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.

A catalytic triad – Lys-Asn-Asp – Is essential for the catalysis of the methyl transfer in plant cation-dependent O-methyltransferases

“…few exceptions, CCoAOMTs from plants show conserved position specificity towards the meta-hydroxyl group of catechols consistent with the vanilloid types of substitution patterns observed in plants (Anterola and Lewis, 2002;Wils et al, 2013;Widiez et al, 2012).…”

Functional characterization of a plastidal cation-dependent O-methyltransferase from the liverwort Plagiochasma appendiculatum

“…2A). The deduced PaOMT1 sequence shared respectively 40.7%, 40.4%, 49.7% and 40.79% identity with McPFOMT (AY145521.1) (Ibdah et al, 2003), At4g26220 (AY056313.1) (Wils et al, 2013), MsCCoAOMT (AAC28973.1) (Ferrer et al, 2005) and VpOMT4 (JF344740) (Widiez et al, 2011) (Fig. 2B).…”

“…Class I OMTs include a group of low molecular weight (23-29 kDa) and Mg 2+ dependent enzymes that specifically methylate the coenzyme-A linked esters of the lignin biosynthetic pathway, caffeoyl-CoA (3) and 5-hydroxyferuloyl-CoA, and are generally referred to as caffeoyl CoA OMTs (CCoAOMTs) (Inoue et al, 1998). Several plant species, such as Mesembryanthemum crystallinum (Ibdah et al, 2003), Arabidopsis thaliana (Raes et al, 2003), rice (Lee et al, 2008), poplar (Tsai et al, 2006) and Vanilla planifolia (Widiez et al, 2011), also harbor genes encoding CCoAOMT-like enzymes able to methylate various flavonoids, anthocyanins, coumarins and aromatic esters (Hamberger et al, 2007). Although CCoAOMT-like enzymes display a broad substrate specificity, they tend to exhibit distinct substrate preferences; thus, the M. crystallinum enzyme PFOMT acts on a spectrum of substrates which share a vicinal dihydroxyl structure, but prefers the phenylpropanoids caffeic acid esters and flavonols, and in particular quercetagetin (Ibdah et al, 2003).…”

“…Although CCoAOMT-like enzymes display a broad substrate specificity, they tend to exhibit distinct substrate preferences; thus, the M. crystallinum enzyme PFOMT acts on a spectrum of substrates which share a vicinal dihydroxyl structure, but prefers the phenylpropanoids caffeic acid esters and flavonols, and in particular quercetagetin (Ibdah et al, 2003). In contrast, the most effective substrate for the enzymes VpOMT4 and VpOMT5 is the flavone tricetin (Widiez et al, 2011). CCoAOMT-like enzymes can be distinguished from the lignin-specific CCoAOMTs not only through their differential substrate specificity, but also by clear differences in their polypeptide sequence .…”

“…Thus, in vivo, their activity results in vanilloid substitutions (Davin et al, 2008;Widiez et al, 2011). CCoAOMT-like enzymes involved in para-O-methylation (an isovanilloid methylation pattern) have been identified in some …”