Steviol glycoside biosynthesis pathway gene expression profiling of transformed and non-transformed plant leaf tissues of Stevia rebaudiana (Bertoni)

Priya Singh Amol S. Phule Heena Tabassum Minal Wani   

Open Access   

Published:  Dec 12, 2025

DOI: 10.7324/JABB.2026.250346
Abstract

Stevia rebaudiana Bertoni, a member of the Asteraceae family, is recognized for its sweetened leaves, which are remarkably sweeter than sucrose by 200–300 times. This astounding property is because of steviol glycosides (SGs), a class of diterpenoid secondary metabolites that primarily consist of stevioside and rebaudioside A. These compounds are formed through a specific SG biosynthetic pathway that contains several key genes. In the present work, gene expression profiling of 15 core genes of SG biosynthetic pathway, along with metabolite analysis, was conducted in three groups of S. rebaudiana plants: In vitro regenerated non-transformed plantlets (NP), in vitro regenerated transformed plantlets (TP) via hairy root cultures using Rhizobium rhizogenes mediated transformation, and control plants (CP). Quantitative real?time polymerase chain reaction results showed that in NP and TP there was upregulation of 13 genes. Both NP and TP showed downregulated SrDXR and SrCDPS in comparison to CP, whereas SrUGT74G1 had higher expression in NP than TP. High-performance liquid chromatography chromatographic studies on SGs showed that stevioside content followed the order TP > NP > CP. These findings demonstrate that transformation enhances SG biosynthesis and support the use of genetically modified S. rebaudiana lines for increased natural sweetener production. Further studies are warranted to elucidate regulatory mechanisms and optimize metabolic engineering approaches.


Keyword:     Stevia rebaudiana Secondary metabolites Steviol glycosides pathway Gene expression Quantitative real?time polymerase chain reaction High-performance liquid chromatography


Citation:

Singh P, Phule AS, Tabassum H, Wani M. Steviol glycoside biosynthesis pathway gene expression profiling of transformed and non-transformed plant leaf tissues of Stevia rebaudiana (Bertoni). J Appl Biol Biotech 2025. Article in Press. http://doi.org/10.7324/JABB.2026.250346

Copyright: Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license.

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Reference

1. Sharma S, Gupta S, Kumari D, Kothari SL, Jain R, Kachhwaha S. Exploring plant tissue culture and steviol glycosides production in Stevia rebaudiana (Bert.) Bertoni: A review. Agriculture. 2023;13(2):475. https://doi.org/10.3390/agriculture13020475

2. Hajihashemi S, Geuns JM, Ehsanpour AA. Gene transcription of steviol glycoside biosynthesis in Stevia rebaudiana Bertoni under polyethylene glycol, paclobutrazol and gibberellic acid treatments in vitro. Acta Physiol Plantarum. 2013;35:2009-14. https://doi.org/10.1007/s11738-013-1226-9

3. Brandle JE, Telmer PG. Steviol glycoside biosynthesis. Phytochemistry. 2007;68(14):1855-63. https://doi.org/10.1016/j.phytochem.2007.02.010

4. Thakur K, Ashrita, Sood A, Kumar P, Kumar D, Warghat AR. Steviol glycoside accumulation and expression profiling of biosynthetic pathway genes in elicited in vitro cultures of Stevia rebaudiana. In Vitro Cell Dev Biol Plant. 2021;57:214-24. https://doi.org/10.1007/s11627-020-10151-3

5. Zhou X, Gong M, Lv X, Liu Y, Li J, Du G, et al. Metabolic engineering for the synthesis of steviol glycosides: Current status and future prospects. Appl Microbiol Biotechnol. 2021;105(13):5367-81. https://doi.org/10.1007/s00253-021-11419-3

6. Ceunen S, Werbrouck S, Geuns JM. Stimulation of steviol glycoside accumulation in Stevia rebaudiana by red LED light. J Plant Physiol. 2012;169(7):749-52. https://doi.org/10.1016/j.jplph.2012.01.006

7. Eslami-Firouzabadi A, Karimi M, Abbasi-Surki A, Shafeinia A, Derikvand-Moghadam F. Optimising the rate and stages of application of nitrogen fertiliser for stevia under greenhouse conditions. South African Journal of Plant and Soil 2023;40:58-63.

8. Olas B. Stevia rebaudiana Bertoni and its secondary metabolites: Their effects on cardiovascular risk factors. Nutrition. 2022;99:111655.

9. Abdel-Aal RA, Abdel-Rahman MS, Al Bayoumi S, Ali LA. Effect of stevia aqueous extract on the antidiabetic activity of saxagliptin in diabetic rats. J Ethnopharmacol. 2021;265:113188. https://doi.org/10.1016/j.jep.2020.113188

10. Bugliani M, Tavarini S, Grano F, Tondi S, Lacerenza S, Giusti L, et al. Protective effects of Stevia rebaudiana extracts on beta cells in lipotoxic conditions. Acta Diabetol. 2022;59:113-126. https://doi.org/10.1007/s00592-021-01793-9

11. Peteliuk V, Rybchuk L, Bayliak M, Storey KB, Lushchak O. Natural sweetener Stevia rebaudiana: Functionalities, health benefits and potential risks. EXCLI J. 2021;20:1412. https://doi.org/10.17179/excli2021-4211

12. Abdullah S, Mohamad Fauzi NY, Khalid AK, Osman M. Effect of gamma rays on seed germination, survival rate and morphology of Stevia rebaudiana hybrid. Malays J Fundam Appl Sci. 2021;17(5):543-9. https://doi.org/10.11113/mjfas.v17n5.2157

13. Álvarez-Robles MJ, López-Orenes A, Ferrer MA, Calderón AA. Methanol elicits the accumulation of bioactive steviol glycosides and phenolics in Stevia rebaudiana shoot cultures. Ind Crops Prod. 2016;87:273-9. https://doi.org/10.1016/j.indcrop.2016.04.054

14. Kahrizi D, Ghaheri M, Yari Z, Yari K, Bahraminejad S. Investigation of different concentrations of MS media effects on gene expression and steviol glycosides accumulation in Stevia rebaudiana Bertoni. Cell Mol Biol. 2018;64(2):23-7. https://doi.org/10.14715/cmb/2018.64.2.11

15. Singh P, Labade D, Chote M, Deshmukh P, Panchal B, Deshpande J, et al. Efficient regeneration of Stevia rebaudiana Bertoni transformants through hairy root culture technique. J Appl Bot Food Qual. 2025;98:22-8. https://doi.org/10.5073/JABFQ.2025.098.003

16. Pan H, Xiao L, Tang K, Xia H, Li Y, Jia H, et al. Screening UDP-glycosyltransferases for effectively transforming stevia glycosides: Enzymatic synthesis of glucosylated derivatives of rubusoside. J Agric Food Chem. 2022;70(48):15178-88. https://doi.org/10.1021/acs.jafc.2c06185

17. Yu J, Tao Y, Pan H, Lin L, Sun J, Ma R, et al. Mutation of Stevia glycosyltransferase UGT76G1 for efficient biotransformation of rebaudioside E into rebaudioside M. J Funct Foods. 2022;92:105033. https://doi.org/10.1016/j.jff.2022.105033

18. Ghaheri M, Kahrizi D, Bahrami G, Mohammadi-Motlagh HR. Study of gene expression and steviol glycosides accumulation in Stevia rebaudiana Bertoni under various mannitol concentrations. Mol Biol Rep. 2019;46(1):7-16. https://doi.org/10.1007/s11033-018-4250-4

19. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods. 2001;25(4):402-8. https://doi.org/10.1006/meth.2001.1262

20. Herath V, Gayral M, Adhikari N, Miller R, Verchot J. Genome-wide identification and characterization of Solanum tuberosum BiP genes reveal the role of the promoter architecture in BiP gene diversity. Sci Rep. 2020;10(1):11327. https://doi.org/10.1038/s41598-020-68407-2

21. Phule AS, Barbadikar KM, Maganti SM, Seguttuvel P, Subrahmanyam D, Babu MP, et al. RNA-seq reveals the involvement of key genes for aerobic adaptation in rice. Sci Rep. 2019;9(1):5235. https://doi.org/10.1038/s41598-019-41703-2

22. Ye J, Jin CF, Li N, Liu MH, Fei ZX, Dong LZ, et al. Selection of suitable reference genes for qRT-PCR normalisation under different experimental conditions in Eucommia ulmoides Oliv. Sci Rep. 2018;8(1):15043. https://doi.org/10.1038/s41598-018-33342-w

23. Sarmiento-López LG, López-Meyer M, Sepúlveda-Jiménez G, Cárdenas L, Rodríguez-Monroy M. Photosynthetic performance and stevioside concentration are improved by the arbuscular mycorrhizal symbiosis in Stevia rebaudiana under different phosphate concentrations. PeerJ. 2020;8:e10173. https://doi.org/10.7717/peerj.10173

24. Libik-Konieczny M, Michalec-Warzecha ?, Dziurka M, Zastawny O, Konieczny R, Rozp?dek P, et al. Steviol glycosides profile in Stevia rebaudiana Bertoni hairy roots cultured under oxidative stress-inducing conditions. Appl Microbiol Biotechnol. 2020;104:5929-41. https://doi.org/10.1007/s00253-020-10661-5

25. Sanchéz-Cordova ÁD, Capataz-Tafur J, Barrera-Figueroa BE, López-Torres A, Sanchez-Ocampo PM, García-López E, et al. Rhizobium rhizogenes-mediated transformation enhances steviol glycosides production and growth in Stevia rebaudiana plantlets. Sugar Tech. 2019;21(3):398-406. https://doi.org/10.1007/s12355-018-0681-4

26. Zheng J, Zhuang Y, Mao HZ, Jang IC. Overexpression of SrDXS1 and SrKAH enhances steviol glycosides content in transgenic Stevia plants. BMC Plant Biol. 2019;19:1-6. https://doi.org/10.1186/s12870-018-1600-2

27. Nasrullah N, Ahmad J, Saifi M, Shah IG, Nissar U, Quadri SN, et al. Enhancement of diterpenoid steviol glycosides by co-overexpressing SrKO and SrUGT76G1 genes in Stevia rebaudiana Bertoni. PLoS One. 2023;18(2):e0260085. https://doi.org/10.1371/journal.pone.0260085

28. Gachon CM, Langlois-Meurinne M, Saindrenan P. Plant secondary metabolism glycosyltransferases: The emerging functional analysis. Trends Plant Sci. 2005;10(11):542-9.

29. Tiwari P, Sangwan RS, Sangwan NS. Plant secondary metabolism linked glycosyltransferases: An update on expanding knowledge and scopes. Biotechnol Adv. 2016;34(5):714-39. https://doi.org/10.1016/j.biotechadv.2016.03.006

30. Bednarek PT, Or?owska R. Plant tissue culture environment as a switch-key of (EPI) genetic changes. Plant Cell Tissue Organ Cult. 2020;140(2):245-57. https://doi.org/10.1007/s11240-019-01724-1

31. Kajla M, Roy A, Singh IK, Singh A. Regulation of the regulators: Transcription factors controlling biosynthesis of plant secondary metabolites during biotic stresses and their regulation by miRNAs. Front Plant Sci. 2023;14:1126567.

32. Kim MJ, Zheng J, Liao MH, Jang IC. Overexpression of Sr UGT-76G1 in Stevia alters major steviol glycosides composition towards improved quality. Plant Biotechnol J. 2019;17(6):1037-47. https://doi.org/10.1111/pbi.13035

33. Abdelsalam NR, Botros WA, Khaled AE, Ghonema MA, Hussein SG, Ali HM, et al. Comparison of uridine diphosphate-glycosyltransferase UGT76G1 genes from some varieties of Stevia rebaudiana Bertoni. Sci Rep. 2019;9(1):8559. https://doi.org/10.1038/s41598-019-44989-4

34. Singh S, Murmu S, Das AB, Haider ZA, Banerjee M. Establishment of root-to-root culture and evaluation of phytochemicals in Rhizobium rhizogenes transformed roots of Stevia rebaudiana. J Pharmacogn Phytochem. 2017;6(6S):49-54.

35. Bayraktar M, Naziri E, Karabey F, Akgun IH, Bedir E, Röck-Okuyucu B, et al. Enhancement of stevioside production by using biotechnological approach in in vitro culture of Stevia rebaudiana. Int J Secondary Metab. 2018;5(4):362-74. https://doi.org/10.21448/ijsm.496724

36. Fazili MA, Bashir I, Ahmad M, Yaqoob U, Geelani SN. In vitro strategies for the enhancement of secondary metabolite production in plants: A review. Bull Natl Res Centre. 2022;46(1):35. https://doi.org/10.1186/s42269-022-00717-z

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