A dominant role of transcriptional regulation during the evolution of C4 photosynthesis in Flaveria species

C ₄ photosynthesis exemplifies convergent evolution of complex traits. Herein, we construct chromosome-scale genome assemblies and perform multi-omics analysis for five Flaveria species, which represent evolutionary stages from C ₃ to C ₄ photosynthesis. Chromosome-scale genome sequence analyses reveal a gradual increase in genome size during the evolution of C ₄ photosynthesis attributed to the expansion of transposable elements. Systematic annotation of genes encoding C ₄ enzymes and transporters identify additional copies of three C ₄ enzyme genes through retrotranspositions in C ₄ species. C ₄ genes exhibit elevated mRNA and protein abundances, reduced protein-to-RNA ratios, and comparable translation efficiencies in C ₄ species, highlighting a critical role of transcriptional regulation in C ₄ evolution. Furthermore, we observe an increased abundance of ethylene response factor (ERF) transcription factors and cognate cis -regulatory elements associated with C ₄ genes regulation. Altogether, our study provides valuable genomic resources for the Flaveria genus and sheds lights on evolutionary and regulatory mechanisms underlying C ₄ photosynthesis.