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The global challenge of feeding an ever-increasing population to maintain food security requires novel approaches to increase crop yields. Photosynthesis, the fundamental energy and material basis for plant life on Earth, is highly responsive to environmental conditions. Evaluating the operational status of the photosynthetic mechanism provides insights into plants' capacity to adapt to their surroundings. Despite immense effort, photosynthesis still falls short of its theoretical maximum efficiency, indicating significant potential for improvement. In this review, we provide background information on the various genetic aspects of photosynthesis, explain its complexity, and survey relevant genetic engineering approaches employed to improve the efficiency of photosynthesis. We discuss the latest success stories of gene-editing tools like CRISPR-Cas9 and synthetic biology in achieving precise refinements in targeted photosynthesis pathways, such as the Calvin-Benson cycle, electron transport chain, and photorespiration. We also discuss the genetic markers crucial for mitigating the impact of rapidly changing environmental conditions, such as extreme temperatures or drought, on photosynthesis and growth. This review aims to pinpoint optimization opportunities for photosynthesis, discuss recent advancements, and address the challenges in improving this critical process, fostering a globally food-secure future through sustainable food crop production. [ABSTRACT FROM AUTHOR]

Summary: CO2 release in the light (RL) and its presumed source, oxidative pentose phosphate pathways, were found to be insensitive to CO2 concentration.The oxidative pentose phosphate pathways form glucose 6‐phosphate (G6P) shunts that bypass the nonoxidative pentose phosphate reactions of the Calvin–Benson cycle. Using adenosine diphosphate glucose and uridine diphosphate glucose as proxies for labeling of G6P in the stroma and cytosol respectively, it was found that only the cytosolic shunt was active.Uridine diphosphate glucose, a proxy for cytosolic G6P, and 6‐phosphogluconate (6PG) were significantly less labeled than Calvin–Benson cycle intermediates in the light. But ADP glucose, a proxy for stromal G6P, is labeled to the same degree as Calvin–Benson cycle intermediates and much greater than 6PG. A metabolically inert pool of sedoheptulose bisphosphate can slowly equilibrate keeping the label in sedoheptulose lower than in other stromal metabolites. Finally, phosphorylation of fructose 6‐phosphate (F6P) in the cytosol can allow some unlabeled carbon in cytosolic F6P to dilute label in phosphenolpyruvate.The results clearly show that there is oxidative pentose phosphate pathway activity in the cytosol that provides a shunt around the nonoxidative pentose phosphate pathway reactions of the Calvin–Benson cycle and is not strongly CO2‐sensitive. [ABSTRACT FROM AUTHOR]

The Calvin-Benson cycle (CBC) evolved over 2 billion years ago but has been subject to massive selection due to falling atmospheric carbon dioxide, rising atmospheric oxygen and changing nutrient and water availability. In addition, large groups of organisms have evolved carbon-concentrating mechanisms (CCMs) that operate upstream of the CBC. Most previous studies of CBC diversity focused on Rubisco kinetics and regulation. Quantitative metabolite profiling provides a top-down strategy to uncover inter-species diversity in CBC operation. CBC profiles were recently published for twenty species including terrestrial C 3 species, terrestrial C 4 species that operate a biochemical CCM, and cyanobacteria and green algae that operate different types of biophysical CCM. Distinctive profiles were found for species with different modes of photosynthesis, revealing that evolution of the various CCMs was accompanied by co-evolution of the CBC. Diversity was also found between species that share the same mode of photosynthesis, reflecting lineage-dependent diversity of the CBC. Connectivity analysis uncovers constraints due to pathway and thermodynamic topology, and reveals that cross-species diversity in the CBC is driven by changes in the balance between regulated enzymes and in the balance between the CBC and the light reactions or end-product synthesis. • Calvin-Benson cycle metabolites were profiled in 20 species. • Calvin-Benson cycle has co-evolved with CO 2 -concentrating mechanisms. • Substantial diversity between species with the same photosynthesis mode. [ABSTRACT FROM AUTHOR]

The global challenge of feeding an ever-increasing population to maintain food security requires novel approaches to increase crop yields. Photosynthesis, the fundamental energy and material basis for plant life on Earth, is highly responsive to environmental conditions. Evaluating the operational status of the photosynthetic mechanism provides insights into plants’ capacity to adapt to their surroundings. Despite immense effort, photosynthesis still falls short of its theoretical maximum efficiency, indicating significant potential for improvement. In this review, we provide background information on the various genetic aspects of photosynthesis, explain its complexity, and survey relevant genetic engineering approaches employed to improve the efficiency of photosynthesis. We discuss the latest success stories of gene-editing tools like CRISPR-Cas9 and synthetic biology in achieving precise refinements in targeted photosynthesis pathways, such as the Calvin-Benson cycle, electron transport chain, and photorespiration. We also discuss the genetic markers crucial for mitigating the impact of rapidly changing environmental conditions, such as extreme temperatures or drought, on photosynthesis and growth. This review aims to pinpoint optimization opportunities for photosynthesis, discuss recent advancements, and address the challenges in improving this critical process, fostering a globally food-secure future through sustainable food crop production.

ABSTRACT One of the current promising solutions to address problems of CO2 emissions is the development of biological carbon fixation strategies. This strategy leads to higher chemical biosynthetic productivity through improved biological CO2 fixation. In a previous study, we introduced the Calvin-Benson Bassham (CBB) genes of the photosynthetic bacterium Cereibacter sphaeroides into Escherichia coli to develop a strain capable of endogenous CO2 recycling by heterologous expression of the CBB genes. However, the heterologous expression of recombinant proteins in E. coli is often hampered by inclusion bodies (IB), which are protein aggregates. Various factors contribute to IB formation, including host cell metabolism, protein synthesis, transformation machinery, and target protein properties. In this study, we investigated the influence of environmental conditions, particularly culture temperature, on IB formation and CO2 recycling in the CBB strain. As a result, by reducing the culture temperature from 37°C to 30°C, a significant suppression of IB formation was achieved, resulting in a remarkable decrease in CO2 release by approximately 5.76 times. In addition, interestingly, an enhancement in the accumulation of pyruvate by approximately 2.3 times was observed at the same time. These results demonstrate the simultaneous improvements in CO2 recycling and the synthesis of organic acids achieved through temperature control. Based on the findings of this study, we believe that temperature control would be a promising approach to increasing chemical biosynthetic productivity as well as biological CO2 fixation activity in integrated autotrophic biorefinery strategies. IMPORTANCE In a previous study, we successfully engineered Escherichia coli capable of endogenous CO2 recycling through the heterologous expression of the Calvin-Benson Bassham genes. Establishing an efficient gene expression environment for recombinant strains is crucial, on par with the importance of metabolic engineering design. Therefore, the primary objective of this study was to further mitigate greenhouse gas emissions by investigating the effects of culture temperature on the formation of inclusion bodies (IB) and CO2 fixation activity in the engineered bacterial strain. The findings demonstrate that lowering the culture temperature effectively suppresses IB formation, enhances CO2 recycling, and concurrently increases the accumulation of organic acids. This temperature control approach, without adding or modifying compounds, is both convenient and efficient for enhancing CO2 recycling. As such, additional optimization of various environmental parameters holds promise for further enhancing the performance of recombinant strains efficiently.

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. [ABSTRACT FROM AUTHOR]

Summary: Recently, we reported estimates of anaplerotic carbon flux through the oxidative pentose phosphate pathway (OPPP) in chloroplasts into the Calvin–Benson cycle. These estimates were based on intramolecular hydrogen isotope analysis of sunflower leaf starch. However, the isotope method is believed to underestimate the actual flux at low atmospheric CO2 concentration (Ca).Since the OPPP releases CO2 and reduces NADP+, it can be expected to affect leaf gas exchange under both rubisco‐ and RuBP‐regeneration‐limited conditions. Therefore, we expanded Farquhar‐von Caemmerer–Berry models to account for OPPP metabolism. Based on model parameterisation with values from the literature, we estimated OPPP‐related effects on leaf carbon and energy metabolism in the sunflowers analysed previously.We found that flux through the plastidial OPPP increases both above and below Ca ≈ 450 ppm (the condition the plants were acclimated to). This is qualitatively consistent with our previous isotope‐based estimates, yet gas‐exchange‐based estimates are larger at low Ca.We discuss our results in relation to regulatory properties of the plastidial and cytosolic OPPP, the proposed variability of CO2 mesophyll conductance, and the contribution of day respiration to the A/Ci curve drop at high Ca. Furthermore, we critically examine the models and parameterisation and derive recommendations for follow‐up studies. [ABSTRACT FROM AUTHOR]

The concerted regulation of chloroplast biosynthetic pathways and NADPH extrusion via malate valve depends on f and m thioredoxins (Trxs). The finding that decreased levels of the thiol-peroxidase 2-Cys peroxiredoxin (Prx) suppress the severe phenotype of Arabidopsis mutants lacking NADPH-dependent Trx reductase C (NTRC) and Trxs f uncovered the central function of the NTRC-2-Cys-Prx redox system in chloroplast performance. These results suggest that Trxs m are also regulated by this system; however, the functional relationship between NTRC, 2-Cys Prxs, and m-type Trxs is unknown. To address this issue, we generated Arabidopsis thaliana mutants combining deficiencies in NTRC, 2-Cys Prx B, Trxs m1, and m4. The single trxm1 and trxm4 mutants showed a wild-type phenotype, growth retardation being noticed only in the trxm1m4 double mutant. Moreover, the ntrc-trxm1m4 mutant displayed a more severe phenotype than the ntrc mutant, as shown by the impaired photosynthetic performance, altered chloroplast structure, and defective light-dependent reduction in the Calvin–Benson cycle and malate-valve enzymes. These effects were suppressed by the decreased contents of 2-Cys Prx, since the quadruple ntrc-trxm1m4-2cpb mutant displayed a wild-type-like phenotype. These results show that the activity of m-type Trxs in the light-dependent regulation of biosynthetic enzymes and malate valve is controlled by the NTRC-2-Cys-Prx system. [ABSTRACT FROM AUTHOR]

Rubisco is possibly the most important enzyme on Earth, certainly in terms of amount. This review describes the initial reports of ribulose 1,5-bisphosphate carboxylating activity. Discoveries of core concepts are described, including its quaternary structure, the requirement for post-translational modification, and its role as an oxygenase as well as a carboxylase. Finally, the requirement for numerous chaperonins for assembly of rubisco in plants is described. [ABSTRACT FROM AUTHOR]

Chloroplast ribulose-5-phosphate-3-epimerase (RPE) is a critical enzyme involved in the Calvin-Benson cycle and oxidative pentose phosphate pathways in higher plants. Three Arabidopsis rpe mutants with reduced level of RPE were identified through their high NPQ (nonphotochemical quenching) phenotype upon illumination, and no significant difference of plant size was found between these rpe mutants and WT (wild type) plants under growth chamber conditions. A decrease in RPE expression to a certain extent leads to a decrease in CO2 fixation, Vcmax and Jmax. Photosynthetic linear electron transport was partially inhibited and activity of ATP synthase was also decreased in the rpe mutants, but the levels of thylakoid protein complexes and other Calvin-Benson cycle enzymes in rpe mutants were not affected. These results demonstrate that some degree of reduction in RPE expression decreases carbon fixation in chloroplasts, which in turn feedback inhibits photosynthetic electron transport and ATP synthase activity due to the photosynthetic control. Taken together, this work provides evidence that RPE plays an important role in the Calvin-Benson cycle and influences the photosynthetic capacity of chloroplasts. [ABSTRACT FROM AUTHOR]

Plant carbon metabolism and climate change: elevated CO 2 and temperature impacts on photosynthesis, photorespiration and respiration. Another signal at glucose H SP 2 sp reflects H isotope fractionation by chloroplast phosphoglucose isomerase (PGI) and associated shifts of the reaction catalysed by PGI from kinetic to equilibrium conditions. Keywords: Calvin-Benson cycle; carbon metabolism; CO2 fertilization; glucose-6-phosphate shunt; hydrogen stable isotopes; oxidative pentose phosphate pathway; photosynthesis; respiration EN Calvin-Benson cycle carbon metabolism CO2 fertilization glucose-6-phosphate shunt hydrogen stable isotopes oxidative pentose phosphate pathway photosynthesis respiration 1310 1314 5 07/18/22 20220815 NES 220815 Introduction Despite significant research efforts, the question of whether rising atmospheric CO SB 2 sb concentration ( I C i SB a sb ) affects leaf respiration remains unanswered (Gonzàlez-Meler I et al i ., 2004; Way I et al i ., 2015; Dusenge I et al i ., 2019). [Extracted from the article]

The concerted regulation of chloroplast biosynthetic pathways and NADPH extrusion via malate valve depends on f and m thioredoxins (Trxs). The finding that decreased levels of the thiol-peroxidase 2-Cys peroxiredoxin (Prx) suppress the severe phenotype of Arabidopsis mutants lacking NADPH-dependent Trx reductase C (NTRC) and Trxs f uncovered the central function of the NTRC-2-Cys-Prx redox system in chloroplast performance. These results suggest that Trxs m are also regulated by this system; however, the functional relationship between NTRC, 2-Cys Prxs, and m-type Trxs is unknown. To address this issue, we generated Arabidopsis thaliana mutants combining deficiencies in NTRC, 2-Cys Prx B, Trxs m1, and m4. The single trxm1 and trxm4 mutants showed a wild-type phenotype, growth retardation being noticed only in the trxm1m4 double mutant. Moreover, the ntrc-trxm1m4 mutant displayed a more severe phenotype than the ntrc mutant, as shown by the impaired photosynthetic performance, altered chloroplast structure, and defective light-dependent reduction in the Calvin–Benson cycle and malate-valve enzymes. These effects were suppressed by the decreased contents of 2-Cys Prx, since the quadruple ntrc-trxm1m4-2cpb mutant displayed a wild-type-like phenotype. These results show that the activity of m-type Trxs in the light-dependent regulation of biosynthetic enzymes and malate valve is controlled by the NTRC-2-Cys-Prx system.

Summary: Stable isotope abundances convey valuable information about plant physiological processes and underlying environmental controls. Central gaps in our mechanistic understanding of hydrogen isotope abundances impede their widespread application within the plant and biogeosciences.To address these gaps, we analysed intramolecular deuterium abundances in glucose of Pinus nigra extracted from an annually resolved tree‐ring series (1961–1995).We found fractionation signals (i.e. temporal variability in deuterium abundance) at glucose H1 and H2 introduced by closely related metabolic processes. Regression analysis indicates that these signals (and thus metabolism) respond to drought and atmospheric CO2 concentration beyond a response change point. They explain ≈ 60% of the whole‐molecule deuterium variability. Altered metabolism is associated with below‐average yet not exceptionally low growth.We propose the signals are introduced at the leaf level by changes in sucrose‐to‐starch carbon partitioning and anaplerotic carbon flux into the Calvin–Benson cycle. In conclusion, metabolism can be the main driver of hydrogen isotope variation in plant glucose. [ABSTRACT FROM AUTHOR]

Summary: As the central carbon uptake pathway in photosynthetic cells, the Calvin–Benson cycle is among the most important biochemical cycles for life on Earth. A carbon flux of anaplerotic origin (i.e. through the chloroplast‐localized oxidative branch of the pentose phosphate pathway) into the Calvin–Benson cycle was proposed recently.Here, we measured intramolecular deuterium abundances in leaf starch of Helianthus annuus grown at varying ambient CO2 concentrations, Ca. Additionally, we modelled deuterium fractionations expected for the anaplerotic pathway and compared modelled with measured fractionations.We report deuterium fractionation signals at H1 and H2 of starch glucose. Below a Ca change point, these signals increase with decreasing Ca consistent with modelled fractionations by anaplerotic flux. Under standard conditions (Ca = 450 ppm corresponding to intercellular CO2 concentrations, Ci, of 328 ppm), we estimate negligible anaplerotic flux. At Ca = 180 ppm (Ci = 140 ppm), more than 10% of the glucose‐6‐phosphate entering the starch biosynthesis pathway is diverted into the anaplerotic pathway.In conclusion, we report evidence consistent with anaplerotic carbon flux into the Calvin–Benson cycle in vivo. We propose the flux may help to: maintain high levels of ribulose 1,5‐bisphosphate under source‐limited growth conditions to facilitate photorespiratory nitrogen assimilation required to build‐up source strength; and counteract oxidative stress. [ABSTRACT FROM AUTHOR]

When isotopes of carbon are fed to photosynthesizing leaves, metabolites of the Calvin-Benson cycle (CBC) are rapidly labeled initially, but then the rate of labeling slows considerably, raising questions about the integration of the CBC within leaf metabolism. We have used 2-h time courses of labeling of Camelina sativa leaf metabolites to test models of 12Cwashout when the CO2 source is rapidly switched to 13CO2. Fitting exponential functions to the time course of CBC metabolites, we found evidence for three temporally distinct processes contributing to the labeling but none for metabolically inactive pools. We next modeled the data of all metabolites by 13C isotopically nonstationary metabolic flux analysis, testing a variety of flux networks. In the model that best explains measured data, three processes determine CBC metabolite labeling. First is fixation of incoming 13CO2; second is dilution by weakly labeled carbon in cytosolic glucose reentering the CBC following oxidative pentose phosphate pathway reactions, which forms a shunt bypassing much of the CBC. Third, very weakly labeled carbon from the vacuole further dilutes the labeling. This model predicts the shunt proceeds at about 5% of the rate of net CO2 fixation and explains the three phases of labeling. In showing the interconnection of three compartments, we have drawn a more complete picture of how carbon moves through photosynthetic metabolism in a way that integrates the CBC, cytosolic sugar pools, glucose-6-phosphate shunt, and vacuolar sugars into a single system. [ABSTRACT FROM AUTHOR]

C4 photosynthesis concentrates CO2 around Rubisco in the bundle sheath, favouring carboxylation over oxygenation and decreasing photorespiration. This complex trait evolved independently in >60 angiosperm lineages. Its evolution can be investigated in genera such as Flaveria (Asteraceae) that contain species representing intermediate stages between C3 and C4 photosynthesis. Previous studies have indicated that the first major change in metabolism probably involved relocation of glycine decarboxylase and photorespiratory CO2 release to the bundle sheath and establishment of intercellular shuttles to maintain nitrogen stoichiometry. This was followed by selection for a CO2-concentrating cycle between phospho enol pyruvate carboxylase in the mesophyll and decarboxylases in the bundle sheath, and relocation of Rubisco to the latter. We have profiled 52 metabolites in nine Flaveria species and analysed 13CO2 labelling patterns for four species. Our results point to operation of multiple shuttles, including movement of aspartate in C3–C4 intermediates and a switch towards a malate/pyruvate shuttle in C4-like species. The malate/pyruvate shuttle increases from C4-like to complete C4 species, accompanied by a rise in ancillary organic acid pools. Our findings support current models and uncover further modifications of metabolism along the evolutionary path to C4 photosynthesis in the genus Flaveria. [ABSTRACT FROM AUTHOR]

The Calvin-Benson cycle of carbon dioxide fixation in chloroplasts is controlled by light-dependent redox reactions that target specific enzymes. Of the regulatory members of the cycle, our knowledge of sedoheptulose-1,7-bisphosphatase (SBPase) is particularly scanty, despite growing evidence for its importance and link to plant productivity. To help fill this gap, we have purified, crystallized, and characterized the recombinant form of the enzyme together with the better studied fructose-1,6-bisphosphatase (FBPase), in both cases from the moss Physcomitrella patens (Pp). Overall, the moss enzymes resembled their counterparts from seed plants, including oligomeric organization-PpSBPase is a dimer, and PpFBPase is a tetramer. The two phosphatases showed striking structural homology to each other, differing primarily in their solvent-exposed surface areas in a manner accounting for their specificity for seven-carbon (sedoheptulose) and six-carbon (fructose) sugar bisphosphate substrates. The two enzymes had a similar redox potential for their regulatory redox-active disulfides (-310 mV for PpSBPase vs. -290 mV for PpFBPase), requirement for Mg(2+) and thioredoxin (TRX) specificity (TRX f > TRX m). Previously known to differ in the position and sequence of their regulatory cysteines, the enzymes unexpectedly showed unique evolutionary histories. The FBPase gene originated in bacteria in conjunction with the endosymbiotic event giving rise to mitochondria, whereas SBPase arose from an archaeal gene resident in the eukaryotic host. These findings raise the question of how enzymes with such different evolutionary origins achieved structural similarity and adapted to control by the same light-dependent photosynthetic mechanism-namely ferredoxin, ferredoxin-thioredoxin reductase, and thioredoxin.

Improving photosynthesis is a promising avenue to increase crop yield. This will be aided by better understanding of natural variance in photosynthesis. Profiling of Calvin–Benson cycle (CBC) metabolites provides a top-down strategy to uncover interspecies diversity in CBC operation. In a study of four C4 and five C3 species, principal components analysis separated C4 species from C3 species and also separated different C4 species. These separations were driven by metabolites that reflect known species differences in their biochemistry and pathways. Unexpectedly, there was also considerable diversity between the C3 species. Falling atmospheric CO2 and changing temperature, nitrogen, and water availability have driven evolution of C4 photosynthesis in multiple lineages. We propose that analogous selective pressures drove lineage-dependent evolution of the CBC in C3 species. Examples of species-dependent variation include differences in the balance between the CBC and the light reactions, and in the balance between regulated steps in the CBC. Metabolite profiles also reveal conserved features including inactivation of enzymes in low irradiance, and maintenance of CBC metabolites at relatively high levels in the absence of net CO2 fixation. These features may be important for photosynthetic efficiency in low light, fluctuating irradiance, and when stomata close due to low water availability. [ABSTRACT FROM AUTHOR]

Fructose-1,6-biphosphate aldolase (FBA) is a multifunctional enzyme in plants, which participates in the process of Calvin-Benson cycle, glycolysis and gluconeogenesis. Despite the importance of FBA genes in regulating plant growth, development and abiotic stress responses, little is known about their roles in cotton. In the present study, we performed a genome-wide identification and characterization of FBAs in Gossypium hirsutum. Totally seventeen GhFBA genes were identified. According to the analysis of functional domain, phylogenetic relationship, and gene structure, GhFBA genes were classified into two subgroups. Furthermore, nine GhFBAs were predicted to be in chloroplast and eight were located in cytoplasm. Moreover, the promoter prediction showed a variety of abiotic stresses and phytohormone related cis-acting elements exist in the 2k up-stream region of GhFBA. And the evolutionary characteristics of cotton FBA genes were clearly presented by synteny analysis. Moreover, the results of transcriptome and qRT-PCR analysis showed that the expression of GhFBAs were related to the tissue distribution, and further analysis suggested that GhFBAs could respond to various abiotic stress and phytohormonal treatments. Overall, our systematic analysis of GhFBA genes would not only provide a basis for the understanding of the evolution of GhFBAs, but also found a foundation for the further function analysis of GhFBAs to improve cotton yield and environmental adaptability.

Fructose-1,6-biphosphate aldolase (FBA) is a multifunctional enzyme in plants, which participates in the process of Calvin-Benson cycle, glycolysis and gluconeogenesis. Despite the importance of FBA genes in regulating plant growth, development and abiotic stress responses, little is known about their roles in cotton. In the present study, we performed a genome-wide identification and characterization of FBAs in Gossypium hirsutum. Totally seventeen GhFBA genes were identified. According to the analysis of functional domain, phylogenetic relationship, and gene structure, GhFBA genes were classified into two subgroups. Furthermore, nine GhFBAs were predicted to be in chloroplast and eight were located in cytoplasm. Moreover, the promoter prediction showed a variety of abiotic stresses and phytohormone related cis -acting elements exist in the 2k up-stream region of GhFBA. And the evolutionary characteristics of cotton FBA genes were clearly presented by synteny analysis. Moreover, the results of transcriptome and qRT-PCR analysis showed that the expression of GhFBAs were related to the tissue distribution, and further analysis suggested that GhFBAs could respond to various abiotic stress and phytohormonal treatments. Overall, our systematic analysis of GhFBA genes would not only provide a basis for the understanding of the evolution of GhFBAs , but also found a foundation for the further function analysis of GhFBAs to improve cotton yield and environmental adaptability. [ABSTRACT FROM AUTHOR]

Summary: Thioredoxins (TRXs) are ubiquitous disulfide oxidoreductases structured according to a highly conserved fold. TRXs are involved in a myriad of different processes through a common chemical mechanism. Plant TRXs evolved into seven types with diverse subcellular localization and distinct protein target selectivity. Five TRX types coexist in the chloroplast, with yet scarcely described specificities. We solved the crystal structure of a chloroplastic z‐type TRX, revealing a conserved TRX fold with an original electrostatic surface potential surrounding the redox site. This recognition surface is distinct from all other known TRX types from plant and non‐plant sources and is exclusively conserved in plant z‐type TRXs. We show that this electronegative surface endows thioredoxin z (TRXz) with a capacity to activate the photosynthetic Calvin–Benson cycle enzyme phosphoribulokinase. The distinct electronegative surface of TRXz thereby extends the repertoire of TRX–target recognitions. Significance Statement: The high‐resolution crystal structure of thioredoxin z (TRXz) from the model alga Chlamydomonas reinhardtii confirms that TRXz is generally structured as a canonical redox TRX but exposes a type‐specific electronegative surface around its active site. The ability of TRXz to interact with its target proteins is determined by this surface and reveals a compatibility with Calvin–Benson phosphoribulokinase that proved to be activated by TRXz in vitro. TRXz hence appears as a novel player in photosynthesis. [ABSTRACT FROM AUTHOR]

Plastid metabolism is critical in both photoautotrophic and heterotrophic plant cells. In chloroplasts, fructose-1,6-bisphosphate aldolase (FBA) catalyses the formation of both fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate within the Calvin–Benson cycle. Three Arabidopsis genes, AtFBA1 – AtFBA3 , encode plastidial isoforms of FBA, but the contribution of each isoform is unknown. Phylogenetic analysis indicates that FBA1 and FBA2 derive from a recently duplicated gene, while FBA3 is a more ancient paralog. fba1 mutants are phenotypically indistinguishable from the wild type, while both fba2 and fba3 have reduced growth. We show that FBA2 is the major isoform in leaves, contributing most of the measurable activity. Partial redundancy with FBA1 allows both single mutants to survive, but combining both mutations is lethal, indicating a block of photoautotrophy. In contrast, FBA3 is expressed predominantly in heterotrophic tissues, especially the leaf and root vasculature, but not in the leaf mesophyll. We show that the loss of FBA3 affects plastidial glycolytic metabolism of the root, potentially limiting the biosynthesis of essential compounds such as amino acids. However, grafting experiments suggest that fba3 is dysfunctional in leaf phloem transport, and we suggest that a block in photoassimilate export from leaves causes the buildup of high carbohydrate concentrations and retarded growth. [ABSTRACT FROM AUTHOR]

Phosphoglucoisomerase (PGI) isomerizes fructose 6-phosphate (F6P) and glucose 6-phosphate (G6P) in starch and sucrose biosynthesis. Both plastidic and cytosolic isoforms are found in plant leaves. Using recombinant enzymes and isolated chloroplasts, we have characterized the plastidic and cytosolic isoforms of PGI. We have found that the Arabidopsis plastidic PGI Km for G6P is three-fold greater compared to that for F6P and that erythrose 4-phosphate is a key regulator of PGI activity. Additionally, the Km of spinach plastidic PGI can be dynamically regulated in the dark compared to the light and increases by 200% in the dark. We also found that targeting Arabidopsis cytosolic PGI into plastids of Nicotiana tabacum disrupts starch accumulation and degradation. Our results, in combination with the observation that plastidic PGI is not in equilibrium, indicates that PGI is an important regulatory enzyme that restricts flow and acts as a one-way valve preventing backflow of G6P into the Calvin-Benson cycle. We propose the PGI may be manipulated to improve flow of carbon to desired targets of biotechnology.

Summary: Experimental research into guard cell metabolism has revealed the roles of the accumulation of various metabolites in guard cell function, but a comprehensive understanding of their metabolism over the diel cycle is still incomplete due to the limitations of current experimental methods. In this study we constructed a four‐phase flux balance model of guard cell metabolism to investigate the changes in guard cell metabolism over the diel cycle, including the day and night and stomatal opening and closing. Our model predicted metabolic flexibility in guard cells of C3 plants, showing that multiple metabolic processes can contribute to the synthesis and metabolism of malate and sucrose as osmolytes during stomatal opening and closing. Our model showed the possibility of guard cells adapting to varying light availability and sucrose uptake from the apoplast during the day by operating in a mixotrophic mode with a switch between sucrose synthesis via the Calvin–Benson cycle and sucrose degradation via the oxidative pentose phosphate pathway. During stomatal opening, our model predicted an alternative flux mode of the Calvin–Benson cycle with all dephosphorylating steps diverted to diphosphate–fructose‐6‐phosphate 1‐phosphotransferase to produce inorganic pyrophosphate, which is used to pump protons across the tonoplast for the accumulation of osmolytes. An analysis of the energetics of the use of different osmolytes in guard cells showed that malate and Cl− are similarly efficient as the counterion of K+ during stomatal opening. Significance Statement: This work presents a four‐phase metabolic model for predicting guard cell metabolism over the diel cycle. The model predicted an alternative flux mode of the Calvin–Benson cycle that maximises the production of inorganic pyrophosphate during stomatal opening. While multiple metabolic processes were shown to be important in synthesising and metabolising osmolytes in guard cells of different experimental systems, our model demonstrated that these processes can operate simultaneously and at different rates depending on the conditions. [ABSTRACT FROM AUTHOR]

Phosphoglucoisomerase (PGI) isomerizes fructose 6-phosphate (F6P) and glucose 6-phosphate (G6P) in starch and sucrose biosynthesis. Both plastidic and cytosolic isoforms are found in plant leaves. Using recombinant enzymes and isolated chloroplasts, we have characterized the plastidic and cytosolic isoforms of PGI. We have found that the Arabidopsis plastidic PGI K m for G6P is three-fold greater compared to that for F6P and that erythrose 4-phosphate is a key regulator of PGI activity. Additionally, the K m of spinach plastidic PGI can be dynamically regulated in the dark compared to the light and increases by 200% in the dark. We also found that targeting Arabidopsis cytosolic PGI into plastids of Nicotiana tabacum disrupts starch accumulation and degradation. Our results, in combination with the observation that plastidic PGI is not in equilibrium, indicates that PGI is an important regulatory enzyme that restricts flow and acts as a one-way valve preventing backflow of G6P into the Calvin-Benson cycle. We propose the PGI may be manipulated to improve flow of carbon to desired targets of biotechnology. [ABSTRACT FROM AUTHOR]

ABSTRACT Most secondary nonphotosynthetic eukaryotes have retained residual plastids whose physiological role is often still unknown. One such example is Euglena longa, a close nonphotosynthetic relative of Euglena gracilis harboring a plastid organelle of enigmatic function. By mining transcriptome data from E. longa, we finally provide an overview of metabolic processes localized to its elusive plastid. The organelle plays no role in the biosynthesis of isoprenoid precursors and fatty acids and has a very limited repertoire of pathways concerning nitrogen-containing metabolites. In contrast, the synthesis of phospholipids and glycolipids has been preserved, curiously with the last step of sulfoquinovosyldiacylglycerol synthesis being catalyzed by the SqdX form of an enzyme so far known only from bacteria. Notably, we show that the E. longa plastid synthesizes tocopherols and a phylloquinone derivative, the first such report for nonphotosynthetic plastids studied so far. The most striking attribute of the organelle could be the presence of a linearized Calvin-Benson (CB) pathway, including RuBisCO yet lacking the gluconeogenetic part of the standard cycle, together with ferredoxin-NADP+ reductase (FNR) and the ferredoxin/thioredoxin system. We hypothesize that the ferredoxin/thioredoxin system activates the linear CB pathway in response to the redox status of the E. longa cell and speculate on the role of the pathway in keeping the redox balance of the cell. Altogether, the E. longa plastid defines a new class of relic plastids that is drastically different from the best-studied organelle of this category, the apicoplast. IMPORTANCE Colorless plastids incapable of photosynthesis evolved in many plant and algal groups, but what functions they perform is still unknown in many cases. Here, we study the elusive plastid of Euglena longa, a nonphotosynthetic cousin of the familiar green flagellate Euglena gracilis. We document an unprecedented combination of metabolic functions that the E. longa plastid exhibits in comparison with previously characterized nonphotosynthetic plastids. For example, and truly surprisingly, it has retained the synthesis of tocopherols (vitamin E) and a phylloquinone (vitamin K) derivative. In addition, we offer a possible solution of the long-standing conundrum of the presence of the CO2-fixing enzyme RuBisCO in E. longa. Our work provides a detailed account on a unique variant of relic plastids, the first among nonphotosynthetic plastids that evolved by secondary endosymbiosis from a green algal ancestor, and suggests that it has persisted for reasons not previously considered in relation to nonphotosynthetic plastids.

Increasing evidence has revealed that the enzymes of several biological pathways assemble into larger supramolecular structures called super-complexes. Indeed, those such as association of the mitochondrial respiratory chain complexes play an essential role in respiratory activity and promote metabolic fitness. Dynamically assembled super-complexes are able to alternate between participating in large complexes and existing in a free state. However, the functional significance of the super-complexes is not entirely clear. It has been proposed that the organization of respiratory enzymes into super-complexes could reduce oxidative damage and increase metabolism efficiency. There are several protein complexes that have been revealed in the plant chloroplast, yet little research has been focused on the formation of super-complexes in this organelle. The photosystem I and light-harvesting complex I super-complex’s structure suggests that energy absorbed by light-harvesting complex I could be efficiently transferred to the PSI core by avoiding concentration quenching. Here, we will discuss in detail core complexes of photosystem I and II, the chloroplast ATPase the chloroplast electron transport chain, the Calvin–Benson cycle and a plastid localized purinosome. In addition, we will also describe the methods to identify these complexes.

Background With limited agricultural land and increasing human population, it is essential to enhance overall photosynthesis and thus productivity. Oxygenic photosynthesis begins with light absorption, followed by excitation energy transfer to the reaction centres, primary photochemistry, electron and proton transport, NADPH and ATP synthesis, and then CO2 fixation (Calvin–Benson cycle, as well as Hatch–Slack cycle). Here we cover some of the discoveries related to this process, such as the existence of two light reactions and two photosystems connected by an electron transport 'chain' (the Z-scheme), chemiosmotic hypothesis for ATP synthesis, water oxidation clock for oxygen evolution, steps for carbon fixation, and finally the diverse mechanisms of regulatory processes, such as 'state transitions' and 'non-photochemical quenching' of the excited state of chlorophyll a. Scope In this review, we emphasize that mathematical modelling is a highly valuable tool in understanding and making predictions regarding photosynthesis. Different mathematical models have been used to examine current theories on diverse photosynthetic processes; these have been validated through simulation(s) of available experimental data, such as chlorophyll a fluorescence induction, measured with fluorometers using continuous (or modulated) exciting light, and absorbance changes at 820 nm (ΔA820) related to redox changes in P700, the reaction centre of photosystem I. Conclusions We highlight here the important role of modelling in deciphering and untangling complex photosynthesis processes taking place simultaneously, as well as in predicting possible ways to obtain higher biomass and productivity in plants, algae and cyanobacteria. [ABSTRACT FROM AUTHOR]

Under the current atmospheric conditions, oxygenic photosynthesis requires photorespiration to operate. In the presence of low CO2/O2 ratios, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) performs an oxygenase side reaction, leading to the formation of high amounts of 2-phosphoglycolate during illumination. Given that 2-phosphoglycolate is a potent inhibitor of photosynthetic carbon fixation, it must be immediately removed through photorespiration. The core photorespiratory cycle is orchestrated across three interacting subcellular compartments, namely chloroplasts, peroxisomes, and mitochondria, and thus cross-talks with a multitude of other cellular processes. Over the past years, the metabolic interaction of photorespiration and photosynthetic CO2 fixation has attracted major interest because research has demonstrated the enhancement of C3 photosynthesis and growth through the genetic manipulation of photorespiration. However, to optimize future engineering approaches, it is also essential to improve our current understanding of the regulatory mechanisms of photorespiration. Here, we summarize recent progress regarding the steps that control carbon flux in photorespiration, eventually involving regulatory proteins and metabolites. In this regard, both genetic engineering and the identification of various layers of regulation point to glycine decarboxylase as the key enzyme to regulate and adjust the photorespiratory carbon flow. Potential implications of the regulation of photorespiration for acclimation to environmental changes along with open questions are also discussed. [ABSTRACT FROM AUTHOR]

The chloroplast contains three types of peroxiredoxins (PRXs). Recently, 2-CysPRX was associated with thioredoxin (TRX) oxidation-dependent redox regulation. Here, this analysis was expanded to include PRXQ and PRXIIE. Oxidized PRXQ was able to inactivate NADPH malate dehydrogenase and fructose-1,6-bisphosphatase most efficiently in the presence of TRX-m1 and TRX-m4. The inactivation ability of TRXs did not entirely match their reductive activation efficiency. PRXIIE was unable to function as TRX oxidase in enzyme regulation. This conclusion was further supported by the observation that PRXQ adopts the oxidized form by about 50% in leaves, supporting a possible function as a TRX oxidase similar to 2-CysPRX. Results on the oxidation state of photosystem I (P700), plastocyanin, and ferredoxin in intact leaves indicate that each type of PRX has distinct regulatory functions, and that both 2-CysPRX and PRXQ conditionally assist in adjusting the redox state of target proteins for proper activity. [ABSTRACT FROM AUTHOR]

• NO or/and ABA enhanced photosynthesis under PEG-induced drought stress. • PEG-induced loss in activities of photosynthesis related enzymes reduced under NO or/and ABA supply. • RAPD profiles showed consistence results when compared with photosynthetic and stress parameters. • NO alone or together with ABA was more effective than ABA alone to alleviate oxidative stress in B. juncea L. seedlings. The involvement of two extremely important signalling molecules, nitric oxide (NO) and abscisic acid (ABA) has been employed by plants to facilitate the adaptive/tolerate response during stressful conditions. However, the interactive role of exogenously applied NO and ABA is very less studied at physiological, biochemical and molecular levels. The present study therefore, evaluated the effects of individual and simultaneous addition of exogenous NO donor SNP (100μM) and ABA (10μM) on photosynthesis, Calvin-Benson cycle enzymes, S-assimilation enzymes, oxidative stress components, and genotoxicity in Brassica juncea cv. Varuna, exposed to polyethylene glycol (PEG)-induced drought stress. Results showed that a loss induced by PEG was significantly surpassed by the application of NO or/and ABA with PEG for chlorophyll content, net photosynthestic rate (Pn), internal CO 2 concentration (Ci), stomatal conductance (gs), transpiration rate (Tr), maximum photosystem II (PSII) efficiency (Fv/Fm), actual PSII efficiency (ΦPSII), intrinsic PSII efficiency (Fv´/ Fm´), photochemical quenching (qP), non-photochemical quenching (NPQ), electron transport chain (ETC), ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCo), glyceraldehyde-3-phosphate dehydrogenase (GapDH), phosphoribulokinase (PRK), ATP-sulfurylase (ATP-S), and serine acetyltransferase (SAT) activities. The genomic template stability (GTS) (measured as changes in RAPD profiles) was significantly affected and showed varying degrees of DNA polymorphism, highest in PEG and lowest in PEG + NO and PEG + NO + ABA. Furthermore, the changes in RAPD profiles showed consistent results when compared with various photosynthetic and oxidative parameters. Altogether, this study concluded that supplementation of individual NO and together with ABA was more effective than individual ABA in alleviating PEG-induced drought stress in B. juncea L. seedlings. [ABSTRACT FROM AUTHOR]

Summary: 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. Significance Statement: Measurements of photosynthesis focus on leaves, however non‐foliar green tissue can make significant contributions to total plant carbon assimilation. This review evaluate photosynthesis and stomatal behavior in non‐foliar organs with a view to manipulate these processes for improved yield or quality. [ABSTRACT FROM AUTHOR]

Cell compartmentalization allows incompatible chemical reactions and localised responses to occur simultaneously, however, it also requires a complex system of communication between compartments in order to maintain the functionality of vital processes. It is clear that multiple such signals must exist, yet little is known about the identity of the key players orchestrating these interactions or about the role in the coordination of other processes. Mitochondria and chloroplasts have a considerable number of metabolites in common and are interdependent at multiple levels. Therefore, metabolites represent strong candidates as communicators between these organelles. In this context, vitamins and similar small molecules emerge as possible linkers to mediate metabolic crosstalk between compartments. This review focuses on two vitamins as potential metabolic signals within the plant cell, vitamin C (L-ascorbate) and vitamin B1 (thiamin). These two vitamins demonstrate the importance of metabolites in shaping cellular processes working as metabolic signals during acclimation processes. Inferences based on the combined studies of environment, genotype, and metabolite, in order to unravel signaling functions, are also highlighted. [ABSTRACT FROM AUTHOR]

Organisms have evolved effective and distinct adaptive strategies to survive. Stipa grandis is a representative species for studying the grazing effect on typical steppe plants in the Inner Mongolia Plateau. Although phenotypic (morphological and physiological) variations in S. grandis in response to long-term grazing have been identified, the molecular mechanisms underlying adaptations and plastic responses remain largely unknown. Here, we performed a transcriptomic analysis to investigate changes in gene expression of S. grandis under four different grazing intensities. As a result, a total of 2357 differentially expressed genes (DEGs) were identified among the tested grazing intensities, suggesting long-term grazing resulted in gene expression plasticity that affected diverse biological processes and metabolic pathways in S. grandis. DEGs were identified in RNA-Seq and qRT-PCR analyses that indicated the modulation of the Calvin–Benson cycle and photorespiration metabolic pathways. The key gene expression profiles encoding various proteins (e.g., ribulose-1,5-bisphosphate carboxylase/oxygenase, fructose-1,6-bisphosphate aldolase, glycolate oxidase, etc.) involved in these pathways suggest that they may synergistically respond to grazing to increase the resilience and stress tolerance of S. grandis. Our findings provide scientific clues for improving grassland use and protection and identifying important questions to address in future transcriptome studies.

The pentose phosphate pathway (PPP) is divided into an oxidative branch that makes pentose phosphates and a non-oxidative branch that consumes pentose phosphates, though the non-oxidative branch is considered reversible. A modified version of the non-oxidative branch is a critical component of the Calvin–Benson cycle that converts CO2 into sugar. The reaction sequence in the Calvin–Benson cycle is from triose phosphates to pentose phosphates, the opposite of the typical direction of the non-oxidative PPP. The photosynthetic direction is favored by replacing the transaldolase step of the normal non-oxidative PPP with a second aldolase reaction plus sedoheptulose-1,7-bisphosphatase. This can be considered an anabolic version of the non-oxidative PPP and is found in a few situations other than photosynthesis. In addition to the strong association of the non-oxidative PPP with photosynthesis metabolism, there is recent evidence that the oxidative PPP reactions are also important in photosynthesizing cells. These reactions can form a shunt around the non-oxidative PPP section of the Calvin–Benson cycle, consuming three ATP per glucose 6-phosphate consumed. A constitutive operation of this shunt occurs in the cytosol and gives rise to an unusual labeling pattern of photosynthetic metabolites while an inducible shunt in the stroma may occur in response to stress.

Starch comprises nearly linear amylose and branched amylopectin, whilst waxy starches are a special form, containing almost exclusively amylopectin. Modern techniques in plant breeding together with new data from starch biosynthesis research have enabled new food and non-food uses of waxy starches. This paper describes the basic ways of glucose conversion to waxy starch in plants. The recent evidence of ADP-Glc accumulation in cytosol of photosynthetically competent cells proposes a more complex pathway of starch biosynthesis based on a tight interconnection of sucrose and starch metabolic pathways. Also many studies indicate the existence of different pathways for the sucrose-starch conversion process in heterotrophic organs of dicotyledonous and monocotyledonous plants. At least six classes of starch synthases (SS) have been recognised in plants including soluble SS1, SS2, SS3, SS4, SS5, and granule bound SS (GBSS), required for the synthesis of short and long chains of amylopectin, till now. As to amylose (not-present in waxy starches), GBSS is the only starch synthase isoform encoded by the waxy genes situated at independent loci.

Stable isotope labeling with 13 CO 2 coupled with mass spectrometry allows monitoring the incorporation of 13 C into photosynthetic intermediates and is a powerful technique for the investigation of the metabolic dynamics of photosynthesis. We describe here a protocol for 13 CO 2 labeling of large leaved plants and of Arabidopsis thaliana rosette, and a method for quantitative mass spectrometry analyses to uncover the labeling pattern of Calvin-Benson cycle sucrose, and starch synthesis as well as carbon-concentrating mechanism metabolites., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)