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. 2019 Oct 17;10:1267. doi: 10.3389/fpls.2019.01267

Figure 4.

Figure 4

Fluorescence intensity of a pH-insensitive fluorescent DNA-binding protein (mCitrine-LacR) following addition of eATP. (A) The construct encoded nuclear-localized mCitrine-LacR fusion protein as a pH-insensitive fluorescent DNA-binding protein, and SUN2-mApple and CBL1-mApple as INM and PM visual markers. All three genes are under the transcriptional control of the RPS5 promoter; the first contains the 35ter and the last two contain the 3C terminator (3Cter). For readability, the module encoding RPS5pro-mCitrine-LacR is in the orientation shown but it is actually in the opposite orientation (Data Sheet 1, combination Figure 3, in supplement). (Data Sheet 1, combination Figure 4, in supplement). (B) Confocal image (maximum projection; enlargement in Data Sheet 9 in supplement) at time point t1 of homozygous fluorescent-tagged locus 16:101 in nuclei of cells in the root transition zone. Two green dots are visible in most nuclei. Nuclei used for fluorescence intensity analysis (part C) are boxed in white. Right: The mCitrine-LacR fusion protein binds to lacO repeats integrated at locus 16:101 on the bottom arm of chromosome 1. (C) pH-insensitive chromatin tag (mCitrine-LacR): Fluorescence intensity profiles of normalized intensities of all 10 white-boxed nuclei (in part B) are overlaid in one graph together with the calculated average values, to which standard deviation bars were added. Fluorescence intensities of individual nuclei can be viewed in supplementary Data Sheet 8, part Figure 4C. Addition of eATP is indicated with black arrowheads at frames 13-14. Normalized and non-normalized numbers are shown, respectively, in sheets 5 of Tables 10 and 1, in supplement. Using the normalized data, the calculated difference in the magnitude of the drop in fluorescence intensity of SEpHluorinD-LacR (Figure 3D) versus bleaching of mCitrine-LacR (this figure) between the time points 14-16 points in response to eATP is statistically significant (p ≤ 0.05). Fluorescence intensity at genomic location is read in spot objects (1-2.8 μm) capturing punctual fluorescence.