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

Abstract

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.

Keywords: adaptation; evolution; kinetics; photosynthesis; rubisco.

Fig. 1.

The evolutionary history of rubisco in the context of atmospheric CO₂ (%) and O₂ (%) following divergence from the ancestral rubisco-like protein (RLP). Important branch points in the phylogeny at which rubisco diverged into different evolutionary lineages are indicated by gray vertical bars. To provide additional context, the time-period at which the First and Second Great Oxidation events occurred along this evolutionary trajectory are also labeled and referenced as gray vertical bars. Graphics of atmospheric CO₂ and O₂ levels were adapted from the TimeTree resource [http://www.timetree.org; (9)].

Fig. 2.

The extent of molecular evolution in rubisco during the radiation of each taxonomic group. (A) Bar plot depicting the total amount of molecular evolution (substitutions per sequence site) in the nucleotide and protein sequences of Form I rubisco across taxonomic groups. RbcL: the extent of sequence evolution in the RbcL subunit. RbcS: the extent of sequence evolution in the RbcS subunit. RbcS*: the extent of sequence evolution in the RbcS subunit using 1,000 bootstrapped stratified sampling of rbcS/RbcS per species (Materials and Methods). The genome in which rbcL and rbcS genes reside within each group is indicated above the plot (bacterial, plastid, nuclear). Error bars represent ± 1 SD of the mean. (B) Bar plot depicting the percentage ratio (%) of nucleotide or amino acid evolution between each rubisco subunit (rbcL to rbcS and RbcL to RbcS, respectively) in each taxonomic group. The color of each bar is determined by the genome in which the rbcL and rbcS gene resides, following the color scale in A. (C) Bar plot depicting the percentage ratio (%) of nucleotide to amino acid evolution in each rubisco subunit (rbcL to RbcL and rbcS to RbcS, respectively) in each taxonomic group. The color of each bar is the same as described in B. Dashed lines indicate the expected ratio given an rbcL or rbcS sequence evolving in the absence of selection.

Fig. 3.

The extent of molecular evolution in rubisco in the context of other genes. (A) Boxplot of the extent of molecular evolution (substitutions per sequence site) in the nucleotide and protein sequences of the rbcL/RbcL and rbcS/RbcS subunit expressed as a percentile (%) of that measured across all other genes and proteins, respectively. See also SI Appendix, Supplemental File 1 and Table S2. (B) As in A but calculating the percentile (%) extent of rubisco molecular evolution (substitutions per sequence site) relative to only the subset of genes and proteins in each species which encode enzymes. See also SI Appendix, Supplemental File 1 and Table S3. (C) Boxplot of the total amount of molecular evolution (substitutions per sequence site) in the nucleotide and protein sequences of each Calvin–Benson–Bassham cycle enzyme expressed as a percentage (%) of that measured in rbcL/RbcL (100%; red horizontal line) across land plants. Phosphoglycerate kinase: PGK. Glyceraldehyde-3-phosphate dehydrogenase A/B subunit: GAPDH-A/GAPDH-B. Triose phosphate isomerase: TPI. Fructose-bisphosphate aldolase: FBA. Fructose-1,6-bisphosphatase: FBP. Transketolase: TKL. Sedoheptulose-bisphosphatase: SBP. Ribose 5-phosphate isomerase: RPI. Ribulose-p-3-epimerase: RPE. Phosphoribulokinase: PRK. See also SI Appendix, Supplemental File 1 and Tables S4 and S5. The raw data for this figure can be found in SI Appendix, Supplemental File 5.

Fig. 4.

The relationship between rubisco molecular and kinetic evolution in C₃ angiosperms. (A) The relationship between RbcL evolution and its corresponding kinetic trait values. AA Ev.: The extent of RbcL amino acid evolution that has occurred since the last common ancestor at the root of the angiosperm phylogeny. S_C/O: specificity. k_catC: carboxylase turnover per site. k_catC/K_C: carboxylation efficiency. K_C: the Michaelis constant for CO₂. K_C^air: the inferred Michaelis constant for CO₂ in 20.95% O₂. K_O: the Michaelis constant for O₂. K_C/K_O: the ratio of the Michaelis constant for CO₂ compared to O₂. (B) The relationship between the extent of RbcL protein evolution (substitutions per sequence site) and each rubisco kinetic trait in A as assessed using least squares regression models. The raw data can be found in SI Appendix, Supplemental File 7.

Fig. 5.

The relationship between rubisco molecular evolution and CO₂ assimilation in C₃ angiosperms. (A) The relationship between the extent of RbcL evolution and leaf-level CO₂ assimilation. AA Ev.: The extent of RbcL amino acid evolution that has occurred since the most recent common ancestor at the root of the angiosperm phylogeny. A_mass: Photosynthetic rate per unit leaf mass. PNUE_mass: Photosynthetic nitrogen use efficiency rate, calculated as photosynthetic rate per unit leaf mass expressed per unit leaf mass nitrogen content (N_mass; % N). A_area: Photosynthetic rate per unit leaf area. PNUE_area: Photosynthetic nitrogen use efficiency rate, calculated as photosynthetic rate per unit leaf area expressed per unit leaf area nitrogen content (N_area; g m⁻² N). (B) The relationship between the extent of RbcL protein evolution (substitutions per sequence site) and each photosynthetic trait in A evaluated on a mass-basis (A_mass, PNUE_mass) as assessed using least squares regression models. (C) As in B but for each photosynthetic trait evaluated on an area-basis (A_area, PNUE_area). The raw data can be found in SI Appendix, Supplemental File 9.