title: Quantitative analysis of glycerol in dicarboxylic acid-rich cutins provides insights into Arabidopsis cutin structure.
abstract: Cutin is an extracellular lipid polymer that contributes to protective cuticle barrier functions against biotic and abiotic stresses in land plants. Glycerol has been reported as a component of cutin, contributing up to 14% by weight of total released monomers. Previous studies using partial hydrolysis of cuticle-enriched preparations established the presence of oligomers with glycerol-aliphatic ester links. Furthermore, glycerol-3-phosphate 2-O-acyltransferases (sn-2-GPATs) are essential for cutin biosynthesis. However, precise roles of glycerol in cutin assembly and structure remain uncertain. Here, a stable isotope-dilution assay was developed for the quantitative analysis of glycerol by GC/MS of triacetin with simultaneous determination of aliphatic monomers. To provide clues about the role of glycerol in dicarboxylic acid (DCA)-rich cutins, this methodology was applied to compare wild-type (WT) Arabidopsis cutin with a series of mutants that are defective in cutin synthesis. The molar ratio of glycerol to total DCAs in WT cutins was 2:1. Even when allowing for a small additional contribution from hydroxy fatty acids, this is a substantially higher glycerol to aliphatic monomer ratio than previously reported for any cutin. Glycerol content was strongly reduced in both stem and leaf cutin from all Arabidopsis mutants analyzed (gpat4/gpat8, att1-2 and lacs2-3). In addition, the molar reduction of glycerol was proportional to the molar reduction of total DCAs. These results suggest "glycerol-DCA-glycerol" may be the dominant motif in DCA-rich cutins. The ramifications and caveats for this hypothesis are presented.
authors.affiliation: Department of Plant Biology, Michigan State University, 48824-1312, USA. Electronic address: wyang26@msu.edu.
authors.forename: Weili
authors.initials: W
authors.lastname: Yang
authors.affiliation: Department of Plant Biology, Michigan State University, 48824-1312, USA.
authors.forename: Mike
authors.initials: M
authors.lastname: Pollard
authors.affiliation: Department of Plant Biology, Michigan State University, 48824-1312, USA.
authors.forename: Yonghua
authors.initials: Y
authors.lastname: Li-Beisson
authors.affiliation: Department of Plant Biology, Michigan State University, 48824-1312, USA.
authors.forename: John
authors.initials: J
authors.lastname: Ohlrogge
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry; D003998:Dicarboxylic Acids / Q000737:chemistry*; D005227:Fatty Acids; D008401:Gas Chromatography-Mass Spectrometry; D005990:Glycerol / Q000032:analysis* / Q000737:chemistry; D005992:Glycerol-3-Phosphate O-Acyltransferase / Q000378:metabolism; D008563:Membrane Lipids / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D018515:Plant Leaves / Q000737:chemistry; D018547:Plant Stems
chemical_list: D029681:Arabidopsis Proteins; D003998:Dicarboxylic Acids; D005227:Fatty Acids; D008563:Membrane Lipids; C000521:cutin; D005992:Glycerol-3-Phosphate O-Acyltransferase; D005990:Glycerol
keywords: Aliphatic monomers; Arabidopsis thaliana; Brassica napus; Brassicaceae; Glycerol; Heteropolymer; Leaf and stem cutins; Mutants; Stable isotope-dilution assay; Triacetin