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plants are widely utilized in traditional medicine (such as and two important commonly medicinal plants), primarily for their properties in promoting blood circulation, strengthening bones and tendons, and so on. However, the high diversity of species differentiation poses a challenge in accurately identifying the various species without specialized taxonomic knowledge. To screen the candidate barcode sequences of species, we first report the complete chloroplasts (CP) genomes of . and . obtained via high throughput Illumina sequencing and compare them with fourteen previously sequenced species. Furthermore, we collected fresh leaf samples from and (totally 37 samples) and evaluated the discriminatory efficacy of the nuclear DNA Internal Transcribed Spacer 2 (ITS2) fragment through comparative analysis of sequence variations and secondary structures. Finally, to analyze the phylogenetic position of species, we constructed a Maximum Likelihood (ML) phylogenetic tree using CP genome sequences of 46 species from seven genera within the Vitaceae family. The CP genomes of exhibited a typical circular tetramerous structure, including a large single-copy region (LSC) (87,381-88,979 bp), a small single-copy region (SSC) (18,649-19,339 bp), and a pair of inverted repeats (IRa and IRb) (26,288-26,934 bp). The guanine-cytosine content of the CP genomes is 37.35%-37.62%. The codon usage shows a significant preference for end with A/T. Then, the results of nucleotide diversity analysis showed that ten polymorphic hotspots ( M- D-GUC, F- 32, S-GCU- G-UCC, 1, 32- L-UAG, S-UGA- Z, E- L, K- 16, 16, and 22) could be the candidate DNA marker suitable for species. Furthermore, our results demonstrate that the ITS2 sequence could effectively discriminate and , whereas the secondary structure cannot, proving that ITS2 can be used as an efficient barcode fragment to accurately identify the two species. The aim of this study was not only to determine the identification efficiency of the CP genome and ITS2 for and but also to clarify the phylogenetic relationship and screen the candidate DNA marker suitable for species, provide valuable data support for further accurate identification of the genus.

Diels & Gilg (TDG), the family member of Vitaceae, is a traditional herbal medicine in China. The root of TDG can be immediately used after cleaning the muddy soil, and can be dehydrated for dry use. TDG is able to be collected all year round, which is commonly used in the treatment of hepatitis, infantile high fever, snake bite, etc. Based on phytochemistry, the chemical components of TDG are divided into flavonoids, phenolic acids, terpenes, steroids, polysaccharide, and other compounds, showing many pharmacological effects which include anti-tumor, anti-oxidation, anti-inflammatory, antipyretic, analgesic, and immunomodulatory activity, as well as other activities. Currently, TDG involves some problems of the reduction of wild resources, the backward processing methods, and storage difficulties as well as the imperfection of detection methods. Therefore, this review summarizes the literature of the past 20 years, and the purpose of this review is to summarize the recent researches on the phytochemistry, pharmacology, quality control, and clinical application of TDG. The above discussions provide new insights for the future research on TDG.

Background Tetrastigma hemsleyanum Diels et Gilg is a valuable medicinal herb, whose main bioactive constituents are flavonoids. Chilling sensitivity is the dominant environmental factor limiting growth and development of the plants. But the mechanisms of cold sensitivity in this plant are still unclear. Also, not enough information on genes involved in flavonoid biosynthesis in T. hemsleyanum is available to understand the mechanisms of its physiological and pharmaceutical effects. Results The electrolyte leakage, POD activity, soluble protein, and MDA content showed a linear sustained increase under cold stress. The critical period of cold damage in T. hemsleyanum was from 12 h to 48 h. Expression profiles revealed 18,104 differentially expressed genes (DEGs) among these critical time points. Most of the cold regulated DEGs were early-response genes. A total of 114 unigenes were assigned to the flavonoid biosynthetic pathway. Fourteen genes most likely to encode flavonoid biosynthetic enzymes were identified. Flavonols of T. hemsleyanum might play a crucial role in combating cold stress. Genes encoding P AL, 4CL , CHS, ANR, FLS , and LAR were significantly up-regulated by cold stress, which could result in a significant increase in crucial flavonols (catechin, epicatechin, rutin, and quercetin) in T. hemsleyanum. Conclusions Overall, our results show that the expression of genes related to flavonol biosynthesis as well as flavonol content increased in T. hemsleyanum under cold stress. These findings provide valuable information regarding the transcriptome changes in response to cold stress and give a clue for identifying candidate genes as promising targets that could be used for improving cold tolerance via molecular breeding. The study also provides candidate genes involved in flavonoid biosynthesis and may be useful for clarifying the biosynthetic pathway of flavonoids in T. hemsleyanum .

Purpose The utilization of pyrogenic organic matter and organic fertilizers is widespread in agroforestry ecosystems for the purpose of augmenting soil nutrient levels and crop yields. Nonetheless, the impacts of these fertilizers on the yield and quality of Tetrastigma hemsleyanum Diels et Gilg, as well as the underlying mechanisms mediating these processes, remain inadequately understood. Methods In a potting experiment, we studied the effects of pyrogenic organic matter (20 and 40 mg C g −1 soil) and organic fertilizer (5% and 10%) on the yield and quality of T. hemsleyanum . Soil chemical properties (available K, AK; available P, AP; available N, AN; organic matter, OM), soil enzymes (urease, protease, and cellobiohydrolase), plant growth (plant height, stem thick, and fresh root weight), flavonoid-related enzymes (phenylalanine ammonia lyase, PAL; chalcone synthase, CHS; and chalcone isomerase, CHI), and total flavonoids were measured to elucidate the effects of the two fertilizers. Results Compared with the control, plant height, fresh root weight, and total flavonoid concentration (increased by 1.60 and 2.70 times, respectively) were enhanced by low and high biochar applications, whereas organic fertilizer addition only had a positive impact on fresh root weight. Furthermore, biochar supplementation enhanced growth, quality, and total flavonoid content by elevating AK, AP, AN, and OM levels, as well as increasing urease, protease, and CHI activities. Fresh root weight following the addition of organic fertilizer addition was significantly correlated with AK, AP, and protease activity. Conclusions The addition of pyrogenic organic matter addition is found to be a more suitable strategy than organic fertilizer addition for effectively enhancing the yield and quality of T. hemsleyanum . Graphical Abstract

Tetrastigma hemsleyanum Diels et Gilg is a folk herb in Zhejiang Province with anti-inflammatory, antineoplastic, and anti-oxidation effects. Given its pharmacological activity, T. hemsleyanum is known as New “Zhebawei” and included in the medical insurance system of Zhejiang and other provinces. Flavonoids are the most important components of T. hemsleyanum , and their contents are mainly regulated by ultraviolet (UV) radiation. In this study, the total flavonoid contents, flavonoid monomer contents, and flavonoid synthesis related enzyme activities (phenylalanine ammonia–lyase, chalcone synthase, and chalcone isomerase), anti-oxidant enzyme activities (catalase, peroxidase, and superoxide dismutase), and biochemical indicators (malondialdehyde, free amino acid, soluble protein, and soluble sugar) in the leaves (L) and root tubers (R) of T. hemsleyanum with UV treatments were determined. Three kinds of UV radiation (UV-A, UV-B, and UV-C) and six kinds of radiation durations (15 and 30 min, 1, 2, 3, and 5 h) were used. Appropriate doses of UV-B and UV-C radiation (30 min to 3 h) induced eustress, which contributed to the accumulation of flavonoids and improve protective enzyme system activities and bioactive compound contents. Especially, certain results were observed in several special structures of the flavonoid monomer: quercetin contents in L increased by nearly 20 times, isoquercitrin contents in R increased by nearly 34 times; most of flavonoids with glycoside content, such as quercitrin (19 times), baicalin (16 times), and apigenin-7G (13 times), increased multiple times. Compared with the CK group, the flavonoid synthase activities, anti-oxidant enzyme activities, and biochemical substance contents in L and R all increased with UV treatments. This study provides a theoretical foundation for regulating flavonoids by light factors and improving the quality of T. hemsleyanum in production and medical industries.

Warm‐temperate evergreen (WTE) forest represents the typical vegetation type of subtropical China, but how its component species responded to past environmental change remains largely unknown. Here, we reconstruct the evolutionary history of Tetrastigma hemsleyanum, an herbaceous climber restricted to the WTE forest. Twenty populations were genotyped using chloroplast DNA sequences and nuclear microsatellite loci to assess population structure and diversity, supplemented by phylogenetic dating, ancestral area reconstructions and ecological niche modeling (ENM) of the species distributions during the Last Glacial Maximum (LGM) and at present. Lineages in Southwest vs Central‐South‐East China diverged through climate/tectonic‐induced vicariance of an ancestral southern range during the early Pliocene. Long‐term stability in the Southwest contrasts with latitudinal range shifts in the Central‐South‐East region during the early‐to‐mid‐Pleistocene. Genetic and ENM data strongly suggest refugial persistence in situ at the LGM. Pre‐Quaternary environmental changes appear to have had a persistent influence on the population genetic structure of this subtropical WTE forest species. Our findings suggest relative demographic stability of this biome in China over the last glacial–interglacial cycle, in contrast with palaeobiome reconstructions showing that this forest biome retreated to areas of today's tropical South China during the LGM.

Tetrastigma hemsleyanum ( T. hemsleyanum ) is a traditional medicinal plant that is widely used in China. Cultivated T. hemsleyanum usually encounters cold stress, limiting its growth and quality at key developmental stages. APETALA2 (AP2)/ethylene-responsive factor (ERF) transcription factors (TFs) comprise one of the largest gene superfamilies in plants and are widely involved in biotic and abiotic stresses. To reveal the roles of AP2/ERF TFs during T. hemsleyanum development, 70 AP2/ERF TFs were identified in T. hemsleyanum . Among them, 18 and 2 TFs were classified into the AP2 and RAV families, respectively. The other 50 TFs belonged to the ERF family and were further divided into the ERF and (dehydration reaction element binding factor) DREB subfamilies. The ERF subfamily contained 46 TFs, while the DREB subfamily contained 4 TFs. Phylogenetic analysis indicated that AP2/ERF TFs could be classified into five groups, in which 10 conserved motifs were confirmed. Several motifs were group- or subgroup-specific, implying that they were significant for the functions of the AP2/ERF TFs of these clades. In addition, 70 AP2/ERF TFs from the five groups were used for an expression pattern analysis under three low-temperature levels, namely, –4, 0, and 4°C. The majority of these AP2/ERF TFs exhibited a positive response to cold stress conditions. Specifically, ThERF5, ThERF31, ThERF46, and ThERF55 demonstrated a more sensitive response to cold stress. Moreover, AP2/ERF TFs exhibited specific expression patterns under cold stress. Transient overexpression and RNA interference indicated that ThERF46 has a specific tolerance to cold stress. These new insights provide the basis for further studies on the roles of AP2/ERF TFs in cold stress tolerance in T. hemsleyanum .

Tetrastigma bracteolatum: (Wall.) Planch, an indigenous plant of Bangladesh, is traditionally used for pain management. This study aimed to determine its biological activities through an integrated , , and approaches. Crude methanol extract was prepared using the maceration technique and fractionated into different solvent fractions. Antioxidant, thrombolytic, and membrane-stabilizing activities were assessed , while antidiabetic, antidiarrheal, CNS-stimulating, and analgesic activities were performed using Swiss albino mice. Among the fractions, the chloroform-soluble fraction (CSF) exhibited the highest phenolic content and membrane stabilizing activity, while the aqueous-soluble fraction (AQSF) demonstrated strong antioxidant properties. The petroleum ether fraction showed significant cytotoxic activity. The crude methanol extract exhibited potent antidiarrheal and hypoglycemic effects in mice, with efficacy comparable to standard drugs. The extract significantly exhibited CNS stimulating activity as well. Additionally, it demonstrated analgesic activity by significantly prolonging pain response times and reducing chemically induced pain behaviors in mice. docking studies revealed strong binding affinities of the extract's compounds against key biological targets involved in inflammation, pain modulation, and metabolic regulation. Moreover, toxicity predictions indicated that all compounds were non-toxic and free from carcinogenic effects. These findings suggest that possesses promising antioxidant, membrane-stabilizing, antidiarrheal, antihyperglycemic, antidepressant, and analgesic properties, supporting its potential therapeutic applications.

Background Tetrastigma hemsleyanum Diels & Gilg, a plant well-known in China for its use in traditional herbal medicine, is renowned for its remarkable medicinal value in various clinical contexts. Its chloroplast and nuclear genomes have been fully sequenced, providing valuable information for breeding and exploration. However, its mitochondrial genome remains uncharacterized, restricting the broader view of its evolution and its potential utilization. We present the assembled and annotated mitogenome of T. hemsleyanum to enhance the genetic resources for the Tetrastigma genus and pave the way for future in-depth studies of this medicinally significant plant. Results In this study, we assembled the mitochondrial genome of T. hemsleyanum var. “Jinhulu 2” by employing both the Illumina and Nanopore sequencing technologies and performed a comparative analysis with Vitis . The results revealed that the mitogenome is multi-chromosomal in structure, comprising two circular chromosomes with a total length of 809,173 bp and a GC content of 44.35%. It contains 76 annotated functional genes, including 35 protein-coding genes, 37 tRNA genes, 3 rRNA genes, and 1 pseudogene. Further analysis revealed 273 simple sequence repeats (SSRs), 109 tandem repeats, and 8,733 dispersed repeats, suggesting a higher repeat abundance in T. hemsleyanum compared to its closely related species. RNA editing analysis identified 57 sites within the mitochondrial CDS (coding DNA sequence), all showing a precise cytosine-to-uracil conversion, typically resulting in the substitution of hydrophilic amino acids with hydrophobic ones. The selection pressure analysis indicated that most mitochondrial CDS are under purifying selection, with only four genes ( ccmB , ccmFc , ccmFn , and rps10 ) exhibiting positive selection. Notably, positive selection on ccmB was consistently observed across all pairwise species comparisons, highlighting its pivotal role in evolutionary adaptation. In addition, we identified 62 homologous fragments transferred from the chloroplast to the mitochondrial genome, including 15 intact chloroplast-derived tRNA genes. Comparative analysis revealed that the syntenic regions and mitochondrial gene organization are relatively conserved within the Vitaceae family compared to the other species examined. Finally, the phylogenetic tree was found to align closely with established taxonomic relationships among members of the rosid clade and underscores the close evolutionary relationship between Tetrastigma and Vitis . Conclusions This study presents the first complete T. hemsleyanum mitogenome (809,173 bp, two circular chromosomes), revealing distinct codon usage, high repeat abundance (SSRs, tandem, dispersed), chloroplast-derived tRNAs, and conserved Vitaceae synteny. Key findings include C-to-U RNA editing, positive selection on ccmB, and phylogenetic confirmation of its close relationship to Vitis . This study elucidates mitochondrial genetics and provides a foundation for molecular breeding of Tetrastigma .

Tetrastigma hemsleyanum , a traditional Chinese medicinal plant with anti-inflammatory, anti-cancer, and anti-tumor properties, faces increasing abiotic stress due to climate change, agricultural chemicals, and industrialization. This study investigated how three abiotic stress factors influence antioxidant enzyme activity, MDA levels, DPPH free radical scavenging capacity, chlorophyll, carotenoids, active compounds, and gene expression in different T. hemsleyanum strains. The comprehensive evaluation indicates that the ZJWZ strain holds potential as a preferred parental material for future resistance breeding. Furthermore, PAL gene expression was strongly positively correlated with flavonoid and phenol contents, highlighting its role in the stress response through the phenylpropanoid-flavonoid pathway. This study contributes to the standardization of the production and breeding of superior strains of T. hemsleyanum . It also lays the foundation for investigating how plants react to environmental stressors.