title: A single amino acid change within the R2 domain of the VvMYB5b transcription factor modulates affinity for protein partners and target promoters selectivity.
abstract: BACKGROUND
  Flavonoid pathway is spatially and temporally controlled during plant development and the transcriptional regulation of the structural genes is mostly orchestrated by a ternary protein complex that involves three classes of transcription factors (R2-R3-MYB, bHLH and WDR). In grapevine (Vitis vinifera L.), several MYB transcription factors have been identified but the interactions with their putative bHLH partners to regulate specific branches of the flavonoid pathway are still poorly understood.
  
  
  RESULTS
  In this work, we describe the effects of a single amino acid substitution (R69L) located in the R2 domain of VvMYB5b and predicted to affect the formation of a salt bridge within the protein. The activity of the mutated protein (name VvMYB5b(L), the native protein being referred as VvMYB5b(R)) was assessed in different in vivo systems: yeast, grape cell suspensions, and tobacco. In the first two systems, VvMYB5b(L) exhibited a modified trans-activation capability. Moreover, using yeast two-hybrid assay, we demonstrated that modification of VvMYB5b transcriptional properties impaired its ability to correctly interact with VvMYC1, a grape bHLH protein. These results were further substantiated by overexpression of VvMYB5b(R) and VvMYB5b(L) genes in tobacco. Flowers from 35S::VvMYB5b(L) transgenic plants showed a distinct phenotype in comparison with 35S::VvMYB5b(R) and the control plants. Finally, significant differences in transcript abundance of flavonoid metabolism genes were observed along with variations in pigments accumulation.
  
  
  CONCLUSIONS
  Taken together, our findings indicate that VvMYB5b(L) is still able to bind DNA but the structural consequences linked to the mutation affect the capacity of the protein to activate the transcription of some flavonoid genes by modifying the interaction with its co-partner(s). In addition, this study underlines the importance of an internal salt bridge for protein conformation and thus for the establishment of protein-protein interactions between MYB and bHLH transcription factors. Mechanisms underlying these interactions are discussed and a model is proposed to explain the transcriptional activity of VvMYB5(L) observed in the tobacco model.
authors.affiliation: Univ, de Bordeaux, Institut des Sciences de la Vigne et du Vin, UMR 1287 Ecophysiologie et Génomique Fonctionnelle de la Vigne, 210 Chemin de Leysotte, 33882 Villenave d'Ornon, France.
authors.forename: Imène
authors.initials: I
authors.lastname: Hichri
authors.forename: Laurent
authors.initials: L
authors.lastname: Deluc
authors.forename: François
authors.initials: F
authors.lastname: Barrieu
authors.forename: Jochen
authors.initials: J
authors.lastname: Bogs
authors.forename: Ali
authors.initials: A
authors.lastname: Mahjoub
authors.forename: Farid
authors.initials: F
authors.lastname: Regad
authors.forename: Bernard
authors.initials: B
authors.lastname: Gallois
authors.forename: Thierry
authors.initials: T
authors.lastname: Granier
authors.forename: Claudine
authors.initials: C
authors.lastname: Trossat-Magnin
authors.forename: Eric
authors.initials: E
authors.lastname: Gomès
authors.forename: Virginie
authors.initials: V
authors.lastname: Lauvergeat
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism; D005419:Flavonoids / Q000096:biosynthesis / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D020570:Genes, myb; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010641:Phenotype; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D018749:RNA, Plant / Q000235:genetics; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D027843:Vitis / Q000235:genetics* / Q000378:metabolism
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D005419:Flavonoids; D010940:Plant Proteins; D018749:RNA, Plant; D014157:Transcription Factors