Improving crop yield potential: Underlying biological processes and future prospects.

The growing world population and global increases in the standard of living both result in an increasing demand for food, feed and other plant-derived products. In the coming years, plant-based research will be among the major drivers ensuring food security and the expansion of the bio-based economy. Crop productivity is determined by several factors, including the available physical and agricultural resources, crop management, and the resource use efficiency, quality and intrinsic yield potential of the chosen crop. This review focuses on intrinsic yield potential, since understanding its determinants and their biological basis will allow to maximize the plant's potential in food and energy production. Yield potential is determined by a variety of complex traits that integrate strictly regulated processes and their underlying gene regulatory networks. Due to this inherent complexity, numerous potential targets have been identified that could be exploited to increase crop yield. These encompass diverse metabolic and physical processes at the cellular, organ and canopy level. We present an overview of some of the distinct biological processes considered to be crucial for yield determination that could further be exploited to improve future crop productivity.

Genome-wide identification and comparative analyses of key genes involved in C4 photosynthesis in five main gramineous crops.

Compared to C3 species, C4 plants showed higher photosynthetic capacity as well as water and nitrogen use efficiency due to the presence of the C4 photosynthetic pathway. Previous studies have shown that all genes required for the C4 photosynthetic pathway exist in the genomes of C3 species and are expressed. In this study, the genes encoding six key C4 photosynthetic pathway enzymes (ß-CA, PEPC, ME, MDH, RbcS, and PPDK) in the genomes of five important gramineous crops (C4: maize, foxtail millet, and sorghum; C3: rice and wheat) were systematically identified and compared. Based on sequence characteristics and evolutionary relationships, their C4 functional gene copies were distinguished from non-photosynthetic functional gene copies. Furthermore, multiple sequence alignment revealed important sites affecting the activities of PEPC and RbcS between the C3 and C4 species. Comparisons of expression characteristics confirmed that the expression patterns of non-photosynthetic gene copies were relatively conserved among species, while C4 gene copies in C4 species acquired new tissue expression patterns during evolution. Additionally, multiple sequence features that may affect C4 gene expression and subcellular localization were found in the coding and promoter regions. Our work emphasized the diversity of the evolution of different genes in the C4 photosynthetic pathway and confirmed that the specific high expression in the leaf and appropriate intracellular distribution were the keys to the evolution of C4 photosynthesis. The results of this study will help determine the evolutionary mechanism of the C4 photosynthetic pathway in Gramineae and provide references for the transformation of C4 photosynthetic pathways in wheat, rice, and other major C3 cereal crops.

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.

Designing the Crops for the Future; The CropBooster Program.

The realization of the full objectives of international policies targeting global food security and climate change mitigation, including the United Nation's Sustainable Development Goals, the Paris Climate Agreement COP21 and the European Green Deal, requires that we (i) sustainably increase the yield, nutritional quality and biodiversity of major crop species, (ii) select climate-ready crops that are adapted to future weather dynamic and (iii) increase the resource use efficiency of crops for sustainably preserving natural resources. Ultimately, the grand challenge to be met by agriculture is to sustainably provide access to sufficient, nutritious and diverse food to a worldwide growing population, and to support the circular bio-based economy. Future-proofing our crops is an urgent issue and a challenging goal, involving a diversity of crop species in differing agricultural regimes and under multiple environmental drivers, providing versatile crop-breeding solutions within wider socio-economic-ecological systems. This goal can only be realized by a large-scale, international research cooperation. We call for international action and propose a pan-European research initiative, the CropBooster Program, to mobilize the European plant research community and interconnect it with the interdisciplinary expertise necessary to face the challenge.

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.

Biotechnology for Tomorrow's World: Scenarios to Guide Directions for Future Innovation.

Depending on how the future will unfold, today's progress in biotechnology research has greater or lesser potential to be the basis of subsequent innovation. Tracking progress against indicators for different future scenarios will help to focus, emphasize, or de-emphasize discovery research in a timely manner and to maximize the chance for successful innovation. In this paper, we show how learning scenarios with a 2050 time horizon help to recognize the implications of political and societal developments on the innovation potential of ongoing biotechnological research. We also propose a model to further increase open innovation between academia and the biotechnology value chain to help fundamental research explore discovery fields that have a greater chance to be valuable for applied research.

Novel bacterial clade reveals origin of form I Rubisco.

Rubisco sustains the biosphere through the fixation of CO2 into biomass. In plants and cyanobacteria, form I Rubisco is structurally comprised of large and small subunits, whereas all other Rubisco forms lack small subunits. The rise of the form I complex through the innovation of small subunits represents a key, yet poorly understood, transition in Rubisco's evolution. Through metagenomic analyses, we discovered a previously uncharacterized clade sister to form I Rubisco that evolved without small subunits. This clade diverged before the evolution of cyanobacteria and the origin of the small subunit; thus, it provides a unique reference point to advance our understanding of form I Rubisco evolution. Structural and kinetic data presented here reveal how a proto-form I Rubisco assembled and functioned without the structural stability imparted from small subunits. Our findings provide insight into a key evolutionary transition of the most abundant enzyme on Earth and the predominant entry point for nearly all global organic carbon.

A wish list for synthetic biology in photosynthesis research.

This perspective summarizes the presentations and discussions at the ' International Symposium on Synthetic Biology in Photosynthesis Research', which was held in Shanghai in 2018. Leveraging the current advanced understanding of photosynthetic systems, the symposium brain-stormed about the redesign and engineering of photosynthetic systems for translational goals and evaluated available new technologies/tools for synthetic biology as well as technological obstacles and new tools that would be needed to overcome them. Four major research areas for redesigning photosynthesis were identified: (i) mining natural variations of photosynthesis; (ii) coordinating photosynthesis with pathways utilizing photosynthate; (iii) reconstruction of highly efficient photosynthetic systems in non-host species; and (iv) development of new photosynthetic systems that do not exist in nature. To expedite photosynthesis synthetic biology research, an array of new technologies and community resources need to be developed, which include expanded modelling capacities, molecular engineering toolboxes, model species, and phenotyping tools.

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.

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.

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.

A high-throughput transient expression system for rice.

Rice is an important global crop and represents a vital source of calories for many food insecure regions. Efforts to improve this crop by improving yield, nutritional content, stress tolerance, or resilience to climate change are certain to include biotechnological approaches, which rely on the expression of transgenes in planta. The throughput and cost of currently available transgenic expression systems is frequently incompatible with modern, high-throughput molecular cloning methods. Here, we present a protocol for isolating high yields of green rice protoplasts and for PEG-mediated transformation of isolated protoplasts. Factors affecting transformation efficiency were investigated, and the resulting protocol is fast, cheap, robust, high-throughput, and does not require specialist equipment. When coupled to a high-throughput modular cloning system such as Golden Gate, this transient expression system provides a valuable resource to help break the "design-build-test" bottleneck by permitting the rapid screening of large numbers of transgenic expression cassettes prior to stable plant transformation. We used this system to rapidly assess the expression level, subcellular localisation, and protein aggregation pattern of nine single-gene expression cassettes, which represent the essential component parts of the ß-cyanobacterial carboxysome.

Whole plant chamber to examine sensitivity of cereal gas exchange to changes in evaporative demand.

BACKGROUND: Improving plant water use efficiency (WUE) is a major target for improving crop yield resilience to adverse climate change. Identifying genetic variation in WUE usually relies on instantaneous measurements of photosynthesis (An) and transpiration (Tr), or integrative measurements of carbon isotope discrimination, at the leaf level. However, leaf gas exchange measurements alone do not adequately represent whole plant responses, especially if evaporative demand around the plant changes. RESULTS: Here we describe a whole plant gas exchange system that can rapidly alter evaporative demand when measuring An, Tr and intrinsic WUE (iWUE) and identify genetic variation in this response. An was not limited by VPD under steady-state conditions but some wheat cultivars restricted Tr under high evaporative demand, thereby improving iWUE. These changes may be ABA-dependent, since the barley ABA-deficient mutant (Az34) failed to restrict Tr under high evaporative demand. Despite higher Tr, Az34 showed lower An than wild-type (WT) barley because of limitations in Rubisco carboxylation activity. Tr and An of Az34 were more sensitive than WT barley to exogenous spraying with ABA, which restricted photosynthesis via substrate limitation and decreasing Rubisco activation. CONCLUSIONS: Examining whole plant gas exchange responses to altered VPD can identify genetic variation in whole plant iWUE, and facilitate an understanding of the underlying mechanism(s).

Structural and functional analyses of Rubisco from arctic diatom species reveal unusual posttranslational modifications.

The catalytic performance of the major CO2-assimilating enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), restricts photosynthetic productivity. Natural diversity in the catalytic properties of Rubisco indicates possibilities for improvement. Oceanic phytoplankton contain some of the most efficient Rubisco enzymes, and diatoms in particular are responsible for a significant proportion of total marine primary production as well as being a major source of CO2 sequestration in polar cold waters. Until now, the biochemical properties and three-dimensional structures of Rubisco from diatoms were unknown. Here, diatoms from arctic waters were collected, cultivated, and analyzed for their CO2-fixing capability. We characterized the kinetic properties of five and determined the crystal structures of four Rubiscos selected for their high CO2-fixing efficiency. The DNA sequences of the rbcL and rbcS genes of the selected diatoms were similar, reflecting their close phylogenetic relationship. The Vmax and Km for the oxygenase and carboxylase activities at 25 °C and the specificity factors (Sc/o) at 15, 25, and 35 °C were determined. The Sc/o values were high, approaching those of mono- and dicot plants, thus exhibiting good selectivity for CO2 relative to O2 Structurally, diatom Rubiscos belong to form I C/D, containing small subunits characterized by a short ßA-ßB loop and a C-terminal extension that forms a ß-hairpin structure (ßE-ßF loop). Of note, the diatom Rubiscos featured a number of posttranslational modifications of the large subunit, including 4-hydroxyproline, ß-hydroxyleucine, hydroxylated and nitrosylated cysteine, mono- and dihydroxylated lysine, and trimethylated lysine. Our studies suggest adaptation toward achieving efficient CO2 fixation in arctic diatom Rubiscos.

Increasing metabolic potential: C-fixation.

Due to the growing world population, crop yields must increase to meet the rising demand. Crop plants also require adaptation to optimize performance in the changing environments caused by climate change. Improving photosynthetic carbon fixation is a promising, albeit technically challenging, strategy whose potential has only just begun to be considered in breeding programmes. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), a fundamental enzyme of carbon fixation, is extremely inefficient and many strategies to improve photosynthesis focus on overcoming the limitations of this enzyme, either by improving Rubisco activity and regulation or by improving the supply of substrates. Although progress is being made, the need to tailor solutions for each crop and their respective environments has been highlighted. Even so, continuing research will be required to achieve these objectives and to grow crops more sustainably in the future.

Identification of Leaf Promoters for Use in Transgenic Wheat.

Wheat yields have plateaued in recent years and given the growing global population there is a pressing need to develop higher yielding varieties to meet future demand. Genetic manipulation of photosynthesis in elite wheat varieties offers the opportunity to significantly increase yields. However, the absence of a well-defined molecular tool-box of promoters to manipulate leaf processes in wheat hinders advancements in this area. Two promoters, one driving the expression of sedoheptulose-1,7-bisphosphatase (SBPase) and the other fructose-1,6-bisphosphate aldolase (FBPA) from Brachypodium distachyon were identified and cloned into a vector in front of the GUS reporter gene. Both promoters were shown to be functionally active in wheat in both transient assays and in stably transformed wheat plants. Analysis of the stable transformants of wheat (cv. Cadenza) showed that both promoters controlled gus expression throughout leaf development as well as in other green tissues. The availability of these promoters provides new tools for the expression of genes in transgenic wheat leaves and also paves the way for multigene manipulation of photosynthesis to improve yields.

Phenotyping of field-grown wheat in the UK highlights contribution of light response of photosynthesis and flag leaf longevity to grain yield.

Improving photosynthesis is a major target for increasing crop yields and ensuring food security. Phenotyping of photosynthesis in the field is critical to understand the limits to crop performance in agricultural settings. Yet, detailed phenotyping of photosynthetic traits is relatively scarce in field-grown wheat, with previous studies focusing on narrow germplasm selections. Flag leaf photosynthetic traits, crop development, and yield traits were compared in 64 field-grown wheat cultivars in the UK. Pre-anthesis and post-anthesis photosynthetic traits correlated significantly and positively with grain yield and harvest index (HI). These traits included net CO2 assimilation measured at ambient CO2 concentrations and a range of photosynthetic photon flux densities, and traits associated with the light response of photosynthesis. In most cultivars, photosynthesis decreased post-anthesis compared with pre-anthesis, and this was associated with decreased Rubisco activity and abundance. Heritability of photosynthetic traits suggests that phenotypic variation can be used to inform breeding programmes. Specific cultivars were identified with traits relevant to breeding for increased crop yields in the UK: pre-anthesis photosynthesis, post-anthesis photosynthesis, light response of photosynthesis, and Rubisco amounts. The results indicate that flag leaf longevity and operating photosynthetic activity in the canopy can be further exploited to maximize grain filling in UK bread wheat.

Increased SBPase activity improves photosynthesis and grain yield in wheat grown in greenhouse conditions.

To meet the growing demand for food, substantial improvements in yields are needed. This is particularly the case for wheat, where global yield has stagnated in recent years. Increasing photosynthesis has been identified as a primary target to achieve yield improvements. To increase leaf photosynthesis in wheat, the level of the Calvin-Benson cycle enzyme sedoheptulose-1,7-biphosphatase (SBPase) has been increased through transformation and expression of a Brachypodium distachyon SBPase gene construct. Transgenic lines with increased SBPase protein levels and activity were grown under greenhouse conditions and showed enhanced leaf photosynthesis and increased total biomass and dry seed yield. This showed the potential of improving yield potential by increasing leaf photosynthesis in a crop species such as wheat. The results are discussed with regard to future strategies for further improvement of photosynthesis in wheat.This article is part of the themed issue 'Enhancing photosynthesis in crop plants: targets for improvement'.