C4 photosynthetic syndromes have evolved multiple times from ancestral C3 photosynthetic pathways. The genus Flaveria has served as a model group to study the evolution of C4 photosynthesis in eudicotyledons as it contains C3 and C4 species as well as species using intermediate types of photosynthesis (e.g. C3-C4 and C4-like). Carbonic anhydrase catalyzes the interconversion of CO2 and bicarbonate, and multiple forms of the enzyme exist in Flaveria species regardless of their photosynthetic type. In C4 species, a highly expressed β-carbonic anhydrase, βCA3a, catalyzes the first reaction of the C4 pathway in leaf mesophyll cells. To understand the evolution of βCA3a and its co-option into the C4 pathway of Flaveria, βCA3 isoforms and the genes encoding them were examined from C3 and clade A C3-C4, C4-like, and C4 Flaveria species. Phylogenetic analyses indicated the gene encoding a chloroplastic βCA was duplicated in the C3 ancestor of all Flaveria species. In the ancestor of Flaveria clade A C4-like and full C4 species, one of the resulting paralogs was duplicated and the chloroplast transit peptide of one of the two duplicates was lost. The resulting isoform, βCA3a, acquired mesophyll cell-specific expression in C4 Flaveria species through the domestication of a transposable element that harbors the mesophyll expression module1-like enhancer in the βca3a gene distal promoter. Reporter gene assays suggest sequences in a 319 bp proximal promoter region of βca3a act synergistically with this distal region to control high levels of βCA3a expression in C4 Flaveria mesophyll cells.
© The Author(s) 2026. Published by Oxford University Press on behalf of American Society of Plant Biologists.