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.

Purification of Rubisco from Leaves.

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.

Antibody Design for the Quantification of Photosynthetic Proteins and Their Isoforms.

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.

Extraction of Soluble Proteins from Leaves.

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.

Down-regulation of wheat Rubisco activase isoforms expression by virus-induced gene silencing.

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 ß-isoforms encoded by the genes TaRca1 and TaRca2. TaRca1 produces a single transcript from which a short 1ß-isoform is expressed, whereas two alternative transcripts are generated from TaRca2 directing expression of either a long 2α-isoform or a short 2ß-isoform. The 2ß isoform is similar but not identical to 1ß. 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.

European soybean to benefit people and the environment.

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.

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.

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 '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.

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.

Into the Shadows and Back into Sunlight: Photosynthesis in Fluctuating Light.

Photosynthesis is an important remaining opportunity for further improvement in the genetic yield potential of our major crops. Measurement, analysis, and improvement of leaf CO2 assimilation (A) have focused largely on photosynthetic rates under light-saturated steady-state conditions. However, in modern crop canopies of several leaf layers, light is rarely constant, and the majority of leaves experience marked light fluctuations throughout the day. It takes several minutes for photosynthesis to regain efficiency in both sun-shade and shade-sun transitions, costing a calculated 10-40% of potential crop CO2 assimilation. Transgenic manipulations to accelerate the adjustment in sun-shade transitions have already shown a substantial productivity increase in field trials. Here, we explore means to further accelerate these adjustments and minimize these losses through transgenic manipulation, gene editing, and exploitation of natural variation. Measurement andanalysis of photosynthesis in sun-shade and shade-sun transitions are explained. Factors limiting speeds of adjustment and how they could be modified to effect improved efficiency are reviewed, specifically nonphotochemical quenching (NPQ), Rubisco activation, and stomatal responses.

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.

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.

Faster than expected Rubisco deactivation in shade reduces cowpea photosynthetic potential in variable light conditions.

Cowpea is the major source of vegetable protein for rural populations in sub-Saharan Africa and average yields are not keeping pace with population growth. Each day, crop leaves experience many shade events and the speed of photosynthetic adjustment to this dynamic environment strongly affects daily carbon gain. Rubisco activity is particularly important because it depends on the speed and extent of deactivation in shade and recovers slowly on return to sun. Here, direct biochemical measurements showed a much faster rate of Rubisco deactivation in cowpea than prior estimates inferred from dynamics of leaf gas exchange in other species1-3. Shade-induced deactivation was driven by decarbamylation, and half-times for both deactivation in shade and activation in saturating light were shorter than estimates from gas exchange (≤53% and 79%, respectively). Incorporating these half-times into a model of diurnal canopy photosynthesis predicted a 21% diurnal loss of productivity and suggests slowing Rubisco deactivation during shade is an unexploited opportunity for improving crop productivity.

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.

A wiring diagram to integrate physiological traits of wheat yield potential.

As crop yields are pushed closer to biophysical limits, achieving yield gains becomes increasingly challenging and will require more insight into deterministic pathways to yields. Here, we propose a wiring diagram as a platform to illustrate the interrelationships of the physiological traits that impact wheat yield potential and to serve as a decision support tool for crop scientists. The wiring diagram is based on the premise that crop yield is a function of photosynthesis (source), the investment of assimilates into reproductive organs (sinks) and the underlying processes that enable expression of both. By illustrating these linkages as coded wires, the wiring diagram can show connections among traits that may not have been apparent, and can inform new research hypotheses and guide crosses designed to accumulate beneficial traits and alleles in breeding. The wiring diagram can also serve to create an ever-richer common point of reference for refining crop models in the future.