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

Abstract

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 these correlations, and thus the strength of catalytic trade-offs, have been over-estimated due to the presence of phylogenetic signal in the kinetic trait data (Bouvier et al., 2021). We demonstrated that only canonical 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 either incorrect or invalid. As such, we stand by our original conclusions. Specifically, the magnitude of rubisco catalytic trade-offs have been overestimated in previous analyses due to phylogenetic biases, and rubisco kinetic evolution has in fact been more limited by phylogenetic constraint.

Rubisco is evolving for improved catalytic efficiency and CO2assimilation in plants

“…Despite this immense throughput, the enzyme is a surprisingly inefficient catalyst with a modest carboxylase turnover rate of <12 s -1 60 and a competing oxygenase activity that results in the loss of fixed carbon 63,64,86 . This discord presents an evolutionary paradox that has attracted significant attention 15,21,22,63,67,[74][75][76][77][78] , with the prevailing assumption being that rubisco is evolutionarily stagnant. Here we demonstrate that the enzyme is not stagnant, but that it is encoded by one of the slowest evolving genes on Earth.…”

“…We hypothesised that if rubisco was undergoing directional selection for improved catalysis, then orthologs that have experienced the largest extent of molecular evolution would be the most efficient catalysts. To test this hypothesis, a dataset of kinetic measurements from C3 angiosperms 21,22,81 was evaluated in the context of the molecular evolution of RbcL (Figure 4A,B). This analysis focused on RbcL as it is the primary determinant of kinetics [21][22][23][24][25][26][27][28][29][30][31][32] , and because sufficient sequence data for RbcS are unavailable.…”

“…To test this hypothesis, a dataset of kinetic measurements from C3 angiosperms 21,22,81 was evaluated in the context of the molecular evolution of RbcL (Figure 4A,B). This analysis focused on RbcL as it is the primary determinant of kinetics [21][22][23][24][25][26][27][28][29][30][31][32] , and because sufficient sequence data for RbcS are unavailable. This revealed that the more RbcL has evolved from the most recent common ancestral sequence, the better its CO2/O2 specificity (SC/O; 10.1% variance explained), CO2 turnover rate (kcatC; 4.6% variance explained) and carboxylation efficiency (kcatC/KC; 3.8% variance explained) (Figure 4B).…”

“…Within Form I rubisco the active site is located in RbcL 15,19,20 . As a result, interspecific differences in Form I kinetics are primarily attributable to sequence variation in RbcL [21][22][23][24][25][26][27][28][29][30][31][32] . Despite not playing a direct role in catalysis RbcS influences the function of rubisco 33 , and its incorporation in the holoenzyme enables its higher kinetic efficiency 34 .…”

“…Moreover, there is increasing recognition of the importance of both environment 57 and organ-specific 58,59 differences in plant RbcS isoform expression on holoenzyme catalysis. However, even though RbcS influences holoenzyme function, sequence variation in RbcL remains the primary determinant of variation in kinetics [21][22][23][24][25][26][27][28][29][30][31][32] .…”