MdMYB8 is associated with flavonol biosynthesis via the activation of the MdFLS promoter in the fruits of Malus crabapple

Abstract

Flavonols are polyphenolic compounds that play important roles in plant stress resistance and development. They are also valuable components of the human diet. The Malus crabapple cultivar ‘Flame’ provides an excellent model for studying flavonol biosynthesis due to the high flavonol content of its fruit peel. To obtain a more detailed understanding of the flavonol regulatory network involved in fruit development, the transcriptomes of the fruit of the Malus cv. ‘Flame’ from five continuous developmental stages were analyzed using RNA sequencing. A flavonol-related gene module was identified through weighted gene coexpression network analysis (WGCNA), and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that phytohormones are involved in regulating flavonol biosynthesis during fruit development. A putative transcription factor, MdMYB8, was selected for further study through hub gene correlation network analysis and yeast one-hybrid assays. Stable overexpression or RNAi knockdown of MdMYB8 in transgenic ‘Orin’ apple calli resulted in a higher or lower flavonol content, respectively, suggesting that MdMYB8 is a regulator of flavonol biosynthesis. This transcriptome analysis provides valuable data for future studies of flavonol synthesis and regulation.

References

[1] Preuss, A. et al. Arabidopsis thaliana expresses a second functional flavonol synthase. FEBS Lett. 583, 1981-1986 (2009).

[2] Santelia, D. et al. Flavonoids redirect PIN-mediated polar auxin fluxes during root gravitropic responses. J. Biol. Chem. 283, 31218-31226 (2008).

[3] Tian, J. et al. The balance of expression of dihydroflavonol 4-reductase and flavonol synthase regulates flavonoid biosynthesis and red foliage coloration in crabapples. Sci. Rep. 5, 12228 (2015).

[4] Lin, J. K., & Weng, M. S. in The science of flavonoids (ed. Grotewold E.) 213-238 (Springer Science+BusinessMedia, Inc, New York, 2006).

[5] Davies, K. M. et al. Enhancing anthocyanin production by altering competition for substrate between flavonol synthase and dihydroflavonol 4-reductase. Euphytica 131, 259-268 (2003).

[6] Holton, T. A. et al. Cloning and expression of flavonol synthase from Petunia hybrida. Plant J 4, 1003-1010 (1993).

[7] Owens, D. K. et al. Functional analysis of a predicted flavonol synthase gene family in Arabidopsis. Plant Physiol. 147, 1046-1061 (2008).

[8] Czemmel, S. et al. The Grapevine R2R3-MYB transcription factor VvMYBF1 regulates flavonol synthesis in developing grape berries. Plant Physiol. 151, 1513-1530 (2009).

[9] Zhang, C. Y. et al. Cloning, characterization and functional analysis of a flavonol synthase from Vaccinium corymbosum. Trees 30, 1595-1605 (2016).

[10] Espley, R. V. et al. Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10. Plant J 49, 414-427 (2007).

[11] Liu, C. et al. Characterization of a citrus R2R3-MYB transcription factor that regulates the flavonol and hydroxycinnamic acid biosynthesis. Sci. Rep. 10, 6 (2016).

[12] Nan, W. et al. Transcriptomic analysis of red-fleshed apples reveals the novel role of MdWRKY11 in flavonoid and anthocyanin biosynthesis. J. Agric. Food Chem. 66, 7076-7086 (2018).

[13] Zhou, H. et al. Molecular genetics of blood-fleshed peach reveals activation of anthocyanin biosynthesis by NAC transcription factors. Plant J 82, 105-121 (2015).

[14] Stracke, R. et al. Differential regulation of closely related R2R3-MYB transcription factors controls flavonol accumulation in different parts of the Arabidopsis thaliana seedling. Plant J 50, 660-677 (2007).

[15] Zhai, R. et al. Two MYB transcription factors regulate flavonoid biosynthesis in pear fruit (Pyrus bretschneideri Rehd.). J. Exp. Bot. 67, 1275-1284 (2016).

[16] Onik, J. C. et al. Comparative transcriptomic profiling to understand pre- and post-ripening hormonal regulations and anthocyanin biosynthesis in early ripening apple fruit. Molecules 23, E1908 (2018).

[17] Wang, N. et al. MYB12 and MYB22 play essential roles in proanthocyanidin and flavonol synthesis in red-fleshed apple (Malus sieversii f. niedzwetzkyana). Plant J 90, 276-292 (2017).

[18] Tian, J. et al. McMYB12 transcription factors co-regulate proanthocyanidin and anthocyanin biosynthesis in Malus crabapple. Sci. Rep. 7, 43715 (2016).

[19] Jaakola, L. New insights into the regulation of anthocyanin biosynthesis in fruits. Trends Plant Sci 18, 477-483 (2013).

[20] Yang, J. et al. DCGL v2.0: an R package for unveiling differential regulation from differential co-expression. PLoS ONE 8, e79729 (2013).

[21] Zhang, B. & Horvath, S. A general framework for weighted gene co-expression network analysis. Stat. Appl. Genet. Mol. Biol 4, 1544-6115 (2005).

[22] Zhang, A. D. et al. Transcriptome analysis identifies a zinc finger protein regulating starch degradation in kiwifruit. Plant Physiol. 178, 850-863 (2018).

[23] Giovannoni, J. J. Fruit ripening mutants yield insights into ripening control. Curr. Opin. Plant Biol. 10, 283-289 (2007).

[24] Kumar, R. et al. Role of plant hormones and their interplay in development and ripening of fleshy fruits. J. Exp. Bot. 65, 4561-4575 (2014).

[25] Loreti, E. et al. Gibberellins, jasmonate and abscisic acid modulate the sucrose-induced expression of anthocyanin biosynthetic genes in Arabidopsis. New Phytol 179, 1004-1016 (2008).

[26] Jiang, Y. & Joyce, D. C. ABA effects on ethylene production, PAL activity, anthocyanin and phenolic contents of strawberry fruit. Plant Growth Regul 39, 171-174 (2003).

[27] Pérez, A. G. et al. Effect of methyl jasmonate on in vitro strawberry ripening. J. Agric. Food Chem. 45, 3733-3737 (1997).

[28] El-Kereamy, A. et al. Exogenous ethylene stimulates the long-term expression of genes related to anthocyanin biosynthesis in grape berries. Physiol. Plant. 119, 175-182 (2003).

[29] Lai, B. et al. Transcriptomic analysis of Litchi chinensis pericarp during maturation with a focus on chlorophyll degradation and flavonoid biosynthesis. BMC Genomics. 16, 1-18 (2015).

[30] Wang, Z. & Dilley, D. R. Aminoethoxyvinylglycine, combined with ethephon, can enhance red color development without over-ripening apples. HortScience 36, 328-331 (2001).

[31] Li, W. F. et al. Anthocyanin accumulation correlates with hormones in the fruit skin of ‘Red Delicious’ and its four generation bud sport mutants. BMC Plant Biol. 18, 363 (2018).

[32] Kim, J. et al. Analysis of eight phytohormone concentrations, expression levels of ABA biosynthesis genes, and ripening-related transcription factors during fruit development in strawberry. J. Plant Physiol. 239, 52-60 (2019).

[33] Lewis, D. R. et al. Auxin and ethylene induce flavonol accumulation through distinct transcriptional networks. Plant Physiol. 156, 144-164 (2011).

[34] Król, P. et al. Priming of seeds with methyl jasmonate induced resistance to hemi-biotroph Fusarium oxysporum f. sp. lycopersici in tomato via 12-oxo-phytodienoic acid, salicylic acid, and flavonol accumulation. J. Plant Physiol. 179, 122-132 (2015).

[35] Gonzalez, A. et al. Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/MYB transcriptional complex in Arabidopsis seedlings. Plant J 53, 814-827 (2008).

[36] Daccord, N. et al. High-quality de novo assembly of the apple genome and methylome dynamics of early fruit development. Nat. Genet. 49, 1099-1106 (2017).

[37] Trapnell, C. et al. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105-1111 (2009).

[38] Anders, S. et al. HTSeq-a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166-169 (2015).

[39] Mortazavi, A. et al. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat. Methods. 5, 621-628 (2008).

[40] Andino, G. K. et al. Differential gene expression in Varroa jacobsoni mites following a host shift to European honey bees (Apis mellifera). BMC Genomics. 20, 554 (2016).

[41] Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. B 57, 289-300 (1995).

[42] Anders, S. & Huber, W. Differential expression analysis for sequence count data. Genome Biol. 11, R106 (2010).

[43] Mao, X. et al. Automated genome annotation and pathway identification using the KEGG Orthology (KO) as a controlled vocabulary. Bioinformatics 21, 3787-3793 (2005).

[44] Yang, T. et al. The use of RNA sequencing and correlation network analysis to study potential regulators of crabapple leaf color transformation. Plant Cell Physiol 59, 1027-1042 (2018).

[45] Zhan, J. P. et al. RNA sequencing of laser-capture microdissected compartments of the maize kernel identifies regulatory modules associated with endosperm cell differentiation. Plant Cell. 27, 531 (2015).

[46] Langfelder, P. & Horvath, S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinform. 9, 559 (2008).

[47] Revilla, E. & Ryan, J. M. Analysis of several phenolic compounds with potential antioxidant properties in grape extracts and wines by high-performance liquid chromatography-photodiode array detection without sample preparation. J. Chromatogr. A. 881, 461-469 (2000).

[48] Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^−ΔΔCT method . Methods 25, 402-408 (2001).

[49] Fortino, V. et al. BACA: bubble chArt to compare annotations. BMC Bioinform. 16, 37 (2015).