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Slash pine (Pinus elliottii Engelm.) is an important timber and resin species in the United States, China, Brazil and other countries. Understanding the genetic basis of these traits will accelerate its breeding progress. We carried out a genome-wide association study (GWAS), transcriptome-wide association study (TWAS) and weighted gene co-expression network analysis (WGCNA) for growth, wood quality, and oleoresin traits using 240 unrelated individuals from a Chinese slash pine breeding population. We developed high quality 53,229 single nucleotide polymorphisms (SNPs). Our analysis reveals three main results: (1) the Chinese breeding population can be divided into three genetic groups with a mean inbreeding coefficient of 0.137; (2) 32 SNPs significantly were associated with growth and oleoresin traits, accounting for the phenotypic variance ranging from 12.3% to 21.8% and from 10.6% to 16.7%, respectively; and (3) six genes encoding PeTLP, PeAP2/ERF, PePUP9, PeSLP, PeHSP, and PeOCT1 proteins were identified and validated by quantitative real time polymerase chain reaction for their association with growth and oleoresin traits. These results could be useful for tree breeding and functional studies in advanced slash pine breeding program., Competing Interests: The authors have declared that no competing interests exist.

Background: At present, oral antidepressants are commonly used in the clinical treatment of depression. However, the current drug treatment may lead to more serious adverse reactions. Therefore, we focus on Chinese traditional medicine, trying to find an effective and safe alternative or complementary medicine. Crocus sativus (saffron) is a traditional Chinese herbal medicine, which is typically used in the clinic to regulate anxiety, insomnia, amnesia, and other mental disorder. The study aimed to explore the neuroprotective effect of ethanol extract of saffron (EES) on corticosterone (CORT)- induced injury in PC12 cells and further explored its potential mechanism., Methods: The authenticity of saffron and the active components of EES were identified by a water test and ultra-performance liquid chromatography-time of flight mass spectrometry system. The screening of cytotoxicity for PC12 cells was incubated with EES in different concentrations for 24 h, and the protective efficacy of EES on CORT (500 μM) -induced PC12 cell injury, cell viability was assessed by Cell Counting Kit-8 (CCK-8) assay. The differentially expressed genes (DEGs) of EES-protected PC12 cells were analyzed using the RNA-seq method, and the results were analyzed for GO and KEGG enrichment. The results of RNA-seq were verified by qPCR analysis., Results: The saffron was initially identified as authentic in the water test and 10 compounds were identified by Ultra Performance Liquid Chromatography (UPLC)- Mass Spectrometry (MS). The results of CCK-8 demonstrated that EES at concentrations above 640 μg/mL exerted a certain cytotoxic effect, and PC12 cells pretreated with EES (20, 40, and 80 μg/mL) significantly reversed the 500 μM CORT-induced cell death. RNA-seq analysis showed that EES regulated 246 differential genes, which were mainly enriched in the MAPK signaling pathway. Dusp5, Dusp6, Gadd45b, Gadd45G, and Pdgfc were further validated by qPCR. Experimental data showed that the results of qPCR were consistent with RNA-seq., Conclusions: These findings provide an innovative understanding of the molecular mechanism of the protective effect of EES on PC12 cells at the molecular transcription level, and Dusp5, Dusp6, Gadd45b, Gadd45g, and Pdgfc may be potential novel targets for antidepressant treatment., (© 2022. The Author(s).)

Objective: Lemna minor is a plant with a huge repertoire of secondary metabolites. The literature indicates that extracts of Lemna minor have antioxidant, antiradical, immunomodulatory and anti-inflammatory properties. The objective of the present study was to find a suitable technique to extract active compounds from this plant and verify whether these extracts have immunomodulatory activity., Materials and Methods: We grew L. minor on a standard medium with Gamborg B5 and vitamins. We extracted compounds from the plant by maceration and decoction. The phytochemical profile of the extracts was characterized by chromatography, spectrophotometry, and spectroscopy. The extracts were tested on cultures of mononuclear cells from four human subjects. These cells were pulsed with carboxyfluorescein succinimidyl ester, grown in triplicate in standard culture medium without (control) and with increasing concentrations of Lemna extracts. Flow cytometry was used to evaluate cell death and proliferation of the total mononuclear cell population and of CD4+, CD8+, B cell and monocyte populations., Results: The Lemna extracts were not cytotoxic and did not cause cell necrosis or apoptosis in immune cells. At low concentrations, they induced very limited proliferation of CD4+ cells within 48 hours. At high concentrations, they induced proliferation of CD8+ cells and B lymphocytes within 48 hours., Conclusions: Unfortunately, we failed to confirm any immunomodulatory activity of Lemna extracts. Growth and death rates of human immune cells were not significantly affected by adding Lemna extracts to the culture medium.

We successfully constructed a coupled in vitro transcription/translation (cIVTT) system based on wheat germ extract (WGE) for efficient expression from PCR-generated DNA templates in short-time (∼3-h) batch reactions. The productivity of this system under optimized conditions was 85 μg (2.8 nmol) per 1 mL of reaction solution (corresponding to 425 μg per 1 mL of WGE), which was about 9-fold higher than that by the conventional batch method using mRNA as a template. The DNA template concentration required for efficient cIVTT was as low as 2.5 nM, which is much lower than those required for other eukaryotic cIVTT systems to maximize their productivity (30-50 nM). The productivity of the present system with a 2.5 nM template was 80-fold and 4-fold higher than that of a commercially available WGE-based cIVTT system with a 2.5 nM and a 40 nM template, respectively. In addition, the present system functioned well in a liposome (i.e., in an artificial cell) without a loss of productivity. Given that WGE-based systems have the advantage of being suitable for the expression of a broad range of proteins, the present cIVTT system is expected to be widely used in future cell-free synthetic biology., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Root chemicals and the sequences of the internal transcribed spacers (ITSs) were analyzed for 9 Ligularia kanaitzensis and 3 L. subspicata samples collected in northwestern Yunnan and southwestern Sichuan, China. Subspicatins A and C were isolated from two L. kanaitzensis samples. Introgression of genes responsible for these compounds from L. subspicata was suggested by their strong connection with L. subspicata/L. lamarum and the geographical proximity of the samples to L. subspicata. DNA analysis of a set of 27 L. kanaitzensis samples including those analyzed previously showed that they belong to two clades, designated A and B. Together with the presence/absence of furanoeremophilane, the 27 samples were sorted into three groups: clade A/furan, clade B/furan, and clade B/non-furan. The ancestral plant presumably belonged to clade B/non-furan, because furanoeremophilanes are biosynthesized from eremophilan-8-ones. 1β-Angeloyloxyfukinone, a likely intermediate between fukinone and subspicatin C, was isolated for the first time. This finding allowed us to propose plausible biosynthetic pathways of subspicatins A and C., (© 2021 Wiley-VHCA AG, Zurich, Switzerland.)

As members of the pathogenesis-related protein (PR)-2 family, β-1,3-glucanases play pivotal roles in plant defense. Previous study showed that the rice genome contains 16 genes encoding putative β-1,3-glucanases, and the β-1,3-glucanases in subfamily A were deduced to be involved in plant defense. However, there was limited direct evidence. In this study, the expression of rice β-1,3-glucanases Gns2-Gns6 belonging to subfamily A in rice plant infection with Magnaporthe oryzae was investigated, and the enhanced expression of Gns6 during infection confirmed its crucial role in the defense of rice seedlings. Enzymological characterization revealed that Gns6 preferentially hydrolyzed laminarin, pachymaran, and yeast glucan. The β-1,3; 1,6-glucanase Gns6 exhibited a specific activity of 1.2 U/mg with laminarin as the substrate. In addition, Gns6 could hydrolyze laminarin via an endo-type mechanism, yielding a series of oligosaccharides with various degrees of polymerization that are known immune elicitors in plants. Moreover, Gns6 exhibited a significant inhibitory effect against the formation of the germ tubes and appressoria, with potential applications in plant protection. Taken together, this study shows that Gns6 is an essential effector in the defensive response of rice against pathogenic fungi., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Two halophilic archaeal strains, Gai3-2 T and NJ-3-1 T , were isolated from salt lake and saline soil samples, respectively, collected in PR China. The 16S rRNA gene sequences of the two strains were 97.5% similar to each other. Strains Gai3-2 T and NJ-3-1 T had the highest sequence similarities to ' Halobonum tyrrellense ' G22 (96.7 and 97.8%, respectively), and displayed similarities of 91.5-93.5% and 92.3-94.7%, respectively, to Halobaculum members. Phylogenetic analysis revealed that the two strains formed different branches and clustered tightly with ' H. tyrrellense ' G22 and Halobaculum members. The average nucleotide identity (ANI), in silico DNA-DNA hybridization ( is DDH) and amino acid identity (AAI) values between the two strains were 83.1, 26.9 and 77.9%, respectively, much lower than the threshold values proposed as a species boundary. These values between the two strains and ' H. tyrrellense ' G22 (ANI 77.9-78.2%, is DDH 22.5-22.6% and AAI 68.8-69.3%) and Halobaculum members (ANI 77.53-77.63%, is DDH 21.8-22.3% and AAI 68.4-69.4%) were almost identical, and much lower than the recommended threshold values for species delimitation. These results suggested that strains Gai3-2 T and NJ-3-1 T represent two novel species of Halobaculum . Based on phenotypic, chemotaxonomic and phylogenetic properties, strains Gai3-2 T (=CGMCC 1.16080 T =JCM 33550 T ) and NJ-3-1 T (=CGMCC 1.16040 T =JCM 33552 T ) represent two novel species of the genus Halobaculum , for which the name Halobaculum halophilum sp. nov. and Halobaculum salinum sp. nov. are proposed.

Ethnopharmacological Relevance: Gentiana kurroo is a multipurpose critically endangered medicinal herb prescribed as medicine in Ayurveda in India and exhibits various pharmacological properties including anti-cancer activity. The species is rich repository of pharmacologically active secondary metabolites together with secoiridoidal glycosides., Aim of the Study: The study aimed to investigate the chemical diversity in different populations/cytotypes prevailing in G. kurroo to identify elite genetic stocks in terms of optimum accumulation/biosynthesis of desired metabolites and having higher in-vitro cytotoxicity potential in relation to chemotypic diversity., Material and Methods: The wild plants of the species were collected from different ranges of altitudes from the Kashmir Himalayas. For cytological evaluation, the standard meiotic analysis was performed. The standard LC-MS/MS technique was employed for phytochemical analysis based on different marker compounds viz. sweroside, swertiamarin, and gentiopicroside. Different tissues such as root-stock, aerial parts, and flowers were used for chemo-profiling. Further, the methanolic extracts of diploid and tetraploid cytotypes were assessed for cytotoxic activity by using MTT assay against four different human cancer cell lines., Results: The quantification of major bioactive compounds based on tissue- and location-specific comparison, as well as in-vitro cytotoxic potential among extant cytotypes, was evaluated. The comprehensive cytomorphological studies of the populations from NW Himalayas revealed the occurrence of different chromosomal races viz. n = 13, 26. The tetraploid cytotype was hitherto unreported. The tissue-specific chemo-profiling revealed relative dominance of different phytoconstituents in root-stock. There was a noticeable increase in the quantity of the analyzed compounds in relation to increasing ploidy status along the increasing altitudes. The MTT assay of methanolic extracts of diploid and tetraploid cytotypes displayed significant cytotoxicity potential in tetraploids. The root-stock extracts of tetraploids were highly active extracts with IC 50 value ranges from 5.65 to 8.53 μg/mL against HCT-116 colon cancer., Conclusion: The chemical evaluation of major bioactive compounds in diverse cytotypes from different plant parts along different altitudes presented an appreciable variability in sweroside, swertiamarin, and gentiopicroside contents. Additionally, the concentrations of these phytoconstituents varied for cytotoxicity potential among different screened cytotypes. This quantitative difference of active bio-constituents was in correspondence with the growth inhibition percentage of different tested cancer cell lines. Thus, the present investigation strongly alludes towards a prognostic approach for the identification of elite cytotypes/chemotypes with significant pharmacological potential., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Legumes are endowed with an opulent class of proteins called lectins that can detect tenuous variations in carbohydrate structures and bind them reversibly with high affinity and specificity. The genus Canavalia, in the family of Leguminosae, is considered to be an affluent source of lectin. An effort has been made to analyse the sequences encoded by the lectin gene and its carbohydrate binding pockets from three species of Canavalia, including C. virosa, C. rosea, and C. pubescens. Crude seed extract showed highest haemagglutination titer against buffalo RBCs and has high affinity to mannose and trehalose. Amplification of the lectin gene by gene-specific primers showed the presence of an 870 bp amplicon. Physicochemical characterization using various bioinformatic tools showed that the isoelectric point was below 7, suggesting that lectin molecules were acidic. A high aliphatic index and high instability index were observed, which indicated that lectin molecules were stable towards a wide range of temperatures. The occurrence of N-glycosylation sites at two sites was also identified in all three species. Prediction of secondary structure showed that approximately 59.05 %, 56.76 % and 54.88 % of the elements were random coils in the case of C. virosa, C. pubescens and C. rosea, respectively. Comparative modelling of the proteins and docking of hypothetical models with sugar moieties that inhibited the agglutination activity suggested that asparagine, serine, alanine, valine, tyrosine and threonine were the major residues involved in hydrogen bonding and other stacking interactions. This can further provide insights on its prospective antibiosis property., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Mentha longifolia L. is well-known to be one of the most pervasive wild-growing species of the Lamiaceae family, which has extensive beneficial properties in the fields of pharmacology and biological products. In the present study, the correlation between Inter-simple sequence repeat (ISSR) markers and morpho-chemical parameters of twenty different M. longifolia accessions (MLACs) were assessed. The geographic information system (GIS) has been employed to interpret the original habitat of the accessions in Iran. ISSR analysis indicated a remarkable difference in the studied accessions, segregated them into three main groups, constructed by an unweighted pair-group method with arithmetic (UPGMA) and principal coordinate analysis (PCoA). A total of 89 bands were generated by 12 ISSR primers, among which 82 (91.97 %) of them were polymorphic. The cluster analysis based on agro-morphological data scattered MLACs into two main groups. The essential oils (EOs) were analyzed through GC/FID/MS, and four chemotypes were characterized according to the major constituents. Pulegone ranged from 0.17 to 69.50 % was the main oil constituent with the highest content. Also, HPLC-PDA was employed to identify and to quantify the phenolic compounds in the MeOH extracts of MLACs. Heatmap cluster based on phenolic compounds produced three main categories of accessions. The components identified in the extracts were rosmarinic acid, rutin, vanillic acid, ferulic acid, chlorogenic acid, caffeic acid, 3,4-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid, and p-coumaric acid, which among them rosmarinic acid (RA) varied from 39.16 to 261.55 mg/100 g (DW) as a predominant constituent. Subsequently, multiple regression analyses between ISSR fragments and morpho-chemical data illustrated considerable relationships in the plant materials. The high variation and correlation observed in metabolic and phenotypic traits of MLACs establish an adequate source to conduct reserves conservation programs., (© 2021 Wiley-VHCA AG, Zurich, Switzerland.)

Medicinal plants are the basic source of medicinal compounds traditionally used for the treatment of human diseases. Calotropis gigantea is a medicinal plant belonging to the family of Apocynaceae in the plant kingdom and subfamily Asclepiadaceae usually bearing multiple medicinal properties to cure a variety of diseases., Background: The Peptide Mass Fingerprinting (PMF) identifies the proteins from a reference protein database by comparing the amino acid sequence that is previously stored in the database and identified., Objective: The purpose of the study is to identify the peptides having anti-cancerous properties by in silico peptide mass fingerprinting., Methods: The calculation of in silico peptide masses is done through the ExPASy PeptideMass and these masses are used to identify the peptides from the MASCOT online server. Anticancer probability is calculated by iACP server, docking of active peptides is done by CABS-dock the server., Results: The anti-cancer peptides are identified with the MASCOT peptide mass fingerprinting server, the identified peptides are screened and only the anti-cancer are selected. De-novo peptide structure prediction is used for 3D structure prediction by PEP-FOLD 3 server. The docking results confirm strong bonding with the interacting amino acids of the receptor protein of breast cancer BRCA1 which shows the best peptide binding to the active chain, the human leukemia protein docking with peptides shows the accurate binding., Conclusion: These peptides are stable and functional and are the best way for the treatment of cancer and many other deadly diseases., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Nectaroscordum siculum ssp. bulgaricum (Janka) Stearn (Allium siculum subsp. dioscoridis (Sm.) K. Richt.) is a traditional culinary spice from South-East Europe. Studies of N. siculum have focused mainly on the botanical and taxonomic characteristics of this species and there is no data available in the scientific literature about its metabolite profile. Thus, the aim of the current study was metabolite profiling of four wild populations of N. siculum grown in Bulgaria by gas chromatography coupled to mass spectrometry (GC-MS) and subsequent principal component analysis (PCA) of the data obtained. The identified primary metabolites (carbohydrates, amino acids, organic acids and lipids) are initial compounds for the biosynthesis of different plant secondary metabolites, such as polyphenols and flavour compounds with valuable biological activities for humans. The health benefits of the phenolic acids identified in this study have been a prerequisite for the implementation of N. siculum in different food systems in order to increase their quality and biological value.

The pepper-bark tree (Warburgia salutaris) is one of the most highly valued medicinal plant species worldwide. Native to southern Africa, this species has been extensively harvested for the bark, which is widely used in traditional health practices. Illegal harvesting coupled with habitat degradation has contributed to fragmentation of populations and a severe decline in its distribution. Even though the species is included in the IUCN Red List as Endangered, genetic data that would help conservation efforts and future re-introductions are absent. We therefore developed new molecular markers to understand patterns of genetic diversity, structure, and gene flow of W. salutaris in one of its most important areas of occurrence (Mozambique). In this study, we have shown that, despite fragmentation and overexploitation, this species maintains a relatively high level of genetic diversity supporting the existence of random mating. Two genetic groups were found corresponding to the northern and southern locations. Our study suggests that, if local extinctions occurred in Mozambique, the pepper-bark tree persisted in sufficient numbers to retain a large proportion of genetic diversity. Management plans should concentrate on maintaining this high level of genetic variability through both in and ex-situ conservation actions.

We here designed an in vitro selection scheme for obtaining an aptamer with which to rationally construct an artificial riboswitch as its component part. In fact, a nanosized DNA-binding aptamer obtained through this scheme allowed us to easily and successfully create eukaryotic riboswitches that upregulate internal ribosome entry site-mediated translation in response to the ligand (nanosized DNA) in wheat germ extract, a eukaryotic cell-free expression system. The induction ratio of the best riboswitch ligand-dose-dependently increased to 21 at 300 μM ligand. This switching efficiency is much higher than that of the same type of riboswitch with a widely used theophylline-binding aptamer, which was in vitro selected without considering its utility for constructing riboswitches. The selection scheme described here would facilitate obtaining various ligand/aptamer pairs suitable for constructing artificial riboswitches, which could serve as elements of synthetic gene circuits in synthetic biology.

Panax notoginseng , a perennial herb of the genus Panax in the family Araliaceae, has played an important role in clinical treatment in China for thousands of years because of its extensive pharmacological effects. Here, we report a high-quality reference genome of P. notoginseng , with a genome size up to 2.66 Gb and a contig N50 of 1.12 Mb, produced with third-generation PacBio sequencing technology. This is the first chromosome-level genome assembly for the genus Panax . Through genome evolution analysis, we explored phylogenetic and whole-genome duplication events and examined their impact on saponin biosynthesis. We performed a detailed transcriptional analysis of P. notoginseng and explored gene-level mechanisms that regulate the formation of characteristic tubercles. Next, we studied the biosynthesis and regulation of saponins at temporal and spatial levels. We combined multi-omics data to identify genes that encode key enzymes in the P. notoginseng terpenoid biosynthetic pathway. Finally, we identified five glycosyltransferase genes whose products catalyzed the formation of different ginsenosides in P. notoginseng . The genetic information obtained in this study provides a resource for further exploration of the growth characteristics, cultivation, breeding, and saponin biosynthesis of P. notoginseng ., (© 2020 The Authors.)

Background: Masson pine (Pinus massoniana Lamb.), the dominant native coniferous species in southern China, is commercially important for supplying timber and oleoresin. However, knowledge of the genetic variability of masson pine germplasm is still limited. In this study, the genetic diversity and population structure of masson pine germplasm were assessed using 204 wild accessions from 10 main distribution regions using 94,194 core single-nucleotide polymorphisms (SNPs) obtained from transcriptome sequencing data., Results: The average expected heterozygosity was 0.2724, implying abundant genetic diversity within masson pine germplasm. Analysis of molecular variance (AMOVA) revealed that 3.29% of the variation was sourced from genetic differentiation. Structure analysis identified two geographically distinct groups. Discriminant analysis of principal components (DAPC) showed that one of those groups was further divided into two clusters. Sichuan and Chongqing provenance is the geographical origin, which diffused outward along two different lines. Oleoresin yield is reflected in the evolution of the two groups, and exhibits two different trends along the two lines of diffusion. The oleoresin yield may be associated with the genes of chitinase, CYP720B, cytochrome P450, ABC transporter, and AP2/ethylene-responsive transcription factor (ERF) based on SNPs and expression., Conclusions: SNP markers from transcriptome sequencing are highly capable of evaluating genetic diversity within different species, as well as the genetic control of objective traits. The functions of these genes will be verified in future studies, and those genes strongly associated with oleoresin yield will be used to improve yields by means of early genotype selection and genetic engineering.

Ethnopharmacological Relevance: Ashwagandha, also known as Indian Ginseng, is a highly traded medicinal plant, which is used in Ayurveda, Siddha and Unani systems of medicine to improve cognitive function, decrease inflammation, and to counter the ill-effects of aging. Withanolide A and Withaferin A from Ashwagandha were shown to improve immunity and have anti-cancer property, respectively., Aim of the Study: Here, we aimed to create reference DNA barcodes for W. somnifera and to authenticate root and powder samples of Ashwagandha collected from markets., Materials and Methods: Three plant specimen of W. somnifera were collected, and reference DNA barcodes were generated using rbcL, matK, trnH-psbA, and ITS2 DNA barcode markers. Market samples in the form of root (n = 33) and powder (n = 70) were collected and authenticated using ITS2 and trnH-psbA DNA barcodes., Results: Genomic DNA was successfully isolated from all plant specimens and market samples. DNA barcoding showed that 77% of samples were authentic. About 22% of non-authentic samples were powder samples and only 1% were root samples. Among the non-authentic samples, 18% were completely substituted with single species (Mucuna pruriens (L.) DC., Trigonella foenum-graceum L., or Senna auriculata (L.) Roxb.) and 82% were mixed samples containing more than one species. About 63% of the mixed samples contained Ashwagandha as the major ingredient. Furthermore, we identified that six taxonomically divergent plant species from four families were present as adulterants in the mixed samples., Conclusion: DNA barcoding revealed that botanical adulteration in the market samples of Ashwagandha is significant. Powder samples are more prone to adulteration than root samples. The adulterated samples contained plant material that is not related to Ashwagandha, which warrants strict quantity control and market surveillance to derive the true medicinal benefits of this medicinal plant., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Scutellaria baicalensis (S. baicalensis) and Scutellaria barbata (S. barbata) are common medicinal plants of the Lamiaceae family. Both produce specific flavonoid compounds, including baicalein, scutellarein, norwogonin, and wogonin, as well as their glycosides, which exhibit antioxidant and antitumor activities. Here, we report chromosome-level genome assemblies of S. baicalensis and S. barbata with quantitative chromosomal variation (2n = 18 and 2n = 26, respectively). The divergence of S. baicalensis and S. barbata occurred far earlier than previously reported, and a whole-genome duplication (WGD) event was identified. The insertion of long terminal repeat elements after speciation might be responsible for the observed chromosomal expansion and rearrangement. Comparative genome analysis of the congeneric species revealed the species-specific evolution of chrysin and apigenin biosynthetic genes, such as the S. baicalensis-specific tandem duplication of genes encoding phenylalanine ammonia lyase and chalcone synthase, and the S. barbata-specific duplication of genes encoding 4-CoA ligase. In addition, the paralogous duplication, colinearity, and expression diversity of CYP82D subfamily members revealed the functional divergence of genes encoding flavone hydroxylase between S. baicalensis and S. barbata. Analyzing these Scutellaria genomes reveals the common and species-specific evolution of flavone biosynthetic genes. Thus, these findings would facilitate the development of molecular breeding and studies of biosynthesis and regulation of bioactive compounds., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Dalbergia odorifera , a critically endangered tree species, produces heartwood containing a vast variety of flavonoids. This heartwood, also known as Chinese rosewood, has high economic and medicinal value, but its formation takes several decades. In this study, we showed that discolored wood induced by pruning displays similar color, structure, and flavonoids content to those of natural heartwood, suggesting that wounding is an efficient method for inducing flavonoid production in D. odorifera . Transcriptome analysis was performed to investigate the mechanism underlying wounding-induced flavonoids production in D. odorifera heartwood. Wounding upregulated the expression of 90 unigenes, which covered 19 gene families of the phenylpropanoid and flavonoid pathways, including PAL, C4H, 4CL, CHS, CHI, 6DCS, F3'5'H, F3H, FMO, GT, PMAT, CHOMT, IFS, HI4'OMT, HID, IOMT, I2'H, IFR, and I3'H. Furthermore, 47 upregulated unigenes were mapped to the biosynthesis pathways for five signal molecules (ET, JA, ABA, ROS, and SA). Exogenous application of these signal molecules resulted in the accumulation of flavonoids in cell suspensions of D. odorifera , supporting their role in wounding-induced flavonoid production. Insights from this study will help develop new methods for rapidly inducing the formation of heartwood with enhanced medicinal value.

Pine resin, which typically consists of terpenoids, is a natural product used in various industrial applications. Oleoresin can be obtained from the xylem tissue by wounding the stem bark. Pinus massoniana (masson pine) is an important resin-tapping tree species that originated in southern China. Masson pines with different genetic backgrounds typically have different resin-yielding capacities (RYCs). However, the mechanisms underlying high resin yield in masson pines are unclear. The aim of this study was to identify the possible genetic regulation pathways and functional genes that influence the resin yield. In this study, we conducted transcriptomic and metabolomic studies of masson pine secondary xylem with high, medium, and low RYCs. A total of 230,068 unigenes and 3894 metabolites were identified from the tissue of the secondary xylem. Several differentially expressed regulation factors, including WRKY, bHLH, and ERF, and functional genes such as PKc and LRR-RLKs, were identified among these masson pines. The Kyoto Encyclopedia of Genes and Genomes pathways were mainly focused on diterpenoid biosynthesis, plant hormone signal transduction, and ABC transporters. Furthermore, integration of the transcriptomic and metabolomic data indicated that the PKc- and LRR-RLK-related regulatory and metabolic pathways may play critical roles in the biosynthesis of terpenoids. These above results improve our understanding of the biosynthesis mechanism of oleoresin in P. massoniana and facilitate further research work into the functional analysis of these candidate genes.

Autophagy can remodel skeletal muscle in response to exercise. However, excessive autophagy can have adverse effects on skeletal muscle. Although Rhodiola crenulata (R. crenulata) is thought to regulate autophagy, its active ingredients and mechanisms of action remain unclear. In this study, molecular docking and network pharmacology were used to screen for autophagy-related targets of R. crenulata. Subsequently, protein-protein interaction (PPI) analysis was used to find the relationships between the inverse docking targets and autophagy-related targets and therefore highlight the key targets. And then the Database for Annotation, Visualization, and Integrated Discovery (DAVID) database was recruited to explain the functions and enrichment pathways of the target proteins. Finally, the potential targets were validated by immunohistochemistry of a mouse model of exhaustive exercise-induced skeletal muscle injury. We found a network of 15 major constituents of R. crenulata with 30 autophagy-related and 105 inverse-docking targets by molecular docking and network pharmacology. The results of PPI analysis indicated that 16 inverse-docking targets interacted 8 autophagy-related proteins. Further pathway analysis showed that R. crenulata could regulate exercise-induced skeletal muscle autophagy through mammalian target of rapamycin (mTOR), AMP activated protein kinase (AMPK) and Forkhead box protein O (FoxO). The results of our animal experiments indicated that R. crenulata could suppress the expression of Ubiquitin-like protein ATG12 (ATG12), Beclin-1 (BECN1), and Serine/threonine-protein kinase ULK1 (ULK1), while increasing the expression of MTOR, NAD-dependent protein deacetylase sirtuin-1 (SIRT1), and Microtubule-associated protein tau (MAPT). In conclusion, this study demonstrated that R. crenulata may protect skeletal muscle injury induced by exhaustive exercise via regulating the mTOR, AMPK, and FoxO singling pathway.

Virus-induced gene silencing (VIGS) has emerged as a fast and efficient reverse and forward genetics tool to study gene function in model plants as well as in agriculturally important plants. In addition, VIGS approach has been successfully used to provide insights into the role of several genes and regulators involved in plant secondary metabolism. Ashwagandha (Withania somnifera) is an important Indian medicinal plant that accumulates pharmacologically important triterpenoid steroidal lactones, which are collectively termed as withanolides. W. somnifera being a highly recalcitrant plant for genetic transformation, Tobacco rattle virus (TRV)-mediated VIGS was established by our group to facilitate understanding of withanolides' pathway. Here, we describe a detailed procedure to carry out VIGS for gene function studies in W. somnifera.

Wheat germ extract (WGE) is one of the most widely used eukaryotic cell-free translation systems for easy synthesis of a broad range of proteins merely by adding template mRNAs. Its productivity has thus far been improved by removing translational inhibitors from the extract and stabilizing the template with terminal protectors. Nonetheless, there remains room for increasing the yield by designing a terminally protected template with higher susceptibility to translation. Given the fact that a 5' terminal protector is a strong inhibitor of the canonical translation, we herein focused on Cripavirus internal ribosome entry sites (IRESes), which allow for a unique translation initiation from a non-AUG start codon without the help of any initiation factors. We mutated their start codons to enhance the IRES-mediated translation efficiency in WGE. One of the mutants showed considerably higher efficiency, 3-4-fold higher than that of its wild type, and also 3-4-fold higher than the canonical translation efficiency by an IRES-free mRNA having one of the most effective canonical-translation enhancers. Because this mutated IRES is compatible with different types of genes and terminal protectors, we expect it will be widely used to synthesize proteins in WGE., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Artemisia absinthium (wormwood), a common ingredient in absinthe, contains the compound thujone, which is unregulated by the U.S. Drug Enforcement Agency. Thujone can cause an "unregulated legal high" in higher concentrations. The European Union limits thujone from Artemisia species to 35 mg/kg while the U.S. Food and Drug Administration requires less than 10 ppm to be "thujone-free." However, individuals can smoke or ingest A. absinthium in different forms. This study developed a polymerase chain reaction (PCR) high-resolution melt (HRM) assay to detect and identify A. absinthium based on primer specificity, sensitivity, repeatability, and robustness. A triplex assay was performed with three "unregulated legal high" species: Datura stramonium, Merremia tuberosa, and A. absinthium; the PCR HRM assay detected and identified each plant at melt temperatures 77.42 ± 0.20°C, 83.88 ± 0.22°C, and 87.77 ± 0.15°C, respectively. The primer set developed distinguished A. absinthium from a variety of plant species and was successfully triplexed., (© 2019 American Academy of Forensic Sciences.)

Terpene trilactones (TTLs) are the main secondary metabolites of Ginkgo biloba. As one of the rate-limiting enzymes in the mevalonic acid (MVA) pathway of TTL biosynthesis, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) catalyzes the 3-hydroxy-3-methylglutaryl coenzyme A to form MVA. In this study, two cDNA sequences of HMGR genes, namely, GbHMGR2 and GbHMGR3, were cloned from G. biloba. The protein sequences of GbHMGR2 and GbHMGR3, which contain several functional domains, were analyzed. Regulatory elements related to light, hormone, and stress response were detected in the promoter regions of GbHMGR2 and GbHMGR3. The catalytic activity of these genes was verified by a functional complement experiment in yeast. Quantitative real-time PCR (qRT-PCR) showed the distinct expression patterns of the two genes in different organs. The TTL contents in the organs were detected by high-performance liquid chromatography- evaporative light scattering detector. GbHMGR2 and GbHMGR3 were responded to cold, dark, methyl jasmonate (MJ), abscisic acid (ABA), salicylic acid (SA), and ethephon (Eth) treatments. The TTL contents were also regulated by cold, dark, MJ, ABA, SA, and Eth treatment. In conclusion, GbHMGR2 and GbHMGR3 may participate in the MVA pathway of TTL biosynthesis.

Objective: Aconitum plants (Ranunculaceae) exhibit toxicity, and accidental ingestion of the plants has been reported in Japan. Identifying the cause of poisoning is important for emergency medical treatment, and a rapid and simple detection technique is required for the identification of poisoning cause. In the present study, we developed a rapid and simple method for detecting Aconitum plant DNA using a loop-mediated isothermal amplification (LAMP) assay., Results: Specific LAMP primers for Aconitum plants were designed based on the trnL-trnF intergenic spacer region. Using the LAMP primers, the LAMP assay included an initiation reaction of 10 min followed by amplification for 20 min at the isothermal reaction temperature of 65 °C. The LAMP reaction was demonstrated to be specific and highly sensitive to Aconitum plants, given that the assay can be used for 1 pg of purified DNA. Using raw extracted DNA as template, the entire detection procedure from DNA extraction to final detection required only 30 min. Moreover, the protocol identified samples containing approximately 5 mg of Aconitum plants cooked and digested with artificial gastric juice. The currently proposed protocol exhibits good potential as a screening method of Aconitum plant poisoning for emergency medical care.

Plants in the Burseraceae are globally recognized for producing resins and essential oils with medicinal properties and have economic value. In addition, most of the aromatic and non-aromatic components of Burseraceae resins are derived from a variety of terpene and terpenoid chemicals. Although terpene genes have been identified in model plant crops (e.g., Citrus , Arabidopsis ), very few genomic resources are available for non-model groups, including the highly diverse Burseraceae family. Here we report the assembly of a leaf transcriptome of Protium copal , an aromatic tree that has a large distribution in Central America, describe the functional annotation of putative terpene biosynthetic genes and compare terpene biosynthetic genes found in P. copal with those identified in other Burseraceae taxa. The genomic resources of Protium copal can be used to generate novel sequencing markers for population genetics and comparative phylogenetic studies, and to investigate the diversity and evolution of terpene genes in the Burseraceae.

Alpine plants remain the least studied plant communities in terrestrial ecosystems. However, how they adapt to high-altitude environments is far from clear. Here, we used RNA-seq to investigate a typical alpine plant maca (Lepidium meyenii) to understand its high-altitude adaptation at transcriptional and post-transcriptional level. At transcriptional level, we found that maca root significantly up-regulated plant immunity genes in day-time comparing to night-time, and up-regulated abiotic (cold/osmotic) stress response genes in Nov and Dec comparing to Oct. In addition, 17 positively selected genes were identified, which could be involved in mitochondrion. At post-transcriptional level, we found that maca had species-specific characterized alternative splicing (AS) profile which could be influenced by stress environments. For example, the alternative 3' splice site events (A3SS, 39.62%) were predominate AS events in maca, rather than intron retention (IR, 23.17%). Interestingly, besides serine/arginine-rich (SR) proteins and long non-coding RNAs (lncRNAs), a lot of components in nonsense-mediated mRNA decay (NMD) were identified under differential alternative splicing (DAS), supporting AS coupled to NMD as essential mechanisms for maca's stress responses and high-altitude adaptation. Taken together, we first attempted to unveil maca's high-altitude adaptation mechanisms based on transcriptome and post-transcriptome evidence. Our data provided valuable insights to understand the high-altitude adaptation of alpine plants., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Breeding efforts in the American cranberry ( Vaccinium macrocarpon Ait.), a North American perennial fruit crop of great importance, have been hampered by the limited genetic and phenotypic variability observed among cultivars and experimental materials. Most of the cultivars commercially used by cranberry growers today were derived from a few wild accessions bred in the 1950s. In different crops, wild germplasm has been used as an important genetic resource to incorporate novel traits and increase the phenotypic diversity of breeding materials. Vaccinium microcarpum (Turcz. ex Rupr.) Schmalh. and V. oxycoccos L., two closely related species, may be cross-compatible with the American cranberry, and could be useful to improve fruit quality such as phytochemical content. Furthermore, given their northern distribution, they could also help develop cold hardy cultivars. Although these species have previously been analyzed in diversity studies, genomic characterization and comparative studies are still lacking. In this study, we sequenced and assembled the organelle genomes of the cultivated American cranberry and its wild relative, V. microcarpum . PacBio sequencing technology allowed us to assemble both mitochondrial and plastid genomes at very high coverage and in a single circular scaffold. A comparative analysis revealed that the mitochondrial genome sequences were identical between both species and that the plastids presented only two synonymous single nucleotide polymorphisms (SNPs). Moreover, the Illumina resequencing of additional accessions of V. microcarpum and V. oxycoccos revealed high genetic variation in both species. Based on these results, we provided a hypothesis involving the extension and dynamics of the last glaciation period in North America, and how this could have shaped the distribution and dispersal of V. microcarpum . Finally, we provided important data regarding the polyploid origin of V. oxycoccos .

There is considerable potential for the use of DNA barcoding methods to authenticate raw medicinal plant materials, but their application to testing commercial products has been controversial. A simple PCR test targeting species-specific sequences within the nuclear ribosomal internal transcribed spacer (ITS) region was adapted to screen commercial products for the presence of Hypericum perforatum L. material. DNA differing widely in amount and extent of fragmentation was detected in a number of product types. Two assays were designed to further analyse this DNA using a curated database of selected Hypericum ITS sequences: A qPCR assay based on a species-specific primer pair spanning the ITS1 and ITS2 regions, using synthetic DNA reference standards for DNA quantitation and a Next Generation Sequencing (NGS) assay separately targeting the ITS1 and ITS2 regions. The ability of the assays to detect H. perforatum DNA sequences in processed medicines was investigated. Out of twenty different matrices tested, both assays detected H. perforatum DNA in five samples with more than 10³ ITS copies µL -1 DNA extract, whilst the qPCR assay was also able to detect lower levels of DNA in two further samples. The NGS assay confirmed that H. perforatum was the major species in all five positive samples, though trace contaminants were also detected., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

The application of medicinal plants are the most important biotechnological alternative in the treatment of numerous diseases, especially in developing countries, such as Brazil. Among them, we specified some specimens of the genus Cnidoscolus used as phytotherapies, with healing properties, analgesic, anti-inflammatory, antibiotic and diuretic, anticancer, among others. Such effects are possibly associated with the presence of terpenoids, alkaloids, coumarins, flavonoids phenolic compounds, among others. Thus, the objective of this work was to evaluate in the literature the studies on the phytochemical, ethnopharmacological and biotechnological applications of this genus, from 1998 to 2017. Among the sixty-one studies reported in this review, ten species are popularly utilized to pharmacological and/or biotechnological applications. Cnidoscolus aconitifolius and Cnidoscolus chayamansa are the most cited species, which were also supported by either animal or cellular investigations indicating some beneficial pharmacological actions like antioxidant, anti-inflammatory and potential cytotoxic activity. The plant parts of this genus under study are important as sources for the isolation and identification of bioactive molecules with biotechnological applications, among the many diseases treated with this phytotherapy. Given these verdicts, ethnopharmacological approaches are significant systematic tools in the determination of plant species that exhibit medicinal and nutritional purposes. The results presented here should further stimulate the development of validation studies to ensure the safe and effective use of these plant species., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Recent advances in next-generation sequencing have revealed that majority of the plant genome is transcribed into long noncoding RNA (lncRNA). Many lncRNAs function by interacting with proteins and forming regulatory complexes. RNA-protein interactions are vital in controlling core cellular processes like transcription and translation. Therefore, identifying proteins that interact with lncRNAs is the first step to deciphering lncRNA functions. Here, we describe an RNA-protein pull-down assay, which enables the identification of proteins that interact with an RNA under study. As an example, we describe pull-down of proteins interacting with lncRNA ELENA1, which promotes the enrichment of MED19a on PR1 promoter to activate PR1 expression.

Context: Eurycoma longifolia Jack (Simaroubaceae) commonly known as Tongkat Ali is one of the most important plants in Malaysia. The plant extracts (particularly roots) are widely used for the treatment of cough and fever besides having antimalarial, antidiabetic, anticancer and aphrodisiac activities., Objectives: This study assesses the extent of adulteration of E. longifolia herbal medicinal products (HMPs) using DNA barcoding validated by HPLC analysis., Materials and Methods: Chloroplastic rbcL and nuclear ITS2 barcode regions were used in the present study. The sequences generated from E. longifolia HMPs were compared to sequences in the GenBank using MEGABLAST to verify their taxonomic identity. These results were verified by neighbor-joining tree analysis in which branches of unknown specimen are compared to the reference sequences established from this study and other retrieved from the GenBank. The HMPs were also analysed using HPLC analysis for the presence of eurycomanone bioactive marker., Results: Identification using DNA barcoding revealed that 37% of the tested HMPs were authentic while 27% were adulterated with the ITS2 barcode region proven to be the ideal marker. The validation of the authenticity using HPLC analysis showed a situation in which a species which was identified as authentic was found not to contain the expected chemical compound., Discussion and Conclusions: DNA barcoding should be used as the first screening step for testing of HMPs raw materials. However, integration of DNA barcoding with HPLC analysis will help to provide detailed knowledge about the safety and efficacy of the HMPs.

Hydroquinone (HQ) is widely used in food stuffs and is an occupational and environmental pollutant. Although the hepatotoxicity of HQ has been demonstrated both in vitro and in vivo, the prevention of HQ-induced hepatotoxicity has yet to be elucidated. In this study, we focused on the intervention effect of aqueous extracts of Flos lonicerae Japonicae (FLJ) on HQ-induced cytotoxicity. We demonstrated that HQ reduced cell viability in a concentration-dependent manner by administering 160  μ mol/L HQ for 12 h as the positive control of cytotoxicity. The aqueous FLJ extracts significantly increased cell viability and decreased LDH release, ALT, and AST in a concentration-dependent manner compared with the corresponding HQ-treated groups in hepatic L02 cells. This result indicated that aqueous FLJ extracts could protect the cytotoxicity induced by HQ. HQ increased intracellular MDA and LPO and decreased the activities of GSH, GSH-Px, and SOD in hepatic L02 cells. In addition, aqueous FLJ extracts significantly suppressed HQ-stimulated oxidative damage. Moreover, HQ promoted DNA double-strand breaks (DSBs) and the level of 8-hydroxy-2'-deoxyguanosine and apoptosis. However, aqueous FLJ extracts reversed HQ-induced DNA damage and apoptosis in a concentration-dependent manner. Overall, our results demonstrated that the toxicity of HQ was mediated by intracellular oxidative stress, which activated DNA damage and apoptosis. The findings also proved that aqueous FLJ extracts exerted protective effects against HQ-induced cytotoxicity in hepatic L02 cells.

Pancreatic cancer is a leading cause of mortality and morbidity worldwide. Due to drug resistance, and the high toxicity and adverse side effects of existing chemotherapeutic drugs, the current treatment of highly aggressive pancreatic cancer is considered inadequate. Allergen‑removed Rhus verniciflua Stokes (aRVS) has a strong antiproliferative effect in various cancer cells, and due to its low toxicity, it has emerged as an attractive candidate for cancer treatment. However, the potential use of aRVS as a treatment for pancreatic cancer is relatively unexplored. The present study examined the effects of aRVS on the invasion and migration of pancreatic cancer cells, and identified the molecular mechanisms underlying its anticancer effects. aRVS inhibited the Janus kinase/signal transducer and activator of transcription pathway in pancreatic cancer cells, and decreased the protein expression of mucin 4. In addition, it inhibited the activation of focal adhesion kinase and Src signaling, and decreased the expression of matrix metalloproteinase 9, which may reduce the migration and invasion of pancreatic cancer cells. In conclusion, the present study suggested that aRVS may be a potential treatment for aggressive pancreatic cancer.

Proteins belonging to cupin superfamily are known to have critical and diverse physiological functions. However, 7S globulins family, which is also a part of cupin superfamily, were undermined as only seed storage proteins. Structure determination of native protein - Vic_CAPAN from Capsicum annuum - was carried out, and its physiological functions were explored after purifying the protein by ammonium sulfate precipitation followed by size exclusion chromatography. The crystal structure of vicilin determined at 2.16 Å resolution revealed two monomers per asymmetric unit which are juxtaposed orthogonal with each other. Vic_CAPAN consists predominately of β-sheets that folds to form a β-barrel structure commonly called cupin fold. Each monomer of Vic_CAPAN consists of two cupin fold domains, N-terminal and C-terminal, which accommodate two different ligands. A bound ligand was identified at the C-terminal cupin fold in the site presumably conserved for metabolites in the crystal structure. The ligand was confirmed to be salicylic acid through mass spectrometric analysis. A copper-binding site was further observed near the conserved ligand-binding pocket, suggesting possible superoxide dismutase activity of Vic_CAPAN which was subsequently confirmed biochemically. Vicilins from other sources did not exhibit this activity indicating functional specificity of Vic_CAPAN. Discovery of bound salicylic acid, which is a known regulator of antioxidant pathway, and revelation of superoxide dismutase activity suggest that Vic_CAPAN has an important role during oxidative stress. As salicylic acid changes the redox state of cell, it may act as a downstream signal for various pathways involved in plant biotic and abiotic stress rescue., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The authentication and traceability of spices is a major concern for industrials and consumers. We focused on species from Zanthoxylum genera which are used for many different applications by local populations and also for trading as spices (dried pericarps or whole fruits). In this case, literature gives contradictory data about botanical names, and commercial labelling is often confusing. We studied commercial fruits pericarps extracts obtained by supercritical CO 2 and analyzed them by GC/MS. The very complex volatile and semi volatile fractions composition of each extract is described. The barcoding method including molecular biology and phylogenetic analyses was also developed in order to check the commercial botanical identification of the raw material. This is a robust method to identify species in berries samples. We used one genetic marker to identify two Rutaceae clusters, including several species of Zanthoxylum genus. These results indicate that Fagara and Zanthoxylum groups could be considered as two different genera. Combination of chemical analysis and DNA analysis provides an original approach to increase chemical and botanical Zanthoxylum genus knowledge., (© 2018 Wiley-VHCA AG, Zurich, Switzerland.)

Most clinical studies of Cannabis today focus on the contents of two phytocannabinoids: (-)-Δ 9 -trans-tetrahydrocannabinol (Δ 9 -THC) and cannabidiol (CBD), regardless of the fact that the plant contains over 100 additional phytocannabinoids whose therapeutic effects and interplay have not yet been fully elucidated. This narrow view of a complex Cannabis plant is insufficient to comprehend the medicinal and pharmacological effects of the whole plant. In this study we suggest a new ESI-LC/MS/MS approach to identify phytocannabinoids from 10 different subclasses, and comprehensively profile the identified compounds in diverse medical Cannabis plants. Overall, 94 phytocannabinoids were identified and used for profiling 36 of the most commonly used Cannabis plants prescribed to patients in Israel. In order to demonstrate the importance of comprehensive phytocannabinoid analysis before and throughout medical Cannabis clinical trials, treatments, or experiments, we evaluated the anticonvulsant effects of several equally high-CBD Cannabis extracts (50% w/w). We found that despite the similarity in CBD contents, not all Cannabis extracts produced the same effects. This study's approach for phytocannabinoid profiling can enable researchers and physicians to analyze the effects of specific Cannabis compositions and is therefore critical when performing biological, medical and pharmacological-based research using Cannabis.

The Ranunculus species are poorly known as medicinal plants. They have potential toxicity given by the ranunculin and its enzymatic degradation compounds: protoanemonin and anemonin. This paper aims to evaluate the anemonin content of four species: R. bulbosus, R. ficaria, R. sardous and R. sceleratus. The evaluation was performed by TLC and HPLC. There were evaluated two types of extracts hydroalcoholic (HA) and glycerol-ethanol (GE). The most concentrated extract in anemonin was found to be the R. sardous aerial part HA extract: 2.66 mg/ml. The lowest anemonin content is in R. sceleratus: 0.13-0.19 mg/ml. In R. bulbosus aerial part the anemonin content is less than the used HPLC method detection limits (7.68 mg/ml). In all cases the GE extracts are less concentrated in anemonin, being more safely for human administration.

Annatto (Bixa orellana L.) is a tropical American crop, commercially valuable due to its application in the food and cosmetics industries as a natural dye. The wild ancestor of cultivated annatto is B. orellana var. urucurana. Although never cultivated, this variety occurs in open forests and anthropogenic landscapes, and is always associated with riparian environments. In this study, we evaluated the genetic diversity and structure of B. orellana var. urucurana populations in Brazilian Amazonia using 16 microsatellite loci. We used Ecological Niche Modeling (ENM) to characterize the potential geographical range of this variety in northern South America. We analyzed 170 samples from 10 municipalities in the states of Rondônia, Pará and Roraima. A total of 194 alleles was observed, with an average of 12.1 alleles per locus. Higher levels of expected (HE) than observed (HO) heterozygosities were found for all populations. Bayesian analysis, Neighbor-Joining dendrograms and PCAs suggest the existence of three strongly structured groups of populations. A strong and positive correlation between genetic and geographic distances was found, suggesting that genetic differentiation might be caused by geographic isolation. From species distribution modelling, we detected that South Rondônia, Madre di Dios River basin, Llanos de Mojos, Llanos de Orinoco and eastern Ecuador are highly suitable areas for wild annatto to occur, providing additional targets for future exploration and conservation. Climatic adaptation analyses revealed strong differentiation among populations, suggesting that precipitation plays a key role in wild annatto's current and potential distribution patterns., Competing Interests: The authors have declared that no competing interests exist.

Low temperature is a major limiting factor for plant growth and development. Dehydrin proteins are generally induced in response to low-temperature stress. In previous research, a full-length dehydrin gene, PicW2, was isolated from Picea wilsonii and its expression was associated with hardiness to cold. In order to gain insight into the mechanism of low-temperature tolerance by studying its three-dimensional crystal structure, prokaryotically expressed PicW2 dehydrin protein was purified using chitosan-affinity chromatography and gel filtration, and crystallized using the vapour-diffusion method. The crystal grew in a condition consisting of 0.1 M HEPES pH 8.0, 25%(w/v) PEG 3350 using 4 mg ml -1 protein solution at 289 K. X-ray diffraction data were collected from a crystal at 100 K to 2.82 Å resolution. The crystal belonged to space group C121, with unit-cell parameters a = 121.55, b = 33.26, c = 73.39 Å, α = γ = 90.00, β = 109.01°. The asymmetric unit contained one molecule of the protein, with a corresponding Matthews coefficient of 2.87 Å 3  Da -1 and a solvent content of 57.20%. Owing to a lack of structures of homologous dehydrin proteins, molecular-replacement trials failed. Data collection for selenium derivatization of PicW2 and crystal structure determination is currently in progress.

Species adulteration in herbal products (HPs) exposes consumers to health risks. Chemical and morphological methods have their own deficiencies when dealing with the detection of species containing the same active compounds in HPs. In this study, we developed a rapid identification method using the recombinase polymerase amplification (RPA) assay to detect two species, Ginkgo biloba and Sophora japonica (as adulteration), in Ginkgo biloba HPs. Among 36 Ginkgo biloba HP samples, 34 were found to have Ginkgo biloba sequences, and 9 were found to have Sophora japonica sequences. During the authentication process, the RPA-LFS assay showed a higher specificity, sensitivity and efficiency than PCR-based methods. We initially applied the RPA-LSF technique to detect plant species in HPs, demonstrating that this assay can be developed into an efficient tool for the rapid on-site authentication of plant species in Ginkgo biloba HPs.

Background: Nowadays herbal products used in traditional medicine are sold in processed forms and thus morphological authentication is almost impossible. With herbal industry rapidly growing size, consumer safety becomes an important issue that requires special attention. Identification of herbal species in the products is therefore needed., Methods: Sequences from the selected regions (matK, rbcL, trnL and ITS1) were retrieved and analysed. Then the most suitable barcode was assessed for discrimination of T. crispa from closely related species by HRM analysis and used in authentication of commercial products., Results: The ITS1 barcode was found to be the suitable primer as melting data from the HRM assay proved to be capable of distinguishing T. crispa from its related species. The developed protocol was then employed to authenticate medicinal products in powdered form. HRM analysis of all tested samples here revealed that five out of eight products contained not only the indicated species T. crispa but also other Tinospora, that have a high level of morphological similarity., Conclusion: Misrepresentation, poor packaging and inappropriate labeling of the tested medicinal herbal products are thought to be the reason of the results here. Using Bar-HRM with the ITS marker lead to success in authenticating the tested herbal products.

Plants, fungi, and bacteria synthesize the aromatic amino acids: l-phenylalanine, l-tyrosine, and l-tryptophan. Chorismate mutase catalyzes the branch point reaction of phenylalanine and tyrosine biosynthesis to generate prephenate. In Arabidopsis thaliana , there are two plastid-localized chorismate mutases that are allosterically regulated (AtCM1 and AtCM3) and one cytosolic isoform (AtCM2) that is unregulated. Previous analysis of plant chorismate mutases suggested that the enzymes from early plants (i.e. bryophytes/moss, lycophytes, and basal angiosperms) formed a clade distinct from the isoforms found in flowering plants; however, no biochemical information on these enzymes is available. To understand the evolution of allosteric regulation in plant chorismate mutases, we analyzed a basal lineage of plant enzymes homologous to AtCM1 based on sequence similarity. The chorismate mutases from the moss/bryophyte Physcomitrella patens (PpCM1 and PpCM2), the lycophyte Selaginella moellendorffii (SmCM), and the basal angiosperm Amborella trichopoda (AmtCM1 and AmtCM2) were characterized biochemically. Tryptophan was a positive effector for each of the five enzymes examined. Histidine was a weak positive effector for PpCM1 and AmtCM1. Neither tyrosine nor phenylalanine altered the activity of SmCM; however, tyrosine was a negative regulator of the other four enzymes. Phenylalanine down-regulates both moss enzymes and AmtCM2. The 2.0 Å X-ray crystal structure of PpCM1 in complex with the tryptophan identified the allosteric effector site and reveals structural differences between the R- (more active) and T-state (less active) forms of plant chorismate mutases. Molecular insight into the basal plant chorismate mutases guides our understanding of the evolution of allosteric regulation in these enzymes., (© 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Curcumae Longae Rhizoma (Curcuma longa L.) is an important traditional Chinese medicine with multiple beneficial effects. To elucidate the genetic and chemical differences among Curcumae Longae Rhizoma samples, three DNA barcoding markers (rbcL, matK, and ITS-LSU D1/D3) and HPLC fingerprinting were employed in this study. The discriminatory power of rbcL and matK was low, as they only detected one sequence type that showed 100% similarity with more than 20 congeneric species in the Barcode of Life Data Systems (BOLD) database. In contrast, ITS-LSU D1/D3 showed sufficient discriminatory power to precisely identify all of the market samples as C. longa L. in a BLAST search as well as differentiate each sample based on 2-10 ITS-LSU D1/D3 haplotypes with intragenomic variability (mean p-distance: 0.7%, range: 0-2.6%; mean number of differences: 9.6 sites, range: 0-38 sites). HPLC fingerprinting of 13 commercial samples showed a similarity that ranged from 0.769 to 0.996, indicating that the sample quality varied. A cluster analysis based on 5 common peak areas from the HPLC chromatogram resulted in two groups. Group I included 9 samples with a relatively high chemical content, and group II contained 4 samples with a low chemical content. A Mantel test revealed a low correlation (r=0.1721, p=0.047) between genetic and chemical differences. Our findings suggest that the integrated approach of ITS-LSU D1/D3 DNA barcoding and HPLC fingerprinting provides a comprehensive, precise, and convenient method to clarify the genetic and chemical differences in Curcumae Longae Rhizoma.

Poppy seeds (Papaver somniferum L.) belong to tasty food ingredients however, they should be considered also as valuable source of biologically active compounds. Content of selected metabolites, antioxidant and proteinase inhibitory activities were analyzed in vitro in extracts from seeds of fifteen poppy genotypes. Considerable variation in all parameters was detected within the set of analyzed poppy genotypes. The genotype Major expressed the highest antioxidant activity determined by all four methodological approaches (DPPH, ABTS, FRAP, RP). The genotype MS 423 exhibited the highest inhibitory activities against trypsin, thrombin and collagenase. Very specific position among all had the genotype Redy. Its grain extract reached significantly high levels in 9 out of 14 measured parameters (TPC, TFC, TTC, TAC, FRAP, RP, inhibitory activities against trypsin, thrombin, collagenase). Edible grains of poppy are valuable source of natural compounds which may be beneficial in pathological states associated with oxidative stress or increased proteinase activities., (© 2017 Wiley-VHCA AG, Zurich, Switzerland.)

CRISPR/Cas9, emerged as an efficient and powerful gene editing technology, has become the mainstream genome editing technology. Constructing mutants using CRISPR/Cas9 system is of great significance to the functional study and breeding application of useful genes. As the basis of the technology, a method for identification of mutation with efficiency and lower cost is needed. In this report, we studied the factors influencing mutation detected by CEL Ⅰ crude extracts, such as the amount of protein, enzyme incubation time, PCR buffers. Under the optimized conditions, we can integrate the mutation detection steps into one-tube reaction. We used this system to examine the mutation types and frequency of rice stn1 mediated by CRISPR/Cas9. We also used this method to identify different mutation types including homozygous, heterozygous and bi-allelic mutations. The accuracy of this method reached 100% verified by sequencing. Altogether, our results showed that using CELⅠ crude extracts was an efficient and low cost method for identification of CRISPR/Cas9 mediated mutation.

We investigate the utility and scalability of new read cloud technologies to improve the draft genome assemblies of the colossal, and largely repetitive, genomes of conifers. Synthetic long read technologies have existed in various forms as a means of reducing complexity and resolving repeats since the outset of genome assembly. Recently, technologies that combine subhaploid pools of high molecular weight DNA with barcoding on a massive scale have brought new efficiencies to sample preparation and data generation. When combined with inexpensive light shotgun sequencing, the resulting data can be used to scaffold large genomes. The protocol is efficient enough to consider routinely for even the largest genomes. Conifers represent the largest reference genome projects executed to date. The largest of these is that of the conifer Pinus lambertiana (sugar pine), with a genome size of 31 billion bp. In this paper, we report on the molecular and computational protocols for scaffolding the P. lambertiana genome using the library technology from 10× Genomics. At 247,000 bp, the NG50 of the existing reference sequence is the highest scaffold contiguity among the currently published conifer assemblies; this new assembly's NG50 is 1.94 million bp, an eightfold increase., (Copyright © 2017 Crepeau et al.)

Many herbal products have a long history of use, but there are increasing concerns over product efficacy, safety and quality in the wake of recent cases exposing discrepancies between labeling and constituents. When it comes to St. John's wort (Hypericum perforatum L.) herbal products, there is limited oversight, frequent off-label use and insufficient monitoring of adverse drug reactions. In this study, we use amplicon metabarcoding (AMB) to authenticate 78 H. perforatum herbal products and evaluate its ability to detect substitution compared to standard methods using thin-layer chromatography (TLC) and high performance liquid chromatography coupled with mass spectrometry (HPLC-MS). Hypericum perforatum was detected in 68% of the products using AMB. Furthermore, AMB detected incongruence between constituent species and those listed on the label in all products. Neither TLC nor HPLC-MS could be used to unambiguously identify H. perforatum. They are accurate methods for authenticating presence of the target compounds, but have limited efficiency in detecting infrageneric substitution and do not yield any information on other plant ingredients in the products. Random post-marketing AMB of herbal products by regulatory agencies could raise awareness among consumers of substitution and would provide an incentive to manufacturers to increase quality control from raw ingredients to commercialized products.