Name: high-throughput analysis of non-photochemical quenching in crops using pulse amplitude modulated chlorophyll fluorometry
Uploaded: 2022-07-06T12:50:00.000+00:00
Duration: 10 min 8 s
Description: Watch this Scientific Journal Video about High-Throughput Analysis of Non-Photochemical Quenching in Crops Using Pulse Amplitude Modulated Chlorophyll Fluorometry at JoVE.com

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 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.</li>
</ol>
2. Collecting leaf samples from the field
<ol>
	<li>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.</li>
	<li>Fill wells of a 24-well plate up to 1/3rd with distilled water. Label the lid and the side of the plate with the replicates to be sampled.</li>
	<li>Select the youngest fully expanded leaf at the top of the plant to be sampled. Hold the leaf against a rubber stopper.</li>
	<li>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. 
	&#8203;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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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).&#160;</li>
</ol>
3. Preparing samples for analysis
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Repeat steps 3.3 to 3.4 for a third leaf disk from the first position of the 24-well plate.</li>
	<li>Move on to the second position of the 24-well plate and repeat steps 3.3 to 3.5.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#176;C), ~50% relative humidity.</li>
</ol>
4. Measuring of non-photochemical quenching using chlorophyll fluorescence imager
<ol>
	<li>Turn on the imager and open the imaging software. Click Settings &#62; 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.</li>
	<li>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.</li>
	<li>Set the pulse PPFD to 6152, the pulse length to 800 ms, and check the box Take F&#39; &#38; Fm&#39; images with all pulses. Click Insert After to add a second step to the protocol.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Wait until the protocol is finished, click File &#62; 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.</li>
	<li>Open a new file for the next sample by selecting File &#62; New and repeat steps 4.10 to 4.15 until all plates have been measured. 
	&#8203;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</li>
</ol>
5. Processing chlorophyll fluorescence data
<ol>
	<li>Open the .igr file in the imaging software. Export the data by clicking File &#62; Export to Folder to create a new folder with all the necessary files.</li>
	<li>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).</li>
	<li>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.</li>
	<li>Open file ProcessFoFm.m (Supplementary File 2) in MATLAB and run to calculate Fo and Fm values for each leaf disk. Run ProcessNPQdata.m (Supplementary File 3) to calculate NPQ values at each time point.</li>
	<li>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.</li>
	<li>Run create_file_to_process.R to consolidate Fv/Fm 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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>

<ol>
	<li>Blankenship, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Mechanisms of Photosynthesis. , (2021).</li><li>Murchie, E. H., Niyogi, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulation+of+photoprotection+to+improve+plant+photosynthesis.">Manipulation of photoprotection to improve plant photosynthesis.</a> Plant Physiology. 155 (1), 86-92 (2011).</li><li>Horton, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+light+harvesting+and+photoprotection:+molecular+mechanisms+and+physiological+consequences.">Optimization of light harvesting and photoprotection: molecular mechanisms and physiological consequences.</a> Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. 367 (1608), 3455-3465 (2012).</li><li>Slattery, R. A., Ort, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photosynthesis:+photosynthetic+efficiency+improvement.">Photosynthesis: photosynthetic efficiency improvement.</a> Encyclopedia of Biological Chemistry III (Third Edition). , 256-267 (2021).</li><li>Zhu, X. -. G., Ort, D. R., Whitmarsh, J., Long, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+slow+reversibility+of+photosystem+II+thermal+energy+dissipation+on+transfer+from+high+to+low+light+may+cause+large+losses+in+carbon+gain+by+crop+canopies:+a+theoretical+analysis.">The slow reversibility of photosystem II thermal energy dissipation on transfer from high to low light may cause large losses in carbon gain by crop canopies: a theoretical analysis.</a> Journal of Experimental Botany. 55 (400), 1167-1175 (2004).</li><li>Kromdijk, J., 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=Improving+photosynthesis+and+crop+productivity+by+accelerating+recovery+from+photoprotection.">Improving photosynthesis and crop productivity by accelerating recovery from photoprotection.</a> Science. 354 (6314), 857-861 (2016).</li><li>Maxwell, K., Johnson, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlorophyll+fluorescence-a+practical+guide.">Chlorophyll fluorescence-a practical guide.</a> Journal of Experimental Botany. 51 (345), 659-668 (2000).</li><li>Murchie, E. H., Lawson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlorophyll+fluorescence+analysis:+a+guide+to+good+practice+and+understanding+some+new+applications.">Chlorophyll fluorescence analysis: a guide to good practice and understanding some new applications.</a> Journal of Experimental Botany. 64 (13), 3983-3998 (2013).</li><li>Baker, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlorophyll+fluorescence:+a+probe+of+photosynthesis+in+vivo.">Chlorophyll fluorescence: a probe of photosynthesis in vivo.</a> Annual Review of Plant Biology. 59 (1), 89-113 (2008).</li><li>Bilger, W., Bj&#246;rkman, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+xanthophyll+cycle+in+photoprotection+elucidated+by+measurements+of+light-induced+absorbance+changes,+fluorescence+and+photosynthesis+in+leaves+of+Hedera+canariensis.">Role of the xanthophyll cycle in photoprotection elucidated by measurements of light-induced absorbance changes, fluorescence and photosynthesis in leaves of Hedera canariensis.</a> Photosynthesis Research. 25 (3), 173-185 (1990).</li><li>Li, X. -. P., 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+pigment-binding+protein+essential+for+regulation+of+photosynthetic+light+harvesting.">A pigment-binding protein essential for regulation of photosynthetic light harvesting.</a> Nature. 403 (6768), 391-395 (2000).</li><li>Niyogi, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PHOTOPROTECTION+REVISITED:+genetic+and+molecular+approaches.">PHOTOPROTECTION REVISITED: genetic and molecular approaches.</a> Annual Review of Plant Physiology and Plant Molecular Biology. 50 (1), 333-359 (1999).</li><li>Ruban, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonphotochemical+Chlorophyll+fluorescence+quenching:+mechanism+and+effectiveness+in+protecting+plants+from+photodamage.">Nonphotochemical Chlorophyll fluorescence quenching: mechanism and effectiveness in protecting plants from photodamage.</a> Plant physiology. 170 (4), 1903-1916 (2016).</li><li>Krause, G. H., Vernotte, C., Briantais, J. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoinduced+quenching+of+chlorophyll+fluorescence+in+intact+chloroplasts+and+algae.+Resolution+into+two+components.">Photoinduced quenching of chlorophyll fluorescence in intact chloroplasts and algae. Resolution into two components.</a> Biochimica et Biophysica Acta (BBA) - Bioenergetics. 679 (1), 116-124 (1982).</li><li>Nilkens, M., 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=Identification+of+a+slowly+inducible+zeaxanthin-dependent+component+of+non-photochemical+quenching+of+chlorophyll+fluorescence+generated+under+steady-state+conditions+in+Arabidopsis.">Identification of a slowly inducible zeaxanthin-dependent component of non-photochemical quenching of chlorophyll fluorescence generated under steady-state conditions in Arabidopsis.</a> Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1797 (4), 466-475 (2010).</li><li>Krause, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoinhibition+of+photosynthesis.+An+evaluation+of+damaging+and+protective+mechanisms.">Photoinhibition of photosynthesis. An evaluation of damaging and protective mechanisms.</a> Physiologia Plantarum. 74 (3), 566-574 (1988).</li><li>Brooks, M. D., Sylak-Glassman, E. J., Fleming, G. R., Niyogi, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+thioredoxin-like/&#946;-propeller+protein+maintains+the+efficiency+of+light+harvesting+in+Arabidopsis.">A thioredoxin-like/&#946;-propeller protein maintains the efficiency of light harvesting in Arabidopsis.</a> Proceedings of the National Academy of Sciences. 110 (29), 2733-2740 (2013).</li><li>Demmig, B., Bj&#246;rkman, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+effect+of+excessive+light+on+chlorophyll+fluorescence+(77K)+and+photon+yield+of+O2+evolution+in+leaves+of+higher+plants.">Comparison of the effect of excessive light on chlorophyll fluorescence (77K) and photon yield of O2 evolution in leaves of higher plants.</a> Planta. 171 (2), 171-184 (1987).</li><li>Malno&#235;, 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=The+plastid+lipocalin+LCNP+is+required+for+sustained+photoprotective+energy+dissipation+in+Arabidopsis.">The plastid lipocalin LCNP is required for sustained photoprotective energy dissipation in Arabidopsis.</a> The Plant Cell. 30 (1), 196-208 (2018).</li><li>Amstutz, C. 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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+noninvasive+screening+for+perturbations+of+metabolism+and+plant+growth+using+chlorophyll+fluorescence+imaging.">Rapid, noninvasive screening for perturbations of metabolism and plant growth using chlorophyll fluorescence imaging.</a> Plant physiology. 132 (2), 485-493 (2003).</li><li>Nedbal, L., Soukupov&#225;, J., Kaftan, D., Whitmarsh, J., Trt&#237;lek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+imaging+of+chlorophyll+fluorescence+using+modulated+light.">Kinetic imaging of chlorophyll fluorescence using modulated light.</a> Photosynthesis Research. 66 (1), 3-12 (2000).</li><li>Kuhlgert, S., 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=MultispeQ+Beta:+a+tool+for+large-scale+plant+phenotyping+connected+to+the+open+PhotosynQ+network.">MultispeQ Beta: a tool for large-scale plant phenotyping connected to the open PhotosynQ network.</a> Royal Society Open Science. 3 (10), 160592 (2016).</li><li>Cruz, J. 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. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+flexible+non-photochemical+quenching+mechanisms+that+regulate+light+harvesting+in+oxygenic+photosynthesis.">Evolution of flexible non-photochemical quenching mechanisms that regulate light harvesting in oxygenic photosynthesis.</a> Physiology and metabolism. 16 (3), 307-314 (2013).</li><li>Delosme, R., Olive, J., Wollman, F. -. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+light+energy+distribution+upon+state+transitions:+an+in+vivo+photoacoustic+study+of+the+wild+type+and+photosynthesis+mutants+from+Chlamydomonas+reinhardtii.">Changes in light energy distribution upon state transitions: an in vivo photoacoustic study of the wild type and photosynthesis mutants from Chlamydomonas reinhardtii.</a> Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1273 (2), 150-158 (1996).</li><li>Quick, W. P., Stitt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+examination+of+factors+contributing+to+non-photochemical+quenching+of+chlorophyll+fluorescence+in+barley+leaves.">An examination of factors contributing to non-photochemical quenching of chlorophyll fluorescence in barley leaves.</a> Biochimica et Biophysica Acta (BBA) - Bioenergetics. 977 (3), 287-296 (1989).</li><li>Horton, P., Hague, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+induction+of+chlorophyll+fluorescence+in+isolated+barley+protoplasts.+IV.+Resolution+of+non-photochemical+quenching.">Studies on the induction of chlorophyll fluorescence in isolated barley protoplasts. IV. Resolution of non-photochemical quenching.</a> Biochimica et Biophysica Acta (BBA) - Bioenergetics. 932, 107-115 (1988).</li></ol>

The authors report no conflicts of interest

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 field conditions typically experience mild stress, therefore a threshold of FvFm &#62; 0.75 was set for inclusion in accordance with previous studies which chose this as the limit for the beginnings of photosynthetic decline30. Handling of the nasal aspirator filters used to hold leaf disks in their place represents another common source of problems. If nasal aspirator filters are oversaturated, excess water will negatively impact leaf physiology also leading to low quantum efficiencies; the same brand must be used throughout the experiment as different brands can hold different amounts of water. Third, field-grown plants experience a fluctuating environment, with differences in rainfall, temperature, humidity, and insect pressure. The growth stages of the plants also contribute to the efficiency of chlorophyll fluorescence. Chlorotic leaves or extensive pest damage should be avoided when sampling. When making comparisons, consistent choice of leaves at similar developmental stages is vital to reduce experimental noise. Rates of activation and relaxation of non-photochemical quenching can vary with leaf age and plant developmental stage31, as a consequence of differences in balance between light absorption, chloroplast development, energy status, and long-term shading. Therefore, a typical choice is to compare rates from the most recent&#160;fully expanded leaf with mature chloroplasts.
This protocol is designed for analysis of field-grown soybean but can be modified for sampling and measurement of greenhouse-grown material or other higher plants. Several considerations are important when adapting the protocol. First, experimental design should be taken into account to reduce noise and avoid the introduction of bias. In the field, soybean genotypes are commonly grown in plots of 50-100 plants in a row, each plot considered a single biological replicate. Each plot can experience different soil, exposure, and drainage conditions which will impact whole plant physiology. Therefore, selecting an appropriate plot design, such as a random block, can avoid bias and reduce in-field variability. To minimize variation within plots, it is suggested to measure multiple replicates (three to five) and take disks from different plants within a plot. However, when working with greenhouse or chamber-grown material, a single plant may constitute a biological replicate with multiple disks taken from the same leaf. Further, the plant position should be rotated several times a week to prevent bias in the conditions experienced. Second, the actinic light intensities used in this protocol reflect the maximum and minimum light intensities experienced by a soybean canopy on a sunny day in Illinois (W 88&#176;13&#697;42&#698;/N 40&#176;06&#697;31&#698;). Depending on the experimental setup and plant under analysis, it is advisable to adjust the light intensity to the particular growth environment to provide more realistic measuring conditions. Finally, both the sampling procedure and measuring protocol can be adapted for use with other closed PAM fluorometer systems, with the provision that the steps required to operate the measuring software will need to be adjusted for a given device.
A major limitation of the approach is the interpretation of the parameter values calculated by the bi-exponential function. This is particularly important if applying the procedure to other higher plants or algae, as the biological processes contributing to NPQ can vary between species. For example, some mosses and algae contain LHCSR proteins rather than PsbS (for a review see32). Further, the relative importance of biological processes can vary, with state transitions or qT, representing a more important part of the Aq2 component in algae than in higher plants, where up to 80% of light-harvesting complexes can reversibly move between photosystems I and II33. Therefore, it should be considered that the bi-exponential function may not always be the best choice to explain the biological processes governing relaxation kinetics22,29 and this should be assessed empirically for the species of choice. Careful experimentation, including the application of inhibitors and uncouplers34,35 or study of mutants21,23 can be used to provide insight into the processes contributing to NPQ relaxation, but care should be taken in drawing conclusions.
The method presented here is similar to that of McAusland et al.29, which used a closed-PAM imager to assess the photosynthetic performance of chamber-grown plants. However, by placing leaf samples on damp filter paper rather than in 96-well plates, and incubating tissue in custom-made chambers to control the O2 and CO2 concentrations, McAusland et al. were able to perform a wider range of measurements assessing photosynthetic parameters29. The main advantages of the approach presented here include the simplicity of the method, which does not require custom equipment, and the use of 24-well plates for initial sampling of leaf tissue which enables collection of samples from field-grown material, potentially increasing the number of genotypes a researcher is able to screen.
In summary, PAM chlorophyll fluorescence analysis is a powerful technique for the measurement of the efficiency of photosynthesis. However, conventional measurements are done on a per-plant basis, limiting screening capacity either by the time taken or the number of people and equipment available to perform assays. The use of leaf disks in a 96-well plate provides a means to measure many genotypes simultaneously, increasing assay throughput. By facilitating the screening of many genotypes in a short space of time by a single investigator29, it is possible to apply the measurement of NPQ relaxation to screening natural diversity of photosynthesis with the potential to perform genome-wide association studies.

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.

<table><tbody><tr><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>&amp;nbsp;61018-56</td><td>Country of Origin: United States of America</td></tr><tr><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&lt;br/&gt; 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&lt;br/&gt; Country of Origin: United Kingdom</td></tr><tr><td>iHank-Nose 100-Pack of Premium Nasal Aspirator Hygiene Filters</td><td>Amazon</td><td>&amp;nbsp;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></tr><tr><td>Parafilm</td><td>Bemis Company Inc.</td><td>&amp;nbsp;S3-594-6</td><td>Semi -transparent flexible film&lt;br/&gt; 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 parameters were calculated by fitting a bi-exponential function (Table 2). After an initial saturating flash to determine Fv/Fm, leaf disks were exposed to low-light (50 &#181;mol m&#8722;2s&#8722;1) and the measured NPQ was &#60; 1 (Figure 1A). Upon transfer to high-light (2000 &#181;mol m&#8722;2s&#8722;1), NPQ increased reaching a maximum value of 4 after 15 min. A return to low-light caused a rapid initial decrease in NPQ (Aq1) with a time constant of 1.07 min (&#964;q1), followed by a second slower relaxing phase (Aq2) with a time constant of 24.5 min (&#964;q2). Residual NPQ that remained at the end of the protocol (~0.5) was captured by the constant (Aq3).
Figure 1B depicts an example of a failed measurement. Upon transfer to high light there is a minimal increase in NPQ (0.38), which does not relax. Close inspection of the data reveals the leaf disk had a low Fv/Fm value indicative of stress (0.27), and the data should be discarded.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63485/63485fig1v2.jpg" /> 
Figure 1: NPQ measurement data using field-grown soybean. (A) NPQ activation and relaxation kinetics of G. max cv. IA3023. Data are presented as the mean of five biological (separate field plots) and three technical (leaf disks from the same plot) replicates with error bars representing standard deviation. (B) Example of a failed technical replicate (n = 1) using of G. max cv. IA3023. The grey and white bars at the top of the graph indicate periods of illumination with a photosynthetic photon flux density (PPFD) of 2000 &#181;mol m&#8722;2s&#8722;1 (white bar), PPFD of 50 &#181;mol m&#8722;2s&#8722;1 (grey bars). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63485/63485fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Step</td>
			<td>Delay</td>
			<td>Action</td>
			<td>Cycles</td>
			<td>PPFD (&#956;mol m-2s-1 )</td>
		</tr>
		<tr>
			<td>1</td>
			<td>0 s</td>
			<td>Change Actinic</td>
			<td>-</td>
			<td>0</td>
		</tr>
		<tr>
			<td>2</td>
			<td>20 s</td>
			<td>Apply Pulse</td>
			<td>1</td>
			<td>6152</td>
		</tr>
		<tr>
			<td>3</td>
			<td>30 s</td>
			<td>Change Actinic</td>
			<td>-</td>
			<td>50</td>
		</tr>
		<tr>
			<td>4</td>
			<td>150 s</td>
			<td>Apply Pulse</td>
			<td>4</td>
			<td>6152</td>
		</tr>
		<tr>
			<td>5</td>
			<td>30 s</td>
			<td>Change Actinic</td>
			<td>-</td>
			<td>2000</td>
		</tr>
		<tr>
			<td>6</td>
			<td>150 s</td>
			<td>Apply Pulse</td>
			<td>6</td>
			<td>6152</td>
		</tr>
		<tr>
			<td>7</td>
			<td>0 s</td>
			<td>Change Actinic</td>
			<td>-</td>
			<td>50</td>
		</tr>
		<tr>
			<td>8</td>
			<td>150 s</td>
			<td>Apply Pulse</td>
			<td>2</td>
			<td>6152</td>
		</tr>
		<tr>
			<td>9</td>
			<td>5 min</td>
			<td>Apply Pulse</td>
			<td>9</td>
			<td>6152</td>
		</tr>
		<tr>
			<td>10</td>
			<td>20 s</td>
			<td>Change Actinic</td>
			<td>-</td>
			<td>0</td>
		</tr>
	</tbody>
</table>
Table 1: Chlorophyll fluorescence imaging protocol to be used with the imager.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Rep</td>
			<td>Aq1</td>
			<td>&#964;q1 / min</td>
			<td>Aq2</td>
			<td>&#964;q2 / min</td>
			<td>Aq3</td>
			<td>maxNPQ</td>
			<td>R2</td>
		</tr>
		<tr>
			<td>1</td>
			<td>2.00</td>
			<td>1.12</td>
			<td>1.88</td>
			<td>16.62</td>
			<td>0.29</td>
			<td>4.17</td>
			<td>0.9999</td>
		</tr>
		<tr>
			<td>2</td>
			<td>2.14</td>
			<td>1.17</td>
			<td>1.70</td>
			<td>13.68</td>
			<td>0.34</td>
			<td>4.17</td>
			<td>0.9999</td>
		</tr>
		<tr>
			<td>3</td>
			<td>1.82</td>
			<td>0.78</td>
			<td>1.88</td>
			<td>18.42</td>
			<td>0.36</td>
			<td>4.05</td>
			<td>0.9997</td>
		</tr>
		<tr>
			<td>4</td>
			<td>2.05</td>
			<td>1.28</td>
			<td>1.67</td>
			<td>26.89</td>
			<td>0.15</td>
			<td>3.87</td>
			<td>0.9999</td>
		</tr>
		<tr>
			<td>5</td>
			<td>1.72</td>
			<td>1.01</td>
			<td>1.81</td>
			<td>21.22</td>
			<td>0.52</td>
			<td>4.05</td>
			<td>0.9998</td>
		</tr>
		<tr>
			<td>Average</td>
			<td>1.94</td>
			<td>1.07</td>
			<td>1.79</td>
			<td>19.37</td>
			<td>0.33</td>
			<td>4.06</td>
			<td tr=""></td>
		</tr>
		<tr>
			<td>S.D.</td>
			<td>0.17</td>
			<td>0.19</td>
			<td>0.10</td>
			<td>5.02</td>
			<td>0.13</td>
			<td>0.12</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2: Calculated NPQ relaxation parameters for G. max cv. IA3023. Values for Aq1, Aq2, and Aq3 are unitless, values for &#964;q1 and &#964;q2 are reported in min. Abbreviations: MaxNPQ = maximum recorded NPQ value in the light, Rep = biological replicate number corresponding to a single plot, where the bi-exponential function is fit using three technical replicates (leaf disks).&#160;S.D. represents standard deviation.
Supplementary Table 1: Description of equations used in the manuscript. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63485/Supplemntary table 1_Re.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary Table 2: Description of parameter values discussed in the manuscript. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63485/Supplementary table 2 _RE.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary Table 3: Pulse amplitude modulated fluorescence technical specifications. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63485/Supplementary Table 3_RE.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary Table 4: Calculated Fv/Fm and NPQ values of G. max cv. IA3023 leaf disks using the protocol provided. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63485/JOVE_Supplementary_Table_4.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary File 1: A script for mapping leaf disk positions using the data output provided by the imager. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63485/Supplementary File 1 - MapAndLabelDiscs.m" target="_blank">Please click here to download this File.</a>
Supplementary File 2: A script for calculating Fo, Fm, Fm&#39;,and Fv/Fm values per leaf disk using the data output provided by the imager and Supplementary File 1. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63485/Supplementary File 2 - ProcessFoFm.m" target="_blank">Please click here to download this File.</a>
Supplementary File 3: A script for calculating NPQ values per leaf disk at each time point during the protocol using the data output from Supplementary File 2. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63485/Supplementary File 3 - ProcessNPQdata.m" target="_blank">Please click here to download this File.</a>
Supplementary File 4: An R script to annotate the output from Supplementary File 3 with Fv/Fm values calculated by Supplementary file 2. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63485/Supplementary File 4 - create_file_to_process_RE.R" target="_blank">Please click here to download this File.</a>
Supplementary File 5: An R script for re-formatting the data from Supplementary File 4 to allow for fitting a bi-exponential decay function to calculated NPQ values following relaxation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63485/Supplementary File 5 - CF-data-processing_2.R" target="_blank">Please click here to download this File.</a>
Supplementary File 6: A script to fit a bi-exponential decay function to calculate NPQ values and calculate relaxation parameters using the data output from Supplementary File 5. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63485/MatLab_NPQ_5_fit_model_v5 (2).zip" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about High-Throughput Analysis of Non-Photochemical Quenching in Crops Using Pulse Amplitude Modulated Chlorophyll Fluorometry at JoVE.com

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 ...

high-throughput-analysis-non-photochemical-quenching-crops-using

Nanyang Technological University

Research

JoVE Journal

Biology

4.1K Views.  University of Illinois at Urbana-Champaign. Deze methode kan inzicht geven in de genetische variatie in niet-fotochemische blussing die beschikbaar is binnen genetische diversiteitspanels of broedpopulaties. Het belangrijkste voordeel van deze techniek is het potentieel om een groot aantal genotypen binnen één dag te screenen op variatie in niet-fotochemische blussing. Het protocol wordt gepresenteerd voor glycine max of sojabonen, maar kan worden aangepast om andere plantenruimtes te analyseren waaruit liftschijven kunnen worden ver...

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

A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated (WATER-PAM) Fluorometry

Conventional methods for experimental manipulation of microalgae have employed large volumes of culture (20 ml to 5 L), so that the culture can be subsampled throughout the experiment1&#8211;7. Subsampling of large volumes can be problematic for several reasons: 1) it causes variation in the total volume and the surface area:volume ratio of the culture during the experiment; 2) pseudo-replication (i.e., replicate samples from the same treatment flask8) is often employed rather than true replicates (i.e., sampling from replicate treatments); 3) the duration of the experiment is limited by the total volume; and 4) axenic cultures or the usual bacterial microbiota are difficult to maintain during long-term experiments as contamination commonly occurs during subsampling.
The use of microtiter plates enables 1 ml culture volumes to be used for each replicate, with up to 48 separate treatments within a 12.65 x 8.5 x 2.2 cm plate, thereby decreasing the experimental volume and allowing for extensive replication without subsampling any treatment. Additionally, this technique can be modified to fit a variety of experimental formats including: bacterial-algal co-cultures, algal physiology tests, and toxin screening9&#8211;11. Individual wells with an alga, bacterium and/or co-cultures can be sampled for numerous laboratory procedures including, but not limited to: WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry, microscopy, bacterial colony forming unit (cfu) counts and flow cytometry. The combination of the microtiter plate format and WATER-PAM fluorometry allows for multiple rapid measurements of photochemical yield and other photochemical parameters with low variability between samples, high reproducibility and avoids the many pitfalls of subsampling a carboy or conical flask over the course of an experiment.

A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated WATER-PAM Fluorometry

The goal of this procedure is to demonstrate the reproducibility and adaptability of using a microtiter plate format for microalgal screening. This rapid screen combines WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry to measure photosynthetic yield as an indicator of Photosystem II (PSII) health with small volume bacterial-algal co-cultures.

Conventional methods for experimental manipulation of microalgae have employed large volumes of culture (20 ml to 5 L), so that the culture can be ...

Environment

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

Evaluation of Photosynthetic Behaviors by Simultaneous Measurements of Leaf Reflectance and Chlorophyll Fluorescence Analyses

Chlorophyll a fluorescence analysis is widely used to measure photosynthetic behaviors in intact plants, and has resulted in the development of many parameters that efficiently measure photosynthesis. Leaf reflectance analysis provides several vegetation indices in ecology and agriculture, including the photochemical reflectance index (PRI), which can be used as an indicator of thermal energy dissipation during photosynthesis because it correlates with non-photochemical quenching (NPQ). However, since NPQ is a composite parameter, its validation is required to understand the nature of the PRI parameter. To obtain physiological evidence for evaluation of the PRI parameter, we simultaneously measured chlorophyll fluorescence and leaf reflectance in xanthophyll cycle defective mutant (npq1) and wild-type Arabidopsis plants. Additionally, the qZ parameter, which likely reflects the xanthophyll cycle, was extracted from the results of chlorophyll fluorescence analysis by monitoring relaxation kinetics of NPQ after switching the light off. These simultaneous measurements were carried out using a pulse-amplitude modulation (PAM) chlorophyll fluorometer and a spectral radiometer. The fiber probes from both instruments were positioned close to each other to detect signals from the same leaf position. An external light source was used to activate photosynthesis, and the measuring lights and saturated light were provided from the PAM instrument. This experimental system enabled us to monitor light-dependent PRI in the intact plant and revealed that light-dependent changes in PRI differ significantly between the wild type and npq1 mutant. Furthermore, PRI was strongly correlated with qZ, meaning that qZ reflects the xanthophyll cycle. Together, these measurements demonstrated that simultaneous measurement of leaf reflectance and chlorophyll fluorescence is a valid approach for parameter evaluation.

We describe a new technical approach to study photosynthetic responses in higher plants involving simultaneous measurements of chlorophyll a fluorescence and leaf reflectance using a PAM and a spectral radiometer for the detection of signals from the same leaf area in Arabidopsis.

Chlorophyll a fluorescence analysis is widely used to measure photosynthetic behaviors in intact plants, and has resulted in the development of many ...

Bioengineering

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light conditions, which are preferably variable in quantity and quality so that studies under different setups can be conducted. These requirements are met by climatic chambers featuring light emitting diodes (LED) lights, which can &#8211; in contrast to fluorescent lights &#8211; be set to different wavelengths. LEDs are energy conserving and emit virtually no heat even at light intensities, which often constitutes a problem with other light sources. The presented protocol provides a step-by-step guidance of how to program a climatic chamber equipped with variable LED lights as well as describing several approaches for in depth analysis of growth phenotypes. Depending on the experimental set-up various characteristics of the growing plants can be observed and analyzed. Here we describe how to determine fresh weight, leaf area, photosynthetic activity, and stomatal density. We demonstrate that in order to obtain reliable data and draw valid conclusions it is mandatory to use a sufficient number of individuals for statistical evaluation. Taking too few plants for this kind of analysis results in high statistical errors and consequently in less clear interpretations of the data.

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Here, we present a protocol for studying plant growth behavior and especially phenotypes in a reproducible manner. We show how to provide variable and at the same time stable light conditions. Proper analyses depend on sufficient sample numbers and valid statistical evaluations.

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light ...

Evaluation of Photosynthetic Efficiency in Photorespiratory Mutants by Chlorophyll Fluorescence Analysis

Photosynthesis and photorespiration represent the largest carbon fluxes in plant primary metabolism and are necessary for plant survival. Many of the enzymes and genes important for photosynthesis and photorespiration have been well studied for decades, but some aspects of these biochemical pathways and their crosstalk with several subcellular processes are not yet fully understood. Much of the work that has identified the genes and proteins important in plant metabolism has been conducted under highly controlled environments that may not best represent how photosynthesis and photorespiration function under natural and farming environments. Considering that abiotic stress results in impaired photosynthetic efficiency, the development of a high-throughput screen that can monitor both abiotic stress and its impact on photosynthesis is necessary.
Therefore, we have developed a relatively fast method to screen for abiotic stress-induced changes to photosynthetic efficiency that can identify uncharacterized genes with roles in photorespiration using chlorophyll fluorescence analysis and low CO2 screening. This paper describes a method to study changes in photosynthetic efficiency in transferred DNA (T-DNA) knockout mutants in Arabidopsis thaliana. The same method can be used for screening ethyl methanesulfonate (EMS)-induced mutants or suppressor screening. Utilizing this method can identify gene candidates for further study in plant primary metabolism and abiotic stress responses. Data from this method can provide insight into gene function that may not be recognized until exposure to increased stress environments.

We describe an approach to measure changes in photosynthetic efficiency in plants after treatment with low CO2 using chlorophyll fluorescence.

Photosynthesis and photorespiration represent the largest carbon fluxes in plant primary metabolism and are necessary for plant survival. Many of the ...

Autofluorescence Imaging to Evaluate Red Algae Physiology

Red algae (Rhodophyta) contain phycobiliproteins and colonize habitats with dim light, however some (e.g., some Chroothece species) can also develop in full sunshine. Most rhodophytes are red, however some can appear bluish, depending on the proportion of blue and red biliproteins (phycocyanin and phycoerythrin). Different phycobiliproteins can capture light at diverse wavelengths and transmit it to chlorophyll a, which makes photosynthesis under very different light conditions possible. These pigments respond to habitat changes in light, and their autofluorescence can help to study biological processes. Using Chroothece mobilis as a model organism and the spectral lambda scan mode in a confocal microscope, the adaptation of photosynthetic pigments to different monochromatic lights was studied at the cellular level to guess the species' optimal growth conditions. The results showed that, even when the studied strain was isolated from a cave, it adapted to both dim and medium light intensities. The presented method is especially useful for studying photosynthetic organisms that do not grow or grow very slowly under laboratory conditions, which is usually the case for those living in extreme habitats.

The present protocol describes step-by-step autofluorescence imaging and evaluation of phycobiliprotein changes in red algae based on spectral analysis. This is a label-free and non-destructive method to evaluate cellular adaptation to extreme habitats, when only scarce material is available and cells grow slowly, or not at all, under laboratory conditions.

Red algae (Rhodophyta) contain phycobiliproteins and colonize habitats with dim light, however some (e.g., some Chroothece species) can also develop in ...

High-Resolution Imaging of Chlorophyll Biosynthesis in Arabidopsis Seedling

Non-invasive Assay for Chlorophyll Biosynthesis Kinetics Determination during Early Stages of Arabidopsis De-etiolation

Chlorophyll biosynthesis is a hallmark of de-etiolation, one of the most dramatic stages in the plant life cycle. The tightly controlled and highly dynamic process of chlorophyll biosynthesis is triggered during the shift from the dark to the light in flowering plants. At the moment when etiolated seedlings are exposed to the first traces of sunlight, rapid (in order of seconds) conversion of protochlorophyllide into chlorophyllide is mediated by unique light-accepting protein complexes, leading via subsequent metabolic steps to the production of fully functional chlorophyll. Standard techniques for chlorophyll content analysis include pigment extraction from detached plant tissues, which does not apply to studying such fast processes. To investigate chlorophyll kinetics in vivo with high accuracy and spatiotemporal resolution in the first hours after light-induced de-etiolation, an instrument and protocol were developed. Here, we present a detailed procedure designed for statistically robust quantification of chlorophyll in the early stages of Arabidopsis de-etiolation.

Author Spotlight: Non-Invasive High-Resolution Measurement of Chlorophyll Synthesis During De-Etiolation

Here, we describe an advanced tool designed for chlorophyll biosynthesis monitoring during the early stages of Arabidopsis seedling de-etiolation. The novel methodology provides non-invasive real-time chlorophyll fluorescence imaging at high spatial and temporal resolution.

Chlorophyll biosynthesis is a hallmark of de-etiolation, one of the most dramatic stages in the plant life cycle. The tightly controlled and highly ...

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 ...

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

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video