Quantitative analysis by flow cytometry of abscisic acid-inducible gene expression in transiently transformed rice protoplasts.
BACKGROUND
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. Here we sought to establish quantitation by FCM of plant hormone (abscisic acid, ABA)-inducible green fluorescent protein (GFP) expression and to compare the method directly with traditional reporter enzyme assays.


METHODS
GFP, beta-glucuronidase, and luciferase reporter genes driven by ABA-inducible or constitutive promoter constructs were expressed in transiently cotransformed rice protoplasts and reporter activities quantified by FCM (for GFP) or traditional enzyme assays. Treatments included cotransformations with specific ABA signaling effector cDNA constructs (encoding VIVIPAROUS-1, an ABA transcription factor, and ABA-INSENSITIVE1-1, a dominant-negative protein phosphatase regulator) and the ABA agonist lanthanum chloride. Dual-color FCM was also performed on GFP-expressing cells immunodecorated with an mAb recognizing a rice cell surface epitope.


RESULTS
Quantitative analysis of ABA-inducible gene expression by FCM using GFP as reporter gave comparable results to traditional reporter enzyme assays, although the signal-to-noise ratio was less for FCM, which can be a limitation of the method at low promoter strengths. Multiparameter-correlated analysis of ABA-inducible GFP expression with a plasma membrane marker showed no apparent correlation between ABA sensitivity, marked by GFP, and presence of a cell surface arabinogalactan glycoprotein.


CONCLUSIONS
Quantitative FCM of GFP-expressing plant cells is a rapid, robust, reproducible, and value-added method relative to traditional enzymatic reporter gene assays.
Hagenbeek
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Department of Biology, Hong Kong University of Science and Technology, Kowloon, Hong Kong, China.
Rock
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D000040:Abscisic Acid / Q000494:pharmacology*; D005434:Flow Cytometry / Q000379:methods; D015870:Gene Expression / Q000187:drug effects*; D017930:Genes, Reporter; D005966:Glucuronidase / Q000032:analysis; D049452:Green Fluorescent Proteins; D008156:Luciferases / Q000032:analysis; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins / Q000032:analysis; D012275:Oryza / Q000187:drug effects* / Q000235:genetics; D011523:Protoplasts / Q000187:drug effects* / Q000502:physiology; D014170:Transformation, Genetic
D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D049452:Green Fluorescent Proteins; D000040:Abscisic Acid; D008156:Luciferases; D005966:Glucuronidase