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 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.</li>
	<li>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.</li>
	<li>Grow plants in a growth chamber or a greenhouse. Recommended conditions are a light intensity of 140 &#181;mol photons&#183;m-2&#183;s-1 and a photoperiod of 16 h light at 22 &#176;C and 9 h dark at 20 &#176;C for 5 days. 
	NOTE: Watering should not be necessary unless the surface becomes visibly dry.</li>
</ol>
2. Set up of the hydroponic system
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Prepare the hydroponics floaters.
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
3. Transfer of seedlings to hydroponics and plant growth
<ol>
	<li>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.</li>
	<li>Remove the pericarp walls by hand if needed in order to optimize the future expansion of the cotyledons.</li>
	<li>Transfer the seedlings within the seed holders to the polystyrene foam floater. Grow the plants with a light intensity of 140 &#181;mol photons&#183;m-2&#183;s-1, a relative humidity of 65% and a photoperiod of 16 h light at 22 &#176;C and 9 h dark at 20 &#176;C for 2 days.</li>
</ol>
4. Preparation prior to treatment
NOTE: This procedure is for testing 20 chemicals from a small compound library in triplicate, with 100 &#181;M 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.
<ol>
	<li>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.</li>
	<li>Remove the 96-well plate containing the small compounds from the -80 &#176;C freezer. Thaw at room temperature.</li>
	<li>Prepare 80 mL of 10 mM MES-KOH buffer adjusted to a pH of 6.2 with 1 M KOH.</li>
	<li>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 &#181;M 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.</li>
	<li>Transfer 10 &#181;L of each chemical (10 mM in DMSO) into each of the three appropriately labeled tubes. Pipet 10 &#181;L of 10 mM ABA dissolved in DMSO into three tubes and 10 &#181;L of DMSO into the six control tubes. 
	CAUTION: By nature, some compounds may cause serious health effects and users must take appropriate protective measures.</li>
	<li>Add 990 &#181;L 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.</li>
</ol>
5. Set up the thermal imaging camera
<ol>
	<li>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 &#176;C to 25 &#176;C), humidity (50% to 70%) and light quality (110 to 140 &#181;mol photons&#183;m-2&#183;s-1) similar to those used to grow the plants.</li>
	<li>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).</li>
	<li>Adjust the recording settings.
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
6. Plant preparation and treatment
<ol>
	<li>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.</li>
	<li>Evenly distribute the positive and negative control tubes as well as the experimental tubes in the racks to account for position-related bias17.</li>
	<li>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.</li>
	<li>Repeat the following steps for each plant to be treated. The sunflower sprout will always remain in the seed holder.
	<ol>
		<li>Carefully lift the seed holder and rapidly dip the root into the shallow dish containing water.</li>
		<li>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.</li>
		<li>Inset the freshly cut plant into one of the tubes containing the chemicals.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
</ol>
7. Recording
<ol>
	<li>Focus the camera to the surface of the cotyledons by pressing Ctrl + Alt + A.</li>
	<li>Mouse over the red button and click on the Record a Movie option. A new window confirming the recording should open.</li>
	<li>Stop the recording 1-2 hours later. 
	NOTE: The protocol can be paused here.</li>
</ol>
8. Data collection
<ol>
	<li>Go to File | Open | Browse for the correct .SEQ file and open it.</li>
	<li>Stop playing the movie.</li>
	<li>On the left side of the main window, click on add a measurement cursor ROI (3x3 pixels) icon. ROI stands for Region of Interest.</li>
	<li>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.</li>
	<li>Repeat the procedure. All plants should be labeled with two cursors.</li>
	<li>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.</li>
	<li>Mouse over the Statistic Viewer icon and select Temporal Plot. A new window will open.</li>
	<li>Run the movie. A graph will fill with the data.</li>
	<li>In this window, click on the double arrow in the top right corner to open a new menu.</li>
	<li>Click on the Save icon. Save as X and Y values in the plot (.csv). Close the software once data has been exported.</li>
</ol>
9. Data analysis
<ol>
	<li>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.</li>
	<li>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.</li>
</ol>

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The authors have nothing to disclose.

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><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...

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

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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5.9K Views.  Pomona College. Dit protocol maakt gebruik van thermische beeldvorming om nieuwe verbindingen te identificeren die de transpiratie van planten kunnen reguleren. Deze techniek is veelzijdig met betrekking tot de plantensoorten die kunnen worden getest, de aard van de verbinding, en het type beeldvorming. Om het opzetten van de planten voor de teelt, eerst een vier centimeter dikke laag fijn vermiculiet toe te voegen aan standaard 10 bij 20 inch plantenbakken zonder gaten en plaats zaadhouders twee centimeter ...

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

A Rapid Laser Probing Method Facilitates the Non-invasive and Contact-free Determination of Leaf Thermal Properties

Plants can produce valuable substances such as secondary metabolites and recombinant proteins. The purification of the latter from plant biomass can be streamlined by heat treatment (blanching). A blanching apparatus can be designed more precisely if the thermal properties of the leaves are known in detail, i.e., the specific heat capacity and thermal conductivity. The measurement of these properties is time consuming and labor intensive, and usually requires invasive methods that contact the sample directly. This can reduce the product yield and may be incompatible with containment requirements, e.g., in the context of good manufacturing practice. To address these issues, a non-invasive, contact-free method was developed that determines the specific heat capacity and thermal conductivity of an intact plant leaf in about one minute. The method involves the application of a short laser pulse of defined length and intensity to a small area of the leaf sample, causing a temperature increase that is measured using a near infrared sensor. The temperature increase is combined with known leaf properties (thickness and density) to determine the specific heat capacity. The thermal conductivity is then calculated based on the profile of the subsequent temperature decline, taking thermal radiation and convective heat transfer into account. The associated calculations and critical aspects of sample handling are discussed.

A method was developed to determine the specific heat capacity and thermal conductivity of leaf tissue by non-invasive, contact-free near infrared laser probing, which requires less than 1 min per sample.

Plants can produce valuable substances such as secondary metabolites and recombinant proteins. The purification of the latter from plant biomass can be ...

Biochemistry

Semi-High Throughput Screening for Potential Drought-tolerance in Lettuce (Lactuca sativa) Germplasm Collections

This protocol describes a method by which a large collection of the leafy green vegetable lettuce (Lactuca sativa L.) germplasm was screened for likely drought-tolerance traits. Fresh water availability for agricultural use is a growing concern across the United States as well as many regions of the world. Short-term drought events along with regulatory intervention in the regulation of water availability coupled with the looming threat of long-term climate shifts that may lead to reduced precipitation in many important agricultural regions has increased the need to hasten the development of crops adapted for improved water use efficiency in order to maintain or expand production in the coming years. This protocol is not meant as a step-by-step guide to identifying at either the physiological or molecular level drought-tolerance traits in lettuce, but rather is a method developed and refined through the screening of thousands of different lettuce varieties. The nature of this screen is based in part on the streamlined measurements focusing on only three water-stress indicators: leaf relative water content, wilt, and differential plant growth following drought-stress. The purpose of rapidly screening a large germplasm collection is to narrow the candidate pool to a point in which more intensive physiological, molecular, and genetic methods can be applied to identify specific drought-tolerant traits in either the lab or field. Candidates can also be directly incorporated into breeding programs as a source of drought-tolerance traits.

Semi-High Throughput Screening for Potential Drought-tolerance in Lettuce Lactuca sativa Germplasm Collections

This protocol was developed to screen a large germplasm collection of the leafy vegetable lettuce (Lactuca sativa L.) for drought-tolerance, in order to identify a small candidate pool of lettuce for use in physiological, molecular, and genetic studies to identify underlying drought-tolerance traits along with breeding programs.

This protocol describes a method by which a large collection of the leafy green vegetable lettuce (Lactuca sativa L.) germplasm was screened for likely ...

Environment

The Use of High-resolution Infrared Thermography (HRIT) for the Study of Ice Nucleation and Ice Propagation in Plants

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events often have devastating effects on agricultural production and can also play an important role in shaping community structure in natural populations of plants, especially in alpine, sub-arctic, and arctic ecosystems. Therefore, a better understanding of the freezing process in plants can play an important role in the development of methods of frost protection and understanding mechanisms of freeze avoidance. Here, we describe a protocol to visualize the freezing process in plants using high-resolution infrared thermography (HRIT). The use of this technology allows one to determine the primary sites of ice formation in plants, how ice propagates, and the presence of ice barriers. Furthermore, it allows one to examine the role of extrinsic and intrinsic nucleators in determining the temperature at which plants freeze and evaluate the ability of various compounds to either affect the freezing process or increase freezing tolerance. The use of HRIT allows one to visualize the many adaptations that have evolved in plants, which directly or indirectly impact the freezing process and ultimately enables plants to survive frost events.

The Use of High-resolution Infrared Thermography HRIT for the Study of Ice Nucleation and Ice Propagation in Plants

Here we present a protocol that allows one to visualize sites of ice formation and avenues of ice propagation in plants utilizing high resolution infrared thermography (HRIT).

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events ...

RGB and Spectral Root Imaging for Plant Phenotyping and Physiological Research: Experimental Setup and Imaging Protocols

Better understanding of plant root dynamics is essential to improve resource use efficiency of agricultural systems and increase the resistance of crop cultivars against environmental stresses. An experimental protocol is presented for RGB and hyperspectral imaging of root systems. The approach uses rhizoboxes where plants grow in natural soil over a longer time to observe fully developed root systems. Experimental settings are exemplified for assessing rhizobox plants under water stress and studying the role of roots. An RGB imaging setup is described for cheap and quick quantification of root development over time. Hyperspectral imaging improves root segmentation from the soil background compared to RGB color based thresholding. The particular strength of hyperspectral imaging is the acquisition of chemometric information on the root-soil system for functional understanding. This is demonstrated with high resolution water content mapping. Spectral imaging however is more complex in image acquisition, processing and analysis compared to the RGB approach. A combination of both methods can optimize a comprehensive assessment of the root system. Application examples integrating root and aboveground traits are given for the context of plant phenotyping and plant physiological research. Further improvement of root imaging can be obtained by optimizing RGB image quality with better illumination using different light sources and by extension of image analysis methods to infer on root zone properties from spectral data.

An experimental protocol is presented for assessment of soil grown plant root systems with RGB and hyperspectral imaging. Combination of RGB image time series with chemometric information from hyperspectral scans optimizes insights into plant root dynamics.

Better understanding of plant root dynamics is essential to improve resource use efficiency of agricultural systems and increase the resistance of crop ...

A Telemetric, Gravimetric Platform for Real-Time Physiological Phenotyping of Plant&ndash;Environment Interactions

Food security for the growing global population is a major concern. The data provided by genomic tools far exceeds the supply of phenotypic data, creating a knowledge gap. To meet the challenge of improving crops to feed the growing global population, this gap must be bridged.
Physiological traits are considered key functional traits in the context of responsiveness or sensitivity to environmental conditions. Many recently introduced high-throughput (HTP) phenotyping techniques are based on remote sensing or imaging and are capable of directly measuring morphological traits, but measure physiological parameters mainly indirectly.
This paper describes a method for direct physiological phenotyping that has several advantages for the functional phenotyping of plant&#8211;environment interactions. It helps users overcome the many challenges encountered in the use of load-cell gravimetric systems and pot experiments. The suggested techniques will enable users to distinguish between soil weight, plant weight and soil water content, providing a method for the continuous and simultaneous measurement of dynamic soil, plant and atmosphere conditions, alongside the measurement of key physiological traits. This method allows researchers to closely mimic field stress scenarios while taking into consideration the environment&#8217;s effects on the plants&#8217; physiology. This method also minimizes pot effects, which are one of the major problems in pre-field phenotyping. It includes a feed-back fertigation system that enables a truly randomized experimental design at a field-like plant density. This system detects the soil-water-content limiting threshold (&#952;) and allows for the translation of data into knowledge through the use of a real-time analytic tool and an online statistical resource. This method for the rapid and direct measurement of the physiological responses of multiple plants to a dynamic environment has great potential for use in screening for beneficial traits associated with responses to abiotic stress, in the context of pre-field breeding and crop improvement.

A Telemetric, Gravimetric Platform for Real-Time Physiological Phenotyping of Plant&#8211;Environment Interactions

This high-throughput, telemetric, whole-plant water relations gravimetric phenotyping method enables direct and simultaneous real-time measurements, as well as the analysis of multiple yield-related physiological traits involved in dynamic plant&#8211;environment interactions.

Food security for the growing global population is a major concern. The data provided by genomic tools far exceeds the supply of phenotypic data, creating ...

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method (EFM)

Water is a key resource, and the plant water transport system sets limits on maximum growth and drought tolerance. When plants open their stomata to achieve a high stomatal conductance (gs) to capture CO2 for photosynthesis, water is lost by transpiration1,2. Water evaporating from the airspaces is replaced from cell walls, in turn drawing water from the xylem of leaf veins, in turn drawing from xylem in the stems and roots. As water is pulled through the system, it experiences hydraulic resistance, creating tension throughout the system and a low leaf water potential (&Psi;leaf). The leaf itself is a critical bottleneck in the whole plant system, accounting for on average 30% of the plant hydraulic resistance3. Leaf hydraulic conductance (Kleaf = 1/ leaf hydraulic resistance) is the ratio of the water flow rate to the water potential gradient across the leaf, and summarizes the behavior of a complex system: water moves through the petiole and through several orders of veins, exits into the bundle sheath and passes through or around mesophyll cells before evaporating into the airspace and being transpired from the stomata. Kleaf is of strong interest as an important physiological trait to compare species, quantifying the effectiveness of the leaf structure and physiology for water transport, and a key variable to investigate for its relationship to variation in structure (e.g., in leaf venation architecture) and its impacts on photosynthetic gas exchange. Further, Kleaf responds strongly to the internal and external leaf environment3. Kleaf can increase dramatically with irradiance apparently due to changes in the expression and activation of aquaporins, the proteins involved in water transport through membranes4, and Kleaf declines strongly during drought, due to cavitation and/or collapse of xylem conduits, and/or loss of permeability in the extra-xylem tissues due to mesophyll and bundle sheath cell shrinkage or aquaporin deactivation5-10. Because Kleaf can constrain gs and photosynthetic rate across species in well watered conditions and during drought, and thus limit whole-plant performance they may possibly determine species distributions especially as droughts increase in frequency and severity11-14.
We present a simple method for simultaneous determination of Kleaf and gs on excised leaves. A transpiring leaf is connected by its petiole to tubing running to a water source on a balance. The loss of water from the balance is recorded to calculate the flow rate through the leaf. When steady state transpiration (E, mmol &bull; m-2 &bull; s-1) is reached, gs is determined by dividing by vapor pressure deficit, and Kleaf by dividing by the water potential driving force determined using a pressure chamber (Kleaf= E /- &Delta;&psi;leaf, MPa)15.
This method can be used to assess Kleaf responses to different irradiances and the vulnerability of Kleaf to dehydration14,16,17.

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method EFM

We describe a relatively rapid (30 min) and realistic method for simultaneously measurement of leaf hydraulic conductance (Kleaf) and stomatal conductance (gs) for transpiring excised leaves. The method can be modified to measure the light and dehydration responses of Kleaf and gs.

Water is a key resource, and the plant water transport system sets limits on maximum growth and drought tolerance. When plants open their stomata to ...

Using Flatbed Scanners to Collect High-resolution Time-lapsed Images of the Arabidopsis Root Gravitropic Response

Research efforts in biology increasingly require use of methodologies that enable high-volume collection of high-resolution data. A challenge laboratories can face is the development and attainment of these methods. Observation of phenotypes in a process of interest is a typical objective of research labs studying gene function and this is often achieved through image capture. A particular process that is amenable to observation using imaging approaches is the corrective growth of a seedling root that has been displaced from alignment with the gravity vector. Imaging platforms used to measure the root gravitropic response can be expensive, relatively low in throughput, and/or labor intensive. These issues have been addressed by developing a high-throughput image capture method using inexpensive, yet high-resolution, flatbed scanners. Using this method, images can be captured every few minutes at 4,800 dpi. The current setup enables collection of 216 individual responses per day. The image data collected is of ample quality for image analysis applications.

This protocol describes a process for rapid collection of images of Arabidopsis seedlings responding to a gravity stimulus using commercially-available flatbed scanners. The method allows for inexpensive, high-volume capture of high-resolution images amenable for downstream analysis algorithms.

Research efforts in biology increasingly require use of methodologies that enable high-volume collection of high-resolution data. A challenge laboratories ...

Automated Stomatal Response Measurement to Bacterial Invasion in Arabidopsis

Direct Observation and Automated Measurement of Stomatal Responses to Pseudomonas syringae pv. tomato DC3000 in Arabidopsis thaliana

Stomata are microscopic pores found in the plant leaf epidermis. Regulation of stomatal aperture is pivotal not only for balancing carbon dioxide uptake for photosynthesis and transpirational water loss but also for restricting bacterial invasion. While plants close stomata upon recognition of microbes, pathogenic bacteria, such as Pseudomonas syringae pv. tomato DC3000 (Pto), reopen the closed stomata to gain access into the leaf interior. In conventional assays for assessing stomatal responses to bacterial invasion, leaf epidermal peels, leaf discs, or detached leaves are floated on bacterial suspension, and then stomata are observed under a microscope followed by manual measurement of stomatal aperture. However, these assays are cumbersome and may not reflect stomatal responses to natural bacterial invasion in a leaf attached to the plant. Recently, a portable imaging device was developed that can observe stomata by pinching a leaf without detaching it from the plant, together with a deep learning-based image analysis pipeline designed to automatically measure stomatal aperture from leaf images captured by the device. Here, building on these technical advances, a new method to assess stomatal responses to bacterial invasion in Arabidopsis thaliana is introduced. This method consists of three simple steps: spray inoculation of Pto mimicking natural infection processes, direct observation of stomata on a leaf of the Pto-inoculated plant using the portable imaging device, and automated measurement of stomatal aperture by the image analysis pipeline. This method was successfully used to demonstrate stomatal closure and reopening during Pto invasion under conditions that closely mimic the natural plant-bacteria interaction.

Author Spotlight: Advancing Stomatal Research with Automated Aperture Measurement

Here, we present a simple method for direct observation and automated measurement of stomatal responses to bacterial invasion in Arabidopsis thaliana. This method leverages a portable stomatal imaging device, together with an image analysis pipeline designed for leaf images captured by the device.

Stomata are microscopic pores found in the plant leaf epidermis. Regulation of stomatal aperture is pivotal not only for balancing carbon dioxide uptake ...

Immunology and Infection

Relating Stomatal Conductance to Leaf Functional Traits

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, morphological leaf traits have the potential to summarize plants strategies in terms of water use efficiency, growth pattern and nutrient use. The leaf economics spectrum (LES) is a recognized framework in functional plant ecology and reflects a gradient of increasing specific leaf area (SLA), leaf nitrogen, phosphorus and cation content, and decreasing leaf dry matter content (LDMC) and carbon nitrogen ratio (CN). The LES describes different strategies ranging from that of short-lived leaves with high photosynthetic capacity per leaf mass to long-lived leaves with low mass-based carbon assimilation rates. However, traits that are not included in the LES might provide additional information on the species&#39; physiology, such as those related to stomatal control. Protocols are presented for a wide range of leaf functional traits, including traits of the LES, but also traits that are independent of the LES. In particular, a new method is introduced that relates the plants&#8217; regulatory behavior in stomatal conductance to vapor pressure deficit. The resulting parameters of stomatal regulation can then be compared to the LES and other plant functional traits. The results show that functional leaf traits of the LES were also valid predictors for the parameters of stomatal regulation. For example, leaf carbon concentration was positively related to the vapor pressure deficit (vpd) at the point of inflection and the maximum of the conductance-vpd curve. However, traits that are not included in the LES added information in explaining parameters of stomatal control: the vpd at the point of inflection of the conductance-vpd curve was lower for species with higher stomatal density and higher stomatal index. Overall, stomata and vein traits were more powerful predictors for explaining stomatal regulation than traits used in the LES.

Unraveling how physiology and morphology are linked allows a deeper understanding of mechanistic functioning of plant leaves. We present both a procedure to derive parameters of stomatal regulation from stomatal conductance measurements and correlations with traditional functional leaf traits.

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, ...

Potato Resilience Analysis Under Climate-Related Stresses

High Throughput Image-Based Phenotyping for Determining Morphological and Physiological Responses to Single and Combined Stresses in Potato

High throughput image-based phenotyping is a powerful tool to non-invasively determine the development and performance of plants under specific conditions over time. By using multiple imaging sensors, many traits of interest can be assessed, including plant biomass, photosynthetic efficiency, canopy temperature, and leaf reflectance indices. Plants are frequently exposed to multiple stresses under field conditions where severe heat waves, flooding, and drought events seriously threaten crop productivity. When stresses coincide, resulting effects on plants can be distinct due to synergistic or antagonistic interactions. To elucidate how potato plants respond to single and combined stresses that resemble naturally occurring stress scenarios, five different treatments were imposed on a selected potato cultivar (Solanum tuberosum L., cv. Lady Rosetta) at the onset of tuberization, i.e. control, drought, heat, waterlogging, and combinations of heat, drought, and waterlogging stresses. Our analysis shows that waterlogging stress had the most detrimental effect on plant performance, leading to fast and drastic physiological responses related to stomatal closure, including a reduction in the quantum yield and efficiency of photosystem II and an increase in canopy temperature and water index. Under heat and combined stress treatments, the relative growth rate was reduced in the early phase of stress. Under drought and combined stresses, plant volume and photosynthetic performance dropped with an increased temperature and stomata closure in the late phase of stress. The combination of optimized stress treatment under defined environmental conditions together with selected phenotyping protocols allowed to reveal&#160;the dynamics of morphological and physiological responses to single and combined stresses. Here, a useful tool is presented for plant researchers looking to identify plant traits indicative of resilience to several climate change-related stresses.

Author Spotlight: Unraveling Plant Responses to Abiotic Stresses Using the PlantScreen Robotic Platform

We designed an image-based phenotyping protocol to determine the morphological and physiological responses to single and combined heat, drought, and waterlogging treatments. This approach enabled the identification of early, late, and recovery responses at a whole plant level, particularly above-ground parts, and highlighted the necessity of using multiple imaging sensors.

High throughput image-based phenotyping is a powerful tool to non-invasively determine the development and performance of plants under specific conditions ...

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

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