High-Throughput Analysis of Non-Photochemical Quenching in Crops Using Pulse Amplitude Modulated Chlorophyll Fluorometry

This method can provide an insight into the genetic variation in non-photochemical quenching available within genetic diversity panels or breeding populations. The main advantage of this technique is the potential to screen a large number of genotypes for variation in non-photochemical quenching within a single day. The protocol is presented for glycine max or soybean, but can be adapted to analyze other plant spaces from which lift discs can be collected.

For somebody performing the protocol for the first time, the key is handling the lift disc to prevent damage or over-saturating sponges with water. To begin, sample the plants in the field site 30 days after germination. Fill 1/3-level of distilled water in all the wells of a 24-well plate.

Label the lid and the side of the plate with the replicates to be sampled. Hold the youngest full-expanded leaf present at the top of the plant against a rubber stopper. Press a number two Humboldt cork borer through the leaf and twist to cut a disc, avoiding the mid-rib.

Consecutively collect five discs from the same leaf for technical replicates. Push the leaf discs out of the cork borer into a single well of a 24-well plate using a cotton swab and repeat this for a different plant of the same genotype. Check that all leaf discs are floating in the water.

If not, gently move leaf discs sticking to the side of a well into a floating position with a cotton swab. Move on to sampling discs from the following plot. Place the lid and seal with a semi-transparent flexible film.

after completing the sampling in the entire 24-well plate. Store the plate out of direct sunlight in a bag, box, or empty cooler. In a clean laboratory space, tap the lid of the sealed plate to dislodge leaf discs stuck to the lid during transport.

Unwrap the film and remove the lid. Transfer a leaf disc from the first position of the 24-well plate into a fresh 96-well plate with the leaf disc facing flat down at the bottom of the well. Cut a nasal aspirator filter in half.

Dip the resulting filter halfway into water and dab on a paper towel to remove excess liquid. Insert the filter into the well with the leaf disc to maintain humidity. Take a second leaf disc from the first position of the 24-well plate and place it face down in the next available position of the the 96-well plate.

Dip the remaining half of the nasal filter produced in water and dab it on a paper towel before inserting it in the well with the second leaf disc. Tape the top-right corner to help orient the plate in the dark for imaging. Seal plates with a semi-transparent flexible film and wrap the plate in aluminum foil.

Write the plot IDs and plate ID on the aluminum foil. Place plates in a dark box or cabinet for a minimum of 30 minutes for the relaxation of the first two phases of non-photochemical quenching. Use a prolonged dark incubation period of one hour before imaging if long-term phases of non-photochemical quenching are of interest.

Prepare an additional dummy plate for focusing during analysis by placing a leaf disc in each corner of a fresh 96-well plate and one in the center. Secure leaf discs with nasal filters as done with previous plates, seal the plate, and incubate in the dark at room temperature and 50%relative humidity. Turn on the imager and open the imaging software.

Click on Settings followed by Protocol to open a window for the entry of steps in the PAM experimental protocol. Set the technical specifications of the machine provided in the manuscript. Set the program to start with a saturating pulse to measure the maximum quantum efficiency of dark-adapted photosynthesis by entering 20 seconds into the box named After a delay of.

Click the box Apply pulse and enter 1 into the box titled This number of times. Set the pulse PPFD to 6, 152, the pulse length to 800 milliseconds, and check the box Take F prime and FM prime images with all pulses. Check Insert after to add a second step to the protocol.

Enter 30 seconds into the box titled After a delay of. Then, select the option Change actinic and enter 50 into the box titled Actinic PPFD to set light intensity to the chamber to 50 PPFD. Click Insert after to add a new step to the protocol and enter 150 seconds into the box titled After a delay of.

Select Apply pulse and enter 4 into the box titled This number of times to apply measuring pulses every 150 seconds four times in a row while the actinic light is held at 50 PPFD. Adjust the delay and light intensity for each step according to the values provided in the manuscript before saving the protocol as a pcl file in the desired location. Turn off the light and place the dummy plate face down on the sample stage so that the adaxial surface of the leaf points up towards the detector and that the sponge is facing down towards the platform.

Set the sample stage height so that the leaf discs are 140 millimeters above the base of the instrument. Click the connect/disconnect to imager camera and hardware camera icon to start the camera. Click the focus fluorescence symbol, represented by a red-colored, two-sided arrow icon with two green lines at the base, and adjust the lens and exposure to bring the plate into focus.

Click the focus fluorescence icon again to turn off the flashing light. Working in the dark, replace the dummy plate with the plate to be analyzed. Place it face down with the tape on the outside used to orient the top right corner and click the map image camera icon.

Adjust the image exposure by opening or closing the aperture until the bar in the popup window is positioned in the green zone. Click the Try Again button after each adjustment of the aperture until the exposure is correctly adjusted and the instrument takes an image. Right click on the image and select Apply image isolation to block out background signals.

The focused leaf area will be displayed in gray and the background in blue. Select the area or pixels of interest to include only the leaf discs by adjusting the histogram and gamma level from the Modify image pull down menu by right clicking on the image. Right click on the image and select Delete high and low cuts to delete the light blue highlighted areas.

Right click on the image and select Delete strays to remove any pixels not touching the last three other pixels. Any areas on the image that appear as isolated islands will be analyzed separately and included in the final data output. Click the run protocol icon to start the program.

A timer will appear at the bottom of the screen informing you how long the protocol has left to run. Wait until the protocol is finished. Click File and Save as to save the data as a igr file.

Close the window by clicking on the red cross in the top right of the window before starting another sample plate. To export the chlorophyll fluorescence data, open the igr file in the imaging software and click on File followed by Export to Folder to create a new folder with all the necessary files. A typical measurement of non-photochemical quenching in field-grown soybeans showed that after an initial saturating flash to determine Fv/Fm when the leaf discs were exposed to low light of 50 micromoles per meter per second, the non-photochemical quenching was less than one.

Furthermore, upon transfer to high light of 2, 000 micromoles per meter per second, non-photochemical quenching increased up to the maximum value of four after 15 minutes. A failed measurement showed a minimal increase in non-photochemical quenching upon transfer to high light. Orienting plates within the imager and a clearly defined plan for sampling and plating leaf discs is essential to ensure that the data is linked to the correct genotype.

This protocol can quantify the impact of environmental variables such as drought and shedding on non-photochemical quenching or evaluate non-photochemical quenching at different canopy levels to refine crop models.