Crop yields can be improved through making photosynthesis more efficient. The regulation of the CO2-fixing enzyme Rubisco during fluctuating light conditions limits productivity and is a target for improvement. Regulation in low light and darkness by accumulation of Rubisco inhibitors, predominantly via 2-carboxy-D-arabinitol 1-phosphate, has been known for over four decades but an explanation is still lacking for its physiological role and high variability across species. We compiled all published data for dark inhibition of Rubisco in flowering plants and investigated phylogenetic trends. Literature data for 157 species across 14 orders was compared and standardized, categorized into four dark inhibition levels, and analysed in the context of current phylogenetic information. We created a novel resource for Rubisco dark inhibition across flowering plants, highlighting clear gaps and biases in the available data, while also raising further questions on the evolution of this trait. Our work supports better understanding of the enigmatic process of photosynthetic regulation by Rubisco dark and low light inhibition and informs future efforts in enhancing photosynthesis in crops.

Photorespiration secures oxygenic photosynthesis from metabolic constraints arising from the oxygenation reaction of Rubisco in today's oxygen-rich atmosphere. Beyond this protective function, it is vital for primary metabolism, cellular redox balance, and stomatal regulation. Importantly, photorespiration has emerged as a key target for improving CO2 assimilation and biomass production. Among proposed strategies, enhancing photorespiratory efficiency via upregulation of core enzymes has highlighted glycine decarboxylase (GDC) as a promising target, however, it remains unclear whether simultaneous upregulation of multiple GDC components would yield additive or synergistic improvements in plant environmental acclimation. Here, we systematically compared single and double GDC overexpression lines under varying photoperiods, elevated oxygen and high light. GDC upregulation consistently promoted growth across photoperiods and enhanced photosynthetic performance under high light and oxygen, indicating a severe benefit of increased photorespiratory metabolite conversion. Notably, double overexpression lines exhibited enhanced metabolic flexibility beyond that observed in single lines, supporting synergistic effects on carbon assimilation and allocation. Short-term high-light exposure further revealed increased Rubisco activity and efficient partitioning of carbon into sucrose and starch. Collectively, our results demonstrate that coordinated enhancement of GDC activity strengthens photorespiratory efficiency and can generate synergistic improvements in photosynthetic robustness, metabolic robustness, and environmental acclimation under stressful conditions. These findings position multicomponent GDC engineering as a promising strategy for developing crops with improved environmental acclimation capacity.

Distinct physiological and anatomical traits can lead to substantial variation in photosynthetic efficiency among plant varieties, which may, in turn, impact agronomically important traits. We conducted a comprehensive comparative analysis of leaf physiology, anatomy, and biochemistry in Solanum lycopersicum (LEA), a modern inbred variety suited for the processing industry, and Solanum pennellii (Lost, accession LA5240), a drought-tolerant, green-fruited wild species, to investigate differences in photosynthetic performance and stomatal physiology. Lost exhibited higher photosynthetic capacity due to both biochemical and anatomical features. Chlorophyll fluorescence revealed that photosynthesis operates at a higher rate in Lost, due to greater electron sink capacity and efficient electron flow through the photosystems. Lost also showed higher Rubisco content as well as greater chlorophyll a/b ratio and total soluble protein levels than LEA, demonstrating investments in carbon capture relative to light harvesting to support superior photosynthetic performance at higher light intensities. Equal stomatal numbers on the abaxial and adaxial surface for Lost supported its greater leaf thickness and higher photosynthetic capacity, whilst LEA's greater stomatal density on the abaxial surface is typical of commercial broadleaf crops. Grafting experiments demonstrated that LEA scions grafted onto Lost rootstocks displayed improved photosynthesis compared with non-grafted LEA and LEA self-grafted plants, demonstrating successfully transferred enhanced photosynthetic traits from rootstock of Lost to LEA scions. Our study highlights the photosynthetic advantages of Lost and suggests avenues for enhancing tomato productivity through trait transfer.

Modelling crops in silico can identify bottlenecks in photosynthetic metabolism that limit the realization of maximum theoretical crop yields. Resource investment among photosynthetic enzymes in C 3 photosynthesis can be optimized to maximize carbon assimilation via targeted alterations, e.g. by regulating the expression of proteins in the Calvin Benson Bassham (CBB) cycle. In this work, the e-Photosynthesis dynamic model of C 3 metabolism was modified to create a rice-specific version. Species-specific equations for temperature dependences of ribulose-1,6-bisphosphate carboxylase/oxygenase (Rubisco) catalytic properties were combined with leaf-level gas exchange measurements for Oryza sativa cv. IR64 to derive photosynthetic parameters describing CBB cycle activity (Vcmax and J). These were used to re-scale enzyme activities in e-Photosynthesis before identifying redistributions of protein among photosynthetic enzymes that were optimal for CO 2 assimilation at different [CO 2]s. Target sets of enzymes were identified for over-expression to engineer improved photosynthesis under [CO 2] scenarios of limited diffusion, as might be experienced during abiotic stress, current yield potential, and future elevated [CO 2]. These were evaluated using sensitivity analysis that assumed variability around achieved protein fold-changes for photosynthetic improvement reported in the literature. Increases in as few as two to as many as six enzymes would achieve photosynthetic rates upto 28% higher under water stressed conditions. In non-stressed leaves under current and future [CO 2], increases in photosynthesis of upto 22% required over-expression of 4-6 enzymes.

Adaptation to increasing environmental temperatures is essential to plant survival and human food production. Thermal tolerance is controlled by a complex network of factors in plants including but not limited to genetic variation and environmental context. Rubisco activase (Rca) is a key photosynthetic enzyme with low thermal tolerance. Here, we report a large machine learning-directed screen of >1,400 synthetic cassava Rca enzymes which identified mutations that convey increased thermal stability while minimizing introduced mutations. We demonstrate multiple synthetic proteins that maintain activity at 8°C higher than wildtype cassava Rca including a single mutation that retains most activity post heat-shock.

There is an urgent need for increased crop productivity to reduce food insecurity and improve sustainability. Photosynthesis converts sunlight energy into carbohydrates, providing the source of nearly all of humanity's food. Photosynthesis is a key target for improvement, owing to inherent inefficiencies in the biochemical process. Over the last decade of advancements in bioengineering, strategies to increase the efficiency of photosynthesis were tested with proven enhancements to crop yields in field trials. Simple strategies like increasing the content of photosynthetic proteins have reliably increased photosynthesis and productivity in crops, as have more complex strategies such as bypassing photorespiration. While insertion of carbon-concentrating mechanisms into C3 plants remains an engineering challenge, modeling suggests that achieving that would have the greatest gain for crop improvement. This review discusses the many successes in improving photosynthesis achieved over the past decade and quantifies the potential for future engineering targets to increase crop productivity.

A vital crop for sub-Saharan Africa, cowpea productivity, is threatened by climate change, including increased heatwave intensity, duration and frequency. Rubisco activase (Rca) is a key molecular chaperone that maintains Rubisco activity and, due to its thermal sensitivity, is a key target for improving crop climate resilience. We identified and characterised four Rca isoforms in Vigna unguiculata cv IT97K-499-35 in vitro: Rca1β, Rca8α, Rca10α and Rca10β. Cowpea leaf and plant growth traits were also investigated during a 5-d +10°C heatwave that included assessment of Rubisco and Rca activity in leaf extracts and relative changes in the abundance of the four Rca isoforms. Cowpea Rca10α and Rca10β had higher thermal maxima, broader thermal optima and higher rates of ATP hydrolysis and Rubisco reactivation in vitro. In the absence of water deficit, the heatwave caused only mild effects, including increases in leaf temperature and expression of Rca10, small decreases in Rubisco activity and activation state and an unaltered temperature response of Rubisco activation by Rca. The superior Rca10α and Rca10β isoforms offer the prospect of enhancing the thermotolerance of cowpea and other crops in anticipation of more extreme future heatwaves.

Rubisco activity is highly regulated and frequently limits carbon assimilation in crop plants. In the chloroplast, various metabolites can inhibit or modulate Rubisco activity by binding to its catalytic or allosteric sites, but this regulation is complex and still poorly understood. Using rice Rubisco, we characterised the impact of various chloroplast metabolites which could interact with Rubisco and modulate its activity, including photorespiratory intermediates, carbohydrates, amino acids; as well as specific sugar-phosphates known to inhibit Rubisco activity - CABP (2-carboxy-d-arabinitol 1,5-bisphosphate) and CA1P (2-carboxy-d-arabinitol 1-phosphate) through in vitro enzymatic assays and molecular docking analysis. Most metabolites did not directly affect Rubisco in vitro activity under both saturating and limiting concentrations of Rubisco substrates, CO2 and RuBP (ribulose-1,5-bisphosphate). As expected, Rubisco activity was strongly inhibited in the presence of CABP and CA1P. High physiologically relevant concentrations of the carboxylation product 3-PGA (3-phosphoglyceric acid) decreased Rubisco activity by up to 30%. High concentrations of the photosynthetically derived hexose phosphates fructose 6-phosphate (F6P) and glucose 6-phosphate (G6P) slightly reduced Rubisco activity under limiting CO2 and RuBP concentrations. Biochemical measurements of the apparent Vmax and Km for CO2 and RuBP (at atmospheric O2 concentration) and docking interactions analysis suggest that CABP/CA1P and 3-PGA inhibit Rubisco activity by binding tightly and loosely, respectively, to its catalytic sites (i.e. competing with the substrate RuBP). These findings will aid the design and biochemical modelling of new strategies to improve the regulation of Rubisco activity and enhance the efficiency and sustainability of carbon assimilation in rice.

Rubisco is the key enzyme in photosynthesis, catalyzing fixation of carbon dioxide from the atmosphere into energy storage molecules. Several inefficiencies in Rubisco limit the rate of photosynthesis, and, therefore, the growth of the plant. Rubisco is sensitive to light, making deactivation of the enzyme upon sampling likely. Moreover, the indirect methods often used to study its activity make obtaining reliable data difficult. In this Chapter, we describe an approach to generate reliable and repeatable data for Rubisco activities, activation state and abundance in plant leaves. We include methods to sample and extract proteins, minimizing Rubisco degradation and deactivation. We describe radiometric techniques to measure Rubisco activities and calculate its activation state at the time of sampling, and to quantify its abundance.

Antibodies are a valuable research tool, with uses including detection and quantification of specific proteins. By using peptide fragments to raise antibodies, they can be designed to differentiate between structurally similar proteins, or to bind conserved motifs in divergent proteins. Peptide sequence selection and antibody validation are crucial to ensure reliable results from antibody-based experiments. This chapter describes the steps for the identification of peptide sequences to produce protein- or isoform-specific antibodies using recombinant technologies as well as the subsequent validation of such antibodies. The photosynthetic protein Rubisco activase is used as a case study to explain the various steps involved and key aspects to take into consideration.

Protein biochemistry can provide valuable answers to better understand plant performance and responses to the surrounding environment. In this chapter, we describe the process of extracting proteins from plant leaf samples. We highlight the key aspects to take into consideration to preserve protein integrity, from sample collection to extraction and preparation or storage for subsequent analysis of protein abundance and/or enzymatic activities.

Rubisco fixes CO2 through the carboxylation of ribulose 1,5-bisphosphate (RuBP) during photosynthesis, enabling the synthesis of organic compounds. The natural diversity of Rubisco properties represents an opportunity to improve its performance and there is considerable research effort focusing on better understanding the properties and regulation of the enzyme. This chapter describes a method for large-scale purification of Rubisco from leaves. After the extraction of Rubisco from plant leaves, the enzyme is separated from other proteins by fractional precipitation with polyethylene glycol followed by ion-exchange chromatography. This method enables the isolation of Rubisco in large quantities for a wide range of biochemical applications.

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.

Rubisco activase (Rca) is an essential photosynthetic enzyme that removes inhibitors from the catalytic sites of the carboxylating enzyme Rubisco. In wheat, Rca is composed of one longer 46 kDa α-isoform and two shorter 42 kDa β-isoforms encoded by the genes TaRca1 and TaRca2. TaRca1 produces a single transcript from which a short 1β-isoform is expressed, whereas two alternative transcripts are generated from TaRca2 directing expression of either a long 2α-isoform or a short 2β-isoform. The 2β isoform is similar but not identical to 1β. Here, virus-induced gene silencing (VIGS) was used to silence the different TaRca transcripts. Abundance of the transcripts and the respective protein isoforms was then evaluated in the VIGS-treated and control plants. Remarkably, treatment with the construct specifically targeting TaRca1 efficiently decreased expression not only of TaRca1 but also of the two alternative TaRca2 transcripts. Similarly, specific targeting of the TaRca2 transcript encoding a long isoform TaRca2α resulted in silencing of both TaRca2 alternative transcripts. The corresponding protein isoforms decreased in abundance. These findings indicate concomitant down-regulation of TaRca1 and TaRca2 at both transcript and protein levels and may impact the feasibility of altering the relative abundance of Rca isoforms in wheat.

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 carbon dioxide (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.

Europe imports large amounts of soybean that are predominantly used for livestock feed, mainly sourced from Brazil, USA and Argentina. In addition, the demand for GM-free soybean for human consumption is project to increase. Soybean has higher protein quality and digestibility than other legumes, along with high concentrations of isoflavones, phytosterols and minerals that enhance the nutritional value as a human food ingredient. Here, we examine the potential to increase soybean production across Europe for livestock feed and direct human consumption, and review possible effects on the environment and human health. Simulations and field data indicate rainfed soybean yields of 3.1 ± 1.2 t ha−1 from southern UK through to southern Europe (compared to a 3.5 t ha−1 average from North America). Drought-prone southern regions and cooler northern regions require breeding to incorporate stress-tolerance traits. Literature synthesized in this work evidenced soybean properties important to human nutrition, health, and traits related to food processing compared to alternative protein sources. While acknowledging the uncertainties inherent in any modelling exercise, our findings suggest that further integrating soybean into European agriculture could reduce GHG emissions by 37–291 Mt CO2e year−1 and fertiliser N use by 0.6–1.2 Mt year−1, concurrently improving human health and nutrition.

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.

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 V-cmax and J(max) 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.

Significant advances in Rubisco research over the past decade have highlighted the intricate nature of the CO2-fixing enzyme and the complexity of environmental and cellular factors that affect its activity in photosynthetic organisms. This special issue offers comprehensive coverage of all things Rubisco (Fig. 1), from functional diversity to folding and assembly, in vivo regulation of its activity, including the role of its molecular partner, Rubisco activase (Rca), as well as sugar phosphate derivatives that inhibit activity.

ABSTRACT With the development of the digital phenotyping, repeated measurements of agronomic traits over time are easily accessible, notably for morphological and phenological traits. However high throughput methods for estimating physiological traits such as photosynthesis are lacking. This study demonstrates the links of fluorescence and reflectance imaging with photosynthetic traits. Two wheat cultivars were grown in pots in a controlled environment. Photosynthesis was characterised by gas-exchange and biochemical analysis at five time points, from booting to 21 days post anthesis. On the same days imaging was performed on the same pots, at leaf and plant scale, using indoor and outdoor phenotyping platforms, respectively. Five image variables (F v /F m and NDVI at the whole plant level and F v /F m , Φ(II) 532 and Φ(NPQ) 1077 at the leaf scale) were compared to variables from A-Ci and A-Par curves, biochemical analysis, and fluorescence instruments. The results suggested that the image variables are robust estimators of photosynthetic traits, as long as senescence is driving the variability. Despite contrasting cultivar behaviour, linear regression models which account for the cultivar and the interaction effects, further improved the modelling of photosynthesis indicators. Finally, the results highlight the challenge of discriminating functional to cosmetic stay green genotypes using digital imaging. Highlight A temporal and multi-scale study of fluorescence and NDVI imaging used as a proxy for photosynthetic parameters