Arabidopsis Deficient in Cutin Ferulate encodes a transferase required for feruloylation of ω-hydroxy fatty acids in cutin polyester

Abstract

The cuticle is a complex aliphatic polymeric layer connected to the cell wall and covers surfaces of all aerial plant organs. The cuticle prevents nonstomatal water loss, regulates gas exchange, and acts as a barrier against pathogen infection. The cuticle is synthesized by epidermal cells and predominantly consists of an aliphatic polymer matrix (cutin) and intracuticular and epicuticular waxes. Cutin monomers are primarily C(16) and C(18) unsubstituted, ω-hydroxy, and α,ω-dicarboxylic fatty acids. Phenolics such as ferulate and p-coumarate esters also contribute to a minor extent to the cutin polymer. Here, we present the characterization of a novel acyl-coenzyme A (CoA)-dependent acyl-transferase that is encoded by a gene designated Deficient in Cutin Ferulate (DCF). The DCF protein is responsible for the feruloylation of ω-hydroxy fatty acids incorporated into the cutin polymer of aerial Arabidopsis (Arabidopsis thaliana) organs. The enzyme specifically transfers hydroxycinnamic acids using ω-hydroxy fatty acids as acyl acceptors and hydroxycinnamoyl-CoAs, preferentially feruloyl-CoA and sinapoyl-CoA, as acyl donors in vitro. Arabidopsis mutant lines carrying DCF loss-of-function alleles are devoid of rosette leaf cutin ferulate and exhibit a 50% reduction in ferulic acid content in stem insoluble residues. DCF is specifically expressed in the epidermis throughout all green Arabidopsis organs. The DCF protein localizes to the cytosol, suggesting that the feruloylation of cutin monomers takes place in the cytoplasm.

Figure 1.

Identification and biochemical characterization of DCF mutant lines. A, RT-PCR analysis of DCF gene expression in dcf-1 and dcf-2 mutants. B, Ferulic- and p-coumaric acid contents of leaf and stem tissue of dcf mutants compared with the Col-0 and qrt1 wild types. Values represent means ± sd of at least three biological replicates. Significant differences between mutant and parental lines are indicated (Student’s t test; *** P < 0.001, ** P < 0.01, * P < 0.05). FS, Flowering stage; VS, vegetative stage. C, Representative HPLC profiles of phenolics released upon depolymerization of wild-type (Col-0) and dcf mutant derived delipidated residues. mAU, Milliabsorbance units. D, Mass spectrum and chemical structure of compound B corresponding to methyl-ferulate.

Figure 2.

Ferulic- and p-coumaric acid amounts released upon base hydrolysis of total cell wall (CWM) preparations and acid-catalyzed depolymerization of delipidated, carbohydrate-free insoluble residues (IR). Values represent means ± sd of at least three biological replicates. Significant differences between mutant and parental lines are indicated (Student’s t test; ** P < 0.01).

Figure 3.

Lipidic polyester monomer composition of wild-type and dcf mutant leaves. Delipidated residues were depolymerized with methanolic hydrochloric acid, and released monomers were analyzed by GC-MS. Values represent means ± sd of at least three biological replicates. Significant differences between mutant and parental lines are indicated (Student’s t test; * P < 0.05).

Figure 4.

DCF expression in major Arabidopsis organs and at different developmental stages. A, qRT-PCR analysis. Seedlings were grown for 10 d in sterile culture. Leaves were harvested from 5-week-old plants (rosette stage). Other material was derived from 8-week-old flowering plants. Stems were divided into three parts, and siliques were harvested at the indicated time points. The levels of expression are calculated relative to the UBQ10 gene, and values represent means ± sd of three biological replicates. B, DCF_pro:DCF-YFP expression pattern in transgenic Arabidopsis plants. DCF was stably expressed in Arabidopsis as a C-terminal translational YFP fusion protein under the control of the native promoter. YFP fluorescence is displayed in green (a–l), and autofluorescence of stem sections upon UV light excitation is displayed in blue (j–l).

Figure 5.

Subcellular localization of the DCF protein. A and B, Single-plane confocal micrographs of leaf epidermis of DCF_pro:DCF-YFP-expressing transgenic plants (A) and after plasmolysis (B). Bars = 25 μm. C, Subcellular fractionation of protein samples derived from DCF_pro:DCF-HA-expressing transgenic plants. F1, Cytosolic fraction; F2, detergent-solubilized microsomal fraction.

Figure 6.

In vitro hydroxycinnamoyl-CoA transferase activity of recombinant DCF protein. A, SDS-PAGE of purified recombinant DCF protein stained with Coomassie Brilliant Blue and corresponding immunoblot analysis. C, Crude extract; ST, supernatant; E, eluate. B, Temperature and pH profiles of recombinant DCF feruloyl-transferase activity. C and D, HPLC chromatograms of products formed after the reaction of recombinant DCF (D) and control reaction with heat-inactivated enzyme (C) with feruloyl-CoA as acyl donor and 16-hydroxy-palmitic acid as acyl acceptor. AU, Absorbance units. E, Mass spectra and chemical structures of the reaction products, ferulic acid methyl-ester and 16-feruloyl-palmitic acid.