Primary Bitter Taste of Citrus is Linked to a Functional Allele of the 1,2-Rhamnosyltransferase Gene Originating from Citrus grandis.

1,2-Rhamnosyltransferase (1,2RhaT) catalyzes the final step of production of flavanone neohesperidoside (FNH) that is responsible for the primary bitter taste of citrus fruits. In this study, species-specific flavonoid profiles were determined in 87 Citrus accessions by identifying eight main flavanone glycosides (FGs). Accumulation of FNHs was completely correlated to the presence of the 1,2RhaT gene in 87 citrus accessions analyzed using a novel 1,2RhaT-specific DNA marker. Pummelo (Citrus grandis) was identified as the genetic origin for a function allele of 1,2RhaT that underpinned FNH-bitterness in modern citrus cultivars. In addition, genes encoding six MYB and five bHLH transcription factors were shown to coexpress with 1,2RhaT and other flavonoid pathway genes related to FNH accumulation, indicating that these transcription factors may affect the fruit taste of citrus. This study provides a better understanding of bitterness formation in Citrus varieties and a genetic marker for the early selection of nonbitterness lines in citrus breeding programs.

Comparative analysis of volatile and carotenoid metabolites and mineral elements in the flesh of 17 kiwifruit.

Kiwifruit contains abundant nutritive compounds and is highly favored by the consumers worldwide. Therefore, detailed metabolic profiling is important to provide theoretic basis for the improvement of kiwifruit quality. In this study, the levels of volatiles, carotenoids, and mineral elements in the flesh of 17 kiwifruit accessions were evaluated. Acids and esters were the main volatiles in kiwifruit. During these 17 kiwifruit accessions, "Chenhong," three "Jinyan," and two "Guichang" germplasms were specifically rich in aromatic esters, which might be associated with their special taste. The main carotenoids were lutein, ß-carotene, and zeaxanthin, and their levels were also genotype specific, with the green-fleshed "Guichang" having the highest level of carotenoids, and red-fleshed "Fuhong" and "Chenhong" being rich in zeaxanthin. Partial correlation analysis showed that the contents of some mineral elements were significantly correlated with those of specific volatiles and carotenoids, indicating the impacts of mineral elements on the accumulation of volatiles and carotenoids in the kiwifruit flesh. These results indicated that the contents of carotenoids and volatiles seemed to be affected by mineral elements and also provided a new potential method for improving fruit flavor quality in production.

Integrated Transcriptomic and Metabolomic analysis reveals a transcriptional regulation network for the biosynthesis of carotenoids and flavonoids in 'Cara cara' navel Orange.

BACKGROUND: Carotenoids and flavonoids are important secondary metabolites in plants, which exert multiple bioactivities and benefits to human health. Although the genes that encode carotenogenesis and flavonoid biosynthetic enzymes are well characterized, the transcriptional regulatory mechanisms that are related to the pathway genes remain to be investigated. In this study, 'Cara cara' navel orange (CNO) fruit at four development stages were used to identify the key genes and TFs for carotenoids and flavonoids accumulation. RESULTS: In this study, CNO was used to investigate the profiles of carotenoids and flavonoids by a combination of metabolomic and transcriptomic analyses. The important stage for the accumulation of the major carotenoid, lycopene was found to be at 120 days after florescence (DAF). The transcripts of five carotenogenesis genes were highly correlated with lycopene contents, and 16, 40, 48, 24 and 18 transcription factors (TFs) were predicted to potentially bind 1-deoxy-D-xylulose-5-phosphate synthase (DXS1), deoxyxylulose 5-phosphate reductoisomerase (DXR), geranylgeranyl diphosphate synthase (GGPPS2), phytoene synthase (PSY1) and lycopene ß-cyclase (LCYB) promoters, respectively. Narirutin was the most abundant flavonoid in the flesh at the early stages, 60 DAF was the most important stage for the accumulation of flavonoids, and 17, 22, 14, 25, 24 and 16 TFs could potentially bind phenylalanine ammonia-lyase (PAL-1 and PAL-4), 4-Coumarate-CoA ligase (4CL-2 and 4CL-5), chalcone synthase (CHS-1) and chalcone isomerase (CHI) promoters, respectively. Furthermore, both sets of 15 candidate TFs might regulate at least three key genes and contribute to carotenoids/flavonoids accumulation in CNO fruit. Finally, a hierarchical model for the regulatory network among the pathway genes and TFs was proposed. CONCLUSIONS: Collectively, our results suggest that DXS1, DXR, GGPPS2, PSY1 and LCYB genes were the most important genes for carotenoids accumulation, while PAL-1, PAL-4, 4CL-2, 4CL-5, CHS-1 and CHI for flavonoids biosynthesis. A total of 24 TFs were postulated as co-regulators in both pathways directly, which might play important roles in carotenoids and flavonoids accumulation in CNO fruit.

The IAA- and ABA-responsive transcription factor CgMYB58 upregulates lignin biosynthesis and triggers juice sac granulation in pummelo.

In citrus, lignin overaccumulation in the juice sac results in granulation and an unpleasant fruit texture and taste. By integrating metabolic phenotyping and transcriptomic analyses, we found 702 differentially expressed genes (DEGs), including 24 transcription factors (TFs), to be significantly correlated with lignin content. CgMYB58 was further identified as a critical R2R3 MYB TF involved in lignin overaccumulation owing to its high transcript levels in Huanong Red-fleshed pummelo (HR, Citrus grandis) fruits. Transient expression of CgMYB58 led to an increase in the lignin content in the pummelo fruit mesocarp, whereas its stable overexpression significantly promoted lignin accumulation and upregulated 19 lignin biosynthetic genes. Among these genes, CgPAL1, CgPAL2, Cg4CL1, and CgC3H were directly modulated by CgMYB58 through interaction with their promoter regions. Moreover, we showed that juice sac granulation in pummelo fruits could be affected by indole-3-acetic acid (IAA) and abscisic acid (ABA) treatments. In HR pummelo, ABA significantly accelerated this granulation, whereas IAA effectively inhibited this process. Taken together, these results provide novel insight into the lignin accumulation mechanism in citrus fruits. We also revealed the theoretical basis via exogenous IAA application, which repressed the expression of CgMYB58 and its target genes, thus alleviating juice sac granulation in orchards.

Volatile Compounds in Fruit Peels as Novel Biomarkers for the Identification of Four Citrus Species.

The aroma quality of citrus fruit is determined by volatile compounds, which bring about different notes to allow discrimination among different citrus species. However, the volatiles with various aromatic traits specific to different citrus species have not been identified. In this study, volatile profiles in the fruit peels of four citrus species collected from our previous studies were subjected to various analyses to mine volatile biomarkers. Principal component analysis results indicated that different citrus species could almost completely be separated. Thirty volatiles were identified as potential biomarkers in discriminating loose-skin mandarin, sweet orange, pomelo, and lemon, while 17 were identified as effective biomarkers in discriminating clementine mandarins from the other loose-skin mandarins and sweet oranges. Finally, 30 citrus germplasms were used to verify the classification based on ß-elemene, valencene, nootkatone, and limettin as biomarkers. The accuracy values were 90.0%, 96.7%, 96.7%, and 100%, respectively. This research may provide a novel and effective alternative approach to identifying citrus genetic resources.

Metabolic Profiling of Pitaya (Hylocereus polyrhizus) during Fruit Development and Maturation.

Pitaya (Hylocereus polyrhizus) has attracted much interest from consumers as it is a novelty fruit with high nutrient content and a tolerance to drought stress. As a group of attractive pigment- and health-promoting natural compounds, betalains represent a visual feature for pitaya fruit quality. However, little information on the correlation between betalains and relevant metabolites exists so far. Currently, color (Commission International del'Eclairage, CIE) parameters, betalain contents, and untargeted metabolic profiling (gas chromatography-time-of-flight-mass spectrometry, GC⁻MS and liquid chromatography tandem mass spectrometry, LC⁻MS) have been examined on 'Zihonglong' fruits at nine different developmental stages, and the variation character of the metabolite contents was simultaneously investigated between peel and pulp. Furthermore, principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) were used to explore metabolite profiles from the fruit samples. Our results demonstrated that the decrease of amino acid, accompanied by the increase of sugars and organic acid, might contribute to the formation of betalains. Notably, as one of four potential biomarker metabolites, citramalic acid might be related to betalain formation.

Cit1,2RhaT and two novel CitdGlcTs participate in flavor-related flavonoid metabolism during citrus fruit development.

Neohesperidosides are disaccharides that are present in some flavonoids and impart a bitter taste, which can significantly affect the commercial value of citrus fruits. In this study, we identified three flavonoid-7-O-di-glucosyltransferase (dGlcT) genes closely related to 1,2-rhamnosyltransferase (1,2RhaT) in citrus genomes. However, only 1,2RhaT was directly linked to the accumulation of neohesperidoside, as demonstrated by association analysis of 50 accessions and co-segregation analysis of an F1 population derived from Citrus reticulata × Poncirus trifoliata. In transgenic tobacco BY2 cells, over-expression of CitdGlcTs resulted in flavonoid-7-O-glucosides being catalysed into bitterless flavonoid-7-O-di-glucosides, whereas over-expression of Cit1,2RhaT converted the same substrate into bitter-tasting flavonoid-7-O-neohesperidoside. Unlike 1,2RhaT, during citrus fruit development the dGlcTs showed an opposite expression pattern to CHS and CHI, two genes encoding rate-limiting enzymes of flavonoid biosynthesis. An uncoupled availability of dGlcTs and substrates might result in trace accumulation of flavonoid-7-O-di-glucosides in the fruit of C. maxima (pummelo). Past human selection of the deletion and functional mutation of 1,2RhaT has led step-by-step to the evolution of the flavor-related metabolic network in citrus. Our research provides the basis for potentially improving the taste in citrus fruit through manipulation of the network by knocking-out 1,2RhaT or by enhancing the expression of dGlcT using genetic transformation.

Simulation and Experiment on Droplet Formation and Separation for Needle-Type Micro-Liquid Jetting Dispenser.

The needle-type droplet jetting dispenser has wide applications in the field of microelectronic packaging, and for which the good quality of droplet formation and separation is the key to successful dispensing. This paper simulates the droplet jetting process which has been divided into 5 stages named backflow, growth, droplet extension, breakage, and separation, and analyses the combined effects of system parameters, such as pressure, viscosity, needle stroke, and nozzle diameter, on the changes of morphologies of ejected droplets, which is verified by experiments. The simulation and experiment results show that a higher driving pressure is quite suitable for the high-viscosity liquid to form normal droplets by avoiding adhesion. When increasing the needle stroke, the pressure should also be lowered properly to prevent the flow-stream. Besides, the nozzle with a large diameter is much more likely to cause sputtering or satellite-droplet problems. The results have a great significance for guiding the parameter settings of the needle-type dispensing approach.

Largely different carotenogenesis in two pummelo fruits with different flesh colors.

Carotenoids in citrus fruits have health benefits and make the fruits visually attractive. Red-fleshed 'Chuhong' ('CH') and pale green-fleshed 'Feicui' ('FC') pummelo (Citrus maxima (Burm) Merr.) fruits are interesting materials for studying the mechanisms of carotenoid accumulation. In this study, particularly high contents of linear carotenes were observed in the albedo tissue, segment membranes and juice sacs of 'CH'. However, carotenoids, especially ß-carotene and xanthophylls, accumulated more in the flavedo tissue of 'FC' than in that of 'CH'. Additionally, the contents of other terpenoids such as limonin, nomilin and abscisic acid significantly differed in the juice sacs at 150 days postanthesis. A dramatic increase in carotenoid production was observed at 45 to 75 days postanthesis in the segment membranes and juice sacs of 'CH'. Different expression levels of carotenogenesis genes, especially the ζ-carotene desaturase (CmZDS), ß-carotenoid hydroxylase (CmBCH) and zeaxanthin epoxidase (CmZEP) genes, in combination are directly responsible for the largely different carotenoid profiles between these two pummelo fruits. The sequences of eleven genes involved in carotenoid synthesis were investigated; different alleles of seven of eleven genes might also explain the largely different carotenogenesis observed between 'CH' and 'FC'. These results enhance our understanding of carotenogenesis in pummelo fruits.

Hypoxia regulates ABCG2 activity through the activivation of ERK1/2/HIF-1α and contributes to chemoresistance in pancreatic cancer cells.

Pancreatic cancer is a drug resistant hypovascular tumor. Although there are many studies on the mechanism of chemoresistance in pancreatic cancers, studies on the relationship between ABCG2 and chemoresistance during hypoxia of pancreatic cancer are rare. Hypoxia-inducible factor-1 (HIF-1α) is a master regulator of the transcriptional response to oxygen deprivation in cancer cells. The aim of this study was to examine the role of ABCG2 and HIF-1α in mediating chemoresistance during hypoxia in pancreatic cancer. In this study, we detected the expression levels of ABCG2, ERK/phosphorylated-ERK (p-ERK) and HIF-1α by immunohistochemistry in fresh pancreatic cancer and paracarcinoma tissues obtained from 25 patients. The mechanism by which p-ERK1/2 and HIF-1α affect ABCG2s expression was analyzed in the hypoxic cultured human pancreatic cancer cell line Capan-2. ABCG2-mediatedregulation of gemcitabine response under hypoxic conditions in pancreatic cancer cells was observed. It was found that ABCG2, ERK/p-ERK and HIF-1α were overexpressed in cancer tissues. ABCG2, HIF-1α and p-ERK levels were demonstrated to be high during hypoxic conditions in pancreatic cancer cells. Hypoxia induced phosphorylation of ERK1/2 to activate HIF-1α and contribute the ABCG2 expression and mediated gemcitabine chemoresistance in pancreatic cancer cells. Hypoxic conditions induced HIF-1α binding to target gene sequences in the ABCG2 promoter, resulting in increased transcription in pancreatic cancer cells. We demonstrated that hypoxia-induced chemoresistance is due to the regulation of ABCG2 through the activation of ERK1/2/HIF-1α. ABCG2 could serve as a predictor of gemcitabine response and, potentially, as a chemotherapeutic target in pancreatic cancer. Inhibition of ERK1/2 and HIF-1αcould result in increased gemcitabine sensitization in pancreatic cancer with highly expressed ABCG2 cell member protein.

A novel combination of oridonin and valproic acid in enhancement of apoptosis induction of HL-60 leukemia cells.

Oridonin, obtained from the traditional Chinese herbal medicine rabdosia rubescens, exerts potent antitumor activities in cancer cells. Valproic acid (VPA), as a potent histone deacetylase inhibitor (HDACI), also plays an important role in inhibition of proliferation of tumor cells. However, there are no reports so far on the cooperation between oridonin and VPA for anti-leukemic effect. Therefore, in the present study, we undertook experiments to determine whether lower concentration of oridonin in conjunction with lower concentration of VPA would produce even more encouraging synergistic effect than each of them alone, and to clarify its molecular mechanism. The results demonstrated that the lower concentration of oridonin in combination with lower concentration of VPA synergistically inhibited the proliferation of HL-60 cells, and induced obvious caspase-dependent apoptosis through activation of the intrinsic apoptosis pathway, which is involved in the downregulation of Bcl-2/Bax ratio, release of cytochrome c to cytosol and caspase-9 activation, as well as through the extrinsic apoptosis pathway mediated by Fas/FasL and caspase-8 activation. In addition, MAPK signaling pathway was also involved in apoptosis induced by oridonin plus VPA. Furthermore, the combination treatment in vivo remarkably reduced the xenograft tumor size and triggered tumor cell apoptosis. Taken together, the novel combination of oridonin plus VPA exerted synergistic anti-proliferative and apoptosis-inducing effects on human myeloid leukemia cells, and may serve as a potential promising anti-leukemia strategy.

Tenascin-C induces resistance to apoptosis in pancreatic cancer cell through activation of ERK/NF-κB pathway.

As a glycol-protein located in extracellular matrix (ECM), tenascin-C (TNC) is absent in most normal adult tissues but is highly expressed in the majority of malignant solid tumors. Pancreatic cancer is characterized by an abundant fibrous tissue rich in TNC. Although it was reported that TNC's expression increased in the progression from low-grade precursor lesions to invasive cancer and was associated with tumor differentiation in human pancreatic cancer, studies on the relations between TNC and tumor progression in pancreatic cancer were rare. In this study, we performed an analysis to determine the effects of TNC on modulating cell apoptosis and chemo-resistance and explored its mechanisms involving activation in pancreatic cancer cell. The expressions of TNC, ERK1/2/p-ERK1/2, Bcl-xL and Bcl-2 were detected by immunohistochemistry and western blotting. Then the effects of exogenous and endogenous TNC on the regulation of tumor proliferation, apoptosis and gemcitabine cytotoxicity were investigated. The associations among the TNC knockdown, TNC stimulation and expressions of ERK1/2/NF-κB/p65 and apoptotic regulatory proteins were also analyzed in cell lines. The mechanism of TNC on modulating cancer cell apoptosis and drug resistant through activation of ERK1/2/NF-κB/p65 signals was evaluated. The effect of TNC on regulating cell cycle distribution was also tested. TNC, ERK1/2/p-ERK1/2, and apoptotic regulatory proteins Bcl-xL and Bcl-2 were highly expressed in human pancreatic cancer tissues. In vitro, exogenous TNC promoted pancreatic cancer cell growth also mediates basal as well as starved and drug-induced apoptosis in pancreatic cancer cells. The effects of TNC on anti-apoptosis were induced by the activation state of ERK1/2/NF-κB/p65 signals in pancreatic cell. TNC phosphorylate ERK1/2 to induce NF-κB/p65 nucleus translocation. The latter contributes to promote Bcl-xL, Bcl-2 protein expressions and reduce caspase activity, which inhibit cell apoptotic processes. TNC mediated gemcitabine chemo-resistance via modulating cell apoptosis in pancreatic cancer. TNC resulted in the enrichment of pancreatic cancer cells in S-phase with a concomitant decrease in number of cells in G1 phase. The present study indicated TNC in cellular matrix induces an activation of ERK1/2/NF-κB/p65 signaling cascade and thereby mediates resistance to apoptosis in pancreatic cancer. TNC could serve as a diagnostic marker and predictor of gemcitabine response and potentially as a target for chemotherapy of pancreatic cancer.

Downregulation of STAT3 and activation of MAPK are involved in the induction of apoptosis by HNK in glioblastoma cell line U87.

Honokiol [3,5-di-(2-propenyl)-1,1-biphenyl-2,2-diol; HNK], a natural bioactive molecular compound isolated from the Magnolia officinalis, exhibits potent antitumor activity against a variety of human cancer cell lines. However, few studies have reported the antineoplastic effects of HNK on glioblastoma cells. It remains unknown how apoptosis is induced by HNK in glioblastoma cells and through which associated pathway this compound acts. The present study confirmed that HNK inhibited proliferation of glioblastoma cells by inducing a slight G0/G1 phase cell cycle arrest and apoptosis. We demonstrated for the first time that HNK triggered apoptosis of glioblastoma cells through both caspase-independent and caspase-dependent pathways, the latter including the extrinsic pathway and intrinsic pathway. Moreover, the inhibition of STAT3 signaling, ERK1/2 as well as activation of the p38 MAPK signaling pathway may be involved in apoptosis induced by HNK in U87 cells. Our findings suggest that HNK treatment could be a promising therapeutic strategy in human glioblastoma.