The goal of this method is to identify mutants in the photorespiratory pathway using a low CO2 screening. We present a method to identify mutants that are disrupting 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 following sections will provide details on seed preparation and sterilization, plant growth and low CO2 treatment, configure the fluorescence imaging system, measure quantum yield of treated samples, representative results and conclusions. Seed preparation and sterilization. Seed preparation consists of seed imbibing and seed sterilization.
It is important to note that all of these steps are done in a laminar flow hood to keep sterile conditions. All the necessary materials, reagents, and growth medium are autoclaved. The seed lines used are plgg1-1, abcb26, and WT or wild type.
Imbibe seeds in sterile water in a laminar flow hood, then stratify at four degrees Celsius in the dark for two days. Under sterile conditions, prepare 10 milliliters of 50%volume to volume bleach solution and add approximately 20 microliters of Tween 20. Remove water from imbibed seeds, then add one milliliter of bleach solution into the microcentrifuge tube and incubate at room temperature for five minutes.
Remove the bleach solution with a pipette. Rinse the seeds in one milliliter of sterile water to resuspend. Remove the water once the seeds have settled to the bottom.
Resuspend seeds in sterile 0.1%agarose solution. For seed plating, basal medium plates with 1%MS medium with vitamins and 1%agar for cultivation of the test mutants. Cut a 200 microliter pipette tip with a razor blade.
Using a pipette, place one seed into the center of the designated square grid for each test mutant or genotype. Here, a square plate with a one by one centimeter grid is used. This helps to keep uniform distance between each seedling and avoid overlapping, which will be important later in the fluorescence imaging and analysis.
Once the seeds have been plated, wrap surgical tape around the lid to seal, then place it in the growth chamber. Plant growth and low CO2 treatment. Grow plants for seven to nine days at 20 degrees Celsius under an eight-hour light cycle of 120 micromoles per meter square per second and 16 hours of darkness at 18 degrees Celsius.
Check plants on the sixth day to determine if they are large enough for imaging. On the eighth day after plating, expose plants to low CO2. Place treatment plants in a sealed box within the growth chamber with a light intensity of 200 micromoles per meter square per second for 12 hours.
The low CO2 condition was constructed using an airtight transparent container with 100 grams of soda lime placed in the bottom of the container. The container was placed within the same growth chamber as the control. The control plants will remain under 120 micromoles per meter square per second in ambient CO2 for 12 hours.
Configure the fluorescence imaging system. Place a testing plate centered under the camera at a fixed distance in the fluorescence imaging system. Within the instrument software, navigate to the live window and check the box Flashes to switch on non-actinic measuring flashes.
Click on the zoom and focus tools until you see a complete and sharp image. Set the value of EL shutter to zero and adjust the sensitivity to get a fluorescent signal in the range of 200 to 500 digital units. Place a light meter in the same position used to adjust the camera settings.
In the live window, check the box Super to start a saturating pulse lasting for 800 milliseconds. Use the slider to adjust the percentage of relative power for the Super pulse until the light meter reads 6, 000 to 8, 000 micromoles per square meter per second. Measure quantum yield of treated samples.
Directly following treatment, cover plates with aluminum foil for 15 minutes for dark adaptation. Remove foil to measure the quantum yield of photosystem two with a pulse amplitude modified fluorometer. Then place the seedling plate directly under the camera and run the quantum yield protocol found in our GitHub repository.
Download the Quantum Yield Protocol from GitHub. Use a fluorometer software to open the program file by clicking on the folder icon and navigating to the file location. Run the quantum yield protocol by clicking on the red lightning icon.
After the protocol is complete, navigate to the pre-analysis window. Partition the plate into individual seedlings by highlighting all the pixels for each seedling on the plate image. Click Background Exclusion to remove any highlighted background pixels, leaving just the seedling area.
Click 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. In the tab Experiment, click Export, then Numeric.
Click on Numerical Average to generate a text file containing the quantum yield for each seedling. Open the text file and spreadsheet for analysis. The spreadsheet contains the area number, size of the pixels, Fm, Ft, Fq, and QY.First, we need to identify the area number to its corresponding genotype, whether wild type or a test mutant, Results.
Here are the plate pictures of raw and fluorescence images from ambient and low CO2 screening of wild types and test mutants. Each plate is labeled by area number with corresponding fluorescence readings as quantum yield or QY.The data is exported as a text file and can be opened in a spreadsheet for analysis. The dark adapted Fv/Fm quantum yield efficiencies of wild types and mutants are visualized by box and whisker plots.
To test the statistical difference of wild types and test mutants, pairwise T-test was used with a P value of less than 0.05. Here, we use the photorespiratory mutant line with reduced quantum yield Fv/Fm as a test mutant to check the efficiency of our screening method. Our results show that test mutants have significantly lower quantum yield efficiency compared to the wild types.
This indicates that our screening method is able to identify photorespiratory mutants using low CO2 screening.Conclusions. An important thing to remember while using this protocol is to make sure that Arabidopsis seedlings are large enough to measure fluorescence, but not so large that the plants would overlap during imaging. It is preferred to image the seedlings as the first true leaves to try to keep the leaf size and leaf angles uniform.
This protocol can be used as a high throughput screening for seedlings. Imaging of each plate is relatively quick and has the potential to screen well over 1, 000 seedlings per day. Chlorophyll fluorescence analysis allowed us to identify photorespiratory mutants using a low CO2 screening, which is important for maintaining photorespiration efficiency.
This protocol is not limited to Arabidopsis and has the potential use for abiotic stress response experiments.
