Jose F. Moran

Drought induces oxidative stress in pea plants

Pea (Pisum sativum L. cv. Frilene) plants subjected to drought (leaf water potential of ≈-1.3 MPa) showed major reductions in photosynthesis (78‰), transpiration (83‰), and glycolate oxidase (EC 1.1.3.1) activity (44‰), and minor reductions (≈18‰) in the contents of chlorophyll a, carotenoids, and soluble protein. Water stress also led to pronounced decreases (72–85‰) in the activities of catalase (EC 1.11.1.6), dehydroascorbate reductase (EC 1.8.5.1), and glutathione reductase (EC 1.6.4.2), but resulted in the increase (32–42‰) of non-specific peroxidase (EC 1.11.1.7) and superoxide dismutase (EC 1.15.1.1). Ascorbate peroxidase (EC 1.11.1.11) and monodehydroascorbate reductase (EC 1.6.5.4) activities decreased only by 15‰ and the two enzymes acted in a cyclic manner to remove H2O2, which did not accumulate in stressed leaves. Drought had no effect on the levels of ascorbate and oxidized glutathione in leaves, but caused a 25‰ decrease in the content of reduced glutathione and a 67‰ increase in that of vitamin E. In leaves, average concentrations of catalytic Fe, i.e. Fe capable of catalyzing free-radical generation by redox cycling, were estimated as 0.7 to 7 μM (well-watered plants, depending on age) and 16 μM (water-stressed plants); those of catalytic Cu were ≈4.5 μM and 18 μM, respectively. Oxidation of lipids and proteins from leaves was enhanced two- to threefold under stress conditions and both processes were highly correlated. Fenton systems composed of the purported concentrations of ascorbate, H2O2, and catalytic metal ions in leaves produced hydroxyl radicals, peroxidized membrane lipids, and oxidized leaf proteins. It is proposed that augmented levels and decompartmentation of catalytic metals occurring during water stress are responsible for the oxidative damage observed in vivo.

COMPLEXES OF IRON WITH PHENOLIC COMPOUNDS FROM SOYBEAN NODULES AND OTHER LEGUME TISSUES : PROOXIDANT AND ANTIOXIDANT PROPERTIES

The low-molecular-mass fraction of the soybean nodule cytosol contains Fe capable of catalyzing free radical production through Fenton chemistry. A large portion of the pool of catalytic Fe, measured as bleomycin-detectable Fe, was characterized as complexes of Fe with phenolic compounds of three classes: phenolic acids, cinnamic acids, and flavonoids. Many of these compounds, along with other phenolics present in legume tissues, were used for a systematic structure-activity relationship study. All phenolics tested were able to chelate Fe, as judged from their inhibitory effect on site-specific deoxyribose degradation (minus EDTA assay). However, only those having catechol, pyrogallol, or 3-hydroxy-4-carbonyl groupings were potent chelators and reductants of Fe3+ at pH 5.5. The same phenolics promoted oxidative damage to DNA (bleomycin assay) and to deoxyribose (plus EDTA assay), but inhibited linolenic acid peroxidation by chelating and reducing Fe3+ and by neutralizing lipid radicals. Also, phenolics having a pyrogallol nucleus attenuated the free radical-mediated inactivation of glutamine synthetase, which was used as a model system, by chelating Fe2+. It is reasoned that under the microaerobic (10-20 nM O2) and acidic (pH 5.5-6.4) conditions prevailing in nodules, phenolics are likely to act primarily as antioxidants, decreasing oxidative damage to biomolecules.

Iron-dependent oxygen free radical generation in plants subjected to environmental stress: toxicity and antioxidant protection

Iron has a pivotal and dual role in free radical chemistry in all organisms. On the one hand, free Fe can participate in Fenton reactions and catalyze (‘catalytic Fe’) the generation of hydroxyl radical and other toxic oxygen species. On the other hand, Fe is a constituent of the antioxidant enzymes catalase, ascorbate peroxidase, guaiacol peroxidase, and ferro-superoxide dismutase. Protein Fe is Fenton inactive but can be released from proteins upon attack by activated oxygen. Healthy, unstressed plants avoid the interaction of catalytic Fe and peroxides by disposing of Fe in vacuoles and apoplast, by sequestering Fe in ferritin, and by having high levels of antioxidant enzymes and metabolites in most subcellular compartments. However, when plants are exposed to a variety of adverse conditions, including chilling, high light, drought and paraquat, oxidative stress ensues due primarily to the decrease in antioxidant defenses but also to the increase in free radical production mediated by catalytic Fe. The latter accumulates in many stressed plant tissues. Oxidative stress may lead to metabolic dysfunction and ultimately to plant cell death, so it needs to be estimated conveniently by quantifying the oxidation products of lipids (malondialdehyde and other cytotoxic aldehydes), proteins (total carbonyls, methionine sulfoxide, 2-oxohistidine), and DNA (8-hydroxyguanine, 5-hydroxycytosine). Protein oxidation appears to be a more sensitive and precocious marker than is lipid peroxidation, and DNA damage may also prove to be a useful marker for stress studies in plants.

Rice hemoglobins. Gene cloning, analysis, and O2-binding kinetics of a recombinant protein synthesized in Escherichia coli.

Although nonsymbiotic hemoglobins (Hbs) are found in different tissues of dicots and monocots, very little is known about hb genes in monocots and the function of Hbs in nonsymbiotic tissues. We report the cloning and analysis of two rice (Oryza sativa L.) hb genes, hb1 and hb2, that code for plant Hbs. Rice hb1 and hb2 genes contain four exons and three introns, as with all of the known plant hb genes. At least three copies of the hb gene were detected in rice DNA, and analysis of gene expression shows that hb1 and hb2 are expressed in leaves but only hb1 is expressed in roots. A cDNA for rice Hb1 was expressed in Escherichia coli, and the recombinant Hb (rHb1) shows an unusually high affinity for O2 because of a very low dissociation constant. The absorbance spectra of the ferric and deoxyferrous rHb1 indicate that, in contrast to symbiotic Hbs, a distal ligand is coordinated to the ligand-binding site. Mutation of the distal His demonstrates that this residue coordinates the heme Fe of ferric and deoxyferrous rHb1 and stabilizes O2 in oxy-rHb1. The biochemical properties of rice rHb1 suggest that this protein probably does not function to facilitate the diffusion of O2.

N2 Fixation, Carbon Metabolism, and Oxidative Damage in Nodules of Dark-Stressed Common Bean Plants

Common beans (Phaseolus vulgaris L.) were exposed to continuous darkness to induce nodule senescence, and several nodule parameters were investigated to identify factors that may be involved in the initial loss of N2 fixation. After only 1 d of darkness, total root respiration decreased by 76% and in vivo nitrogenase (N2ase) activity decreased by 95%. This decline coincided with the almost complete depletion (97%) of sucrose and fructose in nodules. At this stage, the O2 concentration in the infected zone increased to 1%, which may be sufficient to inactivate N2ase; however, key enzymes of carbon and nitrogen metabolism were still active. After 2 d of dark stress there was a significant decrease in the level of N2ase proteins and in the activities of enzymes involved in carbon and nitrogen assimilation. However, the general collapse of nodule metabolism occurred only after 4 d of stress, with a large decline in leghemoglobin and antioxidants. At this final senescent stage, there was an accumulation of oxidatively modified proteins. This oxidative stress may have originated from the decrease in antioxidant defenses and from the Fe-catalyzed generation of activated oxygen due to the increased availability of catalytic Fe and O2 in the infected region.

Functional Characterization and Expression of a Cytosolic Iron-Superoxide Dismutase from Cowpea Root Nodules

An iron-superoxide dismutase (FeSOD) with an unusual subcellular localization, VuFeSOD, has been purified from cowpea (Vigna unguiculata) nodules and leaves. The enzyme has two identical subunits of 27 kD that are not covalently bound. Comparison of its N-terminal sequence (NVAGINLL) with the cDNA-derived amino acid sequence showed that VuFeSOD is synthesized as a precursor with seven additional amino acids. The mature protein was overexpressed in Escherichia coli, and the recombinant enzyme was used to generate a polyclonal monospecific antibody. Phylogenetic and immunological data demonstrate that there are at least two types of FeSODs in plants. An enzyme homologous to VuFeSOD is present in soybean (Glycine max) and common bean (Phaseolus vulgaris) nodules but not in alfalfa (Medicago sativa) and pea (Pisum sativum) nodules. The latter two species also contain FeSODs in the leaves and nodules, but the enzymes are presumably localized to the chloroplasts and plastids. In contrast, immunoblots of the soluble nodule fraction and immunoelectron microscopy of cryo-processed nodule sections demonstrate that VuFeSOD is localized to the cytosol. Immunoblot analysis showed that the content of VuFeSOD protein increases in senescent nodules with active leghemoglobin degradation, suggesting a direct or indirect (free radical-mediated) role of the released Fe in enzyme induction. Therefore, contrary to the widely held view, FeSODs in plants are not restricted to the chloroplasts and may become an important defensive mechanism against the oxidative stress associated with senescence.

The emerging roles of nitric oxide (NO) in plant mitochondria

In recent years nitric oxide (NO) has been recognized as an important signal molecule in plants. Both, reductive and oxidative pathways and different subcellular compartments appear involved in NO production. The reductive pathway uses nitrite as substrate, which is exclusively generated by cytosolic nitrate reductase (NR) and can be converted to NO by the same enzyme. The mitochondrial electron transport chain is another site for nitrite to NO reduction, operating specifically when the normal electron acceptor, O(2), is low or absent. Under these conditions, the mitochondrial NO production contributes to hypoxic survival by maintaining a minimal ATP formation. In contrast, excessive NO production and concomitant nitrosative stress may be prevented by the operation of NO-scavenging mechanisms in mitochondria and cytosol. During pathogen attacks, mitochondrial NO serves as a nitrosylating agent promoting cell death; whereas in symbiotic interactions as in root nodules, the turnover of mitochondrial NO helps in improving the energy status similarly as under hypoxia/anoxia. The contribution of NO turnover during pathogen defense, symbiosis and hypoxic stress is discussed in detail.

Nitrogen nutrition and antioxidant metabolism in ammonium‐tolerant and ‐sensitive plants

Ammonium nutrition is of interest as an alternative to that of using nitrate. However, the former has been reported as stressful to many plant species especially to some important crops, as most abiotic stresses may trigger oxidative imbalances in plants. In this work, we investigate the response of oxidative metabolism of two plant species, spinach (Spinacia oleracea L. cv. Gigante de invierno) and pea (Pisum sativum L. cv. Rondo), which have distinct tolerance to ammonium. Plants were grown in the presence of 1.5 and 3.0 mM N as ammonium and compared with equivalent nitrate nutrition. The antioxidant enzymes and metabolites as well as oxidative damage to proteins were determined. Protein and amino acid contents in both types of plants were also analysed. Ammonium nutrition in sensitive spinach or in the tolerant pea plants does not alter the redox status of ascorbate and glutathione or the phenolic contents, while no clear effect is seen in the antioxidant enzymes. The results showed that the stress originated from applying ammonium as the only N source is not an oxidative stress, independent of the ammonium tolerance of the plant species studied. Moreover, ammonium stress diminishes oxidative damage to proteins in the spinach plants. The data of the protein oxidation together with those from N metabolism highlight the relation between the stress induced by ammonium and an increased protein turnover.

Molecular analysis of the pathway for the synthesis of thiol tripeptides in the model legume Lotus japonicus.

The thiol tripeptides, glutathione (GSH) and homoglutathione (hGSH), perform multiple roles in legumes, including protection against toxicity of free radicals and heavy metals. The three genes involved in the synthesis of GSH and hGSH in the model legume, Lotus japonicus, have been fully characterized and appear to be present as single copies in the genome. The gamma-glutamylcysteine synthetase (gamma(ecs)) gene was mapped on the long arm of chromosome 4 (70.0 centimorgans [cM]) and consists of 15 exons, whereas the glutathione synthetase (gshs) and homoglutathione synthetase (hgshs) genes were mapped on the long arm of chromosome 1 (81.3 cM) and found to be arranged in tandem with a separation of approximately 8 kb. Both genes consist of 12 exons of exactly the same size (except exon 1, which is similar). Two types of transcripts were detected for the gshs gene, which putatively encode proteins localized in the plastids and cytosol. Promoter regions contain cis-acting regulatory elements that may be involved in the plants response to light, hormones, and stress. Determination of transcript levels, enzyme activities, and thiol contents in nodules, roots, and leaves revealed that gamma(ecs) and hgshs are expressed in all three plant organs, whereas gshs is significantly functional only in nodules. This strongly suggests an important role of GSH in the rhizobia-legume symbiosis.

Depletion of the heaviest stable N isotope is associated with NH4+/NH3 toxicity in NH4+-fed plants

BackgroundIn plants, nitrate (NO3-) nutrition gives rise to a natural N isotopic signature (δ15N), which correlates with the δ15N of the N source. However, little is known about the relationship between the δ15N of the N source and the 14N/15N fractionation in plants under ammonium (NH4+) nutrition. When NH4+ is the major N source, the two forms, NH4+ and NH3, are present in the nutrient solution. There is a 1.025 thermodynamic isotope effect between NH3 (g) and NH4+ (aq) which drives to a different δ15N. Nine plant species with different NH4+-sensitivities were cultured hydroponically with NO3- or NH4+ as the sole N sources, and plant growth and δ15N were determined. Short-term NH4+/NH3 uptake experiments at pH 6.0 and 9.0 (which favours NH3 form) were carried out in order to support and substantiate our hypothesis. N source fractionation throughout the whole plant was interpreted on the basis of the relative transport of NH4+ and NH3.ResultsSeveral NO3--fed plants were consistently enriched in 15N, whereas plants under NH4+ nutrition were depleted of 15N. It was shown that more sensitive plants to NH4+ toxicity were the most depleted in 15N. In parallel, N-deficient pea and spinach plants fed with 15NH4+ showed an increased level of NH3 uptake at alkaline pH that was related to the 15N depletion of the plant. Tolerant to NH4+ pea plants or sensitive spinach plants showed similar trend on 15N depletion while slight differences in the time kinetics were observed during the initial stages. The use of RbNO3 as control discarded that the differences observed arise from pH detrimental effects.ConclusionsThis article proposes that the negative values of δ15N in NH4+-fed plants are originated from NH3 uptake by plants. Moreover, this depletion of the heavier N isotope is proportional to the NH4+/NH3 toxicity in plants species. Therefore, we hypothesise that the low affinity transport system for NH4+ may have two components: one that transports N in the molecular form and is associated with fractionation and another that transports N in the ionic form and is not associated with fractionation.