Junran Hao

Transcript and protein profiling analysis of OTA-induced cell death reveals the regulation of the toxicity response process in Arabidopsis thaliana

Ochratoxin A (OTA) is a toxic isocoumarin derivative produced by various species of mould which mainly grow on grain, coffee, and nuts. Recent studies have suggested that OTA induces cell death in plants. To investigate possible mechanisms of OTA phytotoxicity, both digital gene expression (DGE) transcriptomic and two-dimensional electrophoresis proteomic analyses were used, through which 3118 genes and 23 proteins were identified as being up- or down-regulated at least 2-fold in Arabidopsis leaf in response to OTA treatment. First, exposure of excised Arabidopsis thaliana leaves to OTA rapidly causes the hypersensitive reponse, significantly accelerates the increase of reactive oxygen species and malondialdehyde, and enhances antioxidant enzyme defence responses and xenobiotic detoxification. Secondly, OTA stimulation causes dynamic changes in transcription factors and activates the membrane transport system dramatically. Thirdly, a concomitant persistence of compromised photosynthesis and photorespiration is indicative of a metabolic shift from a highly active to a weak state. Finally, the data revealed that ethylene, salicylic acid, jasmonic acid, and mitogen-activated protein kinase signalling molecules mediate the process of toxicity caused by OTA. Profiling analyses on Arabidopsis in response to OTA will provide new insights into signalling transduction that modulates the OTA phytotoxicity mechanism, facilitate mapping of regulatory networks, and extend the ability to improve OTA tolerance in Arabidopsis.

Ochratoxin A biocontrol and biodegradation by Bacillus subtilis CW 14.

BACKGROUND Ochratoxin A (OTA) is a mycotoxin produced by some Aspergillus and Penicillium species. In this study a strain of Bacillus subtilis was tested for its effects on OTA-producing Aspergillus and OTA degradation. The mechanisms of the effects were also investigated. RESULTS A strain of Bacillus spp. isolated from fresh elk droppings was screened out using the methods described by Guan et al. (Int J Mol Sci 9:1489-1503 (2008)). The 16S rRNA gene sequence suggested that it was B. subtilis CW 14. It could inhibit the growth of the OTA-producing species Aspergillus ochraceus 3.4412 and Aspergillus carbonarius, with inhibition rates of 33.0 and 33.3% respectively. At 6 µg mL(-1) OTA, both viable and autoclaved (121 °C, 20 min) cells of CW 14 bound more than 60% of OTA. In addition, OTA was degraded by the cell-free supernatant of CW 14. By high-performance liquid chromatography, the cell-free supernatant degraded 97.6% of OTA after 24 h of incubation at 30 °C, and no degradation products were produced. The fastest degradation occurred during the first 2 h. In 3 g samples of contaminated maize, 47.1% of OTA was degraded by 50 mL inocula of overnight cultures of CW 14. CONCLUSION These findings indicated that B. subtilis CW 14 could both prevent OTA contamination and degrade OTA in crops.

Loop‐linker PCR: An advanced PCR technique for genome walking

In this article, we developed a novel PCR method, termed loop‐linker PCR, to isolate flanking sequences in transgenic crops. The novelty of this approach is its use of a stem‐loop structure to design a loop‐linker adapter. The adapter is designed to form a nick site when ligated with restricted DNA. This modification not only can prevent the self‐ligation of adapters but also promotes the elongation of the 3′ end of the loop‐linker adapter to generate a stem‐loop structure in the ligation products. Moreover, the suppressive effect of the stem‐loop structure decreases nonspecific amplification and increases the success rate of the approach; all extension products will suppress exponential amplification except from the ligation product that contains the specific primer binding site. Using this method, 442, 1830, 107, and 512 bp left border flanking sequences were obtained from the transgenic maizes LY038, DAS‐59122‐7, Event 3272, and the transgenic soybean MON89788, respectively. The experimental results demonstrated that loop‐linker PCR is an efficient, reliable, and cost‐effective method for identifying flanking sequences in transgenic crops and could be applied for other genome walking applications.

Changes in biosynthesis and metabolism of glutathione upon ochratoxin A stress in Arabidopsis thaliana

Ochratoxin A (OTA) is one of the most toxic mycotoxins, which is toxic to plants and simulates oxidative stress. Glutathione is an important antioxidant in plants and is closely associated with detoxification in cells. We have previously shown that OTA exposure induces obvious expression differences in genes associated with glutathione metabolism. To characterize glutathione metabolism and understand its role in OTA phytotoxicity, we observed the accumulation of GSH in the detached leaves of Arabidopsis thaliana under OTA treatment. OTA stimulated a defense response through enhancing glutathione-S-transferase, glutathione peroxidase, glutathione reductase activities, and the transcript levels of these enzymes were increased to maintain the total glutathione content. Moreover, the level of oxidized glutathione (GSSG) was increased and the ascorbate-glutathione cycle fluctuated in response to OTA. The depletion of glutathione using buthionine sulfoximine (BSO, inhibitor of glutamate-cysteine ligase) had no profound effect on OTA toxicity, as glutathione was regenerated through the ascorbate-glutathione cycle to maintain the total glutathione content. The ROS, MDA and GSH accumulation was significantly affected in the mutant gsh1, gr1 and gpx2 after treatment with OTA, which indicated that glutathione metabolism is directly involved in the oxidative stress response of Arabidopsis thaliana subjected to OTA. In conclusion, date demonstrate that glutathione-associated metabolism is closely related with OTA stress and glutathione play a role in resistance of Arabidopsis subjected to OTA.

iTRAQ Mitoproteome Analysis Reveals Mechanisms of Programmed Cell Death in Arabidopsis thaliana Induced by Ochratoxin A

Ochratoxin A (OTA) is one of the most common and dangerous mycotoxins in the world. Previous work indicated that OTA could elicit spontaneous HR-like lesions formation Arabidopsis thaliana, reactive oxygen species (ROS) play an important role in OTA toxicity, and their major endogenous source is mitochondria. However, there has been no evidence as to whether OTA induces directly PCD in plants until now. In this study, the presence of OTA in Arabidopsis thaliana leaves triggered accelerated respiration, increased production of mitochondrial ROS, the opening of ROS-dependent mitochondrial permeability transition pores and a decrease in mitochondrial membrane potential as well as the release of cytochrome c into the cytosol. There were 42 and 43 significantly differentially expressed proteins identified in response to exposure to OTA for 8 and 24 h, respectively, according to iTRAQ analysis. These proteins were mainly involved in perturbation of the mitochondrial electron transport chain, interfering with ATP synthesis and inducing PCD. Digital gene expression data at transcriptional level was consistent with the cell death induced by OTA being PCD. These results indicated that mitochondrial dysfunction was a prerequisite for OTA-induced PCD and the initiation and execution of PCD via a mitochondrial-mediated pathway.

Lipid Rafts Disruption Increases Ochratoxin A Cytotoxicity to Hepatocytes

Lipid rafts are microdomains in plasma membrane and can mediate cytotoxicity. In this study, the role of lipid rafts in ochratoxin A‐induced toxicity was investigated using Hepatoblastoma Cell Line HepG‐2 cells. Disruption of cholesterol‐containing lipid rafts enhanced Ochratoxin A (OTA) toxicity, as shown by increased lactate dehydrogenase leakage, increased reactive oxygen species level and reduction of superoxide dismutase activity in a time‐dependent manner. Isobaric tags for relative and absolute quantitation‐based proteomics of the cell membranes showed that nearly 85.5% proteins were downregulated by OTA, indicating that OTA inhibited the membrane protein synthesis. Most of altered proteins were involved in Gene Ontology “transport”, “cell adhesion” and “vesicle‐mediated transport”. In conclusion, lipid rafts play a key role in OTA‐induced cytotoxicity. This study provides insight into how OTA toxicity is regulated by the plasma membrane, especially the lipid rafts.

A-T linker adapter polymerase chain reaction for determining flanking sequences by rescuing inverse PCR or thermal asymmetric interlaced PCR products

The polymerase chain reaction (PCR)-based genome walking method has been extensively used to isolate unknown flanking sequences, whereas nonspecific products are always inevitable. To resolve these problems, we developed a new strategy to isolate the unknown flanking sequences by combining A-T linker adapter PCR with inverse PCR (I-PCR) or thermal asymmetric interlaced PCR (TAIL-PCR). The result showed that this method can be efficiently achieved with the flanking sequence from the Arabidopsis mutant and papain gene. Our study provides researchers with an additional method for determining genomic DNA flanking sequences to identify the target band from bulk of bands and to eliminate the cloning step for sequencing.

Effect of ochratoxin A and buthionine sulfoximine on proteome and ascorbate-glutathione cycle enzymes in Arabidopsis thaliana

In this study, proteome and activities of glutathione (GSH)-related enzymes were investigated in detached leaves of Arabidopsis thaliana treated with ochratoxin A (OTA) alone or supplemented with buthionine sulfoximine (BSO, a specific inhibitor of the first step in GSH biosynthesis). A comparative proteomic study using two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS) identified 12 differentially expressed proteins mainly involved in GSH metabolism, energy metabolism, sugar metabolism, and photosynthesis. The treatment with OTA significantly enhanced the activities of glutathione-S-transferase (GST) and glutathione reductase (GR) through up-regulating the corresponding genes (GSTF7, GR1), an the diminishing effect of BSO on them counteracted the results. However, both OTA and BSO decreased the activity of ascorbate peroxidase (APX), and OTA also decreased the monodehydroascorbate reductase (MDHAR) and glutathione peroxidase (GPX) activities. Briefly, the OTA-induced phytotoxicity to the A. thaliana detached leaves was increased slightly by addition of BSO, and the fluctuation in GSH synthesis, GSH metabolism and disorder of cellular metabolism happened.