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Ethylene response factors Pp4ERF24 and Pp12ERF96 regulate blue light-induced anthocyanin biosynthesis in ‘Red Zaosu’ pear fruits by interacting with MYB114

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Abstract

Key message

Pp4ERF24 and Pp12ERF96 fine tune blue light-induced anthocyanin biosynthesis via interacting with PpMYB114 and promoting the interaction between PpMYB114 and PpbHLH3, which enhances the expression of PpMYB114-induced PpUFGT.

Abstract

The red coloration of pear fruit is attributed to anthocyanin accumulation, which is transcriptionally regulated by the MYB-bHLH-WD40 complex. A number of ethylene response factors (ERF) have been identified to regulate anthocyanin biosynthesis in different plants. In pear, several ERF transcription factor genes were identified to be potentially involved in the light-induced anthocyanin biosynthesis according to transcriptome data. But the molecular mechanism of these ERFs underlying the regulation of anthocyanin accumulation is unknown. In this study, exposure of ‘Red Zaosu’ pear, a mutant of ‘Zaosu’ pear, to blue light significantly induced the anthocyanin accumulation by increasing the expression levels of anthocyanin biosynthetic genes. Gene expression analysis confirmed that the expression of Pp4ERF24 and Pp12ERF96 genes were up-regulated in the process of blue light-induced anthocyanin biosynthesis. Yeast two-hybrid and bimolecular fluorescence complementation assay revealed that Pp4ERF24 and Pp12ERF96 interacted with PpMYB114, but not with PpMYB10. Bimolecular fluorescence complementation assay demonstrated that the interaction between these two ERFs and PpMYB114 enhanced the interaction between PpMYB114 and PpbHLH3. Further analysis by dual luciferase assay verified that these two ERFs increased the up-regulation of PpMYB114-mediated PpUFGT expression. Furthermore, co-transformation of Pp12ERF96 with PpMYB114 and PpbHLH3 in tobacco leaves led to enhanced anthocyanin accumulation. Transient overexpression of Pp4ERF24 or Pp12ERF96 alone in ‘Red Zaosu’ pear fruit also induced anthocyanin biosynthesis in pear peel. Our findings provide insights into a mechanism involving the synergistic interaction of ERFs with PpMYB114 to regulate light-dependent coloration and anthocyanin biosynthesis in pear fruits.

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Abbreviations

ERF:

Ethylene response factor

UFGT:

UDP-glucose: flavonoid 3-glucosyltransferase

EBG:

Early biosynthetic gene

LBG:

Late biosynthetic gene

PAL:

Phenylalanine ammonia lyase

CHS:

Chalcone synthase

CHI:

Chalcone isomerase

F3′H:

Flavanone 3′-hydroxylase

DFR:

Dihydroflavonol 4-reductase

ANS:

Anthocyanin synthase

bHLH:

Basic helix-loop-helix

DAFB:

Days after full bloom

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Acknowledgements

This study was supported by the National Natural Science Foundation of China (Grant Nos. 31471852 to YT and 31772272 to SB) and the Earmarked Fund for China Agriculture Research System (CARS-28).

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Author notes

  1. Junbei Ni and Songling Bai contributed equally to this work.

Authors and Affiliations

  1. Department of Horticulture, Zhejiang University, Hangzhou, 310058, Zhejiang, People’s Republic of China

    Junbei Ni, Songling Bai, Yuan Zhao, Ruiyan Tao, Lei Yin & Yuanwen Teng

  2. The Key Laboratory of Horticultural Plant Growth, Development and Quality Improvement, The Ministry of Agriculture of China, Hangzhou, 310058, Zhejiang, People’s Republic of China

    Junbei Ni, Songling Bai, Yuan Zhao, Ruiyan Tao, Lei Yin & Yuanwen Teng

  3. Zhejiang Provincial Key Laboratory of Integrative Biology of Horticultural Plants, Hangzhou, 310058, Zhejiang, People’s Republic of China

    Junbei Ni, Songling Bai, Yuan Zhao, Ruiyan Tao, Lei Yin & Yuanwen Teng

  4. School of Science and Technology, Örebro University, Örebro, Sweden

    Minjie Qian

  5. ACON Biotech (Hangzhou) Co., Ltd., Hangzhou, 310058, Zhejiang, People’s Republic of China

    Ling Gao

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  1. Junbei Ni
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Contributions

JN, SB, and YT conceived and planned the study. RT and LY helped collect samples and extracted the total RNA. JN and YZ completed the qPCR, vector construction, and bioinformatics analysis. JN and LG conducted the Y2H, BiFC, dual luciferase, firefly luciferase complementation imaging assays and the transient transformation assay of gene function in tobacco leaves and ‘Red Zaosu’ fruits. JN, SB, MQ and YT wrote the manuscript. All of the authors read and approved the final manuscript.

Corresponding author

Correspondence to Yuanwen Teng.

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The authors declare that they have no competing interest.

Electronic supplementary material

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Supplementary material 1 (DOCX 14 KB)

Supplementary material 2 (DOCX 13 KB)

Supplementary material 3 (DOCX 14 KB)

11103_2018_802_MOESM4_ESM.pdf

(A) Phylogenetic analysis of ERF proteins from ‘Red Zaosu’ pear (PpERF) and Prunus persica (PpeERF). (B) Pp4ERF24, Pp12ERF96, Pp12ERF100, and Pp1ERF1 expression patterns based on ‘Meirensu’ transcriptome data. Supplementary material 4 (PDF 721 KB)

11103_2018_802_MOESM5_ESM.pdf

(A) A yeast two-hybrid (Y2H) assay revealed that Pp4ERF24, Pp12ERF96, Pp12ERF100, and Pp1ERF1 did not interact with PpMYB10. The pGADT7-T and pGBKT7-53 vectors were used as the positive control, while the AD and MYB10-BD combination served as the negative control. Blue plaques indicated an interaction between two proteins. The basal AbA concentration was 200 ng ml−1. (B) A Y2H assay confirmed that PpMYB114, Pp12ERF96, Pp12ERF100, and Pp1ERF1 underwent an auto-activation. The BD vector was used as the negative control. Supplementary material 5 (PDF 4485 KB)

11103_2018_802_MOESM6_ESM.pdf

Analysis of the Pp4ERF24 and Pp12ERF96 promoters revealed several light-responsive elements. Supplementary material 6 (PDF 713 KB)

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Ni, J., Bai, S., Zhao, Y. et al. Ethylene response factors Pp4ERF24 and Pp12ERF96 regulate blue light-induced anthocyanin biosynthesis in ‘Red Zaosu’ pear fruits by interacting with MYB114. Plant Mol Biol 99, 67–78 (2019). https://doi.org/10.1007/s11103-018-0802-1

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