Fig. 7
PtrJAZ1 binds to PdTT8 to inhibit the transcriptional activation activity of the PdMYB118/PdTT8 complex. PtrCHS1, PtrCHI1, PtrF3H, PtrF3’H, PtrF3’5’H, PtrDFR2 and PtrANS1 are the ABG genes respectively encoding chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase, flavanone 3′-hydroxylase, flavonoid 3′5’-hydroxylase, dihydroflavonol 4-reductase and anthocyanidin synthase in Populus. a PtrJAZ1 represses the transcriptional activation activity of the PdMYB118/PdTT8 complex. Protoplasts isolated from the leaves of transgenic plants overexpressing PdMYB118 were transformed with pGreenII62-SK-PdTT8 alone, or co-transformed with pGreenII62-SK-PdTT8 and pGreenII62-SK-PtrJAZ1. qRT-PCR analysis of ABGs in the transfected protoplasts was performed. The empty vector was used as a negative control and the gene expression level in the control was set to 1. Values are means and standard deviations of three biological replicates (n = 3). ** and ***, significant differences at P < 0.01 and P < 0.001 (Student’s t-test). M, transgenic protoplasts transfected with pGreenII62-SK; T, transgenic protoplasts transfected with pGreenII62-SK-PdTT8; T + J, transgenic protoplasts co-transfected with pGreenII62-SK-PdTT8 and pGreenII62-SK-PtrJAZ1. b BiFC assays to test the interaction of PtrJAZ1 with PdTT8. PtrJAZ1 was fused with the N-terminal fragment of YFP, and PdTT8 was linked to the C-terminal fragment of YFP, respectively. Construct pairs indicated on the left were co-expressed in poplar leaf protoplasts. Scale bar = 10 μm. c-e Transient transcription dual-luciferase assays to show that PtrJAZ1 inhibits the transcriptional activation activity of the MYB118/TT8 complex to the promoters of PtrCHS1 (c), PtrDFR2 (d) and PtrANS1 (e). The effectors (pGreenII62-SK-PdMYB118, pGreenII62-SK-PdTT8, pGreenII62-SK-PdMYB118 + pGreenII62-SK-PdTT8 or pGreenII62-SK-PdMYB118 + pGreenII62-SK-PdTT8 + pGreenII62-SK-PtrJAZ1) and reporters (35S::REN-ProPtrCHS1::LUC, 35S::REN-ProPtrDFR2::LUC or 35S::REN-ProPtrANS1::LUC) were co-expressed in poplar protoplasts. The expression level of REN was used as an internal control. The LUC/REN ratio represents relative activity of the PtrCHS1, PtrDFR2 and PtrANS1 promoters. Error bars represent the SDs from three biological replicates. C, protoplasts transfected with pGreenII62-SK (control); M, protoplasts transfected with pGreenII62-SK-PdMYB118; T, protoplasts transfected with pGreenII62-SK-PdTT8; M + T, protoplasts transfected with pGreenII62-SK-PdMYB118 and pGreenII62-SK-PdTT8; M + T + J, protoplasts transfected with pGreenII62-SK-PdMYB118, pGreenII62-SK-PdTT8 and pGreenII62-SK-PtrJAZ1. Values are means and standard deviations of three biological replicates (n = 3). ***, significant differences at P < 0.001 (Student’s t-test)