The relationship between mTOR gene variants rs2295080 T/G and rs1883965 G/A and the risk of cancer remains inconsistent. The present study is aimed at comprehensively investigating the association between mTOR polymorphisms and susceptibility to cancer.  Genetic polymorphisms in  We conducted a comprehensive assessment using odds ratios (ORs), corresponding 95% confidence intervals (CIs), and in silico tools to evaluate the effect of mTOR variations. Immunohistochemical staining (IHS) and GSEA analysis were used to investigate the expression of mTOR in urinary system cancer. P = 0.023; GT versus TT, OR = 0.88, 95%CI = 0.81–0.96, P = 0.004; GG+GT versus TT, OR = 0.87, 95%CI = 0.78–0.96, P = 0.008), especially for cancers of the urinary system, breast, and blood. Variation rs1883965 G/A was associated with cancer susceptibility, especially for digestive cancer. IHS analysis showed that mTOR was upregulated in prostate and bladder cancer. GSEA revealed that the insulin signaling pathway, lysine degradation pathway, and mTOR signaling pathway were enriched in the high mTOR expression group.  The pooled analysis involved 22 case-control studies including 14,747 cancer patients and 16,399 controls. The rs2295080 T/G polymorphism was associated with the risk of cancer (G-allele versus T-allele, OR = 0.89, 95%CI = 0.80–0.98,  The mTOR rs2295080 T/G polymorphism may be associated with susceptibility of urinary cancer. The expression of mTOR is positively correlated with tumor malignancy in prostate cancer. Homo sapiens, missing phosphatase and tensin homologs of the phosphoinositide 3-kinase (PI3K)/AKT/mTOR signal transduction pathway on chromosome 10 are often activated in various carcinomas. They are involved in different cellular processes that include cell proliferation, angiogenesis, and tumorigenesis [Malignant tumors are a major global public health problem . Over thigenesis –8. Mutatigenesis –11.The mTOR gene, also known as FKBP12 rapamycin-associated protein (FRAP), acts as an essential serine-threonine kinase in signal transduction and participates in the biological processes of cell cycle, survival, and autophagy . The humPrevious studies have evaluated the association of genetic variants of the mTOR gene with the susceptibility to cancer. Results of a meta-analysis that included five eligible studies indicated that the rs2295080 TT genotype was related to increased cancer risk, but not with poor clinical outcome . AnotherIn silico tools and immunohistochemical staining (IHS) analysis were used to investigate the expression of mTOR in three main urinary system cancers.The current study sought to identify all eligible case-control studies to comprehensively assess the association between mTOR variants rs2295080 T/G or rs1883965 G/A and susceptibility to cancer –49. In sThe PMC, Embase, Chinese National Knowledge Infrastructure, and Google Scholar databases were searched for potentially relevant published studies. The search terms were  AND (“cancer” OR “tumor” OR ”carcinoma”) AND (“mutant” OR “variant” OR “variation”). The most recent search update was 01 March 2021. In addition to the databases, we also screened qualified research by checking references from published articles.Studies meeting the following criteria were included: (a) case-control or cohort studies addressing the association between the variation mTOR rs2295080 T/G or rs1883965 G/A and risk of cancer, (b) sufficient data of genotype frequencies to evaluate ORs and 95% CIs, and (c) articles written in English or other languages. The major exclusion criteria were as follows: (a) lack of a control group, (b) insufficient data to calculate ORs, and (c) no relevance to mTOR rs2295080 T/G or rs1883965 G/A variants and cancer risk.P value of Hardy-Weinberg equilibrium (HWE) in controls, and method of genotyping. In the stratification analysis, acute lymphoblastic leukemia and acute myeloid leukemia were classified as blood cancer. Urinary system tumors include BLCA, PRAD, and KIRC. Carcinomas of the digestive system included esophageal squamous cell carcinoma, gastric cancer, and colorectal cancer. The study included Asian populations and was divided into West Asian and East Asian groups.Study characteristics retrieved from eligible studies included the surname of the first author, publication date, origin of participants, type of cancer, source of control, ethnicity, gene distribution of mTOR polymorphisms, Q statistic test. P < 0.05 indicated statistical significance. A P value of heterogeneity < 0.05 indicated heterogeneity among the studies. A random effects model (DerSimonian and Laird) was conducted to calculate the ORs. Otherwise, a fixed effects model (Mantel–Haenszel) was performed. A P value of HWE was evaluated by Fisher's exact test. Subgroup analyses included the type of cancer, ethnicity, source of control, and genotype method. Begg's funnel plot was adopted to evaluate publication bias. The reliability of the included case-control studies was assessed by sensitivity analysis. STATA 11.0 software  was used for all statistical analyses.ORs and 95% CIs were used to evaluate the strength of association between the mTOR variants rs2295080 T/G or rs1883965 G/A and cancer susceptibility. For the rs2295080 T/G variation, allelic comparison refers to G-allele versus (vs.) T-allele. The heterozygous, homozygous, dominant, and recessive models represent GT vs. TT, GG vs. TT, GG+GT vs. TT, and GG vs. GT+TT. For the rs1883965 G/A variation, these five genetic models were A-allele vs. G-allele, AG vs. GG, AA vs. GG, AA+AG vs. GG, and AA and AG+GG. Heterogeneity of studies was investigated by the https://www.ncbi.nlm.nih.gov/snp). The expression of mTOR in human tissues was evaluated by another database (http://gemini.cancer-pku.cn/). Gene expression profiles of mTOR across various types of cancers and paired normal tissues were also assessed. We also employed online databases to detect the expression of mTOR in three main urinary system cancer, gene-gene connection, and overall survival time . We also used TCGA database and GEPIA databases to explore the expression of mTOR in PRAD based on different molecular signature. STRING tools were used to assess the protein-protein crosstalk of mTOR (https://string-db.org/cgi/input.pl). We conducted IHS analyses to investigate mTOR expression of PRAD participants enrolled in our institute according to general standards [We utilized an online database to investigate the MAFs in global and subpopulations . Similar findings were observed for the heterozygous contrast  and dominant models . For breast cancer, individuals with the G-allele had a 21% lower risk of cancer . Similar results were indicated for homozygous contrast , dominant model , and recessive model . Individuals with the G-allele had a 1.24-fold higher risk of blood cancer . Similar results were found in the homozygous contrast  and recessive model . In stratified analysis by ethnicity, East Asians carrying the G-allele had an 8% lower risk of cancer than those with the T-allele . Similar findings were indicated in population-based studies and using a TaqMan assay method. For the rs1883965 G/A variation, individuals carrying A-allele had a 1.12-fold higher likelihood of cancer than those carrying the G-allele  (P = 0.044), heterozygous , and dominant models . Similar results were confirmed in the population-based studies .The strength of the association between mTOR variation rs2295080 T/G or rs1883965 G/A and susceptibility of cancer is shown in = 0.045) . In subgP < 0.05, Figures P < 0.05, P > 0.05, Figures P < 0.05, P > 0.05, P < 0.05, P > 0.05, P < 0.05, P < 0.05), but not in Asian patients , but not in African-American patients . Evaluation of publication bias through Begg's funnel plots did not indicate significant publication bias for all five genetic models of rs2295080 (P > 0.05) or rs1883965 variants (P > 0.05).Sensitivity analysis was conducted by excluding every single study to evaluate their impact on the overall ORs. As described in Figures As a main controller of cell proliferation, mTOR participates in a variety of synthetic metabolic processes including lipogenesis, protein synthesis, and nucleotide biosynthesis. mTOR also inhibits catabolic processes including lysosomal biogenesis and autophagy. Inhibitors of the mTOR signaling pathway have been developed to treat some types of malignant tumors , 53. PreSeveral previous publications have assessed the association of mTOR variants rs2295080 T/G or rs1883965 G/A and susceptibility to cancer. However, the sample size of the included studies was insufficient , 29. In For the rs2295080 T/G SNP, a stratified analysis by cancer type revealed that the T-allele is a risk factor for urinary and breast cancer. This result is consistent with a recent published meta-analysis . HoweverConcerning mTOR expression in urinary cancer based on race, downregulated expression was evident in KIRC patients who were Caucasian, African-American, and Asian. For BLCA, the expression of mTOR was upregulated in Caucasians and African-Americans, but not in Asians. For PRAD, mTOR expression was upregulated in Caucasian patients, but not in African-American patients. The expression profiles of mTOR in Asian PRAD patients could not be acquired from the online database. We further used IHC analysis to investigate the mTOR expression in PRAD participants enrolled from our centers. Compared with paracancerous tissues, the expression of mTOR was upregulated in advanced PRAD.The present study has several limitations. First, according to the inclusion criteria, no case-control study on mTOR rs2295080 T/G and rs1883965 G/A polymorphism was included based on African or Caucasian populations. Further studies on African and Caucasian populations with various tumors are warranted. Second, the sample size of eligible studies for the rs1883965 G/A SNP was insufficient. Studies on many types of cancer including testicular cancer, thyroid carcinoma, thymoma, and lymphoma are very limited. Third, upregulated mTOR expression in advanced PRAD was based on IHS analysis. Further studies are needed to demonstrate whether the mTOR rs2295080 T/G or rs1883965 G/A mutations could affect the expression of mTOR in PRAD. The pathogenesis of cancer is complex, and it is not possible that a single mutation would have a huge impact on the progression. As described in In summary, the present study summarized all eligible data for the genetic relationship between the mTOR variants rs2295080 T/G or rs1883965 G/A and susceptibility to different cancers. Our results revealed that rs2295080 T/G polymorphism was associated with susceptibility of urinary cancer, especially in East Asians. The expression of mTOR was upregulated in BLCA and PRAD patients. GSEA revealed that the insulin signaling pathway, lysine degradation pathway, and mTOR signaling pathway were enriched in the high mTOR expression group. The expression of mTOR was positively correlated with tumor malignancy in prostate cancer subjects.


Crataegus bretschneideri Schneid., with an unclear phylogenic position, is mainly distributed in northeast and inner mongolia area of China. In this study, the complete chloroplast genome sequence of C. bretschneideri was determined by using Illumina high-throughput sequencing method. The chloroplast genome was 159,607 bp in length and consisted of a large single-copy (LSC) region , a small single-copy (SSC) region , separated by a pair of inverted repeat  regions. It comprised a total of 114 unique genes, including 80 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. Phylogenetic analysis based on complete chloroplast genomes indicated that C. bretschneideri was closely related to C. marshallii Eggl in the subfamily Maloideae. This complete chloroplast genome will provide valuable insight into evolution, molecular breeding, and phylogenetic analysis of Crataegus species. Crataegus (hawthorn), a member of the Rosaceae family, is widely distributed throughout temperate regions in the Northern Hemisphere including Eurasia and North America  markers, some researchers suggest that C. bretschneideri is closely related to C. pinnatifida , which would be helpful for evolution, phylogenetic analysis and molecular breeding.Chloroplast genomes are important sources for taxonomic classification and phylogenetic reconstruction of plant species  in Beijing, China. A specimen was deposited at the Herbarium of Beijing Academy of Forestry and Pomology Sciences  under the voucher number BJLGY-2020-SZ002. The total genomic DNA from leaves was extracted using a modified CTAB method  as a reference, and the selected contigs were assembled using Sequencher 4.10 (https://www.genecodes.com/) software tools. Annotation was performed using the Plann  region (87601 bp), a small single-copy (SSC) region (19312 bp), separated by a pair of inverted repeat  regions. The total GC content of complete chloroplast genome, LSC, SSC, IR regions were 36.6%, 34.4%, 30.3% and 42.7%, respectively. The chloroplast DNA of C. bretschneideri comprised a total of 114 unique genes, including 80 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. In these genes, 19 genes were duplicated in the IR regions, 15 genes harbored a single intron, and 2  contained double introns.The cp genome of C. bretschneideri, total 31 complete chloroplast genomes were obtained from Genbank and the sister group Prunoideae was taken as an out group. All chloroplast genome sequences were aligned using MAFFT (Katoh et al. C. bretschneideri was closely related to C. marshallii Eggl, rather than C. pinnatifida in the subfamily Maloideae (C. bretschneideri is a distinct Crataegus species, rather than a variant of C. pinnatifida. The phylogenetic tree can provide reference for the parent selection in hawthorn breeding programme. This complete chloroplast genome can be used for future studies on genetic engineering, population and phylogeny of family Rosaceae.To clarify the phylogenetic position of aloideae . This su


The Orchidaceae family is one of the most diverse among flowering plants and serves as an important research model for plant evolution, especially “evo-devo” study on floral organs. Recently, sequencing of several orchid genomes has greatly improved our understanding of the genetic basis of orchid biology. To date, however, most sequenced genomes are from the Epidendroideae subfamily. To better elucidate orchid evolution, greater attention should be paid to other orchid lineages, especially basal lineages such as Apostasioideae.Apostasia ramifera, a terrestrial orchid species from the Apostasioideae subfamily. The genomes of A. ramifera and other orchids were compared to explore the genetic basis underlying orchid species richness. Genome-based population dynamics revealed a continuous decrease in population size over the last 100 000 years in all studied orchids, although the epiphytic orchids generally showed larger effective population size than the terrestrial orchids over most of that period. We also found more genes of the terpene synthase gene family, resistant gene family, and LOX1/LOX5 homologs in the epiphytic orchids.Here, we present a genome sequence of A. ramifera genome sequence reported here should be a helpful resource for future research on orchid biology.This study provides new insights into the adaptive evolution of orchids. The The online version contains supplementary material available at 10.1186/s12864-021-07852-3. The Orchidaceae family is one of the largest among flowering plants, with many species exhibiting great ornamental value due to their colorful and distinctive flowers. At present, there are more than 28 000 orchid species assigned to 763 genera . AccordiPhalaenopsis equestris) showed evidence of an ancient whole-genome duplication event in the orchid lineage and revealed that expansion of MADS-box genes may be related to the diverse morphology of orchid flowers [Dendrobium officinale, Dendrobium catenatum, Phalaenopsis aphrodite, Apostasia shenzhenica, and Vanilla planifolia, has provided data for further investigations on the genetic mechanisms underlying orchid species richness [The genome sequences of several orchid species have been published recently, thereby greatly improving our understanding of orchid biology and evolution. The first reported orchid genome  genome reported for the Apostasioideae subfamily, with most currently published genomes belonging to the Epidendroideae subfamily. Obtaining genomes for other orchid lineages, especially basal lineages, will greatly facilitate our understanding of orchid evolution. Here, we performed de novo assembly and analysis of the Apostasia ramifera genome sequence, the second Apostasia genome after A. shenzhenica. Comparative genomics were carried out with six other published orchid genomes to provide insight into orchid evolution.The Apostasioideae subfamily consists of terrestrial orchid species . SpeciesA. ramifera was sequenced using the Illumina Hiseq 2000 platform. Sequencing of five libraries with different insert sizes ranging from 250 to 5 000 bp generated more than 57 Gb of clean data, accounting for 156X of the genome sequence  but less than that in P. equestris (62 %) and D. catenatum (78 %). Among the repetitive sequences, transposable elements (TEs) were the most abundant (43.1 %), among which long terminal repeats (LTR) were dominant, accounting for 24.07 % of the total genome  but less than that in V. planifolia (28 279), P. equestris (29 545), and D. catenatum (29 257) , and were significantly enriched in several pathways, such as ‘Ribosome biogenesis in eukaryotes’ (ko03008), ‘mRNA surveillance pathway’ (ko03015) and ‘Plant-pathogen interaction’ (ko04626) , Glycerophospholipid metabolism (ko00564), ‘Flavin adenine dinucleotide binding’ (GO:0050660), and ‘UDP-N-acetylmuramate dehydrogenase activity’ (GO:0008762) , we identified 30 putative MADS-box genes in the A. ramifera genome, fewer than that detected in the other sequenced orchids  [V. planifolia  [D. catenatum (35) [P. equestris, there were fewer members in the A-class, B-class, E-class, and AGL6-class in A. ramifera and V. planifolia , P. aphrodite (109), D. officinale (172), D. catenatum (182), and V. planifolia (86)  Fig. . Thus, tera Fig. .Apostasia, in addition to the small R gene family size, we also discovered lower copy numbers in both the NAC and WRKY gene families    Fig. . We also83) Fig. .LOX1 and LOX5 are involved in the development of lateral roots in Arabidopsis, and loss of these two genes causes a significant increase in lateral root emergence [LOX1 and LOX5 in six published orchid genomes using protein sequences from Arabidopsis as the query, and then constructed a gene tree to elucidate the phylogenetic relationship among these genes. We detected multiple copies of LOX1/LOX5 homologs in the epiphytic orchid genomes  were compared among different orchid lineages.With worldwide distribution, orchids are one of the largest flowering plant families and their extraordinary diversity provides an excellent opportunity to explore plant evolution. Certain evolutionary adaptations in orchids, e.g., pollinium, labella and epiphytism, are proposed to have played key roles in their adaptive evolution and radiation. However, the genetic basis underlying those innovations remains incompletely known. In the current study, we sequenced the genome of B- and E-classes of type II MADS genes in Apostasia and Vanilla. Genes in these classes of type II MADS are involved in floral development [Apostasia flowers, which exhibit radial symmetry and no specialized labellum [B- and E-classes of the type II MADS family were also found in V. planifolia, which has bilaterally symmetrical flower petals and a specialized labellum. These results indicate that members in the B- and E-classes may not contribute to the different flower morphologies found among Apostasioideae and other orchids.Compared with other orchids, we found smaller gene families in the elopment . Furtherlabellum . HoweverMIKC* family are involved in pollen development [MIKC* P-subclass member in the A. ramifera genome. Furthermore, P- and S-subclasses members of MIKC* were identified in A. shenzhenica, while P-subclass genes were lost in P. equestris [P-subclass genes is associated with the evolution of pollinia [P- and S-subclass members have been identified in the genome assembly of P. aphrodite [V. planifolia  . Root ar orchids . Among taenopsis . In landaenopsis  reportedractions , 32, thebidopsis , we propde novo assembly and analysis of the genome of A. ramifera, a terrestrial orchid from the Apostasioideae subfamily. We revealed the population size histories of different orchid species and discovered a continuous decrease in population size from the genomes of these species over the last 100 000 years. In addition, the gene family size and subfamily architecture of TPS genes varied greatly among species from different orchid subfamilies, which may be associated with the adaptive evolution of orchids. Genes associated with pathogen resistance were significantly reduced in the genomes of Apostasia compared with that of other orchids. In Apostasia, we also found genes that were likely involved in the regulation of lateral root development, which is an important trait for terrestrial growth. The A. ramifera genome sequence reported here should be an important resource for further investigations on orchid biology. Comparative genomics analysis of A. ramifera and other orchids should provide new insights into the adaptive evolution of these species.In this study, we performed A. ramifera samples were collected from Jianfeng Mountain, Hainan Province, China. No permission was required to collect these samples. The formal identification of plant material was conducted by Prof. Zhongjian Liu. A voucher specimen of the material was deposited at the National Orchid Conservation Center of China under deposition number Liu.JZ6475. For genome sequencing, we collected fresh leaves from A. ramifera. Extraction of genomic DNA was carried out using the modified cetyltrimethylammonium bromide protocol [Apostasia genome.The protocol . Five DNA. ramifera was 332.24 Mb according to k-mer frequency distribution. Only one peak was observed in the k-mer distribution, indicating high homozygosity of the Apostasia genome. For genome assembly, SOAPdenovo2 [de novo genome assembly.The estimated genome size of Pdenovo2  was usedA. ramifera genome, tandem repeat sequences were identified by TRF software [de novo prediction. Briefly, RepeatMasker [Apostasia genome with a RepBase-derived library of known repeat elements. For de novo prediction, we used RepeatModeler and LTR-FINDER [de novo repetitive element library for the A. ramifera genome. RepeatMasker was then applied to search the genome for TEs with the constructed database. Finally, these results were combined, and the redundant sequences were removed to generate a complete repeat annotation.Repeat sequences consist of tandem repeats, such as small and micro-satellite DNA, and interspersed repeats . In the software . Identifsoftware  and de natMasker  and RepeatMasker  were appR-FINDER  to constV. planifolia draft genome [A. ramifera and V. planifolia genomes. Firstly, we used AUGUSTUS [de novo predicted gene sets for our assembly and the V. planifolia genome (BioProject: PRJNA507095). Protein sets derived from five plant genomes, including Arabidopsis thaliana, Phalaenopsis equestris, Oryza sativa, Sorghum bicolor, and Zea mays, were then applied to search against the Apostasia and Vanilla genomes using TBLASTN with an E-value cutoff of 1e-5 and minimum query coverage of 25 %. GeneWise [V. planifolia were downloaded from NCBI SRA, and were de novo assembled by Trinity software. Vanilla transcripts were applied to annotate the V. planifolia genome using the PASA program. The annotation results derived from different methods were then integrated to generate integrated protein-coding gene sets for A. ramifera and V. planifolia with the MAKER [Because previously published work on the t genome  did not AUGUSTUS  and GlimAUGUSTUS  to generGeneWise  was usedhe MAKER  program.Apostasia genome [Arabidopsis.Non-coding RNAs do not translate into protein sequences but exert significant roles in cellular metabolism, and include microRNAs (miRNAs), transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), and small nuclear RNAs (snRNAs). Here, we applied previously described methods to search for non-coding RNAs in the a genome . The miRa genome  and the a genome . Genes eApostasia genomes was performed by searching their protein-coding regions against sequences derived from publicly available databases, including Gene Ontology (GO) [Functional analysis of the predicted genes in the ogy (GO) , 47, Kyoogy (GO) , SwissProgy (GO) , TrEMBL ogy (GO) , non-redogy (GO) .P. equestris, P. aphrodite, D. officinale, D. catenatum, A. shenzhenica, A. officinalis, and O. sativa, as well as the predicted protein sequences from A. ramifera. To limit the disturbance of alternative splicing variants on gene family clustering, the longest transcript of each gene was selected for analysis. Gene families in which the number of genes from Apostasia (including A. ramifera and A. shenzhenica) was 1.5 times higher than that from other orchids were considered expanded in Apostasia.Gene family clustering was conducted using OrthoFinder with comA. ramifera and 15 other species, including 11 monocots , three eudicots , and the outgroup Amborella trichopoda. Protein sequences that contained less than 50 amino acids were removed from the constructed dataset. The pairwise similarities between protein sequences were calculated through all-against-all BLASTP with cutoff criteria: i.e., (i) E-value < 1e-5, (ii) query coverage > 30 %, (iii) alignment identify > 30 %. The results were then entered into OrthoMCL [O. sativa and B. distachyon divergence time (40–54 million years ago) [P. trichocarpa and A. thaliana divergence time (100–120 million years ago) [To build a high-confidence phylogenetic tree, we constructed a multi-species protein set containing protein sequences from OrthoMCL  (v2.0.9)OrthoMCL  with theOrthoMCL  was usedars ago) , (ii) P.ars ago) , (iii) lars ago) , and (ivars ago) . Gene faA. ramifera using the BWA tool [− 8.Identification of heterozygous loci was performed via a previously described method . BrieflyBWA tool . DuplicaBWA tool  was usedBWA tool . The parMADS-box (Pfam Accession: PF00319), TPS (Pfam Accession: PF01397 and PF03936), NAC (Pfam Accession: PF02365), WRKY (Pfam Accession: PF03106), and R (Pfam Accession: PF00931) genes using HMMER [MADS-box genes in A. thaliana reported in [MADS-box genes identified in A. ramifera and V. planifolia. EvolView [NAC, WRKY and R gene families for the selected species. For the TPS genes, the protein sequences that possessed both Pfam domains and contained more than 500 amino acids were considered as functional genes and used for further analysis. To identify LOX genes, protein sequences of the LOX gene family in A. thaliana  were used to search for homologous genes in orchids. The identified protein sequences of each gene family were aligned using MUSCLE [The hidden Markov model profiles  were appng HMMER  v3.2.1).2.1MADS-EvolView  was usedg MUSCLE  was thenAdditional file 1.


Polygonatum kingianum Coll. et Hemsl. is an important plant in Traditional Chinese Medicine. The extracts from its tubers are rich in polysaccharides and other metabolites such as saponins. It is a well-known concept that growing medicinal plants in semi-arid (or drought stress) increases their natural compounds concentrations. This study was conducted to explore the morpho-physiological responses of P. kingianum plants and transcriptomic signatures of P. kingianum tubers exposed to mild, moderate, and severe drought and rewatering.P. kingianum tuber experiences an increased biosynthesis of sucrose, starch, and carotenoid under drought stress. Drought decreases the flavonoids, phenylpropanoids, gingerol, and lignin biosynthesis. These changes can be reversed by rewatering the P. kingianum plants.The stress effects on the morpho-physiological parameters were dependent on the intensity of the drought stress. The leaf area, relative water content, chlorophyll content, and shoot fresh weight decreased whereas electrolyte leakage increased with increase in drought stress intensity. A total of 53,081 unigenes were obtained; 59% of which were annotated. We observed that 1352 and 350 core genes were differentially expressed in drought and rewatering, respectively. Drought stress driven differentially expressed genes (DEGs) were enriched in phenylpropanoid biosynthesis, flavonoid biosynthesis, starch and sucrose metabolism, and stilbenoid diarylheptanoid and gingerol biosynthesis, and carotenoid biosynthesis pathways. Pathways such as plant-pathogen interaction and galactose metabolism were differentially regulated between severe drought and rewatering. Drought reduced the expression of lignin, gingerol, and flavonoid biosynthesis related genes and rewatering recovered the tubers from stress by increasing the expression of the genes. Increased expression of carotenoid biosynthesis pathway related genes under drought suggested their important role in stress endurance. An increase in starch and sucrose biosynthesis was evident from transcriptomic changes under drought stress. Rewatering recovered the drought affected tubers as evident from the contrasting expression profiles of genes related to these pathways. P. kingianum and expands the knowledge on the effect of drought and rewatering on important pathways. This study also provides a large number of candidate genes that could be manipulated for drought stress tolerance and managing the polysaccharide and secondary metabolites’ contents in P. kingianum.These results provide a transcriptome resource for The online version contains supplementary material available at 10.1186/s12870-021-03297-8. Polygonatum kingianum Coll. et Hemsl. is one of the 71 species included in the family Liliaceae and is grown at the elevation of 700–3600 m. In China, it is distributed in Yunnan, Sichuan, Guangxi, and Guizhou provinces; it has generally a southern distribution [P. kingianum is frequently used for the treatment of muscle fatigue, feebleness, osteoporosis, spleen qi deficiency, stomach yin deficiency, dry cough, blood deficiency, age-related diseases, and diabetes owing to the presence of bioactive compounds [Polygonatum species are used in drinks, snacks, vegetables, staple food, and seasoning in China [P. kingianum rhizome are abundant in alkaloids, flavones, steroid saponins, lignins, amino acids, and polysaccharides [ribution . The Polribution . In the in China . The mosin China . The extcharides . Particucharides , 11.It is a well-known concept that medicinal plants grown under semi-arid conditions result in an increased concentration of medicinally important natural products . The envP. kingianum plants and it is possible that the starch and sucrose metabolism and related pathways might be affected.Drought stress impairs different morphological, physiological, and biochemical traits in plants. Visible effects can be seen by observing the morphology of the plants. Most commonly, it has been reported that drought stress significantly reduces the leaf area, and causes early maturity as well as changes in root and shoot length . The redCapparis spinosa, Ammopiptanthus mongolicus, Eleusine coracana, and Opisthopappus taihangensis explored the effect of drought on plant and/or on the biosynthesis of polysaccharides and secondary metabolites [Polygonatum sibiricum, and P. kingianum have aided in the discovery of genes involved in polysaccharide and steroidal saponins biosynthesis [P. kingianum tubers in response to drought stress and rewatering. Considering the medicinal importance of P. kingianum, the effect of drought stress tolerance on the essential pathways related to polysaccharides and secondary metabolites is highly needed. This knowledge will answer the questions if P. kingianum is affected by drought in similar ways as the widely accepted concept of increasing natural component concentration under stress conditions?Advancements in genomics have enabled the large-scale transcriptome profiling of essential genetic resources that are of commercial and medicinal importance. Researchers are increasingly exploring the species with medicinal importance to understand the effect of drought on secondary metabolites and other related pathways. Recently, transcriptome analyses of abolites –26. Simiynthesis , 28. HowP. kingianum Coll. et Hemsl. which is a red-flower bearing genotype, captures 70% of P. kingianum market, and cultivated over 4000 ha in China. We compared the transcriptome of P. kingianum tubers grown under mild, moderate, and severe drought. We also compared the transcriptomic response of drought-affected P. kingianum tubers with the rewatered tubers and explored the differentially expressed genes and pathways.In this study, we have explored the transcriptomic response of P. kingianum plants are differently affected by the intensity of drought i.e., the effects were severe to less sever for Z2 to Z6 plants.Our results demonstrated that the plants challenged with the different levels of drought responded differently to the applied stress. The plants showed successive drying, wilting, and yellowing of the leaves from Z6, Z4, to Z2 as compared to Z8. The rewatered plants (ZF) showed slight recovery Fig. . The leaP. kingianum tuber transcriptome statistics is presented in Table The transcriptome sequencing of 15 samples belonging to four treatments and control resulted in a total of 98.89 Gb clean data (5.99 Gb/sample with Q30 base percent of 93.74%). The summary of the P. kingianum tubers. Top significantly enriched pathways between Z8 and Z6 were photosynhthesis, flavonoid biosynthesis, starch and sucrose metabolism, photosynthesis-antenna proteins, stilbenoid, diarylheptanoid and gingerol biosynthesis, and phenylpropanoid biosynthesis. Genes that were differentially expressed between Z8 and Z4 were enriched in the same pathways except for starch and sucrose metabolism. Under extreme drought i.e. Z2, we noticed the enrichment of DEGs in phenylpropanoid biosynthesis, photosynthesis, flavonoid biosynthesis, sphingolipid metabolism, carotenoid biosynthesis, phenylalanine metabolism, and stilbenoid, diarylheptanoid and gingerol biosynthesis pathways  had the highest number of differentially expressed genes (DEGs) 5176) as compared to the drought treatments Z4 5,023), Z6 , and Z2   were differentially expressed between drought treatment Z4 and rewatering (ZF) followed by 4002 and 3147 DEGs in Z6vsZF and Z2vsZF, respectively , cinnamoyl-coA reductase , and caeffeoylshikimate esterase  were downregulated in drought treated tubers. The cinnamyl-alcohol dehydrogenases were downregulated in drought stress except for one gene . Suggesting differential regulation of the same genes under the same conditions. Fifteen peroxidases (PODs) were downregulated in at least one drought treatment. Many other genes  3′-monooxygenase, c166621.graph_c0; ferulate-5-hydroxylase (FAH), c154189.graph_c0; norbelladine O-methyltransferase, c144288.graph_c0; peroxiredoxin 6, 1-Cys peroxiredoxin, and c167521.graph_c0; trans-cinnamate 4-monooxygenase), and shikimate O-hydroxycinnamoyltransferases (HSTs) also showed downregulation in drought treatment  after rewatering. Suggesting their function returned to normal in ZF. Beta-glucosidases and cinnamyl-alcohol dehydrogenases showed variable expression in rewatered tubers similar to drought stresses. These observations clearly indicate that overall drought stress has negative effect of the phenylpropanoid biosynthesis pathway, and rewatering recovers the expression of many genes in this pathway Fig. .c107223.graph_c0), which only showed upregulation in Z6. None of the genes were differentially regulated between Z2 and ZF. These expression patterns suggest that although drought significantly affected this pathway, rewatering had a limited role in the modification of the expression of these genes  was downregulated in rewatered tuber. These genes convert generyl-generyl-pp into pre-phytoene-pp, which is then converted to phytoene [Nineteen DEGs were enriched in the carotenoid biosynthesis pathway. Three of four (+)-abscisic acid 8′-hydroxylases were downregulated in drought indicating the decreased production of phasic acid in drought stress. One (+)-abscisic acid 8′-hydroxylase was upregulated and one was downregulated in the rewatered tuber. Six 15-cis-phytoene/all-trans-phytoene synthases (PSYs) were upregulated in at least one drought treatment. A 15-cis-phytoene/all-trans-phytoene synthase (phytoene . Therefoc107223.graph_c0). Another notable observation was the rewatering driven upregulation of a chalcone synthase   was upregulated in drought and downregulated in rewatered tubers, while others were downregulated in at least one drought treatment and upregulated in rewatered tubers. A similar expression pattern was observed for calmodulin gene family members where most showed downregulation in drought and upregulation in rewatered plants, while only one gene (c149788.graph_c0) was upregulated in mild drought stress. Three WRKY33s were expressed; one was downregulated in Z4 and two were downregulated in ZF. A flagellin sensing 2 (FLS2) gene was downregulated in all drought treatments. A heatshock protein coding gene was upregulated (c175581.graph_c0) in rewatered plants. Two cyclic nucleotide-gated channels also differentially expressed in Z2 and ZF. Six members of RPS2 (disease resistance protein) were also differentially expressed in at least one drought treatment or rewatered plants. The expression pattern of these genes was variable. Glycerol kinases were downregulated in drought. One respiratory burst oxidase (Rboh) was upregulated in ZF and one was downregulated in Z2 and Z4 , only one (c177133.graph_c0). Similar expression trend was observed for Bzip and C2H2 TFs. Two AP2 , BES1 , DBB , ERF (c178049.graph_c1), GRAS (c178743.graph_c0), HD-ZIP (c178388.graph_c1), MYB (c173691.graph_c0), NAC (c170859.graph_c0), and a WRKY (c162517.graph_c0) were specifically upregulated in Z6 as compared to Z8. Overall, we observed that 26 TFs were differentially expressed between the drought treated tubers as compared to Z8. Of these, two were upregulated in drought conditions as compared to Z8 (c175445.graph_c0 and c162568.graph_c0), while rest of the 24 TFs were downregulated in drought treated tubers. These changes suggest that drought severely affect the expression of bHLH, bZIP, C2H2, C3H, GATA, HSF, M-type MADs, and WRKYs. Other than these, we observed that twenty-six TFs were specifically differentially expressed between severe drought treated and rewatered plants  of which only 127 were differentially expressed in the four comparisons i.e., Z8 vs Z2, Z8 vs Z4, Z8 vs Z6, and Z2 vs Z8. These TFs belong to 33 different families Fig. . In part2 = 0.77). Overall, the qRT-PCR expression followed the expression pattern of the transcripts, thus confirming the reliability of the transcriptome sequencing.We validated the reliability of the transcriptome sequencing by determining the expression of ten randomly selected genes Fig. . The genP. kingianum plants and specifically on the tubers. A recent study reported that drought might effect rhizome bud dormancy [P. kingianum plants as visible from the reduction in leaf area, relative water content, shoot fresh area, and chlorophyll contents . It is directly associated with temperature, light, and rainfall dynamics in an ecosystem . Limiteddormancy  but the nts Fig. . These cm leaves . The ovee extent . Based oP. kingianum tubers  in other drought treatments is indicative that starch is being degraded in response to drought stress for providing energy. On the other hand, the upregulation of sucrose synthases is indicative of the increase sucrose synthesis under drought conditions. This is a common metabolic response to drought in drought-tolerant genotypes as previously noted in wheat [P. kingianum tubers restrict the sucrose synthesis from D-fructose-6P and instead synthesize sucrose from UDP-glucose by upregulating three of four sucrose synthases. We say this because of the restriction of UDP-glucose degradation to trehalose by the downregulation of trehalose 6-phosphate synthase/phosphatases. This further suggests that in P. kingianum, Suc-Tre6p nexus model exists and regulates sucrose levels in tuber [P. kingianum plants would shed detailed light on the signals and negative feedback regulating agents in this process. Further, it might be the case that the breakdown of cellulose and subsequent biosynthesis of D-glucose was restricted in drought treated plants and returned to normal after rewatering. We suggest this based on the changes in the expression of cellulases and beta-glucosidases and their respective positions within the pathway  [P. kingianum Rboh is suppressed under drought stress, due to which downstream processes such as cell wall reinforcement are affected [Changes in the cell wall are a common response to biotic and abiotic stresses –71. CDPKed roles . Anotheridopsis)  and estaaffected .Pseudomonas syringae [P. kingianum tubers [The downregulation of FLS2 in response to drought stress in Z6, Z4, and Z2 is interesting since FLS2 is known to regulate the immune responses against biotic stresses . Similarsyringae . Future m tubers . Among om tubers .P. kingianum tubers. However, their downregulation in specific to drought treatment in P. kingianum tubers should be further explored since earlier reports proposed that these TFs are activated in plant tissues when under stress. In this regard the upregulation of one bHLH (c176124.graph_c0) could propose its role in defense against the mild drought stress. Similarly, the increased expression of c178195.graph_c0 in severe drought as compared to Z8 and downregulation in rewatered tubers is indicative of their known function as drought stress tolerance regulators [P. kingianum plants. Mild drought in P. kingianum tubers is possibly under the regulation of multiple TFs . There nologies .P. kingianum tubers grown in mild, moderate, and severe drought, and rewatering showed that drought significantly affects lignin, gingerol, and flavonoid biosynthesis. Rewatering of the drought-affected P. kingianum recovers the tubers from drought effects by showing contrasting gene expression profiles. PALs, COMTs, CSE, 4CLs, HSTs, PODs, and CCRs are important target genes for higher lignin production in P. kingianum tubers. Phenylpropanoids biosynthesis in general and Gingerol biosynthesis in specific are negatively affected by drought stress. Carotenoids play an essential role in defense against drought stress in this species. Our transcriptome results propose that P. kingianum increased the biosynthesis of starch and sucrose, and Suc-Tre6p model operates in tubers to regulate sucrose levels under drought stress. Several genes such as FLS2, CMLs, CDPKs, Rboh, and RPS2 that are associated with plant-pathogen interaction are negatively regulated by drought stress. To enhance drought stress tolerance in P. kingianum, genes associated with plant-pathogen interaction, starch and sucrose biosynthesis, and carotenoid biosynthesis pathways are prime targets for specific characterization and genetic modification for drought tolerance. For secondary metabolite and the polysaccharide biosynthesis, genes discussed in phenylpropanoid biosynthesis pathway, starch and sucrose biosynthesis, flavonoid biosynthesis, and galactose biosynthesis should be targeted. Future studies may also elaborate how these pathways jointly work to enable P. kingianum tubers in specific and whole plants to cope with drought stress. In this regard, gene co-expression networks and protein-protein interaction studies will be useful together with candidate gene specific characterizations.The transcriptome comparison of P. kingianum Coll. et Hemsl. plants were grown at the research station of Yunnan University of Traditional Chinese Medicine, Kunming, China. The plant material was obtained from Yunnan University of Traditional Chinese Medicine and the formal identification of the plant material was carried out by Prof Pengzhang Ji. No voucher specimen has been deposited in a genebank and no special permission is needed to study this species. Three years old plants were used for drought stress experiment from January to December 2019. The plants were potted in 40 × 30 cm2 plastic pots filled with the homogenous and sterile mixture of organic fertilizer, humus soil, and sandy soil in a ratio of 2:3:5. The experiment was carried out in a greenhouse to avoid rainy days interference and the average day/night temperature and relative humidity were set 30/22 °C and 60/55%, respectively. Drought stress treatment was applied in three treatments i.e., mild drought (Z6), moderate drought (Z4), and severe drought (Z2). In addition, a control (Z8) treatment was applied for comparison. The soil water content for the four treatments was maintained at 75–80% (Z8), 55–60% (Z6), 35–40% (Z4), and 15–20% (Z2) of the maximum water holding capacity of the potted soil. All other growing conditions were kept standard. Twenty pots per treatment were maintained with one plant per pot. One pot per treatment was kept empty to estimate the amount of evaporated water from the soil surface. Every day once at dusk, the weight of each pot was measured and the moisture content was adjusted according to the treatments [P. kingianum seedlings, a pot was destroyed after 30 and 90 days each and the fresh weight of the seedling was measured, which was used to determine the weighing standard. After completion of drought treatment, tubers were harvested from experimental plants, washed thrice with distilled water, and stored at − 80 °C for further analyses.The red-flower line of eatments . During , et al. [, et al. [Triplicate plants were used for the measurement of morpho-physiological trait evaluation. The leaf area was computed by using LI-COR 3100 leaf area meter  as reported earlier . The rel, et al. . Shoot f, et al. .Total RNA was extracted from the tubers by using Spin Column Plant total RNA Purification Kit . RNA purity check, quantification, and integrity check were done as described previously . FurtherRaw data were first processed through in-house Perl scripts and clean data (clean reads) were obtained. At the same time, Q20, Q30, GC-content, and sequence duplication levels of the clean data were calculated. All the downstream analyses were based on clean data with high quality. These post-sequencing analytical procedures were done as reported earlier . The traGene function annotation was done according to NR (NCBI non-redundant protein sequences); Pfam (Protein family); KOG/COG/eggNOG (Clusters of Orthologous Groups of proteins) , 99; SwiP-value < 0.05 found by DESeq were considered as differentially expressed. The screening conditions for differential genes are |log 2 Fold Change| > = 1, and FDR < 0.05. The overall distribution of FPKM values and the PCC was computed and represented as graph and heatmap, respectively in R (www.r-project.org). We used KOBAS [The levels of gene expression were calculated by RSEM . Differeed KOBAS  softwareed KOBAS .P. kingianum genes were randomly selected to validate the RNA-sequencing results. An Actin7 gene was used as an internal control. The PCR reactions were carried out as reported earlier [Ten  earlier . The pri earlier .Table 2LAnalysis of variance was performed to assess the variation among pairwise samples using the GenStat Statistical Software .Additional file 1: Supplementary Table S1. Differentially expressed genes between treatments Z6 and Z8 in P. kingianum tuber. Supplementary Table S2. Differentially expressed genes between treatments Z4 and Z8 in P. kingianum tuber. Supplementary Table S3. Differentially expressed genes between treatments Z2 and Z8 in P. kingianum tuber. Supplementary Table S4. List of core DEGs that were expressed between drought and control treatments in P. kingianum tuber. Supplementary Table S5. DEGs expressed between extreme drought (Z2) and rewatering (ZF) treatments in P. kingianum tuber.Additional file 2: Supplementary Figure S1. KEGG pathway enrichment with differentially expressed genes between drought treated P. kingianum tubers. The soil water content was Z8 (80%), Z6 (60%), Z4 (40%), and Z2 (20%) of the maximum water holding capacity of the filed soil.


Nephelium lappaceum (N. lappaceum) and Nephelium ramboutan-ake (N. ramboutan-ake) are tropical fruits that gain popularity worldwide due to their tastiness. Currently, their potential to be used as pharmaceutical agents is underestimated. Chronic diseases such as cancer, diabetes and aging have high incidence rates in the modern world. Furthermore, pharmaceutical agents targeting pathogenic microorganisms have been hampered by the growing of antimicrobial resistance threats. The idea of food therapy leads to extensive nutraceuticals research on the potential of exotic fruits such as N. lappaceum and N. ramboutan-ake to act as supplements. Phytochemicals such as phenolic compounds that present in the fruit act as potent antioxidants that contribute to the protective effects against diseases induced by oxidative stress. Fruit residuals such as the peel and seeds hold greater nutraceutical potential than the edible part. This review highlights the antioxidant and biological activities , and chemical contents of different parts of N. lappaceum and N. ramboutan-ake. These fruits contain a diverse and important chemical profile that can alleviate or cure diseases. Nephelium lappaceum and Nephelium ramboutan-ake are lacking.The demand for natural antioxidants is gaining attention because of their high therapeutic value. Antioxidants contribute greatly towards the prevention of disease development. Various studies have shown that antioxidants are able to prevent diseases such as atherosclerotic heart disease , neurodeN. lappaceum and N. ramboutan-ake are tropical fruits in the Sapindaceae family. These trees are mostly located in tropical countries such as Malaysia, Thailand, Indonesia, Singapore, Sri Lanka and Philippines [N. lappaceum has also been cultivated in the warmer region of south China, Taiwan, Australia and Hawaii [N. lappaceum is known as rambutan, and N. ramboutan-ake are known as pulasan by the locals. “Rambutan” is a word originating from the Malay “rambut”, which means “hair”, whereas “pulasan” is a word originating from the Malay “pulas”, means twist [lippines . N. lappd Hawaii . N. lappns twist .N. lappaceum and N. ramboutan-ake can be eaten freshly or be further processed into pickles or jams. The seeds can be roasted and eaten. Apart from that, they also act as traditional medicines. The root decoction of N. lappaceum and N. ramboutan-ake can be used to treat fever. In addition, the leaves and roots can be applied as poultices to relieve headache [N. lappaceum which have been described by locals include vermicide, diarrhoea treatment, and dysentery treatment, while a bark decoction can be used to treat thrush [N. ramboutan-ake is also used as a traditional treatment for scabies and itchiness [Both, headache . Other ft thrush . Accorditchiness .N. lappaceum and N. ramboutan-ake remain unexplored, and should thus receive attention from nutraceutical scientists to discover more potent drug candidates for disease treatments. The bioactive components of fruits and their relative bioactivities serve as a new hope and alternative option to solve the current disease dilemma faced by the medical field. Antioxidants present in fruits have the capability of reducing the risk of developing chronic diseases such as cancer, pathogenic disease, diabetic and aging [Local traditional treatments and food therapy have led to an increased interest in their potential therapeutic effects. The therapeutic effects and bioactivities of fruits and vegetables are mainly contributed by phytochemicals which come from phenolic compounds . Most bind aging ,3,4.N. lappaceum and N. ramboutan-ake. It provides new insight for researchers to further study the potential of N. lappaceum and N. ramboutan-ake to act as nutraceuticals.This article reviews the antioxidant activities, bioactivities and chemical profile in different parts of N. lappaceum are ellagitannins, gallotannins, hydroxycinnamic acids, hydroxybenzoic acids and flavonoids (N. lappaceum [N. lappaceum peel [N. lappaceum [N. lappaceum peel was geraniin (397.28 mg/g), followed by ellagic acid (176.99 mg/g), quercetin (167.37 mg/g), rutin (36.40 mg/g) and corilagin (3.81 mg/g) [N. lappaceum peel [N. lappaceum peel, there is great potential in developing supplements from its peel.The major chemical compounds that are present in the peel of avonoids . Geraniiappaceum . On the eum peel . Hydroxyappaceum . Specifi81 mg/g) . Anthocyeum peel . Pro-viteum peel . Small aeum peel . Given t50 cut off value of geraniin- and geraniin-enriched extracts (2000 mg/kg body weight) had no significant adverse effects to rat’s body weight, water intake, histopathological, haematological and biochemical metabolites. However, a study found that rats experienced diarrhoea and the histopathology stain of the hepatocyte was observed to be ‘foamy’ [N. lappaceum because of its potential to cause liver abnormalities.The oral toxicity of geraniin- and geraniin-enriched extracts revealed that the LD ‘foamy’ . Hence, N. lappaceum are made up of crude fat (37.1–38.9%), protein (11.9–14.1%) and fibre (2.8–6.6%), with a moisture content of 34.1–34.6% and an ash content of 2.6–2.9% [The seeds of 2.6–2.9% . However2.6–2.9% .The major components and composition of the fat have been further analysed by many researchers with different proportions of sub-classes reported. The major fatty acids detected are oleic acid and arachidic acid . The perA study reported that a higher total phenolic content (TPC) was detected in peel, while a higher total flavonoid content (TFC) was determined in seeds . The majN. lappaceum is reported to contain sugar, organic acid and vitamin -2nonenal, and nonanal which are the main contributors to fruit aroma [Various vitamins are also present in the pulp, including ascorbic acid, niacin, riboflavin and thiamine. Ascorbic acid (58.29 mg/100 g) is found to be more abundant in the pulp as compared with the peel and seeds. Niacin, riboflavin and thiamine are present in low concentrations in fresh pulp (0.02 to 0.78 mg/100 g) and dried pulp (0.01 to 0.56 mg/100 g) . Howeverit aroma .N. lappaceum have been subjected to in vitro antioxidants activities evaluation  when treated with peel extract. Moreover, positive signs of antioxidant defence systems such as a reduction in MDA and an increase in superoxide dismutase (SOD) and glutathione peroxidase activity (GSH-Px) were demonstrated in different mice tissues [Geraniin is a potent and major antioxidant found in ompounds . The antompounds ,44. Aparompounds . A study tissues .N. lappaceum [Phenolic compounds correspond to the antioxidant properties exerted by fruits. Therefore, the higher the phenolic content in a fruit, the greater the antioxidant activity exerted. Strong positive correlations are reported between TPC and antioxidant activity in appaceum ,39. The appaceum .N. lappaceum as compared with aqueous extract [N. lappaceum were analysed for TPC and antioxidant activities [50 = 4.94 μg/mL) were shown by the methanol extract as compared with other solvents  . This isN. lappaceum and the growing environment of the tree. Cultivar Sichompu shows higher inhibition of ROS formation as compared with cultivar Rongrien [N. lappaceum exhibits higher activity in all antioxidant assays tested when compared with the sweet varieties [Moreover, antioxidant activities and capabilities can be affected by the cultivar of Rongrien . CultivaRongrien . The bitarieties . This isN. lappaceum peel extract has been tested on a cervical cancer cell line (HeLa), breast cancer cell line (MDA-MB-231) and osteosarcoma cell line (MG-63) [N. lappaceum exhibited anti-proliferative effects on breast cancer cell lines and osteosarcoma cancer cell lines. The yellow peel extract of N. lappaceum showed a better anti-proliferative effect on the breast cancer cell line (IC50 = 5.42 μg/mL) and osteosarcoma cell line (IC50 = 6.97 μg/mL) as compared with the red peel extract.The anti-proliferative effect of  (MG-63) . The stu50 = 292 μg/mL) and seeds (IC50 = 305 μg/mL) did not show high anti-proliferative effects on the cancer cell lines [A recent study revealed that the anti-neoplastic effects of methanol peel extract successfully reduced HepG-2 cancer cell line viability via inducing apoptosis with DNA fragmentation and cell shrinkage. Phytochemical analysis showed that the high anti-neoplastic effects of methanol peel extract were contributed by the high phenolic contents such as coumarin, flavonoid, phenols, saponin and tannin. Other compounds, such as alkaloid, carbohydrate, cardiac glycoside, protein, terpenoid and triterpenoid, were also detected in the methanol extract . HoweverN. lappaceum seeds has an inhibitory effect on HIV-1-reverse transcriptase activity and reduces the proteolytic activities of trypsin as well as α-chymotrypsin through disulphide bonds. The isolated trypsin inhibitor is one of the few inhibitors that can stimulate the production of nitric oxide, which acts as an anti-tumour molecule [A trypsin inhibitor extracted from the seed was found to exhibit a dose-dependent anti-tumour effect on breast cancer cell (MCF-7), HepG-2 and nasopharyngeal carcinoma cell lines (CNE-1 and CNE-2) . In addimolecule .Staphylococcus aureus is inhibited by peel extract [Staphylococcus aureus (MRSA) is also inhibited when treated with methanol peel extract (MIC = 0.4 mg/mL) [Candida sp. [Pseudomonas aeruginosa [The anti-microbial activities of the fruit against different microorganisms are shown in  extract ,52,53,544 mg/mL) . Apart fdida sp. ,53, wherruginosa .Escherichia coli (E. coli) [E. coli when treated with ethanolic peel extract with a zone of inhibition of 15 mm [E. coli and Pseudomonas aeruginosa [Most of the studies using methanol peel extracts did not observe inhibitory effects against E. coli) ,53,54. Hruginosa .N. lappaceum most likely contribute to the antimicrobial properties [Anti-bacterial activities are observed to be correlated to the phenolic content. Methanol extract of the peel consists of higher TPC and thus exert better inhibitory effect as compared with seed extract . Furtheroperties .Streptococcus pyogenes, Bacillus subtilis, and S. aureus. Similar to the peel extract, the most sensitive bacterial strain when treated with seed extract is S. aureus. Moreover, there is a weak anti-bacterial activity against E. coli (6.5 mm) and P. aeruginosa (10 mm). This shows that the anti-bacterial activity of the seeds is not solely dependent on the phenolic content but also the protein content.Nonetheless, some anti-microbial activity could be detected in the seed extract . The aquN. lappaceum was found to exhibit a hypoglycemic effect on carbohydrate enzymes such as α-glucosidase and α-amylase in addition to antioxidant activities [N. Lappaceum can be further developed as a supplement for diabetic patients. The bioactive compounds other than geraniin in the peel also carry hypoglycemic actions. The peel extract shows better inhibition activities of α-glucosidase (ic50 = 0.106 µg/mL) and β-glucosidase (ic50 = 7.02 µg/mL) than geraniin. A-glucosidase breaks down starch to glucose, which contributes to high glucose levels. Thus, the inhibition of α-glucosidase is one of the approaches to induce hypoglycemic. The hypoglycemic actions are reported to have strong positive correlation with radical scavenging activity of the peel extract [Geraniin extracted from the peel of tivities . Apart f extract .N. lappaceum were studied in vivo, in which the reduction in α-glucosidase and α-amylase enzyme activities were observed [N. lappaceum extract showed signs of anti-hyperglycemia such as reduction in blood glucose level and improvement of glucose tolerance. Organ damage such as to the liver, pancreas and kidney was not observed in diabetic rats treated with N. lappaceum peel extract [The hypoglycemic actions of observed ,61. More extract ,61. This extract .N. lappaceum [50 = 38.88 μg/mL) and pulp (IC50 = 43.80 μg/mL) extracts [Anti-aging properties have been discovered in the peel as well the pulp of appaceum . A studyextracts .A pre-treatment with peel extract on HepG-2 cell lines observed a reduction in apoptosis. Clear attenuations of liver and kidney damage were also observed in aging mice treated with the peel extract . The synN. lappaceum play a significant role in anti-aging activity.The seeds of the fruit also contribute to the anti-aging effect by means of other mechanisms . The inhN. ramboutan-ake have been observed to contain phytochemicals [N. ramboutan-ake still remain scarce. Fruit rinds of hemicals , such asN. ramboutan-ake have only been tested in vitro  as compared with N. lappaceum (TPC = 223.75 μg GAE/g). The antioxidants of the pulp may be attributed to vitamins, though fewer phenolic compounds are present in the pulp of N. lappaceum. Interestingly, N. ramboutan-ake shows a strong metal chelating effect of 72.78%.A comparative study conducted on different fruit juices including t juices . This caN. ramboutan-ake is a positive correlation with R2 of 0.738 between TPC and DPPH radical scavenging activity [2 of 0.655 and 0.828 between TPC and antioxidant activities for DPPH and FRAP, respectively, is observed [The relationship between TPC and antioxidant properties of activity . These fobserved .N. ramboutan-ake showed a contrasting result with a weak negative correlation with R2 of −0.062 [N. ramboutan-ake.However, a study conducted on different types of underutilised fruits pulp including f −0.062 . This inN. ramboutan-ake on anti-neoplastic effects is observed where the aqueous fraction of the rind is able to reduce the cell viability of HT-29 , HCT-116 , Ca Ski  and MDA-MB-231  cell lines in a dose-dependent manner [The potential of t manner .N. ramboutan-ake extract involves the dysfunction of mitochondria which is confirmed by the elevated level of Bax protein and caspases. Dysfunction of mitochondria will trigger the cellular apoptosis cell signalling pathways, such as an increased ROS level and a reduction in the RSH level, which triggers apoptosis [Among the cancer cell lines, HT-29 shows a significant decline in cell viability. The rinds contain various phytochemicals that have been demonstrated to induce apoptosis in cancer cell lines. The HT-29 apoptosis induced by poptosis .N. ramboutan-ake extract had been investigated by measuring the cell proliferation effect of ethanolic extract of N. ramboutan-ake on 3T3 (mouse embryonic fibroblasts cell) and 4T1 (mouse breast cancer cell) cell lines. The findings show that N. ramboutan-ake exhibits anti-proliferation toward both cell lines, which indicates that the fruit does not exhibit cytotoxic effect on the cell lines [N. ramboutan-ake possesses important bioactive components that could be further developed as nutraceuticals for the prevention of colon cancer.Moreover, the cytotoxicity level of ll lines . Hence, N. ramboutan-ake peels and seeds are effective to suppress the growth of S. aureus and E. coli. The peel exhibits a better suppression effect on both bacterial strains as compared with the seed extract [Due to the inappropriate overuse and the extensive agriculture use of antibiotics, the rise of antibiotic resistance in bacteria has been occurring at an alarming level . Therefo extract .N. ramboutan-ake has also become a target in the search for potential bioactivities. The seed of N. ramboutan-ake has the potential of preventing diabetes and obesity [N. ramboutan-ake seed protein can bind to the active site of α-amylase, which reduces the binding site of this enzyme and inhibits the hydrolysis of starch to glucose. The most effective gastro-digestive enzyme for protein in N. ramboutan-ake seed is pepsin [N. ramboutan-ake seeds is more susceptible to multiple gastro-digestive enzymes, and therefore has higher inhibitory activity than N. lappaceum seeds.The non-edible part of the  obesity . A studys pepsin . The proN. lappaceum and N. ramboutan-ake. The comparison in N. ramboutan-ake are lagging behind N. lappaceum in many aspects, including anti-aging, anti-neoplastic, antimicrobial and antioxidants. Studies conducted on N. lappaceum have demonstrated its tremendous potential to be further developed into nutraceuticals. However, the beneficial properties of N. ramboutan-ake could be further explored to reveal its true potential in future studies.The present review further summarises the studies conducted on N. lappaceum carries pharmaceutical potential such as alleviating oxidative-stress related diseases. However, continuous effort should be carried out in future to determine the exact related bioactive components towards the development of nutraceuticals.Antioxidant activities and capabilities vary in each part of the fruits. This is because of the difference in phytochemical composition. Generally, the peel and seeds exhibit higher antioxidants activities and bioactivities as there are more bioactive compounds present. Phenolic and flavonoid compounds are responsible for the antioxidant activities and other biological activities. In vitro and in vivo studies indicated that N. ramboutan-ake is still lacking, which results in the underutilization of this fruit. Thus, the bioactive components responsible for the biological activities still remained unknown. Research on N. lappaceum can serve as a reference for enhancing knowledge in N. ramboutan-ake. Furthermore, limited evaluation of in vivo bioactivity remains a major challenge to study the pharmaceutical effects of both fruits. Toxicology and pharmacology of the bioactive compounds from fruits should be studied so that their therapeutic effect towards disease prevention can further be supported. In conclusion, this review has highlighted the potential and importance of N. lappaceum and N. ramboutan-ake. Hence, further research should be carried out in order to volarize the potential of both fruits, which can be used as nutraceuticals or food supplements.Unfortunately, the investigation into the phytochemicals and the in vitro and in vivo biological activities of


Body composition was determined using CT, fecal samples were collected, and shotgun metagenomic sequencing was performed. Gut microbiome results were grouped by diet and adiposity.The objective of this study was to increase understanding of the complex interactions between diet, obesity, and the gut microbiome of adult female non-human primates (NHPs). Subjects consumed either a Western , which was not associated with adiposity. Overall, Western diet-fed animals in the P. copriHIGH group showed greater proportional abundance of B. ovatus, B. faecis, P. stercorea, P. brevis, and Faecalibacterium prausnitzii than those in the Western P. copriLOW group. Western diet P. copriLOW subjects had a greater proportion of Eubacterium siraeum. E. siraeum negatively correlated with P. copri proportional abundance regardless of dietary consumption. In the Western diet group, Shannon diversity was significantly higher in P. copriLOW when compared to P. copriHIGH subjects. Furthermore, gut E. siraeum abundance positively correlated with HDL plasma cholesterol indicating that those in the P. copriLOW population may represent a more metabolically healthy population. Untargeted metabolomics on urine and plasma from Western diet-fed P. copriHIGH and P. copriLOW subjects suggest early kidney dysfunction in Western diet-fed P. copriHIGH subjects.Diet was the main contributor to gut microbiome bacterial diversity. Adiposity within each diet was associated with subtle shifts in the proportional abundance of several taxa. Mediterranean diet-fed NHPs with lower body fat had a greater proportion of P. copri appears to mediate metabolic dysfunction in Western diet-fed NHPs.In summary, the data indicate diet to be the major influencer of gut bacterial diversity. However, diet and adiposity must be considered together when analyzing changes in abundance of specific bacterial taxa. Interestingly, Video abstractThe online version contains supplementary material available at 10.1186/s40168-021-01069-y. Gut microbiome dysbiosis is associated with many adverse health outcomes including multiple sclerosis, cancer, diabetes types 1 and 2), asthma, allergies, inflammatory bowel disease, obesity, and autism  and 2, a. ComposiBacteroides or Prevotella. While both Prevotella and Bacteroides are saccharolytic bacteria, these genera of bacteria tend to inhibit each other, thereby giving way to two separate enterotypes [Bacteroides predominate with protein and animal fat consumption and Prevotella are observed with carbohydrate (fiber) consumption [Prevotella are more common in non-Westernized populations consuming a plant-rich diet or in Western populations with high adherence to a Mediterranean or vegetarian diet [Ruminococcus and Streptococcus abundance, while subjects on a vegetable-based diet showed increased abundance of Roseburia, Lachnospira, and Prevotella [The composition of the human gut microbiome is dominated by the Bacteroidetes and Firmicutes bacterial phyla . Within erotypes . The presumption . Enterician diet . Food anevotella . High-faevotella , 15–17.Helicobacter and Spirochaetes in NHPs compared to humans, albeit these NHPs were consuming standard adult monkey chow that would not compare to humanized dietary patterns and may account for some of the noted differences [The microbiome of non-human primates (NHP) harbors Bacteroidetes, Firmicutes, and Proteobacteria, similar to the human microbiome , 19. Proferences . Taken tLactobacillus, Clostridium, Faecalibacterium, and Oscillospira while decreasing Ruminococcus and Coprococcus representation [This study examines the impact of a translationally relevant humanized Western and Mediterranean dietary patterns on the gut microbiota, and association with obesity, in female middle-aged cynomolgous macaques. Application of these diets has been shown to significantly influence the gut populations of cynomolgous macaques . Mediterentation . Howeverentation . 16S seqentation . Shotgunentation –23. ThisMacaca fascicularis were obtained  and housed in groups of 4 animals per pen with daylight exposure on a 12/12 light/dark cycle. Animals were aged at study initiation by dentition to be approximately 8.8 years old. The subjects were habituated to their social groups and were metabolically characterized for group randomization during a 7-month baseline phase while consuming standard monkey chow . Animal groups were randomized so that no differences in age, body weight (BW), body mass index (BMI), or plasma triglyceride concentrations were observed. No gastrointestinal differences or other health concerns were observed at baseline before animals were randomized to dietary pattern. Animals were then assigned to a dietary pattern (Western n=21 or Mediterranean n=17) for 31 months. All animal manipulations were performed according to the guidelines of state and federal laws, the US Department of Health and Human Services, and the Animal Care and Use Committee of Wake Forest University School of Medicine.Adult female Experimental diets were formulated to be isocaloric with respect to protein, fat, carbohydrates, and cholesterol content. Experimental diets translationally represent humanized Western and Mediterranean dietary patterns. For further details on diet formulation and ingredients, see Supplemental Table 2. BW and BMI reported in Table N=14) or highest (N=15) tertiles underwent microbiome analysis and comprise the sample included in this report. Body composition was measured by computed tomography in anesthetized subjects during various intervals throughout the study. Body fat composition reported in Table Metabolic parameters were measured in each subject and previously reported , 25. BriTotal plasma cholesterol (TPC) and high-density lipoprotein cholesterol (HDL-C) were measured at 24 months from plasma collected after an 18 h fast. TPC and HDL-C levels were determined by the Wake Forest Comparative Medicine Clinical Chemistry and Endocrinology Laboratory using reagents (ACE cholesterol and ACE HDL-C standards) and instrumentation  from Alfa Wasserman Diagnostic Technologies . TPC and HDL-C were standardized to calibrated controls from the Centers for Disease Control and Prevention/National Institutes of Health Lipid Standardization Program . Blood sUrine and fecal samples were collected after 26 months of dietary treatments. Animals were moved to metabolic cages with wire bottoms, and sample cups were checked every 15 min for feces. Fecal samples were immediately placed in sterile tubes under aseptic conditions and stored at −80 °C until further processing. As soon as adequate urine samples were available, they were collected, and stored at −80 °C until analysis.DNA was isolated from 100 mg of frozen feces using the Qiagen DNeasy PowerSoil Pro kit , and metagenomic sequencing was performed by CosmosID Inc. . In brief, DNA libraries were prepared using the Illumina Nextera XT library preparation kit , with a modified protocol , 28. LibUnassembled sequencing reads  as previously described . In briet tests allowing for unequal variance using the Satterthwaite method for degrees of freedom. Principal coordinates analysis (PCoA) of bacterial beta diversity based on the Bray-Curtis dissimilarity matrix using relative abundance was used to distinguish groups. Diversity analyses were performed using species taxonomy level. Comparisons by diet and body weight group were assessed by two-way ANOVA of diet, group, and their interaction, estimating different variance components by diet and group; we used linear contrasts to make pairwise comparisons within diet group if the interaction p value was <0.10. Within the Western diet group, animals were split into P. copriLOW and P. copriHIGH groups based on whether they were below the median, and Welch’s two-sample t test was used to compare microbiota and metabolites shifts between Western diet P. copriHIGH and Western diet P. copriLOW experimental groups. Correlations between gut microbial populations and metabolic parameters were summarized by Pearson correlation and expressed as r. A p value of p<0.05 was considered statistically significant in all analyses with the exception of the evaluation of diet × group interaction in the two-way ANOVA which used alpha=0.10.Metabolic parameters were summarized using means and standard deviations. Microbiome diversity and bacterial populations were compared by diet group using two group Western and Mediterranean dietary pattern drives gut microbiome populations. While dietary pattern consumption had no significant effects on bacterial richness Choa1; Fig. ; Fig. 1aBacteroides (B.) pectinophilus, Clostridium sp. SS2/1, Coprococcus catus, Coprococcus comes, Dorea formicigenerans, Dorea longicatena, Eubacterium (E.) eligens, E. hallii, Faecalibacterium prausnitzii, Lachnospiraceae bacterium 5_1_63FAA, Lachnospiraceae bacterium 6_1_63FAA, Lactobacillus (L.) animalis, L. johnsonii, L. murinus, L. reuteri, Oscillibacter sp. ER4, Prevotella (P.) dentasini, P. stercorea, Roseburia hominis, and Streptococcus lutetiensis  and B. vulgatus  were higher in animals fed the Western diet than the Mediterranean diet. Furthermore, virulence factor gene measurements from the shotgun metagenomics sequencing highlighted elevated B. fragilis .While not a major component of the NHP gut microbiota (≤1%), iet Fig. a, distinlis Fig. d and e aris Fig. d and f iEndolimax nana abundance and reduced Neobalantidium coli abundance when compared with fecal samples obtained from Mediterranean diet-fed NHPs , B. uniformis , and Ruminococcus champaneliensis . Within Mediterranean diet-fed subjects, monkeys with increased body adiposity displayed decreased L. animalis  metabolites  have been linked with autoimmune disease, new onset rheumatoid arthritis, hypertension, and diabetes [P. copri could be dependent on host diet background.Our data indicates that diet composition impacts gut microbiota structure and that a Mediterranean diet pattern is associated with increased gut bacterial diversity in female cynomolgous macaques. In accordance with human diet studies in which subjects with high adherence to a Mediterranean diet display elevated Proteobacteria populations , Mediterbundance . Literatber rich , 38. On diabetes –42. TakeBacteroides abundance is low in NHP subjects, possibly due to the mutually exclusive dominance of Prevotella, monkey subjects consuming a Westernized diet did display elevated B. uniformis and B. vulgatus species when compared with Mediterranean diet-fed animals. In this regard, monkeys responded to translationally relevant diets in a similar pattern to humans  in humans compared to NHP colon pH 5.1 [While overall subjects ;). The dsubjects , which c 6.5-6.9 , 45;) wh 5.5-6.0 ;), whichn pH 5.1 .)P. copri abundance. The P. copriLOW subgroup displayed increased bacterial diversity and elevated E. siraeum (strain: V10Sc8a). Gut E. siraeum abundance in NHP from either diet correlated with elevated HDL-C levels, suggesting a more metabolic healthy subgroup. Devillard et al. demonstrated novel biosynthetic activity of E. siraeum mediating the bioconversion of linoleic acid to form conjugated linoleic acids , E. siraeum negatively correlated with obesity [E. siraeum-enriched P. copriLOW subgroup displaying a healthier enterotype.We showed two distinct enterotypes in Western diet-consuming animals based upon -11-18:2 ;). Consu-11-18:2 ), furthe obesity , giving P. copriHIGH and P. copriLOW subjects indicates potential early stage kidney damage in P. copriHIGH animals. Hypertensive adolescents display elevated urinary excretion of carnitine metabolites [P. copriHIGH animals further associates this enterotype with kidney dysfunction and may contribute to proximal tubule damage by reducing local levels of nitric oxide. Additionally, we observed elevated plasma allantoic acid concentrations in Western diet-fed P. copriHIGH subjects. Elevated plasma allantoic acid was evident in a pre-clinical mouse model of cystic kidney disease [P. copriHIGH may indicate a sub-clinical pro-inflammatory phenotype mediated by this gut microbiota enterotype. Elevated plasma CMPF is observed in gestational diabetes and in type 2 diabetes patients [P. copriHIGH enterotype when compared with Western diet-fed P. copriLOW animals.Our metabolic data in urine and plasma samples obtained from Western diet-fed abolites  which alabolites . Therefoabolites . Hence,  disease . Moreove disease  and in c disease , highlig disease . Elevate disease , giving patients . In micepatients , suggestL. animalis compared to monkeys with more adipose tissue. Several Lactobacillus species, including L. animalis, are considered healthy probiotic commensal organisms. Administration of Lactobacillus for 6 weeks as a probiotic supplement was shown to reduce systolic blood pressure in a controlled, randomized, double-blind trial of smokers [Lactobacillus supplementation in regards to cholesterol lowering potential [In our study, obesity (as a single variable) did not appear to be significantly associated with gut microbiome composition , F. prausnitzii, Roseburia hominis, multiple Dorea sp. , and several Coprococcus species (C. catus and C. comes) to be significantly higher in Mediterranean diet-fed NHP subjects than Western diet consuming animals. Our previous work demonstrated an increase in plasma bacterial modified bioactive compounds in Mediterranean diet-fed NHPs [Lactobacillus sp.  and Clostridium sp. SS2/1 in our Mediterranean diet-fed monkeys. Metabolomic analysis of conventional-housed and germ-free mice have indicated that germ-free mice cannot produce plasma 3-indoxyl sulfate and IPA, demonstrating the reliance on bacteria to produce these metabolites [Literature indicates that benefits , 68. We fed NHPs . Consumpabolites . Furtherabolites . The redabolites –75 may rIt is important to note several key limitations of our current study. This study only includes female NHP; sex differences may exist impacting dietary influences on the gut microbiome and metabolic outcomes in males. Baseline fecal samples were not collected during the metabolic characterization phase while the NHP were on standard monkey chow. Therefore, the data we show in this study does not accurately reflect a response to diet, but reports metabolic and microbiome patterns associated with dietary pattern consumption.P. copri by Western diet administration) may represent a potential model to elucidate signaling differences observed in human Westernized populations with gut P. copri associations with several disease states. Furthermore, our metabolomic data indicates potential deleterious metabolite regulation in the urine and plasma of P. copriHIGH Western diet-fed subjects, suggesting that the P. copriHIGH enterotype in subjects eating a Westernized diet may potentiate the development of metabolic disease.In conclusion, our study demonstrated several similarities and key differences in the gut microbiome mediated by human translationally relevant diet administration. However, the key difference . B. Relative abundance of protozoa species in different fecal samples is visualized by bar plots. Samples are aggregated by diet cohort. Each colored box represents a protist species. The height of a color box represents the relative abundance of that organism within the sample. “Other” represents lower abundance taxa (<5%). C. Relative abundance of fungal species in different fecal samples is visualized by bar plots. Samples are aggregated by diet cohort. Each colored box represents a fungal species. The height of a color box represents the relative abundance of that organism within the sample. “Other” represents lower abundance taxa (<5%). D. A. Relative abundance of phages in different fecal samples is visualized by bar plots. Samples are aggregated by diet cohort. Each colored box represents a bacteriophage species. The height of a color box represents the relative abundance of that organism within the sample. “Other” represents lower abundance taxa (<5%). Supplemental Figure S4. Western diet-fed subjects with low P. copri abundance display elevated plasma gamma-glutamyl amino acid and phosphatidylethanolamine metabolites. Supplemental Figure S5. Obesity regardless of diet does not significantly regulate the gut microbiome. A. Shannon diversity. B. PCoA of bacterial beta diversity based on the Bray-Curtis dissimilarity. C. Relative abundance of bacterial species in different fecal samples is visualized by bar plots. Each bar represents a subject and each colored box a bacterial taxon. The height of a color box represents the relative abundance of that organism within the sample. “Other” represents lower abundance taxa. D. Proportional abundance of Treponema (Genus) in obese and lean NHP regardless of dietary pattern. E. Proportional abundance of Treponema succinifaciens in obese and lean NHP regardless of dietary pattern.Additional file 2.Additional file 3.Additional file 4.