Green-fluorescent protein as a new vital marker in plant cells.
The green-fluorescent protein (GFP) from jellyfish Aequorea victoria has been used as a convenient new vital marker in various heterologous systems. However, it has been problematic to express GFP in higher eukaryotes, especially in higher plants. This paper reports that either a strong constitutive or a heat-shock promoter can direct the expression of GFP which is easily detectable in maize mesophyll protoplasts. In this single-cell system, bright green fluorescence emitted from GFP is visible when excited with UV or blue light even in the presence of blue fluorescence from the vacuole or the red chlorophyll autofluorescence from chloroplasts using a fluorescence microscope. No exogenous substrate, co-factor, or other gene product is required. GFP is very stable in plant cells and shows little photobleaching. Viable cells can be obtained after fluorescence-activated cell sorting based on GFP. The paper further reports that GFP can be detected in intact tissues after delivering the constructs into Arabidopsis leaf and root by microprojectile bombardment. The successful detection of GFP in plant cells relies on the use of a universal transcription enhancer from maize or the translation enhancer from tobacco mosaic virus (TMV) to boost the expression. This new reporter could be used to monitor gene expression, signal transduction, co-transfection, transformation, protein trafficking and localization, protein-protein interaction, cell separation and purification, and cell lineage in higher plants.
Sheen
J
J
Department of Molecular Biology, Massachusetts General Hospital, Boston, USA.
Hwang
S
S
Niwa
Y
Y
Kobayashi
H
H
Galbraith
D W
DW
D000818:Animals; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D015415:Biomarkers; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D005819:Genetic Markers; D049452:Green Fluorescent Proteins; D018869:Heat-Shock Response; D008164:Luminescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D008856:Microscopy, Fluorescence; D018515:Plant Leaves / Q000378:metabolism; D018517:Plant Roots / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D037401:Scyphozoa / Q000235:genetics*; D013050:Spectrometry, Fluorescence; D014162:Transfection; D003313:Zea mays / Q000235:genetics / Q000378:metabolism
D015415:Biomarkers; D005819:Genetic Markers; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins