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CRISPR/Cas9-mediated SlAN2 mutants reveal various regulatory models of anthocyanin biosynthesis in tomato plant

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Abstract

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Combining phenotype and gene expression analysis of the CRISPR/Cas9-induced SlAN2 mutants, we revealed that SlAN2 specifically regulated anthocyanin accumulation in vegetative tissues in purple tomato cultivar ‘Indigo Rose.’

Abstract

Anthocyanins play an important role in plant development and also exhibit human health benefits. The tomato genome contains four highly homologous anthocyanin-related R2R3-MYB transcription factors: SlAN2, SlANT1, SlANT1-like, and SlAN2-like/Aft. SlAN2-like/Aft regulates anthocyanin accumulation in the fruit; however, the genetic function of the other three factors remains unclear. To better understand the function of R2R3-MYB transcription factors, we conducted targeted mutagenesis of SlAN2 in the purple tomato cultivar ‘Indigo Rose’ using clustered regularly interspersed short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9). The SlAN2 mutants had a fruit color and anthocyanin content similar to cv. ‘Indigo Rose,’ while the anthocyanin content and the relative expression levels of several anthocyanin-related genes in vegetative tissues were significantly lower in the SlAN2 mutant relative to cv. Indigo Rose. Furthermore, we found that anthocyanin biosynthesis is controlled by different regulators between tomato hypocotyls and cotyledons. In addition, SlAN2 mutants were shorter, with smaller and lighter fruits than cv. ‘Indigo Rose.’ Our findings further our understanding of anthocyanin production in tomato and other plant species.

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References

  • Albert NW, Davies KM, Lewis DH, Zhang H, Montefiori M, Brendolise C, Boase MR, Ngo H, Jameson PE, Schwinn KE (2014) A conserved network of transcriptional activators and repressors regulates anthocyanin pigmentation in eudicots. Plant Cell 26:962–980

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Boches P, Myers J (2007) The anthocyanin fruit tomato gene (Aft) is associated with a DNA polymorphism in a MYB transcription factor. In: HortScience. Amer Soc Horticultural Science 113 S WEST ST, STE 200, Alexandria, VA 22314–2851 USA, pp 856–856

  • Borevitz JO, Xia Y, Blount JW, Dixon RA, Lamb CJ (2000) Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis. Plant Cell 12:2383–2393

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cao X, Qiu Z, Wang X, Van TG, Liu X, Wang J, Wang X, Gao J, Guo Y, Du Y (2017) A putative R3 MYB repressor is the candidate gene underlying atroviolacium, a locus for anthocyanin pigmentation in tomato fruit. J Exp Bot 68:5745–5758

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Carrasco D, De Lorenzis G, Maghradze D, Revilla E, Bellido A, Failla O, Arroyogarcia R (2015) Allelic variation in the VvMYBA1 and VvMYBA2 domestication genes in natural grapevine populations (Vitis vinifera subsp. sylvestris). Plant Syst Evol 301:1613–1624

    Article  CAS  Google Scholar 

  • Chagne D, Carlisle CM, Blond C, Volz RK, Whitworth CJ, Oraguzie NC, Crowhurst RN, Allan AC, Espley RV, Hellens RP, Gardiner SE (2007) Mapping a candidate gene (MdMYB10) for red flesh and foliage colour in apple. BMC Genomics 8:212

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Colanero S, Perata P, Gonzali S (2018) The atroviolacea gene encodes an R3-MYB protein repressing anthocyanin synthesis in tomato plants. Front Plant Sci 9:830

    Article  PubMed  PubMed Central  Google Scholar 

  • Colanero S, Tagliani A, Perata P, Gonzali S (2019) Alternative splicing in the anthocyanin fruit gene encoding an R2R3 MYB transcription factor affects anthocyanin biosynthesis in tomato fruits. Plant Commun 1:100006

    Article  PubMed  PubMed Central  Google Scholar 

  • Dubos C, Stracke R, Grotewold E, Weisshaar B, Martin C, Lepiniec L (2010) MYB transcription factors in Arabidopsis. Trends Plant Sci 15:573–581

    Article  CAS  PubMed  Google Scholar 

  • Elsm AA, Young JC, Rabalski I (2006) Anthocyanin composition in black, blue, pink, purple, and red cereal grains. J Agric Food Chem 54:4696–4704

    Article  CAS  Google Scholar 

  • Gonzalez A, Zhao M, Leavitt JM, Lloyd AM (2008) Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings. Plant J 53:814–827

    Article  CAS  PubMed  Google Scholar 

  • Gonzali S, Mazzucato A, Perata P (2009) Purple as a tomato: towards high anthocyanin tomatoes. Trends Plant Sci 14:237–241

    Article  CAS  PubMed  Google Scholar 

  • Gould KS (2004) Nature's Swiss army knife: the diverse protective roles of anthocyanins in leaves. Biomed Res Int 2004:314–320

    Google Scholar 

  • Hu D, Sun C, Ma Q, You C, Cheng L, Hao Y (2016) MdMYB1 regulates anthocyanin and malate accumulation by directly facilitating their transport into vacuoles in apples. Plant Physiol 170:1315–1330

    Article  CAS  PubMed  Google Scholar 

  • Iwashina T (2015) Contribution to flower colors of flavonoids including anthocyanins: a review. Nat prod commun 10:529–544

    PubMed  Google Scholar 

  • Jaakola L (2013) New insights into the regulation of anthocyanin biosynthesis in fruits. Trends Plant Sci 18:477–483

    Article  CAS  PubMed  Google Scholar 

  • Jeong S, Gotoyamamoto N, Hashizume K, Kobayashi S, Esaka M (2006) Expression of VvmybA1 gene and anthocyanin accumulation in various grape organs. Am J Enol Vitic 57:507–510

    CAS  Google Scholar 

  • Jian W, Cao H, Yuan S, Liu Y, Lu J, Lu W, Li N, Wang J, Zou J, Tang N (2019) SlMYB75, an MYB-type transcription factor, promotes anthocyanin accumulation and enhances volatile aroma production in tomato fruits. Hortic Res 6:1–15

    Article  CAS  Google Scholar 

  • Kiferle C, Fantini E, Bassolino L, Povero G, Spelt C, Buti S, Giuliano G, Quattrocchio F, Koes R, Perata P (2015) Tomato R2R3-MYB proteins SlANT1 and SlAN2: same protein activity. Diff Roles PloS one 10:e0136365

    Article  CAS  Google Scholar 

  • Larkin MA, Blackshields G, Brown NP, Chenna R, Mcgettigan PA, Mcwilliam H, Valentin F, Wallace IM, Wilm A, Lopez R (2007) Clustal W and clustal X version 2.0. Bioinformatics 23:2947–2948

    Article  CAS  PubMed  Google Scholar 

  • Li Z, Peng R, Tian Y, Han H, Xu J, Yao Q (2016) Genome-wide identification and analysis of the MYB transcription factor superfamily in Solanum lycopersicum. Plant Cell Physiol 57:1657–1677

    Article  CAS  PubMed  Google Scholar 

  • Liakopoulos G, Nikolopoulos D, Klouvatou A, Vekkos K-A, Manetas Y, Karabourniotis G (2006) The photoprotective role of epidermal anthocyanins and surface pubescence in young leaves of grapevine (Vitis vinifera). Ann Bot 98:257–265

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2—ΔΔCT method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  • Ma D, Constabel CP (2019) MYB repressors as regulators of phenylpropanoid metabolism in plants. Trends Plant Sci 24:275–289

    Article  CAS  PubMed  Google Scholar 

  • Mathews H (2003) Activation tagging in tomato identifies a transcriptional regulator of anthocyanin biosynthesis, modification, and transport. Plant Cell Online 15:1689–1703

    Article  CAS  Google Scholar 

  • Mazza G (2007) Anthocyanins and heart health. Annali-Istituto Superiore Di Sanita 43:369

    CAS  Google Scholar 

  • Meng X, Yang D, Li X, Zhao S, Sui N, Meng Q (2015) Physiological changes in fruit ripening caused by overexpression of tomato SlAN2, an R2R3-MYB factor. Plant Physiol Biochem 89:24–30

    Article  CAS  PubMed  Google Scholar 

  • Nakabayashi R, Yonekura-Sakakibara K, Urano K, Suzuki M, Yamada Y, Nishizawa T, Matsuda F, Kojima M, Sakakibara H, Shinozaki K, Michael AJ, Tohge T, Yamazaki M, Saito K (2014) Enhancement of oxidative and drought tolerance in Arabidopsis by overaccumulation of antioxidant flavonoids. Plant J 77:367–379

    Article  CAS  PubMed  Google Scholar 

  • Nicholas K (1997) GeneDoc: analysis and visualization of genetic variation. Embnew News 4:28–30

    Google Scholar 

  • Park SH, Morris JL, Park JE, Hirschi KD, Smith RH (2003) Efficient and genotype-independent Agrobacterium–mediated tomato transformation. J Plant Physiol 160:1253–1257

    Article  CAS  PubMed  Google Scholar 

  • Paz-Ares J, Ghosal D, Wienand U, Peterson P, Saedler H (1987) The regulatory c1 locus of Zea mays encodes a protein with homology to myb proto-oncogene products and with structural similarities to transcriptional activators. The EMBO J 6:3553–3558

    Article  CAS  PubMed  Google Scholar 

  • Pireyre M, Burow M (2015) Regulation of MYB and bHLH transcription factors: a glance at the protein level. Mol Plant 8:378–388

    Article  CAS  PubMed  Google Scholar 

  • Povero G, Gonzali S, Bassolino L, Mazzucato A, Perata P (2011) Transcriptional analysis in high-anthocyanin tomatoes reveals synergistic effect of Aft and atv genes. J Plant Physiol 168:270–279

    Article  CAS  PubMed  Google Scholar 

  • Qiu Z, Wang H, Li D, Yu B, Hui Q, Yan S, Huang Z, Cui X, Cao B (2019) Identification of candidate HY5-dependent and -independent regulators of anthocyanin biosynthesis in tomato. Plant Cell Physiol 60:643–656

    Article  CAS  PubMed  Google Scholar 

  • Qiu Z, Wang X, Gao J, Guo Y, Huang Z, Du Y (2016) The tomato hoffman's anthocyaninless gene encodes a bHLH transcription factor involved in anthocyanin biosynthesis that is developmentally regulated and induced by low temperatures. PloS one 11:e0151067

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Rick C, Cisneros P, Chetelat R, DeVerna J (1994) Abg—a gene on chromosome 10 for purple fruit derived from S. lycopersicoides. Rep Tomato Genet Coop 44:29–30

    Google Scholar 

  • Schreiber G, Reuveni M, Evenor D, Oren-Shamir M, Ovadia R, Sapir-Mir M, Bootbool-Man A, Nahon S, Shlomo H, Chen L, Levin I (2012) ANTHOCYANIN1 from Solanum chilense is more efficient in accumulating anthocyanin metabolites than its Solanum lycopersicum counterpart in association with the ANTHOCYANIN FRUIT phenotype of tomato. TAG Theoretical and applied genetics Theoretische und angewandte Genetik 124:295–307

    Article  CAS  PubMed  Google Scholar 

  • Steyn W, Wand S, Holcroft D, Jacobs G (2002) Anthocyanins in vegetative tissues: a proposed unified function in photoprotection. New Phytol 155:349–361

    Article  CAS  PubMed  Google Scholar 

  • Stracke R, Werber M, Weisshaar B (2001) The R2R3-MYB gene family in Arabidopsis thaliana. Curr Opin Plant Biol 4:447–456

    Article  CAS  PubMed  Google Scholar 

  • Sun C, Deng L, Du M, Zhao J, Chen Q, Huang T, Jiang H, Li C-B, Li C (2019) A transcriptional network promotes anthocyanin biosynthesis in tomato flesh. Mol Plant

  • Torres CA, Davies NM, Yanez JA, Andrews PK (2005) Disposition of selected flavonoids in fruit tissues of various tomato (Lycopersicon esculentum Mill.) genotypes. J Agric Food Chem 53:9536–9543

    Article  CAS  PubMed  Google Scholar 

  • Vimolmangkang S, Han Y, Wei G, Korban SS (2013) An apple MYB transcription factor, MdMYB3, is involved in regulation of anthocyanin biosynthesis and flower development. BMC Plant Biol 13:176

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Xu W, Dubos C, Lepiniec L (2015) Transcriptional control of flavonoid biosynthesis by MYB–bHLH–WDR complexes. Trends Plant Sci 20:176–185

    Article  CAS  PubMed  Google Scholar 

  • Yan S, Chen N, Huang Z, Li D, Zhi J, Yu B, Liu X, Cao B, Qiu Z (2019) Anthocyanin fruit encodes an R2R3-MYB transcription factor, SlAN2-like, activating the transcription of SlMYBATV to fine-tune anthocyanin content in tomato fruit. New Phytol 225:2048–2063

    Article  PubMed  CAS  Google Scholar 

  • Yousuf B, Gul K, Wani AA, Singh P (2016) Health benefits of anthocyanins and their encapsulation for potential use in food systems: a review. Crit Rev Food Sci Nutr 56:2223–2230

    Article  CAS  PubMed  Google Scholar 

  • Yu B, Yan S, Zhou H, Dong R, Lei J, Chen C, Cao B (2018) Overexpression of CsCaM3 improves high temperature tolerance in cucumber. Front Plant Sci 9:797

    Article  PubMed  PubMed Central  Google Scholar 

  • Zhang Y, Butelli E, De Stefano R, Schoonbeek H-j, Magusin A, Pagliarani C, Wellner N, Hill L, Orzaez D, Granell A (2013) Anthocyanins double the shelf life of tomatoes by delaying overripening and reducing susceptibility to gray mold. Curr Biol 23:1094–1100

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang Y, de Stefano R, Robine M, Butelli E, Bulling K, Hill L, Rejzek M, Martin C, Schoonbeek H-j (2015) Different ROS-scavenging properties of flavonoids determine their abilities to extend shelf life of tomato. Plant Physiol 86:S11

    Google Scholar 

  • Zhou H, Linwang K, Wang F, Espley RV, Ren F, Zhao J, Ogutu C, He H, Jiang Q, Allan AC (2019) Activator-type R2R3-MYB genes induce a repressor-type R2R3-MYB gene to balance anthocyanin and proanthocyanidin accumulation. New Phytol 221:1919–1934

    Article  CAS  PubMed  Google Scholar 

  • Zhu H, Zhang T-J, Zheng J, Huang X-D, Yu Z-C, Peng C-L, Chow WS (2018) Anthocyanins function as a light attenuator to compensate for insufficient photoprotection mediated by nonphotochemical quenching in young leaves of Acmena acuminatissima in winter. Photosynthetica 56:445–454

    Article  CAS  Google Scholar 

  • Zimmermann IM, Heim MA, Weisshaar B, Uhrig JF (2004) Comprehensive identification of Arabidopsis thaliana MYB transcription factors interacting with R/B-like BHLH proteins. Plant J 40:22–34

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This study was supported by the National Natural Science Foundation of China (31801863) and the Natural Science Foundation of Guangdong Province (2018A030310211).

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Authors and Affiliations

  1. Key Laboratory of Horticultural Crop Biology and Germplasm Innovation in South China, Ministry of Agriculture, College of Horticulture, South China Agricultural University, Guangzhou, 510642, China

    Junjie Zhi, Dongjing Li, Yinggemei Huang, Shuangshuang Yan, Bihao Cao & Zhengkun Qiu

  2. Guangdong Key Laboratory of New Technology Research of Vegetable, Vegetable Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou, 510640, China

    Xiaoxi Liu

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  1. Junjie Zhi
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Contributions

ZQ and BC conceived and designed the experiments; JZ and XL performed majority of experiments and data analyses. DL, YH, BY, SY, and BC performed experiments and data analyses; ZQ and XL wrote the manuscript; SY and BC provided input and revised the manuscript. All authors read and approved the final manuscript.

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Correspondence to Bihao Cao or Zhengkun Qiu.

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Communicated by Kan Wang.

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Zhi, J., Liu, X., Li, D. et al. CRISPR/Cas9-mediated SlAN2 mutants reveal various regulatory models of anthocyanin biosynthesis in tomato plant. Plant Cell Rep 39, 799–809 (2020). https://doi.org/10.1007/s00299-020-02531-1

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