Characterization of heavy metal, antibiotic pollution, and their resistance genes in paddy with secondary municipal-treated wastewater irrigation.

Secondary municipal-treated wastewater irrigation may introduce residual antibiotics into the agricultural systems contaminated with certain heavy metals, ultimately leading to the coexistence of antibiotics and heavy metals. The coexistence may induce synergistic resistance to both in the microbial community. Here, we investigated the effects of long-term municipal-treated irrigation for rice on the microbiome and resistome. The results showed that the target antibiotics were undetectable in edible grains, and the heavy metal concentrations did not exceed the standard in edible rice grains. Heavy metal resistance genes (MRGs) ruvB and acn antibiotic resistance genes (ARGs) sul1 and sul2 were the dominating resistant genes. The coexistence of antibiotics and heavy metals affected the microbial community and promoted metal and antibiotic resistance. Network analysis revealed that Proteobacteria were the most influential hosts for MRGs, ARGs, and integrons, and co-selection may serve as a potential mechanism for resistance maintenance. MRG czcA and ARG sul1 can be recommended as model genes to study the co-selection of ARGs and MRGs in environments. The obtained results highlight the importance of considering the co-occurrence of heavy metals and antibiotics while developing effective methods to prevent the transmission of ARGs. These findings are critical for assessing the possible human health concerns associated with secondary municipal-treated wastewater irrigation for agriculture and improving the understanding of the coexistence of heavy metals and antibiotics.

Full-length transcriptomics study of Ustiloxins-induced hepatocyte injury.

Ustiloxins is a mycotoxin produced by the metabolism of Rice false smut. Studies have shown that Ustiloxins may be toxic to animals, but there is still a lack of toxicological evidence. The liver, as the main organ for the biotransformation of foreign chemicals, may be the direct target organ of Ustiloxins toxicity. In this study, we found that cell viability decreased in a dose- and time-dependent manner when BNL CL.2 cells were treated with different concentrations of Ustiloxins (0, 5, 10, 20, 30, 40, 60, 80, 100, 150 and 200 µg/mL) for 24 and 48 h. In addition, scanning electron microscope observation showed that the cell membrane of the experimental group was damaged, with the appearance of apoptotic bodies. Moreover, the ROS and GSH levels were significantly increased in cells exposed to Ustiloxins. We analyzed the key action targets of Ustiloxins on hepatocyte injury using full-length transcriptomics. A total of 1099 differentially expressed genes were screened, of which 473 genes were up-regulated, and 626 genes were down-regulated. Besides, we also found that the expression of MCM7 and CDC45 in BNL CL.2 cells treated with Ustiloxins decreased, and the expression of CCl-2, CYP1b1, CYP4f13, and GSTM1 increased according to qRT-PCR. Ustiloxins might change CYP450 and GST-related genes, affect DNA replication and cell cycle, and lead to oxidative stress and liver cell injury.

Insights into antibiotic resistance-related changes in microbial communities, resistome and mobilome in paddy irrigated with reclaimed wastewater.

Reclaimed wastewater (reclaimed wastewater, RWW) from municipal wastewater treatment plants for paddy irrigation is a well-established practice to alleviate water scarcity. However, the reuse may result in the persistent exposure of the paddy to residual antibiotics in RWW. Continuous presence of even low-level antibiotics can exert selective pressure on microbiota, resulting in the proliferation and dissemination of antibiotic resistance genes (ARGs) in paddy. In this study, metagenomic analysis was applied to firstly deciphered the effects of residual antibiotics on microbiome and resistome in constructed mesocosm-scale paddy soils. The diversity and abundance of ARG have remarkably risen with the increasing antibiotic concentration in RWW. Network analysis revealed that 28 genera belonging to six phyla were considered as the potential ARG hosts, and their abundances were enhanced with increasing antibiotic concentrations. A partial least-squares path model indicated that the microbial community was the principal direct driver of the ARG abundance and the resistome alteration in paddy soil under long-term RWW irrigation. Microbes may acquire ARGs via horizontal gene transfer. IntI1 could play an essential role in the propagation and spread of ARGs. Functional analysis suggested that enhanced SOS response and T4SSs (Type IV secretion systems) modules could stimulate horizontal transfer potential and promote the ARG abundance. The obtained results provide a scientific decision for assessing the ecological risk of RWW application.

Response of heavy-metal and antibiotic resistance genes and their related microbe in rice paddy irrigated with treated municipal wastewaters.

Paddy irrigation with secondary effluents from municipal wastewater treatment plants (MWTPs) is a well-established practice to alleviate water scarcity. However, the reuse might lead to more complicated contamination caused by interactions between residual antibiotics in effluents and heavy metals in paddy soil. To date, no information is available for the potential effects of dual stress of heavy metals and antibiotics on heavy-metal resistance genes (MRGs) and antibiotic resistance genes (ARGs). Here, this study investigated the response of heavy metal and antibiotic resistance genes, and related microorganisms to the dual threat of antibiotics and heavy metals under the long-term MWTP effluent irrigation for rice paddy using metagenome. The results showed that there was not a negative effect on rice consumption if MWTP effluent was used to irrigate rice for a long time. The concentration of antibiotics could reshape the ARGs and MRG profiles in rice paddy soil. The findings revealed the co-occurrence of ARGs and MRGs in rice paddy soils, thus highlighting the need for simultaneous elimination of antibiotics and heavy metals to effectively reduce ARGs and MRGs. Acn and sul1 genes encoding Iron and sulfonamides resistance mechanisms are the most abundant MRG and ARG, respectively. Network analysis revealed the possibility that IntI1 plays a role in the co-transmission of MRG and ARG to host microbes, and that Proteobacteria are the most dominant hosts for MRG, ARG, and integrons. The presence of antibiotics in irrigated MWTP effluents has been found to stimulate the proliferation of heavy metal and antibiotic resistances by altering soil microbial communities. This study will enhance our comprehension of the co-selection between ARGs and MRGs, as well as reveal the concealed environmental impacts of combined pollution. The obtained results have important implications for food safety and human health in rice.

Chemistry-specific responses due to rice-microbe interactions in the rhizosphere to counteract mefenacet stress.

The widespread use of herbicides has raised considerable concern with regard to their harmful consequences on plant growth, crop yield and the soil ecological environment. It has been well documented that colonization of rhizobacteria in the plant root system has a positive effect on activation of plant defenses to protect the plant from damage. Using the platform of high-throughput analysis with tandem mass spectrometry and Illumina sequencing, we identified the specific activated rhizobacteria, the key growth stimulating substances and the metabolic pathways involved in seedling stage tolerance to mefenacet stress in rice. The relative abundance of beneficial rhizospheremicrobes such as Acidobacteria and Firmicutes increased with mefenacet treatment, indicating that the rhizosphere recruited some beneficial microbes to resist mefenacet stress. Mefenacet treatment induced alterations in several interlinked metabolic pathways, many of which were related to activation of defense response signaling, especially the indole-3-pyruvate pathway. Indole-3-acetaldehyde and indole-3-ethanol from this pathway may act as flexible storage pools for indole-3-acetic acid (IAA). Our findings also suggest that a significant increase of IAA produced by the enrichment of beneficial rhizospheremicrobes, for example genus Bacillus, alleviated the dwarfing phenomenon observed in hydroponic medium following mefenacet exposure, which may be a key signaling molecule primarily for phytostimulation and phytotolerance in microbe-plant interactions.

[Simultaneous determination of three allergic proteins in rice and products by high performance liquid chromatography-tandem mass spectrometry combined with stable isotope-labeled peptides].

Rice is an important cereal that is consumed as both an energy and protein source by a large proportion of the population worldwide. However, clinical studies have found that rice grains are responsible for cases of severe asthma, eczema, and atopic dermatitis in some adult patients. Several allergenic proteins have been identified and biochemically and immunochemically characterized from rice grains. These include α-amylase/trypsin inhibitors, glyoxalase Ⅰ, and α-globulin. In this study, we proposed an approach for the simultaneous quantification of three allergenic proteins in rice and its products, based on a stable isotope-labeled signature peptide standard and liquid chromatography-tandem mass spectrometry. Samples of rice and products were extracted by a salt solution, hydrolyzed by Lys-C and Trypsin, and purified by C18-SD. The linear ion trap-high resolution mass spectrometry (LTQ-Orbitrap) and Protein Discovery software were used to acquire and identify allergenic proteins in rice samples. In present study, three proteins including seed allergenic protein RAG2, glyoxalase Ⅰ, and 19 kDa globulin were identified. To establish a stable quantitative detection method, the signature peptides selected from the identified enzymatic hydrolysis peptides must have greater abundance and higher specificity as characteristic peptides. Three corresponding signature peptides in rice were screened based on the principles of previous study, and were validated through comparisons of the basic local alignment search tool (BLAST) with the NCBI and UniProt databases. The three signature peptides were successively eluted by liquid chromatography and separated on a Poroshell column. They were then detected by positive electrospray ionization (ESI+) in multiple reaction monitoring mode and quantified by an isotope dilution method. To achieve an improvement in the detection sensitivity and specificity, mass spectrometry parameters, such as the collision energy of three ion pairs of each peptide, were optimized. Three recombinant allergenic proteins and the winged stable isotope-labeled signature peptide standard were synthesized. These were then used to compare the effects of different enzymatic conditions, including hydrolysis solvents containing sodium dodecyl sulfate (SDS) with different contents, as well as the enzymes and their amounts, on the digestion efficiency. The data showed that the digestion efficiency of the three proteins could be improved to 65.7%-97.3% when 1 g/L of the SDS-containing hydrolysis solvent, and the combined digestion strategy of Lys-C and Trypsin, were adopted in the enzymatic process. These results indicate the following inferences: a small amount of SDS (1 g/L) in the enzymatic hydrolysis system is beneficial to complete protein denaturation, a Lys-C and Trypsin combined digestion strategy can complement the shortcomings of the two enzymes and improve the digestion efficiency, and the recoveries of the three proteins was not significantly increased by increasing the amount of enzyme when the ratio of protein to enzyme reached more than 20∶1. The method displayed good linearity in the range of 1-200 nmol/L with the correlation coefficients greater than 0.9972. The limits of detection and limits of quantification of the three proteins were 3 mg/kg and 10 mg/kg, respectively. The average recoveries of the three proteins spiked at three levels in different matrices ranging between 80.6%-103.7%, with the intra-day and inter-day precision less than 11.5%. Due to its high stability, excellent sensitivity, and simple operation, this method presents a wide range of application prospects in the analysis of the three allergenic proteins in different rice and rice food products.

Irrigation with secondary municipal-treated wastewater: Potential effects, accumulation of typical antibiotics and grain quality responses in rice (Oryza sativa L.).

Using secondary treated wastewater to irrigate paddies presents an exposure pathway for antibiotics to enter the terrestrial food chain. To date, there has been no information on the biochemical reactions and antibiotic uptake in rice plants irrigated with secondary treated wastewater. The present study investigated antibiotic uptake and concentration-response trends in rice tissues and evaluated the effects of typical antibiotics (tetracycline, roxithromycin, ofloxacin, and sulfamethoxazole) on rice growth, grain yield and quality, and rice physiobiochemical characters via irrigation using treated wastewater augmented with varying concentrations (0-500 µg/L) in paddies. The results showed that the antibiotic accumulation in rice plants irrigated with treated wastewater was limited, and the studied antibiotics were not detected in rice grains (edible parts). The ability of rice to withstand certain antibiotics and grow in a healthy manner is attributed to the capacity to maintain reasonably normal photosynthesis activity and to elevate antioxidative defenses. The highest antibiotic concentration (500 µg/L) did not reduce the processing quality of the rice grain, but it enhanced the cooking and eating quality. From the obtained results, it can be concluded that secondary treated wastewater for paddy irrigation is an alternative water resource securing protection from the environment and rice grain quality.

Water management affects arsenic uptake and translocation by regulating arsenic bioavailability, transporter expression and thiol metabolism in rice (Oryza sativa L.).

Water management is an economic and effective strategy to reduce arsenic (As) accumulation in rice grains, but little is known about the effect of water management on the migration and transformation of As in the soil-rice system. In this study, the effect of the continually (CF) and intermittent flooding (IF) treatments on the dynamic change of As in the rhizosphere soil-pore water-iron plaque-rice system was systematically investigated using pot experiments. The expressions of genes involved in As uptake and translocation in rice plants under different water management treatments were further examined. Results showed that the total As concentration in brown rice was increased by 50.8% in the CF treatment compared to the IF treatment, and dimethylarsinic acid (DMA) made greater contribution (from 15.5% to 29.2%) to total As increase in brown rice under the CF treatment. The CF treatment increased As bioavailability in the rhizosphere soil and soil pore water, which enhanced As uptake and transport to the xylem in rice plants by inducing the expressions of silicon transporter genes (OsLsi1 and OsLsi2) compared to the IF treatment. Moreover, the CF treatment increased As translocation from roots to shoots by reducing soil available sulfur and phytochelatins (PCs) biosynthesis and vacuolar sequestration in rice roots compared with the IF treatment. The study provides insight into the physiological and molecular mechanisms underlying As uptake and translocation in rice plants under different water regimes, which will be helpful for adopting the irrigation technique to mitigate excessive As accumulation in rice grains and associated health risk to humans.

Insight of low-abundance proteins in rice leaves under Cd stress using combinatorial peptide ligand library technology.

Low-abundance proteins (LAPs) play a very important role in interaction, regulation, and metabolism of plant biological processes. A combinatorial peptide ligand library (CPLL) can solve the problem of high-abundance proteins (HAPs) masking LAPs and enlarging the dynamic range of protein concentrations perfectly and be considered as one of the most advanced approaches for plant proteomics research. In this paper, a proper CPLL method to rice leaf proteins was established for the first time and 1056 proteins were identified in rice leaf extracts, and 624 (59.1%) LAPs were newly detected after CPLL. Based on this technology, we detected the response of rice to Cd stress and analyzed the differential LAPs and the biological significance of misexpressed proteins before and after Cd stress by bioinformatics analysis. An important contribution has also been made to a better understanding of the complex mechanisms by which rice adapts to Cd stress. Graphical abstract.

Modulation of steroid metabolism and xenobiotic biotransformation responses in zebrafish (Danio rerio) exposed to triadimefon.

The widely used fungicide triadimefon (TDF) has been detected in aquatic environments, and appears to disrupt steroid homeostasis; however, the toxic effects on fish reproduction triggered by TDF via the key receptor signaling pathways remain largely unknown. The present study showed that TDF (0.069, 0.138, 0.690 mg/L) exposure not only caused disordered germ cell maturation, but also decreased spawned egg production. In order to better understand this reproductive inhibition, we investigated the effects of TDF based on quantitative PCR, Western blot and mass spectrometry methodology in zebrafish. Due to the preferential accumulation of TDF in the liver, a general pattern of up-regulation of genes involved in biotransformation pathway was observed. A significant increase in abcb4 expression appeared to be responsible for TDF excretion. TDF-induced receptors (AhR2 and PXR) changed many genes involved in steroid metabolism, and subsequent disruptions in steroid homeostasis, which might be the key biological pathway in TDF reproductive toxicity. However, due to the different metabolic demands, the transcript profiles involved in steroid metabolism in zebrafish exhibited a sex-specific expression pattern. For example, the increase in gene expression of ahr2 was accompanied by a reduction in the rate of E2 biosynthesis resulting from the diminished cyp19a1a expression, and in turn led to down-regulation of esr1 and vtg1 in the liver, supporting the anti-estrogenic effect of TDF in male fish. In contrast, the increase in E2 production was accompanied by an increase in Esr1 protein expression caused by TDF and paralleled the increase in ahrr1 expression, suggesting that TDF may induce estrogenic activity through AhR-ER interactions in females. In addition, over-induction of cyp3a65 activity mediated through pxr, which helped to accelerate the transformation from TDF to triadimenol in the liver, appeared to elevate T metabolite rate in females. The down-regulation of fshß transcript in males further suggested that TDF might adversely affect normal gametogenesis and induce reproductive toxicity.

Absolute Quantification of Allergen Glb33 in Rice by Liquid Chromatography-Mass Spectrometry using Two Isotope-Labeled Standard Peptides.

Allergen Glb33 is an important allergen in rice that can cause allergic reactions such as asthma and atopic dermatitis. However, knowledge of the content in rice is sparse. In the present work, an absolute protein quantification method was established for allergen Glb33 in rice samples using liquid chromatography-tandem mass spectrometry. After extraction of allergen Glb33 from rice grains using salt solution, the isotope-labeled peptide internal standard was added to the extract, followed by enzymatic digestion with trypsin. The signature peptide and its isotope-labeled analogue from the tryptic hydrolysates of allergen Glb33 and the internal standard were detected by liquid chromatography-tandem mass spectrometry. The quantitative bias caused by tryptic efficiency and matrix effect was corrected by using two isotope-labeled standard peptides. The method exhibited good linearity in the range of 1-200 nM, with coefficients of determination of R2 > 0.998. A high sensitivity was observed, with a limit of quantification of 0.97 nM. Mean recoveries obtained from different rice matrices ranged from 82.7%-98.1% with precision <8.5% in intraday trials ( n = 6), while mean recoveries were from 75.1%-107.4% with precision <14.6% in interday trials ( n = 14). The developed method was successfully applied to the analysis of allergen Glb33 in 24 different rice cultivars.

Dissipation of two field-incurred pesticides and three degradation products in rice (Oryza sativa L.) from harvest to dining table.

BACKGROUND: High levels of harmful pesticide residues in rice can cause undesirable side effects and are a source of great concern to consumers. Reduction of pesticide residues to provide rice security has thus became an urgent problem. RESULTS: In this study, the effects of commercial and home processing on removal of chlorpyrifos and carbosulfan residues from rice, and the formation of metabolites during processing, were studied. The results showed that 3,5,6-trichloro-2-pyridinol (0.87 mg kg-1 ) and carbofuran (0.43 mg kg-1 ) were the predominant components detected in paddy rice. All detected residues were primarily deposited on the rice hull and bran. Washing twice followed by high-pressure cooking was able to further decrease residues in polished rice with the processing factor value <0.25. Following application of pesticides at the recommended rate and twice the recommended rate, with a preharvest interval of 28 days, changes in residues from harvest to dining table based on efficient processing techniques were investigated. The final residues dropped to below maximum residue levels after washing twice followed by high-pressure cooking. CONCLUSION: This simple cooking process thus reduces the risk of dietary exposure, and it is recommended that it is adopted by all consumers. © 2019 Society of Chemical Industry.

Simultaneous determination of three organotin pesticides in fruits and vegetables by high-performance liquid chromatography/tandem mass spectrometry.

RATIONALE: The presence of organotins in the environment affects food safety, making it important to monitor the levels of organotin pesticides (OTPs) in fruit and vegetable samples. METHODS: In the present study, a simple and low cost method for simultaneous determination of three OTPs (azocyclotin, fenbutatin oxide and triphenyltin hydroxide) in vegetable and fruit samples was developed and validated, based on solid-phase extraction and liquid chromatography/tandem mass spectrometry. RESULTS: Extraction with acetonitrile containing 0.2% formic acid positively affected the recoveries of the three OTPs. Moreover, the simultaneous purification of the three OTPs was the most efficient using mixed-mode cation-exchange cartridges and 5.0% ammonium hydroxide in methanol as eluent, and, in this case, mild matrix effects (-9.3% to 21.6%) were obtained for the three OTPs monitored. The developed method reached limits of quantification of 1 µg kg-1 , and linearity was satisfactory, with correlation coefficients >0.995. A fortification study showed that when spiked at 1.0-50.0 µg kg-1 the mean trueness values were from 72.3 to 110.0% in all matrices (three vegetables and three fruits). The intra-day precision was <14.1%, and the inter-day precision (n = 11) was <18.2%. CONCLUSIONS: The proposed method was successfully applied to the simultaneous analysis of three OTPs in vegetables and fruits.

Premature leaf senescence 3, encoding a methyltransferase, is required for melatonin biosynthesis in rice.

Premature leaf senescence in rice is one of the most common factors affecting the plant's development and yield. Although methyltransferases are involved in diverse biological functions, their roles in rice leaf senescence have not been previously reported. In this study, we identified the premature leaf senescence 3 (pls3) mutant in rice, which led to early leaf senescence and early heading date. Further investigations revealed that premature leaf senescence was triggered by the accumulation of reactive oxygen species. Using physiological analysis, we found that chlorophyll content was reduced in the pls3 mutant leaves, while hydrogen peroxide (H2 O2 ) and malondialdehyde levels were elevated. Consistent with these findings, the pls3 mutant exhibited hypersensitivity to exogenous hydrogen peroxide. The expression of other senescence-associated genes such as Osh36 and RCCR1 was increased in the pls3 mutant. Positional cloning indicated the pls3 phenotype was the result of a mutation in OsMTS1, which encodes an O-methyltransferase in the melatonin biosynthetic pathway. Functional complementation of OsMTS1 in pls3 completely restored the wild-type phenotype. We found leaf melatonin content to be dramatically reduced in pls3, and that exogenous application of melatonin recovered the pls3 mutant's leaf senescence phenotype to levels comparable to that of wild-type rice. Moreover, overexpression of OsMTS1 in the wild-type plant increased the grain yield by 15.9%. Our results demonstrate that disruption of OsMTS1, which codes for a methyltransferase, can trigger leaf senescence as a result of decreased melatonin production.

Isolation, identification, and characterization of Ustilaginoidea virens from rice false smut balls with high ustilotoxin production potential.

Ustilaginoidea (U.) virens grows on rice grains and leads to significant rice yield losses in most of the major rice producing areas. Meanwhile, ustiloxins produced by U. virens are a serious hazard to human health and ecological safety of farmlands. The other key point is that ustiloxins have been regarded as a novel resource with their potential in the treatment of cancers. There is no better way to extract ustiloxins than from pure culture of the high ustilotoxin-producing strains. U. virens has become a key research organism. However, due to the presence of some interference components, it is a certain difficulty in the successful isolation of the strain from the false smut balls. We present here a detailed study based on the separation, screening and identification of high ustiloxins-producing strains of U. virens. Through this study, we got a satisfactory success rate of separation and provided a good solution to the problem of separation. At the same time, this study provides quality resources for researchers interested in ustiloxins as anticancer agents.

Separation of selenium species and their sensitive determination in rice samples by ion-pairing reversed-phase liquid chromatography with inductively coupled plasma tandem mass spectrometry.

A highly sensitive method was developed for the simultaneous separation and determination of organic and inorganic selenium species in rice by ion-pairing reversed-phase chromatography combined with inductively coupled plasma tandem mass spectrometry. To achieve a good separation of these species, a comparison between anion-exchange chromatography and ion-pairing reversed-phase chromatography was performed. The results indicated that ion-pairing reversed-phase chromatography was more suitable due to better separation and higher sensitivity for all analytes. In this case, a StableBond C18 column proved to be more robust or to have a better resolution than other C18 columns, when 0.5 mM tetrabutylammonium hydroxide and 10 mM ammonium acetate at pH 5.5 were used as the mobile phase. Moreover, an excellent sensitivity was obtained in terms of interferences by means of tandem mass spectrometry in the hydrogen mode. The detection limits were 0.02-0.12 µg/L, and recoveries of five selenium species were 75-114%, with relative standard deviations ≤ 9.4%. This method was successfully applied to the analysis of rice samples. Compared with previous studies, the proposed method not only gave comparable results when used for measuring selenium-enriched rice, but it can provide greater sensitivity for the detection of low concentrations of selenium species in rice.

Direct Determination of Six Cytokinin Nucleotide Monophosphates in Coconut Flesh by Reversed-Phase Liquid Chromatography-Tandem Mass Spectrometry.

Coconut contains many uncharacterized cytokinins that have important physiological effects in plants and humans. In this work, a method based on liquid chromatography-tandem mass spectrometry was developed for identification and quantification of six cytokinin nucleotide monophosphates in coconut flesh. Excellent separation was achieved using a low-coverage C18 bonded-phase column with an acidic mobile phase, which greatly improved the retention of target compounds. To enable high-throughput analysis, a single-step solid-phase extraction using mixed-mode anion-exchange cartridges was employed for sample preparation. This proved to be an effective method to minimize matrix effects and ensure high selectivity. The limits of detection varied from 0.06 to 0.3 ng/mL, and the limits of quantification ranged from 0.2 to 1.0 ng/mL. The linearity was statistically verified over 2 orders of magnitude, giving a coefficient of determination (R2) greater than 0.9981. The mean recoveries were from 81 to 108%; the intraday precision (n = 6) was less than 11%; and the interday precision (n = 11) was within 14%. The developed method was applied to the determination of cytokinin nucleotide monophosphates in coconut flesh samples, and four of them were successfully identified and quantified. The results showed that trans-zeatin riboside-5'-monophosphate was the dominant cytokinin, with a concentration of 2.7-34.2 ng/g, followed by N6-isopentenyladenosine-5'-monophosphate (≤12.9 ng/g), while the concentrations of cis-zeatin riboside-5'-monophosphate and dihydrozeatin riboside-5'-monophosphate were less than 2.2 and 4.9 ng/g, respectively.

Quantitation of glutathione S-transferases in rice (Oryza sativa L.) roots exposed to cadmium by liquid chromatography-tandem mass spectrometry using isotope-labeled wing peptides as an internal standard.

BACKGROUND: Plant glutathione S-transferases (GSTs, EC 2.5.1.18) are multifunctional enzymes involved in heavy metal cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione to heavy metals. Previous studies demonstrated that individual rice GSTs were differentially induced by heavy metal exposure at the mRNA transcript level. However, little information is available concerning changes in protein concentration of rice GSTs under heavy metal stress. Because the correlation between changes in protein concentration and gene expression under abiotic stress is poor, direct determination of rice GSTs protein concentrations during cadmium (Cd) exposure is a more effective and reliable approach to explore possible mechanisms of rice Cd translocation and accumulation. RESULTS: This study established an optimized and advanced liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based targeted proteomics assay for quantification of OsGSTF14 and OsGSTU6 proteins in Cd-stressed rice roots. The tryptic signature peptides were chosen as surrogate analytes and winged peptides containing the isotope-labeled signature peptides were used as the internal standards. The signature peptides exhibited good linearity in the range of 0.6-60 and 0.3-30 nM, respectively. The limit of detection and limit of quantification were 4.5 and 14.5 µg/g for OsGSTF14, respectively, and 2.1 and 7.0 µg/g for OsGSTU6. The spiking recoveries rates at low, medium and high levels were in the range of 72.5-93.4%, with intra- and inter-day precisions of 5.5-9.1 and 4.2-10.2%, respectively. CONCLUSIONS: The assay successfully quantified the temporal and dose responses of OsGSTF14 and OsGSTU6 proteins in Cd-stressed rice roots, with good accuracy, precision and high-throughput. This assay will have significant application in developing quantification methods of other proteins in Cd-stressed rice, which may provide more insight into the mechanisms of Cd translocation and accumulation in rice.

Nickel in milled rice (Oryza sativa L.) from the three main rice-producing regions in China.

Nickel (Ni) concentrations in milled rice obtained from China and their variations among different provinces and varieties, as well as associated health risks, were investigated. Results showed that the mean Ni concentration in milled rice was 0.49 ± 0.51 mg/kg, which was much higher than reported in United Kingdom, French and Iranian cereals. There were significant variations (P < 0.05) of Ni concentrations in milled rice among different provinces and among varieties in the same province. According to the dietary risk assessment, the mean values of the target hazard quotient for chronic risk ranged from 1.24 to 1.46 for 2-4, 4-7 and 7-11-year-old children, and all values of margin of exposure for hypersensitivity risk were considerably below 10 for all age groups, indicating that the current dietary exposure to Ni in rice is of concern for 2-11-year-old children and Ni-sensitised individuals. It is essential to establish a continuous monitoring programme to control Ni contamination in rice.

Analysis of ustiloxins in rice using polymer cation exchange cleanup followed by liquid chromatography-tandem mass spectrometry.

Ustiloxins are cyclopeptide mycotoxins produced by the pathogenic fungus Ustilaginoidea virens of rice false smut. Quantification of ustiloxins is essential to assess the food safety of rice infected by rice false smut disease. This paper describes a sensitive method for the simultaneous quantification of ustiloxins A, B, C, D and F in rice grains using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Since notable matrix enhancement effects (21%-78%) occurred for all of the target analytes (except for ustiloxin A), several solid phase extraction materials were tested for their ability to retain ustiloxins from aqueous solutions prior to the LC-MS/MS analysis, including C18 sorbents, polymer anion exchange sorbents resin (PAX), and polymer cation exchange resin (PCX). The PCX resin was adopted due to its higher extraction capability and selectivity for all targets compared to others, and in this case, almost no matrix effects (-5% to 8%) were observed for all of the ustiloxins monitored. The developed method reached limits of quantification of 0.2-2ngg-1, and linearity was statistically verified over two orders of magnitude with regression coefficients (R2)>0.991. The mean recoveries were from 85% to 109%, and the inter-day precisions (n=11) were less than 16%, with intra-day precisions (n=6) within 12%. Analysis of samples showed that ustiloxin A was the dominant species, with the content ranging from 5.5 to 273.8ngg-1, followed by ustiloxin B (≤88.7ngg-1), while concentrations of ustiloxins C, D and F were slightly lower (≤43.2ngg-1). To our knowledge, this is the first report on the determination and analysis of five ustiloxins simultaneously in a single analysis.