Convergence, stability, and thermal adaptation of the rubisco large subunit in plants

Enzymes are adapted to perform optimally in different thermal regimes that would otherwise alter kinetics and stability. Whether adaptive evolution in the photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) also compensates for thermal variation remains uncertain. We examined molecular evolution and modeled the change in free energy of protein folding (ΔΔG, where negative values indicate stabilization) of the rubisco large subunit (RbcL) in four phylogenetically distant plant genera: wood ferns (Dryopteris), sea lavenders (Limonium), pines (Pinus), and viburnums (Viburnum). Using codon evolutionary models in each genus, we observed widespread positive selection and parallel substitution in the catalytic α/β barrel domain. Species with warmer growing seasons had derived amino acids with stronger hydrogen bond contributions to ΔΔG. Protein structure-based modeling showed that the hydrogen bond contribution to stability tracked the growing season temperature of species carrying the derived amino acid. Stronger hydrogen bonds were offset by weaker contributions from hydrophobic solvation interactions, such that total ΔΔG showed no relationship with growing season temperature. In Viburnum, the strength of positive selection differed among biomes, with cold temperate and cloud forest clades showing stronger positive selection. These patterns are consistent with environmental tuning of non-covalent interactions within the enzyme. However, modest effect sizes indicate that other components of the rubisco holoenzyme likely also contribute to its thermal evolution.