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Studies on abscisic acid signal transduction in rice (oryza sativa) protoplasts by quantitative dual-colour flow cytometry

Authors Hagenbeek, Dirk
Issue Date 2001
Summary Abscisic acid (ABA) is one of the major plant hormones. It is especially important for seed development and germination and adaptation to environmental stresses. Many of the plant physiological responses to ABA are the consequences of ABA-inducible or ABA-repressible gene expression. Our knowledge of the ABA signal transduction pathways leading to altered gene expression is far from complete. Two of the known transgenes affecting ABA-regulated gene expression are VIVIPAROUS1 (VPl) and ABA INSENSITIVE1 (ABI1). VP1 encodes a transcriptional activator which activates ABA-inducible genes by binding to a DNA-binding basic leucine zipper protein specific for ABA responsive elements (ABREs). The ABIl gene encodes a serine/threonine protein phosphatase with a negative regulatory role in ABA signalling. Rice protoplasts are a good model system to study the cell biology of ABA transduction. They express introduced DNA constructs, are sensitive to exogenous ABA and pharmacological agents, and they make single cell analyses possible. Most reporter gene activities are quantified analyzed by destructive enzyme assays. Quantifying plant gene expression by flow cytometry (FCM) would allow multidimensional cell parameter analysis on a per-cell basis, thereby providing insight into the cellular mechanisms of plant gene regulation. Therefore we established quantitation by FCM of ABA-inducible green fluorescent protein (GFP) expression and statistically compared the method with traditional reporter enzyme assays. Dual-color FCM of GFP-expressing cells immunodecorated with mAbs recognizing rice cell surface epitopes was successfully performed. We conclude that quantitation by FCM of inducible gene expression in rice protoplasts is a valid method with the advantages over biochemical assays of live cell analysis, sorting of populations, and multiparameter correlative analysis. Interactions between ABA, ABI1, VPl, and lanthanum ions were observed by flow cytometry and traditional reporter gene assays in transiently transformed rice protoplasts. Lanthanum and other trivalent ions, were established as specific agonists of ABA-inducible gene expression. The mechanism of trivalent ion action on ABA signalling is unknown. The findings support a sequential model of effector action in ABA signalling cascades, from ABA, La3+ to ABIl to VP1 to gene expression in a single or partially overlapping pathways. The transient rice protoplast system was further used to show that mAbs recognizing specific carbohydrate epitopes of cell surface molecules had modulating effects on ABA-inducible gene expression. JIM19 inhibited ABA-inducible Em promoter activity about 40% and JIM7 activated it about 35%, suggesting that they might directly or indirectly interact with an ABA receptor complex, changing the receptor's sensitivity to ABA. These mAbs, lanthanum and other trivalent ions might be useful tools to study cellular mechanisms of ABA signal transduction.
Note Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2001
Language English
Format Thesis
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