MYC2 transcription factors bind to sequence motifs called G-boxes (Abe et al. 2003;
Abe et al. 1997; Dombrecht et al. 2007). To investigate the role of the G-boxes in the CYP81D11 promoter, reporter gene constructs with mutated G-boxes were designed.
The CYP81D11 promoter contains two neighboring G-boxes (position –206 to –193) separated by only one base pair; one G-box exhibits the sequence CACGTG and the other is represented by CACATG. They were replaced by TTCAAG and TTCAAA (Abe et al. 1997) as replacement with these sequences creates no other known
gl1 jin1-1 Col-0 JAZ1∆3A
relative expression
transcription factor binding sites (http://www.dna.affrc.go.jp/PLACE/). CYP81D11 promoter:GUS constructs in binary plant vectors were created, either using an 894-bp promoter fragment exhibiting the wild-type sequence or the promoter sequence with mutated G-boxes. Transgenic plants were produced by A. tumefaciens-mediated gene transfer into A. thaliana Col-0 plants. The seedlings of the F1 generation grown on agar plates were used for TIBA and MeJA treatment (Figure 4.11).
0 5000 10000 15000
mG-box WT
rel. GUS activity /µg protein un TIBA MeJA
Figure 4. 11: Average activities of a CYP81D11 WT promoter construct and a construct lacking the G-boxes in response to TIBA and MeJA
(A) Scheme of the distinct CYP81D11 promoter:GUS reporter gene constructs that were used to generate the transgenic lines employed in this experiment. The light green box indicates an 894-bp fragment upstream of the CYP81D11 transcription start. The double G-box (position –206 to –193) is marked in green, while the altered sequence is marked in red; both sequences are indicated. The grey box depicts the 5’ UTR of the CYP81D11 gene and the GUS reporter gene is indicated in blue.
(B) GUS activities in response to TIBA and MeJA treatment obtained from transgenic plants containing the CYP81D11 promoter:GUS constructs depicted in (A). Seedlings were grown for 16 days on MS agar and either sprayed with 100 µm TIBA for 8 h or treated with gaseous MeJA (1 µL/L air) for 24 h. Control plants remained untreated. Whole seedlings were harvested for protein extraction. Each bar represents the average ± SEM of two experiments, each carried out with 19–20 transgenic lines.
The GUS activity in transgenic plants encoding the CYP81D11 WT promoter:GUS construct was inducible to similar extents in response to TIBA and MeJA. Plants containing the CYP81D11 mG-box promoter:GUS construct were not inducible by MeJA. This result indicates that the double G-box is absolutely essential for JA-induced CYP81D11 expression. The non-induced and the TIBA-induced levels were reduced about two times. This leads to the suggestion that the G-boxes play a role as elements for constitutive activation, but not as activated elements in response to TIBA.
The substitution of the G-boxes in the CYP81D11 promoter generated a promoter construct insensitive to JA. To investigate the COI1 dependency of this promoter
A B
construct, transfection experiments in Col-0 and coi1-t protoplasts were performed
Figure 4. 12: Transient expression analysis of distinct CYP81D11 promoter constructs carrying sequence alterations at the site of the as-1-like element and the site of the G-boxes (A) Schematic illustration of the distinct CYP81D11 promoter:firefly luciferase constructs used for protoplast transfection. The light blue box indicates an 894-bp fragment upstream of the CYP81D11 transcription start site; wild-type sequences of the as-1 element (position –243 to –225) and the double G-box (position –206 to –193) are marked in green while the altered sequences are depicted by red boxes.
The grey box marks the 5’ UTR of the CYP81D11 gene and the yellow box indicates the firefly luciferase (FF LUC) reporter gene. The construct referred to as WT contains the 894-bp fragment upstream of the CYP81D11 transcription start site and the 5’ UTR of the CYP81D11 gene. The mas-1 construct contains sequence alterations within the as-1 element (position –243 to –225) while the mG-box construct contains alterations in the sequence of the double G-box (position –206 to –193). The mas-1/mG-box construct exhibits alterations in both elements.
(B) Luciferase activities obtained from transfection of distinct promoter:firefly luciferase constructs in A. thaliana Col-0 and coi1-t protoplasts. Leaves of 7-week-old soil-grown and non-induced plants were used for protoplast isolation. The x-coordinate demonstrates the ratio of the firefly luciferase activity to the internal Renilla luciferase standard.
In protoplasts CYP81D11 is constitutively expressed. Therefore they allow expression analyses in the absence of additional treatments. In coi1-t protoplasts, the activity of the WT promoter construct is strongly reduced in comparison to the Col-0 protoplasts. This demonstrates a similar COI1 dependency in protoplasts as in whole plants. Additionally, the reduced activity of the mG-box construct compared to the WT construct in wild-type protoplasts also reflects the results obtained by TIBA treatments in whole plants.
A comparison of the reporter gene expression of the mG-box construct in Col-0 and coi1-t protoplasts demonstrated the COI1 dependency of this construct. This result leads to the assumption that COI1 carries out its JA-Ile-independent function via another promoter element than the G-boxes which are responsible for JA-Ile-dependent CYP81D11 expression.
Nevertheless, the fact that in coi1-t protoplasts the WT construct and the mG-box construct have the same activity demonstrates that the G-boxes only function in the presence of COI1.
The activities of the mas-1 and the mas-1/mG-box constructs are still strongly reduced in the coi1-t mutant compared to the wild-type protoplasts. Thus, it was assumed that COI1 regulates the CYP81D11 promoter not via the TGA binding site.