Flavonoids are secondary metabolites that accumulate in most plant seeds and are involved in physiological functions such as dormancy or viability. This review presents a current view of the genetic and biochemical control of flavonoid metabolism during seed development. It focuses mainly on proanthocyanidin accumulation in Arabidopsis, with comparisons to other related metabolic and regulatory pathways. These intricate networks and their fine-tuned regulation, once they are determined, should contribute to a better understanding of seed coat development and the control of PA and flavonol metabolism. In addition, flavonoids provide an interesting model to study various biological processes and metabolic and regulatory networks.

SummaryGenetic analyses have demonstrated that together with TTG1, a WD-repeat (WDR) protein, TT2 (MYB), and TT8 (bHLH) are necessary for the correct expression of BANYULS (BAN). This gene codes for the core enzyme of proanthocyanidin biosynthesis in Arabidopsis thaliana seed coat. The interplays of TT2, TT8, and their closest MYB/bHLH relatives, with TTG1 and the BAN promoter have been investigated using a combination of genetic and molecular approaches, both in yeast and in planta. The results obtained using glucocorticoid receptor fusion proteins in planta strongly suggest that TT2, TT8, and TTG1 can directly activate BAN expression. Experiments using yeast two-and three-hybrid clearly demonstrated that TT2, TT8, and TTG1 can form a stable ternary complex. Furthermore, although TT2 and TT8 were able to bind to the BAN promoter when simultaneously expressed in yeast, the activity of the complex correlated with the level of TTG1 expression in A. thaliana protoplasts. In addition, transient expression experiments revealed that TTG1 acts mainly through the bHLH partner (i.e. TT8 or related proteins) and that TT2 cannot be replaced by any other related A. thaliana MYB proteins to activate BAN. Finally and consistent with these results, the ectopic expression of TT2 was sufficient to trigger BAN activation in vegetative parts, but only where TTG1 was expressed. Taken together, these results indicate that TT2, TT8, and TTG1 can form a ternary complex directly regulating BAN expression in planta.

SummaryIn Arabidopsis thaliana, several MYB and basic helix-loop-helix (BHLH) proteins form ternary complexes with TTG1 (WD-Repeats) and regulate the transcription of genes involved in anthocyanin and proanthocyanidin (PA) biosynthesis. Similar MYB-BHLH-WDR (MBW) complexes control epidermal patterning and cell fates. A family of small MYB proteins (R3-MYB) has been shown to play an important role in the regulation of epidermal cell fates, acting as inhibitors of the MBW complexes. However, so far none of these small MYB proteins have been demonstrated to regulate flavonoid biosynthesis. The genetic and molecular analyses presented here demonstrated that Arabidopsis MYBL2, which encodes a R3-MYB-related protein, is involved in the regulation of flavonoid biosynthesis. The loss of MYBL2 activity in the seedlings of two independent T-DNA insertion mutants led to a dramatic increase in the accumulation of anthocyanin. In addition, overexpression of MYBL2 in seeds inhibited the biosynthesis of PAs. These changes in flavonoid content correlate well with the increased level of mRNA of several structural and regulatory anthocyanin biosynthesis genes. Interestingly, transient expression analyses in A. thaliana cells suggested that MYBL2 interacts with MBW complexes in planta and directly modulates the expression of flavonoid target genes. These results are fully consistent with the molecular interaction of MYBL2 with BHLH proteins observed in yeast. Finally, MYBL2 expression studies, including its inhibition by light-induced stress, allowed us to hypothesise a physiological role for MYBL2. Taken together, these results bring new insights into the transcriptional regulation of flavonoid biosynthesis and provide new clues and tools for further investigation of its developmental and environmental regulation.Keywords: flavonoid, transcription, network, MYB, bHLH, TTG1. IntroductionFlavonoids are secondary metabolites that fulfil important biological functions and provide useful metabolic and genetic models for plant research, including the analysis of transcriptional regulation of gene expression (Koes et al., 2005;Lepiniec et al., 2006;Peer and Murphy, 2007;Taylor and Grotewold, 2005;Winkel-Shirley, 2001). Flavonoids are involved in protection against various biotic and abiotic stresses, they play roles in the regulation of plant reproduction and development and act as signalling molecules with the biotic environment. Besides these physiological functions, there is a growing interest in these secondary metabolites due to their potential benefits for human health (Halliwell, 2007; Luceri et al., 2007); therefore, improving our understanding of the regulation of flavonoid biosynthesis is an important objective.Although structural genes can be efficiently targeted for crop improvement, the use of regulatory genes seems to be at least as promising (Bovy et al., 2007; Grotewold et al., 940 ª 2008 The Authors Journal compilation ª 2008 Blackwell Publishing LtdThe Plant Journal (2008Journal ( ) 55, 940-953 doi: 10.1111Journal...

SummaryThe control of TT8 expression was investigated in this study, and it was demonstrated that it constitutes a major regulatory step in the specific activation of the expression of flavonoid structural genes. First, the GUS activity generated in planta from a TT8::uidA construct revealed cell-specific activation of the TT8 promoter consistent with the known involvement of the TT8 bHLH factor in proanthocyanidin, anthocyanin and mucilage biosynthesis. Moreover, the activity of this reporter construct was strongly affected in ttg1, TT2 overexpressers (OE), and PAP1-OE, suggesting interplay between TT2, PAP1, TTG1 and the activation of the TT8 promoter in planta. To further investigate the mechanisms involved, we used 35S::TT2-GR and 35S::TTG1-GR transgenic plants (expressing fusion proteins with the glucocorticoid receptor), as well as one-hybrid experiments, to determine the direct effect of these factors on TT8 expression. Interestingly, in vivo binding of TT2 and PAP1 to the TT8 promoter was dependent on the simultaneous expression of TT8 or the homologous bHLH factors GL3 and EGL3. Consistent with these results, the activity of the TT8::uidA reporter was strongly affected in the seed endothelium of a tt8 mutant. Similarly, a strong decrease in the level of TT8 mRNA was detected in the siliques of a gl3 · egl3 mutant and in plants that express a dominant negative form of the PAP1 protein, suggesting that TT8 expression is controlled by different combinations of MYB and bHLH factors in planta. The importance of this positive feedback mechanism in the strong and specific induction of proanthocyanidin biosynthesis in the seed coat of Arabidopsis thaliana is discussed.

Regulation of protein turnover mediated by ZEITLUPE (ZTL) constitutes an important mechanism of the circadian clock in Arabidopsis thaliana. Here, we report that FLAVIN BINDING, KELCH REPEAT, F-BOX1 (FKF1) and LOV KELCH PROTEIN2 (LKP2) play similar roles to ZTL in the circadian clock when ZTL is absent. In contrast with subtle circadian clock defects in fkf1, the clock in ztl fkf1 has a considerably longer period than in ztl. In ztl fkf1 lkp2, several clock parameters were even more severely affected than in ztl fkf1. Although LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSO-CIATED1 (CCA1) expression levels are lower in ztl than in the wild type, introducing both fkf1 and lkp2 mutations into the ztl mutant dramatically diminished LHY expression without further affecting CCA1 expression. This demonstrates different contributions of ZTL, FKF1, and LKP2 in the regulation of LHY and CCA1 expression. In addition, FKF1 and LKP2 also interacted with TIMING OF CAB EXPRESSION1 (TOC1) and PSEUDO-RESPONSE REGULATOR5 (PRR5), and both proteins were further stabilized in ztl fkf1 and ztl fkf1 lkp2 compared with in ztl. Our results indicate that ZTL, FKF1, and LKP2 together regulate TOC1 and PRR5 degradation and are major contributors to determining the period of circadian oscillation and enhancing robustness.