- Top of page
- Materials and Methods
- Supporting Information
Glucosinolates are found in all members of the family Brassicaceae and are a class of secondary metabolites, of which > 120 different types have been found within plants. Glucosinolates are hydrolyzed by myrosinases following plant wounding (Husebye et al., 2002). Following hydrolysis the breakdown products can have beneficial effects such as preventing cancer in humans and enhancing plant protection (Fahey et al., 2001; Talalay & Fahey, 2001; Tierens et al., 2001). Deleterious effects have also been reported and the types of glucosinolates produced by the plant are important in determining the overall impact (Fenwick & Curtis, 1980; Griffiths et al., 1998; Vaughn et al., 2005). The discovery of a forage cultivar of Brassica napus, Bronowski, with reduced amounts of glucosinolates in its seeds was a major breakthrough in the breeding of modern oilseed crops (Kondra & Stefansson, 1970). Most modern varieties of B. napus are referred to as ‘double lows’ as they have seeds with low glucosinolate and erucic acid. The reduced concentrations of glucosinolates are often a consequence of crossing programs which have used the Bronowski germplasm. The glucosinolate concentration within their seeds has been reduced dramatically from > 100 to < 20 μmol g−1 (Toroser et al., 1995). Moreover, this reduction is thought to be associated with a concomitant decrease in glucosinolate production in leaves (Mithen, 1992; Li et al., 1999). A major goal of B. napus plant breeders is to further reduce the concentration of deleterious glucosinolate within seeds in which the cake is to be used for fodder and yet retain the protective effects of high glucosinolate concentrations in the vegetative organs. For this to be achieved, it is necessary for us to develop a better understanding of the processes underlying glucosinolate biosynthesis and accumulation in leaves and seeds.
The chemical structure of glucosinolates is formed around a common core together with a variable side chain. For each of the three different classes of glucosinolates, the aliphatic, aromatic and indolyl glucosinolates, the side chain is derived from a different type of amino acid precursor (Zukalová & Vašák, 2002; Grubb & Abel, 2006; Halkier & Gershenzon, 2006). The corresponding pathways, involved in glucosinolate metabolism, have been reported for the Brassicaceae (Fig. 1). It is thought that the glucosinolates are synthesized mainly in vegetative organs such as young leaves and silique walls, and then transported actively to embryos through the phloem by unknown transporters; however, synthesis in immature seeds has also been proposed (Toroser et al., 1995; Du & Halkier, 1998; Chen & Halkier, 2000; Chen et al., 2001b; Kliebenstein et al., 2001b). Blocking the transport of glucosinolates from vegetative organs to the seeds could provide a means of reducing glucosinolate concentrations in seeds without affecting other tissues.
Figure 1. Glucosinolate metabolic pathways in Brassicaceae. Pathway A: precursors of aliphatic and aromatic glucosinolates are synthesized by a series of reactions (condensation, isomerization and oxidative decarboxylation mainly), which elongate the side chains of amino acids. Pathway B: formation of basic glucosinolates from amino acids and chain-elongated homoamino acids is catalyzed by a series of enzymes. Pathway C: glucosinolates are translocated from vegetative organs to seeds by the unknown glucosinolate transporter to satisfy the huge demand for glucosinolate in seeds. Pathway D: side chains of basic glucosinolates are modified by oxidation and elimination, etc., to form other modified glucosinolates in leaves and seeds. Pathway E: when plants are injured, glucosinolates are hydrolyzed by myrosinase to the breakdown products that are involved in stress defense. The figure is constructed following several publications (Iqbal et al., 1995; Chen & Andreasson, 2001a; Mikkelsen et al., 2002; Grubb & Abel, 2006; Halkier & Gershenzon, 2006; Padilla et al., 2007; Hansen et al., 2008; Gigolashvili et al., 2009; Mugford et al., 2009; Sawada et al., 2009; Dixon et al., 2010; Sonderby et al., 2010; Yatusevich et al., 2010).
Download figure to PowerPoint
Quantitative trait loci (QTL) have been determined to control different glucosinolates in the leaves and seeds of Arabidopsis. Kliebenstein et al. (2001a), found three tissue-specific QTL for aliphatic glucosinolates in both leaves and seeds and a similar number were found for the indolyl glucosinolates, with only one QTL identified as being active in both organs. These results indicate that genes underlying these QTL seem to be responsible for glucosinolate accumulation in the specific tissue rather than both tissues. Subsequently, the genes AOP2–AOP3 were cloned through fine mapping of these QTL and were demonstrated to be involved in glucosinolate modification (Kliebenstein et al., 2001c). Other gene families involved in the control of glucosinolate metabolism in Arabidopsis have been identified through the analysis of loss-of-function mutations such as the CYP79 gene (Hull et al., 2000; Reintanz et al., 2001; Chen et al., 2003; Mikkelsen et al., 2003). Such results together with comparative analysis between phenotypic QTL and expression QTL (eQTL) has resulted in the proposal of a complex pathway for glucosinolate biosynthesis and regulation in Arabidopsis (Kliebenstein et al., 2006; Wentzell et al., 2007). Numerous unknown metabolic regulatory relationships that are complementary to this network and are based on QTL co-location information have also been predicted using metabolic analysis (Keurentjes et al., 2006). A piece of software, MetaNetwork, has subsequently been developed which facilitates such approaches (Fu et al., 2007).
In addition to the work carried out in Arabidopsis, our current knowledge about the accumulation of glucosinolates has been enhanced by QTL mapping in Brassica. In Brassica oleracea (CC, 2n = 18), four QTL, together with the underlying candidate genes, have been identified as key players in the biosynthesis of multiple glucosinolates in the leaves of this species (Li & Quiros, 2001, 2002, 2003; Li et al., 2003). Recently, up to 22 QTL were identified for the accumulation of glucosinolates in the leaves of B. rapa (AA, 2n = 20) (Lou et al., 2008). In QTL studies of total glucosinolate accumulation in the seeds of B. napus, seven QTL have been identified on several linkage groups (Toroser et al., 1995; Uzunova et al., 1995; Howell et al., 2003; Zhao & Meng, 2003; Quijada et al., 2006). Some common QTL were detected between many of these studies which may be a result of the common ancestry of these populations. Most populations are derived from a shared ancestor with low seed glucosinolate concentration (Sharpe & Lydiate, 2003; Hasan et al., 2008). Interestingly, little work has been done on the genetic variation of glucosinolates in the leaves and other vegetative organs, or on the metabolic network of glucosinolate synthesis in this tetraploid species. It is likely that the regulation of glucosinolate synthesis will be very complex compared with that of Arabidopsis and diploid Brassica crops.
In this study, we have attempted to address this gap in our knowledge of the genetic control of glucosinolate concentration in leaves of B. napus and the metabolic network of glucosinolate synthesis. This has been achieved through the measurement of total glucosinolate and individual glucosinolates in seeds and leaves within a doubled-haploid (DH) mapping population of B. napus.
- Top of page
- Materials and Methods
- Supporting Information
Fig. S1 The distribution of consensus QTL and mQTL on 18 of 19 linkage groups in Brassica napus.
Fig. S2 Demonstration of a complex epistatic network for glucosinolate in seeds.
Table S1Arabidopsis genes that control glucosinolate metabolism used for in silico mapping in our experiments; they were collected from the TAIR website
Table S2 The list of 264 consensus QTL first integrated from 436 significant QTL by meta-analysis
Table S3 The list of metabolite QTL identified by the ‘two-round’ strategy of QTL meta-analysis
Table S4 Epistatic interactions detected for glucosinolate concentration in the leaves and seeds of Brassica napus
Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.