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. 2016 Nov 11;113(47):E7629–E7638. doi: 10.1073/pnas.1612872113

Fig. 3.

Fig. 3.

Effect of FtsZ2-1 overexpression during the constriction of the chloroplast division site on chloroplast division and cell-cycle progression in synchronously cultured C. merolae. (A) Schematic diagram of the culture conditions. The control GFP heat-inducible (GFP) or FtsZ heat-inducible (FtsZ OX) cells were synchronized by a 12-h/12-h light/dark cycle at 42 °C and were heat shocked at 50 °C at hour 14 (during the constriction of the chloroplast division site) for 2 h. (B) Immunoblot analyses showing the change in the levels of the chloroplast-division proteins FtsZ2-1, DRP5B, and PDR1 and the M-phase marker H3S10ph in GFP and FtsZ OX cells. The GFP and FtsZ OX samples were blotted on the same membrane. (C) Quantitative RT-PCR analyses showing the change in the mRNA levels of an S-phase marker (PCNA) and an M-phase marker (CDC20) in GFP (blue trace) and FtsZ OX (red trace) cells. DRP3 was used as the internal control. The error bars indicate the SD (n = 3). The expression levels in the control cells at hour 4 were defined as 1.0. (D) Microscopic images of DAPI-stained GFP and FtsZ OX cells at 16, 20, and 24 h in the synchronous culture. The arrowheads indicate cells that possess a single chloroplast and two nuclei. Magenta, autofluorescence of the chloroplast; cyan, DNA stained with DAPI. The fluorescence and phase-contrast images are overlaid. (Scale bars: 5 μm.) (E) DAPI-stained image of two FtsZ OX cells connected by a long narrow bridge that contains a tube-like structure of the undivided chloroplast. PC, phase contrast; magenta, autofluorescence of the chloroplast; cyan, DNA stained with DAPI. (Scale bar: 5 μm.) (F) Immunofluorescent images of GFP and FtsZ OX cells showing the localization of FtsZ2-1, PDR1, and DRP5B just after the heat shock (hour 16 in synchronous culture) and 8 h later (hour 24 in synchronous culture). (Upper) Cells with one dividing chloroplast and one nucleus at hour 16. (Lower) FtsZ OX cells with one undivided chloroplast and two nuclei at hour 24. Green, immunostained FtsZ2-1, PDR1, or DRP5B; magenta, autofluorescence of the chloroplast; PC, phase-contrast. (Scale bars: 1 μm.) Two independent experiments produced similar results. The results from one experiment are shown.