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 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.
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 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.
1. Growing the plants
2. Set up of the hydroponic system
3. Transfer of seedlings to hydroponics and plant growth
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.
5. Set up the thermal imaging camera
6. Plant preparation and treatment
7. Recording
8. Data collection
9. Data analysis
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.
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 effective18,19 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.
The authors have nothing to disclose.
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).
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 |