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.

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.

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.

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.

Salt stress decreases seedling growth and development but increases quercetin and kaempferol content in Apocynum venetum.

Apocynum venetum L. is a traditional Chinese medicinal herb with great potential to treat angiocardiopathy. Its major medicinal constituents are flavonoids. However, the natural habitats of A. venetum are typically affected by salt stress, which can modify both biomass and accumulation of medicinal compounds. In this study, the effects of salt stress on growth and development of A. venetum, accumulation of flavonoids and expression patterns of genes involved in flavonoid biosynthesis were evaluated. In general, the growth and development of seedlings (seedling height, root length, leaf length, leaf width and seed germination) were inhibited by salt stress. Unlike typical halophytes, there was no optimal NaCl concentration range that promoted growth and development, but seedlings had an elevated DW/FW ratio under salt stress (induced by irrigation with 50, 100, 200 or 400 mm NaCl). Furthermore, quercetin and kaempferol were significantly accumulated in A. venetum seedlings under salt stress, resulting in a balanced content and reduced FW. Moreover, the expression of AvCHS, AvCHI and AvF3GT was inhibited by salt stress; however, AvF3'H, AvF3H and AvFLS, which are involved in the flavonol synthesis pathway, were up-regulated under salt stress, consistent with a decrease in total flavonoids and an increase of flavonols (quercetin and kaempferol). In summary, cultivation of A. venetum in saline soils appeared to be feasible and improved the medicinal quality of A. venetum (quercetin and kaempferol accumulation under salt stress), thus this species can effectively utilize saline soil resources.

Subcellular distribution and chemical forms of lithium in Li-accumulator Apocynum venetum.

Apocynum venetum is a promising species to remediate an emerging environmental contaminant lithium (Li). However, no research has been conducted so far relating Li tolerance mechanism. In order to improve the understanding of Li transportation and detoxification, subcellular accumulation and distribution of different chemical forms of Li was studied in Apocynum venetum. Subcellular Li compartmentalization analysis showed that majority of Li was located in vacuole (45.52-72.65%) and cell wall (14.84-29.02%) under Li treatment. Furthermore, water soluble and ethonal extracted Li (inorganic Li) are the main chemical forms of Li taken up by A. venetum. With the increase of Li concentration in the medium, Li content in all subcellular fractions and proportion of F-ethanol form with high mobility increased. The greatest amount of Li was found in soluble fraction in leaves at 25 mg L-1 Li treatment, followed by soluble fraction in leaves at 2.5 mg L-1. These results suggest that Li compartmentation in leaf vacuoles is important in Li detoxification and Li accumulation of A. venetum.