+tomato+DC3000%5BTitle%5D)+AND+%22Mol+Plant-Microbe+Interact%22%5BJournal%5D)">Identification and characterization of a well-defined series of coronatine biosynthetic mutants of Pseudomonas syringae pv. tomato DC3000.</a> Mol Plant-Microbe Interact. 17, 162-174 (2004).</li>
<li>Katagiri, F., Thilmony, R., He, S. Y. The Arabidopsis thaliana-Pseudomonas syringae interaction. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aKatagiri%2C+F+%22The+Arabidopsis+Book%22">The Arabidopsis Book</a>. Somerville, C. R., Meyerowitz, E. M. , American Society of Plant Biologists. Rockville, MD. (2002).</li>
<li>Ma, S. W., Morris, V. L., Cuppels, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+a+DNA+region+required+for+production+of+the+phytotoxin+coronatine+by+Pseudomonas+syringae+pv.+tomato%5BTitle%5D)+AND+%22Mol+Plant-Microbe+Interact%22%5BJournal%5D)">Characterization of a DNA region required for production of the phytotoxin coronatine by Pseudomonas syringae pv. tomato.</a> Mol Plant-Microbe Interact. 4, 69-74 (1991).</li>
<li>Melotto, M., Underwood, W., Koczan, J., Nomura, K., He, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plant+stomata+function+in+innate+immunity+against+bacterial+invasion%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Plant stomata function in innate immunity against bacterial invasion.</a> Cell. 126, 969-980 (2006).</li>
</ol>

No conflicts of interest declared.

We have presented a straight forward procedure to measure stomatal aperture in intact leaf tissue allowing for an easy assessment of stomatal response to different treatments.
Although we have presented results using the model system Arabidopsis/Pseudomonas syringae, the intact leaf stomatal assay can be potentially performed with any plant-bacterium combination. The protocol can easily be modified to fit growth requirements of other plants and bacterial pathogens. The overall principle and procedure remain the same. In addition, this method may be beneficial to researchers who wish to study functional output of guard cells not only to live microbes, but also to other stimuli and chemical agents under conditions that maintain the leaf's natural environment.
Whole leaf can be difficult to image due to its thickness and uneven topography. This problem can be alleviated by removing the mid vein of the leaf so it lays flat on the slide and using confocal microscopy. However, other types of fluorescence microscope can be used to obtain high resolution images of the leaf surface.

<table><tbody><tr><td>Propidium iodide</td><td>Sigma-Aldrich</td><td>P4864</td><td></td></tr><tr><td>Tryptone</td><td>Fisher Scientific</td><td>BP1421-1</td><td></td></tr><tr><td>Yeast Extract</td><td>Difco Laboratories</td><td>0127-01-7</td><td></td></tr><tr><td>Sodium Chloride</td><td>VWR international</td><td>7647-14-5</td><td></td></tr><tr><td>Agar</td><td>Bio Express</td><td>J637-2500G</td><td></td></tr><tr><td>Plant growth chamber</td><td></td><td></td><td></td></tr><tr><td>Shaker incubator</td><td></td><td></td><td></td></tr><tr><td>Laser scanning confocal microscope</td><td></td><td></td><td></td></tr></tbody></table>

assessing stomatal response to live bacterial cells using whole leaf imaging

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

In this procedure who Leaf imaging is used to assess guard cell movement in response to live bacterial cells. First, the bacteria accultured from a glycerol stock and an inoculum is prepared. Then young healthy leaves are collected and stained with propidium iodide.The stained leaves are cut and the leaf pieces are incubated on the top of a bacterial suspension. Finally, the leaf surface is observed under the microscope and stomatal apertures can be measured. The results obtained using fluorescence microscopy show changes in the stomatal aperture in response to bacteria.The main advantage of this method over existing methods like use of epidermal peels, is that the leaf in contact with bacteria is intact, mimicking the natural conditions for bacterial penetration into the leaves To begin this procedure. So Arabidopsis seeds on a one-to-one, to one V to V to V mixture of growing medium. Find vermiculite amite light grow the plants in a growth chamber and water as needed.The plants are ready to use in four to six weeks when they have young, fully expanded leaves prior to bolting. Two days before starting the experiment, prepare the bacterium culture streak. Pseudomonas ringe from glycerol stock on modified LB medium, and incubate for 24 hours at 28 degrees Celsius.Use fresh culture to prepare the inoculum and always start culture plates from glycerol stocks. As bacteria become less virulent after subculture, use bacteria that grew on the plate to start a 10 milliliter liquid culture of pseudomona singe. In a 50 milliliter erlenmeyer flask incubate the culture overnight at 28 degrees Celsius with vigorous shaking until it reaches optical density or od at 600 nanometers between 0.8 and one.Measure the OD at 600 and harvest the cells by centrifugation at 1, 360 times G for 10 minutes. Resus suspend the cells in distilled water so that the final OD is 0.2. This od corresponds to 10 to the eight colony forming units per milliliter.With the bacteria ready, proceed to prepare the leaves and perform the assay in order to stain the leaves. First, prepare 20 micromolar propidium, iodide, or PI solution in water. 10 milliliters of solution is sufficient to stain three small leaves at a time.Retrieve the plants from the growth chamber and with forceps, detach three young fully expanded leaves. Immerse the whole leaves in the PI solution for five minutes. Then remove the leaves and rinse them briefly with distilled water.Next, place a leaf with the lower surface facing down on a microscope slide. Cut the leaf with a sharp razor blade into four pieces, excluding the mid vein so that the leaf lays flat on the slide to incubate the bacteria with the leaves. Add 300 microliters of bacterial suspension under the leaf pieces on the slide, make sure that the lower surface of the leaf is in contact with the inoculum.Incubate the samples under the same environmental conditions that were used to grow the plants. When it is time to observe leaves under the microscope, transfer the leaf pieces to a new slide with the lower surface facing up. Note that a cover slip is not used when mounting the slides.The same leaf sample can be imaged multiple times during incubation, and a time course may be set up according to the research objectives. Here, a laser scanning confocal microscope is used to examine the leaves, observe the lower surface of the leaves to detect the fluorescence of PI using the same leaf samples, capture images of stoma over time. Save the images for measuring the width of the stomatal aperture.Measure stomatal aperture width of at least 60 stoma for each treatment at each time point. Using the LSM image browser, calculate the average and standard error for the stomatal aperture width. Statistical significance of the results can be calculated using two-tailed paired wise students.T-test here is a typical microscopic image of an arabidopsis leaf surface under a laser scanning confocal microscope. In brightfield view, propidium iodide stains the cell wall of viable cells increasing the visibility of guard cells in addition to allowing for the identification of undamaged cells. A range of stomatal PO openings can be seen in these micrographs from completely closed stoma to wide open stoma.Completely closed stoma are identified by the shape of the guard cells. The aperture width is considered to be zero micrometer for open stoma. The aperture width shown is measured by using a straight line drawn across the widest area of the stoma pore.These are typical experimental results of the live leaf inoculation assay. Arabidopsis leaves were incubated in the dark with three different strains of pseudomonas Cy Ringe, only the bacterium P-S-T-D-C 3000 was able to open dark clo stoma as measured by the aperture width of 60 randomly selected stoma per bacterial strain. This technique has paved a way for researchers in the field of planned bacterium interactions to explore the mechanisms of early response to pathogens in plant species.

assessing-stomatal-response-to-live-bacterial-cells-using-whole-leaf

Research

JoVE Journal

Immunology and Infection

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

Source: Lee, H. et. al., Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection. J. Vis. Exp. (2018)This video demonstrates a method for monitoring bacterial infection and effector protein delivery in plant cells using split superfolder green fluorescent protein (sfGFP) complementation combined with confocal microscopy. The approach enables precise visualization of effector translocation into the host cell cytosol.

Monitoring Bacterial Infection and Effector Protein Delivery in Plant Cells

Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

Visualizing Stromule Frequency with Fluorescence Microscopy

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

Here we describe a hydroponic plant growth assay to quantify species presence and visualize the spatial distribution of bacteria during initial colonization of plant roots and after their transfer into different growth environments.

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

The present protocol describes a method that allows single-cell gene expression analysis on Pseudomonas syringae populations grown within the plant apoplast.

Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue

Several commonly used methods are introduced here to study the membrane trafficking events of a plasma membrane receptor kinase. This manuscript describes detailed protocols including the plant material preparation, pharmacological treatment, and confocal imaging setup.

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells

Here, we present a simple method for direct observation and automated measurement of stomatal responses to bacterial invasion in Arabidopsis thaliana. This method leverages a portable stomatal imaging device, together with an image analysis pipeline designed for leaf images captured by the device.

Direct Observation and Automated Measurement of Stomatal Responses to Pseudomonas syringae pv. tomato DC3000 in Arabidopsis thaliana

This protocol describes a high-throughput approach for evaluating plant ion leakage, peroxidase activity, and callose production in the same sample.

Evaluating Leaf Responses to Microbial Secondary Metabolites Using A High-Throughput Format

This protocol presents a comparative method to assess stomatal and epidermal traits in two distinct species using epidermal peels, clearing techniques, and AI-assisted image analysis. The goal is to enable high-throughput, reproducible quantification of cellular structures underlying stomatal function for agricultural applications and broad plant research.

Characterizing Stomatal and Epidermal Traits Using Peels, Clearing, and AI-Based Image Analysis

Learn whole leaf imaging to assess stomatal responses to live bacterial cells using fluorescence microscopy. This intact leaf method minimizes tissue damage and enables real-time observation of guard cell movement in plant-pathogen interaction studies.

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Explore More Videos