Volume 3, Issue 3, Pages 509-523 (May 2010) The Basic Helix-Loop-Helix Transcription Factor MYC1 Is Involved in the Regulation of the Flavonoid Biosynthesis Pathway in Grapevine  Hichri Imène , Heppel Simon C. , Pillet Jérémy , Léon Céline , Czemmel Stefan , Delrot Serge , Lauvergeat Virginie , Bogs Jochen   Molecular Plant  Volume 3, Issue 3, Pages 509-523 (May 2010) DOI: 10.1093/mp/ssp118 Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 1 Phylogenetic analysis of VvMYC1 and related bHLH proteins. (A) Full-length protein sequence alignment of VvMYC1 and its closest homologs PhAN1, AtTT8, and IpIVS. Identical residues are shown in black and conserved residues in dark gray. The MIR (MYB-Interacting Region), the acidic, and the bHLH domains are labeled. (B) Phylogenetic tree of different bHLH proteins regulating the flavonoid pathway in plants. Analysis was performed with Arabidopsis thaliana TT8, GL3, EGL3, and MYC1, Antirrhinum majus DELILA, Oryza sativa Rc, Petunia hybrida AN1 and JAF13, Zea mays B, Lc, and IN1, Malus domestica bHLH33, Gerbera hybrida MYC1, Perilla frutescens MYC-RP, Ipomoea purpurea Ivory seed bHLH proteins, and Vitis vinifera MYC1 and MYCA1. Phylogenetic analyses were conducted using MEGA4 (Tamura et al., 2007). Full-length protein sequences were aligned with Muscle (Edgar, 2004), and the phylogenetic tree was constructed according to the neighbor-joining method. The bootstrap values out of 2000 retrials are indicated at each branch. The scale bar represents the number of substitutions per site. Molecular Plant 2010 3, 509-523DOI: (10.1093/mp/ssp118) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 2 Developmental and Tissue-Specific Analysis of VvMYC1 Expression by Quantitative PCR (qPCR). (A–C) Transcript levels of VvMYC1 in Vitis vinifera L. cv ‘Shiraz’ during grape berry development. Gene expression is shown relative to expression of VvUbiquitin1 in each sample. (D) Analysis of VvMYC1 transcript accumulation in vegetative organs (roots, stems, and leaves) of V. vinifera L. cv ‘Cabernet Sauvignon’. (E–F) Expression of VvMYC1 in seeds and skins of V. vinifera cv ‘Cabernet Sauvignon’ during grape berry development. Gene expression levels of Cabernet Sauvignon samples were normalized by VvEF1g and VvActin transcript levels. All data are presented as mean of three replicates with error bars indicating ±SD. Molecular Plant 2010 3, 509-523DOI: (10.1093/mp/ssp118) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 3 Sub-Cellular Localization of VvMYC1 Using Transient Expression of YFP–VvMYC1 Fusion Protein in Grape Cell Suspension Protoplasts. (A) YFP fluorescence. (B) Bright field image. Scale bars are indicated in the bright field image. Molecular Plant 2010 3, 509-523DOI: (10.1093/mp/ssp118) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 4 Transactivation Capacity of VvMYC1 and VvMYCA1. (A) Constructs of reporter and effector plasmids. VvMYC1 full-length and partial cDNAs VvMYC1ΔNterm (amino acids 219–701) and VvMYC1ΔCterm (amino acids 1–423) and VvMYCA1 ORF were fused to GAL4 DNA-Binding Domain (DBD). (B) In yeast transactivation assay of the LacZ reporter gene by VvMYC1, VvMYCA1 and two truncated VvMYC1 peptides. Activation of LacZ by the respective constructs was determined by measuring β-galactosidase activity. Each value is the mean of two independent yeast transformations and each experiment included three measurements. Error bars indicate SD. MEL1 UAS, Melibiose 1-GAL4 Upstream Activating Sequence; mp, minimal promoter; pADH1, Alcohol Dehydrogenase 1 promoter. Molecular Plant 2010 3, 509-523DOI: (10.1093/mp/ssp118) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 5 Interaction of VvMYC1 and Various Vitis vinifera MYB Transcription Factors. Yeast two-hybrid experiments have been performed by co-transformation with VvMYC1 or VvMYC1ΔNterm fused to GAL4 DNA-Binding Domain (DBD), and VvMYC1 or the indicated V. vinifera MYB proteins fused to GAL4 Activation Domain (AD): MYC1-AD, VvMyb5a-AD, VvMyb5b-AD, VvMybPA1-AD, and VvMybA1-AD. Yeasts were selected on SD -Ade -His -Leu -Trp medium. Molecular Plant 2010 3, 509-523DOI: (10.1093/mp/ssp118) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 6 VvMYC1 Interacts with Various MYB Transcription Factors to Activate Promoters of Flavonoid Pathway Genes and Regulates Its Own Promoter. The MYB transcription factors and promoter fragments used for transfection of grape cell cultures are indicated. Each transfection contained as internal control the Renilla luciferase plasmid pRluc (Horstmann et al., 2004). The normalized luciferase activity was calculated as the ratio between the Firefly and the Renilla luciferase activity. All values indicate the fold increase relative to the control (–/–), which was the activity of the respective promoter transfected without addition of transcription factors. Each column represents the mean value of three independent experiments with error bars indicating six SEs. Molecular Plant 2010 3, 509-523DOI: (10.1093/mp/ssp118) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 7 Transient Expression of VvMYC1, VvMYBA1, and GFP in Grapevine Suspension Cells after Particle Bombardment. (A) Accumulation of anthocyanins in several transformed grapevine cells. (B) Single transformed grapevine cell accumulating anthocyanins in the vacuole. (C) Same cell as in (B), showing GFP fluorescence in the cytosol. Bar 100 μm in (A); 20 μm in (B) and (C). Molecular Plant 2010 3, 509-523DOI: (10.1093/mp/ssp118) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions