The transcription factor CsS40 negatively regulates TCS1 expression and caffeine biosynthesis in connection to leaf senescence in Camellia sinensis.

Caffeine is considered as one of the most important bioactive components in the popular plant beverages tea, cacao, and coffee, but as a wide-spread plant secondary metabolite its biosynthetic regulation at transcription level remains largely unclear. Here, we report a novel transcription factor Camellia sinensis Senescnece 40 (CsS40) as a caffeine biosynthesis regulator, which was discovered during screening a yeast expression library constructed from tea leaf cDNAs for activation of tea caffeine synthase (TCS1) promoter. Besides multiple hits of the non-self-activation CsS40 clones that bound to and activated TCS1 promoter in yeast-one-hybrid assays, a split-luciferase complementation assay demonstrated that CsS40 acts as a transcription factor to activate the CsTCS1 gene and EMSA assay also demonstrated that CsS40 bound to the TCS1 gene promoter. Consistently, immunofluorescence data indicated that CsS40-GFP fusion was localized in the nuclei of tobacco epidermal cells. The expression pattern of CsS40 in 'Fuding Dabai' developing leaves was opposite to that of TCS1; and knockdown and overexpression of CsS40 in tea leaf calli significantly increased and decreased TCS1 expression levels, respectively. The expression levels of CsS40 were also negatively correlated to caffeine accumulation in developing leaves and transgenic calli of 'Fuding Dabai'. Furthermore, overexpression of CsS40 reduced the accumulation of xanthine and hypoxanthine in tobacco plants, meanwhile, increased their susceptibility to aging. CsS40 expression in tea leaves was also induced by senescence-promoting hormones and environmental factors. Taken together, we showed that a novel senescence-related factor CsS40 negatively regulates TCS1 and represses caffeine accumulation in tea cultivar 'Fuding Dabai'. The study provides new insights into caffeine biosynthesis regulation by a plant-specific senescence regulator in tea plants in connection to leaf senescence and hormone signaling.

Genome-Wide Investigation and Functional Analysis Reveal That CsKCS3 and CsKCS18 Are Required for Tea Cuticle Wax Formation.

Cuticular wax is a complex mixture of very long-chain fatty acids (VLCFAs) and their derivatives that constitute a natural barrier against biotic and abiotic stresses on the aerial surface of terrestrial plants. In tea plants, leaf cuticular wax also contributes to the unique flavor and quality of tea products. However, the mechanism of wax formation in tea cuticles is still unclear. The cuticular wax content of 108 germplasms (Niaowang species) was investigated in this study. The transcriptome analysis of germplasms with high, medium, and low cuticular wax content revealed that the expression levels of CsKCS3 and CsKCS18 were strongly associated with the high content of cuticular wax in leaves. Hence, silencing CsKCS3 and CsKCS18 using virus-induced gene silencing (VIGS) inhibited the synthesis of cuticular wax and caffeine in tea leaves, indicating that expression of these genes is necessary for the synthesis of cuticular wax in tea leaves. The findings contribute to a better understanding of the molecular mechanism of cuticular wax formation in tea leaves. The study also revealed new candidate target genes for further improving tea quality and flavor and cultivating high-stress-resistant tea germplasms.

Preparation of Zn-Gly and Se-Gly and Their Effects on the Nutritional Quality of Tea (Camellia sinensis).

BACKGROUND: Micronutrient malnutrition affects millions of people due to a lack of Zn and Se. METHODS: The process conditions for the manufacture of glycine-chelated sodium selenite (Se-Gly) and zinc sulfate heptahydrate (Zn-Gly) were studied. The effects of ligand concentration, pH, reaction ratio, reaction temperature, and reaction time on fertilizer stability were assessed. The effects of Zn-Gly and Se-Gly on tea plants were determined. RESULTS: Orthogonal experiments showed that the optimal preparation conditions for Zn-Gly (75.80 % Zn chelation rate) were pH 6.0, ligand concentration 4 %, reaction ratio 1:2, reaction time 120 min, reaction temperature 70 ℃. The optimal preparation conditions for Se-Gly (56.75 % Se chelation rate) were pH 6.0, ligand concentration 10%, reaction ratio 2:1, reaction time 40 min, temperature 50 ℃. Each chelate was completely soluble in water and verified by infrared spectroscopy and ultraviolet spectroscopy. CONCLUSIONS: Zn-Gly and Se-Gly increased the Zn and Se content in tea plants, and foliar application was more effective than soil application. Combined application of Zn-Gly and Se-Gly was more effective than Zn-Gly or Se-Gly alone. Our findings suggest that Zn-Gly and Se-Gly provide a convenient method of addressing human Zn and Se deficiency.

Tunable color generation in CdTe-doped silicate glass enabled by ultrashort laser pulses: interior coloring and multilevel encryption.

The launch of the big data era puts forward challenges for information security. Herein, a new kind of silicate glass system co-doped with CdO and ZnTe, capable of achieving the controllable generation of intrinsic color centers (brown and green) and tiny nuclei of CdTe via direct laser writing (DLW), is developed. The controlled growth of CdTe QDs thermally, leads to a permanent color of orange at the cost of accelerated aging of the color centers of brown and green. On the one hand, going beyond traditional 2D surface coloration, the high transparency of the studied bulk medium makes 3D volumetric interior coloration possible. On the other hand, by encoding ciphertext into the tiny nuclei of CdTe, a strategy of color encryption and heat decryption is established, which brings about the merits of expanded storage capacity and improved information security. The demonstration application confirmed the user-defined multiscale interior coloration and an unprecedented multidimensional color-encryption scheme with a high-security level. The present work highlights a great leap in transparent materials for color encryption and hopefully stimulates the development of new color division multiplexing encryptions.

Allelopathy of uncomposted and composted invasive aster (Ageratina adenophora) on ryegrass.

In many areas invaded by Ageratina adenophora, the piles of A. adenophora residue need to be safely treated and economically utilized. To explore a new potential use for these residues, on-site aerobic composting, seed germination test and greenhouse experiment were conducted to compare the phytotoxic allelochemicals in uncomposted and composted A. adenophora plants (UA and CA, respectively) and their influence on ryegrass seed germination and seedling growth. The phytotoxicants 4,7-dimethyl-1-(propan-2-ylidene)-1,4,4a,8a-tetrahydronaphthalene-2,6(1H,7H)-dione (DTD) and 6-hydroxy-5-isopropyl-3,8-dimethyl-4a,5,6,7,8,8a-hexahydronaphthalen-2(1 H)-one (HHO) in UA decreased by 10.09 and 11.01 times in CA on average, respectively. Aqueous extracts of CA increased the seed germination rate, root dehydrogenase activity, leaf chlorophyll content and nitrate reductase activity; those of UA behaved oppositely. Compared with chemical fertilizers (CF), CF + CA promoted plant growth, increased plant nutrient uptake, and resulted in higher soil available nutrients, enzyme activity and microbial biodiversity, whereas CA alone had similar or better influences on plants and soils than CF. The predominant bacterial and fungal composition was the same in the soils supplied with CA and CF + CA. Therefore, on-site aerobic composting eliminated the phytotoxicity of CA and provided a new, simple and economical approach for the potential use of A. adenophora biomass as a plant- and soil-friendly organic fertilizer.

In situ aerobic composting eliminates the toxicity of Ageratina adenophora to maize and converts it into a plant- and soil-friendly organic fertilizer.

Ageratina adenophora has invaded many subtropical and tropical countries and caused tremendous ecological and economic losses. This necessitates a new way to use the debris left after clearing this plant. Therefore, the allelochemicals in fresh and aerobically composted A. adenophora plants (FA and CA, respectively) were compared, and their allelopathy against maize was evaluated. The results showed that CA decreased the allelochemicals (6-hydroxy-5-isopropyl-3,8-dimethyl-4a,5,6,7,8,8a-hexahydronaphthalen-2(1H)-one and 4,7-dimethyl-1-(propan-2-ylidene)-1,4,4a,8a-tetrahydronaphthalene-2,6(1H, 7H)-dione) by over 95% compared to FA. In a seed germination test, CA aqueous solutions improved the seed germination and seedling growth, whereas FA solutions led to opposite results. Chemical fertilizers (CF) plus FA resulted in much lower plant biomass and nutrient uptake than CF in a greenhouse experiment. Compared with CF, CF+CA showed positive effects on maize, soil microbial biomass and diversity and enzyme activities in the field. However, the compositions of the predominant microbes were almost unaffected by the application of CA and CF+CA. These significant findings extended our knowledge regarding the elimination of A. adenophora toxicity against other plants and soil microbes through allelochemical degradation in the composting process. In situ aerobic composting provides a new, simple and economical method to convert A. adenophora into a plant- and soil-friendly organic fertilizer.

Expression of the Clifednamide Biosynthetic Pathway in Streptomyces Generates 27,28-seco-Derivatives.

Polycyclic tetramate macrolactams (PoTeMs) are a group of hybrid PK-NRP natural products having a variable set of carbocyclic rings, a conserved assembly pathway, and diverse bioactivities. We report here the identification of seven new PoTeMs, clifednamides D-J (3-9), along with the known clifednamides A (1) and B (2) through rational pathway refactoring and heterologous expression. Remarkably, clifednamides D (3), G (6), and H (7) feature an unprecedented 27,28-seco skeleton. The cytotoxic activities of compounds 1-9 indicated that the hydroxy group of C-25, the methyl group of C-30, the inner five-membered ring, and the intact macrocycle are all critical for the activities. Meanwhile, the cytochrome P450 enzyme CftS023A and the hydroxylase CftS023E involved in oxidative tailoring of clifednamides were found to decorate the fused 5-6 bicyclic intermediates. Accordingly, the biosynthetic pathway for clifednamides was proposed.

Serum galectin-3 as a biomarker for screening, early diagnosis, prognosis and therapeutic effect evaluation of pancreatic cancer.

Galectin-3 plays an important role in cell-cell adhesion, macrophage activation, angiogenesis, metastasis and apoptosis and is overexpressed in pancreatic cancer. We explored the importance of galectin-3 in the screening, early diagnosis, prognosis and therapeutic effect evaluation of pancreatic cancer. A time-resolved fluorescence immunoassay was performed to detect serum galectin-3 level. Serum samples were collected from healthy controls and patients with pancreatic cancer before and after different treatments, and the relationships between galectin-3 level and clinical parameters were analysed. Among the healthy controls, one individual with an abnormally high concentration of galectin-3 (9.85 µg/L) was diagnosed with pancreatic cancer. Compared to the pre-operative level, galectin-3 concentration significantly decreased in patients with radical excision 1 month after surgery (P < .05), but showed no obvious change in patients who underwent palliative resection. Additionally, among patients with radical excision, carcinoma recurrence rate was significantly higher in those with increased or unchanged galectin-3 level. Retrospective analysis revealed the extraordinarily high value and high specificity of galectin-3 for predicting 3-year survival (P < .001). Thus, galectin-3 may serve as a potential biomarker for the screening and early diagnosis of pancreatic cancer and as an independent prognostic indicator in patients with pancreatic cancer.

Identification of prognostic risk factors for pancreatic cancer using bioinformatics analysis.

BACKGROUND: Pancreatic cancer is one of the most common malignant cancers worldwide. Currently, the pathogenesis of pancreatic cancer remains unclear; thus, it is necessary to explore its precise molecular mechanisms. METHODS: To identify candidate genes involved in the tumorigenesis and proliferation of pancreatic cancer, the microarray datasets GSE32676, GSE15471 and GSE71989 were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) between Pancreatic ductal adenocarcinoma (PDAC) and nonmalignant samples were screened by GEO2R. The Database for Annotation Visualization and Integrated Discovery (DAVID) online tool was used to obtain a synthetic set of functional annotation information for the DEGs. A PPI network of the DEGs was established using the Search Tool for the Retrieval of Interacting Genes (STRING) database, and a combination of more than 0.4 was considered statistically significant for the PPI. Subsequently, we visualized the PPI network using Cytoscape. Functional module analysis was then performed using Molecular Complex Detection (MCODE). Genes with a degree ≥10 were chosen as hub genes, and pathways of the hub genes were visualized using ClueGO and CluePedia. Additionally, GenCLiP 2.0 was used to explore interactions of hub genes. The Literature Mining Gene Networks module was applied to explore the cocitation of hub genes. The Cytoscape plugin iRegulon was employed to analyze transcription factors regulating the hub genes. Furthermore, the expression levels of the 13 hub genes in pancreatic cancer tissues and normal samples were validated using the Gene Expression Profiling Interactive Analysis (GEPIA) platform. Moreover, overall survival and disease-free survival analyses according to the expression of hub genes were performed using Kaplan-Meier curve analysis in the cBioPortal online platform. The relationship between expression level and tumor grade was analyzed using the online database Oncomine. Lastly, the eight snap-frozen tumorous and adjacent noncancerous adjacent tissues of pancreatic cancer patients used to detect the CDK1 and CEP55 protein levels by western blot. CONCLUSIONS: Altogether, the DEGs and hub genes identified in this work can help uncover the molecular mechanisms underlying the tumorigenesis of pancreatic cancer and provide potential targets for the diagnosis and treatment of this disease.

ß2-AR regulates the expression of AKR1B1 in human pancreatic cancer cells and promotes their proliferation via the ERK1/2 pathway.

Psychological stress has been recognized as a well-documented risk factor associated with ß2-adrenergic receptor (ß2-AR) in the development of pancreatic cancer. Aldo-keto reductase 1 member B1 (AKR1B1) is a potential interacting partner of ß2-AR, but the effect of their interaction on pancreatic cancer cells is not known at present. We found a positive correlation between AKR1B1 and ß2-AR expression in pancreatic cancer tissue samples, and co-localization of these proteins in the human pancreatic cancer BXPC-3 cell line. Compared to the controls, the CFPAC-1 and PANC-1 pancreatic cancer cells overexpressing ß2-AR and AKR1B1 respectively showed significantly higher proliferation rates, which is attributed to higher proportion of cells in the S phase and decreased percentage of early apoptotic cells. Furthermore, overexpression of ß2-AR led to a significant increase in the expression of AKR1B1 and phosphorylated extracellular signal-regulated kinase (p-ERK1/2). Overexpression of AKR1B1 significantly decreased ß2-AR levels and increased that of p-ERK1/2. Taken together, ß2-AR directly interacted with and up-regulated AKR1B1 in pancreatic cancer cells, and promoted their proliferation and inhibited apoptosis via the ERK1/2 pathway. Our findings also highlight the ß2-AR-AKR1B1 axis as a potential therapeutic target for pancreatic cancer.

Biosynthesis of the Polycyclic System in the Antifungal HSAF and Analogues from Lysobacter enzymogenes.

The biocontrol agent Lysobacter enzymogenes produces polycyclic tetramate macrolactams (PoTeMs), including the antifungal HSAF. To elucidate the biosynthesis of the cyclic systems, we identified eleven HSAF precursors/analogues with zero, one, two, or three rings through heterologous expression of the HSAF gene cluster. A series of combinatorial gene expression and deletion experiments showed that OX3 is the "gatekeeper" responsible for the formation of the first 5-membered ring from lysobacterene A, OX1 and OX2 are responsible for formation of the second ring but with different selectivity, and OX4 is responsible for formation of the 6-membered ring. In vitro experiments showed that OX4 is an NADPH-dependent enzyme that catalyzes the reductive cyclization of 3-dehydroxy alteramide C to form 3-dehydroxy HSAF. Thus, the multiplicity of OX genes is the basis for the structural diversity of the HSAF family, which is the only characterized PoTeM cluster that involves four redox enzymes in the formation of the cyclic system.

[Allelopathic effects of extracts from tuberous roots of Aconitum carmichaeli on three pasture grasses].

The tuberous roots of Aconitum carmichaeli are largely used in traditional Chinese medicine and widely grown in Jiangyou, Sichuan, China. During the growth process, this medicinal plant releases a large amount of allelochemicals into soil, which retard the growth and development of near and late crops. Therefore, a pure culture experiment was thus carried out by seed soaking to study the allelopathic effects of extracts from tuberous roots of A. carmichaeli (ETR) on the seed germination and young seedling growth of Lolium perenne, Trifolium repens, and Medicago sativa, the late pasture grasses after cultivation of A. carmichaeli. The results showed that three pasture grasses varied significantly in seed germination and young seedling growth in response to ETR concentrations. Seed germination of M. sativa was stimulated by low ERT concentration (0.01 x g(-1)), while all of pasture grass seeds germinated poorly in solution with 1.00 g x L(-1). Seed soaking with 1.00 g x L(-1) also inhibited significantly the growth of pasture young seedlings, with M. sativa showing the highest seedling height reduction of 42.05% in seeding height, followed by T. repens (40.21%) and L. perenne with about 11%. Cultivation of L. perenne could thus be beneficial to increase whole land productivity in A. carmichaeli-pasture grass cropping systems. In addition, hydrolysis of protein, starch, and inositol phosphates was blocked and free amino acids, soluble sugars and phosphorus were decreased in seeds by seed soaking with ETR, which could be one of the reason for the inhibition of seed germination. There was a significant reduction in root vigor, nitrate reductase, and chlorophyll after the seed treatment with ETR, indicating the suppression of nutrient uptake, nitrate assimilation, and photosynthesis by allelopathic chemicals in ETR, which could lead to the slow growth rate of pasture grass seedlings.