Abstract: Rubisco is assembled from large subunits (encoded by chloroplast gene rbcL) and small subunits (encoded by the nuclear rbcS multigene family), which are involved in the processes of carbon dioxide fixation in the Calvin cycle of photosynthesis. Although Rubisco has been studied in many plants, the evolutionary divergences among the different rbcS genes are still largely unknown. Here, using a rice closely related wild species, Oryza punctata Kotschy ex Steud, we investigated the differential properties of the rbcS genes in the species. We identified five rbcS genes (OprbcS1 through OprbcS5), OprbcS1 showed a different evolutionary pattern from the remaining four genes in terms of chromosome location, gene structure, and sequence homology. Phylogenetic analysis revealed that plant rbcS1 and other non-rbcS1 genes originated from a common ancient duplication event that occurred at least in seed plants ancestor. RbcS1 was then retained in a few plant lineages, including Oryza, whereas non-rbcS1 was mainly amplified in angiosperms. OprbcS1, OprbcS2–OprbcS4, and OprbcS5 were prominently expressed in stems and seeds, young leaves, and mature leaves, respectively. The yeast two-hybrid assay detected a significant decrease in the interaction between OprbcS1 and OprbcL compared to the other four pairs of proteins (OprbcS2–OprbcS5 and OprbcL). We propose that OprbcS1 might be assigned a divergent function that was predominantly specific to nonphotosynthetic organs, whereas OprbcS2–OprbcS5, having different affinity in the assembly process of Rubisco, might be subfunctionalized in photosynthetic organs. This study not only deepens our understanding of the fine assembly of Rubisco, but also sheds some light on future de novo domestication of wild rice.

Key words: functional divergence, protein–protein interaction, Rubisco, subfunctionalization, wild rice