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 carbon dioxide (CO2) concentration near the Rubisco, the enzyme responsible for the first step of CBB cycle, drawing inspiration from various photosynthetic organisms, and conclude this section by examining ways to enhance CO2 delivery into leaves. Moving beyond individual processes, we discuss two approaches to identifying key targets for photosynthesis improvement: systems modeling and the study of natural variation. Finally, we revisit some of the strategies mentioned above to provide a holistic view of the improvements, analyzing their impact on nitrogen use efficiency and on canopy photosynthesis.

Impact of pod and seed photosynthesis on seed filling and canopy carbon gain in soybean.

There is a limited understanding of the carbon assimilation capacity of nonfoliar green tissues and its impact on yield and seed quality since most photosynthesis research focuses on leaf photosynthesis. In this study, we investigate the photosynthetic efficiency of soybean (Glycine max) pods and seeds in a field setting and evaluate its effect on mature seed weight and composition. We demonstrate that soybean pod and seed photosynthesis contributes 13% to 14% of the mature seed weight. Carbon assimilation by soybean pod and seed photosynthesis can compensate for 81% of carbon loss through the respiration of the same tissues, and our model predicts that soybean pod and seed photosynthesis contributes up to 9% of the total daily carbon gain of the canopy. Chlorophyll fluorescence (CF) shows that the operating efficiency of photosystem II in immature soybean seeds peaks at the 10 to 100 mg seed weight stage, while that of immature pods peaks at the 75 to 100 mg stage. This study provides quantitative information about the efficiency of soybean pod and seed photosynthesis during tissue development and its impact on yield.

Short- and long-term warming events on photosynthetic physiology, growth, and yields of field grown crops.

Global temperatures are rising from increasing concentrations of greenhouse gases in the atmosphere associated with anthropogenic activities. Global warming includes a warmer shift in mean temperatures as well as increases in the probability of extreme heating events, termed heat waves. Despite the ability of plants to cope with temporal variations in temperature, global warming is increasingly presenting challenges to agroecosystems. The impact of warming on crop species has direct consequences on food security, therefore understanding impacts and opportunities to adapt crops to global warming necessitates experimentation that allows for modification of growth environments to represent global warming scenarios. Published studies addressing crop responses to warming are extensive, however, in-field studies where growth temperature is manipulated to mimic global warming are limited. Here, we provide an overview of in-field heating techniques employed to understand crop responses to warmer growth environments. We then focus on key results associated with season-long warming, as expected with rising global mean temperatures, and with heat waves, as a consequence of increasing temperature variability and rising global mean temperatures. We then discuss the role of rising temperatures on atmospheric water vapor pressure deficit and potential implications for crop photosynthesis and productivity. Finally, we review strategies by which crop photosynthetic processes might be optimized to adapt crops to the increasing temperatures and frequencies of heat waves. Key findings from this review are that higher temperatures consistently reduce photosynthesis and yields of crops even as atmospheric carbon dioxide increases, yet potential strategies to minimize losses from high-temperature exist.

Exploring 3D leaf anatomical traits for C4 photosynthesis: chloroplast and plasmodesmata pit field size in maize and sugarcane.

Volume and surface area of chloroplasts and surface area of plasmodesmata pit fields are presented for two C4 species, maize and sugarcane, with respect to cell surface area and cell volume. Serial block face scanning electron microscopy (SBF-SEM) and confocal laser scanning microscopy with the Airyscan system (LSM) were used. Chloroplast size estimates were much faster and easier using LSM than with SBF-SEM; however, the results were more variable than SBF-SEM. Mesophyll cells were lobed where chloroplasts were located, facilitating cell-to-cell connections while allowing for greater intercellular airspace exposure. Bundle sheath cells were cylindrical with chloroplasts arranged centrifugally. Chloroplasts occupied c. 30-50% of mesophyll cell volume, and 60-70% of bundle sheath cell volume. Roughly 2-3% of each cell surface area was covered by plasmodesmata pit fields for both bundle sheath and mesophyll cells. This work will aid future research to develop SBF-SEM methodologies with the aim to better understand the effect of cell structure on C4 photosynthesis.

Transgenic strategies to improve the thermotolerance of photosynthesis.

Warming driven by the accumulation of greenhouse gases in the atmosphere is irreversible over at least the next century, unless practical technologies are rapidly developed and deployed at scale to remove and sequester carbon dioxide from the atmosphere. Accepting this reality highlights the central importance for crop agriculture to develop adaptation strategies for a warmer future. While nearly all processes in plants are impacted by above optimum temperatures, the impact of heat stress on photosynthetic processes stand out for their centrality. Here, we review transgenic strategies that show promise in improving the high-temperature tolerance of specific subprocesses of photosynthesis and in some cases have already been shown in proof of concept in field experiments to protect yield from high temperature-induced losses. We also highlight other manipulations to photosynthetic processes for which full proof of concept is still lacking but we contend warrant further attention. Warming that has already occurred over the past several decades has had detrimental impacts on crop production in many parts of the world. Declining productivity presages a rapidly developing global crisis in food security particularly in low income countries. Transgenic manipulation of photosynthesis to engineer greater high-temperature resilience holds encouraging promise to help meet this challenge.

Evaluation of the effects of elevated CO2 concentrations on the growth of cassava storage roots by destructive harvests and ground penetrating radar scanning approaches.

Cassava (Manihot esculenta Crantz) production will need to be improved to meet future food demands in Sub-Saharan Africa. The selection of high-yielding cassava cultivars requires a better understanding of storage root development. Additionally, since future production will happen under increasing atmospheric CO2 concentrations ([CO2 ]), cultivar selection should include responsiveness to elevated [CO2 ]. Five farmer-preferred African cassava cultivars were grown for three and a half months in a Free Air CO2 Enrichment experiment in central Illinois. Compared to ambient [CO2 ] (~400 ppm), cassava storage roots grown under elevated [CO2 ] (~600 ppm) had a higher biomass with some cultivars having lower storage root water content. The elevated [CO2 ] stimulation in storage root biomass ranged from 33% to 86% across the five cultivars tested documenting the importance of this trait in developing new cultivars. In addition to the destructive harvests to obtain storage root parameters, we explored ground penetrating radar as a nondestructive method to determine storage root growth across the growing season.

Alternative pathway to photorespiration protects growth and productivity at elevated temperatures in a model crop.

Adapting crops to warmer growing season temperatures is a major challenge in mitigating the impacts of climate change on crop production. Warming temperatures drive greater evaporative demand and can directly interfere with both reproductive and vegetative physiological processes. Most of the world's crop species have C3 photosynthetic metabolism for which increasing temperature means higher rates of photorespiration, wherein the enzyme responsible for fixing CO2 fixes O2 instead followed by an energetically costly recycling pathway that spans several cell compartments. In C3 crops like wheat, rice and soybean, photorespiration translates into large yield losses that are predicted to increase as global temperature warms. Engineering less energy-intensive alternative photorespiratory pathways into crop chloroplasts drives increases in C3 biomass production under agricultural field conditions, but the efficacy of these pathways in mitigating the impact of warmer growing temperatures has not been tested. We grew tobacco plants expressing an alternative photorespiratory pathway under current and elevated temperatures (+5 °C) in agricultural field conditions. Engineered plants exhibited higher photosynthetic quantum efficiency under heated conditions than the control plants, and produced 26% (between 16% and 37%) more total biomass than WT plants under heated conditions, compared to 11% (between 5% and 17%) under ambient conditions. That is, engineered plants sustained 19% (between 11% and 21%) less yield loss under heated conditions compared to non-engineered plants. These results support the theoretical predictions of temperature impacts on photorespiratory losses and provide insight toward the optimisation strategies required to help sustain or improve C3 crop yields in a warming climate.

Increased bundle-sheath leakiness of CO2 during photosynthetic induction shows a lack of coordination between the C4 and C3 cycles.

Use of a complete dynamic model of NADP-malic enzyme C4 photosynthesis indicated that, during transitions from dark or shade to high light, induction of the C4 pathway was more rapid than that of C3 , resulting in a predicted transient increase in bundle-sheath CO2 leakiness (ϕ). Previously, ϕ has been measured at steady state; here we developed a new method, coupling a tunable diode laser absorption spectroscope with a gas-exchange system to track ϕ in sorghum and maize through the nonsteady-state condition of photosynthetic induction. In both species, ϕ showed a transient increase to > 0.35 before declining to a steady state of 0.2 by 1500 s after illumination. Average ϕ was 60% higher than at steady state over the first 600 s of induction and 30% higher over the first 1500 s. The transient increase in ϕ, which was consistent with model prediction, indicated that capacity to assimilate CO2 into the C3 cycle in the bundle sheath failed to keep pace with the rate of dicarboxylate delivery by the C4 cycle. Because nonsteady-state light conditions are the norm in field canopies, the results suggest that ϕ in these major crops in the field is significantly higher and energy conversion efficiency lower than previous measured values under steady-state conditions.

Perspectives on improving light distribution and light use efficiency in crop canopies.

Plant stands in nature differ markedly from most seen in modern agriculture. In a dense mixed stand, plants must vie for resources, including light, for greater survival and fitness. Competitive advantages over surrounding plants improve fitness of the individual, thus maintaining the competitive traits in the gene pool. In contrast, monoculture crop production strives to increase output at the stand level and thus benefits from cooperation to increase yield of the community. In choosing plants with higher yields to propagate and grow for food, humans may have inadvertently selected the best competitors rather than the best cooperators. Here, we discuss how this selection for competitiveness has led to overinvestment in characteristics that increase light interception and, consequently, sub-optimal light use efficiency in crop fields that constrains yield improvement. Decades of crop canopy modeling research have provided potential strategies for improving light distribution in crop canopies, and we review the current progress of these strategies, including balancing light distribution through reducing pigment concentration. Based on recent research revealing red-shifted photosynthetic pigments in algae and photosynthetic bacteria, we also discuss potential strategies for optimizing light interception and use through introducing alternative pigment types in crops. These strategies for improving light distribution and expanding the wavelengths of light beyond those traditionally defined for photosynthesis in plant canopies may have large implications for improving crop yield and closing the yield gap.

Removal of redox-sensitive Rubisco Activase does not alter Rubisco regulation in soybean.

Rubisco activase (Rca) facilitates the catalytic repair of Rubisco, the CO2-fixing enzyme of photosynthesis, following periods of darkness, low to high light transitions or stress. Removal of the redox-regulated isoform of Rubisco activase, Rca-α, enhances photosynthetic induction in Arabidopsis and has been suggested as a strategy for the improvement of crops, which may experience frequent light transitions in the field; however, this has never been tested in a crop species. Therefore, we used RNAi to reduce the Rca-α content of soybean (Glycine max cv. Williams 82) below detectable levels and then characterized the growth, photosynthesis, and Rubisco activity of the resulting transgenics, in both growth chamber and field conditions. Under a 16 h sine wave photoperiod, the reduction of Rca-α contents had no impact on morphological characteristics, leaf expansion rate, or total biomass. Photosynthetic induction rates were unaltered in both chamber-grown and field-grown plants. Plants with reduced Rca-α content maintained the ability to regulate Rubisco activity in low light just as in control plants. This result suggests that in soybean, Rca-α is not as centrally involved in the regulation of Rca oligomer activity as it is in Arabidopsis. The isoform stoichiometry supports this conclusion, as Rca-α comprises only ~ 10% of the Rubisco activase content of soybean, compared to ~ 50% in Arabidopsis. This is likely to hold true in other species that contain a low ratio of Rca-α to Rca-ß isoforms.

In vivo evidence for a regulatory role of phosphorylation of Arabidopsis Rubisco activase at the Thr78 site.

Arabidopsis Rubisco activase (Rca) is phosphorylated at threonine-78 (Thr78) in low light and in the dark, suggesting a potential regulatory role in photosynthesis, but this has not been directly tested. To do so, we transformed an rca-knockdown mutant largely lacking redox regulation with wild-type Rca-ß or Rca-ß with Thr78-to-Ala (T78A) or Thr78-to-Ser (T78S) site-directed mutations. Interestingly, the T78S mutant was hyperphosphorylated at the Ser78 site relative to Thr78 of the Rca-ß wild-type control, as evidenced by immunoblotting with custom antibodies and quantitative mass spectrometry. Moreover, plants expressing the T78S mutation had reduced photosynthesis and quantum efficiency of photosystem II (ϕPSII) and reduced growth relative to control plants expressing wild-type Rca-ß under all conditions tested. Gene expression was also altered in a manner consistent with reduced growth. In contrast, plants expressing Rca-ß with the phospho-null T78A mutation had faster photosynthetic induction kinetics and increased ϕPSII relative to Rca-ß controls. While expression of the wild-type Rca-ß or the T78A mutant fully rescued the slow-growth phenotype of the rca-knockdown mutant grown in a square-wave light regime, the T78A mutants grew faster than the Rca-ß control plants at low light (30 µmol photons m-2 s-1) and in a fluctuating low-light/high-light environment. Collectively, these results suggest that phosphorylation of Thr78 (or Ser78 in the T78S mutant) plays a negative regulatory role in vivo and provides an explanation for the absence of Ser at position 78 in terrestrial plant species.

Arabidopsis plants expressing only the redox-regulated Rca-α isoform have constrained photosynthesis and plant growth.

Rubisco activase (Rca) facilitates the release of sugar-phosphate inhibitors from the active sites of Rubisco and thereby plays a central role in initiating and sustaining Rubisco activation. In Arabidopsis, alternative splicing of a single Rca gene results in two Rca isoforms, Rca-α and Rca-ß. Redox modulation of Rca-α regulates the function of Rca-α and Rca-ß acting together to control Rubisco activation. Although Arabidopsis Rca-α alone less effectively activates Rubisco in vitro, it is not known how CO2 assimilation and plant growth are impacted. Here, we show that two independent transgenic Arabidopsis lines expressing Rca-α in the absence of Rca-ß ('Rca-α only' lines) grew more slowly in various light conditions, especially under low light or fluctuating light intensity, and in a short day photoperiod compared to wildtype. Photosynthetic induction was slower in the Rca-α only lines, and they maintained a lower rate of CO2 assimilation during both photoperiod types. Our findings suggest Rca oligomers composed of Rca-α only are less effective in initiating and sustaining the activation of Rubisco than when Rca-ß is also present. Currently there are no examples of any plant species that naturally express Rca-α only but numerous examples of species expressing Rca-ß only. That Rca-α exists in most plant species, including many C3 and C4 food and bioenergy crops, implies its presence is adaptive under some circumstances.

High sink strength prevents photosynthetic down-regulation in cassava grown at elevated CO2 concentration.

Cassava has the potential to alleviate food insecurity in many tropical regions, yet few breeding efforts to increase yield have been made. Improved photosynthetic efficiency in cassava has the potential to increase yields, but cassava roots must have sufficient sink strength to prevent carbohydrates from accumulating in leaf tissue and suppressing photosynthesis. Here, we grew eight farmer-preferred African cassava cultivars under free-air CO2 enrichment (FACE) to evaluate the sink strength of cassava roots when photosynthesis increases due to elevated CO2 concentrations ([CO2]). Relative to the ambient treatments, elevated [CO2] treatments increased fresh (+27%) and dry (+37%) root biomass, which was driven by an increase in photosynthesis (+31%) and the absence of photosynthetic down-regulation over the growing season. Moreover, intrinsic water use efficiency improved under elevated [CO2] conditions, while leaf protein content and leaf and root cyanide concentrations were not affected. Overall, these results suggest that higher cassava yields can be expected as atmospheric [CO2] increases over the coming decades. However, there were cultivar differences in the partitioning of resources to roots versus above-grown biomass; thus, the particular responses of each cultivar must be considered when selecting candidates for improvement.

Soybean photosynthetic and biomass responses to carbon dioxide concentrations ranging from pre-industrial to the distant future.

Increasing atmospheric carbon dioxide concentration ([CO2]) directly impacts C3 plant photosynthesis and productivity, and the rate at which [CO2] is increasing is greater than initially predicted by worst-case scenario climate models. Thus, it is increasingly important to assess the physiological responses of C3 plants, especially those that serve as important crops, to [CO2] beyond the mid-range levels used in traditional experiments. Here, we grew the C3 crop soybean (Glycine max) at eight different [CO2] levels spanning subambient (340 ppm) to the highest level thought plausible (~2000 ppm) in chambers for 5 weeks. Physiological development was delayed and plant height and total leaf area increased at [CO2] levels higher than ambient conditions, with very little difference in these parameters among the elevated [CO2] treatments >900 ppm. Daily photosynthesis initially increased with rising [CO2] but began to level off at ~1000 ppm CO2. Similar results occurred in biomass accumulation. Thus, as [CO2] continues to match or exceed the worst-case emission scenarios, these results indicate that carbon gain, growth, and potentially yield increases will diminish, thereby ultimately constraining the positive impact that continuing increases in atmospheric [CO2] could have on crop productivity and global terrestrial carbon sinks.

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.

Bile Acid Sodium Symporter BASS6 Can Transport Glycolate and Is Involved in Photorespiratory Metabolism in Arabidopsis thaliana.

Photorespiration is an energy-intensive process that recycles 2-phosphoglycolate, a toxic product of the Rubisco oxygenation reaction. The photorespiratory pathway is highly compartmentalized, involving the chloroplast, peroxisome, cytosol, and mitochondria. Though the soluble enzymes involved in photorespiration are well characterized, very few membrane transporters involved in photorespiration have been identified to date. In this work, Arabidopsis thaliana plants containing a T-DNA disruption of the bile acid sodium symporter BASS6 show decreased photosynthesis and slower growth under ambient, but not elevated CO2 Exogenous expression of BASS6 complemented this photorespiration mutant phenotype. In addition, metabolite analysis and genetic complementation of glycolate transport in yeast showed that BASS6 was capable of glycolate transport. This is consistent with its involvement in the photorespiratory export of glycolate from Arabidopsis chloroplasts. An Arabidopsis double knockout line of both BASS6 and the glycolate/glycerate transporter PLGG1 (bass6, plgg1) showed an additive growth defect, an increase in glycolate accumulation, and reductions in photosynthetic rates compared with either single mutant. Our data indicate that BASS6 and PLGG1 partner in glycolate export from the chloroplast, whereas PLGG1 alone accounts for the import of glycerate. BASS6 and PLGG1 therefore balance the export of two glycolate molecules with the import of one glycerate molecule during photorespiration.

The Impacts of Fluctuating Light on Crop Performance.

Rapidly changing light conditions can reduce carbon gain and productivity in field crops because photosynthetic responses to light fluctuations are not instantaneous. Plant responses to fluctuating light occur across levels of organizational complexity from entire canopies to the biochemistry of a single reaction and across orders of magnitude of time. Although light availability and variation at the top of the canopy are largely dependent on the solar angle and degree of cloudiness, lower crop canopies rely more heavily on light in the form of sunflecks, the quantity of which depends mostly on canopy structure but also may be affected by wind. The ability of leaf photosynthesis to respond rapidly to these variations in light intensity is restricted by the relatively slow opening/closing of stomata, activation/deactivation of C3 cycle enzymes, and up-regulation/down-regulation of photoprotective processes. The metabolic complexity of C4 photosynthesis creates the apparently contradictory possibilities that C4 photosynthesis may be both more and less resilient than C3 to dynamic light regimes, depending on the frequency at which these light fluctuations occur. We review the current understanding of the underlying mechanisms of these limitations to photosynthesis in fluctuating light that have shown promise in improving the response times of photosynthesis-related processes to changes in light intensity.

Chlorophyll Can Be Reduced in Crop Canopies with Little Penalty to Photosynthesis.

The hypothesis that reducing chlorophyll content (Chl) can increase canopy photosynthesis in soybeans was tested using an advanced model of canopy photosynthesis. The relationship among leaf Chl, leaf optical properties, and photosynthetic biochemical capacity was measured in 67 soybean (Glycine max) accessions showing large variation in leaf Chl. These relationships were integrated into a biophysical model of canopy-scale photosynthesis to simulate the intercanopy light environment and carbon assimilation capacity of canopies with wild type, a Chl-deficient mutant (Y11y11), and 67 other mutants spanning the extremes of Chl to quantify the impact of variation in leaf-level Chl on canopy-scale photosynthetic assimilation and identify possible opportunities for improving canopy photosynthesis through Chl reduction. These simulations demonstrate that canopy photosynthesis should not increase with Chl reduction due to increases in leaf reflectance and nonoptimal distribution of canopy nitrogen. However, similar rates of canopy photosynthesis can be maintained with a 9% savings in leaf nitrogen resulting from decreased Chl. Additionally, analysis of these simulations indicate that the inability of Chl reductions to increase photosynthesis arises primarily from the connection between Chl and leaf reflectance and secondarily from the mismatch between the vertical distribution of leaf nitrogen and the light absorption profile. These simulations suggest that future work should explore the possibility of using reduced Chl to improve canopy performance by adapting the distribution of the "saved" nitrogen within the canopy to take greater advantage of the more deeply penetrating light.

Economical synthesis of 14C-labeled aminolevulinic acid for specific in situ labeling of plant tetrapyrroles.

The application of metabolic radiolabeling techniques to plant tetrapyrroles, i.e., chlorophyll and hemes, is complicated by the difficulty of obtaining sufficient quantities of radiolabeled aminolevulinic acid (ALA). ALA, the first committed intermediate in the tetrapyrrole biosynthetic pathway, is inconvenient to synthesize chemically and is generally not produced in significant quantities in biological systems. Radiolabeled ALA is therefore usually quite expensive and available only in limited quantities. Here, we describe bulk biosynthesis and purification of 14C-labeled ALA from 14C glycine. We first cloned ALA synthase (ALAS) from Rhodobacter sphaeroides into an expression vector for expression and purification as a fusion with maltose-binding protein. We then used the purified ALAS to synthesize ALA in vitro from 14C-labeled glycine and succinyl-coenzyme A. Finally, we used ion exchange chromatography to separate the ALA product from the crude reaction. We achieved conversion and recovery efficiencies of 80-90%, and chlorophyll radiolabeling experiments with the 14C ALA product revealed no detectable non-specific incorporation into proteins. The ability to economically produce robust quantities of 14C ALA using common methodologies provides a new tool for working with tetrapyrroles, which includes both hemes and chlorophylls and their respective binding proteins. This tool allows the specific detection and quantification of the tetrapyrrole of interest from standard acrylamide gels or hybridization transfer membranes via radiographic imaging, which enables a wide array of experiments involving spatial and temporal resolution of the movement of pigments as they are synthesized, incorporated into their target binding proteins, and eventually degraded.

Carbon assimilation in crops at high temperatures.

Global temperatures are rising, and higher rates of temperature increase are projected over land areas that encompass the globe's major agricultural regions. In addition to increased growing season temperatures, heat waves are predicted to become more common and severe. High temperatures can inhibit photosynthetic carbon gain of crop plants and thus threaten productivity, the effects of which may interact with other aspects of climate change. Here, we review the current literature assessing temperature effects on photosynthesis in key crops with special attention to field studies using crop canopy heating technology and in combination with other climate variables. We also discuss the biochemical reactions related to carbon fixation that may limit crop photosynthesis under warming temperatures and the current strategies for adaptation. Important progress has been made on several adaptation strategies demonstrating proof-of-concept for translating improved photosynthesis into higher yields. These are now poised to test in important food crops.