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.

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.

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.

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.

Characterization of the phytochelatins and their derivatives in rice exposed to cadmium based on high-performance liquid chromatography coupled with data-dependent hybrid linear ion trap orbitrap mass spectrometry.

RATIONALE: The identification and quantification of phytochelatins (PCs) and their derivatives are important to understand their roles in plant growth and development. A method couplling high-performance liquid chromatography with hybrid linear ion trap Orbitrap mass spectrometry (HPLC-LTQ/Orbitrap) was developed to screen PCs that have the same characteristic product ions. This approach was used for the fragmentation pattern analysis of glutathione (GSH) and PC standards, which allowed identification of the fragmentation pathways of their derivatives isolated from rice roots, stems and leaves. METHODS: In this study, we developed a method to detect and identify PCs and their derivatives in rice based on HPLC/LTQ-Orbitrap. Spectrum interpretation and MS/MS fragmentation patterns of PCs provide sufficient information to discover the novel PC derivatives. This approach includes precursor ion scan and product ion scan to detect and character the novel PC derivatives. RESULTS: Based on HCD-MS/MS fragmentation patterns, four PCs and 18 PC derivatives were identified. Among them, seven PC derivatives, i.e., iso-PC2 (Asn), iso-PC3 (Asn), iso-PC2 (Cys), des-γGlu-iso-PC3 (Ser), des-Cys-iso-PC2 (Glu), des-Cys-iso-PC3 (Glu) and des-Cys-iso-PC4 (Glu), have not been previously reported. This method was validated by profiling GSH, PCs and PC derivatives in rice. Preliminary results revealed that PCs and their derivatives, except GSH, are markedly induced by Cd treatment. CONCLUSIONS: The HPLC/LTQ-Orbitrap method was successfully developed for the identification of PCs and their derivatives. The C-terminal linked to Gly is replaced with Glu, Ser, Asn, Gln or Cys, thereby creating a family of chemicals that share several structural properties. This technique could be particularly useful for investigators studying plant metabolomics. Copyright © 2016 John Wiley & Sons, Ltd.

Profiling of phytohormones and their major metabolites in rice using binary solid-phase extraction and liquid chromatography-triple quadrupole mass spectrometry.

A high-throughput method was developed using liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) for the profiling and quantification of 43 phytohormones and their major metabolites, including auxins, abscisic acid, jasmonic acid, salicylic acid, cytokinins and gibberellins in a single sample extract. Considerable matrix effects (MEs) were observed (with most ME values in the range of 29%-84%, but maximum MEs of more than 115%, even up to 206%, existed) in sample extracts for most of the compounds studied. The application of the proposed binary solid-phase extraction using polymer anion and polymer cation exchange resins, was performed to purify 25 acidic and 18 alkaline phytohormones and their major metabolites prior to the LC-MS/MS analysis, which markedly reduced the MEs to acceptable levels, with ME values in the range of ±15%. Moreover, all of the isomers of cytokinins and their metabolites were fully separated on a sub-2µm particle C18 reverse-phase column with the optimized mobile phase consisting of methanol and 5mM ammonium formate. The method showed good linearity for all 43 analytes with regression coefficients (R(2))>0.991. Limits of detection ranged from 0.19 to 7.57 fmol for auxin, gibberellins, abscisic acid and their metabolites, 29.7 fmol for jasmonic acid, 18.1 fmol for salicylic acid, and from 0.03 to 0.31 fmol for cytokinins and their metabolites. The mean recoveries for all of the analytes were from 70.7 to 118.5%, and the inter-day precisions (n=6) were less than 18.7%, with intra-day precisions (n=6) within 25.4%. Finally, 20 compounds were successfully quantified in rice sample profiles using the proposed method, which will greatly facilitate the understanding of hormone-related regulatory networks that influence rice growth and development. To our knowledge, there are limited reports that measure this level of phytohormone species in rice samples using a single analysis.

A novel method for the simultaneous analysis of seven biothiols in rice (Oryza sativa L.) using hydrophilic interaction chromatography coupled with electrospray tandem mass spectrometry.

Analysis of biothiols is still problematic, due to their high polarity, oxidation sensitivity and time-consuming sample preparation. In this paper, a direct, rapid and sensitive method was developed for simultaneous quantification of unbound cysteine (Cys), glutathione (GSH) and phytochelatins (PCs) in rice leaf, stem and root samples by hydrophilic interaction chromatography coupled with electrospray tandem mass spectrometry (HILIC-MS/MS). Homogenized samples were extracted with water containing 50mM dithiothreitol, without derivatization and further clean-up, and the extracts were injected directly onto an Xbridge Amide-HILIC column (3.5µm, 150mm×2.1mm i.d.). The best chromatographic separation and MS sensitivity was achieved using a linear gradient elution with 10mM aqueous ammonium formate and acetonitrile as the mobile phase. In MS/MS mode the detection limit (S/N≥3) of seven biothiols was 3-105nM. Good linearities were observed (r>0.995) with linear dynamic range at least over three orders of magnitude. Recoveries for most analytes were within the range of 77-128%, with relative standard deviations less than 18.2%. The intra-day precision (n=7) was 6.1-11.7%, and the inter-day precision over 15 d (n=15) was 8.5-16.3% for all biothiols. The optimized HILIC-MS/MS method was applied to study the influence of different cadmium (Cd) concentrations (0, 1 and 50µM) on contents of Cys, GSH and PC2-6 in rice tissue. With increasing Cd concentrations in nutrient solutions, contents of PC2-4 in rice roots increased but contents of Cys and GSH decreased. Contents of PC2-4 in both rice leafs and stems increased markedly at high dose Cd (50µM) treatment compared with controls, compared with low Cd concentrations (1µM). However, both PC5 and PC6 were not detected throughout the stress experiment.

Simultaneous determination of triazine herbicides in rice by high-performance liquid chromatography coupled with high resolution and high mass accuracy hybrid linear ion trap-orbitrap mass spectrometry.

A method was developed for the simultaneous determination of 10 triazine herbicides (cyanazine, simazine, simetryn, metribuzin, atrazine, ametryn, terbuthylazine, prometryn, terbutryn, and dimethametryn) in rice samples by high resolution and high mass accuracy hybrid linear ion trap-Orbitrap mass spectrometer. After extraction with acetonitrile and evaporation, the herbicides were redissolved in n-hexane and purified on a Florisil solid-phase extraction column. All compounds were separated within 12 min, producing more than 11 data points for each herbicide and high mass accuracy quantified ions which the mass errors of absolute value were less than 1.9 ppm in pure solution and 2.1 ppm in the matrix-matched standards solution. The method was validated in terms of the limits of detection and the limits of quantification. The linearity was satisfactory, with a correlation coefficient of >0.9975. Precision and recovery studies were evaluated at three concentration levels for Japonica, Indica, and Glutinous rice matrix. The mean recoveries obtained for all analytes in spiked Xiushui 03, Liangyoupeijiu, and Taihunuo rice samples were 83.3-99.0%, 82.0-99.7%, and 84.2-99.4%, respectively, with relative standard deviation in range 1.7-10.6%, 1.2-10.7%, and 1.9-11.6% for spiked rice samples, respectively. The intra-day precision (n=5) for the 10 herbicides in rice samples spiked at an intermediate level was between 2.8% and 7.9%, and the inter-day precision over 10 days (n=10) was between 5.5% and 15.9%.

Simultaneous determination of 15 phenylurea herbicides in rice and corn using HPLC with fluorescence detection combined with UV decomposition and post-column derivatization.

A method was developed for the simultaneous determination of 15 phenylurea herbicides (fenuron, tebuthiuron, metoxuron, monuron, chlortoluron, fluometuron, isoproturon, diuron, monolinuron, metobromuron, buturon, siduron, linuron, chlorbromuron, and neburon) in rice and corn samples by HPLC with fluorescence detection combined with UV decomposition and post-column derivatization. After extraction with acetonitrile and evaporation, the herbicides were redissolved in n-hexane and purified on a Florisil solid-phase extraction column. HPLC separation was carried out on a C18 column with water-acetonitrile gradient elution. UV decomposition was carried out under a 254-nm UV lamp. The method was evaluated in terms of the limits of detection and quantification. The linearity was satisfactory, with a correlation coefficient of >0.9980. Precision and recovery studies were evaluated at three concentration levels for each matrix. Good precision was obtained, with relative standard deviation in the range 1.5-9.6% for spiked rice samples and 0.9-9.9% for spiked corn samples. Recovery (n=6) ranged between 75.3% and 104.3% for rice and between 75.0% and 105.1% for corn. The intra-day precision (n=5) for the 15 herbicides in rice and corn samples spiked at an intermediate level was between 1.5% and 7.1%, and the inter-day precision over 10 days (n=10) was between 6.4% and 15.6%.

[Determination of trace mercury in rice by microwave digestion-hydride generation-atomic fluorescence spectrometry].

It was reported in this paper that trace mercury in rice was determined by microwave digestion-hydride generation-atomic fluorescence spectrometry. The microwave digestion of samples was used, and the optimum conditions for the digestion were developed. The relative factors of hydride generation atomic fluorescence spectrometry for the determination were tested and discussed. The temperature and the relative humidity were discussed too. The detection limit was 0.005 ng x mL(-1). Mercury in reference materials (rice flour, GBW 08508) was determined also by the described method. The result obtained was in good agreement with standard value. The relative standard deviation (RSD) was 2.1%, and the recovery of mercury in rice samples was 95.2%-106.4%.