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Cuticular wax biosynthesis in petunia petals: cloning and characterization of an alcohol-acyltransferase that synthesizes wax-esters

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Abstract

The surface of plants is covered by cuticular wax, which contains a mixture of very long-chain fatty acid (VLCFA) derivatives. This wax surface provides a hydrophobic barrier which reduces non-stomatal water loss. One component of the cuticular wax is the alkyl esters, which typically contain a VLCFA esterified to an alcohol of a similar length. As part of an EST project, we recently identified an acyltransferase with 19% sequence identity (amino acid) to a bacterial ‘bifunctional’ wax-ester synthase/diacylglycerol acyltransferase (WS/DGAT). Northern analysis revealed that this petunia homologue was expressed predominantly within the petals. The cDNA encoding the WS/DGAT homologue was introduced into a yeast strain deficient in triacylglycerol biosynthesis. The expressed protein failed to restore triacylglycerol biosynthesis, indicating that it lacked DGAT activity. However, isoamyl esters of fatty acids were detected, which suggested that the petunia cDNA encoded a wax-synthase. Waxes were extracted from petunia petals and leaves. The petal wax extract was rich in VLCFA esters of methyl, isoamyl, and short-to-medium straight chain alcohols (C4–C12). These low molecular weight wax-esters were not present in leaf wax. In-vitro enzymes assays were performed using the heterologously expressed protein and 14C-labelled substrates. The expressed protein was membrane bound, and displayed a preference for medium chain alcohols and saturated very long-chain acyl-CoAs. In fact, the activity would be sufficient to produce most of the low molecular wax-esters present in petals, with methyl-esters being the exception. This work is the first characterization of a eukaryotic protein from the WS/DGAT family.

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Abbreviations

BHT:

Butlylated hydroxytoluene

BSA:

Bovine serum albumin

BCA:

Bicinchonic acid

BSTFA:

N,O-bis-(trimethylsilyl)-trifluoroacetamide

CoA:

Coenzyme A

DAG:

Diacylglycerol

DGAT:

Diacylglycerol acyltransferase

EST:

Expressed sequence tag

FAMES:

Fatty-acid methyl esters

MGAT:

Monoacylglycerol acyltransferase

TAG:

Triacylglycerides

TMSC:

Trimethylchlorosilane

TMS:

Trimethylsilyl

VCLFA:

Very long chain fatty acid

VLC:

Very long chain

WS:

Wax synthase

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Acknowledgments

This work was funded by Dow Chemical Company (Midland, MI, USA) and Dow Agrosciences LLC (Indiapolis, IN, USA). J. Hilliard was sponsored by a National Science Foundation summer internship program. The nucleotide sequence corresponding to the PhWS1 cDNA has been deposited in GenBank (DQ093641).

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Authors and Affiliations

  1. Donald Danforth Plant Science Center, 975 N Warson Road, St Louis, MO, 63132, USA

    Andrew King, Jeong-Won Nam, Jixiang Han, Josh Hilliard & Jan G. Jaworski

  2. CNAP, Department of Biology, University of York, Heslington, York, YO10 5DD, UK

    Andrew King

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  1. Andrew King
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King, A., Nam, JW., Han, J. et al. Cuticular wax biosynthesis in petunia petals: cloning and characterization of an alcohol-acyltransferase that synthesizes wax-esters. Planta 226, 381–394 (2007). https://doi.org/10.1007/s00425-007-0489-z

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