A new system for fast and quantitative analysis of heterologous gene expression in plants.
• 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.
INRA AgroParisTech, IJPB, UMR 1318, INRA centre de Versailles, Versailles, France.
J
J
Thévenin
C
C
Dubos
W
W
Xu
J
J
Le Gourrierec
Z
Z
Kelemen
F
F
Charlot
F
F
Nogué
L
L
Lepiniec
B
B
Dubreucq
D017360:Arabidopsis / Q000235:genetics; D029681:Arabidopsis Proteins / Q000378:metabolism; D001665:Binding Sites; D019068:Bryopsida / Q000235:genetics*; D015870:Gene Expression; D018506:Gene Expression Regulation, Plant; D005821:Genetic Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008957:Models, Genetic; D046912:Multiprotein Complexes / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011523:Protoplasts / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012639:Seeds / Q000235:genetics; D014158:Transcription, Genetic; D014170:Transformation, Genetic
D029681:Arabidopsis Proteins; D046912:Multiprotein Complexes; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins