Journal Article

Identification and Expression of Cotton (<i>Gossypium hirsutum</i> L.) Plastidial Carbonic Anhydrase

Chau V. Hoang, Heath G. Wessler, Andrea Local, Rickie B. Turley, Robert C. Benjamin and Kent D. Chapman

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 40, issue 12, pages 1262-1270
Published in print January 1999 | ISSN: 0032-0781
Published online January 1999 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/oxfordjournals.pcp.a029514
Identification and Expression of Cotton (Gossypium hirsutum L.) Plastidial Carbonic Anhydrase

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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 β-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 LacZa fusion proteins in E. coil. 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.

Keywords: cotton (Gossypium hirsutum L.); Enzyme activity; Plant gene expression; Plastidial carbonic anhydrase (EC 4.2.1.1); Sulfonamide inhibitors

Journal Article.  0 words. 

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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