Random GFP :: cDNA fusions enable visualization of subcellular structures in cells of Arabidopsis at a high frequency

We describe a general approach for identifying components of subcellular st ructures in a multicellular organism by exploiting the ability to generate thousands of independent transformants in Arabidopsis thaliana. A library o f Arabidopsis cDNAs was constructed so that the cDNAs were inserted at the 3' end of the green fluorescent protein (GFP) coding sequence. The library was introduced en masse into Arabidopsis by Agrobacterium-mediated transfor mation. Fluorescence imaging of 5,700 transgenic plants indicated that appr oximate to 2% of lines expressed a fusion protein with a different subcellu lar distribution than that of soluble GFP, About half of the markers identi fied were targeted to peroxisomes or other subcellular destinations by non- native coding sequence (i.e., out-of-frame cDNAs). This observation suggest s that some targeting signals are of sufficiently law information content t hat they can be generated frequently by chance, The potential of the approa ch for identifying markers with unique dynamic processes is demonstrated by the identification of a GFP fusion protein that displays a cell-cycle regu lated change in subcellular distribution. Our results indicate that screeni ng GFP-fusion protein libraries is a useful approach for identifying and vi sualizing components of subcellular structures and their associated dynamic s in higher plant cells.