Methyl jasmonate and β-cyclodextrin shake hands to boost withaferin A production from the hairy root culture of Withania somnifera

doi:10.21203/rs.3.rs-2211216/v1

Download PDF

Research Article

Methyl jasmonate and β-cyclodextrin shake hands to boost withaferin A production from the hairy root culture of Withania somnifera

https://doi.org/10.21203/rs.3.rs-2211216/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

For the first time, an effort was made to study potential effects of leaf and stem explants of three genotypes of Withania somnifera (L.) Dunal (i.e., G1, G2, and G3) for hairy root induction via applying four strains of Agrobacterium rhizogenes (i.e., LBA 9402, A4, and ATTC 15834) and A. tumefaciens (i.e., C58C1). Furthermore, to improve withaferin A production, the superior treatment was subjected to elicitation with β-cyclodextrin (β-CD) and methyl jasmonate (MeJA) (alone or in combination) after 48 h exposure time. Among 24 treatments resulted from multiplying three main factors, the superiority of “G3/ATCC/LEAF” treatment was verified in terms of “induction rate” and “number of induced roots” alongside “growth curve”, and utilized subsequently for elicitation assay. The highest amounts of withaferin A were quantified for both hairy root sample groups of “0.5 mM β-CD + 100 µM MeJA” (9.57 mg/g DW) and “5.0 mM β-CD + 100 µM MeJA” (17.45 mg/g DW), with the fold change values of 6.84-fold and 12.46-fold compared to control, respectively. Similarly, either individual or simultaneous applications of both elicitors, apart from some fluctuations, could overall boost transcript magnitudes of the four selected genes of HMGR, SQS, SMT-1, and SDS/CYP710A involved in withanolides biosynthetic pathway as compared with control. The results, altogether, demonstrated the superiority of leaf explant and Agropine-type strain of ATCC 15834 for hairy root induction, followed by synergistic effect of exogenous application of both MeJA and β-CD on the in vitro production of withaferin A, and possibly other types of withanolides in the hairy root culture of W. somnifera.

Hairy root

Withania somnifera

β-cyclodextrin

Elicitation

Withaferin A

Gene expression

Since time immemorial, broad spectrum of medicinal plants have been utilized by human being as a reliable repository to cope with a range of various diseases (Petrovska 2012), which is mainly attributed to the incredible therapeutics features thereof, resulted from the availability of diverse naturally occurring products, also called “plant secondary metabolites” (PSMs). Among various plant species utilized by human being for centuries, some members of the genus Withania spp. have had a long application history of more than 3,000 years either in Ayurvedic or indigenous medicine, as reviewed comprehensively by (Khan et al. 2021; Maurya 2010; Mirjalili et al. 2009b).

The genus Withania spp. (family Solanaceae) encompasses ~ 61 known species across the globe (List 2013), of which only W. somnifera and W. coagulans are economically important and widely cultivated (Khan et al. 2021; Mirjalili et al. 2009b), mainly owing to possessing a broad spectrum of secondary metabolites including Alcoholic, Alkaloidal compounds and most importantly withanolides. The withanolides, as a class of naturally occurring C28-steroidal lactone triterpenoids built on an ergostane skeleton functionalized at C-22 and C-26, have been structurally studied widely, as approximately 360 new naturally occurring withanolides have been extracted and characterized from August 1996 to March 2010 (Chen et al. 2011). Among such a structurally diverse naturally occurring constituents, withanolide A, withanolide B, withanon, and predominantly withaferin A seem to be more interesting in different research areas with the emphasis on medicinal applications (Berghe et al. 2012; Dutta et al. 2019; Maurya 2010; Palliyaguru et al. 2016; Sultana et al. 2021).

On the other hand, resembling many PSMs, withanolides accumulation is in small quantities in planta, and in vivo production thereof is inevitably accompanied by destruction of the plant from its habitat, could be undoubtedly led to a significant decline in its natural gene pool, biodiversity, and in the worst cases extinction may take place. Alternatively, plant cell, tissue, and organ culture including hairy roots have been recently introduced as a sustainable, controllable, and eco-friendly solution and subsequent industrial production of PSMs of high value (Dhar et al. 2015; Ochoa-Villarreal et al. 2016). Of different biotechnological approaches, hairy root culture has been suggested as a robust and sustainable resource for the production of various PSMs including withanolides (Dehdashti et al. 2017; Dehdashti et al. 2020; Mirjalili et al. 2009a; Mirjalili et al. 2011; Mishra et al. 2013; Murthy et al. 2008; Namdeo and Ingawale 2021; Pandey et al. 2017; Pandey et al. 2016; Praveen and Murthy 2012; Praveen and Murthy 2013; Saxena et al. 2017; Sivanandhan et al. 2015; Sivanandhan et al. 2013; Sivanandhan et al. 2012b; Thilip et al. 2019; Thilip et al. 2015), mainly due to the following advantages: i) rapid growth in hormone-free medium; ii) ease of storage; iii) the ability to synthesize a wide range of chemical compounds (Namdeo and Ingawale 2021; Sevón and Oksman-Caldentey 2002).

Furthermore, to ameliorate the production of PSMs from the hairy root cultures, many of biotic/abiotic elicitors have been suggested and utilized widely, as reviewed by (Halder et al. 2019). Similarly, the production of withanolides in the hairy roots of W. somnifera has been augmented through applying various elicitors including salicylic acid (SA), methyl jasmonate (MeJA), cell homogenate of the fungus Piriformospora indica (CHP), and seaweeds, which are usually coincident with some alterations in transcript levels of the genes involved in biosynthetic pathway of withanolides, (Pandey et al. 2016; Saxena et al. 2017; Sivanandhan et al. 2015; Sivanandhan et al. 2013; Thilip et al. 2019). Besides, among various elicitors utilized in vitro, Cyclodextrins, alone and/or in combination with other elicitors have been proved as a useful vehicle to improve production of PSMs, as reviewed comprehensively by (Cardillo et al. 2021).

Despite some efforts towards “in vitro’ production of withanolides, particularly using hairy root, “simultaneous” consequences of applying different factors such as “genotypes”, “tissues”, and “Agrobacterium strains” have not been studied. Hence, an endeavor was made here to explore the potential interaction effects of these three major factors not only in terms of “induction rate” and “number of induced roots”, but also for production of the most important class of withanolides namely “Withaferin A”. Meanwhile, no information is publicly available about assessing the potential synergistic/antagonistic consequences of simultaneous application of methyl jasmonate (MeJA) and Cyclodextrins, as the two contemporary elicitors utilized widely. Hence, both individual and co-application of MeJA and β-Cyclodextrin (β-CD) were exogenously applied to scrutinize the hypothesis whether simultaneous application of both aforesaid elicitors are able to ameliorate Withaferin A accumulation in vitro?

Plant material

The seeds of three individual genotypes of W. somnifera (nominated as G1, G2, and G3) were kindly provided by the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Germany. The seeds, upon receiving, were initially surface sterilized by 70% ethanol for 15 seconds, washed subsequently thrice with distilled water, sterilized again with sodium hypochlorite (5%) for 7 minutes and washed again with distilled water thrice. To achieve sterilized plant materials, the resultant sterilized seeds were germinated in a MS medium (Murashige 2009) supplemented with 30 g sucrose and 5.7 grams agar per liter (pH = 5.5–5.8). All the media, in the following, were kept in darkness for 72 hours, transferred then into a growth chamber with 16 hours of light and 8 hours of darkness at temperatures of 22–26°C. The explants from leaf and root tissues of the resultant 4-week seedlings were used for “induction rate” and “number of induced roots”, through different strains of Agrobacterium rhizogenesis and A. tumefaciens.

Transformation of W. somnifera via A. rhizogenes and A. tumefaciens

To induce hairy root, three different strains of A. rhizogenes including LBA 9402 (Prepared from plasmid bank College of Agriculture, Tarbiat Modarres University), A4, and ATTC 15834 (both prepared by National Institute of Genetic Engineering and Biotechnology, NIGEB, Iran) followed by an A. tumefaciens strain of C58C1 (Donated by Houshang Alizadeh University of Tehran) were employed. Each strain was grown in darkness in the phase-log phase (OD₆₀₀ = 0.5–0.8) at 28°C on a shaker at 180 rpm in LB liquid medium containing 50 mg/L of rifampins. Then, the W. somnifera explants (i.e., stems and leaves) were initially prepared from 4-week seedlings, wounded by scalpel, and individually co-cultured with suspension of each strain. Inoculated explants were transferred into MS medium containing L-arginine and incubated for 3 days at ambient temperature of 25–26°C. To abolish additional bacteria, in the following, all the inoculated explants were washed thrice with sterilized distilled water containing 500 mg/L cefotaxime, transferred then into MS medium containing 3% sucrose, 0.75% agar, and 500 mg/L cefotaxime. All the cultures were subsequently sub-cultured at intervals of 12–14 days once. In some cases, after 6–8 days hairy roots were appeared from the wounded places. The roots of non-inoculated explants were also used as control.

Identification of W. somnifera hairy roots by PCR

Each bacterial strain followed by the resultant hairy roots were confirmed via PCR based on amplifying rol C gene, using the forward (5՜-CTCCTGACATCAAACTCGTC-3՜) and reverse (5՜-TGCTTCGAGTTATGGGTACA-3՜) primers. The total genomic DNA of the resultant hairy roots followed by the roots of intact plant materials (as negative control) was extracted from a CTAB-based protocol of (Khan et al. 2007), while the isolation of plasmid DNA from A. rhizogenes (as positive control) was taken place according to the procedure explained by (Bulgakov et al. 1998). PCR reaction was taken place using Bio-Rad Thermocycler, as each mixture contained 400 ng genomic DNA, 10 µL PCR master mix, and 25 pmol of each primer, followed by adding appropriate amounts of distilled water up to 20 µL as final volume. Thermal amplification conditions for PCR reaction included denaturing at 94 ºC for 5 min, a 32-reapeated cycle of 94 ºC for 20 s, 58 ºC for 30 s, and 72 ºC for 30 min, followed by 5 min at 72 ºC as the final extension. The amplicons were electrophoresed on a 1.5% agarose gel system and subsequently visualized based on a gel red-based system under UV conditions and photographed lastly using a Bio-Rad Gel Doc.

Elicitor Treatment With Methyl Jasmonate And βcyclodextrin

Subsequent to the selection of the best hairy root cell line (i.e., “G3/ATCC/LEAF”; see results section for details), elicitation was taken place. In this sense, root tip tissue (5 cm) from the hairy root line of G3/ATCC/LEAF was transferred into 100-mL Erlenmeyer flasks containing 25 mL of ½ MS medium in a rotary shaker at 25°C in darkness for 5 weeks. After 5 weeks, hairy roots were moved to fresh ½ MS media before treatment with each elicitor. The elicitors included MeJA (100 µM; Sigma Chemical Co., Mo, USA), β-CD (0.5 and 5 mM), and a combination of both elicitors (i.e., “100 µM MeJA + 0.5 mM of β-CD” and “100 µM MeJA + 5 mM β-CD”). Each elicitor solution was individually filter-sterilized and subsequently added to the corresponding flasks containing 35-days old hairy root line of G3/ATCC/LEAF and 50 mL of hormone-free ½ MS liquid medium with 5 gr L^− 1 sucrose. Lastly, all the treated hairy root-containing flasks followed by non-treated one (as control) were allowed to incubate for 48 h in darkness at 25°C with three biological replicates.

Rna Extraction And Cdna Synthesis

Sampling was individually taken place at Zero and 48 h upon treatment with the aforesaid elicitors, carefully collected, and each one was divided into two fragments: the first one was about 100–150 mg and utilized for RNA extraction, while the rest was lyophilized for HPLC analysis. The LiCl-based method (Liao et al. 2004) was applied to extract total RNA from ~ 100 mg of all the fine powder of both treated and non-treated hairy roots. Putative genomic DNA contamination was removed through treatment with DNase I (RNase- Free DNase Set, Fermentase), and both RNA quality and quantity were confirmed by NanoDrop® ND-1000 spectrophotometer as well as agarose gel electrophoresis. First strand cDNA synthesis was taken place based on Takara cDNA Synthesis Kit instruction, by means of Oligo-dT primers. The cDNA samples were lastly diluted into a 500 ng/µL working concentration and stored at -20°C for short-term and − 80°C for long-term, until further analyses.

Primer Design And Quantitative Real Time Pcr (Qpcr)

Gene expression analysis was applied to quantitatively measure the expression patterns of four selected genes (i.e., HMGR: 3-hydroxy-3-methylglutaryl coenzyme A reductase, SMT-1: Sadenosyl-methionine-sterol-C-methyltransferase, SQS: Squalene synthase, and SDS/CYP710A: Sterol-22-desaturase) involved in the withanolide biosynthetic pathway (Table 1), as utilized previously (Saxena et al. 2017). A real-time PCR system (Rotor-Gene Q) was employed to perform qRT-PCR assay, in a total reaction volume of 20 µL containing 8 µL EvaGreen Master Mix (containing EvaGreen Dye, Solis BioDyn, Germany), 1.5 µL of diluted cDNA, 0.25 µM of each primer in conjunction with adding PCR-grade water. The qPCR was carried out based on a thermal program of 15 min at 95°C, 40 cycles of 20 s at 96°C, 20 s at the specific annealing temperature for each primer, and 20 s at 72°C. According to (Saxena et al. 2017), the Cyclophyllin (CYP) gene was used as an internal reference (housekeeping) gene, and the expression amount of the control was set as the reference value. Differential expression of the studied genes were calculated through the ΔΔCt method, as for a given treatment, the fold change value comparing to the control was computed as 2^−ΔΔCt, where ΔΔCt = (Ct_{target gene} – Ct _{reference gene})_{the sample} – (Ct_{target gene} – Ct_{reference gene})_control, as described by (Livak and Schmittgen 2001). Notably, to prevent any unbiased results, for each gene of interest, the mean value of the amplifications (equivalent to PCR efficiency; automatically calculated using QIAGEN’s real-time PCR machine, Rotor-Gene Q) was applied instead of “2” in the aforesaid formula (Nasiri et al. 2016). All the PCR experiments were repeated thrice, and the quantitative data were recorded as the mean ± standard error (mean ± SE).

Table 1

List of primer sequences and related characteristics utilized in this study
Gene	Accession No.	Forward Primer	Reverse Primer	Amplicon size (bp)	Tm °C
HMGR	HQ293119.1	5′-GTCGTTCCCCTTCCCCTAAA-3′	5′-ATGAGGGAGAGAATAGCGGC-3′	187	60
SMT-1	GQ921847.1	5′-CAAATGACGCAGAGCAGAAA-3′	5′-AGGCAAGTACCATGAAACAGG-3′	167	60
SQS	GU181386.1	5′-AGACATGCGGAGAAGCAGAT-3′	5′-ATCACGAAGCTCAACAGGA-3′	121	60
SDS/CYP710A	KC008574.1	5′-ACATTGCCGGAGAAGAATTCC-3′	5′-TTCCTCTTGCCTCTCTTCCA-3′	152	60
CYP	GR923728.1	5′-ACGGTTCTCAGTTCTTCATCTG-3′	5′-ACAGCCTCAGCCTTCTTAATC-3′	112	60

Note: HMGR 3-hydroxy-3-methylglutaryl coenzyme A reductase, SMT-1 Sadenosyl-methionine-sterol-C-methyltransferase, SQS Squalene synthase, SDS/CYP710A Sterol-22-desaturase, and CYP Cyclophyllin.

Metabolites Extraction And Hplc Analysis

Extraction and quantification of withaferin A in the hairy root/leaf samples of W. somnifera was performed according to the earlier protocol of (Sivanandhan et al. 2012a). For each sample, ~ 200 mg of the fine powder of lyophilized hairy root/leaf samples were weighed and subjected subsequently to methanol-assisted extraction. The resultant extracts were evaporated to dryness, dissolved subsequently in 1.0 mL of methanol (HPLC grade), and filtered through nylon syringe filter (0.45 µm) for quantification of Withaferin A. The HPLC experiment was performed using High Performance Liquid Chromatography apparatus (Knauer AZURA® HPLC System) at Barij Essential Pharmaceutical Company, Kashan, Iran. The separation was occurred using a reverse-phase column C18 (25 cm) with methanol:water (65:35) as an eluent at a flow rate of 1.5 mL/min, alongside an injection volume of 20 µL and running time of 20 min. Detection of withaferin A was taken place via applying UV detector at the wavelength of 227 nm (Pandey et al. 2016). The standard of withaferin A was prepared from Sigma Company, and quantification thereof was calculated through a standard curve based on three serial dilutions of 50, 100, and 200 µg of the purified withaferin A dissolved in methanol.

Statistical analysis

The hairy root establishment was conducted in the factorial experiment based on a completely randomized design (CRD) with three independent factors of Strain (S), Explant (E), and Genotype (G) in three replications. Both “induction rate” and “number of induced roots” were employed as two individual dependent criteria, and one-way analysis of variance (ANOVA) was applied to statistically determine potential significant differences among the factors. Additional two separate ANOVA analyses were applied to compare the elicited and non-elicited hairy roots in terms of either Withaferin A production or the expression magnitudes of the four genes under study (defined as p < 0.05). All the statistical analyses were carried out by SPSS software, and the mean values were compared via Duncan’s multiple range test (DMRT) at 5% (p ≤ 0.05) probability level. Furthermore, to make discrimination among 24 diverse treatments as well as to determine the most powerful treatment(s) for elicitation assay, a hierarchical cluster analysis (HCA) combined with heatmap visualization was applied using ClustVis web-tool (Metsalu and Vilo 2015). Lastly, the probable correlations among the relative gene expression data and magnitudes of withaferin A were calculated via the ‘‘corrplot’’ function and subsequently plotted via the statistical computing Software Rstudio (www.rstudio.com) (Nasiri et al. 2016).

Establishment of hairy roots

For all the 24 treatments, the initiation of hairy roots was observed over 8–16 days upon inoculation with all four bacterial strains, depending on explants, bacterial strains, and genotypes under study. In general, little amounts of callus formation was initially formed and quickly turned into hairy root formation. Each treatment about establishing hairy roots exhibited various behavior, as detailed below.

According to the ANOVA results (Table 2), for both features of “induction rate” and “number of induced roots”, the interaction among three main factors (i.e., G*S*E interaction) was statistically significant (p < 0.0001, p < 0.05, respectively). Thus, the result of this part was only explained. Based on the mean comparison results for “induction rate”, all the 24 treatments were placed in 12 groups, of which both treatments of “G2/ATCC/LEAF” and “G3/ATCC/LEAF” with the same value of 86.67% were the superior, while G2/A4/LEAF treatment possessed the least amount of 10.00% (Fig. 2A). For the second criterion of “number of induced roots”, again 12 separated groups were acquired, “G3/ATCC/LEAF” followed by “G3/ATCC/STEM” were the first (8.27) and second (7.43) top treatments, while both “G2/A4/STEM” and “G2/A4/LEAF” treatments were together located in the lowest orders (1.23, and 1.07, respectively; Fig. 2B). Notably, to “simultaneously” make discrimination among 24 diverse treatments as well as to determine the most powerful treatment(s) for elicitation assay, a clustered heat map was applied. In this sense, all the 24 treatments were clustered into three major groups, of which only four treatments of G2/ATCC/LEAF, G2/ATCC/STEM, G3/ATCC/LEAF, and G3/ATCC/STEM were located in the first group, with the maximum levels of both traits of “induction rate”, and “number of induced roots” (Fig. 2C). As a result, all these four superior cell lines were nominated for estimation of the next feature of “growth curve” of hairy roots.

Table 2

The ANOVA results to study the potential impacts of explants, bacterial strains, and genotypes on “*the number of induced roots*” and “*induction rate* (%)” of *W. somnifera* hairy roots.
Source of variation	DF	Mean Square (number of induced roots)	Mean Square (induction rate)
G	2	16.58234**	704.1667**
S	3	56.05241**	6187.037**
E	1	0.178006^ns	88.88889^ns
*GS**	6	3.427887**	1080.093**
*GE**	2	1.214172**	184.7222**
*SE**	3	0.238554^ns	381.4815**
*GSE*	6	0.366943*	260.6482**
Error	48	0.12566	27.77778

Note: G, S, and E designate genotype, strain, and explant, respectively.

**: Significant at the 0.01 probability level; *: Significant at the 0.05 probability level; ns: Not significant.

By now, different strains of A. rhizogenes followed by various explants have been employed to assess their ability to induce hairy roots in W. somnifera (Namdeo and Ingawale 2021; Shasmita et al. 2018; Tripathi et al. 2018). For instance, among three Agrobacterium strains of A4, MTCC532, and ATCC 15834 applied to induce hairy roots in leaf and shoot tips of W. somnifera, upon applying four various protocols, the leaf tissue inoculated with ATCC 15834 was selected as the superior one in terms of hairy root induction frequency, agreeing with the current results (Dehdashti et al. 2017). Furthermore, the same authors indicated that the number of induced roots from shoot tips was a little more than leaf tissue (Dehdashti et al. 2017), while in our current study, leaf tissue gave better results in terms of the same feature. At the study of (Sivanandhan et al. 2013), transformation rate was as high as 90% in the leaf explants infected with R1000 strain, followed by generating of 28.2 hairy roots per explant upon 12 days of culture. Using three strains of R1000, MTCC 2364 and MTCC 532 to induce hairy root of leaf explants of W. somnifera, R1000 was the most powerful, and among the various conditions tested, the highest transformation rate (93.3%) was acquired for the same candidate in response to sonication (15 s) and heat treatment (41 ºC for 5 min) (Thilip et al. 2015). Considering the preference of opines, the A. rhizogenes strains are usually detached into five Agropine, Nopaline, Mannopine, Octopine, and Cucumopine groups, of which Agropine-type strains like R1000 and ATCC 15834 have been applied widely, actually owing to a possessing high efficacy for hairy root induction (Dehdashti et al. 2017; Namdeo and Ingawale 2021).

Identification Of Hairy Root By Pcr

To confirm the resultant transformed hairy roots obtained from both leaf and stem tissues of three genotpes of W. somnifera inoculated with all the three different strains of A. rhizogenes (i.e., ATCC15834, A4, and LBA9402) and one strain of A. tumefaciens (C58C1), PCR assay was conducted using rol C gene. In this context, subsequent to amplifying rol C gene, an expected size value of 629 bp was observed for all the transformed hairy roots, while no amplicon was observed in non-transformed in roots belonging to the in vitro sterilized plants, corroborating the successful generation of the resultant hairy roots (Fig. 3). It is argued that Rol genes paly fundamental functions in hairy root induction as well as production of diverse PSMs (Wojciechowska et al. 2020), of which rol C gene seems to possess an accessory role (Pal et al. 2013).

Growth Curve Of Hairy Root Lines

As mentioned above, the four treatments of G2/ATCC/LEAF, G2/ATCC/STEM, G3/ATCC/LEAF, and G3/ATCC/STEM were selected as the most relevant candidates among the others, and subsequently utilized for calculation of growth index (GI). During nine successive weeks, hairy root samples were checked to measure growth ratio and subsequent GI of hairy roots in terms of the fresh weight (FW). As could be seen in Fig. 3, all the samples nearly completed the corresponding logarithmic phase in the 6th week, reaching subsequently to a steady phase. After nine successive weeks, however, only G3/ATCC/LEAF was able to keep its steady phase, while the others failed, as each one experienced a decline. Therefore, only G3/ATCC/LEAF was lastly selected for elicitation process. Considering the GI quantities obtained at 9th week (Fig. 4), all the four clones were ascendingly ranked as G3/ATCC/LEAF, G3/ATCC/STEM, G2/ATCC/LEAF, and G2/ATCC/STEM with the values of 10.18 g FW, 5.4 g FW, 4.9 g FW, and 4.55 g FW, respectively.

Comparison of Withaferin A production at both “ in vivo ” and “elicitation-assisted in vitro” conditions

The withaferin A quantities either in hairy root lines or leaves of three genotypes were measured by HPLC. Based on ANOVA results, there was a significant difference among control (non-elicited hairy root samples), all the five hairy root sample groups treated with five different elicitations, and three genotypes of W. somnifera (only leaf tissue) in terms of withaferin A accumulation (Table 3; p < 0.0001). In this sense, the mean production of withaferin A ranged from 0.33 mg/g DW for G3 to 17.45 mg/g DW for “5.0 mM β-CD + 100 µM MeJA” treatment (Fig. 5). Locking more carefully, the simultaneous action of both MeJA and β-CD at two levels of β-CD (5.0 and 0.5 mM) resulted in the maximum accumulation of withaferin A (17.45 and 9.57 mg/g DW, respectively), as a significant difference was undertaken compared to the control as well as other treatments (p < 0.05). Notably, the treated hairy root clones with “5.0 mM β-CD + 100 µM MeJA” possessed statistically higher amounts than that of “0.5 mM β-CD + 100 µM MeJA” (p < 0.05). The three remaining treated sample groups of “100 µM MeJA”, “5.0 mM β-CD”, and “0.5 mM β-CD” could produce lower amounts of withaferin A (i.e., 3.31, 3.25, and 2.02 mg/g DW, respectively), which were not significantly different from control (1.40 mg/g DW), G1 (0.35 mg/g DW), G2 (0.57 mg/g DW), and G3 (0.33 mg/g DW) (Fig. 5).

Table 3

The ANOVA results to study potential differences of Withaferin A production among five elicited and control groups of hairy roots followed by the leaf tissue of three different genotypes of *W. somnifera* namely G1, G2, and G3.
Source of variation	DF	Mean Square (Withaferin A production)
Treatment	8	98.12191558**
Error	18	6.855444519
**: Significant at the 0.01 probability level

In the earlier works, similar “elicitor-assisted enhancements” have been recorded towards accumulation of various withanolides including withaferin A under in vitro conditions. Upon treatments of hairy roots (via inoculation of leaf explants of W. somnifera with A. rhizogenes strain LBA 9402) with three different MeJA concentrations of 10, 15, 20 µM over a time series of 2, 4, 6, 8 h, the highest amounts of withanolide accumulation was observed at 20 µM MeJA treatment for 4 h (Saxena et al. 2017). Comparing to the control, the production of withanolide A, withaferin A, withanoside IV, withanoside V were ameliorated up to 13.21 (5.6 times), 5.275 (3.9 times), 0.1929 (2.7 times) and 0.161 mg/g DW (3.2 times), respectively (Saxena et al. 2017). Elicitation with Piriformospora indica (CHP; concentrations of 1%, 3% and 5% over 24, 48 and 72 h) increased withanolide A, withaferin A, withanoside IV, and withanoside V amounts versus control, with the maximum levels of 6.37 (2.7 times), 3.28 (2.5 times), 0.171 (2.34 times) and 0.147 mg/g DW (2.9 times) (3% CHP; after 48 h) (Saxena et al. 2017). At the study of (Pandey et al. 2016), A. rhizogenes-mediated transformation of sterol glycosyltransferase–4 (Ws-Sgtl4) gene induce hairy roots from the leaf tissue of W. somnifera, and subsequently elicited with salicylic acid (SA; 1.0 mM) and MeJA (100 µM) over 0, 3, 6, 9, 12, 24, and 48 h exposure time. Based on the results, the maximum amounts of withanolide-A in both T2 and T3 hairy root clones were reached after 12 h treatments with MeJA (respectively, 23- and 20-fold than control) and 9 h elicitation with SA (respectively, 39- and 32-fold than control) (Pandey et al. 2016). Individual applications of SA (0.0, 50, 100, 150, and 200 µM) and MeJA (0.0, 5.0, 10.0, 15.0, and 20.0 µM) in the hairy roots derived from R1000 infected leaf explants of W. somnifera over a time series of 2, 4, 6, 8 h could lead to remarkable growth in withanolides accumulation rather than control (Sivanandhan et al. 2013). Upon elicitation with MeJA and SA, the maximum quantities of withanolide A (114.38 mg/g DW, 50-fold; 132.44 mg/g DW, 58-fold higher than control), withanone (69.89 mg/g DW, 38-fold; 84.35 mg/g DW, 46-fold), and withaferin A (57.46 mg/g DW, 34-fold; 70.72 mg/g DW, 42 times) were detected at 15 µM MeJ and 150 µM SA for 4 h after elicitation, respectively (Sivanandhan et al. 2013). Based on the results of (Thilip et al. 2015), without applying any elicitation, the ratios of both withaferin A and withanolide A were recorded as 6.17 mg/g DW and 3.82 mg/g DW. Such a high levels of both compounds was concluded to be because of potential consequences of T-DNA integration on the secondary metabolite production (Bansal et al. 2014; Bulgakov 2008; Thilip et al. 2015). As the last example, the production of withanolides was improved in the leaf explant-derived hairy roots of W. somnifera upon treatment with the extract of the seaweeds of Gracilaria edulis and Sargassum wightii over a time series of 0.0, 24, 48, 72 h exposure time (Sivanandhan et al. 2015). After 40 days of culture with 48 h exposure time (as the best time), considering 50% G. edulis extract, the contents of withanolide A, withaferin A, and withanone were respectively recorded as 5.23, 2.24, and 4.83 mg/g DW, while for S. wightii (60%), the values of 143 3.78, 1.94 and 2.56 mg/g DW were respectively detected. Overall, comparing to the control, the obtained withanolides magnitudes were substantially in higher levels of 2.32- to 2.66-fold in the hairy root culture (Sivanandhan et al. 2015).

Comparing the aforesaid records to our results, the acquired withanferin A magnitudes were in high levels of 6.84-fold and 12.46-fold values for the joint application of both MeJA and β-CD, that is “0.5 mM β-CD + 100 µM MeJA” and “5.0 mM β-CD + 100 µM MeJA”. The results indicated that simultaneous exogenous application of both MeJA and β-CD seems to be accompanied by a synergistic impact on the accumulation of withaferin A, albeit both MeJA and β-CD could also lonely improve production of this metabolite as compared with the control, concluding that elicitation could be an appropriate vehicle to ameliorate accumulation of Withaferin A in vitro.

Effect Of Elicitation On The Expression Levels Of Genes Responsible For Withanolides Biosynthesis

The expression profiles of four selected genes involved in withanolides biosynthetic pathway were studied for the hairy roots treated with five different elicitations namely MeJA (100 µM), β-CD (0.5 and 5.0 mM) and combination of both (i.e., 0.5 mM β-CD + 100 µM MeJA and 5.0 mM β-CD + 100 µM MeJA) after 48 h exposure compared to control (Fig. 6a-d). In general, the expression levels of all the four selected genes enhanced in all the five elicitation classes after 48 h exposure time as compared with control (Fig. 5a-d). Details for each gene are as follows.

HMGR

Based on the ANOVA results (Table 4), none of five different elicitations was able to significantly affect positively/negatively affect the expression levels of HMGR throughout the experimental treatments (p > 0.5). However, the expression levels of HMGR were somewhat upregulated in all the five different treatments, of which only the treatment with 0.5 mM β-CD was statistically different from control, with the fold change value of 4.76 (Fig. 6a).

Table 4

The ANOVA results to study potential effects of elicitation on the expression levels of four selected genes responsible for withanolides biosynthesis among five elicited and control groups of hairy roots of *W. somnifera*.
Source of variation	DF	Mean Square (HMGR)	Mean Square (SQS)	Mean Square (SMT-1)	Mean Square (SDS/CYP710A)
Treatment	5	6.179084225^ns	8.975956*	709.9715**	8.006453**
Error	12	2.65858545	2.780751	51.37018	1.463422
*: Significant at the 0.01 probability level; : Significant at the 0.05 probability level; ns: Not significant.

SQS

Unlike HMGR, the elicitation could significantly augment the expression levels of the second gene of interest SQS (Table 4; p < 0.5). In this sense, with the exception of “0.5 mM β-CD + 100 µM MeJA” treatment, the sample groups treated with the four remaining elicitations statistically exhibited substantial differences as compared with control in terms of the transcript levels of SQS gene (p < 0.05; Fig. 6b).

SMT-1

Regarding the third gene of interest, SMT-1, a significant difference was again observed among all the treated and non-treated samples (Table 4; p < 0.0001). Interestingly, co-treatment with MeJA (100 µM) and β-CD at both levels (i.e., 0.5 and 5.0 mM) coincided with the maximum transcript magnitudes of the same gene (31.45, and 33.55 fold change, respectively), as significant differences were undertaken compared to the control alongside the three remaining elicited sample groups (p < 0.05; Fig. 6c).

SDS/CYP710A

The expression pattern of the last gene of interest, SDS/CYP710A, was statistically significant over all the elicited/non-elicited samples (Table 4; p < 0.01). In this context, a number of fluctuations were detected, of which three separate sample groups elicited discretely with “0.5 mM β-CD”, “0.5 mM β-CD + 100 µM MeJA”, and “100 µM MeJA” with the fold change values of 4.59, 3.35, and 5.07 were statistically different rather than control (Fig. 6d). However, the two remaining sample groups treated individually with “5.0 mM β-CD”, and “5.0 mM β-CD + 100 µM MeJA” with the transcript quantities of 1.44 and 2.88 exhibited no significant differences as compared with control (Fig. 6d).

Correlation between relative gene expression and in vitro production of Withaferin A

Among different correlations calculated between four selected genes and withaferin A levels over the five different elicited treatments as well as control group, only the expression pattern of SMT-1 gene was significantly associated with withaferin A production (ρ = 0.912; p-value = 0.00000), while for the rest three genes and the same secondary metabolite no obvious relationship was deduced (Fig. 7). Meanwhile, among the four genes under study, only HMGR exhibited an association either with SDS (ρ = 0.518; p-value = 0.0275) and SQS genes (ρ = 0.629; p-value = 0.0051), while the remaining correlations were not statistically significant.

As mentions above, the transcript levels of all the four selected genes enhanced in all the five elicitation groups after 48 h exposure time as compared with control, agreeing with the earlier works in hairy root cultures of W. somnifera (Saxena et al. 2017; Sivanandhan et al. 2015). Comparing to control, apart from some fluctuations, simultaneous and/or individual exogenous applications of both MeJA and β-CD could lead to a growth in transcript magnitudes of the four selected genes involved in withanolides biosynthetic pathway. Most importantly, upon applying “0.5 mM β-CD + 100 µM MeJA” and “5.0 mM β-CD + 100 µM MeJA”, a substantial difference was occurred transcript levels of SMT-1 gene comparing to control, suggesting synergistic effects of both elicitors. Resembling to this, the synergistic effect of MeJA and cyclodextrin elicitors on taxane production in Taxus x media cell cultures could led to an increase in the expression of genes involved in the taxol biosynthetic pathway, followed by taxanes accumulation including taxol (Sabater-Jara et al. 2014).

To sum up, here, the potential effects of three main factors vis. “genotypes”, “tissues”, and “Agrobacterium strains” were pursued in hairy root induction of W. somnifera. Considering “induction rate” and “number of induced roots” followed by growth curve, the superiority of “G3/ATCC/LEAF” treatment was verified, and utilized then for elicitation study. Simultaneous action of both MeJA and β-CD at two levels of β-CD (5.0 and 0.5 mM) resulted in the maximum accumulation of withaferin A (17.45 and 9.57 mg/g DW, respectively) either compared to the control or to the remaining three treatments, or even compared to the leaves tissues of three genotypes of W. somnifera. In fact, comparing to the control, the acquired withanferin A magnitudes were in high levels of 6.84-fold and 12.46-fold values for “0.5 mM β-CD + 100 µM MeJA” and “5.0 mM β-CD + 100 µM MeJA”, respectively. Furthermore, application of both elicitors, apart from some oscillations, could enhance transcript amounts of four selected genes involved in withanolides biosynthetic pathway. Overall, the current results demonstrated the synergistic effect of exogenous application of both MeJA and β-CD on the in vitro production of withaferin A, and possibly other types of withanolides in the hairy root culture of W. somnifera.

Bansal M, Kumar A & Sudhakara Reddy M (2014) Influence of Agrobacterium rhizogenes strains on hairy root induction and ‘bacoside A’production from Bacopa monnieri (L.) Wettst. Acta physiologiae plantarum 36(10):2793-2801
Berghe WV, Sabbe L, Kaileh M, Haegeman G & Heyninck K (2012) Molecular insight in the multifunctional activities of Withaferin A. Biochemical pharmacology 84(10):1282-1291
Bulgakov VP (2008) Functions of rol genes in plant secondary metabolism. Biotechnology advances 26(4):318-324
Bulgakov VP, Khodakovskaya MV, Labetskaya NV, Chernoded GK & Zhuravlev YN (1998) The impact of plant rolC oncogene on ginsenoside production by ginseng hairy root cultures. Phytochemistry 49(7):1929-1934
Cardillo A, Perassolo M, Giulietti A & Rodriguez Talou J (2021) Cyclodextrins: a tool in plant cell and organ culture bioprocesses for the production of secondary metabolites. Plant Cell, Tissue and Organ Culture (PCTOC) 146(1):1-19
Chen L-X, He H & Qiu F (2011) Natural withanolides: an overview. Natural product reports 28(4):705-740
Dehdashti SM, Acharjee S, Kianamiri S & Deka M (2017) An efficient Agrobacterium rhizogenes-mediated transformation protocol of Withania somnifera. Plant Cell, Tissue and Organ Culture (PCTOC) 128(1):55-65
Dehdashti SM, Acharjee S, Nomani A & Deka M (2020) Production of pharmaceutical active recombinant globular adiponectin as a secretory protein in Withania Somnifera hairy root culture. Journal of Biotechnology 323:302-312
Dhar N, Razdan S, Rana S, Bhat WW, Vishwakarma R & Lattoo SK (2015) A decade of molecular understanding of withanolide biosynthesis and in vitro studies in Withania somnifera (L.) Dunal: prospects and perspectives for pathway engineering. Frontiers in plant science 6:1031
Dutta R, Khalil R, Green R, Mohapatra SS & Mohapatra S (2019) Withania somnifera (Ashwagandha) and withaferin A: Potential in integrative oncology. International journal of molecular sciences 20(21):5310
Halder M, Sarkar S & Jha S (2019) Elicitation: A biotechnological tool for enhanced production of secondary metabolites in hairy root cultures. Engineering in life sciences 19(12):880-895
Khan MI, Maqsood M, Saeed RA, Alam A, Sahar A, Kieliszek M, Miecznikowski A, Muzammil HS & Aadil RM (2021) Phytochemistry, Food Application, and Therapeutic Potential of the Medicinal Plant (Withania coagulans): A Review. Molecules 26(22):6881
Khan S, Qureshi MI, Alam T & Abdin M (2007) Protocol for isolation of genomic DNA from dry and fresh roots of medicinal plants suitable for RAPD and restriction digestion. African Journal of Biotechnology 6(3):175
Liao Z, Chen M, Guo L, Gong Y, Tang F, Sun X & Tang K (2004) Rapid isolation of high‐quality total RNA from Taxus and Ginkgo. Preparative biochemistry and biotechnology 34(3):209-214
List P (2013) The Plant List. Version 1.1. Published on the Internet
Livak KJ & Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. methods 25(4):402-408
Maurya R (2010) Chemistry and pharmacology of Withania coagulans: an Ayurvedic remedy. Journal of pharmacy and pharmacology 62(2):153-160
Metsalu T & Vilo J (2015) ClustVis: a web tool for visualizing clustering of multivariate data using Principal Component Analysis and heatmap. Nucleic acids research 43(W1):W566-W570
Mirjalili HM, Fakhr‐Tabatabaei SM, Bonfill M, Alizadeh H, Cusido RM, Ghassempour A & Palazon J (2009a) Morphology and withanolide production of Withania coagulans hairy root cultures. Engineering in Life Sciences 9(3):197-204
Mirjalili M, Moyano E, Bonfill M, Cusido R & Palazon J (2011) Overexpression of the Arabidopsis thaliana squalene synthase gene in Withania coagulans hairy root cultures. Biologia plantarum 55(2):357-360
Mirjalili MH, Moyano E, Bonfill M, Cusido RM & Palazón J (2009b) Steroidal lactones from Withania somnifera, an ancient plant for novel medicine. Molecules 14(7):2373-2393
Mishra S, Sangwan RS, Bansal S & Sangwan NS (2013) Efficient genetic transformation of Withania coagulans (Stocks) Dunal mediated by Agrobacterium tumefaciens from leaf explants of in vitro multiple shoot culture. Protoplasma 250(2):451-458
Murashige T (2009) sKOOG F.(1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15(4):13-497
Murthy HN, Dijkstra C, Anthony P, White DA, Davey MR, Power JB, Hahn EJ & Paek KY (2008) Establishment of Withania somnifera hairy root cultures for the production of withanolide A. Journal of integrative plant biology 50(8):975-981
Namdeo AG & Ingawale DK (2021) Ashwagandha: Advances in plant biotechnological approaches for propagation and production of bioactive compounds. Journal of Ethnopharmacology 271:113709
Nasiri J, Naghavi MR, Alizadeh H & Moghadam MRF (2016) Seasonal-based temporal changes fluctuate expression patterns of TXS, DBAT, BAPT and DBTNBT genes alongside production of associated taxanes in Taxus baccata. Plant cell reports 35(5):1103-1119
Ochoa-Villarreal M, Howat S, Hong S, Jang MO, Jin Y-W, Lee E-K & Loake GJ (2016) Plant cell culture strategies for the production of natural products. BMB reports 49(3):149
Pal A, Swain SS, Mukherjee AK & Chand PK (2013) Agrobacterium pRi T^ sub L^-DNA rolB and T^ sub R^-DNA Opine Genes Transferred to the Spiny Amaranth (Amaranthus spinosus L.), A Nutraceutical Crop. Food technology and Biotechnology 51(1):26
Palliyaguru DL, Singh SV & Kensler TW (2016) Withania somnifera: from prevention to treatment of cancer. Molecular nutrition & food research 60(6):1342-1353
Pandey V, Ansari WA, Misra P & Atri N (2017) Withania somnifera: Advances and implementation of molecular and tissue culture techniques to enhance its application. Frontiers in Plant Science 8:1390
Pandey V, Srivastava R, Akhtar N, Mishra J, Mishra P & Verma PC (2016) Expression of Withania somnifera steroidal glucosyltransferase gene enhances withanolide content in hairy roots. Plant Molecular Biology Reporter 34(3):681-689
Petrovska BB (2012) Historical review of medicinal plants’ usage. Pharmacognosy reviews 6(11):1
Praveen N & Murthy H (2012) Synthesis of withanolide A depends on carbon source and medium pH in hairy root cultures of Withania somnifera. Industrial Crops and Products 35(1):241-243
Praveen N & Murthy H (2013) Withanolide A production from Withania somnifera hairy root cultures with improved growth by altering the concentrations of macro elements and nitrogen source in the medium. Acta physiologiae plantarum 35(3):811-816
Sabater‐Jara AB, Onrubia M, Moyano E, Bonfill M, Palazón J, Pedreño MA & Cusidó RM (2014) Synergistic effect of cyclodextrins and methyl jasmonate on taxane production in Taxus x media cell cultures. Plant biotechnology journal 12(8):1075-1084
Saxena P, Ahlawat S, Ali A, Khan S & Abdin MZ (2017) Gene expression analysis of the withanolide biosynthetic pathway in hairy root cultures of Withania somnifera elicited with methyl jasmonate and the fungus Piriformospora indica. Symbiosis 71(2):143-154
Sevón N & Oksman-Caldentey K-M (2002) Agrobacterium rhizogenes-mediated transformation: root cultures as a source of alkaloids. Planta Medica 68(10):859-868
Shasmita, Rai MK & Naik SK (2018) Exploring plant tissue culture in Withania somnifera (L.) Dunal: in vitro propagation and secondary metabolite production. Critical reviews in biotechnology 38(6):836-850
Sivanandhan G, Arun M, Mayavan S, Rajesh M, Jeyaraj M, Dev GK, Manickavasagam M, Selvaraj N & Ganapathi A (2012a) Optimization of elicitation conditions with methyl jasmonate and salicylic acid to improve the productivity of withanolides in the adventitious root culture of Withania somnifera (L.) Dunal. Applied biochemistry and biotechnology 168(3):681-696
Sivanandhan G, Arunachalam C, Selvaraj N, Sulaiman AA, Lim YP & Ganapathi A (2015) Expression of important pathway genes involved in withanolides biosynthesis in hairy root culture of Withania somnifera upon treatment with Gracilaria edulis and Sargassum wightii. Plant Physiology and Biochemistry 91:61-64
Sivanandhan G, Kapil Dev G, Jeyaraj M, Rajesh M, Arjunan A, Muthuselvam M, Manickavasagam M, Selvaraj N & Ganapathi A (2013) Increased production of withanolide A, withanone, and withaferin A in hairy root cultures of Withania somnifera (L.) Dunal elicited with methyl jasmonate and salicylic acid. Plant Cell, Tissue and Organ Culture (PCTOC) 114(1):121-129
Sivanandhan G, Rajesh M, Arun M, Jeyaraj M, Dev GK, Manickavasagam M, Selvaraj N & Ganapathi A (2012b) Optimization of carbon source for hairy root growth and withaferin A and withanone production in Withania somnifera. Natural Product Communications 7(10):1934578X1200701005
Sultana T, Okla MK, Ahmed M, Akhtar N, Al-Hashimi A, Abdelgawad H & Haq I-u- (2021) Withaferin A: From Ancient Remedy to Potential Drug Candidate. Molecules 26(24):7696
Thilip C, Mehaboob VM, Varutharaju K, Faizal K, Raja P, Aslam A & Shajahan A (2019) Elicitation of withaferin-A in hairy root culture of Withania somnifera (L.) Dunal using natural polysaccharides. Biologia 74(8):961-968
Thilip C, Soundar Raju C, Varutharaju K, Aslam A & Shajahan A (2015) Improved Agrobacterium rhizogenes-mediated hairy root culture system of Withania somnifera (L.) Dunal using sonication and heat treatment. 3 Biotech 5(6):949-956
Tripathi N, Shrivastava D, Mir BA, Kumar S, Govil S, Vahedi M & Bisen PS (2018) Metabolomic and biotechnological approaches to determine therapeutic potential of Withania somnifera (L.) Dunal: A review. Phytomedicine 50:127-136
Wojciechowska M, Owczarek A, Kiss AK, Grąbkowska R, Olszewska MA & Grzegorczyk-Karolak I (2020) Establishment of hairy root cultures of Salvia bulleyana Diels for production of polyphenolic compounds. Journal of biotechnology 318:10-19

Download PDF

Version 1

posted

You are reading this latest preprint version

Methyl jasmonate and β-cyclodextrin shake hands to boost withaferin A production from the hairy root culture of Withania somnifera

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Plant material

Elicitor Treatment With Methyl Jasmonate And βcyclodextrin

Rna Extraction And Cdna Synthesis

Primer Design And Quantitative Real Time Pcr (Qpcr)

Statistical analysis

Results And Discussion

Establishment of hairy roots

Identification Of Hairy Root By Pcr

Growth Curve Of Hairy Root Lines

Effect Of Elicitation On The Expression Levels Of Genes Responsible For Withanolides Biosynthesis

Conclusion

References

Status:

Version 1