Fig. 6

TaSPL7/15 regulate vernalization through binding the regulatory elements of VRN1 and VRN3. a Key TFs enrichment of the putative vernalization-related regulatory elements. nK4-dACRs represents distal ACRs without H3K4me3; K4-dACRs represents distal ACRs neighboring to H3K4me3; P, promoter (2kb upstream of TSS). b The expression of TaSPL7/15 were increased after vernalization in axillary bud and shoot apex. c Genome coverage image showing that TaSPL7 and TaSPL15 bind to the promoter of VRN1 and distal ACRs of VRN3. P1~P4 are the fragments selected for the following experiments. d-e EMSA showing the binding of TaSPL7A to VRN1 promoter (d) and VRN3 distal ACRs (e). P1-1 and P1-2 are the two SPL binding sites within the VRN1 promoter (Fig. S8d). f-g Validation of TaSPL7/15 binding and activation activity in wheat protoplasts (f) and tobacco leaf (g) using dual luciferase Reporter assay. Here, we selected some fragments for verification. P1~P4 are the ACRs marked in Fig. 6c, and P5 is the ACR marked in Fig. S8c. Statistical analyses are shown in the bottom panel and “*” indicates p < 0.05, “**” indicates p < 0.01 in Student’s t-tests. The relative activity was calculated by comparing the LUC/REN value of SPL to the LUC/REN value of GFP. h Induced expression of VRN1 and VRN3 were repressed in spl7-aabbdd spl15-aabbdd hexa-mutants. Fold changes represent the ratio of VRN1 and VRN3 expression ((7 days after vernalization) / (no vernalization)). Samples for RT-qPCR were obtained from plants grown 2 weeks (day for 16h at 22°C, night for 8h at 19°C) after germination and 7 days in greenhouse (day for 16h at 22°C, night for 8h at 19°C) after vernalization (plants grown 2 weeks in green house and vernalization for 3 weeks). All samples were taken between 9AM and 12AM (China Standard Time). i The late flowering phenotype of spl7-aabbdd spl15-aabbdd hexa-mutants (84 days after germination). j Statistics of heading time. Statistical analyses are shown in the bottom panel and “*” indicates p < 0.05, “**” indicates p < 0.01 in Student’s t-tests