Enhanced production of pinosylvin stilbene with aging of Pinus strobus callus and nematicidal activity of callus extracts against pinewood nematodes.

Pinosylvin stilbenes are phenolic compounds mainly occurring in the Pinaceae family. We previously reported that the accumulation of two pinosylvin stilbene compounds, dihydropinosylvin methyl ether (DPME) and pinosylvin monomethyl ether (PME), in Pinus strobus trees was highly enhanced by infection with pine wood nematodes (PWNs: Bursaphelenchus xylophilus), and these two compounds showed strong nematicidal activity against PWNs. In this work, we established a system of pinosylvin stilbene (DPME and PME) production via the in vitro culture of P. strobus calli, and we examined the nematicidal activity of callus extracts. Calli were induced from the culture of mature zygotic embryos of P. strobus. Optimized growth of calli was obtained in 1/2 Litvay medium with 1.0 mg/L 2,4-D and 0.5 mg/L BA. DPME and PME accumulation did not occur in nonaged (one-month-old) calli but increased greatly with prolonged callus culture. The concentrations of DPME and PME in three-month-old dark-brown calli were 6.4 mg/g DW and 0.28 mg/g DW, respectively. The effect of methyl jasmonate treatment on the accumulation of DPME and PME was evaluated in cell suspension culture of P. strobus. However, the treatment appeared to show slight increase of DPME accumulation compared to callus browning. A test solution prepared from crude ethanol extracts from aged calli (three months old) containing 120 µg/ml DPME and 5.16 µg/ml PME treated with PWNs resulted in 100% immobilization of the adult PWNs and 66.7% immobilization of the juvenile PWNs within 24 h. However, nonaged callus extracts did not show any nematicidal activity against juvenile PWNs and showed less than 20% nematicidal activity against adult PWNs. These results indicate that pinosylvin stilbenes can be effectively produced by prolonged culture of P. strobus calli, can be isolated using simple ethanolic extraction, and are applicable as beneficial eco-friendly compounds with nematicidal activity against PWNs.

Enhanced accumulation of pinosylvin stilbenes and related gene expression in Pinus strobus after infection of pine wood nematode.

Pine wood nematodes (PWNs; Bursaphelenchus xylophilus) infect pine trees and cause serious pine wilt disease. Eastern white pine (Pinus strobus) has resistance to PWN. However, the detailed defense mechanisms of P. strobus against PWN are not well known. When P. strobus plants were infected with PWNs, the accumulation of stilbenoids, dihydropinosylvin monomethyl ether (DPME) and pinosylvin monomethyl ether (PME) was increased remarkably. Both DPME and PME had high nematicidal activity. The nematicidal activity of the two compounds was resulted in a developmental stage-dependent manner. Pinosylvin monomethyl ether was more toxic to adult PWNs than juveniles, whereas DPME was found more toxic to juvenile PWNs than the adults. The genes involved in PME and DPME biosynthesis such as phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase (4CL), pinosylvin synthase (STS) and pinosylvin O-methyltransferase (PMT) were isolated using de novo sequencing of the transcriptome in P. strobus. In addition, transcription factors (TFs; bHLH, MYB and WRKY) related to stilbene biosynthesis were isolated. qPCR analyses of the selected genes (PAL, 4CL, STS and PMT) including TFs (bHLH, MYB and WRKY) revealed that the expression level of the selected genes highly enhanced after PWN infection. Our results suggest that pinosylvin-type stilbenoid biosynthesis is highly responsive to PWN infection and plays an important role in PWN resistance of P. strobus trees.

Enhanced monoterpene emission in transgenic orange mint (Mentha × piperita f. citrata) overexpressing a tobacco lipid transfer protein (NtLTP1).

MAIN CONCLUSION: Overexpression of the tobacco lipid transfer protein (NtLTP1) gene in transgenic orange mint resulted in enhanced accumulation of monoterpenes in the cavity of head cells of glandular trichomes, which resulted in enhanced emission of monoterpenes from transgenic orange mints. Plants in the genus Mentha (Lamiaceae) produce volatile oils that accumulate in peltate glandular trichomes in the aerial parts of plants. A lipid transfer protein (NtLTP1) in tobacco showed glandular trichome-specific expression and supported the secretion of diterpenoid lipids from head cells of glandular trichomes (Choi et al., Plant J 70:480-491,2012). Here, we constructed transgenic orange mint (Mentha × piperita f. citrata) overexpressing the tobacco NtLTP1 gene via Agrobacterium-mediated transformation. Transgenic lines of orange mint overexpressing NtLTP1 were confirmed by genomic PCR and RT-PCR. Immunoblotting analysis using an NtLTP1 polyclonal antibody showed clear dark spots at the position of the lipid exudates from tobacco glandular trichomes and the squeezed out lipids from the glandular trichomes of transgenic orange mint. Heads of glandular trichomes in transgenic plants overexpressing the NtLTP1 gene showed a larger diameter than those of the wild-type control. The enhanced size of trichome heads in transgenic orange mint was confirmed by scanning electron microscopy. Volatile components were extracted from wild-type and transgenic orange mint by solid-phase microextraction (SPME) and analyzed by headspace-gas chromatography-mass spectrometry (HS/GC/MS). Linalyl acetate was the most abundant component among the eleven identified monoterpenes in the volatile compounds extracted from both the wild-type and transgenic lines of orange mint. Overexpression of NtLTP1 in transgenic orange mint plants resulted in enhanced emission of volatile monoterpenoids compared with that of volatile monoterpenoids in the wild-type control plants.

Transcriptomic Analysis of Kalopanax septemlobus and Characterization of KsBAS, CYP716A94 and CYP72A397 Genes Involved in Hederagenin Saponin Biosynthesis.

Kalopanax septemlobus, commonly named the castor aralia tree, is a highly valued woody medicinal tree belonging to the family Araliaceae. Kalopanax septemlobus contains approximately 15 triterpenoid saponins primarily constituted of hederagenin aglycones. Hederagenin is a representative precursor for hemolytic saponin in plants. In the present study, transcriptome analysis was performed to discover genes involved in hederagenin saponin biosynthesis in K. septemlobus. De novo assembly generated 82,698 unique sequences, including 17,747 contigs and 64,951 singletons, following 454 pyrosequencing. Oxidosqualene cyclases (OSCs) are enzymes that catalyze the formation of diverse triterpene skeletons from 2,3-oxidosqualene. Heterologous expression of an OSC sequence in yeast revealed that KsBAS is a ß-amyrin synthase gene. Cytochrome P450 genes (CYPs) make up a supergene family in the plant genome and play a key role in the biosynthesis of sapogenin aglycones. In total, 95 contigs and 110 singletons annotated as CYPs were obtained by sequencing the K. septemlobus transcriptome. By heterologous expression in yeast, we found that CYP716A94 was ß-amyrin 28-oxidase involved in oleanolic acid production from ß-amyrin, and CYP72A397 was oleanolic acid 23-hydroxylase involved in hederagenin production from oleanolic acid. Engineered yeast co-expressing KsBAS, CYP716A94 and CYP72A397 produced hederagenin. Kalopanax septemlobus CYP72A397 is a novel CYP enzyme that synthesizes hederagenin aglycone from oleanolic acid as a single product. In conclusion, we characterized three genes participating in sequential steps for hederagenin biosynthesis from ß-amyrin, which are likely to play a major role in hederagenin saponin biosynthesis in K. septemlobus.

ß-Amyrin synthase (EsBAS) and ß-amyrin 28-oxidase (CYP716A244) in oleanane-type triterpene saponin biosynthesis in Eleutherococcus senticosus.

Siberian ginseng (Eleutherococcus senticosus) is a woody medical shrub belonging to the Araliaceae family. E. senticosus contains various types of saponins, including oleanane, noroleanane, lupane, and 3,4-secolupane types, depending on the aglycone structure. Oleanane-type triterpenes are the major saponin components in E. senticosus. Two enzymes (ß-amyrin synthase and ß-amyrin 28-oxidase) are essential for oleanane-type saponin biosynthesis from 2,3-oxidosqualene. In the present study, two full-length cDNAs encoding EsBAS and CYP716A244 were isolated based on transcriptomics analysis of plant leaves. Both ß-amyrin synthase (EsBAS) and ß-amyrin 28-oxidase (CYP716A244), isolated from E. senticosus, were functionally characterised. ß-amyrin production was confirmed by heterologous expression of the EsBAS gene in yeast and tobacco. Oleanolic acid production was confirmed by co-expression of both EsBAS and CYP716A244 in engineered yeast and transgenic tobacco.

Transcriptomic analysis of Siberian ginseng (Eleutherococcus senticosus) to discover genes involved in saponin biosynthesis.

BACKGROUND: Eleutherococcus senticosus, Siberian ginseng, is a highly valued woody medicinal plant belonging to the family Araliaceae. E. senticosus produces a rich variety of saponins such as oleanane-type, noroleanane-type, 29-hydroxyoleanan-type, and lupane-type saponins. Genomic or transcriptomic approaches have not been used to investigate the saponin biosynthetic pathway in this plant. RESULT: In this study, de novo sequencing was performed to select candidate genes involved in the saponin biosynthetic pathway. A half-plate 454 pyrosequencing run produced 627,923 high-quality reads with an average sequence length of 422 bases. De novo assembly generated 72,811 unique sequences, including 15,217 contigs and 57,594 singletons. Approximately 48,300 (66.3%) unique sequences were annotated using BLAST similarity searches. All of the mevalonate pathway genes for saponin biosynthesis starting from acetyl-CoA were isolated. Moreover, 206 reads of cytochrome P450 (CYP) and 145 reads of uridine diphosphate glycosyltransferase (UGT) sequences were isolated. Based on methyl jasmonate (MeJA) treatment and real-time PCR (qPCR) analysis, 3 CYPs and 3 UGTs were finally selected as candidate genes involved in the saponin biosynthetic pathway. CONCLUSIONS: The identified sequences associated with saponin biosynthesis will facilitate the study of the functional genomics of saponin biosynthesis and genetic engineering of E. senticosus.

The involvement of ß-amyrin 28-oxidase (CYP716A52v2) in oleanane-type ginsenoside biosynthesis in Panax ginseng.

Panax species are the most popular medicinal herbs. The root of these plants contains pharmacologically active triterpene saponins, also known as ginsenosides, compounds that are divided into dammarane- and oleanane-type triterpenes. Two CYP716A subfamily genes (CYP716A47 and CYP716A53v2) were recently characterized, encoding an enzyme catalyzing the hydroxylation of dammarane-type triterpenes in Panax ginseng. Herein, we report that one CYP716A subfamily gene (CYP716A52v2) isolated from P. ginseng encodes a ß-amyrin 28-oxidase, which is suggested to modify ß-amyrin into oleanolic acid, a precursor of an oleanane-type saponin (mainly ginsenoside Ro) in P. ginseng. The ectopic expression of both PNY1 and CYP716A52v2 in recombinant yeast resulted in erythrodiol and oleanolic acid production, respectively. In vitro enzymatic activity assays biochemically confirmed that CYP716A52v2 catalyzed the oxidation of ß-amyrin to produce oleanolic acid, and the chemical structure of the oleanolic acid product was confirmed using gas chromatography-mass spectrometry (GC/MS). Transgenic P. ginseng plants were generated via Agrobacterium tumefaciens-mediated transformation: the overexpression of CYP716A52v2 greatly increased the content of oleanane-type ginsenoside (ginsenoside Ro), whereas RNA interference against CYP716A52v2 markedly reduced it. Furthermore, the levels of other dammarene-type ginsenosides were not affected in these transgenic lines. These results indicate that CYP716A52v2 is a ß-amyrin 28-oxidase that plays a key role in the biosynthesis of oleanane-type triterpenes in P. ginseng.

Cytochrome P450 CYP716A53v2 catalyzes the formation of protopanaxatriol from protopanaxadiol during ginsenoside biosynthesis in Panax ginseng.

Ginseng (Panax ginseng C.A. Meyer) is one of the most popular medicinal herbs, and the root of this plant contains pharmacologically active components, called ginsenosides. Ginsenosides, a class of tetracyclic triterpene saponins, are synthesized from dammarenediol-II after hydroxylation by cytochrome P450 (CYP) and then glycosylation by a glycosyltransferase. Protopanaxadiol synthase, which is a CYP enzyme (CYP716A47) that catalyzes the hydroxylation of dammarenediol-II at the C-12 position to yield protopanaxadiol, was recently characterized. Here, we isolated two additional CYP716A subfamily genes (CYP716A52v2 and CYP716A53v2) and determined that the gene product of CYP716A53v2 is a protopanaxadiol 6-hydroxylase that catalyzes the formation of protopanaxatriol from protopanaxadiol during ginsenoside biosynthesis in P. ginseng. Both CYP716A47 and CYP716A53v2 mRNAs accumulated ubiquitously in all organs of ginseng plants. In contrast, CYP716A52v2 mRNA accumulated only in the rhizome. Methyl jasmonate (MeJA) treatment resulted in the obvious accumulation of CYP716A47 mRNA in adventitious roots. However, neither CYP716A52v2 nor CYP716A53v2 mRNA was affected by MeJA treatment during the entire culture period. The ectopic expression of CYP716A53v2 in recombinant WAT21 yeast resulted in protopanaxatriol production after protopanaxadiol was added to the culture medium. In vitro enzymatic activity assays revealed that CYP716A53v2 catalyzed the oxidation of protopanaxadiol to produce protopanaxatriol. The chemical structures of the protopanaxatriol products were confirmed using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC/APCIMS). Our results indicate that the gene product of CYP716A53v2 is a protopanaxadiol 6-hydroxylase that produces protopanaxatriol from protopanaxadiol, which is an important step in the formation of dammarane-type triterpene aglycones in ginseng saponin biosynthesis.