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Gechev T.,Plovdiv University | Gechev T.,Institute of Molecular Biology and Biotechnologies | Mehterov N.,Plovdiv University | Mehterov N.,Institute of Molecular Biology and Biotechnologies | And 2 more authors.
Methods in Enzymology | Year: 2013

A genetic approach is described to isolate mutants more tolerant to oxidative stress. A collection of T-DNA activation tag Arabidopsis thaliana mutant lines was screened for survivors under conditions that trigger H 2O2-induced cell death. Oxidative stress was induced by applying the catalase (CAT) inhibitor aminotriazole (AT) in the growth media, which results in decrease in CAT enzyme activity, H2O2 accumulation, and subsequent plant death. One mutant was recovered from the screening and named oxr1 (oxidative stress resistant 1). The location of the T-DNA insertion was identified by TAIL-PCR. Oxr1 exhibited lack of cell death symptoms and more fresh weight and chlorophyll content compared to wild type. The lack of cell death correlated with more prominent induction of anthocyanins synthesis in oxr1. These results demonstrate the feasibility of AT as a screening agent for the isolation of oxidative stress-tolerant mutants and indicate a possible protective role for anthocyanins against AT-induced cell death. The chapter includes protocols for ethyl methanesulfonate mutagenesis, mutant screening using AT, T-DNA identification by TAIL-PCR, CAT activity measurements, and determination of malondialdehyde, chlorophyll, and anthocyanins. © 2013 Elsevier Inc.

Qureshi M.K.,University of Groningen | Sujeeth N.,University of Groningen | Sujeeth N.,Plovdiv University | Gechev T.S.,Plovdiv University | And 2 more authors.
Acta Physiologiae Plantarum | Year: 2013

Plants use programmed cell death (PCD) as a tool in their growth and development. PCD is also involved in defense against different kinds of stresses including pathogen attack. In both types of PCD, reactive oxygen species (ROS) play an important role. ROS is not only a toxic by-product but also acts as signaling molecule that can trigger defense mechanisms in plant. Earlier transcriptome studies indicated the activation of ROS responsive and/or generating genes. Eight genes were selected with a potential role in ROS-induced PCD pathway and one of them, encoding the zinc finger protein ZAT11 (Zinc Arabidopsis thaliana11) exhibited an altered cell death phenotype. Two independent zat11 mutants in a loh2 (LAG one homologue2) genetic background showed enhanced tolerance to paraquat-induced oxidative stress and PCD; whereas, these double mutants exhibited cell death triggered by Alternaria alternata f. sp. lycopersici-toxin (AAL-toxin) or 3-aminotriazole (AT). This indicates that ZAT11 is involved in an intricate oxidative stress-induced PCD network, and the final outcome depends on ZAT11 interactions with other players specific for the particular types of oxidative stress. © 2013 Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków.

Petrov V.,Plovdiv University | Petrov V.,Institute of Molecular Biology and Biotechnologies | Schippers J.,Max Planck Institute of Molecular Plant Physiology | Schippers J.,University of Potsdam | And 8 more authors.
Plant OMICS | Year: 2013

The ability of some chemical compounds to cause oxidative stress offers a fast and convenient way to study the responses of plants to reactive oxygen species (ROS). In order to unveil potential novel genetic players of the ROS-regulatory network, a population of ~2,000 randomly selected Arabidopsis thaliana T-DNA insertion mutants was screened for ROS sensitivity/resistance by growing seedlings on agar medium supplemented with stress-inducing concentrations of the superoxide-eliciting herbicide methyl viologen or the catalase inhibitor 3-amino-triazole. A semi-robotic setup was used to capture and analyze images of the chemically treated seedlings which helped interpret the screening results by providing quantitative information on seedling area and healthy-to-chlorotic tissue ratios for data verification. A ROS-related phenotype was confirmed in three of the initially selected 33 mutant candidates, which carry T-DNA insertions in genes encoding a Ring/Ubox superfamily protein, ABI5 binding protein 1 (AFP1), previously reported to be involved in ABA signaling, and a protein of unknown function, respectively. In addition, we identified six mutants, most of which have not been described yet, that are related to growth or chloroplast development and show defects in a ROS-independent manner. Thus, semi-automated image capturing and phenotyping applied on publically available T-DNA insertion collections adds a simple means for discovering novel mutants in complex physiological processes and identifying the genes involved.

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