cDNA cloning, gene expression and subcellular localization of anthocyanin 5-aromatic acyltransferase from Gentiana triflora.
Acylation of anthocyanins with hydroxycinnamic acid derivatives is one of the most important and less under-stood modification reactions during anthocyanin biosynthesis. Anthocyanin aromatic acyltransferase catalyses the transfer of hydroxycinnamic acid moieties from their CoA esters to the glycosyl groups of anthocyanins. A full-length cDNA encoding the anthocyanin 5-aromatic acyltransferase (5AT) (EC 2.3.1.153) that acylates the glucose bound at the 5-position of anthocyanidin 3,5-diglucoside was isolated from petals of Gentiana triflora on the basis of the amino acid sequence of the purified enzyme. The isolated full-length cDNA had an open reading frame of 469 amino acids and the calculated molecular weight was 52,736. The deduced amino acid sequence contains consensus motifs that are conserved among the putative acyl CoA-mediated acyltransferases, and this indicates that 5AT is a member of a proposed superfamily of multi-functional acyltransferases (St-Pierre et al. (1998) Plant J. 14, 703-713). The cDNA was expressed in Escherichia coli and yeast, and confirmed to encode 5AT. The enzymatic characteristics of the recombinant 5AT were consistent with those of the native gentian 5AT. Immunoblot analysis using specific antibodies to 5AT showed that the 5AT protein is present in petals, but not in sepals, stems or leaves of G. triflora. RNA blot analysis showed that the 5AT gene is expressed only in petals and that its expression is temporally regulated during flower development coordinately with other anthocyanin biosynthetic genes. Immunohistochemical analysis demonstrated that the 5AT protein is specifically expressed in the outer epidermal cells of gentian petals and that it is localized mainly in the cytosol.
Fujiwara
H
H
Institute for Fundamental Research, Suntory Ltd, Osaka, Japan.
Tanaka
Y
Y
Yonekura-Sakakibara
K
K
Fukuchi-Mizutani
M
M
Nakao
M
M
Fukui
Y
Y
Yamaguchi
M
M
Ashikari
T
T
Kusumi
T
T
D000217:Acyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000872:Anthocyanins / Q000378:metabolism; D000906:Antibodies; D017360:Arabidopsis / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D016384:Consensus Sequence; D017124:Conserved Sequence; D018076:DNA, Complementary; D007700:Kinetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D018547:Plant Stems; D010944:Plants / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology
D000872:Anthocyanins; D000906:Antibodies; D018076:DNA, Complementary; D011994:Recombinant Proteins; D000217:Acyltransferases; C109044:hydroxycinnamoyl-CoA anthocyanidin 3,5-diglucoside 5-O-glucoside-6'''-O-hydroxycinnamoyltransferase