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.

Producing fast and active Rubisco in tobacco to enhance photosynthesis.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) performs most of the carbon fixation on Earth. However, plant Rubisco is an intrinsically inefficient enzyme given its low carboxylation rate, representing a major limitation to photosynthesis. Replacing endogenous plant Rubisco with a faster Rubisco is anticipated to enhance crop photosynthesis and productivity. However, the requirement of chaperones for Rubisco expression and assembly has obstructed the efficient production of functional foreign Rubisco in chloroplasts. Here, we report the engineering of a Form 1A Rubisco from the proteobacterium Halothiobacillus neapolitanus in Escherichia coli and tobacco (Nicotiana tabacum) chloroplasts without any cognate chaperones. The native tobacco gene encoding Rubisco large subunit was genetically replaced with H. neapolitanus Rubisco (HnRubisco) large and small subunit genes. We show that HnRubisco subunits can form functional L8S8 hexadecamers in tobacco chloroplasts at high efficiency, accounting for ∼40% of the wild-type tobacco Rubisco content. The chloroplast-expressed HnRubisco displayed a ∼2-fold greater carboxylation rate and supported a similar autotrophic growth rate of transgenic plants to that of wild-type in air supplemented with 1% CO2. This study represents a step toward the engineering of a fast and highly active Rubisco in chloroplasts to improve crop photosynthesis and growth.

A guide to photosynthetic gas exchange measurements: Fundamental principles, best practice and potential pitfalls.

Gas exchange measurements enable mechanistic insights into the processes that underpin carbon and water fluxes in plant leaves which in turn inform understanding of related processes at a range of scales from individual cells to entire ecosytems. Given the importance of photosynthesis for the global climate discussion it is important to (a) foster a basic understanding of the fundamental principles underpinning the experimental methods used by the broad community, and (b) ensure best practice and correct data interpretation within the research community. In this review, we outline the biochemical and biophysical parameters of photosynthesis that can be investigated with gas exchange measurements and we provide step-by-step guidance on how to reliably measure them. We advise on best practices for using gas exchange equipment and highlight potential pitfalls in experimental design and data interpretation. The Supporting Information contains exemplary data sets, experimental protocols and data-modelling routines. This review is a community effort to equip both the experimental researcher and the data modeller with a solid understanding of the theoretical basis of gas-exchange measurements, the rationale behind different experimental protocols and the approaches to data interpretation.

Exploring natural genetic diversity in a bread wheat multi-founder population: Dual imaging of photosynthesis and stomatal kinetics.

A better understanding of crop phenotype under dynamic environmental conditions will help inform the development of new cultivars with superior adaptation to constantly changing field conditions. Recent research has shown that optimising photosynthetic and stomatal conductance traits holds promise for improved crop performance. However, standard phenotyping tools such as gas-exchange systems are limited by their throughput. In this work, a novel approach based on a bespoke gas-exchange chamber allowing combined measurement of the quantum yield of photosystem II (PSII) with an estimation of stomatal conductance via thermal imaging, was used to phenotype a range of bread wheat (Triticum aestivum L.) genotypes, that were a sub-set of a multi-founder experimental population. Datasets were further supplemented by measurement of photosynthetic capacity and stomatal density. First, we showed that measurement of stomatal traits using our dual imaging system compared to standard IRGA methods showed good agreement between the two methods (R2=0.86) for the rapidity of stomatal opening (Ki), with the dual-imager method resulting in less intra-genotype variation. Using the dual-imaging methods, and traditional approaches we found broad and significant variation in key traits, including photosynthetic CO2 uptake at saturating light and ambient CO2 concentration (Asat), photosynthetic CO2 uptake at saturating light and elevated CO2 concentration (Amax), the maximum velocity of Rubisco for carboxylation (Vcmax), time for stomatal opening (Ki), and leaf evaporative cooling. Anatomical analysis revealed significant variation in flag leaf adaxial stomatal density. Associations between traits highlighted significant relationships between leaf evaporative cooling, leaf stomatal conductance under low (gsmin) and high (gsmax) light intensity, and the operating efficiency of PSII (Fq'/Fm'), highlighting the importance of stomatal conductance and stomatal rapidity in maintaining optimal leaf temperature for photosynthesis in wheat. Additionally, gsmin and gsmax were positively associated, indicating that potential combination of preferable traits (i.e. inherently high gsmax, low Ki and maintained leaf evaporative cooling) are present in wheat. This work highlights for the first time the effectiveness of thermal imaging in screening dynamic stomatal conductance in a large panel of wheat genotypes. The wide phenotypic variation observed suggested the presence of exploitable genetic variability in bread wheat for dynamic stomatal conductance traits and photosynthetic capacity for targeted optimisation within future breeding programs.

C4 monocots and C4 dicots exhibit rapid photosynthetic induction response in contrast to C3 plants.

Considering the prevalence of ever-changing conditions in the natural world, investigation of photosynthetic responses in C4 plants under fluctuating light is needed. Here, we studied the effect of dynamic illumination on photosynthesis in totally 10 C3, C3-C4 intermediate, C4-like and C4 dicots and monocots at CO2 concentrations of 400 and 800 µmol mol-1. C4 and C4-like plants had faster photosynthetic induction and light-induced stomatal dynamics than C3 plants at 400 µmol mol-1, but not at 800 µmol mol-1 CO2, at which the CO2 supply rarely limits photosynthesis. C4 and C4-like plants had a higher water use efficiency than C3 plants at both CO2 concentrations. There were positive correlations between photosynthetic induction and light-induced stomatal response, together with CO2 compensation point, which was a parameter of the CO2-concentrating mechanism of C4 photosynthesis. These results clearly show that C4 photosynthesis in both monocots and dicots adapts to fluctuating light conditions more efficiently than C3 photosynthesis. The rapid photosynthetic induction response in C4 plants can be attributed to the rapid stomatal dynamics, the CO2-concentrating mechanism or both.

Photosynthesis - beyond the leaf.

Although leaves are considered the main site for photosynthesis, other green nonfoliar tissues can carry out considerable amounts of photosynthetic carbon assimilation. With photosynthesis, a potential target for improving crop productivity, physiology and contribution of nonfoliar tissues to overall plant carbon acquisition is gaining increasing attention. This review will provide an overview of nonfoliar photosynthesis, the role of stomata in these tissues and methodologies for quantification and the contribution to overall carbon gain.

Longer epidermal cells underlie a quantitative source of variation in wheat flag leaf size.

The wheat flag leaf is the main contributor of photosynthetic assimilates to developing grains. Understanding how canopy architecture strategies affect source strength and yield will aid improved crop design. We used an eight-founder population to investigate the genetic architecture of flag leaf area, length, width and angle in European wheat. For the strongest genetic locus identified, we subsequently created a near-isogenic line (NIL) pair for more detailed investigation across seven test environments. Genetic control of traits investigated was highly polygenic, with colocalisation of replicated quantitative trait loci (QTL) for one or more traits identifying 24 loci. For QTL QFll.niab-5A.1 (FLL5A), development of a NIL pair found the FLL5A+ allele commonly conferred a c. 7% increase in flag and second leaf length and a more erect leaf angle, resulting in higher flag and/or second leaf area. Increased FLL5A-mediated flag leaf length was associated with: (1) longer pavement cells and (2) larger stomata at lower density, with a trend for decreased maximum stomatal conductance (Gsmax ) per unit leaf area. For FLL5A, cell size rather than number predominantly determined leaf length. The observed trade-offs between leaf size and stomatal morphology highlight the need for future studies to consider these traits at the whole-leaf level.

Excised leaves show limited and species-specific effects on photosynthetic parameters across crop functional types.

Photosynthesis is increasingly becoming a recognized target for crop improvement. Phenotyping photosynthesis-related traits on field-grown material is a key bottleneck to progress here due to logistical barriers and short measurement days. Many studies attempt to overcome these challenges by phenotyping excised leaf material in the laboratory. To date there are no demonstrated examples of the representative nature of photosynthesis measurements performed on excised leaves relative to attached leaves in crops. Here, we tested whether standardized leaf excision on the day prior to phenotyping affected a range of common photosynthesis-related traits across crop functional types using tomato (C3 dicot), barley (C3 monocot), and maize (C4 monocot). Potentially constraining aspects of leaf physiology that could be predicted to impair photosynthesis in excised leaves, namely leaf water potential and abscisic acid accumulation, were not different between attached and excised leaves. We also observed non-significant differences in spectral reflectance and chlorophyll fluorescence traits between the treatments across the three species. However, we did observe some significant differences between traits associated with gas exchange and photosynthetic capacity across all three species. This study represents a useful reference for those who perform measurements of this nature and the differences reported should be considered in associated experimental design and statistical analyses.

The impact of growth at elevated [CO2] on stomatal anatomy and behavior differs between wheat species and cultivars.

The ability of plants to respond to changes in the environment is crucial to their survival and reproductive success. The impact of increasing the atmospheric CO2 concentration (a[CO2]), mediated by behavioral and developmental responses of stomata, on crop performance remains a concern under all climate change scenarios, with potential impacts on future food security. To identify possible beneficial traits that could be exploited for future breeding, phenotypic variation in morphological traits including stomatal size and density, as well as physiological responses and, critically, the effect of growth [CO2] on these traits, was assessed in six wheat relative accessions (including Aegilops tauschii, Triticum turgidum ssp. Dicoccoides, and T. turgidum ssp. dicoccon) and five elite bread wheat T. aestivum cultivars. Exploiting a range of different species and ploidy, we identified key differences in photosynthetic capacity between elite hexaploid wheat and wheat relatives. We also report differences in the speed of stomatal responses which were found to be faster in wheat relatives than in elite cultivars, a trait that could be useful for enhanced photosynthetic carbon gain and water use efficiency. Furthermore, these traits do not all appear to be influenced by elevated [CO2], and determining the underlying genetics will be critical for future breeding programmes.

Guard Cell Starch Degradation Yields Glucose for Rapid Stomatal Opening in Arabidopsis.

Starch in Arabidopsis (Arabidopsis thaliana) guard cells is rapidly degraded at the start of the day by the glucan hydrolases α-AMYLASE3 (AMY3) and ß-AMYLASE1 (BAM1) to promote stomatal opening. This process is activated via phototropin-mediated blue light signaling downstream of the plasma membrane H+-ATPase. It remains unknown how guard cell starch degradation integrates with light-regulated membrane transport processes in the fine control of stomatal opening kinetics. We report that H+, K+, and Cl- transport across the guard cell plasma membrane is unaltered in the amy3 bam1 mutant, suggesting that starch degradation products do not directly affect the capacity to transport ions. Enzymatic quantification revealed that after 30 min of blue light illumination, amy3 bam1 guard cells had similar malate levels as the wild type, but had dramatically altered sugar homeostasis, with almost undetectable amounts of Glc. Thus, Glc, not malate, is the major starch-derived metabolite in Arabidopsis guard cells. We further show that impaired starch degradation in the amy3 bam1 mutant resulted in an increase in the time constant for opening of 40 min. We conclude that rapid starch degradation at dawn is required to maintain the cytoplasmic sugar pool, clearly needed for fast stomatal opening. The conversion and exchange of metabolites between subcellular compartments therefore coordinates the energetic and metabolic status of the cell with membrane ion transport.

Time-series transcriptomics reveals a BBX32-directed control of acclimation to high light in mature Arabidopsis leaves.

The photosynthetic capacity of mature leaves increases after several days' exposure to constant or intermittent episodes of high light (HL) and is manifested primarily as changes in chloroplast physiology. How this chloroplast-level acclimation to HL is initiated and controlled is unknown. From expanded Arabidopsis leaves, we determined HL-dependent changes in transcript abundance of 3844 genes in a 0-6 h time-series transcriptomics experiment. It was hypothesized that among such genes were those that contribute to the initiation of HL acclimation. By focusing on differentially expressed transcription (co-)factor genes and applying dynamic statistical modelling to the temporal transcriptomics data, a regulatory network of 47 predominantly photoreceptor-regulated transcription (co-)factor genes was inferred. The most connected gene in this network was B-BOX DOMAIN CONTAINING PROTEIN32 (BBX32). Plants overexpressing BBX32 were strongly impaired in acclimation to HL and displayed perturbed expression of photosynthesis-associated genes under LL and after exposure to HL. These observations led to demonstrating that as well as regulation of chloroplast-level acclimation by BBX32, CRYPTOCHROME1, LONG HYPOCOTYL5, CONSTITUTIVELY PHOTOMORPHOGENIC1 and SUPPRESSOR OF PHYA-105 are important. In addition, the BBX32-centric gene regulatory network provides a view of the transcriptional control of acclimation in mature leaves distinct from other photoreceptor-regulated processes, such as seedling photomorphogenesis.

Stomata on the abaxial and adaxial leaf surfaces contribute differently to leaf gas exchange and photosynthesis in wheat.

Although stomata are typically found in greater numbers on the abaxial surface, wheat flag leaves have greater densities on the adaxial surface. We determine the impact of this less common stomatal patterning on gaseous fluxes using a novel chamber that simultaneously measures both leaf surfaces. Using a combination of differential illuminations and CO2 concentrations at each leaf surface, we found that mesophyll cells associated with the adaxial leaf surface have a higher photosynthetic capacity than those associated with the abaxial leaf surface, which is supported by an increased stomatal conductance (driven by differences in stomatal density). When vertical gas flux at the abaxial leaf surface was blocked, no compensation by adaxial stomata was observed, suggesting each surface operates independently. Similar stomatal kinetics suggested some co-ordination between the two surfaces, but factors other than light intensity played a role in these responses. Higher photosynthetic capacity on the adaxial surface facilitates greater carbon assimilation, along with higher adaxial stomatal conductance, which would also support greater evaporative leaf cooling to maintain optimal leaf temperatures for photosynthesis. Furthermore, abaxial gas exchange contributed c. 50% to leaf photosynthesis and therefore represents an important contributor to overall leaf gas exchange.

The impact of slow stomatal kinetics on photosynthesis and water use efficiency under fluctuating light.

Dynamic light conditions require continuous adjustments of stomatal aperture. The kinetics of stomatal conductance (gs) is hypothesized to be key to plant productivity and water use efficiency (WUE). Using step-changes in light intensity, we studied the diversity of light-induced gs kinetics in relation to stomatal anatomy in five banana genotypes (Musa spp.) and modeled the impact of both diffusional and biochemical limitations on photosynthesis (A). The dominant A limiting factor was the diffusional limitation associated with gs kinetics. All genotypes exhibited a strong limitation of A by gs, indicating a priority for water saving. Moreover, significant genotypic differences in gs kinetics and gs limitations of A were observed. For two contrasting genotypes, the impact of differential gs kinetics was further investigated under realistic diurnally fluctuating light conditions and at the whole-plant level. Genotype-specific stomatal kinetics observed at the leaf level was corroborated at whole-plant level by transpiration dynamics, validating that genotype-specific responses are still maintained despite differences in gs control at different locations in the leaf and across leaves. However, under diurnally fluctuating light conditions the impact of gs speediness on A and intrinsic (iWUE) depended on time of day. During the afternoon there was a setback in kinetics: absolute gs and gs responses to light were damped, strongly limiting A and impacting diurnal iWUE. We conclude the impact of differential gs kinetics depended on target light intensity, magnitude of change, gs prior to the change in light intensity, and particularly time of day.

The role of photosynthesis related pigments in light harvesting, photoprotection and enhancement of photosynthetic yield in planta.

Photosynthetic pigments are an integral and vital part of all photosynthetic machinery and are present in different types and abundances throughout the photosynthetic apparatus. Chlorophyll, carotenoids and phycobilins are the prime photosynthetic pigments which facilitate efficient light absorption in plants, algae, and cyanobacteria. The chlorophyll family plays a vital role in light harvesting by absorbing light at different wavelengths and allowing photosynthetic organisms to adapt to different environments, either in the long-term or during transient changes in light. Carotenoids play diverse roles in photosynthesis, including light capture and as crucial antioxidants to reduce photodamage and photoinhibition. In the marine habitat, phycobilins capture a wide spectrum of light and have allowed cyanobacteria and red algae to colonise deep waters where other frequencies of light are attenuated by the water column. In this review, we discuss the potential strategies that photosynthetic pigments provide, coupled with development of molecular biological techniques, to improve crop yields through enhanced light harvesting, increased photoprotection and improved photosynthetic efficiency.

Natural variation in stomatal dynamics drives divergence in heat stress tolerance and contributes to seasonal intrinsic water-use efficiency in Vitis vinifera (subsp. sativa and sylvestris).

Stomata control CO2 uptake for photosynthesis and water loss through transpiration, thus playing a key role in leaf thermoregulation, water-use efficiency (iWUE), and plant productivity. In this work, we investigated the relationship between several leaf traits and hypothesized that stomatal behavior to fast (i.e. minutes) environmental changes co-determines, along with steady-state traits, the physiological response of grapevine to the surrounding fluctuating environment over the growing season. No relationship between iWUE, heat stress tolerance, and stomatal traits was observed in field-grown grapevine, suggesting that other physiological mechanisms are involved in determining leaf evaporative cooling capacity and the seasonal ratio of CO2 uptake (A) to stomatal conductance (gs). Indeed, cultivars that in the field had an unexpected combination of high iWUE but low sensitivity to thermal stress displayed a quick stomatal closure to light, but a sluggish closure to increased vapor pressure deficit (VPD) levels. This strategy, aiming both at conserving water under a high to low light transition and in prioritizing evaporative cooling under a low to high VPD transition, was mainly observed in the cultivars Regina and Syrah. Moreover, cultivars with different known responses to soil moisture deficit or high air VPD (isohydric versus anisohydric) had opposite behavior under fluctuating environments, with the isohydric cultivar showing slow stomatal closure to reduced light intensity but quick temporal responses to VPD manipulation. We propose that stomatal behavior to fast environmental fluctuations can play a critical role in leaf thermoregulation and water conservation under natural field conditions in grapevine.

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.

Natural genetic variation in photosynthesis: an untapped resource to increase crop yield potential?

Raising crop yield potential is a major goal to ensure food security for the growing global population. Photosynthesis is the primary determinant of crop productivity and any gain in photosynthetic CO2 assimilation per unit of leaf area (A) has the potential to increase yield. Significant intraspecific variation in A is known to exist in various autotrophic organs that represent an unexploited target for crop improvement. However, the large number of factors that influence photosynthetic rates often makes it difficult to measure or estimate A under dynamic field conditions (i.e. fluctuating light intensities or temperatures). This complexity often results in photosynthetic capacity, rather than realized photosynthetic rates being used to assess natural variation in photosynthesis. Here we review the work on natural variation in A, the different factors determining A and their interaction in yield formation. A series of drawbacks and perspectives are presented for the most common analyses generally used to estimate A. The different yield components and their determination based on different photosynthetic organs are discussed with a major focus on potential exploitation of various traits for crop improvement. To conclude, an example of different possibilities to increase yield in wheat through enhancing A is illustrated.

Photosynthesis in non-foliar tissues: implications for yield.

Photosynthesis is currently a focus for crop improvement. The majority of this work has taken place and been assessed in leaves, and limited consideration has been given to the contribution that other green tissues make to whole-plant carbon assimilation. The major focus of this review is to evaluate the impact of non-foliar photosynthesis on carbon-use efficiency and total assimilation. Here we appraise and summarize past and current literature on the substantial contribution of different photosynthetically active organs and tissues to productivity in a variety of different plant types, with an emphasis on fruit and cereal crops. Previous studies provide evidence that non-leaf photosynthesis could be an unexploited potential target for crop improvement. We also briefly examine the role of stomata in non-foliar tissues, gas exchange, maintenance of optimal temperatures and thus photosynthesis. In the final section, we discuss possible opportunities to manipulate these processes and provide evidence that Triticum aestivum (wheat) plants genetically manipulated to increase leaf photosynthesis also displayed higher rates of ear assimilation, which translated to increased grain yield. By understanding these processes, we can start to provide insights into manipulating non-foliar photosynthesis and stomatal behaviour to identify novel targets for exploitation in continuing breeding programmes.

Light, power, action! Interaction of respiratory energy- and blue light-induced stomatal movements.

Although the signalling pathway of blue light (BL)-dependent stomatal opening is well characterized, little is known about the interspecific diversity, the role it plays in the regulation of gas exchange and the source of energy used to drive the commonly observed increase in pore aperture. Using a combination of red and BL under ambient and low [O2 ] (to inhibit respiration), the interaction between BL, photosynthesis and respiration in determining stomatal conductance was investigated. These findings were used to develop a novel model to predict the feedback between photosynthesis and stomatal conductance under these conditions. Here we demonstrate that BL-induced stomatal responses are far from universal, and that significant species-specific differences exist in terms of both rapidity and magnitude. Increased stomatal conductance under BL reduced photosynthetic limitation, at the expense of water loss. Moreover, we stress the importance of the synergistic effect of BL and respiration in driving rapid stomatal movements, especially when photosynthesis is limited. These observations will help reshape our understanding of diurnal gas exchange in order to exploit the dynamic coordination between the rate of carbon assimilation (A) and stomatal conductance (gs ), as a target for enhancing crop performance and water use efficiency.

Photosynthesis and crop productivity are enhanced by glucose-functionalised carbon dots.

From global food security to textile production and biofuels, the demands currently made on plant photosynthetic productivity will continue to increase. Enhancing photosynthesis using designer, green and sustainable materials offers an attractive alternative to current genetic-based strategies and promising work with nanomaterials has recently started to emerge. Here we describe the in planta use of carbon-based nanoparticles produced by low-cost renewable routes that are bioavailable to mature plants. Uptake of these functionalised nanoparticles directly from the soil improves photosynthesis and also increases crop production. We show for the first time that glucose functionalisation enhances nanoparticle uptake, photoprotection and pigment production, unlocking enhanced yields. This was demonstrated in Triticum aestivum 'Apogee' (dwarf bread wheat) and resulted in an 18% increase in grain yield. This establishes the viability of a functional nanomaterial to augment photosynthesis as a route to increased crop productivity.