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The ethylene response factor MdERF1B regulates anthocyanin and proanthocyanidin biosynthesis in apple

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Abstract

Key message

The regulator MdERF1B in the apple (Malus × domestica) ethylene pathway mainly acts on MdMYB9 and MdMYB11 to regulate anthocyanin and proanthocyanidin accumulation.

Abstract

Dietary anthocyanins and proanthocyanidins (PAs) have health benefits for humans, and are associated with decreased risks of coronary heart disease and cancer. Ethylene can enhance reddening of apple (Malus × domestica), but the regulatory mechanism is poorly understood. In this study, an ethylene response factor (ERF), MdERF1B, was identified and functionally characterized. ‘Orin’ calli overexpressing MdERF1B were generated and then analyzed by quantitative reverse transcription-PCR. Compared with the control calli, the MdERF1B-overexpressing calli showed increased expression levels of MdACO1, MdERF1, and MdERF3 in the ethylene pathway and MdCHS, MdCHI, MdF3H, MdDFR, MdANS, MdLAR, MdANR, MdMYB9 and MdMYB11 in the flavonoid pathway. As a result, the levels of anthocyanins and PAs were significantly increased in the MdERF1B-overexpressing calli. MdERF1B interacted with MdMYB9, MdMYB1, and MdMYB11 proteins in yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays. Furthermore, in yeast one-hybrid and electrophoretic mobility shift assays, MdERF1B also bound to the promoters of MdMYB9, MdMYB1, and MdMYB11. In a luciferase reporter assay, MdERF1B mainly activated proMdMYB9 and proMdMYB11, promoting their expression levels. This was in agreement with MdERF1B’s overexpression in calli, which barely affected MdMYB1 expression. Taken together, our findings provide an insight into the regulatory mechanisms in the ethylene pathway that increase anthocyanin and PA accumulation in apple.

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Abbreviations

PAs:

Proanthocyanidins

PAL:

Phenylalanine ammonia-lyase

CHS:

Chalcone synthase

CHI:

Chalcone isomerase

F3H:

Flavanone 3-hydroxylase

DFR:

Dihydroflavonol 4-reductase

ANS:

Anthocyanidin synthase

UFGT:

Glycosyltransferase

LAR:

Leucoanthocyanidin reductase

ANR:

Anthocyanidin reductase

FLS:

Flavonol synthase

GFP:

Green fluorescent protein

GST:

Glutathione S-transferase

His:

Histidine

Anti:

Antibody

MS:

Murashige and Skoog

YFP:

Yellow fluorescent protein

Trp:

Tryptophan

Leu:

Leucine

Ade:

Adenine

CDS:

Coding DNA sequence

3-AT:

3-Amino-1,2,4-triazole

BiFC:

Bimolecular fluorescence complementation assay

DAFB:

Days after full bloom

EMSA:

Electrophoretic mobility shift assay

TFs:

Transcription factors

DMACA:

4-Dimethylaminocinnamaldehyde

qRT-PCR:

Quantitative reverse transcription-PCR

LUC:

Luciferase

ET:

Ethylene

6-BA:

6-Benzylaminopurine

2,4-D:

2,4-Dichlorophenoxyacetic acid

JA:

Jasmonate

MBW:

MYB–bHLH–WD40

MeJA:

Methyl jasmonate

bHLH:

Basic helix–loop–helix

Y1H:

Yeast one-hybrid

Y2H:

Yeast two-hybrid

4-MUG:

4-Methylumbellifery-β-d-glucuronide hydrate

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Acknowledgements

This work was supported by the National Key Research Project (2016YFC0501505) and the National Natural Science Foundation of China (31572091, 31730080). We thank Shujing Wu Laboratories for providing the vectors. We thank Lesley Benyon, PhD, and Jennifer Smith, PhD, from Liwen Bianji, Edanz Group China (http://www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.

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Authors and Affiliations

  1. State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai’an, Shandong, China

    Jing Zhang, Haifeng Xu, Nan Wang, Shenghui Jiang, Hongcheng Fang, Zongying Zhang, Guanxian Yang, Yicheng Wang, Mengyu Su, Lin Xu & Xuesen Chen

  2. College of Horticulture Science and Engineering, Shandong Agricultural University, Tai’an, Shandong, China

    Jing Zhang, Haifeng Xu, Nan Wang, Shenghui Jiang, Hongcheng Fang, Zongying Zhang, Guanxian Yang, Yicheng Wang, Mengyu Su, Lin Xu & Xuesen Chen

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  1. Jing Zhang
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  2. Haifeng Xu
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  3. Nan Wang
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  4. Shenghui Jiang
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  10. Lin Xu
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  11. Xuesen Chen
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Contributions

XC and JZ: conceived and designed the experiments. JZ and HX: analyzed the data. NW, SJ, HF, ZZ, GY, YW, MS and LX: contributed reagents/materials/analysis tools. JZ: wrote the paper.

Corresponding author

Correspondence to Xuesen Chen.

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Table S1 Primers used for qRT-PCR analysis. (TIF 325 KB)

Table S2 Primers used for cloning CDSs and promoters. (TIF 412 KB)

11103_2018_770_MOESM3_ESM.tif

Fig. S1 RAA motifs in proMdMYB1, proMdMYB9 and proMdMYB11. Transcriptional start sites (ATG) are indicated with +1. (TIF 133 KB)

Fig. S2 Alignment of MdERF1B and AtERF1 protein sequences. (TIF 1389 KB)

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Zhang, J., Xu, H., Wang, N. et al. The ethylene response factor MdERF1B regulates anthocyanin and proanthocyanidin biosynthesis in apple. Plant Mol Biol 98, 205–218 (2018). https://doi.org/10.1007/s11103-018-0770-5

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