Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 139, Issue 3, pp 467–478 | Cite as

Molecular mechanism of MYB111 and WRKY40 involved in anthocyanin biosynthesis in red-fleshed apple callus

  • Tianliang Zhang
  • Haifeng Xu
  • Guanxian Yang
  • Nan Wang
  • Jing Zhang
  • Yicheng Wang
  • Shenghui Jiang
  • Hongcheng Fang
  • Zongying Zhang
  • Xuesen ChenEmail author
Original Article


The WRKY transcription factors play key roles in plant growth and abiotic stress responses; however, the molecular mechanisms behind their involvement in anthocyanin biosynthesis are still unclear. In our study, we identified a Leu zipper motif and a WRKY domain in MdWRKY40 protein. Phylogenetic tree analysis showed that MdWRKY40, AtWRKY18 and AtWRKY40 were on the same evolutionary branch and were Group IIa WRKY proteins. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that MdWRKY40 could interact with itself to form homodimers. Overexpressing MdMYB111 in red-fleshed callus inhibited the expression of MdANS and decreased the anthocyanin content. EMSA assay showed that MdMYB111 and MdWRKY40 could bind the MRE and the W box, respectively, in the MdANS promoter. Overexpressing MdWRKY40 in red-fleshed callus did not affect the expression of MdANS or the anthocyanin content. However, overexpressing MdWRKY40 in callus overexpressing MdMYB111 weakened the inhibitory effect of MdMYB111 on anthocyanin biosynthesis. Knocking out the Leu zipper motif of MdWRKY40 (LLSMdWRKY40) prevented its self-interaction, and knocking out C-x5-C sequence of MdWRKY40 (LCSMdWRKY40) prevented it from binding to W box. It did not weaken the inhibitory effect of MdMYB111 on anthocyanin biosynthesis when overexpressing LCSMdWRKY40 or LLSMdWRKY40 in callus overexpressing MdMYB111. Thus, MdMYB111 and MdWRKY40 may play important roles in the anthocyanin biosynthetic pathway.

Key message

MdWRKY40 interacts with itself to form homodimers by the Leu zipper motif at the N-terminal, and it binds two W boxes distantly separated in the MdANS promoter in the presence of C-x5-C sequence. MdMYB111 binds the MRE in the looped region induced by MdWRKY40. In addition, it weakens the inhibitory effect of MdMYB111 on expression of MdANS and anthocyanin biosynthesis when overexpressing MdWRKY40 in callus overexpressing MdMYB111.


Molecular mechanism MdMYB111 and MdWRKY40 Homodimers Anthocyanin biosynthesis Red-fleshed apple callus 



Anthocyanin synthase


UDP-glucose:flavonoid 3-glucosyltransferase


Activation domain


Binding domain


Electrophoretic mobility shift assays


Yeast two-hybrid


No tryptophan


No leucine


No histidine


No adenine


Bimolecular fluorescence complementation


MYB-recognition element


Yellow fluorescent protein



We thank Emma Tacken, PhD, and Lesley Benyon, PhD, from Liwen Bianji, Edanz Group China (, for editing the English text of a draft of this manuscript.

Author contributions

Tianliang Zhang and Haifeng Xu have contributed equally to this work. Xuesen Chen and Tianliang Zhang: conceived and designed the experiments. Tianliang Zhang and Haifeng Xu: performed the experiments. Tianliang Zhang: analyzed the data. Haifeng Xu, Guanxian Yang, Jing Zhang, Nan Wang, Yicheng Wang, Shenghui Jiang, Hongcheng Fang, Zongying Zhang: contributed reagents/materials/analysis tools. Tianliang Zhang and Xuesen Chen: wrote the paper.


This work was supported by the National Natural Science Foundation of China (31572091 and 31730080) and the National Key Research and Development Project (SQ2016YFSF030011).

Compliance with ethical standards

Conflict of interest

The authors declare no competing financial interests.

Supplementary material

11240_2019_1637_MOESM1_ESM.tif (2.8 mb)
Supplementary material 1 Supplementary Figure S1. Removal of MdWRKY40 Leu zipper motif using overlap PCR (TIFF 2896 kb)
11240_2019_1637_MOESM2_ESM.tif (8 mb)
Supplementary material 2 Supplementary Figure S2. Recombinant plasmid construction (TIFF 8216 kb)
11240_2019_1637_MOESM3_ESM.tif (5.9 mb)
Supplementary material 3 Table S1 Primer list (TIFF 6031 kb)


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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Tianliang Zhang
    • 1
    • 2
  • Haifeng Xu
    • 1
    • 2
  • Guanxian Yang
    • 1
    • 2
  • Nan Wang
    • 1
    • 2
  • Jing Zhang
    • 1
    • 2
  • Yicheng Wang
    • 1
    • 2
  • Shenghui Jiang
    • 1
    • 2
  • Hongcheng Fang
    • 1
    • 2
  • Zongying Zhang
    • 1
    • 2
  • Xuesen Chen
    • 1
    • 2
    Email author
  1. 1.State Key Laboratory of Crop BiologyShandong Agricultural UniversityTai’anChina
  2. 2.Collaborative Innovation Center of Fruit & Vegetable Quality and Efficient ProductionTai’anChina

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