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.

The Calvin-Benson-Bassham cycle in C4 and Crassulacean acid metabolism species.

The Calvin-Benson-Bassham (CBB) cycle is the ancestral CO2 assimilation pathway and is found in all photosynthetic organisms. Biochemical extensions to the CBB cycle have evolved that allow the resulting pathways to act as CO2 concentrating mechanisms, either spatially in the case of C4 photosynthesis or temporally in the case of Crassulacean acid metabolism (CAM). While the biochemical steps in the C4 and CAM pathways are known, questions remain on their integration and regulation with CBB cycle activity. The application of omic and transgenic technologies is providing a more complete understanding of the biochemistry of C4 and CAM species and will also provide insight into the CBB cycle in these plants. As the global population increases, new solutions are required to increase crop yields and meet demands for food and other bioproducts. Previous work in C3 species has shown that increasing carbon assimilation through genetic manipulation of the CBB cycle can increase biomass and yield. There may also be options to improve photosynthesis in species using C4 photosynthesis and CAM through manipulation of the CBB cycle in these plants. This is an underexplored strategy and requires more basic knowledge of CBB cycle operation in these species to enable approaches for increased productivity.

Feeding the world: impacts of elevated [CO2] on nutrient content of greenhouse grown fruit crops and options for future yield gains.

Several long-term studies have provided strong support demonstrating that growing crops under elevated [CO2] can increase photosynthesis and result in an increase in yield, flavour and nutritional content (including but not limited to Vitamins C, E and pro-vitamin A). In the case of tomato, increases in yield by as much as 80% are observed when plants are cultivated at 1000 ppm [CO2], which is consistent with current commercial greenhouse production methods in the tomato fruit industry. These results provide a clear demonstration of the potential for elevating [CO2] for improving yield and quality in greenhouse crops. The major focus of this review is to bring together 50 years of observations evaluating the impact of elevated [CO2] on fruit yield and fruit nutritional quality. In the final section, we consider the need to engineer improvements to photosynthesis and nitrogen assimilation to allow plants to take greater advantage of elevated CO2 growth conditions.

Glyceraldehyde-3-phosphate dehydrogenase subunits A and B are essential to maintain photosynthetic efficiency.

In plants, glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) reversibly converts 1,3-bisphosphoglycerate to glyceraldehyde-3-phosphate coupled with the reduction of NADPH to NADP+. The GAPDH enzyme that functions in the Calvin-Benson cycle is assembled either from 4 glyceraldehyde-3-phosphate dehydrogenase A (GAPA) subunit proteins forming a homotetramer (A4) or from 2 GAPA and 2 glyceraldehyde-3-phosphate dehydrogenase B (GAPB) subunit proteins forming a heterotetramer (A2B2). The relative importance of these 2 forms of GAPDH in determining the rate of photosynthesis is unknown. To address this question, we measured the photosynthetic rates of Arabidopsis (Arabidopsis thaliana) plants containing reduced amounts of the GAPDH A and B subunits individually and jointly, using T-DNA insertion lines of GAPA and GAPB and transgenic GAPA and GAPB plants with reduced levels of these proteins. Here, we show that decreasing the levels of either the A or B subunits decreased the maximum efficiency of CO2 fixation, plant growth, and final biomass. Finally, these data showed that the reduction in GAPA protein to 9% wild-type levels resulted in a 73% decrease in carbon assimilation rates. In contrast, eliminating GAPB protein resulted in a 40% reduction in assimilation rates. This work demonstrates that the GAPA homotetramer can compensate for the loss of GAPB, whereas GAPB alone cannot compensate fully for the loss of the GAPA subunit.

Increased sedoheptulose-1,7-bisphosphatase content in Setaria viridis does not affect C4 photosynthesis.

Sedoheptulose-1,7-bisphosphatase (SBPase) is one of the rate-limiting enzymes of the Calvin cycle, and increasing the abundance of SBPase in C3 plants provides higher photosynthetic rates and stimulates biomass and yield. C4 plants usually have higher photosynthetic rates because they operate a biochemical CO2-concentrating mechanism between mesophyll and bundle sheath cells. In the C4 system, SBPase and other enzymes of the Calvin cycle are localized to the bundle sheath cells. Here we tested what effect increasing abundance of SBPase would have on C4 photosynthesis. Using green foxtail millet (Setaria viridis), a model C4 plant of NADP-ME subtype, we created transgenic plants with 1.5 to 3.2 times higher SBPase content compared to wild-type plants. Transcripts of the transgene were found predominantly in the bundle sheaths suggesting the correct cellular localization of the protein. The abundance of ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit was not affected in transgenic plants overexpressing SBPase, and neither was leaf chlorophyll content or photosynthetic electron transport parameters. We found no association between SBPase content in S. viridis and saturating rates of CO2 assimilation. Moreover, a detailed analysis of CO2 assimilation rates at different CO2 partial pressures, irradiances, and leaf temperatures showed no improvement of photosynthesis in plants overexpressing SBPase. We discuss the potential implications of these results for understanding the role of SBPase in regulation of C4 photosynthesis.

Rieske FeS overexpression in tobacco provides increased abundance and activity of cytochrome b6 f.

Photosynthesis is fundamental for plant growth and yield. The cytochrome b6 f complex catalyses a rate-limiting step in thylakoid electron transport and therefore represents an important point of regulation of photosynthesis. Here we show that overexpression of a single core subunit of cytochrome b6 f, the Rieske FeS protein, led to up to a 40% increase in the abundance of the complex in Nicotiana tabacum (tobacco) and was accompanied by an enhanced in vitro cytochrome f activity, indicating a full functionality of the complex. Analysis of transgenic plants overexpressing Rieske FeS by the light-induced fluorescence transients technique revealed a more oxidised primary quinone acceptor of photosystem II (QA ) and plastoquinone pool and faster electron transport from the plastoquinone pool to photosystem I upon changes in irradiance, compared to control plants. A faster establishment of qE , the energy-dependent component of nonphotochemical quenching, in transgenic plants suggests a more rapid buildup of the transmembrane proton gradient, also supporting the increased in vivo cytochrome b6 f activity. However, there was no consistent increase in steady-state rates of electron transport or CO2 assimilation in plants overexpressing Rieske FeS grown in either laboratory conditions or field trials, suggesting that the in vivo activity of the complex was only transiently increased upon changes in irradiance. Our results show that overexpression of Rieske FeS in tobacco enhances the abundance of functional cytochrome b6 f and may have the potential to increase plant productivity if combined with other traits.

Improving plant productivity by re-tuning the regeneration of RuBP in the Calvin-Benson-Bassham cycle.

The Calvin-Benson-Bassham (CBB) cycle is arguably the most important pathway on earth, capturing CO2 from the atmosphere and converting it into organic molecules, providing the basis for life on our planet. This cycle has been intensively studied over the 50 yr since it was elucidated, and it is highly conserved across nature, from cyanobacteria to the largest of our land plants. Eight out of the 11 enzymes in this cycle catalyse the regeneration of ribulose-1-5 bisphosphate (RuBP), the CO2 acceptor molecule. The potential to manipulate RuBP regeneration to improve photosynthesis has been demonstrated in a number of plant species, and the development of new technologies, such as omics and synthetic biology provides exciting future opportunities to improve photosynthesis and increase crop yields.

Field-grown ictB tobacco transformants show no difference in photosynthetic efficiency for biomass relative to the wild type.

In this study, four tobacco transformants overexpressing the inorganic carbon transporter B gene (ictB) were screened for photosynthetic performance relative to the wild type (WT) in field-based conditions. The WT and transgenic tobacco plants were evaluated for photosynthetic performance to determine the maximum rate of carboxylation (Vc, max), maximum rate of electron transport (Jmax), the photosynthetic compensation point (Γ*), quantum yield of PSII (ΦPSII), and mesophyll conductance (gm). Additionally, all plants were harvested to compare differences in above-ground biomass. Overall, transformants did not perform better than the WT on photosynthesis-, biomass-, and leaf composition-related traits. This is in contrast to previous studies that have suggested significant increases in photosynthesis and yield with the overexpression of ictB, although not widely evaluated under field conditions. These findings suggest that the benefit of ictB is not universal and may only be seen under certain growth conditions. While there is certainly still potential benefit to utilizing ictB in the future, further effort must be concentrated on understanding the underlying function of the gene and in which environmental conditions it offers the greatest benefit to crop performance. As it stands at present, it is possible that ictB overexpression may be largely favorable in controlled environments, such as greenhouses.

Suppression of metabolite shuttles for export of chloroplast and mitochondrial ATP and NADPH increases the cytosolic NADH:NAD+ ratio in tobacco leaves in the dark.

The communication between chloroplasts and mitochondria, which depends on the inter-organellar exchange of carbon skeletons, energy, and reducing equivalents, is essential for maintaining efficient respiratory metabolism and photosynthesis. We devised a multi-transgene approach to manipulate the leaf energy and redox balance in tobacco (Nicotiana tabacum) while monitoring the in vivo cytosolic redox status of NAD(H) using the biosensor c-Peredox-mCherry. Our strategy involved altering the shuttling capacity of the chloroplast by (1) increasing the chloroplast malate valve capacity by overexpression of the chloroplast malate valve transporter pOMT from Arabidopsis (AtpOMT1) while (2) reducing the activity of the chloroplast triose-phosphate/3-phosphoglycerate shuttle by knocking down the cytosolic NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (NtGAPC). This was accompanied by (3) alterations to the export of reducing equivalents in the mitochondria by knocking down the mitochondrial malate dehydrogenase (NtmMDH) and (4) an increased expression of the mitochondrial fission regulator FIS1A from Arabidopsis (AtFIS1A). The multi-transgene tobacco plants were analysed in glasshouse conditions and showed significant increases in the cytosolic NADH:NAD+ in the dark when transcript levels for NtGAPC or NtmMDH were knocked down. In addition, principal component analysis and Spearman correlation analyses showed negative correlations between average transcript levels for the gene targets and parameters related to chlorophyll fluorescence and plant growth. Our results highlight the importance of the shuttling of energy and reducing equivalents from chloroplasts and mitochondria to support photosynthesis and growth and suggest an important role for the dual 2-oxoglutarate/malate and oxaloacetate/malate transporter (pOMT).

Insights Into the Regulation of the Expression Pattern of Calvin-Benson-Bassham Cycle Enzymes in C3 and C4 Grasses.

C4 photosynthesis is characterized by the compartmentalization of the processes of atmospheric uptake of CO2 and its conversion into carbohydrate between mesophyll and bundle-sheath cells. As a result, most of the enzymes participating in the Calvin-Benson-Bassham (CBB) cycle, including RubisCO, are highly expressed in bundle-sheath cells. There is evidence that changes in the regulatory sequences of RubisCO contribute to its bundle-sheath-specific expression, however, little is known about how the spatial-expression pattern of other CBB cycle enzymes is regulated. In this study, we use a computational approach to scan for transcription factor binding sites in the regulatory regions of the genes encoding CBB cycle enzymes, SBPase, FBPase, PRK, and GAPDH-B, of C3 and C4 grasses. We identified potential cis-regulatory elements present in each of the genes studied here, regardless of the photosynthetic path used by the plant. The trans-acting factors that bind these elements have been validated in A. thaliana and might regulate the expression of the genes encoding CBB cycle enzymes. In addition, we also found C4-specific transcription factor binding sites in the genes encoding CBB cycle enzymes that could potentially contribute to the pathway-specific regulation of gene expression. These results provide a foundation for the functional analysis of the differences in regulation of genes encoding CBB cycle enzymes between C3 and C4 grasses.

Stimulating photosynthetic processes increases productivity and water-use efficiency in the field.

Previous studies have demonstrated that the independent stimulation of either electron transport or RuBP regeneration can increase the rate of photosynthetic carbon assimilation and plant biomass. In this paper, we present evidence that a multigene approach to simultaneously manipulate these two processes provides a further stimulation of photosynthesis. We report on the introduction of the cyanobacterial bifunctional enzyme fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase or the overexpression of the plant enzyme sedoheptulose-1,7-bisphosphatase, together with the expression of the red algal protein cytochrome c6, and show that a further increase in biomass accumulation under both glasshouse and field conditions can be achieved. Furthermore, we provide evidence that the stimulation of both electron transport and RuBP regeneration can lead to enhanced intrinsic water-use efficiency under field conditions.

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.

Overexpression of the Rieske FeS protein of the Cytochrome b6f complex increases C4 photosynthesis in Setaria viridis.

C4 photosynthesis is characterised by a CO2 concentrating mechanism that operates between mesophyll and bundle sheath cells increasing CO2 partial pressure at the site of Rubisco and photosynthetic efficiency. Electron transport chains in both cell types supply ATP and NADPH for C4 photosynthesis. Cytochrome b6f is a key control point of electron transport in C3 plants. To study whether C4 photosynthesis is limited by electron transport we constitutively overexpressed the Rieske FeS subunit in Setaria viridis. This resulted in a higher Cytochrome b6f content in mesophyll and bundle sheath cells without marked changes in the abundances of other photosynthetic proteins. Rieske overexpression plants showed better light conversion efficiency in both Photosystems and could generate higher proton-motive force across the thylakoid membrane underpinning an increase in CO2 assimilation rate at ambient and saturating CO2 and high light. Our results demonstrate that removing electron transport limitations can increase C4 photosynthesis.

Stability of wheat grain yields over three field seasons in the UK.

Ensuring food security in a changing climate is a major contemporary challenge and requires development of climate-resilient crops that perform well under variable environments. The hypothesis that yield stability in suboptimal conditions is linked to yield penalties in optimal conditions was investigated in field-grown wheat in the UK. The phenotypic responses, rate of wheat crop development, and final grain yield to varying sowing date, rainfall, air temperature, and radiation patterns were studied for a panel of 61 elite commercial wheat cultivars grown in the UK in 2012, 2013, and 2014. Contrasting climatic patterns, particularly rainfall accumulation and distribution over the season, influenced the relative performance of the cultivars affecting the duration of grain development stage and impacting on productivity. Indices for crop productivity, yield stability, and performance under suboptimal conditions revealed four cultivars with a combination of stable and high relative grain yields over the three seasons: Gladiator, Humber, Mercato, and Zebedee. Genetic similarity between cultivars partially explained yield performance in the contrasting seasons. The year of release of the cultivars correlated with grain yield but not with yield stability, supporting the contention that breeding for yield potential does not select for climate resilience and yield stability of crops. Further analysis of the outstanding cultivars may unravel target traits for breeding efforts aimed at increasing wheat yield potential and stability in the changing climate.

Genotypic, Developmental and Environmental Effects on the Rapidity of gs in Wheat: Impacts on Carbon Gain and Water-Use Efficiency.

Stomata are the primary gatekeepers for CO2 uptake for photosynthesis and water loss via transpiration and therefore play a central role in crop performance. Although stomatal conductance (gs ) and assimilation rate (A) are often highly correlated, studies have demonstrated an uncoupling between A and gs that can result in sub-optimal physiological processes in dynamic light environments. Wheat (Triticum aestivum L.) is exposed to changes in irradiance due to leaf self-shading, moving clouds and shifting sun angle to which both A and gs respond. However, stomatal responses are generally an order of magnitude slower than photosynthetic responses, leading to non-synchronized A and gs responses that impact CO2 uptake and water use efficiency ( iWUE). Here we phenotyped a panel of eight wheat cultivars (estimated to capture 80% of the single nucleotide polymorphism variation in North-West European bread wheat) for differences in the speed of stomatal responses (to changes in light intensity) and photosynthetic performance at different stages of development. The impact of water stress and elevated [CO2] on stomatal kinetics was also examined in a selected cultivar. Significant genotypic variation was reported for the time constant for stomatal opening (Ki, P = 0.038) and the time to reach 95% steady state A (P = 0.045). Slow gs opening responses limited A by ∼10% and slow closure reduced iWUE, with these impacts found to be greatest in cultivars Soissons, Alchemy and Xi19. A decrease in stomatal rapidity (and thus an increase in the limitation of photosynthesis) (P < 0.001) was found during the post-anthesis stage compared to the early booting stage. Reduced water availability triggered stomatal closure and asymmetric stomatal opening and closing responses, while elevated atmospheric [CO2] conditions reduced the time for stomatal opening during a low to high light transition, thus suggesting a major environmental effect on dynamic stomatal kinetics. We discuss these findings in terms of exploiting various traits to develop ideotypes for specific environments, and suggest that intraspecific variation in the rapidity of stomatal responses could provide a potential unexploited breeding target to optimize the physiological responses of wheat to dynamic field conditions.

Feeding the world: improving photosynthetic efficiency for sustainable crop production.

A number of recent studies have provided strong support demonstrating that improving the photosynthetic processes through genetic engineering can provide an avenue to improve yield potential. The major focus of this review is on improvement of the Calvin-Benson cycle and electron transport. Consideration is also given to how altering regulatory process may provide an additional route to increase photosynthetic efficiency. Here we summarize some of the recent successes that have been observed through genetic manipulation of photosynthesis, showing that, in both the glasshouse and the field, yield can be increased by >40%. These results provide a clear demonstration of the potential for increasing yield through improvements in photosynthesis. In the final section, we consider the need to stack improvement in photosynthetic traits with traits that target the yield gap in order to provide robust germplasm for different crops across the globe.

Overexpressing the H-protein of the glycine cleavage system increases biomass yield in glasshouse and field-grown transgenic tobacco plants.

Photorespiration is essential for C3 plants, enabling oxygenic photosynthesis through the scavenging of 2-phosphoglycolate. Previous studies have demonstrated that overexpression of the L- and H-proteins of the photorespiratory glycine cleavage system results in an increase in photosynthesis and growth in Arabidopsis thaliana. Here, we present evidence that under controlled environment conditions an increase in biomass is evident in tobacco plants overexpressing the H-protein. Importantly, the work in this paper provides a clear demonstration of the potential of this manipulation in tobacco grown in field conditions, in two separate seasons. We also demonstrate the importance of targeted overexpression of the H-protein using the leaf-specific promoter ST-LS1. Although increases in the H-protein driven by this promoter have a positive impact on biomass, higher levels of overexpression of this protein driven by the constitutive CaMV 35S promoter result in a reduction in the growth of the plants. Furthermore in these constitutive overexpressor plants, carbon allocation between soluble carbohydrates and starch is altered, as is the protein lipoylation of the enzymes pyruvate dehydrogenase and alpha-ketoglutarate complexes. Our data provide a clear demonstration of the positive effects of overexpression of the H-protein to improve yield under field conditions.

Optimizing photorespiration for improved crop productivity.

In C3 plants, photorespiration is an energy-expensive process, including the oxygenation of ribulose-1,5-bisphosphate (RuBP) by ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and the ensuing multi-organellar photorespiratory pathway required to recycle the toxic byproducts and recapture a portion of the fixed carbon. Photorespiration significantly impacts crop productivity through reducing yields in C3 crops by as much as 50% under severe conditions. Thus, reducing the flux through, or improving the efficiency of photorespiration has the potential of large improvements in C3 crop productivity. Here, we review an array of approaches intended to engineer photorespiration in a range of plant systems with the goal of increasing crop productivity. Approaches include optimizing flux through the native photorespiratory pathway, installing non-native alternative photorespiratory pathways, and lowering or even eliminating Rubisco-catalyzed oxygenation of RuBP to reduce substrate entrance into the photorespiratory cycle. Some proposed designs have been successful at the proof of concept level. A plant systems-engineering approach, based on new opportunities available from synthetic biology to implement in silico designs, holds promise for further progress toward delivering more productive crops to farmer's fields.