Integrated genomic analysis revealed a dominant role of transcriptional regulation during the evolution of C4 photosynthesis in Flaveria species

Abstract C4 photosynthesis represents a remarkable example of convergent evolution of complex traits. In this study, we present an integrated genomic analysis of the genus Flaveria, which includes species at different evolutionary stages from C3 to C4 photosynthesis. The chromosome-scale genome sequences of five Flaveria species, representing C3, intermediate, and C4 types, reveal a gradual increase in genome size attributed to the expansion of transposable elements (TEs). Through a combination of gene phylogeny and transcript abundance analysis, we systematically identified genes encoding C4 enzymes and transporters. We further found that C4 species acquired additional copies of genes encoding three C4 enzymes through retrotranspositions. Moreover, we observed an increased abundance of ERF cis-regulatory elements and ERF transcriptional factors associated with the regulation of C4 genes in C4 species compared to non-C4 species. Notably, C4 genes exhibited elevated both RNA and protein levels but decreased protein-to-RNA ratios in C4 species. Additionally, codon usage patterns are conserved among the five Flaveria species. All these results suggest a predominant role of transcriptional regulation in the evolution of C4 photosynthesis. Our comprehensive dataset also provides valuable resources to study the evolution and regulatory mechanisms underlying C4 photosynthesis.