Imaging plant cell death: GFP-Nit1 aggregation marks an early step of wound and herbicide induced cell death.
BACKGROUND
A great deal is known about the morphological endpoints of plant cell death, but relatively little is known about its sequence of events and/or its execution at the biochemical level. Live cell imaging using GFP-tagged markers is a powerful way to provide dynamic portraits of a cellular process that can in turn provide a descriptive foundation valuable for future biochemical and genetic investigations.


RESULTS
While characterizing a collection of random GFP-protein fusion markers we discovered that mechanical wounding induces rapid aggregation of a GFP-Nitrilase 1 fusion protein in Arabidopsis cells directly abutting wound sites. Time-lapse imaging of this response shows that the aggregation occurs in cells that subsequently die 30-60 minutes post-wounding, indicating that GFP-Nit1 aggregation is an early marker of cell death at wound sites. Time-lapse confocal imaging was used to characterize wound-induced cell death using GFP-Nit1 and markers of the nucleus and endoplasmic reticulum. These analyses provide dynamic portraits of well-known death-associated responses such as nuclear contraction and cellular collapse and reveal novel features such as nuclear envelope separation, ER vesiculation and loss of nuclear-lumen contents. As a parallel system for imaging cell death, we developed a chemical method for rapidly triggering cell death using the herbicides bromoxynil or chloroxynil which cause rapid GFP-Nit1 aggregation, loss of nuclear contents and cellular collapse, but not nuclear contraction, separating this response from others during plant cell death.


CONCLUSIONS
Our observations place aggregation of Nitrilase 1 as one of the earliest events associated with wound and herbicide-induced cell death and highlight several novel cellular events that occur as plant cells die. Our data create a detailed descriptive framework for future investigations of plant cell death and provide new tools for both its cellular and biochemical analysis.
Cutler
Sean R
SR
Department of Botany, University of Toronto, 25 Willcocks St., Toronto, Ontario, M5S 3B2, Canada. cutler@botany.utoronto.ca
Somerville
Chris R
CR
D000619:Aminohydrolases / Q000032:analysis* / Q000235:genetics; D017360:Arabidopsis / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D029681:Arabidopsis Proteins / Q000032:analysis* / Q000235:genetics; D015415:Biomarkers; D016923:Cell Death / Q000502:physiology*; D002467:Cell Nucleus / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure; D004721:Endoplasmic Reticulum / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics; D006540:Herbicides / Q000633:toxicity*; D021621:Imaging, Three-Dimensional; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009570:Nitriles / Q000633:toxicity; D009685:Nuclear Envelope / Q000502:physiology / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000032:analysis
D029681:Arabidopsis Proteins; D015415:Biomarkers; D006540:Herbicides; D009570:Nitriles; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C014356:benzonitrile; D000619:Aminohydrolases; C021867:nitrilase; C006826:bromoxynil