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>
	<li>Basu, S., Ramegowda, V., Kumar, A., Pereira, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+adaptation+to+drought+stress.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gate+control:+guard+cell+regulation+by+microbial+stress.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revisiting+the+Plant's+Dilemma.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+high-throughput+screening+in+biomedical+research.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increasing+the+delivery+of+next+generation+therapeutics+from+high+throughput+screening+libraries.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+genetic+interrogation+of+natural+variation+uncovers+a+molecule+that+is+glycoactivated.">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/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 ...

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