Triterpene and Flavonoid Biosynthesis and Metabolic Profiling of Hairy

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Triterpene and flavonoid biosynthesis and metabolic profiling of hairy roots, adventitious roots, and seedling roots of Astragalus membranaceus Yun Ji Park, Aye Aye Thwe, Xiaohua Li, Yeon Jeong Kim, Jae Kwang Kim, Valan Arasu Mariadhas, Naif Abdullah Al-Dhabi, and Sang Un Park J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 24 Sep 2015 Downloaded from http://pubs.acs.org on September 25, 2015

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Journal of Agricultural and Food Chemistry

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Triterpene and flavonoid biosynthesis and metabolic profiling of hairy roots,

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adventitious roots, and seedling roots of Astragalus membranaceus

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Yun Ji Park,† Aye Aye Thwe,† Xiaohua Li,† Yeon Jeong Kim,† Jae Kwang Kim,‡ Mariadhas

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Valan Arasu,§ Naif Abdullah Al-Dhabi,§,* Sang Un Park†,⊥,*

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†

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Daejeon, 305-764, Korea

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‡

Department of Crop Science, Chungnam National University, 99 Daehak-ro, Yuseong-gu,

Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National

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University, Incheon, 406-772, Korea

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§

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College of Science, King Saud University, P. O. Box 2455, Riyadh 11451, Saudi Arabia

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⊥

14

Saudi Arabia

Department of Botany and Microbiology, Addiriyah Chair for Environmental Studies,

Visiting Professor Program (VPP), King Saud University, P.O. Box 2455, Riyadh 11451,

15 16

*

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N. A. Al-Dhabi:

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Department of Botany and Microbiology, Addiriyah Chair for Environmental Studies,

19

College of Science, King Saud University, P. O. Box 2455, Riyadh 11451, Saudi Arabia.

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Phone: +966-1- 467-5829; Fax: +966-1- 469-7204; E-mail:[email protected]

21

S.U. Park:

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Department of Crop Science, Chungnam National University, 99 Daehak-ro, Yuseong-gu,

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Daejeon, 305-764, Korea.Phone: +82-42-821-5730. Fax: +82-42-822-2631. E-mail:

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[email protected]

To whom correspondence should be addressed

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ACS Paragon Plus Environment


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ABSTRACT

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Astragalus membranaceus, an important traditional Chinese herb with various

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medical applications. Astragalosides (ASTs), calycosin and calycosin-7-O-β-D-glucoside

29

(CG) are the primary metabolic components in A. membranaceus roots. The dried roots of A.

30

membranaceus have various medicinal properties. Present study aimed to investigate the

31

expression levels of genes related to the biosynthetic pathways of ASTs, calycosin, and CG in

32

order to investigate the differences between seedling roots (SRs), adventitious roots (ARs),

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and hairy roots (HRs) using quantitative real-time polymerase chain reaction (qRT-PCR).

34

qRT-PCR study revealed that the transcription level of genes involved in the AST

35

biosynthetic pathway was the lowest in ARs and showed similar patterns in HRs and SRs.

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Moreover, most genes involved in the synthesis of calycosin and CG exhibited the highest

37

expression levels in SRs. High-performance liquid chromatography (HPLC) analysis

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indicated that the expression level of the genes correlated with the content of ASTs, calycosin,

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and CG in the three different types of roots. ASTs were the most abundant in SRs. CG

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accumulation was greater than calycosin accumulation in ARs and HRs, while the opposite

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was true in SRs. Additionally, 43 metabolites were identified using gas chromatography-time-

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of-flight mass spectrometry (GC-TOF-MS). The principal component analysis (PCA)

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documented the differences among SRs, ARs, and HRs. PCA comparatively differentiated

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among the three samples. The results of PCA showed that HRs was distinct from ARs and

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SRs based on the dominant amouts of sugars and clusters derived from closely simmilar

46

biochemical pathways. Also, ARs had higher concentration of phenylalanine, a precursor for

47

the phenylpropanoid biosynthetic pathway, as well as CG. TCA cycle intermediates levels

48

including succinic acid and citric acid indicated higher amount in SRs than in others.


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KEYWORDS: Astragalus membranaceus, Astragalosides, Calycosin, calycosin-7-O-β-D-

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glucoside

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INTRODUCTION

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Astragalus membranaceus, a traditional Chinese herb belonged to Fabaceae family is

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popularly used as a traditional medicine in Korea, Japan, China, and other Asian countries.

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The dried roots of A. membranaceus, called “Hwang Qi”, have commonly used in the

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treatment of chronic illness 1. Many modern pharmacological studies have shown that A.

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membranaceus has a wide range of potential therapeutic applications, including anti-

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oxidative properties 2, immunostimulant activity 3, hypotensive effects 4, antibacterial and

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antiviral properties 5, 6, and antimutagenic activity 7. Many biologically active constituents of

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A. membranaceus roots, including flavonoids, alkaloide, saponins, phenolic compounds,

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triterpene and polysaccharides, have been identified 8, 9. Among them, astragalosides (ASTs),

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comprising a class of cycloartane triterpenoid-type glycosides, include astragaloside I (AST-

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I), astragaloside II (AST-II), astragaloside III (AST-III), and astragaloside IV (AST-IV) as the

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primary constituents in A. membranaceus roots

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glucoside (CG) are two other major isoflavones isolated from this herb

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commonly used in the treatment of diabetes and cardiovascular disease in traditional

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medicine system 13. Calycosin and CG protect endothelial cells from hypoxia-induced barrier

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impairment

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inhibit lecithin peroxidation induced by hydroxyl radical-generation 11. Thus, the components

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of A. membranaceus, which have been well characterized, have a variety of important

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biological functions that support the use of this traditional Chinese herb.

72

10

. Calycosin and calycosin-7-O-β-D-

14

11, 12

. ASTs are

, exhibit natural anti-inflammatory and anti-osteoarthritic activities

15

, and

Triterpenes, which comprise one of the largest groups of terpenoids, consist of 30

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carbons with number of

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the mevalonate pathway cytosolic system (Figure 1A) 16. Triterpene synthesis initiates from

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condensation of two acetyl-CoA molecules, proceeds through condensation of one

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dimethylallyl diphosphate (DMAPP) and two isoprenyl diphosphate (IPP) molecules, and

oxygen atoms adhered and are linked from a C5 isoprene unit of


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17

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results in farnesyl diphosphate (FPP), followed by ramification into sterol and triterpene

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The first diversifying step in triterpenoid biosynthesis is the cyclization of 2,3-oxidosqualene,

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which is catalyzed by oxidosqualene cyclase (OSC); this step enables the synthesis of more

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than 100 variations of triterpenoids in plants

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synthase (CAS) and lanosterol synthase (LAS), are involved in both sterol biosynthesis and

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triterpenoid biosynthesis in higher plants

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oxidosqualene from FPP, are the main backbone for the synthesis of ursane, oleanane,

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dammarane and lupeol which are the major triterpenoid skeletons 18, 20.

.

18

. Several OSCs, including cycloartenol

19

. Squalene synthase (SS) synthesizes 2,3-

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In the plant system isoflavones calycosin and CG are metabolically produced via the

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phenylpropanoid pathway (Figure 1B). Phenylalanine ammonia-lyase (PAL) is the starting

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enzyme which converts L-phenylalanine into trans-cinnamic acid.

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hydroxylase (C4H) and 4-coumarate:coenzyme A-ligase (4CL) subsequently acts on trans-

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cinnamic acid to produce and p-coumarate CoA ester respectively 21. After that, p-coumaroyl-

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CoA condensed with three molecules of malonyl-CoA to synthesize chalcone by the catalytic

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action

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trihydroxyflavanone) was produced using the isomerization reaction of chalcone isomerase

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(CHI). In species that synthesize isoflavones, the enzyme isoflavone synthase (IFS), which

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catalyzes formation of all isoflavones, has two roles: diverting naringenin into genistein

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production and catalyzing daidzein 22. A methyl group is then added to daidzein by isoflavone

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O-methyltransferase (IOMT) to form formononetin

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hydroxylation of isoflavone 3′-hydroxylase (I3′H), and this compound can be modified to CG

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by calycosin 7-O-glucosyltransferase (UCGT) 24.

of

chalcone

synthase

(CHS).

In

the

next

reaction,

Further, Cinnamate-4-

naringenin

(5,7,4′-

23

. Finally, calycosin is synthesized by

99

Through these well-characterized pathways, the common components of A.

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membranaceus are synthesized and promote the beneficial effects of the herb. In addition to



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these well-known synthetic pathways, A. membranaceus is also a good candidate for the

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natural product isolation due to its long history in traditional medicine and abundance of

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beneficial compounds. Commonly called “Hwang Qi”, the traditional medicine from A.

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membranaceus is prepared from the dried roots of the plant, and studies have characterized

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the phytochemistry, pharmacology, and clinical applications of the preparation 25. However,

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to the best of our knowledge, no studies have compared the contents and expression profiles

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of genes involved in biosynthetic pathways between seedling roots (SRs), adventitious roots

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(ARs), and hairy roots (HRs) of A. membranaceus.

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In vitro root culture is the well known method for the bulk level production of novel

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plant metabolites with nutraceutical, pharmaceutical, and industrial applications 26, 27. In vitro

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culturing of ARs with the supply of optimized concentration of

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comparatively produce valuable secondary metabolites

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culture systems has indicated that effective culture systems can promote large-scale

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multiplication, strain improvement, and species conservation 28. Genetically transformed HR

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cultures have several beneficial properties, including rapid growth, genetic and biochemical

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stability, relatively low-cost culture obligation, and growth in hormone-free medium

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Additionally, HRs derived from Agrobacterium rhizogenes by infecting into plants exhibit

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multi-enzyme biosynthetic potential similar to or greater than the parent plants 30.

phytohormone

27

. Establishment of AR suspension

29

.

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Metabolomics provides biochemical snapshot of an organism’s phenotype. This

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technique provide the chance to for the in-depth identification of markers for the individual

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metabolite with specific to species, cultivar, certain stages of development, or conditions such

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as disease, stress, or daily and seasonal changes respectively

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chromatography and mass spectrometry (GC-MS) has allowed for the identification and

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robust quantification of several hundred metabolites within a single extract. Recently, several


31

. A combination of gas

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metabolomics studies have done for the classification of diverse biological samples varying

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in status, origin, and quality using GC-time of flight (TOF)-MS 32-34. However, no metabolite

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profiles of the A. membranaceus have been reported to date.

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In this study, we aimed to compare the expression levels of genes related to AST,

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calycosin, and CG biosynthetic pathways among SRs, ARs, and HRs. Moreover, ASTs,

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calycosin, and CG extracted from these three different types of root cultures of A.

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membranaceus were quantified using high-performance liquid chromatography (HPLC).

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These results demonstrated an efficient root culture system for production of secondary

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metabolites in A. membranaceus. In addition, the levels of hydrophilic metabolites were

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analyzed from different root materials using GC-TOF-MS.

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MATERIALS AND METHODS

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Induction of HRs and ARs

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Hairy roots were initiated from excised leaves of A. membranaceus after being

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infected with the Agrobacterium rhizogenes R1000. Briefly, the excised leaves (1.0 × 1.0 cm)

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were washed thoroughly with distilled water and dipped into the A. rhizogenes for 10 min.

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After that immediately the leaves were collected and kept in the sterile filter paper for the

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removal of the liquid and then placed on the autoclaved sterile solidified MS medium 35.

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After that the samples were kept in the incubator at 25°C and maintained in the dark for two

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days. After that, the explants tissue was cultivated in the hormone-free MS medium for 3-4

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weeks. Upon emergence of the hairy roots, the hairy roots were removed from the flask and

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cultivated in the MS medium containing 200 mg/L timentin at 25°C under dark condition.

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Further, the roots were routinely cultivated by transferring it into the fresh medium to collect

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the fresh hairy roots. Rapidly growing adventitious roots were carefully collected and

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initiated from leaf explants of A. membranaceus maintained on agar-solidified MS media

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containing 1.0 mg/L 1-naphthaleneacetic acid (NAA) as described by Thwe et al 36. Seedling

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roots excised from A. membranaceus plantlets were grown in vitro.

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The above obtained three types of root cultures were collected and further

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subcultured in hormone-free MS liquid medium, cultivated at 25°C on a shaker (100 rpm)

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under dark condition for 24 h. After 20 days of incubation, the hairy roots were harvested for

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the extraction of total RNA, and analysis of phytochemicals. The harvested samples were

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immediately stored in -80°C for further molecular and chemical analysis.

157 158

Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR)


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On the basis of the sequences of AmAACT, AmIDI, AmSS, AmPAL1, Am4CL,

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AmCHS1, AmCHI, and AmIOMT, we designed real-time PCR primers using the Primer3

161

website (http://frodo.wi.mit.edu/primer3/). Total RNA was extracted from each collected

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samples using modified trizol method. The first strand of cDNA was synthesized using 1µg

163

of total RNA according to the manufacturer’s method (ReverTra ace, Toyobo, Japan). The

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reverse transcribed cDNA products were used as templates for gene expression analysis with

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gene-specific primers. Real-time PCR products were tested for specificity of fragment sizes,

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melting curves, and sequences by PCR, qRT-PCR, and cloning into a T-Blunt vector for

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sequencing, respectively. The housekeeping phosphoribosyl transferase gene (NCBI

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GenBank Accession number KF355974) was used to normalize the relative expression of the

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targeted genes. qRT-PCR was performed on a CFX96 real time system (BIO-RAD

170

Laboratories, USA) with the 2X Real-Time PCR Smart mix (BioFACT, Korea) under

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following conditions: 95℃ for 15minutes, followed by 40cycles of 95℃ for 15 seconds,

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annealing for 15seconds at 55℃, and elongation for 20seconds at 72℃. Three replications for

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each sample were used in the real-time analyses.

174 175

HPLC analysis

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The materials were freeze-dried and ground into a fine powder. Briefly, 100-mg

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samples were extracted with 1.5 mL MeOH and sonicated for 60 min. The samples were

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vortexed every 20 min during sonication. After centrifugation at 4000 rpm at room

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temperature for 10 min, the supernatant was filtered through a 0.45-µm PTFE syringe filter

180

(Advantec DISMIC-13HP; Toyo Roshi Kaisha, Ltd., Tokyo, Japan) for HPLC analysis.

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HPLC-grade methanol (MeOH) was purchased from J. T. Baker (Phillipsburg, NJ, USA).

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Formic acid was purchased from Sam Chun Pure Chemical Co., Ltd. (Gyeonggi, Korea).

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AST I, AST II, AST III, AST IV, calycosin, and CG were obtained from ChromaDex (Irvine,



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CA, USA). HPLC analysis was performed using a C18 column (250 × 4.6 mm, 5 µm; RStech;

185

Daejeon, Korea). The flow rate was maintained at 1.0 mL/min, the column was maintained at

186

30°C, and the injection volume was 20 µL. The mobile phases consisted of 0.3% formic acid

187

in water (A) and acetonitrile (B), and a gradient elution protocol was used. The ultraviolet

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(UV) detection wavelength was 260 nm for isoflavonoids. The drift tube temperature for

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evaporating light scattering detection (ELSD) was 70°C, and the nebulizing gas flow rate was

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3.0 L/min. The concentration of each compound was determined using a standard curve. All

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samples were analyzed in triplicate.

192 193

Metabolite analysis using GC-TOF-MS

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Analytical grade reagents and chemicals used in this experiment. Chloroform and methanol

195

were

196

trimethylsilyltrifluoroacetamide (MSTFA) and ribitol and were procured from Sigma

197

Chemical Co. (St. Louis, MO, USA). Pyridine and methoxyamine hydrochloride were

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procured from Thermo Fisher Scientific (Pittsburgh, PA, USA). The extraction of polar

199

metabolite was performed as described by Kim et al., 2007. The sample preparation methods,

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chemicals and reagents, esterification procedures, GC-TOFMS instrument operating

201

conditions, analytical methods for the separation of the samples and the scanning and the

202

detection of the compounds rang were as implemented as described in our previous research

203

paper Kim et al.

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identification was performed by comparison with reference compounds and the use of an in-

205

house library.

procured

from

J.T.

Baker

(Phillipsburg,

USA).

N-methyl-N-

34

. ChromaTOF software was used to assist with peak location. Peak

206 207

NJ,

Statistical analysis


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The data for gene expression and each constituent were analyzed using Statistic

209

al Analysis System software (SAS version 9.2). Treatment means were compared usin

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g Duncan’s multiple range test. Quantification data acquired from GC-TOF-MS were

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subjected to principal component analysis (PCA) (SIMCA-P version 13.0; Umetrics, Umea,

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Sweden) to evaluate differences among groups of multivariate data. The data file was scaled

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with unit wariance scaling for the multivariate analysis. The PCA output consisted of score

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plots to visualize the contrast between different samples and loading plots to explain the

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cluster separation.

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RESULTS AND DISCUSSION

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Transcription levels of genes related to AST biosynthetic pathways in different root

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cultures of A. membranaceus

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To investigate the mechanisms controlling AST biosynthesis in different root cultures

220

of A. membranaceus, the expression levels of genes encoding AST-synthesizing enzymes

221

were analyzed in SRs, ARs, and HRs. The expression levels of AmAACT, AmHMGS,

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AmHMGR1, AmHMGR2, AmHMGR3, AmMK, AmPMK, AmMVD, AmIDI, AmFPS, AmSS,

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AmCAS, and AmSE are shown in Figure 2. The expression patterns of AmAACT, AmFPS,

224

AmCAS, and AmSE were similar, with observably higher expression in HRs than in SRs.

225

Additionally, AmHMGRs (AmHMGR1, AmHMGR2, and AmHMGR3) exhibited lower

226

expression in SRs than in HRs. In contrast, AmHMGS, AmMK, AmPMK, AmMVD, AmIDI,

227

and AmSS exhibited high expression in SRs, but were moderately expressed in HRs.

228

Although the transcripts encoding all of these enzymes were expressed in ARs, ARs exhibited

229

the lowest expression levels for these genes.

230 231

Quantitative analysis of ASTs



232

A total of four ASTs (AST I, AST II, AST III, and AST IV) were extracted and

233

quantified in three different root cultures (Table 1). All of the identified compounds were

234

found to be present at high levels in SRs (AST I: 2532.87 µg/g dry wt, AST II: 1342.25 µg/g

235

dry wt, AST III: 1712.40 µg/g dry wt, and AST IV: 620.97 µg/g dry wt). ARs exhibited the

236

least AST I (177.34 µg/g dry wt), AST II (189.63 µg/g dry wt), and AST III (280.38 µg/g dry

237

wt) and moderate levels of AST IV (120.84 µg/g dry wt). The levels of these compounds

238

were relatively higher in HRs than in ARs (AST I: 785.87 µg/g dry wt, AST II: 391.35 µg/g

239

dry wt, AST III: 378.92 µg/g dry wt), whereas the level of AST IV was slightly lower in HRs

240

(119.44 µg/g dry wt) than in ARs.

241 242

Expression analysis of calycosin and CG biosynthetic genes in different root cultures of

243

A. membranaceus

244

To investigate the control of calycosin and CG biosynthesis, we examined the

245

expression levels of genes related to calycosin biosynthesis in ARs, HRs, and SRs using qRT-

246

PCR. The expression levels of AmPAL, AmC4H, Am4CL, AmCHS, AmCHR, AmCHI, AmIFS,

247

AmI3′H, and AmUCGT are shown in Figure 3. The expression levels of these genes, with the

248

exception of AmUCGT, were the highest in SRs, demonstrating that AmUCGT exhibited a

249

different expression profile than the other genes. The expression of AmUCGT was extremely

250

low compared to that of other genes and was the highest in ARs. The expression patterns of

251

AmPAL, AmC4H, Am4Cl, AmCHR, and AmI3′H were similar, with observably higher

252

expression in ARs than in HRs. However, the expression levels of AmCHS, AmCHI, and

253

AmIFS were higher in ARs than in HRs.

254 255 256

Calycosin and CG contents in different root cultures Next, we assessed the contents of two different flavonoids, calycosin and CG in the


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three types of roots. As shown in Table 2, CG and calycosin accumulated at 5.64 and 3.56

258

µg/g dry wt, respectively, in ARs. The levels of CG and calycosin in HRs were produced 2.84

259

and 2.93 µg/g dry wt, respectively. However, calycosin (5.70 (µg/g dry wt) was more

260

abundant than CG (3.10 (µg/g dry wt) in SRs; this was correlated with low expression of

261

AmUCGT, which converts calycosin to CG. Although AmUCGT transcript levels were

262

relatively higher in ARs than in HRs, the CG content was high in HRs.

263

264

GC-TOF-MS analysis of polar metabolites in different root cultures

265

GC-TOF-MS was used to identify and quantify low-molecular-weight hydrophilic

266

compounds in A. membranaceus samples. We were able to identify 43 compounds including

267

13 organic acids, 12 amino acids, 6 sugar alcohols, 5 sugars, 3 carbohydrates and 1 amine in

268

these samples (Supplementary 1). The corresponding retention times agreed with our

269

previous data

270

calculations of all analytes were based on the peak area ratios relative to that of the IS.

34

. Quantification was performed using selected ions, and the quantitative

271

PCA was used to explore the data structure obtained by GC-TOF-MS. PCA reduces

272

the dimensionality of complex data sets and thereby facilitate the visualization of inherent

273

patterns in the data. PCA transforms the original variables, using an orthogonal linear

274

transformation, to a new set of uncorrelated variables known as principal components (PCs).

275

As shown in Figure 4, the first and second PCs of the PCA score plot represented 58.4% and

276

34.4% of the total variance of the samples, respectively. The variance of the differences

277

among roots was successfully captured in the two PCs. The first PC resolved the metabolite

278

profiles of HRs and other roots. The metabolomes between SRs and ARs were separated by

279

PC 2. The loading plot of the PCA revealed the magnitude and direction of correlation of the



280

original variables with PCs. Correlation analysis is a common statistical method used to

281

establish relationships between metabolite signals belonging to a biological system 37. Sugars

282

such as glucose, fructose, mannose, and sucrose were clustered in the left of the loading plot,

283

indicating that the level of sugars in HRs was higher than that in other roots. In plant systems,

284

the building blocks for secondary metabolites are derived from primary metabolism. In this

285

study, free glucose, a building block of CG, was higher in HRs, whereas CG content was

286

lower in HRs than in SRs and ARs (Supplementary 2). The loading plot of PC 2 indicated

287

that a higher amount of phenylalanine was present in ARs than in SRs and HRs and TCA-

288

cycle intermediates such as citric acid and succinic acid were higher in SRs than in others.

289

Phenylalanine is a major amino acid donor for the synthesis of CG. Thus metabolomics can

290

assist in dissecting the mechanisms that regulate the conversion of primary metabolites into

291

secondary metabolites in plants.

292

The objective of the present study was to examine an efficient root culture system for

293

ASTs, calycosin, and CG accumulation and the levels of hydrophilic metabolites in different

294

root types of A. membranaceus. The results indicate that the highest amounts of ASTs were

295

produced in SRs of this plant with high expression levels of genes associated with this

296

biosynthetic pathway. Also, we observed that ARs and HRs accumulate CG higher than

297

calycosin, but SRs showed it reversely. Totally, 43 metabolites were distinguished among

298

three samples of A. membranaceus. We demonstrated that HRs were clearly separate from

299

other root cultures because of the high levels of sugars and clusters derived from closely

300

related biochemical pathways.

301

In vitro root culture has been considered as an alternative method to produce

302

secondary metabolites based on a year-round cultivation and high-level quality and quantity

303

38

. Many researches demonstrated new possibilities using in vitro cultures for accumulation of


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useful phytochemicals in plants. Development of an AR culure system could promote large-

305

scale multiplication, strain improvement, and species conservation 28, but optimal condition is

306

necessary to boost root biomass and secondary metabolites 39-42. AR cultures from many plant

307

species including Rhphanus sativus

308

javanica

309

Total saponin and flavonoid contents of 40-d-old AR derived from A. membranaceus were

310

shown higher than that of 1-yr-old roots under field cultivation

311

remarkable experimental tool to investigate the various sides of biosynthesis related to

312

biologically effective compounds. Numerous successful examples for HR cultures have done

313

based on phytochemical production from medicinal plants. M. Du et al

314

AST IV and polysaccharides are produced in HR of A. membrananceus on a large scale. Even

315

though we demonstrated that SRs contain the largest amounts of ASTs, ARs or HRs of A.

316

membranaceus are great materials for ASTs, calycosin, and CG production possibly using

317

optimal conditions for yield.

46

43

, Panax notoginseng

44

, Cornus capitata

45

, and Rhus

have been conducted and cultured for production of valuable phytochemicals 27.

318


47

. HR cultures offer a

48

have reported that


319

320

ABBREVIATIONS

321

AST, astrogaloside; CG, calycosin-7-O-β-D-glucoside; SR, seedling root; AR, adventitious

322

root; HR, hairy root; qRT-PCR, quantitative real-time polymerase chain reaction; HPLC,

323

high-performance liquid chromatography; GC-TOF-MS, gas chromatography-time-of-light

324

mass spectrometry; PCA, principal component analysis; DMAPP, dimethylallyl diphosphate;

325

IPP, isoprenyl diphosphate; FPP, farnesyl diphosphate; OSC, oxidosqualene cyclase; CAS,

326

cycloartenol synthase; LAS, lanosterol synthase; SS, squalene synthase; PAL, phenylalanine

327

ammonia-lyase; C4H, cinnamate-4-hydroxylase; 4CL, 4-coumarate:coenzyme A-ligase; CHS,

328

chalcone synthase; CHR, chalcone reductase; CHI, chalcone isomerase; IFS, isoflavone

329

synthase; IOMT, isoflavone O-methyltransferase; I3’H, isoflavone 3’-hydroxylase; UCGT,

330

calycosin 7-O-glucosyltransferase; ELSD, evaporating light scattering detection; TMS,

331

trimethylsilyl; MSTFA, N-methyl-N-(TMS)-trifluoroacetamide; AACT, acetoacetyl-CoA

332

thiolase; HMGS, HMG-CoA synthase; HMGR, HMG-CoA reductase; MK, mevalonate

333

kinase; PMK, phosphomevalonate kinase; MVD, mevalonate diphosphate decarboxylase; IDI,

334

isopentenyl diphosphate isomerase; FPS, farnesyl diphosphate synthase.

335

336

Funding

337

The Project was full financially supported by King Saud University, through Vice Deanship

338

of Research Chairs.

339 340

Notes

341

The authors declare no competing financial interest.


Page 16 of 32

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485

Table 1. AST contents in different types of roots of A. membranaceus. (μg/g dry weight).

486

Each value is from three determinations ± SD. AR, adventitious root; SR, seedling root; HR,

487

hairy root. Compound

AR

HR

SR

AST I

177.34 ± 6.72

785.87 ± 37.56

2532.87 ± 61.91

AST II

189.63 ± 3.32

391.35 ± 12.63

1342.25 ± 2.84

AST III

280.38 ± 8.58

378.92 ± 42.06

1712.40 ± 111.69

AST IV

120.84 ± 5.93

119.44 ± 2.35

620.97 ± 5.46

488 489



490 491

Table 2. Analysis of isoflavone contents, including calycosin and CG contents, in different

492

types of roots from A. membranaceus. Each value is from three determinations ± SD. AR,

493

adventitious root; SR, seedling root; HR, hairy root. Compound

AR

HR

SR

calycosin

3.56 ± 0.54

2.93 ± 1.04

5.70 ± 0.11

CG

5.64 ± 0.12

7.14 ± 3.43

3.10 ± 0.62

494 495


Page 24 of 32

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496 497

Figure legends

498

Figure 1. Putative scheme of biosynthetic pathway leading to (A) astragaloside and (B)

499

calycosin and calycosing-7-O-β-D-glucoside. AACT, acetoacetyl-CoA thiolase; HMGS,

500

HMG-CoA synthase; HMGR, HMG-CoA reductase; MK, mevalonate kinase; PMK,

501

phosphomevalonate kinase; MVD, mevalonate diphosphate decarboxylase; IDI, isopentenyl

502

diphosphate isomerase; FPS, farnesyl diphosphate synthase; DMAPP, dimethylallyl

503

diphosphate; IPP, isoprenyl diphosphate; FPP, farnesyl diphosphate; SS, squalene synthase;

504

PAL,

505

coumarate:coenzyme A-ligase; CHS, chalcone synthase; CHR, chalcone reductase; CHI,

506

chalcone isomerase; IFS, isoflavone synthase; IOMT, isoflavone O-methyltransferase; I3’H,

507

isoflavone 3’-hydroxylase; UCGT, calycosin 7-O-glucosyltransferase.

phenylalanine

ammonia-lyase;

C4H,

cinnamate-4-hydroxylase;

4CL,

4-

508

509

Figure 2. Accumulation of genes related to astragaloside biosynthetic in three different types

510

of roots. AR, adventitious root; SR, seedling root; HR, hairy root. The height of each bar and

511

the error bars indicate the means and standard errors, respectively, from 3 independent

512

measurements.

513 514

Figure 3. Expression levels of genes related to the calycosin and calycosin-7-O-β-D-

515

glucoside biosynthetic pathways. (A) AmPAL, (B) AmC4H, (C) Am4CL, (D) AmCHS, (E)

516

AmCHR, (F) AmCHI, (G) AmIFS, (H) AmI3′H, and (I) AmUCGT gene expression levels in

517

the three types of roots. AR, adventitious root; SR, seedling root; HR, hairy root. The height

518

of each bar and the error bars indicate the means and standard errors, respectively, from 3

519

independent measurements.



520 521 522

Figure 4. Scores (A) and loading plots (B) of principal components 1 and 2 from the PCA

523

results obtained from polar metabolite data for different types of roots from A. membranaceus.

524

AR, adventitious root; SR, seedling root; HR, hairy root.

525


Page 26 of 32

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526

527 528

Figure 1

529



530

531 532

Figure 2

533


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Page 29 of 32


534

535 536

Figure 3

537



Page 30 of 32

538

A

539 540

B

541 542

Figure 4


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543


Table of Contents Graphics

544

545

Triterpene and flavonoid biosynthesis and metabolic profiling of hairy roots,

546

adventitious roots, and seedling roots of Astragalus membranaceus

547 548 549

Yun Ji Park,† Aye Aye Thwe,† Xiaohua Li,† Yeon Jeong Kim,† Jae Kwang Kim,‡ Mariadhas

550

Valan Arasu,§ Naif Abdullah Al-Dhabi,§,* Sang Un Park†,⊥,*

551

552 553



554


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Triterpene and Flavonoid Biosynthesis and Metabolic Profiling of Hairy

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