Comprehensive analysis of the laccase gene family in tea plant highlights its roles in development and stress responses.

BACKGROUND: Laccase (LAC) is the pivotal enzyme responsible for the polymerization of monolignols and stress responses in plants. However, the roles of LAC genes in plant development and tolerance to diverse stresses are still largely unknown, especially in tea plant (Camellia sinensis), one of the most economically important crops worldwide. RESULTS: In total, 51 CsLAC genes were identified, they were unevenly distributed on different chromosomes and classified into six groups based on phylogenetic analysis. The CsLAC gene family had diverse intron-exon patterns and a highly conserved motif distribution. Cis-acting elements in the promoter demonstrated that promoter regions of CsLACs encode various elements associated with light, phytohormones, development and stresses. Collinearity analysis identified some orthologous gene pairs in C. sinensis and many paralogous gene pairs among C. sinensis, Arabidopsis and Populus. Tissue-specific expression profiles revealed that the majority of CsLACs had high expression in roots and stems and some members had specific expression patterns in other tissues, and the expression patterns of six genes by qRT‒PCR were highly consistent with the transcriptome data. Most CsLACs showed significant variation in their expression level under abiotic (cold and drought) and biotic (insect and fungus) stresses via transcriptome data. Among them, CsLAC3 was localized in the plasma membrane and its expression level increased significantly at 13 d under gray blight treatment. We found that 12 CsLACs were predicted to be targets of cs-miR397a, and most CsLACs showed opposite expression patterns compared to cs-miR397a under gray blight infection. Additionally, 18 highly polymorphic SSR markers were developed, these markers can be widely used for diverse genetic studies of tea plants. CONCLUSIONS: This study provides a comprehensive understanding of the classification, evolution, structure, tissue-specific profiles, and (a)biotic stress responses of CsLAC genes. It also provides valuable genetic resources for functional characterization towards enhancing tea plant tolerance to multiple (a)biotic stresses.

Fatty acid export protein BnFAX6 functions in lipid synthesis and axillary bud growth in Brassica napus.

Sugar is considered as the primary regulator of plant apical dominance, whereby the outgrowth of axillary buds is inhibited by the shoot tip. However, there are some deficiencies in this theory. Here, we reveal that Fatty Acid Export 6 (BnFAX6) functions in FA transport, and linoleic acid or its derivatives acts as a signaling molecule in regulating apical dominance of Brassica napus. BnFAX6 is responsible for mediating FA export from plastids. Overexpression of BnFAX6 in B. napus heightened the expression of genes involved in glycolysis and lipid biosynthesis, promoting the flow of photosynthetic products to the biosynthesis of FAs (including linoleic acid and its derivatives). Enhancing expression of BnFAX6 increased oil content in seeds and leaves and resulted in semi-dwarf and increased branching phenotypes with more siliques, contributing to increased yield per plant relative to wild-type. Furthermore, decapitation led to the rapid flow of the carbon from photosynthetic products to FA biosynthesis in axillary buds, consistent with the overexpression of BnFAX6 in B. napus. In addition, free FAs, especially linoleic acid, were rapidly transported from leaves to axillary buds. Increasing linoleic acid in axillary buds repressed expression of a key transcriptional regulator responsible for maintaining bud dormancy, resulting in bud outgrowth. Taken together, we uncovered that BnFAX6 mediating FA export from plastids functions in lipid biosynthesis and in axillary bud dormancy release, possibly through enhancing linoleic acid level in axillary buds of B. napus.

The high-affinity transporter BnPHT1;4 is involved in phosphorus acquisition and mobilization for facilitating seed germination and early seedling growth of Brassica napus.

BACKGROUND: Seed germination and seedling establishment are two of the most critical phases in plant development. However, the molecular mechanisms underlying the effect of phosphorus on seed germination and post-germinated growth of oilseed rape are unclear so far. Here, we report the role of BnPHT1;4 in seed germination and early seedling development of Brassica napus. RESULTS: Our results show that BnPHT1;4 is preferentially expressed in cotyledons of early developing seedlings. Overexpression of BnPHT1;4 in oilseed rape promoted seed germination and seedling growth. Expression levels of the genes related to ABA and GA biosynthesis and signaling were significantly altered in BnPHT1;4 transgenic seedlings. Consequently, active GA level was up-regulated, whereas ABA content was down-regulated in BnPHT1;4 transgenic seedlings. Furthermore, exogenous GA could promote seed germination of wild type, while exogenous ABA could partially recover the advanced-germination phenotype of BnPHT1;4 transgenic seeds. Total phosphorus content in cotyledons of the transgenic seedlings was decreased more rapidly than that in wild type when Pi was supplied or deficient, and Pi contents in shoots and roots of the BnPHT1;4 transgenic plants were higher than those in wild type under high and low Pi conditions. CONCLUSIONS: Our data suggest that the high-affinity transporter BnPHT1;4 is involved in phosphorus acquisition and mobilization for facilitating seed germination and seedling growth of Brassica napus by modulating ABA and GA biosynthesis.

The ARF7 and ARF19 Transcription Factors Positively Regulate PHOSPHATE STARVATION RESPONSE1 in Arabidopsis Roots.

PHOSPHATE STARVATION RESPONSE1 (PHR1) is a key regulatory component of the response to phosphate (Pi) starvation. However, the regulation of PHR1 in this response remains poorly understood. Here, we report that PHR1 is a target of the transcription factors AUXIN RESPONSE FACTOR7 (ARF7) and ARF19 and is positively regulated by auxin signaling in Arabidopsis (Arabidopsis thaliana) roots. PHR1 expression was induced by exogenous auxin and suppressed by auxin transport inhibitors in Arabidopsis roots. In the PHR1 promoter, three auxin-response elements, which are bound directly by ARF7 and ARF19, were shown to be essential for PHR1 expression. The arf7, arf19, and arf7 arf19 mutants showed down-regulated expression of PHR1 and downstream Pi starvation-induced genes in roots; they also exhibited defective Pi uptake in roots and overaccumulation of anthocyanin in shoots. The induction of lateral root formation in response to low Pi and to exogenous auxin was decreased in the phr1 mutant, whereas the expression of LATERAL ORGAN BOUNDARIES-DOMAIN16 (LBD16) and LBD29 was not changed significantly. PHR1 acted independently of LBD16 and LBD29 in the regulation of lateral root formation in response to low Pi. Under low-Pi conditions, lateral root impairment in the arf7 arf19 mutant was partially rescued by constitutive expression of PHR1, demonstrating that reduced PHR1 expression contributed to the arf7 arf19 phenotype. In addition to PHR1, other genes encoding MYB-CC members also were targets of ARF7 and ARF19. Our work thus reveals a mechanism coordinating auxin signaling and the PHR1 regulon in Arabidopsis responses to Pi deficiency.

Spectral changes of cosine-Gaussian-correlated Schell-model beams with rectangular symmetry scattered on a deterministic medium.

Based on the theory of first-order Born approximation, analytical expressions for rectangular cosine-Gaussian Schell-model (RCGSM) beams scattered on a deterministic medium in the far zone are derived. In terms of the analytical formula obtained, the changes of a RCGSM beam's scattered spectrum are numerically investigated. Results show that several parameters (including the scattering directions sx and sy, effective radius σ of the scattering medium, the initial beam's correlation widths δx and δy, and line width Γ0 of the incident spectrum) closely influence the distributions of the normalized scattered spectrum in the far zone. These features of a RCGSM beam scattered spectrum can be used to obtain information about the structure of a deterministic medium.

A Brassica napus PHT1 phosphate transporter, BnPht1;4, promotes phosphate uptake and affects roots architecture of transgenic Arabidopsis.

Phosphorus (P) is one of the essential nutrient elements for plant development. In this work, BnPht1;4 gene, encoding a phosphate transporter of PHT1 family, was isolated from Brassica napus. BnPht1;4 possesses the major characteristic of PHT1 high-affinity Pi transporters in plants, such as plasma-membrane localization and 12 transmembrane-spanning domains. Quantitative reverse-transcription PCR analysis and promoter activity assay showed BnPht1;4 was inert in plants under Pi sufficient conditions. However, expression of this gene was remarkably enhanced in roots under Pi deficient conditions. Interestingly, under low Pi conditions, its promoter activity is impaired in tips of elongated roots, suggesting that the high-affinity Pi transporter may be not involved in low Pi response at root tip area. The experimental results also indicated that BnPht1;4 induction by Pi deficiency is dependent on the existence of sugar. In 35S:BnPht1;4 transgenic Arabidopsis, the increase of Pi availability resulted in the change of root architecture under Pi deficient conditions, showing longer primary roots and lower lateral root density than that of wild type. By cis-element analysis, two P1BS and two W-box elements were found in BnPht1;4 promoter. Yeast one-hybrid assay indicated that PHR1 could bind to the BnPht1;4 promoter. P1BS elements in BnPht1;4 promoter are essential for BnPht1;4 induction in Pi starvation response. Furthermore, WRKY75 could bind to the BnPht1;4 promoter, in which W-box elements are important for this binding. These results indicated BnPht1;4 may be dually controlled by two family regulators under low Pi responses. Thus, our data on the regulative mechanism of high-affinity Pi transporter in Pi starvation response will be valuable for B. napus molecular agriculture.

Novel Camellia sinensis O-Methyltransferase Regulated by CsMADSL1 Specifically Methylates EGCG in Cultivar "GZMe4".

Epigallocatechin-3-O-(4-O-methyl)gallate (EGCG4″Me) in Camellia sinensis possesses numerous beneficial biological activities. However, the germplasm rich in EGCG4″Me and the O-methyltransferase responsible for EGCG4″Me biosynthesis are poorly understood. Herein, the content of EGCG3″Me and EGCG4″Me in the shoots of 13 cultivars was analyzed to demonstrate that EGCG4″Me is characteristically accumulated in the "GZMe4" cultivar but not in the other 12 cultivars. A novel O-methyltransferase (CsOMTL1) was identified from "GZMe4" using RNA-Seq and correlation analysis. Using the recombinant enzyme, EGCG4″Me was synthesized in vitro. Overexpression of CsOMTL1 via Agrobacterium-mediated genetic transformation caused constitutive accumulation of EGCG4″Me in C. sinensis callus. Moreover, the transcription factor CsMADSL1 localized in the nucleus activated the transcription of CsOMTL1 and specifically interacted with its promoter. Hence, our study identified a novel O-methyltransferase that characteristically catalyzes the synthesis of EGCG4″Me and a positive regulator of EGCG4″Me synthesis in "GZMe4", which might provide a strategy for the breeding of a tea cultivar rich in EGCG4″Me.

Integrated physiological, metabolite and proteomic analysis reveal the glyphosate stress response mechanism in tea plant (Camellia sinensis).

Glyphosate residues can tremendously impact the physiological mechanisms of tea plants, thus threatening tea security and human health. Herein, integrated physiological, metabolite, and proteomic analyses were performed to reveal the glyphosate stress response mechanism in tea plant. After exposure to glyphosate (≥1.25 kg ae/ha), the leaf ultrastructure was damaged, and chlorophyll content and relative fluorescence intensity decreased significantly. The characteristic metabolites catechins and theanine decreased significantly, and the 18 volatile compounds content varied significantly under glyphosate treatments. Subsequently, tandem mass tags (TMT)-based quantitative proteomics was employed to identify the differentially expressed proteins (DEPs) and to validate their biological functions at the proteome level. A total of 6287 proteins were identified and 326 DEPs were screened. These DEPs were mainly catalytic, binding, transporter and antioxidant active proteins, involved in photosynthesis and chlorophyll biosynthesis, phenylpropanoid and flavonoid biosynthesis, sugar and energy metabolism, amino acid metabolism, and stress/defense/detoxification pathway, etc. A total of 22 DEPs were validated by parallel reaction monitoring (PRM), demonstrating that the protein abundances were consistent between TMT and PRM data. These findings contribute to our understanding of the damage of glyphosate to tea leaves and molecular mechanism underlying the response of tea plants to glyphosate.

Characterization of two O-methyltransferases involved in the biosynthesis of O-methylated catechins in tea plant.

Tea is known for having a high catechin content, with the main component being (-)-epigallocatechin gallate (EGCG), which has significant bioactivities, including potential anti-cancer and anti-inflammatory activity. The poor intestinal stability and permeability of EGCG, however, undermine these health-improving benefits. O-methylated EGCG derivatives, found in a few tea cultivars in low levels, have attracted considerable interest due to their increased bioavailability. Here, we identify two O-methyltransferases from tea plant: CsFAOMT1 that has a specific O-methyltransferase activity on the 3''-position of EGCG to generate EGCG3''Me, and CsFAOMT2 that predominantly catalyzes the formation of EGCG4″Me. In different tea tissues and germplasms, the transcript levels of CsFAOMT1 and CsFAOMT2 are strongly correlated with the amounts of EGCG3''Me and EGCG4''Me, respectively. Furthermore, the crystal structures of CsFAOMT1 and CsFAOMT2 reveal the key residues necessary for 3''- and 4''-O-methylation. These findings may provide guidance for the future development of tea cultivars with high O-methylated catechin content.

Purification of MDI Isomers Using Dynamic Falling Film Melt Crystallization: Experiment and Molecular Simulation.

In this work, the isomer mixture of 4,4'-diphenylmethane diisocyanate (MDI) and 2,4'-MDI was separated and purified by dynamic falling film melt crystallization, and 99.3% purity and 50.8% yield of 4,4'-MDI could be obtained under optimized conditions. The separation mechanism was simulated by density functional theory (DFT) and molecular dynamics (MD) simulation. Results showed that compared with 2,4'-MDI, 4,4'-MDI molecules could form a more stable and symmetrical crystal structure due to their stronger charge density symmetry and electrostatic potential energy. Furthermore, the separation phenomenon and the formation of the crystal structure were observed according to the radial distribution function (RDF) and orientation correlation function obtained from MD simulation. Finally, the attachment energy (AE) model was used to observe and compare different crystal surfaces; it was proposed that the aggregation of 4,4'-MDI was attributed to the polar attraction between isocyanate groups according to the results of the orientation correlation function. It was also observed that compared with 2,4'-MDI, 4,4'-MDI molecules on the (110) crystal surface were easier to form crystal structures.

Characterization of Cuticular Wax in Tea Plant and Its Modification in Response to Low Temperature.

Cuticular wax ubiquitously covers the outer layer of plants and protects them against various abiotic and biotic stresses. Nevertheless, the characteristics of cuticular wax and its role in cold resistance in tea plants remain unclear. In our study, cuticular wax from different tissues, cultivars, and leaves during different spatio-temporal growth stages were characterized and compared in tea plants. The composition, distribution pattern, and structural profile of cuticular wax showed considerable tissue specificity, particularly in petals and seeds. During the spatial development of tea leaves, total wax content increased from the first to fifth leaf in June, while a decreasing pattern was observed in September. Additionally, the total wax content and number of wax compounds were enhanced, and the wax composition significantly varied with leaf growth from June to September. Ten cultivars showed considerable differences in total wax content and composition, such as the predominance of saturated fatty acids and primary alcohols in SYH and HJY cultivars, respectively. Correlation analysis suggested that n-hexadecanoic acid is positively related to cold resistance in tea plants. Further transcriptome analysis from cold-sensitive AJBC, cold-tolerant CYQ, and EC 12 cultivars indicated that the inducible expression of wax-related genes was associated with the cold tolerance of different cultivars in response to cold stress. Our results revealed the characterization of cuticular wax in tea plants and provided new insights into its modification in cold tolerance.

Rh(iii)-catalyzed spiroannulation of 3-arylquinoxalin-2(1H)-ones with alkynes: practical access to spiroquinoxalinones.

The Rh(iii)-catalyzed synthesis of spiroquinoxalinone derivatives from 3-arylquinoxalin-2(1H)-ones and alkynes via a C-H functionalization/[3 + 2] annulation sequence has been developed. This method, featuring low catalyst loading, was amenable to Gram scale synthesis and tolerated a variety of functional groups and substitution patterns on the aryl rings, providing the target products in good to excellent yields.

Synthesis of novel caffeic acid derivatives and their protective effect against hydrogen peroxide induced oxidative stress via Nrf2 pathway.

AIM: This study was aimed to synthesize novel caffeic acid derivatives and evaluate their potential applications for the treatment of oxidative stress associated disease. MAIN METHODS: Caffeic acid sulfonamide derivatives were synthesized by coupling sulfonamides to the backbone of caffeic acid and fully characterized by melting point test, FT-IR, MS, NMR, UV-vis and n-octanol-water distribution assay. Their free radical scavenging ability was evaluated using DPPH assay and cytotoxicity against A549 cells were determined by MTT assay. The protective effect of these derivatives against hydrogen peroxide (H2O2) induced oxidative injury was assessed in A549 cells from cell viability, production of reactive oxygen species (ROS) and malondialdehyde (MDA), alternation of antioxidase activities, and expressions of Nrf2 and its target genes. KEY FINDINGS: Six novel caffeic acid sulfonamide derivatives were obtained. The derivatives showed better liphophilicity than the parent caffeic acid. CASMZ, CAST and CASQ exhibited similar DPPH scavenging capability as caffeic acid, while the protection of hydroxyl groups on the benzene ring with acetyl groups caused decrease in radical scavenging activity. No inhibitory effect on the proliferation of A549 cells were observed up to a concentration of 50 µM. Pre-treatment of cells with these derivatives strongly inhibited H2O2 induced decrease of cell viability, reduced the production of ROS and MDA, promoted antioxidase activities, and further upregulated the expression of Nrf2 and its target genes. SIGNIFICANCE: Caffeic acid sulfonamide derivatives were synthesized with simple reactions under mild conditions. They might protect cells from H2O2-induced oxidative injury via Nrf2 pathway.

Transcriptome profiling analysis reveals the role of silique in controlling seed oil content in Brassica napus.

Seed oil content is an important agronomic trait in oilseed rape. However, the molecular mechanism of oil accumulation in rapeseeds is unclear so far. In this report, RNA sequencing technique (RNA-Seq) was performed to explore differentially expressed genes in siliques of two Brassica napus lines (HFA and LFA which contain high and low oil contents in seeds, respectively) at 15 and 25 days after pollination (DAP). The RNA-Seq results showed that 65746 and 66033 genes were detected in siliques of low oil content line at 15 and 25 DAP, and 65236 and 65211 genes were detected in siliques of high oil content line at 15 and 25 DAP, respectively. By comparative analysis, the differentially expressed genes (DEGs) were identified in siliques of these lines. The DEGs were involved in multiple pathways, including metabolic pathways, biosynthesis of secondary metabolic, photosynthesis, pyruvate metabolism, fatty metabolism, glycophospholipid metabolism, and DNA binding. Also, DEGs were related to photosynthesis, starch and sugar metabolism, pyruvate metabolism, and lipid metabolism at different developmental stage, resulting in the differential oil accumulation in seeds. Furthermore, RNA-Seq and qRT-PCR data revealed that some transcription factors positively regulate seed oil content. Thus, our data provide the valuable information for further exploring the molecular mechanism of lipid biosynthesis and oil accumulation in B. nupus.

[Immune response for phase I clinical trial of a hepatitis B immunogenic complex therapeutic vaccine, YIC].

OBJECTIVE: A hepatitis B immunogenic complex therapeutic vaccine, yeast-derived recombinant HBsAg combined with human anti-HBs immunoglobulin (YIC), was evaluated for safety and immune response in phase I clinical trial. METHODS: The subtypes IgG1, IgG2, IgG3 and IgG4 of serum anti-HBs collected from 20 immunized subjects were analyzed by ELISA. The lymphocyte proliferation assay was carried out in five subjects and was analyzed by 3H-thymidine incorporation. The assays for IFNgamma, IL-2, IL-4, IL-6, IL-10 and TNFalpha were measured using Human Cytometric Bead Array Kit with FACSCalibur. RESULTS: The results showed that the subtypes of anti-HBs antibodies induced by 30, 60 and 90 microg YIC-immunized groups among all of the adult volunteers (20/20) were IgG1 and IgG3. The level of IgG1 was higher than that of IgG3 in each volunteer but the strength was different from each other. The rHBsAg-stimulated lymphocyte proliferation induced by three injections of 90 microg of YIC showed that the stimulation index was more than 2.0 in four out of the five individuals (4/5), ranging from 2.70 to 4.75. PHA-stimulated lymphocyte proliferation was not related to rHBsAg-stimulated lymphocyte proliferation. In the 60 microg YIC-immunized group there was no significant difference between the levels of IFNgamma, IL-2, IL-4, IL-6 and IL-10 at day 0 and day 42. At day 71, in comparison to day 0, the level of IFNgamma was higher in all eight subjects studied (P = 0.015) and the level of IL-2 was also increased in seven out of eight subjects (P = 0.002). In contrast, the levels of IL-4, IL-6, IL-10 and TNFalpha showed no significant difference in all the subjects (P-values: 0.298, 0.976, 0.202 and 0.996). CONCLUSION: Our results indicate that this hepatitis B immunogenic complex therapeutic vaccine (YIC) can induce a potent anti-HBs response.

Homogeneous preparation of cellulose acetate propionate (CAP) and cellulose acetate butyrate (CAB) from sugarcane bagasse cellulose in ionic liquid.

Cellulose acetate butyrate (CAB) and cellulose acetate propionate (CAP) were prepared homogeneously in a 1-allyl-3-methylimidazolium chloride (AmimCl) ionic liquid system from sugarcane bagasse (SB). The reaction temperature, reaction time, and molar ratio of butyric (propionic) anhydride/anhydroglucose units in the cellulose affect the butyryl (B) or propionyl (P) content of CAB or CAP samples. The (13)C NMR data revealed the distribution of the substituents of CAB and CAP. The thermal stability of sugar cane bagasse cellulose was found by thermogravimetric analysis to have decreased after chemical modification. After reaction, the ionic liquid was effectively recycled and reused. This study provides a new way for high-value-added utilization of SB and realizing the objective of turning waste into wealth.

Vaccination with recombinant HBsAg-HBIG complex in healthy adults.

A therapeutic vaccine for viral hepatitis B composed of yeast-derived recombinant HBsAg complexed to human anti-HBs immunoglobulin (yeast-derived-immunogenic complex, YIC) with alum as the adjuvant was evaluated for safety. In stage 1, 22 healthy Chinese adult volunteers were vaccinated with three doses of 30 microg, 60 microg or 90 microg of HBsAg in YIC at 4-week intervals. In stage 2, nine volunteers received 90 microg of HBsAg in YIC for six injections. All immunizations were well tolerated. Renal, liver function and other blood chemistry tests remained within normal range. All recipients developed serum anti-HBs, the highest being 1000 mIU/ml, and the subtypes of anti-HBs were IgG1 and IgG3. The serum levels of interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) were increased, while no significant increase was observed in interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10) or tumor necrosis factor-alpha (TNF-alpha). These results indicate that this complex is safe and can induce a potent anti-HBs response.