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. 2021 Nov 3;1(1):14.
doi: 10.1186/s43897-021-00018-5.

The PyPIF5-PymiR156a-PySPL9-PyMYB114/MYB10 module regulates light-induced anthocyanin biosynthesis in red pear

Hainan Liu #  1   2 , Qun Shu #  3 , Kui Lin-Wang  4 , Andrew C Allan  4   5 , Richard V Espley  4 , Jun Su  3 , Maosong Pei  1   2 , Jun Wu  6
Affiliations

The PyPIF5-PymiR156a-PySPL9-PyMYB114/MYB10 module regulates light-induced anthocyanin biosynthesis in red pear

Hainan Liu et al. Mol Hortic. .

Abstract

Some cultivars of pear (Pyrus L.) show attractive red fruit skin due to anthocyanin accumulation. This pigmentation can be affected by environmental conditions, especially light. To explore the light-induced regulation network for anthocyanin biosynthesis and fruit coloration in pear, small RNA libraries and mRNA libraries from fruit skins of 'Yunhongyihao' pear were constructed to compare the difference between bagging and debagging treatments. Analysis of RNA-seq of fruit skins with limited light (bagged) and exposed to light (debagged), showed that PyPIF5 was down-regulated after bag removal. PymiR156a was also differentially expressed between bagged and debagged fruit skins. We found that PyPIF5 negatively regulated PymiR156a expression in bagged fruits by directly binding to the G-box motif in its promoter. In addition, PymiR156a overexpression promoted anthocyanin accumulation in both pear skin and apple calli. We confirmed that PymiR156a mediated the cleavage of PySPL9, and that the target PySPL9 protein could form heterodimers with two key anthocyanin regulators (PyMYB114/PyMYB10). We proposed a new module of PyPIF5-PymiR156a-PySPL9-PyMYB114/MYB10. When the bagged fruits were re-exposed to light, PyPIF5 was down-regulated and its inhibitory effect on PymiR156a was weakened, which leads to degradation of the target PySPL, thus eliminating the blocking effect of PySPL on the formation of the regulatory MYB complexes. Ultimately, this promotes anthocyanin biosynthesis in pear skin.

Keywords: Anthocyanin; PyMYB114/MYB10; PyPIF5; PySPL9; PymiR156a; Red-skinned pear.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Phenotype of “Yunhongyihao” and sequencing of sRNAs and RNA in bagged and debagged fruits. A Phenotype of “Yunhongyihao” at 4, 8, and 10 days after bag removal for bagged (B1, B2, B3) and debagged (D1, D2, D3) fruits. B Venn diagram showing the number of DE-miRNAs (i) and DEGs (ii). C The number of upregulated and downregulated DE-miRNAs and DEGs. D Cluster analysis of the expression levels of DE-miRNAs (i) and DEGs (ii)
Fig. 2
Fig. 2
PyPIF5 protein suppresses PymiR156a expression by binding G-box in promoter. A The expression profile of PyPIF5. B Overexpression and suppression of PyPIF5. C Expression pattern analysis PymiR156a and PySPL9 in inoculated pear. D Dual-luciferase assay for the transcriptional activation of PyPIF5. E Electrophoretic mobility shift assay for examining the DNA-binding property of PyPIF5. As the progressively increase of Cold probe concentration, the accumulation of Free probe increases. The addition of mutant probes further confirmed that PyPIF5 can specifically bind to the G-box sequence within PymiR156a promoter
Fig. 3
Fig. 3
The expression profile of PymiR156a and its targets analysis. A The expression profile of PymiR156a in bagged and debagged fruits skins. B Predicted target sites on PySPLs of PymiR156a. C Phylogenetic analysis of SPLs from pear, apple and Arabidopsis thaliana. D Sequence logo of the SBP domain of PySPL proteins (by weblogo.berkeley.edu). The conserved zinc finger structures (Zn-1 and Zn-2) and the nuclear localization signal (NLS) are indicated. E Expression profile analysis of PySPLs. F Subcellular localization of PySPL9 protein. G Verification of cleavage events of PymiR156a on PySPL
Fig. 4
Fig. 4
Overexpression and suppression of PymiR156a. A Overexpression and suppression of PymiR156a in apple calli (i), and Green fluorescence detection (ii). B Overexpression and suppression of PymiR156a in Cv. “Zaosu”. C Overexpression of PymiR156a in Arabidopsis seedling. D Anthocyanin content in apple calli, inoculated pear skin and Arabidopsis seedling (nmol/100 mg fresh weight)
Fig. 5
Fig. 5
The interaction between PymiR156a-targeted PySPL9 and PyMYB114/10. A The interaction between PySPL9 and PyMYB114/10 verified by yeast two-hybrid assays. DDO: double dropout SD medium (lacking leucine and tryptophan). QDO/X-α-gal: quadruple dropout SD medium (lacking adenine, histidine, leucine and tryptophan) containing X-α-gal. B Detection of β galactosidase activity. C Firefly Luciferase Complementation Imaging (LCI) assay for protein-protein interactions
Fig. 6
Fig. 6
A negative regulatory model of PyPIF5 on PymiR156a-mediated light-induced anthocyanin biosynthesis in pear. In the light PyPIF5 does not maintain repression of the expression of PymiR156a, therefore SPL mRNA is cleaved, and SPL proteins decline in the complex
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