Cloning and in silico characterization of cinnamyl alcohol dehydrogenase gene involved in lignification of Tall fescue (Festuca arundinacea Schreb.).

Tall fescue, a promising temperate forage grass of Himalayan region, possesses extraordinary property of rapid growth with high biomass production, but its poor digestibility due to higher lignin content limits its utilization in livestock feeding. The lignification in Tall fescue is under the control of enzymatic cascade of different regulatory enzymes. Cinnamyl alcohol dehydrogenase (CAD) is a crucial regulatory enzyme that catalyzes the last step of monolignol biosynthesis and is a potential candidate for altering the content and types of lignin, and hence increasing the digestibility of fodder crops. Hence, the present investigation was conducted on isolation, cloning and characterization of CAD gene from Tall fescue. Isolation and amplification of CAD gene resulted in an amplicon of 1521 bp. The CAD gene sequence was submitted to NCBI database with an accession number MW442831. Translation of the CAD gene sequence exhibited an ORF of 361 amino acids. The deduced CAD protein was predicted to be hydrophobic, acidic and thermally stable with molecular formula C1712H2734N460O520S23, molecular mass of 38.82 kDa, theoretical pI of 5.60 and 3 strong transmembrane helices. The CAD protein was predicted to have a dimer forming behavior with putative NAD(P) binding site between amino acids 48 and 301, putative substrate-binding site between amino acids 48 and 301, catalytic zinc-binding site between amino acids 48 and 164 and structural zinc-binding site between amino acid residue 101 and 115. A conserved 189GLGGVG194 motif is the binding site for NADP(H). The conserved motif pattern of CAD's zinc catalytic center was found to be 69GHEVVGEV(X)EVG(X)2V83. The zinc-binding site was found to be conserved between amino acid 89 and 115 and was found to be 89G(X)2VG(X)G(X)2VGXC(X)2C(X)2C(X)5QYC115. The deciphered sequence and putative protein information might be useful in subsequent research in lignin bioengineering for enhanced digestibility, biomass conversion as well as impact of lignin on cell wall mechanics.

Contrasting patterns of nucleotide polymorphism suggest different selective regimes within different parts of the PgiC1 gene in Festuca ovina L.

BACKGROUND: Phosphoglucose isomerase (PGI, EC 5.3.1.9) is an essential metabolic enzyme in all eukaryotes. An earlier study of the PgiC1 gene, which encodes cytosolic PGI in the grass Festuca ovina L., revealed a marked difference in the levels of nucleotide polymorphism between the 5' and 3' portions of the gene. METHODS: In the present study, we characterized the sequence polymorphism in F. ovina PgiC1 in more detail and examined possible explanations for the non-uniform pattern of nucleotide polymorphism across the gene. RESULTS: Our study confirms that the two portions of the PgiC1 gene show substantially different levels of DNA polymorphism and also suggests that the peptide encoded by the 3' portion of PgiC1 is functionally and structurally more important than that encoded by the 5' portion. Although there was some evidence of purifying selection (dN/dS test) on the 5' portion of the gene, the signature of purifying selection was considerably stronger on the 3' portion of the gene (dN/dS and McDonald-Kreitman tests). Several tests support the action of balancing selection within the 5' portion of the gene. Wall's B and Q tests were significant only for the 5' portion of the gene. There were also marked peaks of nucleotide diversity, Tajima's D and the dN/dS ratio at or around a PgiC1 codon site (within the 5' portion of the gene) that a previous study had suggested was subject to positive diversifying selection. CONCLUSIONS: Our results suggest that the two portions of the gene have been subject to different selective regimes. Purifying selection appears to have been the main force contributing to the relatively low level of polymorphism within the 3' portion of the sequence. In contrast, it is possible that balancing selection has contributed to the maintenance of the polymorphism within the 5' portion of the gene.

Use of two grasses for the phytoremediation of aqueous solutions polluted with terbuthylazine.

The capacity of two grasses, tall fescue (Festuca arundinacea) and orchardgrass (Dactylis glomerata), to remove terbuthylazine (TBA) from polluted solutions has been assessed in hydroponic cultures. Different TBA concentrations (0.06, 0.31, 0.62, and 1.24 mg/L) were chosen to test the capacity of the two grasses to resist the chemical. Aerial biomass, effective concentrations (to cause reductions of 10, 50, and 90% of plant aerial biomass) and chlorophylls contents of orchardgrass were found to be more affected. Tall fescue was found to be more capable of removing the TBA from the growth media. Furthermore, enzymes involved both in the herbicide detoxification and in the response to herbicide-induced oxidative stress were investigated. Glutathione S-transferase (GST, EC. 2.5.1.18) and ascorbate peroxidase (APX, EC. 1.11.1.11) of tall fescue were found to be unaffected by the chemical. GST and APX levels of orchardgrass were decreased by the treatment. These negative modulations exerted by the TBA on the enzyme of orchardgrass explained its lower capacity to cope with the negative effects of the TBA.

Evidence for Positive Selection within the PgiC1 Locus in the Grass Festuca ovina.

The dimeric metabolic enzyme phosphoglucose isomerase (PGI, EC 5.3.1.9) plays an essential role in energy production. In the grass Festuca ovina, field surveys of enzyme variation suggest that genetic variation at cytosolic PGI (PGIC) may be adaptively important. In the present study, we investigated the molecular basis of the potential adaptive significance of PGIC in F. ovina by analyzing cDNA sequence variation within the PgiC1 gene. Two, complementary, types of selection test both identified PGIC1 codon (amino acid) sites 200 and 173 as candidate targets of positive selection. Both candidate sites involve charge-changing amino acid polymorphisms. On the homology-modeled F. ovina PGIC1 3-D protein structure, the two candidate sites are located on the edge of either the inter-monomer boundary or the inter-domain cleft; examination of the homology-modeled PGIC1 structure suggests that the amino acid changes at the two candidate sites are likely to influence the inter-monomer interaction or the domain-domain packing. Biochemical studies in humans have shown that mutations at several amino acid sites that are located close to the candidate sites in F. ovina, at the inter-monomer boundary or the inter-domain cleft, can significantly change the stability and/or kinetic properties of the PGI enzyme. Molecular evolutionary studies in a wide range of other organisms suggest that PGI amino acid sites with similar locations to those of the candidate sites in F. ovina may be the targets of positive/balancing selection. Candidate sites 200 and 173 are the only sites that appear to discriminate between the two most common PGIC enzyme electromorphs in F. ovina: earlier studies suggest that these electromorphs are implicated in local adaptation to different grassland microhabitats. Our results suggest that PGIC1 sites 200 and 173 are under positive selection in F. ovina.

Evidence for alternative splicing mechanisms in meadow fescue (Festuca pratensis) and perennial ryegrass (Lolium perenne) Rubisco activase gene.

Rubisco activase is required to regulate the catalytic activity of Rubisco in plants, in an ATP-dependent manner. One or two Rubisco activase proteins have been identified in different plant species. In some species, the two isoforms are the products of alternative splicing of the Rubisco activase gene. The aim of this study was to confirm that Lolium perenne and Festuca pratensis plants have two isoforms of Rubisco activase and that they are the products of alternative splicing of common pre-mRNA. Protein gel blot analyses indicated that L. perenne and F. pratensis leaves contained two Rubisco activase proteins. Sequence analysis of cDNA and genomic DNA showed that differential splicing generated two mRNAs that differed in sequence only in the inclusion of 48 bp. The insertion contains a stop codon leading to the synthesis of a shorter polypeptide. Under the conditions of our experiment, the shorter splicing variant of L. perenne and F. pratensis Rubisco activase gene was preferentially produced. Any further studies concerning Rubisco activase genes in L. perenne and/or F. pratensis plants should take into consideration the mechanism of its expression.

Comparison between Agrobacterium-mediated and direct gene transfer using the gene gun.

Agrobacterium-mediated transformation and direct gene transfer using the gene gun (microparticle -bombardment) are the two most widely used methods for plant genetic modification. The Agrobacterium method has been successfully practiced in dicots for many years, but only recently have efficient protocols been developed for grasses. Microparticle bombardment has evolved as a method delivering exogenous nucleic acids into plant genome and is a commonly employed technique in plant science. Here these two systems are compared for transformation efficiency, transgene integration, and transgene expression when used to transform tall fescue (Festuca arundinacea Schreb.). The tall fescue transformation protocols lead to the production of large numbers of fertile, independent transgenic lines.

Ascorbic acid enhances the accumulation of polycyclic aromatic hydrocarbons (PAHs) in roots of tall fescue (Festuca arundinacea Schreb.).

Plant contamination by polycyclic aromatic hydrocarbons (PAHs) is crucial to food safety and human health. Enzyme inhibitors are commonly utilized in agriculture to control plant metabolism of organic components. This study revealed that the enzyme inhibitor ascorbic acid (AA) significantly reduced the activities of peroxidase (POD) and polyphenol oxidase (PPO), thus enhancing the potential risks of PAH contamination in tall fescue (Festuca arundinacea Schreb.). POD and PPO enzymes in vitro effectively decomposed naphthalene (NAP), phenanthrene (PHE) and anthracene (ANT). The presence of AA reduced POD and PPO activities in plants, and thus was likely responsible for enhanced PAH accumulation in tall fescue. This conclusion is supported by the significantly enhanced uptake of PHE in plants in the presence of AA, and the positive correlation between enzyme inhibition efficiencies and the rates of metabolism of PHE in tall fescue roots. This study provides a new perspective, that the common application of enzyme inhibitors in agricultural production could increase the accumulation of organic contaminants in plants, hence enhancing risks to food safety and quality.

Expression of a Trichoderma reesei ß-1,4 endo-xylanase in tall fescue modifies cell wall structure and digestibility and elicits pathogen defence responses.

An endo-xylanase from Trichoderma reesei (xyn2) has been expressed in tall fescue targeted to the vacuole, apoplast or Golgi, constitutively under the control of the rice actin promoter, and to the apoplast under the control of a senescence enhanced gene promoter. Constitutive xylanase expression in the vacuole, apoplast, and golgi, resulted in only a small number of plants with low enzyme activities and in reduced plant growth in apoplast, and golgi targeted plants. Constitutive expression in the apoplast also resulted in increased levels of cell wall bound hydroxycinnamic acid monomers and dimers, but no significant effect on cell wall xylose or arabinose content. In situ constitutive xylanase expression in the Golgi also resulted in increased ferulate dimers. However, senescence induced xylanase expression in the apoplast was considerably higher and did not affect plant growth or the level of monomeric hydroxycinnamic acids or lignin in the cell walls. These plants also showed increased levels of ferulate dimers, and decreased levels of xylose with increased levels of arabinose in their cell walls. While the release of cell wall hydroxycinnamic acids on self digestion was enhanced in these plants in the presence of exogenously applied ferulic acid esterase, changes in cell wall composition resulted in decreases in both tissue digestibility and cellulase mediated sugar release. In situ detection of H(2)O(2) production mediated by ethylene release in leaves of plants expressing apoplast xylanase could be leading to increased dimerisation. High-level xylanase expression in the apoplast also resulted in necrotic lesions on the leaves. Together these results indicate that xylanase expression in tall fescue may be triggering plant defence responses analogous to foliar pathogen attack mediated by ethylene and H(2)O(2).

Glutathione peroxidases in Lolium multiflorum and Festuca arundinacea: Activity, susceptibility to herbicides and characteristics.

To evidence a possible mechanism of defense toward oxidative stress induced by herbicides in plants, an investigation was carried on the activity of glutathione peroxidase (GPX) in Italian ryegrass (Lolium multiflorum) and in Festuca (Festuca arundinacea) in response to atrazine (6-chloro-N-ethyl-N'-isopropyl-1,3,5-triazine-2,4-diamine) and fluorodifen (4-nitrophenyl α,α,α-trifluoro-2-nitro-p-tolyl ether). In general, the herbicide treatments significantly induced GPX activity in the shoots of Italian ryegrass, whereas inhibited it in Festuca. These opposite behaviors are examined, taking into account the accumulation and persistence of the two herbicides in the plants, and they are discussed in terms of GPX counteraction to oxidative stress in the first case, and of a lower detoxification rate unable to prevent a deleterious effect on the GPX activity in the second case. Further information on the properties of Italian ryegrass and Festuca GPX were achieved by purification and isolation of the enzymes, performed by protein liquid chromatography and by electrophoretic analyses. GPX of both the plants were found to be heterodimer with multiple function in showing also glutathione S-transferase (GST) activity.

Geographic and molecular variation in a natural plant transgene.

A PCR based survey of Festuca ovina plants from populations around the southern part of the Baltic Sea demonstrates both geographic and molecular variation in the enzyme gene PgiC2, horizontally transferred from a Poa-species. Our results show that PgiC2-a natural functional nuclear transgene-is not a local ephemeral phenomenon but is present in a very large number of individuals. We find also that its frequency is geographically variable and that it appears in more than one molecular form. The chloroplast variation in the region does not indicate any distinct subdivision due to different colonization routes after the last glaciation. Our data illustrate the geographic and molecular variation that may occur in natural populations with a polymorphic, unfixed transgene affected by diverse kinds of mutational and evolutionary processes.

Festuca arundinacea, glutathione S-transferase and herbicide safeners: a preliminary case study to reduce herbicidal pollution.

The expression of glutathione S-transferase (GST) activity in Festuca arundinacea was investigated in response to the following herbicide safeners: benoxacor, cloquintocet-mexyl, fenchlorazol-ethyl, fenclorim, fluxofenim and oxabetrinil. All the above compounds enhanced the GST activity tested towards the "model" substrate 1-chloro-2,4-dinitrobenzene (CDNB). Assays of GST activity towards the herbicides terbuthylazine (N(2)-tert-butyl-6-chloro-N(4)-ethyl-1,3,5-triazine-2,4-diamine) and butachlor (N-butoxymethyl-2-chloro-2',6'-diethylacetanilide) as substrates also showed the ability of the safeners to enhance the enzyme activity towards both these herbicides, with the exception of cloquintocet-mexyl for the enzyme activity towards butachlor. As a consequence of the above effects at a macro-scale level, decreased herbicide accumulation and persistence were ascertained in response to the addition of the safener benoxacor to both terbuthylazine and butachlor treatments. These results are discussed in terms of capacity of benoxacor to induce herbicide detoxification in Festuca arundinacea with a view to utilizing them in reducing herbicide pollution.

The SNF1-type serine-threonine protein kinase SAPK4 regulates stress-responsive gene expression in rice.

BACKGROUND: Plants respond to extracellularly perceived abiotic stresses such as low temperature, drought, and salinity by activation of complex intracellular signaling cascades that regulate acclimatory biochemical and physiological changes. Protein kinases are major signal transduction factors that have a central role in mediating acclimation to environmental changes in eukaryotic organisms. In this study, we characterized the function of the sucrose nonfermenting 1-related protein kinase2 (SnRK2) SAPK4 in the salt stress response of rice. RESULTS: Translational fusion of SAPK4 with the green fluorescent protein (GFP) showed subcellular localization in cytoplasm and nucleus. To examine the role of SAPK4 in salt tolerance we generated transgenic rice plants with over-expression of rice SAPK4 under control of the CaMV-35S promoter. Induced expression of SAPK4 resulted in improved germination, growth and development under salt stress both in seedlings and mature plants. In response to salt stress, the SAPK4-overexpressing rice accumulated less Na+ and Cl- and showed improved photosynthesis. SAPK4-regulated genes with functions in ion homeostasis and oxidative stress response were identified: the vacuolar H+-ATPase, the Na+/H+ antiporter NHX1, the Cl- channel OsCLC1 and a catalase. CONCLUSION: Our results show that SAPK4 regulates ion homeostasis and growth and development under salinity and suggest function of SAPK4 as a regulatory factor in plant salt stress acclimation. Identification of signaling elements involved in stress adaptation in plants presents a powerful approach to identify transcriptional activators of adaptive mechanisms to environmental changes that have the potential to improve tolerance in crop plants.

Functional analysis of a fungal endophyte stress-activated MAP kinase.

The ability of fungi to sense and respond rapidly to environmental stress is crucial for their survival in the wild. One of the most important pathways involved in this response is the stress-activated MAP (mitogen-activated protein) kinase pathway. We report here on the isolation of the stress-activated MAP kinase, sakA, from the fungal endophyte Epichloë festucae. Complementation of the stress sensitivity and cell cycle defects of an Schizosaccharomyces pombe sty1Delta mutant with sakA confirmed it encodes a functional MAP kinase. Analysis of an E. festucae DeltasakA mutant revealed sakA is essential for growth under conditions of temperature and osmotic stress in culture, and for sensitivity to the fungicide fludioxonil. However, the DeltasakA mutant shows no increased sensitivity to hydrogen peroxide. Given sakA can rescue the sty1Delta mutant from sensitivity to oxidative stress, SakA has the potential to sense and transduce oxidative stress signals. The DeltasakA mutant is also defective in conidia formation, suggesting a role for SakA in asexual development of E. festucae. The detection of elevated hydrogen peroxide production in the DeltasakA mutant suggests there may be a link between MAP kinase and ROS (reactive oxygen species) signalling pathways in E. festucae.

Origin and timing of the horizontal transfer of a PgiC gene from Poa to Festuca ovina.

A segregating second locus, PgiC2, for the enzyme phosphoglucose isomerase (PGIC) is found in the grass sheep's fescue, Festuca ovina. We have earlier reported that a phylogenetic analysis indicates that PgiC2 has been horizontally transferred from the reproductively separated grass genus Poa. Here we extend our analysis to include intron and exon information on 27 PgiC sequences from 18 species representing five genera, and confirm our earlier finding. The origin of PgiC2 can be traced to a group of closely interrelated, polyploid and partially asexual Poa species. The sequence most similar to PgiC2 is found in Poa palustris with a divergence, based on synonymous substitutions, of only 0.67%. This value suggests that the transfer took place less than 600,000 years ago (late Pleistocene), at a time when most extant Poa and Festuca species already existed.

Changes in plasmalemma K+Mg2+ -ATPase dephosphorylating activity and H+ transport in relation to seasonal growth and freezing tolerance of Festuca pratensis Huds.

Changes in plasmalemma K(+)Mg(2+)-ATPase dephosphorylating activity and H(+) transport were examined in freezing-tolerant and non-tolerant genotypes of the perennial grass species Festuca pratensis Huds. Enzyme activity and DeltamuH(+) were measured in plasmalemma fractions isolated from basal nodes and roots. Three types of experiments were undertaken: (i) a field experiment, utilizing the seasonal growth and cessation cycle of a perennial plant; (ii) a cold acclimation experiment in hydroponics; and (iii) an instant freezing test. A specific fluctuation in K(+)Mg(2+)-ATPase activity was found throughout the seasonal growth of the plants (i). The K(+)Mg(2+)-ATPase activity peaks for both the basal node and the root plasmalemma were determined early in the spring before the renewal of growth. The lowest activity values in roots occurred at the time approaching flowering, and in basal nodes at the transition into the growth cessation. The K(+)Mg(2+)-ATPase activity was approximately 50% lower in the basal node plasmalemma of freezing-tolerant plants than of non-tolerant ones, when assessed at the optimal growth stage in hydroponics. In hydroponics (ii) and in the freezing test (iii), temperature stress was followed by a more pronounced change in the level of K(+)Mg(2+)-ATPase activity than in that of H(+) transport, and this change was more clearly differentiated in the basal node plasmalemma of contrasting genotypes than in the roots. Stress response was manifested differently in freezing-tolerant and non-tolerant plants at cold acclimation (4-2 degrees C) and at freezing (-8 degrees C) temperatures. Proton transport regulation via coupled changes in the hydrolysed ATP/transported proton ratio, as an attribute of freezing-tolerant plants, is discussed.

Expression of a fungal ferulic acid esterase increases cell wall digestibility of tall fescue (Festuca arundinacea).

In the cell walls of forage grasses, ferulic acid is esterified to arabinoxylans and participates with lignin monomers in oxidative coupling pathways to generate ferulate-polysaccharide-lignin complexes that cross-link the cell wall. Such cross-links hinder cell wall degradation by ruminant microbes, reducing plant digestibility. In this study, genetically modified Festuca arundinacea plants were produced expressing an Aspergillus niger ferulic acid esterase (FAEA) targeted to the vacuole. The rice actin promoter proved to be effective for FAEA expression, as did the cauliflower mosaic virus (CaMV) 35S and maize ubiquitin promoters. Higher levels of expression were, however, found with inducible heat-shock and senescence promoters. Following cell death and subsequent incubation, vacuole-targeted FAEA resulted in the release of both monomeric and dimeric ferulic acids from the cell walls, and this was enhanced several fold by the addition of exogenous endo-1,4-beta-xylanase. Most of the FAEA-expressing plants showed increased digestibility and reduced levels of cell wall esterified phenolics relative to non-transformed plants. It is concluded that targeted FAEA expression is an effective strategy for improving wall digestibility in Festuca and, potentially, other grass species used for fodder or cellulosic ethanol production.

Expression of the bacteriophage T4 lysozyme gene in tall fescue confers resistance to gray leaf spot and brown patch diseases.

Tall fescue (Festuca arundinacea Schreb.) is an important turf and forage grass species worldwide. Fungal diseases present a major limitation in the maintenance of tall fescue lawns, landscapes, and forage fields. Two severe fungal diseases of tall fescue are brown patch, caused by Rhizoctonia solani, and gray leaf spot, caused by Magnaporthe grisea. These diseases are often major problems of other turfgrass species as well. In efforts to obtain tall fescue plants resistant to these diseases, we introduced the bacteriophage T4 lysozyme gene into tall fescue through Agrobacterium-mediated genetic transformation. In replicated experiments under controlled environments conducive to disease development, 6 of 13 transgenic events showed high resistance to inoculation of a mixture of two M. grisea isolates from tall fescue. Three of these six resistant plants also displayed significant resistance to an R. solani isolate from tall fescue. Thus, we have demonstrated that the bacteriophage T4 lysozyme gene confers resistance to both gray leaf spot and brown patch diseases in transgenic tall fescue plants. The gene may have wide applications in engineered fungal disease resistance in various crops.

Glutathione S-transferases in Festuca arundinacea: identification, characterization and inducibility by safener benoxacor.

Over recent years it has emerged how certain no crop-species can be employed in phytoremediating contaminated soils or preventing herbicide pollution; in this contest Festuca arundinacea was investigated. Shoots of Festuca were submitted to fast protein liquid chromatography in order to identify their glutathione S-transferases (GST; EC 2.5.1.18), by a combination of anionic, affinity and RP-HPLC chromatography. The chromatographic procedure revealed satisfactory yield and four GSTs were identified: they were named FaGST I, FaGST II, FaGST III and FaGST IV. Among these, significant differences were observed in the chromatographic behaviours, structure, activity toward a "model" substrate, 1-chloro-2,4-dinitrobenzene, and responsiveness to the herbicide safener benoxacor. FaGST I showed the highest activity toward the above substrate, and this activity was up-regulated by the herbicide safener. Therefore, FaGST I was purified till homogeneity and was determined to be an heterodimer consisting of two subunits of 28.0 and 27.2kDa. Each subunit of FaGST I was further characterized by means of LC-ESI-MS/MS and immunoblotting analysis, which revealed that both the subunits belong to the tau subclass.

Identifying, cloning and structural analysis of differentially expressed genes upon Puccinia infection of Festuca rubra var. rubra.

Differentially expressed genes in response to rust infection (Puccinia sp.) in creeping red fescue (Festuca rubra var. rubra) were identified and quantified using the mRNA differential display technique. The differentially induced genes were identified as homologs of mitogen-activated protein kinase (MAPK) 3 of Arabidopsis thaliana, stem rust resistance protein Rpg1 of barley and Hsp70 of Spinacia oleracea. The change in the steady state expression levels of these genes in response to rust infection was tested by Northern blot analysis and further quantified by real-time PCR. A steady accumulation of transcripts in the course of rust infection was observed. Full-length transcript of a fescue MPK-3 was obtained by RACE PCR. Its corresponding cDNA encodes a protein with a predicted MW of 42.5 kDa which was mapped onto the structural model of homologs MAPK to illustrate the corresponding MAPK signature motifs. This study, for the first time, presents evidence on the rust infection dependent metabolic pathways in creeping red fescue.

Simultaneous overexpression of both CuZn superoxide dismutase and ascorbate peroxidase in transgenic tall fescue plants confers increased tolerance to a wide range of abiotic stresses.

To mitigate the oxidative damage inflicted by biotic or abiotic stresses, plants have evolved complex anti-oxidative defense mechanisms that involve induction of antioxidant and anti-oxidative enzymes, such as superoxide dismutase (SOD) and ascorbate peroxidase (APX). To determine whether overexpression of the genes encoding copper-zinc SOD (CuZnSOD) and APX in plants is capable of decreasing reactive oxygen species (ROS) produced in response to abiotic stresses, we generated transgenic tall fescue plants expressing the CuZnSOD and APX genes in chloroplasts under the control of the oxidative stress-inducible promoter, sweet potato peroxidase anionic 2 (SWPA2). Transgenic plants were generated by Agrobacterium-mediated genetic transformation, and genotypes were confirmed by DNA blot analysis. Transgenic plants were exposed to several ROS-generating abiotic stresses, such as methyl viologen (MV), H(2)O(2), and the heavy metals copper, cadmium, and arsenic, and their tolerance was evaluated. High levels of CuZnSOD and APX gene transcripts in the transgenic plants under these treatments suggested that the transgenes were functionally expressed. Compared to transgenic plants, higher amounts of ROS were generated in the leaves of control plants exposed to abiotic stresses, resulting in increased thiobarbituric acid reactive substances (TBARS), ion leakage, and chlorophyll degradation. These parameters were significantly lower in transgenic plants. Enzyme activity assays and native polyacrylamide gel electrophoresis (PAGE) showed that total SOD and APX were highly active in transgenic plants under the abiotic stresses examined. We conclude that one of the mechanisms of increased anti-oxidative defense in transgenic tall fescue plants is overexpression of the CuZnSOD and APX genes, which are utilized in scavenging ROS and thus provide improved tolerance to abiotic stresses.