Methyl Viologen-induced Oxidative Stress Research Articles

Arabidopsis transcription factor ANAC102 predominantly expresses a nuclear protein and acts as a negative regulator of methyl viologen-induced oxidative stress responses.

Plants, being sessile organisms, constantly need to respond to environmental stresses, often leading to the accumulation of reactive oxygen species (ROS). While ROS can be harmful, they also act as second messengers guiding plant growth and stress responses. Because chloroplasts are sensitive to environmental changes and are both a source and a target of ROS during stress conditions, they are important in conveying environmental changes to the nucleus, where acclimation responses are coordinated to maintain organellar and overall cellular homeostasis. ANAC102 has previously been established as a regulator of β-cyclocitral-mediated chloroplast-to-nucleus signaling, protecting plants against photooxidative stress. However, debates persist about where ANAC102 is located-in chloroplasts or in the nucleus. Our study, utilizing the genomic ANAC102 sequence driven by its native promoter, establishes ANAC102 primarily as a nuclear protein, lacking a complete N-terminal chloroplast-targeting peptide. Moreover, our research reveals the sensitivity of plants overexpressing ANAC102 to severe superoxide-induced chloroplast oxidative stress. Transcriptome analysis unraveled a dual role of ANAC102 in negatively and positively regulating genome-wide transcriptional responses to chloroplast oxidative stress. Through the integration of published data and our own study, we constructed a comprehensive transcriptional network, which suggests that ANAC102 exerts direct and indirect control over transcriptional responses through downstream transcription factor networks, providing deeper insights into the ANAC102-mediated regulatory landscape during oxidative stress.

A dual role for glutathione transferase U7 in plant growth and protection from methyl viologen-induced oxidative stress.

Plant glutathione S-transferases (GSTs) are glutathione-dependent enzymes with versatile functions, mainly related to detoxification of electrophilic xenobiotics and peroxides. The Arabidopsis (Arabidopsis thaliana) genome codes for 53 GSTs, divided into seven subclasses; however, understanding of their precise functions is limited. A recent study showed that class II TGA transcription factors TGA2, TGA5, and TGA6 are essential for tolerance of UV-B-induced oxidative stress and that this tolerance is associated with an antioxidative function of cytosolic tau-GSTs (GSTUs). Specifically, TGA2 controls the expression of several GSTUs under UV-B light, and constitutive expression of GSTU7 in the tga256 triple mutant is sufficient to revert the UV-B-susceptible phenotype of tga256. To further study the function of GSTU7, we characterized its role in mitigation of oxidative damage caused by the herbicide methyl viologen (MV). Under non-stress conditions, gstu7 null mutants were smaller than wild-type (WT) plants and delayed in the onset of the MV-induced antioxidative response, which led to accumulation of hydrogen peroxide and diminished seedling survival. Complementation of gstu7 by constitutive expression of GSTU7 rescued these phenotypes. Furthermore, live monitoring of the glutathione redox potential in intact cells with the fluorescent probe Grx1-roGFP2 revealed that GSTU7 overexpression completely abolished the MV-induced oxidation of the cytosolic glutathione buffer compared with WT plants. GSTU7 acted as a glutathione peroxidase able to complement the lack of peroxidase-type GSTs in yeast. Together, these findings show that GSTU7 is crucial in the antioxidative response by limiting oxidative damage and thus contributes to oxidative stress resistance in the cell.

Preparation of the Enzymatic Hydrolysates from Chlorella vulgaris Protein and Assessment of Their Antioxidant Potential Using Caenorhabditis elegans.

This study aimed to assess the antioxidant potential of Chlorella vulgaris protein-derived enzymatic hydrolysate using Caenorhabditis elegans. Protein extraction was performed using an alkali solution after complete C. vulgaris swelling and hydrolysis using four commercial proteases (alcalase, neutrase, protamex, and flavourzyme). The results showed that the flavourzyme hydrolysates exhibited the strongest antioxidant activity both in vitro and in vivo. Under the optimum conditions of the enzymatic hydrolysis, the half-maximal effective concentration of the hydrolysates for superoxide and hydroxyl radicals was 0.323mg/mL and 0.139mg/mL, respectively. The hydrolysates could significantly extend the lifespan, improve the resistance to methyl viologen-induced oxidative stress, reduce the levels of reactive oxygen species, and enhance the activity of catalase and superoxide dismutase in C. elegans.

Enhanced Tolerance to Methyl Viologen-Mediated Oxidative Stress via AtGR2 Expression From Chloroplast Genome.

Owing to their sessile life habit, plants are continuously subjected to a broad range of environmental stresses. During periods of (a)biotic stresses, reactive oxygen species (ROS) levels can rise excessively, leading to oxidative stress. Glutathione reductase (GR) plays an important role in scavenging the ROS and maintenance of redox potential of the cell during oxidative stress. To enhance ROS scavenging capacity, and hence stress tolerance, the Arabidopsis thalianaGR2 (AtGR2) gene was expressed from the tobacco plastid (chloroplast) genome, the main source of ROS production in plant photosynthetic tissues, in this study. Leaves of transplastomic tobacco plants had about seven times GR activity and 1.5 times total glutathione levels compared to wild type. These transplastomic tobacco plants showed no discernible phenotype and exhibited more tolerance to methyl viologen-induced oxidative stress than wild-type control plants. The results indicate that introducing AtGR2 in chloroplasts is an efficient approach to increase stress tolerance. This study also provides evidence that increasing antioxidant enzyme via plastid genome engineering is an alternative to enhance plant’s tolerance to stressful conditions.

Condensed tannins are inducible antioxidants and protect hybrid poplar against oxidative stress.

Condensed tannins (CTs) have been studied extensively as potential defenses against pests and pathogens, and for their beneficial effects on human health. They are known to possess high in vitro antioxidant capacity, but whether they can function as in planta antioxidants for protection against oxidative stress has not been previously tested. Here, we show that stress induction of CTs in poplar (Populus) is matched closely by an increase in antioxidant activity under both high light and nitrogen deficiency. We also investigate the effects of CTs as in vivo antioxidants directly, using transgenic poplar plants which overexpress poplar MYB transcription factors that regulate the CT pathway. These transgenics have 50-fold higher CT concentrations than controls, and and also have dramatically higher antioxidant activity. High-CT and control poplar leaves were exposed to methyl viologen for 24 h. Chlorophyll fluorescence was used to measure maximum quantum efficiency of photosystem II photochemistry (Fv/Fm), and leaf discs were stained with 3,3'-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) to assess hydrogen peroxide and superoxide levels. After methyl viologen exposure, high-CT transgenics retained higher Fv/Fm ratios and accumulated less hydrogen peroxide and superoxide than the controls. Our findings indicate that high-CT concentrations protect poplar against methyl viologen-induced oxidative stress and suggest a broader function of CTs than previously supposed.

Biochemical and functional characterization of OsCSD3, a novel CuZn superoxide dismutase from rice.

Superoxide dismutases (SODs, EC 1.15.1.1) belong to an important group of antioxidant metalloenzymes. Multiple SODs exist for scavenging of reactive oxygen species (ROS) in different cellular compartments to maintain an intricate ROS balance. The present study deals with molecular and biochemical characterization of CuZn SOD encoded by LOC_Os03g11960 (referred to as OsCSD3), which is the least studied among the four rice isozymes. The OsCSD3 showed higher similarity to peroxisomal SODs in plants. The OsCSD3 transcript was up-regulated in response to salinity, drought, and oxidative stress. Full-length cDNA encoding OsCSD3 was cloned and expressed in Escherichia coli and analyzed for spectral characteristics. UV (ultraviolet)-visible spectroscopic analysis showed evidences of d-d transitions, while circular dichroism analysis indicated high β-sheet content in the protein. The OsCSD3 existed as homodimer (∼36 kDa) with both Cu2+ and Zn2+ metal cofactors and was substantially active over a wide pH range (7.0-10.8), with optimum pH of 9.0. The enzyme was sensitive to diethyldithiocarbamate but insensitive to sodium azide, which are the characteristics features of CuZn SODs. The enzyme also exhibited bicarbonate-dependent peroxidase activity. Unlike several other known CuZn SODs, OsCSD3 showed higher tolerance to hydrogen peroxide and thermal inactivation. Heterologous overexpression of OsCSD3 enhanced tolerance of E. coli sod double-knockout (ΔsodA ΔsodB) mutant and wild-type strain against methyl viologen-induced oxidative stress, indicating the in vivo function of this enzyme. The results show that the locus LOC_Os03g11960 of rice encodes a functional CuZn SOD with biochemical characteristics similar to the peroxisomal isozymes.

OsDCL1a activation impairs phytoalexin biosynthesis and compromises disease resistance in rice.

MicroRNAs (miRNAs) are small non-coding RNAs that act as post-transcriptional regulators of gene expression via sequence-specific cleavage or translational repression of target transcripts. They are transcribed as long single-stranded RNA precursors with unique stem-loop structures that are processed by a DICER-Like (DCL) ribonuclease, typically DCL1, to produce mature miRNAs. Although a plethora of miRNAs have been found to be regulated by pathogen infection in plants, the biological function of most miRNAs remains largely unknown. Here, the contribution of OsDCL1 to rice immunity was investigated. Activation-tagged Osdcl1a (Osdcl1a-Ac) rice mutants were examined for resistance to pathogen infection. mRNA and small RNA deep sequencing, quantitative real-time PCR (RT-qPCR) and stem-loop reverse tanscripion-PCR (RT-PCR) were used to examine DCL1a-mediated alterations in the rice transcriptome. Rice diterpene phytoalexins were quantified by liquid chromatography-tandem mass spectrometry (LC-MSMS). Accumulation of O2·- was determined by nitroblue tetrazolium (NBT) staining. dcl1a-Ac mutants exhibit enhanced susceptibility to infection by fungal pathogens which was associated with a weaker induction of defence gene expression. Comparison of the mRNA and miRNA transcriptomes of dcl1a-Ac and wild-type plants revealed misregulation of genes involved in detoxification of reactive oxygen species. Consequently, dcl1a-Ac plants accumulated O2·- in their leaves and were more sensitive to methyl viologen-induced oxidative stress. Furthermore, dcl1a-Ac plants showed downregulation of diterpenoid phytoalexin biosynthetic genes, these genes also being weakly induced during pathogen infection. Upon pathogen challenge, dcl1a-Ac plants failed to accumulate major diterpenoid phytoalexins. OsDCL1a activation resulted in marked alterations in the rice miRNAome, including both upregulation and downregulation of miRNAs. OsDCL1a activation enhances susceptibility to infection by fungal pathogens in rice. Activation of OsDCL1a represses the pathogen-inducible host defence response and negatively regulates diterpenoid phytoalexin production. These findings provide a basis to understand the molecular mechanisms through which OsDCL1a mediates rice immunity.

Anastatica hierochuntica, an Arabidopsis Desert Relative, Is Tolerant to Multiple Abiotic Stresses and Exhibits Species-Specific and Common Stress Tolerance Strategies with Its Halophytic Relative, Eutrema (Thellungiella) salsugineum.

The search for novel stress tolerance determinants has led to increasing interest in plants native to extreme environments – so called “extremophytes.” One successful strategy has been comparative studies between Arabidopsis thaliana and extremophyte Brassicaceae relatives such as the halophyte Eutrema salsugineum located in areas including cold, salty coastal regions of China. Here, we investigate stress tolerance in the desert species, Anastatica hierochuntica (True Rose of Jericho), a member of the poorly investigated lineage III Brassicaceae. We show that A. hierochuntica has a genome approximately 4.5-fold larger than Arabidopsis, divided into 22 diploid chromosomes, and demonstrate that A. hierochuntica exhibits tolerance to heat, low N and salt stresses that are characteristic of its habitat. Taking salt tolerance as a case study, we show that A. hierochuntica shares common salt tolerance mechanisms with E. salsugineum such as tight control of shoot Na+ accumulation and resilient photochemistry features. Furthermore, metabolic profiling of E. salsugineum and A. hierochuntica shoots demonstrates that the extremophytes exhibit both species-specific and common metabolic strategies to cope with salt stress including constitutive up-regulation (under control and salt stress conditions) of ascorbate and dehydroascorbate, two metabolites involved in ROS scavenging. Accordingly, A. hierochuntica displays tolerance to methyl viologen-induced oxidative stress suggesting that a highly active antioxidant system is essential to cope with multiple abiotic stresses. We suggest that A. hierochuntica presents an excellent extremophyte Arabidopsis relative model system for understanding plant survival in harsh desert conditions.

The Tryptophan-Rich Sensory Protein (TSPO) is Involved in Stress-Related and Light-Dependent Processes in the Cyanobacterium Fremyella diplosiphon.

The tryptophan-rich sensory protein (TSPO) is a membrane protein, which is a member of the 18 kDa translocator protein/peripheral-type benzodiazepine receptor (MBR) family of proteins that is present in most organisms and is also referred to as Translocator protein 18 kDa. Although TSPO is associated with stress- and disease-related processes in organisms from bacteria to mammals, full elucidation of the functional role of the TSPO protein is lacking for most organisms in which it is found. In this study, we describe the regulation and function of a TSPO homolog in the cyanobacterium Fremyella diplosiphon, designated FdTSPO. Accumulation of the FdTSPO transcript is upregulated by green light and in response to nutrient deficiency and stress. A F. diplosiphon TSPO deletion mutant (i.e., ΔFdTSPO) showed altered responses compared to the wild type (WT) strain under stress conditions, including salt treatment, osmotic stress, and induced oxidative stress. Under salt stress, the FdTSPO transcript is upregulated and a ΔFdTSPO mutant accumulates lower levels of reactive oxygen species (ROS) and displays increased growth compared to WT. In response to osmotic stress, FdTSPO transcript levels are upregulated and ΔFdTSPO mutant cells exhibit impaired growth compared to the WT. By comparison, methyl viologen-induced oxidative stress results in higher ROS levels in the ΔFdTSPO mutant compared to the WT strain. Taken together, our results provide support for the involvement of membrane-localized FdTSPO in mediating cellular responses to stress in F. diplosiphon and represent detailed functional analysis of a cyanobacterial TSPO. This study advances our understanding of the functional roles of TSPO homologs in vivo.

Molecular Cloning and Biochemical Characterization of the Iron Superoxide Dismutase from the Cyanobacterium Nostoc punctiforme ATCC 29133 and Its Response to Methyl Viologen-Induced Oxidative Stress.

Superoxide dismutase (SOD) detoxifies cell-toxic superoxide radicals and constitutes an important component of antioxidant machinery in aerobic organisms, including cyanobacteria. The iron-containing SOD (SodB) is one of the most abundant soluble proteins in the cytosol of the nitrogen-fixing cyanobacterium Nostoc punctiforme ATCC 29133, and therefore, we investigated its biochemical properties and response to oxidative stress. The putative SodB-encoding open reading frame Npun_R6491 was cloned and overexpressed in Escherichia coli as a C-terminally hexahistidine-tagged protein. The purified recombinant protein had a SodB specific activity of 2560±48 U/mg protein at pH 7.8 and was highly thermostable. The presence of a characteristic iron absorption peak at 350nm, and its sensitivity to H2O2 and azide, confirmed that the SodB is an iron-containing SOD. Transcript level of SodB in nitrogen-fixing cultures of N. punctiforme decreased considerably (threefold) after exposure to an oxidative stress-generating herbicide methyl viologen for 4 h. Furthermore, in-gel SOD activity analysis of such cultures grown at increasing concentrations of methyl viologen also showed a loss of SodB activity. These results suggest that SodB is not the primary scavenger of superoxide radicals induced by methyl viologen in N. punctiforme.

Simultaneous expression of regulatory genes associated with specific drought-adaptive traits improves drought adaptation in peanut.

Adaptation of crops to drought-prone rain-fed conditions can be achieved by improving plant traits such as efficient water mining (by superior root characters) and cellular-level tolerance mechanisms. Pyramiding these drought-adaptive traits by simultaneous expression of genes regulating drought-adaptive mechanisms has phenomenal relevance in improving stress tolerance. In this study, we provide evidence that peanut transgenic plants expressing Alfalfa zinc finger 1 (Alfin1), a root growth-associated transcription factor gene, Pennisetum glaucum heat-shock factor (PgHSF4) and Pea DNA helicase (PDH45) involved in protein turnover and protection showed improved tolerance, higher growth and productivity under drought stress conditions. Stable integration of all the transgenes was noticed in transgenic lines. The transgenic lines showed higher root growth, cooler crop canopy air temperature difference (less CCATD) and higher relative water content (RWC) under drought stress. Low proline levels in transgenic lines substantiate the maintenance of higher water status. The survival and recovery of transgenic lines was significantly higher under gradual moisture stress conditions with higher biomass. Transgenic lines also showed significant tolerance to ethrel-induced senescence and methyl viologen-induced oxidative stress. Several stress-responsive genes such as heat-shock proteins (HSPs), RING box protein-1 (RBX1), Aldose reductase, late embryogenesis abundant-5 (LEA5) and proline-rich protein-2 (PRP2), a gene involved in root growth, showed enhanced expression under stress in transgenic lines. Thus, the simultaneous expression of regulatory genes contributing for drought-adaptive traits can improve crop adaptation and productivity under water-limited conditions.

EcbZIP60, a basic leucine zipper transcription factor from Eleusine coracana L. improves abiotic stress tolerance in tobacco by activating unfolded protein response pathway

The basic leucine zippers (bZIPs) are one of the largest families of transcription factors that have been demonstrated to play diverse roles in plant growth development. These proteins are known to regulate many cellular responses in plants under stress conditions. In this study, a stress-responsive transcription factor belonging to bZIP family was cloned from finger millet (Eleusine coracana) and functionally validated in tobacco. The expression of EcbZIP60 was highly upregulated under drought, osmotic, salt and methyl viologen-induced stress in finger millet. Constitutive expression of EcbZIP60 in tobacco resulted in reduced growth of plants under normal growth conditions. However, the transgenic plants showed improved tolerance to drought stress with higher stomatal conductance and photosynthesis, resulting in improved growth. The transgenic plants also showed increased tolerance to salinity and methyl viologen-induced oxidative stress and to endoplasmic reticulum stress inducers dithiothreitol and tunicamycin. The transgenic plants showed significant upregulation of unfolded protein-responsive pathway genes such as BiP1, CRT1 and PDIL both under normal and stress conditions, although dehydrin group of genes did not show any upregulation. The results demonstrate that improved tolerance of EcbZIP60-expressing transgenic tobacco to diverse stresses could be through unfolded protein-responsive signalling pathway.

Proteome-wide dataset generated by iTRAQ-3DLCMS/MS technique for studying the role of FerB protein in oxidative stress in Paracoccus denitrificans.

3DLC protein- and peptide-fractionation technique combined with iTRAQ-peptide labeling and Orbitrap mass spectrometry was employed to quantitate Paracoccus dentirificans total proteome with maximal coverage. This resulted in identification of 24,948 peptides representing 2627 proteins (FDR<0.01) in P. dentirificans wild type and ferB mutant strains grown in the presence or absence of methyl viologen as an oxidative stressor. The data were generated for assessment of FerB protein role in oxidative stress as published by Pernikářová et al.; proteomic responses to a methyl viologen-induced oxidative stress in the wild type and FerB mutant strains of P. denitrificans, J. Proteomics 2015;125:68–75. Dataset is supplied in the article.

Proteomic responses to a methyl viologen-induced oxidative stress in the wild type and FerB mutant strains of Paracoccus denitrificans

FerB is a cytoplasmic flavoprotein from the soil bacterium Paracoccus denitrificans with a putative role in defense against oxidative stress. To further explore this hypothesis, we compared protein variations upon methyl viologen treatment in wild-type and FerB mutant strains by a quantitative proteomic analysis based on iTRAQ-3DLC–MS/MS analysis. The proteins showing the most prominent increase in abundance were assigned to carbon fixation and sulfur assimilatory pathways. By employing these proteins as indirect markers, oxidative stress was found to be 15% less severe in the wild-type than in the FerB-deficient mutant cells. Oxidative stress altered the levels of proteins whose expression is dependent on the transcriptional factor FnrP. The observed down-regulation of the fnrP regulon members, most notably that of nitrous oxide reductase, was tentatively explained by an oxidative degradation of the [4Fe–4S] center of FnrP leading to a protein form which no longer activates transcription. While the level of FerB remained relatively constant, two proteins homologous to FerB accumulated during oxidative stress. When their genes were expressed in Escherichia coli, neither of the protein products contained a bound flavin, whereas they both had a high activity of flavin reductase, one preferentially utilizing NADH and the other NADPH.

Overexpression of ShCHL P in tomato improves seedling growth and increases tolerance to salt, osmotic, and oxidative stresses

Geranylgeranyl reductase (CHL P) catalyzes the reduction of geranylgeranyl diphosphate to phytyl diphosphate and provides phytol for both chlorophyll (Chl) and tocopherol (TP) synthesis. Our previous study has found that the Solanum habrochaites CHL P (ShCHL P) gene was repressed by cold stress. In this study, we functionally characterized this gene with respect to abiotic stress tolerance. ShCHL P is expressed highly in leaves and stems, and barely in roots. Also, its expression was suppressed by low and high temperatures, drought, salt, and oxidative stresses. Transgenic tomato plants overexpressing ShCHL P showed increased levels of Chl and α-TP in leaves. In contrast, Chl and α-TP contents were reduced in the co-suppression plants, which exhibited chlorosis. These results confirmed the previous findings that CHL P is essential for Chl and TP synthesis in plants. Moreover, the ShCHL P overexpression and suppression lines showed improved and inhibited early seedling growth under normal, salt, and osmotic stress conditions, respectively, as compared with the wild type. Surprisingly, both overexpression and suppression of CHL P in transgenic tomato enhanced tolerance to methyl viologen-induced oxidative stress. These results indicate that tomato CHL P plays an important role in response to abiotic stress through regulation of Chl and TP synthesis. CHL P might be a good candidate gene for genetic improvement of plant growth under abiotic stress conditions in tomato.

Glutathione reductase a unique enzyme: molecular cloning, expression and biochemical characterization from the stress adapted C4 plant, Pennisetum glaucum (L.) R. Br.

The generation of excess reactive oxygen species (ROS) is one of the most common consequences of abiotic stress on plants. Glutathione reductase (GR, E.C. 1.6.4.2) and allied enzymes of the ascorbate-glutathione cycle play a crucial role to maintain the homeostatic redox balance in the cellular environment. GR plays an essential role in upholding the reduced glutathione pool under stress conditions. In the present study, a full-length GR cDNA and corresponding genomic clone was isolated from Pennisetum glaucum (L.) R. Br. The PgGR cDNA, encodes a 497-amino acid peptide with an estimated molecular mass of ~53.5 kDa. The PgGR peptide exhibits 54-89% sequence homology with GR from other plants and is cytoplasmic in nature. The PgGR enzyme was purified to near homogeneity, the recombinant protein being relatively thermostable and displaying activity in a broad range of temperature, pH and substrate concentrations. The PgGR transcript level was differentially regulated by heat, cold, salinity and methyl viologen-induced oxidative stress. The heterologously expressed PgGR protein in E. coli showed an improved protection against metal- and methyl viologen-induced oxidative stress. Our overall finding underscores the role of PgGR gene that responds to multiple abiotic stresses and provides stress tolerance in the experimental model (E. coli) which can be potentially used for the improvement of crops under abiotic stress conditions.

Abiotic stress-induced accumulation of raffinose in Arabidopsis leaves is mediated by a single raffinose synthase (RS5, At5g40390)

BackgroundThe sucrosylgalactoside oligosaccharide raffinose (Raf, Suc-Gal1) accumulates in Arabidopsis leaves in response to a myriad of abiotic stresses. Whilst galactinol synthases (GolS), the first committed enzyme in Raf biosynthesis are well characterised in Arabidopsis, little is known of the second biosynthetic gene/enzyme raffinose synthase (RS). Conflicting reports suggest the existence of either one or six abiotic stress-inducible RSs (RS-1 to -6) occurring in Arabidopsis. Indirect evidence points to At5g40390 being responsible for low temperature-induced Raf accumulation in Arabidopsis leaves.ResultsBy heterologously expressing At5g40390 in E.coli, we demonstrate that crude extracts synthesise Raf in vitro, contrary to empty vector controls. Using two independent loss-of-function mutants for At5g40390 (rs 5–1 and 5–2), we confirm that this RS is indeed responsible for Raf accumulation during low temperature-acclimation (4°C), as previously reported. Surprisingly, leaves of mutant plants also fail to accumulate any Raf under diverse abiotic stresses including water-deficit, high salinity, heat shock, and methyl viologen-induced oxidative stress. Correlated to the lack of Raf under these abiotic stress conditions, both mutant plants lack the typical stress-induced RafS activity increase observed in the leaves of wild-type plants.ConclusionsCollectively our findings point to a single abiotic stress-induced RS isoform (RS5, At5g40390) being responsible for Raf biosynthesis in Arabidopsis leaves. However, they do not support a single RS hypothesis since the seeds of both mutant plants still contained Raf, albeit at 0.5-fold lower concentration than seeds from wild-type plants, suggesting the existence of at least one other seed-specific RS. These results also unambiguously discount the existence of six stress-inducible RS isoforms suggested by recent reports.

Nitric oxide mediates cold‐ and dehydration‐induced expression of a novel MfHyPRP that confers tolerance to abiotic stress

Hybrid proline-rich proteins (HyPRPs) are cell wall-localized proteins, and are frequently responsive to environmental stresses. The coding sequence of a HyPRP cDNA was isolated from Medicago falcata, a forage crop that shows cold and drought tolerance. The predicted MfHyPRP contains a proline-rich domain at N-terminus after the signal peptide and a conserved eight-cysteine motif at the C-terminus. Higher level of MfHyPRP transcript was observed in leaves than in stems and roots under control conditions, while more MfHyPRP transcript was induced in leaves and stems than in roots after cold treatment. Levels of MfHyPRP transcript and MfHyPRP protein in leaves were induced by cold, dehydration, abscisic acid (ABA), hydrogen peroxide (H2 O2) and nitric oxide (NO), but not responsive to salt stress. The cold- or dehydration-induced expression of MfHyPRP was blocked by scavenger of NO, but not affected by inhibitor of ABA biosynthesis or scavenger of H2 O2. The results indicated that NO, but not ABA and H2 O2, was essential in the cold- and dehydration-induced expression of MfHyPRP. Overexpression of MfHyPRP in tobacco led to increased tolerance to freezing, chilling and osmotic stress as well as methyl viologen-induced oxidative stress. The increased cold and osmotic stress tolerance was proposed to be associated with improved protection against oxidative damages. It is suggested that NO mediates cold- and dehydration-induced expression of MfHyPRP that confers tolerance to abiotic stress.

The antioxidative defense system is involved in the delayed senescence in a wheat mutant tasg1

Wheat, which is the most important food crop worldwide, is a cereal that presents considerable potential for increased yield. A new wheat (Triticum aestivum L.) mutant tasg1 with delayed leaf senescence was constructed using ethyl methane sulfonate as a mutagen. Natural senescence in tasg1 was distinctly delayed in the field, as indicated by the slower progression of chlorophyll degradation, net photosynthetic rate than its wild type. Further, the malondialdehyde and the hydrogen peroxide content was lower and antioxidative enzyme activity higher in tasg1 than those in its wild type during both natural senescence and methyl viologen-induced oxidative stress. The results suggest that tasg1 is a functional stay-green wheat mutant with the Type B (in which senescence initiates on schedule, but progresses at a rate lower than that in the respective WTs) or Type A (in which senescence initiates late but proceeds at a normal rate) and B combination and that the competence of the antioxidant defense system is one of the most important mechanisms underlying the expression of the stay-green phenotype.

Enhanced tolerance to methyl viologen-induced oxidative stress and high temperature in transgenic potato plants overexpressing the CuZnSOD, APX and NDPK2 genes

Oxidative stress is a major threat for plants exposed to various environmental stresses. Previous studies found that transgenic potato plants expressing both copper zinc superoxide dismutase (CuZnSOD) and ascorbate peroxidase (APX) (referred to as SSA plants), or nucleoside diphosphate kinase 2 (NDPK2) (SN plants), showed enhanced tolerance to methyl viologen (MV)-induced oxidative stress and high temperature. This study aimed to develop transgenic plants that were more tolerant of oxidative stress by introducing the NDPK2 gene into SSA potato plants under the control of an oxidative stress-inducible peroxidase (SWPA2) promoter to create SSAN plants. SSAN leaf discs and whole plants showed enhanced tolerance to MV, as compared to SSA, SN or non-transgenic (NT) plants. SSAN plants sprayed with 400 µM MV exhibited about 53 and 83% less visible damage than did SSA and SN plants, respectively. The expression levels of the CuZnSOD, APX and NDPK2 genes in SSAN plants following MV treatment correlated well with MV tolerance. SOD, APX, NDPK and catalase antioxidant enzyme activities were also increased in MV-treated SSAN plants. In addition, SSAN plants were more tolerant to high temperature stress at 42°C, exhibiting a 6.2% reduction in photosynthetic activity as compared to plants grown at 25°C. In contrast, the photosynthetic activities of SN and SSA plants decreased by 50 and 18%, respectively. These results indicate that the simultaneous overexpression of CuZnSOD, APX and NDPK2 is more effective than single or double transgene expression for developing plants with enhanced tolerance to various environmental stresses.