Exploration of jasmonate signalling via automated and standardized transient expression assays in tobacco cells

• Published in: The Plant journal : for cell and molecular biology Vol. 44; no. 6; pp. 1065 - 1076
• Main Authors: Sutter, V. de, Vanderhaeghen, R, Lammertyn, F, Vanhoutte, I, Karimi, M, Inze, D, Goossens, A, Hilson, P
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01-12-2005; Blackwell Science; Wiley
• Subjects: alkaloid biosynthesis; Arabidopsis; Arabidopsis - cytology; Arabidopsis - genetics; Arabidopsis - metabolism; Arabidopsis thaliana; automation; biochemical pathways; Biological and medical sciences; Cell physiology; Cells, Cultured; Cloning, Molecular - methods; cultured cells; Cyclopentanes - pharmacology; Fundamental and applied biological sciences. Psychology; Gateway recombinational cloning; gene expression; Gene Expression Profiling - methods; Gene Expression Profiling - standards; Gene Expression Regulation, Plant; Genes, Reporter; genetic vectors; jasmonic acid; Life Sciences; Luciferases, Firefly - analysis; Methyltransferases - metabolism; Molecular and cellular biology; molecular cloning; Nicotiana - cytology; Nicotiana - genetics; Nicotiana - metabolism; Nicotiana tabacum; nicotine; Nicotine - biosynthesis; Origin Recognition Complex - metabolism; Oxylipins; plant biochemistry; plant genetics; Plants, Genetically Modified - cytology; Plasmids - metabolism; protoplast transient expression assay; protoplasts; Protoplasts - metabolism; Robotics; Signal Transduction; tobacco; tobacco BY‐2; Transcription Factors - metabolism; Transfection - methods; Gateway recombinational cloning; Arabidopsis; EC 1.13.12.7; Biosynthesis; Nicotiana tabacum; Arabidopsis thaliana; Alkaloid; alkaloid biosynthesis; automation; Gene; Cruciferae; Dicotyledones; Protoplast; EC 2.1.1.53; Angiospermae; Development; Spermatophyta; tobacco BY-2; Solanaceae; protoplast transient expression assay; Transcription factor
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1111/j.1365-313X.2005.02586.x

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/Ca(2+) 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.