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 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.
Photosynthesis consists of light absorption, primary electron transfer, energy stabilization, and the synthesis and transport of photosynthetic products1. Understanding each step is vital to guide efforts to increase crop photosynthetic efficiency. Light affects the rate of photosynthesis, requiring balancing energy supply, in the form of photons, with demand for reducing equivalents. When supply exceeds demand, for example under high-light or during reduced CO2 fixation caused by stomatal closure, build-up of reducing power increases the probability of reactive oxygen species formation with the potential to damage the photosynthetic apparatus and impair electron transport. Therefore, to prevent damage, plants have developed several photo-protective mechanisms, including detoxification of reactive oxygen species and non-photochemical quenching of the excited chlorophyll states (NPQ)2.
Maintaining high rates of photosynthesis is challenging under a field environment. Seasonal and diurnal changes, along with environmental fluctuations such as wind-induced leaf movements and transient cloud cover, cause shifts in the amount and intensity of light received by plants for photosynthesis3. NPQ dissipates excess light energy and can help prevent photo-damage while allowing for sustained rates of photosynthesis at high-light4. However, prolonged NPQ during high- to low-light transitions continues to dissipate energy that could be used for carbon reduction5. As a result, speeding up the relaxation of NPQ can increase the efficiency of photosynthesis6, making NPQ relaxation an attractive target for crop improvement.
Pulse amplitude modulated chlorophyll fluorescence (PAM) analysis can be used to calculate NPQ from measurable parameters (Supplementary Table 1 and Supplementary Table 2)7,8,9. This article focuses on determining NPQ relaxation rates in field-grown plants for the purpose of screening natural variation in germplasm. However, PAM chlorophyll fluorometry analysis can also be used for a wide variety of purposes, applied to species ranging from algae to higher plants, and is reviewed elsewhere7,8,9.
In a dark-adapted leaf or cell, photosystem II (PSII) reaction centers are open to receive electrons and there is no NPQ. Switching on a low-intensity measuring light elicits chlorophyll fluorescence while avoiding electron transport through PSII. The recorded minimum fluorescence in this dark-adapted state is described by the parameter Fo. Applying a high-intensity light pulse to a dark-adapted leaf can rapidly reduce the first stable electron acceptor pool of quinones bound to the quinone A site. This temporarily blocks electron transfer capacity in PSII reaction centers, which are then said to be closed and unable to receive electrons from water-splitting. By using a short pulse duration, there is insufficient time to stimulate NPQ. The resulting chlorophyll fluorescence is equivalent to the maximum value obtainable in the absence of NPQ, or maximum fluorescence, Fm. The difference between minimal and maximal fluorescence is referred to as variable fluorescence, Fv. The maximum photochemical quantum yield of photosystem II (Fv/Fm) is calculated from these two parameters using the following equation:
Fv/Fm = (Fm-Fo)/Fm
This can provide an important indicator of photosystem function and stress. Turning on an actinic (photosynthetic) light stimulates non-photochemical quenching, and subsequent application of a saturating flash allows for the measurement of light-adapted maximal fluorescence, Fm'. By comparing the difference between dark and light-adapted maximum fluorescence, NPQ can be calculated according to the Stern-Volmer equation10:
NPQ = Fm/Fm' – 1
In higher plants, NPQ has been described as consisting of at least five distinct components, including qE, qT, qZ, qI and qH. The precise mechanisms involved in NPQ are not fully understood; however, qE is considered to be the major component of NPQ in most plants. Crucial factors for full engagement of qE have been found to include the build-up of a proton gradient across the thylakoid membrane, the activity of photosystem II subunit S11,12, and de-epoxidated xanthophylls, antheraxanthin, lutein, and in particular zeaxanthin13. qE relaxes the fastest of any NPQ component (< 2 min)14, and reversible activation of qE is therefore particularly important for adaptation to shifting light intensities. A second slower phase of NPQ relaxation (~2-30 min) encompasses both qT, related to state transitions, and qZ, involving interconversion of zeaxanthin to violaxanthin15. Slow relaxing (> 30 min) of NPQ may include both photoinhibitory quenching (qI)16 and processes independent of photodamage17,18, such as qH, which is sustained quenching in the peripheral antennae of PSII mediated by a plastid lipocalin protein19,20.
NPQ increases during exposure to high light. Subsequent transfer to low light can result in downregulation of NPQ. The decay of fast, intermediate, and slow relaxing phases can be captured in the parameters of a bi-exponential function15,21,22,23
NPQ = Aq1(-t/τ1) + Aq2(-t/τ2) + Aq3
The theoretical basis for the bi-exponential function is based on the assumption of first-order utilization of hypothetical quenchers, including qE (Aq1), the combined relaxation of qZ and qT (Aq2), with the corresponding time-constants τq1 and τq2, and long-term NPQ, which includes qI and photodamage independent processes (Aq3). As such, the bi-exponential function provides a more realistic representation of the multiple connected biological processes involved in quenching chlorophyll fluorescence compared to a simpler Hill equation which lacks a theoretical basis24.
NPQ can be measured using a variety of commercially available PAM fluorometers25,26, from simple hand-held devices27 to more advanced closed systems28. However, a limitation of several of these approaches is a relatively low throughput, which makes screening large collections of plants challenging without multiple devices and a team of researchers. To address this issue, McAusland et al. developed a procedure based on excised leaf tissue and used it to identify differences in chlorophyll fluorescence between two wheat cultivars29. The attraction of this approach is that imaging leaf disks, taken from multiple plants with a single device, can facilitate screening hundreds of genotypes within a day. This makes it possible to assess variation in NPQ relaxation as part of genome wide association studies, or for screening breeding populations with the potential to increase crop photosynthetic efficiency and ultimately yield.
Building on the findings of McAusland et al.29, we use PAM chlorophyll fluorescence analysis of leaf disks for high-throughput screening of NPQ relaxation rates in Glycine max (G. max; soybean). This protocol uses the CF Imager25, which is comparable to other commercially available closed-PAM systems, such as the popular FluorCam26. With a dark room for adaptation of samples, users can image 96-well plates, Petri dishes, and small plants. The key advantage of this approach is the increase in throughput afforded by using leaf disks compared to sequential analysis of individual plants. Herein we present representative results, and a method for sampling, measuring, and analysis of NPQ in field-grown plants.
1. Seed planting
2. Collecting leaf samples from the field
3. Preparing samples for analysis
4. Measuring of non-photochemical quenching using chlorophyll fluorescence imager
5. Processing chlorophyll fluorescence data
Figure 1A depicts a typical measurement of NPQ in field-grown soybean. Plants were grown in Urbana, IL (latitude 40.084604°, longitude -88.227952°) during summer 2021, with seeds planted on June 5th. 2021. The leaf discs were sampled after 30 days of planting seeds, and measurements were made with the protocol provided (Table 1). Fv/Fm and NPQ values were calculated for each leaf disk (Supplementary Table 4) and NPQ relaxation parameters were calculated by fitting a bi-exponential function (Table 2). After an initial saturating flash to determine Fv/Fm, leaf disks were exposed to low-light (50 µmol m−2s−1) and the measured NPQ was < 1 (Figure 1A). Upon transfer to high-light (2000 µmol m−2s−1), NPQ increased reaching a maximum value of 4 after 15 min. A return to low-light caused a rapid initial decrease in NPQ (Aq1) with a time constant of 1.07 min (τq1), followed by a second slower relaxing phase (Aq2) with a time constant of 24.5 min (τq2). Residual NPQ that remained at the end of the protocol (~0.5) was captured by the constant (Aq3).
Figure 1B depicts an example of a failed measurement. Upon transfer to high light there is a minimal increase in NPQ (0.38), which does not relax. Close inspection of the data reveals the leaf disk had a low Fv/Fm value indicative of stress (0.27), and the data should be discarded.
Figure 1: NPQ measurement data using field-grown soybean. (A) NPQ activation and relaxation kinetics of G. max cv. IA3023. Data are presented as the mean of five biological (separate field plots) and three technical (leaf disks from the same plot) replicates with error bars representing standard deviation. (B) Example of a failed technical replicate (n = 1) using of G. max cv. IA3023. The grey and white bars at the top of the graph indicate periods of illumination with a photosynthetic photon flux density (PPFD) of 2000 µmol m−2s−1 (white bar), PPFD of 50 µmol m−2s−1 (grey bars). Please click here to view a larger version of this figure.
Step | Delay | Action | Cycles | PPFD (μmol m-2s-1 ) |
1 | 0 s | Change Actinic | – | 0 |
2 | 20 s | Apply Pulse | 1 | 6152 |
3 | 30 s | Change Actinic | – | 50 |
4 | 150 s | Apply Pulse | 4 | 6152 |
5 | 30 s | Change Actinic | – | 2000 |
6 | 150 s | Apply Pulse | 6 | 6152 |
7 | 0 s | Change Actinic | – | 50 |
8 | 150 s | Apply Pulse | 2 | 6152 |
9 | 5 min | Apply Pulse | 9 | 6152 |
10 | 20 s | Change Actinic | – | 0 |
Table 1: Chlorophyll fluorescence imaging protocol to be used with the imager.
Rep | Aq1 | τq1 / min | Aq2 | τq2 / min | Aq3 | maxNPQ | R2 |
1 | 2.00 | 1.12 | 1.88 | 16.62 | 0.29 | 4.17 | 0.9999 |
2 | 2.14 | 1.17 | 1.70 | 13.68 | 0.34 | 4.17 | 0.9999 |
3 | 1.82 | 0.78 | 1.88 | 18.42 | 0.36 | 4.05 | 0.9997 |
4 | 2.05 | 1.28 | 1.67 | 26.89 | 0.15 | 3.87 | 0.9999 |
5 | 1.72 | 1.01 | 1.81 | 21.22 | 0.52 | 4.05 | 0.9998 |
Average | 1.94 | 1.07 | 1.79 | 19.37 | 0.33 | 4.06 | |
S.D. | 0.17 | 0.19 | 0.10 | 5.02 | 0.13 | 0.12 |
Table 2: Calculated NPQ relaxation parameters for G. max cv. IA3023. Values for Aq1, Aq2, and Aq3 are unitless, values for τq1 and τq2 are reported in min. Abbreviations: MaxNPQ = maximum recorded NPQ value in the light, Rep = biological replicate number corresponding to a single plot, where the bi-exponential function is fit using three technical replicates (leaf disks). S.D. represents standard deviation.
Supplementary Table 1: Description of equations used in the manuscript. Please click here to download this Table.
Supplementary Table 2: Description of parameter values discussed in the manuscript. Please click here to download this Table.
Supplementary Table 3: Pulse amplitude modulated fluorescence technical specifications. Please click here to download this Table.
Supplementary Table 4: Calculated Fv/Fm and NPQ values of G. max cv. IA3023 leaf disks using the protocol provided. Please click here to download this Table.
Supplementary File 1: A script for mapping leaf disk positions using the data output provided by the imager. Please click here to download this File.
Supplementary File 2: A script for calculating Fo, Fm, Fm',and Fv/Fm values per leaf disk using the data output provided by the imager and Supplementary File 1. Please click here to download this File.
Supplementary File 3: A script for calculating NPQ values per leaf disk at each time point during the protocol using the data output from Supplementary File 2. Please click here to download this File.
Supplementary File 4: An R script to annotate the output from Supplementary File 3 with Fv/Fm values calculated by Supplementary file 2. Please click here to download this File.
Supplementary File 5: An R script for re-formatting the data from Supplementary File 4 to allow for fitting a bi-exponential decay function to calculated NPQ values following relaxation. Please click here to download this File.
Supplementary File 6: A script to fit a bi-exponential decay function to calculate NPQ values and calculate relaxation parameters using the data output from Supplementary File 5. Please click here to download this File.
Careful choice and handling of leaf disks are critical to obtain reliable measurements of NPQ. First, damage to the tissue, such as rough handling with tweezers, will introduce stress, resulting in low values for the maximum quantum efficiency of photosynthesis. Non-stressed plants typically have Fv/Fm values of around 0.8318, with significant declines indicating a reduction in photosynthetic performance9. However, plants grown under field conditions typically experience mild stress, therefore a threshold of FvFm > 0.75 was set for inclusion in accordance with previous studies which chose this as the limit for the beginnings of photosynthetic decline30. Handling of the nasal aspirator filters used to hold leaf disks in their place represents another common source of problems. If nasal aspirator filters are oversaturated, excess water will negatively impact leaf physiology also leading to low quantum efficiencies; the same brand must be used throughout the experiment as different brands can hold different amounts of water. Third, field-grown plants experience a fluctuating environment, with differences in rainfall, temperature, humidity, and insect pressure. The growth stages of the plants also contribute to the efficiency of chlorophyll fluorescence. Chlorotic leaves or extensive pest damage should be avoided when sampling. When making comparisons, consistent choice of leaves at similar developmental stages is vital to reduce experimental noise. Rates of activation and relaxation of non-photochemical quenching can vary with leaf age and plant developmental stage31, as a consequence of differences in balance between light absorption, chloroplast development, energy status, and long-term shading. Therefore, a typical choice is to compare rates from the most recent fully expanded leaf with mature chloroplasts.
This protocol is designed for analysis of field-grown soybean but can be modified for sampling and measurement of greenhouse-grown material or other higher plants. Several considerations are important when adapting the protocol. First, experimental design should be taken into account to reduce noise and avoid the introduction of bias. In the field, soybean genotypes are commonly grown in plots of 50-100 plants in a row, each plot considered a single biological replicate. Each plot can experience different soil, exposure, and drainage conditions which will impact whole plant physiology. Therefore, selecting an appropriate plot design, such as a random block, can avoid bias and reduce in-field variability. To minimize variation within plots, it is suggested to measure multiple replicates (three to five) and take disks from different plants within a plot. However, when working with greenhouse or chamber-grown material, a single plant may constitute a biological replicate with multiple disks taken from the same leaf. Further, the plant position should be rotated several times a week to prevent bias in the conditions experienced. Second, the actinic light intensities used in this protocol reflect the maximum and minimum light intensities experienced by a soybean canopy on a sunny day in Illinois (W 88°13ʹ42ʺ/N 40°06ʹ31ʺ). Depending on the experimental setup and plant under analysis, it is advisable to adjust the light intensity to the particular growth environment to provide more realistic measuring conditions. Finally, both the sampling procedure and measuring protocol can be adapted for use with other closed PAM fluorometer systems, with the provision that the steps required to operate the measuring software will need to be adjusted for a given device.
A major limitation of the approach is the interpretation of the parameter values calculated by the bi-exponential function. This is particularly important if applying the procedure to other higher plants or algae, as the biological processes contributing to NPQ can vary between species. For example, some mosses and algae contain LHCSR proteins rather than PsbS (for a review see32). Further, the relative importance of biological processes can vary, with state transitions or qT, representing a more important part of the Aq2 component in algae than in higher plants, where up to 80% of light-harvesting complexes can reversibly move between photosystems I and II33. Therefore, it should be considered that the bi-exponential function may not always be the best choice to explain the biological processes governing relaxation kinetics22,29 and this should be assessed empirically for the species of choice. Careful experimentation, including the application of inhibitors and uncouplers34,35 or study of mutants21,23 can be used to provide insight into the processes contributing to NPQ relaxation, but care should be taken in drawing conclusions.
The method presented here is similar to that of McAusland et al.29, which used a closed-PAM imager to assess the photosynthetic performance of chamber-grown plants. However, by placing leaf samples on damp filter paper rather than in 96-well plates, and incubating tissue in custom-made chambers to control the O2 and CO2 concentrations, McAusland et al. were able to perform a wider range of measurements assessing photosynthetic parameters29. The main advantages of the approach presented here include the simplicity of the method, which does not require custom equipment, and the use of 24-well plates for initial sampling of leaf tissue which enables collection of samples from field-grown material, potentially increasing the number of genotypes a researcher is able to screen.
In summary, PAM chlorophyll fluorescence analysis is a powerful technique for the measurement of the efficiency of photosynthesis. However, conventional measurements are done on a per-plant basis, limiting screening capacity either by the time taken or the number of people and equipment available to perform assays. The use of leaf disks in a 96-well plate provides a means to measure many genotypes simultaneously, increasing assay throughput. By facilitating the screening of many genotypes in a short space of time by a single investigator29, it is possible to apply the measurement of NPQ relaxation to screening natural diversity of photosynthesis with the potential to perform genome-wide association studies.
The authors have nothing to disclose.
This work is supported by the research project Realizing Increased Photosynthetic Efficiency (RIPE) that is funded by the Bill & Melinda Gates Foundation, Foundation for Food and Agriculture Research, and the U.K. Foreign, Commonwealth & Development Office under grant number OPP1172157.
24 well tissue culture plate | Fisher Scientific | FB012929 | Country of Origin: United States of America |
96 well tissue culture plate | Fisher Scientific | FB012931 | Country of Origin: United States of America |
Aluminum foil | Antylia Scientific | 61018-56 | Country of Origin: United States of America |
Black marker pen | Sharpie | SAN30001 | Country of Origin: United States of America |
CF imager | Technologica Ltd. | N/A | chlorophyll fluorescence imager Country of Origin: United Kingdom |
Cork-borer, 7mm | Humboldt Mfg Co | H9665 | Country of Origin: United States of America |
FluorImager V2.305 Software | Technologica Ltd. | N/A | imaging software Country of Origin: United Kingdom |
iHank-Nose 100-Pack of Premium Nasal Aspirator Hygiene Filters | Amazon | B07P6XCTGV | Country of Origin: United States of America |
Marker stakes | John Henry Company | KN0151 | Country of Origin: United States of America |
Paper scissors | VWR | 82027-596 | Country of Origin: United States of America |
Parafilm | Bemis Company Inc. | S3-594-6 | Semi -transparent flexible film Country of Origin: United States of America |
Solid rubber stoppers | Fisher Scientific | 14-130M | Country of Origin: United States of America |