Genome-wide identification of tea plant (Camellia sinensis) BAHD acyltransferases reveals their role in response to herbivorous pests.

BACKGROUND: BAHD acyltransferases are among the largest metabolic protein domain families in the genomes of terrestrial plants and play important roles in plant growth and development, aroma formation, and biotic and abiotic stress responses. Little is known about the BAHDs in the tea plant, a cash crop rich in secondary metabolites. RESULTS: In this study, 112 BAHD genes (CsBAHD01-CsBAHD112) were identified from the tea plant genome, with 85% (98/112) unevenly distributed across the 15 chromosomes. The number of BAHD gene family members has significantly expanded from wild tea plants to the assamica type to the sinensis type. Phylogenetic analysis showed that they could be classified into seven subgroups. Promoter cis-acting element analysis revealed that they contain a large number of light, phytohormones, and stress-responsive elements. Many members displayed tissue-specific expression patterns. CsBAHD05 was expressed at more than 500-fold higher levels in purple tea leaves than in green tea leaves. The genes exhibiting the most significant response to MeJA treatment and feeding by herbivorous pests were primarily concentrated in subgroups 5 and 6. The expression of 23 members of these two subgroups at different time points after feeding by tea green leafhoppers and tea geometrids was examined via qPCR, and the results revealed that the expression of CsBAHD93, CsBAHD94 and CsBAHD95 was significantly induced after the tea plants were subjected to feeding by both pricking and chewing pests. Moreover, based on the transcriptome data for tea plants being fed on by these two pests, a transcriptional regulatory network of different transcription factor genes coexpressed with these 23 members was constructed. CONCLUSIONS: Our study provides new insights into the role of BAHDs in the defense response of tea plants, and will facilitate in-depth studies of the molecular function of BAHDs in resistance to herbivorous pests.

De novo full length transcriptome analysis of a naturally caffeine-free tea plant reveals specificity in secondary metabolic regulation.

Tea plants are crops with economic, health and cultural value. Catechin, caffeine and theanine are the main secondary metabolites of taste. In the process of germplasm collection, we found a resource in the Sandu Aquatic Autonomous County of Guizhou (SDT) that possessed significantly different characteristic metabolites compared with the cultivar 'Qiancha 1'. SDT is rich in theobromine and theophylline, possesses low levels of (-)-epicatechin-3-gallate, (-)-epigallocatechin-3-gallate, and theanine content, and is almost free of caffeine. However, research on this tea resource is limited. Full-length transcriptome analysis was performed to investigate the transcriptome and gene expression of these metabolites. In total, 78,809 unique transcripts were obtained, of which 65,263 were complete coding sequences. RNA-seq revealed 3415 differentially expressed transcripts in the tender leaves of 'Qiancha 1' and 'SDT'. Furthermore, 2665, 6231, and 2687 differentially expressed transcripts were found in different SDT tissues. These differentially expressed transcripts were enriched in flavonoid and amino acid metabolism processes. Co-expression network analysis identified five modules associated with metabolites and found that genes of caffeine synthase (TCS) may be responsible for the low caffeine content in SDT. Phenylalanine ammonia lyase (PAL), glutamine synthetase (GS), glutamate synthase (GOGAT), and arginine decarboxylase (ADC) play important roles in the synthesis of catechin and theanine. In addition, we identified that ethylene resposive factor (ERF) and WRKY transcription factors may be involved in theanine biosynthesis. Overall, our study provides candidate genes to improve understanding of the synthesis mechanisms of these metabolites and provides a basis for molecular breeding of tea plant.

Efficient and simple simultaneous adsorption removal of multiple aflatoxins from various liquid foods.

In this study, a polydopamine modified nanofibers membrane (PDA-PS NFsM) was prepared and evaluated as the adsorbent for simultaneous removal of a variety of aflatoxins in various liquid foods, including edible oil, soy sauce and milk, rice vinegar and liquor. The removal efficiency for every single aflatoxin from all samples involved above was more than 76.5% within 1 h at 25 °C, except the liquors with higher ethanol content, for which the efficiency was lower. Moreover, PDA-PS NFsM can be removed directly after the adsorption process without any subsequent separation. The results suggested that the adsorption mechanism of the aflatoxins onto PDA-PS NFsM was chemisorption-based spontaneous endothermic reaction and aflatoxins were adsorbed by electrostatic interaction, hydrogen bonding and π-π interaction. This study confirmed that the PDA-PS NFsM has a good practical application potential in the simultaneous removal of a variety of aflatoxins from various liquid foods.

Efficient, rapid and simple adsorption method by polydopamine polystyrene nanofibers mat for removal of multi-class antibiotic residues in environmental water.

Polydopamine polystyrene nanofibers mat (PDA-PS NFsM) was prepared as an adsorbent for the simultaneous removal of multiple antibiotic residues in environmental water for the first time. PDA-PS NFsM can directly be used to adsorb 18 antibiotic residues belonging to 8 classes without any pretreatment of water samples. The adsorption process was completed within 5 min. All antibiotics could be removed at the same time, and the removal rate of each target was above 85%. Moreover, the used PDA-PS NFsM can be easily separated from the environmental water by taking out directly, and can be reused for 10 times after simple regeneration. The thermodynamic and kinetic properties of PDA-PS NFsM adsorption of antibiotic residues were further investigated consequently. It was found that the adsorptions of PDA-PS NFsM to the targets were spontaneous and endothermic process (ΔG<0, ΔH>0, ΔS>0). The results of adsorption kinetic experiments illustrated that the adsorption process was rapid, the adsorption equilibrium of which can be reached in 5 min. Adsorption isotherm experiments proved that the adsorption process of PDA-PS NFsM was consistent with Langmuir adsorption (R2 > 0.994), and the maximum adsorption capacity of PDA-PS NFsM towards all targets were 123.76 mg g-1. The developed method is rapid and simple, and can efficiently adsorb and remove a variety of antibiotics in environmental water, which has good practical application prospect.

[Determination of tetracycline and fluoroquinolone residues in fish by polydopamine nanofiber mat based solid phase extraction combined with ultra performance liquid chromatography-tandem mass spectrometry].

Tetracyclines and fluoroquinolones are common antibacterial drugs used in aquaculture, and their residues may pose a risk to human health. The low concentration of drug residues and complex matrixes such as fats and proteins in aquatic products necessitate the urgent development of efficient sample pretreatment methods. Solid phase extraction (SPE) is the most common sample pretreatment method, in which the core is an adsorbent. Compared with traditional SPE adsorbents, nanofiber mat (NFsM) has more interaction sites because of their large specific surface area. Furthermore, NFsMs modified with specific functional groups can significantly improve the extraction efficiency of tetracyclines and fluoroquinolones. Polydopamine (PDA) is spontaneously synthesized by the oxidative self-polymerization of dopamine-hydrochloride in alkaline solutions (pH>7.5). Because of its rich amino and catechol groups, PDA can form π-π stacking, electrostatic attraction, hydrophobic interaction, and hydrogen bonding interactions with target molecules. By exploiting the above advantages, polystyrene (PS) NFsM, as a template, was prepared by the electrostatic spinning method, and PDA-PS NFsM was obtained by functional modification of PDA through self-polymerization. Fourier transform infrared spectroscopy (FT-IR) and field-emission scanning electron microscopy (FESEM) were used to characterize the synthesized PS NFsM and PDA-PS NFsM. It was proved that PDA was successfully modified on the PS NFsM, with the SEM images revealing a rough outer core shell structure and an inner honeycomb structure. Subsequently, the handmade SPE column with PDA-PS NFsM was completed. A novel and efficient screening analytical method based on PDA-PS NFsM for the simultaneous determination of three tetracyclines (tetracycline (TET), chlortetracycline (CTC), and oxytetracycline (OTC)) and three fluoroquinolones (enrofloxacin (ENR), ciprofloxacin (CIP), and norfloxacin (NOR)) in fish by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was established. The SPE procedure was optimized to develop an efficient method for sample preparation. Evaluate parameters including the amount of NFsM usage, ionic strength, flow rate of the sample solution, composition of eluent, and breakthrough volume were investigated. Only (20±0.1) mg of PDA-PS NFsM was sufficient to completely adsorb the targets, and the analytes retained on NFsM could be eluted by 1 mL of formic acid-ethyl acetate (containing 20% methanol) (1∶99, v/v). The residues were redissolved in 0.1 mL 10% methanol aqueous solution containing 0.2% formic acid. In addition, no adjustment of the pH and ionic strength of the sample solutions was required, and the breakthrough volume was 50 mL. The limits of detection (LODs) and limits of quantification (LOQs) of the six target compounds were measured at 3 times and 10 times the signal-to-noise ratio (S/N), respectively. The LODs and LOQs were 0.3-1.5 µg/kg and 1.0-5.0 µg/kg, respectively. The linear ranges of the six target compounds were LOQ-1000 µg/kg, and the coefficient of determination (R2) was greater than 0.999. To evaluate the accuracy and precision, blank spiked samples at three levels (low, medium, and high) were prepared for the recovery experiments, and each level with six parallel samples (n=6). The recoveries ranged from 94.37% to 102.82%, with intra-day and inter-day relative standard deviations of 2.38% to 8.06% and 4.10% to 9.10%, respectively. To evaluate the purification capacity of PDA-PS NFsM, the matrix effects before and after SPE were calculated and compared. Matrix effects before SPE were -12.98% to -38.68%. After the completion of SPEbased on PDA-PS NFsM, the matrix effect of each target analyte was significantly reduced to -2.15% to -7.36%, which proved the significant matrix removal capacity of PDA-PS NFsM. Finally, the practicality of this method was evaluated by using it to analyze real samples. This SPE method based on PDA-PS NFsM is efficient, practical, and environmentally friendly, and it has great potential for use in the routine monitoring of drug residues in fish.

Sodium 4-styrenesulfonate functionalized nanofibers mat as 96-well plate solid-phase extraction adsorbent for quantitative determination of multiple ß-agonists residues in pork samples.

In this work, sodium 4-styrenesulfonate functionalized polyacrylonitrile nanofibers mat (SS/PAN NFM) was firstly prepared and applied as 96-well plate solid-phase extraction adsorbent for quantitative determination of seven ß-agonists residues in pork samples. The functional modification endowed the SS/PAN NFM with superior adsorption performance for target ß-agonists. The adsorption process is spontaneous (ΔG < 0), the initial adsorption rate can reach 6.03-9.09 mg/g/min and the maximum adsorption capacity is calculated to be 48.3 mg/g at 298 K. Moreover, SS/PAN NFM can be reused for 12 times without degradation in adsorption capability. Combined with UPLC-MS/MS, the limits of detection can reach 0.006-0.24 µg/kg, the recoveries ranged from 87.2% to 111% and the relative standard deviations of intra-day and inter-day precisions were in the scope of 1.75%-11.6% and 5.08%-13.5%, respectively. The obtained results fully demonstrated the practicability of this method in preventing the hazard of ß-agonists residues.

Genome-wide identification and expression analysis of SABATH methyltransferases in tea plant (Camellia sinensis): insights into their roles in plant defense responses.

SABATH methyltransferases convent plant small-molecule metabolites into volatile methyl esters, which play important roles in many biological processes and defense reactions in plants. In this study, a total of 32 SABATH genes were identified in the Camellia sinensis var. sinensis (CSS) genome, which were renamed CsSABATH1 to CsSABATH32. Genome location annotation suggested that tandem duplication was responsible for the expansion of SABATH genes in tea plant. Multiple sequence alignment and phylogenetic analysis showed that the CsSABATHs could be classified into three groups (I, II and III), which were also supported by gene structures and conserved motifs analysis. Group II contained only two CsSABATH proteins, which were closely related to PtIAMT, AtIAMT and OsIAMT. The group III SABATH genes of tea plant exhibited expansion on the CSS genome compared with Camellia sinensis var. assamica (CSA) genome. Based on RNA-seq data, the CsSABATHs exhibited tissue-specific expression patterns, and the members with high expression in buds and young leaves were also obviously upregulated after MeJA treatment. The expression of many transcription factors was significantly correlated with that of different members of the CsSABATH gene family, suggesting a potential regulatory relationship between them. Quantitative real-time PCR (qPCR) expression analysis showed that CsSABATHs could respond to exogenous JA, SA and MeSA treatments in tea plants. RNA-seq data analysis and qPCR validation suggested that CsSABATH8, 11, 16, 25, 29 and 32 might play a special role in plant defense against insect herbivory. These results provide references for evolutionary studies of the plant SABATH family and the exploration of the potential roles of CsSABATHs in tea plant defense responses.

Rapid, simple and green solid phase extraction based on polyaniline nanofibers-mat for detecting non-steroidal anti-inflammatory drug residues in animal-origin food.

In this study, polyaniline modified polyacrylonitrile nanofibers mat (PANI NFsM) was prepared as a novel adsorbent for the solid-phase extraction (SPE) of non-steroidal anti-inflammatory drug residues in meat or egg samples. The solvent extracts of samples were simply diluted with water to perform the SPE, and then the eluent was directly analyzed. Significant reduction of the matrix effect was obtained after SPE using only 5 mg of PANI NFsM. The entire sample preparation time is 5-10 times lower than the existing methods. The limits of detection of the target analytes ranging from 0.6 to 12.2 µg kg-1 had already met the demand of food safety monitoring by only 1 g sample. The recoveries ranged from 85.18% to 107.31%, with the intra-day and inter-day relative standard deviations of 2.74% to 16.01%, revealing satisfactory accuracy and precision. Finally, real samples analyses were applied to verify the practicability of the method.

Simultaneous extraction of four plant growth regulators residues in vegetable samples using solid phase extraction based on thiol-functionalized nanofibers mat.

A simple and environmentally friendly sample pretreatment method was established for extraction of four plant growth regulators (PGRs) residues in vegetable samples. In order to effectively extract targets from complex sample matrices, three types of polar groups (COOH, NH2, or SH) functionalized polyacrylonitrile nanofibers mats (PAN NFsM) were prepared and evaluated, respectively. The superior -SH functionalized PAN NFsM was further selected as a novel adsorbent of solid phase extraction (SPE). Under optimal conditions, the proposed method could achieve high sensitivity (0.2-2 ng g-1) and acceptable recovery (88.94%-106.42%) by consuming lower amount of adsorbent (only 5 mg) and organic reagent (only 1.4 mL), and performing much fewer steps than current methods. In addition, the prepared SH-PAN NFsM possessed excellent reusability which could be reused for 6 times without degradation in adsorption capability. These results presented the great potential of the established method for environmental-friendly and efficiently detecting PGRs residues.

A nanofiber mat prepared from sulfonated polyaniline for solid-phase extraction of fluoroquinolones from water and biological fluids prior to their quantitation by UPLC-MS/MS.

A bifunctional sulfonated polyaniline nanofiber mat (NFM) was synthesized by oxidative polymerization and by using polyacrylonitrile nanofiber mat (NFM) as the template. The adsorption capacity of the NFM for fluoroquinolones (FQs) is distinctly improved and the adsorption was a spontaneous process. According to theoretical calculations, hydrogen bonding and ion-exchange interaction are the two major kinds of interaction mechanisms between adsorbent and FQs. The adsorption and desorption properties for FQs were systematically evaluated by adsorption isotherms and by thermodynamic and kinetic studies. The results indicate that the NFM is a viable sorbent for FQs because of rapid mass transfer and good adsorption/desorption efficiency. The NFM is re-usable for at least 20 cycles without decline in performance. Following desorption of the FQs with 10% (V/V) formic acid/acetonitrile, they were quantified by UPLC with MS/MS detection. The sorbent was applied to the solid phase extraction of the FQs norfloxacin, ciprofloxacin, ofloxacin, enrofloxacin, danofloxacin, pefloxacin, marbofloxacin, lomefloxacin and difloxacin from water and biological fluids. Figures of merit include (a) low limits of detection (0.5-1.5 ng L-1 for water, 0.016-0.052 µg L-1 for urine and serum), (b) high recoveries from spiked samples (82.3%-109.4%) with low relative standard deviations (1.1%-12.3%); (c) short extraction times (2 min), and (d) low adsorbent dosage (4 mg). Graphical abstractSchematic representation of a bi-functional sulfonated polyaniline nanofiber mat (NFM) for solid phase extraction (SPE) of fluoroquinolones (FQs) in water, urine and serum.

Simple, efficient, and eco-friendly sample preparation for simultaneous determination of paracetamol and chloramphenicol in meat.

We have developed and validated a simple, eco-friendly, and reliable method to simultaneously determine paracetamol and chloramphenicol in meat with ultra performance liquid chromatography and tandem mass spectrometry. The samples were firstly extracted with ethylenediaminetetraacetic acid-McIlvaine's buffer and then purified by solid-phase extraction by using a novel core-shell polyaniline/polyacrylonitrile nanofibers mat. Compared with existing methods for the two analytes, the proposed method was simplified greatly with much fewer sample preparation steps, consumed much less time (< 2 min per sample) and organic solvent (0.7 mL per sample). Low detection levels (0.15-0.20 µg/kg for paracetamol, 0.01 µg/kg for chloramphenicol) with good precision and recoveries of the target compounds were obtained. The proposed method was applied to determine the residual paracetamol and chloramphenicol in pork, chicken, and beef samples, and the test results were consistent with those using the Chinese national standard methods, which demonstrates the reliability and practicality of the new method.

High-Throughput and High-Efficient Micro-solid Phase Extraction Based on Sulfonated-Polyaniline/Polyacrylonitrile Nanofiber Mats for Determination of Fluoroquinolones in Animal-Origin Foods.

We herein describe a high-throughput 96-well plate micro-solid phase extraction sample preparation technique based on novel sulfonated-polyaniline/polyacrylonitrile nanofiber mats (sulfonated-PANI/PAN NFMs) for multiresidue detection of fluoroquinolones (FQs) in various animal-origin food samples. Through the double-modification of polyaniline and sulfonic acid, the resulting functionalized sulfonated-PANI/PAN NFMs present high extraction efficiency for FQs. Compared with the existing methods, this approach demonstrates its advantages of being suitable for more sample matrices (milk, animal muscle, liver, kidney, and egg), lower sample amount (0.5 g), lower sorbent requirement (5.0 mg), lower volume of organic solvent (0.7 mL), shorter time (0.2 min per sample), and high sensitivity (0.012-0.06 µg·kg-1). In addition, sulfonated-PANI/PAN NFMs possess excellent reusability which could be reused 10 times without an obvious decrease in extraction efficiency. Combined with ultra performance liquid chromatography-tandem mass spectrometry, the novel sample preparation technique can be expected as an efficient method for routine trace FQ residue monitoring in animal-origin food samples.

Lowering greening of cookies made from sunflower butter using acidic ingredients and effect on reducing capacity, tryptophan and protein oxidation.

Lowering of greening formed from oxidized chlorogenic acid (CGA) and amino groups, and favoured at alkaline pH, was investigated using acidic ingredients (sour cream, buttermilk, yoghurt, and honey) in sunflower butter cookies. Cookies with maple syrup added were used as a positive control. Changes in greening intensity, greening reactants (total phenols, CGA, protein), antioxidant capacity, tryptophan and Schiff base fluorescence were measured. Percentage greening, pH and aw of cookies followed the same order: maple syrup > sour cream ≥ buttermilk > yoghurt > honey. pH was positively correlated with greening intensity (r = 0.77) and negatively correlated with CGA (r = -0.96). Total phenolic content, antioxidant capacity, tryptophan and Schiff bases were similar among cookies. The results suggest it is possible to decrease greening by minimizing storage time and using acidic ingredients. Minimal greening with acidic ingredients can extend the application of sunflower butter as a baking ingredient without loss of free radical-scavenging capacity, or higher protein oxidation.

Chlorogenic acid oxidation-induced greening of sunflower butter cookies as a function of different sweeteners and storage conditions.

Sunflower butter use as an allergen-free alternative to tree and legume nut butter in baking is limited by chlorogenic acid induced greening that occurs at alkaline pH. Limited information is available on controlling this greening in a food matrix. This study examined how different liquid sweeteners and relative humidity influenced greening of sunflower butter cookies. Doughs had similar initial pH (7.52-7.66) which increased to 8.44-9.13 after baking as ranked: xylitol>maple syrup>corn syrup>honey>agave syrup. Cookies made with maple syrup had the highest moisture and greening corresponding with lowest free chlorogenic acid. The % greening followed the same trend as greening intensity, and was positively correlated (r=0.9101) with chlorogenic-lysine adduct content. Our findings provide an ingredient solution to controlling greening, as results demonstrate that greening can be promoted with high relative humidity storage, and use of high moisture and pH ingredients. Unwanted greening can be inhibited by simply changing the liquid sweetener.

Ionically dispersed Fe(ii)-N and Zn(ii)-N in porous carbon for acidic oxygen reduction reactions.

Inspired by the zinc protoporphyrin found in red blood cells during heme production, we have developed a novel type of bimetallic Fe, Zn/N/C catalyst with high metal loading (Fe 1.2 wt% and Zn 1.7 wt%), demonstrating high activity and high stability for oxygen reduction processes in acidic electrolytes.

The Multivesicular Bodies (MVBs)-Localized AAA ATPase LRD6-6 Inhibits Immunity and Cell Death Likely through Regulating MVBs-Mediated Vesicular Trafficking in Rice.

Previous studies have shown that multivesicular bodies (MVBs)/endosomes-mediated vesicular trafficking may play key roles in plant immunity and cell death. However, the molecular regulation is poorly understood in rice. Here we report the identification and characterization of a MVBs-localized AAA ATPase LRD6-6 in rice. Disruption of LRD6-6 leads to enhanced immunity and cell death in rice. The ATPase activity and homo-dimerization of LRD6-6 is essential for its regulation on plant immunity and cell death. An ATPase inactive mutation (LRD6-6E315Q) leads to dominant-negative inhibition in plants. The LRD6-6 protein co-localizes with the MVBs marker protein RabF1/ARA6 and interacts with ESCRT-III components OsSNF7 and OsVPS2. Further analysis reveals that LRD6-6 is required for MVBs-mediated vesicular trafficking and inhibits the biosynthesis of antimicrobial compounds. Collectively, our study shows that the AAA ATPase LRD6-6 inhibits plant immunity and cell death most likely through modulating MVBs-mediated vesicular trafficking in rice.

The DnaJ OsDjA7/8 is essential for chloroplast development in rice (Oryza sativa).

DnaJ proteins belong to chaperones of Hsp40 family that ubiquitously participate in various cellular processes. Previous studies have shown chloroplast-targeted DnaJs are involved in the development of chloroplast in some plant species. However, little is known about the function of DnaJs in rice, one of the main staple crops. In this study, we characterized a type I DnaJ protein OsDjA7/8. We found that the gene OsDjA7/8 was expressed in all collected tissues, with a priority in the vigorous growth leaf. Subcellular localization revealed that the protein OsDjA7/8 was mainly distributed in chloroplast. Reduced expression of OsDjA7/8 in rice led to albino lethal at the seedling stage. Transmission electron microscopy observation showed that the chloroplast structures were abnormally developed in the plants silenced for OsDjA7/8. In addition, the transcriptional expression of the genes tightly associated with the development of chloroplast was deeply reduced in the plants silenced for OsDjA7/8. Collectively, our study reveals that OsDjA7/8 encodes a chloroplast-localized protein and is essential for chloroplast development and differentiation in rice.