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Planta

, Volume 249, Issue 1, pp 95–111 | Cite as

Functional characterization of an oxidosqualene cyclase (PdFRS) encoding a monofunctional friedelin synthase in Populus davidiana

  • Jung Yeon Han
  • Chang-Ho Ahn
  • Prakash Babu Adhikari
  • Subramanyam Kondeti
  • Yong Eui ChoiEmail author
Original Article
Part of the following topical collections:
  1. Terpenes and Isoprenoids

Abstract

Main conclusion

An oxidosqualene cyclase (PdFRS) from Populus davidiana was characterized as a monofunctional friedelin synthase by its heterologous expression in yeast and overexpression in plants.

Triterpenes are one of the largest classes of plant chemical compounds composed of three terpene units, which form the basic skeleton of all sterols and saponins. Friedelin (friedelan-3-one), a pentacyclic triterpene, occurs in many plant families and is particularly present in rich amounts in cork tissues from trees. The biosynthesis of friedelin occurs through the oxidosqualene cyclase (OSC) enzyme that generates friedelin from 2,3-oxidosqualene after the maximum rearrangement of a triterpene skeleton. Populus davidiana is called Korean aspen and grows in northern East Asia. From 57,322 unique sequences generated from the P. davidiana transcriptome database, one complete coding sequence (PdFRS) was obtained from a contig, which showed 74% identity to Betula platyphylla β-amyrin synthase and 73% identity with friedelin synthase from Maytenus ilicifolia. The open reading frame (ORF) region of the PdFRS sequence was 2280 bp long and composed a 759 amino acid protein with a predicted molecular mass of 87.81 kDa. qPCR analysis revealed that methyl jasmonate treatments strongly upregulated PdFRS gene expression and resulted in enhanced friedelin accumulation in leaves. Heterologous expression of the PdFRS gene in yeast resulted in the production of friedelin triterpene as a single product, which was confirmed by comparison with the mass fragmentation pattern from an authentic friedelin standard by GC/MS analysis. Transgenic P. davidiana overexpressing the PdFRS gene was constructed via Agrobacterium-mediated transformation. Overexpression of PdFRS in transgenic P. davidiana lines resulted in enhanced friedelin production.

Keywords

Populus davidiana Friedelin synthase Triterpene synthase Oxidosqualene cyclase 

Abbreviations

GC/MS

Gas chromatography–mass spectrometry

OSC

Oxidosqualene cyclase

ORF

Open reading frame

PdFRS

Populus davidiana friedelin synthase gene

qPCR

Quantitative polymerase chain reaction

RT-PCR

Reverse transcription-polymerase chain reaction

Introduction

Triterpenes are the cyclic and acyclic chemical compounds consisting of six isoprene units. Cyclic triterpenes are further categorized based on their number of triterpene rings. Triterpenoids play crucial roles in living organisms as membrane constituents (Nes and Heftmann 1981), hormones (steroids in plants and animals) (Wenzel and Emick 1956), and defense compounds (saponin in plants) (González-Coloma et al. 2011).

Friedelin (friedelan-3-one) is a pentacyclic triterpene and has been reported not only in numerous higher plants but also in lower plants such as algae, lichens, and mosses (Chandler and Hooper 1979). Friedelin is a common constituent of cork and/or stem barks from different plant species, such as Quercus suber (Ghosh et al. 2010; Castola et al. 2005; Moiteiro et al. 2006), Salix tetrasperma (El-Shazly et al. 2012), Calophyllum pinetorum (Alarcón et al. 2008), Drypetes tessmanniana (Kuete et al. 2010), Prunus turfosa (Sainsbury 1970), Pterocarpus erinaceus (Noufou et al. 2012), Terminalia avicennioides (Mann et al. 2011), etc. Friedelin is present in leaves from Maytenus aquifolium (Corsino et al. 2000), rhizomes from Polygonum bistorta (Manoharan et al. 2005), flowers from Mammea siamensis (Subhadhirasakul and Pechpongs 2005), roots from Cannabis sativa (Slatkin et al. 1971), and in forest (Kong et al. 2008) and crop field (Dong et al. 2014) soils. The compound is also one of the major constituents in leaf epicuticular wax from Kalanchoe daigremontiana (van Maarseveen and Jetter 2009) and Calluna vulgaris (Szakiel et al. 2013) and in fruit epicuticular wax from grape (Nordby and McDonald 1994). There are many reports on the antimicrobial and antifungal activities of friedelin (Ghosh et al. 2010; Parveen et al. 2010; Mokoka et al. 2013; Pretto et al. 2004). The positive relationship between soil friedelin levels and the soil microbial community has also been reported (Dong et al. 2014).

The first committed step in triterpenoid diversification is the cyclization of epoxysqualene into various triterpenes catalyzed by different oxidosqualene cyclases (OSCs) (Xu et al. 2004). Friedelin is a unique pentacyclic triterpene with the highest number of rearrangements (Fig. 1). The biosynthesis of the compound commences with the protonation of oxidosqualene followed by subsequent cyclization, several rearrangements, and, finally, deprotonation (Fig. 1). Various types of OSCs from plants have been cloned and characterized by heterologous expression in yeast (Thimmappa et al. 2014). Only a few studies have been performed on genes involved in friedelin biosynthesis. The first report on the isolation of this type of gene (KdFRS) was reported in K. daigremontiana (Wang et al. 2010). Through heterologous expression in yeast, KdFRS is a multifunctional enzyme that produces friedelin together with small amounts of β-amyrin and taraxerol (Wang et al. 2010). Recently, a monofunctional friedelin synthase (MiFRS) was isolated from Maytenus ilicifolia, which produced friedelin as a single product upon its heterologous expression in yeast (Souza-Moreira et al. 2016; Alves et al. 2018).
Fig. 1

Pentacyclic friedelin formation biosynthetic pathway through oxidosqualene protonation, cyclization, several rearrangements, and, finally, deprotonation

Populus (poplars, cottonwoods, or aspens) is a model system to study tree plant biology and biotechnology. P. davidiana (Korean aspen) is native to northern East Asia (Korea, China, and Russia) (Lee et al. 2011). The occurrence of friedelin in Populus was reported in P. yunnanensis roots that contained friedelin, 21 alpha-hydroxyfriedelan-3-one, and 21 alpha-acetoxyfriedelan-3-one (Chen and Xu 1990). However, there is no information regarding the presence of friedelin in P. davidiana plants nor has a friedelin synthase gene been characterized in Populus species.

In this work, we isolated an OSC gene (PdFRS) from the P. davidiana transcriptome, and characterized the PdFRS gene as a monofunctional friedelin synthase by demonstrating its heterologous expression in yeast and functional analysis in plants. Construction of a transgenic P. davidiana line overexpressing the PdFRS gene was achieved via Agrobacterium-mediated transformation. We confirmed that the overexpression of PdFRS in transgenic P. davidiana resulted in enhanced production of the triterpene friedelin.

Materials and methods

Plant materials and in vitro culture of plants

An approximately 20-year-old superior clone (Odea 19) of P. davidiana (Koo et al. 2007) has been growing in a clonal plantation situated at Yeongju-si in the province of Gyeongsangbuk-do, South Korea. This genotype was clonally propagated by cutting of horizontally extended roots of trees in a greenhouse at Kangwon National University. In addition, these plants clonally propagated the P. davidiana plants mentioned in Park et al. (2017).

To analyze the effect of methyl jasmonate treatment (MeJA) on the transcriptional activities of PdFRS and friedelin accumulation in leaves, the detached leaves from aseptically grown plants were inoculated in liquid WPM medium (Lloyd and McCown 1980) with 1% sucrose and various concentrations of MeJA (0, 5, 10, and 20 μM) for 24 h. All tissues were immediately frozen in liquid nitrogen and stored at − 80 °C until use. Approximately ten leaflets were inoculated in a Petri dish containing medium. Three replicates were performed with more than ten leaves per replicate.

Isolation of PdFRS sequences from transcriptome sequences

We previously registered transcriptome sequences for P. davidiana by 454 pyrosequencing (Park et al. 2017). The transcriptome sequence information was registered in the NCBI Sequence Read Archive (SRA accession no. SRR3123353). Basic Local Alignment Search Tool (BLAST) analysis of 57,322 unique sequences (15,217 contigs and 42,105 singletons) from the P. davidiana transcriptome led to the selection of the putative OSC sequences.

Phylogenetic analysis of selected mRNA sequences

The deduced PdFRS amino acid sequences in this experiment and those of other plants obtained from DDBJ/GenBank/EMBL were analyzed phylogenetically. The analysis was performed using the neighbor-joining method with MEGA 6.0 (Tamura et al. 2013). A bootstrap with 1000 replicates was used to estimate the strength of the nodes in the tree (Felsenstein 1985).

RT-PCR analysis

To analyze the organ-specific expression of the PdFRS gene, total RNAs were isolated from old to young leaves, petioles, stems, and roots from P. davidiana. Total RNAs were isolated from the leaves of transgenic and non-transgenic P. davidiana to observe the expression of PdFRS among transgenic lines. The RNAs were reverse transcribed by the ImProm-II Reverse Transcription System (Promega, Madison, WI, USA). The first-strand cDNA was used as a template for RT-PCR analysis that was performed with the following conditions: (1) 96 °C for 5 min, (2) 30 cycles of 96 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min, and (3) a final 10-min extension at 72 °C. The following primers were used for RT-PCR: (1) PdFRS, 5′-ATG TGG AGG CTA AAG ATT GCA GAG-3′ (forward primer) and 5′-TCA AAG CTT CTT GGA AGG CAA TG-3′ (reverse primer); (2) nptII gene, 5′-ATC GGG AGC GGC GAT ACC GTA-3′ (forward primer) and 5′-AG GCT ATT CGG CTA TGA CTG-3′ (reverse primer), and (3) the sequence between the CaMV 35S promoter and PdFRS, 5′-GTT CAC CAC CGA TAA TGA GA-3′ (forward primer) and 5′-TCA AAG CTT CTT GGA AGG CAA TG-3′ (reverse primer). β-Actin (5′-CGT GAT CTT ACA GAT AGC TTC ATG A-3′ and 5′-AGA GAA GCT AAG ATT GAT CCT CC-3′) was used as the control to check for RNA integrity and the accuracy of loading.

qPCR analysis

mRNA was isolated from control to MeJA-treated leaves followed by reverse transcription with the ImProm-II Reverse Transcription System (Promega, Madison, WI, USA). qPCR was performed using a Qiagen Rotor Gene Q Real-time PCR system with an SYBR Green PCR kit (Qiagen, Germany). The primers for qPCR were 5′-CCC TCC TGA GTA CCG CAA AG-3′ and 5′-CCA CCA CGG TCA AGA ATC CA-3′. The amplification conditions for the real-time PCRs were 95 °C for 5 min, followed by 40 cycles of 95 °C for 5 s and 60 °C for 10 s. The qPCR data are presented as the average relative quantities ± SE from three replicates. The expression value for each gene was calculated using the \( - \Delta \Delta C_{\text{T}} \) method (Livak and Schmittgen 2001). The P. davidiana 18S rRNA gene was used for normalization. The data presented are the mean ± SE of the transcripts obtained from at least three independent experiments.

Ectopic expression of PdFRS gene in yeast

The PdFRS ORF sequence was submitted to NCBI under accession number KY931453.1. The PdFRS cDNA primers for gene isolation were 5′-ATG TGG AGG CTA AAG ATT GCA GAG-3′ (forward) and 5′-TCA AAG CTT CTT GGA AGG CAA TG-3′ (reverse). The PCR conditions were 25 cycles at 94 °C for 40 s, 50 °C for 40 s, and 72 °C for 2 min. The PCR products were cloned into the pYES2.1/V5-HIS-TOPO expression vector (Invitrogen, USA) under the control of the GAL1 promoter in the sense orientation and the recombinant vector was transformed into Escherichia coli. The nucleotide sequence of the inserted DNA was confirmed by sequencing. The erg7 mutant (MATa erg7 ura3−1 trp1−1) was kindly provided by F. Karst, Colmar, France (Karst and Lacroute 1977). Cells were grown to the early stationary phase at 30 °C in YPD medium containing 1% yeast extract (Oxoid, Basingstoke, UK), 2% peptone (Oxoid), 2% glucose (Merck, Darmstadt, Germany), and 20 mg/ml ergosterol [ethanol:Tween-80 (1:1; v/v)]. The erg7 yeast was transformed with the pYES2.1/V5-HIS-TOPO vector containing the PdFRS coding sequence using an electroporation method. The culture conditions, induction by galactose, and preparation of the triterpene monoalcohol fraction have been described previously (Han et al. 2006).

Construction of transgenic P. davidiana overexpressing PdFRS gene

The PdFRS open reading frame (GenBank accession number KY931453.1) was isolated by PCR. The 5′-ATG TGG AGG CTA AAG ATT GCA GAG-3′ forward primer and 5′-TCA AAG CTT CTT GGA AGG CAA TG-3′ reverse primer were used to amplify the PdFRS gene. To generate the overexpression vector, the PdFRS gene ORF sequence was cloned into the GATEWAY vector pCR8/GW/TOPO (Invitrogen, Carlsbad, CA) and then transferred to the pK2WG7 destination vector, in which PdFRS was driven by the 35S CaMV promoter. The vector was inserted into Agrobacterium tumefaciens GV3101 competent cells and transformed into P. davidiana plants using the same methods as Li et al. (2017) except for using the leaf segments for the initial culture materials maintained in vitro (Park et al. 2017).

GC/MS analysis

Root barks, MeJA-treated leaves, and leaves from transgenic and non-transgenic plants were air-dried at 50 °C in a drying oven. Milled powder (200 mg) from each of the samples was soaked in 100% methanol (1 ml) and sonicated for 30 min at a constant frequency of 20 kHz at 25 °C. The supernatant obtained by centrifugation was subsequently filtered using a SepPak C-18 Cartridge (Waters).

The extracts from the plant samples and engineered yeasts were analyzed by gas chromatography/mass spectrometry (GC/MS). For the GC/MS analysis, an aliquot of the solution was analyzed using a gas chromatograph (Agilent 7890A) linked to an inert MSD system (Agilent 5975C) with a Triple-Axis detector and equipped with an HP-5MS capillary column (30 m × 0.25 mm, film thickness 0.25 mm). The injection temperature was 250 °C and the column temperature was programmed as follows: 50 °C for 5 min, increased to 150 °C at a rate of 5 °C min−1 followed by a hold at 150 °C for 5 min, increased to 300 °C at a rate of 5 °C min−1, and then hold for 20 min. The carrier gas was helium and flow rate was 1.2 ml min−1. The interface temperature was 300 °C with a split injection (10:1). The temperature of the ionization chamber was 250 °C and ionization was performed by electron impact at 70 eV.

In the GC analysis of root bark extracts, all the prominent chromatogram peaks were identified by comparison with a library database and authentic standards. In the extracts from engineered yeasts, MeJA-treated leaves, and transgenic and non-transgenic leaves, the friedelin peak was identified by comparing the retention times and mass spectra with the authentic standards. The friedelin and α- and β-amyrin standard chemicals for GC–MS analysis were purchased from Sigma-Aldrich Inc. (Saint Louis, MO, USA).

Results

Identification of triterpene in P. davidiana

Secondary metabolites from P. davidiana root barks were analyzed by GC/MS. Three types of triterpenes (friedelin, β-amyrin, and α-amyrin) in root bark extracts were identified by the comparison of authentic standard compounds (Fig. 2). A friedelin peak at a retention time of 38.5 min was correctly matched with that of the friedelin standard (Fig. 2a, b). The mass fragmentation pattern of the peak contained m/z at 69, 95, 109, 205, 273, 302, and 426, which is the same pattern as authentic friedelin (Fig. 2c). β-Amyrin (oleanane skeleton) and α-amyrin (ursane skeleton) were also found at retention times of 35.7 and 36.5 min, respectively (Fig. 2a). The retention time and mass fraction patterns of these two triterpene compounds also corresponded with authentic β-amyrin and α-amyrin standards, respectively (Fig. 2d, e). The other compounds marked as 1–5 with Arabic numerals in Fig. 1a were vitamin E (1), campesterol (2), β-sitosterol (3), cycloartenol (4), and 5 stigmast-4-en-3-one (5), which were identified by comparison with GC library compounds. Two more unidentified pentacyclic triterpene peaks (6 and 7) in Fig. 2a were detected at 41.4 and 43.7 min retention times, respectively; these peaks were closely similar to the mass fragmentation pattern of friedelin, with prominent 205 m/z ions that are typically shown by friedelin and its derivatives. However, the heaviest ions among the two peaks were 440 and 442 m/z, respectively.
Fig. 2

Triterpene analysis of root bark extracts from P. davidiana by GC/MS analyses. a TIC chromatogram of root bark extracts from P. davidiana. The Arabic numerals on the chromatogram peaks are for vitamin E (1), campesterol (2), β-sitosterol (3), cycloartenol (4), 5 stigmast-4-en-3-one (5), and friedelin derivatives (6 and 7). Arrows indicate β-amyrin, α-amyrin, and friedelin. TIC of authentic friedelin (b), β-amyrin (c), and α-amyrin (d) standards. Mass spectra for friedelin (e), β-amyrin (f), and α-amyrin (g) together with those for the authentic standards

Isolation of the PdFRS gene and sequence analysis

We previously reported the transcriptome analysis of a cDNA library constructed from mRNA extracted from in vitro cultured plants or P. davidiana using the GS FLX Titanium platform (Park et al. 2017). In this work, we performed a BLAST analysis to seek OSC genes from 10,283 contigs and 42,105 singletons registered to the NCBI Sequence Read Archive (SRA, accession no. SRR3123353). One contig (ID number: EPT001TT0851C000767) with 10 reads and 12 singleton sequences was annotated as an OSC sequence by the BLAST analysis (Table 1). A complete coding sequence (PdFRS) was obtained from a contig (EPT001TT0851C000767) that had 73% identity (0.0 e value) with friedelin synthase from M. ilicifolia, 70% identity (0.0 e value) with friedelin synthase from K. daigremontiana, and 75% identity (0.0 e value) with β-amyrin synthase from B. platyphylla. Among 12 other singletons, four singletons were grouped as friedelin synthase with 73–77% identity. Among these four singletons, two (JDOEMO002HQSFJ and JDOEMO001BL8PO) shared 97–98% identity with PdFRS, indicating that they are the same gene as PdFRS (Table 1). Four other singletons showed high identity (71–85%) with β-amyrin synthase from B. platyphylla and Aralia elata. The remaining four singletons were clearly classified into the OSC sequence with a 0.0 e value and encoded cycloartenol synthase, which is involved in phytosterol biosynthesis.
Table 1

EST sequences showing homology to OSC genes isolated from P. dividina plants

Gene name

 

Typical EST ID

Read number

GenBank accession no.

Homology to

Accession no.

e value

Gene name (species name)

Identity (%)

Friedelin synthase

 Contig

EPT001TT0851C000767

10

KU297271

Friedelin synthase, MiFRS (Maytenus ilicifolia)

73

KX147270.1

0.0

 Singleton

JDOEMO002HQSFJ

1

 

Friedelin synthase, MiFRS (Maytenus ilicifolia)

76

KX147270.1

1e−147

 Singleton

JDOEMO001BL8PO

1

 

Friedelin synthase, MiFRS (Maytenus ilicifolia)

74

KX147270.1

8e−111

 Singleton

JDOEMO001CD8MC

1

 

Friedelin synthase, MiFRS (Maytenus ilicifolia)

77

KX147270.1

2e−110

 Singleton

JDOEMO001BI59Q

1

 

Friedelin synthase, MiFRS (Maytenus ilicifolia)

77

KX147270.1

6e−125

β- or α-amyrin synthase

 Singleton

JDOEMO002H6Q33

1

 

β-amyrin synthase, OSBPY (Betula platyphylla)

85

AB055512.1

6e−107

 Singleton

JDOEMO001DP2VY

1

 

β-amyrin synthase, OSBPY (Betula platyphylla)

73

AB055512.1

4e−104

 Singleton

JDOEMO001EHO4 J

1

 

β-amyrin synthase, AeAS (Aralia elata)

71

HM219225.1

1e−84

 Singleton

JDOEMO001AFKUY

1

 

β-amyrin synthase, OSBPY (Betula platyphylla)

75

AB055512.1

9e−79

Cycloartenol synthase

 Singleton

JDOEMO002GZ3L7

1

 

Cycloartenol synthase, CASBPX1 (Betula platyphylla)

85

AB055509.1

0.0

 Singleton

JDOEMO002GXZRL

1

 

Cycloartenol synthase, CASBPX2 (Betula platyphylla)

83

AB055510.1

0.0

 Singleton

JDOEMO002H6Q33

1

 

Cycloartenol synthase, CASBPX1 (Betula platyphylla)

85

AB055509.1

0.0

 Singleton

JDOEMO001EXED8

1

 

Cycloartenol synthase, CASBPX2 (Betula platyphylla)

80

AB055510.1

0.0

An OSC sequence (PdFRS, GenBank accession number KY931453.1) with the full ORF region was obtained from a contig (EPT001TT0851C000767) that was 2280 bp long and led to a 759 amino acid protein with a predicted molecular mass of 87.81 kDa. The maximum-likelihood phylogenetic tree revealed that PdFRS was closely related within a clade of the β-amyrin synthase group, and showed a close evolutionary relationship with friedelin synthases, such as K. daigremontiana KdFRS and M. ilicifolia MiFRS (Fig. 3). The deduced amino acid sequences of PdFRS had 68 and 73% identity with KdFRS and MiFRS, respectively. However, this sequence also had 73 and 72% identity with β-amyrin synthases from B. platyphylla and Lotus japonicus, respectively. The multiple sequence alignment of PdFRS with other oxidosqualene cyclases revealed that the sequence contains four conserved QW motifs, an OSC-specific MXCH/YR motif, a DCTAE motif with a catalytic aspartic acid residue, and the pentacyclic synthase-specific SF residue motif (Fig. 4). An enzyme pocket was predicted using fpocket2 (Le Guilloux et al. 2009), which detected 47 different residues among which only Leu480 was found to be highly and uniquely conserved in friedelin synthases, but not in almost all other OSC sequences. Other pocket site residues with absolute conservation in friedelin synthase, but less so in other OSCs were Gly530, Lys726, and Asn551 in the order of their unique conservation (Fig. 5).
Fig. 3

Maximum-likelihood phylogenetic analysis of 111 characterized plant OSCs. OSCs known to synthesize friedelin at varying proportions are marked with pink color. Other OSC colors are marked as blue: BAS, rec: CAS, purple: LUS, brown: TAS, green: LAS, and black: miscellaneous OSCs

Fig. 4

Multiple sequence alignment of OSC sequences known to synthesize friedelin at varying proportions. Identical residues are shaded black and similar residues are shaded gray. Four QW motifs known to stabilize OSC structure are underlined in black, the DCTAE motif containing the catalytic Asp residue is underlined in red, and the OSC-specific MXCH/YR motif and pentacyclic synthase-specific SF residue motif are underlined in green. Asterisks represent the residue positions conserved in KdFRS and MiFRS, the only known FRSs to synthesize only friedelin as the end product

Fig. 5

Sequence alignment of representative FRS, BAS, and TAS sequences. The residues that are identical to FRS are dotted, while the overall identical residues are boxed. Blue arrows show missing residue positions in FRSs known to synthesize only friedelin. Red arrows show the uniquely conserved pocket site residue positions in FRSs

qPCR analysis of PdFRS expression

PdFRS transcripts were detected in all organs (young leaf, mature leaf, petiole, stem bark, and root), though the expression levels were different, with the highest expression in the roots (Fig. 6a). The quantitative order of PdFRS mRNA accumulation was root > stem > petiole > leaf.
Fig. 6

PdFRS gene expression and friedelin accumulation in P. davidiana plants. a RT-PCR analysis of the PdFRS gene in various organs from P. davidiana plants. b qPCR analysis of PdFRS gene in P. davidiana leaves after treatment with different concentrations (0, 5, 10, and 20 μM) of MeJA for 24 h. c Accumulation of friedelin in leaves from P. davidiana plants after treatment with different concentrations (0, 5, 10, and 20 μM) of MeJA for 24 h. The data presented are the mean ± SE of the transcripts obtained from at least three repeated experiments

To investigate the change in PdFRS gene transcriptional activities by stress and elicitor treatment, leaves from in vitro cultured P. davidiana plants were treated to various concentrations (0, 5, 10, and 20 μM) of MeJA for 24 h. qPCR analysis revealed that PdFRS gene transcriptional activity was highly activated by MeJA treatment and was a maximum at 10-μM MeJA treatment for 24 h (Fig. 6b). Friedelin content in the leaves after MeJA treatment was analyzed by GC/MS. Friedelin accumulation in the leaves was clearly enhanced by MeJA treatment. The friedelin content was highest for the 10-μM MeJA treatment for 24 h (Fig. 6c).

Heterologous expression of the PdFRS gene in yeast

The PdFRS gene ORF region was inserted into the pYES2.1 expression vector and the plasmid was inserted into an ERG7-deficient yeast mutant that lacks a functional lanosterol synthase gene (Karst and Lacroute 1977). Methanol extracts from transgenic yeast expressing PdFRS were analyzed by GC/MS. The total ion chromatogram (TIC) for the yeast extracts revealed a new product at a 38.5 min retention time (Fig. 7a). This peak was not detected in the extracts from control yeast expressing the empty vector (Fig. 7b). This new peak in transgenic yeast showed the same retention time as the authentic friedelin standard compound (Fig. 7a, b). The mass fragmentation pattern of the peak in transgenic yeast contained m/z at 95, 205, 273, 302, and 426 (Fig. 7c), which showed the same pattern as authentic friedelin (Fig. 7d). No other additional new triterpenoid peaks were detected in the chromatograms for the yeast extract expressing PdFRS cDNA, indicating the monofunctional activity of the PdFRS enzyme.
Fig. 7

GC analysis of yeast extracts overexpressing the P. davidiana friedelin synthase gene. a Chromatogram for the control yeast with empty vector as a control. b Chromatogram for the transformed yeast with the PdFRS gene. c GC chromatograph for the authentic friedelin standard. d Mass spectra for friedelin in the transformed yeast with a retention time of 38.5 min. e Mass spectra for the authentic friedelin standard

Construction of transgenic P. davidiana plants overexpressing PdFRS

Transgenic P. davidiana plants overexpressing PdFRS driven by CaMV35 promoters were constructed by Agrobacterium-mediated transformation (Fig. 8a). Adventitious shoots were formed from leaf segments cultured on WPM medium (Lloyd and McCown 1980) with 2.0-mg/l BA (Fig. 8b); kanamycin-resistant shoots were obtained on medium with 50-mg/l kanamycin (Fig. 8c, d). Five independent transgenic lines of P. davidiana shoots produced roots on medium with 100-mg/l kanamycin (Fig. 8e). Insertion of the nptII gene into transgenic plants was confirmed by PCR with genomic DNA (Fig. 8f). Moreover, PCR was performed for the detection of the sequence between the CaMV 35S promoter and PdFRS region to confirm the integrity of the construction of the overexpressing PdFRS gene in the transgenic lines. All five transgenic lines showed obvious PCR-amplified bands for the CaMV 35S promoter and PdFRS region, but not the non-transgenic plants (Fig. 8f).
Fig. 8

Detection and expression analysis of introduced genes in transgenic P. davidiana co-overexpressing PdFRS. a T-DNA region of the plasmid for PdFRS gene overexpression. RB right T-DNA border, Nter terminator region of the nopaline synthase gene, nptII neomycin phosphotransferase gene, Npro promoter region of the nopaline synthase gene, P35S CaMV 35S promoter sequence, PdFRS cDNA sequences encoding the friedelin enzyme from P. davidiana, T35S CaMV 35S terminator sequence, LB left T-DNA border. be Construction of transgenic P. davidiana overexpressing PdFRS. Adventitious shoot formation on leaf segments on WPM medium with 1.0-mg/l benzyladenine (b). Kanamycin-resistant shoot formation on medium with 50-mg/l kanamycin (c, d). Five transgenic plants surviving on medium with 100-mg/l kanamycin (e). f Genomic DNA PCR for the nptII gene and the P35S-PdFRS sequence between the CaMV 35S promoter and PdFRS for the detection of the introduced genes in the transgenic P. davidiana lines. g RT-PCR analysis of the introduced PdFRS gene in the transgenic lines (Tr1, Tr2, Tr3, Tr4, and Tr5). 18S rRNA was used as an internal control. A non-transgenic (NT) plant was used as a negative control for the introduced genes

Leaves from five different transgenic and non-transgenic (NT) P. davidiana plant lines were used in the RT-PCR analysis to confirm the overexpression of the PdFRS gene. All transgenic lines showed enhanced PdFRS gene transcription, though they showed different patterns of expression among the transgenic lines (Fig. 8g). Weak PCR amplification of PdFRS mRNA was detected in the non-transgenic P. davidiana (Fig. 8g).

Enhanced friedelin accumulation in transgenic P. davidiana overexpressing PdFRS

To analyze the friedelin content in both transgenic and non-transgenic (control) P. davidiana plants, leaf extracts were analyzed by GC/MS. The total ion chromatogram (TIC) for the extracts revealed that the P. davidiana transgenic lines clearly showed enhanced production of the friedelin triterpene compared to the non-transgenic plants (Fig. 9). The height of the friedelin peak at a retention time of 38.5 min in leaf extracts from all three transgenic lines was clearly enhanced (Fig. 9a–g). The friedelin contents in the leaves from transgenic P. davidiana corresponded to the expression of the PdFRS gene in the transgenic lines (Figs. 8g, 9h). Transgenic line 3 showed the highest friedelin content (29.8 μg/g DW) in leaves. The friedelin content in the leaves from non-transgenic (control) plants was 6.7-μg/g DW (Fig. 9h).
Fig. 9

GC/MS analyses for friedelin in leaf extracts from both non-transgenic (control) and transgenic (lines 1–5) P. davidiana overexpressing PdFRS. a TIC chromatogram of leaf extracts from non-transgenic P. davidiana. bf TIC chromatograms for leaf extracts from three transgenic P. davidiana lines (Tr1–5). g Chromatogram for the authentic friedelin standard. h Concentration of friedelin in leaves among transgenic and non-transgenic lines. The data presented are the mean ± SE of the friedelin concentration obtained from at least three repeated experiments

Discussion

Occurrence and biological activity of friedelin

Friedelin is an active ingredient from cork tissues in oak tree (Castola et al. 2005; Moiteiro et al. 2006; Ghosh et al. 2010). The friedelin derivative cerin (2α-hydroxyfriedelan-3-one) is also particularly rich in oak cork tissues (Ghosh et al. 2010). Friedelin is also present in epicuticular waxes in K. daigremontiana leaves (van Maarseveen and Jetter 2009) and grapefruit (Nordby and McDonald 1994). Although there are no reports on the presence of the triterpene friedelin in P. davidiana, relative species in the same genus (P. yunnanensis) contained friedelin, 21 alpha-hydroxyfriedelan-3-one, and 21 alpha-acetoxyfriedelan-3-one (Chen and Xu 1990). In this work, we demonstrated the presence of friedelin and other two triterpenes (α- and β-amyrin) in P. davidiana, for the first time, together with the functional analysis of the PdFRS gene isolated from this plant as a friedelin synthase gene.

Isolation and characterization of the PdFRS gene

The production of triterpene skeletons in plants is determined by the activities of OSC enzymes. Through BLAST analysis of the transcriptome sequences obtained from a cDNA library extracted from P. davidiana using pyrosequencing, only one contig with ten reads obtained from 10,283 contigs was annotated as an OSC enzyme (PdFRS); this sequence was functionally determined to be friedelin synthase in the present work. Thus, the sequences coding for friedelin synthase may be the major OSC sequences in P. davidiana. Among the other 12 OSC sequences obtained from 42,105 singletons, four sequences showed high similarity with the PdFRS gene, while another four sequences were grouped with β-amyrin synthase and showed 64–85% identity; these sequences may be involved in the production of other triterpenes such as α- and β-amyrin triterpene. We demonstrated the presence of α- and β-amyrin triterpenes in root barks from P. davidiana. The remaining four sequences were classified as cycloartenol synthases with a 0 e value and involved in phytosterol biosynthesis. In Populus species, several triterpenes were found in P. tremuloides (Abramovitch and Micetic 1963; Fernandez et al. 2001), P. tremula (Roshchin et al. 1986), and P. x euramericana (Xu et al. 2010).

The phylogenetic analysis revealed that PdFRS is grouped with β-amyrin synthase sequences and has 73–75% identity with other β-amyrin synthase from B. platyphylla, L. japonicas, and M. truncatula. However, PdFRS also displays 70% identity with friedelin synthase from K. daigremontiana and 73% identity with friedelin synthase from M. ilicifolia. Thus, simple phylogenetic analysis of PdFRS with other known OSC genes is not adequate to determine the particular enzyme before functional analysis of the gene.

The enzyme active sites (pocket site residues) in friedelin synthase were predicted using fpocket2 (Le Guilloux et al. 2009). Leu480 was uniquely conserved in friedelin synthases and not present in almost all other OSC sequences. Souza-Moreira et al. (2016) substituted the leucine 482 residue in MiFRS from M. ilicifolia with threonine (Leu482Thr). The resulting triterpenes from the modified enzymes led to the production of β-amyrin alone. Therefore, they concluded that Leu482 is important for friedelin production (Souza-Moreira et al. 2016). The residue at position 482 in the KdFRS enzyme from P. davidiana also had a leucine, making it similar to two other friedelin synthases (MiFRS and KdFRS). Other pocket site residues with absolute conservation in FRS but less so in other OSCs were Gly530, Asn551, and Lys726. The analysis indicated that the additional three pocket site residues (Gly530, Asn551, and Lys726) may be involved in proper FRS functioning. One OSC sequence annotated as glutinol synthase (KdGLS) or ADK35124.1 apparently had all of the pocket site residues (Leu480, Gly530, Asn551, and Lys726) and is a multifunctional protein that produces friedelin as its second highest compound after glutinol (Wang et al. 2010).

Effect of MeJA treatment on the expression of PdFRS gene and friedelin accumulation

Plants produce MeJA in response to biotic and abiotic stresses, such as herbivory and wounding, and the production of MeJA evokes plant defense systems (Baldwin 1998; Cheong and Choi 2003). In addition, MeJA has been used as an effective elicitor to stimulate the biosynthesis of secondary metabolites (Namdeo 2007). MeJA treatment not only enhanced PdFRS mRNA accumulation but also enhanced friedelin accumulation in leaves from P. davidiana plants. These results indicate that the expression of the PdFRS gene is highly due to MeJA treatment, which fosters friedelin accumulation in plants.

Monofunctional friedelin production by ectopic expression of PdFRS in yeast

The PdFRS gene ORF region was heterologously expressed in yeast. GC/MS analysis of methanol extracts from transgenic yeast expressing PdFRS revealed that a new peak in the transgenic yeast showed the same retention time as the authentic friedelin standard compound; additional new triterpenoid peaks were not detected in the chromatograms from yeast extracts expressing PdFRS cDNA. An OSC enzyme for friedelin synthase (KdFRS) was first isolated from K. daigremontiana by Wang et al. (2010). However, heterologous expression of KdFRS in yeast resulted in a multifunctional enzyme forming friedelin together with the small amounts of β-amyrin and taraxerol. Recently, a monofunctional friedelin synthase (MiFRS) was isolated from M. ilicifolia, and heterologous expression of MiFRS in yeast resulted in the production of a single friedelin product (Souza-Moreira et al. 2016). We first reported the friedelin synthase in Populus species, and the PdFRS gene in P. davidiana encodes a monofunctional friedelin synthase.

Enhanced friedelin production in transgenic P. davidiana overexpressing PdFRS

In transgenic P. davidiana lines overexpressing PdFRS, the friedelin content in the leaves ranged from 15.6 to 29.8 μg/g DW. In non-transgenic plants, the triterpene in the leaves was only detected at 6.7 μg/g DW. This result demonstrates that the PdFRS gene is truly involved in friedelin production in plants and that overexpression of PdFRS in P. davidiana significantly stimulated friedelin accumulation.

Friedelin has many biological roles, including antimicrobial and antifungal activities (Ghosh et al. 2010; Kuete et al. 2010; Tamokou et al. 2009; Tchakam et al. 2012) and allelopathic actions (Santos et al. 2008; Ghosh et al. 2010). Thus, enhanced friedelin accumulation in P. davidiana plants may have increased their resistance to pathogens. There were also positive relationships between friedelin and the microbial community composition in soils under different land uses and seasons (Dong et al. 2014).

Friedelin showed antiulcerogenic activity (Queiroga et al. 2000; Antonisamy et al. 2015) and potent analgesic and antipyretic effects (Antonisamy et al. 2011). Friedelin showed effective antioxidant and free radical scavenging in both in vitro and in vivo studies (Sunil et al. 2013). Thus, transgenic P. davidiana with enhanced production of friedelin might also have a better medicinal value.

Author contribution statement

YEC designed the research and wrote the paper. JYH performed the Populus yeast transformation and analysis of the phytochemicals by GC/MS. CHA and PBA performed the Populus genetic transformation and analyzed the RT-PCR and qPCR experiments. All authors read and approved the manuscript.

Notes

Acknowledgements

This work was supported by the Rural Development Administration, Republic of Korea [Next-Generation Bio-Green 21 Program (PJ01344401 and PJ01369103)].

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Jung Yeon Han
    • 1
  • Chang-Ho Ahn
    • 1
  • Prakash Babu Adhikari
    • 1
  • Subramanyam Kondeti
    • 1
  • Yong Eui Choi
    • 1
    Email author
  1. 1.Department of Forest Resources, College of Forest and Environmental SciencesKangwon National UniversityChuncheonRepublic of Korea

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