This work is supported by the research project Realizing Increased Photosynthetic Efficiency (RIPE) that is funded by the Bill &#38; Melinda Gates Foundation, Foundation for Food and Agriculture Research, and the U.K. Foreign, Commonwealth &#38; Development Office under grant number OPP1172157.

1. Seed planting
<ol>
	<li>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.</li>
	<li>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 &#176;C. 
	NOTE: For the purpose of screening genetic diversity, it is expected that multiple different genotypes are grown and c...

<ol>
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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+atypical+short-chain+dehydrogenase-reductase+functions+in+the+relaxation+of+photoprotective+qH+in+Arabidopsis.">An atypical short-chain dehydrogenase-reductase functions in the relaxation of photoprotective qH in Arabidopsis.</a> Nature Plants. 6 (2), 154-166 (2020).</li><li>Dall'Osto, L., Cazzaniga, S., Wada, M., Bassi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+origin+of+a+slowly+reversible+fluorescence+decay+component+in+the+Arabidopsis+npq4+mutant.">On the origin of a slowly reversible fluorescence decay component in the Arabidopsis npq4 mutant.</a> Philosophical Transactions of the Royal Society B: Biological Sciences. 369 (1640), 20130221 (2014).</li><li>Chekanov, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-photochemical+quenching+in+the+cells+of+the+carotenogenic+chlorophyte+Haematococcus+lacustris+under+favorable+conditions+and+under+stress.">Non-photochemical quenching in the cells of the carotenogenic chlorophyte Haematococcus lacustris under favorable conditions and under stress.</a> Biochimica et Biophysica Acta (BBA) - General Subjects. 1863 (10), 1429-1442 (2019).</li><li>Allorent, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dual+strategy+to+cope+with+high+light+in+Chlamydomonas+reinhardtii.">A dual strategy to cope with high light in Chlamydomonas reinhardtii.</a> The Plant Cell. 25 (2), 545-557 (2013).</li><li>Holzwarth, A. R., Lenk, D., Jahns, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+analysis+of+non-photochemical+chlorophyll+fluorescence+quenching+curves:+I.+Theoretical+considerations.">On the analysis of non-photochemical chlorophyll fluorescence quenching curves: I. Theoretical considerations.</a> Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1827 (6), 786-792 (2013).</li><li>Barbagallo, R. P., Oxborough, K., Pallett, K. E., Baker, N. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+environmental+photosynthetic+imaging+reveals+emergent+phenotypes.">Dynamic environmental photosynthetic imaging reveals emergent phenotypes.</a> Cell Systems. 2 (6), 365-377 (2016).</li><li>McAusland, L., Atkinson, J. A., Lawson, T., Murchie, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+procedure+utilising+chlorophyll+fluorescence+imaging+to+phenotype+dynamic+photosynthesis+and+photoprotection+in+leaves+under+controlled+gaseous+conditions.">High throughput procedure utilising chlorophyll fluorescence imaging to phenotype dynamic photosynthesis and photoprotection in leaves under controlled gaseous conditions.</a> Plant Methods. 15 (1), 109 (2019).</li><li>Woo, N. S., Badger, M. R., Pogson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid,+non-invasive+procedure+for+quantitative+assessment+of+drought+survival+using+chlorophyll+fluorescence.">A rapid, non-invasive procedure for quantitative assessment of drought survival using chlorophyll fluorescence.</a> Plant Methods. 4 (1), 27 (2008).</li><li>Bielczynski, L. W., &#321;&#261;cki, M. K., Hoefnagels, I., Gambin, A., Croce, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leaf+and+plant+age+affects+photosynthetic+performance+and+photoprotective+capacity.">Leaf and plant age affects photosynthetic performance and photoprotective capacity.</a> Plant Physiology. 175 (4), 1634-1648 (2017).</li><li>Niyogi, K. K., Truong, T. 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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 Fv/Fm values of around 0.8318, with significant declines indicating a reduction in photosynthetic performance9. However, plants grown under ...

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 elsewhere7,8,9.
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 Fo. 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, Fm. The difference between minimal and maximal fluorescence is referred to as variable fluorescence, Fv. The maximum photochemical quantum yield of photosystem II (Fv/Fm) is calculated from these two parameters using the following equation:
Fv/Fm = (Fm-Fo)/Fm
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, Fm&#39;. By comparing the difference between dark and light-adapted maximum fluorescence, NPQ can be calculated according to the Stern-Volmer equation10:
NPQ = Fm/Fm&#39; - 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 S11,12, and de-epoxidated xanthophylls, antheraxanthin, lutein, and in particular zeaxanthin13. qE relaxes the fastest of any NPQ component (&#60; 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 (&#62; 30 min) of NPQ may include both photoinhibitory quenching (qI)16 and processes independent of photodamage17,18, such as qH, which is sustained quenching in the peripheral antennae of PSII mediated by a plastid lipocalin protein19,20.
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 function15,21,22,23
NPQ = Aq1(-t/&#964;1) + Aq2(-t/&#964;2) + 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 &#964;q1 and &#964;q2, 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 fluorometers25,26, 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.

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			<td>24 well tissue culture plate</td>
			<td>Fisher Scientific</td>
			<td>FB012929</td>
			<td>Country of Origin: United States of America</td>
		</tr>
		<tr>
			<td>96 well tissue culture plate</td>
			<td>Fisher Scientific</td>
			<td>FB012931</td>
			<td>Country of Origin: United States of America</td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>Antylia Scientific</td>
			<td>&#160;61018-56</td>
			<td>Country of Origin: United States of America</td>
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			<td>Black marker pen</td>
			<td>Sharpie</td>
			<td>SAN30001</td>
			<td>Country of Origin: United States of America</td>
		</tr>
		<tr>
			<td>CF imager</td>
			<td>Technologica Ltd.</td>
			<td>N/A</td>
			<td>chlorophyll fluorescence imager 
			Country of Origin: United Kingdom</td>
		</tr>
		<tr>
			<td>Cork-borer, 7mm</td>
			<td>Humboldt Mfg Co</td>
			<td>H9665</td>
			<td>Country of Origin: United States of America</td>
		</tr>
		<tr>
			<td>FluorImager V2.305 Software</td>
			<td>Technologica Ltd.</td>
			<td>N/A</td>
			<td>imaging software 
			Country of Origin: United Kingdom</td>
		</tr>
		<tr>
			<td>iHank-Nose 100-Pack of Premium Nasal Aspirator Hygiene Filters</td>
			<td>Amazon</td>
			<td>&#160;B07P6XCTGV</td>
			<td>Country of Origin: United States of America</td>
		</tr>
		<tr>
			<td>Marker stakes</td>
			<td>John Henry Company</td>
			<td>KN0151</td>
			<td>Country of Origin: United States of America</td>
		</tr>
		<tr>
			<td>Paper scissors</td>
			<td>VWR</td>
			<td>82027-596</td>
			<td>Country of Origin: United States of America</td>
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			<td>Parafilm</td>
			<td>Bemis Company Inc.</td>
			<td>&#160;S3-594-6</td>
			<td>Semi -transparent flexible film 
			Country of Origin: United States of America</td>
		</tr>
		<tr>
			<td>Solid rubber stoppers</td>
			<td>Fisher Scientific</td>
			<td>14-130M</td>
			<td>Country of Origin: United States of America</td>
		</tr>
	</tbody>
</table>

high-throughput analysis of non-photochemical quenching in crops using pulse amplitude modulated chlorophyll fluorometry

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.

Figure 1A depicts a typical measurement of NPQ in field-grown soybean. Plants were grown in Urbana, IL (latitude 40.084604&#176;, longitude -88.227952&#176;) 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). Fv/Fm and NPQ values were calculated for each leaf disk (Supplementary Table 4) and NPQ relaxation par...

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

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.

Photosynthesis is not optimized in modern crop varieties, and therefore provides an opportunity for improvement. Speeding up the relaxation of ...

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.

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