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概要

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

Abstract

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.

Introduction

Abiotic stress conditions commonly seen in farmer'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'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 uncharacterized^2,3.

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 process^5,6,7. 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 chloroplast^5,6.

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.

Protocol

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).

1. Seed imbibation and stratification
   NOTE: The seed lines used are plgg1-1 (salk_053463), abcb26 (salk_085232), and wild type (WT, Col-0).
   1. Dispense the seeds into 1.5 mL microcentrifuge tubes. Imbibe the seeds in sterile water in a laminar flow hood and stratify at 4 °C in the dark for 2 days.
2. Seed sterilization
   1. Under sterile conditions, prepare 10 mL of 50% (v/v) bleach solution and add approximately 20 µL 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.
   2. Remove the bleach solution with a pipette.
   3. 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.
   4. Resuspend the seeds in sterile 0.1% agarose solution.
3. Seed plating
   1. Make a 1 L volume of Murashige & 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.
      1. 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.
      2. Autoclave at 121 °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 & Skoog Basal Medium plates contain 1% MS medium with vitamins and 1% agar for the cultivation of the test mutants.
   2. Cut a 200 µL 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 µL 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.
   3. 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.

2. Plant growth and low CO₂ treatment

1. Grow the plants for 7-9 days at 20 °C under an 8 h light cycle of 120 µmol·m⁻²s⁻¹ and 16 h of darkness at 18 °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.
2. Low CO₂ treatment
   1. After the 7-9 days, place four of the eight plates from the ambient growth chamber conditions into photorespiratory conditions of 20 °C, continuous light levels of 200 µmol·m⁻²s⁻¹, and low CO₂ for 12 h.
   2. 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 µmol·m⁻²s⁻¹ in ambient CO₂ for 12 h.

3. Configuring the fluorescence imaging system

1. 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.
2. Within the instrument's software, navigate to the Live Window and check the box Flashes to switch on non-actinic measuring flashes.
3. 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.
4. Set the value of El. Shutter に 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.
5. Place a light meter in the same position used to adjust the camera settings.
6. 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.
7. Use the slider to adjust the percentage of relative power for the Super pulse until the light meter reads 6,000-8,000 µmol·m⁻²s⁻¹.

4. Design the quantum yield program

1. 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.
2. Define the following global variables according to the configured light settings:
   Shutter = 0
   Sensitivity = 40
   Super = 65
3. Include the time-step for logging data:
   TS = 20ms
4. 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.
5. Record the Fo measurements into data tables.
   <0,F0period..F0duration>=>mfmsub
   <0s>->checkpoint,"startFo"
   <F0duration – F0period>=>checkpoint,"endFo";
   Where < , > represents the set of fluorescence values indexed by time; => 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.
6. Collect and store the Fm measurement by sampling fluorescence after a saturating pulse.
   PulseDuration=960ms; ##
   a1=F0duration + 40ms
   a2 = a1 + 480ms;
   <a1>=>SatPulse(PulseDuration);
   <a1 + 80ms, a1 + 160ms.a1 + PulseDuration>=>mfmsub
   <a1 + PulseDuration + 80ms>=>mfmsub;
   <a2>=>checkpoint,"startFm"
   <a1 + PulseDuration – 80ms>=>checkpoint,"endFm"
   <a2 + 80ms>=>checkpoint,"timeVisual";
   ​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.

5. Measuring the quantum yield of the treated samples

1. 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.
2. 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’s software to open the program file by clicking on the folder icon and navigating to the file location.
3. Run the quantum yield program by clicking on the red lightning icon.
4. 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.
5. 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.
6. 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.

6. Opening the data file

1. 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.

Representative Results

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 < 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.

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. Please click here to view a larger version of this figure.

Figure 2: Dark-adapted F_v/F_m 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 F_v/F_m 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. Please click here to view a larger version of this figure.

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 > 44, p < 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. Please click here to view a larger version of this figure.

Supplemental Table S1: Compiled fluorescent data from all seedling plates used in the experiment. Please click here to download this File.

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. Please click here to download this File.

Discussion

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.

Materials

1.5 mL microcentrifuge tube	VWR	10810-070	container for seed sterilization
agarose	VWR	9012-36-6	chemical used to suspend seeds for ease of plating
Arabidopsis thaliana seeds (abcb26)	ABRC, ordered through TAIR www.arabidopsis.org	SALK_085232	arabidopsis seeds used as experimental group
Arabidopsis thaliana seeds (plgg1-1)	ABRC, ordered through TAIR www.arabidopsis.org	SALK_053469C	parental arabidopsis seeds
Arabidopsis thaliana seeds (WT)	ABRC, ordered through TAIR www.arabidopsis.org	Col-0	arabidopsis wild type seeds used as a control group
bleach	clorox	generic bleach	chemical used to sterilize seeds
Carbolime absorbent	Medline products	S232-104-001	CO2 absorbent
Closed FluorCam	Photon Systems Instruments	FC 800-C	Fluorescence imager
FluoroCam FC 800-C	Photon Systems Instruments	Closed FluorCam FC 800-C/1010-S	Fluorescence imager
FluoroCam7	Photon Systems Instruments	Closed FluorCam FC 800-C/1010-S	Fluorescence image analysis software
Gelzan (plant agar)	Phytotech labs	71010-52-1	chemical used to solidify MS media as plates
glass flask 1 L	Fisherbrand	FB5011000	container for making and autoclaving MS media
growth chamber	caron	7317-50-2	growth chamber used to grow plants
Murashige & Skoog Basal Medium with Vitamins & 1.0 g/L MES (MS)	Phytotech labs	M5531	growth media for arabidopsis seedlings
potassium Hydroxide (KOH)	Phytotech labs	1310-58-3	make as 1 M solution for ph adjustment
spider lights	Mean Well Enterprises	XLG-100-H-AB	lights used in the light assay
Square Petri Dish with Grid, sterile	Simport Scientific	D21016	used to hold MS media for arabidopsis seedlings
surgical tape	3M	1530-1	tape used to seal plates
tween 20	biorad	9005-64-5	surfactant used to assist seed sterilization