Identification and expression of cotton (Gossypium hirsutum L.) plastidial carbonic anhydrase.
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 LacZ alpha 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 bighest 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.
Hoang
C V
CV
Department of Biological Sciences, University of North Texas, Denton 76203-5220, USA.
Wessler
H G
HG
Local
A
A
Turley
R B
RB
Benjamin
R C
RC
Chapman
K D
KD
D000595:Amino Acid Sequence; D001483:Base Sequence; D002256:Carbonic Anhydrases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D017343:Genes, Plant; D003368:Gossypium / Q000201:enzymology* / Q000235:genetics*; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D018087:Plastids / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D018744:DNA, Plant; D007527:Isoenzymes; D002256:Carbonic Anhydrases