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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

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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.

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Acknowledgments

We thank Dr. Richard A. Dixon for supplying some of the OMT substrates. We acknowledge NIH grant S10RR025424 to Nilgun Tumer for a confocal microscope.

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Authors and Affiliations

  1. Department of Plant Biology and Pathology, School of Environmental and Biological Sciences, Rutgers University, 59 Dudley Road, New Brunswick, NJ, 08901, USA

    Thomas Widiez, Qing Yan, Michael Lawton, Daphna Havkin-Frenkel & Faith C. Belanger

  2. Center for Advanced Food Technology, School of Environmental and Biological Sciences, Rutgers University, New Brunswick, NJ, 08901, USA

    Thomas G. Hartman

  3. Aromatic, Medicinal and Spice Crops Unit, Newe Ya’ar Research Center, Agricultural Research Organization, Ramat Yishay, Israel

    Nativ Dudai

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  1. Thomas Widiez
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  2. Thomas G. Hartman
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  4. Qing Yan
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Correspondence to Faith C. Belanger.

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Widiez, T., Hartman, T.G., Dudai, N. et al. 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. Plant Mol Biol 76, 475–488 (2011). https://doi.org/10.1007/s11103-011-9772-2

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  • DOI: https://doi.org/10.1007/s11103-011-9772-2

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