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It has been 65 years since the Calvin–Benson cycle was first formulated. In this paper, the development of the concepts that are critical to the cycle is traced and the contributions of Calvin, Benson, and Bassham are discussed. Some simplified views often found in text books such as ascending paper chromatography and the use of the “lollipop” for short labeling are discussed and further details given. Key discoveries that underpinned elucidation of the cycle such as the importance of sedoheptulose phosphate and ribulose 1,5-bisphosphate are described. The interchange of ideas between other researchers working on what is now called the pentose phosphate pathway and the development of the ideas of Calvin and Benson are explored while the gluconeogenic aspects of the cycle are emphasized. Concerns raised about anomalies of label distribution in glucose are considered. Other carbon metabolism pathways associated with the Calvin–Benson cycle are also described. Finally, there is a section describing the rift between Calvin and Benson.

Background:Photosynthetic microalgae are emerging as potential biomass feedstock for sustainable production of biofuels and value-added bioproducts. CO2 biomitigation through these organisms is considered as an eco-friendly and promising alternative to the existing carbon sequestration methods. Nonetheless, the inherent relatively low photosynthetic capacity of microalgae has hampered the practical use of this strategy for CO2 biomitigation applications.Results:Here, we demonstrate the feasibility of improving photosynthetic capacity by the genetic manipulation of the Calvin cycle in the typical green microalga Chlorella vulgaris. Firstly, we fused a plastid transit peptide to upstream of the enhanced green fluorescent protein (EGFP) and confirmed its expression in the chloroplast of C. vulgaris. Then we introduced the cyanobacterial fructose 1,6-bisphosphate aldolase, guided by the plastid transit peptide, into C. vulgaris chloroplast, leading to enhanced photosynthetic capacity (~1.2-fold) and cell growth. Molecular and physiochemical analyses suggested a possible role for aldolase overexpression in promoting the regeneration of ribulose 1,5-bisphosphate in the Calvin cycle and energy transfer in photosystems.Conclusions:Our work represents a proof-of-concept effort to enhance photosynthetic capacity by the engineering of the Calvin cycle in green microalgae. Our work also provides insights into targeted genetic engineering toward algal trait improvement for CO2 biomitigation uses.

There are four forms of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) found in nature. Forms I, II, and III catalyse the carboxylation and oxygenation of ribulose 1,5-bisphosphate, while form IV, also called the Rubisco-like protein (RLP), does not catalyse either of these reactions. There appear to be six different clades of RLP. Although related to bona fide Rubisco proteins at the primary sequence and tertiary structure levels, RLP from two of these clades is known to perform other functions in the cell. Forms I, II, and III Rubisco, along with form IV (RLP), are thought to have evolved from a primordial archaeal Rubisco. Structure/function studies with both archaeal form III (methanogen) and form I (cyanobacterial) Rubisco have identified residues that appear to be specifically involved with interactions with molecular oxygen. A specific region of all form I, II, and III Rubisco was identified as being important for these interactions.

• In plants, rubisco activase (Rca) regulates rubisco by removing inhibitory molecules such as ribulose-1,5-bisphosphate (RuBP). In cyanobacteria, a homologous protein (activase-like cyanobacterial protein, ALC), contains a distinctive C-terminal fusion resembling the small-subunit of rubisco. Although cyanobacterial rubisco is believed to be less sensitive to RuBP inhibition, the ALC is widely distributed among diverse cyanobacteria. • Using microscopy, biochemistry and molecular biology, the cellular localization of the ALC, its effect on carboxysome/cell ultrastructure in Fremyella diplosiphon, and its function in vitro were studied. Bioinformatic analysis uncovered evolutionary relationships between the ALC and rubisco. • ALC localizes to carboxysomes and exhibits ATPase activity. Furthermore, the ALC induces rubisco aggregation in a manner similar to that of another carboxysomal protein, M35, and this activity is affected by ATP. An alc deletion mutant showed modified cell morphology when grown under enriched CO₂ and impaired regulation of carboxysome biogenesis, without affecting growth rate. Carbamylation of Fremyella recombinant rubisco was inhibited by RuBP, but this inhibition was not relieved by the ALC. • The ALC does not appear to function like a canonical Rca; instead, it exerts an effect on the response to CO₂ availability at the level of a metabolic module, the carboxysome, through rubisco network formation, and carboxysome organization.

Low atmospheric CO₂ in recent geological time led to the evolution of carbon-concentrating mechanisms (CCMs) such as C₄ photosynthesis in >65 terrestrial plant lineages. We know little about the impact of low CO₂ on the Calvin–Benson cycle (CBC) in C₃ species that did not evolve CCMs, representing >90% of terrestrial plant species. Metabolite profiling provides a top-down strategy to investigate the operational balance in a pathway. We profiled CBC intermediates in a panel of C₄ (Zea mays, Setaria viridis, Flaveria bidentis, and F. trinervia) and C₃ species (Oryza sativa, Triticium aestivum, Arabidopsis thaliana, Nicotiana tabacum, and Manihot esculenta). Principal component analysis revealed differences between C₄ and C₃ species that were driven by many metabolites, including lower ribulose 1,5-bisphosphate in C₄ species. Strikingly, there was also considerable variation between C₃ species. This was partly due to different chlorophyll and protein contents, but mainly to differences in relative levels of metabolites. Correlation analysis indicated that one contributory factor was the balance between fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase, phosphoribulokinase, and Rubisco. Our results point to the CBC having experienced different evolutionary trajectories in C₃ species since the ancestors of modern plant lineages diverged. They underline the need to understand CBC operation in a wide range of species.

Low N decreases net photosynthetic rate (Pn), but a tolerant wheat cultivar maintained relatively high Pn by maintaining carboxylation capacity through enhanced Rubisco activation and sustained electron transport. Abstract Studying the response of photosynthesis to low nitrogen (N) and the underlying physiological mechanism can provide a theoretical basis for breeding N-efficient cultivars and optimizing N management. We conducted hydroponic experiments using two wheat (Triticum aestivum) cultivars, Zaoyangmai (low N sensitive) and Yangmai158 (low N tolerant), with either 0.25 mM N as a low N (LN) treatment or 5 mM N as a control. Under LN, a decrease in net photosynthetic rate (Pn) was attributed to reduction in the maximum Rubisco carboxylation rate, which then accelerated a reduction in the maximum ribulose-1,5-bisphosphate regeneration rate, and the reduction in Pn was 5-35% less in Yangmai158 than in Zaoyangmai. Yangmai158 maintained a 10-25% higher Rubisco concentration, especially in the upper leaves, and up-regulated Rubisco activase activity compared with Zaoyangmai to increase the Rubisco activation to sustain Rubisco carboxylation under LN conditions. In addition, Yangmai158 increased electron flux to the photorespiratory carbon oxidation cycle and alternative electron flux to maintain a faster electron transport rate and avoid photodamage. In conclusion, the LN-tolerant cultivar showed enhanced Rubisco activation and sustained electron transport to maintain a greater photosynthetic capacity under LN conditions.

The Calvin cycle is the initial pathway of photosynthetic carbon fixation, and several of its reaction steps are suggested to exert rate-limiting influence on the growth of higher plants. Plastid fructose 1,6-bisphosphate aldolase (aldolase, EC 4.1.2.13) is one of the nonregulated enzymes comprising the Calvin cycle and is predicted to have the potential to control photosynthetic carbon flux through the cycle. In order to investigate the effect of overexpression of aldolase, this study generated transgenic tobacco (Nicotiana tabacumL. cv Xanthi) expressingArabidopsisplastid aldolase. Resultant transgenic plants with 1.4–1.9-fold higher aldolase activities than those of wild-type plants showed enhanced growth, culminating in increased biomass, particularly under high CO₂ concentration (700 ppm) where the increase reached 2.2-fold relative to wild-type plants. This increase was associated with a 1.5-fold elevation of photosynthetic CO₂ fixation in the transgenic plants. The increased plastid aldolase resulted in a decrease in 3-phosphoglycerate and an increase in ribulose 1,5-bisphosphate and its immediate precursors in the Calvin cycle, but no significant changes in the activities of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) or other major enzymes of carbon assimilation. Taken together, these results suggest that aldolase overexpression stimulates ribulose 1,5-bisphosphate regeneration and promotes CO₂ fixation. It was concluded that increased photosynthetic rate was responsible for enhanced growth and biomass yields of aldolase-overexpressing plants.

Photorespiration is indispensable for oxygenic photosynthesis since it detoxifies and recycles 2-phosphoglycolate (2PG), which is the primary oxygenation product of Rubisco. However, C₄ plant species typically display very low rates of photorespiration due to their efficient biochemical carbon-concentrating mechanism. Thus, the broader relevance of photorespiration in these organisms remains unclear. In this study, we assessed the importance of a functional photorespiratory pathway in the C₄ plant Flaveria bidentis using knockdown of the first enzymatic step, namely 2PG phosphatase (PGLP). The isolated RNAi lines showed strongly reduced amounts of PGLP protein, but distinct signs of the photorespiratory phenotype only emerged below 5% residual PGLP protein. Lines with this characteristic were stunted in growth, had strongly increased 2PG content, exhibited accelerated leaf senescence, and accumulated high amounts of branched-chain and aromatic amino acids, which are both characteristics of incipient carbon starvation. Oxygen-dependent gas-exchange measurements consistently suggested the cumulative impairment of ribulose-1,5-bisphosphate regeneration with increased photorespiratory pressure. Our results indicate that photorespiration is essential for maintaining high rates of C₄ photosynthesis by preventing the 2PG-mediated inhibition of carbon utilization efficiency. However, considerably higher 2PG accumulation can be tolerated compared to equivalent lines of C₃ plants due to the differential distribution of specific enzymatic steps between the mesophyll and bundle sheath cells.

Biological fixation of atmospheric CO₂ via the Calvin–Benson–Bassham cycle has massive ecological impact and offers potential for industrial exploitation, either by improving carbon fixation in plants and autotrophic bacteria, or by installation into new hosts. A kinetic model of the Calvin–Benson–Bassham cycle embedded in the central carbon metabolism of the cyanobacterium Synechocystis sp. PCC 6803 was developed to investigate its stability and underlying control mechanisms. To reduce the uncertainty associated with a single parameter set, random sampling of the steady-state metabolite concentrations and the enzyme kinetic parameters was employed, resulting in millions of parameterized models which were analyzed for flux control and stability against perturbation. Our results show that the Calvin cycle had an overall high intrinsic stability, but a high concentration of ribulose 1,5-bisphosphate was associated with unstable states. Low substrate saturation and high product saturation of enzymes involved in highly interconnected reactions correlated with increased network stability. Flux control, that is the effect that a change in one reaction rate has on the other reactions in the network, was distributed and mostly exerted by energy supply (ATP), but also by cofactor supply (NADPH). Sedoheptulose 1,7-bisphosphatase/fructose 1,6-bisphosphatase, fructose- bisphosphate aldolase, and transketolase had a weak but positive effect on overall network flux, in agreement with published observations. The identified flux control and relationships between metabolite concentrations and system stability can guide metabolic engineering. The kinetic model structure and parameterizing framework can be expanded for analysis of metabolic systems beyond the Calvin cycle.

In C₃ plants, CO₂ assimilation is limited by ribulose 1,5-bisphosphate (RuBP) regeneration rate at high CO₂. RuBP regeneration rate in turn is determined by either the chloroplast electron transport capacity to generate NADPH and ATP or the activity of Calvin cycle enzymes involved in regeneration of RuBP. Here, transgenic tobacco (Nicotiana tabacum 'W38') expressing an antisense gene directed at the transcript of either the Rieske iron-sulfur protein of the cytochrome (Cyt) b₆/f complex or the δ-subunit of chloroplast ATP synthase have been used to investigate the effect of a reduction of these complexes on chloroplast electron transport rate (ETR). Reductions in δ-subunit of ATP synthase content did not alter chlorophyll, Cyt b₆/f complex, or Rubisco content, but reduced ETR estimated either from measurements of chlorophyll fluorescence or CO₂ assimilation rates at high CO₂. Plants with low ATP synthase content exhibited higher nonphotochemical quenching and achieved higher ETR per ATP synthase than the wild type. The proportional increase in ETR per ATP synthase complex was greatest at 35°C, showing that the ATP synthase activity can vary in vivo. In comparison, there was no difference in the ETR per Cyt b₆/f complex in plants with reduced Cyt b₆/f content and the wild type. The ETR decreased more drastically with reductions in Cyt b₆/f complex than ATP synthase content. This suggests that chloroplast ETR is more limited by Cyt b₆/f than ATP synthase content and is a potential target for enhancing photosynthetic capacity in crops.