A new system for fast and quantitative analysis of heterologous gene expression in plants

Abstract

Summary• 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 GatewayÒ 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.

Endosperm breakdown in Arabidopsis requires heterodimers of the basic helix-loop-helix proteins ZHOUPI and INDUCER OF CBP EXPRESSION 1

“…S3). We tested the activation of the At3g08900 promoter by ZOU, ICE1 or SCRM-D and by combinations of ZOU with ICE or SCRM-D in a Physcomitrella patens protoplast system (Thévenin et al, 2012), and confirmed that only combinations of ZOU with ICE or SCRM-D could activate promoter activity to levels above background (supplementary material Fig. S3).…”

“…Moss culture, protoplast preparation, vector pBS TPp-A and -B recombination, protoplast transformation and flow cytometry measurement were carried out as described (Thévenin et al, 2012) (see supplementary material Fig. S3).…”

Nuclear retention of the transcription factor NLP7 orchestrates the early response to nitrate in plants

“…For promoter transactivation assays, Arabidopsis protoplasts were prepared from cell cultures by a sucrose gradient after 3 h of cell wall digestion as in Thomine et al 39 polyethylene glycol (PEG)-mediated transfection was performed using 10 mg of plasmid DNA. Plasmids were obtained from M. Lepetit (pNRT2.1:LUC) 21 and from B. Dubreucq (pACT:LEC2) 40 . The p35S:NLP7 construct was obtained by subcloning into pBS 7 .…”

MYB118 Represses Endosperm Maturation in Seeds of Arabidopsis

“…Promoters previously cloned into the pDONR207 entry vector (see above) were transferred into the destination vector pBS TPp-B (Thévenin et al, 2012) by LR recombination.…”