This research was funded by the Louisiana Board of Regents (AWD-AM210544).

1. Seed preparation and sterilization
NOTE: Seed preparation consists of seed imbibing and seed sterilization. It is important to note that all these steps are to be carried out in a laminar flow hood to maintain sterile conditions. All necessary materials, reagents, and growth media must be autoclaved (see the Table of Materials).
<ol>
	<li>Seed imbibation and stratification 
	NOTE: The seed lines used are plgg1-1 (salk_053463), abcb26 (salk_085232), and wild type (WT, Col-0).
	<ol>
		<li>Dispense the seeds into 1.5 mL microcentrifuge tubes. Imbibe the seeds in sterile water in a laminar flow hood and stratify at 4 &#176;C in the dark for 2 days.</li>
	</ol>
	</li>
	<li>Seed sterilization
	<ol>
		<li>Under sterile conditions, prepare 10 mL of 50% (v/v) bleach solution and add approximately 20 &#181;L of Tween 20. For seed sterilization, remove water from the imbibed seeds, add 1 mL of bleach solution into the microcentrifuge tubes, and incubate at room temperature for 5 min.</li>
		<li>Remove the bleach solution with a pipette.</li>
		<li>Rinse the seeds in 1 mL of sterile water to resuspend. Remove the water once the seeds have settled to the bottom. Repeat step 1.6 and step 1.7 seven times.</li>
		<li>Resuspend the seeds in sterile 0.1% agarose solution.</li>
	</ol>
	</li>
	<li>Seed plating
	<ol>
		<li>Make a 1 L volume of Murashige &#38; Skoog Basal Medium with vitamins and 1.0 g/L of 2-(N-morphonio)ethanesulfonic acid (MS) by adding 5.43 g of the powder to 500 mL of distilled water. Adjust the pH between 5.6 to 5.8 using potassium hydroxide (KOH). Fill to 1 L using distilled water.
		<ol>
			<li>Split the liquid solution into 500 mL and place each half into a 1 L flask containing a magnetic stir bar. Add 5 g of agar powder to obtain a 1% agar w/v solution for each flask.</li>
			<li>Autoclave at 121 &#176;C and 15 psi for 30 min; then, place at room temperature while slowly stirring. When cooled, pour 25 mL of MS agar into a square Petri dish. Allow it to solidify. 
			NOTE: These Murashige &#38; Skoog Basal Medium plates contain 1% MS medium with vitamins and 1% agar for the cultivation of the test mutants.</li>
		</ol>
		</li>
		<li>Cut a 200 &#181;L pipette tip with a razor blade. Place one seed in the center of the designated square grid for each test mutant or genotype (1 cm2 square) of a square MS plate (Figure 1) using a 200 &#181;L micropipette. 
		NOTE: The 1 cm x 1 cm square grid helps to keep a uniform distance between the seedlings and avoid overlapping, which will be important in later fluorescence imaging and analysis.</li>
		<li>Once the seeds have been plated, wrap with surgical tape around the lid to seal, and place it in the growth chamber in conditions as described below.</li>
	</ol>
	</li>
</ol>
2. Plant growth and low CO2 treatment
<ol>
	<li>Grow the plants for 7-9 days at 20 &#176;C under an 8 h light cycle of 120 &#181;mol&#183;m&#8722;2s&#8722;1 and 16 h of darkness at 18 &#176;C. Check the plants on the 6th day to determine if they are large enough for imaging. On the 8th day after plating, expose the plants to low CO2.</li>
	<li>Low CO2 treatment
	<ol>
		<li>After the 7-9 days, place four of the eight plates from the ambient growth chamber conditions into photorespiratory conditions of 20 &#176;C, continuous light levels of 200 &#181;mol&#183;m&#8722;2s&#8722;1, and low CO2 for 12 h.</li>
		<li>Construct the low CO2 condition using an airtight transparent container with 100 g of soda lime placed in the bottom of the container. Place the container within the same growth chamber as the control. Keep the control plants under 120 &#181;mol&#183;m&#8722;2s&#8722;1 in ambient CO2 for 12 h.</li>
	</ol>
	</li>
</ol>
3. Configuring the fluorescence imaging system
<ol>
	<li>Place a testing plate (prepared according to section 1 and section 2) centered under the camera at a fixed distance in the fluorescence imaging system.</li>
	<li>Within the instrument&#39;s software, navigate to the Live Window and check the box Flashes to switch on non-actinic measuring flashes.</li>
	<li>Click on the Zoom and Focus tools until a complete and sharp image is visible. To this end, use a stage or a shelf to adjust the distance between the plants and the camera. Keep the zoom, focus, and distance from the camera constant for the entire experiment.</li>
	<li>Set the value of El. Shutter to 0 and adjust the Sensitivity to get a fluorescent signal in the range of 200-500 digital units. 
	NOTE: A lower El. Shutter value (0-1) will ensure the measuring flashes are non-actinic, while a higher value (2) will improve the resolution of the image.</li>
	<li>Place a light meter in the same position used to adjust the camera settings.</li>
	<li>In the Live Window, check the box Super to start a saturating pulse lasting for 800 ms. Remember to check the box every time for a new pulse.</li>
	<li>Use the slider to adjust the percentage of relative power for the Super pulse until the light meter reads 6,000-8,000 &#181;mol&#183;m&#8722;2s&#8722;1.</li>
</ol>
4. Design the quantum yield program
<ol>
	<li>Import standard operating tools for the imaging system. 
	Include default.inc 
	Include light.inc 
	NOTE: The program syntax used for reference in this experiment is for a FluorCam imaging system.</li>
	<li>Define the following global variables according to the configured light settings: 
	Shutter = 0 
	Sensitivity = 40 
	Super = 65</li>
	<li>Include the time-step for logging data: 
	TS = 20ms</li>
	<li>Collect the Fo measurement by sampling fluorescence in a dark-adapted state. 
	F0duration = 2s; 
	F0period = 200ms; 
	NOTE: F0duration defines the time range over which the dark-adapted fluorescence is recorded. F0period defines the time interval over which the dark-adapted fluorescence measurement is repeated.</li>
	<li>Record the Fo measurements into data tables. 
	&#60;0,F0period..F0duration&#62;=&#62;mfmsub 
	&#60;0s&#62;-&#62;checkpoint,&#34;startFo&#34; 
	&#60;F0duration - F0period&#62;=&#62;checkpoint,&#34;endFo&#34;; 
	Where &#60; , &#62; represents the set of fluorescence values indexed by time; =&#62; stores measurements in a system file; mfmsub represents the entire data set for the experiment; and checkpoint creates a subset for data of specific measurements such as startFo.</li>
	<li>Collect and store the Fm measurement by sampling fluorescence after a saturating pulse. 
	PulseDuration=960ms; ## 
	a1=F0duration + 40ms 
	a2 = a1 + 480ms; 
	&#60;a1&#62;=&#62;SatPulse(PulseDuration); 
	&#60;a1 + 80ms, a1 + 160ms.a1 + PulseDuration&#62;=&#62;mfmsub 
	&#60;a1 + PulseDuration + 80ms&#62;=&#62;mfmsub; 
	&#60;a2&#62;=&#62;checkpoint,&#34;startFm&#34; 
	&#60;a1 + PulseDuration - 80ms&#62;=&#62;checkpoint,&#34;endFm&#34; 
	&#60;a2 + 80ms&#62;=&#62;checkpoint,&#34;timeVisual&#34;; 
	&#8203;Where a1 and a2 are variables to coordinate the sampling time with the saturating pulse; a1 represents the starting time of the Fm measurement; and a2 represents the mid-point time of the pulse.</li>
</ol>
5. Measuring the quantum yield of the treated samples
<ol>
	<li>Directly following treatment, cover the plates with aluminum foil for 15 min for dark adaptation. Remove the foil to measure the quantum yield of photosystem II with a pulse amplitude-modified fluorometer. Then, place the seedling plate directly under the camera and run the quantum yield protocol found in the GitHub repository.</li>
	<li>Download the quantum_yield protocol from GitHub (https://github.com/South-lab/fluorescent-screen) or use a similarly designed program from section 4. Use the fluorometer&#8217;s software to open the program file by clicking on the folder icon and navigating to the file location.</li>
	<li>Run the quantum yield program by clicking on the red lightning icon.</li>
	<li>After the protocol is complete, navigate to the preanalysis window. Partition the plate into individual seedlings by using the Selection Tools to highlight all the pixels for each seedling on the plate image. Click Background exclusion to remove any highlighted background pixels, leaving just the seedling area.</li>
	<li>Click on Analyze to generate fluorescence data for each seedling on the plate image. Manually adjust the fluorescence value range to display consistent minimum and maximum values among all plates.</li>
	<li>Click on Numerical-Average from the tab Experiment | Export | Numeric. Select All data and by column and click Ok to generate a text file containing QY measurements for each seedling.</li>
</ol>
6. Opening the data file
<ol>
	<li>Open the text file in a spreadsheet for analysis. Of the headings showing area number, size of pixels, Fm, Ft, Fq, and QY, identify the area number for the core genotype (WT or test mutant) and QYmax. Perform a pairwise t-test with respect to wild type to determine significance. Data are interpreted as significantly different when p-values are below 0.05.</li>
</ol>

<ol><li>Peterhansel, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Photorespiration%5BTitle%5D)+AND+%22Arabidopsis+Book%22%5BJournal%5D)">Photorespiration.</a> Arabidopsis Book. 8, 0130(2010).</li>
<li>Bordych, C., Eisenhut, M., Pick, T. R., Kuelahoglu, C., Weber, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Co-expression+analysis+as+tool+for+the+discovery+of+transport+proteins+in+photorespiration%5BTitle%5D)+AND+%22Plant+Biology%22%5BJournal%5D)">Co-expression analysis as tool for the discovery of transport proteins in photorespiration.</a> Plant Biology. 15 (4), 686-693 (2013).</li>
<li>Eisenhut, M., Pick, T. R., Bordych, C., Weber, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Towards+closing+the+remaining+gaps+in+photorespiration--the+essential+but+unexplored+role+of+transport+proteins%5BTitle%5D)+AND+%22Plant+Biology%22%5BJournal%5D)">Towards closing the remaining gaps in photorespiration--the essential but unexplored role of transport proteins.</a> Plant Biology. 15 (4), 676-685 (2013).</li>
<li>Walker, B. J., VanLoocke, A., Bernacchi, C. J., Ort, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+costs+of+photorespiration+to+food+production+now+and+in+the+future%5BTitle%5D)+AND+%22Annual+Review+of+Plant+Biology%22%5BJournal%5D)">The costs of photorespiration to food production now and in the future.</a> Annual Review of Plant Biology. 67 (1), 107-129 (2016).</li>
<li>South, P. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bile+acid+sodium+symporter+BASS6+can+transport+glycolate+and+is+involved+in+photorespiratory+metabolism+in+Arabidopsis+thaliana%5BTitle%5D)+AND+%22Plant+Cell%22%5BJournal%5D)">Bile acid sodium symporter BASS6 can transport glycolate and is involved in photorespiratory metabolism in Arabidopsis thaliana.</a> Plant Cell. 29 (4), 808-823 (2017).</li>
<li>Pick, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PLGG1%2C+a+plastidic+glycolate+glycerate+transporter%2C+is+required+for+photorespiration+and+defines+a+unique+class+of+metabolite+transporters%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">PLGG1, a plastidic glycolate glycerate transporter, is required for photorespiration and defines a unique class of metabolite transporters.</a> Proceedings of the National Academy Sciences of the United States of America. 110 (8), 3185-3190 (2013).</li>
<li>Kuhnert, F., Schlüter, U., Linka, N., Eisenhut, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transport+proteins+enabling+plant+photorespiratory+metabolism%5BTitle%5D)+AND+%22Plants%22%5BJournal%5D)">Transport proteins enabling plant photorespiratory metabolism.</a> Plants. 10 (5), 880(2021).</li>
<li>Badger, M. R., Fallahi, H., Kaines, S., Takahashi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chlorophyll+fluorescence+screening+of+Arabidopsis+thaliana+for+CO2+sensitive+photorespiration+and+photoinhibition+mutants%5BTitle%5D)+AND+%22Funct+Plant+Biology%22%5BJournal%5D)">Chlorophyll fluorescence screening of Arabidopsis thaliana for CO2 sensitive photorespiration and photoinhibition mutants.</a> Funct Plant Biology. 36 (11), 867-873 (2009).</li>
<li>Ogawa, T., Sonoike, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Screening+of+mutants+using+chlorophyll+fluorescence%5BTitle%5D)+AND+%22Journal+of+Plant+Research%22%5BJournal%5D)">Screening of mutants using chlorophyll fluorescence.</a> Journal of Plant Research. 134 (4), 653-664 (2021).</li>
<li>Kleffmann, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Arabidopsis+thaliana+chloroplast+proteome+reveals+pathway+abundance+and+novel+protein+functions%5BTitle%5D)+AND+%22Current+Biology%22%5BJournal%5D)">The Arabidopsis thaliana chloroplast proteome reveals pathway abundance and novel protein functions.</a> Current Biology. 14 (5), 354-362 (2004).</li>
<li>Hempel, J. J. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Molecular+characterization+of+the+plastid-localized+ABC+protein+TAP1+in+Arabidopsis+thaliana%22">Molecular characterization of the plastid-localized ABC protein TAP1 in Arabidopsis thaliana</a>. , University of Stavanger. Ph.D. thesis (2018).</li>
</ol>

The authors have no competing financial interests or conflicts of interest.

The experimental methods outlined in this paper come with some advantages and limitations. One advantage is that this method can screen many plant seedlings, although some precautions must be taken to prevent contamination of the plant media plate during the plating and growing process. Therefore, it is critical to seal the Arabidopsis plates with surgical tape. Another advantage of this experiment is that it has a shorter 12 h photorespiratory stress period compared to the previously published work8. The reduction in treatment time was to better distinguish differences in Fv/Fm between the WT and selected mutants. Treatment time may need to be varied depending on the severity of the phenotype in mutant strains and the level of CO2 in the experimental setup. One limitation, however, is that this method requires a leaf area large enough for the fluorometer to measure and for Fv/Fm values to be calculated. Adjusting the initial growing time of tested seedlings will ensure that the fluorometer imager has adequate leaf area to measure for each seedling without the leaves overlapping during imaging. The seedlings must be imaged at the first true leaves to keep the leaf size and leaf angles uniform. The resolution of the image may be improved by increasing the shutter speed and sensitivity of the camera. Neighboring pixels will be better distinguished; however, increasing the sensitivity too much will overexpose the camera and lead to false fluorescence maximums. Optimization and troubleshooting may be necessary to balance the resolution and fluorescence values. 
The ability to screen for photosynthetic mutants in a standardized procedure is important. It can allow for a consistent, high-throughput method to identify photorespiration mutants with genes of interest for photosynthesis and photorespiration metabolism. Overall, screening for Fv/Fm in this analysis takes approximately 30 s per plate of plant seedlings, allowing for the screening of over 1,000 seedlings per day. Chlorophyll fluorescence analysis allows for the identification of photorespiratory mutants using a low CO2 screening, which is important for maintaining photorespiratory efficiency. Given the large number of unknown transporters theorized to be in the photorespiration pathway3, this method can be used to quickly evaluate the effect the genotype has on photosynthetic efficiency. Additionally, fluorometers can measure other aspects of photosynthetic efficiency such as non-photochemical quenching and photosystem II operating efficiency. These additional protocols take longer per seedling plate but could be used for further and more sensitive analysis as opposed to this quick high-throughput screen of mutants. This method is not limited to Arabidopsis and can also be adjusted to screen for T-DNA mutants in a range of other abiotic stress conditions such as high and low temperatures, high light stress, and drought conditions to identify genes important for photosynthesis and photorespiration. Any additional modifications would need to be optimized on an individual basis.

Abiotic stress conditions commonly seen in farmer&#39;s fields can negatively impact crop yields by reducing photosynthetic efficiency. Detrimental environmental conditions such as heat waves, climate change, drought, and soil salinity can cause abiotic stresses that alter CO2 availability and reduce a plant&#39;s response to high light stress. The two largest terrestrial carbon fluxes are photosynthesis and photorespiration, which are essential for plant growth and crop yields. Many of the important proteins and enzymes involved in these processes have been characterized under laboratory conditions and identified at the genetic level1. Although much progress has been made in understanding photosynthesis and photorespiration, many steps, including transport between plant organelles, remain uncharacterized2,3.
Photorespiration, the second largest carbon flux in plants after photosynthesis, begins when the enzyme Rubisco fixes oxygen instead of carbon dioxide to ribulose 1,5 bisphosphate (RuBP), generating the inhibitory compound 2-phosphoglycolate (2PG)1. To minimize the inhibitory effects of 2PG and to recycle the previously fixed carbon, C3 plants have evolved the multi-organellar process of photorespiration. Photorespiration converts two molecules of 2PG into one molecule of 3-phosphoglycerate (3PGA), which can re-enter the C3 carbon fixation cycle1. Thus, photorespiration only converts 75% of the previously fixed carbon from the generation of 2PG and consumes ATP in the process. As a result, the process of photorespiration is a significant 10%-50% drag on the photosynthetic process, depending on water availability and growing season temperatures4.
The enzymes involved in photorespiration have been an area of research focus for decades, but only a small number of transport proteins have been characterized at the genetic level, even though at least 25 transport steps are involved in the process5,6,7. The two transport proteins that are directly involved in the movement of carbon generated in the photorespiration process are the plastidic glycolate/glycerate transporter PLGG1 and the bile acid sodium symporter BASS6, both of which are involved in the export of glycolate from the chloroplast5,6.
Under ambient [CO2], Rubisco fixes an oxygen molecule to RuBP approximately 20% of the time1. When plants are subjected to low [CO2], rates of photorespiration increase, making low [CO2] an ideal environment to test for mutants that may be important under elevated photorespiration stress. Testing additional putative chloroplast transport protein T-DNA lines under low CO2 for 24 h and measuring changes to chlorophyll fluorescence led to the identification of bass6-1 plant lines that demonstrated a photorespiration mutant phenotype5. Further characterization demonstrated that BASS6 is a glycolate transporter in the inner membrane of the chloroplast.
This paper describes in detail a protocol similar to what was initially used to identify BASS6 as a photorespiration transporter, which came from a list of putative transport proteins located within the chloroplast membrane8 This protocol can be used in a high-throughput experiment characterizing Arabidopsis T-DNA mutants or EMS-generated mutant plants as a way to identify genes important for maintaining photosynthetic efficiency under a range of abiotic stresses such as heat, high light stress, drought, and CO2 availability. Screening plant mutants using chlorophyll fluorescence has been used in the past to rapidly identify genes important for primary metabolism9. With as much as 30% of the Arabidopsis genome containing genes that code for proteins of unknown or poorly characterized function, stress-induced analysis of photosynthetic efficiency could provide insight into molecular functions not observed under controlled conditions in mutant plants10. The goal of this method is to identify mutants of the photorespiratory pathway using a low CO2 screening. We present a method to identify mutants that disrupt photorespiration after exposure to low CO2. An advantage of this method is that it is a high-throughput screening for seedlings that can be done in a relatively short period of time. The video protocol sections provide details on seed preparation and sterilization, plant growth and low CO2 treatment, the configuration of the fluorescence imaging system, the measurement of quantum yield of the treated samples, representative results, and conclusions.

<table><tbody><tr><td>1.5 mL microcentrifuge tube</td><td>VWR</td><td>10810-070</td><td>container for seed sterilization</td></tr><tr><td>agarose</td><td>VWR</td><td>9012-36-6</td><td>chemical used to suspend seeds for ease of plating</td></tr><tr><td>&lt;em&gt;Arabidopsis thaliana&lt;/em&gt; seeds (&lt;em&gt;abcb26&lt;/em&gt;)</td><td>ABRC, ordered through TAIR www.arabidopsis.org</td><td>SALK_085232</td><td>arabidopsis seeds used as experimental group</td></tr><tr><td>&lt;em&gt;Arabidopsis thaliana&lt;/em&gt; seeds (&lt;em&gt;plgg1-1&lt;/em&gt;)</td><td>ABRC, ordered through TAIR www.arabidopsis.org</td><td>SALK_053469C</td><td>parental arabidopsis seeds&amp;nbsp;</td></tr><tr><td>&lt;em&gt;Arabidopsis thaliana&lt;/em&gt; seeds (WT)</td><td>ABRC, ordered through TAIR www.arabidopsis.org</td><td>Col-0</td><td>arabidopsis wild type seeds used as a control group</td></tr><tr><td>&amp;nbsp;bleach&amp;nbsp;</td><td>clorox</td><td>generic bleach&amp;nbsp;</td><td>chemical used to sterilize seeds</td></tr><tr><td>Carbolime absorbent</td><td>Medline products</td><td>S232-104-001</td><td>CO&lt;sub&gt;2&lt;/sub&gt; absorbent</td></tr><tr><td>Closed FluorCam</td><td>Photon Systems Instruments</td><td>FC 800-C</td><td>Fluorescence imager</td></tr><tr><td>FluoroCam FC 800-C</td><td>Photon Systems Instruments</td><td>Closed FluorCam FC 800-C/1010-S</td><td>Fluorescence imager</td></tr><tr><td>FluoroCam7</td><td>Photon Systems Instruments</td><td>Closed FluorCam FC 800-C/1010-S</td><td>Fluorescence image analysis software</td></tr><tr><td>Gelzan (plant agar)</td><td>Phytotech labs</td><td>71010-52-1</td><td>chemical used to solidify MS media as plates&amp;nbsp;</td></tr><tr><td>glass flask 1 L</td><td>Fisherbrand</td><td>FB5011000</td><td>container for making and autoclaving MS media</td></tr><tr><td>growth chamber</td><td>caron</td><td>7317-50-2</td><td>growth chamber used to grow plants</td></tr><tr><td>Murashige &amp;amp; Skoog Basal Medium with Vitamins &amp;amp; 1.0 g/L MES (MS)</td><td>Phytotech labs</td><td>M5531&amp;nbsp;</td><td>growth media for arabidopsis seedlings&amp;nbsp;</td></tr><tr><td>potassium Hydroxide (KOH)</td><td>Phytotech labs</td><td>1310-58-3</td><td>make as 1 M solution for ph adjustment</td></tr><tr><td>spider lights</td><td>Mean Well Enterprises</td><td>XLG-100-H-AB</td><td>lights used in the light assay&amp;nbsp;</td></tr><tr><td>Square Petri Dish with Grid, sterile</td><td>Simport Scientific</td><td>D21016</td><td>used to hold MS media for arabidopsis seedlings</td></tr><tr><td>surgical tape</td><td>3M</td><td>1530-1</td><td>tape used to seal plates</td></tr><tr><td>tween 20</td><td>biorad</td><td>&amp;nbsp;9005-64-5</td><td>surfactant used to assist seed sterilization</td></tr></tbody></table>

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.

The results show plate images of raw and fluorescence images from ambient and low CO2 screening of WT and test mutants. Each plantlet is labeled by area number, with corresponding fluorescence readings given as QY. The data are exported as a text file and can be opened in a spreadsheet for analysis (see Supplemental Table S1). Mutant lines plgg1-1 and abcb26 were selected to demonstrate the positive and negative identification of genes associated with photorespiratory stress. PLGG1 codes for the first transporter in the pathway following the oxygenation of RuBP6, while ABCB26 is thought to code for an antigen transporter not known to be involved in photorespiration11. The dark-adapted Fv/Fm QY efficiencies of WT and mutants are visualized by box and whisker plots. To test the statistical difference between the WT and test mutants, a pairwise t-test was used with a p-value &#60; 0.05. Here, we used photorespiratory mutant lines with reduced QY Fv/Fm as test mutants to check the efficiency of the screening method. The results show that the test mutants have a significantly lower QY efficiency than WT. Figure 3B shows a significant reduction in the ratio of variable to maximal fluorescence (Fv/Fm) for plgg1-1 but not abcb26 at low CO2. This result is consistent with the role of PLGG1 as a transporter involved in photorespiration, while abcb26 does not demonstrate a phenotype different from the WT control under low CO2 conditions. Thus, this screening method can identify photorespiratory mutants using low CO2 screening.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63801/63801fig01.jpg" /> 
Figure 1: Example schematic of the seed plate layout. Two technical replicates are shown with six seeds placed in a row. WT control seeds placed above mutant seeds. Abbreviation: WT = wild type. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63801/63801fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63801/63801fig02.jpg" /> 
Figure 2: Dark-adapted Fv/Fm images of 9-day-old seedlings. The wild-type control is compared to T-DNA mutant lines at ambient and low CO2. The color scale represents the average Fv/Fm of each seedling. Scale bar = 1 cm. Abbreviations: Fv/Fm = ratio of variable to maximal fluorescence; WT = wild type; plgg1-1 = plastidal glycolate/glycerate translocator 1; abcb26 = ATP-binding cassette B26. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63801/63801fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63801/63801fig03.jpg" /> 
Figure 3: Fluorescence observations. Maximal quantum yield (Fv/Fm) measurements made on seedlings at (A) ambient and (B) low CO2 conditions. The boxes represent the range between the inner quartiles, the lines within the boxes represent the medians, and the whiskers represent the maximal and minimal observations. * Indicates significant difference based on a pairwise t-test relative to WT (n &#62; 44, p &#60; 0.05; Supplemental Table S2). Abbreviations: Fv/Fm = ratio of variable to maximal fluorescence; WT = wild type; plgg1-1 = plastidal glycolate/glycerate translocator 1; abcb26 = ATP-binding cassette B26. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63801/63801fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Table S1: Compiled fluorescent data from all seedling plates used in the experiment. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63801/63801_Table S1.xlsx" target="_blank">Please click here to download this File.</a>
Supplemental Table S2: Statistics are reported from a t-test between wild type and both mutant genotypes. Mutants are considered significantly different from wild type when the p-value is lower that 0.05. Table A represents t-tests performed on plants grown in ambient CO2, while Table B represents t-tests performed on plants grown in low CO2. t-Test: two-sample assuming equal variances. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63801/63801_SuppTable S2.xlsx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Evaluation of Photosynthetic Efficiency in Photorespiratory Mutants by Chlorophyll Fluorescence Analysis at JoVE.com

Evaluation of Photosynthetic Efficiency in Photorespiratory Mutants by Chlorophyll Fluorescence Analysis

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 ...

evaluation-photosynthetic-efficiency-photorespiratory-mutants

Nanyang Technological University

Research

JoVE Journal

Biology

1.9K Views.  Louisiana State University. Het doel van deze methode is om mutanten in de fotorespiratoire route te identificeren met behulp van een lage CO2-screening. We presenteren een methode om mutanten te identificeren die de fotorespiratie verstoren na blootstelling aan lage CO2. Een voordeel van deze methode is dat het een high throughput screening is voor zaailingen die in een relatief korte tijd kan worden gedaan.De volgende secties geven details over zaadvoorbereiding en sterilisatie, plantengroei en lage CO2-behande...

Video: Evaluation of Photosynthetic Efficiency in Photorespiratory Mutants by Chlorophyll Fluorescence Analysis

How to Quantify the Fraction of Photoactivated Fluorescent Proteins in Bulk and in Live Cells

Photoactivatable and -convertible fluorescent proteins (PA-FPs) have been used in fluorescence live-cell microscopy for analyzing the dynamics of cells and protein ensembles. Thus far, no method has been available to quantify in bulk and in live cells how many of the PA-FPs expressed are photoactivated to fluoresce.
Here, we present a protocol involving internal rulers, i.e., genetically coupled spectrally distinct (photoactivatable) fluorescent proteins, to ratiometrically quantify the fraction of all PA-FPs expressed in a cell that are switched on to be fluorescent. Using this protocol, we show that different modes of photoactivation yielded different photoactivation efficiencies. Short high-power photoactivation with a confocal laser scanning microscope (CLSM) resulted in up to four times lower photoactivation efficiency than hundreds of low-level exposures applied by CLSM or a short pulse applied by widefield illumination. While the protocol has been exemplified here for (PA-)GFP and (PA-)Cherry, it can in principle be applied to any spectrally distinct photoactivatable or photoconvertible fluorescent protein pair and any experimental set-up.

Here, we present a protocol that involves genetically coupled spectrally distinct photoactivatable and fluorescent proteins. These fluorescent protein chimeras permit quantification of the PA-FP fraction that is photoactivated to be fluorescent, i.e., the photoactivation efficiency. The protocol reveals that different modes of photoactivation yield different photoactivation efficiencies.

Photoactivatable and -convertible fluorescent proteins (PA-FPs) have been used in fluorescence live-cell microscopy for analyzing the dynamics of cells ...

Biochemistry

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

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

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871, who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory, and examples describing applications of the method are provided.

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...

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 ...

Measuring Photophysiology of Attached Stage of Colacium sp. by a Cuvette-Type Fast Repetition Rate Fluorometer

Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II (PSII) photophysiology and primary productivity. Although FRRf can measure PSII absorption cross-section (&#963;PSII), maximum photochemical efficiency (Fv/Fm), effective photochemical efficiency (Fq&#8242;/Fm&#8242;), and non-photochemical quenching (NPQNSV) for various eukaryotic algae and cyanobacteria, almost all FRRf studies to date have focused on phytoplankton. Here, the protocol describes how to measure PSII photophysiology of an epizoic alga Colacium sp.&#160;Ehrenberg 1834 (Euglenophyta), in its attached stage (attached to zooplankton), using cuvette-type FRRf. First, we estimated the effects of substrate zooplankton (Scapholeberis mucronata O.F. M&#252;ller 1776, Cladocera, Daphniidae) on baseline fluorescence and &#963;PSII, Fv/Fm, Fq&#8242;/Fm&#8242;, and NPQNSV of planktonic Colacium sp. To validate this methodology, we recorded photophysiology measurements of attached Colacium sp. on S. mucronata and compared these results with its planktonic stage. Representative results showed how the protocol could determine the effects of calcium (Ca) and manganese (Mn) on Colacium sp. photophysiology and identify the various effects of Mn enrichment between attached and planktonic stages. Finally, we discuss the adaptability of this protocol to other periphytic algae.

Measuring Photophysiology of Attached Stage of Colacium sp. by a Cuvette-Type Fast Repetition Rate Fluorometer

Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II photophysiology and primary productivity. Here we describe a protocol to measure PSII photophysiology of epizoic alga, Colacium sp. on substrate zooplankton using cuvette-type FRRf.

Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II (PSII) photophysiology and primary productivity. Although FRRf ...

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.

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 ...

Observation of Photobehavior in Chlamydomonas reinhardtii

For the survival of the motile phototrophic microorganisms, being under proper light conditions is crucial. Consequently, they show photo-induced behaviors (or photobehavior) and alter their direction of movement in response to light. Typical photobehaviors include photoshock (or photophobic) response and phototaxis. Photoshock is a response to a sudden change in light intensity (e.g., flash illumination), wherein organisms transiently stop moving or move backward. During phototaxis, organisms move toward the light source or in the opposite direction (called positive or negative phototaxis, respectively). The unicellular green alga Chlamydomonas reinhardtii is an excellent organism to study photobehavior because it rapidly changes its swimming pattern by modulating the beating of cilia (a.k.a., flagella) after photoreception. Here, various simple methods are shown to observe photobehaviors in C. reinhardtii. Research on C. reinhardtii&#39;s photobehaviors has led to the discovery of common regulatory mechanisms between eukaryotic cilia and channelrhodopsins, which may contribute to a better understanding of ciliopathies and the development of new optogenetics methods.

Observation of Photobehavior in Chlamydomonas reinhardtii

Most swimming photoautotrophic organisms show photo-induced behavioral changes (photobehavior). The present protocol observes the said photobehavior in the model organism Chlamydomonas reinhardtii.

For the survival of the motile phototrophic microorganisms, being under proper light conditions is crucial. Consequently, they show photo-induced ...

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 ...

Evaluation of Photosynthetic Efficiency in Photorespiratory Mutants by Chlorophyll Fluorescence Analysis

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