Effects of vitro sucrose on quality components of tea plants (Camellia sinensis) based on transcriptomic and metabolic analysis.

BACKGROUND: Tea plants [Camellia sinensis (L.) O. Kuntze] can produce one of the three most widely popular non-alcoholic beverages throughout the world. Polyphenols and volatiles are the main functional ingredients determining tea's quality and flavor; however, the biotic or abiotic factors affecting tea polyphenol biosynthesis are unclear. This paper focuses on the molecular mechanisms of sucrose on polyphenol biosynthesis and volatile composition variation in tea plants. RESULTS: Metabolic analysis showed that the total content of anthocyanins, catechins, and proanthocyanidins(PAs) increased with sucrose, and they accumulated most significantly after 14 days of treatment. Transcriptomic analysis revealed 8384 and 5571 differentially expressed genes in 2-day and 14-day sucrose-treated tea plants compared with control-treated plants. Most of the structural genes and transcription factors (TFs) involved in polyphenol biosynthesis were significantly up-regulated after 2d. Among these transcripts, the predicted genes encoding glutathione S-transferase (GST), ATP-binding cassette transporters (ABC transporters), and multidrug and toxic compound extrusion transporters (MATE transporters) appeared up regulated. Correspondingly, ultra-performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-QQQ-MS/MS) analysis revealed that the content of non-galloylated catechins and oligomeric PAs decreased in the upper-stem and increased in the lower-stem significantly, especially catechin (C), epicatechin (EC), and their oligomeric PAs. This result suggests that the related flavonoids were transported downward in the stem by transporters. GC/MS data implied that four types of volatile compounds, namely terpene derivatives, aromatic derivatives, lipid derivatives, and others, were accumulated differently after in vitro sucrose treatment. CONCLUSIONS: Our data demonstrated that sucrose regulates polyphenol biosynthesis in Camellia sinensis by altering the expression of transcription factor genes and pathway genes. Additionally, sucrose promotes the transport of polyphenols and changes the aroma composition in tea plant.

Overcoming ABCB1-mediated multidrug resistance by transcription factor BHLHE40.

Multidrug resistance (MDR) hinders treatment efficacy in cancer therapy. One typical mechanism contributing to MDR is the overexpression of permeability-glycoprotein (P-gp) encoded by ATP-binding cassette subfamily B member 1 (ABCB1). Basic helix-loop-helix family member e40 (BHLHE40) is a well-known transcription factor that has pleiotropic effects including the regulation of cancer-related processes. However, whether BHLHE40 regulates MDR is still unknown. Chromatin immunoprecipitation-seq study revealed BHLHE40 occupancy in the promoter of ABCB1 gene. Adriamycin (ADM)-resistant human chronic myeloid leukemia cells (K562/A) and human breast cancer cells (MCF-7/A) were established. BHLHE40 expression was downregulated in the ADM-resistant cell lines. Overexpression of BHLHE40 resensitized resistant cells to ADM, promoted cell apoptosis in vitro and suppressed tumor growth in vivo, whereas BHLHE40 knockdown induced resistance to ADM in parental cells. Moreover, we found that BHLHE40 regulated drug resistance by directly binding to the ABCB1 promoter (-1605 to -1597) and inactivating its transcription. In consistence, the expression of BHLHE40 was negatively correlated with ABCB1 in various cancer cells, while positively with cancer cell chemosensitivity and better prognosis of patients with breast cancer. The study reveals the role of BHLHE40 as a transcriptional suppressor on the expression of ABCB1, major ABC transporter in chemoresistance. The findings extend the function of BHLHE40 in tumor progression and provides a novel mechanism for the reversal of multidrug resistance.

Modeling faults among e-bike-related fatal crashes in China.

OBJECTIVES: This article aims to model fault in e-bike fatal crashes in a county-level city in China. METHOD: Three-year crash data are retrieved from the crash reports (2012-2014) from the Taixing Police Department. A mixed logit model is introduced to explore significant factors associated with fault assignment, as well as accounting for similarity among fault assignment and heterogeneity within unobserved variables. RESULTS: The modeling results indicate some interesting new findings. First, precrash behaviors of both drivers and e-bike riders are found to be significant to fault assignment. Second, bike lane and median type are significantly associated with e-bike rider fault commitment. Third, specific groups of e-bike riders (low-educated and older) and drivers (heavy good vehicles) are more likely to be at fault in e-bike crashes. Last, crash location and the built environment have significant correlations with faulty behaviors of e-bike riders. CONCLUSIONS: Safety countermeasures are proposed including (1) the deployment of traffic design and control elements including physically separated bike lanes, medians, video surveillance systems for e-bike riders, and left-turning treatments for nonmotorists (e.g., a 2-step e-bike left turning); (2) the amendment of the current traffic regulations on drunk e-bike riders and child e-bike passengers; (3) the development of a license system for specific e-bike rider groups (older and low-educated) and a safety campaign for drivers (to increase safety awareness when parking on-street or driving heavy good vehicles). Some interesting future research topics are also suggested: e-bike riders' behaviors at unsignalized intersections and mid-block openings, e-bike safety in suburban areas, and an in-depth study of the effect of the built environment on e-bike safety.

Characterization and Expression Profiling of Camellia sinensis Cinnamate 4-hydroxylase Genes in Phenylpropanoid Pathways.

Cinnamate 4-hydroxylase (C4H), a cytochrome P450-dependent monooxygenase, participates in the synthesis of numerous polyphenoid compounds, such as flavonoids and lignins. However, the C4H gene number and function in tea plants are not clear. We screened all available transcriptome and genome databases of tea plants and three C4H genes were identified and named CsC4Ha, CsC4Hb, and CsC4Hc, respectively. Both CsC4Ha and CsC4Hb have 1518-bp open reading frames that encode 505-amino acid proteins. CsC4Hc has a 1635-bp open reading frame that encodes a 544-amino acid protein. Enzymatic analysis of recombinant proteins expressed in yeast showed that the three enzymes catalyzed the formation of p-coumaric acid (4-hydroxy trans-cinnamic acid) from trans-cinnamic acid. Quantitative real-time PCR (qRT-PCR) analysis showed that CsC4Ha was highly expressed in the 4th leaf, CsC4Hb was highly expressed in tender leaves, while CsC4Hc was highly expressed in the young stems. The three CsC4Hs were induced with varying degrees by abiotic stress treatments. These results suggest they may have different subcellular localization and different physiological functions.