Perspectives on improving photosynthesis to increase crop yield.

Improving photosynthesis, the fundamental process by which plants convert light energy into chemical energy, is a key area of research with great potential for enhancing sustainable agricultural productivity and addressing global food security challenges. This perspective delves into the latest advancements and approaches aimed at optimizing photosynthetic efficiency. Our discussion encompasses the entire process, beginning with light harvesting and its regulation and progressing through the bottleneck of electron transfer. We then delve into the carbon reactions of photosynthesis, focusing on strategies targeting the enzymes of the Calvin-Benson-Bassham (CBB) cycle. Additionally, we explore methods to increase CO2 concentration near the Rubisco, the enzyme responsible for the first step of CBB cycle, drawing inspiration from various photosynthetic organisms, and conclude this section by examining ways to enhance CO2 delivery into leaves. Moving beyond individual processes, we discuss two approaches to identifying key targets for photosynthesis improvement: systems modeling and the study of natural variation. Finally, we revisit some of the strategies mentioned above to provide a holistic view of the improvements, analyzing their impact on nitrogen use efficiency and on canopy photosynthesis.

Kinetic modeling identifies targets for engineering improved photosynthetic efficiency in potato (Solanum tuberosum cv. Solara).

Potato (Solanum tuberosum) is a significant non-grain food crop in terms of global production. However, its yield potential might be raised by identifying means to release bottlenecks within photosynthetic metabolism, from the capture of solar energy to the synthesis of carbohydrates. Recently, engineered increases in photosynthetic rates in other crops have been directly related to increased yield - how might such increases be achieved in potato? To answer this question, we derived the photosynthetic parameters Vcmax and Jmax to calibrate a kinetic model of leaf metabolism (e-Photosynthesis) for potato. This model was then used to simulate the impact of manipulating the expression of genes and their protein products on carbon assimilation rates in silico through optimizing resource investment among 23 photosynthetic enzymes, predicting increases in photosynthetic CO2 uptake of up to 67%. However, this number of manipulations would not be practical with current technologies. Given a limited practical number of manipulations, the optimization indicated that an increase in amounts of three enzymes - Rubisco, FBP aldolase, and SBPase - would increase net assimilation. Increasing these alone to the levels predicted necessary for optimization increased photosynthetic rate by 28% in potato.

Regulation of Rubisco activity in crops.

Efficient plant acclimation to changing environmental conditions relies on fast adjustments of the transcriptome, proteome, and metabolome. Regulation of enzyme activity depends on the activity of specific chaperones, chemical post-translational modifications (PTMs) of amino acid residues, and changes in the cellular and organellar microenvironment. Central to carbon assimilation, and thus plant growth and yield, Rubisco activity is regulated by its chaperone Rubisco activase (Rca) and by adjustments in the chloroplast stroma environment. Focused on crops, this review highlights the main PTMs and stromal ions and metabolites affecting Rubisco and Rca in response to environmental stimuli. Rca isoforms differ in regulatory properties and heat sensitivity, with expression changing according to the surrounding environment. Much of the physiological relevance of Rubisco and Rca PTMs is still poorly understood, though some PTMs have been associated with Rubisco regulation in response to stress. Ion and metabolite concentrations in the chloroplast change in response to variations in light and temperature. Some of these changes promote Rubisco activation while others inhibit activation, deactivate the enzyme, or change the rates of catalysis. Understanding these regulatory mechanisms will aid the development of strategies to improve carbon fixation by Rubisco under rapidly changing environments as experienced by crop plants.

Dynamics of Rubisco regulation by sugar phosphate derivatives and their phosphatases.

Regulating the central CO2-fixing enzyme Rubisco is as complex as its ancient reaction mechanism and involves interaction with a series of cofactors and auxiliary proteins that activate catalytic sites and maintain activity. A key component among the regulatory mechanisms is the binding of sugar phosphate derivatives that inhibit activity. Removal of inhibitors via the action of Rubisco activase is required to restore catalytic competency. In addition, specific phosphatases dephosphorylate newly released inhibitors, rendering them incapable of binding to Rubisco catalytic sites. The best studied inhibitor is 2-carboxy-d-arabinitol 1-phosphate (CA1P), a naturally occurring nocturnal inhibitor that accumulates in most species during darkness and low light, progressively binding to Rubisco. As light increases, Rubisco activase removes CA1P from Rubisco, and the specific phosphatase CA1Pase dephosphorylates CA1P to CA, which cannot bind Rubisco. Misfire products of Rubisco's complex reaction chemistry can also act as inhibitors. One example is xylulose-1,5-bisphosphate (XuBP), which is dephosphorylated by XuBPase. Here we revisit key findings related to sugar phosphate derivatives and their specific phosphatases, highlighting outstanding questions and how further consideration of these inhibitors and their role is important for better understanding the regulation of carbon assimilation.

A 'wiring diagram' for sink strength traits impacting wheat yield potential.

Identifying traits for improving sink strength is a bottleneck to increasing wheat yield. The interacting processes determining sink strength and yield potential are reviewed and visualized in a set of 'wiring diagrams', covering critical phases of development (and summarizing known underlying genetics). Using this framework, we reviewed and assembled the main traits determining sink strength and identified research gaps and potential hypotheses to be tested for achieving gains in sink strength. In pre-anthesis, grain number could be increased through: (i) enhanced spike growth associated with optimized floret development and/or a reduction in specific stem-internode lengths and (ii) improved fruiting efficiency through an accelerated rate of floret development, improved partitioning between spikes, or optimized spike cytokinin levels. In post-anthesis, grain, sink strength could be augmented through manipulation of grain size potential via ovary size and/or endosperm cell division and expansion. Prospects for improving spike vascular architecture to support all rapidly growing florets, enabling the improved flow of assimilate, are also discussed. Finally, we considered the prospects for enhancing grain weight realization in relation to genetic variation in stay-green traits as well as stem carbohydrate remobilization. The wiring diagrams provide a potential workspace for breeders and crop scientists to achieve yield gains in wheat and other field crops.

A 'wiring diagram' for source strength traits impacting wheat yield potential.

Source traits are currently of great interest for the enhancement of yield potential; for example, much effort is being expended to find ways of modifying photosynthesis. However, photosynthesis is but one component of crop regulation, so sink activities and the coordination of diverse processes throughout the crop must be considered in an integrated, systems approach. A set of 'wiring diagrams' has been devised as a visual tool to integrate the interactions of component processes at different stages of wheat development. They enable the roles of chloroplast, leaf, and whole-canopy processes to be seen in the context of sink development and crop growth as a whole. In this review, we dissect source traits both anatomically (foliar and non-foliar) and temporally (pre- and post-anthesis), and consider the evidence for their regulation at local and whole-plant/crop levels. We consider how the formation of a canopy creates challenges (self-occlusion) and opportunities (dynamic photosynthesis) for components of photosynthesis. Lastly, we discuss the regulation of source activity by feedback regulation. The review is written in the framework of the wiring diagrams which, as integrated descriptors of traits underpinning grain yield, are designed to provide a potential workspace for breeders and other crop scientists that, along with high-throughput and precision phenotyping data, genetics, and bioinformatics, will help build future dynamic models of trait and gene interactions to achieve yield gains in wheat and other field crops.

A procedure to introduce point mutations into the Rubisco large subunit gene in wild-type plants.

Photosynthetic inefficiencies limit the productivity and sustainability of crop production and the resilience of agriculture to future societal and environmental challenges. Rubisco is a key target for improvement as it plays a central role in carbon fixation during photosynthesis and is remarkably inefficient. Introduction of mutations to the chloroplast-encoded Rubisco large subunit rbcL is of particular interest for improving the catalytic activity and efficiency of the enzyme. However, manipulation of rbcL is hampered by its location in the plastome, with many species recalcitrant to plastome transformation, and by the plastid's efficient repair system, which can prevent effective maintenance of mutations introduced with homologous recombination. Here we present a system where the introduction of a number of silent mutations into rbcL within the model plant Nicotiana tabacum facilitates simplified screening via additional restriction enzyme sites. This system was used to successfully generate a range of transplastomic lines from wild-type N. tabacum with stable point mutations within rbcL in 40% of the transformants, allowing assessment of the effect of these mutations on Rubisco assembly and activity. With further optimization the approach offers a viable way forward for mutagenic testing of Rubisco function in planta within tobacco and modification of rbcL in other crops where chloroplast transformation is feasible. The transformation strategy could also be applied to introduce point mutations in other chloroplast-encoded genes.

Rubiscosome gene expression is balanced across the hexaploid wheat genome.

Functional and active Rubisco is essential for CO2 fixation and is a primary target for engineering approaches to increasing crop yields. However, the assembly and maintenance of active Rubisco are dependent on the coordinated biosynthesis of at least 11 nuclear-encoded proteins, termed the 'Rubiscosome'. Using publicly available gene expression data for wheat (Triticum aestivum L.), we show that the expression of Rubiscosome genes is balanced across the three closely related subgenomes that form the allohexaploid genome. Each subgenome contains a near complete set of homoeologous genes and contributes equally to overall expression, both under optimal and under heat stress conditions. The expression of the wheat thermo-tolerant Rubisco activase isoform 1ß increases under heat stress and remains balanced across the subgenomes, albeit with a slight shift towards greater contribution from the D subgenome. The findings show that the gene copies in all three subgenomes need to be accounted for when designing strategies for crop improvement.

Phenotypic variation in photosynthetic traits in wheat grown under field versus glasshouse conditions.

Recognition of the untapped potential of photosynthesis to improve crop yields has spurred research to identify targets for breeding. The CO2-fixing enzyme Rubisco is characterized by a number of inefficiencies, and frequently limits carbon assimilation at the top of the canopy, representing a clear target for wheat improvement. Two bread wheat lines with similar genetic backgrounds and contrasting in vivo maximum carboxylation activity of Rubisco per unit leaf nitrogen (Vc,max,25/Narea) determined using high-throughput phenotyping methods were selected for detailed study from a panel of 80 spring wheat lines. Detailed phenotyping of photosynthetic traits in the two lines using glasshouse-grown plants showed no difference in Vc,max,25/Narea determined directly via in vivo and in vitro methods. Detailed phenotyping of glasshouse-grown plants of the 80 wheat lines also showed no correlation between photosynthetic traits measured via high-throughput phenotyping of field-grown plants. Our findings suggest that the complex interplay between traits determining crop productivity and the dynamic environments experienced by field-grown plants needs to be considered in designing strategies for effective wheat crop yield improvement when breeding for particular environments.

High-throughput phenotyping of physiological traits for wheat resilience to high temperature and drought stress.

Interannual and local fluctuations in wheat crop yield are mostly explained by abiotic constraints. Heatwaves and drought, which are among the top stressors, commonly co-occur, and their frequency is increasing with global climate change. High-throughput methods were optimized to phenotype wheat plants under controlled water deficit and high temperature, with the aim to identify phenotypic traits conferring adaptative stress responses. Wheat plants of 10 genotypes were grown in a fully automated plant facility under 25/18 °C day/night for 30 d, and then the temperature was increased for 7 d (38/31 °C day/night) while maintaining half of the plants well irrigated and half at 30% field capacity. Thermal and multispectral images and pot weights were registered twice daily. At the end of the experiment, key metabolites and enzyme activities from carbohydrate and antioxidant metabolism were quantified. Regression machine learning models were successfully established to predict plant biomass using image-extracted parameters. Evapotranspiration traits expressed significant genotype-environment interactions (G×E) when acclimatization to stress was continuously monitored. Consequently, transpiration efficiency was essential to maintain the balance between water-saving strategies and biomass production in wheat under water deficit and high temperature. Stress tolerance included changes in carbohydrate metabolism, particularly in the sucrolytic and glycolytic pathways, and in antioxidant metabolism. The observed genetic differences in sensitivity to high temperature and water deficit can be exploited in breeding programmes to improve wheat resilience to climate change.

An isoleucine residue acts as a thermal and regulatory switch in wheat Rubisco activase.

The regulation of Rubisco, the gatekeeper of carbon fixation into the biosphere, by its molecular chaperone Rubisco activase (Rca) is essential for photosynthesis and plant growth. Using energy from ATP hydrolysis, Rca promotes the release of inhibitors and restores catalytic competence to Rubisco-active sites. Rca is sensitive to moderate heat stress, however, and becomes progressively inhibited as the temperature increases above the optimum for photosynthesis. Here, we identify a single amino acid substitution (M159I) that fundamentally alters the thermal and regulatory properties of Rca in bread wheat (Triticum aestivum L.). Using site-directed mutagenesis, we demonstrate that the M159I substitution extends the temperature optimum of the most abundant Rca isoform by 5°C in vitro, while maintaining the efficiency of Rubisco activation by Rca. The results suggest that this single amino acid substitution acts as a thermal and regulatory switch in wheat Rca that can be exploited to improve the climate resilience and efficiency of carbon assimilation of this cereal crop as temperatures become warmer and more volatile.

Heat-induced changes in the abundance of wheat Rubisco activase isoforms.

The Triticum aestivum (wheat) genome encodes three isoforms of Rubisco activase (Rca) differing in thermostability, which could be exploited to improve the resilience of this crop to global warming. We hypothesized that elevated temperatures would cause an increase in the relative abundance of heat-stable Rca1ß. Wheat plants were grown at 25° C : 18°C (day : night) and exposed to heat stress (38° C : 22°C) for up to 5 d at pre-anthesis. Carbon (C) assimilation, Rubisco activity, CA1Pase activity, transcripts of Rca1ß, Rca2ß, and Rca2α, and the quantities of the corresponding protein products were measured during and after heat stress. The transcript of Rca1ß increased 40-fold in 4 h at elevated temperatures and returned to the original level after 4 h upon return of plants to control temperatures. Rca1ß comprised up to 2% of the total Rca protein in unstressed leaves but increased three-fold in leaves exposed to elevated temperatures for 5 d and remained high at 4 h after heat stress. These results show that elevated temperatures cause rapid changes in Rca gene expression and adaptive changes in Rca isoform abundance. The improved understanding of the regulation of C assimilation under heat stress will inform efforts to improve wheat productivity and climate resilience.

Hybrid Cyanobacterial-Tobacco Rubisco Supports Autotrophic Growth and Procarboxysomal Aggregation.

Much of the research aimed at improving photosynthesis and crop productivity attempts to overcome shortcomings of the primary CO2-fixing enzyme Rubisco. Cyanobacteria utilize a CO2-concentrating mechanism (CCM), which encapsulates Rubisco with poor specificity but a relatively fast catalytic rate within a carboxysome microcompartment. Alongside the active transport of bicarbonate into the cell and localization of carbonic anhydrase within the carboxysome shell with Rubisco, cyanobacteria are able to overcome the limitations of Rubisco via localization within a high-CO2 environment. As part of ongoing efforts to engineer a ß-cyanobacterial CCM into land plants, we investigated the potential for Rubisco large subunits (LSU) from the ß-cyanobacterium Synechococcus elongatus (Se) to form aggregated Rubisco complexes with the carboxysome linker protein CcmM35 within tobacco (Nicotiana tabacum) chloroplasts. Transplastomic plants were produced that lacked cognate Se Rubisco small subunits (SSU) and expressed the Se LSU in place of tobacco LSU, with and without CcmM35. Plants were able to form a hybrid enzyme utilizing tobacco SSU and the Se LSU, allowing slow autotrophic growth in high CO2 CcmM35 was able to form large Rubisco aggregates with the Se LSU, and these incorporated small amounts of native tobacco SSU. Plants lacking the Se SSU showed delayed growth, poor photosynthetic capacity, and significantly reduced Rubisco activity compared with both wild-type tobacco and lines expressing the Se SSU. These results demonstrate the ability of the Se LSU and CcmM35 to form large aggregates without the cognate Se SSU in planta, harboring active Rubisco that enables plant growth, albeit at a much slower pace than plants expressing the cognate Se SSU.

The relative abundance of wheat Rubisco activase isoforms is post-transcriptionally regulated.

Diurnal rhythms and light availability affect transcription-translation feedback loops that regulate the synthesis of photosynthetic proteins. The CO2-fixing enzyme Rubisco is the most abundant protein in the leaves of major crop species and its activity depends on interaction with the molecular chaperone Rubisco activase (Rca). In Triticum aestivum L. (wheat), three Rca isoforms are present that differ in their regulatory properties. Here, we tested the hypothesis that the relative abundance of the redox-sensitive and redox-insensitive Rca isoforms could be differentially regulated throughout light-dark diel cycle in wheat. While TaRca1-ß expression was consistently negligible throughout the day, transcript levels of both TaRca2-ß and TaRca2-α were higher and increased at the start of the day, with peak levels occurring at the middle of the photoperiod. Abundance of TaRca-ß protein was maximal 1.5 h after the peak in TaRca2-ß expression, but the abundance of TaRca-α remained constant during the entire photoperiod. The redox-sensitive TaRca-α isoform was less abundant, representing 85% of the redox-insensitive TaRca-ß at the transcript level and 12.5% at the protein level. Expression of Rubisco large and small subunit genes did not show a consistent pattern throughout the diel cycle, but the abundance of Rubisco decreased by up to 20% during the dark period in fully expanded wheat leaves. These results, combined with a lack of correlation between transcript and protein abundance for both Rca isoforms and Rubisco throughout the entire diel cycle, suggest that the abundance of these photosynthetic enzymes is post-transcriptionally regulated.

Photoprotection and optimization of sucrose usage contribute to faster recovery of photosynthesis after water deficit at high temperatures in wheat.

Plants are increasingly exposed to events of elevated temperature and water deficit, which threaten crop productivity. Understanding the ability to rapidly recover from abiotic stress, restoring carbon assimilation and biomass production, is important to unravel crop climate resilience. This study compared the photosynthetic performance of two Triticum aestivum L. cultivars, Sokoll and Paragon, adapted to the climate of Mexico and UK, respectively, exposed to 1-week water deficit and high temperatures, in isolation or combination. Measurements included photosynthetic assimilation rate, stomatal conductance, in vitro activities of Rubisco (EC 4.1.1.39) and invertase (INV, EC 3.2.1.26), antioxidant capacity and chlorophyll a fluorescence. In both genotypes, under elevated temperatures and water deficit (WD38°C), the photosynthetic limitations were mainly due to stomatal restrictions and to a decrease in the electron transport rate. Chlorophyll a fluorescence parameters clearly indicate differences between the two genotypes in the photoprotection when subjected to WD38°C and showed faster recovery of Paragon after stress relief. The activity of the cytosolic invertase (CytINV) under these stress conditions was strongly related to the fast photosynthesis recovery of Paragon. Taken together, the results suggest that optimal sucrose export/utilization and increased photoprotection of the electron transport machinery are important components to limit yield fluctuations due to water shortage and elevated temperatures.

Photosynthesis across African cassava germplasm is limited by Rubisco and mesophyll conductance at steady state, but by stomatal conductance in fluctuating light.

Sub-Saharan Africa is projected to see a 55% increase in food demand by 2035, where cassava (Manihot esculenta) is the most widely planted crop and a major calorie source. Yet, cassava yield in this region has not increased significantly for 13 yr. Improvement of genetic yield potential, the basis of the first Green Revolution, could be realized by improving photosynthetic efficiency. First, the factors limiting photosynthesis and their genetic variability within extant germplasm must be understood. Biochemical and diffusive limitations to leaf photosynthetic CO2 uptake under steady state and fluctuating light in 13 farm-preferred and high-yielding African cultivars were analyzed. A cassava leaf metabolic model was developed to quantify the value of overcoming limitations to leaf photosynthesis. At steady state, in vivo Rubisco activity and mesophyll conductance accounted for 84% of the limitation. Under nonsteady-state conditions of shade to sun transition, stomatal conductance was the major limitation, resulting in an estimated 13% and 5% losses in CO2 uptake and water use efficiency, across a diurnal period. Triose phosphate utilization, although sufficient to support observed rates, would limit improvement in leaf photosynthesis to 33%, unless improved itself. The variation of carbon assimilation among cultivars was three times greater under nonsteady state compared to steady state, pinpointing important overlooked breeding targets for improved photosynthetic efficiency in cassava.

Rubisco and carbon-concentrating mechanism co-evolution across chlorophyte and streptophyte green algae.

Green algae expressing a carbon-concentrating mechanism (CCM) are usually associated with a Rubisco-containing micro-compartment, the pyrenoid. A link between the small subunit (SSU) of Rubisco and pyrenoid formation in Chlamydomonas reinhardtii has previously suggested that specific RbcS residues could explain pyrenoid occurrence in green algae. A phylogeny of RbcS was used to compare the protein sequence and CCM distribution across the green algae and positive selection in RbcS was estimated. For six streptophyte algae, Rubisco catalytic properties, affinity for CO2 uptake (K0.5 ), carbon isotope discrimination (δ13 C) and pyrenoid morphology were compared. The length of the ßA-ßB loop in RbcS provided a phylogenetic marker discriminating chlorophyte from streptophyte green algae. Rubisco kinetic properties in streptophyte algae have responded to the extent of inducible CCM activity, as indicated by changes in inorganic carbon uptake affinity, δ13 C and pyrenoid ultrastructure between high and low CO2 conditions for growth. We conclude that the Rubisco catalytic properties found in streptophyte algae have coevolved and reflect the strength of any CCM or degree of pyrenoid leakiness, and limitations to inorganic carbon in the aquatic habitat, whereas Rubisco in extant land plants reflects more recent selective pressures associated with improved diffusive supply of the terrestrial environment.

Variation in key leaf photosynthetic traits across wheat wild relatives is accession dependent not species dependent.

The wild relatives of modern wheat represent an underutilized source of genetic and phenotypic diversity and are of interest in breeding owing to their wide adaptation to diverse environments. Leaf photosynthetic traits underpin the rate of production of biomass and yield and have not been systematically explored in the wheat relatives. This paper identifies and quantifies the phenotypic variation in photosynthetic, stomatal, and morphological traits in up to 88 wheat wild relative accessions across five genera. Both steady-state measurements and dynamic responses to step changes in light intensity are assessed. A 2.3-fold variation for flag leaf light and CO2 -saturated rates of photosynthesis Amax was observed. Many accessions showing higher and more variable Amax , maximum rates of carboxylation, electron transport, and Rubisco activity when compared with modern genotypes. Variation in dynamic traits was also significant; with distinct genus-specific trends in rates of induction of nonphotochemical quenching and rate of stomatal opening. We conclude that utilization of wild relatives for improvement of photosynthesis is supported by the existence of a high degree of natural variation in key traits and should consider not only genus-level properties but variation between individual accessions.

Overexpression of ca1pase Decreases Rubisco Abundance and Grain Yield in Wheat.

Rubisco catalyzes the fixation of CO2 into organic compounds that are used for plant growth and the production of agricultural products, and specific sugar-phosphate derivatives bind tightly to the active sites of Rubisco, locking the enzyme in a catalytically inactive conformation. 2-carboxy-d-arabinitol-1-phosphate phosphatase (CA1Pase) dephosphorylates such tight-binding inhibitors, contributing to the maintenance of Rubisco activity. Here, we investigated the hypothesis that overexpressing ca1pase would decrease the abundance of Rubisco inhibitors, thereby increasing the activity of Rubisco and enhancing photosynthetic performance and productivity in wheat (Triticum aestivum). Plants of four independent wheat transgenic lines overexpressing ca1pase showed up to 30-fold increases in ca1pase expression compared to the wild type. Plants overexpressing ca1pase had lower numbers of Rubisco tight-binding inhibitors and higher Rubisco activation state than the wild type; however, there were 17% to 60% fewer Rubisco active sites in the four transgenic lines than in the wild type. The lower Rubisco content in plants overexpressing ca1pase resulted in lower initial and total carboxylating activities measured in flag leaves at the end of the vegetative stage and lower aboveground biomass and grain yield measured in fully mature plants. Hence, contrary to what would be expected, ca1pase overexpression decreased Rubisco content and compromised wheat grain yields. These results support a possible role for Rubisco inhibitors in protecting the enzyme and maintaining an adequate number of Rubisco active sites to support carboxylation rates in planta.

During photosynthetic induction, biochemical and stomatal limitations differ between Brassica crops.

Interventions to increase crop radiation use efficiency rely on understanding of how biochemical and stomatal limitations affect photosynthesis. When leaves transition from shade to high light, slow increases in maximum Rubisco carboxylation rate and stomatal conductance limit net CO2 assimilation for several minutes. However, as stomata open intercellular [CO2 ] increases, so electron transport rate could also become limiting. Photosynthetic limitations were evaluated in three important Brassica crops: Brassica rapa, Brassica oleracea and Brassica napus. Measurements of induction after a period of shade showed that net CO2 assimilation by B. rapa and B. napus saturated by 10 min. A new method of analyzing limitations to induction by varying intercellular [CO2 ] showed this was due to co-limitation by Rubisco and electron transport. By contrast, in B. oleracea persistent Rubisco limitation meant that CO2 assimilation was still recovering 15 min after induction. Correspondingly, B. oleracea had the lowest Rubisco total activity. The methodology developed, and its application here, shows a means to identify the basis of variation in photosynthetic efficiency in fluctuating light, which could be exploited in breeding and bioengineering to improve crop productivity.