Functional analysis of the ScAG and ScAGL11 MADS-box transcription factors for anthocyanin biosynthesis and bicolour pattern formation in Senecio cruentus ray florets

Abstract

Cineraria (Senecio cruentus) is an ornamental plant with pure colour and bicolour cultivars, widely used for landscaping. Anthocyanin biosynthesis influences coloration patterns in cineraria. However, how anthocyanins accumulate and distribute in cineraria is poorly understood. This study investigated the molecular mechanisms underlying anthocyanin biosynthesis and bicolour formation in cineraria using pure colour and bicolour cultivars. Transcriptome and gene expression analysis showed that five genes, ScCHS2, ScF3H1, ScDFR3, ScANS, and ScbHLH17, were inhibited in the white cultivar and colourless regions of bicolour cultivars. In contrast, two MADS-box genes, ScAG and ScAGL11, showed significantly higher expression in the colourless regions of bicolour cultivars. ScAG and ScAGL11 were localized in the nucleus and co-expressed with the bicolour trait. Further functional analysis verified that ScAG inhibits anthocyanin accumulation in tobacco (Nicotiana tabacum). However, virus-induced gene silencing (VIGS) experiments showed that silencing of ScAG and ScAGL11 increases anthocyanin content in cineraria leaves. Similar results were observed when ScAG and ScAGL11 were silenced in the cineraria capitulum, accompanied by the smaller size of the colourless region, specifically in the ScAG/ ScAGL11-silenced plants. The expression of ScCHS2, ScDFR3, and ScF3H1 increased in silenced cineraria leaves and capitulum. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation experiments demonstrated that ScAG interacts with ScAGL11. Moreover, ScAG directly inhibited the transcription of ScF3H1 while ScAGL11 inhibited ScDFR3 expression by binding to their promoters separately. The findings reported herein indicate that ScAG and ScAGL11 negatively regulate anthocyanin biosynthesis in cineraria ray florets, and their differential expression in ray florets influences the bicolour pattern appearance.

References

[1] Jin X, Huang H, Wang L et al. Transcriptomics and metabolite analysis reveals the molecular mechanism of anthocyanin biosynthesis branch pathway in different Senecio cruentus cultivars. Front Plant Sci. 2016; 7: 1307.

[2] Iwashina T . Contribution to flower colors of flavonoids including anthocyanins: a review. Nat Prod Rep. 2015; 10: 529-44.

[3] Yoshidam K, Mori M, Konda T . Blue flower color development by anthocyanins: from chemical structure to cell physiology. Nat Prod Rep. 2009; 26: 884-915.

[4] Xu W, Dubos C, Lepiniec L . Transcriptional control of flavonoid biosynthesis by MYB-bHLH-WDR complexes. Trends Plant Sci. 2015; 20: 176-85.

[5] Wang L, Tang W, Hu Y et al. A MYB/bHLH complex regulates tissue-specific anthocyanin biosynthesis in the inner pericarp of red-centred kiwifruit Actinidia chinensis cv. HongYang . Plant J. 2019; 99: 359-78.

[6] An JP, Wang XF, Zhang XW et al. An apple MYB transcription factor regulates cold tolerance and anthocyanin accumulation and undergoes MIEL1-mediated degradation. Plant Biotechnol J. 2020; 18: 337-53.

[7] Albert NW, Griffiths AG, Cousins GR et al. Anthocyanin leaf markings are regulated by a family of R2R3-MYB genes in the genus Trifolium . New Phytol. 2015; 205: 882-93.

[8] Yuan YW, Sagawa JM, Frost L et al. Transcriptional control of floral anthocyanin pigmentation in monkeyflowers (Mimulus) . New Phytol. 2014; 204: 1013-27.

[9] Yamagishi M, Shimoyamada Y, Nakatsuka T et al. Two R2R3-MYB genes, homologs of petunia AN2, regulate anthocyanin biosyntheses in flower tepals, tepal spots and leaves of Asiatic hybrid lily. Plant Cell Physiol. 2010; 51: 463-74.

[10] Yamagishi M, Toda S, Tasaki K . The novel allele of the LhMYB12 gene is involved in splatter-type spot formation on the flower tepals of Asiatic hybrid lilies (Lilium spp.) . New Phytol. 2014; 201: 1009-20.

[11] Suzuki K, Suzuki T, Nakatsuka T et al. RNA-seq-based evaluation of bicolor tepal pigmentation in Asiatic hybrid lilies (Lilium spp.) . BMC Genomics. 2016; 17: 611.

[12] Dong QX, Zhao H, Huang Y et al. FtMYB18 acts as a negative regulator of anthocyanin/proanthocyanidin biosynthesis in Tartary buckwheat . Plant Mol Biol. 2020; 104: 309-25.

[13] Wan SZ, Li CF, Ma XD et al. PtrMYB57 contributes to the negative regulation of anthocyanin and proanthocyanidin biosynthesis in poplar . Plant Cell Rep. 2017; 36: 1263-76.

[14] Hui Z, Wang KL, Wang F et al. Activator-type R2R3-MYB genes induce a repressor-type R2R3-MYB gene to balance anthocyanin and proanthocyanidin accumulation. New Phytol. 2019; 221: 1919-34.

[15] Mahmood K, Xu ZH, El-Kereamy A et al. The Arabidopsis transcription factor ANAC032 represses anthocyanin biosynthesis in response to high sucrose and oxidative and abiotic stresses. Front Plant Sci. 2016; 7: 1548.

[16] Su S, Xiao W, Guo W et al. The CYCLOIDEA-RADIALIS module regulates petal shape and pigmentation, leading to bilateral corolla symmetry in Torenia fournieri (Linderniaceae) . New Phytol. 2017; 215: 1582-93.

[17] Saedler H, Becker A, Winter KU et al. MADS-box genes are involved in floral development and evolution. Acta Biochim Pol. 2001; 48: 351-8.

[18] Kapazoglou A, Engineer C, Drosou V et al. The study of two barley type I-like MADS-box genes as potential targets of epigenetic regulation during seed development. BMC Plant Biol. 2012; 12: 166.

[19] Elitzur T, Yakir E, Quansah L et al. Banana MaMADS transcription factors are necessary for fruit ripening and molecular tools to promote shelf-life and food security. Plant Physiol. 2016; 171: 380-91.

[20] Alvarez-Buylla ER, Pelaz S, Gold SE et al. An ancestral MADS-box gene duplication occurred before the divergence of plants and animals. Proc Natl Acad Sci USA. 2000; 97: 5328-33.

[21] Kaufmann K, Melzer R, Theissen G . MIKC-type MADS domain proteins: structural modularity, protein interactions and network evolution in land plants. Gene. 2005; 347: 183-98.

[22] Zahn LM, Feng B, Ma H . Beyond the ABC-model: regulation of floral homeotic genes. Adv Bot Res. 2006; 44: 163-207.

[23] Li C, Wang Y, Xu L et al. Genome-wide characterization of the MADS-box gene family in radish (Raphanus sativus L.) and assessment of its roles in flowering and floral organogenesis . Front Plant Sci. 2016; 7: 1390.

[24] Jaakola L, Poole M, Jones MO et al. A SQUAMOSA MADS box gene involved in the regulation of anthocyanin accumulation in bilberry fruits. Plant Physiol. 2010; 153: 1619-29.

[25] Otani M, Aoyagi K, Nakano M . Suppression of B function by chimeric repressor gene-silencing technology (CRES-T) reduces the petaloid tepal identity in transgenic Lilium sp. PLoS One. 2020; 15: e023717.

[26] Lalusin AG, Nishita K, Kim SH et al. A new MADS-box gene (IbMADS10) from sweet potato (Ipomoea batatas (L.) Lam) is involved in the accumulation of anthocyanin . Mol Gen Genomics. 2006; 275: 44-54.

[27] Qi X, Liu C, Song L et al. PaMADS7, a MADS-box transcription factor, regulates sweet cherry fruit ripening and softening. Plant Sci. 2020; 301: 110634.

[28] Lu WJ, Chen JX, Ren XC et al. One novel strawberry MADS-box transcription factor FaMADS1 alpha acts as a negative regulator in fruit ripening. Sci Hortic. 2018; 227: 124-31.

[29] Ma G, Zou Q, Shi X et al. Ectopic expression of the AaFUL1 gene identified in Anthurium andraeanum affected floral organ development and seed fertility in tobacco . Gene. 2019; 696: 197-205.

[30] Wu R, Wang T, McGie T et al. Overexpression of the kiwifruit SVP3 gene affects reproductive development and suppresses anthocyanin biosynthesis in petals, but has no effect on vegetative growth, dormancy, or flowering time. J Exp Bot. 2014; 65: 4985-95.

[31] Wu J, Zhao G, Yang Y et al. Identification of differentially expressed genes related to coloration in red/green mutant pear (Pyrus communis L.) . Tree Genet Genomes. 2013; 9: 75-83.

[32] Feng X, An Y, Zheng J et al. Proteomics and SSH analyses of ALA-promoted fruit coloration and evidence for the involvement of a MADS-box gene, MdMADS1 . Front Plant Sci. 2016; 7: 1615.

[33] Li BJ, Zheng BQ, Wang JY et al. New insight into the molecular mechanism of colour differentiation among floral segments in orchids. Commun Biol. 2020; 3: 89.

[34] Li Y, Liu Y, Qi F et al. Establishment of virus-induced gene silencing system and functional analysis of ScbHLH17 in Senecio cruentus . Plant Physiol Biochem. 2020; 147: 272-9.

[35] Yamagishi M, Akagi K . Morphology and heredity of tepal spots in Asiatic and oriental hybrid lilies (Lilium spp.) . Euphytica. 2013; 194: 325-34.

[36] Bai YH, Pattanaik S, Patra B et al. Flavonoid-related basic helix-loop-helix regulators, NtAn1a and NtAn1b, of tobacco have originated from two ancestors and are functionally active. Planta. 2011; 234: 363-75.

[37] Lu S, Zhang Y, Zhu K et al. The citrus transcription factor CsMADS6 modulates carotenoid metabolism by directly regulating carotenogenic genes. Plant Physiol. 2018; 176: 2657-76.

[38] Lu S, Ye J, Zhu K et al. A fruit ripening-associated transcription factor CsMADS5 positively regulates carotenoid biosynthesis in citrus. J Exp Bot. 2021; 72: 3028-43.

[39] Noor SH, Saito M, Yamada E et al. Double flower formation induced by silencing of C-class MADS-box genes. Sci Hortic. 2014; 178: 1-7.

[40] Zhang T, Zhao Y, Juntheikki I et al. Dissecting functions of SEPALLATA-like MADS box genes in patterning of the pseudanthial inflorescence of Gerbera hybrida . New Phytol. 2017; 216: 939-54.

[41] Nguyen CT, Tran GB, Nguyen NH et al. The MYB-bHLH-WDR interferers (MBWi) epigenetically suppress the MBW’s targets. Biol Cell. 2019; 111: 284-91.

[42] Li J, Ma N, An YY et al. FcMADS9 of fig regulates anthocyanin biosynthesis . Sci Hortic. 2021; 278: 109820.

[43] Zhang Z, Shi Y, Ma Y et al. The strawberry transcription factor FaRAV1 positively regulates anthocyanin accumulation by activation of FaMYB10 and anthocyanin pathway genes. Plant Biotechnol J. 2020; 18: 2267-79.

[44] Lou D, Xiong C, Lin A et al. SlBBX20 interacts with the COP9 signalosome subunit SlCSN5-2 to regulate anthocyanin biosynthesis by activating SlDFR expression in tomato. Hortic Res. 2021; 8: 163.

[45] Jiang GX, Li Z, Song Y et al. LcNAC13 physically interacts with LcR1MYB1 to coregulate anthocyanin biosynthesis-related genes during litchi fruit ripening. Biomol Ther. 2019; 9: 135.

[46] Gu Z, Zhu J, Hao Q et al. A novel R2R3-MYB transcription factor contributes to petal blotch formation by regulating organ-specific expression of PsCHS in tree peony (Paeonia suffruticosa) . Plant Cell Physiol. 2018; 60: 599-611.

[47] Naing AH, Park DY, Park KI et al. Differential expression of anthocyanin structural genes and transcription factors determines coloration patterns in gerbera flowers. 3 Biotech. 2018; 8: 393.

[48] Iida S, Morita Y, Choi JD et al. Genetics and epigenetics in flower pigmentation associated with transposable elements in morning glories. Adv Biophys. 2004; 38: 141-59.

[49] Liu XJ, Chuang YN, Chiou CY et al. Methylation effect on chalcone synthase gene expression determines anthocyanin pigmentation in floral tissues of two Oncidium orchid cultivars. Planta. 2012; 236: 401-9.

[50] Cheng J, Liao L, Zhou H et al. A small indel mutation in an anthocyanin transporter causes variegated colouration of peach flowers. J Exp Bot. 2015; 66: 7227-39.

[51] Deguchi A, Tatsuzawa F, Hosokawa M et al. Tobacco streak virus (strain dahlia) suppresses post-transcriptional gene silencing of flavone synthase II in black dahlia cultivars and causes a drastic flower color change. Planta. 2015; 242: 663-75.

[52] Martins TR, Berg JJ, Blinka S et al. Precise spatio-temporal regulation of the anthocyanin biosynthetic pathway leads to petal spot formation in Clarkia gracilis (Onagraceae) . New Phytol. 2013; 197: 958-69.

[53] Yuan YW, Rebocho AB, Sagawa JM et al. Competition between anthocyanin and flavonol biosynthesis produces spatial pattern variation of floral pigments between Mimulus species . Proc Natl Acad Sci USA. 2016; 113: 2448-53.

[54] Hsu CC, Chen YY, Tsai WC et al. Three R2R3-MYB transcription factors regulate distinct floral pigmentation patterning in Phalaenopsis spp. Plant Physiol. 2015; 168: 175-91.

[55] Martins TR, Jiang P, Rausher MD . How petals change their spots: cis-regulatory re-wiring in Clarkia (Onagraceae) . New Phytol. 2017; 216: 510-8.

[56] Yamagishi M, Sakai M . The MicroRNA828/ MYB12 module mediates bicolor pattern development in Asiatic hybrid lily (Lilium spp.) flowers . Front Plant Sci. 2020; 11: 590791.

[57] Dreni L, Kater MM . MADS reloaded: evolution of the AGAMOUS subfamily genes. New Phytol. 2014; 201: 717-32.

[58] Yu D, Kotilainen M, Pollanen E et al. Organ identity genes and modified patterns of flower development in Gerbera hybrida (Asteraceae) . Plant J. 1999; 17: 51-62.

[59] Garceau DC, Batson MK, Pan IL . Variations on a theme in fruit development: the PLE lineage of MADS-box genes in tomato (TAGL1) and other species. Planta. 2017; 246: 313-21.

[60] Kramer EM, Jaramillo MA, VSD S . Patterns of gene duplication and functional evolution during the diversification of the AGAMOUS subfamily of MADS box genes in angiosperms. Genetics. 2004; 166: 1011-23.

[61] Liljegren SJ, Ditta GS, Eshed Y et al. SHATTERPROOF MADS-box genes control seed dispersal in Arabidopsis . Nature. 2000; 404: 766-70.

[62] Boss PK, Vivier M, Matsumoto S et al. A cDNA from grapevine (Vitis vinifera L.), which shows homology to AGAMOUS and SHATTERPROOF, is not only expressed in flowers but also throughout berry development . Plant Mol Biol. 2001; 45: 541-53.

[63] Tadiello A, Pavanello A, Zanin D et al. A PLENA-like gene of peach is involved in carpel formation and subsequent transformation into a fleshy fruit. J Exp Bot. 2009; 60: 651-61.

[64] Gimenez E, Pineda B, Capel J et al. Functional analysis of the Arlequin mutant corroborates the essential role of the ARLEQUIN/ TAGL1 gene during reproductive development of tomato . PLoS One. 2010; 5: e14427.

[65] Itkin M, Seybold H, Breitel D et al. TOMATO AGAMOUS-LIKE 1 is a component of the fruit ripening regulatory network . Plant J. 2009; 60: 1081-95.

[66] Vrebalov J, Pan IL, AJM A et al . Fleshy fruit expansion and ripening are regulated by the tomato SHATTERPROOF gene TAGL1 . Plant Cell. 2009; 21: 3041-62.

[67] Gimenez E, Dominguez E, Pineda B et al. Transcriptional activity of the MADS box ARLEQUIN/ TOMATO AGAMOUS-LIKE1 gene is required for cuticle development of tomato fruit . Plant Physiol. 2015; 168: 1036-48.

[68] Hou JH, Gao ZH, Zhang Z et al. Isolation and characterization of an AGAMOUS homologue PmAG from the Japanese apricot (Prunus mume Sieb. Et Zucc.) . Plant Mol Biol Rep. 2011; 29: 473-80.

[69] Lu C, Pu Y, Liu Y et al. Comparative transcriptomics and weighted gene co-expression correlation network analysis (WGCNA) reveal potential regulation mechanism of carotenoid accumulation in Chrysanthemum × morifolium . Plant Physiol Biochem. 2019; 142: 415-28.

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

[71] Masuda K, Xu ZJ, Takahashi S et al. Peripheral framework of carrot cell nucleus contains a novel protein predicted to exhibit a long α-helical domain. Exp Cell Res. 1997; 232: 173-81.

[72] Kumar S, Stecher G, Tamura K . MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016; 33: 1870-4.

[73] Edgar RC . MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004; 32: 1792-7.

[74] Hellens RP, Allan AC, Friel EN et al. Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants. Plant Methods. 2005; 1: 1-14.

[75] Horsch RB, Klee HJ . Rapid assay of foreign gene expression in leaf discs transformed by Agrobacterium tumefaciens: role of T-DNA borders in the transfer process . Proc Natl Acad Sci USA. 1986; 83: 4428-32.

[76] Yan H, Fu DQ, Zhu BZ et al. Sprout vacuum-infiltration: a simple and efficient agroinoculation method for virus-induced gene silencing in diverse solanaceous species. Plant Cell Rep. 2012; 31: 1713-22.