A single amino acid change within the R2 domain of the VvMYB5b transcription factor modulates affinity for protein partners and target promoters selectivity
1 Univ. de Bordeaux, Institut des Sciences de la Vigne et du Vin (ISVV), UMR 1287 Ecophysiologie et Génomique Fonctionnelle de la Vigne (EGFV), 210 Chemin de Leysotte, 33882 Villenave d'Ornon, France
2 INRA, ISVV, UMR 1287 EGFV, 33882 Villenave d'Ornon, France
3 ENITAB, ISVV, UMR 1287 EGFV, 33882 Villenave d'Ornon, France
4 Department of Horticulture, Oregon State University, Corvallis, Oregon 97331, USA
5 Dienstleistungszentrum Landlicher Raum (DLR) Rheinpfalz, Breitenweg 71, Viticulture and Enology group, D-67435 Neustadt/W, Germany
6 Fachhochschule Bingen, Berlinstr. 109, 55411 Bingen am Rhein, Germany
7 Université de Toulouse, INP-ENSAT Toulouse, Génomique et Biotechnologie des Fruits, Avenue de l'Agrobiopole BP 32607, 31326 Castanet-Tolosan, France
8 Chimie et Biologie des Membranes et des Nanoobjets, UMR CNRS 5248, Bâtiment B14bis, Allée Geoffroy de Saint Hilaire, Université Bordeaux, 33600 Pessac, France
BMC Plant Biology 2011, 11:117 doi:10.1186/1471-2229-11-117Published: 23 August 2011
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.
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 VvMYB5bL, the native protein being referred as VvMYB5bR) was assessed in different in vivo systems: yeast, grape cell suspensions, and tobacco. In the first two systems, VvMYB5bL 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 VvMYB5bR and VvMYB5bL genes in tobacco. Flowers from 35S::VvMYB5bL transgenic plants showed a distinct phenotype in comparison with 35S::VvMYB5bR and the control plants. Finally, significant differences in transcript abundance of flavonoid metabolism genes were observed along with variations in pigments accumulation.
Taken together, our findings indicate that VvMYB5bL 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 VvMYB5L observed in the tobacco model.