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Transcriptional Profiling and Molecular Characterization of Astragalosides, Calycosin, and Calycosin-7‑O‑β‑D‑glucoside Biosynthesis in the Hairy Roots of Astragalus membranaceus in Response to Methyl Jasmonate Pham Anh Tuan,†,⊥ Eunsook Chung,‡,⊥ Aye Aye Thwe,† Xiaohua Li,† Yeon Bok Kim,∥ Valan Arasu Mariadhas,§ Naif Abdullah Al-Dhabi,§ Jai-Heon Lee,*,‡ and Sang Un Park*,†,∥ †

Department of Crop Science, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 305-764, Korea Department of Genetic Engineering, Dong-A University, Busan 604-714, Korea ∥ Herbal Crop Research Division, Department of Herbal Crop Research, National Institute of Horticultural & Herbal Science, Bisanro 92, Eumseong, Chungbuk 369-873, Korea § Department of Botany and Microbiology, Addiriyah Chair for Environmental Studies, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia ‡

S Supporting Information *

ABSTRACT: We used the next-generation Illumina/Solexa HiSeq2000 platform on RNA analysis to investigate the transcriptome of Astragalus membranaceus hairy roots in response to 100 μM methyl jasmonate (MeJA). In total, 77 758 230 clean reads were assembled into 48 636 transcripts (average length of 1398 bp), which were clustered into 23 658 loci (genes). Of these, 19 940 genes were annotated by BLASTx searches. In addition, DESeq analysis showed that 2127 genes were upregulated, while 1247 genes were down-regulated by MeJA. Seventeen novel astragaloside (AST) biosynthetic genes and seven novel calycosin and calycosin-7-O-β-D-glucoside (CG) biosynthetic genes were isolated. The accumulation of ASTs, calycosin, and CG increased significantly in MeJA-treated hairy roots compared with control hairy roots. Our findings will provide a valuable resource for molecular characterization of AST, calycosin, and CG biosynthetic pathways and may lead to new approaches to maximize their production and biomass productivity in the hairy roots of A. membranaceus. KEYWORDS: astragaloside, Astragalus membranaceus, calycosin-7-O-β-D-glucoside, calycosin, methyl jasmonate, transcriptional profiling

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INTRODUCTION Astragalus membranaceus, a plant belonging to the family Fabaceae, has a long history of use as a traditional medicine in China, Japan, Korea, and other Asian areas. The dried root of A. membranaceus, known as “Huang Qi” is one of the most important Chinese traditional herbs and has been used for debility, chronic illness, and spleen deficiency.1 Pharmacological studies of A. membranaceus crude extract have found that it possesses a wide range of biological properties, such as antiinflammatory activity,2 antibacterial and antiviral properties,1,3 antihyperglycemic activity,4 and immunostimulant effects.5 More than 100 compounds have been identified in A. membranaceus, including triterpene saponins, flavonoids, and polysaccharides.6,7 Of these, astragalosides (ASTs), including AST I, AST II, AST III, and AST IV, calycosin, and calycosin-7O-β-D-glucoside (CG) are believed to be the major active constituents of A. membranaceus and are well studied.8−11 ASTs have various therapeutic effects and are used clinically in the treatment of diabetes and cardiovascular disease.12−14 Calycosin and CG also possess diverse bioactivities, such as protecting endothelial cells from hypoxia-induced barrier impairment,15 inhibiting high glucose-induced mesangial cell proliferation,16 and antiviral activities against coxsackie virus B3.17 © XXXX American Chemical Society

Its long history in traditional medicine and richness in beneficial compounds make A. membranaceus a good model for assessing the applications of biotechnology in improving natural product production. Several approaches to producing secondary metabolites in hairy root cultures of A. membranaceus have been studied and developed.18−20 However, to our knowledge, the gene expression profiles of the metabolic pathways in the hairy root cultures of A. membranaceus have not been investigated. Treatment with methyl jasmonate (MeJA), a signaling molecule the regulates many physiological processes in plants, can stimulate the biosynthesis of plant secondary metabolites, such as terpenoid indole alkaloids in Catharanthus roseus cells,21 flavonoids in Scutellaria baicalensis cells,22 and phenylpropanoids in Brassica rapa leaves.23 Therefore, application of MeJA is one of the most frequently used tools in the investigation and engineering of natural bioproduction in plants. RNA-sequencing (RNA-seq) technology, which uses highthroughput sequencing technology called next-generation Received: April 12, 2015 Revised: June 4, 2015 Accepted: June 14, 2015

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DOI: 10.1021/acs.jafc.5b01822 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

independent hairy root replicates were pooled for further analyses. All samples were frozen in liquid nitrogen immediately after harvesting and then stored at −80 °C or freeze-dried for total RNA isolation or HPLC analysis. RNA Isolation and Illumina Sequencing. The samples were ground into powder using liquid nitrogen, and total RNA was isolated using a Plant Total RNA Mini Kit (Geneaid, Taiwan) according to the manufacturer’s instructions. The quality and concentration of total extracted RNA was determined by 1% agarose gel electrophoresis and spectrophotometer analysis in a NanoVue Plus spectrophotometer (GE Healthcare Bio-Science Crop, USA). mRNA was purified from total RNA using Sera-Mag Magnetic Oligo(dT) beads (Illumina, San Diego, CA, USA). cDNA synthesis, library construction, and DNA sequencing were performed using Illumina/Solexa HiSeq2000 platform by SEEDERS Company (Daejeon, Korea). The resulting highquality reads were then deposited in the Short Read Archive at the National Center for Biotechnology Information (NCBI) with the accession number SRR923811. De Novo Assembly and Functional Annotation. Reads obtained using an Illumina sequencer were filtered and de novo assembled using Velvet and Oases at high k-mers of 57, 59.31 A. membranaceus transcripts were searched against the plant genome information in Phytozome (http://www.phytozome.net/) with a wide range of E-value cutoff, from 1 × 10−100 to 1 × 10−6. The numbers of annotated genes stabilize around cutoff values of 1 × 10−6 to 1 × 10−10 (data not shown). Therefore, we chose a default cutoff of 1 × 10−10 for best blast hit number. To assign function to each gene, gene ontology (GO) analysis and clusters of orthologous groups (COG) analyses were performed. To gain an overview of gene pathway networks, genes were mapped according to the Kyoto Encyclopedia of Genes and Genomes (KEGG). Genes encoding transcription factors were identified using the Plant Transcription Factor Database (http:// planttfdb.cbi.edu.cn/). Differentially Expressed Gene Analysis. Differential expression analysis was performed based on the negative-binomial distribution, as described by Anders and Huber.32 Raw counts data for the control and MeJA-treated hairy roots were compared to determine the log2-fold change in abundance of each transcript. Differentially expressed genes were those having log2-fold change of ≤ −1 or ≥1 and a false discovery rate of ≤0.01. Identification of Genes Related to AST, Calycosin, and CG Biosynthetic Pathways. Genes involved in the AST, calycosin, and CG biosynthetic pathways were searched for using functional annotation data based on the candidate gene names. In addition, the AST, calycosin, and CG biosynthetic genes of Arabidopsis obtained from The Arabidopsis Information Resource (TAIR) were used as queries to search for homologous sequences in A. membranaceus transcriptome database. Following this, each search sequence was confirmed by using the BLAST program in the National Center for Biotechnology Information GenBank database. Quantitative Real-Time PCR. On the basis of the sequences of A. membranaceus 1-deoxy-D-xylulose 5-phosphate synthase (AmDXS), (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (AmHDS), acetoacetyl-coenzyme A thiolase (AmAACT), isopentenyl diphosphate isomerase (AmIDI), squalene synthase (AmSS), phenylalanine ammonia lyase (AmPAL1), cinnamic acid 4-hydroxylase (Am4CL), chalcone synthase (AmCHS1), chalcone isomerase (AmCHI), and isoflavone O-methyltransferase (AmIOMT), we designed real-time PCR primers using the Primer3 Web site (http://bioinfo.ut.ee/ primer3-0.4.0/primer3/; Table S10, Supporting Information). Realtime PCR products were tested for specificity of fragment sizes, melting curves, and sequences by PCR, real-time PCR, and cloning into a T-Blunt vector for sequencing, respectively. The expression levels of these genes were normalized and then analyzed by relative quantification using the A. membranaceus adenine phosphoribosyl transferase housekeeping gene (NCBI Genbank accession number KF355974) as a reference. qRT-PCR was performed by previous paper.33 Three replications for each sample were used in the real-time analyses.

sequencing (NGS), is a powerful and cost-efficient tool for discovering gene expression, novel genes, and differentially expressed genes. Due to its higher accuracy and dynamic range, RNA-seq is replacing other methods for quantifying gene expression, including microarray and serial analysis of gene expression (SAGE). RNA-seq is very sensitive and allows more precise quantification of differential gene expression, as well as the detection of low-abundance transcripts. Furthermore, RNAseq is useful in nonmodel plants that lack a reference genome because the focus of sequencing is the coding regions. Therefore, the transcriptomes of various nonmodel agricultural crops have been assessed using RNA-seq, including olive,24 chestnut,25 and tea.26 For model agriculture crops such as Zea mays and soybean, RNA-seq provides breeders with new tools and methodologies to discover genome-wide expression, to isolate novel transcripts, and to explore alternative RNA splicing events, which allow great steps forward in plant breeding.27−29 To investigate the transcriptional profiling in A. membranaceus hairy roots in response to MeJA, we used the nextgeneration Illumina/Solexa HiSeq2000 platform for RNA analysis (RNA-seq). The assembled and annotated transcriptome sequences provide a valuable resource for the molecular characterization of secondary metabolite biosynthesis in A. membranaceus. In addition, we identified most genes related to AST, calycosin, and CG biosynthesis in A. membranaceus. Transcriptional regulation of AST, calycosin, and CG biosynthetic genes and changes in AST, calycosin, and CG accumulation were investigated in the hairy roots of A. membranaceus treated with MeJA. These data may establish new approaches to maximize AST, calycosin, and CG production and biomass productivity in the hairy roots of A. membranaceus.

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MATERIALS AND METHODS

Establishment of Hairy Root Transformation and MeJA Treatment. Excised cotyledons and leaves of A. membranaceus from 20-day-old seedlings were used as the explant materials for cocultivation with wild-type Agrobacterium rhizogenes strain R1000. The excised explants were dipped into a liquid A. rhizogenes culture in inoculation medium for 10 min, blotted dry on sterile filter paper, and incubated in the dark at 25 °C on agar-solidified half-strength (1/2) MS medium. After 2 days of cocultivation, the explant tissues were thoroughly washed with sterilized distilled water and transferred to a hormone-free 1/2 MS medium containing cefotaxime (500 mg/L). The hairy roots were induced from the wound sites after 20 days of inoculation. Genomic DNA was isolated from several different fastgrowing hairy root lines using the method described by Edwards et al.30 Polymerase chain reaction (PCR) analysis was then performed using primers that detect rol A, B, C, and D genes to confirm the integration of A. rhizogenes open reading frames into the recipient plant genome (data not shown). For the next steps, approximately 200 mg of fresh hairy roots was transferred to 30 mL of 1/2 MS liquid medium in 100 mL flasks. Hairy root cultures were maintained at 25 °C on a gyratory shaker (100 rpm) in a growth chamber under standard cool white fluorescent tubes with a flux rate of 35 μmol s−1 m−2 and a 16-h photoperiod. For MeJA treatments, hairy root samples were treated with MeJA (Sigma) after 3 weeks of culture in 30 mL of 1/2 MS liquid medium. Hairy root samples were harvested 24 h after MeJA treatment. In another study (unpublished data), we found that the production of AST, calycosin, and CG in hairy roots treated with 100 μM MeJA was higher than that in the hairy roots treated with other concentrations of MeJA. Therefore, the hairy roots treated with 100 μM MeJA was chosen for the further transcriptome analysis. Untreated hairy roots were used as the control. The entire experiment was repeated three times, and three B


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Journal of Agricultural and Food Chemistry Table 1. Summary of Sequences from A. membranaceus Transcriptome after Illumina Sequencing control hairy roots_1

control hairy roots_2

MeJA-treated hairy roots_1

MeJA-treated hairy roots_2

total

25 122 120 101 2 537 334 120

98 257 776 101 9 924 035 376

25 122 120 78.23 1 965 385 644

98 257 776 79.95 7 856 836 134

19 973 663 90.02 1 798 088 737

77 758 230 90.81 7 061 762 722

Raw Data number of reads mean length (bp) total base pairs

24 006 768 101 2 424 683 568

number of reads mean length (bp) total base pairs

24 006 768 82.00 1 968 502 825

number of reads mean length (bp) total base pairs

18 905 452 91.74 1 734 455 319

24 006 768 101 2 424 683 568

25 122 120 101 2 537 334 120 Trimmed Data 24 006 768 25 122 120 77.09 82.49 1 850 604 616 2 072 343 049 Paired-End Data 18 905 452 19 973 663 89.46 92.02 1 691 227 010 1 837 991 656

HPLC Analysis. The materials were freeze-dried and ground into a fine powder. AST I, AST II, AST III, AST IV, calycosin, and CG were analyzed by HPLC.34,35 The concentration of each compound was determined using a standard curve. All samples were analyzed in triplicate. Statistical Analysis. The data for gene expression and AST, calycosin, and CG content were analyzed using Statistical Analysis System software (SAS version 9.2). Treatment means were compared using Duncan’s multiple range test.

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RESULTS AND DISCUSSION Illumina Sequencing and De Novo Sequence Assembly. cDNA libraries were constructed from A. membranaceus hairy roots treated with 100 μM MeJA for 24 h and from untreated hairy roots and sequenced. In total, 98 257 776 reads of 101 bp from 9 924 035 376 bp were obtained from the Illumina/Solexa HiSeq2000 platform (Table 1). Of these, 77 758 230 paired-end clean reads, with a mean length of 90.81 bp, were obtained from 98 257 776 trimmed reads. The clean reads were assembled into 48 636 transcripts (average length of 1398 bp), which were then clustered into 23 658 loci using Velvet and Oases at high k-mers of 57, 59 (Table 2).31 Because

Figure 1. Distribution of transcript and gene numbers based on transcript and gene length, respectively.

predicted A. membranaceus genes. GO analysis categorized 19 940 genes as biological process, cellular component, or molecular function (Figure 2). Each gene may have more than one GO term and be assigned to different GO categories. The dominant groups within the biological process, cellular component, and molecular function categories were cellular process (8402 members), cell part (13 182 members), and nucleotide binding (3748 members), respectively. A total of 17 189 genes were aligned to 25 categories of the COG database (Figure 3). The R category “general function prediction only” was the largest group (3505 members), followed by the T category “signal transduction mechanisms” (1996 members) and the S category “function unknown”, which has no concrete assignment (1537 members). In addition, 4969 genes were mapped to 116 KEGG pathways (Table S2, Supporting Information). The biosynthesis of plant hormones, phenylpropanoids, terpenoids, and steroids were the three largest groups with 282, 210, and 184 identified genes, respectively. The analyses provided excellent material for the molecular characterization of genes related to secondary metabolite biosynthetic pathways in A. membranaceus. Differentially Expressed Gene Analysis in A. membranaceus Hairy Roots in Response to MeJA. The differentially expressed gene (DEG) analysis between MeJAtreated and control hairy roots was performed using the DESeq method.32 Genes were determined to be differentially expressed if they had a log2-fold change of ≤ −1 or ≥1 and a false discovery rate of ≤0.01. A total of 3374 genes were found to be differentially expressed in MeJA-treated hairy roots compared with control hairy roots. Of these, 2127 genes were upregulated and 1247 genes were down-regulated by MeJA. BLASTx searches of 3374 DEGs against the plant genome

Table 2. Summary of Sequence Assembly from the A. membranaceus Transcriptome number of sequences minimum length (bp) maximum length (bp) mean length (bp) annotated sequences full-length coding sequences

transcript

locus

48 636 200 14 974 1 398 40 904 21 899

23 658 200 11 135 1 161 19 940 10 311

our main goal was to study the nonredundant set of mRNA sequences for A. membranaceus, the longest or highest coverage transcript for each locus was chosen to produce 23 658 nonredundant cDNAs (genes), with an average length of 1161 bp, containing 10 311 full-length coding sequences. Distribution of the number of transcripts and genes organized by transcript length and gene length, respectively, are shown in Figure 1. Functional Annotation. A total of 40 904 transcripts clustering into 19 940 genes were annotated by searching against the plant genome information using Phytozome (http://www.phytozome.net/) with an E-value cutoff of 1 × 10−10 (Table S1, Supporting Information). Subsequently, GO and COG analyses were performed to classify the function of C


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Figure 2. GO analysis of genes based on biological process, cellular component, and molecular function categories.

revealed that 667 up-regulated genes and 238 down-regulated genes could be mapped to 88 and 76 KEGG pathways, respectively (Tables S5 and S6, Supporting Information, respectively). Of these, MeJA up-regulated a large numbers of genes belonging to the biosynthesis of plant hormone group (71 genes), the biosynthesis of terpenoids and steroids group (44 genes), and the biosynthesis of phenylpropanoids group (34 genes) in A. membranaceus hairy roots. The application of MeJA stimulates the transcription of biosynthetic genes and accumulation of secondary metabolites in plants.36−38 The annotated differentially expressed genes are an important and valuable resource for the identification of novel MeJAresponding genes and for the investigation of specific MeJAregulated pathways in A. membranaceus. Transcriptional Regulation of Transcription Factors in A. membranaceus Hairy Roots in Response to MeJA. Transcription factors are important elements in regulating plant responses to a range of abiotic and biotic stresses by controlling the expression patterns of genes involved in signaling pathways.39,40 In the A. membranaceus transcriptome, 3784 genes encoding transcription factors were annotated (Table S7, Supporting Information). bHLH, NAC, WRKY, MYB-related, and bZIP are the five largest families of transcription factors and have 339, 292, 287, 281, and 227 members, respectively (Figure 4). Of the 3784 identified transcription factors, 395 were up-regulated and 250 were down-regulated by MeJA in A. membranaceus hairy roots (Tables S8 and S9, Supporting Information, respectively). A high number of transcription factors regulated by MeJA suggested that transcription factors play various roles in molecular mechanisms in response to MeJA as well as in the defense mechanisms of A. membranaceus hairy roots. Moreover, information about transcription factors will be helpful in investigating their roles in secondary metabolite biosynthesis in response to stresses. MeJA Increases Transcription Levels of Genes Related to the AST Biosynthetic Pathway in A. membranaceus Hairy Roots. Triterpene ASTs are built from isopentenyl diphosphate, which is supplied from the cytosol mevalonic acid

Figure 3. COG functional classification of genes. (A) RNA processing and modification; (B) chromatin structure and dynamics; (C) energy production and conversion; (D) cell cycle control, cell division, and chromosome partitioning; (E) amino acid transport and metabolism; (F) nucleotide transport and metabolism; (G) carbohydrate transport and metabolism; (H) coenzyme transport and metabolism; (I) lipid transport and metabolism; (J) translation, ribosomal structure, and biogenesis; (K) transcription; (L) replication, recombination, and repair; (M) cell wall/membrane/envelope biogenesis; (N) cell motility; (O) posttranslational modification, protein turnover, and chaperones; (P) inorganic ion transport and metabolism; (Q) secondary metabolite biosynthesis, transport, and catabolism; (R) general function prediction only; (S) function unknown; (T) signal transduction mechanisms; (U) intracellular trafficking, secretion, and vesicular transport; (V) defense mechanisms; (W) extracellular structures; (Y) nuclear structure; (Z) cytoskeleton.

information using Phytozome (http://www.phytozome.net/) with an E-value cutoff of 1 × 10−10 revealed that 2920 DEGs, including 1852 up-regulated genes and 1068 down-regulated genes, were annotated (Tables S3 and S4, Supporting Information, respectively). Furthermore, the KEGG analysis D


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AST biosynthetic pathway (Table 3). In the MEP pathway, fulllength coding sequences of AmDXS, 2-C-methyl-D-erythritol 4phosphate cytidylyltransferase (AmMCT), 4-(cytidine 5′phospho)-2-C-methyl-D-erithritol kinase (AmCMK), AmHDS, and (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (AmHDR) and partial-length coding sequences of 1-deoxy-Dxylulose 5-phosphate reductoisomerase (AmDXR) were found. Entire genes that are involved in the MVA pathway were isolated, including AmAACT, 3-hydroxy-3-methylglutaryl coenzyme A synthase (AmHMGS), 3-hydroxy-3-methylglutaryl-coenzyme A reductase (AmHMGR1, AmHMGR2, AmHMGR3), mevalonate kinase (AmMVK), phosphomevalonate kinase (AmPMK1, AmPMK2), mevalonate diphosphate decarboxylase (AmMVD), and AmIDI. In addition, full-length coding sequence of farnesyl diphosphate synthase (AmFDS), an enzyme that functions in the initial step of triterpene formation in the MVA pathway and MEP pathway, was also found in the A. membranaceus transcriptome library. These 17 novel AST biosynthetic genes of A. membranaceus share high similarity and identity with other orthologous genes (data not shown) and have been submitted to Genbank at the NCBI.

Figure 4. Numbers of genes encoding transcription factors.

(MVA) pathway and the plastid methylerythritol phosphate (MEP) pathway (Figure 5). On the basis of the functional annotation of the A. membranaceus transcriptome library, 17 novel genes were identified that encode enzymes related to the

Figure 5. Proposed AST biosynthetic pathway in plants. AACT, acetoacetyl-CoA thiolase; CMK, 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol kinase; DXR, 1-deoxy-D-xylulose 5-phosphate reductoisomerase; DXS, 1-deoxy-D-xylulose 5-phosphate synthase; FDS, farnesyl diphosphate synthase; HDR, (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase; HDS, (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; HMGS, 3-hydroxy-3-methylglutaryl-CoA synthase; IDI, isopentenyl diphosphate isomerase; MCT, 2-Cmethyl-D-erythritol 4-phosphate cytidylyltransferase; MDS, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; MVD, mevalonate diphosphate decarboxylase; MVK, mevalonate kinase; PMK, phosphomevalonate kinase; SS, squalene synthase ; DMAPP, dimethylallyl diphosphate; IPP, isopentenyl diphosphate. E


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Journal of Agricultural and Food Chemistry Table 3. Candidate Genes for AST Biosynthesis in the A. membranaceus Transcriptome gene name

gene ID

accession number (NCBI)

1-deoxy-D-xylulose 5-phosphate synthase (AmDXS)a,b 1-deoxy-D-xylulose 5-phosphate reductoisomerase (AmDXR)a 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (AmMCT)a,b 4-(cytidine 5′-phospho)-2-C-methyl-D-erithritol kinase (AmCMK)a,b (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (AmHDS)a,b (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (AmHDR)a,b acetoacetyl-coenzyme A thiolase (AmAACT)a,b 3-hydroxy-3-methylglutaryl coenzyme A synthase (AmHMGS)a,b 3-hydroxy-3-methylglutaryl-coenzyme A reductase (AmHMGR1)a,b AmHMGR2a,b AmHMGR3a,b mevalonate kinase (AmMVK)a,b phosphomevalonate kinase (AmPMK1)a,b AmPMK2a,b mevalonate diphosphate decarboxylase (AmMVD)a,b isopentenyl diphosphate isomerase (AmIDI)a farnesyl diphosphate synthase (AmFDS)a,b squalene synthase (AmSS)

Locus_19738 Locus_12894

KF355950 KF355951

Locus_9577

a

control hairy roots (RPKM)

MeJA-treated hairy roots (RPKM)

fold change

62 776

1 724 836

27.81 1.08

KF355952

97

100

1.03

Locus_28629

KF355953

69

136

1.97

Locus_3604

KF355954

829

2 365

2.85

Locus_21871

KF355955

1 179

2 588

2.20

Locus_600 Locus_212

KF355956 KF355957

5 817 1 251

18 711 3 780

3.22 3.02

Locus_920

KF355958

2 401

9 314

3.88

Locus_4326 Locus_19060 Locus_32998 Locus_2082 Locus_8364 Locus_1511 Locus_732 Locus_1394 Locus_17877

KF355959 KF355960 KF355961 KF355962 KF355963 KF355964 KF355965 KF355966 HQ829974

500 331 472 494 47 349 2 571 1 921 1 225

1 611 1 929 2 903 1 072 4 686 3 936 25 244 15 097 18 025

3.22 5.83 6.15 2.17 99.70 11.28 9.82 7.86 14.71

The genes identified in this study. bThe full-length coding sequence genes.

RPKM values showed that most genes related to the AST biosynthetic pathway were up-regulated by the application of MeJA in A. membranaceus hairy roots. AmPMK2, AmDXS, and AmSS exhibited the highest increases in expression in MeJAtreated hairy roots, with fold changes of 99.7, 27.81, and 14.71, respectively (Table 3). Expression levels of five selected genes were validated by qRT-PCR (Figure 6A). Although the foldchanges of these genes were different than those detected by RNA-seq, their expression profiles were consistent with the RNA-seq. The same hairy root materials as those used for RNA-seq and qRT-PCR were used for HPLC analysis of AST I, AST II, AST III, and AST IV (Figure 6B). The accumulation of AST I, AST II, AST III, and AST IV was significantly increased by 2.98-, 2.85-, 2.30-, and 1.57-fold, respectively, in MeJAtreated hairy roots compared with control hairy roots. These results suggest that the accumulation of ASTs was tightly regulated by the expression levels of genes involved in the MVA and MEP pathways in A. membranaceus hairy roots in response to MeJA. Furthermore, large amounts of AST I, AST II, AST III, and AST IV were found in both control (2386.06, 774.85, 401.52, and 124.24 μg/g, respectively) and MeJA-treated (7104.02, 2205.2, 924.98, and 194.77 μg/g, respectively) hairy roots. These amounts were much higher than those of the natural roots of A. membranaceus.10 Hairy roots of A. membranaceus may, therefore, be a good material for the production of ASTs. MeJA Increases Transcription Levels of Genes Related to Calycosin and CG Biosynthetic Pathways in A. membranaceus Hairy Roots. AmPAL1, AmC4H1, AmCHS1, chalcone reductase (AmCHR), AmCHI, isoflavone synthase (AmIFS), and isoflavone 3′-hydroxylase (AmI3′H), which are involved in calycosin and CG biosynthetic pathways (Figure 7),9 were also found in the A. membranaceus transcriptome library. In addition, seven new full-length coding

Figure 6. Expression levels of five selected AST biosynthetic genes determined by qRT-PCR (A) and AST content (B) in 100 μM MeJAtreated and untreated hairy roots. The height of each bar and the error bars show the mean and standard error, respectively, from three independent measurements. The letters a and b indicate significant differences at the 5% level by Duncan’s multiple range test.

F


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According to fold change values of RNA-seq, with the exception of AmIOMT, all calycosin and CG biosynthetic genes were up-regulated by MeJA in A. membranaceus hairy roots (Table 4). Expression levels of AmUCGT, AmCHS1, and AmPAL3, with fold changes of 7.07, 3.79, and 2.89, respectively, increased the most in MeJA-treated hairy roots compared with control hairy roots. qRT-PCR of five selected genes was performed to confirm the gene expression profiles analyzed by RNA-seq (Figure 8A). The higher expression levels of AmPAL1, Am4CL, AmCHS1, and AmCHI and the lower expression level of AmIOMT detected in MeJA-treated hairy roots by qRT-PCR supported the reliability of RNA-seq analyses. The higher expression levels of calycosin and CG biosynthetic genes were correlated with their higher accumulation in MeJA-treated hairy roots compared with control hairy roots (Figure 8B). Specifically, MeJA-treated hairy roots contained 23.61 μg/g of CG, whereas untreated hairy roots contained 17.75 μg/g. Untreated hairy roots did not accumulate calycosin, but hairy roots treated with MeJA contained 0.54 μg/g of calycosin. These data may not only broaden our understanding of the molecular mechanisms involved in the calycosin and CG biosynthetic pathways in A. membranaceus but also aid in their metabolic engineering in plants. A. membranaceus, one of the most important Chinese traditional herbs, has a wide range of natural products.6,7 MeJA application increased the expression levels of most genes related to AST, calycosin, and CG biosynthesis and their accumulation. Therefore, MeJA-treated hairy roots of A. membranaceus may be an excellent material for the production of ASTs, calycosin, and CG because of their biochemical stability and rapid growth rate.41,42 On the other hand, although AST, calycosin, and CG were identified a long time ago, the complete biosynthetic pathway and biosynthetic genes of AST, calycosin, and CG are still largely unknown. Recently, based on the hypotheses that genes involved in secondary metabolite biosynthesis are often coordinately regulated at the transcriptional level, coexpression analysis has been widely used to identify new candidate genes in plants. There are numerous examples of successful gene discovery studies using this strategy such as the isolation of two glycosyltransferases involved in anthocyanin modification in Arabidopsis thaliana,43 and the last

Figure 7. Proposed calycosin and CG biosynthetic pathways in plants. PAL, phenylalanine ammonia lyase; C4H, cinnamate-4-hydroxylase; 4CL, 4-coumaroyl CoA ligase; CHS, chalcone synthase; CHR, chalcone reductase; CHI, chalcone isomerase; IFS, isoflavone synthase; IOMT, isoflavone O-methyltransferase; I3′H, isoflavone 3′hydroxylase; UCGT, UDP-glucose:calycosin-7-O-glucosyltransferase.

sequences (AmPAL2, AmPAL3, AmC4H2, Am4CL, AmCHS2, AmIOMT, and UDP-glucose:calycosin 7-O-glucosyltransferase (AmUCGT)) were identified (Table 4). BLAST searches of the NCBI Genbank database showed that they exhibited high homology to other orthologous genes (date not shown). The seven new calycosin and CG biosynthetic genes of A. membranaceus have been deposited into the NCBI Genbank (accession numbers shown in Table 4).

Table 4. Candidate Genes for Calycosin and CG Biosynthesis in the A. membranaceus Transcriptome gene name phenylalanine ammonia-lyase (AmPAL1) AmPAL2a,b AmPAL3a,b cinnamate 4-hydroxylase (AmC4H1) AmC4H2a,b 4-coumaroyl:CoA-ligase (Am4CL)a,b chalcone synthase (AmCHS1) AmCHS2a,b chalcone reductase (AmCHR) chalcone isomerase (AmCHI) isoflavone synthase (AmIFS) isoflavone O-methyltransferase (AmIOMT)a,b isoflavone-3′-hydroxylase (AmI3′H) UDP-glucose:calycosin 7-O-glucosyltransferase (AmUCGT)a,b a

gene ID

accession number (NCBI)

control hairy roots (RPKM)

MeJA-treated hairy roots (RPKM)

fold change

Locus_27697 Locus_440 Locus_28451 Locus_4902 Locus_15996 Locus_858 Locus_1134 Locus_5993 Locus_9056 Locus_17766 Locus_138 Locus_2606 Locus_39 Locus_30758

EF567076 KF355967 KF355968 HQ339960 KF355969 KF355970 JQ048940 KF355971 HM357239 DQ205407 JF912329 KF355972 JQ609280 KF355973

5 268 2 016 715 3 548 27 465 3 526 1 559 1 097 17 888 2 610 26 250 1 788 15 660 365

7 547 2 301 2 069 7 540 62 687 5 750 5 908 2 717 22 132 6 663 39 575 1 728 16 214 2 582

1.43 1.14 2.89 2.13 2.28 1.63 3.79 2.48 1.24 2.55 1.51 0.97 1.04 7.07

The genes identified in this study. bThe full-length coding sequence genes. G


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transcriptome sequences provide a valuable resource for molecular characterization of ASTs, calycosin, and CG as well as other secondary metabolite biosynthetic pathways in A. membranaceus. The up-regulated biosynthesis of ASTs, calycosin, and CG in MeJA-treated hairy roots may lead to new approaches to maximize their production and biomass productivity in the hairy roots of A. membranaceus.

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ASSOCIATED CONTENT

S Supporting Information *

Table S1, the annotation of transcripts; Table S2, the KEGG pathway analysis of genes; Table S3, the annotation of genes up-regulated by MeJA; Table S4, the annotation of genes down-regulated by MeJA; Table S5, KEGG pathway analysis of genes up-regulated by MeJA; Table S6, the KEGG pathway analysis of genes down-regulated by MeJA; Table S7, the annotation of genes encoding transcription factors; Table S8, the annotation of transcription factor genes up-regulated by MeJ; Table S9, the annotation of transcription factor genes down-regulated by MeJA; Table S10, primers used for qRTPCR. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b01822.

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Figure 8. Expression levels of five selected calycosin and CG biosynthetic genes determined by qRT-PCR (A) and calycosin and CG content (B) in 100 μM MeJA-treated and untreated hairy roots. The height of each bar and the error bars show the mean and standard error, respectively, from three independent measurements. The letters a and b indicate significant differences at the 5% level by Duncan’s multiple range test.

AUTHOR INFORMATION

Corresponding Authors

*J. Lee. Phone: + 82-51-200-7592. Fax: +82-51-200-7505. Email: [email protected]. *S. U. Park. Phone: +82-42-821-5730. Fax: +82-42-822-2631. E-mail: [email protected]. Author Contributions ⊥

P.A. Tuan and E. Chung contributed equally to this work.

Funding

four missing steps of the seco-iridoid biosynthesis pathway in Catharanthus roseus.44 The RNA-sequencing transcriptional profiling of A. membranaceus hairy roots in response to MeJA in this study will provide useful information to identify novel genes putatively involved in the downstream steps of the AST, calycosin, and CG biosynthetic pathways using coexpression analysis strategy. Functional food, which contains numerous compounds beneficial to health, recently has attracted much interest. Therefore, much research has focused on developing efficient strategies to optimize the production of beneficial compounds for health in plants without gene modification or breeding. Among them, application of elicitors has been reported to efficiently affect the natural product accumulation in plants.21−23 Here, we reported that application of MeJA significantly increased the accumulation of AST, calycosin, and CG in the hairy roots of A. membranaceus. This finding may provide a new method to develop functional food products having high concentrations of AST, calycosin, and CG in the future. In conclusion, we identified 23 658 genes by high-throughput sequencing technology. There were 19 940 annotated genes, 4969 genes mapped to 116 KEGG pathways, and 3784 genes encoding transcription factors. RNA-seq analysis revealed that 2127 genes were up-regulated and 1247 genes were downregulated by 100 μM MeJA in A. membranaceus hairy roots. In addition, we isolated 24 novel genes involved in the biosynthesis of ASTs, calycosin, and CG, chemical markers of the quality of A. membranaceus. The assembled and annotated

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2015R1A2A2A01004054). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED

AACT, acetoacetyl-CoA thiolase; CMK, 4-(cytidine 5′diphospho)-2-C-methyl-D-erythritol kinase; DXR, 1-deoxy-Dxylulose 5-phosphate reductoisomerase; DXS, 1-deoxy-Dxylulose 5-phosphate synthase; FDS, farnesyl diphosphate synthase; HDR, (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase; HDS, (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; HMGS, 3-hydroxy-3-methylglutaryl-CoA synthase; IDI, isopentenyl diphosphate isomerase; MCT, 2-C-methyl-Derythritol 4-phosphate cytidylyltransferase; MDS, 2-C-methylD-erythritol 2,4-cyclodiphosphate synthase; MVD, mevalonate diphosphate decarboxylase; MVK, mevalonate kinase; PMK, phosphomevalonate kinase; SS, squalene synthase; DMAPP, dimethylallyl diphosphate; IPP, isopentenyl diphosphate; PAL, phenylalanine ammonia lyase; C4H, cinnamate-4-hydroxylase; 4CL, 4-coumaroyl CoA ligase; CHS, chalcone synthase; CHR, chalcone reductase; CHI, chalcone isomerase; IFS, isoflavone synthase; IOMT, isoflavone O-methyltransferase; I3′H, isoflavone 3′-hydroxylase; UCGT, UDP-glucose:calycosin-7-Oglucosyltransferase H


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roots of Astragalus membranaceus in bioreactors. Biotechnol. Lett. 2003, 25, 1853−1856. (20) Ionkova, I.; Momekov, G.; Proksch, P. Effects of cycloartane saponins from hairy roots of Astragalus membranaceus Bge., on human tumor cell targets. Fitoterapia 2010, 81, 447−451. (21) Rischer, H.; Orešič, M.; Seppänen-Laakso, T.; Katajamaa, M.; Lammertyn, F.; Diaz, W. A.; Montagu, M. V.; Inzé, D.; OksmanCaldentey, K. M.; Goossens, A. Gene-tometabolite networks for terpenoid indole alkaloid biosynthesis in Catharanthus roseus cells. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 5614−5619. (22) Xu, H.; Park, N. I.; Li, X.; Kim, Y. K.; Lee, S. Y.; Park, S. U. Molecular cloning and characterization of phenylalanine ammonialyase, cinnamate 4-hydroxylase and genes involved in flavone biosynthesis in Scutellaria baicalensis. Bioresour. Technol. 2010, 101, 9715−9722. (23) Liang, Y. S.; Kim, H. K.; Lefeber, A. W.; Erkelens, C.; Choi, Y. H.; Verpoorte, R. Identification of phenylpropanoids in methyl jasmonate treated Brassica rapa leaves using two-dimensional nuclear magnetic resonance spectroscopy. J. Chromatogr. A 2006, 1112, 148− 155. (24) Alagna, F.; D’Agostino, N.; Torchia, L.; Servili, M.; Rao, R.; Pietrella, M.; Giuliano, G.; Chiusano, M. L.; Baldoni, L.; Perrotta, G. Comparative 454 pyrosequencing of transcripts from two olive genotypes during fruit development. BMC Genomics 2009, 10, No. 399. (25) Barakat, A.; DiLoreto, D. S.; Zhang, Y.; Smith, C.; Baier, K.; Powell, W. A.; Wheeler, N.; Sederoff, R.; Carlson, J. E. Comparison of the transcriptomes of American chestnut (Castanea dentata) and Chinese chestnut (Castanea mollissima) in response to the chestnut blight infection. BMC Plant Biol. 2009, 9, No. 51. (26) Shi, C. Y.; Yang, H.; Wei, C. L.; Yu, O.; Zhang, Z. Z.; Jiang, C. J.; Sun, J.; Li, Y. Y.; Chen, Q.; Xia, T.; Wan, X. C. Deep sequencing of the Camellia sinensis transcriptome revealed candidate genes for major metabolic pathways of tea-specific compounds. BMC Genomics 2011, 12, No. 131. (27) Pérez-de-Castro, A. M.; Vilanova, S.; Cañizares, J.; Pascual, L.; Blanca, J. M.; Díez, M. J.; Prohens, J.; Picó, B. Application of genomic tools in plant breeding. Curr. Genomics 2012, 13, 179−195. (28) Li, P.; Ponnala, L.; Gandotra, N.; Wang, L.; Si, Y.; Tausta, S. L.; Kebrom, T. H.; Provart, N.; Patel, R.; Myers, C. R.; Reidel, E. J.; Turgeon, R.; Liu, P.; Sun, Q.; Nelson, T.; Brutnell, T. P. The developmental dynamics of the maize leaf transcriptome. Nat. Genet. 2010, 42, 1060−1067. (29) Severin, A. J.; Woody, J. L.; Bolon, Y. T.; Joseph, B.; Diers, B. W.; Farmer, A. D.; Muehlbauer, G. J.; Nelson, R. T.; Grant, D.; Specht, J. E.; Graham, M. A.; Cannon, S. B.; May, G. D.; Vance, C. P.; Shoemaker, R. C. RNA-Seq Atlas of Glycine max: A guide to the soybean transcriptome. BMC Plant Biol. 2010, 10, No. 160. (30) Edwards, K.; Johnstone, C.; Thompson, C. A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res. 1991, 19, 1349. (31) Schulz, M. H.; Zerbino, D. R.; Vingron, M.; Birney, E. Oases: Robust de novo RNAseq assembly across the dynamic range of expression levels. Bioinformatics 2012, 28, 1086−1092. (32) Anders, S.; Huber, W. Differential expression analysis for sequence count data. Genome Biol. 2010, 11, No. R106. (33) Tuan, P. A.; Thwe, A. A.; Kim, Y. B.; Kim, J. K.; Kim, S. J.; Lee, S.; Chung, S. O.; Park, S. U. Effects of white, blue, and red lightemitting diodes on carotenoid biosynthetic gene expression levels and carotenoid accumulation in sprouts of tartary buckwheat (Fagopyrum tataricum Gaertn.). J. Agric. Food Chem. 2013, 61, 12356−12361. (34) Thwe, A. A.; Mai, N. T. T.; Li, X.; Kim, Y.; Kim, Y. B.; Uddin, M. R.; Kim, Y. S.; Bae, H.; Kim, H. H.; Lee, M. Y.; Park, S. U. Production of astragaloside and flavones from adventitious root cultures of Astragalus membranaceus var mongholicus. Plant Omics J. 2012, 5 (5), 466−470. (35) Pan, H.; Fang, C.; Zhou, T.; Wang, Q.; Chen, J. Accumulation of calycosin and its 7-O-beta-D-glucoside and related gene expression in seedlings of Astragalus membranaceus Bge. var. mongholicus (Bge.)

REFERENCES

(1) Rios, J. L.; Waterman, P. G. A review of the pharmacology and toxicology of Astragalus. Phytother. Res. 1997, 11, 411−418. (2) Zhang, W. J.; Hufnag, P.; Binder, B. R.; Wojta, J. Antiinflammatory activity of astragaloside IV is mediated by inhibition of NF-kappaB activation and adhesion molecule expression. J. Thromb. Haemost. 2003, 90, 904−914. (3) Tang, L.; Liu, Y.; Wang, Y.; Long, C. Phytochemical analysis of an antiviral fraction of Radix astragali using HPLC-DAD-ESIMS/MS. J. Nat. Med. 2010, 64, 182−186. (4) Zhang, Y. D.; Wang, Y. L.; Shen, J. P. Effects of Astragalous saponins on antiinflammation and antihypertension. Acta Pharmacol. Sin. 1984, 19, 333−337. (5) Bedir, E.; Pugh, N.; Calis, I.; Pasco, D. S.; Khan, I. A. Immunostimulatory effects of cycloartane-type triterpene glycosides from Astragalus species. Biol. Pharm. Bull. 2000, 23, 834−837. (6) Wu, F.; Chen, X. A review of pharmacological study on Astragalus membranaceus (Fisch.) Bge. Zhong Yao Cai 2004, 27, 232−234. (7) Song, Z. H.; Ji, Z. N.; Lo, C. K.; Dong, T. T.; Zhao, K. J.; Li, O. T.; Haines, C. J.; Kung, S. D.; Tsim, K. W. Chemical and biological assessment of a traditional Chinese herbal decoction prepared from Radix Astragali and Radix Angelicae Sinensis: Orthogonal array design to optimize the extraction of chemical constituents. Planta Med. 2004, 70, 1222−1227. (8) Xu, R. Y.; Nan, P.; Yang, Y.; Pan, H.; Zhou, T.; Chen, J. Ultraviolet irradiation induces accumulation of isoflavonoids and transcription of genes of enzymes involved in the calycosin-7-O-β-dglucoside pathway in Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao. Physiol. Plant. 2011, 142, 265−273. (9) Pan, H.; Wang, Y.; Zhang, Y.; Zhou, T.; Fang, C.; Nan, P.; Wang, X.; Li, X.; Wei, Y.; J, C. Phenylalanine ammonia lyase functions as a switch directly controlling the accumulation of calycosin and calycosin7-O-b-D-glucoside in Astragalus membranaceus var. mongholicus plants. J. Exp. Bot. 2008, 59, 3027−3037. (10) Kwon, H. J.; Hwang, J.; Lee, S. K.; Park, Y. D. Astragaloside content in the periderm, cortex, and xylem of Astragalus membranaceus root. J. Nat. Med. 2013, 67, 850−855. (11) Lai, P. K.; Chan, J. Y.; Cheng, L.; Lau, C. P.; Han, S. Q.; Leung, P. C.; Fung, K. P.; Lau, C. B. Isolation of anti-inflammatory fractions and compounds from the root of Astragalus membranaceus. Phytother. Res. 2012, 27, 581−587. (12) Han, L.; Chen, K. J. Progress of experimental pharmacologic study on the effect of Astragalus on the cardiac vascular system. Chin. J. Integr. Med. 2000, 20, 234−237. (13) Huang, C. L.; Lu, Y. P. Effect of Astragalus injection on insulin resistance in auxiliary treating patients with diabetes mellitus type 2. Chin. J. Integr. Med. 2003, 23, 779−780. (14) Liu, Z. Q.; Li, Q. Z.; Qin, G. J. Effect of Astragalus injection on platelet function and plasma endothelin in patients with early stage diabetic nephropathy. Chin. J. Integr. Med. 2001, 21, 274−276. (15) Fan, Y.; Wu, D. Z.; Gong, Y. Q.; Zhou, R. Y.; Hu, Z. B. Effects of calycosin on the impairment of barrier function induced by hypoxia in human umbilical vein endothelial cells. Eur. J. Pharmacol. 2003, 481, 33−40. (16) Tang, D.; He, B.; Zheng, Z. G.; Wang, R. S.; Gu, F.; Duan, T. T.; Cheng, H. Q.; Zhu, Q. Inhibitory effects of two major isoflavonoids in Radix Astragali on high glucose-induced mesangial cells proliferation and AGEs-induced endothelial cells apoptosis. Planta Med. 2011, 77, 729−732. (17) Chen, L.; Li, Z.; Tang, Y.; Cui, X.; Luo, R.; Guo, S.; Zheng, Y.; Huang, C. Isolation, identification and antiviral activities of metabolites of calycosin-7-O-β-D-glucopyranoside. J. Pharm. Biomed. Anal. 2011, 56, 382−389. (18) Hirotani, M.; Zhou, Y.; Rut aTsutomu Furuya, H. Cycloartane triterpene glycosides from the hairy root cultures of Astragalus membranaceus. Phytochemistry 1994, 37, 1403−1407. (19) Du, M.; Wu, X. J.; Ding, J.; Hu, Z. B.; White, K. N.; BranfordWhite, C. J. Astragaloside IV and polysaccharide production by hairy I


Article

Journal of Agricultural and Food Chemistry Hsiao induced by low temperature stress. Plant Cell Rep. 2007, 26, 1111−1120. (36) Palazon, J.; Cusido Rosa, M.; Bonfill, M.; Mallol, A.; Moyano, E.; Morales, C.; Pinol, M. T. Elicitation of different Panax ginseng transformed root phenotypes for an improved ginsenoside production. Plant Physiol. Biochem. 2003, 41, 1019−1025. (37) Komaraiah, P.; Reddy, G. V.; Reddy, P. S.; Raghavendra, A. S.; Ramakrishna, S. V.; Reddanna, P. Enhanced production of antimicrobial sesquiterpenes and lipoxygenase metabolites in elicitortreated hairy root cultures of Solanum tuberosum. Biotechnol. Lett. 2003, 25, 593−597. (38) Hayashi, H.; Huang, P.; Inoue, K. Up-regulation of soyasaponin biosynthesis by methyl jasmonate in cultured cells of Glycyrrhiza glabra. Plant Cell Physiol. 2003, 44, 404−411. (39) Singh, K.; Foley, R. C.; Oñate-Sánchez, L. Transcription factors in plant defense and stress responses. Curr. Opin. Plant Biol. 2002, 5, 430−436. (40) Glazebrook, J. Genes controlling expression of defense responses in Arabidopsis - 2001 status. Curr. Opin. Plant Biol. 2001, 4, 301−308. (41) Georgiev, M. I.; Pavlov, A. I.; Bley, T. Hairy root type plant in vitro systems as sources of bioactive substances. Appl. Microbiol. Biotechnol. 2007, 74, 1175−1185. (42) Srivastava, S.; Srivastava, A. K. Hairy root culture for massproduction of high value secondary metabolites. Crit. Rev. Biotechnol. 2007, 27, 29−43. (43) Yonekura-Sakakibara, K.; Fukushima, A.; Nakabayashi, R.; Hanada, K.; Matsuda, F.; Sugawara, S.; Inoue, E.; Kuromori, T.; Ito, T.; Shinozaki, K.; Wangwattana, B.; Yamazaki, M.; K, S. Two glycosyltransferases involved in anthocyanin modification delineated by transcriptome independent component analysis in Arabidopsis thaliana. Plant J. 2012, 69, 154−167. (44) Miettinen, K.; Dong, L.; Navrot, N.; Schneider, T.; Burlat, V.; Pollier, J.; Woittiez, L.; van der Krol, S.; Lugan, R.; Ilc, T.; Verpoorte, R.; Oksman-Caldentey, K. M.; Martinoia, E.; Bouwmeester, H.; Goossens, A.; Memelink, J.; Werck-Reichhart, D. The seco-iridoid pathway from Catharanthus roseus. Nat. Commun. 2014, 5, No. 3606.

J


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