A guide to photosynthetic gas exchange measurements: Fundamental principles, best practice and potential pitfalls.

Gas exchange measurements enable mechanistic insights into the processes that underpin carbon and water fluxes in plant leaves which in turn inform understanding of related processes at a range of scales from individual cells to entire ecosytems. Given the importance of photosynthesis for the global climate discussion it is important to (a) foster a basic understanding of the fundamental principles underpinning the experimental methods used by the broad community, and (b) ensure best practice and correct data interpretation within the research community. In this review, we outline the biochemical and biophysical parameters of photosynthesis that can be investigated with gas exchange measurements and we provide step-by-step guidance on how to reliably measure them. We advise on best practices for using gas exchange equipment and highlight potential pitfalls in experimental design and data interpretation. The Supporting Information contains exemplary data sets, experimental protocols and data-modelling routines. This review is a community effort to equip both the experimental researcher and the data modeller with a solid understanding of the theoretical basis of gas-exchange measurements, the rationale behind different experimental protocols and the approaches to data interpretation.

The inverse relationship between solar-induced fluorescence yield and photosynthetic capacity: benefits for field phenotyping.

Improving photosynthesis is considered a promising way to increase crop yield to feed a growing population. Realizing this goal requires non-destructive techniques to quantify photosynthetic variation among crop cultivars. Despite existing remote sensing-based approaches, it remains a question whether solar-induced fluorescence (SIF) can facilitate screening crop cultivars of improved photosynthetic capacity in plant breeding trials. Here we tested a hypothesis that SIF yield rather than SIF had a better relationship with the maximum electron transport rate (Jmax). Time-synchronized hyperspectral images and irradiance spectra of sunlight under clear-sky conditions were combined to estimate SIF and SIF yield, which were then correlated with ground-truth Vcmax and Jmax. With observations binned over time (i.e. group 1: 6, 7, and 12 July 2017; group 2: 31 July and 18 August 2017; and group 3: 24 and 25 July 2018), SIF yield showed a stronger negative relationship, compared with SIF, with photosynthetic variables. Using SIF yield for Jmax (Vcmax) predictions, the regression analysis exhibited an R2 of 0.62 (0.71) and root mean square error (RMSE) of 11.88 (46.86) µmol m-2 s-1 for group 1, an R2 of 0.85 (0.72) and RMSE of 13.51 (49.32) µmol m-2 s-1 for group 2, and an R2 of 0.92 (0.87) and RMSE of 15.23 (30.29) µmol m-2 s-1 for group 3. The combined use of hyperspectral images and irradiance measurements provides an alternative yet promising approach to characterization of photosynthetic parameters at plot level.

The effect of increasing temperature on crop photosynthesis: from enzymes to ecosystems.

As global land surface temperature continues to rise and heatwave events increase in frequency, duration, and/or intensity, our key food and fuel cropping systems will likely face increased heat-related stress. A large volume of literature exists on exploring measured and modelled impacts of rising temperature on crop photosynthesis, from enzymatic responses within the leaf up to larger ecosystem-scale responses that reflect seasonal and interannual crop responses to heat. This review discusses (i) how crop photosynthesis changes with temperature at the enzymatic scale within the leaf; (ii) how stomata and plant transport systems are affected by temperature; (iii) what features make a plant susceptible or tolerant to elevated temperature and heat stress; and (iv) how these temperature and heat effects compound at the ecosystem scale to affect crop yields. Throughout the review, we identify current advancements and future research trajectories that are needed to make our cropping systems more resilient to rising temperature and heat stress, which are both projected to occur due to current global fossil fuel emissions.

Estimating photosynthetic traits from reflectance spectra: A synthesis of spectral indices, numerical inversion, and partial least square regression.

The lack of efficient means to accurately infer photosynthetic traits constrains understanding global land carbon fluxes and improving photosynthetic pathways to increase crop yield. Here, we investigated whether a hyperspectral imaging camera mounted on a mobile platform could provide the capability to help resolve these challenges, focusing on three main approaches, that is, reflectance spectra-, spectral indices-, and numerical model inversions-based partial least square regression (PLSR) to estimate photosynthetic traits from canopy hyperspectral reflectance for 11 tobacco cultivars. Results showed that PLSR with inputs of reflectance spectra or spectral indices yielded an R2 of ~0.8 for predicting V cmax and J max , higher than an R2 of ~0.6 provided by PLSR of numerical inversions. Compared with PLSR of reflectance spectra, PLSR with spectral indices exhibited a better performance for predicting V cmax (R2 = 0.84 ± 0.02, RMSE = 33.8 ± 2.2 µmol m-2 s-1 ) while a similar performance for J max (R2 = 0.80 ± 0.03, RMSE = 22.6 ± 1.6 µmol m-2 s-1 ). Further analysis on spectral resampling revealed that V cmax and J max could be predicted with ~10 spectral bands at a spectral resolution of less than 14.7 nm. These results have important implications for improving photosynthetic pathways and mapping of photosynthesis across scales.

Plot-level rapid screening for photosynthetic parameters using proximal hyperspectral imaging.

Photosynthesis is currently measured using time-laborious and/or destructive methods which slows research and breeding efforts to identify crop germplasm with higher photosynthetic capacities. We present a plot-level screening tool for quantification of photosynthetic parameters and pigment contents that utilizes hyperspectral reflectance from sunlit leaf pixels collected from a plot (~2 m×2 m) in <1 min. Using field-grown Nicotiana tabacum with genetically altered photosynthetic pathways over two growing seasons (2017 and 2018), we built predictive models for eight photosynthetic parameters and pigment traits. Using partial least squares regression (PLSR) analysis of plot-level sunlit vegetative reflectance pixels from a single visible near infra-red (VNIR) (400-900 nm) hyperspectral camera, we predict maximum carboxylation rate of Rubisco (Vc,max, R2=0.79) maximum electron transport rate in given conditions (J1800, R2=0.59), maximal light-saturated photosynthesis (Pmax, R2=0.54), chlorophyll content (R2=0.87), the Chl a/b ratio (R2=0.63), carbon content (R2=0.47), and nitrogen content (R2=0.49). Model predictions did not improve when using two cameras spanning 400-1800 nm, suggesting a robust, widely applicable and more 'cost-effective' pipeline requiring only a single VNIR camera. The analysis pipeline and methods can be used in any cropping system with modified species-specific PLSR analysis to offer a high-throughput field phenotyping screening for germplasm with improved photosynthetic performance in field trials.

High-throughput field phenotyping using hyperspectral reflectance and partial least squares regression (PLSR) reveals genetic modifications to photosynthetic capacity.

Spectroscopy is becoming an increasingly powerful tool to alleviate the challenges of traditional measurements of key plant traits at the leaf, canopy, and ecosystem scales. Spectroscopic methods often rely on statistical approaches to reduce data redundancy and enhance useful prediction of physiological traits. Given the mechanistic uncertainty of spectroscopic techniques, genetic modification of plant biochemical pathways may affect reflectance spectra causing predictive models to lose power. The objectives of this research were to assess over two separate years, whether a predictive model can represent natural and imposed variation in leaf photosynthetic potential for different crop cultivars and genetically modified plants, to assess the interannual capabilities of a partial least square regression (PLSR) model, and to determine whether leaf N is a dominant driver of photosynthesis in PLSR models. In 2016, a PLSR analysis of reflectance spectra coupled with gas exchange data was used to build predictive models for photosynthetic parameters including maximum carboxylation rate of Rubisco (V c,max ), maximum electron transport rate (J max ) and percentage leaf nitrogen ([N]). The model was developed for wild type and genetically modified plants that represent a wide range of photosynthetic capacities. Results show that hyperspectral reflectance accurately predicted V c,max, J max and [N] for all plants measured in 2016. Applying these PLSR models to plants grown in 2017 resulted in a strong predictive ability relative to gas exchange measurements for V c,max, but not for J max, and not for genotypes unique to 2017. Building a new model including data collected in 2017 resulted in more robust predictions, with R2 increases of 17% for V c,max . and 13% J max . Plants generally have a positive correlation between leaf nitrogen and photosynthesis, however, tobacco with reduced Rubisco (SSuD) had significantly higher [N] despite much lower V c,max. The PLSR model was able to accurately predict both lower V c,max and higher leaf [N] for this genotype suggesting that the spectral based estimates of V c,max and leaf nitrogen [N] are independent. These results suggest that the PLSR model can be applied across years, but only to genotypes used to build the model and that the actual mechanism measured with the PLSR technique is not directly related to leaf [N]. The success of the leaf-scale analysis suggests that similar approaches may be successful at the canopy and ecosystem scales but to use these methods across years and between genotypes at any scale, application of accurately populated physical based models based on radiative transfer principles may be required.

Hyperspectral Proximal Sensing for Estimating Photosynthetic Capacities at Leaf and Canopy Scales.

Agronomists, plant breeders, and plant biologists have been promoting the need to develop high-throughput methods to measure plant traits of interest for decades. Measuring these plant traits or phenotypes is often a bottleneck since skilled personnel, resources, and ample time are required. Additionally, plant phenotypic traits from only a select number of breeding lines or varieties can be quantified because the "gold standard" measurement of a desired trait cannot be completed in a timely manner. As such, numerous approaches have been developed and implemented to better understand the biology and production of crops and ecosystems. In this chapter, we explain one of the recent approaches leveraging hyperspectral measurements to estimate different aspects of photosynthesis. Notably, we outline the use of hyperspectral radiometer and imaging to rapidly estimate two of the rate-limiting steps of photosynthesis: the maximum rate of the carboxylation of Rubisco (Vcmax) and the maximum rate of electron transfer or regeneration of RuBP (Jmax).

Emerging approaches to measure photosynthesis from the leaf to the ecosystem.

Measuring photosynthesis is critical for quantifying and modeling leaf to regional scale productivity of managed and natural ecosystems. This review explores existing and novel advances in photosynthesis measurements that are certain to provide innovative directions in plant science research. First, we address gas exchange approaches from leaf to ecosystem scales. Leaf level gas exchange is a mature method but recent improvements to the user interface and environmental controls of commercial systems have resulted in faster and higher quality data collection. Canopy chamber and micrometeorological methods have also become more standardized tools and have an advanced understanding of ecosystem functioning under a changing environment and through long time series data coupled with community data sharing. Second, we review proximal and remote sensing approaches to measure photosynthesis, including hyperspectral reflectance- and fluorescence-based techniques. These techniques have long been used with aircraft and orbiting satellites, but lower-cost sensors and improved statistical analyses are allowing these techniques to become applicable at smaller scales to quantify changes in the underlying biochemistry of photosynthesis. Within the past decade measurements of chlorophyll fluorescence from earth-orbiting satellites have measured Solar Induced Fluorescence (SIF) enabling estimates of global ecosystem productivity. Finally, we highlight that stronger interactions of scientists across disciplines will benefit our capacity to accurately estimate productivity at regional and global scales. Applying the multiple techniques outlined in this review at scales from the leaf to the globe are likely to advance understanding of plant functioning from the organelle to the ecosystem.

Hyperspectral Leaf Reflectance as Proxy for Photosynthetic Capacities: An Ensemble Approach Based on Multiple Machine Learning Algorithms.

Global agriculture production is challenged by increasing demands from rising population and a changing climate, which may be alleviated through development of genetically improved crop cultivars. Research into increasing photosynthetic energy conversion efficiency has proposed many strategies to improve production but have yet to yield real-world solutions, largely because of a phenotyping bottleneck. Partial least squares regression (PLSR) is a statistical technique that is increasingly used to relate hyperspectral reflectance to key photosynthetic capacities associated with carbon uptake (maximum carboxylation rate of Rubisco, Vc,max ) and conversion of light energy (maximum electron transport rate supporting RuBP regeneration, Jmax ) to alleviate this bottleneck. However, its performance varies significantly across different plant species, regions, and growth environments. Thus, to cope with the heterogeneous performances of PLSR, this study aims to develop a new approach to estimate photosynthetic capacities. A framework was developed that combines six machine learning algorithms, including artificial neural network (ANN), support vector machine (SVM), least absolute shrinkage and selection operator (LASSO), random forest (RF), Gaussian process (GP), and PLSR to optimize high-throughput analysis of the two photosynthetic variables. Six tobacco genotypes, including both transgenic and wild-type lines, with a range of photosynthetic capacities were used to test the framework. Leaf reflectance spectra were measured from 400 to 2500 nm using a high-spectral-resolution spectroradiometer. Corresponding photosynthesis vs. intercellular CO2 concentration response curves were measured for each leaf using a leaf gas-exchange system. Results suggested that the mean R 2 value of the six regression techniques for predicting Vc,max (Jmax ) ranged from 0.60 (0.45) to 0.65 (0.56) with the mean RMSE value varying from 47.1 (40.1) to 54.0 (44.7) µmol m-2 s-1. Regression stacking for Vc,max (Jmax ) performed better than the individual regression techniques with increases in R 2 of 0.1 (0.08) and decreases in RMSE by 4.1 (6.6) µmol m-2 s-1, equal to 8% (15%) reduction in RMSE. Better predictive performance of the regression stacking is likely attributed to the varying coefficients (or weights) in the level-2 model (the LASSO model) and the diverse ability of each individual regression technique to utilize spectral information for the best modeling performance. Further refinements can be made to apply this stacked regression technique to other plant phenotypic traits.