The Plant Journal (2006) 47, 224–237 doi: 10.1111/j.1365-313X.2006.02786.x Genetical metabolomics of flavonoid biosynthesis in Populus: a case study Kris Morreel1, Geert Goeminne1,Ve´ ronique Storme1, Lieven Sterck1, John Ralph2, Wouter Coppieters3, Peter Breyne4, Marijke Steenackers4, Michel Georges3, Eric Messens1 and Wout Boerjan1,* 1Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology (VIB), Ghent University, B-9052 Gent, Belgium, 2United States Dairy Forage Research Center, Agricultural Research Service, United States Department of Agriculture and Department of Forestry, University of Wisconsin, Madison, WI 53706, USA, 3Department of Genetics, Faculty of Veterinary Medicine, University of Lie` ge, B-4000 Lie` ge, Belgium, and 4The Research Institute for Nature and Forest (INBO), B-9500 Geraardsbergen, Belgium Received 30 January 2006; accepted 7 March 2006. *For correspondence (fax þ32 9 3313809; e-mail [email protected]). Summary Genetical metabolomics [metabolite profiling combined with quantitative trait locus (QTL) analysis] has been proposed as a new tool to identify loci that control metabolite abundances. This concept was evaluated in a case study with the model tree Populus. Using HPLC, the peak abundances were analyzed of 15 closely related flavonoids present in apical tissues of two full-sib poplar families, Populus deltoides cv. S9-2 · P. nigra cv. Ghoy and P. deltoides cv. S9-2 · P. trichocarpa cv. V24, and correlation and QTL analysis were used to detect flux control points in flavonoid biosynthesis. Four robust metabolite quantitative trait loci (mQTL), associated with rate-limiting steps in flavonoid biosynthesis, were mapped. Each mQTL was involved in the flux control to one or two flavonoids. Based on the identities of the affected metabolites and the flavonoid pathway structure, a tentative function was assigned to three of these mQTL, and the corresponding candidate genes were mapped. The data indicate that the combination of metabolite profiling with QTL analysis is a valuable tool to identify control points in a complex metabolic pathway of closely related compounds. Keywords: poplar, flavonoids, metabolite profiling, correlation analysis, mQTL, genetical metabolomics. Introduction Metabolite profiling has gained much attention as a reveal flux-regulating control points, either to all pathway powerful functional genomics tool to unravel gene function intermediates or to a subset of intermediates only. Import- (Fiehn et al., 2000; Goodacre et al., 2004; Sumner et al., antly, and in contrast to other complex traits, such as 2003). For example, comparative metabolite profiling of morphological or physiological traits, the molecular struc- wild-type and mutant plants has shown that mutations in ture of the analyzed metabolites and the knowledge of the single genes can affect the concentrations of a wide variety pathway architecture may already suggest the function of of metabolites (Rohde et al., 2004). In addition, metabolite the gene underlying the mQTL. Hence, when the genome profiling of plants grown under various environmental sequence of the studied organism is available, the identifi- conditions has revealed groups of co-regulated metabolites cation of candidate genes with the predicted functions (Weckwerth and Fiehn, 2002). Metabolite levels are thus should be possible. controlled by both genetic and environmental factors. Poplar (Populus sp.) has become the model of choice for The question arises as to whether metabolite concentra- molecular genetic research on trees (Boerjan, 2005). Genetic tions can be considered as quantitative traits in a search for maps have been created (Cervera et al., 2001, 2004; Yin metabolite quantitative trait loci (mQTL) that control their et al., 2004), and sequencing of the Populus genome has abundance, and whether QTL analyses of the concentrations recently been completed (http://www.ornl.gov/ipgc/ and of all intermediates in a given biochemical pathway can http://genome.jgi-psf.org/Poptr1/Poptr1.home.html). The 224 ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd Journal compilation ª 2006 The Authors phenylalanine cinnamic acid p-coumaric acid PAL C4H GENERAL PHENYLPROPANOID 4CL 4CL cinnamoyl-CoA p-coumaroyl-CoA caffeoyl-CoA BIOSYNTHESIS HCTC3H HCT OH CHS CHS CHS OH QTL OH FLAVONOID HOHO HO OH HO OH BIOSYNTHESIS ª CHALCONES 2006 Blackwell Publishing Ltd, OH O OH O OH O pinocembrin chalcone naringenin chalcone eriodictyol chalcone MeO O CHI CHI CHI OH 5 OH O OMT ? pinostrobin 3 QTL 3' OH OH 2' 4' B HO O HO 8 O 5' HO O 7 / 8 7 2 6' A FLAVANONES 6 3 F3´H 5 4 FS FS FS OH OH O OH O OH O OH OH The Plant Journal pinocembrin naringenin eriodictyol22 HO O HO O F3´H HO O FLAVONES F3H F3H F3H OH OH O OH OH O OH OH O chrysin apigenin 66 luteolin DIHYDRO- HO O HO O HO O , (2006), AT ? FLAVONOLS F3´H OH QTL OH OH OH O OH O OH O Pinobanksinpinobanksin 1 HO O aromadendrin taxifolin 47 Genetical metabolomics in poplar , 224–237 O FLS FLS FLS OH OH O 15 OH OH O CH3 pinobanksin 3-acetate 44 HO O HO O HO O F3OGlcT F4´H ? F3´H OH OH OH OH CA FRT OH O OH O OH O OH galangin 9 kaempherol1010 quercetin1111 HO O FLAVONOLS F3OMT F3OMT F3OMT OH CA CA OH OH O OH O rutinoside HO O HO O HO O rutin 1212 O O O OH O CH OH O CH OH O CH 3 3 225 galangin 3-methyl ether3 13 kaempherol3-methyl ether quercetin3-methyl ether1414 226 Kris Morreel et al. genus Populus, which consists of approximately 30 species phenylpropanoids and flavonoids. The metabolite profiles (Cervera et al., 2005), presents a rich diversity of flavonoids were different for the three parents, both qualitatively and in the young leaves, buds and bud exudates, the composi- quantitatively. Characteristic chromatogram peaks of each tion of which is characteristic for each species (Greenaway parent could be traced back in the chromatograms of their et al., 1992). In these apical tissues, flavonoids are thought to respective progeny, indicating the inheritance of parent- function both as sunscreens and as defense compounds specific compounds in the offspring. Based on analysis of (Christensen et al., 1998; Dixon et al., 2002). Because the the 15 most abundant chromatogram peaks, the mean overall structure of the flavonoid pathway is well known broad-sense heritability was shown to vary between 0.55 (Figure 1) and different flavonoids have distinct UV/visible and 0.82, depending on the quantification method used absorption characteristics (Markham and Mabry, 1975), (Experimental procedures). flavonoid biosynthesis is an ideal model to evaluate the Because flavonoids are abundantly present in apical feasibility of detecting mQTL controlling the metabolite tissues of Populus (Greenaway et al., 1992) and have levels in a pathway. QTL analyses of the concentrations of characteristic UV/visible absorption spectra, and because closely related metabolites, belonging to a complex, multi- the structure of the flavonoid biosynthetic pathway is well branched pathway, have not been performed in any plant described, this pathway lends itself as an excellent model to species to date. evaluate the feasibility of genetical metabolomics of a In this pilot study, a QTL analysis was carried out on the complex biosynthetic pathway. A total of 29 flavonoids concentrations of the major flavonoids present in apical could be clearly distinguished in all individuals of family 001. tissues of two F1 mapping families of poplar that share a The chromatograms of family 002 revealed 39 flavonoids. common female parent. QTL analyses of 15 shared flavo- Thirteen of these 39 flavonoids were undetectable in 25–50% noids revealed four robust mQTL that control flavonoid of the family 002 individuals, and for some of them chi- levels, two on linkage group (LG) XIII, one on LG III, and one squared tests hinted at a 1:1 or 1:3 Mendelian segregation. on LG IV. The chemical structure of the flavonoids, coupled However, no significant mQTL could be detected for any of with current knowledge of the pathway architecture and the 13 compounds using single-trait QTL analysis of popu- in silico mapping of candidate genes, allowed the tentative lation 87002, suggesting that these flavonoids were below assignment of a function to three of these mQTL: the mQTL the detection limit rather than absent in part of the family. on LG III might be involved in the committed step to Of the 26 flavonoid peaks that were present in all flavonoid biosynthesis, i.e. chalcone synthase (CHS), and individuals of family 002, spiking indicated that 15 of them the two mQTL on LG XIII might act at branch points within were also found in family 001. Because of the restricted the pathway, namely acetylation of the 3-O position and number of traits that are accepted by MultiQTL (see below), methylation of the 7-O position in the production of pino- and to be able to compare the results for both families 001 banksin 3-acetate and pinostrobin respectively. and 002 that share the common female parent P. deltoides cv S9-2, all subsequent analyses were focused on these 15 common peaks (Figure 2). Results HPLC metabolite profiles Flavonoid concentration distributions To determine whether metabolite profiles were inherited The concentration distributions of all 15 flavonoids present from parents to offspring, the aromatic compounds present in both families were unimodal and in most cases skewed to in apical tissues of the F1 families 001 and 002 (Experimental the right. Most of these flavonoids were clearly present in procedures), and of their parents, Populus deltoides cv. S9- the common P. deltoides cv. S9-2 parent, as expected, but 2, P. nigra cv. Ghoy and P. trichocarpa cv. V24, were analyzed often not detected in the P. nigra cv. Ghoy or in P.