The Unique Role of the ECERIFERUM2-LIKE Clade of the BAHD Acyltransferase Superfamily in Cuticular Wax Metabolism

Abstract

The elongation of very-long-chain fatty acids is a conserved process used for the production of many metabolites, including plant cuticular waxes. The elongation of precursors of the most abundant cuticular wax components of some plants, however, is unique in requiring ECERIFERUM2-LIKE (CER2-LIKE) proteins. CER2-LIKEs are a clade within the BAHD superfamily of acyltransferases. They are known to be required for cuticular wax production in both Arabidopsis and maize based on mutant studies. Heterologous expression of Arabidopsis and rice CER2-LIKEs in Saccharomyces cerevisiae has demonstrated that they modify the chain-length specificity of elongation when paired with particular condensing enzymes. Despite sequence homology, CER2-LIKEs are distinct from the BAHD superfamily in that they do not appear to use acyl transfer activity to fulfill their biological function. Here, we review the discovery and characterization of CER2-LIKEs, propose several models to explain their function, and explore the importance of CER2-LIKE proteins for the evolution of plant cuticles.

Keywords: BAHD acyltransferase; cuticle; eceriferum; elongation; evolution; very-long-chain fatty acids.

Figure 1

Schematic of cuticular wax biosynthesis in Arabidopsis. Very-long-chain fatty acids (VLCFAs) are lengthened in two-carbon increments (black), and derivatized by one of two pathways. The acyl-reduction pathway produces primary alcohols, a portion of which are esterified to fatty acids to make wax esters (blue). The alkane-forming pathway produces aldehyde intermediates, from which a carbonyl group is lost to form an alkane; alkanes may subsequently undergo mid-chain oxidation to produce secondary alcohols and ketones (red). CER: ECERIFERUM; WSD: bifunctional wax ester synthase/diacylglycerol acyltransferase; MAH1: mid-chain alkane hydroxylase.

Figure 2

Schematic of fatty acid elongation. Malonyl-CoA (blue) and an acyl-CoA primer (red) are condensed by a ketoacyl-CoA synthase (KCS) to produce a β-ketoacyl-CoA. The β-ketoacyl-CoA is reduced by a β-ketoacyl-CoA reductase (KCR) to yield a β-hydroxyacyl-CoA, which in turn is dehydrated by a β-hydroxyacyl-CoA dehydratase (HCD/PAS2) to produce an enoyl-CoA. The enoyl-CoA is reduced by an enoyl-CoA reductase (ECR/CER10) to give an acyl-CoA that is two carbons longer than the initial acyl-CoA used as a primer. The acyl-CoA product can then be used as a primer for the same reaction sequence, allowing for repeated elongation of VLCFAs in two-carbon units.

Figure 3

Simplified schematic of BAHD acyl transfer activity. First (I), the acyl acceptor is deprotonated by a catalytic histidine residue on the BAHD. Second (II), the electronegative acyl acceptor carries out a nucleophilic attack on the carbonyl carbon of the acyl-CoA, the acyl donor. This is proposed to form a tetrahedral intermediate (not shown). Finally (III), the new ester bond between the acyl donor and acceptor is created with the release of CoA.

Figure 4

Models of paired CER6-CER2 activity. (A) Active site extension. A hypothetical acyl-CoA binding site in CER2 could physically extend the substrate-binding pocket of CER6; (B) Allosteric modulation. Protein-protein interaction between CER2 and CER6 could modify the structure of the substrate-binding pocket of CER6, enabling it to accept longer acyl chains; (C) Substrate binding and concentration. CER2 could provide acyl-CoAs to the fatty acid elongase; (D) Thioesterase activity. CER2 could act as a thioesterase, providing free fatty acid to load onto the active-site cysteine of CER6.

Figure 5

(A) Phylogenetic reconstruction of inferred gene duplications (red boxes) and losses (grey) in the smallest clade of BAHD acyltransferases that includes all five Arabidopsis CER2-LIKE homologs and their closest relatives (a large clade of Selaginella homologs). Some inferred losses affect the entire clades noted. Biochemically characterized CER2-LIKEs are in green text; (B) Phylogram of the BAHD acyltransferases recovered using Arabidopsis CER2-LIKE homologs. The subclade in blue was used for the tree-reconciliation analysis (A). Characterized BAHD acyltransferases from Arabidopsis are numbered, clockwise, and marked on the tree with orange circles; (1) At5g48930, HCT/HYDROXYCINNAMOYL-COA SHIKIMATE/QUINATE HYDROXYCINNAMOYL TRANSFERASE [62]; (2) At2g19070, SHT/SPERMIDINE HYDROXYCINNAMOYLTRANSFERASE [63]; (3) At5g41040, ASFT/ALIPHATIC SUBERIN FERULOYL TRANSFERASE [64]; (4) At3g48720, DCF/DEFICIENT IN CUTIN FERULATE [32]; (5) At5g63560, FACT/FATTY ALCOHOL:CAFFEOYL-COA CAFFEOYL TRANSFERASE [65]; (6) At1g65450, GLC/GLAUCE [66]; (7) At4g31910, DRL/DWARF AND ROUND LEAF 1 [67]/BAT1/BR-RELATED-ACYLTRANSFERASE [68]/PIZ/PIZZA [69]; (8) At3g29670, PMAT2/PHENOLIC GLUCOSIDE MALONYLTRANSFERASE 2 [70]; (9) At5g67160, EPS2/ENHANCED PSEUDOMONAS SUSCEPTIBILITY 1 [71]. The full gene tree (with complete gene identifiers and scale) is provided in the supplemental information.