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Ectopic overexpression of WsSGTL1, a sterol glucosyltransferase gene in Withania somnifera, promotes growth, enhances glycowithanolide and provides tolerance to abiotic and biotic stresses

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Overexpression of sterol glycosyltransferase (SGTL1) gene of Withania somnifera showing its involvement in glycosylation of withanolide that leads to enhanced growth and tolerance to biotic and abiotic stresses.

Abstract

Withania somnifera is widely used in Ayurvedic medicines for over 3000 years due to its therapeutic properties. It contains a variety of glycosylated steroids called withanosides that possess neuroregenerative, adaptogenic, anticonvulsant, immunomodulatory and antioxidant activities. The WsSGTL1 gene specific for 3β-hydroxy position has a catalytic specificity to glycosylate withanolide and sterols. Glycosylation not only stabilizes the products but also alters their physiological activities and governs intracellular distribution. To understand the functional significance and potential of WsSGTL1 gene, transgenics of W. somnifera were generated using Agrobacterium tumefaciens-mediated transformation. Stable integration and overexpression of WsSGTL1 gene were confirmed by Southern blot analysis followed by quantitative real-time PCR. The WsGTL1 transgenic plants displayed number of alterations at phenotypic and metabolic level in comparison to wild-type plants which include: (1) early and enhanced growth with leaf expansion and increase in number of stomata; (2) increased production of glycowithanolide (majorly withanoside V) and campesterol, stigmasterol and sitosterol in glycosylated forms with reduced accumulation of withanolides (withaferin A, withanolide A and withanone); (3) tolerance towards biotic stress (100 % mortality of Spodoptera litura), improved survival capacity under abiotic stress (cold stress) and; (4) enhanced recovery capacity after cold stress, as indicated by better photosynthesis performance, chlorophyll, anthocyanin content and better quenching regulation of PSI and PSII. Our data demonstrate overexpression of WsSGTL1 gene which is responsible for increase in glycosylated withanolide and sterols, and confers better growth and tolerance to both biotic and abiotic stresses.

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Acknowledgments

We are grateful to the Director, CSIR-National Botanical Research Institute, Lucknow, for the facilities provided. SS is thankful to CSIR for the award of Senior Research Fellowship. PM is thankful to the Department of Biotechnology, New Delhi, for the financial support provided through the project No. GAP 231225.

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  1. Tissue Culture and Plant Transformation Laboratory, CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow, 226001, India

    Syed Saema, Ruchi Singh, Abhishek Niranjan & Pratibha Misra

  2. Department of Bioscience, Integral University, Lucknow, India

    Syed Saema & Iffat Zareen Ahmad

  3. Department of Biotechnology, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow, India

    Laiq ur Rahman

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  1. Syed Saema
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Correspondence to Pratibha Misra.

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Author contribution statement

Conceived and designed the experiments: SS PM LR, Corrected the manuscript: PM SS, Performed the experiments: SS RS, Analyzed the data: SS LR AN IZA. Wrote the paper: SS.

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Communicated by H. S. Judelson.

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299_2015_1879_MOESM1_ESM.tif

WsSGTL1 gene expression analysis in T1 transgenics of W.somnifera. a PCR analysis for the detection of NPTII gene (L to R); WT-wild type, C-nontransgenic control, L1 to L7 transgenics, M-100 bp ladder. b RT-PCR analysis for the detection of WsSGTL1 expression; WT, L1, L3, L4, L6 & L7-transgenics. c. Relative expression of WsSGTL1 gene by real time PCR. d Southern blot analysis showing the stable integration of NPTII gene in T1 progeny of transgenic W. somnifera overexpressing WsSGTL1 gene; Lane 1-WT, Lane 2-L1, Lane 3-L3 and Lane 4-L6. (TIFF 1698 kb)

299_2015_1879_MOESM2_ESM.tif

Morphological characterization of T1 transgenics of W.somnifera. a 8-week-old seedlings growing in the pot. b 4-month-old plants in the pot. c Morphological difference in the leaf size (L to R) of the transgenic lines and WT. (TIFF 4098 kb)

299_2015_1879_MOESM3_ESM.tif

Chlorophyll Fluorescence Imaging for maximum photochemical quantum yield (Fv/Fm) for WT and WsSGTL1 transgenic lines (L1, L2 and L3) of W. somnifera. a-d Before cold stress. e–h After 1 h of cold treatment (0 °C). i-l After recovery of 10 days. The false color code depicted on the right side of the images ranges from 0.000 (black) to 1.000 (pink) (TIFF 4548 kb)

299_2015_1879_MOESM4_ESM.tif

Light response curves for energy fluxes of PSII and PSI for WT and L1 before cold stress, immediate after 1 h of cold treatment (0 °C) and on 10th day of recovery. a-c Photochemical quantum yield for PSII Y(II). d-f Quantum yield of non-light-induced non-photochemical fluorescence quenching for PSII, Y(NO). g-i Quantum yield of light-induced non-photochemical fluorescence quenching for PSII, Y(NPQ). j-l ETR of PSII (ETRII) (M–O) Photochemical quantum yield for PSI Y(I). p-r Quantum yield of non-photochemical energy dissipation in PSI due to donor side limitation, Y(ND). s-u Quantum yield of non-photochemical energy dissipation in PSI due to acceptor side limitation Y(NA). v-x ETR of PSI (ETRI).Values are average ± SEs of three to five replicates (TIFF 1271 kb)

299_2015_1879_MOESM5_ESM.tif

Light response curves for energy fluxes of PSII and PSI for WT and L3 before cold stress, immediate after 1 h of cold treatment (0 °C) and on 10th day of recovery. a-c Photochemical quantum yield for PSII Y(II). d-f Quantum yield of non-light-induced non-photochemical fluorescence quenching for PSII, Y(NO). g-i Quantum yield of light-induced non-photochemical fluorescence quenching for PSII, Y(NPQ). j-l ETR of PSII (ETRII) (M–O) Photochemical quantum yield for PSI Y(I). p-r Quantum yield of non-photochemical energy dissipation in PSI due to donor side limitation, Y(ND). s-u Quantum yield of non-photochemical energy dissipation in PSI due to acceptor side limitation Y(NA). v-x ETR of PSI (ETRI).Values are average ± SEs of three to five replicates. (TIFF 1309 kb)

List of primers used in study. (DOC 37 kb)

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Saema, S., Rahman, L.u., Singh, R. et al. Ectopic overexpression of WsSGTL1, a sterol glucosyltransferase gene in Withania somnifera, promotes growth, enhances glycowithanolide and provides tolerance to abiotic and biotic stresses. Plant Cell Rep 35, 195–211 (2016). https://doi.org/10.1007/s00299-015-1879-5

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