Functional characterization of two p-coumaroyl ester 3'-hydroxylase genes from coffee tree: evidence of a candidate for chlorogenic acid biosynthesis.
Chlorogenic acid (5-CQA) is one of the major soluble phenolic compounds that is accumulated in coffee green beans. With other hydroxycinnamoyl quinic acids (HQAs), this compound is accumulated in particular in green beans of the cultivated species Coffea canephora. Recent work has indicated that the biosynthesis of 5-CQA can be catalyzed by a cytochrome P450 enzyme, CYP98A3 from Arabidopsis. Two full-length cDNA clones (CYP98A35 and CYP98A36) that encode putative p-coumaroylester 3'-hydroxylases (C3'H) were isolated from C. canephora cDNA libraries. Recombinant protein expression in yeast showed that both metabolized p-coumaroyl shikimate at similar rates, but that only one hydroxylates the chlorogenic acid precursor p-coumaroyl quinate. CYP98A35 appears to be the first C3'H capable of metabolising p-coumaroyl quinate and p-coumaroyl shikimate with the same efficiency. We studied the expression patterns of both genes on 4-month old C. canephora plants and found higher transcript levels in young and in highly vascularized organs for both genes. Gene expression and HQA content seemed to be correlated in these organs. Histolocalization and immunolocalization studies revealed similar tissue localization for caffeoyl quinic acids and p-coumaroylester 3'-hydroxylases. The results indicated that HQA biosynthesis and accumulation occurred mainly in the shoot tip and in the phloem of the vascular bundles. The lack of correlation between gene expression and HQA content observed in some organs is discussed in terms of transport and accumulation mechanisms.
Mahesh
Venkataramaiah
V
Laboratoire de Génomique et Qualité du café, IRD, UMR 1097 DGPC, 911 Avenue Agropolis, BP 64501, 34394, Montpellier cedex 5, France.
Million-Rousseau
Rachel
R
Ullmann
Pascaline
P
Chabrillange
Nathalie
N
Bustamante
José
J
Mondolot
Laurence
L
Morant
Marc
M
Noirot
Michel
M
Hamon
Serge
S
de Kochko
Alexandre
A
Werck-Reichhart
Danièle
D
Campa
Claudine
C
D000595:Amino Acid Sequence; D002726:Chlorogenic Acid / Q000378:metabolism*; D040503:Coffea / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004952:Esters / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011801:Quinic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism; D016415:Sequence Alignment
D004952:Esters; D010940:Plant Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C472707:caffeoylquinic acid; D011801:Quinic Acid; D002726:Chlorogenic Acid; D003577:Cytochrome P-450 Enzyme System