Regulation of stilbene biosynthesis in Vitis vinifera by the transcription factors VvMYB14 and VvMYB15
Funded by the Federal Ministry of Education and Research and the Italian project AGER-SERRES, 2010-2015.
This project was carried out as part of a doctoral thesis by Janine Höll
Summary
Stilbenes are polyphenols that play an essential role in the plant defense system in the protection against biotic (e.g. attack by herbivores, microbes) and abiotic (e.g. UV radiation) stress. Stilbene biosynthesis occurs in only a few plant species, with grapevine representing one of the main producers. The basic building block of most stilbenes is trans-resveratrol, which is formed using precursors from the phenylpropanoid reaction pathway. This condensation reaction is catalyzed by stilbene synthase (STS). So far, stilbene synthases from different plants have been isolated and partially characterized, but the transcriptional regulation of stilbene biosynthesis was unknown for a long time. In this project, two R2R3-MYB transcription factors involved in the regulation of stilbene biosynthesis in Vitis vinifera (grapevine) were isolated and characterized. The two MYB transcription factors were named VvMYB14 and VvMYB15. Using transient promoter expression analysis in grapevine and in the heterologous tobacco system, a specific activation of the stilbene synthase promoter by the two transcription factors could be demonstrated. Crucially, however, ectopic expression of the regulator VvMYB15 in different grapevine model systems (leaf infiltration, hairy roots and in vitro plants) led to an induction of stilbene biosynthesis, which was consequently reflected in an increase in stilbene production. These metabolite data could be confirmed by LC-Q-TOF-MS analysis and furthermore various simple and oligomeric stilbene derivatives with diverse modifications could be identified by this methodology.
To obtain further information on the regulation of stilbene synthase by the two transcription factors VvMYB14 and VvMYB15, their behavior was examined in relation to different environmental conditions and developmental factors. Both transcription factors and the tested stilbene synthases responded with increased expression under stress. The analysis included cold stress, addition of hormones, wounding and lack of light. In addition, a comparative gene expression analysis during different growth phases in grapes showed an increase in the expression of both regulators and the tested stilbene synthases at the beginning of ripening ("véraison") in the berry skin and in the seeds. This was also reflected in an increased concentration of stilbenes in the ripe berry. In addition, a gene expression analysis was carried out in various non-stressed wine tissues and different wine varieties, which showed a consistent profile of VvMYB14/15 and STS expression and stilbene content in all samples examined. These analyses of the influence of developmental changes, environmental factors and tissue specificity detected a high basal level of expression of both transcription factors and stilbene biosynthesis, which can be increased many times over under the influence of stress. Furthermore, a divergent expression behavior of the two transcription factors under various conditions became apparent in the course of the investigations, which may indicate a divergent regulatory function of VvMYB14 and VvMYB15.
In the course of this work, two R2R3-MYB transcription factors were identified and functionally characterized for the first time, which were able to induce stilbene biosynthesis in wine.
Publications:
Höll et al (2013): The R2R3-MYB transcription factors MYB14 and MYB15 regulate stilbene biosynthesis in Vitis vinifera. In: The Plant cell 25. 10.1105/tpc.113.117127.
Höll Janine (2014): The R2R3 MYB Transcription Factors VvMYB14 and VvMYB15 as Regulators of the Stilbene Biosynthesis in Vitis vinifera. Dissertation