A dominant role of transcriptional regulation during the evolution of C(4) 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.