Regulation of Rubisco activity by interaction with chloroplast metabolites.

Rubisco activity is highly regulated and frequently limits carbon assimilation in crop plants. In the chloroplast, various metabolites can inhibit or modulate Rubisco activity by binding to its catalytic or allosteric sites, but this regulation is complex and still poorly understood. Using rice Rubisco, we characterised the impact of various chloroplast metabolites which could interact with Rubisco and modulate its activity, including photorespiratory intermediates, carbohydrates, amino acids; as well as specific sugar-phosphates known to inhibit Rubisco activity - CABP (2-carboxy-d-arabinitol 1,5-bisphosphate) and CA1P (2-carboxy-d-arabinitol 1-phosphate) through in vitro enzymatic assays and molecular docking analysis. Most metabolites did not directly affect Rubisco in vitro activity under both saturating and limiting concentrations of Rubisco substrates, CO2 and RuBP (ribulose-1,5-bisphosphate). As expected, Rubisco activity was strongly inhibited in the presence of CABP and CA1P. High physiologically relevant concentrations of the carboxylation product 3-PGA (3-phosphoglyceric acid) decreased Rubisco activity by up to 30%. High concentrations of the photosynthetically derived hexose phosphates fructose 6-phosphate (F6P) and glucose 6-phosphate (G6P) slightly reduced Rubisco activity under limiting CO2 and RuBP concentrations. Biochemical measurements of the apparent Vmax and Km for CO2 and RuBP (at atmospheric O2 concentration) and docking interactions analysis suggest that CABP/CA1P and 3-PGA inhibit Rubisco activity by binding tightly and loosely, respectively, to its catalytic sites (i.e. competing with the substrate RuBP). These findings will aid the design and biochemical modelling of new strategies to improve the regulation of Rubisco activity and enhance the efficiency and sustainability of carbon assimilation in rice.

Purification of Rubisco from Leaves.

Rubisco fixes CO2 through the carboxylation of ribulose 1,5-bisphosphate (RuBP) during photosynthesis, enabling the synthesis of organic compounds. The natural diversity of Rubisco properties represents an opportunity to improve its performance and there is considerable research effort focusing on better understanding the properties and regulation of the enzyme. This chapter describes a method for large-scale purification of Rubisco from leaves. After the extraction of Rubisco from plant leaves, the enzyme is separated from other proteins by fractional precipitation with polyethylene glycol followed by ion-exchange chromatography. This method enables the isolation of Rubisco in large quantities for a wide range of biochemical applications.

Regulation of Rubisco activity in crops.

Efficient plant acclimation to changing environmental conditions relies on fast adjustments of the transcriptome, proteome, and metabolome. Regulation of enzyme activity depends on the activity of specific chaperones, chemical post-translational modifications (PTMs) of amino acid residues, and changes in the cellular and organellar microenvironment. Central to carbon assimilation, and thus plant growth and yield, Rubisco activity is regulated by its chaperone Rubisco activase (Rca) and by adjustments in the chloroplast stroma environment. Focused on crops, this review highlights the main PTMs and stromal ions and metabolites affecting Rubisco and Rca in response to environmental stimuli. Rca isoforms differ in regulatory properties and heat sensitivity, with expression changing according to the surrounding environment. Much of the physiological relevance of Rubisco and Rca PTMs is still poorly understood, though some PTMs have been associated with Rubisco regulation in response to stress. Ion and metabolite concentrations in the chloroplast change in response to variations in light and temperature. Some of these changes promote Rubisco activation while others inhibit activation, deactivate the enzyme, or change the rates of catalysis. Understanding these regulatory mechanisms will aid the development of strategies to improve carbon fixation by Rubisco under rapidly changing environments as experienced by crop plants.

Photoinhibition of Photosystem I Provides Oxidative Protection During Imbalanced Photosynthetic Electron Transport in Arabidopsis thaliana.

Photosynthesis involves the conversion of sunlight energy into stored chemical energy, which is achieved through electron transport along a series of redox reactions. Excess photosynthetic electron transport might be dangerous due to the risk of molecular oxygen reduction, generating reactive oxygen species (ROS) over-accumulation. Avoiding excess ROS production requires the rate of electron transport to be coordinated with the capacity of electron acceptors in the chloroplast stroma. Imbalance between the donor and acceptor sides of photosystem I (PSI) can lead to inactivation, which is called PSI photoinhibition. We used a light-inducible PSI photoinhibition system in Arabidopsis thaliana to resolve the time dynamics of inhibition and to investigate its impact on ROS production and turnover. The oxidation state of the PSI reaction center and rates of CO2 fixation both indicated strong and rapid PSI photoinhibition upon donor side/acceptor side imbalance, while the rate of inhibition eased during prolonged imbalance. PSI photoinhibition was not associated with any major changes in ROS accumulation or antioxidant activity; however, a lower level of lipid oxidation correlated with lower abundance of chloroplast lipoxygenase in PSI-inhibited leaves. The results of this study suggest that rapid activation of PSI photoinhibition under severe photosynthetic imbalance protects the chloroplast from over-reduction and excess ROS formation.

Drought increases cowpea (Vigna unguiculata [L.] Walp.) susceptibility to cowpea severe mosaic virus (CPSMV) at early stage of infection.

The physiological and biochemical responses of a drought tolerant, virus-susceptible cowpea genotype exposed to drought stress (D), infected by Cowpea severe mosaic virus (CPSMV) (V), and to these two combined stresses (DV), at 2 and 6 days post viral inoculation (DPI), were evaluated. Gas exchange parameters (net photosynthesis, transpiration rate, stomatal conductance, and internal CO2 partial pressure) were reduced in D and DV at 2 and 6 DPI compared to control plants (C). Photosynthesis was reduced by stomatal and biochemical limitations. Water use efficiency increased at 2 DPI in D, DV, and V, but at 6 DPI only in D and DV compared to C. Photochemical parameters (effective quantum efficiency of photosystem II and electron transport rate) decreased in D and DV compared to C, especially at 6 DPI. The potential quantum efficiency of photosystem II did not change, indicating reversible photoinhibition of photosystem II. In DV, catalase decreased at 2 and 6 DPI, ascorbate peroxidase increased at 2 DPI, but decreased at 6 DPI. Hydrogen peroxide increased at 2 and 6 DPI. Peroxidase increased at 6 DPI and chitinase at 2 and 6 DPI. ß-1,3-glucanase decreased in DV at 6 DPI compared to V. Drought increased cowpea susceptibility to CPSMV at 2 DPI, as verified by RT-PCR. However, at 6 DPI, the cowpea plants overcome this effect. Likewise, CPSMV increased the negative effects of drought at 2 DPI, but not at 6 DPI. It was concluded that the responses to combined stresses are not additive and cannot be extrapolated from the study of individual stresses.

Dissipation of excess photosynthetic energy contributes to salinity tolerance: a comparative study of salt-tolerant Ricinus communis and salt-sensitive Jatropha curcas.

The relationships between salt tolerance and photosynthetic mechanisms of excess energy dissipation were assessed using two species that exhibit contrasting responses to salinity, Ricinus communis (tolerant) and Jatropha curcas (sensitive). The salt tolerance of R. communis was indicated by unchanged electrolyte leakage (cellular integrity) and dry weight in leaves, whereas these parameters were greatly affected in J. curcas. The leaf Na+ content was similar in both species. Photosynthesis was intensely decreased in both species, but the reduction was more pronounced in J. curcas. In this species biochemical limitations in photosynthesis were more prominent, as indicated by increased C(i) values and decreased Rubisco activity. Salinity decreased both the V(cmax) (in vivo Rubisco activity) and J(max) (maximum electron transport rate) more significantly in J. curcas. The higher tolerance in R. communis was positively associated with higher photorespiratory activity, nitrate assimilation and higher cyclic electron flow. The high activity of these alternative electron sinks in R. communis was closely associated with a more efficient photoprotection mechanism. In conclusion, salt tolerance in R. communis, compared with J. curcas, is related to higher electron partitioning from the photosynthetic electron transport chain to alternative sinks.