2Department of Molecular Ecology, Max Planck Institute for Chemical Ecology, Jena, Germany
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.
Fluorescence microscopy became indispensable to the biological sciences because of its high sensitivity, the selectivity with which it could be used to analyze signals, and its non-destructive character, which made it possible to carry out in vivo experiments for real-time analyses (1). A large variety of fluorescent dyes have been developed to detect and localize biomolecules in living cells and to visualize organelles, membrane structures, cell compartments, and specific tissues. The discovery of fluorescent proteins (2) has enabled the production of fusion proteins and promoter-reporter fusions that can be localized in living cells and tissues, both in animals and plants.
Fluorescent dyes and proteins are designed to be excited with a broad spectrum of light sources ranging from 330-nm [e.g., of dansyl cadaverine used to label proteins by transaminidation (3)], to 700-nm excitation wavelengths (e.g., of Alexa 700 used to label antibodies and detect proteins in cells). Correspondingly, emitted lights can range from 400 to 750 nm for various fluorophores. However, plant tissues are known to possess high levels of autofluorescence in chloroplasts and to accumulate various fluorescent secondary metabolites, for example, phenolics and alkaloids, in their vacuolar compartments (4). In chloroplasts, endogenous autofluorescence arises from a variety of biomolecules, including chlorophyll, carotene, and xanthophyll. Chlorophyll, the major contributor to autofluorescence, has an absorption band in the blue region of the visible spectrum, which produces a significant amount of fluorescence at wavelengths >600 nm when excited with wavelengths between 420 and 460 nm (5,6). Therefore, the presence of photosynthetic pigments severely restricts the usefulness of fluoroimaging in plants. With the development and progress in confocal laser scanning microscopy, imaging with green cells and tissues became possible (7); however, efficient fluorescence imaging with larger intact portions of the green tissues remains difficult.
Previously, it has been shown that chlorophyll bleaching herbicide isoxaflutole (IFT) can facilitate GFP detection in transgenic soybean tissues in vitro (8). Here, we demonstrate that removing chlorophyll and xanthophyll pigments with a post-transcriptional gene silencing method (9) can be another inexpensive alternative means of conducting fluorescence imaging in green plant parts. Specifically, we show that transiently silencing phytoene desaturase in the leaves, stems, and floral tissues by virus-induced gene silencing (VIGS) allows for the visualization of a GFP-sporamin fusion protein expressed in the vascular bundles of tobacco leaves.Material and methods Plant material and GFP-fusion protein transformation
GFP-SPOR plants were obtained by transforming Nicotiana attenuata plants (10) with the binary plant transformation vector pRESC2SPOR, carrying a fusion of the green fluorescent protein (eGFP) gene and the sweet potato sporamin coding sequence under the control of SUC2 promoter from Arabidopsis thaliana (11). To construct pRESC2SPOR (11.4 kb), the described expression cassette was PCR-amplified with the primers SUC1–31 (5′-GCGGCGTCTAGAGCATGCAAAATAGCACACC-3′) and SUC2–33 (5′-GCGGCGGGTCACCATTACACATCGGTAGGTTTG-3′) and plasmid pGTV-Spor (provided by Norbert Sauer, Molecular Plant Physiology, Universität Erlangen-Nürnberg, Erlangen, Germany) (11) as a template, using high-fidelity Phusion DNA polymerase (Finnzymes, Woburn, MA, USA). The obtained PCR product (2.2 kb) was digested with XbaI (all restriction enzymes provided by New England BioLabs GmbH, Frankfurt, Germany) and BstEII and cloned in the vector pRESC200 (12) cut with the same enzymes (9.2 kb). The GFP-SPOR–transformed N. attenuata plants were cultivated in the glasshouse to produce seeds; these were germinated together with wild-type (WT) seeds in petri dishes on Gamborg's B5 medium (13) (Gold Biotechnology, St. Louis, MO, USA), supplemented with 35 mg/L hygromycin (transgenic line only; Duchefa, Haarlem, The Netherlands), as described in Reference 10,.Plant cultivation and VIGS silencing
Freshly germinated 10-day-old seedlings were transplanted from petri dishes to Teku pots (Pöppelmann GmbH & Co. KG, Lohne, Germany) with soil and cultivated for an additional 10 days before being transferred to larger 1-L pots with soil. After 5 days, the seedling were agro-infiltrated on the three youngest expanded leaves with Agrobacterium tumefaciens GV3101 culture carrying pTVPD silencing vector or empty vector (EV) pTV00 (14), together with A. tumefaciens GV3101 carrying plasmid pBINTRA6, which contains an essential intron-disrupted RNA1 of tobacco rattle virus (TRV) (plasmids provided by Sir David Baulcombe, Professor of Botany, Royal Society Research Professor Department of Plant Sciences, University of Cambridge, Cambridge, UK) (14), in a 1:1 ratio, all dispensed in 5 mM MgCl2/5 mM MES (Sigma-Aldrich Chemie GmbH, Munich, Germany) buffer as described in References (14) and (15). pTV00 vector contained no insert (14); pTVPD (D. Baulcombe, personal communication) consisted of pTV00 carrying between its SalI and ApaI sites a 206-bp fragment of the N. benthamiana pds gene (positions 1–206 in GenBank AJ571700) in an antisense orientation. Infiltrated plants were maintained in the dark and under high humidity for 2 days before being uncovered and maintained at 20–22°C with a 16/8-h light/dark regime and light intensities between 400 and 1000 µmol m−2 s−1.