Quantitative analysis by flow cytometry of abscisic acid-inducible gene expression in transiently transformed rice protoplasts.

摘要: 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 regulation. Here we sought to establish quantitation FCM hormone (abscisic acid, ABA)-inducible green fluorescent protein (GFP) and compare method directly with traditional reporter enzyme assays. Materials Methods GFP, β-glucuronidase, luciferase genes driven ABA-inducible or constitutive promoter constructs were expressed in transiently cotransformed rice protoplasts activities quantified (for GFP) assays. Treatments included cotransformations specific ABA signaling effector cDNA (encoding VIVIPAROUS-1, an transcription factor, ABA-INSENSITIVE1-1, dominant-negative phosphatase regulator) agonist lanthanum chloride. Dual-color was also performed GFP-expressing cells immunodecorated mAb recognizing cell surface epitope. Results Quantitative using GFP as gave comparable results assays, although signal-to-noise ratio less for FCM, which can be limitation at low strengths. Multiparameter-correlated plasma membrane marker showed no apparent correlation between sensitivity, marked GFP, presence arabinogalactan glycoprotein. Conclusions Quantitative is rapid, robust, reproducible, value-added relative enzymatic Cytometry 45:170–179, 2001. © 2001 Wiley-Liss, Inc.