Hedyotis diffusa, Curcuma zedoaria (Christm.) Rosc., Astragalus membranaceus (Fisch.) Bunge, and Glycyrrhiza uralensis Fisch. Our findings revealed that ADQ significantly inhibited cell proliferation in both parental and chemo-resistant breast cancer cells, but with little cytotoxcity effects on the normal cells. Besides, ADQ was found to facilitate the G2/M arresting and apoptosis induction effects of paclitaxel. Network pharmacology and bioinformatics analysis further demonstrated that ADQ yielded 132 candidate compounds and 297 potential targets, and shared 22 putative targets associating with breast cancer chemoresponse. Enrichment analysis and experimental validation demonstrated that ADQ might improve breast cancer chemosensitivity via inhibiting caveolin-1, which further triggered expression changes of cell cycle-related proteins p21/cyclinB1 and apoptosis-associated proteins PARP1, BAX and Bcl-2. Besides, ADQ enhanced in vivo paclitaxel chemosensitivity on breast cancer. Our study not only uncovers the novel function and mechanisms of ADQ in chemosensitizing breast cancer at least partly via targeting caveolin-1, but also sheds novel light in utilizing network pharmacology in Chinese Medicine research.Chinese formulas have been paid increasing attention in cancer multidisciplinary therapy due to their multi-targets and multi-substances property. Here, we aim to investigate the anti-breast cancer and chemosensitizing function of Ai Du Qing (ADQ) formula made up of  Breast cancer is the most common female malignancy and one of the leading causes of cancer-related deaths, with 252,710 new cases of invasive breast cancer and 40,610 breast cancer-related deaths diagnosed in women in the United States in 2017 . ChemothHedyotis diffusa , Chen (Minister) herb Curcuma zedoaria (Christm.) Rosc. , Zuo (adjuvant) herb Astragalus membranaceus (Fisch.) Bunge , and Shi (courier) herb Glycyrrhiza uralensis Fisch . Each herb in the formula can inhibit cancer growth via cell cycle arrest, apoptosis induction, and immune regulation  has attracted worldwide attention from clinicians and researchers due to its multi-target and multi-substance characteristics and good safety profile . In contgulation . In addigulation . HoweverIn the past decade, multi-omic technologies including genomics, transcriptomic, proteomics, metabolomics, and serum pharmacokinetics have been developed for the high-throughput screening and identification of targets involved in TCM formulas . Howeverin vitro and in vivo. To this end, we investigated the anti-cancer and chemosensitizing functions of ADQ extracts in this disease. The results showed that ADQ effectively and safely enhanced breast cancer chemosensitivity in vitro and in vivo. To determine the mechanism of action, we constructed a “drug–target–disease” network among ADQ components (drug), ADQ targets (target), and genes in breast cancer chemoresistance (disease). The results of the network analysis and biological experimental findings suggested that ADQ mainly targets CAV1 to induce chemosensitizing effects. The results of this study not only provide scientific evidence to support the application of ADQ formula in the treatment of breast cancer but also highlight the novel role of network pharmacology in the modernization of TCM.The current study was designed to determine the preclinical efficacy of ADQ against breast cancer 18 Column  with the SecurityGuard Cartridge System  was applied for HPLC analysis. The mobile phases consisted of acetonitrile (A) and 0.05% (v/v) phosphoric acid (B) using a gradient program of 15% A in 0–23 min, 15–38% A in 23–40 min, 38% A in 40–50 min, 38–61% A in 50–60 min, and 61% in 60–75 min. The flow rate was 1.0 mL/min and the column temperature was set to 30°C The DAD detector was set at 216, 236, 260, 276, and 308 nm. P-coumaric acid, calycosin-7-glucoside, liquiritin, glycyrrhizic acid, and curcumol were prepared and diluted with methanol for the preparation of standard solutions. A volume of 10 μL of these solutions was analyzed with HPLC, and the calibration curves were finally established. For the preparation of sample solutions, 0.1 g ADQ was dissolved in 20 mL methanol. After sonicating for 60 min, the sample solution was filtrated through a 0.2 μm membrane filter for HPLC analysis.For ADQ preparation, BHSSC, EZ, HQ, and GC were mixed at a 1:1:1:1 ratio and then subjected to a grinding machine. The mixture was extracted with 95% alcohol by reflux extraction for 1 h and repeated three times. Then the supernatants were concentrated by rotary evaporation and evaporated to dryness in a water bath to obtain raw ethanol extract powder. The production ratio was calculated as 7.2–9.6%. For quality control analysis, the Agilent 1260 System  with DAD was applied for HPLC analysis. The Agilent C1. The cells were maintained in DMEM supplemented with 10% FBS, 1% penicillin, 1% streptomycin (Gibco), 40 U/L insulin , 40 U/mL heparin (Sigma), and 1% non-essential amino acids .The human breast cancer cell lines MDA-MB-231 and MCF-7 were purchased from the American Type Culture Collection . MDA-MB-231/T and MCF-7/T cells were derived from parental cells by gradually increasing paclitaxel (taxol) treatments for 6 months in the laboratory. MDA-MB-231, MCF-7, MDA-MB-231/T, and MCF-7/T cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin . Primary human mammary epithelial cells (HUMECs) and its Ready Medium  were purchased from Gibco, and human umbilical vein endothelial cells (HUVECs) were purchased from the National Infrastructure of Cell Line Resource3 cells/well were seeded into 6-well plates. After cell attachment, paclitaxel or ADQ was added to the wells alone or in combination for 4 h. Then the cells were cultured with fresh medium for 2 weeks. The colonies were fixed in 4% paraformaldehyde, stained with Coomassie Blue, photographed, and counted under a microscope.After the indicated drug treatment, cell numbers were counted using trypan blue exclusion on a Cellometer Mini device . Experiments were performed in triplicate. For the colony formation assay, cells at a density of 1 × 10For the drug efflux assay, MDA-MB-231 or MCF-7 cells were pretreated with ADQ for 24 h, followed by incubation with epirubicin for 60 min at 37°C. After dug exposure and washing, the cells were released in drug-free medium for 90 min and harvested for flow cytometry analysis. For cell cycle analysis, cells were fixed in ice-cold 70% ethanol at -20°C overnight. Then cells were washed with phosphate-buffered saline, stained with 50 mg/mL propidium iodide (Sigma), and dissolved in 100 mg/L RNase A (Sigma). For apoptosis analysis, the cells were stained with the Annexin V-FITC Apoptosis Staining/Detection Kit . All flow cytometry analyses were conducted with FACSAria SORP (BD Biosciences) and analyzed by Modifit LT or FlowJo software.p-H2AX expression, cells were incubated with 4% paraformaldehyde and 0.2% triton X-100 for 10 min. Following blocking in goat serum for 60 min, the samples were co-incubated with primary antibodies against p-H2AX  at 4°C overnight and subsequently labeled with fluorescence-conjugated secondary antibodies for 2 h at room temperature. Then DAPI was applied for nuclear staining, and the signals were detected by fluorescence microscopy . With regard to Hoechst 33528 detection, cells were seeded at 60–70% confluency in 6-well plates, and then treated with the indicated drug for 48 h. Then Hoechst 33258 staining was conducted according to the manufacturer’s instructions.For measurements of phosphorylated histone 2 the TCMID3, and the BATMAN-TCM4. The ingredients were screened according to drug likeness (DL) and OB values, and the ingredients were retained if DL ≥ 0.18 and OB ≥ 30, a criterion suggested by the TCMSP database. The ingredient–target networks were constructed for these herbs using Cytoscape software (version 3.2.1).The chemical ingredients were collected from TCM databases including the TCMSP Database5, and then analyzed with the GEO2R online analysis tool6. The dataset GSE41112 includes 24 tumors of breast cancer patients with chemotherapy and 37 without chemotherapy, and the GSE87455 dataset includes human breast cancer samples with “no treatment” (n = 122) and “chemo only” (n = 83) groups. The DEGs were screened with P ≤ 0.05 and fold control (FC) ≥ 1.5 criteria, delivered to the Search Tool for the Retrieval of Interacting Genes/Protines (STRING) database to evaluate the PPI information, and also submitted to the DAVID7 for enrichment analysis. The significant enrichment analysis of DEGs was assessed based on the GO and KEGG8.Gene expression data were retrieved from the NCBI GEO databaseThe pcDNA 3.1(+)-CAV1 was provided by Vigene Company  and transfected into cells using Lipofectamine 2000 . After 24 h, the transfected cells were passaged and selected for 2 weeks with 10 μg/mL puromycin (Invitrogen). Pooled populations of positive cells were used for subsequent experiments. Negative control cell lines were generated by transfecting cells with scrambled plasmids. The small interfering RNAs (siRNAs) targeting CAV1 or scrambled siRNAs were purchased from Invitrogen (Carlsbad) and transfected using the X-tremeGENE siRNA transfection reagent  according to the manufacturer’s instructions.To determine the protein concentration, cells were lysed in RIPA buffer (Sigma) containing a protease inhibitor mixture (Roche Diagnostics). The protein concentration was measured with the bicinchoninic acid assay . Quantified protein lysates (15 μg) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and resolved on 12% polyacrylamide gels. Then the proteins were transferred onto a PVDF membrane . The membrane was probed with primary antibodies including CAV1, p21, cyclin B1, cleaved poly (ADP-ribose) polymerase (PARP), Bcl2-associated X protein (BAX), B-cell lymphoma 2 (Bcl-2), p53, and phosphorylated p53 , and β-actin  at 4°C overnight. After washing three times with Tris-buffered saline and 0.05% Tween-20, the membrane was incubated with secondary anti-rabbit or anti-mouse antibodies for 2 h at room temperature. The signals were visualized using the ECL Advance Western Blotting Detection Reagent  and quantified with FlowJo software.n = 6 mice, total of 60 glands), respectively. Paclitaxel was given by intraperitoneal injection at 10 mg/kg every 3 days, and ADQ was given by oral gavage at 100 mg/kg once a day. The body weight and tumor volumes were recorded throughout the whole experimental period.All animal procedures were performed in accordance with institutional guidelines for the care and use of laboratory animals approved by the Animal Care and Use Committee of Guangzhou University of Chinese Medicine and the National Institutes of Health guide for the care and use of laboratory animals. The mouse mammary tumor virus (MMTV)-PyMT mouse model of breast cancer spontaneously develops 100% multiple and luminal-like breast tumors from normal mammary epitheliums by 8–12 weeks, similar to the pathological processes and characteristics in human breast cancer . In thisHematoxylin and eosin staining and IHC were conducted according to the protocol provided by t-test or one-way analysis of variance was used to determine the significance of the data between groups. P-values less than 0.05 were considered statistical significant.The data are shown as the mean ± standard deviation. The two-tailed Student’s p-coumaric acid (peak 1), calycosin-7-glucoside (peak 2), liquiritin (peak 3), glycyrrhizic acid (peak 4), and curcumol (peak 5) in ADQ was compared among different batches . Based on an established HPLC method, good linearity of five compounds was achieved with a correlation coefficient of 2R≥ 0.9995 (Supplementary Table S1). The retention times of these compounds were determined to be 19.8, 23.9, 25.8, 50.7, and 69.5 min, respectively. The contents of the five compounds in the ADQ samples were also determined (Supplementary Table S2). We evaluated the influence of ADQ on the proliferation of breast cancer cell lines including MDA-MB-231 and MCF-7, as well as their derived paclitaxel-resistant cell lines MDA-MB-231/T and MCF-7/T. Significant inhibition of growth was observed in both parental and resistant cells at 24 h , 48 h , and 72 h . IC50 of ADQ was shown in Figure 1D for the indicated cell lines. Specifically, the IC50 values of ADQ at 48 h for MDA-MB-231, MDA-MB-231/T, MCF-7, and MCF-7/T were 49.809, 57.789, 65.799, and 70.964 μg/mL, respectively, suggesting that ADQ had similar suppressive effects on both sensitive and resistant breast cancer cells. To determine the cytotoxic effects of ADQ on normal cells, we also investigated its effects on HUMECs and HUVECs, and found that ADQ did not have cytotoxic inhibitory effects on both types of normal cells . These findings indicated that ADQ exerted selective toxic effects on breast cancer cells.For quality control of ADQ, the chromatographic fingerprinting of ADQ was conducted and quantitative analysis of Figure 1 and Supplementary Figure S2, the tested concentrations of taxol and ADQ were set according to their IC50 at 48 h. ADQ significantly enhanced paclitaxel-induced death in MDA-MB-231 and MCF-7 cells. Interestingly, ADQ administration at 50 μg/mL also caused a clear reduction in the number of resistant breast cancer cells . We also evaluated the long-term inhibitory effects of ADQ on the colony formation capabilities of breast cancer cells with or without paclitaxel. The results showed that the combination group exhibited a substantial reduction in the colony numbers of parental MDA-MB-231 and MCF-7 cells . In addition, the growth of paclitaxel-resistant MDA-MB-231/T and MCF-7/T cells was greatly suppressed in the presence of ADQ . The drug efflux assay revealed that ADQ could enhance epirubicin influx into breast cancer cells, as shown by the increased fluorescence intensity of epirubicin in ADQ-treated cells . These results indicated that ADQ could chemosensitize both paclitaxel-sensitive and paclitaxel-resistant human breast cancer cells.To determine the synergistic activities of ADQ with paclitaxel in breast cancer, MDA-MB-231 and MCF-7 cells were treated with ADQ and paclitaxel for 48 h. As presented in Figure 3A, the flow cytometry results revealed that paclitaxel or ADQ alone could induce G2/M checkpoint arrest in both breast cancer cell lines. In addition, ADQ and paclitaxel combination increased G2/M arrest by 26% in MDA-MB-231 cells and by 66% in MCF-7 cells . Furthermore, the G2/M population of MDA-MB-231/T and MCF-7/T cells was also arrested by ADQ with a 56 and 63% increase, respectively . Previous studies have shown that perturbation of the G2/M transition was largely due to DNA damage, and p-H2AX could be a marker for monitoring DNA damage . Furthermore, the expression of p-H2AX in MDA-MB-231/T and MCF-7/T cells was also significantly enhanced following ADQ administration, indicating that the chemosensitizing effects of ADQ might be attributed to DNA damage-induced G2/M checkpoint arrest .Uncontrolled cell mitosis represents one of the hallmarks of cancer. Thus, we used PI staining to examine the effects of ADQ on the cell cycle distribution of both paclitaxel-sensitive and paclitaxel-resistant cells. As labeled in A damage . The immFigure 4A, the percentage of early and late apoptotic events in MDA-MB-231 and MCF-7 cells reached about 20 and 25%, respectively, after exposure to 50 ng/mL paclitaxel. Interestingly, when ADQ was administrated with paclitaxel concurrently, the percentage of MDA-MB-231 and MCF-7 apoptotic cells was increased to approximately 60% and 51%, respectively. Notably, ADQ was also capable of inducing apoptosis in paclitaxel-resistant breast cancer cells. The percentage of apoptotic cells in MDA-MB-231/T reached 45% following ADQ treatment after 48 h, and MCF-7/T cells reached 30% . Based on flow cytometry results, Hoechst 33258 staining was utilized to observe the morphological changes of apoptotic cells by fluorescence imaging . Together, these findings confirmed that ADQ promoted paclitaxel-induced apoptosis in breast cancer cells.Apoptosis is another important mechanism that causes cell death induced by chemotherapy drugs . To dete imaging . In paclFigure 5A), 14 compounds in EZ targeting 41 genes , 21 compounds in HQ targeting 222 genes , and 94 compounds in GC targeting 241 genes . The “candidate active compounds” are listed in Supplementary Table S3. Overall, our results showed that ADQ yielded 132 candidate compounds and 297 potential targets after eliminating all duplicates . Specifically, the network included 429 nodes and 2874 ingredient–target interactions, of which 132 candidate compounds had a median of 10 target correlations, suggesting the existence of complex correlations among different compounds and targets.ADQ consists of four herbs including BHSSC, EZ, HQ, and GC. To establish the ingredient–target network of ADQ, we screened candidate compounds for (OB ≥ 30%) and DL (DL ≥ 0.1) in each herb. There were 12 compounds in BHSSC targeting 225 genes . The Venn diagram analysis showed that ADQ shared 22 putative targets with the two datasets . These hub genes included CAV1, HIF1A, CCNB1, BAX, BCL2, PARP1, ERBB3, MCL1, PRKCA, CDCA7, CDKN1A, CDKN2A, TOP1, TOP2A, SELE, ATP5B, RUNX2, BIRC5, ATF2, RUNX1T1, MT2A, and EIF5B , among which CAV1 was the key target with the largest node size according to “Degree” in the node size mapping . We further extracted the 22 significant targets to construct the PPI containing 22 nodes and 71 edges based on the STRING database, and the PPI enrichment P-value of these hub genes was 2.22 × 10-16, indicating that they were at least partially biologically connected . All of these findings suggested that CAV1 might be one of the the most likely mechanisms affecting ADQ regulation network.To determine the pharmacological mechanisms of ADQ against chemoresistance, the DEGs of breast cancer patients before and after chemotherapy were extracted from GSE41112 and GSE87455 microarray sets. A total of 3286 genes in GSE5764 and 4877 genes in GSE87455 were identified using the GEO2R analysis tool ; in the molecular function category were enzyme binding, protein binding, and transcription factor binding ; and in the biological process category were cell death, cell proliferation, and cell differentiation . In Figure 8D, KEGG analysis (P < 0.05) indicated that multiple cancer-related pathways were significantly involved in the mechanisms of ADQ including focal adhesion, apoptosis, cell cycle, p53, HIF-1, ErbB, phosphoinositide 3-kinase (PI3K)/Akt, Janus kinase/signal transducer and activator of transcription, mammalian target of rapamycin, NF-kappa B, and tumor necrosis factor signaling pathways. Notably, It was various targets in p53 signaling were tightly associated with ADQ pharmacological action . P53 signaling can be stimulated by a number of stress signals including DNA damage, oxidative stress, and activated oncogenes, consequently leading to cell cycle arrest and apoptosis . This phenomenon was consistent with the oncogenic and tumor suppressor roles of CAV1 in breast cancer development . Then we determined if CAV1 overexpression abrogated the anti-cancer and chemosensitizing effects induced by ADQ in breast cancer cells. As shown in Figure 10A, ADQ synergistically interacted with paclitaxel to suppress MCF-7 cell proliferation, whereas CAV1 overexpression obviously abrogated the synergistic effects of ADQ with paclitaxel in suppressing breast cancer growth (p ≤ 0.01). To determine if CAV1 signaling was involved in ADQ chemosensitizing activity, the indicated proteins were analyzed . Western blot analysis revealed that ADQ alone could inhibit CAV1 expression, accompanied by the increased expression of p21 and decreased expression of cyclin B1. In addition, the expression of cleaved PARP1 and BAX were enhanced by ADQ, whereas Bcl-2 expression was decreased following ADQ treatment. Notably, CAV1 overexpression resulted in decreased p21 and increased cyclin B1 expression. Furthermore, CAV1 overexpression inhibited apoptosis, as shown by the decreased expression of cleaved PARP1 and BAX and increased Bcl-2 expression. Cell cycle analysis further revealed that CAV1 overexpression relieved the G2/M arresting effects of ADQ from 68.08 to 57.08% in MCF-7 cells . CAV1 overexpression also significantly reduced the apoptotic events induced by paclitaxel with ADQ treatment from 47.89 to 36.78% . These data showed that ADQ inhibited CAV1 to improve breast cancer chemosensitivity.We continued to validate whether the chemosensitizing activity of ADQ was pression . In thiselopment . To direCAV1high MDA-MB-231 cells, CAV1 knockdown did not significantly aggravate the inhibitory ability of ADQ in the presence of paclitaxel, suggesting that ADQ might target CAV1 to chemosensitize breast cancer . Similarly, siCAV1 did not significantly change the expression levels of p21, cyclin B1, PARP1, BAX, and Bcl-2 when administrated together with paclitaxel and ADQ , nor were significant changes observed in cell cycle distribution and apoptotic cells . Furthermore, MDA-MB-231/T and MCF-7/T showed strong CAV1 expression compared with their parental cell lines . In paclitaxel-resistant breast cancer cell models, both CAV1 silencing and ADQ treatment resulted in the significant suppression of cell proliferation, but CAV1 silencing did not increase the effects of ADQ, namely inhibition of proliferation, cell cycle arrest, or apoptosis induction in MDA-MB-231/T and MCF-7/T cells . These findings demonstrated that ADQ could target CAV1 to induce cell cycle arrest and apoptosis, thereby chemosensitizing paclitaxel-resistant breast cancer cells.In in vivo efficacy of ADQ on breast cancer with the MMTV-PyMT transgenic mouse model, which spontaneously develops 100% multiple and luminal-like breast tumors from normal mammary epitheliums by 8–12 weeks, similar to the pathological processes and characteristics in human breast cancer . The results of the in vivo experiments revealed that ADQ alone could significantly inhibit breast tumor growth  and reduce tumor volume , while with little influence on mouse body weight . Meanwhile, ADQ alone caused an increase in the apoptotic ratio and a decrease in ki67 expression, further demonstrating its tumor suppressive functions . The results also revealed that the combination of paclitaxel and ADQ exerted stronger inhibitory effects on overall tumor growth than paclitaxel alone . Notably, ADQ did not lead to significant weight loss throughout the experiment, whereas a slight reduction of body weight was found in the paclitaxel-treated mice . Furthermore, both H&E staining and the TUNEL assay revealed that the synergistic use of paclitaxel and ADQ resulted in significant increases of apoptosis in tumor tissues, accompanied by the decreased expression of Ki67 and CAV1 . These results indicate that ADQ is a potential adjuvant drug for breast cancer treatment with good safety.We finally evaluated the t cancer . Therefoin vivo findings further confirmed the anti-breast cancer and chemosensitizing effects of ADQ with good safety. Therefore, it is of great value to explore the effects and mechanisms of ADQ in breast cancer.With increasing attention being paid to multi-target strategies for cancer treatment, TCM has become a valuable resource. In this study, we investigated the anti-cancer and chemosensitizing functions of ADQ extracts in breast cancer. Our findings revealed that ADQ significantly inhibited the growth of breast cancer cells by cell cycle arrest and apoptosis induction. In particular, ADQ did not cause cytotoxic effects in normal cells. Similar findings have also been shown for other herbs including Huangqi, Ginseng, Banzhilian, Huachansu injection, TJ-48, Shenqi fuzheng injection, and Kanglaite injection . NotablySupplementary Figure S5). However, although ADQ administration led to CAV1 inhibition, the expression of p53 and p-p53 (ser15) did not significantly change, indicating that ADQ-induced p21 and apoptosis activation might not be related to p53 . With regard to the genetic status of p53, MDA-MB-231 was mutated and MCF-7 was wild type. Because the degradation speed of mutated p53 was significantly reduced, p53 expression was highly elevated in MDA-MB-231 cells compared with MCF-7 cells ; however, ADQ had little effect on p53 expression in both breast cancer cells , indicating that the susceptibility of MDA-MB-231 or MCF-7 cells to ADQ was not related to p53 genetic status. Overall, network pharmacology has provided us with a highly efficient strategy to identify key targets and their complex correlation in the development of TCM formulas.The development of network pharmacology has shifted our traditional view from the “one drug, one target” model to the “drug–target network” . BioinfoCAV1 plays dual roles in the progression of breast, lung, cervical, gastric, glioma liver, and prostate cancers . During The results of our study demonstrated that ADQ improved the cancer chemoresponse, and that CAV1 was at least in part responsible for these chemosensitizing effects. Our work has great implications for the discovery of breast cancer therapeutic targets by integrating bioinformatics and network pharmacology followed by experimental validation. This study not only provides experimental evidence and molecular mechanisms that may facilitate the safe and effective therapeutic use of herbal medicines for breast cancer, and may lead to CAV1-based therapeutic strategies for mammary malignancies. However, preclinical studies are needed to confirm its chemosensitizing effects and active ingredients.NW and ZW conducted the design of the experiments and wrote the manuscript. FZ contributed to the revised manuscript. NW conducted network pharmacology analysis and bioinformatics analysis. BY, XL, and XZ contributed to the drug preparation and quality control of ADQ. SW, YZ, and SL carried out cell culture and molecular biology experiments. ZH, HP, and YL conducted flow cytometry analysis.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Acupuncture affects synaptic plasticity, causes an increase in neurotrophic factors, and results in neuroprotection, cell proliferation, antiapoptosis, antioxidant activity, anti-inflammation, and maintenance of the blood-brain barrier.In this article, we review signal transduction pathways through which acupuncture treats nervous system diseases. We electronically searched the databases, including PubMed, MEDLINE, clinical Key, the Cochrane Library, and the China National Knowledge Infrastructure from their inception to December 2018 using the following MeSH headings and keywords alone or in varied combination: acupuncture, molecular, signal transduction, genetic, cerebral ischemic injury, cerebral hemorrhagic injury, stroke, epilepsy, seizure, depression, Alzheimer's disease, dementia, vascular dementia, and Parkinson's disease. Acupuncture treats nervous system diseases by increasing the brain-derived neurotrophic factor level and involves multiple signal pathways, including p38 MAPKs, Raf/MAPK/ERK 1/2, TLR4/ERK, PI3K/AKT, AC/cAMP/PKA, ASK1-JNK/p38, and downstream CREB, JNK, m-TOR, NF- Acupuncture is a form of therapy practiced for more than 3000 years in Asia. Medical doctors practice acupuncture under the guidance of meridian theory to achieve “de qi” status . To perfNowadays, signal transduction has been applied for explaining acupuncture mechanisms. The signal transduction pathway of acupuncture has been mentioned with respect to many diseases, including neurological , cardiovWe electronically searched the databases, including PubMed, MEDLINE, clinical Key, the Cochrane Library, and the China National Knowledge Infrastructure from their inception to December 2018 using the following MeSH headings and keywords alone or in varied combination: acupuncture, molecular, signal transduction, genetic, cerebral ischemic injury, cerebral hemorrhagic injury, stroke, epilepsy, seizure, depression, Alzheimer's disease (AD), dementia, vascular dementia (VD), and Parkinson's disease (PD). In addition, we used Boolean operators  to narrow or widen search results. All articles written in English or Chinese were manually screened, and relevant studies were identified. We included additional articles after performing a manual review of the reference lists of identified studies or review articles. Excluded articles included those with unavailable full text, those written in other languages, those not mainly related to the mechanism of the signal transduction pathway, or those with limited details of experimental methods or results. Flowchart of the search processes was as shown in Ischemic injury of the brain, also known as cerebral infarction, is a crucial health issue in the modern world because of its associated disability and socioeconomic burden. Acupuncture has shown beneficial effects on ischemic stroke rehabilitation by exerting the antiapoptosis effect on the ischemic area, promoting neurogenesis and cell proliferation, and regulating cerebral blood flow , 22. A rAcupuncture causes an increase in the expression of neurotrophic factors, such as BDNF and glial-derived neurotrophic factor (GDNF), in the central nervous system (CNS), exerts a neuroprotective effect on hypoxic-ischemic insults, and results in neurogenesis after the reconstruction phase , 26. In The mitogen-activated protein kinase (MAPK) family includes ERK1/2, JNK, and p38 MAPK proteins. In animals, the MAPK family is triggered by growth factors, stress, or an inflammatory environment and regulates cell functions, such as proliferation, division, differentiation, survival, and apoptosis. EA can trigger the MAPK family. ERK is believed to mediate reperfusion injury by inhibiting inflammatory reactions and promoting cell proliferation and growth . HoweverκB) cells, p38 MAPK, and myeloid differentiation primary response 88 (MYD88), as well as simultaneously reducing cytokine tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) levels [Environmental stresses and inflammatory cytokines activate p38 MAPKs and induce apoptosis and inflammation . In the ) levels .2+/calmodulin-dependent protein kinase (CaMK) [The p38 MAPK pathway activates the expression of CREB protein and reduces the apoptosis of ischemic neural cells. Acupuncture on GV16, GV20, GV24, ST36, and HT7 also triggered the CREB pathway in the hippocampus and improved cognitive impairment in an animal model –51. The e (CaMK) , 50, 52.e (CaMK) . A studyBeing a cell cycle initiator, PI3K/AKT pathways are essential for cell survival . HoweverBecause of the balance between Raf/MAPK/ERK1/2 and PI3K/AKT systems, some studies have included the pretreatment protocol , 65. EA κB is another protein complex related to cell survival. Some studies have demonstrated that EA regulates the NF-κB-mediated apoptosis pathway and provides neuroprotection [NF-otection , 67.κB. Therefore, acupuncture results in cell proliferation, antiapoptosis, neuroprotection, and BBB maintenance. The most frequently chosen acupoints include GV20, GV14, and ST36. The mechanisms and main results of identified articles are summarized in Acupuncture improved neurogenic defects and cognitive impairment in a cerebral ischemic/reperfusion animal model. In summary, acupuncture not only increases the levels of neurotrophic factors but also modulates signaling pathways, such as Raf/MAPK/ERK1/2 and PI3K/AKT and downstream CREB and NF-β, and NF-κB, prevents apoptosis by reducing the expression of p53 protein, and promotes neurogenesis by increasing the levels of BDNF and nerve growth factors [Hemorrhagic stroke is less common than ischemic stroke. The causes of hemorrhagic stroke include high blood pressure, brain trauma, aneurysms, arteriovenous malformations, and brain tumors. In cerebral hemorrhagic injury, blood vessel spasms and oxidative stress caused by ischemia and reperfusion cause an injury to neural cells. Acupuncture could improve the hypoperfusion status and hematoma absorption, reduce brain edema, and promote neurogenesis in the brain . Thus, s factors .α and NF-κB levels [Acupuncture increased the expression of endogenous GDNF and inhibited the early expression of VEGF, thus regulating nerve remodeling after cerebral hemorrhagic injury . At the B levels , 74. Li B levels . AntiapoB levels , 77. HowTaken together, acupuncture could improve neurogenic disability and reduce brain edema by increasing caveolin-1/matrix metalloproteinase levels and inducing antiapoptosis through the activation of the Bcl-2 pathway in a cerebral hemorrhagic model. The mechanisms and main results of identified articles are summarized in Seizure is an abrupt, spontaneous, excessive, or synchronous neuronal activity in the brain that leads to various uncontrolled shaking movements or loss of consciousness. Seizure attack affects 8%–10% of the general population in their lifetimes. The recurrence of seizure results in epileptic syndrome, which affects 2%–3% of the general population . Epileptγ-aminobutyric acid neurotransmission, including the upregulation of glutamic acid decarboxylase 67 (GAD67), is a self-protective and anticonvulsive mechanism [β, TNF-α, and cyclooxygenase-2 (COX-2) levels in the hippocampus of an epileptic rat model [In addition to the use of medication, some studies have reported that acupuncture reduced the frequency of seizures and improved the quality of life –82. Someechanism , 84. Acuechanism . Acupuncechanism . Inflammechanism . Acupuncat model , 89. Wanat model , 91. Yanat model .α level and reducing pPKCε and pERk1/2 levels in a kainic acid-induced rat model [α, pERK, pp38, pJNK, and pNFκB levels [With regard to molecular pathways, acupuncture on the auricular acupoint suppressed transient receptor potential ankyrin 1 (TRPA1) pathways by increasing the phosphorylated protein kinase C (pPKC)-at model . Liao etB levels . Yang etB levels . The uprB levels , 97.Taken together, acupuncture exerts the antiepileptic effect by changing anatomical, neurotransmitter, inflammatory cytokines and molecular level. With respect to signal transduction, acupuncture reduces seizure frequency by suppressing TRPA1/pERK and TLR4/ERK pathways and activating the PI3K/Akt pathway. Furthermore, acupuncture augments the antiapoptosis process and provides neuroprotection by increasing the GRP78 level and reducing the CHOP level. The mechanisms and main results of identified articles are summarized in Depressive disorders are common psychiatric disorders that affect approximately 17% of people in their lifetimes. A study reported that 12%–20% of depressed patients experience treatment-resistant depression, resulting in a considerable social burden . In addiκB protein and COX-2 levels [Acupuncture treats depression by regulating neurotransmitters, neuroinflammatory cytokines, the hypothalamus–pituitary–adrenal axis, and the hypothalamus–pituitary–sex gland axis . Further2 levels .Antidepressants alleviate the symptoms of depression by activating the MAPK/ERK pathway, which increases ERK1/2 and p-ERK1/2 expression. Many studies have reported that acupuncture activates the MAPK/ERK pathway and downstream CREB pathway and elevates BDNF production –114. TheMolecular studies have reported that acupuncture plays a role in the neuroendocrine model of depression. Lu et al. demonstrated that acupuncture could relieve the symptoms of depression and increase cortisol, PKA, and PKC levels . Oh et aκB pathway is the mechanism through which acupuncture treats depression. The aforementioned pathways cause cell survival, differentiation, antiapoptosis, and synaptic plasticity of neurons, thus alleviating depression symptoms and improving learning/memory dysfunction [The results of the Gene Ontology functional term and Kyoto Encyclopedia of Genes and Genomes database analysis indicated that the regulation of the Toll-like receptor signaling pathway, nucleotide-binding oligomerization domain-like receptor signaling pathway, MAPK/ERK pathway, PI3K/Akt pathway, neurotrophin signaling pathway, TNF pathway, and NF-function –124.κB pathways. Because of the aforementioned mechanism, we observed an increase in neuron growth factor levels, neurogenesis, and antiapoptosis accompanied by the alleviation of depression symptoms. The mechanisms and main results of identified articles are summarized in In summary, acupuncture can treat depression by upregulating MAPK/ERK and AC–cAMP–PKA–CREB pathways and downregulating JNK and NF-AD is a progressive neurodegenerative disease that is presented with dementia, memory loss, disorientation, personality disorder, mood swings, behavior disturbance, and language problems. Because of patients' cognitive decline, they withdraw from their family and society . Risk faβ deposits, upregulation of BDNF expression, and regulation of cell proliferation and neural plasticity in the brain [In addition to medication, acupuncture has been reported to improve cognitive function and the global clinical status of patients with AD without causing major adverse effects , 128. Mehe brain –131. Acuhe brain , 132.β deposits in the brain disturb BDNF signaling pathways, such as Ras/ERK, PI3K/Akt, and PKA/cAMP, which regulate BDNF expression and cause AD development [β deposits in the brain, elevates the BDNF level, and exerts a neuroprotective effect on CNS cells [Aelopment , 134. AcNS cells , 136. LiNS cells .2+ amplitude pathway and signal homeostasis [β-induced AD model, acupuncture on GV20 and BL23 reduced the level of peroxisome proliferator-activated receptor-γ (PPAR-γ) level and the deposition of Tau protein, thus reducing neuroinflammation [The p38 MAPKs are activated by environmental stresses and inflammatory cytokines and induce apoptosis and inflammation. In an AD animal model, acupuncture could improve cognitive impairment by reducing p38 MAPK levels, thus reducing neuroinflammation in the CNS , 138. Soeostasis . In an A RBL2 gene in humans and serves as a tumor suppressor signal. Acupuncture on CV17, CV12, CV6, SP10, and ST36 elevated the p130 level, caused cell proliferation in the brain, and treated dementia and aging-related diseases in SAMP10 mice [Acupuncture regulated cell cycle and aging in an AD model. N-myc downregulated gene 2 (NDRG2) encodes a cytoplasmic protein that may play a role in neurite outgrowth. Wang et al. demonstrated that EA on GV20 suppressed the astrocyte NDRG2 expression and glial fibrillary acidic protein level, thereby treating memory impairment of amyloid precursor protein/presenilin-1 double transgenic mice . P130, kP10 mice . TelomerP10 mice . AcupuncP10 mice –145.β-secretase gene transcription and β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) protein levels in AD through the sirtuin 1 (SIRT1)-PPARγ-proliferator-activated receptor γ coactivator 1 (PGC-1) pathway [β 25–35 suppresses mitochondrial biogenesis by inactivating the AMP-activated protein kinase (AMPK)–SIRT1–PGC-1α pathway in hippocampal neurons [β deposits, suppressing autophagy process and relieving cognition deficits [γ- PGC-1 pathway, and elevated ATP level. Because of the aforementioned mechanism, acupuncture balances brain metabolism and improves cognition impairment of AD mice [γ–PGC-1 suppresses BACE1 expression, thus reducing Aβ production in the hippocampus and improving cognitive decline in SAMP8 mice [Metabolic stress modulates  pathway . Aβ 25–3 neurons . Therefodeficits . Acupuncdeficits , 150. Fo AD mice , 151. FuMP8 mice .β deposits in the brain, thereby improving learning ability and memory in AD models. The most commonly chosen acupoints were GV20 and the Sanjiao regimen . Acupuncture regulates cell cycle and aging by modulating NDRG2 and P130 expression, telomerase activity, and Bcl-2/Bax balance. Many studies have reported that acupuncture on GV14 and BL23 modulates brain energy metabolism impairment and treats cognitive impairment. The mechanisms and main results of identified articles are summarized in In summary, acupuncture treats AD by regulating neurotransmitter release, elevating the neurotrophic factor level, and exerting anti-inflammatory effects. Thus, many molecular signaling pathways involved in acupuncture were reported in the AD model, including the BDNF–TrkB pathway, the cholinergic system–CREB–BDNF pathway, G-protein regulation, and the p38 MAPK family. The aforementioned pathways are believed to exert antiapoptosis and anti-inflammatory effects and reduce AVD, which accounts for 15% of dementia cases, is the second most common cause of dementia after AD. Multiple and recurrent ischemia of the brain caused by ischemia or hemorrhage has been found to be the main causes of VD . Althougα levels [Acupuncture can improve the scores on the Mini-Mental Status Examination, the revised Hasegawa's dementia scale, and activities of daily living examination for VD patients , 172. Frα levels . AcupuncAcupuncture exerted antioxidant and antiapoptosis effects in VD models. Zhu et al. reported that acupuncture on GV20 and ST36 inactivated the apoptosis signal-regulating kinase 1 (ASK1)–JNK/p38 pathway and elevated thioredoxin-1 and thioredoxin reductase-1 levels . The p38Taken together, acupuncture treats VD by activating MAPK/ERK and ASK1–JNK/p38 pathways; increasing CREB, mTOR, and Bcl-2 levels; and reducing the Bax level. In addition, through the aforementioned mechanism, acupuncture exerts an effect on antioxidant activity, antiapoptosis, and synaptic plasticity. The most commonly chosen acupoints were GV20, GV24, and ST36. The mechanisms and main results of identified articles are summarized in PD is a chronic neural degenerative disorder that mainly affects the motor system. Patients with PD experience shaking, rigidity, and walking difficulty. In advanced stages of the disease, behavioral disturbance, depression, poor sleep, and cognitive dysfunction are noted . TreatmeBoth manual acupuncture and EA help alleviate some motor symptoms in patients with PD and some nonmotor symptoms, such as psychiatric disorders, sleep disorders, and gastrointestinal symptoms. Acupuncture also improved the therapeutic efficacy of levodopa, lowering the necessary dosage –177. Redα, and IL-1β levels. The regulation of cytokines reduces the neuroinflammation of the SN and alleviates PD symptoms [α signaling pathways and reduces neuronal excitotoxicity in the hippocampus [In light of signal transduction, EA at 2 Hz on GV16 and LR3 inactivate the ERK 1/2 signaling pathway and p38/MAPK signaling pathway, causing an increase in tyrosine hydroxylase–positive neurons and a decrease in COX-2, TNF-symptoms , 159. Acsymptoms –162. Lu pocampus .α-syn, caused recovery of DA neurons in the SN, and improved motor function of an animal model without any notable adverse effect [Rapamycin, an inhibitor of mTOR, is a potent inducer of autophagy and has an effect on PD . Howevere effect .α, IL-6, and IL-1β, induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), thus suppressing the neuroinflammatory process [Oxidative stress and inflammation both contribute to the neural toxicity and development of PD . Many st process . The nuc process . Similar process .κB and Nrf2/HO-1 pathways [κB protein expression and downregulated 26S proteasome protein expression in rotenone-induced PD rats [PD symptoms were relieved through the modification of TLR/NF-pathways . EA on G PD rats . P53 pla PD rats . P53 dys PD rats . Park et PD rats .At the gene level, Choi et al. demonstrated that EA regulated gene expression in the striatum and exerted a neuroprotective effect on MPTP parkinsonism mice , 191. Yeα signaling pathway. Acupuncture also regulates apoptosis by balancing the Bcl-2 and m-TOR-independent pathway. The most chosen acupoints include GB34, LR3, and GV16. Moreover, high-frequency EA (100 Hz) on ST36 and SP6 reduces neuroinflammation through the Nrf2–ARE pathway. The mechanisms and main results of identified articles are summarized in In summary, acupuncture improved motor dysfunction and memory of PD. These effects were accompanied by the regulation of gene expression. Acupuncture modulates neuroinflammation by inactivating ERK 1/2 and p38/MAPK signaling pathway and reduces neuronal excitotoxicity through the pPKA/pPKC/CaMKIIκB, and Bcl-2/Bax balance. We summarized the common signal transduction pathways through which acupuncture treats nervous system diseases (Acupuncture treats nervous system diseases through many signal transduction pathways. Besides increasing the neurotrophic factors level, acupuncture influences pathways including p38 MAPKs, Raf/MAPK/ERK1/2, TLR4/ERK, PI3K/AKT, AC/cAMP/PKA, ASK1–JNK/p38, and downstream CREB, JNK, m-TOR, NF-diseases . Through


Citrullus lanatus) is one of the most important food crops in the Cucurbitaceae family. Diversification after domestication has led cultivated watermelons to exhibit diverse fruit flesh colors, including red, yellow, and orange. Recently, there has been increased interest in red-fleshed watermelons because they contain the antioxidant cis-isomeric lycopene. We performed whole genome resequencing (WGRS) of 24 watermelons with different flesh colors to identify single-nucleotide polymorphisms (SNPs) related to high lycopene content. The resequencing data revealed 203,894–279,412 SNPs from read mapping between inbred lines and the 97103 reference genome. In total, 295,065 filtered SNPs were identified, which had an average polymorphism information content of 0.297. Most of these SNPs were intergenic (90.1%) and possessed a transversion (Tv) rate of 31.64%. Overall, 2,369 SNPs were chosen at 0.5 Mb physical intervals to analyze genetic diversity across the 24 inbred lines. A neighbor-joining dendrogram and principal coordinate analysis (PCA) based on the 2,369 SNPs revealed that the 24 inbred lines could be grouped into high and low lycopene-type watermelons. In addition, we analyzed SNPs that could discriminate high lycopene content, red-fleshed watermelon from low lycopene, yellow or orange watermelon inbred lines. For validation, 19 SNPs (designated as WMHL1–19) were chosen randomly, and cleavage amplified polymorphic sequence (CAPS) markers were designed. Genotyping of the above 24 lines and 12 additional commercial cultivars using WMHL1–19 CAPS markers resulted in match rates of over 0.92 for most validated markers in correlation with the flesh color phenotypes. Our results provide valuable genomic information regarding the high lycopene content phenotype of red-fleshed cultivated watermelons, and the identified SNPs will be useful for the development of molecular markers in the marker-assisted breeding of watermelons with high lycopene content.Cultivated watermelon ( Citrullus lanatus) is one of the most important horticultural crops in the Cucurbitaceae family. Watermelon originated from north Africa more than 4000 years ago and is now grown throughout the world for its fleshy and sweet fruit [Watermelon , it is now possible to identify potential molecular markers from the informed selection of genes.During the 1980s , marker-Guo et al.  developeThe carotenoid biosynthesis pathway and its involvement in fruit color has been well characterized in tomato and citrus , 16. Carc and C (dominant to c), respectively. Henderson [Y (dominant to y and yo), y (yellow), and yo (dominant to y), respectively. Furthermore, an interaction between the C and I loci was found to result in red flesh, where the C locus is either allele C or c, which is inhibited when the I locus is in the homozygous recessive allelic (i) form [lycopene beta-cyclase (LCYB) gene, and its expression between red and canary yellow watermelon, and developed the Clcyb.600 marker, which perfectly co-segregated with flesh color phenotypes. Recently, Zhu et al. [Poole  reportedenderson  reported(i) form . Althoug(i) form , 29, 30 u et al.  ruled ouSeveral QTLs associated with flesh color have been detected by various researchers. Two QTLs have been identified for red flesh color on chromosomes 2 and 8 . RecentlIn total, 24 watermelon inbred lines were obtained from two domestic seed companies in Korea (Hyundai and Asia Seed Company) for WGRS analysis. The collected inbred lines possessed diverse fruit characteristics, including shape (circular and elliptic), fruit ground color , stripes , and flesh color , as indicated in As listed in Statistical analysis for color parameters were conducted using an SPSS 19.0 statistical software package. Significance was analyzed using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test at the P < 0.05 level. The carotenoid contents among lines in the red, yellow, and orange flesh color groups were compared by calculating the standard error of the mean.http://tgsol.seeders.co.kr/, Korea). The generated Illumina paired-end sequence reads for each inbred line were represented approximately 20× genome coverage considering the reference watermelon (97103) genome size (350 Mb), as previously described [WGRS of inbred watermelon lines was performed through NGS using the Hiseq 2000 and Nextseq  systems at Seeders company  or intergenic (other genomic) SNP variants. Genic SNP variants were further categorized into coding sequences of exon and intron. The polymorphism information content (PIC), an index of polymorphism, was calculated for SNP variants from the 24 watermelon lines . The abor) in MEGA 7.0.14 software [http://www.circos.ca/). The physical location of major QTLs for various fruit trait-related characteristics, including flesh color and lycopene content, were obtained from previous reports [To elucidate the genetic relationship between 24 inbred watermelon lines, a principal component analysis (PCA) was performed based on a subset of the total SNPs using the package SNPRelate in R . Pairwissoftware . SNPs, i reports , 42, 43 AccI, AluI, ApeKI, AvaI, BamHI, BbsI, BsrI, Bsu36I, DpnI, EcoNI, EcoRI, Hind-III, MluI, MspI, NdeI, NruI, PstI, SacI, SalI, SpeI, XbaI, and XhoI). Following prediction of the enzyme site, primers were designed for candidate CAPS sequences using Primer3Plus software with the following criteria: oligo length of 18–24 bp, GC content of 40–60%, Tm of 55–60°C, and amplicon length of 100–500 bp.Based on the preliminary visual assessment and a cross-validation by estimation of color coordinates, the 24 inbred lines were classified according to the color of their fruit flesh . Among d2O to a 12 μL volume. Amplifications were conducted with an initial denaturation step of 95°C for 3 min, which was followed by 40 cycles of 30 s at 95°C, 30 s at 58°C, 30 s at 72°C; and a final cycle of 10 min at 72°C. For the enzyme digestion of PCR amplicons, a 15 μL reaction mixture was used containing: 12 μL of PCR product, 1.5 μL of 10× enzyme buffer, 1 U of restriction enzyme, and ddH20 to a final volume of 15 μL, which was then incubated at 37°C for 2–4 h. Digested products were analyzed by electrophoresis in a 3% metaphor agarose gel  containing 1× Tris-Borate-EDTA (TBE) buffer, followed by fragment separation at 180 V for 25 min. After the digestion of PCR products and subsequent separation of fragments in agarose gel, the match rate between SNP information and CAPS genotyping was analyzed, as described in a previous study [For experimental validation of CAPS markers, each PCR was carried out in a 12 μL reaction volume containing 100 ng of template DNA, 1 μL of 100 μM primers, 1.2 μL of 10× PCR buffer, 1 μL of 10 mM dNTP, and 0.08 μL 5 U/μL Tag, and ddHus study .Cross hybridization between different inbred lines results in genetic populations with different carotenoid profiles, and consequently, the flesh color variations often seen in the pericarp tissues of watermelon fruit . To inveStatistically significant (P < 0.05) differences were observed for all color parameters . All theab) values and carotenoid contents revealed substantial variation among red, yellow, and orange inbred lines  to 41.9 × 106 (line 842), with an average length of 101 bp per inbred line. The total length of reads ranged between 3,518,799,499 bp (line 834) and 4,236,980,805 bp (line 842) (NGS-based WGRS strategies have been widely applied for genetic studies of important traits in many crops . To undeine 842) .http://www.ncbi.nlm. nih.gov/bioproject/ PRJNA516776).After the trimming of raw reads, the ratio of the total length of raw reads over the trimmed reads  was calculated, and varied from 59.8% (54) to 66.46% (843) , wherebySNP markers are co-dominant, highly informative, and polymorphic. These attributes make SNPs effective DNA-based molecular markers for studying genome association and genetic diversity in crop breeding . After tThe rates of transitions (Ts) and transversions (Tv) were 68.36% and 31.64%, respectively. The SNP PIC values ranged from 0.1 to 0.38, with an average value of 0.297. Most SNPs were in intergenic regions (90.1%), with the remaining (9.9%) located within genic regions. Most (70.7%) SNPs within genic regions were in introns rather than exons. To determine the possible linked functions of SNPs located within genic regions, we searched the corresponding transcripts of genic SNPs. Accordingly, we identified 7712 non-redundant transcripts, and Gene Ontology (GO) annotations were simultaneously performed. Based on GO analysis, these transcripts were classified into “cellular components (79%)”, “molecular functions (73%)”, and “biological processes (72%)”, and were annotated to “primarily located in intracellular components, nucleus, and cytoplasmic part”; “proteins involved in enzyme binding, protein binding, and DNA binding”; and “involved in cellular and metabolic processes and protein metabolism”, respectively .PIC indicates the usefulness of a marker for detecting polymorphism. Multi-allelic molecular markers, such as RAPD, and AFLP, usually have PIC values that range between 0.5 and 1.0. In contrast, SNPs are bi-allelic in nature, possess a co-dominant inheritance pattern, and have lower PIC values, which can range between 0 and 0.5 . SNPs wiTo determine the genetic relationships between the 24 different flesh-colored lines, a NJ tree was constructed using 2369 selected SNPs . AccordiY, y, yo, C, and I) [O), two , or three  alleles, respectively. Thus, genetic relationships within watermelon inbred lines, as inferred from genome-wide SNPs  , 28, sug, and I) , skin co, and I) , or stri, and I) , have beide SNPs , would nide SNPs . With noide SNPs : clusteride SNPs . These rWe also determined the genome-wide distribution frequency of SNPs in three typical inbred lines with red (803: Cluster-IA), yellow (835: Cluster-IIA), and orange (840: Cluster-IIB) colors, grouped separately in both the PCA and NJ tree . As showThe analysis of genetic diversity between 24 inbred lines revealed that the SNPs were able to generally distinguish between red flesh type with high lycopene content, and non-red flesh type, such as yellow or orange, with low lycopene cultivars. To validate the SNPs detected during resequencing , we randWe also noted some discrepancies between SNP information (WMHL11 and WMHL17), which can be explained by a failure of restriction digestion or technical errors during resequencing, as observed in a previous study on watermelons . With thin silico expression profiling of these genes was performed using comparative watermelon transcript data (BioProject: PRJNA338036) from the same genes available in various fruit developmental stages of red-fleshed (LSW177) and pale-yellow-fleshed (COS) cultivated watermelons. The results revealed that these SNP-carrying genes presented preferential and stage-specific expression between LSW177 and COS. At 10 DPA, these genes were generally upregulated in pale yellow fleshed COS compared with LSW177. At the 26 DPA ripening stage, almost all genes displayed robust, uniform upregulated expression of their transcripts in both COS and LSW177, relative to other developmental stages. At the 18, 34, and 42 DPA stages, almost all SNP-carrying genes were significantly upregulated in the red flesh of LSW177 compared to that in COS , stripe pattern (wsb6-11), and flesh color (Clcyb.600 and Lcyb) , 30, 52.lycopene β-cyclase (LCYB) gene (Cycl.600 and Lcyb) were identified [LCYB gene , where all SNPs from these genes were found to be cosegregated with the flesh color and high-lycopene phenotypes of the inbred lines used in this study. As listed in LCYB at 8,721,803 (Cla005007: Unknown Protein) and 8,923,655 bp (Cla005012: Kinesin-like protein), with PICs of 0.37 and 0.38, respectively. The obtained SNP from kinesin-like protein  was confirmed to be cosegregated with the flesh color and high lycopene content upon validation  of flesh color (FC3.1) . A QTL oIn the present study, genome resequencing revealed significant genetic variations among different flesh-colored inbred lines in watermelons. By utilizing resequencing data, we identified numerous SNPs associated with flesh-color characteristics among all the inbred lines, which will be useful for the rapid development of diagnostic markers for MAS and fine mapping. Analysis of genetic diversity using 2369 SNPs mined on a genome-wide scale demonstrated practical significance, whereby two groups were found in the 24 inbred lines. Upon validation, the selected SNP/CAPS were able to distinguish between inbred lines with high (red) and low (yellow/orange) lycopene contents, and the results were correlated with the flesh color phenotype of each individual. Overall, for the first time, polymorphic SNPs were discovered in watermelons with high lycopene content. The SNPs identified from the current WGRS approach in watermelon may be utilized in population genetics, QTL mapping, and genomics in order to assist in the breeding of watermelons with high lycopene content.S1 FigPhotographs of longitudinal cross-sections of representative individuals for the flesh color categories of 24 inbred lines used in WGRS (A) and 12 commercial cultivars used in CAPS validation (B).(PDF)Click here for additional data file.S2 FigIdentified transcripts are annotated into functional categories of cellular component, molecular function and biological process as indicated in the figure.(PDF)Click here for additional data file.S3 FigA) Determination of the optimal K-value  based on five independent runs with burn-in period of 10,000 iterations followed by 10,000 Monte Carlo Markov Chain (MCMC) iterations according to a previous study [B) Population structure for the 24 watermelon inbred lines when K = 3. Each individual inbred line is indicated by a narrow vertical bar, which is partitioned into red, blue and green zones in proportion to the association coefficiencies to the 3 populations, such as the high lycopene content of cluster I comprising two other sub-clusters (A and B) and the low lycopene content of cluster II and III populations. The color and shape schemes are the same as those for the neighbor-joining (NJ) analysis in (us study . The rat(TIF)Click here for additional data file.S4 Figgreen asterisk”.The name of validated CAPS markers (WMHL) are shown below their corresponding gel pictures. Numbers in lanes represents the DNA sample names of the inbred lines  and comm(PDF)Click here for additional data file.S5 Figin silico expression profiling are indicated at the top and bottom of the heat map, respectively. A combined table showing the gene ID, marker name and gene description. The color scale at the top indicate up (red) or down (green) regulated expression.DPA and watermelon lines used for (TIF)Click here for additional data file.S6 FigLCYB gene (Cycl.600 and Lcyb) by Bang et al. [Upper panel shows the position of SNP hotspots of different chromosomes. Lower panel describes the schematic representation of genetic linkage map for markers on chromosome 4 (WII04E08-38 to WII04EBsaHI-6) and Chromosome 3 (BVWS00048 to WII03E09-92) linked to flesh color (FC4.1 and FC3.1) or lycopene content (LCY4.1) according to previous studies . A CAPS g et al. , 30 mark(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.


Arabidopsis, tomato, rice, and maize, the potential roles of OPRs in wheat are not fully understood. Here, forty-eight putative OPR genes were found and classified into five subfamilies, with 6 in sub. I, 4 in sub. II, 33 in sub. III, 3 in sub. IV, and 2 in sub. V. Similar gene structures and conserved protein motifs of TaOPRs in wheat were identified in the same subfamilies. An analysis of cis-acting elements in promoters revealed that the functions of OPRs in wheat were mostly related to growth, development, hormones, biotic, and abiotic stresses. A total of 14 wheat OPR genes were identified as tandem duplicated genes, while 37 OPR genes were segmentally duplicated genes. The expression patterns of TaOPRs were tissue- and stress-specific, and the expression of TaOPRs could be regulated or induced by phytohormones and various stresses. Therefore, there were multiple wheat OPR genes, classified into five subfamilies, with functional diversification and specific expression patterns, and to our knowledge, this was the first study to systematically investigate the wheat OPR gene family. The findings not only provide a scientific foundation for the comprehensive understanding of the wheat OPR gene family, but could also be helpful for screening more candidate genes and breeding new varieties of wheat, with a high yield and stress resistance.The 12-oxo-phytodienoic acid reductases (OPRs), which belong to the old yellow enzyme (OYE) family, are flavin mononucleotide (FMN)-dependent oxidoreductases with critical functions in plants. Despite the clear characteristics of growth and development, as well as the defense responses in  Jasmonates (JAs) are a kind of lipid-derived signaling molecules in plants and function in response to various stresses, as well as plant growth and development ,2. JasmoOPR genes belong to the OYE family and are classified as FMN-dependent oxidoreductases, with multiple subfamilies in plants [OPR genes were classified into seven conserved subfamilies. Furthermore, two subfamilies in dicots (sub. I and II) and five subfamilies (sub. IV) in monocots were discovered [Arabidopsis thaliana were divided into two groups, group I (OPRI) and group II (OPRII) [AtOPR1 and AtOPR2 (OPRI) preferentially catalyze -12-oxophytodienoic acid , which is not involved in the JA biosynthetic process. The AtOPR3 belonging to OPRII can convert the 9S,13S-OPDA to oxo-2(2’(Z)-pentenyl)-cyclopentane-1-octanoic acid (OPC-8:0), and OPC-8:0 is the precursor of JA biosynthesis [n plants . The Oxiscovered . In prev (OPRII) . The AtOynthesis ,11. ThenArabidopsis, the OPRs can be activated by wounding, pathogens, cadmium ion, and hormone signaling molecules, such as JA, abscisic acid (ABA), salicylic acid (SA), and ethylene (ET) [Arabidopsis and Nicotiana tabacum L. plants presented tissue-specific patterns [AtOPR1 was expressed in young seeds, while AtOPR2 was detected in pollen, and both AtOPR1 and AtOPR2 were found in roots [AtOPR3 was expressed in various tissues of the Arabidopsis [AtOPR3 (OPRII). Thus, AtOPR3 can be localized in the peroxisome. However, this specific peroxisomal target sequence was not discovered in OPRI [OPR and the subsequent signaling pathway [OPR may exhibit multi-functions, owing to the substrate specificity, subcellular location, and differential expression in different tissues and specific stresses [ZmOPR1 and ZmOPR2 defenses against pathogens were induced by SA and chitooligosaccharides, not by wounding, while ZmOPR7 and ZmOPR8 were induced by wounding, as well as crosstalk with signaling molecules, such as JA, ABA, and SA [OPR genes belonging to sub. I and sub. II have been reported, and they could enhance wheat tolerance to abiotic stresses  through the ABA-dependent pathway or the JA biosynthesis pathway [To date, the biochemical and physiological functions of OPRs have been reported in dicots and monocots. In dicot ene (ET) ,14. The patterns . AtOPR1 in roots . In addibidopsis . A perox in OPRI ,17. The  pathway . An indistresses . For ins, and SA . It was  pathway ,22.OPR gene family in wheat. In this study, the wheat OPR gene family was identified via a genome-wide search. Then, the putative 48 wheat OPR genes were systematically analyzed, including the gene phylogenetic relationship, gene structure, protein conserved domain, chromosome localization, cis-acting regulatory elements, and expression profiles, to indicate the evolutionary and functional features of these genes. In addition, the relative expression of five OPR genes from five different subfamilies (2 tissues and eight stresses for each gene) were determined using qRT-PCR to better comprehend the OPR functions in growth and development, as well as in stress responses. This work could contribute to the selection of more candidate genes for further functional study of OPR genes in order to improve resistance against various stresses.The functions of OPRI and OPRII have been well characterized in dicot plants ,23,24,25Oxidored_FMN conserved domain via Pfam and the NCBI-CD database. From the TAIR database, 3 Arabidopsis OPRs were obtained, and 8 maize OPRs and 13 rice OPRs were explored and compared against NCBI by the key word, 12-oxo-phytodienoic acid reductase. Arabidopsis, maize, and rice OPR gene IDs were available in A total of 55 TaOPRs were identified by local BLASTP using HMM profiles, and at last, 48 putative TaOPRs were confirmed by detecting the TaOPRII-B1, TaOPRIII-D13, and TaOPRIII-B9). In sub. I, the TaOPRI-D2 and TaOPRI-A3 were predicted to be localized in mitochondrial, and the other four TaOPRI proteins were localized in the chloroplast. In sub. III, IV, and V, a total of 17 OPRs were predicted to be localized in mitochondria, and 19 OPRs were localized in cytoplasms . On the basis of the classification of other plants and the amino acid structure of OPRs [To further investigate the phylogenetic relationships and predict the classification of the wheat OPR gene family in the Arabidopsis of sub. I and sub. II was different [OPR genes, and the OPR subfamily, generated later, evolved some new subgroup-specific functions after divergence [OPR family genes were diverse in different subfamilies. While there were conserved domains in OPRs, the gene functions might be significantly different. This was consistent with the OPRs in maize [In addition, TaOPRI-B1, TaOPRI-B2, TaOPRI-D2, TaOPRI-A3, TaOPRI-B3, and TaOPRI-D3 were closely related to AtOPR1, AtOPR2, ZmOPR5, and OsOPR11 and were clustered in sub. I, and TaOPRII-A1, TaOPRII-B1, TaOPRII-D1, and TaOPRII-B2 were tightly linked to AtOPR3, ZmOPR7, ZmOPR8, and OsOPR7 and were clustered in sub. II. Wheat sub. III was clustered with ZmOPR1-3, and OsOPR1-6. Wheat sub. IV was tightly related to ZmOPR4, ZmOPR6, OsOPR9, and OsOPR10. Wheat sub. V was clustered with OsOPR8, OsOPR12, and OsOPR13. In previous studies, the physiological biochemical character of the ifferent ,16,26. Hin maize  and riceOPR genes, the conserved protein blocks of the 48 TaOPRs were aligned using ClustalW, and the maximum-likelihood method was used to construct a phylogenetic tree b. We fouOPR genes  and 7  during evolution.According to the available wheat genome annotation information, a total of 48 wheat lization . There wOPR genes. A total of 14 wheat OPR genes were identified as tandem duplicated genes and located in chromosomes 1  and 7 . Additionally, 4 groups of two tandem duplicated genes were located in chromosomes 1D, 7A, 7B, and 7D, and 2 groups of three tandem duplicated genes were located in chromosomes 1A and 1B (OPR genes) were segmentally duplicated genes, including 30 pairs (36 OPR genes) of homoeologous genes, and were distributed in different chromosomes  of three-leaf-stage seedlings were used to determine the tissue-specific expression patterns by qRT-PCR b. Based OPR genes showed differential expression patterns under drought and heat stresses , under 8 stress treatments, including abiotic stresses , hormonal treatments , and biotic stress (aphid). The relative expression of TaOPR genes in five subfamilies  showed various expression profiles  were down-regulated under salt stress, and the TaOPRII-B1, TaOPRIII-B7, TaOPRIV-A1, and TaOPRV-B1 were negatively regulated by aphid treatment. Nevertheless, some genes in different subfamilies exhibited opposite expression patterns under drought, heat, wounding, MeJA, ABA, and SA treatments. To further ascertain the biological functions of profiles . OverallArabidopsis [Camellia sinensis [OPR genes were identified from maize, and a series of analyses revealed that various OPR genes were located in the cytoplasm or peroxisome, performing different functions, and were differentially regulated in response to stress-associated hormones, wounding, or pathogen infections [OPR genes were identified, and the expression profiles obviously showed tissue-specific and stress-specific elements, such as hormones, wounding, and other abiotic or biotic stresses [OPR genes, but the previous work of TaOPR1 and TaOPR2 could provide an insight on functions of OPRs [OPRs are multigene families and can be identified in many plants . While pbidopsis , tomato bidopsis , maize [bidopsis , rice [1bidopsis , and Camsinensis , they hafections . In ricestresses . In whea of OPRs ,22.OPR genes within a subfamily showed a similar exon/intron structure, indicating that the evolution might not only affect the gene function, but also the gene structure [OPR gene family [TaOPPRIII-B10, TaOPPRIII-D10, TaOPPRIII-A11, TaOPPRIII-B11, and TaOPPRIII-D11) which lacked motif 1, 5, and 8, and they presented converse expression patterns with other genes possessing the whole motifs. The similar trends were found for most of the other genes that lacked motifs. Therefore, the diversity of protein structure probably contributed to diverse expression patterns. As a result of the selection pressures during genome evolution in wheat, the OPR genes within the same subfamily gradually formed various gene structures or protein motifs in order to respond to various conditions [In this study, a total of 48 TaOPRs were identified from wheat and characterized. The 48 TaOPRs were clustered into five subfamilies (named as sub. I-V), which is consistent with Li et al. (2009). According to the phylogenetic and gene structure analysis, most of the tructure ,29. Subfe family . Analogonditions .Arabidopsis) were conducted to investigate the evolution of the OPR family. We found 13 and 8 proteins in monocot rice and maize, respectively, and 3 OPR proteins in dicot Arabidopsis. Thus, the number of OPR proteins in wheat (48 TaOPRs) was much higher than that in rice, maize. and Arabidopsis. The reason might be attributed to the allohexaploid genome and complex evolution in wheat [Comparisons of wheat OPR proteins with other plant species  could enhance salinity tolerance via an ABA-dependent signaling pathway, and TaOPR2 (sub. II) was involved in the JA biosynthesis pathway and could be induced by various stresses  [TaOPR2 could rescue the male sterility phenotype of Arabidopsis mutant opr3, thereby it played a vital role in the male development of wheat. It has been reported, in several studies, that OPRs participated in the JA signaling pathway to defend against phloem-feeding insects, such as aphids and brown planthoppers [Arabidopsis can modulate the stomatal closure to defend against drought stress by affecting the production of OPDA (cis-12-oxo-phytodienoic acid), a precursor of OPR, and this process was closely related to JA and ABA [Arabidopsis [Based on the predictive analysis of cis-acting regulatory elements, the OPRs might involve regulating various biological processes. In our study, the wheat OPRs were closely associated with the stimulation of phytohormones, including MeJA, ABA, SA, GA, IAA, and ET, as well as responses to various stresses . Thus, it could be speculated that the wheat tritici) ,22. In athoppers ,37. More and ABA . Supportbidopsis ,38,39. TOPR genes in wheat were diverse in different tissues and developmental periods, which could be inferred from the analyses of qRT-PCR and public transcriptome data. In Arabidopsis, the two OPR genes (AtOPR1 and AtOPR3) from different subfamilies were proved to act in different biological processes or defense responses with tissue-specific expression patterns. The AtOPR1 were mainly expressed in young rosettes, while the AtOPR3 were predominantly expressed in young flowers [AtOPR3 in subfamily II were male-sterile, and the OPDA (precursor of AtOPR3), when combined with ABA, could inhibit seed germination [AtOPR3 was shown to function in the defense against nematodes through the JA and auxin signaling pathways in Arabidopsis [OPR homoeologous genes, such as TaOPRIII-13B with TaOPRIII-13A and TaOPRIII-13D. These diverse gene expression patterns of OPR among homoeologous genes might be attributed to the loss or gain of gene function during the wheat evolution process [TaOPRII-1 was high in all the five tissues. Likewise, the TaOPRIII-7 was highly expressed in root and leaf. Thus, it can be inferred that expression of wheat OPR genes exhibit a strongly tissue-specific pattern, which is in accordance with previous studies on Arabidopsis, rice, and maize [The expression profiles of  flowers ,16. The mination . Besidesbidopsis . These r process . In thisnd maize ,16,20.Arabidopsis and tomato [TaOPRI-B2 (sub. I) and TaOPRII-B1 (sub. II) were up-regulated when challenged with abiotic stresses  and phytohormones (MeJA and ABA). However, the expression of TaOPRIII-B7 (subfamily III), TaOPRIV-A1 (subfamily IV), and TaOPRV-B1 (subfamily V) decreased under abiotic  and biotic (aphid) stresses. The expressions of TaOPRII-B1 in our trial (TaOPR2 (the same gene with TaOPRII-B1) under drought, salt, wounding, and MeJA treatments [TaOPR1 in subfamily I was up-regulated when challenged with salt stress, however in our study, the TaOPRI-B2 (not the same gene as TaOPR1) was significantly down-regulated. As stated above, even if OPRs belonged to the same subfamily, they might exhibit inconsistent expression patterns under some stimulations. Beyond that, wheat varieties, as well as nutrient composition and contents, were varied between these investigations, and whether they influenced the responses remained inconclusive. More explorations were needed to illuminate the detailed mechanisms and other potential causes. Above all, the OPRs played multiple and vital roles in many biological processes of plants. For more example, the OPR3 (OPRII) was essential for the embryo development of tomato [OPR7 and OPR8 (OPRII) could regulate the development and defensive responses of maize through the JA signaling pathway, and strong developmental defects were found in double mutant opr7 opr8 (OPRII), such as the formation of a feminized tassel and the extreme elongation of ear shanks [The functions of OPRs in plant growth and development, as well as the defense responses to biotic and abiotic stresses, have been thoroughly investigated in d tomato ,26. In tur trial  were coneatments . Exceptieatments  proved tf tomato . Moreover shanks . The OPROPR1  could participate in defensive responses to abiotic and biotic stresses  as well as the stimulations of phytohormones  [OPR genes of subfamilies III, IV, and V in wheat have not been documented. The comprehensive analysis of the qRT-PCR results and public RNA-seq databases demonstrated that the OPR genes in sub. III, IV, and V could be induced by multiple stresses, and more investigations were needed to crosslink their multi-functions and detailed mechanisms.The biological functions of OPRs in subfamilies III, IV, and V were less understood in comparison with those of subfamilies I and II. Rice  and ET) ,44,45. B and ET) . To our Arabidopsis thaliana (At), monocot Oryza sativa (Os), Zea mays (Zm), and Triticum aestivum (Ta) were obtained from public databases . At first, a local protein database was constructed using all the wheat protein sequences, which were downloaded from the Ensembl Plants of wheat (ftp://ftp.ensemblgenomes.org/pub/plants/release-42/fasta/triticum_aestivum). Then, the HMM profile of the family, Oxidored_FMN (PF00724), was downloaded from PFAM (http://pfam.xfam.org/), and this was used to search and compare the data with the local protein database, using the hmmsearch tool of the HMMER3.0 software. All the obtained protein sequences were verified by submitting them to the PFAM databases (http://pfam.xfam.org/) and NCBI Batch CD-search database (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) to confirm the structural integrity of the Oxidored_FMN domain [https://web.expasy.org/protparam/) [http://www.cbs.dtu.dk/services/TargetP/).The protein sequences of dicot N domain . Finallytparam/) . The subhttp://molevol.cmima.csic.es/castresana/Gblocks_server.html), which eliminated divergent regions and poorly aligned regions. The ProtTest v3.4 was used to select the best-fit model of amino acid substitution for tree-contracting analyses. The maximum-likelihood (ML) phylogenetic tree was constructed based on the conserved blocks and best-fit model using MEGA 7.0 software with 1000 bootstrap replications. Multiple alignments of the conserved TaOPR protein sequences were performed using ClustalW tool. The conserved protein blocks were selected using Gblocks Server (TaOPR were acquired from Ensembl plants (http://plants.ensembl.org/Triticum_aestivum/Info/Index), and the exon-intron structures were displayed by searching the Gene Structure Display Server (GSDS) (http://gsds.cbi.pku.edu.cn/) [http://meme-suite.org/) [The CDS and genomic sequences of edu.cn/) . The prote.org/) .cis-acting regulatory elements via PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). Virous cis-acting regulatory elements of all TaOPR transcripts were displayed in a diagram . Gene duplications of TaOPR were analyzed using the Multiple Collinearity Scan toolkit (MCScanx) (http://chibba.pgml.uga.edu/mcscan2/), and the E-value was 1 × 10−10. Tandem duplication of events were defined as two or more adjacent homologous genes located on one chromosome without any intervening gene, while segmentally duplicated genes were generated through polyploidy and experienced chromosome rearrangements [TaOPR genes was visualized by the CIRCOS program.To map the locations of putative ngements . The chrTaOPR genes in different stress challenges and tissues, the expression data were obtained from WheatExp (https://wheat.pw.usda.gov/WheatExp/) [TaOPR genes were presented in To analyze the expression profiles of eatExp/) ,52 and tSchizaphis graminum) were placed on leaves for 12 h and 24 h of feeding [The wheat cultivar “KN199” was grown in a controlled climate chamber  under the following conditions: 25 °C/14 h light and 20 °C/10 h dark. The three-leaf-stage seedlings were used for tissue-specific analysis (leaf and root) and different stress treatments. Stress treatments included abiotic stress, hormones, and biotic stress. To induce abiotic stresses , seedlings were treated with a Hoagland liquid medium containing 200 mM NaCl for 1 h and 6 h , 20% PEG6000 for 1 h and 6 h (drought stress), and 40 °C for 1 h and 6 h (heat stress) , respect feeding . SeedlinTaOPRI-B2, TaOPRII-B1, TaOPRIII-B7, TaOPRIV-A1, TaOPRV-B1, and TaActin, used in this work, were presented in The total RNA was extracted using the Takara RNA extraction Kit , following the Instructions for Users. The first cDNA strand was synthesized using the Roche Transcriptor First Strand cDNA Synthesis Kit . The qRT-PCR was performed using the CWBIO FastSYBR Mixture  in a Roche LightCycler 480 System . A 20 μL reaction system was used, which can be described as follows: 10 μL 2×UltraSYBR Mixture (2×), 0.4 μL of forward primer (10.0 μmol/L), 0.4 μL of reverse primer (10.0 μmol/L), 1 μL (200 ng) of cDNA, and 8.2 μL of RNase-free water. The reaction procedure was completed in accordance with the following program: 95 °C for 10 min; 35 cycles of 95 °C for 15 s, 60 °C for 1 min; 95 °C for 15 s, 60 °C for 1 min, 95 °C for 15 s, and 60 °C for 15 s. Primer sequences for the OPR gene family for the first time in this study. A total of 48 TaOPRs were obtained and were classified into subfamilies I–V after systematic investigations. Tandem and segmental duplications contributed to the expansion of the OPR gene family, and segmental duplication tended to play the predominant role. The wheat OPRs were involved in crucial processes, such as plant growth and development, phytohormone-mediated metabolism, and defensive responses to various abiotic and biotic stresses. The expression patterns of TaOPR genes were tissue-specific and had stress-responsive diversity. This study could not only provide a scientific foundation for the comprehensive understanding of the wheat OPR gene family, but was also be helpful for screening more candidate genes and breeding new varieties of wheat with a high yield and stress resistance.We identified and characterized the wheat


Scientific Reports 10.1038/s41598-018-25030-6, published online 04 May 2018Correction to: The PDF and HTML versions of this Article contain extensive typographical errors in the reference citations in the Discussion section.In the fourth paragraph,7,8,11–14,17,19. In contrast, women in our study were most often injured at home and the workplace. A universal finding in studies of work-related eye injuries is that 55% to 91% of patients were not using protective eye wear at the time of injury20–23.”“For the young-adult and middle-aged males in our study, activity risk factors for major ocular trauma were metal-working, driving, social behaviours such as fighting, and participation in dangerous sports or hobbies, similar to findings from previous reportsshould read:7,8,11–14,17,20. In contrast, women in our study were most often injured at home and the workplace. A universal finding in studies of work-related eye injuries is that 55% to 91% of patients were not using protective eye wear at the time of injury21–24.”“For the young-adult and middle-aged males in our study, activity risk factors for major ocular trauma were metal-working, driving, social behaviours such as fighting, and participation in dangerous sports or hobbies, similar to findings from previous reportsAdditionally,24.”“Most patients involved in traffic accidents are young adolescents riding motorcycles, a common occurrence in Taiwanshould read:25.”“Most patients involved in traffic accidents are young adolescents riding motorcycles, a common occurrence in TaiwanIn the fifth paragraph,25, thereby continuing to be exposed to high risks of ocular trauma over time. Second, a law implemented in 1997 enforcing motorcyclists to wear helmets has reduced the risk and severity of head and/or eye injuries decade by decade, particularly among young males26,27.”“By contrast, middle-age laborers engaging in high-risk work, such as construction, manufacturing, or agriculture, have elongated their working ages and are now retiring later than beforeshould read:26, thereby continuing to be exposed to high risks of ocular trauma over time. Second, a law implemented in 1997 enforcing motorcyclists to wear helmets has reduced the risk and severity of head and/or eye injuries decade by decade, particularly among young males27,28.”“By contrast, middle-age laborers engaging in high-risk work, such as construction, manufacturing, or agriculture, have elongated their working ages and are now retiring later than beforeAdditionally,23; and avoiding risky behaviours, such as drinking alcohol before driving or not following safety procedures for operating machinery. Education regarding these safety measures is essential to reduce the incidence and severity of ocular injuries. Evidence from our current study and previous studies indicate that children were usually injured at home or at school28–30; patients aged 60 years or older usually had ocular trauma because of a fall at home31.”“The risk of ocular trauma related to working with hazardous materials or involvement in a traffic accident can be greatly reduced by such measures as using safety (laminated) glass in windscreens; wearing helmets, safety goggles, seat belts, and other protective gear appropriate to the high-risk activityshould read:24; and avoiding risky behaviours, such as drinking alcohol before driving or not following safety procedures for operating machinery. Education regarding these safety measures is essential to reduce the incidence and severity of ocular injuries. Evidence from our current study and previous studies indicate that children were usually injured at home or at school29–31; patients aged 60 years or older usually had ocular trauma because of a fall at home32.”“The risk of ocular trauma related to working with hazardous materials or involvement in a traffic accident can be greatly reduced by such measures as using safety (laminated) glass in windscreens; wearing helmets, safety goggles, seat belts, and other protective gear appropriate to the high-risk activityIn the sixth paragraph,19,32”“By contrast, in studies of “all severities” of ocular trauma presenting to the emergency department, closed-globe injuries were more prevalent, with open-globe injuries reported to represent 6.3% of cases in northern Taiwan and 14.2% in Koreashould read:19,20”“By contrast, in studies of “all severities” of ocular trauma presenting to the emergency department, closed-globe injuries were more prevalent, with open-globe injuries reported to represent 6.3% of cases in northern Taiwan and 14.2% in KoreaIn paragraph seven,11,13,28,30,31,33. On the other hand, posterior segment injuries, such as vitreous haemorrhage, retinal injury, and optic nerve injury, were seen less frequently in our study, but were associated with a worse final visual acuity14,28.”“In this study, anterior segment injuries were common and included corneal/scleral laceration or limbal wound dehiscence, hyphaemia, and lens injury, correlating with results reported in our previous study and other published studiesshould read:11,13,29,31–33. On the other hand, posterior segment injuries, such as vitreous haemorrhage, retinal injury, and optic nerve injury, were seen less frequently in our study, but were associated with a worse final visual acuity14,29.”“In this study, anterior segment injuries were common and included corneal/scleral laceration or limbal wound dehiscence, hyphaemia, and lens injury, correlating with results reported in our previous study and other published studiesIn the eighth paragraph,et al.’s study19, and 33% of patients in May et al.’s study20.”“Subsequent procedures were performed in 15.4–18.2% of the patients in the two periods of this study, 9.6% of patients in Oum should read:et al.’s study20, and 33% of patients in May et al.’s study21.”“Subsequent procedures were performed in 15.4–18.2% of the patients in the two periods of this study, 9.6% of patients in Oum In the ninth paragraph,4,7,8,11–14,16,28,31. Factors that have been documented to correlate with the visual prognosis after trauma include visual acuity immediately after the injury, presence of an afferent pupillary defect, type and mechanism of injury, location and extent of penetrating wounds, and presence of lens damage, vitreous haemorrhage, retinal detachment, intraocular foreign body, or endophthalmitis14,28,38.”“In our previous study and other published studies, the visual outcome after major ocular trauma was generally unsatisfactoryshould read:4,7,8,11–14,16,29,32. Factors that have been documented to correlate with the visual prognosis after trauma include visual acuity immediately after the injury, presence of an afferent pupillary defect, type and mechanism of injury, location and extent of penetrating wounds, and presence of lens damage, vitreous haemorrhage, retinal detachment, intraocular foreign body, or endophthalmitis14,29,38.”“In our previous study and other published studies, the visual outcome after major ocular trauma was generally unsatisfactoryFinally, in the tenth paragraph,et al.11, but far less favourable than those in Tsai et al.’s report32 consisting of “all severities” of ocular trauma treated in the Emergency Department, in which 45.7% of patients had good final visual acuities of 20/40 or better, 24.9% had moderate visual acuities of 20/200 to 20/50, and 11.3% had poor visual acuities of 20/400 or worse.”“The visual outcome profile of our patients was similar to another report of hospitalized ocular trauma patients in Taiwan by Chang should read:et al.11, but far less favourable than those in Tsai et al.’s report19 consisting of “all severities” of ocular trauma treated in the Emergency Department, in which 45.7% of patients had good final visual acuities of 20/40 or better, 24.9% had moderate visual acuities of 20/200 to 20/50, and 11.3% had poor visual acuities of 20/400 or worse.”“The visual outcome profile of our patients was similar to another report of hospitalized ocular trauma patients in Taiwan by Chang