|Hoang, C - UNIV. OF NORTH TEXAS|
|Wessler, H - UNIV. OF NORTH TEXAS|
|Local, A - UNIV. OF NORTH TEXAS|
|Benjamin, R - UNIV. OF NORTH TEXAS|
|Chapman, K - UNIV. OF NORTH TEXAS|
Submitted to: Plant And Cell Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 15, 1999
Publication Date: N/A
Interpretive Summary: Carbonic anhydrase (CA) is an enzyme which catalyzes an increased rate of equilibration between CO2 and HCO3. This protein is usually associated with providing substrates to produce sugars during photosynthesis, and in the green alga Chlamydomnas, operates as a carbon dioxide concentrating mechanism. Other metabolic functions are likely, but as yet, not characterized. In this study, two CA clones have been isolated and characterized from dark-grown cotton cotyledons. These clones show high similarity to other CA from plants and include the plastid transit peptide which is responsible for directing the protein to and into the plastid. As would be expected the high levels of mRNA encoding CA are found in mature leaves, however, the highest levels are found in dark-grown (non photosynthesizing) cotyledons. The function of CA in non photosynthesizing is yet to be understood and may function in fatty acid metabolism, which is srapidly occurring in 48 h dark-grown seedlings.
Technical Abstract: Four carbonic anhydrase (CA) cDNA clones were isolated from a 48 h dark- grown cotton (Gossypium hirsutum L.) seedling 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 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 5 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 the highest in the cotyledons of dark- grown cotton seedlings. Collectively, our data indicate the presence of a plastid-localized CA in cotyledons of germinated cottonseeds, suggesting a role for CA in postgerminative growth.