Nanoridges that characterize the surface morphology of flowers require the synthesis of cutin polyester

Abstract

Distinctive nanoridges on the surface of flowers have puzzled plant biologists ever since their discovery over 75 years ago. Although postulated to help attract insect pollinators, the function, chemical nature, and ontogeny of these surface nanostructures remain uncertain. Studies have been hampered by the fact that no ridgeless mutants have been identified. Here, we describe two mutants lacking nanoridges and define the biosynthetic pathway for 10,16-dihydroxypalmitate, a major cutin monomer in nature. Using gene expression profiling, two candidates for the formation of floral cutin were identified in the model plant Arabidopsis thaliana: the glycerol-3-phosphate acyltransferase 6 (GPAT6) and a member of a cytochrome P450 family with unknown biological function (CYP77A6). Plants carrying null mutations in either gene produced petals with no nanoridges and no cuticle could be observed by either scanning or transmission electron microscopy. A strong reduction in cutin content was found in flowers of both mutants. In planta overexpression suggested GPAT6 preferentially uses palmitate derivatives in cutin synthesis. Comparison of cutin monomer profiles in knockouts for CYP77A6 and the fatty acid omega-hydroxylase CYP86A4 provided genetic evidence that CYP77A6 is an in-chain hydroxylase acting subsequently to CYP86A4 in the synthesis of 10,16-dihydroxypalmitate. Biochemical activity of CYP77A6 was demonstrated by production of dihydroxypalmitates from 16-hydroxypalmitate, using CYP77A6-expressing yeast microsomes. These results define the biosynthetic pathway for an abundant and widespread monomer of the cutin polyester, show that the morphology of floral surfaces depends on the synthesis of cutin, and identify target genes to investigate the function of nanoridges in flower biology.

Fig. 1.

Flower morphology and surface characteristics of WT and mutants (gpat6 and cyp77a6). (A) Permeability to toluidine blue of the epidermis of flowers of WT and T-DNA insertional mutants for CYP77A6 and GPAT6. (B–D) Examination of petal abaxial epidermis using SEM (B, WT; C, gpat6–1; and D, cyp77a6–1). (E and F) Higher magnification of sepal surface showing a close-up of nanoridges under SEM (E, WT; F, gpat6–1). (G and H) Cross section of petals viewed under TEM (G, WT; H, gpat6–1). (Scale bars: A, 0.5 mm; B–D, 10 μm; E and F, 5 μm; G and H, 0.5 μm.) Similar SEM observations were made also for cyp77a6–2 and gpat6–2 mutants (see Fig. S2).

Fig. 2.

GPAT6 provides C16-based monomers for polyester synthesis. (A) Polyester monomer content in flowers of WT and gpat6–1 and gpat6–2 mutants. (B) GPAT6 overexpression in Arabidopsis stems increases C16 monomers. Data are mean with 95% CI (n = 9). Asterisks denote statistically significant difference between WT and both mutant lines (P < 0.001, t test). DHP: 10,16-dihydroxypalmitate. DCA, α, ω-dicarboxylic acids; FA, fatty acids.

Fig. 3.

Polyester monomer profile for flowers of cyp77a6 and cyp86a4 mutants. (A) Polyester monomer content in WT and cyp77a6–1 and cyp77a6–2 mutants. (B) Polyester monomer content in WT and cyp86a4–1 and cyp86a4–2 mutants. Data are mean with 95% CI (n = 4 for cyp86a4 mutants). Asterisks denote statistically significant difference between WT and both mutant lines (P < 0.001, t test). DHP: 10,16-dihydroxypalmitate. DCA, α, ω-dicarboxylic acids; FA, fatty acids.

Fig. 4.

Production of dihydroxypalmitate isomers by CYP77A6-expressing yeasts. Microsomes from CYP77A6-expressing Saccharomyces cerevisiae were incubated with 16-hydroxypalmitic acid (S), in the absence (A) or the presence (B) of NADPH. The mixture of dihydroxypalmitates (P), which included the 10,16-dihydroxy isomer, was produced only in the presence of NADPH.

Fig. 5.

The biosynthetic pathway of 10,16-dihydroxypalmitate in Arabidopsis. (A) Enzymes. CYP86A4 and CYP77A6 are identified in this study. The HOTHEAD oxidase has been shown to be involved in dicarboxylic acid formation (22). Diacid formation could also be catalyzed by a single cytochrome P450 (36). GPAT6 and long-chain acyl-CoA synthetases could act upstream or downstream of fatty acid oxidases (17), so R is unknown and may be H or SCoA or glycerolipid. (B) Changes in C16 cutin monomers and total cutin content in the knockout mutants.