Evaluation of Suitable Reference Genes for Quantitative Real-Time PCR in Various Tissues of Apocynum venetum.

Apocynum venetum L. is an economically valuable plant with tolerance to drought and salinity. Its leaves are utilized in tea production and pharmaceuticals, while the stem bark serves as a high-quality fiber material. To gain insights into the gene expression patterns of A. venetum using quantitative real-time PCR (qRT-PCR), it is crucial to identify appropriate reference genes. This study selected nine candidate genes, including α-tubulin (TUA), ß-tubulin (TUB), actin (ACT), cyclophilin (CYP), elongation factor-1α (EF-1α), the B family of regulatory subunits of protein phosphatase (PPP2R2, PPP2R3, and PPP2R5), and phosphoglycerate kinase (PGK), to determine the most appropriate reference genes in the leaf, stem, and root tissues of A. venetum. A comprehensive ranking by geNorm, NormFinder, BestKeeper, and RefFinder software and Venn diagrams was used to screen more stable reference genes in different tissues. The two most stable reference genes were CYP and TUA in leaves, PGK and PPP2R3 in stems, and TUA and EF-1α in roots, respectively. The relative expression values of the four genes involved in proline metabolism under polyethylene glycol treatment were used to validate the screened reference genes, and they exhibited highly stable expression levels. These findings represent the first set of stable reference genes for future gene expression studies in A. venetum. They significantly contribute to enhancing the accuracy and reliability of gene expression analyses in this economically important plant species.

Transcriptome analysis reveals how cadmium promotes root development and accumulates in Apocynum venetum, a promising plant for greening cadmium-contaminated soil.

Cadmium (Cd) contamination poses a substantial threat the environment, necessitating effective remediation strategies. Phytoremediation emerges as a cost-efficient and eco-friendly approach for reducing Cd levels in the soil. In this study, the suitability of A. venetum for ameliorating Cd-contaminated soils was evaluated. Mild Cd stress promoted seedling and root growth, with the root being identified as the primary tissue for Cd accumulation. The Cd content of roots ranged from 0.35 to 0.55 mg/g under treatment with 10-50 µM CdCl2·2.5 H2O, and the bioaccumulation factor ranged from 28.78 to 84.43. Transcriptome sequencing revealed 20,292 unigenes, and 7507 nonredundant differentially expressed genes (DEGs) were identified across five comparison groups. DEGs belonging to the "MAPK signaling pathway-plant," "monoterpenoid biosynthesis," and "flavonoid biosynthesis pathway" exhibited higher expression levels in roots compared to stems and leaves. In addition, cytokinin-related DEGs, ROS scavenger genes, such as P450, glutathione-S-transferase (GST), and superoxide dismutase (SOD), and the cell wall biosynthesis-related genes, CSLG and D-GRL, were also upregulated in the root tissue, suggesting that Cd promotes root development. Conversely, certain ABC transporter genes, (e.g, NRAMP5), and some vacuolar iron transporters, predominantly expressed in the roots, displayed a strong correlation with Cd content, revealing the mechanism underlying the compartmentalized storage of Cd in the roots. KEGG enrichment analysis of DEGs showed that the pathways associated with the biosynthesis of flavonoids, lignin, and some terpenoids were significantly enriched in the roots under Cd stress, underscoring the pivotal role of these pathways in Cd detoxification. Our study suggests A. venetum as a potential Cd-contaminated phytoremediation plant and provides insights into the molecular-level mechanisms of root development promotion and accumulation mechanism in response to Cd stress.

Apocynum venetum, a medicinal, economical and ecological plant: a review update.

Apocynum venetum L. is an important medicinal perennial rhizome plant with good ecological and economic value. Its leaves have many pharmacological effects such as anti-inflammatory, anti-depression, anti-anxiolytic, etc., while its fibers have the title of "king of wild fibers". Furthermore, it was suitable for the restoration of degraded saline soil in arid areas. An increasing studies have been published in the past years. A scientometric analysis was used to analyze the publications of Apocynum venetum L. to clearly review the pharmacology, fiber application of Apocynum venetum L. and the potential value with its similar species (Apocynum pictum Schrenk) to the environment.

Genome-Wide Investigation of the NAC Transcription Factor Family in Apocynum venetum Revealed Their Synergistic Roles in Abiotic Stress Response and Trehalose Metabolism.

NAC (NAM, ATAF1/2, and CUC2) transcription factors (TFs) are one of the most prominent plant-specific TF families and play essential roles in plant growth, development and adaptation to abiotic stress. Although the NAC gene family has been extensively characterized in many species, systematic analysis is still relatively lacking in Apocynum venetum (A. venetum). In this study, 74 AvNAC proteins were identified from the A. venetum genome and were classified into 16 subgroups. This classification was consistently supported by their gene structures, conserved motifs and subcellular localizations. Nucleotide substitution analysis (Ka/Ks) showed the AvNACs to be under the influence of strong purifying selection, and segmental duplication events were found to play the dominant roles in the AvNAC TF family expansion. Cis-elements analysis demonstrated that the light-, stress-, and phytohormone-responsive elements being dominant in the AvNAC promoters, and potential TFs including Dof, BBR-BPC, ERF and MIKC_MADS were visualized in the TF regulatory network. Among these AvNACs, AvNAC58 and AvNAC69 exhibited significant differential expression in response to drought and salt stresses. The protein interaction prediction further confirmed their potential roles in the trehalose metabolism pathway with respect to drought and salt resistance. This study provides a reference for further understanding the functional characteristics of NAC genes in the stress-response mechanism and development of A. venetum.

Physiological and Transcriptional Responses of Apocynum venetum to Salt Stress at the Seed Germination Stage.

Apocynum venetum is a semi-shrubby perennial herb that not only prevents saline-alkaline land degradation but also produces leaves for medicinal uses. Although physiological changes during the seed germination of A. venetum in response to salt stress have been studied, the adaptive mechanism to salt conditions is still limited. Here, the physiological and transcriptional changes during seed germination under different NaCl treatments (0-300 mmol/L) were examined. The results showed that the seed germination rate was promoted at low NaCl concentrations (0-50 mmol/L) and inhibited with increased concentrations (100-300 mmol/L); the activity of antioxidant enzymes exhibited a significant increase from 0 (CK) to 150 mmol/L NaCl and a significant decrease from 150 to 300 mmol/L; and the content of osmolytes exhibited a significant increase with increased concentrations, while the protein content peaked at 100 mmol/L NaCl and then significantly decreased. A total of 1967 differentially expressed genes (DEGs) were generated during seed germination at 300 mmol/L NaCl versus (vs.) CK, with 1487 characterized genes (1293 up-regulated, UR; 194 down-regulated, DR) classified into 11 categories, including salt stress (29), stress response (146), primary metabolism (287), cell morphogenesis (156), transcription factor (TFs, 62), bio-signaling (173), transport (144), photosynthesis and energy (125), secondary metabolism (58), polynucleotide metabolism (21), and translation (286). The relative expression levels (RELs) of selected genes directly involved in salt stress and seed germination were observed to be consistent with the changes in antioxidant enzyme activities and osmolyte contents. These findings will provide useful references to improve seed germination and reveal the adaptive mechanism of A. venetum to saline-alkaline soils.

Characterization of Boeremia exigua causing stem necrotic lesions on Luobuma in northwest China.

Luobuma (Apocynum venetum, Poacynum pictum, and P. hendersonni) are perennial herbs widely used in the textile and medical industries and ecological restoration. In the summer of 2020, reddish-brown or off-white sunken shape necrotic lesions were observed on the stems and shoots of seven Luobuma ecotypes grown in the field in Yuzhong County, Gansu province of China, which is a limiting factor that affects the growth, function and application of Luobuma. To make clear whether the new symptoms were caused by a novel pathogen, a combined research in field and greenhouse was conducted. Based on the morphological and molecular analysis results, the pathogen causing the necrotic lesions was identified as Boeremia exigua var. rhapontica. The incidence and disease index of the seven ecotypes in the field ranged from 11.49 to 33.68% and 6.63 to 23.01, respectively, from 2020 to 2021. The results showed that the disease severity gradually increased with the growing season. According to the pathogenicity analysis of the eight ecotypes in the greenhouse, the ecotypes Pp-BMK and Pp-BMH were susceptible, while ecotype Pp-BMQ was resistant to Boeremia exigua var. rhapontica infection. Thus, the present study provides a theoretical basis for preventing and controlling the stem and leaf necrotic lesions disease on Luobuma by planting resistant varieties/ecotypes. To our knowledge, this is the first report of stem necrotic lesions and leaf spots on Luobuma caused by B. exigua var. rhapontica.

Total Flavonoids Extracts of Apocynum L. from the Ili River Valley Region at Different Harvesting Periods and Bioactivity Analysis.

In the current study, the total content from two Apocynum species leaves (Apocynum venetum and Apocynum hendersonii) collected from the Ili River Valley Region were extracted, and their bioactivities were investigated. The results showed a significant variation in the total flavonoid contents in the leaf samples collected at different periods (June, July, August, and September), with the highest content in August (60.11 ± 0.38 mg RE/g DW for A. venetum and 56.56 ± 0.24 mg RE/g DW for A. hendersonii), and the lowest in June (22.36 ± 0.05 mg RE/g DW for A. venetum and 20.79 ± 0.02 mg RE/g DW for A. hendersonii). The total flavonoid content was comparably higher in A. venetum than in A. hendersonii. Leaves extracts from the two species demonstrated strong bioactivity, which positively correlated with the total flavonoid contents. The anti-oxidative activity of A. venetum was higher than that of A. hendersonii in tandem with its higher flavonoid contents; the antibacterial activity, however, was conversely opposite. Furthermore, a total of 83 flavonoid metabolites were identified in the two species based on UPLC-ESI-MS/MS, out of which 24 metabolites were differentially accumulated. The variability in these metabolites might be the reason for the different bioactivities displayed by the two species. The present study provides insight into the optimal harvest time for Apocynum species planted in the major distribution area of the Ili River Valley and the specific utilization of A. venetum and A. hendersonii.

Isoquercitrin from Apocynum venetum L. produces an anti-obesity effect on obese mice by targeting C-1-tetrahydrofolate synthase, carbonyl reductase, and glutathione S-transferase P and modification of the AMPK/SREBP-1c/FAS/CD36 signaling pathway in mice in vivo.

In the present study, mice with high-fat-diet-induced obesity were used in investigating the anti-obesity effects of an aqueous extract and isoquercitrin from Apocynum venetum L. The aqueous extract and the signal molecule isoquercitrin significantly reduced the body weight gain, food intake, water consumption, and fasting blood glucose, plasma triglyceride and total cholesterol levels of the obese mice. Furthermore, the mechanism of action of isoquercitrin was explored through RT-PCR analyses and uptake experiments of adenosine 5'-monophosphate-activated protein kinase (AMPK) and sterol regulatory-element binding protein (SREBP-1c) inhibitors and glucose. The indexes of SREBP-1c, fatty acid synthase (FAS), stearoyl-CoA desaturase-1 (SCD), and cluster of differentiation 36 (CD36) in obese mice significantly increased but returned to normal levels after the administration of isoquercitrin. Meanwhile, the anti-obesity effect of isoquercitrin was diminished by the inhibitors of AMPK and SREBP-1c. In addition, intestinal glucose uptake in normal mice was significantly inhibited after the oral administration of isoquercitrin. Moreover, 2D gel electrophoresis based proteome-wide cellular thermal shift assay (CETSA) showed that the potential target proteins of isoquercitrin were C-1-tetrahydrofolate synthase, carbonyl reductase, and glutathione S-transferase P. These results suggested that isoquercitrin produces an anti-obesity effect by targeting the above-mentioned proteins and regulating the AMPK/SREBP-1c signaling pathway and potentially prevents obesity and obesity-related metabolic disorders.

Structural characterization and immunoregulatory activity of a neutral polysaccharide from the roots of Apocynum venetum L.

The structural characteristics and immunoregulatory activities of neutral heteropolysaccharide (AVRP-N) separated from the roots of Apocynum venetum L. were extensively investigated. The results showed that the weight average molecular mass (Mw) of AVRP-N was 6.430 × 103 Da. Moreover, the backbone is composed of natural acetylated (1 â†’ 4)-ß-D-Man and (1 â†’ 5)-α-L-Ara domains. The mannan is composed of →4)-ß-D-Manp-(1→, →4)-ß-D-Glcp-(1→, and the terminal group α-D-Galp-(1→ attached to →4,6)-ß-D-Manp-(1→ at O-6. Araban is composed of →5)-α-L-Araf-(1→; the terminal group α-L-Araf-(1→attached to→2,3,5)-α-L-Araf-(1→ at O-2, O-3 and →3,5)-α-L-Araf-(1→ at O-3. In addition, the senior structure shows that AVRP-N has a triple-helix conformation. Furthermore, AVRP-N exhibited immunomodulatory effects, which could significantly regulate the proliferation of mouse splenic lymphocytes by enhancing the secretion of the cytokines (IFN-γ, IL-2, IL-4, and IL-10). Our results provide new structural and immunoregulatory information for natural polysaccharides derived from Apocynum venetum L.

The complete chloroplast genome and phylogenetic relationship of Apocynum pictum (Apocynaceae), a Central Asian shrub and second-class national protected species of western China.

Apocynum pictum of the dogbane family, Apocynaceae, is a perennial semi-shrub species of ecological, medicinal, and economic value. It is mainly distributed in semi-arid, saline-alkaline, and desert regions of Xinjiang, Qinghai, and Gansu of western China and adjacent regions from Kazakhstan and Mongolia. Here, we reported the complete chloroplast (cp) genome of A. pictum for the first time, and we found that it had a circular structure with an estimated length of 150,749 bp and a GC content of 38.3%. The cp genome was composed of a large single copy (LSC), a single small single copy (SSC), and two inverted repeat (IR) regions, which were 81,888 bp, 17,251 bp and 25,805 bp long, respectively. The cp genome of A. pictum encoded 134 genes and contained 66 simple sequence repeats (SSRs). A comparative analysis with other cp genomes from Apocynaceae indicated that the cp genome of A. pictum was very conserved, except for subtle differences occurring in the protein-coding genes accD, ndhF, rpl22, rpl32, rpoC2, ycf1 and ycf2. A phylogenetic reconstruction showed that A. pictum and A. venetum were sister species, forming a strongly supported clade with Trachelospermum. Interestingly, nucleotide substitution ratios (Ka/Ks) between A. pictum and A. venetum on accD and ndhF were >1.0, suggesting positive selective pressure on these genes. Our result enriches the genomic resources for the diverse dogbane family and provides critical molecular resources to develop future studies on ecological adaptation to desert habitats in Apocynum.

The chloroplast genome sequence and phylogenetic analysis of Apocynum venetum L.

Apocynum venetum L. (Apocynaceae) is valuable for its medicinal compounds and fiber content. Native A. venetum populations are threatened and require protection. Wild A. venetum resources are limited relative to market demand and a poor understanding of the composition of A. venetum at the molecular level. The chloroplast genome contains genetic markers for phylogenetic analysis, genetic diversity evaluation, and molecular identification. In this study, the entire genome of the A. venetum chloroplast was sequenced and analyzed. The A. venetum cp genome is 150,878 bp, with a pair of inverted repeat regions (IRA and IRB). Each inverted repeat region is 25,810 bp, which consist of large (LSC, 81,951 bp) and small (SSC, 17,307 bp) single copy areas. The genome-wide GC content was 38.35%, LSC made up 36.49%, SSC made up 32.41%, and IR made up 43.3%. The A. venetum chloroplast genome encodes 131 genes, including 86 protein-coding genes, eight ribosomal RNA genes, and 37 transfer RNA genes. This study identified the unique characteristics of the A. venetum chloroplast genome, which will help formulate effective conservation and management strategies as well as molecular identification approaches for this important medicinal plant.

Genome wide characterization of R2R3 MYB transcription factor from Apocynum venetum revealed potential stress tolerance and flavonoid biosynthesis genes.

MYB transcription factors are crucial in regulating stress tolerance and expression of major genes involved in flavonoid biosynthesis. The functions of MYBs is well explored in a number of plants, yet no study is reported in Apocynum venetum. We identified a total of 163 MYB candidates, that comprised of 101 (61.96%) R2R3, 6 3R, 1 4R and 55 1R. Syntenic analysis of A. venetum R2R3 (AvMYBs) showed highest orthologous pairs with Vitis vinifera MYBs followed by Arabidopsis thaliana among the four species evaluated. Thirty segmental duplications and 6 tandem duplications were obtained among AvMYB gene pairs signifying their role in the MYB gene family expansion. Nucleotide substitution analysis (Ka/Ks) showed the AvMYBs to be under the influence of strong purifying selection. Expression analysis of selected AvMYBs under low temperature and cadmium stresses resulted in the identification of AvMYB48, AvMYB97, AvMYB8, AvMYB4 as potential stress responsive genes and AvMYB10 and AvMYB11 in addition, proanthocyanidin biosynthesis regulatory genes which is consistent with their annotated homologues in Arabidopsis. Tissue specific expression profile analysis of the AvMYBs further supported the qPCR analysis result. MYBs with higher transcript levels in root, stem and leaf like AvMYB4 for example, was downregulated under the stresses and such with low transcript level such as AvMYB48 which had low transcript in the leaf was upregulated under both stresses. Transcriptome and phylogenetic analyses suggested AvMYB42 as a potential regulator of anthocyanin biosynthesis. Thus, this study provided valuable information on AvR2R3-MYB gene family with respect to stress tolerance and flavonoid biosynthesis.

Apocynum venetum leaf extract alleviated doxorubicin-induced cardiotoxicity through the AKT/Bcl-2 signaling pathway.

BACKGROUND: Doxorubicin (DOX) is a broad-spectrum anti-tumor drug that has been associated with cardiotoxicity. Plant extracts have been shown to confer protection against DOX-induced cardiotoxicity. Apocynum venetum L. belongs to the Apocynaceae family. Flavonoid extracted from Apocynum venetum L. possess various biological effects, such as lowering blood pressure levels, sedation, diuresis, anti-aging, and improving immunity. PURPOSE: This study investigated the mechanism by which dry leaf extract of Apocynum venetum L. (AVLE) alleviates DOX-induced cardiomyocyte apoptosis. METHODS: HPLC-MS/MS and HPLC methods were used to analyze the components of AVLE. The effects of DOX and AVLE on apoptosis of H9c2 and HMC cells were assessed using the MTT assay. Calcein AM/PI, TUNEL, and flow cytometry were carried out to determine the effects of AVLE on DOX-induced apoptosis. The effect of AVLE on DOX-induced oxidative stress in cardiomyocytes was investigated using ELISA test. Mito-Tracker Red CMXRos, JC-1, and RT-qPCR assays were performed to evaluate the impact of AVLE on DOX-induced cardiomyocyte mitochondrial activity and membrane permeability. Western blot assay was carried out to determine the activation of multiple signaling molecules, including phosphorylated-protein kinase B (p-AKT), Cytochrome c, Bcl-2 family, and caspase family in the apoptosis pathway. The AKT inhibitor was used to block AKT/Bcl-2 signaling pathway to investigate the role of AKT in the protection conferred by AVLE against DOX-induced cardiotoxicity. RESULTS: A total of 8 compounds, including rutin, hyperoside, isoquercetin, unidentified compounds, myricetin, quercetin, quercetin-3-O-glucuronide and kaempferol, were detected in AVLE. Of note, DOX suppressed lactate dehydrogenase (LDH) levels, aggravated oxidative stress, and promoted cardiomyocyte apoptosis. It also upregulated the mRNA expression levels of voltage-dependent anion channel 1 (VDAC1), adenosine nucleotide transporter 1 (ANT1), and cyclophilin D (CYPD), while suppressing mitochondrial activity and mitochondrial membrane permeability. Treatment with DOX altered the expression levels of apoptosis-associated proteins, Bcl-2 and Bax. However, AVLE treatment alleviated DOX-induced effects on cardiomyocytes. In addition, application of AKT inhibitors promoted DOX-induced apoptosis and reversed the inhibitory effects of AVLE on DOX-induced apoptosis. CONCLUSIONS: AVLE confer cardio protection by suppressing oxidative stress and apoptosis of cardiomyocytes via AKT/Bcl-2 signaling pathway.

[Comprehensive analysis of components in Chinese medicines derived from Apocynum venetum and Poacynum pictum in Xinjiang based on HPLC fingerprint and chemometrics].

This study established high-performance liquid chromatography(HPLC) fingerprints of Chinese medicines derived from Apocynum venetum and Poacynum pictum in Xinjiang and explored their composition differences with the combination of content determination, similarity analysis, cluster analysis and principal component analysis. The HPLC conditions included Phenomenex Kinetex C_(18) column(4.6 mm ×100 mm, 2.6 µm), acetonitrile-0.01% trifluoroacetic acid aqueous solution as mobile phase, gradient elution, flow rate of 0.6 mL·min~(-1), detection wavelength of 281 nm and column temperature of 25 ℃. The content of chlorogenic acid, quercetin-3-O-sophoroside, rutin, hyperin, isoquercitrin, trifolin and astragalin was determined in 31 batches of medicinal materials, and fingerprint research and chemometric analysis were performed with Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine(Version 2004 A) and SPSS 21.0. In the Chinese Pharmacopoeia 2020, the quality of Apocyni Veneti Folium is controlled by character identification, microscopic identification, thin layer chromatography identification and quantitative determination of hyperin. There were 21 common peaks of A. venetum and P. pictum in the HPLC fingerprints, 5 of which were identified as chlorogenic acid, hyperin, isoquercitrin, trifolin and astragalin, with their content also determined. Except for 3 batches of medicinal materials, the similarity of other 28 batches was higher than 0.83, indicating good similarity. Two categories were formed in the cluster analysis based on content determination, which showed that some differences existed in similarities between different regions of Xinjiang. The medicinal materials were ranked by quality with principal component analysis, and the results indicated that the top 15 all came from northern Xinjiang. The quality difference of A. venetum and P. pictum had a correlation with the place of origin. This study provides a reference for the analysis and evaluation of A. venetum and P. pictum from different habitats and the selection of introduction and cultivation areas.

Comparative transcriptome analysis reveals the molecular mechanism of salt tolerance in Apocynum venetum.

Apocynum venetum is a traditional Chinese medicinal herb with tolerance to various abiotic stresses, especially, salinity. However, only a few studies have investigated the salt-tolerant mechanism of this non-halophyte under salt stress at phenotypic and physiological levels. To explore the molecular mechanism of salinity tolerance in A. venetum, the global transcriptome profiles of seedling leaves under different salt-stress durations, using 200 mM NaCl, were analyzed. De novo assembly of approximately 715 million high-quality reads and approximately 105.61 Gb sequence data was performed. In total, 2822 differentially expressed genes (DEGs) were identified. DEGs were significantly enriched in flavonoid metabolism-related pathways such as "flavonoid biosynthesis" and "phenylpropanoid biosynthesis". Most of these DEGs were downregulated under salt stress. However, genes encoding the non-selective cation channels and antioxidants were upregulated under salt stress, whereas most cell wall-related DEGs were downregulated. Consequently, the concentration of flavonoids decreased, whereas that of Na+ increased with exposure time. Thus, we hypothesized that the accumulation of Na+ in the leaves, which resulted in reduced flavonoid concentration under salt stress, directly led to a decrease in the salt tolerance of A. venetum. This was verified by overexpressing four flavonoid synthesis pathway genes in Arabidopsis. The transgenic plants showed higher salt tolerance than the wild-type plants due to the accumulation of total flavonoids. These physiological and transcriptome analyses of A. venetum revealed major molecular underpinnings contributing to the responses of A. venetum to salt stress, thereby improving our understanding of the molecular mechanisms underlying salt tolerance in A. venetum and plants in general. The findings serve as a basis for functional studies on and engineering strategies for plant salinity tolerance.

Overexpression of an Apocynum venetum flavonols synthetase gene confers salinity stress tolerance to transgenic tobacco plants.

Soil salinity is a major limiting factor for agricultural production, threatening food security worldwide. A thorough understanding of the mechanisms underlying plant responses is required to effectively counter its deleterious effects on crop productivity. Total flavonoid accumulation reportedly improves salinity tolerance in many crops. Therefore, we isolated the full-length cDNA of a flavonol synthetase (FLS) gene from Apocynum venetum (AvFLS). The gene contained a 1008-bp open reading frame encoding a protein composed of 335 amino acid residues. Multiple sequence alignment showed that the AvFLS protein was highly homologous to FLSs from other plants. AvFLS was expressed in leaves, stems, roots, flowers, and germinated seeds. Expression pattern analysis revealed that AvFLS was significantly induced by salinity stress. AvFLS overexpression in tobacco positively affected the development and growth of transgenic plants under salinity stress: root and seedling growth were inhibited to a lesser extent, while seed germination rate increased. Additionally, the overexpression of AvFLS under salinity stress resulted in an increase in total flavonoid content (1.63 mg g-1 in wild-type samples and 4.63 mg g-1 on average in transgenic samples), which accompanied the increase in the activity of antioxidant enzymes and inhibited the production of reactive oxygen species. Further, AvFLS-overexpressing transgenic tobacco plants absorbed more K+ than wild type plants, leading to an increased K+/Na+ ratio, which in turn contributed to the maintenance of Na+/K+ homeostasis. These findings suggest that an AvFLS-induced increase in total flavonoid content enhanced plant salinity tolerance, implying the importance of AvFLS gene responses to salinity stress.

Quercetin alleviates seed germination and growth inhibition in Apocynum venetum and Apocynum pictum under mannitol-induced osmotic stress.

Quercetin is one of the main flavonoids in the human diet and mainly found in different plant tissues, including seeds, flowers, leaves, stems, and roots. However, its biological function in plant tissues, especially in seeds, is unknown. In this study, the seed germination and subsequent seedling growth of Apocynum pictum and A. venetum under osmotic stress (400 mmol L-1 mannitol) supplemented with 5 µmol L-1 quercetin were evaluated after 7, 14, and 21 days of germination. Results showed that quercetin improved the germination percentage and seed vigor, as indicated by the higher germination energy, shoot length, root length, dry weight, fresh weight, and chlorophyll content in A. pictum and A. venetum seedlings under the mannitol compared with those under the mannitol alone. Quercetin decreased H2O2 and O2- production and cell membrane damage, and mostly increased the gene expression of superoxide dismutase, peroxidase, catalase, chalcone synthase and flavonol synthase in A. pictum and A. venetum seedlings under the mannitol compared with those under the mannitol alone. In addition, the germination energy of A. pictum was 21.57% higher than that of A. venetum, and the gene expression of key enzymes in quercetin biosynthesis in A. pictum was mostly higher than that in A. venetum after 1 and 7 days of germination. These results indicated that quercetin was an effective anti-osmotic agent that alleviated the adverse effect of mannitol-induced osmotic stress on seed germination and seed vigor, and A. pictum seeds were more osmotic resistant than A. venetum seeds.

Comprehensive analysis of components in Chinese medicines derived from Apocynum venetum and Poacynum pictum in Xinjiang based on HPLC fingerprint and chemometrics / 中国中药杂志

This study established high-performance liquid chromatography(HPLC) fingerprints of Chinese medicines derived from Apocynum venetum and Poacynum pictum in Xinjiang and explored their composition differences with the combination of content determination, similarity analysis, cluster analysis and principal component analysis. The HPLC conditions included Phenomenex Kinetex C_(18) column(4.6 mm ×100 mm, 2.6 μm), acetonitrile-0.01% trifluoroacetic acid aqueous solution as mobile phase, gradient elution, flow rate of 0.6 mL·min~(-1), detection wavelength of 281 nm and column temperature of 25 ℃. The content of chlorogenic acid, quercetin-3-O-sophoroside, rutin, hyperin, isoquercitrin, trifolin and astragalin was determined in 31 batches of medicinal materials, and fingerprint research and chemometric analysis were performed with Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine(Version 2004 A) and SPSS 21.0. In the Chinese Pharmacopoeia 2020, the quality of Apocyni Veneti Folium is controlled by character identification, microscopic identification, thin layer chromatography identification and quantitative determination of hyperin. There were 21 common peaks of A. venetum and P. pictum in the HPLC fingerprints, 5 of which were identified as chlorogenic acid, hyperin, isoquercitrin, trifolin and astragalin, with their content also determined. Except for 3 batches of medicinal materials, the similarity of other 28 batches was higher than 0.83, indicating good similarity. Two categories were formed in the cluster analysis based on content determination, which showed that some differences existed in similarities between different regions of Xinjiang. The medicinal materials were ranked by quality with principal component analysis, and the results indicated that the top 15 all came from northern Xinjiang. The quality difference of A. venetum and P. pictum had a correlation with the place of origin. This study provides a reference for the analysis and evaluation of A. venetum and P. pictum from different habitats and the selection of introduction and cultivation areas.

Cost-effectiveness analysis of combining traditional Chinese medicine in the treatment of hypertension: compound Apocynum tablets combined with Nifedipine sustained-release tablets vs Nifedipine sustained-release tablets alone.

BACKGROUND: We evaluated the long-term cost-effectiveness of antihypertensive traditional Chinese medicines (TCMs) and to compare the cost-effectiveness of a combined treatment consisting of compound Apocynum tablets and Nifedipine sustained-release tablets with the cost-effectiveness of treatment with Nifedipine sustained-release tablets alone. METHODS: A Markov model was used to simulate the potential incremental cost-effectiveness per quality-adjusted life year (QALY) to be gained from compound Apocynum tablets and Nifedipine sustained-release tablets compared with Nifedipine sustained-release tablets alone. Model parameter estimates were informed by previously published studies. The direct medical costs of outpatients with hypertension were estimated from the health care provider's perspective. A 5% annual discount rate was applied to both costs and QALYs. RESULTS: TCMs combined with Nifedipine sustained-release tablets group generated a total 20-year cost of 11,517.94 RMB (US $1739.87), whereas Nifedipine sustained-release tablets alone group resulted in a 20-year cost of 7253.71 RMB (US $1095.73). TCMs combined with Nifedipine sustained-release tablets group resulted in a generation of 12.69 QALYs, whereas Nifedipine sustained-release tablets alone group resulted in 12.50. The incremental cost-utility ratio was 22,443.32 RMB (US $3390.23) per QALY. Considering the threshold of 1 GDP per capita in China in 2018 (US $9764.95), the combination of compound Apocynum tablets and Nifedipine sustained-release tablets was a cost-effective strategy. One-way and probabilistic sensitivity analysis showed unchanged results over an acceptable range. CONCLUSIONS: Combining Traditional Chinese Medicines with chemical medicines is more cost-effective strategy in the treatment of hypertension.

De novo transcriptome assembly and population genetic analyses of an important coastal shrub, Apocynum venetum L.

BACKGROUND: Apocynum venetum L. is an important medicinal plant that is mainly distributed in the coastal areas and northwest of China. In addition to its high medical and economic value, its adaptation to saline-alkali and coastal saline lands makes A. venetum an ideal candidate for use in vegetation restoration. To date, the study of A. venetum has been limited in the northwest region of China, little attention has been paid to the genetic diversity and population structure of A. venetum populations in the coastal region. Here, we performed transcriptome sequencing of total RNA from A. venetum leaves and developed efficient expressed sequence tag-simple sequence repeat (EST-SSR) markers for analyzing the genetic diversity and population structure of A. venetum in the coastal region. RESULTS: A total of 86,890 unigenes were generated after de novo assembly, and 68,751 of which were successfully annotated by searching against seven protein databases. Furthermore, 14,072 EST-SSR loci were detected and 10,243 primer pairs were successfully designed from these loci. One hundred primer pairs were randomly selected and synthesized, twelve primer pairs were identified as highly polymorphic and further used for population genetic analysis. Population genetic analyses showed that A. venetum exhibited low level of genetic diversity (mean alleles per locus, NA = 3.3; mean expected heterozygosity, HE = 0.342) and moderate level of genetic differentiation among the populations (genetic differentiation index, FST = 0.032-0.220) in the coastal region. Although the contemporary (mean mc = 0.056) and historical (mean mh = 0.106) migration rates among the six A. venetum populations were moderate, a decreasing trend over the last few generations was detected. Bayesian structure analysis clustered six populations into two major groups, and genetic bottlenecks were found to have occurred in two populations (QG, BH). CONCLUSIONS: Using novel EST-SSR markers, we evaluated the genetic variation of A. venetum in the coastal region and determined conservation priorities based on these findings. The large dataset of unigenes and SSRs identified in our study, combining samples from a broader range, will support further research on the conservation and evolution of this important coastal plant and its related species.