Molecular mechanism of MYB111 and WRKY40 involved in anthocyanin biosynthesis in red-fleshed apple callus
- 165 Downloads
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.
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.
KeywordsMolecular mechanism MdMYB111 and MdWRKY40 Homodimers Anthocyanin biosynthesis Red-fleshed apple callus
Electrophoretic mobility shift assays
Bimolecular fluorescence complementation
Yellow fluorescent protein
We thank Emma Tacken, PhD, and Lesley Benyon, PhD, from Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.
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.
- Ballester AR, Molthoff J, de Vos R, te Lintel HB, Orzaez D, Fernandezmoreno JP, Tripodi P, Grandillo S, Martin C, Heldens J, Ykema M, Granell A, Bovy A (2010) Biochemical molecular analysis of pink tomatoes, deregulated expression of the gene encoding transcription factor SlMYB12 leads to pink tomato fruit color. Plant Physiol 152:71–84PubMedPubMedCentralGoogle Scholar
- Cook NC, Samman S (1996) Flavonoids-chemistry, metabolism, cardiop rotective effects, and dietary sources. J Nutr Biochem 7:66–76Google Scholar
- Hartmann U, Sagasser M, Mehrtens F, Stracke R, Weisshaar B (2005) Differential combinatorial interactions of cis-acting elements recognized by R2R3-MYB, BZIP, and BHLH factors control light-responsive and tissue-specific activation of phenylpropanoid biosynthesis genes. Plant Mol Biol 57(2):155–171PubMedGoogle Scholar
- Ji XH, Wang YT, Zhang R, Wu SJ, An MM, Li M, Wang CZ, Chen XL, Zhang YM, Chen XS (2015) Effect of auxin, cytokinin and nitrogen on anthocyanin biosynthesis in callus cultures of redfleshed apple (Malus sieversii f. niedzwetzkyana). Plant Cell Tissue Organ Cult 120:325–337Google Scholar
- Kenneth JL, Thomas DS (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408Google Scholar
- Kim SY, Lee JR, Kim SR (2006) Characterization of an apple anthocyanidin synthase gene in transgenic tobacco. Plants J Plant Biol 49(4):326–330Google Scholar
- Kondo S, Hiraoka K, Kobayashi S, Honda C, Terahara N (2002) Changes in the expression of anthocyanin biosynthetic genes during apple development. J Am Soc Hortic Sci 127:971–976Google Scholar
- Li HH, Liu X, An JP, Hao YJ, Wang XF, You CX (2017) Cloning and elucidation of the functional role of apple MdLBD13 in anthocyanin biosynthesis and nitrate assimilation. Plant Cell Tissue Organ Cult 130:47–59Google Scholar
- Sun JJ, Wang YC, Chen XS, Gong XJ, Wang N, Ma L, Qiu YF, Wang YL, Feng SQ (2017) Effects of methyl jasmonate and abscisic acid on anthocyanin biosynthesis in callus cultures of red-fleshed apple. Plant Cell Tissue Organ Cult 130:227–237Google Scholar
- Wang LK, Bolitho K, Grafton K, Kortstee A, Karunairetnam S, McGhie TK, Espley RV, Hellens RP, Allan AC (2010) An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae. BMC Plant Biol 10:50Google Scholar
- Wang N, Zhang ZY, Jiang SH, Xu HF, Wang YC, Feng SQ, Chen XS (2016) Synergistic effects of light and temperature on anthocyanin biosynthesis in callus cultures of red-fleshed apple. Plant Cell Tissue Organ Cult 127:217–227Google Scholar
- Xie XB, Zhao J, Hao YJ, Fang CB, Wang Y (2017) The ectopic expression of apple MYB1 and bHLH3 differentially activates anthocyanin biosynthesis in tobacco. Plant Cell Tissue Organ Cult 131:183–194Google Scholar
- Zhang WS, Li X, Zheng JT, Wang GY, Sun CD, Ferguson IB, Chen KS (2008) Bioactive components and antioxidant capacity of Chinese bayberry (Myrica rubra Sieb. and Zucc.) fruit in relation to fruit maturity and postharvest storage. Eur Food Res Technol 227(4):1091–1097Google Scholar