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. 2018 Aug 10;30(9):2197–2213. doi: 10.1105/tpc.18.00293

Figure 2.

Figure 2.

Inducible Overexpression of ANAC087 and ANAC046 Leads to Ectopic Cell Death.

(A) The pPASPA3-ToIM marker showed ectopic expression and cell death in the epidermis distal of the LRC 24 h after induction of the p35S-SMB-GR (right, indicated by an asterisk). In the mock treatment (left), only regular cell death in the LRC was observed. The eGFP signal is shown in green in the cytoplasm, whereas the mRFP signal is shown in magenta in the vacuole. Merge of both signals indicates vacuolar collapse, a hallmark of cell death (arrowheads). Bars = 50 µm.

(B) Inducible overexpression of ANAC087-GFP and ANAC046-GFP (induced with estradiol [estr]) using the chimeric XVE TF and the pRPS5A promoter led to intense ectopic Evans Blue staining. Shown is the root of a representative 5-d-old T3 seedling; four homozygous lines were analyzed in T3 for ANAC087-GFP and three lines for ANAC046-GFP. In the control line, where only NLS-GFP is inducibly expressed, no ectopic Evans Blue staining was observed. Photographs show the time point of 24 h after induction. Evans Blue is shown in blue. Bars = 50 µm.

(C) As indicated by RT-qPCR analysis, 24 h induction by estradiol caused strong expression of ANAC087 and ANAC046 in the pRPS5A:XVE>>ANAC087-GFP and pRPS5A:XVE>>ANAC046-GFP line, respectively. The error bars show the se of means of three independent biological replicates (three independent treatments and three technical repeats each). t test; **P < 0.01 and ****P < 0.0001.

(D) Macroscopic images of estradiol-induced overexpression lines of ANAC087 and ANAC046 followed over time showed a different response for both TFs. Six days after estradiol treatment, XVE-ANAC087 seedlings arrested root growth, while the shoot survived, whereas the XVE-ANAC046 seedlings showed complete growth arrest and death of the seedling. In contrast, wild-type seedlings were not visually affected by the estradiol treatment. Arrowheads indicate root tips at the beginning (0 d estradiol) and the end (6 d estradiol) of the estradiol treatment. Representative full time courses are shown in Supplemental Movie 3.