Molecular evolution of rbcL in three gymnosperm families: identifying adaptive and coevolutionary patterns

The chloroplast-localized ribulose-1, 5-biphosphate carboxylase/oxygenase (Rubisco), the primary enzyme responsible for autotrophy, is instrumental in the continual adaptation of plants to variations in the concentrations of CO2. The large subunit (LSU) of Rubisco is encoded by the chloroplast rbcL gene. Although adaptive processes have been previously identified at this gene, characterizing the relationships between the mutational dynamics at the protein level may yield clues on the biological meaning of such adaptive processes. The role of such coevolutionary dynamics in the continual fine-tuning of RbcL remains obscure.

The gene rbcL has experienced bursts of adaptations in response to the changing concentration of CO2 in the atmosphere. These adaptations have emerged as a result of a continuous dynamic of mutations, many of which may have involved innovation of functional Rubisco features. Analysis of the protein structure and the functional implications of such mutations put forward the conclusion that this evolutionary scenario has been possible through a complex interplay between adaptive mutations, often structurally destabilizing, and compensatory mutations. Our results unearth patterns of evolution that have likely optimized the Rubisco activity and uncover mutational dynamics useful in the molecular engineering of enzymatic activities. Reviewers: This article was reviewed by Prof. Christian Blouin (nominated by Dr W Ford Doolittle), Dr Endre Barta (nominated by Dr Sandor Pongor), and Dr Nicolas Galtier.

We used the timescale and phylogenetic analyses to investigate and search for processes of adaptive evolution in rbcL gene in three gymnosperm families, namely Podocarpaceae, Taxaceae and Cephalotaxaceae. To understand the relationships between regions identified as having evolved under adaptive evolution, we performed coevolutionary analyses using the software CAPS. Importantly, adaptive processes were identified at amino acid sites located on the contact regions among the Rubisco subunits and on the interface between Rubisco and its activase. Adaptive amino acid replacements at these regions may have optimized the holoenzyme activity. This hypothesis was pinpointed by evidence originated from our analysis of coevolution that supported the correlated evolution between Rubisco and its activase. Interestingly, the correlated adaptive processes between both these proteins have paralleled the geological variation history of the concentration of atmospheric CO2. Conclusions: The gene rbcL has experienced bursts of adaptations in response to the changing concentration of CO2 in the atmosphere. These adaptations have emerged as a result of a continuous dynamic of mutations, many of which may have involved innovation of functional Rubisco features. Analysis of the protein structure and the functional implications of such mutations put forward the