The identification of cutin synthase: formation of the plant polyester cutin

Abstract

A hydrophobic cuticle consisting of waxes and the polyester cutin covers the aerial epidermis of all land plants, providing essential protection from desiccation and other stresses. We have determined the enzymatic basis of cutin polymerization through characterization of a tomato extracellular acyltransferase, CD1, and its substrate, 2-mono(10,16-dihydroxyhexadecanoyl)glycerol. CD1 has in vitro polyester synthesis activity and is required for cutin accumulation in vivo, indicating that it is a cutin synthase.

Figure 1. CD1 is a GDSL-motif lipase/hydrolase family protein that localizes to the nascent cuticle

(a) Fruits of the M82 wild type tomato cultivar and the cd1 mutant on the day of harvest at the fully ripe stage and 14 days after storage at room temperature. Scale bars = 1 cm. (b) Light microscopy showing the cuticle of M82 and cd1 ripe fruit stained with Sudan Red 7b. Scale bars = 50 µm. (c–d) TEM immunolocalization of CD1 in the cuticle of 15 days post-anthesis (DPA) M82 fruits, over the periclinal cell wall of an epidermal cell (c) and in the anticlinal peg of cuticle between two adjacent epidermal cells (d). Scale bars = 500 nm. Cut, cuticle; Cyt, cytoplasm; ACW, anticlinal cell wall; PCW, periclinal cell wall; PM, plasma membrane; Vac, vacuole. Gold particles are highlighted with yellow circles in the cuticle or red circles elsewhere, and magnified areas of each image are shown in the right panels.

Figure 2. The identification of 2-MHG in the soluble surface lipids of cd1 fruit

(a) GC-MS chromatograms of the cd1 mutant and M82 wild type TMS-derivatized extracts. The total ion chromatograms and several single ion chromatograms corresponding to characteristic fragments of 2-MHG are shown. Inset is an enlargement of the region surrounding the 2-MHG peak. For reference, several of the wax compounds common to both mutant and wild type are labeled. (b) Mass spectrum from the 2-MHG peak found in the cd1 extract, with interpretation of the spectrum inset. The spectrum of synthetic 2-MHG is shown in Supplementary Fig. 10. For more detailed discussion of the spectrum of 2-MHG, see ref. .

Figure 3. Acyltransferase activity of CD1

(a) Proposed model for CD1 catalyzed cutin biosynthesis by transfer of the hydroxyacyl group from 2-MHG to the growing polymer. For simplicity, ester linkage via the primary hydroxyl is shown, although linkage via the secondary hydroxyl can also occur in cutin. (b) MALDI-TOF positive ion spectra of lipid products from in vitro assays with 2-MHG substrate and purified recombinant CD1 enzyme.