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The role of MrbHLH1 and MrMYB1 in regulating anthocyanin biosynthetic genes in tobacco and Chinese bayberry (Myrica rubra) during anthocyanin biosynthesis

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Abstract

Anthocyanins, being important for both plant functions and human health, were transcriptionally regulated by the MYB–bHLH–WD40 transcription complex. The key MYB regulator for Chinese bayberry (Myrica rubra), MrMYB1, has been characterized in previous studies, while the specific bHLH partner(s) are unknown. In this study, MrbHLH1 and MrbHLH2 were isolated based on their homology to known plant bHLHs involved in anthocyanin biosynthesis regulation. Coordinate expression of MrbHLH1 with MrMYB1 and the anthocyanin biosynthetic genes was observed during fruit development, while MrbHLH2 showed a weaker correlation. Further transient assays in tobacco leaves suggested that MrbHLH1, but not MrbHLH2, was associated with MrMYB1 and triggered significant anthocyanin production. The lack of function of the MrbHLH2 in anthocyanin biosynthesis regulation suggested that different MrbHLH genes within the same phylogenic subfamily have different functions. Overexpression of MrMYB1 and MrbHLH1 in tobacco confirmed the crucial role of MrMYB1–MrbHLH1 in anthocyanin biosynthesis and all of the structural genes from NtCHS were up-regulated by the complex. Dual luciferase assays, however, indicated that MrMYB1 and MrbHLH1 selectively activated five of the eight promoters of biosynthetic genes from bayberry (MrCHI, MrF3′H, MrDFR1, MrANS, MrUFGT), although expression levels of all eight biosynthetic genes including MrCHS and downstream genes were coordinately increased during fruit ripening. Moreover, the interaction between MrbHLH1 and MrMYB1 was confirmed by yeast two-hybrid assay. In conclusion, MrbHLH1, but not MrbHLH2, was the essential partner of MrMYB1 during anthocyanin biosynthesis regulation in tobacco and bayberry, however, the biosynthetic genes in these two species responded differently to the MrMYB1–MrbHLH1 complex.

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Abbreviations

AbA:

Aureobasidin A

ACE:

ACGT-containing element

ANS:

Anthocyanidin synthase

bHLH:

basic Helix-Loop-Helix

BQ:

Biqi

CHI:

Chalcone isomerase

CHS:

Chalcone synthase

CTAB:

Cetyltrimethylammonium bromide

DAFB:

Days after full bloom

DFR:

Dihydroflavonol 4-reductase

F3′5′H:

Flavonoid 3′,5′-hydroxylase

F3H:

Flavanone 3-hydroxylase

F3′H:

Flavonoid 3′-hydroxylase

LDOX:

Leucoanthocyanidin dioxygenase

LUC:

Firefly luciferase

MBW:

MYB–bHLH–WD40

MCS:

Multiple cloning sites

MRE:

MYB recognized element

OMT:

O-methyltransferase

ORF:

Open reading frame

Q-PCR:

Real-time quantitative PCR

RACE:

Rapid amplification of cDNA ends

REN:

Renilla luciferase

SD:

Synthetic dropout

TF:

Transcription factor

UFGT:

UDP glucose: flavonoid 3-O-glucosyltransferase

UTR:

Untranslated region

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Acknowledgments

We thank Prof. Don Grierson from University of Nottingham (UK) for his kind discussion, suggestion, and efforts in language editing. This research was supported by the National High Technology Research and Development Program of China [2013AA102606], the National Natural Science Foundation of China [31071781], the Program of International Science and Technology Cooperation [2011DFB31580], the Special Scientific Research Fund of Agricultural Public Welfare Profession of China [201203089].

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Authors and Affiliations

  1. Laboratory of Fruit Quality Biology, The State Agriculture Ministry Laboratory of Horticultural Plant Growth, Development and Quality Improvement, Zhejiang University, Hangzhou, 310058, People’s Republic of China

    Xiao-Fen Liu, Xue-Ren Yin, Yan-Na Shi, Yu-Ji Huang, Chang-Jie Xu & Kun-Song Chen

  2. The New Zealand Institute of Plant and Food Research, Private Bag 92169, Auckland, New Zealand

    Andrew C. Allan, Kui Lin-Wang & Ian B. Ferguson

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  1. Xiao-Fen Liu
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  2. Xue-Ren Yin
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  3. Andrew C. Allan
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  4. Kui Lin-Wang
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  5. Yan-Na Shi
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  8. Chang-Jie Xu
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  9. Kun-Song Chen
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Correspondence to Chang-Jie Xu.

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X.-F. Liu and X.-R. Yin have contributed equally to this work.

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Liu, XF., Yin, XR., Allan, A.C. et al. The role of MrbHLH1 and MrMYB1 in regulating anthocyanin biosynthetic genes in tobacco and Chinese bayberry (Myrica rubra) during anthocyanin biosynthesis. Plant Cell Tiss Organ Cult 115, 285–298 (2013). https://doi.org/10.1007/s11240-013-0361-8

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