Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Mar 1;68(7):1689-1696.
doi: 10.1093/jxb/erx031.

Salicylic acid interferes with GFP fluorescence in vivo

Affiliations

Salicylic acid interferes with GFP fluorescence in vivo

Jennifer de Jonge et al. J Exp Bot. .

Abstract

Fluorescent proteins have become essential tools for cell biologists. They are routinely used by plant biologists for protein and promoter fusions to infer protein localization, tissue-specific expression and protein abundance. When studying the effects of biotic stress on chromatin, we unexpectedly observed a decrease in GFP signal intensity upon salicylic acid (SA) treatment in Arabidopsis lines expressing histone H1-GFP fusions. This GFP signal decrease was dependent on SA concentration. The effect was not specific to the linker histone H1-GFP fusion but was also observed for the nucleosomal histone H2A-GFP fusion. This result prompted us to investigate a collection of fusion proteins, which included different promoters, subcellular localizations and fluorophores. In all cases, fluorescence signals declined strongly or disappeared after SA application. No changes were detected in GFP-fusion protein abundance when fluorescence signals were lost indicating that SA does not interfere with protein stability but GFP fluorescence. In vitro experiments showed that SA caused GFP fluorescence reduction only in vivo but not in vitro, suggesting that SA requires cellular components to cause fluorescence reduction. Together, we conclude that SA can interfere with the fluorescence of various GFP-derived reporter constructs in vivo. Assays that measure relocation or turnover of GFP-tagged proteins upon SA treatment should therefore be evaluated with caution.

Keywords: Arabidopsis; GFP fusion proteins; fluorescence microscopy; fluorescent protein; histones; salicylic acid..

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
SA-dependent decrease of GFP signal in roots of plants expressing Histone H1 and H1.2 GFP fusion proteins. Seedlings of transgenic lines expressing pH1.1:H1.1-GFP (A-B), pH1.2:H1.2-GFP (C-D) or pH1.3:H1.3-GFP fusion constructs were either control treated (A, C and E labeled as Mock) or treated with 50 µM SA (B, D and F labeled as 50 µM SA). Images were taken after 1 h of incubation. Scale bar = 50 µm.
Fig. 2.
Fig. 2.
SA-dependent decrease of GFP signal is concentration dependent. Seedlings of transgenic lines expressing pH1.1:H1.1-GFP, pH1.2:H1.2-GFP or p35S:H2A-GFP fusion constructs were treated with 0 µM (first column), 50 µM (second column), 100 µM (third column) or 200 µM SA (last column). Images were taken after 30 min of incubation. Compare the partial loss of GFP signal after 50 µM to the complete loss after 200 µM SA. Scale bar, 50 µm.
Fig. 3.
Fig. 3.
SA-dependent decrease of fluorescent protein signal is independent of the promoter or cellular localization of the fusion proteins. Transgenic lines expressing different GFP fusion constructs were mock-treated (upper images) or treated with 50 µM SA (lower images) for 1 h. Constructs used were: p35S:U2B”-GFP (A and F), pMSI1:MSI1-GFP (B and G), p35S:Tubulin‐A-GFP (C and H), pRPS5a:mDII-VENUS (D and I) and pUBQ10:RFP (E and J). Note that all reporter lines showed a similar sensitivity to SA. Scale bar, 50 µm.
Fig. 4.
Fig. 4.
SA-dependent decrease of fluorescent protein signal with time. Seedlings of transgenic lines expressing pH1.1:H1.1-GFP, pH1.2:H1.2-GFP, p35S:H2A-GFP and p35S:U2B”-GFP fusion constructs were treated with 50 µM SA. Images were taken every hour after the start of incubation. Compare the partial loss of GFP signal after 1 h to the complete loss after 24 h. Scale bar, 50 µm.
Fig. 5.
Fig. 5.
GFP fusion protein abundance is unaffected by SA treatment. Immunoblot analysis from mock-treated and 200 µM SA-treated plants using anti-GFP antibodies. Samples were taken after 1 h. (A) 10% SDS-PAGE of 18 µg total protein from pH1:H1-GFP (~64 kDa), pH1.3:H1.3-GFP (~50 kDa), pH1.2:H1.2-GFP (~64 kDa) and Col-0 control plants. (B) 12% SDS-PAGE of 10 µg protein from p35S:H2A-GFP (~32 kDa), p35S:U2B”-GFP (~52 kDa), p35S:Tubulin‐A-GFP (~75 kDa) and Col-0 control.
Fig. 6.
Fig. 6.
In vitro fluorescence of EGFP remains stable in the presence of SA. Fluorescence of recombinant EGFP protein either mock-treated (grey diamonds) or treated with 200 µM SA (black squares) was measured over a time course of 6 h. Each data point represents the average of three replicates. Error bars indicate SE.

References

    1. Ascenzi R, Gantt JS. 1997. A drought-stress-inducible histone gene in Arabidopsis thaliana is a member of a distinct class of plant linker histone variants. Plant Molecular Biology 34, 629–641. - PubMed
    1. Boudonck K, Dolan L, Shaw PJ. 1999. The movement of coiled bodies visualized in living plant cells by the green fluorescent protein. Molecular Biology of the Cell 10, 2297–2307. - PMC - PubMed
    1. Boursiac Y, Boudet J, Postaire O, Luu DT, Tournaire-Roux C, Maurel C. 2008. Stimulus-induced downregulation of root water transport involves reactive oxygen species-activated cell signalling and plasma membrane intrinsic protein internalization. The Plant Journal 56, 207–218. - PubMed
    1. Brunoud G, Wells DM, Oliva M, et al. 2012. A novel sensor to map auxin response and distribution at high spatio-temporal resolution. Nature 482, 103–106. - PubMed
    1. Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. 1994. Green fluorescent protein as a marker for gene expression. Science 263, 802–805. - PubMed

Publication types

MeSH terms