Phylogeny, Genetic Diversity and Population Structure of Fritillaria cirrhosa and Its Relatives Based on Chloroplast Genome Data.

Fritillaria cirrhosa and its relatives have been utilized in traditional Chinese medicine for many years and are under priority protection in China. Despite their medicinal and protective value, research on their phylogeny, genetic diversity, and divergence remains limited. Here, we investigate the chloroplast genome variation architecture of 46 samples of F. cirrhosa and its relatives collected from various regions, encompassing the majority of wild populations across diverse geographical areas. The results indicate abundant variations in 46 accessions including 1659 single-nucleotide polymorphisms and 440 indels. Six variable markers (psbJ, ndhD, ycf1, ndhG, trnT-trnL, and rpl32-trnL) were identified. Phylogenetic and network analysis, population structure analysis, and principal component analysis showed that the 46 accessions formed five clades with significant divergence, which were related to their geographical distribution. The regions spanning from the southern Hengduan Mountains to the Qinghai-Tibet Plateau exhibited the highest levels of genetic diversity. F. cirrhosa and its relatives may have suffered a genetic bottleneck and have a relatively low genetic diversity level. Moreover, geographical barriers and discrete patches may have accelerated population divergence. The study offers novel perspectives on the phylogeny, genetic diversity, and population structure of F. cirrhosa and its relatives, information that can inform conservation and utilization strategies in the future.

[Identification of HSP70 gene family members in Fritillaria cirrhosa and expression analysis in different tissues under high temperature stress].

The heat shock protein 70 family contains the stress proteins ubiquitous in plants. These proteins are involved in the responses to different abiotic stress conditions and have highly conserved gene sequences. However, little is known about the molecular mechanisms of Fritillaria cirrhosa in response to high-temperature stress. Here, 26 HSP70s, FcHSP70-1 to FcHSP70-26, were identified from the transcriptome data of root, bulb, stem, leaf, and fruit samples of F. cirrhosa. The proteins encoded by FcHSP70s had the lengths ranging from 560 aa to 944 aa, with the molecular weight of 61.64-100.01 kDa and the theoretical isoelectric point between 5.00 and 6.59. The secondary structural elements of HSP70s were mainly random coils and α-helixes. Subcellular localization prediction revealed that FcHSP70s were distributed in mitochondria, chloroplasts, nuclei, endoplasmic reticulum, and cytoplasm. The phylogenetic tree showed that 7 members of the HSP70 family belonged to the Dnak subfamily and 19 members belonged to the HSP110/SSE subfamily. In addition, the qRT-PCR results showed that the expression of FcHSP70-5, FcHSP70-8, FcHSP70-17, FcHSP70-18, and FcHSP70-23 in F. cirrhosa was significantly up-regulated at 35 ℃, which indicated that these genes might play a role in the response to high temperature stress. In addition, compared with other tissues, stems and leaves were sensitive to high temperature stress, with the expression of 18 genes up-regulated by 18.18 and 8.03 folds on average, respectively. These findings provide valuable information about the molecular mechanism of HSP70s of F. cirrhosa in response to high temperature stress.

Physiological, cytological and multi-omics analysis revealed the molecular response of Fritillaria cirrhosa to Cd toxicity in Qinghai-Tibet Plateau.

Fritillaria cirrhosa, an endangered plant endemic to plateau regions, faces escalating cadmium (Cd) stress due to pollution in the Qinghai-Tibet Plateau. This study employed physiological, cytological, and multi-omics techniques to investigate the toxic effects of Cd stress and detoxification mechanisms of F. cirrhosa. The results demonstrated that Cd caused severe damage to cell membranes and organelles, leading to significant oxidative damage and reducing photosynthesis, alkaloid and nucleoside contents, and biomass. Cd application increased cell wall thickness by 167.89% in leaves and 445.78% in bulbs, leading to weight percentage of Cd increases of 76.00% and 257.14%, respectively. PER, CESA, PME, and SUS, genes responsible for cell wall thickening, were significantly upregulated. Additionally, the levels of metabolites participating in the scavenging of reactive oxygen species, including oxidized glutathione, D-proline, L-citrulline, and putrescine, were significantly increased under Cd stress. Combined multi-omics analyses revealed that glutathione metabolism and cell wall biosynthesis pathways jointly constituted the detoxification mechanism of F. cirrhosa in response to Cd stress. This study provides a theoretical basis for further screening of new cultivars for Cd tolerance and developing appropriate cultivation strategies to alleviate Cd toxicity.

An experimental study on the visual identification of Fritillaria ussuriensis based on LAMP and nucleic acid colloidal gold technique.

Fritillaria ussuriensis Maxim is one of the traditional Chinese valuable herbs, which is the dried bulb of Fritillaria, a plant of the lily family. The identification of authenticity about F. ussuriensis is still technically challenging. In this study, visual identification was performed by ring-mediated isothermal amplification and nucleic acid colloidal gold techniques. Firstly, multiple sequence comparative analysis was performed by DNAMAN to find the differential sites of F. ussuriensis and its mixed pseudo-products, and the specific identification primers of F. ussuriensis were designed. Genomic DNA was extracted by the modified CTAB method, and the reaction system and reaction conditions were optimized to construct LAMP for the visual detection of F. ussuriensis, meanwhile, the genuine product was cloned and the extracted plasmid was sequenced. The specificity and sensitivity were detected, and also verified by nucleic acid colloidal gold method, and 20 commercially available samples were tested. The extracted DNA met the requirements of the experiment, and the genuine F. ussuriensis PCR product titrated on a test strip showed two bands on the T and C lines, while the counterfeit and negative control showed only one band on the C line, which matched the LAMP results. The specificity was 100 %, and the sensitivity of LAMP assay was up to 0.01 ng µL-1, while that of colloidal gold assay was 0.1 ng µL-1, thus the LAMP assay had high sensitivity. 14 out of 20 commercially available samples of F. ussuriensis were qualified, and 6 were unqualified, and the results of the two methods of identification were consistent. In this study, the combined detection method of LAMP and colloidal gold for nucleic acid was established to be specific, rapid, precise and visualized, which can provide a new technical idea for the detection of F. ussuriensis.

Biomod2 modeling for predicting the potential ecological distribution of three Fritillaria species under climate change.

The Fritillaria species ranked as a well-known traditional medicine in China and has become rare due to excessive harvesting. To find reasonable strategy for conservation and cultivation, identification of new ecological distribution of Fritillaria species together with prediction of those responses to climate change are necessary. In terms of current occurrence records and bioclimatic variables, the suitable habitats for Fritillaria delavayi, Fritillaria taipaiensis, and Fritillaria wabuensis were predicted. In comparison with Maxent and GARP, Biomod2 obtained the best AUC, KAPPA and TSS values of larger than 0.926 and was chosen to construct model. Temperature seasonality was indicated to put the greatest influence on Fritillaria taipaiensis and Fritillaria wabuensis, while isothermality was of most importance for Fritillaria delavayi. The current suitable areas for three Fritillaria species were distributed in south-west China, accounting for approximately 17.72%, 23.06% and 20.60% of China's total area, respectively. During 2021-2100 period, the suitable habitats of F. delavayi and F. wabuensis reached the maximum under SSP585 scenario, while that of F. taipaiensis reached the maximum under SSP126 scenario. The high niche overlap among three Fritillaria species showed correlation with the chemical composition (P ≤ 0.05), while no correlation was observed between niche overlap and DNA barcodes, indicating that spatial distribution had a major influence on chemical composition in the Fritillaria species. Finally, the acquisition of species-specific habitats would contribute to decrease in habitat competition, and future conservation and cultivation of Fritillaria species.

Isolation and identification of two pathogens causing leaf spot of Fritillaria taipaiensis P. Y. Li. in China.

Fritillaria taipaiensis P. Y. Li is one of the biological sources for Fritillariae Cirrhosae Bulbus. Its bulbs are widely used for treating respiratory diseases such as pneumonia, bronchitis and influenza. Cultivated F. taipaiensis suffers from many diseases during its growing season. Leaf spot is a destructive disease that is increasingly affecting F. taipaiensis and can cause an incidence of up to 30% in severe cases. Leaf spot inhibits the growth of F. taipaiensis by causing disease spots on the surface of leaves. In severe cases, these spots can result in leaf desiccation and blackspot formation at the lesion site, leading to a decrease in photosynthesis. Leaf spot has shown little benefit, and it can even result in a reduced yield of bulbs and the death of plants. According to previous studies, Alternaria alternata has been identified as the pathogen of leaf spot in many medicinal plants, but the main pathogens of the leaf spot of F. taipeiensis remains uncertain. In this paper, five isolates from diseased leaves of F. taipaiensis were isolated and purified and the pathogenicity test showed that isolates B-5 and B-7 induced leaf spot symptoms on healthy F. taipaiensis leaves. Integrating multiple phylogenetic analyses of rDNA using Internal transcribed spacer region (ITS), Beta-tubulin (TUB2), RNA polymerase II second largest subunit (RPB2) and Translation elongation factor 1-alpha (TEF1-a) primers, strain B-5 and strain B-7 were eventually identified as Didymella segeticola and A. alternata. This is also the first report on the pathogens that cause leaf spot in F. taipaiensis in China.

Chromosome Fissions and Fusions Act as Barriers to Gene Flow between Brenthis Fritillary Butterflies.

Chromosome rearrangements are thought to promote reproductive isolation between incipient species. However, it is unclear how often, and under what conditions, fission and fusion rearrangements act as barriers to gene flow. Here we investigate speciation between two largely sympatric fritillary butterflies, Brenthis daphne and Brenthis ino. We use a composite likelihood approach to infer the demographic history of these species from whole-genome sequence data. We then compare chromosome-level genome assemblies of individuals from each species and identify a total of nine chromosome fissions and fusions. Finally, we fit a demographic model where effective population sizes and effective migration rate vary across the genome, allowing us to quantify the effects of chromosome rearrangements on reproductive isolation. We show that chromosomes involved in rearrangements experienced less effective migration since the onset of species divergence and that genomic regions near rearrangement points have a further reduction in effective migration rate. Our results suggest that the evolution of multiple rearrangements in the B. daphne and B. ino populations, including alternative fusions of the same chromosomes, have resulted in a reduction in gene flow. Although fission and fusion of chromosomes are unlikely to be the only processes that have led to speciation between these butterflies, this study shows that these rearrangements can directly promote reproductive isolation and may be involved in speciation when karyotypes evolve quickly.

Morphological variability of wild-growing crown imperial (Fritillaria imperialis L.) germplasm in central region of Iran-implications for in-situ conservation initiatives.

BACKGROUND: Crown imperial (Fritillaria imperialis L.) is a threatened bulbous plant which has great ornamental and medicinal values and importance. In the present study, a total of 100 specimens of wild-growing F. imperialis from 10 natural areas of Markazi province, Iran, representing one of the main centers of genetic diversity of this species, were evaluated using 37 phenotypic attributes during April 2021. RESULTS: High level of genetic variation within populations (75%) and low levels of genetic variation among populations (25%) was revealed. The highest coefficient of variation (CV) was found in leaf trichome (82.00%) and then margin of crown leaves (80.44%). In addition, flower color (CV = 50.86%), flower number (CV = 44.61%), peduncle diameter (CV = 33.44%), and plant length (CV = 32.55%)-all important from an ornamental point of view- showed relatively high CV values. The CV was the lowest for flower shape, filament color, bulb shape, bulblet number, and floral scent. Ward cluster analysis identified two main clusters, containing 14 and 86 specimens, respectively. The first group consisted mainly of specimens from the adjacent Shahbaz and Rasvand populations. According to the principal component analysis (PCA), the first six components of data accounted for 88.36% of total variance. The Shahbaz-1, Shahbaz-2, Shahbaz-6, Shahbaz-7, Shahbaz-9, and Bolagh-8 specimens showed the highest variation and were separated from others, which they can be used further in breeding programs, while Sarchal-2, Bolagh-3, and Chepeqli-4 specimens showed the lowest variability. Moreover, the studied populations were clustered into four distinct groups, each including populations that were geographically close to one another. CONCLUSIONS: Although the examined specimens revealed high genetic diversity herein, the results indicated that wild-growing populations of F. imperialis are still at risk suffering from overcollection in the most of studied areas, especially in Deh-Sad and Tureh.

Transcriptome Analysis of CYP450 Family Members in Fritillaria cirrhosa D. Don and Profiling of Key CYP450s Related to Isosteroidal Alkaloid Biosynthesis.

Fritillaria cirrhosa D. Don (known as Chuan-Bei-Mu in Chinese) can synthesize isosteroidal alkaloids (ISA) with excellent medicinal value, and its bulb has become an indispensable ingredient in many patented drugs. Members of the cytochrome P450 (CYP450) gene superfamily have been shown to play essential roles in regulating steroidal alkaloids biosynthesis. However, little information is available on the P450s in F. cirrhosa. Here, we performed full-length transcriptome analysis and discovered 48 CYP450 genes belonging to 10 clans, 25 families, and 46 subfamilies. By combining phylogenetic trees, gene expression, and key F. cirrhosa ISA content analysis, we presumably identify seven FcCYP candidate genes, which may be hydroxylases active at the C-22, C-23, or C-26 positions in the late stages of ISA biosynthesis. The transcript expression levels of seven FcCYP candidate genes were positively correlated with the accumulation of three major alkaloids in bulbs of different ages. These data suggest that the candidate genes are most likely to be associated with ISA biosynthesis. Finally, the subcellular localization prediction of FcCYPs and transient expression analysis within Nicotiana benthamiana showed that the FcCYPs were mainly localized in the chloroplast. This study presents a systematic analysis of the CYP450 gene family in F. cirrhosa and provides a foundation for further functional characterization of the CYPs involved in ISA biosynthesis.

Primary Investigation of Phenotypic Plasticity in Fritillaria cirrhosa Based on Metabolome and Transcriptome Analyses.

Phenotypic plasticity refers to the adaptability of an organism to a heterogeneous environment. In this study, the differential gene expression and compositional changes in Fritillaria cirrhosa during phenotypic plasticity were evaluated using transcriptomic and metabolomic analyses. The annotation profiles of 1696 differentially expressed genes from the transcriptome between abnormal and normal phenotypes revealed that the main annotation pathways were related to the biosynthesis of amino acids, ABC transporters, and plant-pathogen interactions. According to the metabolome, the abnormal phenotype had 36 upregulated amino acids, including tryptophan, proline, and valine, which had a 3.77-fold higher relative content than the normal phenotype. However, saccharides and vitamins were found to be deficient in the abnormal phenotypes. The combination profiles demonstrated that phenotypic plasticity may be an effective strategy for overcoming potential stress via the accumulation of amino acids and regulation of the corresponding genes and transcription factors. In conclusion, a pathogen attack on F. cirrhosa may promote the synthesis of numerous amino acids and transport them into the bulbs through ABC transporters, which may further result in phenotypic variation. Our results provide new insights into the potential mechanism of phenotypic changes.

Integrative analysis of the steroidal alkaloids distribution and biosynthesis of bulbs Fritillariae Cirrhosae through metabolome and transcriptome analyses.

BACKGROUND: Bulbus Fritillariae Cirrhosae (BFC) is an endangered high-altitude medicine and food homology plant with anti-tumor, anti-asthmatic, and antitussive activities as it contains a variety of active ingredients, especially steroidal alkaloids. Bulbus Fritillariae Thunbergia (BFT) is another species of Fritillaria that grows at lower altitude areas. Production of plant-derived active ingredients through a synthetic biology strategy is one of the current hot topics in biological research, which requires a complete understanding of the related molecular pathways. Our knowledge of the steroidal alkaloid biosynthesis in Fritillaria species is still very limited. RESULTS: To promote our understanding of these pathways, we performed non-target metabolomics and transcriptome analysis of BFC and BFT. Metabolomics analysis identified 1288 metabolites in BFC and BFT in total. Steroidal alkaloids, including the proposed active ingredients of Fritillaria species peimine, peimisine, peiminine, etc., were the most abundant alkaloids detected. Our metabolomics data also showed that the contents of the majority of the steroidal alkaloids in BFC were higher than in BFT. Further, our comparative transcriptome analyses between BFC and BFT identified differentially expressed gene sets among these species, which are potentially involved in the alkaloids biosynthesis of BFC. CONCLUSION: These findings promote our understanding of the mechanism of steroidal alkaloids biosynthesis in Fritillaria species.

The genome sequence of the lesser marbled fritillary, Brenthis ino, and evidence for a segregating neo-Z chromosome.

The lesser marbled fritillary, Brenthis ino (Rottemburg, 1775), is a species of Palearctic butterfly. Male Brenthis ino individuals have been reported to have between 12 and 14 pairs of chromosomes, a much-reduced chromosome number than is typical in butterflies. Here, we present a chromosome-level genome assembly for Brenthis ino, as well as gene and transposable element annotations. The assembly is 411.8 Mb in length with a contig N50 of 9.6 Mb and a scaffold N50 of 29.5 Mb. We also show evidence that the male individual from which we generated HiC data was heterozygous for a neo-Z chromosome, consistent with inheriting 14 chromosomes from one parent and 13 from the other. This genome assembly will be a valuable resource for studying chromosome evolution in Lepidoptera, as well as for comparative and population genomics more generally.

Improved chromosome-level genome assembly of the Glanville fritillary butterfly (Melitaea cinxia) integrating Pacific Biosciences long reads and a high-density linkage map.

BACKGROUND: The Glanville fritillary (Melitaea cinxia) butterfly is a model system for metapopulation dynamics research in fragmented landscapes. Here, we provide a chromosome-level assembly of the butterfly's genome produced from Pacific Biosciences sequencing of a pool of males, combined with a linkage map from population crosses. RESULTS: The final assembly size of 484 Mb is an increase of 94 Mb on the previously published genome. Estimation of the completeness of the genome with BUSCO indicates that the genome contains 92-94% of the BUSCO genes in complete and single copies. We predicted 14,810 genes using the MAKER pipeline and manually curated 1,232 of these gene models. CONCLUSIONS: The genome and its annotated gene models are a valuable resource for future comparative genomics, molecular biology, transcriptome, and genetics studies on this species.

Identification and profiling of conserved microRNAs in different developmental stages of crown imperial (Fritillaria imperialis L.) using high-throughput sequencing.

BACKGROUND: Novel strategies for improvement of ornamental plants and their properties relay on miRNA control of differential plant gene expression modulation. Still, in response to the same abiotic stresses, some conserved miRNA families show different expression patterns in different plant species. In parallel, the use of deep sequencing technologies reveals new levels of complexity of regulatory networks in plants through identification of new miRNAs. METHODS AND RESULTS: Fritillaria imperialis plants were collected from their natural habitats in Koohrang, Chaharmahal va Bakhtiari, Iran. Several tissues including stamen, pistil, petal, sepal, leaf, stem, bulb and fruit were collected during three developmental stages (stem elongation, flower development and seed head stages). Using RNAseq and qRT-PCR approach, this research revealed 21 conserved miRNAs, matching 15 miRNA families, in Fritilaria imperialis. CONCLUSIONS: The expression of seven conserved miRNAs (Fim-miR156b, Fim-miR159, Fim-miR166a-5p, Fim-miR169d-5p, Fim-miR171c, Fim-miR393 and Fim-miR396e-3p) was further investigated in different tissues and three developmental stages, suggesting different roles for these miRNAs during growth and development of crown imperial. Gained knowledge from this research can open the door to find efficient ways to secure crown imperial survival, preservation and utilization and if proven useful may be applied in other plant species as well.

Genome-wide transcriptional analysis unveils the molecular basis of organ-specific expression of isosteroidal alkaloids biosynthesis in critically endangered Fritillaria roylei Hook.

Fritillaria roylei Hook. is a critically endangered high altitude Himalayan medicinal plant species with rich source of pharmaceutically active structurally diverse steroidal alkaloids. Nevertheless, except few marker compounds, the chemistry of the plant remains unexplored. Therefore, in the current study, transcriptome sequencing efforts were made to elucidate isosteroidal alkaloids biosynthesis by creating first organ-specific genomic resource using bulb, stem, and leaf tissues derived from natural populations of Indian Himalayan region. Overall, 349.9 million high quality paired-end reads obtained using NovaSeq 6000 platform were assembled (de novo) into 82,848 unigenes and 31,061 isoforms. Functional annotation and organ specific differential expression (DE) analysis identified 2488 significant DE transcripts, grouped into three potential sub-clusters (sub-cluster I: 728 transcripts; sub-cluster II: 446 transcripts and Sub-cluster III: 1314 transcripts). Subsequently, pathway enrichment (GO, KEGG) and protein-protein network analysis revealed significantly higher enrichment of phenyl-propanoid and steroid backbone including terpenoid, sesquiterpenoid and triterpenoid biosynthesis in bulb. Additionally, upregulated expression of cytochrome P450, UDP-dependent Glucuronosyltransferase families and key transcription factor families (FAR1, bHLH, GRAS, C2H2, TCP and MYB) suggests 'bulb' as a primary site of MVA mediated isosteroidal alkaloids biosynthesis. The comprehensive elucidation of molecular insights in this study is a first step towards the understanding of isosteroidal alkaloid biosynthesis pathway in F. roylei. Furthermore, key genes and regulators identified here can facilitate metabolic engineering of potential bioactive compounds at industrial scale.

Simultaneous identification of Fritillariae cirrhosae bulbus and its common adulterants in one reaction by multiplex ligation-dependent probe amplification and high-resolution melting curve assay.

Fritillariae cirrhosae bulbus, a well-known and precious medicinal and edible herb in China, causes remarkable effects on swelling and relieving cough, with fewer side effects than other congeneric medicine. It has been subject to various cheaper congeneric adulteration because of its high price and limited production. In this paper, a rapid, high throughput, sensitive and efficient technique was described for simultaneous identification of F. cirrhosae bulbus and its common adulterants by employing multiplex ligation-dependent probe amplification coupled with high-resolution melting (MLPA-HRM) curve assay in their internal transcribed spacer 1 (ITS1) regions. This assay was highly sensitive with a detection limit of 0.19 ng genomic DNA, and highly specific with no cross-reaction with common adulterants. Mixed sample analysis showed as low as 10% adulteration can be detected from F. cirrhosae bulbus in one MLPA-HRM reaction. Overall, the method described in this paper is well suited for detecting adulteration in F. cirrhosae bulbus.

Comparative transcriptome analysis infers bulb derived in vitro cultures as a promising source for sipeimine biosynthesis in Fritillaria cirrhosa D. Don (Liliaceae, syn. Fritillaria roylei Hook.) - High value Himalayan medicinal herb.

Fritillaria cirrhosa D. Don (Liliaceae, syn. Fritillaria roylei Hook.) is a critically endangered medicinal herb of immense importance due to its pharmaceutical bioactive compound, especially sipeimine, used for the treatment of chronic respiratory disorders. However, the industrial demand for sipeimine solely depends on its endangered natural habitat. Therefore; there is an utmost need for its biodiversity conservation as well as for the sustainable utilization of phytochemicals. Plant cell culture and transcriptomics-based molecular bioprospection of key regulatory genes involved in sipeimine biosynthesis as such will play a crucial role in exploring the unexplored traits, that are in supply crisis or nearly in extinction stage. De novo comparative transcriptome sequencing of the bulb (in vivo), callus, and regenerated plantlets (in vitro) resulted in more than 150 million high-quality paired-end clean reads that assembled into final 31,428 transcripts. Functional annotation and unigenes classification with multiple public databases such as KEGG, Refseq, Uniprot, TAIR, GO, and COG, etc. along with chemical structures and functional biocatalytic activity analysis of different steroidal alkaloids facilitated the identification of 30 unigenes specific to sipeimine biosynthesis. Additionally, ABC transporters and TFs like bHLH, MYC, MYB, and WRKY suggests their possible role in metabolite translocation and regulation in vivo as well as in vitro tissues. Differential gene expression and quantitative analysis revealed that the MVA pathway probably the predominant route for 5C intermediate (IPP & DMAPP) biosynthesis. Further, the genes involved in the downstream biosynthesis pathway viz. SQLE, CAS1, SMT1, SMO1, SMO2, SC5DL, DHCR7, DHCR24, CYP710A, 3ß-HSD, CYP90D2, and CYP374A6 shown similar expression pattern with RNA-Seq and qRT-PCR findings. The positive correlation between higher expression of proposed biosynthetic pathway genes and relatively higher accumulation of sipeimine in differentiated naturally grown bulb tissues (in vivo), undifferentiated cells (callus), and de-differentiated tissues i.e. regenerated plantlets (in vitro) has been evident from the present study. Comprehensive genomic resources created in F. cirrhosa will provide strong evidence of bulb derived in vitro culture as an alternative promising source for steroidal alkaloids biosynthesis and metabolite upscaling through genetic and metabolic engineering.

Mechanisms of Fritillariae Thunbergii Flos in lung cancer treatment from a systems pharmacology perspective.

ETHNOPHARMACOLOGICAL RELEVANCE: Fritillariae Thunbergii Flos (FTF) included in the Chinese Pharmacopoeia (1977 Edition) is a Chinese medicinal herb traditionally used to treat bronchitis. In recent years, it has been applied in the treatment of lung cancer. However, the molecular mechanism remains largely unknown. METHODS: The screening of bioactive compounds, acquisition of drug targets, network construction, and experimental validation in vivo were combined to explored the mechanism of FTF in the treatment of lung carcinoma with regards to systems pharmacology. RESULTS: The network Lung Cancer Pathway consisted of 114 nodes (44 compounds and 70 potential targets) and 361 edges, as well as modules that included inflammatory response, angiogenesis, negative regulation of the apoptotic process, and positive regulation of cell proliferation and migration. It was examined by conducting experiments that involved the administration of ethanol-based extracts of FTF in Lewis lung carcinoma mice. The extracts exerted excellent anti-lung cancer effects in vivo by significantly inhibiting tumor proliferation, thereby extending the survival period of tumor-bearing mice. Moreover, FTF induced the downregulation of PIK3CG, Bcl-2, eNOS, VEGF, p-STAT3, and STAT3 genes in tumor-bearing mice. CONCLUSIONS: The findings of the present study verify the therapeutic effects and mechanism of FTF on lung cancer and provide a theoretical basis to support the comprehensive utilization of FTF resources.

Resistance of Fritillaria imperialis to freezing stress through gene expression, osmotic adjustment and antioxidants.

Plant survival in response to freezing stress depends on the efficient activation of tolerance mechanisms. Fritillaria imperialis exposure to freezing stress enhanced signalling molecules Ca2+ and H2O2 along with overexpression of Ca2+ signalling proteins (Ca2+ dependent protein kinases, CPK), followed by upregulation of NHX1 (Na+/H+ antiporter), LEA (late embryogenesis abundant proteins) and P5CS (1-pyrroline-5-carboxylate synthetase). Overexpression of OsCNGC6 was responsible for high accumulation Ca2+, Na+ and K+. The NHX1 gene product transported Na+ to vacuoles and increased cytosolic K+ content to re-establish ionic homeostasis under stress conditions. The reduced water potential of leaves was due to high accumulation of osmolytes and ions. No changes were observed in relative water content of leaves, which might be correlated with overexpression of the LEA gene, which protects against dehydration. High accumulation of H2O2 under freezing stress was responsible for activation of antioxidant systems involving SOD, phenols, anthocyanins, catalase and ascorbate peroxidase. Photosynthesis, suppressed in freezing-stressed plants, returned to normal levels after termination of freezing stress. Taken together, our findings suggest that Fritillaria efficiently tolerated freezing stress through induction of signalling mechanisms and overexpression of cold stress-responsive genes, and prevention of cold-induced water stress, oxidative stress and photosynthetic damage.

Comparison of the abilities of universal, super, and specific DNA barcodes to discriminate among the original species of Fritillariae cirrhosae bulbus and its adulterants.

Fritillariae cirrhosae bulbus is a famous type of traditional Chinese medicine used for cough relief and eliminating phlegm. The medicine originates from dried bulbs of five species and one variety of Fritillaria. Recently, immature bulbs from other congeneric species, such as F. ussuriensis, have been sold as adulterants of Fritillariae cirrhosae bulbus in medicine markets owing to the high price and limited availability of the genuine medicine. However, it is difficult to accurately identify the bulbs from different original species of Fritillariae cirrhosae bulbus and its adulterants based on traditional methods, although such medicines have different prices and treatment efficacies. The present study adopted DNA barcoding to identify these different species and compared the discriminatory power of super, universal, and specific barcodes in Fritillaria. The results revealed that the super-barcode had strong discriminatory power (87.5%). Among universal barcodes, matK provided the best species resolution (87.5%), followed by ITS (62.5%), rbcL (62.5%), and trnH-psbA (25%). The combination of these four universal barcodes provided the highest discriminatory power (87.5%), which was equivalent to that of the super-barcode. Two plastid genes, ycf1 and psbM-psbD, had much better discriminatory power (both 87.5%) than did other plastid barcodes, and were suggested as potential specific barcodes for identifying Fritillaria species. Phylogenetic analyses indicated that F. cirrhosa was not a "good" species that was composed of multiple lineages, which might have affected the evaluation of the discriminatory ability. This study revealed that the complete plastid genome, as super barcode, was an efficient and reliable tool for identifying the original species of Fritillariae cirrhosae bulbus and its adulterants.