Joo Hee Chung

GDSL lipase-like 1 regulates systemic resistance associated with ethylene signaling in Arabidopsis

Systemic resistance is induced by necrotizing pathogenic microbes and non-pathogenic rhizobacteria and confers protection against a broad range of pathogens. Here we show that Arabidopsis GDSL LIPASE-LIKE 1 (GLIP1) plays an important role in plant immunity, eliciting both local and systemic resistance in plants. GLIP1 functions independently of salicylic acid but requires ethylene signaling. Enhancement of GLIP1 expression in plants increases resistance to pathogens including Alternaria brassicicola, Erwinia carotovora and Pseudomonas syringae, and limits their growth at the infection site. Furthermore, local treatment with GLIP1 proteins is sufficient for the activation of systemic resistance, inducing both resistance gene expression and pathogen resistance in systemic leaves. The PDF1.2-inducing activity accumulates in petiole exudates in a GLIP1-dependent manner and is fractionated in the size range of less than 10 kDa as determined by size exclusion chromatography. Our results demonstrate that GLIP1-elicited systemic resistance is dependent on ethylene signaling and provide evidence that GLIP1 may mediate the production of a systemic signaling molecule(s).

GDSL LIPASE1 Modulates Plant Immunity through Feedback Regulation of Ethylene Signaling

A GDSL lipase regulates ethylene-dependent pathogen resistance and ethylene-salicylic acid signaling interactions in plants. Ethylene is a key signal in the regulation of plant defense responses. It is required for the expression and function of GDSL LIPASE1 (GLIP1) in Arabidopsis (Arabidopsis thaliana), which plays an important role in plant immunity. Here, we explore molecular mechanisms underlying the relationship between GLIP1 and ethylene signaling by an epistatic analysis of ethylene response mutants and GLIP1-overexpressing (35S:GLIP1) plants. We show that GLIP1 expression is regulated by ethylene signaling components and, further, that GLIP1 expression or application of petiole exudates from 35S:GLIP1 plants affects ethylene signaling both positively and negatively, leading to ETHYLENE RESPONSE FACTOR1 activation and ETHYLENE INSENSITIVE3 (EIN3) down-regulation, respectively. Additionally, 35S:GLIP1 plants or their exudates increase the expression of the salicylic acid biosynthesis gene SALICYLIC ACID INDUCTION-DEFICIENT2, known to be inhibited by EIN3 and EIN3-LIKE1. These results suggest that GLIP1 regulates plant immunity through positive and negative feedback regulation of ethylene signaling, and this is mediated by its activity to accumulate a systemic signal(s) in the phloem. We propose a model explaining how GLIP1 regulates the fine-tuning of ethylene signaling and ethylene-salicylic acid cross talk.

Nontargeted Metabolite Profiling in Compatible Pathogen-Inoculated Tobacco (Nicotiana tabacum L. cv. Wisconsin 38) Using UPLC-Q-TOF/MS

A biphasic reactive oxygen species (ROS) production has previously been observed in tobacco at 1 and 48 h after inoculation with the hemibiotrophic compatible pathogen, Phytophthora parasitica var. nicotianae (Ppn). To characterize the response of tobacco to biphasically produced ROS concerning the propagation of Ppn, ultraperformance liquid chromatography-quadrupole-time of flight/ mass spectrometry (UPLC-Q-TOF/MS) based metabolic profiling combined with multivariate statistical analysis was performed. Among the nonredundant 355 mass ions in ESI+ mode and 345 mass ions in ESI- mode that were selected as significantly changed by Ppn inoculation (|p(corr)| > 0.6 on S-plot of orthogonal partial least-squares discriminant analysis (OPLS-DA), fold-change > 2, and p < 0.05 in the independent two-sample t test), 76 mass ions were identified on the basis of their accurate mass ions and MS/MS spectra. Phenolic amino acids, phenylpropanoids, hydroxycinnamic acid amides, linoleic acid, linolenic acid, lysophospholipids, glycoglycerolipids, and trioxidized phospholipids were identified as having changed after Ppn inoculation. On the basis of their quantitative changes, the metabolic responses occurring at each phase of ROS production after Ppn inoculation were investigated in this study.

GDSL lipase 1 regulates ethylene signaling and ethylene-associated systemic immunity in Arabidopsis

Arabidopsis GDSL lipase 1 (GLIP1) has been shown to modulate systemic immunity through the regulation of ethylene signaling components. Here we demonstrate that the constitutive triple response mutant ctr1‐1 requires GLIP1 for the ethylene response, gene expression, and pathogen resistance. The glip1‐1 mutant was defective in induced resistance following primary inoculation of necrotrophic pathogens, whereas GLIP1‐overexpressing plants showed resistance to multiple pathogens. Necrotrophic infection triggered the downregulation of EIN3 and the activation of ERF1 and SID2 in a GLIP1‐dependent manner. These results suggest that GLIP1 positively and negatively regulates ethylene signaling, resulting in an ethylene‐associated, necrotroph‐induced immune response.

Proteomic and biochemical analyses reveal the activation of unfolded protein response, ERK-1/2 and ribosomal protein S6 signaling in experimental autoimmune myocarditis rat model

BackgroundTo investigate the molecular and cellular pathogenesis underlying myocarditis, we used an experimental autoimmune myocarditis (EAM)-induced heart failure rat model that represents T cell mediated postinflammatory heart disorders.ResultsBy performing unbiased 2-dimensional electrophoresis of protein extracts from control rat heart tissues and EAM rat heart tissues, followed by nano-HPLC-ESI-QIT-MS, 67 proteins were identified from 71 spots that exhibited significantly altered expression levels. The majority of up-regulated proteins were confidently associated with unfolded protein responses (UPR), while the majority of down-regulated proteins were involved with the generation of precursor metabolites and energy metabolism in mitochondria. Although there was no difference in AKT signaling between EAM rat heart tissues and control rat heart tissues, the amounts and activities of extracellular signal-regulated kinase (ERK)-1/2 and ribosomal protein S6 (rpS6) were significantly increased. By comparing our data with the previously reported myocardial proteome of the Coxsackie viruses of group B (CVB)-mediated myocarditis model, we found that UPR-related proteins were commonly up-regulated in two murine myocarditis models. Even though only two out of 29 down-regulated proteins in EAM rat heart tissues were also dysregulated in CVB-infected rat heart tissues, other proteins known to be involved with the generation of precursor metabolites and energy metabolism in mitochondria were also dysregulated in CVB-mediated myocarditis rat heart tissues, suggesting that impairment of mitochondrial functions may be a common underlying mechanism of the two murine myocarditis models.ConclusionsUPR, ERK-1/2 and S6RP signaling were activated in both EAM- and CVB-induced myocarditis murine models. Thus, the conserved components of signaling pathways in two murine models of acute myocarditis could be targets for developing new therapeutic drugs or methods aimed at treating enigmatic myocarditis.

Quantitative analysis of heterocyclic amines in urine by liquid chromatography coupled with tandem mass spectrometry

A sensitive, reproducible, and rapid analytical method for the analysis of trace-level heterocyclic amines (HCAs) that are expected to have high levels of human exposure was developed. Liquid-liquid extraction (LLE) with dichloromethane (DCM) followed by solid-phase extraction (SPE) was carried out. Liquid extraction with DCM under basic conditions was efficient in extracting HCAs from urine samples. For further purification, mixed mode cationic exchange (MCX) cartridges were applied to eliminate the remaining interferences after liquid extraction. Separation and quantification were performed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in selected reaction monitoring (SRM) mode. The overall recoveries ranged between 71.0% and 113.6% with relative standard deviations (RSDs) of 5.1% to 14.7% for the entire procedure. The limits of detection (LODs) and limits of quantification (LOQs) of the proposed analytical method were in the ranges of 0.04 to 0.10 ng/ml and 0.15 to 0.36 ng/ml, respectively. This method was applied to the analysis of monitoring in urine samples for Korean school children, and the results demonstrated that the method can be used for the trace determination of HCAs in urine samples.

Metabolic survey of defense responses to a compatible hemibiotroph, Phytophthora parasitica var. nicotianae, in ethylene signaling-impaired tobacco.

Reactive oxygen species (ROS) and ethylene play an important role in determining the resistance or susceptibility of plants to pathogen attack. A previous study of the response of tobacco cultivar ( Nicotiana tabacum L. cv. Wisconsin 38) to a compatible hemibiotroph, Phytophthora parasitica var. nicotianae (Ppn) showed that biphasic bursts of ROS and ethylene are positively associated with disease severity. The levels of ethylene and ROS might influence the susceptibility of plants to pathogens, with changing levels of metabolite related to disease resistance or susceptibility. In this study, to obtain more detailed information on the interaction of ROS and ethylene signaling related to resistance and/or susceptibility of plants to pathogen, Ppn-induced metabolic profiles from wild type (WT) and ethylene signaling-impaired transgenic plants that expressed Ein3 antisense (Ein3-AS) were compared using ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). Nonredundant mass ions (576 in ESI+ mode and 336 in ESI- mode) were selected, and 56 mass ions were identified on the basis of their accurate mass ions and MS/MS spectra. Two-way hierarchical clustering analysis of the selected mass ions revealed that nicotine and phenylpropanoid-polyamine conjugates, such as caffeoyl-dihydrocaffeoyl-spermidine, dicaffeoyl-spermidine, caffeoyl-feruloyl-spermidine, and two bis(dihydrocaffeoyl)-spermine isomers, and their intermediates, such as arginine and putrecine, were present at lower levels in Ein3-AS transgenic plants during Ppn interaction than in WT, whereas galactolipid and oxidized free fatty acid levels were higher in Ein3-AS transgenic plants. Taken together, these results reveal a function for ethylene signaling in tobacco defense responses during Ppn interaction.

Inter-Laboratory Validation of Method to Determine Residual Enrofloxacin in Chicken Meat

An inter-laboratory study was performed to evaluate the performance of a method developed for the quantification of enrofloxacin in chicken meat. Liquid-liquid extraction combined with a clean-up procedure based on solid-phase extraction followed by a liquid chromatography-tandem mass spectrometric method was used by three individual laboratories. All the investigated results of calibration curves and limits of quantification were within the acceptable range for regulatory testing of enrofloxacin. The three laboratories received blind a certified reference material to analyze in triplicate and assess using statistical analysis. From the results, no statistical differences were found between the laboratories in the precision of the method. Additionally, all the results of the z-score, which is an indication of fixed interval bias criteria for accuracy from the laboratories, fell within the allowable limits (±2σ). Based on this proficiency testing by inter-laboratory comparisons, the analytical method including the sample preparation step was proven to be applicable.

Development of chemical ionization method in a GC-TOF mass spectrometer for accurate mass and isotope ratio measurement

An accurate mass and isotope ratio were determined using a gas chromatography/time of flight mass spectrometer in CI positive mode for the identification of unknown metabolites. High mass tune was used to improve the ion intensity of . Chromatographic resolution and dynamic range enhancement were performed to obtain more reliable accurate masses and correct isotope abundance ratios. Average absolute errors of mass and isotope ratios for 24 reference metabolite -TMS (trimethylsilyl) derivatives were 6.8 ppm, 1.5% of (M+1/M ratio) and 1.7% of (M+2/M ratio), respectively. The correct formulas of twenty one compound were retrieved within top-2 hit from the heuristic algorithm for elemental composition using each accurate mass and isotope abundance ratio.