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

• Published in: Plant and cell physiology 1999-12, Vol.40 (12), p.1262-1270
• Main Authors: Hoang, C.V. (University of North Texas, Denton (USA)), Wessler, H.G, Local A, Turley, R.B, Benjamin, R.C, Chapman, K.D
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Base Sequence; CARBONATE DEHYDRATASE; CARBONATE DESHYDRATASE; CARBONATO DESHIDRATASA; Carbonic Anhydrases - genetics; Carbonic Anhydrases - isolation & purification; Carbonic Anhydrases - metabolism; Cloning, Molecular; cotton (Gossypium hirsutum L.); DNA Primers - genetics; DNA, Complementary - genetics; DNA, Plant - genetics; Enzyme activity; Escherichia coli - genetics; EXPRESION GENICA; EXPRESSION DES GENES; GENE EXPRESSION; Genes, Plant; Gossypium - enzymology; Gossypium - genetics; GOSSYPIUM HIRSUTUM; IDENTIFICACION; IDENTIFICATION; Isoenzymes - genetics; Isoenzymes - isolation & purification; Isoenzymes - metabolism; Molecular Sequence Data; Plant gene expression; Plastidial carbonic anhydrase (EC 4.2.1.1); Plastids - enzymology; Sequence Homology, Amino Acid; Sulfonamide inhibitors
• ISSN: 0032-0781; 1471-9053
• DOI: 10.1093/oxfordjournals.pcp.a029514

Four carbonic anhydrase (CA) cDNA clones were isolated from a 48 h dark-grown cotton (Gossypium hirsutum L.) seedling cDNA library. Nucleotide sequence analysis revealed two different CA isoforms designated GhCA1 and GhCA2. The encoded polypeptides possess N-terminal serine/threonine-rich regions indicative of plastid transit peptides, and approximately 80% sequence identity to other plant plastidial beta-CAs. The GhCA1 cDNA encodes a nearly complete 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 LacZalpha fusion proteins in E. coli. Lysates of these strains contained 9-fold higher levels of CA activity as compared to untransformed controls and this activity was inhibited by CA-specific inhibitors. Sulfanilamide, acetazolamide, ethoxyzolamide, each at 10 mM, inhibited recombinant CA activity approximately 50%, 65%, and 75%, respectively. In plant tissue homogenates these inhibitors reduced CA activity by 50%, 70%, and 95%, respectively. Although CA activity was highest in extracts of mature cotton leaves, probing total RNA with GhCA1 revealed CA transcript levels to be highest in the cotyledons of dark-grown cotton seedlings. Collectively, our data indicate the presence of a plastid-localized CA in cotyledons of germinated seeds, suggesting a role for CA in postgerminative growth