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It has been reported that overproduction of Rubisco activase (RCA) in rice (Oryza sativa L.) decreased Rubisco content, resulting in declining photosynthesis. We examined the effects of RCA levels on Rubisco content using transgenic rice with overexpressed or suppressed RCA under the control of different promoters of the RCA and Rubisco small subunit (RBCS) genes. All plants were grown hydroponically with different N concentrations (0.5, 2.0 and 8.0 mM-N). In RCA overproduced plants with > 2-fold RCA content (RCA-HI lines), a 10%–20% decrease in Rubisco content was observed at 0.5 and 2.0 mM-N. In contrast, at 8.0 mM-N, Rubisco content did not change in RCA-HI lines. Conversely, in plants with 50%–60% increased RCA content (RCA-MI lines), Rubisco levels remained unchanged, regardless of N concentration. Such effects on Rubisco content were independent of the promoter that was used. In plants with RCA suppression to < 10% of the wild-type RCA content, Rubisco levels were increased at 0.5 mM-N, but were unchanged at 2.0 and 8.0 mM-N. Thus, the effects of the changes in RCA levels on Rubisco content depended on N supply. Moreover, RCA overproduction was feasible without a decrease in Rubisco content, depending on the degree of RCA production.

The uncertainty of future climate change is placing pressure on cropping systems to continue to provide stable increases in productive yields. To mitigate future climates and the increasing threats against global food security, new solutions to manipulate photosynthesis are required. This review explores the current efforts available to improve carbon assimilation within plant chloroplasts by engineering Rubisco, which catalyzes the rate-limiting step of CO2 fixation. Fixation of CO2 and subsequent cycling of 3-phosphoglycerate through the Calvin cycle provides the necessary carbohydrate building blocks for maintaining plant growth and yield, but has to compete with Rubisco oxygenation, which results in photorespiration that is energetically wasteful for plants. Engineering improvements in Rubisco is a complex challenge and requires an understanding of chloroplast gene regulatory pathways, and the intricate nature of Rubisco catalysis and biogenesis, to transplant more efficient forms of Rubisco into crops. In recent times, major advances in Rubisco engineering have been achieved through improvement of our knowledge of Rubisco synthesis and assembly, and identifying amino acid catalytic switches in the L-subunit responsible for improvements in catalysis. Improving the capacity of CO2 fixation in crops such as rice will require further advances in chloroplast bioengineering and Rubisco biogenesis.

RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE (Rubisco) produces pyruvate in the chloroplast through β-elimination of the aci-carbanion intermediate. Here we show that this side reaction supplies pyruvate for isoprenoid, fatty acid and branched-chain amino acid biosynthesis in photosynthetically active tissue. 13C labelling studies of intact Arabidopsis plants demonstrate that the total carbon commitment to pyruvate is too large for phosphoenolpyruvate to serve as a precursor. Low oxygen stimulates Rubisco carboxylase activity and increases pyruvate production and flux through the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway, which supplies the precursors for plastidic isoprenoid biosynthesis. Metabolome analysis of mutants defective in phosphoenolpyruvate or pyruvate import and biochemical characterization of isolated chloroplasts further support Rubisco as the main source of pyruvate in chloroplasts. Seedlings incorporated exogenous, 13C-labelled pyruvate into MEP pathway intermediates, while adult plants did not, underscoring the developmental transition in pyruvate sourcing. Rubisco β-elimination leading to pyruvate constituted 0.7% of the product profile in in vitro assays, which translates to 2% of the total carbon leaving the Calvin–Benson–Bassham cycle. These insights solve the “pyruvate paradox”, improve the fit of metabolic models for central metabolism and connect the MEP pathway directly to carbon assimilation.

Site turnover rate (k cat) of Rubisco was measured in intact leaves of different plants. Potato (Solanum tuberosum L.) and birch (Betula pendula Roth.) leaves were taken from field-growing plants. Sunflower (Helianthus annuus L.), wild type (wt), Rubisco-deficient (-RBC), FNR-deficient (-FNR), and Cyt b 6 f deficient (-CBF) transgenic tobacco (Nicotiana tabacum L.) were grown in a growth chamber. Rubisco protein was measured with quantitative SDS-PAGE and FNR protein content with quantitative immunoblotting. The Cyt b 6 f level was measured in planta by maximum electron transport rate and the photosystem I (PSI) content was assessed by titration with far-red light. The CO2 response of Rubisco was measured in planta with a fast-response gas exchange system at maximum ribulose 1,5-bisphosphate concentration. Reaction site k cat was calculated from V m and Rubisco content. Biological variation of k cat was significant, ranging from 1.5 to 4 s−1 in wt, but was >6 s−1 at 23 °C in -RBC leaves. The lowest k cat of 0.5 s−1 was measured in -FNR and -CBF plants containing sufficient Rubisco but having slow electron transport rates. Plotting k cat against PSI per Rubisco site resulted in a hyperbolic relationship where wt plants are on the initial slope. A model is suggested in which Rubisco Activase is converted into an active ATP-form on thylakoid membranes with the help of a factor related to electron transport. The activation of Rubisco is accompanied by the conversion of the ATP-form into an inactive ADP-form. The ATP and ADP forms of Activase shuttle between thylakoid membranes and stromally-located Rubisco. In normal wt plants the electron transport-related activation of Activase is rate-limiting, maintaining 50-70% Rubisco sites in the inactive state.

Wheat is the second most important direct source of food calories in the world. After considerable improvement during the Green Revolution, increase in genetic yield potential appears to have stalled. Improvement of photosynthetic efficiency now appears a major opportunity in addressing the sustainable yield increases needed to meet future food demand. Effort, however, has focused on increasing efficiency under steady-state conditions. In the field, the light environment at the level of individual leaves is constantly changing. The speed of adjustment of photosynthetic efficiency can have a profound effect on crop carbon gain and yield. Flag leaves of wheat are the major photosynthetic organs supplying the grain of wheat, and will be intermittently shaded throughout a typical day. Here, the speed of adjustment to a shade to sun transition in these leaves was analysed. On transfer to sun conditions, the leaf required about 15 min to regain maximum photosynthetic efficiency. In vivo analysis based on the responses of leaf CO2 assimilation (A) to intercellular CO2 concentration (ci) implied that the major limitation throughout this induction was activation of the primary carboxylase of C3 photosynthesis, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). This was followed in importance by stomata, which accounted for about 20% of the limitation. Except during the first few seconds, photosynthetic electron transport and regeneration of the CO2 acceptor molecule, ribulose-1,5-bisphosphate (RubP), did not affect the speed of induction. The measured kinetics of Rubisco activation in the sun and de-activation in the shade were predicted from the measurements. These were combined with a canopy ray tracing model that predicted intermittent shading of flag leaves over the course of a June day. This indicated that the slow adjustment in shade to sun transitions could cost 21% of potential assimilation. This article is part of the themed issue ‘Enhancing photosynthesis in crop plants: targets for improvement’.

To understand the effect of heat and drought on three major cereal crops, the physiological and biochemical (i.e., metabolic) factors affecting photosynthesis were examined in rice, wheat, and maize plants grown under long-term water deficit (WD), high temperature (HT) and the combination of both stresses (HT-WD). Diffusional limitations to photosynthesis prevailed under WD for the C species, rice and wheat. Conversely, biochemical limitations prevailed under WD for the C species, maize, under HT for all three species, and under HT-WD in rice and maize. These biochemical limitations to photosynthesis were associated with Rubisco activity that was highly impaired at HT and under HT-WD in the three species. Decreases in Rubisco activation were unrelated to the amount of Rubisco and Rubisco activase (Rca), but were probably caused by inhibition of Rca activity, as suggested by the mutual decrease and positive correlation between Rubisco activation state and the rate of electron transport. Decreased Rubisco activation at HT was associated with biochemical limitation of net CO assimilation rate ( ). Overall, the results highlight the importance of Rubisco as a target for improving the photosynthetic performance of these C (wheat and rice) and C (maize) cereal crops under increasingly variable and warmer climates.

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. Here, 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.

Carboxysomes are bacterial microcompartments, whose structural features enable the encapsulated Rubisco holoenzyme to operate in a high-CO 2 environment. Consequently, Rubiscos housed within these compartments possess higher catalytic turnover rates relative to their plant counterparts. This particular enzymatic property has made the carboxysome, along with associated transporters, an attractive prospect to incorporate into plant chloroplasts to increase future crop yields. To date, two carboxysome types have been characterized, the α-type that has fewer shell components and the β-type that houses a faster Rubisco. While research is underway to construct a native carboxysome in planta , work investigating the internal arrangement of carboxysomes has identified conserved Rubisco amino acid residues between the two carboxysome types which could be engineered to produce a new, hybrid carboxysome. In theory, this hybrid carboxysome would benefit from the simpler α-carboxysome shell architecture while simultaneously exploiting the higher Rubisco turnover rates in β-carboxysomes. Here, we demonstrate in an Escherichia coli expression system, that the Thermosynechococcus elongatus Form IB Rubisco can be imperfectly incorporated into simplified Cyanobium α-carboxysome-like structures. While encapsulation of non-native cargo can be achieved, T. elongatus Form IB Rubisco does not interact with the Cyanobium carbonic anhydrase, a core requirement for proper carboxysome functionality. Together, these results suggest a way forward to hybrid carboxysome formation.

Rubisco activase (Rca) facilitates the release of sugar‐phosphate inhibitors at Rubisco catalytic sites during CO2 fixation. Most plant species express two Rca isoforms, the larger Rca‐α and the shorter Rca‐β, either by alternative splicing from a single gene or expression from separate genes. The mechanism of Rubisco activation by Rca isoforms has been intensively studied in C3 plants. However, the functional role of Rca in C4 plants where Rubisco and Rca are located in a much higher [CO2] compartment is less clear. In this study, we selected four C4 bioenergy grasses and the model C4 grass setaria (Setaria viridis) to investigate the role of Rca in C4 photosynthesis. All five C4 grass species contained two Rca genes, one encoding Rca‐α and the other Rca‐β, which were positioned closely together in the genomes. A variety of abiotic stress‐related motifs were identified in the Rca‐α promoter of each grass, and while the Rca‐β gene was constantly highly expressed at ambient temperature, Rca‐α isoforms were expressed only at high temperature but never surpassed 30% of Rca‐β content. The pattern of Rca‐α induction on transition to high temperature and reduction on return to ambient temperature was the same in all five C4 grasses. In sorghum (Sorghum bicolor), sugarcane (Saccharum officinarum), and setaria, the induction rate of Rca‐α was similar to the recovery rate of photosynthesis and Rubisco activation at high temperature. This association between Rca‐α isoform expression and maintenance of Rubisco activation at high temperature suggests that Rca‐α has a functional thermo‐protective role in carbon fixation in C4 grasses by sustaining Rubisco activation at high temperature. Activation of Rubisco, the enzyme responsible for carboxylation in photosynthesis, by Rubisco activase (Rca) has been well studied in C3 but not in C4 plants. We examined the role of Rca in four C4 bioenergy grasses and a model C4 grass under various conditions. Each species contained genes for both the large (Rca‐α) and short (Rca‐β) Rca forms, but while Rca‐β was highly expressed in all conditions tested, Rca‐α was only expressed at high temperature. Rca‐α induction upon transition to high temperature matched recovery of photosynthesis and Rubisco activation, suggesting Rca‐α provides a thermoprotective role in C4 grass photosynthesis.

Carboxysomes are membrane-free organelles for carbon assimilation in cyanobacteria. The carboxysome consists of a proteinaceous shell that structurally resembles virus capsids and internal enzymes including ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the primary carbon-fixing enzyme in photosynthesis. The formation of carboxysomes requires hierarchical self-assembly of thousands of protein subunits, initiated from Rubisco assembly and packaging to shell encapsulation. Here we study the role of Rubisco assembly factor 1 (Raf1) in Rubisco assembly and carboxysome formation in a model cyanobacterium, Synechococcus elongatus PCC7942 (Syn7942). Cryo-electron microscopy reveals that Raf1 facilitates Rubisco assembly by mediating RbcL dimer formation and dimer–dimer interactions. Syn7942 cells lacking Raf1 are unable to form canonical intact carboxysomes but generate a large number of intermediate assemblies comprising Rubisco, CcaA, CcmM, and CcmN without shell encapsulation and a low abundance of carboxysome-like structures with reduced dimensions and irregular shell shapes and internal organization. As a consequence, the Raf1-depleted cells exhibit reduced Rubisco content, CO₂-fixing activity, and cell growth. Our results provide mechanistic insight into the chaperone-assisted Rubisco assembly and biogenesis of carboxysomes. Advanced understanding of the biogenesis and stepwise formation process of the biogeochemically important organelle may inform strategies for heterologous engineering of functional CO₂-fixing modules to improve photosynthesis.