Rapid, Microscale, Acetyl Bromide-Based Method for High-Throughput Determination of Lignin Content in Arabidopsis thaliana XUE FENG CHANG, † RICHARD CHANDRA, † THOMAS BERLETH, § AND RODGER P. BEATSON* ,# Department of Wood Science, The University of British Columbia, 2900-2424 Main Mall, Vancouver, BC, Canada V6T 1Z4, Department of Cell and Systems Biology, University of Toronto, 25 Willcocks Street, Toronto, Ontario, Canada M5S 3B2, Chair of Advanced Papermaking, British Columbia Institute of Technology, 3700 Willingdon Avenue, Burnaby, BC, Canada V5G 3H2 The acetyl bromide method has been modified to enable the rapid microscale determination of lignin content in Arabidopsis with the goal of determining the genes that control lignin in plants. Modifications include reduction in sample size, use of a microball mill, adoption of a modified rapid method of extraction, use of an ice-bath to stabilize solutions and reduction in the volume of solutions. The microscale method was shown to be rapid, accurate and precise with values in agreement with those determined by the full-scale acetyl bromide method. The extinction coefficient for Arabidopsis lignin, dissolved using acetyl bromide, was determined to be 23.35 g -1 L cm -1 at 280 nm. This value is independent of the Arabidopsis accession, environmental growth conditions and is insensitive to lignin structure. The newly developed method can be used to determine lignin content in the inflorescence stems of Arabidopsis for mapping of lignin-related genes. KEYWORDS: Lignin determination; Arabidopsis; acetyl bromide; microscale; high-throughput; extinction coefficient; extraction INTRODUCTION Lignin is a major chemical component of plants and the second most abundant natural polymer after cellulose (1). It is found mainly in the thickened secondary cell wall, strengthening stem and vascular tissue, allowing upward growth and permitting water and minerals to be conducted through the xylem under negative pressure (2). Lignin is a complex aromatic polymer mainly comprised of three phenylpropanoid units, p-hydroxy- phenyl (H), guaiacyl (G), and syringyl (S) (3). Although lignin provides a renewable source of phenolic polymers, its presence in plants is often detrimental for other applications. High lignin content limits the digestibility of forage crops by cattle (4), inhibits the enzyme hydrolysis process in the biofuel industry (5) and adversely affects chemical pulping and bleaching processes (6, 7). These concerns and interests of agriculture and industry have stimulated the study of genes governing lignin content in plants. Arabidopsis thaliana provides a model system for the study of the genes governing lignin content in angiosperms. Full genome sequence comparison, as well as expression data and genetic studies have demonstrated high conservation of major signaling and biosynthesis pathways across the angiosperms, which include all major crop plants and hardwood trees (8–10). Signaling and biosynthesis pathways are amenable to genetic dissection in Arabidopsis, because the short growth cycle, small size, and small genome, facilitate growth and analysis of large numbers of genetic variants at low costs (11, 12). There is abundant genetic and phenotypic variation among naturally occurring populations of Arabidopsis. This natural diversity has been used for the mapping of Quantitative Trait Loci (QTL) and, based on this, for the cloning of relevant genes in processes such as flowering time, fruit size and root growth (13, 14). Also QTLs affecting lignification and cell wall digestibility have been mapped in Arabidopsis using established recombinant inbred lines (15). Such results can potentially be extrapolated to crop plants (16) and trees and thus may be used to custom design plants for specific end-uses. The task of identifying genes that determine lignin content in Arabidopsis illustrates the need for a small-scale high- throughput protocol for lignin quantification. First, to reveal natural variation, lignin content has to be determined across a large number of Arabidopsis local varieties, called accessions. Then, once accessions with divergent lignin content have been identified, new mapping populations can be established from a cross of selected accessions. To provide sufficient genetic * Address correspondence to this author at Chair of Advanced Papermaking, British Columbia Institute of Technology, 3700 Will- ingdon Ave., Burnaby, BC, Canada V5G 3H2 [e-mail Rodger_Beatson@ bcit.ca; telephone (604) 432-8951; fax (604) 432-9572]. † The University of British Columbia. § University of Toronto. # British Columbia Institute of Technology. J. Agric. Food Chem. 2008, 56, 6825–6834 6825 10.1021/jf800775f CCC: $40.75  2008 American Chemical Society Published on Web 07/31/2008