Role of Glycerol and Acyltransferases in Biosynthesis and Structure of Plant Extracellular Lipid Polymers

Michigan State University, East Lansing MI

Investigators

Abstract

Cutin and suberin are complex, extracellular lipid polymers that provide a critical barrier between plants and their environment and supply protection from biotic and abiotic stress. In addition, cutin is essential for maintaining organ identity, suberin synthesis is a fundamental wound response and both polymers have structural roles. Despite these essential functions and although cutin and suberin are the most abundant lipid polymers of plants, their complex structure and insolubility have resulted in a dearth of knowledge about their synthesis and assembly. The principal investigator recently characterized Arabidopsis stem and leaf polyesters and identified the omega-dicarboxylic fatty acid derived from linoleic acid (18:2-DCA) as the major aliphatic component. In addition, a glycerol-3-phosphate acyltransferase (GPAT5) was found to be required for suberin accumulation in Arabidopsis roots and seeds. Ectopic over-expression of this acyltransferase greatly alters stem epidermal lipid production resulting in surface monoacylglycerol and fatty acid accumulation. These unexpected results bring to light the role of glycerolipids in surface lipid metabolism and have led to a new working hypothesis for the synthesis and assembly of plant surface lipid polymers. Rather than exporting monomers as previously assumed, it is now hypothesized that plants assemble polyester precursors as acylglycerols inside the cell before export. Testing this new model is a major objective of this project. First, it is important to have a clearer understanding of the chemical components of polyesters. Are Arabidopsis leaf and stem polyesters composed largely of polyacylglycerols? Initial emphasis will be on determining covalent linkages between glycerol and carboxylate monomers. In addition, in situ depolymerization and determination of the amount of glycerol and the proportion of its free and esterified hydroxyls are proposed. The project will also determine the structure of lipids containing this 18:2-DCA using spectroscopic and other chemical analytical methods. A second major focus is to understand how glycerol acyltransferases participate in polyester synthesis. The acyl donor and acceptor substrate specificity for GPAT5 acyltransferase will be established in the project. Finally, initial analysis suggests several other GPAT isoforms may also be involved in synthesis of surface lipids. These enzymes will be characterized by substrate specificity, reverse-genetic and gain-of-function experiments in order to establish the biochemical function(s) of GPAT acyltransferases and their role in biosynthesis of cutin and suberin. Broader Impacts: Better understanding of lipid polyesters may also open the door to strategies to genetically manipulate their barrier or structural properties in ways beneficial for agriculture. Cutin and suberin research have been hampered not only by the complex nature of the polymers but also by a lack of analytical techniques; e.g. an analysis of Arabidopsis cutin composition was not achieved until recently and most mutants purported to be altered in cutin have not been analyzed chemically. This project proposes to develop improved biochemical as well as chemical analytical methods for characterization of surface lipid structures and their biosynthesis. Graduate and undergraduate students and postdocs trained in this project will be part of a team effort where chemical and molecular genetic approaches take place side-by-side in the same laboratory and often at the same bench. Participants will gain multi-disciplinary experience including chemistry, analytical biochemistry, isotope labeling, molecular genetics, plant transformation and mutant analysis.