Enhanced fluorescence imaging in chlorophyll-suppressed tobacco tissues using virus-induced gene silencing of the phytoene desaturase gene

Abstract

Fluorescence imaging in plants is unusually challenging because of the large amounts of photosynthetic pigments contained in green plant tissues. For example, chlorophyll can obstruct the penetration of light and has high levels of autofluorescence at wavelengths that are often used for fluorescence imaging. Until now, mostly confocal laser scanning microscopy or the use of non-green parts of the plants, typically roots, have been used to overcome these limitations. We constructed tobacco (Nicotiana attenuata) plants expressing GFP-sporamin fusion polypeptide in their vascular tissues. As expected, it was not possible to visualize GFP fluorescence in tobacco leaves or stems using a stereomicroscope and filters specific for GFP detection; however, GFP fluorescence was readily detectable when virus-induced gene silencing (VIGS) was used to transiently silence the phytoene desaturase (PDS) gene in order to bleach chlorophyll-containing tissues. This method is an inexpensive alternative to confocal laser scanning microscopy for the detection of GFP fusion proteins or promoter-GFP reporter fusions in plant leaves.

Keywords

• chlorophyll
• fluorescence microscopy
• green fluorescence protein
• GFP
• phytoene desaturase
• tobacco plant