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

Summary

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

Abstract

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.

Introduction

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 "decisions" are made are poorly understood³. Introducing bioactive molecules into plants can modulate their physiology and help in probing new mechanisms of regulation.

Protocol

1. Growing the plants

1. 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.
2. Place the seed holders (see Table of Materials) 2 cm apart in the plant trays.
3. Fill seed holders with vermiculite.
4. 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 ....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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