Article Figures & Data
Figures
- Figure 1.
GUS Expression in Arabidopsis Flowers.
(A) Flower from a plant containing the promoter of STK fused to the GUS coding sequence, showing ovule- and septum-specific GUS expression.
(B) Aspecific GUS expression throughout the entire flower from a plant containing only the 5′-flanking region without intron region in the 5′UTR of STK fused to the GUS coding sequence.
- Figure 2.
Schematic Representation of the STK Promoter.
(A) Promoter/intron region with the two regions (a and b) that were binding BPC1 in the yeast one-hybrid screen. ATG, start codon.
(B) Sequence of the region of the STK promoter that contains 12 GA-rich regions (shaded, numbered 1 to 12). The underlined sequences are the oligonucleotide sequences used in the EMSA experiments. The location of the exon is displayed in bold.
- Figure 3.
Analysis of BPC1 Binding to GA Elements.
EMSA competition assays using labeled element 4 as a probe. The molar excess of the competitor is indicated above the figures.
(A) Unlabeled element 4 is used as a competitor (lane 1 is free probe). A 100-fold excess of unlabeled element 4 out competes the labeled probe completely. Lane 7 is a control using the unrelated maltose binding protein, no band shift is observed.
(B) Competition assays using unlabeled element 4 (lanes 2 to 4), element 2 (lanes 5 to 7), element 9 (lanes 8 to 10), and element 12 (lanes 11 to 13) as a competitor, showing that elements 4, 12, and 9 are bound by BPC1, whereas element 2 is not.
(C) Competition assay using mutated element 12 (nucleotide 7, A to T) as a competitor. The affinity of BPC1 for the mutated element is completely lost because of this mutation
(D) Alignment of the different elements that were able to bind BPC1 and were not able to bind BPC1, leading to the shown consensus.
(E) Point mutations compared with the consensus, which lead to the decrease in binding capacity of BPC1. *, sequence as present in element 2 in Figure 3D; **, sequence as present in element 6 in Figure 3D.
- Figure 4.
TPM Analysis of BPC1 Interaction with the STK Promoter.
DNA end-to-end distance (E to E) measured in TPM experiments. The trace shows the variations in time of the DNA end-to-end distance before and after addition of the protein. The arrow indicates the time of addition of protein. The black line shows the average value of the DNA end-to-end distance before and after addition of protein.
(A) The DNA fragment used is 1413 bp long and contains BPC1 binding sites 1 through 12; the distance between box 12 and the digoxigenin label is 240 bp, whereas the distance between site 1 and the biotin label is 320 bp. The end-to-end distance after addition of BPC1 is reduced by ∼50 to 60 nm.
(B) DNA fragment used contains only boxes 4 and 12; the distance between boxes 4 and 12 is approximately like the distance in the other DNA fragment. The same holds for the distance between these boxes and the digoxigenin and biotin labels. The addition of BPC1 does not change the DNA end-to-end distance of the DNA.
(C) Control experiment using the unrelated MBP. The DNA fragment is the same as used in the experiment represented in Figure 4A. No alteration of the DNA end-to-end distance after the addition of the MBP was observed.
- Figure 5.
STK and BPC1 Expression Analysis in Wild-Type and bpc1 Mutant Arabidopsis Flowers by in Situ Hybridization.
(A) In situ hybridization on very young flowers showing BPC1 expression in the floral meristem and floral organ primordia.
(B) Expression of BPC1 in a flower showing expression in all floral organs and especially in ovules.
(C) Expression of BPC1 in ovules.
(D) In situ hybridization on a bpc1 mutant flower using the BPC1 probe, showing no expression.
(E) and (F) In situ hybridization on bpc1 mutant flowers showing STK-specific expression like in wild-type flowers.
s, septum; st, stamen; p, pistil; o, ovule. Bars in (A) and (C) = 20 μm; bars in (B) and (D) to (F) = 40 μm.
- Figure 6.
STK Expression Analysis in Wild-Type and bpc1 Mutant Arabidopsis Flowers by Real-Time RT-PCR.
Error bars represent standard deviations calculated on five different replicas. STK is approximately three times upregulated in the bpc1 mutant with respect to the wild type.
