title: Functional characterization of two new members of the caffeoyl CoA O-methyltransferase-like gene family from Vanilla planifolia reveals a new class of plastid-localized O-methyltransferases.
abstract: Caffeoyl CoA O-methyltransferases (OMTs) have been characterized from numerous plant species and have been demonstrated to be involved in lignin biosynthesis. Higher plant species are known to have additional caffeoyl CoA OMT-like genes, which have not been well characterized. Here, we identified two new caffeoyl CoA OMT-like genes by screening a cDNA library from specialized hair cells of pods of the orchid Vanilla planifolia. Characterization of the corresponding two enzymes, designated Vp-OMT4 and Vp-OMT5, revealed that in vitro both enzymes preferred as a substrate the flavone tricetin, yet their sequences and phylogenetic relationships to other enzymes are distinct from each other. Quantitative analysis of gene expression indicated a dramatic tissue-specific expression pattern for Vp-OMT4, which was highly expressed in the hair cells of the developing pod, the likely location of vanillin biosynthesis. Although Vp-OMT4 had a lower activity with the proposed vanillin precursor, 3,4-dihydroxybenzaldehyde, than with tricetin, the tissue specificity of expression suggests it may be a candidate for an enzyme involved in vanillin biosynthesis. In contrast, the Vp-OMT5 gene was mainly expressed in leaf tissue and only marginally expressed in pod hair cells. Phylogenetic analysis suggests Vp-OMT5 evolved from a cyanobacterial enzyme and it clustered within a clade in which the sequences from eukaryotic species had predicted chloroplast transit peptides. Transient expression of a GFP-fusion in tobacco demonstrated that Vp-OMT5 was localized in the plastids. This is the first flavonoid OMT demonstrated to be targeted to the plastids.
authors.affiliation: Department of Plant Biology and Pathology, School of Environmental and Biological Sciences, Rutgers University, 59 Dudley Road, New Brunswick, NJ 08901, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Widiez
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Hartman
authors.forename: Nativ
authors.initials: N
authors.lastname: Dudai
authors.forename: Qing
authors.initials: Q
authors.lastname: Yan
authors.forename: Michael
authors.initials: M
authors.lastname: Lawton
authors.forename: Daphna
authors.initials: D
authors.lastname: Havkin-Frenkel
authors.forename: Faith C
authors.initials: FC
authors.lastname: Belanger
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D015723:Gene Library; D049452:Green Fluorescent Proteins / Q000032:analysis; D008780:Methyltransferases / Q000032:analysis / Q000302:isolation & purification / Q000502:physiology*; D008969:Molecular Sequence Data; D010802:Phylogeny; D010940:Plant Proteins / Q000032:analysis / Q000302:isolation & purification / Q000502:physiology*; D018087:Plastids / Q000737:chemistry*; D016415:Sequence Alignment; D031669:Vanilla / Q000737:chemistry*
chemical_list: D010940:Plant Proteins; D049452:Green Fluorescent Proteins; D008780:Methyltransferases; C059825:caffeoyl-CoA O-methyltransferase