title: Exploration of jasmonate signalling via automated and standardized transient expression assays in tobacco cells.
abstract: Although sequence information and genome annotation are improving at an impressive pace, functional ontology is still non-existent or rudimentary for most genes. In this regard, transient expression assays are very valuable for identification of short functional segments in particular pathways, because they can be performed rapidly and at a scale unattainable in stably transformed tissues. Vectors were constructed and protocols developed for systematic transient assays in plant protoplasts. To enhance throughput and reproducibility, protoplast treatments were performed entirely by a liquid-handling robot in multiwell plates, including polyethylene glycol/Ca2+ cell transfection with plasmid mixtures, washes and lysis. All transcriptional readouts were measured using a dual firefly/Renilla luciferase assay, in which the former was controlled by a reporter promoter and the latter by the 35S CaMV promoter, which served as internal normalization standard. The automated protocols were suitable for transient assays in protoplasts prepared from cell cultures of Nicotiana tabacum Bright Yellow-2 and Arabidopsis thaliana. They were implemented in a screen to discover potential regulators of genes coding for key enzymes in nicotine biosynthesis. Two novel tobacco transcription factors were found, NtORC1 and NtJAP1, that positively regulate the putrescine N-methyltransferase (PMT) promoter. In addition, combinatorial tests showed that these two factors act synergistically to induce PMT transcriptional activity. The development and use of high-throughput plant transient expression assays are discussed.
authors.affiliation: Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology, Ghent University, Technologiepark 927, B-9052 Gent, Belgium.
authors.forename: Valerie
authors.initials: V
authors.lastname: De Sutter
authors.forename: Rudy
authors.initials: R
authors.lastname: Vanderhaeghen
authors.forename: Sofie
authors.initials: S
authors.lastname: Tilleman
authors.forename: Freya
authors.initials: F
authors.lastname: Lammertyn
authors.forename: Isabelle
authors.initials: I
authors.lastname: Vanhoutte
authors.forename: Mansour
authors.initials: M
authors.lastname: Karimi
authors.forename: Dirk
authors.initials: D
authors.lastname: Inzé
authors.forename: Alain
authors.initials: A
authors.lastname: Goossens
authors.forename: Pierre
authors.initials: P
authors.lastname: Hilson
mesh_terms: D017360:Arabidopsis / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods; D003517:Cyclopentanes / Q000494:pharmacology*; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards; D018506:Gene Expression Regulation, Plant; D017930:Genes, Reporter; D049409:Luciferases, Firefly / Q000032:analysis; D008780:Methyltransferases / Q000378:metabolism; D009538:Nicotine / Q000096:biosynthesis; D051738:Origin Recognition Complex / Q000378:metabolism; D054883:Oxylipins; D030821:Plants, Genetically Modified / Q000166:cytology; D010957:Plasmids / Q000378:metabolism; D011523:Protoplasts / Q000378:metabolism; D012371:Robotics; D015398:Signal Transduction; D014026:Nicotiana / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection / Q000379:methods
chemical_list: D003517:Cyclopentanes; D051738:Origin Recognition Complex; D054883:Oxylipins; D014157:Transcription Factors; D009538:Nicotine; C011006:jasmonic acid; D049409:Luciferases, Firefly; D008780:Methyltransferases; C112598:putrescine N-methyltransferase