DEV01584 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Comparison of folds of tissue in the hinge region of (A) control flowers of A.majus and (B) A. majus flowers expressing MIXTA under the control of the CaMV35S promoter. The folds in the hinge region (arrowed) are present in both untransformed wild-type flowers (A) and in flowers ectopically expressing MIXTA (B), suggesting that MIXTA is a relatively weak promoter of mesophyll cell expansion because its ectopic expression does not counteract the adaxial activity of AmMYBML1 in the petal mesophyll, which creates the folds on the hinge.

• Supplemental Figure 2 -

  Fig. S2. Protoplast transfection assays to determine whether DIV, DEF or GLO are likely to interact directly with the AmMYBML1 promoter. Histogram of levels of GUS activity driven by the activity of the AmMYBML1 promoter in tobacco protoplast transfection assays. The activity of the AmMYBML1 promoter alone and the effects of the plasmids expressing DIV, DEF and GLO, DEF-VP16 and GLO, and combinations of these effectors are shown with standard errors. The longest AmMYBML1 promoter fragment was fused to the gene encoding GUS to make a reporter of AmMYBML1 promoter activity. cDNA sequences encoding DIV, DEF and GLO were cloned between the double CaMV 35S promoter and terminator sequences in pJIT60 (Guerineau and Mullineaux, 1993) to provide effector plasmids. One additional effector plasmid was made in which sequences encoding the activation domain of the VP16 transcriptional activator from Herpes Simplex Virus were cloned behind the DEF cDNA to make a DEF-VP16 translational fusion protein. Transfection of tobacco protoplasts (Negrutiu et al., 1987) was used to measure GUS reporter activity as a function of the different effector proteins. Equal amounts of DNA were included in each assay using pJIT60 (containing the CaMV35S promoter and terminator sequences) to equalise the DNA content of each assay and to control for any titration effects provided by the CaMV35S promoter. Alone, the AmMYBML1 promoter drove significant GUS activity in tobacco protoplasts. Expression of DIV caused a reduction in GUS activity by about 35%. Addition of DEF and GLO together resulted in a 47% inhibition of GUS activity compared with protoplasts with no effectors, although expression of DEF or GLO alone did not reduce GUS activity significantly compared with controls. The combination of DIV, DEF and GLO reduced the activity of the AmMYBML1 promoter yet further (66% reduction). This repression was not alleviated by inclusion of a strong transcriptional activation domain fused to DEF. Assays with the short (223bp) AmMYBML1 promoter showed no effect of DIV on GUS activity (data not shown).