Characterization and expression of the maize β-carbonic anhydrase gene repeat regions

In maize, carbonic anhydrase (CA; EC 4.2.1.1) catalyzes the first reaction of the C4 photosynthetic pathway; it catalyzes the hydration of CO2 to bicarbonate and provides an inorganic carbon source for the primary carboxylation reaction catalyzed by phosphoenolpyruvate (PEP) carboxylase. The β-CA isozymes from maize, as well as other agronomically important NADP-malic enzyme (NADP-ME) type C4 crops, have remained relatively uncharacterized but differ significantly from the β-CAs of other C4 monocot species primarily due to transcript length and the presence of repeat sequences. This research confirmed earlier findings of repeat sequences in maize CA transcripts, and demonstrated that the gene encoding these transcripts is also composed of repeat sequences. One of the maize CA genes was sequenced and found to encode two domains, with distinct groups of exons corresponding to the repeat regions of the transcript. We have also shown that expression of a single repeat region of the CA transcript produced active enzyme that associated as a dimer and was composed primarily of α-helices, consistent with that observed for other plant CAs. As the presence of repeat regions in the CA gene is unique to NADP-ME type C4 monocot species, the implications of these findings in the context of the evolution of the location and function of this C4 pathway enzyme are strongly suggestive of CA gene duplication resulting in an evolutionary advantage and a higher photosynthetic efficiency.