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 -hydroxyacids and ␣, -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. P lant surface lipids fulfill critical roles in the control of water and gas exchange, as protection from pathogens and UV radiation, as structural components, and to prevent cell fusions during organogenesis (1-3). These lipids are organized into the cuticle, a complex hydrophobic layer that covers the epidermis of plant leaves and other aerial organs and therefore is one of the largest biological interfaces in nature. The framework of the cuticle layer is provided by cutin, a plant-specific polyester composed of omega-substituted fatty acids and glycerol monomers (1, 4, 5). This insoluble polyester matrix is embedded and covered with waxes, a mixture of fatty acid derivatives that is easily extractable in organic solvents and has thus been more amenable to study than cutin (2, 6). Suberin is another type of cell-wall-associated lipid polymer, the most well known form being cork. It is composed of aliphatic and aromatic domains and is found in roots, the periderm of stems, and other tissues where it functions to restrict movement of water and ions across cell walls (1,7,8). Suberin also differs from cutin in that it is usually deposited abutting the inner face of the primary cell wall, whereas cutin is deposited at the outer face.Although cutin, one of the most abundant lipid polymers of nature, is the structural polymer of the plant cutic...

Disruption of Glycosylphosphatidylinositol-Anchored Lipid Transfer Protein Gene Altered Cuticular Lipid Composition, Increased Plastoglobules, and Enhanced Susceptibility to Infection by the Fungal Pathogen Alternaria brassicicola

“…8). These results are consistent with similar observations on the Arabidopsis gpat4/gpat8 mutant lacking glycerol-3-phosphate acyltransferase (GPAT4 and GPAT8) activity, which is essential for cutin biosynthesis (Li et al, 2007). It has been reported that Ser esterases with cutinolytic activities were only expressed during initial contact between A. brassicicola conidia and cutin on host surfaces that did not contain wax (Fan and Kö ller, 1998), which also supports our observations.…”

A comparative transcriptomic approach to understanding the formation of cork

“…The enrichment in the 'suberin' GO term is in agreement with the upregulation in cork oak outer bark of the putative orthologs of wellknown Arabidopsis or potato enzymes with a key role in the biosynthesis of suberin (Table S6A). Specifically, we found upregulation in cork of putative orthologs to the Arabidopsis GPAT5, CYP86A1, CYP86B1 and AtHHT/ASFT, and of putative orthologs to the potato CYP86A33 and FHT Li et al 2007;Höfer et al 2008;Serra et al 2009Serra et al , 2010Compagnon et al 2009;Gou et al 2009;Molina et al 2009). Aside from suberin, phellem imperviousness has been attributed to the associated waxes, which in cork consist mainly of isoprenoids such as triterpenes and sterols (Castola et al 2005).…”

Highlights of recent progress in plant lipid research

“…Li et al (O5) studied a double knock-out mutant gpat4/gpat8 affected in glycerol-3-phosphate acyltransferases (GPATs), and showed a strong reduction in cutin monomers and a steep increase in cuticle permeability. Overexpression of any of these acyltransferases in Arabidopsis increased up to 80% the content of specific C16 and/or C18 cutin monomers in leaves and stems [37]. These results identify the first acyltransferases involved in cutin synthesis.…”