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SUMMARY Photosynthetic inefficiencies limit the productivity and sustainability of crop production and the resilience of agriculture to future societal and environmental challenges. Rubisco is a key target for improvement as it plays a central role in carbon fixation during photosynthesis and is remarkably inefficient. Introduction of mutations to the chloroplast‐encoded Rubisco large subunit rbc L is of particular interest for improving the catalytic activity and efficiency of the enzyme. However, manipulation of rbc L is hampered by its location in the plastome, with many species recalcitrant to plastome transformation, and by the plastid's efficient repair system, which can prevent effectivemore » maintenance of mutations introduced with homologous recombination. Here we present a system where the introduction of a number of silent mutations into rbc L within the model plant Nicotiana tabacum facilitates simplified screening via additional restriction enzyme sites. This system was used to successfully generate a range of transplastomic lines from wild‐type N. tabacum with stable point mutations within rbc L in 40% of the transformants, allowing assessment of the effect of these mutations on Rubisco assembly and activity. With further optimization the approach offers a viable way forward for mutagenic testing of Rubisco function in planta within tobacco and modification of rbc L in other crops where chloroplast transformation is feasible. The transformation strategy could also be applied to introduce point mutations in other chloroplast‐encoded genes.« less

Rubisco sustains the biosphere through the fixation of CO₂ into biomass. In plants and cyanobacteria, form I Rubisco is structurally comprised of large and small subunits, whereas all other Rubisco forms lack small subunits. The rise of the form I complex through the innovation of small subunits represents a key, yet poorly understood, transition in Rubisco's evolution. Here, through metagenomic analyses, we discovered a previously uncharacterized clade sister to form I Rubisco that evolved without small subunits. This clade diverged before the evolution of cyanobacteria and the origin of the small subunit; thus, it provides a unique reference point tomore » advance our understanding of form I Rubisco evolution. Structural and kinetic data presented here reveal how a proto-form I Rubisco assembled and functioned without the structural stability imparted from small subunits. Our findings provide insight into a key evolutionary transition of the most abundant enzyme on Earth and the predominant entry point for nearly all global organic carbon.« less

The antiquity and global abundance of the enzyme, RuBisCO, attests to the crucial and longstanding role it has played in the biogeochemical cycles of Earth over billions of years. The counterproductive oxygenase activity of RuBisCO has persisted over billions of years of evolution, despite its competition with the carboxylase activity necessary for carbon fixation, yet hypotheses regarding the selective pressures governing RuBisCO evolution have been limited to speculation. Here we report the resurrection and biochemical characterization of ancestral RuBisCOs, dating back to over one billion years ago (Gyr ago). Our findings provide an ancient point of reference revealing divergent evolutionarymore » paths taken by eukaryotic homologues towards improved specificity for CO₂, versus the evolutionary emphasis on increased rates of carboxylation observed in bacterial homologues. Consistent with these distinctions, in vivo analysis reveals the propensity of ancestral RuBisCO to be encapsulated into modern-day carboxysomes, bacterial organelles central to the cyanobacterial CO₂ concentrating mechanism.« less

Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyzes carboxylation of ribulose-1,5- bisphosphate, the first in a series of reactions leading to the incorporation of atmospheric CO2 into biomass. Rubisco requires Rubisco activase (RCA), an AAA? ATPase that reactivates Rubisco by remodelling the conformation of inhibitor-bound sites. RCA is regulated by the ratio of ADP:ATP, with the precise response potentiated by redox regulation of the alpha-isoform. Measuring the effects of ADP on the activation of Rubisco by RCA using the wellestablished photometric assay is problematic because of the adenine nucleotide requirement of 3-phosphoglycerate (3- PGA) kinase. Described here is a novel assay for measuring RCAmore » activity in the presence of variable ratios of ADP:ATP. The assay couples the formation of 3-PGA from ribulose 1,5-bisphosphate and CO₂ to NADH oxidation through cofactor-dependent phosphoglycerate mutase, enolase, PEP carboxylase and malate dehydrogenase. The assay was used to determine the effects of Rubisco and RCA concentration and ADP:ATP ratio on RCA activity, and to measure the activation of a modified Rubisco by RCA. Variations of the basic assay were used to measure the activation state of Rubisco in leaf extracts and the activity of purified Rubisco. The assay can be automated for high-throughput processing by conducting the reactions in two stages.« less