The transcription factor EMISSION OF BENZENOIDS II activates the MYB ODORANT1 promoter at a MYB binding site specific for fragrant petunias
Version of Record online: 1 JUL 2011
© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd
The Plant Journal
Volume 67, Issue 5, pages 917–928, September 2011
How to Cite
Van Moerkercke, A., Haring, M. A. and Schuurink, R. C. (2011), The transcription factor EMISSION OF BENZENOIDS II activates the MYB ODORANT1 promoter at a MYB binding site specific for fragrant petunias. The Plant Journal, 67: 917–928. doi: 10.1111/j.1365-313X.2011.04644.x
- Issue online: 30 AUG 2011
- Version of Record online: 1 JUL 2011
- Accepted manuscript online: 18 MAY 2011 02:29AM EST
- Received 14 March 2011; revised 4 May 2011; accepted 16 May 2011; published online 1 July 2011.
Figure S1. GUS activity in petals of transgenic petunia lines. (a) Box-plot representing GUS activities in petals of transgenic lines. Values are normalised for protein content. (b) Normalised GUS activity of each individual transgenic line used for generating the box-plot in (a). The number of independent transformants is indicated between brackets. nd, not detected.
Figure S2. The ODO1 promoter drives tissue-specific and developmental expression of the uidA (GUS) reporter in stable transformants. Histochemical staining of a representative stable line, transformed with the M19:GUS reporter construct. Staining of a stable CaMV 35S:GUS reporter line is shown for comparison.
Figure S3. Structure of the ODO1 promoter of P. hybrida cv. Mitchell. The structure of the 2kbp fragment of the ODO1 promoter of Mitchell is shown. The primers used to generate the 5′-deletion reporter constructs are indicated by arrows. F: forward primer, R: reverse primer.
Figure S4. Activity of the 1.2 kbp ODO1 promoters of P. hybrida cultivars Mitchell, V26 and R27 in Mitchell petals. Petunia hybrida cv. Mitchell petals were separately infiltrated with A. tumefaciens harbouring M12:GUS, V12:GUS and R12:GUS. Flowers were co-infiltrated with CaMV 35S:LUC-harbouring A. tumefaciens to enable normalisation, and incubated for 48 h prior to enzymatic analyses (Average ± SE; n = 4). Letters indicate significant differences (ANOVA, P < 0.05 according to least significant difference post-hoc analysis.
Figure S5. Trans-activation of the 1.9 kbp and 1.2 kbp ODO1 promoter fragments by EOBII in N. benthamiana leaves. A. tumefaciens harbouring a reporter construct (M19:GUS or M12:GUS), the effector construct 35S:EOBII or 35S:LUC were mixed in the ratio 5:5:2 and co-infiltrated in leaves of N. benthamiana. Leaves were incubated 48 h prior to GUS and LUC analyses (Average ± SE, n = 6). Co-infiltration with CaMV 35S:LUC-containing A. tumefaciens enabled normalisation.
Figure S6. Electrophoretic mobility shift assay (EMSA) using EOBII and wild type (M) and two mutated (m1 and m2) probes. A mobility shift is seen with purified recombinant GST-EOBII (arrow). As a control protein, purified recombinant GST-SnRK2.4 was used (Boudsock et al., 2004). Cold competitors are unlabelled probes and are identical to the respective labelled probes. Proteins and probes for each lane are indicated above the gel.
Figure S7.Luciferase expression by Agrobacterium tumefaciens GV3101 cured of plasmid pMP90. Tobacco leaves were infiltrated with Agrobacterium tumefaciens GV3101 containing plasmid pMP90 [GV3101(pMP90)] or A. tumefaciens cured of plasmid pMP90 (GV3101), both harbouring a CaMV 35S:LUC construct. Leaves were co-infiltrated with A. tumefaciens GV3101 harbouring a construct with a 35S-driven, intron-containing uidA, to enable normalisation (Average ± SE, n = 6).
Table S1. Primers used to generate the promoter reporter constructs.
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