Name: Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus (Video) | JoVE Journal
Uploaded: 2020-01-30T15:00:00.000+00:00
Duration: 7 min 8 s
Description: Discover a thermal imaging method for screening compounds that regulate plant transpiration in Helianthus annuus. This protocol enables researchers to identify novel regulators of stomatal behavior and water use efficiency.

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5 minutes

The work was supported by Pomona College Start-up Funds and Hirsch Research Initiation Grants Fund (to FJ) as well as the Pomona College Molecular Biology Program through the Stellar Summer Research Assistant Program (to KG).

1. Growing the plants
<ol>
	<li>Add a 4 cm-thick layer of fine vermiculite to standard 10 in. x 20 in. (254 mm x 501 mm) plant trays with no holes.</li>
	<li>Place the seed holders (see Table of Materials) 2 cm apart in the plant trays.</li>
	<li>Fill seed holders with vermiculite.</li>
	<li>Place a sunflower seed with its pointed end down in each seed holder, pushing down so half of the seed remains exposed. 
	NOTE: A sunflower seed is asymmetrical and the pointed end from where the radicle will emerge should point downwards. Proper seed placement is important as the reorientation of root and stem are not possible within the seed holders. The rounded end of the seed should extend past the top of the seed holder.</li>
	<li>Once the seeds are in place, cover them with an additional 2 cm-thick layer of fine vermiculite. Water by misting from above. The surface should remain wet after an hour. If that is the case, cover the trays with lids.</li>
	<li>Grow plants in a growth chamber or a greenhouse. Recommended conditions are a light intensity of 140 &#181;mol photons&#183;m-2&#183;s-1 and a photoperiod of 16 h light at 22 &#176;C and 9 h dark at 20 &#176;C for 5 days. 
	NOTE: Watering should not be necessary unless the surface becomes visibly dry.</li>
</ol>
2. Set up of the hydroponic system
<ol>
	<li>Find an appropriately sized container suitable for growing plants hydroponically. The size of the container should be adapted to the space available in the growth chamber or greenhouse. A minimal depth of 15 cm is recommended.</li>
	<li>Fill the container with distilled water and add general hydroponics fertilizer as indicated by the manufacturer. The resulting hydroponic solution should be aerated and in constant movement, which can be achieved by using air and water pumps.</li>
	<li>Prepare the hydroponics floaters.
	<ol>
		<li>Cut a sheet of 2 cm-thick expanded polystyrene foam (see Table of Materials) to the dimensions of the container. The sheet should cover most of the surface of the container in order to limit the growth of algae. A wood burning tool is effective for cutting polystyrene foam and is versatile enough for this protocol. 
		CAUTION: Fumes or vapor released during hot cutting of polystyrene foam are serious health hazards. Use proper respiratory protection. Users can also satisfy ventilation requirements by cutting the foam under a fume hood.</li>
		<li>Make holes (1-2 cm in diameter) in the polystyrene foamsheet using a wood burning tool. The distance between the centers of two holes can be adjusted to the needs of the experiment. However, a minimal distance of 2.5 cm is recommended.</li>
	</ol>
	</li>
</ol>
3. Transfer of seedlings to hydroponics and plant growth
<ol>
	<li>Gently pull out 5-day old seedlings from the vermiculite and transfer immediately to a container filled with water for 30 min. This step will remove excess vermiculite and soften the remaining pericarps. The emerging primary root should be visible.</li>
	<li>Remove the pericarp walls by hand if needed in order to optimize the future expansion of the cotyledons.</li>
	<li>Transfer the seedlings within the seed holders to the polystyrene foam floater. Grow the plants with a light intensity of 140 &#181;mol photons&#183;m-2&#183;s-1, a relative humidity of 65% and a photoperiod of 16 h light at 22 &#176;C and 9 h dark at 20 &#176;C for 2 days.</li>
</ol>
4. Preparation prior to treatment
NOTE: This procedure is for testing 20 chemicals from a small compound library in triplicate, with 100 &#181;M ABA in 10 mM MES-KOH (pH = 6.2) and 10 mM MES-KOH (pH = 6.2) containing 1% (v/v) dimethyl sulfoxide (DMSO) as positive and negative controls, respectively.
<ol>
	<li>Ensure that there are enough plants ready for treatment. Plants ready for treatment should be mature enough to have fully unfolded cotyledons, but young enough that lateral root proliferation is minimal. To do a standard screening, 69 such plants are needed.</li>
	<li>Remove the 96-well plate containing the small compounds from the -80 &#176;C freezer. Thaw at room temperature.</li>
	<li>Prepare 80 mL of 10 mM MES-KOH buffer adjusted to a pH of 6.2 with 1 M KOH.</li>
	<li>Label cap-less 2 mL microtubes. Prepare at least six tubes for the negative control treatment (10 mM MES-KOH (pH = 6.2) containing 1% (v/v) DMSO). Use three tubes for ABA treatment (100 &#181;M ABA in 10 mM MES-KOH pH = 6.2), positive control). Use the remaining 60 tubes to analyze the effect of the 20 chemicals in triplicate.</li>
	<li>Transfer 10 &#181;L of each chemical (10 mM in DMSO) into each of the three appropriately labeled tubes. Pipet 10 &#181;L of 10 mM ABA dissolved in DMSO into three tubes and 10 &#181;L of DMSO into the six control tubes. 
	CAUTION: By nature, some compounds may cause serious health effects and users must take appropriate protective measures.</li>
	<li>Add 990 &#181;L of 10 mM MES-KOH (pH = 6.2) to each of the 69 tubes. Dispense the MES buffer with enough force to mix the chemical with the MES buffer but be very careful not to use so much force that the chemicals and MES buffer squirt out of the tube. Alternatively, vortex at low speed.</li>
</ol>
5. Set up the thermal imaging camera
<ol>
	<li>Mount the thermal imaging camera on a copy stand. Connect all cables to a laptop. 
	NOTE: The recording is done under conditions of temperature (20 &#176;C to 25 &#176;C), humidity (50% to 70%) and light quality (110 to 140 &#181;mol photons&#183;m-2&#183;s-1) similar to those used to grow the plants.</li>
	<li>Turn on the camera then the laptop and open the thermal imaging analysis software. 
	NOTE: The subsequent instructions for recording apply to a specific software used (see Table of Materials).</li>
	<li>Adjust the recording settings.
	<ol>
		<li>Mouse over the record red button at the top of the central window. A dropdown menu will appear. Click on the wrench icon Record Settings.</li>
		<li>Select the appropriate record mode and options. The record periodically option, with one frame captured per minute and a manual stop could be used. Note the file destination where the software will save the video. Close the Record Settings window.</li>
	</ol>
	</li>
</ol>
6. Plant preparation and treatment
<ol>
	<li>Place the cap-less tubes containing the chemicals in tube racks. Alternatively, a polystyrene foam sheet can be cut and poked with a wood burning tool as described in step 2.3 to make a custom tube rack. The diameter of each hole should be very close to the external diameter of the capless tubes in order to hold them firmly. 
	NOTE: The field of view of the camera is a limiting factor that should be taken into consideration when deciding on how the cap-less tubes will be held in place.</li>
	<li>Evenly distribute the positive and negative control tubes as well as the experimental tubes in the racks to account for position-related bias17.</li>
	<li>Prepare the following materials next to the plants grown hydroponically: microdissection scissors, a shallow dish with water, delicate task wipes, the 69 cap-less tubes containing the different chemicals.</li>
	<li>Repeat the following steps for each plant to be treated. The sunflower sprout will always remain in the seed holder.
	<ol>
		<li>Carefully lift the seed holder and rapidly dip the root into the shallow dish containing water.</li>
		<li>Cut the primary root underwater to prevent cavitation. The cut should occur 0.8-1 cm underneath the most basal end of the seed holder.</li>
		<li>Inset the freshly cut plant into one of the tubes containing the chemicals.</li>
		<li>If there are any drops of water on the cotyledons, gently dab them dry with a delicate task wipe. 
		NOTE: These four steps must be done as quickly as possible (10 min or less) to prevent inconsistencies in the kinetics study.</li>
	</ol>
	</li>
	<li>Move the plants under the thermal imaging camera and ensure that all plants are within the field of view of the camera. Adjust camera height and racks position as necessary.</li>
</ol>
7. Recording
<ol>
	<li>Focus the camera to the surface of the cotyledons by pressing Ctrl + Alt + A.</li>
	<li>Mouse over the red button and click on the Record a Movie option. A new window confirming the recording should open.</li>
	<li>Stop the recording 1-2 hours later. 
	NOTE: The protocol can be paused here.</li>
</ol>
8. Data collection
<ol>
	<li>Go to File | Open | Browse for the correct .SEQ file and open it.</li>
	<li>Stop playing the movie.</li>
	<li>On the left side of the main window, click on add a measurement cursor ROI (3x3 pixels) icon. ROI stands for Region of Interest.</li>
	<li>Mouse over the center of a cotyledon of the first plant and left-click once. The cursor 1 is now in place. Label the second cotyledon of the first plant by repeating the procedure. The order of the labels should be noted.</li>
	<li>Repeat the procedure. All plants should be labeled with two cursors.</li>
	<li>Click on the Edit ROIs icon. In the main window, left click and hold on the top left corner and scroll to the bottom right corner to select all the ROIs.</li>
	<li>Mouse over the Statistic Viewer icon and select Temporal Plot. A new window will open.</li>
	<li>Run the movie. A graph will fill with the data.</li>
	<li>In this window, click on the double arrow in the top right corner to open a new menu.</li>
	<li>Click on the Save icon. Save as X and Y values in the plot (.csv). Close the software once data has been exported.</li>
</ol>
9. Data analysis
<ol>
	<li>Open the .csv file using a data analysis software (e.g., Microsoft Excel). Note that the three first columns (A to C) provide information about the frame number, the absolute time and the relative time. The remaining columns give the temperature of each ROI over time.</li>
	<li>Decide on the nature of the statistical tool to be used; this decision depends on different factors including the experimental design. 
	NOTE: In our example, a standard score, or z-score, is calculated for each sample based on population mean and population standard deviation. For each sample, a p-value is then calculated from the z-score. This method allows the confirmation of the positive and negative controls as well as the identification of new compounds to be tested further.</li>
</ol>

<ol><li>Basu, S., Ramegowda, V., Kumar, A., Pereira, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plant+adaptation+to+drought+stress%5BTitle%5D)+AND+%22F1000Research%22%5BJournal%5D)">Plant adaptation to drought stress.</a> F1000Research. 5, (2016).</li>
<li>McLachlan, D. H., Kopischke, M., Robatzek, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gate+control%3A+guard+cell+regulation+by+microbial+stress%5BTitle%5D)+AND+%22The+New+Phytologist%22%5BJournal%5D)">Gate control: guard cell regulation by microbial stress.</a> The New Phytologist. 203 (4), 1049-1063 (2014).</li>
<li>Leung, J., Bazihizina, N., Mancuso, S., Valon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Revisiting+the+Plant%27s+Dilemma%5BTitle%5D)+AND+%22Molecular+Plant%22%5BJournal%5D)">Revisiting the Plant's Dilemma.</a> Molecular Plant. 9 (1), 7-9 (2016).</li>
<li>Macarron, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+high-throughput+screening+in+biomedical+research%5BTitle%5D)+AND+%22Nature+Reviews+Drug+Discovery%22%5BJournal%5D)">Impact of high-throughput screening in biomedical research.</a> Nature Reviews Drug Discovery. 10 (3), 188-195 (2011).</li>
<li>Wigglesworth, M. J., Murray, D. C., Blackett, C. J., Kossenjans, M., Nissink, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increasing+the+delivery+of+next+generation+therapeutics+from+high+throughput+screening+libraries%5BTitle%5D)+AND+%22Current+Opinion+in+Chemical+Biology%22%5BJournal%5D)">Increasing the delivery of next generation therapeutics from high throughput screening libraries.</a> Current Opinion in Chemical Biology. 26, 104-110 (2015).</li>
<li>Zhao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemical+genetic+interrogation+of+natural+variation+uncovers+a+molecule+that+is+glycoactivated%5BTitle%5D)+AND+%22Nature+Chemical+Biology%22%5BJournal%5D)">Chemical genetic interrogation of natural variation uncovers a molecule that is glycoactivated.</a> Nature Chemical Biology. 3 (11), 716-721 (2007).</li>
<li>Park, S. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Abscisic+acid+inhibits+type+2C+protein+phosphatases+via+the+PYR%2FPYL+family+of+START+proteins%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins.</a> Science. 324 (5930), 1068-1071 (2009).</li>
<li>Ma, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulators+of+PP2C+phosphatase+activity+function+as+abscisic+acid+sensors%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Regulators of PP2C phosphatase activity function as abscisic acid sensors.</a> Science. 324 (5930), 1064-1068 (2009).</li>
<li>Cao, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+ABA-mimicking+ligand+that+reduces+water+loss+and+promotes+drought+resistance+in+plants%5BTitle%5D)+AND+%22Cell+Research%22%5BJournal%5D)">An ABA-mimicking ligand that reduces water loss and promotes drought resistance in plants.</a> Cell Research. 23 (8), 1043-1054 (2013).</li>
<li>Okamoto, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activation+of+dimeric+ABA+receptors+elicits+guard+cell+closure%2C+ABA-regulated+gene+expression%2C+and+drought+tolerance%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Activation of dimeric ABA receptors elicits guard cell closure, ABA-regulated gene expression, and drought tolerance.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (29), 12132-12137 (2013).</li>
<li>Rodriguez, P. L., Lozano-Juste, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unnatural+agrochemical+ligands+for+engineered+abscisic+acid+receptors%5BTitle%5D)+AND+%22Trends+in+Plant+Science%22%5BJournal%5D)">Unnatural agrochemical ligands for engineered abscisic acid receptors.</a> Trends in Plant Science. 20 (6), 330-332 (2015).</li>
<li>Kim, T. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemical+genetics+reveals+negative+regulation+of+abscisic+acid+signaling+by+a+plant+immune+response+pathway%5BTitle%5D)+AND+%22Current+Biology%22%5BJournal%5D)">Chemical genetics reveals negative regulation of abscisic acid signaling by a plant immune response pathway.</a> Current Biology. 21 (11), 990-997 (2011).</li>
<li>Ito, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+Abscisic+Acid+Antagonists+Identified+with+Chemical+Array+Screening%5BTitle%5D)+AND+%22ChemBioChem%22%5BJournal%5D)">Novel Abscisic Acid Antagonists Identified with Chemical Array Screening.</a> ChemBioChem. 16 (17), 2471-2478 (2015).</li>
<li>Ye, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Novel+Chemical+Inhibitor+of+ABA+Signaling+Targets+All+ABA+Receptors%5BTitle%5D)+AND+%22Plant+Physiology%22%5BJournal%5D)">A Novel Chemical Inhibitor of ABA Signaling Targets All ABA Receptors.</a> Plant Physiology. 173 (4), 2356-2369 (2017).</li>
<li>Takeuchi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Designed+abscisic+acid+analogs+as+antagonists+of+PYL-PP2C+receptor+interactions%5BTitle%5D)+AND+%22Nature+Chemical+Biology%22%5BJournal%5D)">Designed abscisic acid analogs as antagonists of PYL-PP2C receptor interactions.</a> Nature Chemical Biology. 10 (6), 477-482 (2014).</li>
<li>Rauf, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Progress+in+modification+of+sunflower+oil+to+expand+its+industrial+value%5BTitle%5D)+AND+%22Journal+of+the+Science+of+Food+and+Agriculture%22%5BJournal%5D)">Progress in modification of sunflower oil to expand its industrial value.</a> Journal of the Science of Food and Agriculture. 97 (7), 1997-2006 (2017).</li>
<li>Caraus, I., Alsuwailem, A. A., Nadon, R., Makarenkov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detecting+and+overcoming+systematic+bias+in+high-throughput+screening+technologies%3A+a+comprehensive+review+of+practical+issues+and+methodological+solutions%5BTitle%5D)+AND+%22Briefings+in+Bioinformatics%22%5BJournal%5D)">Detecting and overcoming systematic bias in high-throughput screening technologies: a comprehensive review of practical issues and methodological solutions.</a> Briefings in Bioinformatics. 16 (6), 974-986 (2015).</li>
<li>Costa, J. M., Grant, O. M., Chaves, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thermography+to+explore+plant-environment+interactions%5BTitle%5D)+AND+%22Journal+of+Experimental+Botany%22%5BJournal%5D)">Thermography to explore plant-environment interactions.</a> Journal of Experimental Botany. 64 (13), 3937-3949 (2013).</li>
<li>Merlot, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+infrared+thermal+imaging+to+isolate+Arabidopsis+mutants+defective+in+stomatal+regulation%5BTitle%5D)+AND+%22The+Plant+Journal%22%5BJournal%5D)">Use of infrared thermal imaging to isolate Arabidopsis mutants defective in stomatal regulation.</a> The Plant Journal. 30 (5), 601-609 (2002).</li>
</ol>

The authors have nothing to disclose.

The number of compounds that can be tested on a given day mostly depends on (i) the environmentally controlled space available to grow the plants and to perform the screen, as well as (ii) the number of individuals who can be involved in step 6 of the protocol. We recommend the use of three experimental replicates to consolidate the interpretation of the results after statistical treatment. In a typical day, one to two individuals can screen 60 compounds in triplicates without difficulty by testing for example [60 chemic...

Stress tolerance in plants is a polygenic trait influenced by a variety of molecular, cellular, developmental and physiological features and mechanisms1. Plants in a fluctuating environment need to continuously modulate their stomatal movements to balance the photosynthetic demand for carbon while maintaining sufficient water and preventing pathogen invasion2; however, the mechanisms by which these trade-off &#34;decisions&#34; are made are poorly understood3. Introducing bioactive molecules into plants can modulate their physiology and help in probing new mechanisms of regulation.
The large-scale screening of small molecules is an effective strategy used in anti-cancer drug discovery and pharmacological assays to test the physiological effects of hundreds to thousands of molecules in a short period of time4,5. In plant biology, high-throughput screening has showed its effectiveness for example in the identification of the synthetic molecule pyrabactin6, as well as the discovery of the long-sought receptor of abscisic acid (ABA)7,8. Since then, agonists and antagonists of ABA receptors, and small molecules able to modulate the expression of ABA-inducible reporter genes have been identified9,10,11,12,13,14,15. High-throughput screening approaches currently available to identify small compounds that can modulate stomatal aperture have some drawbacks: (i) protocols revolving around the ABA signaling pathway may prevent the identification of novel ABA-independent mechanisms, and (ii) in vivo strategies used for the identification of bioactive small molecules rely primarily on their physiological effects on seed germination or seedling growth, and not on the regulation of plant transpiration per se.
Additionally, while there are many ways to treat plants with bioactive molecules, most of them are not well suited for a large-scale study of stomatal movement. Briefly, the three most common techniques are foliar application by spraying or dipping, treatment of the root system, and root irrigation. Foliar application is not compatible with the most common and rapid methodologies to measure stomatal aperture since the presence of droplets at the leaf surface interfere with large-scale data collection. The major limitations of root irrigation are the large sample volume requirements, the potential retention of the compounds by elements in the rhizosphere, and the reliance on active root uptake.
Here, we present a large-scale method to identify new compounds regulating plant transpiration that does not necessarily involve ABA- or known drought-responsive mechanisms and allows for efficient and reliable treatment of plants. In this system, Helianthus annuus plants are treated using a root feeding approach that consists of cutting the primary root of seedlings grown hydroponically and dipping the cut site into the sample solution. Once treated, the effect of each compound on the transpiration of plants is measured using an infrared thermal imaging camera. Since a major determinant of leaf surface temperature is the rate of evaporation from the leaf, thermal imaging data can be directly correlated to stomatal conductance. The relative change in foliar temperature following chemical treatment thus provides a direct means to quantify the plant transpiration.
H. annuus is one of the five largest oilseed crops in the world16 and discoveries made directly on this plant may facilitate future transfers of technology. In addition, H. annuus seedlings have large and flat cotyledons, as well a thick primary root, which was ideal for the development of this protocol. However, this method can be readily adapted to other plants and a variety of compounds.
This protocol can be used to effectively identify molecules able to trigger stomatal closure or promote stomatal opening, which has major implications for understanding the signals that regulate stomatal conductance and plant adaptation to environmental stresses.

<table><tbody><tr><td>1020 plastic growing trays without drain holes</td><td></td><td></td><td>Standard 10 x 20 inch trays</td></tr><tr><td>2.0 mL microtubes, capless</td><td>Genesee Scientific</td><td>22-283NC</td><td></td></tr><tr><td>Abscisic acid (ABA)</td><td>Sigma-Aldrich</td><td>A1049</td><td></td></tr><tr><td>Air pump</td><td>Active Aqua</td><td>AAPA7.8L</td><td>2 Outlets, 3W, 7.8 L/min</td></tr><tr><td>Airstones</td><td></td><td></td><td></td></tr><tr><td>Chemical compound library</td><td>MicroSource Discovery</td><td></td><td>Natural Product Collection</td></tr><tr><td>Creative Versa-Tool (wood burning tool)</td><td>Nasco</td><td>9724549</td><td></td></tr><tr><td>Dimethylsulfoxide (DMSO), plant cell culture tested</td><td>Sigma-Aldrich</td><td>D4540</td><td></td></tr><tr><td>Dwarf Sunspot Sunflower seeds</td><td>Outsidepride.com</td><td></td><td></td></tr><tr><td>Erythrosin B</td><td>Sigma-Aldrich</td><td>200964</td><td></td></tr><tr><td>Hydroponics fertilizer set (FloraBloom, FloraGrow, FloraMicro)</td><td>General Hydroponics</td><td>GL51GH1421.31.11</td><td></td></tr><tr><td>Kimwipes Delicate Task Wipers</td><td>Kimberly-Clark Professional</td><td>34155</td><td></td></tr><tr><td>Laptop</td><td>Dell</td><td></td><td></td></tr><tr><td>MES hydrate</td><td>Sigma-Aldrich</td><td>M2933</td><td></td></tr><tr><td>Microdissection scissors</td><td></td><td></td><td></td></tr><tr><td>Microsoft Excel</td><td>Microsoft</td><td></td><td></td></tr><tr><td>Potassium hydroxide (KOH)</td><td>Sigma-Aldrich</td><td>P5958</td><td></td></tr><tr><td>ResearchIR Software</td><td>FLIR</td><td></td><td></td></tr><tr><td>R-Tech Rigid Polystyrene Foam Board</td><td>Insulfoam</td><td></td><td></td></tr><tr><td>Seedholders</td><td>Araponics</td><td>N/A</td><td></td></tr><tr><td>Super Tub (plastic utility tub)</td><td>Maccourt</td><td>ST3608</td><td>36 x 24 x 8 inch tub used for hydroponics</td></tr><tr><td>T450sc LWIR (Long-Wave Infrared) Handheld Thermal Imaging Camera</td><td>FLIR</td><td>FLIR-T62101</td><td>Comes with required charging cable and USB cable needed to connect to laptop</td></tr><tr><td>Vermiculite</td><td></td><td></td><td></td></tr><tr><td>Water filter</td><td>SunSun</td><td>HW-304B Pro Canister Filter</td><td></td></tr></tbody></table>

identification of novel regulators of plant transpiration by large-scale thermal imaging screening in helianthus annuus

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water deficit or pathogens plays a crucial role. Identifying small molecules that regulate stomatal movement can therefore contribute to understanding the physiological basis by which plants adapt to their environment. Large-scale screening approaches that have been used to identify regulators of stomatal movement have potential limitations: some rely heavily on the abscisic acid (ABA) hormone signaling pathway, therefore excluding ABA-independent mechanisms, while others rely on the observation of indirect, long-term physiological effects such as plant growth and development. The screening method presented here allows the large-scale treatment of plants with a library of chemicals coupled with a direct quantification of their transpiration by thermal imaging. Since evaporation of water through transpiration results in leaf surface cooling, thermal imaging provides a non-invasive approach to investigate changes in stomatal conductance over time. In this protocol, Helianthus annuus seedlings are grown hydroponically and then treated by root feeding, in which the primary root is cut and dipped into the chemical being tested. Thermal imaging followed by statistical analysis of cotyledonary temperature changes over time allows for the identification of bioactive molecules modulating stomatal aperture. Our proof-of-concept experiments demonstrate that a chemical can be carried from the cut root to the cotyledon of the sunflower seedling within 10 minutes. In addition, when plants are treated with ABA as a positive control, an increase in leaf surface temperature can be detected within minutes. Our method thus allows the efficient and rapid identification of novel molecules regulating stomatal aperture.

An experiment using the red dye Erythrosine B (0.8 kDa) demonstrates the ability of chemicals to be visibly absorbed through a cut root into the cotyledons of a sunflower seedling within 10 minutes (Figure 1).
When plants are treated with ABA, an increase in leaf temperature is detected in sunflower cotyledons within minutes. This increase in leaf temperature is associated with a decrease in stomata...

We provide a method for identifying modulators of foliar transpiration by large-scale screening of a compound library.

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water ...

identification-novel-regulators-plant-transpiration-large-scale

Research

JoVE Journal

Biology

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

We describe a relatively rapid (30 min) and realistic method for simultaneously measurement of leaf hydraulic conductance (Kleaf) and stomatal conductance (gs) for transpiring excised leaves. The method can be modified to measure the light and dehydration responses of Kleaf and gs.

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method (EFM)

This protocol was developed to screen a large germplasm collection of the leafy vegetable lettuce (Lactuca sativa L.) for drought-tolerance, in order to identify a small candidate pool of lettuce for use in physiological, molecular, and genetic studies to identify underlying drought-tolerance traits along with breeding programs.

Semi-High Throughput Screening for Potential Drought-tolerance in Lettuce (Lactuca sativa) Germplasm Collections

Here we present a protocol that allows one to visualize sites of ice formation and avenues of ice propagation in plants utilizing high resolution infrared thermography (HRIT).

The Use of High-resolution Infrared Thermography (HRIT) for the Study of Ice Nucleation and Ice Propagation in Plants

A method was developed to determine the specific heat capacity and thermal conductivity of leaf tissue by non-invasive, contact-free near infrared laser probing, which requires less than 1 min per sample.

A Rapid Laser Probing Method Facilitates the Non-invasive and Contact-free Determination of Leaf Thermal Properties

This high-throughput, telemetric, whole-plant water relations gravimetric phenotyping method enables direct and simultaneous real-time measurements, as well as the analysis of multiple yield-related physiological traits involved in dynamic plant–environment interactions.

A Telemetric, Gravimetric Platform for Real-Time Physiological Phenotyping of Plant&ndash;Environment Interactions

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

We designed an image-based phenotyping protocol to determine the morphological and physiological responses to single and combined heat, drought, and waterlogging treatments. This approach enabled the identification of early, late, and recovery responses at a whole plant level, particularly above-ground parts, and highlighted the necessity of using multiple imaging sensors.

High Throughput Image-Based Phenotyping for Determining Morphological and Physiological Responses to Single and Combined Stresses in Potato

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

Large-Scale Screens of Metagenomic Libraries

Large-scale immunodetection of target proteins across the entire primate brain is possible by employing novel tissue embedding and sectioning methods combined with the use of creative apparatus for batch staining of multiple free-floating sections at a given time.

Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain

Discover a thermal imaging method for screening compounds that regulate plant transpiration in Helianthus annuus. This protocol enables researchers to identify novel regulators of stomatal behavior and water use efficiency.

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

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