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. 2016 Feb 15;7:10654. doi: 10.1038/ncomms10654

Figure 8. Transactivation assays of putative downstream gene promoters by the GAME9 and SlMYC2 transcription factors.

Figure 8

(a) The capacity of GAME9 to transactivate 12 different promoters of candidate downstream genes was evaluated in protoplasts prepared from tobacco Bright Yellow-2 (BY-2) cells (n=4). A validation experiment (n=8) was performed, confirming the transactivation of the C5-SD promoter (Supplementary Fig. 2). Values in the y-axis are normalized fold-changes relative to protoplasts co-transfected with the reporter constructs and a pCaMV35S:GUS (GUS) control plasmid. For the normalization procedure, see Materials and Methods. Details for each promoter are provided in Supplementary Table 5. Student's t-test was used to assess whether the transgenic lines significantly differ from wild-type plants: *P value<0.05; ***P value<0.001. GAME1: UDP-galactosyltransferase; GAME2: UDP-xylosyltransferase; GAME4: CYP88D; GAME6: CYP72A; GAME7: CYP72A; GAME17: UDP-glucosyltransferase; GAME18: UDP-glucosyltransferase; HMGR1: HMG CoA reductase 1; HMGR2: HMG CoA reductase 2; SQMO: Squalene monooxygenase; SMO1: Methylsterol monooxygenase 2-2-like; C5-SD: Δ(7)-sterol-C5(6)-desaturase; GAME9: Ethylene responsive transcription factor. (b) Transactivation of SGA-related gene promoters with GAME9, SlMYC2 and the combination of both. (c) Transactivation of C5-SD promoter with deletions (d) and/or mutations (m). The -, (+) and+symbols point to activation of the promoter by GAME9/SlMYC2:—no activation (LUC activity below 1.5); (+) limited activation (LUC between 1.5 and 2-fold);+strong promoter activation (LUC activity larger than 2-fold). (d) In vitro binding of the GAME9 and SlMYC2 proteins to the G- and GCC-boxes located in the promoters of the C5-SD and SSR2 genes. Electrophoretic mobility shift assays (EMSA) were performed with probes containing a GCC- or a G-box were separated on a 1% agarose gel without additional treatment (lane 1) or with 200 ng of the purified protein (lane 2). Controls: a sequence containing a mutated version of the box was loaded (lane 3) and incubated with protein (lane 4).