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WsSGTL1 gene from Withania somnifera, modulates glycosylation profile, antioxidant system and confers biotic and salt stress tolerance in transgenic tobacco

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Abstract

Glycosylation of sterols, catalysed by sterol glycosyltransferases (SGTs), improves the sterol solubility, chemical stability and compartmentalization, and helps plants to adapt to environmental changes. The SGTs in medicinal plants are of particular interest for their role in the biosynthesis of pharmacologically active substances. WsSGTL1, a SGT isolated from Withania somnifera, was expressed and functionally characterized in transgenic tobacco plants. Transgenic WsSGTL1-Nt lines showed an adaptive mechanism through demonstrating late germination, stunted growth, yellowish-green leaves and enhanced antioxidant system. The reduced chlorophyll content and chlorophyll fluorescence with decreased photosynthetic parameters were observed in WsSGTL1-Nt plants. These changes could be due to the enhanced glycosylation by WsSGTL1, as no modulation in chlorophyll biogenesis-related genes was observed in transgenic lines as compared to wildtype (WT) plants. Enhanced accumulation of main sterols like, campesterol, stigmasterol and sitosterol in glycosylated form was observed in WsSGTL1-Nt plants. Apart from these, other secondary metabolites related to plant’s antioxidant system along with activities of antioxidant enzymes (SOD, CAT; two to fourfold) were enhanced in WsSGTL1-Nt as compared to WT. WsSGTL1-Nt plants showed significant resistance towards Spodoptera litura (biotic stress) with up to 27 % reduced larval weight as well as salt stress (abiotic stress) with improved survival capacity of leaf discs. The present study demonstrates that higher glycosylation of sterols and enhanced antioxidant system caused by expression of WsSGTL1 gene confers specific functions in plants to adapt under different environmental challenges.

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Abbreviations

NTPH:

Nicotiana tabacum cv. Petit Havana

UGT:

Uridine diphosphate glycosyltransferase

WsSGTL1:

Sterol glucosyltransferase gene (Clone1) of Withania somnifera

WsSGTL1-Nt :

WsSGTL1 expressing transgenic plants of Nicotiana tabacum

WT:

Wild type

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Acknowledgments

The Director, Council of Scientific and Industrial Research, National Botanical Research Institute, is gratefully acknowledged by authors for the provided best facilities and moral support. PM is thankful to the Department of Biotechnology (Project No. GAP 231225), Govt. of India, for providing financial support to carry out the research work and CSIR-NMITLI project for identification of WsSGTL1 gene. VP is thankful to CSIR for the award of Senior Research Fellowship.

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  1. Council of Scientific and Industrial Research - National Botanical Research Institute, Rana Pratap Marg, Lucknow, 226 001, India

    Vibha Pandey, Abhishek Niranjan, K. Chandrashekhar, Manoj K. Mishra, Prabodh K. Trivedi & Pratibha Misra

  2. Department of Botany, Faculty of Science, Banaras Hindu University, Varanasi, 221005, India

    Vibha Pandey, Neelam Atri & Manoj K. Mishra

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  1. Vibha Pandey
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  2. Abhishek Niranjan
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Correspondence to Pratibha Misra.

Electronic supplementary material

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425_2014_2046_MOESM1_ESM.tif

Supplementary Fig. S1 Stomatal conductance, gs (a) and chlorophyll quantification (b) from leaves of 3-month-old WsSGTL1-Nt (L1, L2, L3) and WT plants. Data are expressed as mean ± SD of three independent experiments. Different letters indicate significantly different values in a particular tissue (DMRT, P ≤ 0.05) and ANOVA significant at P ≤ 0.01 (TIFF 124 kb)

425_2014_2046_MOESM2_ESM.tif

Supplementary Fig. S2 Expression analysis of some nuclear encoded genes related to chlorophyll biogenesis in WsSGTL1-Nt and WT. No significant difference was observed in the expression of these genes, which suggested that stress adaptation is not associated with chlorophyll biogenesis while in WT were relatively lower, i.e. 46.7, 3.9 μg g−1 DW and 11 mg g−1 DW, respectively. Three independent experiments were performed (TIFF 253 kb)

425_2014_2046_MOESM3_ESM.tif

Supplementary Fig. S3 Average seed (100 seeds) weight (a) was calculated for seeds of WsSGTL1-Nt and WT, showing no significant difference in yield. Also, no significant difference was observed during cotyledon emergence (b) of WsSGTL1-Nt and WT seeds. Data are expressed as mean ± SD of three independent experiments. Different letters indicate significantly different values in a particular tissue (DMRT, P ≤ 0.05) and ANOVA significant at P ≤ 0.01 (TIFF 74 kb)

425_2014_2046_MOESM4_ESM.tif

Supplementary Fig. S4 HPLC profiling of seed extract of WsSGTL1-Nt (L1, L2 and L3 lines) and wild type plants illustrating the more glycosylated sterols in WsSGTL1-Nt. Standards of 1: campesterol; 2: stigmasterol; 3: sitosterol (a) before hydrolysis (b) after hydrolysis (c) (TIFF 393 kb)

425_2014_2046_MOESM5_ESM.tif

Supplementary Fig. S5 HPLC profiling of leaf extract of WsSGTL1-Nt (L1, L2 and L3 lines) and WT showing enhanced accumulation of phenolics in transgenic lines as compared to wild type. a Standards (1: Gallic Acid; 2: Protocetechric Acid; 3:Chlorogenic Acid; 4: Coffeic Acid; 5: Rutin; 6: Ferulic Acid; 7: Quercetin; 8: Kaempherol). b–e L1, L2 and L3 lines of WsSGTL1-Nt (b, c, d) and wild type plants (e) (TIFF 226 kb)

425_2014_2046_MOESM6_ESM.tif

SupplementaryFig. S6 Quantitative estimation of some phenolics which may serve as substrates (resulted in glycosylated products). HPLC quantification reveals no significant change in accumulation of gallic acid (GA) metabolites in WsSGTL1-Nt and WT, while amount of other secondary metabolites like, kempherol (K), ferulic acid (FA) and caffeic acid (CA) decreased significantly in correspondence to their enhanced glycosylated form in WsSGTL1-Nt as compared to WT plants (TIFF 108 kb)

425_2014_2046_MOESM7_ESM.tif

Supplementary Fig. S7 Quantification of chlorophyll contents; chlorophyll-A, chlorophyll-B, carotenoid in the leaf-disc of WsSGTL1-Nt and WT were carried out during 0 to 6 day of 200 mM NaCl treatment, showing enhanced salt tolerance capacity of transgenic lines as lesser chlorosis in WsSGTL1-Nt. Data are expressed as mean ± SD of three independent experiments. Different letters indicate significantly different values in a particular tissue (DMRT, P ≤ 0.05) and ANOVA significant at P ≤ 0.01 (TIFF 143 kb)

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Pandey, V., Niranjan, A., Atri, N. et al. WsSGTL1 gene from Withania somnifera, modulates glycosylation profile, antioxidant system and confers biotic and salt stress tolerance in transgenic tobacco. Planta 239, 1217–1231 (2014). https://doi.org/10.1007/s00425-014-2046-x

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