Methyl Viologen-induced Oxidative Stress Research Articles

Isolation and characterization of the Arabidopsis heat‐intolerant 2 (hit2) mutant reveal the essential role of the nuclear export receptor EXPORTIN1A (XPO1A) in plant heat tolerance

*The Arabidopsis heat-intolerant 2 (hit2) mutant was isolated on the basis of its impaired ability to withstand moderate heat stress (37 degrees C). Determination of the genetic mutation that underlies the hit2 thermosensitive phenotype allowed better understanding of the mechanisms by which plants cope with heat stress. *Genetic analysis revealed that hit2 is a single recessive mutation. Map-based cloning was used to identify the hit2 locus. The response of hit2 to other types of heat stress was also investigated to characterize the protective role of HIT2. *hit2 was defective in basal but not in acquired thermotolerance. hit2 was sensitive to methyl viologen-induced oxidative stress, and the survival of hit2 seedlings in response to heat stress was affected by light conditions. The mutated locus was located at the EXPORTIN1A (XPO1A) gene, which encodes a nuclear transport receptor. Two T-DNA insertion lines, xpo1a-1 and xpo1a-3, exhibited the same phenotypes as hit2. *The results provide evidence that Arabidopsis XPO1A is dispensable for normal plant growth and development but is essential for thermotolerance, in part by mediating the protection of plants against heat-induced oxidative stress.

Silencing approach using Poly (ADP-ribose) polymerase gene to improve drought stress tolerance in maize

Current and predicted climatic conditions, such as prolonged drought and heat episodes affect plant growth and yield, cause annual losses estimated at billions of dollars and pose a serious challenge for agricultural production worldwide, (Boyer 1982, Mittler 2006). Progress in generating transgenic crops with enhanced tolerance to abiotic stress has nevertheless been slow. The complex field environment with its heterogenic conditions, abiotic stress combinations, and global climatic changes are but a few of the challenges of modern agriculture. A combination of approaches has contributed significantly towards improving abiotic stress tolerance in both the greenhouse and the field (Hirayama and Shinozaki 2010). However, the increasing unpredictability and vagaries of rainfall and temperature variations consequent to global climate change, the dwindling availability of irrigation water and phosphate fertilizer, the escalating population in developing countries indicate that new ways of improving maize yield stability and stress tolerance under suboptimal conditions must be found and employed. A novel strategy to improve abiotic stress tolerance in Arabidopsis and Brassica has been reported by De Block et al., (2005). In their study, poly(ADP-ribosyl) ation activity was downregulated and transgenic plants became tolerant to a broad range of abiotic stresses such as drought, high light and heat. The researchers noted that stress tolerance was obtained by maintaining energy homeostasis due to reduced stress-induced energy consumption by prevention of NADP breakdown. Under abiotic stress conditions, plants overexpressing hairpin RNAi against Arabidopsis APP gene preserve their energy homeostasis without an overactivation of the mitochondrial respiration and avoiding the production of reactive oxygen species. Hence, plants with a lowered PARP activity appear tolerant to multiple stresses. Furthermore, a genome-wide transcript analysis of stressed anti PARP2 transgenic Arabidopsis (hpAt-PARP2) plants revealed that the induction of specific ABA signalling pathways steered increased levels of the cyclic nucleotide ADP-ribose (cADPR) thereby contributing towards tolerance to abiotic stresses (Vanderauwera et al., 2007). Indeed, modulation of poly(ADPribosyl) ation (PAR) reaction by an Arabidopsis thaliana ADP-ribose/NADH pyrophosphohydrolase, AtNUDX7 led to plants becoming tolerant to oxidative stress (Ogawa et al., 2009). Under oxidative stress, AtNUDX7 serves to maintain NAD+ levels by supplying ATP via nucleotide recycling from free ADP-ribose molecules and thus regulates the defence mechanisms against oxidative DNA damage via modulation of the PAR reaction. Furthermore, energy use efficiency is characterized by an epigenetic component that can be directed through selection to increase yield on top of heterosis (Hauben et al., 2009). Arabidopsis findings have previously been found to be directly applicable to commercial crop improvement (Nelson et al., 2005). Therefore, engineering crop plants for high NAD+ regeneration by an efficient 8 upregulation of the NAD+ salvage pathway or by a reduced NAD+ consumption under stress conditions, is a valuable approach to enhance overall stress tolerance in crops. The aim of this study was to assess a similar approach in the model species Zea mays through silencing maize PARP1 gene, a homolog of the Arabidopsis APP gene. This was achieved by establishing tropical maize regeneration system from immature embryo through somatic embryogenesis as a prerequisite for effective genetic transformation mediated by Agrobacterium. Our next goal was to engineer two hairpin constructs targeting maize PARP1 gene within the specific region at the 5’-end of the gene because silencing of the Arabidopsis APP gene using hairpin RNA constructs proved to be a valuable approach to obtain drought tolerance in Arabidopsis and canola (De Block et al., 2005). However, hairpin constructs are not so stable in extreme temperatures in the greenhouse which results in problems with stability in subsequent progenies (Szittya et al., 2003). Therefore, we aimed at evaluating the utility of artificial micro- RNAs (amiRNA) to silence the maize PARP1 gene. This technology is expected to be more effective and stable than the hairpin RNA approach because temperature does not affect the accumulation of microRNA (Szittya et al., 2003). Three amiRNAs were designed targeting maize PARP1 gene. As a complementary approach to the amiRNA and RNAi transgene technology we sought for mutants in the maize PARP1 gene in the Uniform Mu collections mutagenized by the transposon Mutator. Transgene technology and efficient transformation are important to fully exploit a species as a model for functional genomics studies. We developed an efficient and standardized maize transformation method based on the following criteria: (1) Agrobacterium tumefaciens co-cultivation was preferred over particle bombardment because usually intact T-DNA’s are transferred at low copy number, (2) a maize inbred line was favoured over a hybrid line because homogeneous progenies allow transgene testing already in T1 or T2, (3) Gateway vectors were optimized for use in monocots and provide a toolbox for rapid gene cloning. And finally, the transgenic maize plants overexpressing amiRNA constructs against maize PARP1 gene were evaluated for drought and Methyl Viologen-induced oxidative stress tolerance.

Molecular cloning and characterization of genes encoding Pennisetum glaucum ascorbate peroxidase and heat-shock factor: Interlinking oxidative and heat-stress responses

The recent genetic and biochemical studies reveal a considerable overlap among cellular processes in response to heat and oxidative stress stimuli in plants suggesting an intimate relationship between the heat-shock response and oxidative stress responses. Pennisetum glaucum (Pg) seedlings were exposed to heat stress (42 degrees C for 0.5, 1.0 and 24h) and a mixture of RNA from all the heat stressed seedlings was used to prepare cDNA. Full-length cDNA clones encoding for cytoplasmic ascorbate peroxidase 1 (PgAPX1) and heat-shock factor (PgHSF) were isolated by screening heat stress-specific cDNA library using corresponding EST sequences as radioactive probes. These full-length cDNAs were expressed in E. coli and their recombinant proteins were purified to near homogeneity. The recombinant PgAPX1 preferred ascorbate but did not accept guaiacol as a reducing substrate. Over-expression of PgAPX1 protects E. coli cells against methyl viologen-induced oxidative stress. Sequence analysis of PgAPX1 promoter identified a number of putative stress regulatory cis-elements including a heat-shock element (HSE). Heat-shock transcription factors (HSFs) play a central role in mediating these overlapping cellular processes. Gel shift analysis and competition with specific and non-specific unlabeled DNA probes showed a specific interaction between HSE of PgAPX1 and the PgHSF protein. Expression analysis of PgHSF in Pennisetum showed maximum increase in transcript level in response to heat stress within 30 min of exposure and slowed down at subsequent time points of heat stress, indicating a typical characteristic of HSF in terms of early responsiveness. Expression of PgAPX1 significantly increased under heat-stress condition; however, the maximum expression observed at 24h of heat stress. In gel activity of PgAPX1 in Pennisetum plants also showed an increase in response to heat stress (42 degrees C) being maximum at 24h and these trends are in conformity with the expression pattern of PgAPX1. Expression patterns and interactive specificity of HSF with HSE (PgAPX1) suggest a probable vital interlink in heat and oxidative stress signaling pathways that plays a significant role in comprehending the underlying mechanisms in plant abiotic stress tolerance.

Chlamydomonas reinhardtii, a model system for functional validation of abiotic stress responsive genes

Stress tolerance is a multigenic character and there are many stress responsive genes, which are stress specific. Although many of these have been cloned, their functional significance remains fragmentary. Hence it is important to identify the relevant stress genes involved in altering the metabolism for adaptation. Overexpression is one of the several approaches and Chlamydomonas is a suitable system to study the functional relevance of stress genes. Stress responses can only be assessed on prior exposure to sublethal induction stress. In this study the acclimation response of Chlamydomonas was assessed for different abiotic stresses using physiological screens like chlorophyll stability, membrane damage, cell viability, accumulation of free radicals, survival and recovery growth. We demonstrate that Chlamydomonas responds to diverse stresses and is a potential system to study the relevance of stress genes. The relevance of choline oxidase A (codA), a key enzyme in glycinebetaine biosynthesis, was examined by developing transformants expressing codA gene from Arthrobacter globiformis. Southern positive transformants showed enhanced accumulation of glycinebetaine. The transformants also showed enhanced growth under salinity, high light coupled with methylviologen-induced oxidative stress, high temperature and cold stress. However the transgenics were not tolerant to PEG-mediated simulated osmotic stress, LiCl, menadione and UV stress. Increased cell survival and decreased chlorophyll degradation in transformants under acclimated conditions further confirmed the relevance of codA in imparting stress tolerance. Our results indicated that the relevance of stress responsive genes can be efficiently validated for diverse abiotic stresses using Chlamydomonas system.

Lysine Decarboxylase Expression by Vibrio vulnificus Is Induced by SoxR in Response to Superoxide Stress

Lysine decarboxylase expression by Vibrio vulnificus, which is up-regulated by CadC in response to acid stress, is also induced by SoxR in response to superoxide stress. SoxR binds to the promoter region of the cadBA operon, coding for a lysine-cadaverine antiporter (CadB) and a lysine decarboxylase (CadA). The induction of cadBA transcription by SoxR is independent of CadC. Cadaverine, which neutralizes the external medium, also appears to scavenge superoxide radicals, since increasing cellular cadaverine by elevating the gene dosage of cadBA significantly diminished the induction of Mn-containing superoxide dismutase under methyl viologen-induced oxidative stress. Consistently, a lack of cadaverine caused by mutation in cadA resulted in low tolerance to oxidative stress compared with that of the wild type.

Screening of inbred lines to develop a thermotolerant sunflower hybrid using the temperature induction response (TIR) technique: a novel approach by exploiting residual variability

Plants, when exposed to sub-lethal stress (induction stress), develop the ability to withstand severe temperatures and this phenomenon is often referred to as acquired thermotolerance. Earlier it was reported that induction stress alters gene expression and brings greater adaptation to heat stress and that the genetic variability in thermotolerance is only seen upon induction stress. Based on this concept, the temperature induction response (TIR) technique has been developed to identify thermotolerant lines. By following the TIR technique, sunflower hybrid KBSH-1 parents were screened for high temperature tolerance. Seedlings of parental lines including CMS 234 A, CMS 234 B and 6 D-1 showed considerable genetic variability for thermotolerance and it was attributed to the expression of existing residual variability for stress responses. Thus, the existing variability forms the basis for identifying thermotolerant lines. The identified parental inbred lines were selected and established in the field and crossed to get F1 hybrid seeds. The KBSH-1 hybrid developed from selected variants of parental lines was compared with the original KBSH-1 for thermotolerance. The selected KBSH-1 was more tolerant compared with the original hybrid both at the seedling as well as at the plant level. The physiological and molecular basis of thermotolerance was studied in the KBSH-1 original and the hybrid developed from selected variants of parental lines. The selected hybrid exhibited high tolerance to Menadione (naphthoquinone)-induced oxidative stress. Even the methyl viologen-induced oxidative stress damage was relatively less in the selected hybrid population. The selected hybrid also showed enhanced expression of the heat shock proteins HSP 90 and HSP 104 and also accumulated higher levels of the heat shock transcription factor HSFA.

Cloning, Expression, and Characterization of Two Manganese Superoxide Dismutases from Caenorhabditis elegans

Two genes encoding manganese superoxide dismutase (sod-2 and sod-3) have been identified in the nematode Caenorhabditis elegans. Each gene is composed of five exons, and intron positions are identical; however, intron sizes and sequences are not the same. The predicted protein sequences are 86.3% homologous (91.8% conservative), and the cDNAs are only 75.2% homologous. Both deduced protein sequences contain the expected N-terminal mitochondrial transit peptides. Reverse transcriptase polymerase chain reaction analysis shows that both genes are expressed under normal growth conditions and that their RNA transcripts are trans-spliced to the SL-1 leader sequence. The latter result together with Northern blot analysis indicate that both genes have mono-cistronic transcripts. The sod-3 gene was mapped to chromosome X, and the location of sod-2 was confirmed to be chromosome I. Polymerase chain reaction was used to amplify the cDNA regions encoding the predicted mature manganese superoxide dismutase proteins and each was cloned and expressed to high levels in Escherichia coli cells deficient in cytosolic superoxide dismutases. Both proteins were shown to be active in E. coli, providing similar protection against methyl viologen-induced oxidative stress. The expressed enzymes, which were not inhibited by hydrogen peroxide or cyanide, are dimeric, show quite different electrophoretic mobilities and isoelectric points, but exhibit comparable specific activities.