Singlet oxygen ( 1 O 2 ) production is associated with stress signalling. Here, using Arabidopsis as a model system, we study the effects of the accumulation of 8-hydroxyguanosine (8-oxoG), a major product of 1 O 2 -mediated RNA oxidation. We show that 8-oxoG can accumulate in vivo when 1 O 2 is produced in the cytoplasm.Conditions for such production include the application of RB in the light, dark-to-light transitions in the flu mutant, or subjecting plants to combined dehydration/light exposure. Transcriptomes of these treatments displayed a significant overlap with transcripts stimulated by the cytosolic 80S ribosomal translation inhibitors, cycloheximide and homoharringtonine. We demonstrate that 8-oxoG accumulation correlates with a decrease in RNA translatability, resulting in the rapid decrease of the levels of labile gene repressor elements such as IAA1 and JAZ1 in a proteasome-dependent manner. Indeed, genes regulated by the labile repressors of the jasmonic acid signalling pathway were induced by cycloheximide, RB or dehydration/light treatment independently of the hormone. The results suggest that 1 O 2 , by oxidizing RNA, attenuated cellular translatability and caused specific genes to be released from the repression of their cognate short half-life repressors. The findings here describe a novel means of gene regulation via the direct interaction of 1 O 2 with RNA.
Drought induces osmotic stress in roots, a condition simulated by the application of high-molecular-weight polyethylene glycol. Osmotic stress results in the reduction of Arabidopsis thaliana root growth and production of 1O2 from an unknown non-photosynthetic source. Reduced root growth can be alleviated by application of the 1O2 scavenger histidine. Here we examined the possibility that 1O2 production involves Russell reactions occurring among the enzymatic products of lipoxygenases, the fatty acid hydroperoxides. Lipoxygenase (LOX) activity was measured for purified soybean (Glycine max) LOX1 and in crude Arabidopsis root extracts using linoleic acid as substrate. Formation of the 13(S)-Hydroperoxy-9(Z),11(E)-octadecadienoic acid product was inhibited by salicylhdroxamic acid, which is a lipoxygenase inhibitor, but not by histidine, whereas 1O2 production was inhibited by both. D2O, which specifically extends the half-life of 1O2, augmented the lipoxygenase-dependent generation of 1O2, as expected from a Russell-type reaction. The addition of linoleic acid to roots stimulated 1O2 production and inhibited growth, suggesting that the availability of lipoxygenase substrate is a rate-limiting step. Indeed, water stress rapidly increased linoleic and linolenic acids by 2.5-fold in roots. Mutants with root-specific microRNA repression of lipoxygenases showed downregulation of lipoxygenase protein and activity. The lines with downregulated lipoxygenase displayed significantly less 1O2 formation, improved root growth in osmotic stress, and an altered transcriptome response compared to wild type. The results show that lipoxygenases can serve as an enzymatic source of ‘dark’ 1O2 during osmotic stress and demonstrate a role for 1O2 in defining the physiological response.
Regression bugs occur whenever software functionality that previously worked as desired stops working, or no longer works as expected. Code changes, such as bug fixes or new feature work, may result in a regression bug. Regression bugs are an annoying and painful phenomena in the software development process, requiring a great deal of effort to localize, effectively hindering team progress. In this paper we present Regression Detective, a method which assists the developer locating source code segments that caused a given regression bug. Unlike some of the existing tools, our approach doesn't require an automated test suite or executing past versions of the system. It is highly scalable to millions of loc systems. The developer, who has no prior knowledge of the code or the bug, reproduces the bug according to the steps described in the bug database. We evaluated our approach with bugs from leading open source projects (Eclipse, Tomcat, Ant). In over 90% of the cases, the developer only has to examine 10-20 lines of code in order to locate the bug, regardless of the code base size.
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