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A beta-glucosidase gene from Stevia rebaudiana may be involved in the steviol glycosides catabolic pathway

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Abstract

We herein report the preparation of a full-length raucaffricine-O-beta-d-glucosidase gene of stevia rebaudiana Bertoni (named SrRG1, GenBank accession number MK920450). Sequence analysis indicated SrRG1 consists of a 1650 bp open reading frame encoding a protein of 549 amino acids. Its deduced amino acid sequence showed a high identity of 82% with a raucaffricine-O-beta-d-glucosidase from H. annuus of glycoside hydrolase family 1. The expression pattern analyzed by real-time quantitative PCR showed no significant difference among different tissues, developmental stages, and cultivars under normal growth conditions. Furthermore, the gene function of SrRG1 was preliminarily studied by agrobacterium-mediated transformation on instantaneous expression. In the test of agrobacterium-mediated transformation on instantaneous expression, it was observed that overexpression of SrRG1 increased the accumulation of steviol content and decreased the major components and total SGs contents. Such results demonstrated that SrRG1 may participate in the steviol glycosides catabolic pathway. However, the effect of silencing construct infiltration on steviol and SGs content was not significant and its expression pattern was constitutive, which most probably, attributed the hydrolysis of SGs to the secondary activity of SrRG1. This study firstly identified the bate-glucosidase in stevia and advances our understanding of steviol glycosides hydrolyzation.

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References

  1. Bendtsen JD, Nielsen H, Heijne GV, Brunak S (2004) Improved prediction of signal peptides: signalp 30. J Mol Biol 340(4):783–795. https://doi.org/10.1016/j.jmb.2004.05.028

    Article  CAS  PubMed  Google Scholar 

  2. Brandle JE, Richman A, Swanson AK, Chapman BP (2002) Leaf Ests from Stevia rebaudiana: a resource for gene discovery in diterpene synthesis. Plant Mol Biol 50:613–622. https://doi.org/10.1023/A:1019993221986

    Article  CAS  PubMed  Google Scholar 

  3. Falcao HG, Handa CL, Silva MBR, de Camargo AC, Shahidi F, Kurozawa LE, Ida EI (2018) Soybean ultrasound pre-treatment prior to soaking affects beta-glucosidase activity, isoflavone profile and soaking time. Food Chem 269:404–412. https://doi.org/10.1016/j.foodchem.2018.07.028

    Article  CAS  PubMed  Google Scholar 

  4. Geerlings A, Ibanez MM, Memelink J, Van Der Heijden R, Verpoorte R (2000) Molecular cloning and analysis of strictosidine beta-d-glucosidase, an enzyme in terpenoid indole alkaloid biosynthesis in Catharanthus roseus. J Biol Chem 275:3051–3056

    Article  CAS  PubMed  Google Scholar 

  5. Gerasimenko I, Sheludko Y, Ma X, Stockigt J (2002) Heterologous expression of a Rauvolfia cDNA encoding strictosidine glucosidase, a biosynthetic key to over 2000 monoterpenoid indole alkaloids. Eur J Biochem 269:2204–2213. https://doi.org/10.1046/j.1432-1033.2002.02878.x

    Article  CAS  PubMed  Google Scholar 

  6. Goyal SK, Samsher GRK (2010) Stevia (Stevia rebaudiana) a bio-sweetener: a review. Int J Food Sci Nutr 61:1–10. https://doi.org/10.3109/09637480903193049

    Article  CAS  PubMed  Google Scholar 

  7. Guleria P, Yadav SK (2013) Agrobacterium mediated transient gene silencing (AMTS) in Stevia rebaudiana: insights into steviol glycoside biosynthesis pathway. PLoS ONE 8:e74731. https://doi.org/10.1371/journal.pone.0074731

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Horton P, Park KJ, Obayashi T, Fujita N, Harada H, Adams-Collier C, Nakai K (2007) WoLF PSORT: protein localization predictor. Nucleic Acids Res 35:W585–W587. https://doi.org/10.1093/nar/gkm259

    Article  PubMed  PubMed Central  Google Scholar 

  9. Ketudat Cairns JR, Esen A (2010) beta-Glucosidases. Cell Mol Life Sci 67:3389–3405. https://doi.org/10.1007/s00018-010-0399-2

    Article  CAS  PubMed  Google Scholar 

  10. Ketudat Cairns JR, Mahong B, Baiya S, Jeon JS (2015) beta-Glucosidases: Multitasking, moonlighting or simply misunderstood? Plant Sci 241:246–259. https://doi.org/10.1016/j.plantsci.2015.10.014

    Article  CAS  PubMed  Google Scholar 

  11. Ko JA, Kim YM, Ryu YB, Ieong HJ, Park TS, Wee YJ, Kim JS, Kim D, Lee WS (2012) Mass production of rubusoside using a novel stevioside-specific beta-glucosidase from Aspergillus aculeatus. J Agric Food Chem 60:6210–6216. https://doi.org/10.1021/jf300531e

    Article  CAS  PubMed  Google Scholar 

  12. Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol Biol Evol 35:1547–1549. https://doi.org/10.1093/molbev/msy096

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods Mol Biol 25:402–408. https://doi.org/10.1006/meth.2001.1262

    Article  CAS  Google Scholar 

  14. Nielsen H (2017) Predicting Secretory Proteins with SignalP. Methods Mol Biol 1611:59–73. https://doi.org/10.1007/978-1-4939-7015-5_6

    Article  CAS  PubMed  Google Scholar 

  15. Nomura T, Quesada AL, Kutchan TM (2008) The new beta-d-glucosidase in terpenoid-isoquinoline alkaloid biosynthesis in Psychotria ipecacuanha. J Biol Chem 283:34650–34659

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Pankoke H, Buschmann T, Muller C (2013) Role of plant beta-glucosidases in the dual defense system of iridoid glycosides and their hydrolyzing enzymes in Plantago lanceolata and Plantago major. Phytochemistry 94:99–107. https://doi.org/10.1016/j.phytochem.2013.04.016

    Article  CAS  PubMed  Google Scholar 

  17. Ruppert M, panjikar S, Barleben L, Stockigt J (2006) Heterologous expression, purification, crystallisation and preliminary Xray analysis of raucaffricine glucosidase a plant enzyme specifically involved in Rauvolvia alkaloid biosynthesis. Structural Biology and Crystallization Communications 62:257–260. https://doi.org/10.1107/S174430910600457X

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Richman A, Swanson A, Humphrey T, Chapman R, Mcgarvey B, Pocs R, Brandle J (2005) Functional genomics uncovers three glucosyltransferases involved in the synthesis of the major sweet glucosides of Stevia rebaudiana. Plant J 41:56–67. https://doi.org/10.1111/j.1365-313X.2004.02275.x

    Article  CAS  PubMed  Google Scholar 

  19. Richman AS, Gijzen M, Starratt AN, Yang Z, Brandle JE (1999) Diterpene synthesis in Stevia rebaudiana: recruitment and up-regulation of key enzymes from the gibberellin biosynthetic pathway. Plant J 19:411–421. https://doi.org/10.1046/j.1365-313X.1999.00531.x

    Article  CAS  PubMed  Google Scholar 

  20. Uniprot C (2015) UniProt: a hub for protein information. Nucleic Acids Res 43:D204–D212. https://doi.org/10.1093/nar/gku989

    Article  CAS  Google Scholar 

  21. Vassão DG, Wielsch N, Gomes AMMM, Gebauer-Jung S, Hupfer Y, Svatoš A, Gershenzon J (2018) Plant Defensive beta-Glucosidases Resist Digestion and Sustain Activity in the Gut of a Lepidopteran Herbivore. Front Plant Sci 9:1389. https://doi.org/10.3389/fpls.2018.01389

    Article  PubMed  PubMed Central  Google Scholar 

  22. Wang ZL, Wang JP, Jiang MH, Wei YT, Pang H, Wei H, Huang RB, Du LQ (2015) Selective production of rubusoside from stevioside by using the sophorose activity of β-glucosidase from Streptomyces sp. GXT6. Appl Microbiol Biotechnol 99:9663–9674. https://doi.org/10.1007/s00253-015-6802-z

    Article  CAS  PubMed  Google Scholar 

  23. Warzecha H, Obitz P, Stockigt J (1999) Purification, partial amino acid sequence and structure of the product of raucaffricine-O-beta-D-glucosidase from plant cell cultures of Rauwolfia serpentina. Phytochemistry 50:1099–1109. https://doi.org/10.1016/S0031-9422(98)00689-X

    Article  CAS  PubMed  Google Scholar 

  24. Yang YH, Huang SZ, Han YL, Yuan HY, Gu CS, Zhao YH (2014) Base substitution mutations in uridinediphosphate-dependent glycosyltransferase 76G1 gene of Stevia rebaudiana causes the low levels of rebaudioside A: mutations in UGT76G1, a key gene of steviol glycosides synthesis. Plant Physiol Biochem 80:220–225. https://doi.org/10.1016/j.plaphy.2014.04.005

    Article  CAS  PubMed  Google Scholar 

  25. Yoshiara LY, Madeira TB, De Camargo AC, Shahidi F, Ida EI (2018) Multistep Optimization of beta-Glucosidase Extraction from Germinated Soybeans (Glycine max L Merril) and Recovery of Isoflavone Aglycones. Foods. https://doi.org/10.3390/foods7070110

    Article  PubMed  PubMed Central  Google Scholar 

  26. Zhou Y, Zeng LT, Gui JD, Liao YY, Li JL, Tang JC, Meng Q, Dong F, Yang ZY (2017) Functional characterizations of beta-glucosidases involved in aroma compound formation in tea (Camellia sinensis). Food Res Int 96:206–214. https://doi.org/10.1016/j.foodres.2017.03.049

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The work was mainly supported by the Natural Science Foundation of Jiangsu Province (BK20160600) and the Young Science Foundation Project of the National Natural Science Foundation of China (31601371).

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Authors and Affiliations

  1. Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing, 210014, China

    Yongheng Yang, Menglan Hou, Ting Zhang, Yuming Sun, Yongxia Zhang, Suzhen Huang, Xiaoyang Xu & Haiyan Yuan

  2. The Jiangsu Provincial Platform for Conservation and Utilization of Agricultural Germplasm, Nanjing, 210014, China

    Yongheng Yang, Menglan Hou, Ting Zhang, Yuming Sun, Yongxia Zhang, Suzhen Huang, Xiaoyang Xu & Haiyan Yuan

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  1. Yongheng Yang
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  2. Menglan Hou
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  4. Yuming Sun
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  5. Yongxia Zhang
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  6. Suzhen Huang
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Contributions

YY designed the experiments. YY and MH carried out the sequence analysis and agrobacterium-mediated transformation on instantaneous expression. YY drafted the manuscript. MH carried out the HPLC analysis. XX carried out cDNA cloning and vectors constructing. TZ and YS participated and advised in manuscript development. YZ carried out sequence analysis. HY and SH advised in manuscript correction. All authors read and approved the final submission.

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Correspondence to Xiaoyang Xu or Haiyan Yuan.

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Yang, Y., Hou, M., Zhang, T. et al. A beta-glucosidase gene from Stevia rebaudiana may be involved in the steviol glycosides catabolic pathway. Mol Biol Rep 47, 3577–3584 (2020). https://doi.org/10.1007/s11033-020-05450-2

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  • DOI: https://doi.org/10.1007/s11033-020-05450-2

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