Integrated metabolome and transcriptome analyses reveal the molecular mechanism underlying dynamic metabolic processes during taproot development of Panax notoginseng.

BACKGROUND: Panax notoginseng (Burk) F. H. Chen is one of the most famous Chinese traditional medicinal plants. The taproot is the main organ producing triterpenoid saponins, and its development is directly linked to the quality and yield of the harvested P. notoginseng. However, the mechanisms underlying the dynamic metabolic changes occurring during taproot development of P. notoginseng are unknown. RESULTS: We carried out metabolomic and transcriptomic analyses to investigate metabolites and gene expression during the development of P. notoginseng taproots. The differentially accumulated metabolites included amino acids and derivatives, nucleotides and derivatives, and lipids in 1-year-old taproots, flavonoids and terpenoids in 2- and 3-year-old taproots, and phenolic acids in 3-year-old taproots. The differentially expressed genes (DEGs) are related to phenylpropanoid biosynthesis, metabolic pathway and biosynthesis of secondary metabolites at all three developmental stages. Integrative analysis revealed that the phenylpropanoid biosynthesis pathway was involved in not only the development of but also metabolic changes in P. notoginseng taproots. Moreover, significant accumulation of triterpenoid saponins in 2- and 3-year-old taproots was highly correlated with the up-regulated expression of cytochrome P450s and uridine diphosphate-dependent glycosyltransferases genes. Additionally, a gene encoding RNase-like major storage protein was identified to play a dual role in the development of P. notoginseng taproots and their triterpenoid saponins synthesis. CONCLUSIONS: These results elucidate the molecular mechanism underlying the accumulation of and change relationship between primary and secondary metabolites in P. notoginseng taproots, and provide a basis for the quality control and genetic improvement of P. notoginseng.

EbMYBP1, a R2R3-MYB transcription factor, promotes flavonoid biosynthesis in Erigeron breviscapus.

Erigeron breviscapus, a traditional Chinese medicinal plant, is enriched in flavonoids that are beneficial to human health. While we know that R2R3-MYB transcription factors (TFs) are crucial to flavonoid pathway, the transcriptional regulation of flavonoid biosynthesis in E. breviscapus has not been fully elucidated. Here, EbMYBP1, a R2R3-MYB transcription factor, was uncovered as a regulator involved in the regulation of flavonoid accumulation. Transcriptome and metabolome analysis revealed that a large group of genes related to flavonoid biosynthesis were significantly changed, accompanied by significantly increased concentrations of the flavonoid in EbMYBP1-OE transgenic tobacco compared with the wild-type (WT). In vitro and in vivo investigations showed that EbMYBP1 participated in flavonoid biosynthesis, acting as a nucleus-localized transcriptional activator and activating the transcription of flavonoid-associated genes like FLS, F3H, CHS, and CHI by directly binding to their promoters. Collectively, these new findings are advancing our understanding of the transcriptional regulation that modulates the flavonoid biosynthesis.

Phytochemical profiles of edible flowers of medicinal plants of Dendrobium officinale and Dendrobium devonianum.

The discovery of new edible flowers that are nontoxic, innocuous flowers having human health benefits, surveys of their phytochemicals and utilization are of great scientific and commercial interest. Dendrobium officinale and Dendrobium devonianum are precious Traditional Chinese Medicine. During the massive commercial cultivation, a lot of flowers were produced and certified as edible flowers, and the phytochemical profiles and bioactivities warrant evaluate. The present study aimed to investigate the phytochemicals and antioxidative activities in flowers of D. officinale (DOF) and D. devonianum (DDF). In total, 474 metabolites were identified using a widely targeted metabonomics method, 16 amino acids and 6 flavonoids were measured using high-performance liquid chromatography (HPLC), and 8 fatty acids were detected using gas chromatography-mass spectrometry (GC-MS). Both flowers contained various amino acids, including 7 essential amino acids, diverse flavonoids, especially quercetin, kaempferol and their derivatives, and high levels of methyl linoleate and methyl linolenate. The relative levels of quercetin, kaempferol and their glycosides were higher in DDF than in DOF, whereas the relative levels of several flavonoids C-glycosides were high in DOF. Ethanol extracts of both DOF and DDF showed antioxidative capacities including the scavenging of 1,1-diphenyl-2-picrylhydrazyl and hydroxyl radicals. Both edible flowers contained flavonoids, amino acids, and fatty acids and have antioxidative activities, which should be explored for use in functional foods and pharmaceuticals.

Identification of two UDP-glycosyltransferases involved in the main oleanane-type ginsenosides in Panax japonicus var. major.

MAIN CONCLUSION: Two UDP-glycosyltransferases from Panax japonicus var. major were identified, and the biosynthetic pathways of three oleanane-type ginsenosides (chikusetsusaponin IVa, ginsenoside Ro, zingibroside R1) were elucidated. Chikusetsusaponin IVa and ginsenoside Ro are primary active components formed by stepwise glycosylation of oleanolic acid in five medicinal plants of the genus Panax. However, the key UDP-glycosyltransferases (UGTs) in the biosynthetic pathway of chikusetsusaponin IVa and ginsenoside Ro are still unclear. In this study, two UGTs (PjmUGT1 and PjmUGT2) from Panax japonicus var. major involved in the biosynthesis of chikusetsusaponin IVa and ginsenoside Ro were identified based on bioinformatics analysis, heterologous expression and enzyme assays. The results show that PjmUGT1 can transfer a glucose moiety to the C-28 carboxyl groups of oleanolic acid 3-O-ß-D-glucuronide and zingibroside R1 to form chikusetsusaponin IVa and ginsenoside Ro, respectively. Meanwhile, PjmUGT2 can transfer a glucose moiety to oleanolic acid 3-O-ß-D-glucuronide and chikusetsusaponin IVa to form zingibroside R1 and ginsenoside Ro. This work uncovered the biosynthetic mechanism of chikusetsusaponin IVa and ginsenoside Ro, providing the rational production of valuable saponins through synthetic biology strategy.

Transcriptome analysis of Panax zingiberensis identifies genes encoding oleanolic acid glucuronosyltransferase involved in the biosynthesis of oleanane-type ginsenosides.

MAIN CONCLUSION: Oleanolic acid glucuronosyltransferase (OAGT) genes synthesizing the direct precursor of oleanane-type ginsenosides were discovered. The four recombinant proteins of OAGT were able to transfer glucuronic acid at C-3 of oleanolic acid that yields oleanolic acid 3-O-ß-glucuronide. Ginsenosides are the primary active components in the genus Panax, and great efforts have been made to elucidate the mechanisms underlying dammarane-type ginsenoside biosynthesis. However, there is limited information on oleanane-type ginsenosides. Here, high-performance liquid chromatography analysis demonstrated that oleanane-type ginsenosides (particularly ginsenoside Ro and chikusetsusaponin IV and IVa) are the abundant ginsenosides in Panax zingiberensis, an extremely endangered Panax species in southwest China. These ginsenosides are derived from oleanolic acid 3-O-ß-glucuronide, which may be formed from oleanolic acid catalyzed by an unknown oleanolic acid glucuronosyltransferase (OAGT). Transcriptomic analysis of leaves, stems, main roots, and fibrous roots of P. zingiberensis was performed, and a total of 46,098 unigenes were obtained, including all the identified homologous genes involved in ginsenoside biosynthesis. The most upstream genes were highly expressed in the leaves, and the UDP-glucosyltransferase genes were highly expressed in the roots. This finding indicated that the precursors of ginsenosides are mainly synthesized in the leaves and transported to different parts for the formation of particular ginsenosides. For the first time, enzyme activity assay characterized four genes (three from P. zingiberensis and one from P. japonicus var. major, another Panax species with oleanane-type ginsenosides) encoding OAGT, which particularly transfer glucuronic acid at C-3 of oleanolic acid to form oleanolic acid 3-O-ß-glucuronide. Taken together, our study provides valuable genetic information for P. zingiberensis and the genes responsible for synthesizing the direct precursor of oleanane-type ginsenosides.

Transcriptome analysis of Panax vietnamensis var. fuscidicus discovers putative ocotillol-type ginsenosides biosynthesis genes and genetic markers.

BACKGROUND: P. vietnamensis var. fuscidiscus, called "Yesanqi" in Chinese, is a new variety of P. vietnamensis, which was first found in Jinping County, the southern part of Yunnan Province, China. Compared with other Panax plants, this species contains higher content of ocotillol-type saponin, majonoside R2. Despite the pharmacological importance of ocotillol-type saponins, little is known about their biosynthesis in plants. Hence, P. vietnamensis var. fuscidiscus is a suitable medicinal herbal plant species to study biosynthesis of ocotillol-type saponins. In addition, the available genomic information of this important herbal plant is lacking. RESULTS: To investigate the P. vietnamensis var. fuscidiscus transcriptome, Illumina HiSeq™ 2000 sequencing platform was employed. We produced 114,703,210 clean reads, assembled into 126,758 unigenes, with an average length of 1,304 bp and N50 of 2,108 bp. Among these 126,758 unigenes, 85,214 unigenes (67.23%) were annotated based on the information available from the public databases. The transcripts encoding the known enzymes involved in triterpenoid saponins biosynthesis were identified in our Illumina dataset. A full-length cDNA of three Squalene epoxidase (SE) genes were obtained using reverse transcription PCR (RT-PCR) and the expression patterns of ten unigenes were analyzed by reverse transcription quantitative real-time PCR (RT-qPCR). Furthermore, 15 candidate cytochrome P450 genes and 17 candidate UDP-glycosyltransferase genes most likely to involve in triterpenoid saponins biosynthesis pathway were discovered from transcriptome sequencing of P. vietnamensis var. fuscidiscus. We further analyzed the data and found 21,320 simple sequence repeats (SSRs), 30 primer pairs for SSRs were randomly selected for validation of the amplification and polymorphism in 13 P. vietnamensis var. fuscidiscus accessions. Meanwhile, five major triterpene saponins in roots of P. vietnamensis var. fuscidicus were determined using high performance liquid chromatography (HPLC) and evaporative light scattering detector (ELSD). CONCLUSIONS: The genomic resources generated from P. vietnamensis var. fuscidiscus provide new insights into the identification of putative genes involved in triterpenoid saponins biosynthesis pathway. This will facilitate our understanding of the biosynthesis of triterpenoid saponins at molecular level. The SSR markers identified and developed in this study show genetic diversity for this important crop and will contribute to marker-assisted breeding for P. vietnamensis var. fuscidiscus.

Isolation and characterization of thermo-acidophilic endospore-forming bacteria from the concentrated apple juice-processing environment.

Forty-five thermo-acidophilic, spore-forming bacteria were isolated from a concentrated apple juice-processing environment. All of them were Gram-positive, rod shaped, and strictly aerobic that most likely belong to the genus of Alicyclobacillus. A fast identification method-16S rDNA PCR-RFLP was used to identify them. The results indicated that at the similarity level of 87%, apple juice isolates strains of 1-4 and 1-2-4 clustered with the reference strain of A. acidoterrstris DSM 3922T, and 4-2-1, S-22 and 5-1 with A. cycloheptanicus DSM 4006T, respectively. The other tested strains were different from all the reference strains in this study and may be new species of Alicyclobacillus genus or the other. In order to confirm this conclusion, we selected 7 16S rDNA PCR-RFLP identified strains and 5 type strains of Alicyclobacillus genus, carried on 51 kinds of phenotypic characteristics and analysis the data by unweighted pair group method with arithmetic mean (UPGMA). The results showed that the similarity degree between every two strains was lower than 80%. It also suggested that they may be different from each other and the unidentified strains may be new species. In addition, spoilage effects of them on 12 Brix apple juice were also studied. The result suggested that all 19 tested bacterial strains caused apple juice to become turbid, form a precipitate and off odor at varying rates when incubated at 37 degrees C up to 12 days. It suggested that these bacteria are associated with the spoilage of apple juice during storage.