Gene Silencing of BnTT10 Family Genes Causes Retarded Pigmentation and Lignin Reduction in the Seed Coat of Brassica napus

Abstract

Yellow-seed (i.e., yellow seed coat) is one of the most important agronomic traits of Brassica plants, which is correlated with seed oil and meal qualities. Previous studies on the Brassicaceae, including Arabidopsis and Brassica species, proposed that the seed-color trait is correlative to flavonoid and lignin biosynthesis, at the molecular level. In Arabidopsis thaliana, the oxidative polymerization of flavonoid and biosynthesis of lignin has been demonstrated to be catalyzed by laccase 15, a functional enzyme encoded by the AtTT10 gene. In this study, eight Brassica TT10 genes (three from B. napus, three from B. rapa and two from B. oleracea) were isolated and their roles in flavonoid oxidation/polymerization and lignin biosynthesis were investigated. Based on our phylogenetic analysis, these genes could be divided into two groups with obvious structural and functional differentiation. Expression studies showed that Brassica TT10 genes are active in developing seeds, but with differential expression patterns in yellow- and black-seeded near-isogenic lines. For functional analyses, three black-seeded B. napus cultivars were chosen for transgenic studies. Transgenic B. napus plants expressing antisense TT10 constructs exhibited retarded pigmentation in the seed coat. Chemical composition analysis revealed increased levels of soluble proanthocyanidins, and decreased extractable lignin in the seed coats of these transgenic plants compared with that of the controls. These findings indicate a role for the Brassica TT10 genes in proanthocyanidin polymerization and lignin biosynthesis, as well as seed coat pigmentation in B. napus.

Molecular Mapping and QTL for Expression Profiles of Flavonoid Genes in Brassica napus

“…In Arabidopsis thaliana , the flavonoid biosynthesis pathway has been characterized mainly using different tt mutants, which have transparent and colorless testa (seed coats) (Holton and Cornish, 1995; Devic et al, 1999; Wan et al, 2002; Xie et al, 2003; Baudry et al, 2006; Lepiniec et al, 2006; Routaboul et al, 2006; Cheng, 2013; Saito et al, 2013). The present study showed that TT10 and AHA10 were involved in seed color formation of rapeseed, but these genes have yet to be successfully used in rapeseed breeding programs (Fu et al, 2007; Stein et al, 2013; Zhang et al, 2013). The flavonoid biosynthesis pathways of Brassica species are much more complex than those of A. thaliana (Supplementary Figure S3); in addition to consisting of more synthesis-related genes, this pathway is also involved in multi-loci interactions, which have been shown to be involved in the formation of seed coat color in B .…”

“…In addition, TT2 (R2R3-MYB), TT8 (bHLH), and TTG1 (WDR) modulate proteins, including DFR, LDOX, BAN, and TT12, thereby affecting PA production, and form a complex called MBW (MYB-bHLH-WD40) in the flavonoid pathway (Baudry et al, 2004, 2006; Lepiniec et al, 2006). Previous studies have proposed TTG1, TT8, TT10, TT12 , and AHA10 as candidate genes involved in seed coat color formation in Brassica species (Xie et al, 2003; Fu et al, 2007; Chai et al, 2009; Li et al, 2012; Stein et al, 2013; Zhang et al, 2013; Padmaja et al, 2014). Therefore, we predict that the candidate genes bZIP25, MYC1 , and MYB51 are involved in the flavonoid biosynthesis pathway through different regulator networks in rapeseed (Figure 5).…”

“…A09 that accounted for 40–60% of the phenotypic variance of seed coat color (Somers et al, 2001; Liu et al, 2005; Badani et al, 2006; Fu et al, 2007; Xiao et al, 2007; Rahman et al, 2010; Zhang et al, 2011). Candidate genes involved in seed coat color determination, such as TT10 and AHA10 , have still not successfully been used in rapeseed breeding programs aimed at producing seeds with a particular coat color (Fu et al, 2007; Stein et al, 2013; Zhang et al, 2013). Efforts to breed yellow-seeded B. napus have been largely unsuccessful, since seed coat color is a typical quantitative trait under polygenic control (Rahman, 2001; Liu et al, 2005; Badani et al, 2006) that is influenced by factors such as maternal effects and the environment (Deynze et al, 1993).…”

LACCASE5 Is Required for Lignification of the Brachypodium distachyon Culm

“…Clades I and II gathered 14 and three B. distachyon laccases, respectively, and some Arabidopsis proteins of unknown functions. Clade III comprised three B. distachyon laccases with AtLAC15 and BnTT10 laccase proteins involved in proanthocyanidin polymerization (Pourcel et al, 2005;Zhang et al, 2013). This result suggests that other members of clade III might also catalyze the oxidation of flavonoids.…”

“…1). In addition, we incorporated in this tree some other laccases reported to catalyze the oxidative polymerization of various phenolics, such as SofLAC from sugarcane (Cesarino et al, 2013), ZmLAC3 from maize (Zea mays; Caparrós-Ruiz et al, 2006), PtLAC3 from Populus trichocarpa (Ranocha et al, 2002), GaLAC1 from Gossypium arboretum (Wang et al, 2008), and Brassica napus Transparent Testa10 (BnTT10; Zhang et al, 2013;Supplemental Table S2). The resulting phylogeny comprised four large clades (Fig.…”