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

C ₄ 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 C ₃ to C ₄ photosynthesis. The chromosome-scale genome sequences of five Flaveria species, representing C ₃ , intermediate, and C ₄ 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 C ₄ 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 C ₄ genes in C ₄ species compared to non-C ₄ species. Notably, C ₄ genes exhibited elevated both RNA and protein levels but decreased protein-to-RNA ratios in C ₄ 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 C ₄ photosynthesis. Our comprehensive dataset also provides valuable resources to study the evolution and regulatory mechanisms underlying C ₄ photosynthesis.