Rubisco is evolving for improved catalytic efficiency and CO2 assimilation in plants.

Rubisco is the primary entry point for carbon into the biosphere. However, rubisco is widely regarded as inefficient leading many to question whether the enzyme can adapt to become a better catalyst. Through a phylogenetic investigation of the molecular and kinetic evolution of Form I rubisco we uncover the evolutionary trajectory of rubisco kinetic evolution in angiosperms. We show that rbcL is among the 1% of slowest-evolving genes and enzymes on Earth, accumulating one nucleotide substitution every 0.9 My and one amino acid mutation every 7.2 My. Despite this, rubisco catalysis has been continually evolving toward improved CO2/O2 specificity, carboxylase turnover, and carboxylation efficiency. Consistent with this kinetic adaptation, increased rubisco evolution has led to a concomitant improvement in leaf-level CO2 assimilation. Thus, rubisco has been slowly but continually evolving toward improved catalytic efficiency and CO2 assimilation in plants.

Evolution: Unearthing missing links in the origin of our planet's primary carbon-fixing enzyme.

Form I rubisco underpins life on Earth, but the origins of its anomalous and highly complex multimeric structure have long proven elusive. New research helps to unravel this enigma by uncovering key missing links in the enzyme's evolutionary history.

Brassicaceae display variation in efficiency of photorespiratory carbon-recapturing mechanisms.

Carbon-concentrating mechanisms enhance the carboxylase efficiency of Rubisco by providing supra-atmospheric concentrations of CO2 in its surroundings. Beside the C4 photosynthesis pathway, carbon concentration can also be achieved by the photorespiratory glycine shuttle which requires fewer and less complex modifications. Plants displaying CO2 compensation points between 10 ppm and 40 ppm are often considered to utilize such a photorespiratory shuttle and are termed 'C3-C4 intermediates'. In the present study, we perform a physiological, biochemical, and anatomical survey of a large number of Brassicaceae species to better understand the C3-C4 intermediate phenotype, including its basic components and its plasticity. Our phylogenetic analysis suggested that C3-C4 metabolism evolved up to five times independently in the Brassicaceae. The efficiency of the pathway showed considerable variation. Centripetal accumulation of organelles in the bundle sheath was consistently observed in all C3-C4-classified taxa, indicating a crucial role for anatomical features in CO2-concentrating pathways. Leaf metabolite patterns were strongly influenced by the individual species, but accumulation of photorespiratory shuttle metabolites glycine and serine was generally observed. Analysis of phosphoenolpyruvate carboxylase activities suggested that C4-like shuttles have not evolved in the investigated Brassicaceae. Convergent evolution of the photorespiratory shuttle indicates that it represents a distinct photosynthesis type that is beneficial in some environments.

Response to Tcherkez and Farquhar: Rubisco adaptation is more limited by phylogenetic constraint than by catalytic trade-off.

Rubisco is the primary entry point for carbon into the biosphere. It has been widely proposed that rubisco is highly constrained by catalytic trade-offs due to correlations between the enzyme's kinetic traits across species. In previous work, we have shown that the strength of these correlations, and thus the strength of catalytic trade-offs, have been overestimated due to the presence of phylogenetic signal in the kinetic trait data (Bouvier et al., 2021). We demonstrated that only the trade-offs between the Michaelis constant for CO2 and carboxylase turnover, and between the Michaelis constants for CO2 and O2 were robust to phylogenetic effects. We further demonstrated that phylogenetic constraints have limited rubisco adaptation to a greater extent than the combined action of catalytic trade-offs. Recently, however, our claims have been contested by Tcherkez and Farquhar (2021), who have argued that the phylogenetic signal we detect in rubisco kinetic traits is an artefact of species sampling, the use of rbcL-based trees for phylogenetic inference, laboratory-to-laboratory variability in kinetic measurements, and homoplasy of the C4 trait. In the present article, we respond to these criticisms on a point-by-point basis and conclusively show that all are unfounded. As such, we stand by our original conclusions. Namely, although rubisco kinetic evolution has been limited by biochemical trade-offs, these are not absolute and have been previously overestimated due to phylogenetic biases. Instead, rubisco adaptation has in fact been more limited by phylogenetic constraint.

Rubisco Adaptation Is More Limited by Phylogenetic Constraint Than by Catalytic Trade-off.

Rubisco assimilates CO2 to form the sugars that fuel life on earth. Correlations between rubisco kinetic traits across species have led to the proposition that rubisco adaptation is highly constrained by catalytic trade-offs. However, these analyses did not consider the phylogenetic context of the enzymes that were analyzed. Thus, it is possible that the correlations observed were an artefact of the presence of phylogenetic signal in rubisco kinetics and the phylogenetic relationship between the species that were sampled. Here, we conducted a phylogenetically resolved analysis of rubisco kinetics and show that there is a significant phylogenetic signal in rubisco kinetic traits. We re-evaluated the extent of catalytic trade-offs accounting for this phylogenetic signal and found that all were attenuated. Following phylogenetic correction, the largest catalytic trade-offs were observed between the Michaelis constant for CO2 and carboxylase turnover (∼21-37%), and between the Michaelis constants for CO2 and O2 (∼9-19%), respectively. All other catalytic trade-offs were substantially attenuated such that they were marginal (<9%) or non-significant. This phylogenetically resolved analysis of rubisco kinetic evolution also identified kinetic changes that occur concomitant with the evolution of C4 photosynthesis. Finally, we show that phylogenetic constraints have played a larger role than catalytic trade-offs in limiting the evolution of rubisco kinetics. Thus, although there is strong evidence for some catalytic trade-offs, rubisco adaptation has been more limited by phylogenetic constraint than by the combined action of all catalytic trade-offs.

C4 anatomy can evolve via a single developmental change.

C4 photosynthesis is a complex trait that boosts productivity in warm environments. Paradoxically, it evolved independently in numerous plant lineages, despite requiring specialised leaf anatomy. The anatomical modifications underlying C4 evolution have previously been evaluated through interspecific comparisons, which capture numerous changes besides those needed for C4 functionality. Here, we quantify the anatomical changes accompanying the transition between non-C4 and C4 phenotypes by sampling widely across the continuum of leaf anatomical traits in the grass Alloteropsis semialata. Within this species, the only trait that is shared among and specific to C4 individuals is an increase in vein density, driven specifically by minor vein development that yields multiple secondary effects facilitating C4 function. For species with the necessary anatomical preconditions, developmental proliferation of veins can therefore be sufficient to produce a functional C4 leaf anatomy, creating an evolutionary entry point to complex C4 syndromes that can become more specialised.