Name: identification of novel regulators of plant transpiration by large-scale thermal imaging screening in helianthus annuus
Uploaded: 2020-01-30T15:00:00
Description: Watch this Scientific Journal Video about Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus at JoVE.com

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 ...

<ol>
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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abscisic+acid+inhibits+type+2C+protein+phosphatases+via+the+PYR/PYL+family+of+START+proteins.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulators+of+PP2C+phosphatase+activity+function+as+abscisic+acid+sensors.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ABA-mimicking+ligand+that+reduces+water+loss+and+promotes+drought+resistance+in+plants.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+dimeric+ABA+receptors+elicits+guard+cell+closure,+ABA-regulated+gene+expression,+and+drought+tolerance.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unnatural+agrochemical+ligands+for+engineered+abscisic+acid+receptors.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+genetics+reveals+negative+regulation+of+abscisic+acid+signaling+by+a+plant+immune+response+pathway.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Abscisic+Acid+Antagonists+Identified+with+Chemical+Array+Screening.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Chemical+Inhibitor+of+ABA+Signaling+Targets+All+ABA+Receptors.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designed+abscisic+acid+analogs+as+antagonists+of+PYL-PP2C+receptor+interactions.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+modification+of+sunflower+oil+to+expand+its+industrial+value.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+and+overcoming+systematic+bias+in+high-throughput+screening+technologies:+a+comprehensive+review+of+practical+issues+and+methodological+solutions.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermography+to+explore+plant-environment+interactions.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+infrared+thermal+imaging+to+isolate+Arabidopsis+mutants+defective+in+stomatal+regulation.">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 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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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

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...

Watch this Scientific Journal Video about Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus at JoVE.com

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

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

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 ...

This protocol uses thermal imaging to identify new compounds able to regulate plant transpiration. This technique is versatile in regard to the plant species that can be tested, the nature of the compound, and the type of imaging. To set up the plants for cultivation, first add a four centimeter thick layer of fine vermiculite to standard 10 by 20 inch plant trays with no holes and place seed holders two centimeters apart in the trays.Fill the seed holders with vermiculite and place one sunflower seed pointed end down into each holder so that half of the seed remains exposed. When all of the seeds have been plated, cover them with an additional two centimeters of fine vermiculite and mist with water from above. After one hour, cover the trays with lids and place the plants in a growth chamber in a greenhouse.To set up the hydroponics system, fill an appropriately sized container suitable for growing plants hydroponically with distilled water and add general hydroponics fertilizer as indicated by the manufacturer. Then use air and water pumps to aerate the resulting hydroponics solution with constant movement. To prepare the hydroponics floaters, cut a sheet of two centimeter thick expanded polystyrene foam to the dimensions of the container and use wood burning tool to make one to two centimeter diameter holes in the foam.Then place the floaters into the hydroponics system. Five days after planting, gently pull the seedlings out of the vermiculite. Immediately place the seedlings in a container of water for 30 minutes to remove excess vermiculite and to soften the remaining pericarps.When the emerging primary roots are visible, remove the pericarps by hand as necessary. Transfer the seedlings within the seed holders into the prepared polystyrene foam floaters in the hydroponics system. Grow the plants in hydroponics for two days.Let the chemicals from the small compound library thaw at room temperature. Label six capless two milliliter microtubes for the negative control treatment, three tubes for the ABA treatment, and the final 60 tubes for analysis of the effects of the 20 chemicals of interest in triplicate. Add 10 microliters of each chemical into each of the test tubes, 10 microliters of 10 millimolar ABA and dimethyl sulfoxide into the three ABA tubes, and 10 microliters of dimethyl sulfoxide into the six control tubes.Then carefully mix 990 microliters of 10 millimolar MES potassium hydroxide into each tube. Place the tubes into a tube rack with the positive and negative control and experimental tubes evenly distributed within the rack to account for position related bias. To set up the thermal imaging camera, first mount the camera on a copy stand and connect all of the cables to a laptop.Turn on the camera before turning on the laptop and open the thermal imaging analysis software. To adjust the recording parameters, roll the mouse over the Record button to allow selection of the wrench icon under Record Settings and select the appropriate record mode and options. For treatment of the seedlings, carefully lift the seed holder and rapidly dip the root into the shallow dish containing water.Cut the primary root of each seedling under water 0.8 to one centimeter beneath the most basal end of the seed holder to prevent capitation and insert the freshly cut plant into one tube of chemicals. When all of the seedlings have been transferred, place the plants under the thermal imaging camera and confirm that all of the plants are within the field of view of the camera. To focus the camera on the surface of the cotyledons, press Control Alt A and click Record a Movie.A new window confirming the recording will open. After one to two hours, stop the recording. For data analysis, open the correct seq files and pause the movie.Click the add a measurement cursor region of interest three by three pixels icon and move the mouse over the center of a cotyledon of the first plant. Left-click on the image to label the cotyledon. Label the second cotyledon of the first plant in the same manner.When all of the plants have been labeled with two cursors, click the edit regions of interest icon and left-click hold and scroll to select all of the regions of interest. Next, roll the mouse over the statistic viewer icon and select temporal plot. A new window will open.Run the movie, a graph will fill with the data. Then in the graph window, click the double arrow to open a new menu and click the save icon to save the data. The use of the red dye erythrosine B demonstrates the ability of chemicals to be visibly absorbed through a cut root into the cotyledons of a sunflower seedling within 10 minutes.When plants are treated with ABA, an increase in leaf temperature is detected in sunflower cotyledons within 30 minutes that is associated with the decrease in the stomatal aperture and the stomatal conductance. In addition, an increased foliar temperature is observed 15 minutes after treatment with 10 micromolar ABA and 20 minutes after treatment with five micromolar ABA demonstrating that the measurement of leaf temperature by thermal imaging is a good proxy for measuring stomatal aperture and conductance. In this representative experiment, a standard score-based statistical treatment allow the identification of chemicals that promote stomatal closure or opening.To measure the effects of compounds on transpiration, it is important to grow plants under optimal conditions of light, humidity, and temperature. This protocol can be applied to most dicots. It can also be used to evaluate the effect of new compounds on photosynthetic performance, for example by measuring chlorophyll fluorescence.

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