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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photorespiration.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-expression+analysis+as+tool+for+the+discovery+of+transport+proteins+in+photorespiration.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+closing+the+remaining+gaps+in+photorespiration--the+essential+but+unexplored+role+of+transport+proteins.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+costs+of+photorespiration+to+food+production+now+and+in+the+future.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bile+acid+sodium+symporter+BASS6+can+transport+glycolate+and+is+involved+in+photorespiratory+metabolism+in+Arabidopsis+thaliana.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PLGG1,+a+plastidic+glycolate+glycerate+transporter,+is+required+for+photorespiration+and+defines+a+unique+class+of+metabolite+transporters.">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., Schl&#252;ter, U., Linka, N., Eisenhut, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport+proteins+enabling+plant+photorespiratory+metabolism.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlorophyll+fluorescence+screening+of+Arabidopsis+thaliana+for+CO2+sensitive+photorespiration+and+photoinhibition+mutants.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+of+mutants+using+chlorophyll+fluorescence.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arabidopsis+thaliana+chloroplast+proteome+reveals+pathway+abundance+and+novel+protein+functions.">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://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular characterization of the plastid-localized ABC protein TAP1 in Arabidopsis thaliana. , (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</s...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>Arabidopsis thaliana seeds (abcb26)</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>Arabidopsis thaliana seeds (plgg1-1)</td>
			<td>ABRC, ordered through TAIR www.arabidopsis.org</td>
			<td>SALK_053469C</td>
			<td>parental arabidopsis seeds&#160;</td>
		</tr>
		<tr>
			<td>Arabidopsis thaliana 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>&#160;bleach&#160;</td>
			<td>clorox</td>
			<td>generic bleach&#160;</td>
			<td>chemical used to sterilize seeds</td>
		</tr>
		<tr>
			<td>Carbolime absorbent</td>
			<td>Medline products</td>
			<td>S232-104-001</td>
			<td>CO2 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&#160;</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 &#38; Skoog Basal Medium with Vitamins &#38; 1.0 g/L MES (MS)</td>
			<td>Phytotech labs</td>
			<td>M5531&#160;</td>
			<td>growth media for arabidopsis seedlings&#160;</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&#160;</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>&#160;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....

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

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1.8K Views.  Louisiana State University. We describe an approach to measure changes in photosynthetic efficiency in plants after treatment with low CO2 using chlorophyll fluorescence. 

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

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical shock or electrical stimulation is available for study of various aspects of seizure activity and behavior. All flies, including wild-type, will undergo seizure-like activity if stimulated at a high enough voltage. Seizure like activity is an all-or-nothing response and each genotype has a specific seizure threshold. The seizure threshold of a specific genotype of fly can be altered either by treatment with a drug or by genetic suppression or enhancement. The threshold is easily measured by electrophysiology. Seizure-like activity can be induced via high frequency electrical stimulation delivered directly to the brain and recorded through the dorsal longitudinal muscles (DLMs) in the thorax. The DLMs are innervated by part of the giant fiber system. Starting with low voltage, high frequency stimulation, and subsequently raising the voltage in small increments, the seizure threshold for a single fly can be measured.

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Using high frequency electrical stimulation, seizure-like activity can be induced in Drosophila. This activity is easily recorded from the giant fiber system.

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical ...

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells possess several lines of defense against damage to biologically relevant molecules like nucleic acids, lipids and proteins. In addition to organelle dynamics (fusion/fission/motility/inheritance) and tightly controlled protease activity, the degradation of surplus, damaged or compromised organelles by autophagy (cellular &#8216;self-eating&#8217;) has received much attention from the scientific community. The regulation of autophagy is quite complex and depends on genetic and environmental factors, many of which have so far not been elucidated. Here a novel method is presented that allows the convenient study of autophagy in the filamentous fungus Penicillium chrysogenum. It is based on growth of the fungus on so-called &#8216;starvation pads&#8217; for stimulation of autophagy in a reproducible manner. Samples are directly assayed by microscopy and evaluated for autophagy induction / progress. The protocol presented here is not limited for use with P. chrysogenum and can be easily adapted for use in other filamentous fungi.

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

A convenient and powerful method for studying autophagy in Penicillium chrysogenum by using starvation pads (mixtures of agarose and tap water in a microscope slide containing a central cavity) is presented here.

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

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 ...

Fluorescence Lifetime Macro Imager for Biomedical Applications

This paper presents a new photoluminescence lifetime imager designed to map the molecular oxygen (O2) concentration in different phosphorescent samples ranging from solid-state, O2-sensitive coatings to live animal tissue samples stained with soluble O2-sensitive probes. In particular, the nanoparticle-based near-infrared probe NanO2-IR, which is excitable with a 625 nm light-emitting diode (LED) and emits at 760 nm, was used. The imaging system is based on the Timepix3 camera (Tpx3Cam) and the opto-mechanical adaptor, which also houses an image intensifier. O2 phosphorescence lifetime imaging microscopy (PLIM) is commonly required for various studies, but current platforms have limitations in their accuracy, general flexibility, and usability.
The system presented here is a fast and highly sensitive imager, which is built on an integrated optical sensor and readout chip module, Tpx3Cam. It is shown to produce high-intensity phosphorescence signals and stable lifetime values from surface-stained intestinal tissue samples or intraluminally stained fragments of the large intestine and allows the detailed mapping of tissue O2 levels in about 20 s or less. Initial experiments on the imaging of hypoxia in grafted tumors in unconscious animals are also presented. We also describe how the imager can be re-configured for use with O2-sensitive materials based on Pt-porphyrin dyes using a 390 nm LED for the excitation and a bandpass 650 nm filter for emission. Overall, the PLIM imager was found to produce accurate quantitative measurements of lifetime values for the probes used and respective two-dimensional maps of the O2 concentration. It is also useful for the metabolic imaging of ex vivo tissue models and live animals.

This paper describes the use of a new, fast optical imager for the macroscopic photoluminescence lifetime imaging of long decay emitting samples. The integration, image acquisition, and analysis procedures are described, along with the preparation and characterization of the sensor materials for the imaging and the application of the imager in studying biological samples.

This paper presents a new photoluminescence lifetime imager designed to map the molecular oxygen (O2) concentration in different phosphorescent samples ...