These authors contributed equally to this work.
A new system for fast and quantitative analysis of heterologous gene expression in plants
Article first published online: 24 OCT 2011
© 2011 INRA. New Phytologist © 2011 New Phytologist Trust
Volume 193, Issue 2, pages 504–512, January 2012
How to Cite
Thévenin, J., Dubos, C., Xu, W., Le Gourrierec, J., Kelemen, Z., Charlot, F., Nogué, F., Lepiniec, L. and Dubreucq, B. (2012), A new system for fast and quantitative analysis of heterologous gene expression in plants. New Phytologist, 193: 504–512. doi: 10.1111/j.1469-8137.2011.03936.x
- Issue published online: 20 DEC 2011
- Article first published online: 24 OCT 2011
- Received: 21 July 2011, Accepted: 13 September 2011
- Physcomitrella patens;
- transcription factor
- •Large-scale analysis of transcription factor–cis-acting element interactions in plants, or the dissection of complex transcriptional regulatory mechanisms, requires rapid, robust and reliable systems for the quantification of gene expression.
- •Here, we describe a new system for transient expression analysis of transcription factors, which takes advantage of the fast and easy production and transfection of Physcomitrella patens protoplasts, coupled to flow cytometry quantification of a fluorescent protein (green fluorescent protein). Two small-sized and high-copy Gateway® vectors were specifically designed, although standard binary vectors can also be employed.
- •As a proof of concept, the regulation of BANYULS (BAN), a key structural gene involved in proanthocyanidin biosynthesis in Arabidopsis thaliana seeds, was used. In P. patens, BAN expression is activated by a complex composed of three proteins (TT2/AtMYB123, TT8/bHLH042 and TTG1), and is inhibited by MYBL2, a transcriptional repressor, as in Arabidopsis. Using this approach, two new regulatory sequences that are necessary and sufficient for specific BAN expression in proanthocyanidin-accumulating cells were identified.
- •This one hybrid-like plant system was successfully employed to quantitatively assess the transcriptional activity of four regulatory proteins, and to identify their target recognition sites on the BAN promoter.