Widely targeted metabolomics reveals differences in metabolites of Paeonia lactiflora cultivars.

INTRODUCTION: Paeonia lactiflora contains diverse active constituents and exhibits various pharmacological activities. However, only partial identification of biologically active substances from P. lactiflora has been achieved using low-throughput techniques. Here, the roots of P. lactiflora, namely, Fenyunu (CK), Dafugui (DFG), and Red Charm (HSML), were studied. The primary and secondary metabolites were investigated using ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESIMS/MS). METHODS: The chemical compounds and categories were detected using broadly targeted UPLC-MS/MS. Principal component analysis (PCA), orthogonal partial least-squares discriminant analysis (OPLS-DA), and hierarchical clustering analysis (HCA) were carried out for metabolites of different varieties of P. lactiflora. RESULTS: A total of 1237 compounds were detected and classified into 11 categories. HCA, PCA, and OPLS-DA of these metabolites indicated that each variety of P. lactiflora was clearly separated from the other groups. Differential accumulated metabolite analysis revealed that the three P. lactiflora varieties contained 116 differentially activated metabolites (DAMs) involved in flavonoid, flavone, and flavonol metabolism. KEGG pathway analysis revealed that, in 65 pathways, 336 differentially abundant metabolites (DMs) were enriched in the CK and DFG groups; moreover, the type and content of terpenoids were greater in the CK group than in the DFG group. The CK and HSML groups contained 457 DMs enriched in 61 pathways; the type and amount of flavonoids, terpenoids, and tannins were greater in the CK group than in the HSML group. The DFG and HSML groups contained 497 DMs enriched in 65 pathways; terpenoids and alkaloids were more abundant in the HSML variety than in the DFG variety. CONCLUSIONS: A total of 1237 compounds were detected, and the results revealed significant differences among the three P. lactiflora varieties. Among the three P. lactiflora varieties, phenolic acids and flavonoids composed the largest and most diverse category of metabolites, and their contents varied greatly. Therefore, CK is suitable for medicinal plant varieties, and DFG and HSML are suitable for ornamental plant varieties. Twelve proanthocyanidin metabolites likely determined the differences in color among the three varieties.

Electroacupuncture Improves Neuronal Damage and Mitochondrial Dysfunction Through the TRPC1 and SIRT1/AMPK Signaling Pathways to Alleviate Parkinson's Disease in Mice.

Parkinson's disease (PD) is a neurodegenerative disease that mainly manifests as cognitive decline and motor dysfunction, the treatment of which is still a major challenge in the clinical field. Acupuncture therapy has been shown in many studies to enhance the body's own immunity and disease resistance. This study mainly discusses the specific mechanism underlying electroacupuncture intervention in improving PD. Male C57BL/6 mice were intraperitoneally injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to induce a mouse PD model, and the chorea trembling control area of the head of PD mice was treated by electroacupuncture. Western blotting was used to detect the expression of related proteins in mouse pathological samples; TUNEL measured neuronal apoptosis levels; Nissl staining observed neuronal damage; immunofluorescence and immunohistochemistry were used to detect the expression of Iba-1, TH, and α-syn in substantia nigra denser (SN). The expression levels of oxidative stress factors and inflammatory factors were measured by kits. Flow cytometry measured mitochondrial membrane potential and Ca2+ levels. MPTP intraperitoneal injection induced an increase in inflammatory factors in PD mice and promoted the oxidative stress response, and the inflammatory response was alleviated after electroacupuncture treatment. Electroacupuncture intervention effectively alters the decrease in oxidative stress levels and alleviates neuronal damage in PD mice. Electroacupuncture improves mitochondrial dysfunction induced by MPTP in PD mice by activating the SIRT1/AMPK signaling pathway. We also confirmed that knocking down TRPC1 can inhibit the SIRT1/AMPK signaling pathway, weaken the Ca2+ content in mouse neuronal tissue, and promote cell apoptosis. Electroacupuncture improves neuronal damage and alleviates PD in mice through the TRPC1 and SIRT1/AMPK signaling pathways. In addition, electroacupuncture therapy can improve MPTP-induced mitochondrial dysfunction in PD mice and alleviate the PD process.

Profiling of widely targeted metabolomics for the identification of chemical composition in epidermis, xylem and pith of Gleditsiae Spina.

Gleditsiae Spina, the thorn of Gleditsia sinensis Lam., has a long history of being used as a traditional medicine in East Asian countries. However, only a few biologically active substances have been identified from it. In this study, the epidermis, xylem and pith of Gleditsiae Spina, respectively Gs-E, Gs-X and Gs-P, were studied. We used a widely targeted metabolomics method to investigate the chemical composition of Gs-E, Gs-X and Gs-P. A total of 728 putative metabolites were identified from Gleditsiae Spina, including 211 primary metabolites and 517 secondary metabolites. These primary and secondary metabolites could be categorized into more than 10 different classes. Flavonoids, phenolic acids, lipids, amino acids and derivatives, and organic acids constituted the main metabolite groups. Multivariate statistical analysis showed that the Gs-E, Gs-X and Gs-P samples could be clearly separated. Differential accumulated metabolite (DAM) analysis revealed that more than half of the DAMs exhibited the highest relative concentrations in Gs-E, and most of the DAMs showed the lowest relative concentrations in Gs-X. Moreover, 11 common differential primary metabolites and 79 common differential secondary metabolites were detected in all comparison groups. These results further our understanding of chemical composition and metabolite accumulation of Gleditsiae Spina.

Electroacupuncture promotes the recovery of rats with spinal cord injury by suppressing the Notch signaling pathway via the H19/EZH2 axis.

BACKGROUND: Spinal cord injury (SCI) is a life-changing event with an extremely poor prognosis. In our preliminary studies, electroacupuncture (EA) was found to promote the repair of SCI, which was closely related to the Notch signaling pathway. Therefore, in the present study, we hypothesized that EA protects against SCI by inhibiting the Notch signaling pathway and sought to investigate the underlying molecular mechanisms. METHODS: Rat and cell models of SCI were established. The expression of long non-coding RNA H19 was measured by real-time quantitative polymerase chain reaction. The expression levels of EZH2, Notch1, Notch3, Notch4, Hes1, and PS1 protein were measured by western blot. Cell apoptosis and viability were analyzed using flow cytometry and Cell Counting Kit-8 assays, respectively. The expressions of glial fibrillary acidic protein (GFAP) and nestin were detected by immunofluorescence staining. RESULTS: The expressions of H19, EZH2, and GFAP were significantly increased after SCI but were inhibited by EA; in contrast, nestin expression was significantly decreased by SCI but was restored by EA. Moreover, oxygen-glucose deprivation (OGD) treatment elevated the expression of H19, EZH2, and Notch-related factors as well as apoptosis in PC-12 cells, while suppressing cell viability. Suppressing H19 alleviated the effects of OGD on cell viability and apoptosis, and inhibited the expression of EZH2 and Notch-related factors expression; these effects were reversed by EZH2 overexpression. Finally, EA promoted the recovery of SCI rats and neural stem cell (NSC) proliferation by inhibiting the Notch signaling pathway, which was reversed by H19 overexpression. CONCLUSIONS: Our results demonstrated that EA promotes the recovery of SCI rats and increases the proliferation and differentiation of NSCs by suppressing the Notch signaling pathway via modulating the H19/EZH2 axis.

Widely Targeted Metabolomics Analysis of Different Parts of Salsola collina Pall.

Salsola collina Pall has a long history of being used as a traditional medicine to treat hypertension, headache, insomnia, constipation and vertigo. However, only a few biologically active substances have been identified from S. collina. Here, the shoots and roots of S. collina, namely L-Sc and R-Sc, were studied. The primary and secondary metabolites were investigated using ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS). A total of 637 putative metabolites were identified and these metabolites were mainly classified into ten different categories. Correlation analysis, hierarchical clustering analysis, principal component analysis and orthogonal partial least squares discriminant analysis of metabolites showed that the L-Sc samples could be clearly separated from the R-Sc samples. Differential accumulated metabolite analysis revealed that most of differential primary metabolites were significantly lower in the L-Sc than in the R-Sc. Conversely, the major differential secondary metabolites had higher levels in the L-Sc than in the R-Sc. Further analysis indicated that the flavonoids were the major putative antioxidant components and most of putative antioxidant components exhibited higher relative concentrations in the L-Sc than the R-Sc. These results improve our understanding of metabolite accumulation and provide a reference for the study of medicinal value in S. collina.

Genomic analyses of diverse wild and cultivated accessions provide insights into the evolutionary history of jujube.

The Chinese jujube (Ziziphus jujuba Mill.), a member of the Rhamnaceae family, is an important perennial fruit tree crop of substantial economic, ecological and nutritional value, and is also used as a traditional herbal medicine. Here, we report the resequencing of 493 jujube accessions, including 202 wild and 291 cultivated accessions at >16× depth. Our population genomic analyses revealed that the Shanxi-Shaanxi area of China was jujube's primary domestication centre and that jujube was then disseminated into East China before finally extending into South China. Divergence events analysis indicated that Ziziphus acidojujuba and Ziziphus jujuba diverged around 2.7 Mya, suggesting the interesting possibility that a long pre-domestication period may have occurred prior to human intervention. Using the large genetic polymorphism data set, we identified a 15-bp tandem insertion in the promoter of the jujube ortholog of the POLLEN DEFECTIVE IN GUIDANCE 1 (POD1) gene, which was strongly associated with seed-setting rate. Integrating genome-wide association study (GWAS), transcriptome data, expression analysis and transgenic validation in tomato, we identified a DA3/UBIQUITIN-SPECIFIC PROTEASE 14 (UBP14) ortholog, which negatively regulate fruit weight in jujube. We also identified candidate genes, which have likely influenced the selection of fruit sweetness and crispness texture traits among fresh and dry jujubes. Our study not only illuminates the genetic basis of jujube evolution and domestication and provides a deep and rich genomic resource to facilitate both crop improvement and hypothesis-driven basic research, but also identifies multiple agriculturally important genes for this unique perennial tree fruit species.

A simple, rapid and efficient method for essential element supplementation based on seed germination.

Fertilizer application is typically used to increase the availability of essential elements. In this study, an improved method for essential element supplementation based on seed germination was established. Solutions of essential elements (Fe2+, Zn2+, Cu2+, Mn2+, SeO32+, or I- or their combination) were applied to germinating soybean seeds, and the contents of the essential elements in the soybean sprouts were analyzed by atomic absorption spectroscopy. Compared with the control (seeds treated with water), the contents of iron, zinc, copper, manganese, selenium, and iodine in soybean sprouts produced by germinating seeds treated with solutions containing 10 mM essential elements were approximately 10-2000 times higher. Moreover, treatment with essential element solution increased the total antioxidant capacity and content of total thiols in the soybean sprouts. This rapid and simple technique can be used to improve nutrition for humans and livestock in regions deficient in essential elements.

Phased secondary small interfering RNAs in Panaxnotoginseng.

BACKGROUND: Recent results demonstrated that either non-coding or coding genes generate phased secondary small interfering RNAs (phasiRNAs) guided by specific miRNAs. Till now, there is no studies for phasiRNAs in Panax notoginseng (Burk.) F.H. Chen (P. notoginseng), an important traditional Chinese herbal medicinal plant species. METHODS: Here we performed a genome-wide discovery of phasiRNAs and its host PHAS loci in P. notoginseng by analyzing small RNA sequencing profiles. Degradome sequencing profile was used to identify the trigger miRNAs of these phasiRNAs and potential targets of phasiRNAs. We also used RLM 5'-RACE to validate some of the identified phasiRNA targets. RESULTS: After analyzing 24 small RNA sequencing profiles of P. notoginseng, 204 and 90 PHAS loci that encoded 21 and 24 nucleotide (nt) phasiRNAs, respectively, were identified. Furthermore, we found that phasiRNAs produced from some pentatricopeptide repeat-contain (PPR) genes target another layer of PPR genes as validated by both the degradome sequencing profile and RLM 5'-RACE analysis. We also found that miR171 with 21 nt triggers the generations of 21 nt phasiRNAs from its conserved targets. CONCLUSIONS: We validated that some phasiRNAs generated from PPRs and TASL genes are functional by targeting other PPRs in trans. These results provide the first set of PHAS loci and phasiRNAs in P. notoginseng, and enhance our understanding of PHAS in plants.

Isolation and functional characterization of a cold responsive phosphatidylinositol transfer-associated protein, ZmSEC14p, from maize (Zea may L.).

KEY MESSAGE: A Sec14-like protein, ZmSEC14p , from maize was structurally analyzed and functionally tested. Overexpression of ZmSEC14p in transgenic Arabidopsis conferred tolerance to cold stress. Sec14-like proteins are involved in essential biological processes, such as phospholipid metabolism, signal transduction, membrane trafficking, and stress response. Here, we reported a phosphatidylinositol transfer-associated protein, ZmSEC14p (accession no. KT932998), isolated from a cold-tolerant maize inbred line using the cDNA-AFLP approach and RACE-PCR method. Full-length cDNA that consisted of a single open reading frame (ORF) encoded a putative polypeptide of 295 amino acids. The ZmSEC14p protein was mainly localized in the nucleus, and its transcript was induced by cold, salt stresses, and abscisic acid (ABA) treatment in maize leaves and roots. Overexpression of ZmSEC14p in transgenic Arabidopsis conferred tolerance to cold stress. This tolerance was primarily displayed by the increased germination rate, root length, plant survival rate, accumulation of proline, activities of antioxidant enzymes, and the reduction of oxidative damage by reactive oxygen species (ROS). ZmSEC14p overexpression regulated the expression of phosphoinositide-specific phospholipase C, which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) and generates second messengers (inositol 1,4,5-trisphosphate and 1,2-diacylglycerol) in the phosphoinositide signal transduction pathways. Moreover, up-regulation of some stress-responsive genes such as CBF3, COR6.6, and RD29B in transgenic plants under cold stress could be a possible mechanism for enhancing cold tolerance. Taken together, this study strongly suggests that ZmSEC14p plays an important role in plant tolerance to cold stress.

Expression and functional analyses of EXO70 genes in Arabidopsis implicate their roles in regulating cell type-specific exocytosis.

During exocytosis, Golgi-derived vesicles are tethered to the target plasma membrane by a conserved octameric complex called the exocyst. In contrast to a single gene in yeast and most animals, plants have greatly increased number of EXO70 genes in their genomes, with functions very much unknown. Reverse transcription-polymerase chain reactions were performed on all 23 EXO70 genes in Arabidopsis (Arabidopsis thaliana) to examine their expression at the organ level. Cell-level expression analyses were performed using transgenic plants carrying ß-glucuronidase reporter constructs, showing that EXO70 genes are primarily expressed in potential exocytosis-active cells such as tip-growing and elongating cells, developing xylem elements, and guard cells, whereas no expression was observed in cells of mature organs such as well-developed leaves, stems, sepals, and petals. Six EXO70 genes are expressed in distinct but partially overlapping stages during microspore development and pollen germination. A mutation in one of these genes, EXO70C1 (At5g13150), led to retarded pollen tube growth and compromised male transmission. This study implies that multiplications of EXO70 genes may allow plants to acquire cell type- and/or cargo-specific regulatory machinery for exocytosis.

Combination of reversible male sterility and doubled haploid production by targeted inactivation of cytoplasmic glutamine synthetase in developing anthers and pollen.

Reversible male sterility and doubled haploid plant production are two valuable technologies in F(1)-hybrid breeding. F(1)-hybrids combine uniformity with high yield and improved agronomic traits, and provide self-acting intellectual property protection. We have developed an F(1)-hybrid seed technology based on the metabolic engineering of glutamine in developing tobacco anthers and pollen. Cytosolic glutamine synthetase (GS1) was inactivated in tobacco by introducing mutated tobacco GS genes fused to the tapetum-specific TA29 and microspore-specific NTM19 promoters. Pollen in primary transformants aborted close to the first pollen mitosis, resulting in male sterility. A non-segregating population of homozygous doubled haploid male-sterile plants was generated through microspore embryogenesis. Fertility restoration was achieved by spraying plants with glutamine, or by pollination with pollen matured in vitro in glutamine-containing medium. The combination of reversible male sterility with doubled haploid production results in an innovative environmentally friendly breeding technology. Tapetum-mediated sporophytic male sterility is of use in foliage crops, whereas microspore-specific gametophytic male sterility can be applied to any field crop. Both types of sterility preclude the release of transgenic pollen into the environment.