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. 2015 Sep 11;10(12):e1078064. doi: 10.1080/15592324.2015.1078064

RNAi-mediated gene silencing of WsSGTL1 in W.somnifera affects growth and glycosylation pattern

Syed Saema 1,2, Laiq ur Rahman 3, Abhishek Niranjan 1, Iffat Zareen Ahmad 2, Pratibha Misra 1,*
PMCID: PMC4854344  PMID: 26357855

Abstract

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.

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

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 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 pFGC1008-WsSGTL1 vector has been confirmed by double digestion with Asc1 and Spe1 enzyme (Fig. 1C).

Figure 1.

Figure 1.

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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:λHindIII (C).

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 2.

Figure 2.

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Agrobacterium mediated transformation of W.somnifera (pFGC-WsSGTL1 gene). Leaf explant (A). Shoot induction on selection medium (B&C). Necrosed shoot (D).

Gene expression analysis

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

Figure 3.

Figure 3.

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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).

Increase in withanolide content

To check the effect of WsSGTL1 silencing in the developed transgenic lines, withanolide 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 transformed lines L1, L2 and L3 was 1.75, 1.8 and fold2-, respectively, to that of the WT and vector transformed plants. While the increase in withanolide A content of transformed lines 1.5, 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 respective transgenic as 3.8, 4.3 and 4.fold4- respectively than WT (Fig. 4B), Fig. S1

Figure 4.

Figure 4.

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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).

Synthesis of glycosylated sterols in transgenic plants

The effect of silencing of WsSGTL1 gene of W.somnifera on glycosylation of sterol content was analyzed for β sitosterol, stigmasterol and campesterol before and after acid hydrolysis from leaves of young shoot of the transgenic, WT and vector transformed plants. Differences in the 2 values of sterols were taken into account as glycosylated sterols. The level of glycosylated campesterol (0.9–1.2 µg/g DW), glycosylated β-sitosterol (0.41–3.6 µg/g DW) and glycosylated stigmasterol (1.0–1.8 µg/g DW) was decreased in transgenic lines as compared to WT and vector transformed plants as 26.27 µg/g DW, 52.222 µg/g DW and 26.81 µg/g DW, respectively (Fig. 5A-C), Fig. S2

Figure 5.

Figure 5.

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Quantitative estimation of glycosylation of sterols in WT, Wildtype; C, Control vector transformed transgenic and transgenic lines (L1, L2 and L3). Free sterols were measured by breaking bond between sterols and sugar moiety after acid hydrolysis of extract and compared with free sterols before hydrolysis (black bar represents sterols before hydrolysis and gray bar represents sterols after hydrolysis) Difference between 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 wild-type and transgenic plants (*, P< 0.05; **, P < 0.01; ***, P <0.001).

Effect of silencing on growth

Significant consequences of silencing of WsSGTL1 gene has been observed in W.somnifera. The transgenic obtained were unable to grow further and got necrosed (Fig. 2D).

Discussion

Biological effects of glycosylation in plant cells are particularly interesting in the case of phytohormones, the key regulators of plant growth and development.35 Recently, overexpression of WsSGTL1 gene of W.somnifera in N. tabacum.36 and A.thaliana.32 has revealed that it affects glycosylation and better plant growth than WT plants. However, the biological functions of WsSGTL1 in W.somnifera remain largely unknown . In the present study, using the available information and known sequence for WsSGTL1 from W.somnifera we successfully cloned, a partial cDNA fragment for WsSGTL1 from W.somnifera to silence the WsSGTL1 gene and studied their expression and its effect on withanolide, glycowithanolide viz. withanoside V and glycosylated campesterol, glycosylated stigmasterol and glycosylated β sitosterol content in the transgenics.

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 3β- 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.

Our data clearly indicates that reduction in activity of WsSGTL1 gene causes the growth arrest as the regenerated transgenic showed poor growth than that of WT plants and were unable to survive. This supported the assumption that the overexpression of this gene in A.thalliana promotes better growth and development of transgenic seedlings.32 Itkin et al.41 has reported the retardation in growth of tomato due to downregulation of glycoalkaloid metabolism (GAME1) gene. Moreover, M. Truncatula growth is severely affected by alterations in glycosylation of the saponin hederagenin.42 In oat, it was seen that incomplete glycosylation of the triterpene glycoside (i.e., saponin) avenacin resulted in degeneration of the epidermis and altered root hair development.43 And, Wang et al.44 showed the overexpression of a glycosyltransferase-deficient mutant or targeted disruption of LH3 by siRNA in cells resulted in abnormal cell morphology followed by cell death. Growth ceasation of transgenic W.somnifera can be correlated with low accumulation of glycosylated sito 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 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.

Plant materials

The material of Withania somnifera chemotype NMITLI-101, used in the present study was collected from the germplasm being maintained at CSIR-National Botanical Research Institute, Lucknow, India.

Strains and plasmids

The cloning host was Escherichia coli DH5α and the strain of A.tumefaciens used for transformation of W. somnifera was GV3101. The binary vector pFGC1008 was used for cloning the target sequence to generate the binary vector pFGC1008-WsSGTL1 containing the gene in sense and antisense direction which was isolated from W. Somnifera and empty vector without gene was taken as control vector.

Media and culture conditions

E. coli DH5α was grown in Luria Broth (LB) medium at 37°C. E. coli transformants were selected on Luria Agar (LA) containing chloramphenicol (30 mg/l). A. tumefaciens strain was grown in YEB (Yeast Extract and Beef) medium containing rifampicin (25 mg/l) at 28°C. The A. tumefaciens transformants harbouring the plasmid WsSGTL1 gene were selected on YEB + gentamycin (40 mg/l) + rifampicin (25 mg/l) + chloramphenicol (30 mg/l).

RNAi construct preparation

Total RNA was extracted from leaves of W. somnifera, using the Spectrum Plant Total RNA kit (SIGMA) and the first strand cDNA was synthesized using the Revert AID First Strand cDNA synthesis kit (Fermentas), according to the manufacturer's instructions. The partial cDNA fragment of the WsSGTL1 (DQ356887.1) was amplified with primers WsSGTL1(F1) and WsSGTL1(R1) containing restriction sites. The PCR parameters were: initial denaturation at 94°C for 2 min, followed by 35 cycles of 94°C for 30 sec, 62°C for 30 sec, 72°C for 1 min 30 sec and final extension at 72°C for 5 min. 652 bp cDNA fragment of WsSGTL1 gene was selected for designing a small interfering RNA (SiRNA) construct in pFGC1008 vector. The amplified partial cDNA fragment containing restriction sites was used in designing RNAi construct for silencing WsSGTL1 gene. It was amplified using primers containing restriction sites for Asc1 at 5′ and Swa1 at 3′ end to clone in sense direction and using primer containing restriction sites for Spe1 at 5′ and BamH1 at 3′ end to clone in antisense direction into pFGC 1008 vector. The resulting pFGC1008-WsSGTL1 vector was confirmed by digesting with Asc1 and Spe 1restriction enzyme. A 360 bp GUS-intron was harboured between sense and antisense fragment making it an intron-containing hairpin RNA (ihpRNA) construct. The pFGC1008-WsSGTL1 construct was containing CaMV 35S as promoter and octopine synthase (OCS) as terminator. The construct also contained hygromycin phosphotransferase (HPT11) gene as plant selectable marker.

Agrobacterium tumefaciens culture and transformation

Prepared constructs of A. tumefaciens to be used for silencing in W.somnifera explants were grown into 5 ml Nutrient Broth (NB) medium (primary culture) containing gentamycin (40 mg/l), rifampicin (25 mg/l) and chloramphenicol (30 mg/l). A.tumefaciens mediated transformation of W.somnifera was carried out as per earlier reported protocol.34

Selection and generation of transformants

Fresh and green callus was excised from the explants, while the brown portion was removed. The excised callus was cultured on the medium as reported earlier.34 Selection of transformants was done on 7 mg/ml hygromycin. The pH of all the media was adjusted to 5.8 before adding agar and autoclaved at 121ºC for 20 min. All the media were solidified with 0.8% (w v−1) agar. Hygromycin, cefotaxime and acetosyringone were added after filter sterilization through a 0.45-μm PVDF membrane (Millex HV; Merck-Millipore, Billerica, USA) to the autoclaved media. Cultures were incubated at 25 ± 2ºC under 3 klux light through fluorescent tubes for 16-h light: 8-h dark photo-cycle at a light intensity of 50 to 60 µmol m−2 s−1. All the phytohormones and antibiotics used for the preparation of media were from M/s Sigma Aldrich, USA. Regenerated shoots containing 2 or more leaves (5 weeks old) were taken for further studies.

PCR confirmation of T-DNA integration in W.somnifera

To check the integration of transformed T-DNA in W.somnifera, PCR was conducted using various integration fragment specific primers with leaf genomic DNA of independant T0 transgenic lines and wild type normal (WT) W.somnifera, isolated by DNeasy Plant Minikit, Qiagen. Initially six independent T0 transgenic lines were confirmed for presence of HPT11 gene and later 3 independent transgenic lines were taken up for further analysis. For detection of HPT11 gene, PCR amplification was conducted with the HPTI1F and HPT11R gene primers (Table S1) (annealing temperature 60°C; product size 300 bp). To confirm the integration of sense fragment, specific primer sets SiSF1 and SiSR1 (Table S1, annealing temperature 55°C), SiAF1 and SiAR1 (Table S1) to confirm the integration of antisense fragment (annealing temperature 55°C). PCR reactions were set up with the following thermal profile: 94ºC for 5 min, followed by 35 cycles at 94ºC for 30 sec, 60ºC (for HPT11 gene) or 55ºC (for sense and anti sense insertion of WsSGTL1 gene) for 15 sec, 72ºC for 45 sec and 72ºC for 5 min as final extension step. The amplified products were analyzed by 1% agrose gel electrophoresis.

Semi-quantitative and qRT-PCR analysis

RNA was isolated from the leaves of T0 transformants lines and WT W.somnifera by using Spectrum Plant Total RNA kit (Sigma-Aldrich, St. Louis, MO, US) which was subsequently treated with RNase-free DNase (Thermo Fisher Scientific, Vilnius, Lithuania). RNA was subjected to reverse transcription to generate first-strand cDNA using oligo dT primers (Thermo Fisher Scientific).

To analyze the expression pattern of T0 transformants and WT, were subjected to semi- quantitative PCR analysis using gene-specific primers WsSGTL1F2 and WsSGTL1R2 (Table S1). To equalize, ubiquitin gene primers of Nicotiana tabacum were used (Table S1) and analysis was carried out using PCR Master-mix (Thermo Fisher Scientific) with the following cycle conditions: 94°C for 2 min, 26 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 45s, followed by a final 5min extension at 72°C. Three independent experiments using 3 biological replications were performed.

Relative expression of WsSGTL1 transcript was analyzed in transgenic, control vector transformed transgenic and WT W.somnifera plants by quantitative real time PCR (qRT-PCR) using WsSGTL1F3, WsSGTL1R3 and ubiquitin F2, ubiquitin R2 primers of N.tabacum (Table S1). qRT-PCR was performed in 20 μl for set of selected genes using Power SYBR Green PCR Master Mix (ABI, USA). After obtaining ct value for each reaction, the fold change was calculated by using Delta-Delta ct method. Three independent experiments were conducted using 3 biological replications.

Extraction of Secondary Plant Metabolites and Hplc Analysis

Withanolides and Withanoside V

The leaves of young shoots of T0 transgenic lines, control vector transformed transgenic and WT W. somnifera (500 mg, 5 weeks old) were crushed and dried into liquid nitrogen. The sample was extracted overnight in 10 ml of methanol water (25:75 v/v) at room temperature on orbital shaker and filtered. The filtrate was collected and the residue was extracted twice at 4 h intervals with the same amount of extracts. The filtrates were pooled and extracted with n-hexane (3 × 30 ml). The n-hexane fraction was discarded and methanol-water fraction was further extracted with chloroform (3 × 30 mL). The chloroform fractions were pooled and was concentrated up to dryness (39). The residue was dissolved into HPLC grade methanol (2 ml) and filtered through 0.45 μM nylon filter (Millipore). The solution was further diluted to 10 folds and injected into HPLC.

Separation for qualitative and quantitative analysis of the withanolides were performed by HPLC-PDA with a Shimadzu (Japan) LC-10A system comprising an LC-10AT dual-pump system, a SPD-10A PDA detector (operated at 227 nm), and Rheodyne injection valve with 20-μL sample loop. Compounds were separated on a (Merck) RP-C18 column (4.6 mm × 250 mm, 5-μm pore size) protected by a guard column containing the same packing. The mobile phase prepared from 0.1% (v/v) acetic acid in HPLC-grade water (component A) and 0.1% (v/v) acetic acid in HPLC-grade methanol (component B) in gradient mode. Before use, the components were filtered through 0.45-μm nylon filters and de-aerated in an ultrasonic bath. The gradient was from 40–60% B in 0–30 min, hold up for 2 min, then from 60% to 75% B in 32 min to 45 min, 75% to 95% B in 45min to 54 min and 100% in 54min to 55 min. The flow rate was 0.6 ml min-1. Data were integrated by Shimadzu class VP series software and results were obtained by comparison with the standards. Results are mean values from 3 replicate analyses of the same sample. All samples and solutions were filtered through 0.45-μm nylon filters before analysis by HPLC. Standard compounds, viz., withaferin A, withanolide A, withanone and withanoside V (Sigma) were accurately weighed (10 mg separately) and dissolved in 10 ml methanol to prepare stock solution of 1 mg/ml. These stock solutions were subsequently diluted to prepare solutions with concentrations in the range of 0.5 mg/ml to 50 mg/ml. These working standard solutions were used for quantification of samples.

Sterols

Dried leaf samples (100 mg) were homogenized in 1 ml of methanol (80% v/v) followed by incubation at 40°C for 2 h. Glycosylated and nonglycosylated sterols were analyzed quantitatively from acid-hydrolysed or non-hydrolysed extracts of silenced WsSGTL1 and WT plants, respectively. For acid hydrolysis, leaf samples were homogenized in 1 ml of ethyl acetate in 1% HCl and incubated at 90°C for 2 h. Acid hydrolysis of extract was required to obtain free sterols by breaking of bonds between sterols and sugar moiety. Homogenous extracts obtained were dissolved in high-performance liquid chromatography (HPLC)-grade methanol and drained through 0.2-μm filter (Millipore, India) before conducting HPLC analysis. Free sterols were quantified before and after hydrolysis, and the difference between hydrolysed and nonhydrolysed sterols would be due to the glycosylation action of WsSGTL1.

For the estimation of sterols the above HPLC system was used. Sterols (β sitosterol, stigmasterol and campesterol) were separated by isocratic solution of acetonitrile and water (95:5, v/v) at flow rate of 2 ml/min at 34°C for 40 minutes run time as These 3 are most common phytosterols.45 The scanning of sterols was performed at 202 nm. Standard compounds, viz., β sitosterol, stigmasterol and campesterol (Sigma) were accurately weighed (10 mg separately) and dissolved in 10 ml methanol to prepare stock solution of 1 mg/ml. These stock solutions were subsequently diluted to prepare solutions with concentrations in the range of 0.5–50 mg/ml. These working standard solutions were used for quantification in samples.

Parameters like specificity, linearity, peak purity, precision and accuracy, limits of quantification and detection and robustness were followed to quantify all compounds by HPLC. All data were integrated by Shimadzu class VP series software and results were obtained by comparison with standards. The results were the mean values from 3 independent experiments with 3 biological replicates.

Data collection and statistical analysis

Growth and development were recorded by visual observations. Each experiment was repeated 3 times using 3 biological replicates. The values of data are mean ± standard error of 3 replicates. All statistical analyses were performed by using ANOVA-INDOSTAT software.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed

Supplemental Material

Supplemental data for this article can be accessed on the publishers website.publisher's website

Supplemental Figures and Table

Funding

PM is thankful to the Department of Biotechnology, New Delhi, for the financial support provided through the project No. GAP 231225. Department of Biotechnology, New Delhi, provided financial support through the project No. GAP 231225. The authors 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.

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