RESEARCH PAPER Plant Signaling & Behavior 10:12, e1078064; December 2015; © 2015 Taylor & Francis Group, LLC

RNAi-mediated gene silencing of WsSGTL1 in W.somnifera affects growth and glycosylation pattern Syed Saema1,2, Laiq ur Rahman3, Abhishek Niranjan1, Iffat Zareen Ahmad2, and Pratibha Misra1,* 1

Council of Scientific and Industrial Research - National Botanical Research Institute; Lucknow; Uttar Pradesh, India; 2Department of Bioscience; Integral University; Lucknow, Uttar Pradesh, India; 3Council of Scientific and Industrial Research - Central Institute of Medicinal and Aromatic Plants; Lucknow, Uttar Pradesh, India

Keywords: Agrobacterium tumefaciens, Glycosylation, RNAi, Sterols, Transgenic, WsSGTL1 Abbreviations: WT, Wild type; WsSGTL1, sterol glucosyltransferase gene of W.somnifera (clone1); RNAi, RNA interference; SGs, sterol glucosides; HPLC, High Performance Liquid Chromatography

Sterol glycosyltransferases (SGTs) belong to family 1 of glycosyltransferases (GTs) and are enzymes responsible for synthesis of sterol–glucosides (SGs) in many organisms. WsSGTL1 is a SGT of Withania somnifera that has been found associated with plasma membranes. However its biological function in W.somnifera is largely unknown. In the present study, we have demonstrated through RNAi silencing of WsSGTL1 gene that it performs glycosylation of withanolides and sterols resulting in glycowithanolides and glycosylated sterols respectively, and affects the growth and development of transgenic W.somnifera. For this, RNAi construct (pFGC1008-WsSGTL1) was made and genetic transformation was done by Agrobacterium tumefaciens. HPLC analysis depicts the reduction of withanoside V (the glycowithanolide of W.somnifera) and a large increase of withanolides (majorly withaferin A) content. Also, a significant decrease in level of glycosylated sterols has been observed. Hence, the obtained data provides an insight into the biological function of WsSGTL1 gene in W.somnifera.

Introduction Withania somnifera, also known as ‘Ashwagandha’, ‘Indian ginseng’ and ‘winter cherry’, is a perennial medicinal plant of traditional Ayurvedic and Unani system of medicine and found in many countries for immense therapeutic properties of its different parts.1,2. Several investigations have illustrated the pharmacological importance of its withanolides and glycowithanolides.3-9 More than 80 compounds including alkaloids and steroids have been identified by metabolic profiling from this plant.2,10-12 Pharmacological properties of W. somnifera include anti-hyperglycemic, immunomodulatory, neuropharmacological, musculotropic, hepatoprotective, cardioprotective, chemoprotective, radiosensitizing activities along with anti-aging, macrophage-activating, morphine tolerance and dependence-inhibiting, diuretic, hypocholesterolemic, rejuvenating, aphrodisiac, hemopoetic effects.2-4,13-17 Withanolides are a group of naturally occurring steroids based on ergostane nucleus and characterized by a lactone-containing side chain.18 Involvement of steroid nucleus, side chain and additional ring formation are known for their structural diversity. The withanosides (saponins) are mainly comprised of withanolides with one or more glucose units attached to C-3 or C-27 positions.19,20 Withanolide biogenesis and accumulation is limited to specific genera of Solanaceae family, among

them Withania shows maximum production of withanolide in more than 200 diversified forms, with or without functional groups.21-23 The biosynthetic pathway of withanolides, their function in W. somnifera and metabolic step(s) leading to their glyco-transformations are unknown. However, it has been demonstrated that precursor molecules governing withanolide biosynthesis could be isoprenoids. Isoprenoids produced during isoprenogenesis, is governed by 2 independent pathways; the classical cytosolic mevalonate (MVA) pathway and plastid localized non-mevalonate pathway, also called deoxyxylulose pathway (DOXP) or methyl erythreitol pathway (MEP), which ultimately leads to biosynthesis of 24-methylene cholesterol. 5,21-25 The study showed that glycosylation of these withanolides to withanosides and sitoindosides was catalyzed by GTs.25 Glycosylation of sterols by sterol glycosyltransferase (SGTs) genes performs crucial role in regulating cellular homeostasis, lipid metabolism, enhanced water solubility, stress tolerance and in development events. 26-28 The growing cell wall is dynamically modified by enzymes that change the structure of pectins and hemicelluloses, thereby altering their interactions with each other and with cellulose. Growth cessation is correlated with reduced expression of genes that promote wall loosening and changes in matrix polysaccharides that lead to a less extensible cell wall.29 SGs are the primers for cellulose synthesis in cotton fibers, and

*Correspondence to: Pratibha Misra; Email: pratibhafl[email protected] Submitted: 07/10/2015; Revised: 07/21/2015; Accepted: 07/22/2015 http://dx.doi.org/10.1080/15592324.2015.1078064

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SGTs are thought to be involved in SG synthesis. 30 GhSGT2 of cotton similar to WsSGT may have important functions in cellulose biosynthesis.31 Recent study on SGT gene of Withania somnifera (WsSGTL1) in Arabidopsis shows that it can enhance salt tolerance, heat tolerance and cold accumulation ability in transgenic Arabidopsis plants.32 To investigate the role of WsSGT in W.somnifera, silencing of WsSGTL1 gene in W.somnifera has been done in present study. Since, RNAi based on hairpin RNA (hpRNA) strategy has been reported as more efficient for gene silencing.33 that is why, we preferred to go with RNAi for the reduction of WsSGTL1 expression in W.somnifera. For this, a partial cDNA fragment of WsSGTL1 encoding gene was isolated. The isolated fragment of WsSGTL1 was employed to design a RNAi construct (pFGC1008-WsSGTL1). The construct was used for

transformation of W.somnifera through Agrobacterium tumefaciens. The regenerated WsSGTL1-silenced plantlets were analyzed for WsSGTL1 expression. Withanolide, withanoside V and glycosylated sterol contents were examined through HPLC analysis to understand the potential role of WsSGTL1 in W.somnifera.

Results Phylogenetic analysis of WsSGTL1 and integration of WsSGTL1 gene in pFGC1008 vector Phylogenetic analysis of WsSGTL1 was performed based on the alignment of their full-length amino acid sequences with closely related sterol glucosyltransferases from plants. The Phylogram of WsSGTL1 was generated using MEGA5.0 software with the Neighbor–Joining algorithm. The phylogram shows WsSGTL1 falls within the same clade constituting the SGT’s from Solanum lycopersicum and Nicotiana sylvestris. (Fig.1A). The integration of WsSGTL1 gene inside pFGC1008 vector was successful in sense and antisense direction (Fig. 1B). The construction of pFGC1008WsSGTL1 vector has been confirmed by double digestion with Asc1 and Spe1 enzyme (Fig. 1C). Transformation and regeneration Cloned construct was mobilized into GV3101 strain of A. tumefaciens through electroporation. Agrobacterium tumefaciens mediated genetic transformation was carried out for the introduction of RNAi cassette of WsSGTL1 gene into W.somnifera as reported earlier.34 (Fig. 2A-C). Minimum inhibitory concentration of hygromycin was optimized for plant selection, found that 8mgl-1 or above hygromycin was lethal for regeneration. Therefore, the selection of transformed callus and shoots was made on 7 mgl-1 hygromycin. Antibiotic resistant transformants were further confirmed through PCR analysis.

Figure 1. Phylogenetic analysis of Withania somnifera sterol glucosyltransferase (DQ356887.1, ACCESSION: ABC96116) was done by alignment of their full-length coding sequences in the context of closely related SGT proteins. The sequences were downloaded from NCBI database based on BLASTx results. The Phylogram was generated using MEGA5.0 software with the maximum likelihood algorithm. Scale bar indicates 0.05 amino acid substitutions per site (A). Schematic diagram of T-DNA region (B). Double digestion with Asc1 and Spe1 to confirm positive clone of WsSGTL1 fragment in pFGC-1008; M:lHindIII (C).

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Gene expression analysis The integration of T-DNA region of ihpRNAi construct in W.somnifera was conducted through PCR, semi-quantitative

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RT-PCR and qRT-PCR analysis using gene specific primers. PCR analysis of the total DNA extracted from the transgenic lines confirmed the presence of HPT11 gene (Fig. 3A) and both sense and antisense WsSGTL1 gene in 3 lines (L1, L2 and L3) as shown in (Fig. 3B). These shoots were selected for further studies. Semi-quantitative PCR and qRT- PCR was carried out to assess the expression of WsSGTL1 gene in transgenic lines (Fig. 3C and D). It was observed that both vector transformed and control WT plants behaved similarly as the relative expression of WsSGTL1 gene was similar in both, whereas it was significantly reduced in all the 3 respective transgenic lines. A maximum reduction of 90% was observed as compared to WT and vector transformed plants (Fig 3D). Increase in withanolide content To check the effect of WsSGTL1 silencing in the develFigure 2. Agrobacterium mediated transformation of W.somnifera (pFGC-WsSGTL1 gene). Leaf explant (A). oped transgenic lines, withanolide Shoot induction on selection medium (B&C). Necrosed shoot (D). contents were measured in young shoots of transformed as well as WT and vector transformed plants through HPLC analysis. Increase in withaferin A content of Effect of silencing on growth transformed lines L1, L2 and L3 was 1.75, 1.8 and fold2-, respecSignificant consequences of silencing of WsSGTL1 gene has tively, to that of the WT and vector transformed plants. While been observed in W.somnifera. The transgenic obtained were the increase in withanolide A content of transformed lines 1.5, unable to grow further and got necrosed (Fig. 2D). 2.5 and fold2- respectively to that of WT plants. Amount of withanone was also increased as 1.6, 1.8 and 1.fold5- in putative transgenic lines than WT lines as indicated in (Fig. 4A). However, a decrease in withanoside V content was observed in respecDiscussion tive transgenic as 3.8, 4.3 and 4.fold4- respectively than WT (Fig. 4B), Fig. S1 Biological effects of glycosylation in plant cells are particularly interesting in the case of phytohormones, the key regulators of Synthesis of glycosylated sterols in transgenic plants plant growth and development.35 Recently, overexpression of The effect of silencing of WsSGTL1 gene of W.somnifera on WsSGTL1 gene of W.somnifera in N. tabacum.36 and A.thaliglycosylation of sterol content was analyzed for b sitosterol, stig- ana.32 has revealed that it affects glycosylation and better plant masterol and campesterol before and after acid hydrolysis from growth than WT plants. However, the biological functions of leaves of young shoot of the transgenic, WT and vector trans- WsSGTL1 in W.somnifera remain largely unknown . In the presformed plants. Differences in the 2 values of sterols were taken ent study, using the available information and known sequence into account as glycosylated sterols. The level of glycosylated for WsSGTL1 from W.somnifera we successfully cloned, a partial campesterol (0.9–1.2 mg/g DW), glycosylated b-sitosterol cDNA fragment for WsSGTL1 from W.somnifera to silence the (0.41–3.6 mg/g DW) and glycosylated stigmasterol (1.0–1.8 mg/ WsSGTL1 gene and studied their expression and its effect on g DW) was decreased in transgenic lines as compared to WT and withanolide, glycowithanolide viz. withanoside V and glycosyvector transformed plants as 26.27 mg/g DW, 52.222 mg/g DW lated campesterol, glycosylated stigmasterol and glycosylated b and 26.81 mg/g DW, respectively (Fig. 5A-C), Fig. S2 sitosterol content in the transgenics.

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Figure 3. Molecular characterization of W.somnifera plants transformed with RNAi construct. Genomic DNA PCR for the detection of HPTII gene (L to R); M-100 bp ladder, L1 to L6 transgenics, WT-wild type (A). Genomic DNA PCR amplification indicating the integration of sense and antisense WsSGTL1 fragment (B). Semi quantitative -PCR analysis indicating the decrease in expression of WsSGTL1 gene (C). Relative Expression of WsSGTL1 gene by qRT- PCR of WT; C as control: transformed with pFGC1008 vector alone and transgenic lines (L1,L2,L3) (D).

Figure 4. Withanolides in leaf extract of WT,Wildtype; C, Control vector transformed transgenic and transgenic lines (L1, L2 and L3). Withanolide contents (A). Withanoside V (B). Results are mean § SE of 3 independent experiments. Asterisks indicate that mean values are significantly different between WT and transgenic plants (*, P< 0.05; **, P < 0.01; ***, P < 0.001).

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An isolated partial WsSGTL1 gene specific fragment of 652 bp was used in making RNAi construct (pFGC1008-WsSGTL1). The developed construct was used for transformation of W.somnifera leaf explants following the protocol of Pandey et al.34 The transgenic were observed with suppressed WsSGTL1 gene expression (maximum of 90% reduction) than WT and vector transformed plants. Based on transcript level of WsSGTL1 silenced plants, L1 line showing maximum decrease in relative expression of WsSGTL1 gene was observed with increase in withanolide content thereafter L2 and L3 line. HPLC analysis of transgenics indicated that withaferin A content increases (maximum fold2-) and withanoside V content decreases (maximum to 4.fold4-). In W. somnifera, withanosides are the glycosylated forms of steroidal lactones which are synthesized through the action of GTs.12,25,37,38 Moreover WsSGTL1 gene is specific for 3b- hydroxy position has a catalytic specificity to glycosylate withanolide and sterols.39 Hence the conversion of withanolide into glycosylated product was observed significantly less as silencing of the gene in transgenics might have brought about the ceasing of glycosylation reaction, suggesting the regulatory role of WsSGTL1 gene in glycosylation. As glycosylation of several metabolites related to withanolide synthesis revealed the involvement of the WsSGTs in the biosynthesis of therapeutically important glycosylated withanolides.39,40 WsSGTL1 gene may glycosylate sterols has been further documented by the experiments conducted in this study through estimation of glycosylated sterols by HPLC analysis, where decrease in glycosylated sterols has been observed than Wild type plants. This indicated that the partial cDNA sequence used in the present RNAi strategy for silencing the gene was found effective in regulating the glycosylted products.

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sterol and other sterol glucoside which is required for cellulose biosynthesis as Peng et al.30 reported that SGs reportedly have primary functions in cellulose biosynthesis in cotton fibers. Li et al. 31 suggested that GhSGT2 may have important functions in cellulose biosynthesis in cotton fibers and also demonstrate that activity of GhSGT2 is similar to SGTL1 as it share same conserved domain. The present study provides an insight that WsSGTL1 gene is a regulatory gene for glycowithanolide biosynthesis and glycosylation of sterols in addition to growth and development of plant. Resolving the glycosylation steps and eventually the entire withanolide biosynthetic pathway at the molecular level should be a main target for future WsSGT research.

Materials and Methods Phylogenetic analysis Phylogenetic analysis of Withania somnifera sterol glucosyltransFigure 5. Quantitative estimation of glycosylation of sterols in WT, Wildtype; C, Control vector transformed ferase (DQ356887.1, ACCESS transgenic and transgenic lines (L1, L2 and L3). Free sterols were measured by breaking bond between sterION: ABC96116) was done by ols and sugar moiety after acid hydrolysis of extract and compared with free sterols before hydrolysis (black alignment of their full-length codbar represents sterols before hydrolysis and gray bar represents sterols after hydrolysis) Difference between ing sequences in the context of free sterols before and after hydrolysis resulted in glycosylated amount.. Data are expressed as mean § SE of 3 independent experiments. Asterisks indicate that mean values are significantly different between wildclosely related SGT proteins. The type and transgenic plants (*, P< 0.05; **, P < 0.01; ***, P