doi:

DOI: 10.3724/SP.J.1006.2018.00185

Acta Agronomica Sinica (作物学报) 2018/44:2 PP.185-196

An R2R3-MYB Transcription Factor GmMYB184 Regulates Soybean Isoflavone Synthesis


Abstract:
Isoflavones comprise a group of secondary metabolites produced almost exclusively by plants in the legume family, including soybean[Glycine max (L.) Merr.]. They play vital roles in plant defense and have many beneficial effects on human health. Isoflavone content is controlled by multiple genes and complex metabolic networks. The modification of certain structural genes in the isoflavone pathway by genetic engineering has been unable to significantly improve isoflavone content. The identification and application of transcription factors specific to the isoflavone pathway may effectively resolve this problem. An R2R3-type transcription factor related to isoflavone content, GmMYB184, was cloned and its function was identified. Subcellular localization study confirmed the nuclear localization of GmMYB184. The expression profile of GmMYB184 was similar to that of IFS2 (isoflavone synthase 2), which was consistent with the isoflavone accumulation pattern. In addition, GmMYB184 and IFS2 mainly expressed in roots and mature seeds, and the expression level increased by seeds maturing. Glutathione (GSH) induction expression analysis showed that both GmMYB184 and IFS2 were induced by GSH, indicating they could be involved in similar biological processes. To examine whether GmMYB184 could regulate the expression of isoflavone biosynthesis-related genes, a dual luciferase reporter gene assay was performed, showing that GmMYB184 could increase the expression of IFS2 and CHS8 (chalcone synthase 8) to five and seven folds, respectively. Finally, to further verify the function of GmMYB184 during isoflavone biosynthesis, we constructed and then transformed overexpression vector and RNAi vector for GmMYB184 to soybean hairy roots respectively. RNAi silencing of GmMYB184 in hairy roots resulted in reduction of isoflavones. However, overexpression of GmMYB184 was not sufficient to increase isoflavone contents in hairy roots. Taken together, these results provide a theoretical foundation for the molecule mechanism explanation and genetic improvement of isoflavone content in soybean.

Key words:soybean,isoflavone content,GmMYB184,transcription factor,functional identification

ReleaseDate:2018-03-08 10:13:39



[1] Yu O, McGonigle B. Metabolic engineering of isoflavone biosynthesis. Adv Agron 2005, 86:147-190

[2] Cederroth C R, Nef S. Soy, phytoestrogens and metabolism:a review. Mol Cell Endocrinol 2009, 304:30-42

[3] Rochfort S, Panozzo J. Phytochemicals for health, the role of pulses. J Agric Food Chem 2007, 55:7981-7994

[4] Dixon R A. Natural products and plant disease resistance. Nature 2001, 411:843-847

[5] Graham T L, Graham M Y. Signaling in soybean phenylpropanoid responses (dissection of primary, secondary, and conditioning effects of light, wounding, and elicitor treatments). Plant Physiol, 1996, 110:1123-1133

[6] Hammerschmidt R. PHYTOALEXINS:what have we learned after 60 years? Annu Rev Phytopathol, 1999, 37:285-306

[7] Gutierrez-Gonzalez J J, Wu X, Gillman J D, Lee J D, Zhong R, Yu O, Shannon G, Ellersieck M, Nguyen H T, Sleper D A. Intricate environment-modulated genetic networks control isoflavone accumulation in soybean seeds. BMC Plant Biol, 2010, 10:105

[8] Dixon R A, Steele C L. Flavonoids and isoflavonoids:a gold mine for metabolic engineering. Trends Plant Sci, 1999, 4:394-400

[9] Zernova O V, Lygin A V, Widholm J M, Lozovaya V V. Modification of isoflavones in soybean seeds via expression of multiple phenolic biosynthetic genes. Plant Physiol Biochem, 2009, 47:769-777

[10] 易金鑫, 徐照龙, 王峻峰, 张大勇, 何晓兰, 朱虹润, 马鸿翔. GmCHS8和GmIFS2基因共同决定大豆中异黄酮的积累. 作物学报, 2011, 37:571-578 Yi J X, Xu Z L, Wang J F, Zhang D Y, He X L, Zhu H R, Ma H X. The GmCHS8 and GmIFS2 genes together determine the accumulation of isoflavones in soybeans. Acta Agron Sin, 2011, 37:571-578(in Chinese with English abstract)

[11] Butelli E, Titta L, Giorgio M, Mock H P, Matros A, Peterek S, Schijlen E G, Hall R D, Bovy A G, Luo J, Martin, C. Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors. Nat Biotechnol, 2008, 26:1301-1308

[12] Cutanda-Perez M C, Ageorges A, Gomez C, Vialet S, Terrier N, Romieu C, Torregrosa L. Ectopic expression of VlmybA1 in grapevine activates a narrow set of genes involved in anthocyanin synthesis and transport. Plant Mol Biol, 2009, 69:633-648

[13] Hichri I, Barrieu F, Bogs J, Kappel C, Delrot S, Lauvergeat V. Recent advances in the transcriptional regulation of the flavonoid biosynthetic pathway. J Exp Bot, 2011, 62:2465-2483

[14] Zhou M L, Hou H L, Zhu X M, Shao J R, Wu Y M, Tang Y X. Soybean transcription factor GmMYBZ2 represses catharanthine biosynthesis in hairy roots of Catharanthus roseus. Appl Microbiol Biotechnol, 2011, 91:1095-1105

[15] Chen Y H, Yang X Y, He K, Liu M H, Li J G, Gao Z F, Lin Z Q, Zhang Y F, Wang X X, Qiu X M, Shen Y P, Zhang L, Deng X H, Luo J C, Deng X W, Chen Z L, Gu H Y, Qu L J. The MYB transcription factor superfamily of Arabidopsis:expression analysis and phylogenetic comparison with the rice MYB family. Plant Mol Biol, 2006, 60:107-124

[16] Jin H, Martin C. Multifunctionality and diversity within the plant MYB-gene family. Plant Mol Biol, 1999, 41:577-585

[17] Stracke R, Werber M, Weisshaar B. The R2R3-MYB gene family in Arabidopsis thaliana. Curr Opin Plant Biol, 2001, 4:447-456

[18] Du H, Yang S S, Liang Z, Feng B R, Liu L, Huang Y B, Tang Y X. Genome-wide analysis of the MYB transcription factor superfamily in soybean. BMC Plant Biol, 2012, 12:106

[19] Chu S, Wang J, Zhu Y, Liu S, Zhou X, Zhang H, Wang C E, Yang W, Tian Z, Cheng H, Yu D Y. An R2R3-type MYB transcription factor, GmMYB29, regulates isoflavone biosynthesis in soybean. PLoS Genet, 2017, 13:e1006770

[20] Liu X, Yuan L, Xu L, Xu Z, Huang Y, He X, Ma H, Yi J, Zhang D. Over-expression of GmMYB39 leads to an inhibition of the isoflavonoid biosynthesis in soybean (Glycine max L.). Plant Biotechnol Rep, 2013, 7:445-455

[21] Yan J, Wang B, Zhong Y, Yao L, Cheng L, Wu T. The soybean R2R3 MYB transcription factor GmMYB100 negatively regulates plant flavonoid biosynthesis. Plant Mol Biol, 2015, 89:35-48

[22] Robbins M P, Hartnoll J, Morris P. Phenylpropanoid defence responses in transgenic Lotus corniculatus 1. Glutathione elicitation of isoflavan phytoalexins in transformed root cultures. Plant Cell Rep, 1991, 10:59-62

[23] Shelton D, Stranne M, Mikkelsen L, Pakseresht N, Welham T, Hiraka H, Tabata S, Sato S, Paquette S, Wang T L, Martin C, Bailey P. Transcription factors of Lotus:regulation of isoflavonoid biosynthesis requires coordinated changes in transcription factor activity. Plant Physiol, 2012, 159:531-547

[24] Dubos C, Stracke R, Grotewold E, Weisshaar B, Martin C, Lepiniec L. MYB transcription factors in Arabidopsis. Trends Plant Sci, 2010, 15:573-581

[25] Higgins D G, Thompson J D, Gibson T J. Using CLUSTAL for multiple sequence alignments. Method Enzymol, 1996, 266:383-402

[26] Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6:Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol, 2013, 30:2725-2729

[27] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods, 2001, 25:402-408

[28] Sun J M, Sun B L, Han F X, Yan S R, Yang H, Kikuchi A. Rapid HPLC method for determination of 12 isoflavone components in soybean seeds. Agric Sci China, 2011, 10:70-77

[29] Chung E S, Kim K M, Lee J H. Molecular cloning and characterization of a soybean GmMBY184 induced by abiotic stresses. J Plant Biotechnol, 2012, 39:175-181

[30] Ishida T, Kurata T, Okada K, Wada T. A genetic regulatory network in the development of trichomes and root hairs. Annu Review Plant Biol, 2008, 59:365-386

[31] Nadeau J A. Stomatal development:new signals and fate determinants. Curr Opin Plant Biol, 2009, 12:29-35

[32] Wang X, Niu Q W, Teng C, Li C, Mu J, Chua N H, Zuo J. Overexpression of PGA37/MYB118 and MYB115 promotes vegetative-to-embryonic transition in Arabidopsis. Cell Res, 2009, 19:224-235

[33] Muller D, Schmitz G, Theres K. Blind homologous R2R3 Myb genes control the pattern of lateral meristem initiation in Arabidopsis. Plant Cell, 2006, 18:586-597

[34] Mu R L, Cao Y R, Liu Y F, Lei G, Zou H F, Liao Y, Wang H W, Zhang W K, Ma B, Du J Z, Yuan M, Zhang J S, Chen S Y. An R2R3-type transcription factor gene AtMYB59 regulates root growth and cell cycle progression in Arabidopsis. Cell Res, 2009, 19:1291-1304

[35] Abe H, Yamaguchi-Shinozaki K, Urao T, Iwasaki T, Hosokawa D, Shinozaki K. Role of Arabidopsis MYC and MYB homologs in drought-and abscisic acid-regulated gene expression. Plant Cell, 1997, 9:1859-1868

[36] Mengiste T, Chen X, Salmeron J, Dietrich R. The BOTRYTIS SUSCEPTIBLE1 gene encodes an R2R3MYB transcription factor protein that is required for biotic and abiotic stress responses in Arabidopsis. Plant Cell, 2003, 15:2551-2565

[37] Borevitz J O, Xia Y, Blount J, Dixon R A, Lamb C. Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis. Plant Cell, 2000, 12:2383-2394

[38] Sablowski R W, Moyano E, Culianez-Macia F A, Schuch W, Martin C, Bevan M. A flower-specific Myb protein activates transcription of phenylpropanoid biosynthetic genes. EMBO J, 1994, 13:128-137

[39] Solano R, Nieto C, Avila J, Canas L, Diaz I, Paz-Ares J. Dual DNA binding specificity of a petal epidermis-specific MYB transcription factor (MYB.Ph3) from Petunia hybrida. EMBO J, 1995, 14:1773-1784

[40] Yu O, Shi J, Hession A O, Maxwell C A, McGonigle B, Odell J T. Metabolic engineering to increase isoflavone biosynthesis in soybean seed. Phytochemistry, 2003, 63:753-763

[41] Grotewold E, Chamberlin M, Snook M, Siame B, Butler L, Swenson J, Maddock S, St Clair G, Bowen B. Engineering secondary metabolism in maize cells by ectopic expression of transcription factors. Plant Cell, 1998, 10:721-740

[42] Holl J, Vannozzi A, Czemmel S, D'Onofrio C, Walker A R, Rausch T, Lucchin M, Boss P K, Dry I B, Bogs J. The R2R3-MYB transcription factors MYB14 and MYB15 regulate stilbene biosynthesis in Vitis vinifera. Plant Cell, 2013, 25:4135-4149

[43] Dhaubhadel S, Gijzen M, Moy P, Farhangkhoee M. Transcriptome analysis reveals a critical role of CHS7 and CHS8 genes for isoflavonoid synthesis in soybean seeds. Plant Physiol, 2007, 143:326-338