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

Summary

The protocol introduces a high-throughput method for measuring the relaxation of non-photochemical quenching by pulse amplitude modulated chlorophyll fluorometry. The method is applied to field-grown Glycine max and can be adapted to other species to screen for genetic diversity or breeding populations.

Abstract

Photosynthesis is not optimized in modern crop varieties, and therefore provides an opportunity for improvement. Speeding up the relaxation of non-photochemical quenching (NPQ) has proven to be an effective strategy to increase photosynthetic performance. However, the potential to breed for improved NPQ and a complete understanding of the genetic basis of NPQ relaxation is lacking due to limitations of oversampling and data collection from field-grown crop plants. Building on previous reports, we present a high-throughput assay for analysis of NPQ relaxation rates in Glycine max (soybean) using pulse amplitude modulated (PAM) chlorophyll fluorometry. Leaf disks are sampled from field-grown soybeans before transportation to a laboratory where NPQ relaxation is measured in a closed PAM-fluorometer. NPQ relaxation parameters are calculated by fitting a bi-exponential function to the measured NPQ values following a transition from high to low light. Using this method, it is possible to test hundreds of genotypes within a day. The procedure has the potential to screen mutant and diversity panels for variation in NPQ relaxation, and can therefore be applied to both fundamental and applied research questions.

Introduction

Photosynthesis consists of light absorption, primary electron transfer, energy stabilization, and the synthesis and transport of photosynthetic products¹. Understanding each step is vital to guide efforts to increase crop photosynthetic efficiency. Light affects the rate of photosynthesis, requiring balancing energy supply, in the form of photons, with demand for reducing equivalents. When supply exceeds demand, for example under high-light or during reduced CO₂ fixation caused by stomatal closure, build-up of reducing power increases the probability of reactive oxygen species formation with the potential to damage the photosynthetic apparatus and impair electron transport. Therefore, to prevent damage, plants have developed several photo-protective mechanisms, including detoxification of reactive oxygen species and non-photochemical quenching of the excited chlorophyll states (NPQ)².

Maintaining high rates of photosynthesis is challenging under a field environment. Seasonal and diurnal changes, along with environmental fluctuations such as wind-induced leaf movements and transient cloud cover, cause shifts in the amount and intensity of light received by plants for photosynthesis³. NPQ dissipates excess light energy and can help prevent photo-damage while allowing for sustained rates of photosynthesis at high-light⁴. However, prolonged NPQ during high- to low-light transitions continues to dissipate energy that could be used for carbon reduction⁵. As a result, speeding up the relaxation of NPQ can increase the efficiency of photosynthesis⁶, making NPQ relaxation an attractive target for crop improvement.

Pulse amplitude modulated chlorophyll fluorescence (PAM) analysis can be used to calculate NPQ from measurable parameters (Supplementary Table 1 and Supplementary Table 2)^7,8,9. This article focuses on determining NPQ relaxation rates in field-grown plants for the purpose of screening natural variation in germplasm. However, PAM chlorophyll fluorometry analysis can also be used for a wide variety of purposes, applied to species ranging from algae to higher plants, and is reviewed elsewhere^7,8,9.

In a dark-adapted leaf or cell, photosystem II (PSII) reaction centers are open to receive electrons and there is no NPQ. Switching on a low-intensity measuring light elicits chlorophyll fluorescence while avoiding electron transport through PSII. The recorded minimum fluorescence in this dark-adapted state is described by the parameter F_o. Applying a high-intensity light pulse to a dark-adapted leaf can rapidly reduce the first stable electron acceptor pool of quinones bound to the quinone A site. This temporarily blocks electron transfer capacity in PSII reaction centers, which are then said to be closed and unable to receive electrons from water-splitting. By using a short pulse duration, there is insufficient time to stimulate NPQ. The resulting chlorophyll fluorescence is equivalent to the maximum value obtainable in the absence of NPQ, or maximum fluorescence, F_m. The difference between minimal and maximal fluorescence is referred to as variable fluorescence, F_v. The maximum photochemical quantum yield of photosystem II (F_v/F_m) is calculated from these two parameters using the following equation:

F_v/F_m = (F_m-F_o)/F_m

This can provide an important indicator of photosystem function and stress. Turning on an actinic (photosynthetic) light stimulates non-photochemical quenching, and subsequent application of a saturating flash allows for the measurement of light-adapted maximal fluorescence, F_m'. By comparing the difference between dark and light-adapted maximum fluorescence, NPQ can be calculated according to the Stern-Volmer equation¹⁰:

NPQ = F_m/F_m' - 1

In higher plants, NPQ has been described as consisting of at least five distinct components, including qE, qT, qZ, qI and qH. The precise mechanisms involved in NPQ are not fully understood; however, qE is considered to be the major component of NPQ in most plants. Crucial factors for full engagement of qE have been found to include the build-up of a proton gradient across the thylakoid membrane, the activity of photosystem II subunit S^11,12, and de-epoxidated xanthophylls, antheraxanthin, lutein, and in particular zeaxanthin¹³. qE relaxes the fastest of any NPQ component (< 2 min)¹⁴, and reversible activation of qE is therefore particularly important for adaptation to shifting light intensities. A second slower phase of NPQ relaxation (~2-30 min) encompasses both qT, related to state transitions, and qZ, involving interconversion of zeaxanthin to violaxanthin¹⁵. Slow relaxing (> 30 min) of NPQ may include both photoinhibitory quenching (qI)¹⁶ and processes independent of photodamage^17,18, such as qH, which is sustained quenching in the peripheral antennae of PSII mediated by a plastid lipocalin protein^19,20.

NPQ increases during exposure to high light. Subsequent transfer to low light can result in downregulation of NPQ. The decay of fast, intermediate, and slow relaxing phases can be captured in the parameters of a bi-exponential function^15,21,22,23

NPQ = Aq1^(-t/τ1) + Aq2^(-t/τ2) + Aq3

The theoretical basis for the bi-exponential function is based on the assumption of first-order utilization of hypothetical quenchers, including qE (Aq1), the combined relaxation of qZ and qT (Aq2), with the corresponding time-constants τq1 and τq2, and long-term NPQ, which includes qI and photodamage independent processes (Aq3). As such, the bi-exponential function provides a more realistic representation of the multiple connected biological processes involved in quenching chlorophyll fluorescence compared to a simpler Hill equation which lacks a theoretical basis²⁴.

NPQ can be measured using a variety of commercially available PAM fluorometers^25,26, from simple hand-held devices²⁷ to more advanced closed systems²⁸. However, a limitation of several of these approaches is a relatively low throughput, which makes screening large collections of plants challenging without multiple devices and a team of researchers. To address this issue, McAusland et al. developed a procedure based on excised leaf tissue and used it to identify differences in chlorophyll fluorescence between two wheat cultivars²⁹. The attraction of this approach is that imaging leaf disks, taken from multiple plants with a single device, can facilitate screening hundreds of genotypes within a day. This makes it possible to assess variation in NPQ relaxation as part of genome wide association studies, or for screening breeding populations with the potential to increase crop photosynthetic efficiency and ultimately yield.

Building on the findings of McAusland et al.²⁹, we use PAM chlorophyll fluorescence analysis of leaf disks for high-throughput screening of NPQ relaxation rates in Glycine max (G. max; soybean). This protocol uses the CF Imager²⁵, which is comparable to other commercially available closed-PAM systems, such as the popular FluorCam²⁶. With a dark room for adaptation of samples, users can image 96-well plates, Petri dishes, and small plants. The key advantage of this approach is the increase in throughput afforded by using leaf disks compared to sequential analysis of individual plants. Herein we present representative results, and a method for sampling, measuring, and analysis of NPQ in field-grown plants.

Protocol

1. Seed planting

1. Choose a field site with fertile, well-drained, but not sandy soil, and with a pH of nearly 6.5. Mark out 1.2 m row plots with 0.75 m spacing by scoring the ground with a hoe.
2. Plant 50 seeds/m of G. max cv. IA3023 at 3 cm depth along each plot at the beginning of the growing season when soil temperatures are between 25 to 30 °C.
   NOTE: For the purpose of screening genetic diversity, it is expected that multiple different genotypes are grown and compared. Plant 2-5 rows per genotype arranged in a random block design. Consideration should be given to whether the climatic conditions suit the growth of soybeans, including soil type, temperature, and day length.

2. Collecting leaf samples from the field

1. Sample the plants in the field site 30 days after germination.
   NOTE: After 30 days soybean plants will be in the vegetative phase. The number of days post-germination before sampling is dependent on the biological question being addressed.
2. Fill wells of a 24-well plate up to 1/3^rd with distilled water. Label the lid and the side of the plate with the replicates to be sampled.
3. Select the youngest fully expanded leaf at the top of the plant to be sampled. Hold the leaf against a rubber stopper.
4. Press a #2 Humboldt cork borer through the leaf and twist to cut a disk while avoiding the midrib. Consecutively collect 5 disks from the same leaf for technical replicates. Take approximately 30% more leaf disks for each plot than required in case leaf tissue is damaged in transit or from sampling.
   ​NOTE: The number of biological replicates (plots or plants) and the number of technical replicates (leaf disks from the same plant or plot) can vary depending on the experimental design.
5. Push the leaf disks out of the cork borer into a single well of a 24-well plate using a cotton swab. Check that all leaf disks are floating in water. If not, gently move leaf disks sticking to the side of a well into a floating position with a cotton swab.
6. Move on to the next plot and repeat steps 2.3 to 2.5. Push the leaf disks out of the cork borer into a separate well in a 24-well plate using a cotton swab. Repeat this step to collect a third biological replicate.
7. Repeat step 2.6 until a complete 24-well plate has been sampled. Place a lid on and seal with a semi-transparent, flexible film. Store the plate out of direct sunlight, in a bag, box, or empty cooler (no ice). 

3. Preparing samples for analysis

1. Return to a clean laboratory space after sampling. Tap the lid of the sealed plate to dislodge leaf disks stuck to the lid during transport. Unwrap the film and remove the lid.
2. Transfer a leaf disk from the first position of the 24-well plate into a fresh 96-well plate, with the leaf disk facing flat down at the bottom of the well.
3. 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 disk to maintain humidity.
4. Take a second leaf disk from the first position of the 24-well plate and place face down in the next available position of the 96-well plate. Dip the remaining half of the nasal filter produced in step 3.3 in water and dab on a paper towel before inserting it in the well with the second leaf disk.
5. Repeat steps 3.3 to 3.4 for a third leaf disk from the first position of the 24-well plate.
6. Move on to the second position of the 24-well plate and repeat steps 3.3 to 3.5.
7. Place the lid on the plate when all wells have leaf disks and nasal aspirator filters inserted. Tape the top right corner to help orient the plate in the dark for imaging.
8. 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.
9. Place plates in a dark box or cabinet for a minimum of 30 min, to allow for relaxation of the first two phases of NPQ (qE, qT, qZ). Use a longer, dark incubation period of 1 h before imaging if long-term phases of NPQ are of interest.
10. Prepare an additional dummy plate for focusing during analysis. To do this, place a leaf disk in each of the corners of a fresh 96-well plate and one in the center. Secure leaf disks with nasal filters as done with previous plates. Seal the plate and incubate in the dark at room temperature (24 °C), ~50% relative humidity.

4. Measuring of non-photochemical quenching using chlorophyll fluorescence imager

1. Turn on the imager and open the imaging software. Click Settings > Protocol to open a window for the entry of steps in the PAM experimental protocol. The technical specifications of the machine are provided in Supplementary Table 3.
2. Set the program to start with a saturating pulse to measure the maximum quantum efficiency of dark-adapted photosynthesis by entering 20 s into the box: After a delay of. Click the box Apply Pulse and enter 1 into the box: This number of times.
3. Set the pulse PPFD to 6152, the pulse length to 800 ms, and check the box Take F' & Fm' images with all pulses. Click Insert After to add a second step to the protocol.
4. Enter 30 s into the box: After a delay of. Select the option Change actinic and enter 50 into the box: Actinic PPFD, to set light intensity in the chamber to 50 PPFD.
5. Click Insert After to add a new step to the protocol. Enter 150 s into the box: After a delay of, select Apply Pulse and enter 4 into the box: This number of times, to apply measuring pulses every 150 s, 4x in a row while the actinic light is held at 50 PPFD.
6. Complete information of the protocol is provided in Table 1, use steps 4.2 and 4.3 to change the light intensity and steps 4.4 and 4.5 to enter cycles of saturating pulses. Adjust the delay and light intensity for each step according to values provided in Table 1. Save your protocol as a .pcl file in a known location.
7. Turn off the light, place the dummy plate on the sample stage, and set the sample stage height so that the leaf disks are 140 mm above the base of the instrument. A dummy plate is used as light will repeatedly be flashed onto the plate when focusing; this will require re-dark adaption of any samples to be measured and may cause photodamage.
8. 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. Adjust the lens and exposure to bring the plate into focus.
9. 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.
10. Click the Map Image camera icon. Adjust the image exposure by opening or closing the aperture until the bar in the pop-up window is positioned in the green zone.
11. 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 grey and the background in blue.
12. Select the area/pixels of interest to include only the leaf disks by adjusting the histogram and gamma level from the modify image pull-down menu by right-clicking on the image.
13. Right-click on the image and select Delete High and Low Cuts (Color Map) to delete the light blue highlighted area. Right-click on the image and select Delete Strays (Heavy) to remove any pixels that are not touching at least three other pixels.
    NOTE: Any areas on the image that appear as isolated islands will be analyzed separately and included in the final data output. Image isolation and removal of stray pixels result in clean, comprehensible data.
14. 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.
15. Wait until the protocol is finished, click File > Save As, and save the data as an .igr file. Close the window by clicking on the red-cross in the top right of the window before you start running another sample plate.
16. Open a new file for the next sample by selecting File > New and repeat steps 4.10 to 4.15 until all plates have been measured.
    ​NOTE: It is advisable to measure plates within a period of 4 h or less to minimize the potential impact of circadian regulation on the results

5. Processing chlorophyll fluorescence data

1. Open the .igr file in the imaging software. Export the data by clicking File > Export to Folder to create a new folder with all the necessary files.
2. Copy the following three MATLAB files into the resulting folder: MapAndLabelDiscs.m (Supplementary File 1), ProcessFoFm.m (Supplementary File 2), and ProcessNPQdata.m (Supplementary File 3), and the R file: create_file_to_process.R (Supplementary File 4).
3. Open MapAndLabelDiscs.m (Supplementary File 1) in MATLAB and run. Save the map of numbered leaf disks generated in a pop-up window as a .png file to check leaf disk numbering later.
4. Open file ProcessFoFm.m (Supplementary File 2) in MATLAB and run to calculate F_o and F_m values for each leaf disk. Run ProcessNPQdata.m (Supplementary File 3) to calculate NPQ values at each time point.
5. Open the file create_file_to_process.R (Supplementary File 4) in Rstudio and add the date into the code on line 5. Add the plate number to line 8 in create_file_to_process.R.
6. Run create_file_to_process.R to consolidate F_v/F_m values and NPQ data into one file which is named after the data with the suffix -cf-summary.csv. Check leaf disk numbering in the cf-summary file from step 5.5 using the .png file from step 5.3. Add-in information relating to plot number and accession.
7. Open R script CF-data-processing_2.R (Supplementary File 5) and change line 13 to the working directory path. Change line 16 in CF-data-processing_2.R to the name of the file from step 5.5.
8. Change line 48 in CF-data-processing_2.R to the name of an output file. Run script CF-data-processing_2.R to re-format the data for curve fitting.
9. Open the script MatLab_NPQ_5_fit_model_v5.m (Supplementary File 6) in MATLAB and change line 5 to the name of the output file from step 5.8. Change line 85 in MatLab_NPQ_5_fit_model_v5.m to a new output file name. Run script MatLab_NPQ_5_fit_model_v5.m to calculate NPQ relaxation parameters.

Results

Figure 1A depicts a typical measurement of NPQ in field-grown soybean. Plants were grown in Urbana, IL (latitude 40.084604°, longitude -88.227952°) during summer 2021, with seeds planted on June 5th. 2021. The leaf discs were sampled after 30 days of planting seeds, and measurements were made with the protocol provided (Table 1). F_v/F_m and NPQ values were calculated for each leaf disk (Supplementary Table 4) and NPQ relaxation par...

Discussion

Careful choice and handling of leaf disks are critical to obtain reliable measurements of NPQ. First, damage to the tissue, such as rough handling with tweezers, will introduce stress, resulting in low values for the maximum quantum efficiency of photosynthesis. Non-stressed plants typically have F_v/F_m values of around 0.83¹⁸, with significant declines indicating a reduction in photosynthetic performance⁹. However, plants grown under ...

Materials

Name	Company	Catalog Number	Comments
24 well tissue culture plate	Fisher Scientific	FB012929	Country of Origin: United States of America
96 well tissue culture plate	Fisher Scientific	FB012931	Country of Origin: United States of America
Aluminum foil	Antylia Scientific	61018-56	Country of Origin: United States of America
Black marker pen	Sharpie	SAN30001	Country of Origin: United States of America
CF imager	Technologica Ltd.	N/A	chlorophyll fluorescence imager Country of Origin: United Kingdom
Cork-borer, 7mm	Humboldt Mfg Co	H9665	Country of Origin: United States of America
FluorImager V2.305 Software	Technologica Ltd.	N/A	imaging software Country of Origin: United Kingdom
iHank-Nose 100-Pack of Premium Nasal Aspirator Hygiene Filters	Amazon	B07P6XCTGV	Country of Origin: United States of America
Marker stakes	John Henry Company	KN0151	Country of Origin: United States of America
Paper scissors	VWR	82027-596	Country of Origin: United States of America
Parafilm	Bemis Company Inc.	S3-594-6	Semi -transparent flexible film Country of Origin: United States of America
Solid rubber stoppers	Fisher Scientific	14-130M	Country of Origin: United States of America