Identification and expression of cotton (Gossypium hirsutum L.) plastidialcarbonic anhydrase

Fear carbonic anhydrase (CA) cDNA clones were isolated from a 48 h dark-gro wn cotton (Gossypium hirsutum L.) seedling cDNA library, Nucleotide sequenc e analysis revealed, two different CA isoforms designated GhCA1 and GhCA2. The encoded polypeptides possess N-terminal serine/threonine-rich regions i ndicative of plastid transit peptides, and approximately 80% sequence ident ity to other plant plastidial beta-CAs, The GhCA1 cDNA encodes a nearly com plete preprotein of 323 amino acids with a molecular mass of 34.9 kDa and a predicted mature protein of 224 amino acids with a molecular mass of 24.3 kDa. Eleven nucleotide differences within ORFs of GhCA1 and GhCA2 result in 5 conservative amino acid substitutions. The 3' GhCA2 untranslated region contains five additional substitutions and one single nucleotide addition. GhCA1 clones, nearly full-length or with 70% of the transit peptide deleted , were expressed as LacZa fusion proteins in E. coli. Lysates of these stra ins contained 9-fold higher levels of CA activity as compared to untransfor med controls and this activity was inhibited by CA-specific inhibitors. Sul fanilamide, acetazolamide, ethoxyzolamide, each at 10 mM, inhibited recombi nant CA activity approximately 50%, 65%, and 75%, respectively. In plant ti ssue homogenates these inhibitors reduced CA activity by 50%, 70%, and 95%, respectively. Although CA activity was highest in extracts of mature cotto n leaves, probing total RNA with GhCA1 revealed CA transcript levels to be highest in, the cotyledons of dark-grown cotton seedlings. Collectively, ou r data indicate the presence of a plastid-localized CA in cotyledons of ger minated seeds, suggesting a role for GA in postgerminative growth.