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.

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 time^4,5. 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 identified^{9,10,11,12,13,14,15}. 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.

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 rounded end of the seed should extend past the top of the seed holder.
5. 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.
6. Grow plants in a growth chamber or a greenhouse. Recommended conditions are a light intensity of 140 µmol photons·m^-2·s^-1 and a photoperiod of 16 h light at 22 °C and 9 h dark at 20 °C for 5 days.
   NOTE: Watering should not be necessary unless the surface becomes visibly dry.

2. Set up of the hydroponic system

1. 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.
2. 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.
3. Prepare the hydroponics floaters.
   1. 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.
   2. 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.

3. Transfer of seedlings to hydroponics and plant growth

1. 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.
2. Remove the pericarp walls by hand if needed in order to optimize the future expansion of the cotyledons.
3. Transfer the seedlings within the seed holders to the polystyrene foam floater. Grow the plants with a light intensity of 140 µmol photons·m^-2·s^-1, a relative humidity of 65% and a photoperiod of 16 h light at 22 °C and 9 h dark at 20 °C for 2 days.

4. Preparation prior to treatment

NOTE: This procedure is for testing 20 chemicals from a small compound library in triplicate, with 100 µM 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.

1. 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.
2. Remove the 96-well plate containing the small compounds from the -80 °C freezer. Thaw at room temperature.
3. Prepare 80 mL of 10 mM MES-KOH buffer adjusted to a pH of 6.2 with 1 M KOH.
4. 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 µM 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.
5. Transfer 10 µL of each chemical (10 mM in DMSO) into each of the three appropriately labeled tubes. Pipet 10 µL of 10 mM ABA dissolved in DMSO into three tubes and 10 µL of DMSO into the six control tubes.
   CAUTION: By nature, some compounds may cause serious health effects and users must take appropriate protective measures.
6. Add 990 µL 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.

5. Set up the thermal imaging camera

1. 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 °C to 25 °C), humidity (50% to 70%) and light quality (110 to 140 µmol photons·m^-2·s^-1) similar to those used to grow the plants.
2. 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).
3. Adjust the recording settings.
   1. 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.
   2. 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.

6. Plant preparation and treatment

1. 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.
2. Evenly distribute the positive and negative control tubes as well as the experimental tubes in the racks to account for position-related bias¹⁷.
3. 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.
4. Repeat the following steps for each plant to be treated. The sunflower sprout will always remain in the seed holder.
   1. Carefully lift the seed holder and rapidly dip the root into the shallow dish containing water.
   2. 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.
   3. Inset the freshly cut plant into one of the tubes containing the chemicals.
   4. 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.
5. 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.

7. Recording

1. Focus the camera to the surface of the cotyledons by pressing Ctrl + Alt + A.
2. Mouse over the red button and click on the Record a Movie option. A new window confirming the recording should open.
3. Stop the recording 1-2 hours later.
   NOTE: The protocol can be paused here.

8. Data collection

1. Go to File | Open | Browse for the correct .SEQ file and open it.
2. Stop playing the movie.
3. On the left side of the main window, click on add a measurement cursor ROI (3x3 pixels) icon. ROI stands for Region of Interest.
4. 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.
5. Repeat the procedure. All plants should be labeled with two cursors.
6. 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.
7. Mouse over the Statistic Viewer icon and select Temporal Plot. A new window will open.
8. Run the movie. A graph will fill with the data.
9. In this window, click on the double arrow in the top right corner to open a new menu.
10. Click on the Save icon. Save as X and Y values in the plot (.csv). Close the software once data has been exported.

9. Data analysis

1. 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.
2. 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.

Representative 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 stomatal aperture and stomatal conductance. Increased foliar temperature is observed 15 min after treatment with 10 µM ABA (p-value = 0.02) and 20 min with 5 µM ABA (p-value = 0.003) (Figure 2). Overall, these results show that measurements of leaf temperature by thermal imaging is a good proxy for measuring stomatal aperture and conductance.

Figure 3 shows a proof of concept experiment using a subset of 20 chemicals from the NatProd Collection with positive (100 µM ABA) and negative controls. In this representative experiment, a standard-score-based statistical treatment allows the identification of chemicals promoting stomatal closure or, while the assay should be optimized for that specific purpose, chemicals promoting stomatal opening. In the given example, a heat-map visualization of the standard scores allows the rapid identification of chemicals #02 and #16 as potential candidates.

Figure 4 summarizes the important steps of the workflow.

Figure 1: Effectiveness of the cut root feeding approach. (A) A seedling fed for 1 h with Erythrosine B in 10 mM MES-KOH (pH = 6.2) is visibly red (right image) compared to the control (left image). The images were taken after the cut root feeding followed by an overnight incubation in absolute ethanol to remove the natural plant pigments. Bar = 10 mm. (B) Accumulation of Erythrosine B in cotyledons over time. Erythrosine B can be detected by spectrophotometry in plant extracts from cotyledons 8 min (p-value = 0.032) after the transfer of cut-root sunflower seedlings to the dye. Error bars indicate SEM. * indicates p-value < 0.05 (n = 3). Please click here to view a larger version of this figure.

Figure 2: Relationships between leaf temperature, stomatal aperture and conductance to show sensitivity of the experimental design. (A) Representative image showing differences in leaf temperature between 100 µM ABA-treated (+ABA) and non-treated (Control) sunflower seedlings after 30 min visualized by thermal imaging. (B) Left: plants treated with 100 µM ABA for 30 min show an increased temperature compared to control plants (* indicates p-value < 0.01), n = 3). Right: measurements of stomatal aperture on epidermal peels from the same plants show a decrease in stomatal aperture (width/length) (* indicates p-value < 0.01, n = 3, number of stomata per plant ≈ 162). (C) Leaf conductance measured with a leaf porometer and coupled with leaf temperature measurements show that there is a strong correlation (Pearson's coefficient = -0.89, n = 6) between leaf surface temperature and stomatal conductance. Plants treated with 100 µM ABA for 30 min show an increased temperature and decreased conductance compared to control plants (n = 6). (D) Dose-response study shows reduced leaf temperature in plants treated with ABA concentrations as low as 5 µM after 20 min of treatment (p-value = 0.0037, n = 3). Error bars indicate SEM. Please click here to view a larger version of this figure.

Figure 3: Representative results from the screening of 20 chemical compounds. (A) Heatmap of Z-scores reflecting plant responses to 20 compounds tested in triplicates. Dark red and dark blue indicate confidence level of >99% for stomatal closure and opening, respectively. Six plants were treated with DMSO (control), three were treated with 100 µM ABA, and other plants were treated with 100 µM of chemical in triplicate. Plants responding to compound 16 (C#16) show a stomatal closure similar to that observed in ABA-treated plants. Two plants out of three treated with compound 02 (C#02) show a significant increase in stomatal opening. (B) Kinetics of the response of plants to compounds 02 and 16. Average changes in temperature over time are shown for plants responding to control treatment (n = 6), 100 µM ABA (n = 3) or 100 µM of each compound (n = 3). Error bars indicate SEM. Changes in temperature are consistently statistically significant after 10 min of ABA treatment (p-value = 0.026, n = 3), 15 min of treatment with C#16 (p-value = 0.030, n = 3) and 71 min of C#02 (p-value = 0.044, n = 3) compared to control. Fluctuations shared by all the samples is background noise due to the dynamic control of ambient temperature in the growth chamber. Please click here to view a larger version of this figure.

Figure 4: Summary of the screening workflow. Note that the images represent important steps and are independent from each other. Please click here to view a larger version of this figure.

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 chemicals + 6 negative (DMSO) controls + 3 positive (ABA) controls] in the morning, midday and afternoon.

This method relies on healthy seedlings with fully developed cotyledons. As the imaging occurs from above, an ideal seedling should show an angle of 90° between the hypocotyl and the cotyledonary blade in order to collect as much information as possible. This angle is mainly regulated by light and should therefore be optimized by adjusting the growing conditions. Our results show that it takes around 10 min for a chemical to reach the cotyledons and a few more minutes to respond to a chemical such as ABA. This observation makes step 6.4 the most time-sensitive step in the protocol. It is therefore critical to treat all the plants in a given assay in less than 15 min to avoid discrepancies between the plant responses. Among the external factors that passively affect foliar temperature measurements, ventilation is likely to introduce position-related biases or significant variability among replicates. Users should exercise caution by controlling ventilation flows and limit position-related biases by randomly distributing the samples before recording. To account for other potential factors, recording should be done under similar temperature, humidity, and light conditions to those used to grow the plants since any changes in these conditions may affect stomata closure and/or foliar temperature. Finally, a compound able to modulate stomatal closure should be evaluated for its toxicity. This holds particularly true if the compound triggers stomatal closure, as it is known to be an indirect consequence of intense stress experienced by the plant.

By providing an effective delivery method of bioactive molecules and a method to directly measure plant transpiration, this protocol addresses some of the drawbacks associated with current screening approaches, as mentioned in the introduction. Our protocol is not exclusive to sunflower seedlings and can be applied to most dicots with a hypocotyl to cotyledon angle of 90°. Thermal imaging of Arabidopsis cotyledons is effective^18,19 and our protocol could therefore be adapted to seedlings with similarly small cotyledons. In addition, chlorophyll fluorescence imaging could be used to measure photosynthetic performance in combination. While less time-effective, measurements of the transpiration-driven accumulation in cotyledons of Erythrosine B added to each chemical could potentially be used to evaluate transpiration rates if a thermal imaging camera is not available. In all, this large-scale screening method efficiently evaluates plant foliar response to bioactive molecules and is readily adaptable to a variety of applications.

Materials

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