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Improving diosgenin production and its biosynthesis in Trigonella foenum-graecum L. hairy root cultures
Industrial Crops & Products 145 (2020) 112075
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Improving diosgenin production and its biosynthesis in Trigonella foenumgraecum L. hairy root cultures
T
Farnaz Zolfagharia, Sajad Rashidi-Monfareda,*, Ahmad Moienib, Davar Abedinia, Amin Ebrahimic a
Agricultural Biotechnology Department, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran Genetics and Plant Breeding Department, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran c Agronomy and Plant Breeding Department, Faculty of Agriculture, Shahrood University of Technology, Semnan, Iran b
ARTICLE INFO
ABSTRACT
Keywords: Agrobacterium rhizogenes Diosgenin Gene expression Hairy roots Fenugreek
Induction of hairy roots stemmed from Agrobacterium rhizogenes transformation, is a key step to reach mass production of secondary metabolites in medicinal plants. Accordingly, this study is aimed to investigate potential impact of different A. rhizogenes strains, including ATCC15834, R1000, A4 and C58, on diosgenin biosynthesis in high- “Boshruyeh” (23.8 mg/gDW) and low “Hamadan” (6.4 mg/gDW) diosgenin-producing genotypes of fenugreek. Interestingly, comparing the metabolite extracted from both leaves and hairy roots of these genotypes suggested that hairy roots are promising platform to produce scalable amount of diosgenin, producing 143.96 mg/gDW in high diosgenin producing hairy roots (A4-mediated hairy roots induction in “Boshruyeh” (BA4)), while 23.8 mg/gDW in high diosgenin producing genotype. Transcript abundance of 10 genes involved in diosgenin biosynthesis pathway as well as three rate-limiting genes; i.e 1-deoxy-D-xylulose-5-phosphate synthase (DXS), 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) and 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, functioning in plastid 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway were measured. Comparison of the transcription and metabolite analysis revealed that the more expression of Δ24-reductase gene, the higher accumulation of diosgenin in the hairy roots. In high diosge nin producing hairy roots, the expression of Δ24-reductase gene were significantly up-regulated (8-folds). Moreover, integration of metabolite and transcript results revealed that A4 has a strong capacity in boosting diosgenin accumulation in the both genotypes. These results may lead to the next step in production of diosgenin in scalable and commercial levels.
1. Introduction Trigonella foenum-graecum, is one of the world-appealing medicinal plant belonging to the Fabaceae family that has been extensively used from ancient time to the current era. This plant is an annual dicotyledonous legume with 0.7 of C-value, which is about 1.5 fold higher than that is found in Lotus corniculatus, as a model legume (Chaudhary et al., 2018; Martin et al., 2011). Fenugreek possesses a number of valuable phytochemicals, including alkaloids, steroids, flavonoids, saponins lysine-rich proteins, and volatile oils. Among these, diosgenin is a wellknown bioactive compound, which received a considerable attention owing to its potential feature in anticancer and anti-aging activities (Raju and Mehta, 2008; Smith, 2003). Moreover, this metabolite is broadly utilized in pharmaceutical industry as a precursor of synthetic steroidal drugs such as testosterone, progesterone, norethisterone and glucocorticoids (Chaudhary et al., 2018; Murlidhar and Goswami,
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2012). Diosgenin is also known as a naturally occurring phytochemical found in foodstuffs as a controlling agent for cancer chemoprevention (Raju and Mehta, 2008). Consequently, providing a reliable and affordable platform so as to scale-up this precious compound is desired. More recently, Aminkar and co-workers measured the diosgenin content in several genotypes collected from different region of Iran, and realized that there are a considerable differences among these samples (Aminkar et al., 2018). Until now, a plethora of precious metabolites have been discovered and clinically verified; nonetheless, the main concern to utilize such compounds are the less-abundance and being tissue-specific of these metabolites in the given plants (Murthy et al., 2014). For many known and unknown reasons, plants often produce secondary metabolites in low quantities. As a consequence, a number of strategies have widely been recommended to enhance the amount of valuable compounds (Dörnenburg and Knorr, 1995; Murthy et al., 2014; Qian et al., 2005).
Corresponding author. E-mail address: [email protected] (S. Rashidi-Monfared).
https://doi.org/10.1016/j.indcrop.2019.112075 Received 19 August 2019; Received in revised form 24 December 2019; Accepted 27 December 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.
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In this regard, transformed (hairy) roots could be the stepping-stone in term of achieving sufficient medicinal metabolites. In addition, this strategy is a scalable and affordable as compared with other biotechnological means (Bourgaud et al., 2001; Ibañez et al., 2016). The neoplastic tissues resulted in Agrobacterium rhizogenes characterized by high growth rate, genetic stability and growth in hormone free media producing comparable metabolites than intact plants (Srivastava and Srivastava, 2007). The rol oncogenes including rolA, B, C and D, carrying on the Ri plasmid, play a fundamental role in hairy root induction (Costantino et al., 1994; Nilsson and Olsson, 1997). Different strains of A. rhizogenes have showed different capability in induction efficiency (Li et al., 2017; Thwe et al., 2016). So far, several strains, including A4, ATCC15834, LBA9402, MAFF03–01724, R-1601, R-1000, and TR105, have been applied in hairy root induction in quite a few number of herbs (Setamam et al., 2014; Thwe et al., 2016). The biosynthesis pathway of diosgenin has been generally constructed and some related enzymes have been experimentally characterized (Sawai et al., 2014; Vaidya et al., 2013). Likewise other group of triterpenes, diosgenin is formed by condensing of C5 units of isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMAPP), catalyzing by isopentenyl diphosphate isomerase (Ramos-Valdivia et al., 1997). This reaction leads to production of a key diosgenin precursor; farnesyl pyrophosphate (Vincken et al., 2007). Isopentenyl diphosphate can be synthesized from both 2-C-methyl-D-erythritol 4phosphate (MEP) and mevalonate (MVA) pathways (Augustin et al., 2011). Laule and co-workers showed a cross-talk between biosynthesis of isoprenoid in cytosolic and plastidial pathways (Laule et al., 2003). Studying the expression rate of important genes can provide new insight into engineering metabolic pathways, and suggesting a reliable and efficient source for scaling up pharmacologically important metabolites. Previously, we investigated the regulation of rate-limiting enzymes and elements involving in metabolite accumulation in known herbs, which spotlighted new areas of interests in medicinal plants (Abedini et al., 2018; Abedini and Rashidi-Monfared, 2018; Kakeshpour et al., 2015; Mastakani et al., 2018; Yang et al., 2015). Herein, the study focused on induction of hairy roots by different strains of A. rhizogenes in T. foenum-graecum L. Additionally, the expression level of several genes involved in diosgenin biosynthesis pathway was investigated and the diosgenin content on the established hairy roots were also measured.
explants of both “Boshruyeh” and “Hamadan” genotypes were wounded, and then each individual explants were immersed in liquid WPM medium containing each strain of A. rhizogenes for 15 min. After that, co-cultured explants were dried on a sterile tissue paper. These samples were consequently incubated under dark condition at 25 ± 1 °C for 2 days. The explants were rinsed with sterile distilled water and cefotaxime antibiotic then placed in WPM medium containing 6 mg l−1 agar-agar for one week to remove the residual bacteria from the explants after incubation. Subsequently, explants were cultivated in a WPM medium possessing cefotaxime (350 mg L−1). After emerging hairy roots, samples were cultured in a hormone-free medium without antibiotic with shaking and darkness conditions at 25 ± 1 °C for one month. Eventually, hairy roots were harvested to further analysis such as assessing the gene expression levels and measuring the diosgenin content. 2.2. Genomic DNA extraction and PCR assay Genomic DNA was extracted for the studied hairy root lines using the Cetyl trimethylammonium bromide (CTAB) method (Murray and Thompson, 1980). To confirm and verify the obtained hairy roots, two pairs of primers were designed from a part of rolB and virD, and then amplified using PCR assay. The program was as follow: initial denaturation at 95 °C for 3 min for 1 cycle, followed with 40 cycles including; 95 °C for 30 s, 54 °C for 30 and elongation for 40 s. After finishing the PCR, PCR products were run on the 1 % of agarose gelelectrophoresis. The sequences of both the primers are listed in the Table 1. 2.3. HPLC analysis The obtained hairy roots were dried using freeze drier and subsequently ground into a fine powder. Then the 200 mg of the each samples, consisting of three replications, were immersed with 20 ml of 96 % ethanol, and were incubated in a sonicator water bath (UltrasonicCleanerXPS240-4 L water baths (Sharpertek, USA)) with a frequency of 60 kHz for 2 h. Afterward, 20 ml of H2SO4 (1 mol/L) was added to the samples and placed at 121 °C for 2 h. Obtained samples were washed three times with n-Hexan, (1 mol/L), NaOH, respectively. The samples were finally rinsed with distilled water. The supernatant phase was removed and the debris washed with deionized water and the upper phase removed again. In order to dehydration of the extracts, dried sodium sulfate (Na2SO4) was added to the samples. The extract was dissolved in 500 μl of acetonitrile, HPLC Grade, and filtered through 0.22 μm filter to remove any small particulates. In this study, diosgenin content was measured using High Performance Liquid Chromatography (HPLC), (Waters USA) composed of a K-1001 pump, SPDM20A PDA detector (K-2501, Knauer, Berlin, Germany) on a C18 column, with flow rate of 0.7 mL/min. The chromatographic separation was carried out using a mixture of acetonitrile and water (90:10) solvent. Five concentrations of diosgenin (Sigma-Aldrich-Germany; CAS Number: 512049) i.e. 100, 150, 300, 500 and 1000 ppm were prepared via dissolving in acetonitrile, then placed in ultrasonic for 30 min. These concentrations were consequently injected in 50 μl volume into the HPLC system and the correlation coefficient (R2) was calculated 0.99 for the data sets.
2. Material and method 2.1. Induction and establishment of hairy roots Previously, the diosgenin content of several genotypes collected from different regions of Iran were studied, and “Boshruyeh” (23.8 mg/ gDW) and “Hamadan” (6.4 mg/gDW) demonstrated high- and lowdiosgenin content, respectively. In the present study, with considering previous results, the hairy roots for “Boshruyeh” and “Hamadan” genotypes (Aminkar et al., 2018) were induced. For seed germination, the fenugreek seeds were washed in tap water for 5 min and then soaked in 70 % of ethanol for 1 min, followed by washing with sterilized water. Seed surface were sterilized by 3 % sodium hypochlorite (NaOCl) for 15 min and washed with sterile distilled water. Finally, the seeds were cultured in petri dishes containing Woody Plant Medium (WPM) medium and placed in darkness condition for 4–5 days. After that, germinated seeds were transferred to a WPM medium with photoperiod of 16−8 h for 10 days. Four A. rhizogenes strains including ATCC15834, R1000, A4 and C58 were utilized to induce hairy roots in fenugreek genotypes. These strains were cultured into 10 ml liquid Luria-Bertani (LB) medium containing 100 mg l−1 of Rifampicin at 28 °C and 224 RCF for 24 h in darkness conditions. The optical density (OD) was about 0.6. All strains were stored in glycerol stock at −80 °C. Hairy roots were induced via different strains of A. rhizogenes including ATCC15834, R1000, A4 and C58. For this purpose, hypocotyl
2.4. Read assembly and phylogenetic construction The reads from several RNAseq projects (With SRA accession nos.: SRX2765627, SRX2765626, SRX2765625, SRX2676952, SRX2676951, SRX2666620, SRX2631114, SRX026992, SRX026991, SRX026990, SRX026989, SRX026988) were downloaded from NCBI database (http://www.ncbi.nlm.nih.gov/sra). After identifying reads with highest similarity to the key genes involved in isopenthyl diphosphate in plastid, including 1-deoxy-D-xylulose-5-phosphate synthase (DXS), 12
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Table 1 The list of primers used in validation of transgenic roots indication and gene expression analysis in induced hairy roots of fenugreek. Primer no.
Primer name
Use
Primer sequence
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
rolB-Forward rolB-Reverse virD-Forward virD-Reverse SQS. Forward SQS. Reverse CAS. Forward CAS. Reverse SEP. Forward SEP. Reverse SMT1. Forward SMT1. Reverse Δ24-reductase. Forward Δ24-reductase. Reverse BGL2. Forward BGL2. Reverse C22-hydroxylase. Forward C22-hydroxylase. Reverse C26-hydroxylase. Forward C26-hydroxylase. Reverse C4-demethylase. Forward C4-demethylase. Reverse Δ7 reductase. Forward Δ7 reductase. Reverse DXR. Forward DXR. Reverse DXS. Forward DXS. Reverse HDR.Forward HDR.Reverse GAPDH. Forward GAPDH. Reverse
Hairy roots indication Hairy roots indication Hairy roots indication Hairy roots indication qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR Reference gene Reference gene
5′-gctcttgcagtgctagattt-3′ 5′-gaaggtgcaagctacctctc-3′ 5′-atgtcgcaaggacgtaagccca-3′ 5′-ggagtctttcagcatggagcaa-3′ 5′-ggctcaaccatgattctcatactg-3′ 5′-tacccactgttccattgctatcc-3′ 5′-aagagagatccaacaccactgc-3′ 5′-tacatgacgacggtattctccc-3′ 5′-ctattctcagaaggaccgatctc-3′ 5′-cagatgcaccagagagtaatcg-3′ 5′-gagaaggctgcagagggtctag-3′ 5′-cacagtaactcatgacaacagcaacct-3′ 5′-aggtgggagatatgctagaatggg-3′ 5′-cattctgtgtgtctccctgcc-3′ 5′-cctggtcctgagagcataacaaatg-3′ 5′-catcagccacaccttgtccttc-3′ 5′-ctatggggtgacgatgctaagatg -3′ 5′-tgctattgccatctttgcttcca-3′ 5′-cgttcctaccacggtgccttt-3′ 5′-ttgagtttgttctcctgctctagcc-3′ 5′-atggaactgacaagggttttcggt-3′ 5′-actgcctacaatgcttctgtctca-3′ 5′-ttgaccgagcgaaaagggatga -3′ 5′-gcaccaagtgaaggattccagac-3′ 5-aggcaccatgacaggagttcttag-3 5-gcaaactagcagcatattcgcgt-3 5-cagtaggggtttatgtgacagttgc-3 5-cctccaatagaaccctcttccact-3 5-ggattgacagtgagcagagaattgg-3 5-catcttccacgaccttgtctgg-3 5′-tatgtttgttgttggtgtcaacgagcaacgaatacaag-3′ 5′-atgttaaatgatgcagcccttccacctctc -3′
deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) and 4-hydroxy3-methylbut-2-enyl diphosphate reductase (HDR) from related species, using the offline BLAST software v.2.7.0 (Altschul et al., 1997), they were then assembled into contigs with the Codon Code Aligner v.5.0.1. program (https://www.codoncode.com/aligner) based on the alignthen assemble strategy. The ORF finder tool (https://www.ncbi.nlm. nih.gov/orffinder/) created the ORF of obtained consensus sequences; the specific primers were consequently designed and confirmed using the Oligo Analyzer v.3.1 (http://eu.idtdna.com/calc/analyzer). Furthermore, similar protein sequences for each consensus sequence were queried in the BLAST search tool. The multiple alignment analysis was carried out by online Clustal omega (https://www.ebi.ac.uk/Tools/ msa/clustalo/) program. Then, phylogeny tree was constructed using the MEGA 7 software (MEGA, PA, USA) based on the NeighbourJoining (NJ) method, with the default options and bootstraps analysis (3000 replicates).
workers (Mohammadi et al., 2019), were used. The first strand cDNA was synthesized with one μg of extracted RNA using HyperscriptTM Reverse Transcriptase (GeneAll Inc, South Korea Cat No.: 601-100). The real-time PCR was carried out in three technical replicates with the BioRad system (Bio-Rad, Hercules, CA, USA) using the SYBR®Green PCR Master Mix 2X (Ampliqon, Denmark (Lot No.: A322701)). The qRT-PCR amplification was performed as follows: pre-denaturation at 94 °C for 10 min, followed by 40 cycles consisting of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s and extension at 72 for 20 s. Moreover, the melting curve was acquired by slowly ramping up the temperature from 65 °C to 95 °C. The GAPDH was selected as an internal reference gene. All collected data were analyzed based on the Livak method (2−ΔΔCt) (Livak and Schmittgen, 2001), and the relative expression was calculated via the Microsoft Excel software (Microsoft Office 2013). The studied primers are listed in the Table 1. 3. Results
2.5. RNA extraction, cDNA synthesis and qRT-PCR assay
3.1. Hairy roots establishment and validation of transgenic roots
Total RNA of each hairy root was extracted using Topaz Kit (Top Plant and Fungi RNA Purification Kit, TOPAZ GENE RESEARCH., cat No.: TGK2004, Iran). The quality and quantity of extracted RNA were assessed by gel electrophoresis and a NanoDrop spectrophotometer (BioTek, EPOCH, serial 121004C, USA). Then, to avoid the contamination of genomic DNA, extracted RNA was treated by RNase-free DNaseI (Thermo Fisher Scientific., Cat No.: EN0521). In this study, the transcript abundance of thirteen genes including Squalene synthase (SQS), Squalene epoxidase (SEP), Cycloartenol synthase (CAS), Δ24-reductase, Sterol-methyltransferase (SMT1), (SMT1), C4-demethylas, Δ7reductase, C22-hydroxylase, C26-hydroxylase, 26-O-beta glycosidase (BGL), DXS, DXR and HDR were measured. Specific primers amplifying DXS, DXR and HDR genes were designed, for the other genes, the primers which had previously been designed by Mohammadi and co-
Hairy roots induction was successfully carried out using different A. rhizogenes strains, i.e. ATCC15834, R1000, A4 and C58. All of the utilized strains induced hairy root in genotypes, high- and low-diosgenin content “Boshruyeh” and “Hamadan”, respectively (Fig. 2). Measuring dry weight of established hairy roots indicated to the strong efficiency of ATCC15834 and R1000 strains in both genotypes; however, A4 had comparatively lower biomass production. HATCC15834 and HR1000 represented the highest biomass weight (1.11 g), and HA4 and BA4 showed the lowest production (0.26 and 0.53 g, respectively) (For more details, see Fig. S1. Supplementary data). Molecular indication was obtained using PCR assay for the transformed status of the hairy root lines. Specific primers amplifying rolB and virD genes were utilized in order to distinguish between transformed and untransformed roots 3
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Fig. 1. Biosynthesis pathway of diosgenin in fenugreek. Mevalonate (MVA) pathway in cytosol: Acetyl-CoA converts to Cycloartenol via multiple reactions (pink box). Cycloartenol converts to either cycloartenol, leading to cholesterol formation (red box) following cholesterol converts to 3β-cholesta-5-en-3, 22-diol (blue box), which ultimately results in diosgenin production. Dashed lines (from Acetyl-CoA to farnesyle diphosphate) indicate multiple steps involved in the pathway (Mohammadi et al., 2019). Methylerythritol 4-phosphate (MEP) pathway in plastid: DXS: 1-deoxy-D-xylulose-5-phosphate synthase; DXR: 1-deoxy-D-xylulose-5-phosphate reductoisomerase; MCT: 2-C-methyl-D-erythritol4-(cytidyl-5-diphosphate) transferase; CMK: 4cytidine 5′-diphospho-2-C-methyl-D-erythritol kinase; MCS: 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase; HDS; hydroxy-2-methyl-2(E)-butenyl 4-diphosphate synthase; HDR: hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase.
(Table 1, no. 1–4). According to the results, after amplification, the expected size for rolB gene, transformed from Ri-plasmid was observed, confirming the transgenic nature of the established hairy roots (Fig. S2A supplementary data). On the other hand, no products were detected using virD primers (Fig. S2-B Supplementary data), illustrating the complete elimination of bacteria from the tissues. In all amplifications, Ri-plasmid and untransformed roots were utilized as positive and negative control, respectively. Therefore, by considering these results the influence of each individual strain of A. rhizogenes on two genotypes in both transcriptomic and metabolomics level were investigated.
of “Hamedan” genotype (Fig. 4). Among the studied strains, A4 illustrated a strong capability in accumulating diosgenin. Taken together, based on the results, to produce a considerable amount of diosgenin, suitable plant hosts as well as promising strain are required. Herein, it is observed that “Boshruyeh” genotype and A4 strain could lead to induce a promising hairy root line, which ultimately results in higher production of diosgenin. Interestingly, analyzing the chromatographic curve stemmed from the plants leave illustrates two relatively same peaks, dioscin and diosgenin; one the other hand, HPLC result of the hairy root extracts showed only single sharp peak indicating diosgenin compound (For more detail, see Fig. S3 in Supplementary data). In addition, comparing diosgenin from wild plant and hairy roots demonstrated that the accumulation of this metabolite in hairy roots is considerably higher than the plant (23.8 mg/gDW in “Boshruyeh” as a high-diosgenin producing genotype), indicating to the robust capacity of the fenugreek hairy roots to scale-up the diosgenin accumulation.
3.2. Diosgenin content in hairy roots of Fenugreek In this study, the amount of diosgenin extracted from various fenugreek hairy root lines were measured. According to the HPLC results, “Boshruyeh” genotype infected with A4 strain showed dominant content of diosgenin (143.96 mg/gDW) as compared with other treatments. On the other hand, “Hamedan” genotype infected with C58 strain demonstrated lower amount of this metabolite (29.9 mg/gDW). In general, the amount of diosgenin in hairy roots stemmed from “Boshruyeh” genotype, except one infected with R1000 strain, was higher than those
3.3. Expression of diosgenin biosynthesis pathway genes To evaluate the potential effects of different strains of A. rhizogenes on transcription of genes functioning in diosgenin pathway, the 4
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expression level of several genes were assessed using real-time PCR in resultant hairy roots. To this end, the following genes were studied: SQS, SEP, CAS, Δ24-reductase, SMT1, C4-demethylas, Δ7-reductase, C22-hydroxylase, C26-hydroxylase and BGL2. Given the results, expression of all genes illustrated a different pattern when the explant transformed with different strains, indicating that each strain has a different pattern of gene regulation in the fenugreek host (Fig. 4). As we expected, the expression level of some determinative genes, i.e. SQS, CAS and Δ24-reductase, in hairy roots associated with “Boshruyeh” genotype were comparatively higher than those in the “Hamdan” genotype, which obviously indicates the importance of genotype for induction of hairy roots. The expression of SQS and CAS in the highest producing diosgenin hairy root (A4-mediated hairy roots induction in “Boshruyeh” (BA4)) up-regulated 4.3 and 6.5 fold higher than those in ATCC15834-mediated hairy roots induction in “Hamdan” (H15834), respectively; in addition, expression of Δ24-reductase in BA4 were 4fold higher than that of in A4-mediated hairy roots in induction “Hamdan”. In general, A4 and R1000 strains caused the higher expression in a majority of genes (Fig. 3). The expression of Δ24-reductase, CAS and SQS in “Boshruyeh”, for instance, which have a great impact on the production of diosgenin, were higher when infected with A4 strain, 5, 5 and 8 folds, respectively. In contrast, in roots infected with ATCC15834 strain, the transcript level of three important upstream genes of the diosgenin biosynthetic pathway, i.e. Δ24-reductase, CAS, SQS, were considerably lower than the expression of the rest of the studied genes. In the diosgenin biosynthetic pathway, there is a branch point where two determinative genes including SMT1 and Δ24-reductase take part, which leads to the production of either diosgenin or phytosterol (24alkyl sterols) (Fig. 1). The expression of Δ24-reductase gene leading to production of diosgenin, was higher in “Boshruyeh” genotype infected with A4 strain. Similarly, the expression of BGL2 gene reached its peak in “Boshruyeh” genotype infected with R1000 strain. This gene is also considered as an important gene, converting the last step of diosgenin biosynthesis. However, due to down-regulation of Δ24-reductase in “Boshruyeh” infected with R1000, the metabolite content in this treatment is relatively low. In other words, down-regulation of Δ24-reductase, as a rate-limiting enzyme, results in lower production of
Fig. 3. The amount of diosgenin content in hairy roots of fenugreek induced by different A. rhizogenes strains, i.e. ATCC15834, R1000, A4 and C58. Two genotypes of fenugreek including “Boshruyeh” and “Hamadan” (identified with B and H, respectively) were used as host plant.
substrates needed for the downstream enzymes involved in diosgenin biosynthesis pathway. Interestingly, comparing transcript abundance of the genes involved in diosgenin biosynthesis in the hairy roots induced by R1000 (HR1000) and A4 (BA4) strains that produce higher amount of diosgenin (Fig. 3), and leaf of two high- and low diosgenin-producing genotypes (Mohammadi et al., 2019) showed increased expression of all genes in hairy root of both genotypes relative to their plant leaves (Figs. 5 and S7). The expression of DXR, DXS and HDR genes, which are key genes regulating terpenoid biosynthesis pathway in plastids were also measured. It is reported that DXS and DXR are key genes at the beginning of the plastid MEP pathway, and HDR is the last rat-limiting enzyme of the pathway (Morrone et al., 2010) (Fig. 1). Herein, the results interestingly demonstrated that these genes are highly expressed in induced hairy roots (Fig. 4). Phylogenetic analysis also revealed that DXR, DXS and HDR genes of fenugreek are closely linked with those in Medicago truncatula, indicating to having a common ancestor for both the barrelclover and fenugreek (Supplementary data S4-6).
Fig. 2. Sequential stages representing hairy root induction in Fenugreek. A) seed germination, B) hairy root induction, C) hairy toots growth after three weeks induction, D) transporting the hairy roots from solid to liquid medium, E) three weeks after culturing in a liquid medium, F) preparation and measurement of fenugreek hairy roots before storing in −80 °C freezer.
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Fig. 4. Relative expression analysis of diosgenin biosynthetic genes in hairy roots of fenugreek induced by different A. rhizogenes strains, i.e. R15834, R1000, A4 and C58. Abbreviations: SQS: Squalene synthase, SEP: Squalene epoxidase, CAS: Cycloartenol synthase, Δ24-reductase, SMT1: Setrol-methyltransferase1, BGL2: 26-O-beta glycosidase, DXS: 1-deoxy-D-xylulose-5-phosphate synthase, DXR: 1-deoxy-D-xylulose 5-phosphate reductoisomerase and HDR: 4-hydroxy-3-methylbut-2-enyl diphosphate reductase. The lines on each bar represent standard deviation. Two genotypes of fenugreek including “Boshruyeh” and “Hamadan” are identified with B and H, respectively. The expression of the genes were relatively compared with control samples. The control sample for Δ24-reductase, SQS, SMT1 was B.C58; for SEP, CAS, and BGL2 was H.15834; for C4-demethylas, Δ7-reductase, C22-Hydroxylase, C26-Hydroxylase was B.15834; for DXS, DXR and HDR was H.A4
promising way, since it is a superior method with several privileges over callus and cell culture (Srivastava and Srivastava, 2007). Previously, we measured diosgenin content of several fenugreek genotypes collected from different regions of Iran. The result showed that “Boshruyeh” and “Hamedan” genotypes have the highest and lowest diosgenin content. In order to understand the impact of different genetic background in hairy root induction and diosgenin biosynthesis, the described genotypes were chosen for hairy root induction in this study. We established hairy root lines using different strains of A. rizhogenes, ATCC15834, R1000, A4 and C58 in two above-mentioned high- and low-diosgenin producing genotypes of fenugreek. All studied strains successfully induced hairy roots in each genotype. It is pointed out that insertion of T-DNA (Ri-plasmid) containing opines and viral genes, causes hairy root induction (Srivastava and Srivastava, 2007).
4. Discussion For centuries, fenugreek has been globally used for its medical and remedy aims. Fenugreek is also considered as a significant source of diosgenin and several precious compounds. Noteworthy, in comparison with yam, this plant can be a suitable alternative in term of diosgenin production, due to possessing shorter life cycle, ease of production and less cost (Chaudhary et al., 2018; Murlidhar and Goswami, 2012). Diosgenin is extensively used as a precursor of various steroid hormones, and is also demonstrated anticancer and antigenic activities, cardioprotective and contraceptive properties (Chaudhary et al., 2018; Raju and Mehta, 2008; Smith, 2003). Nonetheless, an accurate and reliable strategy for enhanced production of diosgenin is required. Induction of hairy roots using A. rizhogenes strains seems to be a 6
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Fig. 5. Comparison of the transcript abundance in the established hairy roots (induced by R1000 and A4 A. rhizogenes strains) and leaf of high and low producing diosgenin (“Boshruyeh” and “Hamadan” identified with B and H letters, respectively). The leaf also identified with “L” letter. Gene abbreviations: SQS: Squalene synthase, SEP: Squalene epoxidase, CAS: Cycloartenol synthase, SMT1: Setrol-methyltransferase1, C22-H: C22-Hydroxylase, C26H: C26-Hydroxylase.
For experimentally confirmation; therefore, presence of rolB and absence of virD is required. Herein, the same results using PCR assay was observed and the results were consistent with the previous reports (Brijwal and Tamta, 2015; Hashemi and Naghavi, 2016). Comparison of diosgenin content in both wild plant and hairy roots demonstrated that the metabolite content in transformed roots is competitively higher than that of in the wild plant. “Boshruyeh” genotype infected with A4, for instance, showed by 6-fold diosgenin compared with plant itself, indicating a higher potential of hairy root source for producing this valuable metabolite. The lowest-diosgenin producing hairy root (HC58) also showed higher metabolite amount as compared with the high producing genotype in plants (“Boshruyeh”, 30 and 23.8 mg/gDW, respectively). The last step of diosgenin formation, hydrolysis of dioscin to diosgenin (Fig. 1), is converted by a beta-glucosidase. When measuring diosgenin content in the wild plant, Aminkar and coworkers showed dioscin pick as same as the diosgenin peak in chromatography (Fig. S3-B, Supplementary data); consequently, Ionic Liquid Based Ultrasonic Assisted Extraction (ILUAE) method were used to optimize the chemically hydrolysis dioscin to diosgenin (Aminkar et al., 2018). However, in the present study, only one sharp pick referring diosgenin was detected (Fig. S3, Supplementary data). Two important oncogenes (rolB and C genes) carried by the T-DNA of Ri plasmids link to each other and positively contribute to root formation (Spena et al., 1987). It has also previously suggested that these genes encode non-specific betaglucosidase enzymes. This change is probably because of the nonespecific beta glucosidase activity of rolB and C genes of A. rhizogenes. Accordingly, the activity of the enzymes accompanying plant betaglucosidase enzymes enhances the conversion of dioscin to diosgenin; thereby increasing diosgenin accumulation in hairy root lines (Estruch et al., 1991; Faiss et al., 1996). In the previous work, Mohammadi and co-workers proposed the most possible pathway of diosgenin biosynthesis and measured the expression rate of all genes involved in the pathway in various genotypes, which had illustrated different amount of diosgenin (Mohammadi et al., 2019). Combining transcript and metabolite analysis revealed that Δ24-reductase gene, which converts cycloartenol to cycloartanol, is the first-committed and rate-limiting enzyme in this pathway. Transcriptional analysis of functionally important genes involved in diosgenin biosynthesis pathway broadened our horizons in accumulation and regulation of an important metabolite in hairy roots. According to the qRT-PCR results, although each individual strain caused different regulatory pattern, A4 and R1000 up-regulated the expression rate of most studied genes. More previously, Thwe and coworkers reported that A4 and R1000 strains have strong potential in increasing transcript level of most metabolic genes for synthesis of cyanidin 3-O-glucoside, rutin and cyanidin 3-O-rutinoside (Thwe et al., 2016). Diosgenin biosynthesis pathway consists of a branch-point,
where cycloartenol could follow either phytostrol or cholesterol production. The diosgenin formation is ultimately occurred from cholesterol pathway (Sawai et al., 2014). Therefore, the expression level of Δ24-reductase, probably play a pivotal role in diosgenin accumulation. On the other hand, SMT1 redirects biosynthesis pathway toward C-24alkyl phytosterols production (Diener et al., 2000). Our results indicate that the transcript level of Δ24-reductase in A4-mediated hairy roots induction is comparatively higher than the other transformed hairy root lines, whereas expression of SMT1 is up-regulated in R1000-mediated hairy roots induction. These results clearly indicate that A4 could be a promising strain for increased production of diosgenin. These results are in consist with our previous researches, which highlighted the relation of a rate-limiting enzyme and metabolite content (Abedini et al., 2018; Yang et al., 2015). Furthermore, it is also assumed that the probable effect of higher production in hairy root lines is related to the up-regulation of the Δ24-reductase gene. As it can be inferred from the Fig. 5, expression of this gene is considerably higher in HR1000 and BA4 hairy roots than that of in the plant’s leaf; thereby increasing the biosynthesis of diosgenin in the hairy roots which also further confirm our previous findings (Mohammadi et al., 2019). These study results revealed that not only Δ24-reductase plays a critical role in directing the metabolic flux toward the diosgenin production, but also the increased availability of Isopentenyl diphosphate (IDP) could be the probable cause of considerable difference of metabolite content between the plant and the hairy roots. The IDP is a central intermediate in the biosynthesis of isoprenoids which is synthesized from two routes: the cytosol mevalonate (MVA) and plastid 2-C-methyl-D-erythritol 4phosphate (MEP) pathways (Lange et al., 2000; Morrone et al., 2010). In plants possessing green chloroplast, MEP pathway contributes to biosynthesis of monoterpenes, diterpenes, carotenoids, and the side chains of chlorophylls and plastoquinone (Phillips et al., 2008). Shortly, this pathway is dedicated to produce precursors for these compounds in the given plant. Analyzing the transcription of three rate-limiting genes, i.e. DXR, DXS and HDR, revealed that these genes are highly expressed in the hairy roots. The enzymes are localized in chloroplast where are involved in the biosynthesis of the basic five-carbon precursors IDP and DMADP. Due to the plastidic IDP precursor is transported to cytosol; thus, their expression in hairy roots might indicate to direct involvement of MEP pathway in diosgenin accumulation. Therefore, in hairy roots lacking of chloroplasts, both MVA and MEP pathways could provide IDP precursor for the diosgenin biosynthesis pathway. In this reason, it is highly likely that hairy roots can produce significantly increased amount of diosgenin metabolites compared with the wild plant. The results presented here strengthen the potential applicability of hairy root as not only a scalable platform but also an affordable strategy in fenugreek; skipping an additional step needed for diosgenin purification. Taken together, it can be firmly concluded that the activity of 7
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MEP enzymes, increased expression of Δ24-reductase gene in hairy roots lines and non-specific beta-glucosidase activity of bacterial genes are the fair justification of higher accumulation of diosgenin in hairy roots. The results could be considered to engineer the metabolic flux toward diosgenin biosynthesis so as to improve the production of diosgenin in hairy root cultures of fenugreek, which have been regarded as promising alternative platform as compared with the plant. We hope that this study opened up a new avenue for commercialized production of diosgenin using hairy root platform.
References Abedini, D., Rashidi-Monfared, S., Abbasi, A.R., 2018. The effects of promoter variations of the N-methylcanadine 1-hydroxylase (CYP82Y1) gene on the noscapine production in opium poppy. Sci. Rep. 8, 4973. https://doi.org/10.1038/s41598-018-23351-0. Abedini, D., Rashidi-Monfared, S., 2018. Co-regulation analysis of co-expressed modules under cold and pathogen stress conditions in tomato. Mol. Biol. Rep. 45, 335–345. https://doi.org/10.1007/s11033-018-4166-z. Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. https://doi.org/10.1093/nar/25.17. 3389. Aminkar, S., Shojaeiyan, A., Rashidi-Monfared, S., Ayyari, M., 2018. Quantitative assessment of diosgenin from different ecotypes of Iranian fenugreek (Trigonella foenum-graecum L.) by high-performance liquid chromatography. Int. J. Hortic. Sci. Technol. 5, 115–121. Augustin, J.M., Kuzina, V., Andersen, S.B., Bak, S., 2011. Molecular activities, biosynthesis and evolution of triterpenoid saponins. Phytochemistry 72, 435–457. https://doi.org/10.1016/j.phytochem.2011.01.015. Bourgaud, F., Gravot, A., Milesi, S., Gontier, E., 2001. Production of plant secondary metabolites: a historical perspective. Plant Sci. 161, 839–851. https://doi.org/10. 1016/S0168-9452(01)00490-3. Brijwal, L., Tamta, S., 2015. Agrobacterium rhizogenes mediated hairy root induction in endangered Berberis aristata DC. SpringerPlus 4, 443. https://doi.org/10.1186/ s40064-015-1222-1. Chaudhary, S., Chaudhary, P.S., Chikara, S.K., Sharma, M.C., Iriti, M., 2018. Review on fenugreek (Trigonella foenum-graecum L.) and its important secondary metabolite diosgenin. Not. Bot. Horti Agrobot. Cluj. 46, 22–31. https://doi.org/10.15835/ nbha46110996. Costantino, P., Capone, I., Cardarelli, M., De Paolis, A., Mauro, M., Trovato, M., 1994. Bacterial plant oncogenes: therol genes’ saga. Genetica 94, 203–211. https://doi.org/ 10.1007/BF01443434. Diener, A.C., Li, H., Zhou, W.-x., Whoriskey, W.J., Nes, W.D., Fink, G.R., 2000. Sterol methyltransferase 1 controls the level of cholesterol in plants. Plant Cell 12, 853–870. https://doi.org/10.1105/tpc.12.6.853. Dörnenburg, H., Knorr, D., 1995. Strategies for the improvement of secondary metabolite production in plant cell cultures. Enzyme Microb. Technol. 17, 674–684. https://doi. org/10.1016/0141-0229(94)00108-4. Estruch, J., Chriqui, D., Grossmann, K., Schell, J., Spena, A., 1991. The plant oncogene rolC is responsible for the release of cytokinins from glucoside conjugates. EMBO J. 10, 2889–2895. Faiss, M., Strnad, M., Redig, P., Doležal, K., Hanuš, J., Van Onckelen, H., Schmülling, T., 1996. Chemically induced expression of the rolC‐encoded β‐glucosidase in transgenic tobacco plants and analysis of cytokinin metabolism: rolC does not hydrolyze endogenous cytokinin glucosides in planta. Plant J. 10, 33–46. https://doi.org/10. 1046/j.1365-313X.1996.10010033.x. Hashemi, S.M., Naghavi, M.R., 2016. Production and gene expression of morphinan alkaloids in hairy root culture of Papaver orientale L. using abiotic elicitors. Plant Cell Tissue Organ Cult. 125, 31–41. https://doi.org/10.1007/s11240-015-0927-8. Ibañez, S., Talano, M., Ontañon, O., Suman, J., Medina, M.I., Macek, T., Agostini, E., 2016. Transgenic plants and hairy roots: exploiting the potential of plant species to remediate contaminants. N. Biotechnol. 33, 625–635. https://doi.org/10.1016/j.nbt. 2015.11.008. Kakeshpour, T., Nayebi, S., Rashidi-Monfared, S., Moieni, A., Karimzadeh, G., 2015. Identification and expression analyses of MYB and WRKY transcription factor genes in Papaver somniferum L. Physiol. Mol. Biol. Plants 21, 465–478. https://doi.org/10. 1007/s12298-015-0325-z. Lange, B.M., Rujan, T., Martin, W., Croteau, R., 2000. Isoprenoid biosynthesis: the evolution of two ancient and distinct pathways across genomes. Proc. Natl. Acad. Sci. U. S. A. 97, 13172–13177. https://doi.org/10.1073/pnas.240454797. Laule, O., Fürholz, A., Chang, H.-S., Zhu, T., Wang, X., Heifetz, P.B., Gruissem, W., Lange, M., 2003. Crosstalk between cytosolic and plastidial pathways of isoprenoid biosynthesis in Arabidopsis thaliana. Proc. Natl. Acad. Sci. U. S. A. 100, 6866–6871. https://doi.org/10.1073/pnas.1031755100. Li, S., Cong, Y., Liu, Y., Wang, T., Shuai, Q., Chen, N., Gai, J., Li, Y., 2017. Optimization of Agrobacterium-mediated transformation in soybean. Front. Plant Sci. 8, 246. https:// doi.org/10.3389/fpls.2017.00246. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using realtime quantitative PCR and the 2− ΔΔCT method. Methods 25, 402–408. https://doi. org/10.1006/meth.2001.1262. Martin, E., Akan, H., Ekici, M., Aytac, Z., 2011. New chromosome numbers in the genus Trigonella L. (Fabaceae) from Turkey. Afr. J. Biotechnol. 10, 116–125. Mastakani, F.D., Pagheh, G., Rashidi-Monfared, S., Shams-Bakhsh, M., 2018. Identification and expression analysis of a microRNA cluster derived from pre-ribosomal RNA in Papaver somniferum L. and Papaver bracteatum L. PLoS One 13, e0199673. https://doi.org/10.1371/journal.pone.0199673. Mohammadi, M., Mashayekh, T., Rashidi-Monfared, S., Ebrahimi, A., Abedini, D., 2019. New insights into diosgenin biosynthesis pathway and its regulation in Trigonella foenum-graecum L. Phytochem. Anal. https://doi.org/10.1002/pca.2887. Morrone, D., Lowry, L., Determan, M.K., Hershey, D.M., Xu, M., Peters, R., 2010. Increasing diterpene yield with a modular metabolic engineering system in E. coli: comparison of MEV and MEP isoprenoid precursor pathway engineering. Appl. Microbiol. Biotechnol. 85, 1893–1906. https://doi.org/10.1007/s00253-009-2219-x. Murlidhar, M., Goswami, T., 2012. A review on the functional properties, nutritional content, medicinal utilization and potential application of fenugreek. J. Food Process.
5. Conclusion The induction of hairy roots in fenugreek considerably increased the diosgenin content as compared with the plant. Based on the carried out experiments, some potential reasons were proposed which probably contribute to the increased accumulation of diosgenin in the hairy roots. Firstly, the chromatic graphs analysis indicate a single sharp pick referring diosgenin compound that may due to the none-specific beta glucosidase activity of rolB and rolC genes of A. rhizogenes, which could convert diocin precursor to diosgenin accompanied by plant beta-glucosidase enzymes. Secondly, the expression of Δ24-reductase, as a ratelimiting enzyme in the pathway, is highly expressed as compared with SMT1 in hairy roots. Two determinative genes, that is, Δ24‐reductase and SMT1, taking part in the branch‐point of diosgenin and C‐24‐alkyl phytosterols production, respectively. The up-regulation of Δ24‐reductase plays an essential role in directing the metabolic flux toward the diosgenin biosynthesis. Thirdly, plastid DXR, DXS and HDR genes are highly expressed in the induced hairy roots that naturally produce the basic five-carbon precursors IDP which is utilized for plastid development. As hairy roots lack differentiated chloroplast, plastidic IDP precursor is transported to cytosol; thus, the expression of the plastid key genes in hairy roots might indicate to direct involvement of MEP pathway in diosgenin accumulation. As a result, the higher availability of IDP stemming from both MVA and MEP pathways can increase the precursor of diosgenin production. By and large, due to the difficult culturing and lower amount of diosgenin in fenugreek plant, it seems that application of hairy root lines could be a promising way to scale-up diosgenin production. CRediT authorship contribution statement Farnaz Zolfaghari: Investigation, Formal analysis, Writing - original draft, Visualization. Sajad Rashidi-Monfared: Conceptualization, Methodology, Data curation, Writing - review & editing, Supervision, Project administration. Ahmad Moieni: Conceptualization, Methodology, Data curation, Writing - review & editing, Supervision, Project administration. Davar Abedini: Investigation, Formal analysis, Writing - original draft, Visualization. Amin Ebrahimi: Writing - review & editing. Declaration of Competing Interest The authors declare that there are no conflicts of interest. Acknowledgments The authors would like to acknowledge Tarbiat Modares University (TMU), Iran, for funding and supporting this work. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.indcrop.2019.112075. 8
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F. Zolfaghari, et al. Technol. 3. Murray, M.G., Thompson, W.F., 1980. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 8, 4321–4326. https://doi.org/10.1093/nar/8.19.4321. Murthy, H.N., Dandin, V.S., Zhong, J.-J., Paek, K.-Y., 2014. Strategies for Enhanced Production of Plant Secondary Metabolites from Cell and Organ Cultures, Production of Biomass and Bioactive Compounds Using Bioreactor Technology. Springer, pp. 471–508. Nilsson, O., Olsson, O., 1997. Getting to the root: the role of the Agrobacterium rhizogenes rol genes in the formation of hairy roots. Physiol. Plant. 100, 463–473. https:// doi.org/10.1111/j.1399-3054.1997.tb03050.x. Phillips, M.A., León, P., Boronat, A., Rodríguez-Concepción, M., 2008. The plastidial MEP pathway: unified nomenclature and resources. Trends Plant Sci. 13, 619–623. https://doi.org/10.1016/j.tplants.2008.09.003. Qian, Z.G., Zhao, Z.J., Xu, Y., Qian, X., Zhong, J., 2005. Highly efficient strategy for enhancing taxoid production by repeated elicitation with a newly synthesized jasmonate in fed‐batch cultivation of Taxus chinensis cells. Biotechnol. Bioeng. 90, 516–521. https://doi.org/10.1002/bit.20460. Raju, J., Mehta, R., 2008. Cancer chemopreventive and therapeutic effects of diosgenin, a food saponin. Nutr. Cancer 61, 27–35. https://doi.org/10.1080/ 01635580802357352. Ramos-Valdivia, A.C., van der Heijden, R., Verpoorte, R., 1997. Elicitor-mediated induction of anthraquinone biosynthesis and regulation of isopentenyl diphosphate isomerase and farnesyl diphosphate synthase activities in cell suspension cultures of Cinchona robusta how. Planta 203, 155–161. https://doi.org/10.1007/ s004250050177. Sawai, S., Ohyama, K., Yasumoto, S., Seki, H., Sakuma, T., Yamamoto, T., Takebayashi, Y., Kojima, M., Sakakibara, H., Aoki, T., 2014. Sterol side chain reductase 2 is a key enzyme in the biosynthesis of cholesterol, the common precursor of toxic steroidal
glycoalkaloids in potato. Plant Cell 26, 3763–3774. https://doi.org/10.1105/tpc.114. 130096. Setamam, N.M., Sidik, N.J., Rahman, Z.A., Zain, C.R., 2014. Induction of hairy roots by various strains of Agrobacterium rhizogenes in different types of Capsicum species explants. BMC Res. Notes 7, 414. https://doi.org/10.1186/1756-0500-7-414. Smith, M.J.A.M.R., 2003. Therapeutic applications of fenugreek. Altern. Med. Rev. 8, 20–27. Spena, A., Schmülling, T., Koncz, C., Schell, J., 1987. Independent and synergistic activity of rol A, B and C loci in stimulating abnormal growth in plants. EMBO J. 6, 3891–3899. Srivastava, S., Srivastava, A.K., 2007. Hairy root culture for mass-production of highvalue secondary metabolites. Crit. Rev. Biotechnol. 27, 29–43. https://doi.org/10. 1080/07388550601173918. Thwe, A., Valan Arasu, M., Li, X., Park, C.H., Kim, S.J., Al-Dhabi, N.A., Park, S.U., 2016. Effect of different Agrobacterium rhizogenes strains on hairy root induction and phenylpropanoid biosynthesis in tartary buckwheat (Fagopyrum tataricum Gaertn). Front. Microbiol. 7, 318. https://doi.org/10.3389/fmicb.2016.00318. Vaidya, K., Ghosh, A., Kumar, V., Chaudhary, S., Srivastava, N., Katudia, K., Tiwari, T., Chikara, S.K., 2013. De novo transcriptome sequencing inTrigonella foenum-graecum L. to identify genes involved in the biosynthesis of diosgenin. Plant Genome 6. https://doi.org/10.3835/plantgenome2012.08.0021. Vincken, J.-P., Heng, L., de Groot, A., Gruppen, H., 2007. Saponins, classification and occurrence in the plant kingdom. Phytochemistry 68, 275–297. https://doi.org/10. 1016/j.phytochem.2006.10.008. Yang, K., Rashidi-Monfared, S., Wang, H., Lundgren, A., Brodelius, P.E., 2015. The activity of the artemisinic aldehyde Δ11 (13) reductase promoter is important for artemisinin yield in different chemotypes of Artemisia annua L. Plant Mol. Biol. 88, 325–340. https://doi.org/10.1007/s11103-015-0284-3.
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Improving diosgenin production and its biosynthesis in Trigonella foenumgraecum L. hairy root cultures
T
Farnaz Zolfagharia, Sajad Rashidi-Monfareda,*, Ahmad Moienib, Davar Abedinia, Amin Ebrahimic a
Agricultural Biotechnology Department, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran Genetics and Plant Breeding Department, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran c Agronomy and Plant Breeding Department, Faculty of Agriculture, Shahrood University of Technology, Semnan, Iran b
ARTICLE INFO
ABSTRACT
Keywords: Agrobacterium rhizogenes Diosgenin Gene expression Hairy roots Fenugreek
Induction of hairy roots stemmed from Agrobacterium rhizogenes transformation, is a key step to reach mass production of secondary metabolites in medicinal plants. Accordingly, this study is aimed to investigate potential impact of different A. rhizogenes strains, including ATCC15834, R1000, A4 and C58, on diosgenin biosynthesis in high- “Boshruyeh” (23.8 mg/gDW) and low “Hamadan” (6.4 mg/gDW) diosgenin-producing genotypes of fenugreek. Interestingly, comparing the metabolite extracted from both leaves and hairy roots of these genotypes suggested that hairy roots are promising platform to produce scalable amount of diosgenin, producing 143.96 mg/gDW in high diosgenin producing hairy roots (A4-mediated hairy roots induction in “Boshruyeh” (BA4)), while 23.8 mg/gDW in high diosgenin producing genotype. Transcript abundance of 10 genes involved in diosgenin biosynthesis pathway as well as three rate-limiting genes; i.e 1-deoxy-D-xylulose-5-phosphate synthase (DXS), 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) and 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, functioning in plastid 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway were measured. Comparison of the transcription and metabolite analysis revealed that the more expression of Δ24-reductase gene, the higher accumulation of diosgenin in the hairy roots. In high diosge nin producing hairy roots, the expression of Δ24-reductase gene were significantly up-regulated (8-folds). Moreover, integration of metabolite and transcript results revealed that A4 has a strong capacity in boosting diosgenin accumulation in the both genotypes. These results may lead to the next step in production of diosgenin in scalable and commercial levels.
1. Introduction Trigonella foenum-graecum, is one of the world-appealing medicinal plant belonging to the Fabaceae family that has been extensively used from ancient time to the current era. This plant is an annual dicotyledonous legume with 0.7 of C-value, which is about 1.5 fold higher than that is found in Lotus corniculatus, as a model legume (Chaudhary et al., 2018; Martin et al., 2011). Fenugreek possesses a number of valuable phytochemicals, including alkaloids, steroids, flavonoids, saponins lysine-rich proteins, and volatile oils. Among these, diosgenin is a wellknown bioactive compound, which received a considerable attention owing to its potential feature in anticancer and anti-aging activities (Raju and Mehta, 2008; Smith, 2003). Moreover, this metabolite is broadly utilized in pharmaceutical industry as a precursor of synthetic steroidal drugs such as testosterone, progesterone, norethisterone and glucocorticoids (Chaudhary et al., 2018; Murlidhar and Goswami,
⁎
2012). Diosgenin is also known as a naturally occurring phytochemical found in foodstuffs as a controlling agent for cancer chemoprevention (Raju and Mehta, 2008). Consequently, providing a reliable and affordable platform so as to scale-up this precious compound is desired. More recently, Aminkar and co-workers measured the diosgenin content in several genotypes collected from different region of Iran, and realized that there are a considerable differences among these samples (Aminkar et al., 2018). Until now, a plethora of precious metabolites have been discovered and clinically verified; nonetheless, the main concern to utilize such compounds are the less-abundance and being tissue-specific of these metabolites in the given plants (Murthy et al., 2014). For many known and unknown reasons, plants often produce secondary metabolites in low quantities. As a consequence, a number of strategies have widely been recommended to enhance the amount of valuable compounds (Dörnenburg and Knorr, 1995; Murthy et al., 2014; Qian et al., 2005).
Corresponding author. E-mail address: [email protected] (S. Rashidi-Monfared).
https://doi.org/10.1016/j.indcrop.2019.112075 Received 19 August 2019; Received in revised form 24 December 2019; Accepted 27 December 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.
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In this regard, transformed (hairy) roots could be the stepping-stone in term of achieving sufficient medicinal metabolites. In addition, this strategy is a scalable and affordable as compared with other biotechnological means (Bourgaud et al., 2001; Ibañez et al., 2016). The neoplastic tissues resulted in Agrobacterium rhizogenes characterized by high growth rate, genetic stability and growth in hormone free media producing comparable metabolites than intact plants (Srivastava and Srivastava, 2007). The rol oncogenes including rolA, B, C and D, carrying on the Ri plasmid, play a fundamental role in hairy root induction (Costantino et al., 1994; Nilsson and Olsson, 1997). Different strains of A. rhizogenes have showed different capability in induction efficiency (Li et al., 2017; Thwe et al., 2016). So far, several strains, including A4, ATCC15834, LBA9402, MAFF03–01724, R-1601, R-1000, and TR105, have been applied in hairy root induction in quite a few number of herbs (Setamam et al., 2014; Thwe et al., 2016). The biosynthesis pathway of diosgenin has been generally constructed and some related enzymes have been experimentally characterized (Sawai et al., 2014; Vaidya et al., 2013). Likewise other group of triterpenes, diosgenin is formed by condensing of C5 units of isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMAPP), catalyzing by isopentenyl diphosphate isomerase (Ramos-Valdivia et al., 1997). This reaction leads to production of a key diosgenin precursor; farnesyl pyrophosphate (Vincken et al., 2007). Isopentenyl diphosphate can be synthesized from both 2-C-methyl-D-erythritol 4phosphate (MEP) and mevalonate (MVA) pathways (Augustin et al., 2011). Laule and co-workers showed a cross-talk between biosynthesis of isoprenoid in cytosolic and plastidial pathways (Laule et al., 2003). Studying the expression rate of important genes can provide new insight into engineering metabolic pathways, and suggesting a reliable and efficient source for scaling up pharmacologically important metabolites. Previously, we investigated the regulation of rate-limiting enzymes and elements involving in metabolite accumulation in known herbs, which spotlighted new areas of interests in medicinal plants (Abedini et al., 2018; Abedini and Rashidi-Monfared, 2018; Kakeshpour et al., 2015; Mastakani et al., 2018; Yang et al., 2015). Herein, the study focused on induction of hairy roots by different strains of A. rhizogenes in T. foenum-graecum L. Additionally, the expression level of several genes involved in diosgenin biosynthesis pathway was investigated and the diosgenin content on the established hairy roots were also measured.
explants of both “Boshruyeh” and “Hamadan” genotypes were wounded, and then each individual explants were immersed in liquid WPM medium containing each strain of A. rhizogenes for 15 min. After that, co-cultured explants were dried on a sterile tissue paper. These samples were consequently incubated under dark condition at 25 ± 1 °C for 2 days. The explants were rinsed with sterile distilled water and cefotaxime antibiotic then placed in WPM medium containing 6 mg l−1 agar-agar for one week to remove the residual bacteria from the explants after incubation. Subsequently, explants were cultivated in a WPM medium possessing cefotaxime (350 mg L−1). After emerging hairy roots, samples were cultured in a hormone-free medium without antibiotic with shaking and darkness conditions at 25 ± 1 °C for one month. Eventually, hairy roots were harvested to further analysis such as assessing the gene expression levels and measuring the diosgenin content. 2.2. Genomic DNA extraction and PCR assay Genomic DNA was extracted for the studied hairy root lines using the Cetyl trimethylammonium bromide (CTAB) method (Murray and Thompson, 1980). To confirm and verify the obtained hairy roots, two pairs of primers were designed from a part of rolB and virD, and then amplified using PCR assay. The program was as follow: initial denaturation at 95 °C for 3 min for 1 cycle, followed with 40 cycles including; 95 °C for 30 s, 54 °C for 30 and elongation for 40 s. After finishing the PCR, PCR products were run on the 1 % of agarose gelelectrophoresis. The sequences of both the primers are listed in the Table 1. 2.3. HPLC analysis The obtained hairy roots were dried using freeze drier and subsequently ground into a fine powder. Then the 200 mg of the each samples, consisting of three replications, were immersed with 20 ml of 96 % ethanol, and were incubated in a sonicator water bath (UltrasonicCleanerXPS240-4 L water baths (Sharpertek, USA)) with a frequency of 60 kHz for 2 h. Afterward, 20 ml of H2SO4 (1 mol/L) was added to the samples and placed at 121 °C for 2 h. Obtained samples were washed three times with n-Hexan, (1 mol/L), NaOH, respectively. The samples were finally rinsed with distilled water. The supernatant phase was removed and the debris washed with deionized water and the upper phase removed again. In order to dehydration of the extracts, dried sodium sulfate (Na2SO4) was added to the samples. The extract was dissolved in 500 μl of acetonitrile, HPLC Grade, and filtered through 0.22 μm filter to remove any small particulates. In this study, diosgenin content was measured using High Performance Liquid Chromatography (HPLC), (Waters USA) composed of a K-1001 pump, SPDM20A PDA detector (K-2501, Knauer, Berlin, Germany) on a C18 column, with flow rate of 0.7 mL/min. The chromatographic separation was carried out using a mixture of acetonitrile and water (90:10) solvent. Five concentrations of diosgenin (Sigma-Aldrich-Germany; CAS Number: 512049) i.e. 100, 150, 300, 500 and 1000 ppm were prepared via dissolving in acetonitrile, then placed in ultrasonic for 30 min. These concentrations were consequently injected in 50 μl volume into the HPLC system and the correlation coefficient (R2) was calculated 0.99 for the data sets.
2. Material and method 2.1. Induction and establishment of hairy roots Previously, the diosgenin content of several genotypes collected from different regions of Iran were studied, and “Boshruyeh” (23.8 mg/ gDW) and “Hamadan” (6.4 mg/gDW) demonstrated high- and lowdiosgenin content, respectively. In the present study, with considering previous results, the hairy roots for “Boshruyeh” and “Hamadan” genotypes (Aminkar et al., 2018) were induced. For seed germination, the fenugreek seeds were washed in tap water for 5 min and then soaked in 70 % of ethanol for 1 min, followed by washing with sterilized water. Seed surface were sterilized by 3 % sodium hypochlorite (NaOCl) for 15 min and washed with sterile distilled water. Finally, the seeds were cultured in petri dishes containing Woody Plant Medium (WPM) medium and placed in darkness condition for 4–5 days. After that, germinated seeds were transferred to a WPM medium with photoperiod of 16−8 h for 10 days. Four A. rhizogenes strains including ATCC15834, R1000, A4 and C58 were utilized to induce hairy roots in fenugreek genotypes. These strains were cultured into 10 ml liquid Luria-Bertani (LB) medium containing 100 mg l−1 of Rifampicin at 28 °C and 224 RCF for 24 h in darkness conditions. The optical density (OD) was about 0.6. All strains were stored in glycerol stock at −80 °C. Hairy roots were induced via different strains of A. rhizogenes including ATCC15834, R1000, A4 and C58. For this purpose, hypocotyl
2.4. Read assembly and phylogenetic construction The reads from several RNAseq projects (With SRA accession nos.: SRX2765627, SRX2765626, SRX2765625, SRX2676952, SRX2676951, SRX2666620, SRX2631114, SRX026992, SRX026991, SRX026990, SRX026989, SRX026988) were downloaded from NCBI database (http://www.ncbi.nlm.nih.gov/sra). After identifying reads with highest similarity to the key genes involved in isopenthyl diphosphate in plastid, including 1-deoxy-D-xylulose-5-phosphate synthase (DXS), 12
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Table 1 The list of primers used in validation of transgenic roots indication and gene expression analysis in induced hairy roots of fenugreek. Primer no.
Primer name
Use
Primer sequence
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
rolB-Forward rolB-Reverse virD-Forward virD-Reverse SQS. Forward SQS. Reverse CAS. Forward CAS. Reverse SEP. Forward SEP. Reverse SMT1. Forward SMT1. Reverse Δ24-reductase. Forward Δ24-reductase. Reverse BGL2. Forward BGL2. Reverse C22-hydroxylase. Forward C22-hydroxylase. Reverse C26-hydroxylase. Forward C26-hydroxylase. Reverse C4-demethylase. Forward C4-demethylase. Reverse Δ7 reductase. Forward Δ7 reductase. Reverse DXR. Forward DXR. Reverse DXS. Forward DXS. Reverse HDR.Forward HDR.Reverse GAPDH. Forward GAPDH. Reverse
Hairy roots indication Hairy roots indication Hairy roots indication Hairy roots indication qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR Reference gene Reference gene
5′-gctcttgcagtgctagattt-3′ 5′-gaaggtgcaagctacctctc-3′ 5′-atgtcgcaaggacgtaagccca-3′ 5′-ggagtctttcagcatggagcaa-3′ 5′-ggctcaaccatgattctcatactg-3′ 5′-tacccactgttccattgctatcc-3′ 5′-aagagagatccaacaccactgc-3′ 5′-tacatgacgacggtattctccc-3′ 5′-ctattctcagaaggaccgatctc-3′ 5′-cagatgcaccagagagtaatcg-3′ 5′-gagaaggctgcagagggtctag-3′ 5′-cacagtaactcatgacaacagcaacct-3′ 5′-aggtgggagatatgctagaatggg-3′ 5′-cattctgtgtgtctccctgcc-3′ 5′-cctggtcctgagagcataacaaatg-3′ 5′-catcagccacaccttgtccttc-3′ 5′-ctatggggtgacgatgctaagatg -3′ 5′-tgctattgccatctttgcttcca-3′ 5′-cgttcctaccacggtgccttt-3′ 5′-ttgagtttgttctcctgctctagcc-3′ 5′-atggaactgacaagggttttcggt-3′ 5′-actgcctacaatgcttctgtctca-3′ 5′-ttgaccgagcgaaaagggatga -3′ 5′-gcaccaagtgaaggattccagac-3′ 5-aggcaccatgacaggagttcttag-3 5-gcaaactagcagcatattcgcgt-3 5-cagtaggggtttatgtgacagttgc-3 5-cctccaatagaaccctcttccact-3 5-ggattgacagtgagcagagaattgg-3 5-catcttccacgaccttgtctgg-3 5′-tatgtttgttgttggtgtcaacgagcaacgaatacaag-3′ 5′-atgttaaatgatgcagcccttccacctctc -3′
deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) and 4-hydroxy3-methylbut-2-enyl diphosphate reductase (HDR) from related species, using the offline BLAST software v.2.7.0 (Altschul et al., 1997), they were then assembled into contigs with the Codon Code Aligner v.5.0.1. program (https://www.codoncode.com/aligner) based on the alignthen assemble strategy. The ORF finder tool (https://www.ncbi.nlm. nih.gov/orffinder/) created the ORF of obtained consensus sequences; the specific primers were consequently designed and confirmed using the Oligo Analyzer v.3.1 (http://eu.idtdna.com/calc/analyzer). Furthermore, similar protein sequences for each consensus sequence were queried in the BLAST search tool. The multiple alignment analysis was carried out by online Clustal omega (https://www.ebi.ac.uk/Tools/ msa/clustalo/) program. Then, phylogeny tree was constructed using the MEGA 7 software (MEGA, PA, USA) based on the NeighbourJoining (NJ) method, with the default options and bootstraps analysis (3000 replicates).
workers (Mohammadi et al., 2019), were used. The first strand cDNA was synthesized with one μg of extracted RNA using HyperscriptTM Reverse Transcriptase (GeneAll Inc, South Korea Cat No.: 601-100). The real-time PCR was carried out in three technical replicates with the BioRad system (Bio-Rad, Hercules, CA, USA) using the SYBR®Green PCR Master Mix 2X (Ampliqon, Denmark (Lot No.: A322701)). The qRT-PCR amplification was performed as follows: pre-denaturation at 94 °C for 10 min, followed by 40 cycles consisting of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s and extension at 72 for 20 s. Moreover, the melting curve was acquired by slowly ramping up the temperature from 65 °C to 95 °C. The GAPDH was selected as an internal reference gene. All collected data were analyzed based on the Livak method (2−ΔΔCt) (Livak and Schmittgen, 2001), and the relative expression was calculated via the Microsoft Excel software (Microsoft Office 2013). The studied primers are listed in the Table 1. 3. Results
2.5. RNA extraction, cDNA synthesis and qRT-PCR assay
3.1. Hairy roots establishment and validation of transgenic roots
Total RNA of each hairy root was extracted using Topaz Kit (Top Plant and Fungi RNA Purification Kit, TOPAZ GENE RESEARCH., cat No.: TGK2004, Iran). The quality and quantity of extracted RNA were assessed by gel electrophoresis and a NanoDrop spectrophotometer (BioTek, EPOCH, serial 121004C, USA). Then, to avoid the contamination of genomic DNA, extracted RNA was treated by RNase-free DNaseI (Thermo Fisher Scientific., Cat No.: EN0521). In this study, the transcript abundance of thirteen genes including Squalene synthase (SQS), Squalene epoxidase (SEP), Cycloartenol synthase (CAS), Δ24-reductase, Sterol-methyltransferase (SMT1), (SMT1), C4-demethylas, Δ7reductase, C22-hydroxylase, C26-hydroxylase, 26-O-beta glycosidase (BGL), DXS, DXR and HDR were measured. Specific primers amplifying DXS, DXR and HDR genes were designed, for the other genes, the primers which had previously been designed by Mohammadi and co-
Hairy roots induction was successfully carried out using different A. rhizogenes strains, i.e. ATCC15834, R1000, A4 and C58. All of the utilized strains induced hairy root in genotypes, high- and low-diosgenin content “Boshruyeh” and “Hamadan”, respectively (Fig. 2). Measuring dry weight of established hairy roots indicated to the strong efficiency of ATCC15834 and R1000 strains in both genotypes; however, A4 had comparatively lower biomass production. HATCC15834 and HR1000 represented the highest biomass weight (1.11 g), and HA4 and BA4 showed the lowest production (0.26 and 0.53 g, respectively) (For more details, see Fig. S1. Supplementary data). Molecular indication was obtained using PCR assay for the transformed status of the hairy root lines. Specific primers amplifying rolB and virD genes were utilized in order to distinguish between transformed and untransformed roots 3
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Fig. 1. Biosynthesis pathway of diosgenin in fenugreek. Mevalonate (MVA) pathway in cytosol: Acetyl-CoA converts to Cycloartenol via multiple reactions (pink box). Cycloartenol converts to either cycloartenol, leading to cholesterol formation (red box) following cholesterol converts to 3β-cholesta-5-en-3, 22-diol (blue box), which ultimately results in diosgenin production. Dashed lines (from Acetyl-CoA to farnesyle diphosphate) indicate multiple steps involved in the pathway (Mohammadi et al., 2019). Methylerythritol 4-phosphate (MEP) pathway in plastid: DXS: 1-deoxy-D-xylulose-5-phosphate synthase; DXR: 1-deoxy-D-xylulose-5-phosphate reductoisomerase; MCT: 2-C-methyl-D-erythritol4-(cytidyl-5-diphosphate) transferase; CMK: 4cytidine 5′-diphospho-2-C-methyl-D-erythritol kinase; MCS: 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase; HDS; hydroxy-2-methyl-2(E)-butenyl 4-diphosphate synthase; HDR: hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase.
(Table 1, no. 1–4). According to the results, after amplification, the expected size for rolB gene, transformed from Ri-plasmid was observed, confirming the transgenic nature of the established hairy roots (Fig. S2A supplementary data). On the other hand, no products were detected using virD primers (Fig. S2-B Supplementary data), illustrating the complete elimination of bacteria from the tissues. In all amplifications, Ri-plasmid and untransformed roots were utilized as positive and negative control, respectively. Therefore, by considering these results the influence of each individual strain of A. rhizogenes on two genotypes in both transcriptomic and metabolomics level were investigated.
of “Hamedan” genotype (Fig. 4). Among the studied strains, A4 illustrated a strong capability in accumulating diosgenin. Taken together, based on the results, to produce a considerable amount of diosgenin, suitable plant hosts as well as promising strain are required. Herein, it is observed that “Boshruyeh” genotype and A4 strain could lead to induce a promising hairy root line, which ultimately results in higher production of diosgenin. Interestingly, analyzing the chromatographic curve stemmed from the plants leave illustrates two relatively same peaks, dioscin and diosgenin; one the other hand, HPLC result of the hairy root extracts showed only single sharp peak indicating diosgenin compound (For more detail, see Fig. S3 in Supplementary data). In addition, comparing diosgenin from wild plant and hairy roots demonstrated that the accumulation of this metabolite in hairy roots is considerably higher than the plant (23.8 mg/gDW in “Boshruyeh” as a high-diosgenin producing genotype), indicating to the robust capacity of the fenugreek hairy roots to scale-up the diosgenin accumulation.
3.2. Diosgenin content in hairy roots of Fenugreek In this study, the amount of diosgenin extracted from various fenugreek hairy root lines were measured. According to the HPLC results, “Boshruyeh” genotype infected with A4 strain showed dominant content of diosgenin (143.96 mg/gDW) as compared with other treatments. On the other hand, “Hamedan” genotype infected with C58 strain demonstrated lower amount of this metabolite (29.9 mg/gDW). In general, the amount of diosgenin in hairy roots stemmed from “Boshruyeh” genotype, except one infected with R1000 strain, was higher than those
3.3. Expression of diosgenin biosynthesis pathway genes To evaluate the potential effects of different strains of A. rhizogenes on transcription of genes functioning in diosgenin pathway, the 4
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expression level of several genes were assessed using real-time PCR in resultant hairy roots. To this end, the following genes were studied: SQS, SEP, CAS, Δ24-reductase, SMT1, C4-demethylas, Δ7-reductase, C22-hydroxylase, C26-hydroxylase and BGL2. Given the results, expression of all genes illustrated a different pattern when the explant transformed with different strains, indicating that each strain has a different pattern of gene regulation in the fenugreek host (Fig. 4). As we expected, the expression level of some determinative genes, i.e. SQS, CAS and Δ24-reductase, in hairy roots associated with “Boshruyeh” genotype were comparatively higher than those in the “Hamdan” genotype, which obviously indicates the importance of genotype for induction of hairy roots. The expression of SQS and CAS in the highest producing diosgenin hairy root (A4-mediated hairy roots induction in “Boshruyeh” (BA4)) up-regulated 4.3 and 6.5 fold higher than those in ATCC15834-mediated hairy roots induction in “Hamdan” (H15834), respectively; in addition, expression of Δ24-reductase in BA4 were 4fold higher than that of in A4-mediated hairy roots in induction “Hamdan”. In general, A4 and R1000 strains caused the higher expression in a majority of genes (Fig. 3). The expression of Δ24-reductase, CAS and SQS in “Boshruyeh”, for instance, which have a great impact on the production of diosgenin, were higher when infected with A4 strain, 5, 5 and 8 folds, respectively. In contrast, in roots infected with ATCC15834 strain, the transcript level of three important upstream genes of the diosgenin biosynthetic pathway, i.e. Δ24-reductase, CAS, SQS, were considerably lower than the expression of the rest of the studied genes. In the diosgenin biosynthetic pathway, there is a branch point where two determinative genes including SMT1 and Δ24-reductase take part, which leads to the production of either diosgenin or phytosterol (24alkyl sterols) (Fig. 1). The expression of Δ24-reductase gene leading to production of diosgenin, was higher in “Boshruyeh” genotype infected with A4 strain. Similarly, the expression of BGL2 gene reached its peak in “Boshruyeh” genotype infected with R1000 strain. This gene is also considered as an important gene, converting the last step of diosgenin biosynthesis. However, due to down-regulation of Δ24-reductase in “Boshruyeh” infected with R1000, the metabolite content in this treatment is relatively low. In other words, down-regulation of Δ24-reductase, as a rate-limiting enzyme, results in lower production of
Fig. 3. The amount of diosgenin content in hairy roots of fenugreek induced by different A. rhizogenes strains, i.e. ATCC15834, R1000, A4 and C58. Two genotypes of fenugreek including “Boshruyeh” and “Hamadan” (identified with B and H, respectively) were used as host plant.
substrates needed for the downstream enzymes involved in diosgenin biosynthesis pathway. Interestingly, comparing transcript abundance of the genes involved in diosgenin biosynthesis in the hairy roots induced by R1000 (HR1000) and A4 (BA4) strains that produce higher amount of diosgenin (Fig. 3), and leaf of two high- and low diosgenin-producing genotypes (Mohammadi et al., 2019) showed increased expression of all genes in hairy root of both genotypes relative to their plant leaves (Figs. 5 and S7). The expression of DXR, DXS and HDR genes, which are key genes regulating terpenoid biosynthesis pathway in plastids were also measured. It is reported that DXS and DXR are key genes at the beginning of the plastid MEP pathway, and HDR is the last rat-limiting enzyme of the pathway (Morrone et al., 2010) (Fig. 1). Herein, the results interestingly demonstrated that these genes are highly expressed in induced hairy roots (Fig. 4). Phylogenetic analysis also revealed that DXR, DXS and HDR genes of fenugreek are closely linked with those in Medicago truncatula, indicating to having a common ancestor for both the barrelclover and fenugreek (Supplementary data S4-6).
Fig. 2. Sequential stages representing hairy root induction in Fenugreek. A) seed germination, B) hairy root induction, C) hairy toots growth after three weeks induction, D) transporting the hairy roots from solid to liquid medium, E) three weeks after culturing in a liquid medium, F) preparation and measurement of fenugreek hairy roots before storing in −80 °C freezer.
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Fig. 4. Relative expression analysis of diosgenin biosynthetic genes in hairy roots of fenugreek induced by different A. rhizogenes strains, i.e. R15834, R1000, A4 and C58. Abbreviations: SQS: Squalene synthase, SEP: Squalene epoxidase, CAS: Cycloartenol synthase, Δ24-reductase, SMT1: Setrol-methyltransferase1, BGL2: 26-O-beta glycosidase, DXS: 1-deoxy-D-xylulose-5-phosphate synthase, DXR: 1-deoxy-D-xylulose 5-phosphate reductoisomerase and HDR: 4-hydroxy-3-methylbut-2-enyl diphosphate reductase. The lines on each bar represent standard deviation. Two genotypes of fenugreek including “Boshruyeh” and “Hamadan” are identified with B and H, respectively. The expression of the genes were relatively compared with control samples. The control sample for Δ24-reductase, SQS, SMT1 was B.C58; for SEP, CAS, and BGL2 was H.15834; for C4-demethylas, Δ7-reductase, C22-Hydroxylase, C26-Hydroxylase was B.15834; for DXS, DXR and HDR was H.A4
promising way, since it is a superior method with several privileges over callus and cell culture (Srivastava and Srivastava, 2007). Previously, we measured diosgenin content of several fenugreek genotypes collected from different regions of Iran. The result showed that “Boshruyeh” and “Hamedan” genotypes have the highest and lowest diosgenin content. In order to understand the impact of different genetic background in hairy root induction and diosgenin biosynthesis, the described genotypes were chosen for hairy root induction in this study. We established hairy root lines using different strains of A. rizhogenes, ATCC15834, R1000, A4 and C58 in two above-mentioned high- and low-diosgenin producing genotypes of fenugreek. All studied strains successfully induced hairy roots in each genotype. It is pointed out that insertion of T-DNA (Ri-plasmid) containing opines and viral genes, causes hairy root induction (Srivastava and Srivastava, 2007).
4. Discussion For centuries, fenugreek has been globally used for its medical and remedy aims. Fenugreek is also considered as a significant source of diosgenin and several precious compounds. Noteworthy, in comparison with yam, this plant can be a suitable alternative in term of diosgenin production, due to possessing shorter life cycle, ease of production and less cost (Chaudhary et al., 2018; Murlidhar and Goswami, 2012). Diosgenin is extensively used as a precursor of various steroid hormones, and is also demonstrated anticancer and antigenic activities, cardioprotective and contraceptive properties (Chaudhary et al., 2018; Raju and Mehta, 2008; Smith, 2003). Nonetheless, an accurate and reliable strategy for enhanced production of diosgenin is required. Induction of hairy roots using A. rizhogenes strains seems to be a 6
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Fig. 5. Comparison of the transcript abundance in the established hairy roots (induced by R1000 and A4 A. rhizogenes strains) and leaf of high and low producing diosgenin (“Boshruyeh” and “Hamadan” identified with B and H letters, respectively). The leaf also identified with “L” letter. Gene abbreviations: SQS: Squalene synthase, SEP: Squalene epoxidase, CAS: Cycloartenol synthase, SMT1: Setrol-methyltransferase1, C22-H: C22-Hydroxylase, C26H: C26-Hydroxylase.
For experimentally confirmation; therefore, presence of rolB and absence of virD is required. Herein, the same results using PCR assay was observed and the results were consistent with the previous reports (Brijwal and Tamta, 2015; Hashemi and Naghavi, 2016). Comparison of diosgenin content in both wild plant and hairy roots demonstrated that the metabolite content in transformed roots is competitively higher than that of in the wild plant. “Boshruyeh” genotype infected with A4, for instance, showed by 6-fold diosgenin compared with plant itself, indicating a higher potential of hairy root source for producing this valuable metabolite. The lowest-diosgenin producing hairy root (HC58) also showed higher metabolite amount as compared with the high producing genotype in plants (“Boshruyeh”, 30 and 23.8 mg/gDW, respectively). The last step of diosgenin formation, hydrolysis of dioscin to diosgenin (Fig. 1), is converted by a beta-glucosidase. When measuring diosgenin content in the wild plant, Aminkar and coworkers showed dioscin pick as same as the diosgenin peak in chromatography (Fig. S3-B, Supplementary data); consequently, Ionic Liquid Based Ultrasonic Assisted Extraction (ILUAE) method were used to optimize the chemically hydrolysis dioscin to diosgenin (Aminkar et al., 2018). However, in the present study, only one sharp pick referring diosgenin was detected (Fig. S3, Supplementary data). Two important oncogenes (rolB and C genes) carried by the T-DNA of Ri plasmids link to each other and positively contribute to root formation (Spena et al., 1987). It has also previously suggested that these genes encode non-specific betaglucosidase enzymes. This change is probably because of the nonespecific beta glucosidase activity of rolB and C genes of A. rhizogenes. Accordingly, the activity of the enzymes accompanying plant betaglucosidase enzymes enhances the conversion of dioscin to diosgenin; thereby increasing diosgenin accumulation in hairy root lines (Estruch et al., 1991; Faiss et al., 1996). In the previous work, Mohammadi and co-workers proposed the most possible pathway of diosgenin biosynthesis and measured the expression rate of all genes involved in the pathway in various genotypes, which had illustrated different amount of diosgenin (Mohammadi et al., 2019). Combining transcript and metabolite analysis revealed that Δ24-reductase gene, which converts cycloartenol to cycloartanol, is the first-committed and rate-limiting enzyme in this pathway. Transcriptional analysis of functionally important genes involved in diosgenin biosynthesis pathway broadened our horizons in accumulation and regulation of an important metabolite in hairy roots. According to the qRT-PCR results, although each individual strain caused different regulatory pattern, A4 and R1000 up-regulated the expression rate of most studied genes. More previously, Thwe and coworkers reported that A4 and R1000 strains have strong potential in increasing transcript level of most metabolic genes for synthesis of cyanidin 3-O-glucoside, rutin and cyanidin 3-O-rutinoside (Thwe et al., 2016). Diosgenin biosynthesis pathway consists of a branch-point,
where cycloartenol could follow either phytostrol or cholesterol production. The diosgenin formation is ultimately occurred from cholesterol pathway (Sawai et al., 2014). Therefore, the expression level of Δ24-reductase, probably play a pivotal role in diosgenin accumulation. On the other hand, SMT1 redirects biosynthesis pathway toward C-24alkyl phytosterols production (Diener et al., 2000). Our results indicate that the transcript level of Δ24-reductase in A4-mediated hairy roots induction is comparatively higher than the other transformed hairy root lines, whereas expression of SMT1 is up-regulated in R1000-mediated hairy roots induction. These results clearly indicate that A4 could be a promising strain for increased production of diosgenin. These results are in consist with our previous researches, which highlighted the relation of a rate-limiting enzyme and metabolite content (Abedini et al., 2018; Yang et al., 2015). Furthermore, it is also assumed that the probable effect of higher production in hairy root lines is related to the up-regulation of the Δ24-reductase gene. As it can be inferred from the Fig. 5, expression of this gene is considerably higher in HR1000 and BA4 hairy roots than that of in the plant’s leaf; thereby increasing the biosynthesis of diosgenin in the hairy roots which also further confirm our previous findings (Mohammadi et al., 2019). These study results revealed that not only Δ24-reductase plays a critical role in directing the metabolic flux toward the diosgenin production, but also the increased availability of Isopentenyl diphosphate (IDP) could be the probable cause of considerable difference of metabolite content between the plant and the hairy roots. The IDP is a central intermediate in the biosynthesis of isoprenoids which is synthesized from two routes: the cytosol mevalonate (MVA) and plastid 2-C-methyl-D-erythritol 4phosphate (MEP) pathways (Lange et al., 2000; Morrone et al., 2010). In plants possessing green chloroplast, MEP pathway contributes to biosynthesis of monoterpenes, diterpenes, carotenoids, and the side chains of chlorophylls and plastoquinone (Phillips et al., 2008). Shortly, this pathway is dedicated to produce precursors for these compounds in the given plant. Analyzing the transcription of three rate-limiting genes, i.e. DXR, DXS and HDR, revealed that these genes are highly expressed in the hairy roots. The enzymes are localized in chloroplast where are involved in the biosynthesis of the basic five-carbon precursors IDP and DMADP. Due to the plastidic IDP precursor is transported to cytosol; thus, their expression in hairy roots might indicate to direct involvement of MEP pathway in diosgenin accumulation. Therefore, in hairy roots lacking of chloroplasts, both MVA and MEP pathways could provide IDP precursor for the diosgenin biosynthesis pathway. In this reason, it is highly likely that hairy roots can produce significantly increased amount of diosgenin metabolites compared with the wild plant. The results presented here strengthen the potential applicability of hairy root as not only a scalable platform but also an affordable strategy in fenugreek; skipping an additional step needed for diosgenin purification. Taken together, it can be firmly concluded that the activity of 7
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MEP enzymes, increased expression of Δ24-reductase gene in hairy roots lines and non-specific beta-glucosidase activity of bacterial genes are the fair justification of higher accumulation of diosgenin in hairy roots. The results could be considered to engineer the metabolic flux toward diosgenin biosynthesis so as to improve the production of diosgenin in hairy root cultures of fenugreek, which have been regarded as promising alternative platform as compared with the plant. We hope that this study opened up a new avenue for commercialized production of diosgenin using hairy root platform.
References Abedini, D., Rashidi-Monfared, S., Abbasi, A.R., 2018. The effects of promoter variations of the N-methylcanadine 1-hydroxylase (CYP82Y1) gene on the noscapine production in opium poppy. Sci. Rep. 8, 4973. https://doi.org/10.1038/s41598-018-23351-0. Abedini, D., Rashidi-Monfared, S., 2018. Co-regulation analysis of co-expressed modules under cold and pathogen stress conditions in tomato. Mol. Biol. Rep. 45, 335–345. https://doi.org/10.1007/s11033-018-4166-z. Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. https://doi.org/10.1093/nar/25.17. 3389. Aminkar, S., Shojaeiyan, A., Rashidi-Monfared, S., Ayyari, M., 2018. Quantitative assessment of diosgenin from different ecotypes of Iranian fenugreek (Trigonella foenum-graecum L.) by high-performance liquid chromatography. Int. J. Hortic. Sci. Technol. 5, 115–121. Augustin, J.M., Kuzina, V., Andersen, S.B., Bak, S., 2011. Molecular activities, biosynthesis and evolution of triterpenoid saponins. Phytochemistry 72, 435–457. https://doi.org/10.1016/j.phytochem.2011.01.015. Bourgaud, F., Gravot, A., Milesi, S., Gontier, E., 2001. Production of plant secondary metabolites: a historical perspective. Plant Sci. 161, 839–851. https://doi.org/10. 1016/S0168-9452(01)00490-3. Brijwal, L., Tamta, S., 2015. Agrobacterium rhizogenes mediated hairy root induction in endangered Berberis aristata DC. SpringerPlus 4, 443. https://doi.org/10.1186/ s40064-015-1222-1. Chaudhary, S., Chaudhary, P.S., Chikara, S.K., Sharma, M.C., Iriti, M., 2018. Review on fenugreek (Trigonella foenum-graecum L.) and its important secondary metabolite diosgenin. Not. Bot. Horti Agrobot. Cluj. 46, 22–31. https://doi.org/10.15835/ nbha46110996. Costantino, P., Capone, I., Cardarelli, M., De Paolis, A., Mauro, M., Trovato, M., 1994. Bacterial plant oncogenes: therol genes’ saga. Genetica 94, 203–211. https://doi.org/ 10.1007/BF01443434. Diener, A.C., Li, H., Zhou, W.-x., Whoriskey, W.J., Nes, W.D., Fink, G.R., 2000. Sterol methyltransferase 1 controls the level of cholesterol in plants. Plant Cell 12, 853–870. https://doi.org/10.1105/tpc.12.6.853. Dörnenburg, H., Knorr, D., 1995. Strategies for the improvement of secondary metabolite production in plant cell cultures. Enzyme Microb. Technol. 17, 674–684. https://doi. org/10.1016/0141-0229(94)00108-4. Estruch, J., Chriqui, D., Grossmann, K., Schell, J., Spena, A., 1991. The plant oncogene rolC is responsible for the release of cytokinins from glucoside conjugates. EMBO J. 10, 2889–2895. Faiss, M., Strnad, M., Redig, P., Doležal, K., Hanuš, J., Van Onckelen, H., Schmülling, T., 1996. Chemically induced expression of the rolC‐encoded β‐glucosidase in transgenic tobacco plants and analysis of cytokinin metabolism: rolC does not hydrolyze endogenous cytokinin glucosides in planta. Plant J. 10, 33–46. https://doi.org/10. 1046/j.1365-313X.1996.10010033.x. Hashemi, S.M., Naghavi, M.R., 2016. Production and gene expression of morphinan alkaloids in hairy root culture of Papaver orientale L. using abiotic elicitors. Plant Cell Tissue Organ Cult. 125, 31–41. https://doi.org/10.1007/s11240-015-0927-8. Ibañez, S., Talano, M., Ontañon, O., Suman, J., Medina, M.I., Macek, T., Agostini, E., 2016. Transgenic plants and hairy roots: exploiting the potential of plant species to remediate contaminants. N. Biotechnol. 33, 625–635. https://doi.org/10.1016/j.nbt. 2015.11.008. Kakeshpour, T., Nayebi, S., Rashidi-Monfared, S., Moieni, A., Karimzadeh, G., 2015. Identification and expression analyses of MYB and WRKY transcription factor genes in Papaver somniferum L. Physiol. Mol. Biol. Plants 21, 465–478. https://doi.org/10. 1007/s12298-015-0325-z. Lange, B.M., Rujan, T., Martin, W., Croteau, R., 2000. Isoprenoid biosynthesis: the evolution of two ancient and distinct pathways across genomes. Proc. Natl. Acad. Sci. U. S. A. 97, 13172–13177. https://doi.org/10.1073/pnas.240454797. Laule, O., Fürholz, A., Chang, H.-S., Zhu, T., Wang, X., Heifetz, P.B., Gruissem, W., Lange, M., 2003. Crosstalk between cytosolic and plastidial pathways of isoprenoid biosynthesis in Arabidopsis thaliana. Proc. Natl. Acad. Sci. U. S. A. 100, 6866–6871. https://doi.org/10.1073/pnas.1031755100. Li, S., Cong, Y., Liu, Y., Wang, T., Shuai, Q., Chen, N., Gai, J., Li, Y., 2017. Optimization of Agrobacterium-mediated transformation in soybean. Front. Plant Sci. 8, 246. https:// doi.org/10.3389/fpls.2017.00246. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using realtime quantitative PCR and the 2− ΔΔCT method. Methods 25, 402–408. https://doi. org/10.1006/meth.2001.1262. Martin, E., Akan, H., Ekici, M., Aytac, Z., 2011. New chromosome numbers in the genus Trigonella L. (Fabaceae) from Turkey. Afr. J. Biotechnol. 10, 116–125. Mastakani, F.D., Pagheh, G., Rashidi-Monfared, S., Shams-Bakhsh, M., 2018. Identification and expression analysis of a microRNA cluster derived from pre-ribosomal RNA in Papaver somniferum L. and Papaver bracteatum L. PLoS One 13, e0199673. https://doi.org/10.1371/journal.pone.0199673. Mohammadi, M., Mashayekh, T., Rashidi-Monfared, S., Ebrahimi, A., Abedini, D., 2019. New insights into diosgenin biosynthesis pathway and its regulation in Trigonella foenum-graecum L. Phytochem. Anal. https://doi.org/10.1002/pca.2887. Morrone, D., Lowry, L., Determan, M.K., Hershey, D.M., Xu, M., Peters, R., 2010. Increasing diterpene yield with a modular metabolic engineering system in E. coli: comparison of MEV and MEP isoprenoid precursor pathway engineering. Appl. Microbiol. Biotechnol. 85, 1893–1906. https://doi.org/10.1007/s00253-009-2219-x. Murlidhar, M., Goswami, T., 2012. A review on the functional properties, nutritional content, medicinal utilization and potential application of fenugreek. J. Food Process.
5. Conclusion The induction of hairy roots in fenugreek considerably increased the diosgenin content as compared with the plant. Based on the carried out experiments, some potential reasons were proposed which probably contribute to the increased accumulation of diosgenin in the hairy roots. Firstly, the chromatic graphs analysis indicate a single sharp pick referring diosgenin compound that may due to the none-specific beta glucosidase activity of rolB and rolC genes of A. rhizogenes, which could convert diocin precursor to diosgenin accompanied by plant beta-glucosidase enzymes. Secondly, the expression of Δ24-reductase, as a ratelimiting enzyme in the pathway, is highly expressed as compared with SMT1 in hairy roots. Two determinative genes, that is, Δ24‐reductase and SMT1, taking part in the branch‐point of diosgenin and C‐24‐alkyl phytosterols production, respectively. The up-regulation of Δ24‐reductase plays an essential role in directing the metabolic flux toward the diosgenin biosynthesis. Thirdly, plastid DXR, DXS and HDR genes are highly expressed in the induced hairy roots that naturally produce the basic five-carbon precursors IDP which is utilized for plastid development. As hairy roots lack differentiated chloroplast, plastidic IDP precursor is transported to cytosol; thus, the expression of the plastid key genes in hairy roots might indicate to direct involvement of MEP pathway in diosgenin accumulation. As a result, the higher availability of IDP stemming from both MVA and MEP pathways can increase the precursor of diosgenin production. By and large, due to the difficult culturing and lower amount of diosgenin in fenugreek plant, it seems that application of hairy root lines could be a promising way to scale-up diosgenin production. CRediT authorship contribution statement Farnaz Zolfaghari: Investigation, Formal analysis, Writing - original draft, Visualization. Sajad Rashidi-Monfared: Conceptualization, Methodology, Data curation, Writing - review & editing, Supervision, Project administration. Ahmad Moieni: Conceptualization, Methodology, Data curation, Writing - review & editing, Supervision, Project administration. Davar Abedini: Investigation, Formal analysis, Writing - original draft, Visualization. Amin Ebrahimi: Writing - review & editing. Declaration of Competing Interest The authors declare that there are no conflicts of interest. Acknowledgments The authors would like to acknowledge Tarbiat Modares University (TMU), Iran, for funding and supporting this work. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.indcrop.2019.112075. 8
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F. Zolfaghari, et al. Technol. 3. Murray, M.G., Thompson, W.F., 1980. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 8, 4321–4326. https://doi.org/10.1093/nar/8.19.4321. Murthy, H.N., Dandin, V.S., Zhong, J.-J., Paek, K.-Y., 2014. Strategies for Enhanced Production of Plant Secondary Metabolites from Cell and Organ Cultures, Production of Biomass and Bioactive Compounds Using Bioreactor Technology. Springer, pp. 471–508. Nilsson, O., Olsson, O., 1997. Getting to the root: the role of the Agrobacterium rhizogenes rol genes in the formation of hairy roots. Physiol. Plant. 100, 463–473. https:// doi.org/10.1111/j.1399-3054.1997.tb03050.x. Phillips, M.A., León, P., Boronat, A., Rodríguez-Concepción, M., 2008. The plastidial MEP pathway: unified nomenclature and resources. Trends Plant Sci. 13, 619–623. https://doi.org/10.1016/j.tplants.2008.09.003. Qian, Z.G., Zhao, Z.J., Xu, Y., Qian, X., Zhong, J., 2005. Highly efficient strategy for enhancing taxoid production by repeated elicitation with a newly synthesized jasmonate in fed‐batch cultivation of Taxus chinensis cells. Biotechnol. Bioeng. 90, 516–521. https://doi.org/10.1002/bit.20460. Raju, J., Mehta, R., 2008. Cancer chemopreventive and therapeutic effects of diosgenin, a food saponin. Nutr. Cancer 61, 27–35. https://doi.org/10.1080/ 01635580802357352. Ramos-Valdivia, A.C., van der Heijden, R., Verpoorte, R., 1997. Elicitor-mediated induction of anthraquinone biosynthesis and regulation of isopentenyl diphosphate isomerase and farnesyl diphosphate synthase activities in cell suspension cultures of Cinchona robusta how. Planta 203, 155–161. https://doi.org/10.1007/ s004250050177. Sawai, S., Ohyama, K., Yasumoto, S., Seki, H., Sakuma, T., Yamamoto, T., Takebayashi, Y., Kojima, M., Sakakibara, H., Aoki, T., 2014. Sterol side chain reductase 2 is a key enzyme in the biosynthesis of cholesterol, the common precursor of toxic steroidal
glycoalkaloids in potato. Plant Cell 26, 3763–3774. https://doi.org/10.1105/tpc.114. 130096. Setamam, N.M., Sidik, N.J., Rahman, Z.A., Zain, C.R., 2014. Induction of hairy roots by various strains of Agrobacterium rhizogenes in different types of Capsicum species explants. BMC Res. Notes 7, 414. https://doi.org/10.1186/1756-0500-7-414. Smith, M.J.A.M.R., 2003. Therapeutic applications of fenugreek. Altern. Med. Rev. 8, 20–27. Spena, A., Schmülling, T., Koncz, C., Schell, J., 1987. Independent and synergistic activity of rol A, B and C loci in stimulating abnormal growth in plants. EMBO J. 6, 3891–3899. Srivastava, S., Srivastava, A.K., 2007. Hairy root culture for mass-production of highvalue secondary metabolites. Crit. Rev. Biotechnol. 27, 29–43. https://doi.org/10. 1080/07388550601173918. Thwe, A., Valan Arasu, M., Li, X., Park, C.H., Kim, S.J., Al-Dhabi, N.A., Park, S.U., 2016. Effect of different Agrobacterium rhizogenes strains on hairy root induction and phenylpropanoid biosynthesis in tartary buckwheat (Fagopyrum tataricum Gaertn). Front. Microbiol. 7, 318. https://doi.org/10.3389/fmicb.2016.00318. Vaidya, K., Ghosh, A., Kumar, V., Chaudhary, S., Srivastava, N., Katudia, K., Tiwari, T., Chikara, S.K., 2013. De novo transcriptome sequencing inTrigonella foenum-graecum L. to identify genes involved in the biosynthesis of diosgenin. Plant Genome 6. https://doi.org/10.3835/plantgenome2012.08.0021. Vincken, J.-P., Heng, L., de Groot, A., Gruppen, H., 2007. Saponins, classification and occurrence in the plant kingdom. Phytochemistry 68, 275–297. https://doi.org/10. 1016/j.phytochem.2006.10.008. Yang, K., Rashidi-Monfared, S., Wang, H., Lundgren, A., Brodelius, P.E., 2015. The activity of the artemisinic aldehyde Δ11 (13) reductase promoter is important for artemisinin yield in different chemotypes of Artemisia annua L. Plant Mol. Biol. 88, 325–340. https://doi.org/10.1007/s11103-015-0284-3.
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