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. Author manuscript; available in PMC: 2020 Nov 13.
Published in final edited form as: Nat Plants. 2018 May 28;4(6):365–375. doi: 10.1038/s41477-018-0160-7

Figure 5. Overexpression of ORE1 and KIR1 induces senescence and cell death symptoms to varying degrees.

Figure 5

a) Tobacco leaf infiltration with Agrobacterium carrying p35S::ORE1-GFP leads to expression of senescence symptoms at three days after infiltration (DAI), and cell death symptoms at 10 DAI, while p35S::KIR1-GFP expression induces cell death symptoms already at 3 DAI. Infiltration with p35S::NLS-GFP plasmid is used as a negative control; the presence of GFP signal at 2DAI (green) in all samples indicates successful transfection. b) Five-day-old Col-0 wild-type (WT) plants and seedlings of a representative line for an estradiol-inducible pRPS5A::XVE≫KIR1-GFP and pRPS5A::XVE≫ORE1-GFP construct directly after and three days after transfer to estradiol-containing medium (DAT). Red arrowheads indicate chlorotic cotyledons, white arrowheads the growth-arrested root tip. c-d) Real-time quantitative PCR reveals an increased accumulation of dPCD- associated transcripts in response to time-course estradiol treatment of 5-day-old XVE≫KIR1-GFP (c) and pRPS5A::XVE≫ORE1-GFP (d) seedlings. Expression values are relative to the reference genes PEX4 and UBL5; a minimum of two biological replicates are shown (n=20 seedlings per replicate). Expression data of estradiol- treated lines is normalized to mock-treated samples. e) Representative Col-0 wild-type and pCEP1::XVE≫KIR1- GFP flowers after estradiol treatment. Whereas the Col-0 stigma appears still viable, the KIR1-GFP stigma appears collapsed (arrowheads). f) Representative Col-0 and pCEP1::XVE≫KIR1-GFP flowers after estradiol treatment and pollination. Whereas silique elongation in WT indicates successful pollination and seed development, in estradiol-treated transgenic lines silique elongation appears severely reduced, and seed set is absent. Box-and-whisker plots are calculated following the Tukey method; white box-and-whisker plots show the silique length (from left to right, mean±SD=14.413±3.504, 15.788±1.762, 13.760±18.04, 4.390±1.207, N=15,8,5,20.), grey box-and-whisker plots show the seeds per silique (from left to right, mean±SD=40.933±18.211, 47.000±13.038, 34.200±9.576, 0.650±2.007, N=15,8,5,20.). white circles and gray rectangles show the outliers. Statistical differences were calculated using two-way ANOVA with multiple comparisons by Fisher’s LSD test. *=p<0.05, **=p<0.01, ****=p<0.0001. Scale bars, 50 µm.