Identification of acyltransferases required for cutin biosynthesis and production of cutin with suberin-like monomers

Abstract

Cutin and suberin are the two major lipid-based polymers of plants. Cutin is the structural polymer of the epidermal cuticle, the waterproof layer covering primary aerial organs and which is often the structure first encountered by phytopathogens. Suberin contributes to the control of diffusion of water and solutes across internal root tissues and in periderms. The enzymes responsible for assembly of the cutin polymer are largely unknown. We have identified two Arabidopsis acyltransferases essential for cutin biosynthesis, glycerol-3-phosphate acyltransferase (GPAT) 4 and GPAT8. Double knockouts gpat4/gpat8 were strongly reduced in cutin and were less resistant to desiccation and to infection by the fungus Alternaria brassicicola. They also showed striking defects in stomata structure including a lack of cuticular ledges between guard cells, highlighting the importance of cutin in stomatal biology. Overexpression of GPAT4 or GPAT8 in Arabidopsis increased the content of C16 and C18 cutin monomers in leaves and stems by 80%. In order to modify cutin composition, the acyltransferase GPAT5 and the cytochrome P450-dependent fatty acyl oxidase CYP86A1, two enzymes associated with suberin biosynthesis, were overexpressed. When both enzymes were overexpressed together the epidermal polyesters accumulated new C20 and C22 omega-hydroxyacids and alpha,omega-diacids typical of suberin, and the fine structure and water-barrier function of the cuticle were altered. These results identify GPATs as partners of fatty acyl oxidases in lipid polyester synthesis and indicate that their cooverexpression provides a strategy to probe the role of cutin composition and quantity in the function of plant cuticles.

Fig. 1.

Permeability of cuticles of GPAT knockouts to toluidine blue. Seedlings were immersed for 2 min in 0.05% toluidine blue-O and rinsed with water.

Fig. 2.

Analysis of the lipid polyester monomer content of 7-week-old Arabidopsis plants. (A) Effect of knocking out GPAT4 and GPAT8 or overexpressing GPAT8 on major stem cutin monomers. Overexpression of GPAT4 (data not shown) gave similar results to GPAT8. (B) Effect of overexpressing the suberin-associated GPAT5 and/or the fatty acyl oxidase CYP86A1 on major stem cutin monomers. Additional lipid polyester compositions are presented in SI Fig. 8. Values are means from four experiments with 95% confidence intervals. Asterisks denote a significant difference with WT (t test, two-sided P < 0.05 at least).

Fig. 3.

Features of epidermal cells of WT and gpat mutant and overexpressor. (A–C) Cuticle of pavement cells of stems as seen by TEM. (Scale bars, 0.5 μm.) (D and E) SEM images of stomata from adaxial surface. (Scale bars, 5 μm.) (F and G) Confocal laser scanning microscopy images of stomata from abaxial surface of 5-week-old leaves stained with Nile red (one single optical section at the stomata surface, fluorescence of dye is represented in green). (Scale bars,10 μm.) (H and I) TEM images of transdermal section of stem guard cells. (Scale bars, 5 μm.) (Insets) Magnified images of outer cuticular ledges. (Scale bars, 0.5 μm.)

Fig. 4.

Water loss of excised rosettes in the dark. Rosettes were excised in the middle of the night period and immediately weighed in the dark every 10 min. Values are means for six rosettes. Error bars are 95% confidence intervals. Experiment was repeated twice with different overexpressor lines and gave similar results.

Fig. 5.

Susceptibility of GPAT KO and overexpressors to A. brassicicola. (A) Symptom developments in the WT and gpat4/gpat8 KO rosette leaves 3 days after inoculation. (B) In planta-formed spores were counted 7 days after inoculation. Three batches of spores from 10 lesions were counted (mean ± SD).