Genome assembly and resequencing analyses provide new insights into the evolution, domestication and ornamental traits of crape myrtle

Crape myrtle(Lagerstroemia indica)is a globally used ornamental woody plant and is the representative species of Lagerstroemia.However,studies on the evolution and genomic breeding of L.indica have been hindered by the lack of a reference genome.Here we assembled the first high-quality genome of L.indica using PacBio combined with Hi-C scaffolding to anchor the 329.14-Mb genome assembly into 24 pseudochromosomes.We detected a previously undescribed independent whole-genome triplication event occurring 35.5 million years ago in L.indica following its divergence from Punica granatum.After resequencing 73 accessions of Lagerstroemia,the main parents of modern crape myrtle cultivars were found to be L.indica and L.fauriei.During the process of domestication,genetic diversity tended to decrease in many plants,but this was not observed in L.indica.We constructed a high-density genetic linkage map with an average map distance of 0.33 cM.Furthermore,we integrated the results of quantitative trait locus(QTL)using genetic mapping and bulk segregant analysis(BSA),revealing that the major-effect interval controlling internode length(IL)is located on chr1,which contains CDL15,CRG98,and GID1b1 associated with the phytohormone pathways.Analysis of gene expression of the red,purple,and white flower-colour flavonoid pathways revealed that differential expression of multiple genes determined the flower colour of L.indica,with white flowers having the lowest gene expression.In addition,BSA of purple-and green-leaved individuals of populations of L.indica was performed,and the leaf colour loci were mapped to chr12 and chr17.Within these intervals,we identified MYB35,NCED,and KAS1.Our genome assembly provided a foundation for investigating the evolution,population structure,and differentiation of Myrtaceae species and accelerating the molecular breeding of L.indica.

An Air‑Rechargeable Zn Battery Enabled by Organic–Inorganic Hybrid Cathode

Self-charging power systems collecting energy harvesting technology and batteries are attracting extensive attention.To solve the disadvantages of the traditional integrated system,such as highly dependent on energy supply and complex structure,an airrechargeable Zn battery based on MoS_(2)/PANI cathode is reported.Benefited from the excellent conductivity desolvation shield of PANI,the MoS_(2)/PANI cathode exhibits ultra-high capacity(304.98 mAh g^(−1) in N_(2) and 351.25 mAh g^(−1) in air).In particular,this battery has the ability to collect,convert and store energy simultaneously by an airrechargeable process of the spontaneous redox reaction between the discharged cathode and O2 from air.The air-rechargeable Zn batteries display a high open-circuit voltage(1.15 V),an unforgettable discharge capacity(316.09 mAh g^(−1) and the air-rechargeable depth is 89.99%)and good air-recharging stability(291.22 mAh g^(−1) after 50 air recharging/galvanostatic current discharge cycle).Most importantly,both our quasi-solid zinc ion batteries and batteries modules have excellent performance and practicability.This work will provide a promising research direction for the material design and device assembly of the next-generation self-powered system.

Positive regulatory role of R2R3 MYBs in terpene biosynthesis in Lilium‘Siberia’

Terpenoids are the main components contributing to the fragrance of Lilium‘Siberia’,and LiTPS2 plays a critical role in the biosynthesis of monoterpenoids.Although the major terpene synthases in Lilium‘Siberia’have been identified,how these TPS genes are transcriptionally regulated remains elusive in this distinguished flower.This study aimed to identify transcription factors that regulate the terpene synthesis in Lilium,and disclose the related underlying transcriptional regulation mechanism.In this study,we identified three R2R3-MYB TFs—LiMYB1,LiMYB305 and LiMYB330,which were involved in regulating the biosynthesis of terpenes in Lilium‘Siberia’.Quantitative real-time PCR showed spatial and temporal expression patterns consistent with the emission patterns of terpene compounds.When LiMYB1,LiMYB305 and LiMYB330were overexpressed in flowers,the release of some main monoterpenes,such as linalool and ocimene,as well as the expression of TPS genes,especially for LiTPS2,were enhanced.A virus-induced gene silencing(VIGS)assay showed that silencing these three LiMYBs decreased the level of monoterpenes by down-regulating the expression of the TPS genes.The yeast one-hybrid and transient expression assays indicated that all three LiMYBs could bind to and activate the promoter of LiTPS2.Moreover,the yeast two-hybrid assay verified that LiMYB1 could interact with LiMYB308 and LiMYB330,indicating their synergistic roles in the regulation of floral terpene biosynthesis.In general,these results indicated that LiMYB1,LiMYB305,and LiMYB330 might play essential roles in terpene biosynthesis in Lilium and would provide a new perspective for the transcriptional regulation of volatile terpenes in flowers.

Photoperiod-and temperature-mediated control of the ethylene response and winter dormancy induction in Prunus mume

Plant dormancy is essential for perennial plant survival.Different genotypes of Prunus mume,including Eumume group and Apricot Mei group,undergo leaf senescence and dormancy at different times.In order to verify the cold resistance of P.mume,freeze resistance evaluation was carried out.Our results showed that Apricot Mei group had a stronger freezing tolerance than Eumume group and that leaf senescence and dormancy of Apricot Mei group occurred at an earlier period before winter.Based on phenotypic data in response to seasonal climate change,the significant candidate regions were selected using GWAS.Furthermore,through KEGG pathway and qRT-PCR analyses,we found that the ethylene-related genes,including PmEIL(Pm002057)and PmERF(Pm004265),were significantly upregulated in‘Songchun’Mei(Apricot Mei group)and downregulated in‘Zaohua Lve’Mei(Eumume group).Ethylene-related genes expression models showed that ethylene may be indirectly involved in the induction of dormancy.The PmEIL and PmERF genes were the core genes of the ethylene signal transduction pathway and were regulated by the exogenous ACC or PZA compounds.For non-dormant or weekly dormant perennial plants,application of ACC was able to induce plant dormancy and thus enhance cold/freeze tolerance.Overall,the expression of the major ethylene genes played a significant role in dormancy induction and freezing tolerance in P.mume;accordingly,application of ACC and PZA compounds were an effective approach for enhancing cold/freeze of tolerance of woody plant.

Transcriptome profiles reveal that gibberellin-related genes regulate weeping traits in crape myrtle

Plant architecture includes vital traits that influence and benefit crops,and economically important trees.Different plant architectures provide natural beauty.Weeping ornamental plants are aesthetically appealing to people.The regulatory mechanism controlling the weeping trait is poorly understood in crape myrtle.To investigate the weeping trait mechanism,transcriptional profiling of different organs in weeping and upright crape myrtle was performed based on phenotype.Phenotypic and histological analyses demonstrated that endodermal cells were absent,and that new shoot phenotypes could be rescued by the GA3 treatment of weeping plants.The transcriptional analysis and coexpression network analysis(WGCNA)of differentially expressed genes indicated that GA synthesis and signal transduction pathways play a role in weeping traits.When the expression level of a negative element of GA signaling,LfiGRAS1,was reduced by virus-induced gene silencing(VIGS),new branches grew in infected plants in a negatively geotropic manner.An integrated analysis implied that GA had a strong influence on weeping crape myrtle by interacting with other factors.This study helps to elucidate the mechanism governing the weeping trait and can improve the efficiency of breeding in Lagerstroemia.

Expansion of PmBEAT genes in the Prunus mume genome induces characteristic floral scent production

Prunus mume is the only plant in the genus Prunus of the Rosaceae family with a characteristic floral scent,and the main component of this scent is benzyl acetate.By contrast,benzyl acetate is not synthesized in Prunus persica flowers.Here,we searched for benzyl alcohol acetyltransferase(BEAT)genes based on genomic data from P.mume and P.persica and found 44 unique PmBEATs in P.mume.These genes,which were mainly detected in clusters on chromosomes,originated from gene duplication events during the species evolution of P.mume,and retroduplication and tandem duplication were the two dominant duplication patterns.The genes PmBEAT34,PmBEAT36 and PmBEAT37,which were generated by tandem duplication,were highly expressed in flowers,and their highest levels were detected during the blooming stage.In vitro,PmBEAT34,PmBEAT3,and PmBEAT37 all had benzyl alcohol acetyltransferase activity that was localized in the cytoplasm.Overexpression of the PmBEAT36 or PmBEAT37 genes increased benzyl acetate production in the petal protoplasts of P.mume,and interference in the expression of these genes slightly decreased the benzyl acetate content.In addition,light and temperature regulated the expression of the PmBEAT34,PmBEAT36 and PmBEAT37 genes.According to these results,we hypothesize that the expansion of the PmBEAT genes in the genome induce the characteristic floral scent of P.mume.

Establishment and Verification of An Efficient Virus-induced Gene Silencing System in Forsythia

To understand the functional identification of large-scale genomic sequences in Forsythia,tobacco rattle virus(TRV)-mediated virus-induced gene silencing(VIGS),suitable for the plant,was explored in this study.The results showed that the TRV-mediated VIGS system could be successfully used in Forsythia for silencing the reporter gene FsPDS(Forsythia phytoene desaturase)using stem infiltration and leaf infiltrationmethods.All the treated plants were pruned below the injection site after 7–15 d infection;the FsPDS was silenced and typical photobleaching symptoms were observed in newly sprouted leaves at the whole-plant level.Meanwhile,this system has been successfully tested and verified through virus detection and qRT-PCR analysis.After the optimization,Forsythia magnesium chelatase subunit H(FsChlH)was silenced successfully in Forsythia using this system,resulting in yellow leaveswith decreased chlorophyll content.The system was stable,highly efficient and had greater rapidity and convenience,which made it suitable to study the function of genes related to physiological pathways such as growth and development,and metabolic regulation in Forsythia.

Research advances in and prospects of ornamental plant genomics

The term‘ornamental plant’refers to all plants with ornamental value,which generally have beautiful flowers or special plant architectures.China is rich in ornamental plant resources and known as the“mother of gardens”.Genomics is the science of studying genomes and is useful for carrying out research on genome evolution,genomic variations,gene regulation,and important biological mechanisms based on detailed genome sequence information.Due to the diversity of ornamental plants and high sequencing costs,the progress of genome research on ornamental plants has been slow for a long time.With the emergence of new sequencing technologies and a reduction in costs since the whole-genome sequencing of the first ornamental plant(Prunus mume)was completed in 2012,whole-genome sequencing of more than 69 ornamental plants has been completed in<10 years.In this review,whole-genome sequencing and resequencing of ornamental plants will be discussed.We provide analysis with regard to basic data from whole-genome studies of important ornamental plants,the regulation of important ornamental traits,and application prospects.

Targeted deletion of floral development genes in Arabidopsis with CRISPR/Cas9 using the RNA endoribonuclease Csy4 processing system

The formation of flowers in higher plants is controlled by complex gene regulatory networks.The study of floral development in Arabidopsis is promoted and maintained by transposon-tagged mutant lines.In this study,we report a CRISPR/Cas9 genome-editing system based on RNA endoribonuclease Csy4 processing to induce high-efficiency and inheritable targeted deletion of transcription factors involved in floral development in Arabidopsis.Using AP1,SVP,and TFL1 as the target genes,multisite and multiple-gene mutations were achieved with a tandemly arrayed Csy4-sgRNA architecture to express multiplexed sgRNAs from a single transcript driven by the Pol II promoter in transgenic lines.Targeted deletions of chromosomal fragments between the first exon and second exon in either one or three genes were generated by using a single binary vector.Interestingly,the efficiency of site-targeted deletion was comparable to that of indel mutation with the multiplexed sgRNAs.DNA sequencing analysis of RT-PCR products showed that targeted deletions of AP1 and TFL1 could lead to frameshift mutations and introduce premature stop codons to disrupt the open-reading frames of the target genes.In addition,no RT-PCR amplified product was acquired after SVPtargeted deletion.Furthermore,the targeted deletions resulted in abnormal floral development in the mutant lines compared to that of wild-type plants.AP1 and SVP mutations increased plant branching significantly,while TFL1 mutant plants displayed a change from indeterminate to determinate inflorescences.Thus,our results demonstrate that CRISPR/Cas9 with the RNA endoribonuclease Csy4 processing system is an efficient tool to study floral development and improve floral traits rapidly and simply.

Genome-wide investigation of the bZIP transcription factor gene family in Prunus mume:Classification, evolution, expression profile and low-temperature stress responses

Prunus mume is an important woody plant that has high ornamental and economic value, widely distributed and used in landscape architecture in East Asia. In plants, basic(region) leucine zipper(bZIP) transcription factors play important regulatory roles in growth, development,dormancy and abiotic stress. To date, bZIP transcription factors have not been systematically studied in P. mume. In this study, 49 bZIP genes were first identified in P. mume, and the PmbZIP family was divided into 12 groups according to the grouping principles for the Arabidopsis thaliana bZIP family. For the first time, we constructed a detailed model of the PmbZIP domains(R-x_(3)–N-(x)_7-R/K-x_(2)-K-x_(6)-L-x_(6)-L-_(6)-L). Phylogenetic and synteny analyses showed that PmbZIPs duplication events might have occurred during the large-scale genome duplication events. A relatively short time of speciation and the finding that 91.84% of the bZIP genes formed orthologous pairs between P. mume and Prunus armeniaca provided evidence of a close relationship. Gene expression patterns were analysed in different tissues and periods, indicating that PmbZIP genes with the same motifs exhibited similar expression patterns. The gene expression results showed that PmbZIP31/36/41 genes played a more prominent role in the response to freezing stress than cold stress. The expression level of almost all subset Ⅲ genes was upregulated under freezing treatment, especially after cold exposure. We analysed the gene expression patterns of PmbZIP12/31/36/41/48 and their responses to low-temperature stress, which provided useful resources for future studies on the cold/freezing-tolerant molecular breeding of P. mume.

Heritage trees and landscape design in urban areas of Rwanda

Trees play a key role in neighborhood landscapes, a belief that has been widely held for millennia in areas beyond Sub-Saharan Africa. Unfortunately, awareness of modern landscape architecture was almost absent in Rwanda until the late 20th century. Today, houses with sur- rounding decorative and amenity plants ate a common feature in Rwanda＇s neighborhood landscapes and, as the villagization of settle- ments progresses, new kinds of landscapes are emerging. This paper explores neighborhood tree planting around human settlements in the country. Remote sensing, photogrammetry, photo interpretation, and plant surveys were the core methods used. The average tree cover frac- tion ranged between 10%-35%. As the result of what is hereafter referred to as the ＂luxury effect,＂ a discrete gradient was detected along which the diversity of ornamental and amenity trees increases with the socio-economic status of neighborhoods： from rural settlements to urban residences via a series of intermediate designs, in which different levels of human-built vegetated areas alternate with non-landscaped spaces. Showy, non-productive amenity trees tend to occur more in wealthy quarters of the inner core of cities while edible ornamentals and other productive neighborhood trees prevail in rural and spontaneous settle- ments. In general, the practice of landscape plant design, in spite of its constant improvement, is still striving to get established as a profession in the country.

Outlier Detection of Mixed Data Based on Neighborhood Combinatorial Entropy

Outlier detection is a key research area in data mining technologies,as outlier detection can identify data inconsistent within a data set.Outlier detection aims to find an abnormal data size from a large data size and has been applied in many fields including fraud detection,network intrusion detection,disaster prediction,medical diagnosis,public security,and image processing.While outlier detection has been widely applied in real systems,its effectiveness is challenged by higher dimensions and redundant data attributes,leading to detection errors and complicated calculations.The prevalence of mixed data is a current issue for outlier detection algorithms.An outlier detection method of mixed data based on neighborhood combinatorial entropy is studied to improve outlier detection performance by reducing data dimension using an attribute reduction algorithm.The significance of attributes is determined,and fewer influencing attributes are removed based on neighborhood combinatorial entropy.Outlier detection is conducted using the algorithm of local outlier factor.The proposed outlier detection method can be applied effectively in numerical and mixed multidimensional data using neighborhood combinatorial entropy.In the experimental part of this paper,we give a comparison on outlier detection before and after attribute reduction.In a comparative analysis,we give results of the enhanced outlier detection accuracy by removing the fewer influencing attributes in numerical and mixed multidimensional data.

Identification of the PmWEEP locus controlling weeping traits in Prunus mume through an integrated genome-wide association study and quantitative trait locus mapping

Weeping Prunus mume(mei)has long been cultivated in East Asia for its specific ornamental value.However,little is known about the regulatory mechanism of the weeping trait in mei,which limits molecular breeding for the improvement of weeping-type cultivars.Here,we quantified the weeping trait in mei using nested phenotyping of 214 accessions and 342 F 1 hybrids.Two major associated loci were identified from the genome-wide association study(GWAS),which was conducted using 3,014,409 single nucleotide polymorphisms(SNPs)derived from resequencing,and 8 QTLs and 55 epistatic loci were identified from QTL mapping using 7,545 specific lengths amplified fragment(SLAF)markers.Notably,an overlapping PmWEEP major QTL was fine mapped within a 0.29 Mb region on chromosome 7(Pa7),and a core SNP locus closely associated with the weeping trait was screened and validated.Furthermore,a total of 22 genes in the PmWEEP QTL region were expressed in weeping or upright mei based on RNA-seq analysis.Among them,only a novel gene(Pm024213)containing a thioredoxin(Trx)domain was found to be close to the core SNP and specifically expressed in buds and branches of weeping mei.Co-expression analysis of Pm024213 showed that most of the related genes were involved in auxin and lignin biosynthesis.These findings provide insights into the regulatory mechanism of the weeping trait and effective molecular markers for molecular-assisted breeding in Prunus mume.

Metabolite profiling and transcriptome analyses reveal novel regulatory mechanisms of melatonin biosynthesis in hickory

Studies have shown that melatonin regulates the expression of various elements in the biosynthesis and catabolism of plant hormones.In contrast,the effects of these different plant hormones on the biosynthesis and metabolism of melatonin and their underlying molecular mechanisms are still unclear.In this study,the melatonin biosynthesis pathway was proposed from constructed metabolomic and transcriptomic libraries from hickory(Carya cathayensis Sarg.)nuts.The candidate pathway genes were further identified by phylogenetic analysis,amino-acid sequence alignment,and subcellular localization.Notably,most of the transcription factor-related genes coexpressed with melatonin pathway genes were hormone-responsive genes.Furthermore,dual-luciferase and yeast one‐hybrid assays revealed that CcEIN3(response to ethylene)and CcAZF2(response to abscisic acid)could activate melatonin biosynthesis pathway genes,a tryptophan decarboxylase coding gene(CcTDC1)and an N-acetylserotonin methyltransferase coding gene(CcASMT1),by directly binding to their promoters,respectively.Our results provide a molecular basis for the characterization of novel melatonin biosynthesis regulatory mechanisms and demonstrate for the first time that abscisic acid and ethylene can regulate melatonin biosynthesis.

Transcriptome and chemical analyses identify candidate genes associated with flower color shift in a natural mutant of Chrysanthemum × morifolium

Flower color variation in Chrysanthemum×morifolium is an important horticultural trait.This study identifies a natural bud sprout mutant of chrysanthemum cultivar Hanluhong(HLH)which normally produces red flowers.In the mutant(hlh),the tip of the petals turn golden yellow and gradually turn yellow from tip to the base.After two years of consecutive propagation through cuttings,the mutant traits were stable.Here,the contents of carotenoids and anthocyanidins were determined by HPLC-MS/MS.Compared to HLH,the hlh displayed significantly higher contents of lutein.Then we comparatively analyzed the transcriptome of the ray florets tissues during three flower developmental stages.Higher number of stage specific DEGs were found in mutant cultivar as compared to wild type.About 200 DEGs were filtered related to the metabolism of flavonoids,carotenoids,terpenoids,and anthocyanins.Combined with weighted gene co-expression network analysis(WGCNA)and qRT-PCR verification,CHI,DFR,ANS and CCD4 genes involved in anthocyanins biosynthesis and carotenoids degradation were identified as candidate genes.Among the transcription factors,MYB,bHLH and WD40 members showed significant expression variations between the two cultivars.These results improve our understanding of flower color variation especially red and yellow color transition in chrysanthemum.

The genomics of ornamental plants: current status and opportunities

With the rapid development of sequencing technologies,followed by the reduction of sequencing cost,numerous ornamental plants have been sequenced,resulting in their genomic studies shifting from gene cloning and marker development to whole genome profiling.A profound understanding of genome structure and function at the whole genome level can not only help to modify ornamental traits,such as fragrance,color and flower shape,through genetic engineering,but also infer the genetic relationship and evolutionary history of ornamental plants via comparative genomics analysis.In this paper,we review the current situation of sequencing strategies and the application of genomics to study the origin and evolution of ornamental plants.We highlight challenges of ornamental plant genomic research.The use of cutting-edge technologies,such as genomics,gene editing and molecular design polymerization breeding,can facilitate our understanding of genetic regulation mechanisms and the germplasm innovation of important traits in ornamental plants.The results can be expected to significantly increase the breeding efficiency of ornamental plants.

Mapping the genetic architecture of developmental modularity in ornamental plants

Developmental modularity,i.e.,coherent organization and function of developmentally related traits,is an emergent property of organismic development and evolution.However,knowledge about how modular variation and evolution are driven genetically is still limited.Here,using ornamental plants as an example,we propose a computational framework to map,visualize and annotate the genetic architecture of trait modularity by integrating modularity theory into system mapping,a statistical model for multifaceted genetic mapping of complex traits.A developmental module can be viewed as an ecosystem,in which the constituting components compete for space and resources or cooperate symbiotically to organize its function and behavior.This interactive process is quantified by mathematical models and evolutionarily interpreted by game theory.The proposed framework can test whether and how genes regulate the coordination of different but interconnected traits through their competition or cooperation to downstream developmental modularity.

Ti_(3)C_(2)T_(x)-TiSe_(2) analogous heterostructure for flexible zinc ion battery

Exploring zinc-free anode materials is one of the effective strategies to get the zinc dendrites problem of flexible zinc ion battery(ZIB)solved.In this work,an analogous heterostructure(AHS)is constructed from the excellent MXene(Ti_(3)C_(2)T_(x))and TiSe_(2) nanosheets.The AHS not only possesses numerous diffu-sion paths and Zn^(2+)storage sites but also possesses a stable conductive network to accelerate charge transfer in the electrode.As a collaborative advantage,electrochemical measurement results show that MXene/TiSe_(2) electrodes display an excellent specific capacity of 177.9 mAh g^(-1) at 0.10 A g^(-1) and a long-term cycling stability of 77.4%capacity retention after 400 cycles.DFT computations further demon-strate the excellent performance of MXene/TiSe_(2) electrodes including desirable electronic conductivity and low Zn^(2+)migration barriers.The assembled flexible ZIB not only delivers a good specific capacity of 42.2μAh cm^(-2) at 0.20 mA cm^(-2) and a competitive energy density of 37.4μWh cm^(-2) but also exhibits excellent flexibility and thermostability.Furthermore,after 400 cycles at 0.60 A g^(-1),flexible ZIB shows a capacity retention of 73.8%.This work gives a successful attempt to design 2D layered materials as Zn metal-free anode for flexible ZIB.

MXene-enhanced environmentally stable organohydrogel ionic diode toward harvesting ultralow-frequency mechanical energy and moisture energy

With the accelerating advancement of distributed sensors and portable electronic devices in the era of big data,harvesting energy from the surrounding environment to power electrical devices has become increasingly attractive.However,most mechanical energy harvesters often require high operating frequencies to function properly.Moreover,for practical applications,the survivability of devices in harsh operating environments is a vital issuewhich must be addressed.Besides,the single-stimulus responsiveness limits their further applications in complex external environments.Here,a pressure and moisture dual-responsive ionic diode consisting of two organohydrogels with opposite charges as an energy harvester is proposed.The organohydrogel ionic diode utilizes the migration of cations and anions to form the depletion zone and followed by an enhancement of the built-in potential along the depletion zone as a result of mechanical stress or humidity,converting ultralow-frequency mechanical energy or moisture energy into electrical energy.Meanwhile,this mechanism is further confirmed by the finite element analysis.With the increased rectification ratio due to the introduction of MXene,the ionic diode exhibits a relatively large output current(∼10.10μA cm^(−2))and power density(∼0.10μW cm^(−2))at a mechanical pressure of 0.01 Hz,outperforming most currently available mechanical energy harvesters.More impressively,the incorporation of ethylene glycol provides the hydrogel ionic diode with excellent temperature tolerance and long-term environmental stability.The organohydrogel ionic diode can also be applied as a moisture-driven power generator and self-powered humidity sensor.This study presents promising prospects for the efficient collection of renewable and sustainable energy and the practical application of hydrogel-based energy harvesters in extreme environments.

Identification of bioactive anti-angiogenic components targeting tumor endothelial cells in Shenmai injection using multidimensional pharmacokinetics

Shenmai injection(SMI)is a well-defined herbal preparation that is widely and clinically used as an adjuvant therapy for cancer.Previously,we found that SMI synergistically enhanced the activity of chemotherapy on colorectal cancer by promoting the distribution of drugs in xenograft tumors.However,the underlying mechanisms and bioactive constituents remained unknown.In the present work,the regulatory effects of SMI on tumor vasculature were determined,and the potential anti-angiogenic components targeting tumor endothelial cells(TECs)were identified.Multidimensional pharmacokinetic profiles of ginsenosides in plasma,subcutaneous tumors,and TECs were investigated.The results showed that the concentrations of protopanaxadiol-type(PPD)ginsenosides(Rb1,Rb2/Rb3,Rc,and Rd)in both plasma and tumors,were higher than those of protopanaxatriol-type(Rg1 and Re)and oleanane-type(Ro)ginsenosides.Among PPD-type ginsenosides,Rd exhibited the greatest concentrations in tumors and TECs after repeated injection.In vivo bioactivity results showed that Rd suppressed neovascularization in tumors,normalized the structure of tumor vessels,and improved the anti-tumor effect of 5-fluorouracil(5 FU)in xenograft mice.Furthermore,Rd inhibited the migration and tube formation capacity of endothelial cells in vitro.In conclusion,Rd may be an important active form to exert the anti-angiogenic effect on tumor after SMI treatment.