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

This protocol uses thermal imaging to identify new compounds able to regulate plant transpiration. This technique is versatile in regard to the plant species that can be tested, the nature of the compound, and the type of imaging. To set up the plants for cultivation, first add a four centimeter thick layer of fine vermiculite to standard 10 by 20 inch plant trays with no holes and place seed holders two centimeters apart in the trays.

Fill the seed holders with vermiculite and place one sunflower seed pointed end down into each holder so that half of the seed remains exposed. When all of the seeds have been plated, cover them with an additional two centimeters of fine vermiculite and mist with water from above. After one hour, cover the trays with lids and place the plants in a growth chamber in a greenhouse.

To set up the hydroponics system, fill an appropriately sized container suitable for growing plants hydroponically with distilled water and add general hydroponics fertilizer as indicated by the manufacturer. Then use air and water pumps to aerate the resulting hydroponics solution with constant movement. To prepare the hydroponics floaters, cut a sheet of two centimeter thick expanded polystyrene foam to the dimensions of the container and use wood burning tool to make one to two centimeter diameter holes in the foam.

Then place the floaters into the hydroponics system. Five days after planting, gently pull the seedlings out of the vermiculite. Immediately place the seedlings in a container of water for 30 minutes to remove excess vermiculite and to soften the remaining pericarps.

When the emerging primary roots are visible, remove the pericarps by hand as necessary. Transfer the seedlings within the seed holders into the prepared polystyrene foam floaters in the hydroponics system. Grow the plants in hydroponics for two days.

Let the chemicals from the small compound library thaw at room temperature. Label six capless two milliliter microtubes for the negative control treatment, three tubes for the ABA treatment, and the final 60 tubes for analysis of the effects of the 20 chemicals of interest in triplicate. Add 10 microliters of each chemical into each of the test tubes, 10 microliters of 10 millimolar ABA and dimethyl sulfoxide into the three ABA tubes, and 10 microliters of dimethyl sulfoxide into the six control tubes.

Then carefully mix 990 microliters of 10 millimolar MES potassium hydroxide into each tube. Place the tubes into a tube rack with the positive and negative control and experimental tubes evenly distributed within the rack to account for position related bias. To set up the thermal imaging camera, first mount the camera on a copy stand and connect all of the cables to a laptop.

Turn on the camera before turning on the laptop and open the thermal imaging analysis software. To adjust the recording parameters, roll the mouse over the Record button to allow selection of the wrench icon under Record Settings and select the appropriate record mode and options. For treatment of the seedlings, carefully lift the seed holder and rapidly dip the root into the shallow dish containing water.

Cut the primary root of each seedling under water 0.8 to one centimeter beneath the most basal end of the seed holder to prevent capitation and insert the freshly cut plant into one tube of chemicals. When all of the seedlings have been transferred, place the plants under the thermal imaging camera and confirm that all of the plants are within the field of view of the camera. To focus the camera on the surface of the cotyledons, press Control Alt A and click Record a Movie.

A new window confirming the recording will open. After one to two hours, stop the recording. For data analysis, open the correct seq files and pause the movie.

Click the add a measurement cursor region of interest three by three pixels icon and move the mouse over the center of a cotyledon of the first plant. Left-click on the image to label the cotyledon. Label the second cotyledon of the first plant in the same manner.

When all of the plants have been labeled with two cursors, click the edit regions of interest icon and left-click hold and scroll to select all of the regions of interest. Next, roll the mouse over the statistic viewer icon and select temporal plot. A new window will open.

Run the movie, a graph will fill with the data. Then in the graph window, click the double arrow to open a new menu and click the save icon to save the data. The use of the red dye erythrosine B demonstrates the ability of chemicals to be visibly absorbed through a cut root into the cotyledons of a sunflower seedling within 10 minutes.

When plants are treated with ABA, an increase in leaf temperature is detected in sunflower cotyledons within 30 minutes that is associated with the decrease in the stomatal aperture and the stomatal conductance. In addition, an increased foliar temperature is observed 15 minutes after treatment with 10 micromolar ABA and 20 minutes after treatment with five micromolar ABA demonstrating that the measurement of leaf temperature by thermal imaging is a good proxy for measuring stomatal aperture and conductance. In this representative experiment, a standard score-based statistical treatment allow the identification of chemicals that promote stomatal closure or opening.

To measure the effects of compounds on transpiration, it is important to grow plants under optimal conditions of light, humidity, and temperature. This protocol can be applied to most dicots. It can also be used to evaluate the effect of new compounds on photosynthetic performance, for example by measuring chlorophyll fluorescence.