Positive Fluorescent Selection Permits Precise, Rapid, and In-Depth Overexpression Analysis in Plant Protoplasts

• Published in: Plant physiology (Bethesda) Vol. 149; no. 3; pp. 1231 - 1239
• Main Authors: Bargmann, Bastiaan O.R, Birnbaum, Kenneth D
• Format: Journal Article
• Language: English
• Published: Rockville, MD American Society of Plant Biologists 01.03.2009
• Subjects: Arabidopsis; Arabidopsis - genetics; Arabidopsis - metabolism; Auxins; Biological and medical sciences; Breakthrough Technologies; cell culture; Cells; Chemical suspensions; chemistry; drug effects; flow cytometry; Fundamental and applied biological sciences. Psychology; gene activation; Gene Expression Regulation, Plant; Gene Expression Regulation, Plant - drug effects; Genes, Reporter; Genetic vectors; genetics; green fluorescent protein; Green Fluorescent Proteins; Green Fluorescent Proteins - metabolism; Indoleacetic Acids; Indoleacetic Acids - chemistry; Indoleacetic Acids - pharmacology; Luminescent Measurements; Luminescent Measurements - methods; Luminescent Proteins; Luminescent Proteins - metabolism; metabolism; methods; Organ Specificity; Organ Specificity - drug effects; pharmacology; Plant physiology and development; Plant Proteins; Plant Proteins - genetics; Plant Proteins - metabolism; Plant roots; Plants; Plasmids; Protoplasts; Protoplasts - drug effects; Protoplasts - metabolism; rapid methods; Red Fluorescent Protein; Reporter genes; roots; screening; signal transduction; Transcription, Genetic; Transcription, Genetic - drug effects; Transfection; Plant physiology; Fluorescent compound; Protoplast; Depth
• ISSN: 0032-0889; 1532-2548; 1532-2548
• DOI: 10.1104/pp.108.133975

Transient genetic modification of plant protoplasts is a straightforward and rapid technique for the study of numerous aspects of plant biology. Recent studies in metazoan systems have utilized cell-based assays to interrogate signal transduction pathways using high-throughput methods. Plant biologists could benefit from new tools that expand the use of cell culture for large-scale analysis of gene function. We have developed a system that employs fluorescent positive selection in combination with flow cytometric analysis and fluorescence-activated cell sorting to isolate responses in the transformed protoplasts exclusively. The system overcomes the drawback that transfected protoplast suspensions are often a heterogeneous mix of cells that have and have not been successfully transformed. This Gateway-compatible system enables high-throughput screening of genetic circuitry using overexpression. The incorporation of a red fluorescent protein selection marker enables combined utilization with widely available green fluorescent protein (GFP) tools. For instance, such a dual labeling approach allows cytometric analysis of GFP reporter gene activation expressly in the transformed cells or fluorescence-activated cell sorting-mediated isolation and downstream examination of overexpression effects in a specific GFP-marked cell population. Here, as an example, novel uses of this system are applied to the study of auxin signaling, exploiting the red fluorescent protein/GFP dual labeling capability. In response to manipulation of the auxin response network through overexpression of dominant negative auxin signaling components, we quantify effects on auxin-responsive DR5::GFP reporter gene activation as well as profile genome-wide transcriptional changes specifically in cells expressing a root epidermal marker.