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Three Paralogous R2R3-MYB Genes Contribute to Delphinidin-Related Anthocyanins Synthesis in Petunia hybrida

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Abstract

Three ANTHOCYANIN SYNTHESIS REGULATOR genes (ASR1–3) that encode R2R3-MYB transcription factors were identified recently from Petunia hybrida. In this study, we conducted additional experiments to characterize their specific function in the regulation of anthocyanin synthesis. The ASR1–3 proteins were localized in the nucleus. Analysis of their regulatory function by transient expression in the petunia corolla and yeast two-hybrid assays showed that the residues Arg at position 51 and Ala at position 102 in the N-terminal domain were essential for the regulatory function of the proteins, and the conserved domain at the C-terminal end was important for activation of the protein. RNA sequencing of overexpression (OE) and RNA-interference transgenic lines confirmed that the ASR proteins specifically induce structural genes of anthocyanin synthesis, including the early biosynthesis genes CHSj, F3H, and F3′5′H-1, the late biosynthesis genes DFR, ANS, RT, MT, AT, and GT, and the anthocyanin-related glutathione S-transferase gene AN9. In addition, a member of the detoxifying efflux carrier family, a DTX35-like gene, was upregulated by ASRs. Determination of anthocyanin/anthocyanidin contents in ASR1-OE petunia lines revealed that ASR1 especially induced the flavonoid 3′,5′-hydroxylase, which was consistent with the abundance of dihydromyricetins and delphinidins, but not cyanidins. The ASR genes were induced under high-intensity light and upregulated the expression of MYBx and MYB27, thereby providing feedback repression of anthocyanin synthesis. An updated model is presented outlining the mechanisms underlying anthocyanin synthesis in petunia.

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Abbreviations

AN:

Anthocyanin

ANS:

Anthocyanidin synthase

AT:

Acylation

bHLH:

Basic helix-loop-helix

4CL:

4-Coumaroyl-CoA ligase

C4H:

Cinnamate 4-hydroxylase

CHI:

Chalcone isomerase

CHS:

Chalcone synthase

DFR:

Dihydroflavonol 4-reductase

DHK:

Dihydrokaempferol

DHM:

Dihydromyricetin

DHQ:

Dihydroquercetin

DPL:

DEEP PURPLE

F3H:

Flavanone 3-hydroxylase

F3′H:

Flavonoid 3′-hydroxylase

F3′5′H:

Flavonoid 3′,5′-hydroxylase

FLS:

Flavonol synthase

GT:

Glucosylation

MT:

Methylation

PAC:

Pale Aleurone Color

PAL:

Phenylalanine ammonia lyase

PAP:

PRODUCTION OF ANTHOCYANIN PIGMENT

PHZ:

PURPLE HAZE

R2R3-MYB:

R2R3 repeat myeloblastosis protein

RT:

Rhamnosylation

SD:

Synthetic dropout medium

TT:

Transparent testa

TTG:

Transparent testa glabra

WDR:

Beta-transducin repeat protein

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Acknowledgements

We thank Dr Ronald Koes and Francesca M. Quattrocchio for the petunia cultivars and Gateway vectors. We thank Robert McKenzie, PhD, from Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.

Funding

This research was supported by the National Natural Science Foundation of China (U1504320) and Financial Project of Henan Province (20188105 and 2019XKYH03).

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Authors and Affiliations

  1. Horticultural Research Institute, Henan Academy of Agricultural Sciences, Zhengzhou, 450002, Henan Province, China

    Zhenzhu Fu, Hui Jiang, Yacong Chao, Xiaoyu Dong, Xin Yuan, Limin Wang, Jing Zhang, Menglan Xu, Huijuan Wang, Yanmin Li, Jie Gao & Hechen Zhang

  2. College of Forestry, Henan Agricultural University, Zhengzhou, 450002, Henan Province, China

    Yacong Chao, Menglan Xu & Jie Gao

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  1. Zhenzhu Fu
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  4. Xiaoyu Dong
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  5. Xin Yuan
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  12. Hechen Zhang
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Contributions

HZ designed the experiments and wrote the manuscript; ZF, HJ, and LW performed the gene searches and RNA-Seq data analysis; XD, and HW cared for the plants; JZ and JG performed DNA and RNA extraction; YC, YL, and MX performed qRT-PCR analysis and generated transgenic petunia plants.

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Correspondence to Hechen Zhang.

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Fu, Z., Jiang, H., Chao, Y. et al. Three Paralogous R2R3-MYB Genes Contribute to Delphinidin-Related Anthocyanins Synthesis in Petunia hybrida. J Plant Growth Regul 40, 1687–1700 (2021). https://doi.org/10.1007/s00344-020-10224-y

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