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. 2020 Apr 20;20:173. doi: 10.1186/s12870-020-02389-1

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© The Author(s) 2020

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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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)