Isolation and characterization of Arabidopsis mutants with enhanced tolerance to oxidative stress

Abstract

We have previously reported a method for isolation of mutants with enhanced tolerance to the fungal AAL toxin and given a detailed characterization of atr1 (AAL toxin resistant, Gechev et al. in Biochem Biophys Res Commun 375:639-644, 2008). Herewith, we report eight more mutants with enhanced tolerance to the AAL toxin. Phenotypic analysis showed that six of the mutants were reduced in size compared with their original background loh2. Furthermore, atr2 showed delayed flowering and senescence. The mutants were also evaluated for oxidative stress tolerance by growing them on ROS-inducing media supplemented with either aminotriazole or paraquat, generating, respectively, H 2 O 2 or superoxide radicals. Oxidative stress, confirmed by induction of the marker genes, HIGH AFFINITY NITRATE TRANSPORTER At1G08090 and HEAT SHOCK PROTEIN 17 At3G46230, inhibited growth of all lines. However, while the original background loh2 developed necrotic lesions and died rapidly on ROS-inducing plant growth media, atr1, atr2, atr7 and atr9 remained green and viable. The tolerance against oxidative stress-induced cell death was confirmed by fresh weight and chlorophyll measurements. Real-time PCR analysis revealed that the expression of the EXTENSIN gene At5G46890, previously shown to be downregulated by aminotriazole in atr1, was repressed in all lines, consistent with the growth inhibition induced by oxidative stress. Taken together, the data indicate a complex link between growth, development and oxidative stress tolerance and indicates that growth inhibition can be uncoupled from oxidative stress-induced cell death.

Oxidative stress provokes distinct transcriptional responses in the stress-tolerant atr7 and stress-sensitive loh2 Arabidopsis thaliana mutants as revealed by multi-parallel quantitative real-time PCR analysis of ROS marker and antioxidant genes

“…When grown on PQ, loh2 seedlings were unable to fully develop their cotyledons, while when grown on AT they expanded their cotyledons but then started yellowing and eventually died without developing any further. The oxidative stress-induced growth inhibition is apparently a common response of all plants and also occurs in atr mutants, as evidenced by their reduction of fresh weight and repression of growth-related genes [11,25]. However, the atr7 mutant eventually overcomes the oxidative stresses and Loh2 and atr7 plants were grown on MS media or MS supplemented with 7 mM AT or 1 mM PQ.…”

“…In order to find mutants perturbed in oxidative stressinduced cell death, a large-scale screen of chemically mutagenized loh2 seeds was carried out. The screening recovered nine atr (AAL-toxin resistant) mutants, five of which were more tolerant to AT-and PQinduced PCD, with one of them, atr7, exhibiting the strongest oxidative stress tolerance [11]. The atr7 mutant tends to have slightly less biomass expressed by fresh weight.…”

“…Some of the other atr mutants have more striking phenotypes in absence of stress. For example, atr2 and atr6 are much smaller than both loh2 and atr7; furthermore, atr2 has delayed senescence and atr6 has altered leaf shape [11]. Crosses between the different atr lines and rough mapping indicated that the mutations in all these lines are in different genes.…”

Molecular mechanisms of desiccation tolerance in the resurrection glacial relic Haberlea rhodopensis

“…RNA isolation, next-generation sequencing (NGS), and quantitative RT-PCR Four total-RNA samples were prepared for mRNA sequencing using the TriZOL method of RNA isolation followed by DNAse treatment [25]. The concentration of the samples was analyzed with a Nanodrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA).…”

Role of Reactive Oxygen Species and Contribution of New Players in Defense Mechanism under Drought Stress in Rice

“…Compared to APX, which is present in different cellular organelles, CAT is present only in peroxisomes and removes excessive amount of ROS generated under stress conditions providing a clue of variation in its activity (de Carvalho, 2008). However, CAT activity can be inhibited by herbicides induced by ROS like methyl-viologen/paraquat (PQ) and 3-aminotriazole (AT) (Gechev et al, 2008;Qureshi et al, 2011). The rice APX plays an important role in development especially at seedling and reproductive stage by scavenging H2O2 under drought and some of the other abiotic stress (Zhang et al, 2013).…”