Loss of the Transit Peptide and an Increase in Gene Expression of an Ancestral Chloroplastic Carbonic Anhydrase Were Instrumental in the Evolution of the Cytosolic C₄ Carbonic Anhydrase in Flaveria

C4 photosynthesis has evolved multiple times from ancestral C3 species. Carbonic anhydrase (CA) catalyzes the reversiblehydration of CO2 and is involved in both C3 and C4 photosynthesis; however, its roles and the intercellular and intracellularlocations of the majority of its activity differ between C3 and C4 plants. To understand the molecular changes underlying theevolution of the C4 pathway, three cDNAs encoding distinct b-CAs (CA1, CA2, and CA3) were isolated from the leaves of theC3 plant Flaveria pringlei. The phylogenetic relationship of the F. pringlei proteins with other embryophyte b-CAs wasreconstructed. Gene expression and protein localization patterns showed that CA1 and CA3 demonstrate high expression inleaves and their products localize to the chloroplast, while CA2 expression is low in all organs examined and encodes acytosolic enzyme. The roles of the F. pringlei enzymes were considered in light of these results, other angiosperm b-CAs, andArabidopsis (Arabidopsis thaliana) “omics” data. All three F. pringlei CAs have orthologs in the closely related C4 plant Flaveriabidentis, and comparisons of ortholog sequences, expression patterns, and intracellular locations of their products indicatedthat CA1 and CA2 have maintained their ancestral role in C4 plants, whereas modifications to the C3 CA3 gene led to theevolution of the CA isoform that catalyzes the first step in the C4 photosynthetic pathway. These changes included the loss ofthe chloroplast transit peptide and an increase in gene expression, which resulted in the high levels of CA activity seen in thecytosol of C4 mesophyll cells.