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Plants differ widely in their growth and tolerance responses to ammonium and urea nutrition, while derived phenotypes seem markedly different from plants grown under nitrate supply. Plant responses to N sources are complex, and the traits involved remain unknown. This work reports a comprehensive and quantitative root proteomic study on the NH4+-tolerant legume Medicago truncatula grown under axenic conditions with either nitrate, NH4+ or urea supply as sole N source by using the iTRAQ method. Sixty-one different proteins among the three N sources were identified. Interestingly, among the proteomic responses, urea nutrition displayed greater similarity to nitrate than to ammonium nutrition. We found remarkable differences in membrane proteins that play roles in sensing the N form, and regulate the intracellular pH and the uptake of N. Also, several groups of proteins were differentially expressed in the C metabolism pathway involved in reorganizing N assimilation. In addition, enzymes related to phenylpropanoid metabolism, including the peroxidases POD2, POD6, POD7 and POD11, which were up-regulated under ammonium nutrition, contributed to the reinforcement of cell walls, as confirmed by specific staining of lignin. Thus, we identified cell wall lignification as an important tolerance mechanism of root cells associated with the stunted phenotype typical of plants grown under ammonium nutrition. This work was supported by the grants AGL2014-52396-P and AGL2017-86293-P from the Spanish Ministry of Economy and Competitiveness-MINECO. RE received a Juan de la Cierva-incorporación grant IJCI-2014-21452. JB is a holder of a PhD fellowship from the Public University of Navarre. The Proteomics Unit of Navarrabiomed is a member of ProteoRed, PRB3-ISCIII-Spain, and is supported by grant PT17/0019/009, of the PE I + D+I 2018-2020 funded by ISCIII and FEDER.

Soybean is a crop of agronomic importance that requires adequate watering during its growth to achieve high production. In this study, we determined physiological, photochemical and metabolic differences in five soybean varieties selected from the parental lines of a nested association mapping population during mild drought. These varieties have been described as high yielding (NE3001, HY1; LD01‐5907, HY2) or drought tolerant (PI518751; HYD1; PI398881, HYD2). Nevertheless, there has been little research on the physiological traits that sustain their high productivity under water‐limited conditions. The results indicate that high‐yielding varieties under drought cope with the shortage of water by enhancing their photoprotective defences and invest in growth and productivity, linked to a higher intrinsic water use efficiency. This is the case of the variety N‐3001 (HY1), with a tolerance strategy involving a faster transition into the reproductive stage to avoid the drought period. The present study highlights the role of the physiological and biochemical adjustments of various soybean varieties to cope with water‐limited conditions. Moreover, the obtained results underscore the fact that the high phenotypic plasticity among soybean phenotypes should be exploited to compensate for the low genetic variability of this species when selecting plant productivity in constrained environments., This work was supported by grants from the Spanish Ministry of Science and Innovation and FEDER funds (AGL2014‐52396) and from the Basque Government (UPV/EHU‐GV IT‐1018‐16; IT‐932‐16). J.B. is a holder of a PhD fellowship from the Public University of Navarre. R.E. received a Juan de la Cierva‐incorporación grant (IJCI‐2014‐21452).

A “box‐in‐box” cocultivation system was used to investigate plant responses to microbial volatile compounds (VCs) and to evaluate the contributions of organic and inorganic VCs (VOCs and VICs, respectively) to these responses. Arabidopsis plants were exposed to VCs emitted by adjacent Alternaria alternata and Penicillium aurantiogriseum cultures, with and without charcoal filtration. No VOCs were detected in the headspace of growth chambers containing fungal cultures with charcoal filters. However, these growth chambers exhibited elevated CO2 and bioactive CO and NO headspace concentrations. Independently of charcoal filtration, VCs from both fungal phytopathogens promoted growth and distinct developmental changes. Plants cultured at CO2 levels observed in growth boxes containing fungal cultures were identical to those cultured at ambient CO2. Plants exposed to charcoal‐filtered fungal VCs, nonfiltered VCs, or superelevated CO2 levels exhibited transcriptional changes resembling those induced by increased irradiance. Thus, in the “box‐in‐box” system, (a) fungal VICs other than CO2 and/or VOCs not detected by our analytical systems strongly influence the plants' responses to fungal VCs, (b) different microorganisms release VCs with distinct action potentials, (c) transcriptional changes in VC‐exposed plants are mainly due to enhanced photosynthesis signaling, and (d) regulation of some plant responses to fungal VCs is primarily posttranscriptional., This work was partially supported by the Comisión Interministerial de Ciencia y Tecnología and Fondo Europeo de Desarrollo Regional (Spain; grants BIO2013‐49125‐C2‐1‐P and BIO2016‐78747‐P) and the Government of Navarra (refs. P1044 AGROESTI and P1004 PROMEBIO).

Carotenoids have traditionally been subscribed to their role as accessory pigments in photosynthesis. However, the large and growing body of literature investigated on the field have revealed that carotenoids fulfil a plethora of essential roles in plants but also in animals and in humans. Recent studies emphasizing the functional role of molecules derived from carotenoids oxidation as β-cyclocitral or dihydroactinidiolide led to a renewed interest, opening a new era for the carotenoids research. This review brings together the knowledge obtained so far regarding diversity and functions of carotenoids, highlighting carotenoids versatility and the remarkable parallel roles of carotenoids in both plants and in animals. Evolutionary aspects and the responses of carotenoids to biotic and abiotic stresses are discussed. Furthermore, we outline the way in which one can understand the environmental regulation to enhance carotenoid content in food. In addition, an up-to-date overview of carotenoids as elements of information storage system for the responses to environmental signals is provided together with suggestions for future directions of research., The authors acknowledge the support of research grants BFU 2010-15021 (co-funded by Feder), AGL2012-39715-CO3-01, UPV/ EHU-GV IT-624-13 and the JAE-Doc-2011-046 fellow from the Spanish National Research Council (CSIC) received by RE.

Indole-3-acetaldoxime (IAOx) is a particularly relevant molecule as an intermediate in the pathway for tryptophan-dependent auxin biosynthesis. The role of IAOx in growth-signalling and root phenotype is poorly studied in cruciferous plants and mostly unknown in non-cruciferous plants. We synthesized IAOx and applied it to M. truncatula plants grown axenically with NO3 -, NH4 + or urea as the sole nitrogen source. During 14 days of growth, we demonstrated that IAOx induced an increase in the number of lateral roots, especially under NH4 + nutrition, while elongation of the main root was inhibited. This phenotype is similar to the phenotype known as superroot previously described in SUR1- and SUR2-defective Arabidopsis mutants. The effect of IAOx, IAA or the combination of both on the root phenotype was different and dependent on the type of N-nutrition. Our results also showed the endogenous importance of IAOx in a legume plant in relation to IAA metabolism, and suggested IAOx long-distance transport depending on the nitrogen source provided. Finally, our results point out to CYP71A as the major responsible enzymes for IAA synthesis from IAOx, while they exclude indole-3-acetaldehyde oxidases. © 2019 Elsevier B.V. This work was supported by the grants AGL2017-86293-P and CGL2017-84723-P from the Spanish Ministry of Economy and Competitiveness (MINECO) , AGL2014-52396-P from the Ministry of Science Innovation and Universities (MICINN) , and IT932-16 from the Basque Government, Spain . JB and PL-G are holders of PhD fellowships from the Public University of Navarre. ACh received a Juan de la Cierva initiation grant FJCI-2016-27905 and RE received a Juan de la Cierva incorporation grant IJCI-2014-21452. This research was also supported by the Basque Government through the BERC 2018-2021 program, and by the Spanish Ministry of Science, Innovation and Universities through the BC3 María de Maeztu excellence accreditation (MDM-2017-0714).

The development of new nitrogen fertilizers is necessary to optimize crop production whilst improving the environmental aspects arising from the use of nitrogenous fertilization as a cultural practice. The use of urease inhibitors aims to improve the efficiency of urea as a nitrogen fertilizer by preventing its loss from the soil as ammonia. However, although the action of urease inhibitors is aimed at the urease activity in soil, their availability for the plant may affect its urease activity. The aim of this work was therefore to evaluate the effect of two urease inhibitors, namely acetohydroxamic acid (AHA) and N-(n-butyl) thiophosphoric triamide (NBPT), on the germination of pea and spinach seeds. The results obtained show that urease inhibitors do not affect the germination process to any significant degree, with the only process affected being imbibition in spinach, thus also suggesting different urease activities for both plants. Our findings therefore suggest an activity other than the previously reported urolytic activity for urease in spinach. Furthermore, of the two inhibitors tested, NBPT was found to be the most effective at inhibiting urease activity, especially in pea seedlings. © 2012 Elsevier GmbH., This work was supported by the Spanish MICIIN (Grant No. AGL2009-13339-CO2-02 [to P.A.T.]). S.C. was supported by a doctoral fellowship from the Public University of Navarre.

7 p., 6 figures, 1 table and references, The widespread use of NO3− fertilization has had a major ecological impact. NH4+ nutrition may help to reduce this impact, although high NH4+ concentrations are toxic for most plants. The underlying tolerance mechanisms are not yet fully understood, although they are thought to include the limitation of C, the disruption of ion homeostasis, and a wasteful NH4+ influx/efflux cycle that carries an extra energetic cost for root cells. In this study, high irradiance (HI) was found to induce a notable tolerance to NH4+ in the range 2.5–10mM in pea plants by inducing higher C availability, as shown by carbohydrate content. This capacity was accompanied by a general lower relative N content, indicating that tolerance is not achieved through higher net N assimilation on C-skeletons, and it was also not attributable to increased GS content or activity in roots or leaves. Moreover, HI plants showed higher ATP content and respiration rates. This extra energy availability is related to the internal NH4+ content regulation (probably NH4+ influx/efflux) and to an improvement of the cell ionic balance. The limited C availability at lower irradiance (LI) and high NH4+ resulted in a series of metabolic imbalances, as reflected in a much higher organic acid content, thereby suggesting that the origin of the toxicity in plants cultured at high NH4+ and LI is related to their inability to avoid large-scale accumulation of the NH4+ ion., This work was supported by the Spanish MICIIN (grant nos. AGL2006-12792-CO2-01 and AGL2009-13339-CO2-02 [to P.A.-T.] and AGL2007-64432/AGR [to J.F.M.]). IA was supported by a doctoral Fellowship from the Public University of Navarre.

6 p., 4 figures, 1 table and bibliography, In plants, it has been proposed that hexacoordinate (class 1) non-symbiotic Hbs (nsHb-1) function in vivo as peroxidases. However, little is known about peroxidase activity of nsHb-1. We evaluated the peroxidase activity of rice recombinant Hb1 (a nsHb-1) by using the guaiacol/H2O2 system at pH 6.0 and compared it to that from horseradish peroxidase (HRP). Results showed that the affinity of rice Hb1 for H2O2 was 86-times lower than that of HRP (Km = 23.3 and 0.27 mM, respectively) and that the catalytic efficiency of rice Hb1 for the oxidation of guaiacol using H2O2 as electron donor was 2838-times lower than that of HRP (kcat/Km = 15.8 and 44 833 mM-1 min-1, respectively). Also, results from this work showed that rice Hb1 is not chemically modified and binds CO after incubation with high H2O2 concentration, and that it poorly protects recombinant Escherichia coli from H2O2 stress. These observations indicate that rice Hb1 inefficiently scavenges H2O2 as compared to a typical plant peroxidase, thus indicating that non-symbiotic Hbs are unlikely to function as peroxidases in planta., This work was financed by SEP-PROMEP (grant number UAEMor-PTC-01-01/PTC23) and Consejo Nacional de Ciencia y Tecnología (CoNaCyT grant number 42873Q), México, to R.A.-P., and by DGI-MICINN, Spain (grant AGL 2007-64432/AGR) to J. F. M. F. V.-M. was a recipient of an undergraduate fellowship from PROMEP and CoNaCyT (IdAP 9891).

Editors: Kapuganti Jagadis Gupta, Abir U. Igamberdiev, Xanthine oxidoreductase (XOR) is among the most-intensively studied enzymes known to participate in the consumption of oxygen in eukaryotic cells. However, it attracted the attention of researchers due its participation in free radical production in vivo, mainly through the production of superoxide radicals. In plants, XOR is a key enzyme involved in purine degradation where it catalyzes the oxidation of hypoxanthine to xanthine and of xanthine to uric acid. Both reactions are accompanied by electron transfer to either NAD+ with simultaneous formation of NADH or to molecular oxygen, which results in formation of superoxide radicals. Characterization of plant XOR mutants and isolated XOR proteins from various plant species provided evidence that the enzyme plays significant role in plant growth, leaf senescence, fruit size, synthesis of nitrogen storage compounds, and plant–pathogen interactions. Moreover, the ability of XOR to carry out redox reactions as NADH oxidase and to produce reactive oxygen species and nitric oxide, together with a possible complementary role in abscisic acid synthesis have raised further attention on the importance of this enzyme. Based on these established and proposed functions, XOR is discussed as regulator of different processes of interest in plant biology and agriculture., The authors acknowledge the support of the research grants AGL2010-16167 to J.F.M. from the Spanish Ministry of Science and Innovation and Bi 1075/5-1 to F.B. by the Deutsche Forschungsgemeinschaft. R.E. received a JAE-Doctor grant from the Spanish Research Council (CSIC).

Phosphate starvation compromises electron fow through the cytochrome pathway of the mitochondrial electron transport chain, and plants commonly respond to phosphate deprivation by increasing fow through the alternative oxidase (AOX). To test whether this response is linked to the increase in nitric oxide (NO) production that also increases under phosphate starvation, Arabidopsis thaliana seedlings were grown for 15 d on media containing either 0 or 1 mM inorganic phosphate. The effects of the phosphate supply on growth, the production of NO, respiration, the AOX level and the production of superoxide were compared for wild-type (WT) seedlings and the nitrate reductase double mutant nia. Phosphate deprivation increased NO production in WT roots, and the AOX level and the capacity of the alternative pathway to consume electrons in WT seedlings; whereas the same treatment failed to stimulate NO production and AOX expression in the nia mutant, and the plants had an altered growth phenotype. The NO donor S-nitrosoglutathione rescued the growth phenotype of the nia mutants under phosphate deprivation to some extent, and it also increased the respiratory capacity of AOX. It is concluded that NO is required for the induction of the AOX pathway when seedlings are grown under phosphate-limiting conditions., This work was supported by research grants (AGL2010-16167; AGL2014-52396-P) from the Spanish Ministry of Economy and Competitiveness (MINECO) to JFM, an FPI fellowship from MINECO (BR), and a Marie Curie Intra-European Fellowship for Career Development within the 7th European Community Framework Programme (KJG and RGR).

16 Pags.- 2 Figs. The definitive version, with all 3 Figs., is available at: http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1399-3054, The thiol tripeptide glutathione (GSH; γGlu-Cys-Gly) is very abundant in legume nodules where it performs multiple functions that are critical for optimal nitrogen fixation. Some legume nodules contain another tripeptide, homoglutathione (hGSH; γGlu-Cys-βAla), in addition to or instead of GSH. We have isolated from a pea (Pisum sativum L.) nodule library a cDNA, GSHS2, that is expressed in nodules but not in leaves. This cDNA was overexpressed in insect cells and its protein product was identified as a highly active and specific hGSH synthetase. The enzyme, the first of this type to be completely purified, is predicted to be a homodimeric cytosolic protein. It shows a specific activity of 3400 nmol hGSH min-1 mg-1 protein with a standard substrate concentration (5 mM β-alanine) and Km values of 1.9 mM for β-alanine and 104 mM for glycine. The specificity constant (Vmax/Km) shows that the pure enzyme is 57.3-fold more specific for β-alanine than for glycine. Southern blot analysis revealed that the gene is present as a single copy in the pea genome and that there are homologous genes in other legumes. We conclude that the synthesis of hGSH in pea nodules is catalysed by a specific hGSH synthetase and not by a GSH synthetase with broad substrate specificity., M.A.M. was the recipient of a predoctoral fellowship from the Basque Government (Spain). This work was supported by grant no. 2FD97-1101 from the Comisión Interministerial de Ciencia y Tecnología and the European Union and by grant no. PB98-0522 from the Dirección General de Investigación Científica (Spain).

Nitric oxide cytotoxicity arises from its rapid conversion to peroxynitrite (ONOO(-)) in the presence of superoxide, provoking functional changes in proteins by nitration of tyrosine residues. The physiological significance of this post-translational modification is associated to tissue injury in animals, but has not been yet clarified in plants. The objective of this study was to establish new approaches that could help to understand ONOO(-) reactivity in plants. A recombinant Fe-superoxide dismutase from cowpea (Vigna unguiculata (L.) Walp.), rVuFeSOD, was the target of the ONOO(-)-generator SIN-1, and the anti-nitrative effect of plant antioxidants and haemoglobins was tested in vitro. Nitration on rVuFeSOD was evaluated immunochemically or as the loss of its enzymatic activity. This assay proved to be useful to test a variety of plant compounds for anti-nitrative capacity. Experimental data confirmed that rice (Oryza sativa L.) haemoglobin-1 (rOsHbI) and cowpea leghaemoglobin-2 exerted a protective function against ONOO(-) by diminishing nitration on rVuFeSOD. Both plant haemoglobins were nitrated by SIN-1. The chelator desferrioxamine suppressed nitration in rOsHbI, indicating that Fe plays a key role in the reaction. The removal of the haem moiety in rOsHbI importantly suppressed nitration, evidencing that this reaction may be self-catalyzed. Among small antioxidants, ascorbate remarkably decreased nitration in all tests. The phenolic compounds caffeic acid, gallic acid, pyrogallol, 4-hydroxybenzoic acid and the flavonoid gossypin also diminished tyrosine nitration and protected rVuFeSOD to different extents. It is concluded that small plant antioxidants, especially ascorbate, and haemoglobins may well play key roles in ONOO(-) homeostasis in vivo., E.U. was the recipient of a Ph.D. fellowship from the Public University of Navarre (UPNa). Financial support was provided by the MICINN/MINECO, Government of Spain (grant AGL2010-16167). A.C.A. and E.T. were supported by the project SABioD (Department of Innovation, Government of Navarra).

Gold nanostructures can undergo plasmonic behavior without need of light couplers like in traditional surface plasmon resonance (SPR) systems. This effect, known as LSPR (localized SPR), can be exploited to develop optical biosensors in simple configuration. In this paper, an LSPR system based on gold lines nanopattern as transducer has been developed by using a common laboratory spectrophotometer as reader and an adapted flow cell. This novel system was applied to anti-immunoglobulin G (IgG) detection, as proof of concept. For this purpose, the transducer surface was covalently biofunctionalized with IgG and incubated with increasing concentrations of anti-IgG. The response was determined by measuring changes in light transmission spectra (¿¿, in nanometers). Contrary to other reported nanopatterns, biosensor features as repeatability, regeneration, and detectability have been deeply studied. Results indicate that the system can act as a robust and regenerable biosensor with a limit of detection of 5.98 ± 1.34 nM.

28 Pags.- 2 Figs. the definitive version is available at: http://onlinelibrary.wiley.com/journal/10.1111/%28ISSN%291399-3054, Reactive oxygen species are a ubiquitous danger for aerobic organisms. This risk is especially elevated in legume root nodules due to the strongly reducing conditions, the high rates of respiration, the tendency of leghemoglobin to autoxidize, the abundance of nonprotein Fe and the presence of several redox proteins that leak electrons to O(2). Consequently, nodules are particularly rich in both quantity and diversity of antioxidant defenses. These include enzymes such as superoxide dismutase (EC 1.15.1.1) and ascorbate peroxidase (EC 1.11.1.11) and metabolites such as ascorbate and thiol tripeptides. Nodule antioxidants have been the subject of intensive molecular, biochemical and functional studies that are reviewed here. The emerging theme is that antioxidants are especially critical for the protection and optimal functioning of N(2) fixation. We hypothesize that this protection occurs at least at two levels: the O(2) diffusion barrier in the nodule parenchyma (inner cortex) and the infected cells in the central zone., Work in the laboratory of M. B. was supported by grants PB98-0522, 2FD97-1101 and HB98-163 from Dirección General de Enseñanza Superior e Investigación Científica, Comisión Interministerial de Ciencia y Tecnología, and the European Union. Work in the laboratory of D. A. D. was supported by National Science Foundation grants DCB-8903254, IBN-9206453 and IBN-9507491.

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.

Non-symbiotic hemoglobin from rice (Oryza sativa L.), OsHb-1, with its rather hexacoordinated than pentacoordinated heme and high affinity for oxygen, may have a particular role in O2 and environmental sensing. Here, the 21 kDa monomeric OsHb-1 was electrochemically studied at graphite electrodes and further probed in analysis of environmental species such as hydrogen peroxide, cyanide, and superoxide. Redox potential of the OsHb-1 heme iron was found to be -136 mV vs. SCE, at pH 6.5, while the rate constant ks for the heterogeneous electron transfer (ET) between graphite and OsHb-1 immobilised in the Nafion membrane at the carbon nanotubes-modified electrodes was below 0.2 s-1. Despite sluggish ET, OsHb-1 efficiently, with current densities exceeding 2 mA cm-2 at ¿0.3 V, electrocatalyzed reduction of O2 starting from the potentials of OsHb-1¿s heme. The bioelectrocatalytic reduction of O2 was inhibited by CN- thus enabling its sensitive, 100 pM analysis. Peroxidase-like activity of OsHb-1 and the reaction of superoxide anion with the heme iron of OsHb-1, in de-oxygenated solutions, were studied and analysed in terms of OsHb-1 reactivity. The results obtained indicate OsHb-1 is a sensitive tool for environmental biosensing and toxicity screening, The work was supported by the Danish Council for Independent Research, Natural Sciences (FNU), Project Number 11-107176, and by the Spanish Ministry of Economy and Competitiveness (Grant No. AGL2010-16167).

12 Pags.- 1 Tabl.- 2 Figs. The definitive version is available at: http://onlinelibrary.wiley.com/journal/10.1111/%28ISSN%291399-0047, Eukaryotic iron superoxide dismutases (FeSODs) are homodimeric proteins that constitute a fundamental protection against free radicals, which can damage essential cellular mechanisms. The protein was cloned and overexpressed in Escherichia coli with an N-terminal His tag. Crystallization experiments of the protein resulted, after several refined screenings, in crystals suitable for X-ray diffraction analysis. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 82.54, b = 48.41, c = 64.28 Å, α = γ = 90, β = 119.66°, and contain one molecule per asymmetric unit. At cryogenic temperatures, the crystals diffracted to a resolution limit of 1.80 Å using synchrotron radiation at the European Synchrotron Radiation Facility (ESRF)., IGM thanks CNIO for a postdoctoral fellowship. JFM thanks CSIC-EU for an I3P postdoctoral contract. Full financial support was obtained through a CNIO internal grant to GM and partial support by grant PB-0522 (DGIC) to MB.

8 Pags., 9 Figs., Three main families of SODs in plants may be distinguished according to the metal in the active center: CuZnSODs, MnSOD, and FeSOD. CuZnSODs have two sub-families localized either in plant cell cytosol or in plastids, the MnSOD family is essentially restricted to mitochondria, and the FeSOD enzyme family has been typically localized into the plastid. Here, we describe, based on a phylogenetic tree and experimental data, the existence of two FeSOD sub-families: a plastidial localized sub-family that is universal to plants, and a cytosolic localized FeSOD sub-family observed in determinate-forming nodule legumes. Anti-cytosolic FeSOD (cyt_FeSOD) antibodies were employed, together with a novel antibody raised against plastidial FeSOD (p_FeSOD). Stress conditions, such as nitrate excess or drought, markedly increased cyt_FeSOD contents in soybean tissues. Also, cyt_FeSOD content and activity increased with age in both soybean and cowpea plants, while the cyt_CuZnSOD isozyme was predominant during early stages. p_FeSOD in leaves decreased with most of the stresses applied, but this isozyme markedly increased with abscisic acid in roots. The great differences observed for p_FeSOD and cyt_FeSOD contents in response to stress and aging in plant tissues reveal distinct functionality and confirm the existence of two immunologically differentiated FeSOD sub-families. The in-gel FeSOD activity patterns showed a good correlation to cyt_FeSOD contents but not to those of p_FeSOD. This indicates that cyt_FeSOD is the main active FeSOD in soybean and cowpea tissues. The diversity of functions associated with the complexity of FeSOD isoenzymes depending of the location is discussed., This work was supported by the Spanish MICINN (Grant Nos. AGL2007-64432/AGR and AGL2010-16167) and by the Government of Navarra (Resolución 57/2007). M.G. and L.P. are grateful for fellowships from the Dept. of Innovation of the Government of Navarra, Spain.

This is published as paper No. 10577, Journal Series, Agriculture Research Division, University of Nebraska-Lincoln., Hemoprotein derivatives of an abundant soybean (Glycine max Merr.) root nodule leghemoglobin, Lba, were studied for their modified spectral characteristics and physical properties. Three modified hemoprotein derivatives of Lba (Lbam1, Lbam2, and Lbam3) were purified by preparative isoelectric focusing. The ferric forms of these pigments were green and exhibited anomalous spectra in the visible region as compared to the Lba3+ forms. These modified pigments showed a hypochromic shift of 10 nm for the charge transfer absorption maximum; however, differences were not apparent in the Soret region. Upon binding with nicotinate, the [alpha] and [beta] bands were shifted significantly into the red region as compared to the Lba3+ nicotinate complex. The three Lbam fractions were reduced by dithionite or by NADH in the presence of riboflavin. Lbam2+ also bound nicotinate and displayed absorption spectra indistinguishable from those of Lba2+ nicotinate. In contrast to Lba2+, Lbam2+ displayed aberrant spectra when bound with either O2 or CO. These complexes exhibited a prominent charge transfer band at approximately 620 nm and failed to exhibit spectra characteristic of Lba2+O2 and Lba2+CO. The protein moiety of these modified pigments was intact because their tyrosine/tryptophan ratios and their amino acid compositions were identical with those of Lba, nor were differences observed in the peptide profiles resulting from trypsin digests of purified Lba and Lbams. Automated Edman degradation of selected peaks further confirmed the intactness of the protein backbone including the absence of deamination. Pyridine hemochromogen for heme from Lbams could be formed, and the spectra displayed distinct differences compared to those of Lba. A new peak at 580 nm and a loss of a peak at 480 nm were observed for all three Lbams., This work was supported by grants from the Nebraska Soybean Development, Utilization and Marketing Board (F.W.W.), The Center for Biotechnology, University of Nebraska-Lincoln (G.S.), and Dirección General de Investigación Científica y TecnolÓgica (grant PB92- 0058; M.B.).

The form of nitrogen nutrition affects many biochemical and physiological processes in plants, leading to markedly different growth responses. Many plant species have been characterized as ammonium tolerant or sensitive. The objective of this work was to assess the range of physiological adaptative responses involved in tolerance of ammonium nutrition at the intra-specific level. Pisum sativum L was selected as a model of tolerance for the study, and four varieties of agricultural importance (Snap-pea, Rondo, Tristar and Eclipse) were grown hydroponically with ammonium or nitrate (0.5, 2.5, 5.0 and 10.0 mM)as the sole nitrogen source. Physiological parameters including photosynthesis, respiration and ammonium contents were analyzed. Activities/expression of key enzymes of the nitrogen metabolism was also determined, for which novel antibodies against glutamate dehydrogenase and glutamine synthetase were obtained. Snap-pea was considered a reference plant for ammonium tolerance, since its biomass accumulation was not affected by nitrogen source. It avoided the physiological effects of ammonium through low growth potential, high respiratory rates and activity of the alternative pathway. A multivariate analysis of the results showed that each of the four varieties had distinct adaptative responses to ammonium nutrition, highlighting the flexibility of response to ammonium nutrition at the species level. A general activation of glutamate dehydrogenase (GDH) was observed, regardless of the degree of tolerance to ammonium, which may be due to GDH being part of a common stress response to carbon deficit rather than an ammonium tolerance mechanism. (C) 2010 Elsevier B.V. All rights reserved., This work was supported by the Spanish Ministry of Science and Innovation (grant nos. AGL2006-12792-CO2-01 and AGL2003-06571-CO2-01 [to PA-T] and AGL2007-64432/AGR [to JFM]), by the Government of Navarra (Res 57/2007 to JFM). MDD-V was supported by a doctoral Fellowship from the Basque Government, Spain.

The use of urea as an N fertilizer has increased to such an extent that it is now the most widely used fertilizer in the world. However, N losses as a result of ammonia volatilization lead to a decrease in its efficiency, therefore different methods have been developed over the years to reduce these losses. One of the most recent involves the use of urea combined with urease inhibitors, such as N-(n-butyl) thiophosphoric triamide (NBPT), in an attempt to delay the hydrolysis of urea in the soil. The aim of this study was to perform an in-depth analysis of the effect that NBPT use has on plant growth and N metabolism. Wheat plants were cultivated in a greenhouse experiment lasting 4 weeks and fertilized with urea and NBPT at different concentrations (0, 0.012, 0.062, 0.125%). Each treatment was replicated six times. A non-fertilized control was also cultivated. Several parameters related with N metabolism were analysed at the end of growth period. NBPT use was found to have visible effects, such as a transitory yellowing of the leaf tips, at the end of the first week of treatment. At a metabolic level, plants treated with the inhibitor were found to have more urea in their tissues and a lower amino acid content, lower glutamine synthetase activity, and lower urease and glutamine synthetase content at the end of the study period, whereas their urease activity seemed to have recovered by this stage, This work was supported by the Spanish MICIIN (grant no. AGL2009-13339-CO2-02 [to P.A.T.]). S.C was supported by a doctoral fellowship from the Public University of Navarre.

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, O2, 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., This work was supported by Deutsche Forschungsgemeinschaft (BA 1177/8-1) (KJG, HB), Deutsche Forschungsgemeinschaft (SFB 567) (WMK), King Saud University in Riyadh, Saudi Arabia (WMK) and Natural Sciences and Engineering Research Council of Canada (AUI). Spanish MICIIN grant no. AGL2010-16167 (JFM), European Science Foundation (ESF) Functional Dynamics in Complex Chemical and Biological Systems visiting grant to KJG and JFM.

13 p., il., tablas y bibliografía, Background: In 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. Results: Several 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. Conclusions: This 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., The authors wish to thank to Gustavo Garijo for technical assistance. This work was supported by the Spanish MICIIN (grant nos. AGL2006-12792-CO2-01 and 02 and AGL2009- 13339-CO2-01 and 02 [to P.A.-T. and C.G.M.] and AGL2007-64432/AGR [to J.F.M.]), by the Portuguese FCT (PTDC/BIA- BEC/099323/2008) and by the Basque Government IT526-10. IA was supported by a postdoctoral Fellowship from the Public University of Navarre. Technical support was provided by SGIker to the UPV/EHU researchers.

8 p., 7 figures, 2 tables and references, The application of urease inhibitors in conjunction with urea fertilizers as a means of reducing N loss due to ammonia volatilization requires an in-depth study of the physiological effects of these inhibitors on plants. The aim of this study was to determine how the urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT) affects N metabolism in pea and spinach. Plants were cultivated in pure hydroponic culture with urea as the sole N source. After 2 weeks of growth for pea, and 3 weeks for spinach, half of the plants received NBPT in their nutrient solution. Urease activity, urea and ammonium content, free amino acid composition and soluble protein were determined in leaves and roots at days 0, 1, 2, 4, 7 and 9, and the NBPT content in these tissues was determined 48 h after inhibitor application. The results suggest that the effects of NBPT on spinach and pea urease activity differ, with pea being most affected by this treatment, and that the NBPT absorbed by the plant caused a clear inhibition of the urease activity in pea leaf and roots. The high urea concentration observed in leaves was associated with the development of necrotic leaf margins, and was further evidence of NBPT inhibition in these plants. A decrease in the ammonium content in roots, where N assimilation mainly takes place, was also observed. Consequently, total amino acid contents were drastically reduced upon NBPT treatment, indicating a strong alteration of the N metabolism. Furthermore, the amino acid profile showed that amidic amino acids were major components of the reduced pool of amino acids. In contrast, NBPT was absorbed to a much lesser degree by spinach plants than pea plants (35% less) and did not produce a clear inhibition of urease activity in this species., This work was supported by the Spanish MICIIN (grant no. AGL2009-13339-CO2-02 [to P.A.T.]). S.C was supported by a doctoral fellowship from the Public University of Navarre.

8 p., 7 figures and bibliography, Photosynthesis provides plant metabolism with reduced carbon (C) but is also the main source of oxidative stress in plants. Likewise, high doses of NH4+ as sole N source have been reported to be toxic for most plants, resulting in reduced plant growth and restricting C availability. The combination of high photosynthetic photon flux densities (PPFD) and NH4+ nutrition may provide higher C availability but could also have a detrimental effect on the plants, therefore the objective of this study is to evaluate whether NH4+ induces photo-oxidative stress that is exacerbated under high light conditions. Pea plants (Pisum sativum cv. sugar-snap) were grown hydroponically with NH4+ (0.5, 2.5, 5 and 10mM) under high (750μmolphotonsm -2s -1) or low PPFD conditions (350μmolphotonsm -2s -1). High PPFD contributes to a higher tolerance to ammonium by pea plants, as it originated higher biomass content due to higher photosynthetic rates. However, a deficit of N (0.5 and 2.5mM NH4+) under high PPFD conditions caused an antioxidant response, as indicated by increased photoprotective pigment and chloroplastic superoxide dismutase contents. Plants grown with higher doses of N and high PPFD showed less need for photoprotection. An increase in the specific leaf weight (SLW) ratio was observed associated not only with high PPFDs but also with the highest NH4+ dose. Overall, these results demonstrate that, despite the activation of some photoprotective responses at high PPFD, there were no photoinhibitory symptoms and a positive effect on NH4+ toxicity, thus suggesting that the harmful effects of NH 4 + are not directly related to the generation of photo-oxidative stress., This work was supported by the Spanish MICIIN (grant nos. AGL2006-12792-CO2-01 and AGL2003-06571-CO2-01 [to P.A.-T.] and AGL2007-64432/AGR [to J.F.M.]), by the Government of Navarra (Res 57/2007 to J.F.M.), and BFU 2007-62637 of MEC and EHU-GV IT-299-07 of the Basque Government to JMB and JIG-P. IA was supported by a doctoral Fellowship from the Public University of Navarre, Spain.

Ferric leghemoglobin reductase (FLbR) is able to reduce ferric leghemoglobin (Lb3+) to ferrous (Lb2+) form. This reaction makes Lb functional in performing its role since only reduced hemoglobins bind O2. FLbR contains FAD as prosthetic group to perform its activity. FLbR-1 and FLbR-2 were isolated from soybean root nodules and it has been postulated that they reduce Lb3+. The existence of Lb2+ is essential for the nitrogen fixation process that occurs in legume nodules; thus, the isolation of FLbR for the study of this enzyme in the nodule physiology is of interest. However, previous methods for the production of recombinant FLbR are inefficient as yields are too low. We describe the production of a recombinant FLbR-2 from Escherichia coli BL21(DE3) by using an overexpression method based on the self-induction of the recombinant E. coli. This expression system is four times more efficient than the previous overexpression method. The quality of recombinant FLbR-2 (based on spectroscopy, SDS-PAGE, IEF, and native PAGE) is comparable to that of the previous expression system. Also, FLbR-2 is purified near to homogeneity in only few steps (in a time scale, the full process takes 3 days). The purification method involves affinity chromatography using a Ni-nitrilotriacetic acid column. Resulting rFLbR-2 showed an intense yellow color, and spectral characterization of rFLbR-2 indicated that rFLbR-2 contains flavin. Pure rFLbR-2 was incubated with soybean Lba and NADH, and time drive rates showed that rFLbR-2 efficiently reduces Lb3+.

Ferric leghemoglobin reductase (FLbR) is able to reduce ferric leghemoglobin (Lb3+) to ferrous (Lb2+) form. This reaction makes Lb functional in performing its role since only reduced hemoglobins bind O2. FLbR contains FAD as prosthetic group to perform its activity. FLbR-1 and FLbR-2 were isolated from soybean root nodules and it has been postulated that they reduce Lb3+. The existence of Lb2+ is essential for the nitrogen fixation process that occurs in legume nodules; thus, the isolation of FLbR for the study of this enzyme in the nodule physiology is of interest. However, previous methods for the production of recombinant FLbR are inefficient as yields are too low. We describe the production of a recombinant FLbR-2 from Escherichia coli BL21(DE3) by using an overexpression method based on the self-induction of the recombinant E. coli. This expression system is four times more efficient than the previous overexpression method. The quality of recombinant FLbR-2 (based on spectroscopy, SDS-PAGE, IEF, and native PAGE) is comparable to that of the previous expression system. Also, FLbR-2 is purified near to homogeneity in only few steps (in a time scale, the full process takes 3 days). The purification method involves affinity chromatography using a Ni-nitrilotriacetic acid column. Resulting rFLbR-2 showed an intense yellow color, and spectral characterization of rFLbR-2 indicated that rFLbR-2 contains flavin. Pure rFLbR-2 was incubated with soybean Lba and NADH, and time drive rates showed that rFLbR-2 efficiently reduces Lb3+.

Superoxide dismutases (SODs; EC 1.15.1.1) are a group of metalloenzymes which are essential to protect cells under aerobic conditions. In biological systems, it has been reported that SODs and other proteins are susceptible to be attacked by peroxynitrite (ONOO(-)) which can be originated from the reaction of nitric oxide with superoxide radical. ONOO(-) is a strong oxidant molecule capable of nitrating peptides and proteins at the phenyl side chain of the tyrosine residues. In the present work, bovine serum albumin (BSA) and recombinant iron-superoxide dismutase from the plant cowpea (Vu_FeSOD) are used as target molecules to estimate ONOO(-) production. The method employs the compound SIN-1, which simultaneously generates *NO and O(2)(-) in aerobic aqueous solutions. First, assay conditions were optimized incubating BSA with different concentrations of SIN-1, and at a later stage, the effect on the tyrosine nitration and catalytic activity of Vu_FeSOD was examined by in-gel activity and spectrophotometric assays. Both BSA and Vu_FeSOD are nitrated in a dose-dependent manner, and, at least in BSA nitration, the reaction seems to be metal catalyzed.

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.

Nitrogen fixation (NF) in legume nodules is very sensitive to environmental constraints. Nodule sucrose synthase (SS; EC 2.4.1.13) has been suggested to play a crucial role in those circumstances because its downregulation leads to an impaired glycolytic carbon flux and, therefore, a depletion of carbon substrates for bacteroids. In the present study, the likelihood of SS being regulated by oxidative signaling has been addressed by the in vivo supply of paraquat (PQ) to nodulated pea plants and the in vitro effects of oxidizing and reducing agents on nodule SS. PQ produced cellular redox imbalance leading to an inhibition of NF. This was preceded by the downregulation of SS gene expression, protein content, and activity. In vitro, oxidizing agents were able to inhibit SS activity and this inhibition was completely reversed by the addition of dithiothreitol. The overall results are consistent with a regulation model of nodule SS exerted by the cellular redox state at both the transcriptional and post-translational levels. The importance of such mechanisms for the regulation of NF in response to environmental stresses are discussed.

Nodule ferric leghemoglobin reductase (FLbR) and leaf dihydrolipoamide reductase (DLDH) belong to the same family of pyridine nucleotide-disulfide oxidoreductases. We report here the cloning, expression, and characterization of a second protein with FLbR activity, FLbR-2, from soybean (Glycine max) nodules. The cDNA is 1,779 bp in length and codes for a precursor protein comprising a 30-residue mitochondrial transit peptide and a 470-residue mature protein of 50 kD. The derived protein has considerable homology with soybean nodule FLbR-1 (93% identity) and pea (Pisum sativum) leaf mitochondria DLDH (89% identity). The cDNA encoding the mature protein was overexpressed in Escherichia coli. The recombinant enzyme showed Km and kcat values for ferric leghemoglobin that were very similar to those of DLDH. The transcripts of FLbR-2 were more abundant in stems and roots than in nodules and leaves. Immunoblots of nodule fractions revealed that an antibody raised against pea leaf DLDH cross-reacted with recombinant FLbR-2, native FLbR-2 of soybean nodule mitochondria, DLDH from bacteroids, and an unknown protein of approximately 70 kD localized in the nodule cytosol. Immunogold labeling was also observed in the mitochondria, cytosol, and bacteroids of soybean nodules. The similar biochemical, kinetic, and immunological properties, as well as the high amino acid sequence identity and mitochondrial localization, draw us to conclude that FLbR-2 is soybean DLDH.

This is journal paper no. 12,643, Agricultural Research Division, University of Nebraska., Nodule ferric leghemoglobin reductase (FLbR) and leaf dihydrolipoamide reductase (DLDH) belong to the same family of pyridine nucleotide-disulfide oxidoreductases. We report here the cloning, expression, and characterization of a second protein with FLbR activity, FLbR-2, from soybean (Glycine max) nodules. The cDNA is 1,779 bp in length and codes for a precursor protein comprising a 30-residue mitochondrial transit peptide and a 470-residue mature protein of 50 kD. The derived protein has considerable homology with soybean nodule FLbR-1 (93% identity) and pea (Pisum sativum) leaf mitochondria DLDH (89% identity). The cDNA encoding the mature protein was overexpressed in Escherichia coli. The recombinant enzyme showed Km and kcat values for ferric leghemoglobin that were very similar to those of DLDH. The transcripts of FLbR-2 were more abundant in stems and roots than in nodules and leaves. Immunoblots of nodule fractions revealed that an antibody raised against pea leaf DLDH cross-reacted with recombinant FLbR-2, native FLbR-2 of soybean nodule mitochondria, DLDH from bacteroids, and an unknown protein of approximately 70 kD localized in the nodule cytosol. Immunogold labeling was also observed in the mitochondria, cytosol, and bacteroids of soybean nodules. The similar biochemical, kinetic, and immunological properties, as well as the high amino acid sequence identity and mitochondrial localization, draw us to conclude that FLbR-2 is soybean DLDH., This work was supported by the National Science Foundation (grant no. OSR-92552255) and the U.S. Department of Agriculture-Cooperative State Research Education and Extension Service (grant no. 95-37305-2441). Access to the BioCad workstation was provided by the Center for Biotechnology at the University of Nebraska, Lincoln, funded through the Nebraska Research Initiative. J.F.M. was the recipient of a postdoctoral contract from the Ministry of Education and Culture (Spain).

This review discusses the major findings from the current studies on rice Hbs. At the molecular level, a family of the nshb genes, consisting of hb1, hb2, hb3, hb4 and hb5, and a single copy of the thb gene exist in Oryza sativa var. indica and O. sativa var. japonica, Hb transcripts coexist in rice organs and Hb polypeptides exist in rice embryonic and vegetative organs and in the cytoplasm of differentiating cells. At the structural level, the crystal structure of rice Hb1 has been elucidated, and the structures of the other rice Hbs have been modeled. Kinetic analysis indicated that rice Hb1 and 2, and possibly rice Hb3 and 4, exhibit a very high affinity for O 2, whereas rice Hb5 and tHb possibly exhibit a low to moderate affinity for O 2. Based on the accumulated information on the properties of rice Hbs and data from the analysis of other plant and non-plant Hbs, it is likely that Hbs play a variety of roles in rice organs, including O 2-transport, O 2-sensing, NO-scavenging and redox-signaling. From an evolutionary perspective, an outline for the evolution of rice Hbs is available. Rice nshb and thb genes vertically evolved through different lineages, rice nsHbs evolved into clade I and clade II lineages and rice nshbs and thbs evolved under the effect of neutral selection. This review also reveals lacunae in our ability to completely understand rice Hbs. Primary lacunae are the absence of experimental information about the precise functions of rice Hbs, the properties of modeled rice Hbs and the cis-elements and trans-acting factors that regulate the expression of rice hb genes, and the partial understanding of the evolution of rice Hbs., We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)

High-performance liquid chromatography (HPLC) with fluorescence detection was used to study thiol metabolism in legume nodules. Glutathione (GSH) was the major non-protein thiol in all indeterminate nodules examined, as well as in the determinate nodules of cowpea (Vigna unguiculata), whereas homoglutathione (hGSH) predominated in soybean (Glycine max), bean (Phaseolus vulgaris), and mungbean (Vigna radiata) nodules. All nodules had greater thiol concentrations than the leaves and roots of the same plants because of active thiol synthesis in nodule tissue. The correlation between thiol tripeptides and the activities of glutathione synthetase (GSHS) and homoglutathione synthetase (hGSHS) in the nodules of eight legumes, and the contrasting thiol contents and activities in alfalfa (Medicago sativa) leaves (98% hGSH, 100% hGSHS) and nodules (72% GSH, 80% GSHS) indicated that the distribution of GSH and hGSH is determined by specific synthetases. Thiol contents and synthesis decreased with both natural and induced nodule senescence, and were also reduced in the senescent zone of indeterminate nodules. Thiols and GSHS were especially abundant in the meristematic and infected zones of pea (Pisum sativum) nodules. Thiols and -glutamylcysteinyl synthetase were also more abundant in the infected zone of bean nodules, but hGSHS was predominant in the cortex. Isolation of full-length cDNA sequences coding for -glutamylcysteinyl synthetase from legume nodules revealed that they are highly homologous to those from other higher plants., This work was supported by the Dirección General de Enseñanza Superior e Investigación Científica (Ministry of Education and Culture, Spain; grant nos. PB98-0522, 2FD97-1101, and HB98-163). M.A.M., J.F.M., I.I.-O., and M.C.R. were the recipients, respectively, of a predoctoral fellowship from the Gobierno Vasco, a postdoctoral contract from the Ministry of Education and Culture, a postdoctoral fellowship from the European Union (Training and Mobility Program), and a predoctoral fellowship from the Ministry of Education and Culture.

40 Pags.- 6 Tabls. The definitive version, with Figs., is available at: http://www.plantphysiol.org/, Nitrate-fed and dark-stressed bean (Phaseolus vulgaris) and pea (Pisum sativum) plants were used to study nodule senescence. In bean, 1 d of nitrate treatment caused a partially reversible decline in nitrogenase activity and an increase in O2 diffusion resistance, but minimal changes in carbon metabolites, antioxidants, and other biochemical parameters, indicating that the initial decrease in nitrogenase activity was due to O2 limitation. In pea, 1 d of dark treatment led to a 96% decline in nitrogenase activity and sucrose, indicating sugar deprivation as the primary cause of activity loss. In later stages of senescence (4 d of nitrate or 2–4 d of dark treatment), nodules showed accumulation of oxidized proteins and general ultrastructural deterioration. The major thiol tripeptides of untreated nodules were homoglutathione (72%) in bean and glutathione (89%) in pea. These predominant thiols declined by approximately 93% after 4 d of nitrate or dark treatment, but the loss of thiol content can be only ascribed in part to limited synthesis by γ-glutamylcysteinyl, homoglutathione, and glutathione synthetases. Ascorbate peroxidase was immunolocalized primarily in the infected and parenchyma (inner cortex) nodule cells, with large decreases in senescent tissue. Ferritin was almost undetectable in untreated bean nodules, but accumulated in the plastids and amyloplasts of uninfected interstitial and parenchyma cells following 2 or 4 d of nitrate treatment, probably as a response to oxidative stress., Supported by grants nos. PB95-0091 and PB98-0522 from the Dirección General de Enseñanza Superior e Investigación Científica (Ministry of Education and Culture, Spain) to M.B.

6 Pags.- 3 Tabls.- 1 Fig., on-exchange high performance liquid chromatography (HPLC) with an Aminex column and photodiode-array detection was used to purify, from soybean root nodules, highly polar phenolic acid conjugates that are recalcitrant to isolation by conventional techniques based on reversed-phase chemistry. HPLC, acid and enzymatic hydrolysis, ultraviolet spectroscopy, high resolution fast atom bombardment mass spectrometry (MS) and single and tandem electrospray MS were used to identify the 4-O-β-glucosides of p-hydroxybenzoic, protocatechuic and vanillic acids (which are reported in legumes for the first time) and the 5-O-β-glucoside of gentisic acid. The four phenolic acids, as well as gallic acid and its methyl ester, were also found in soybean nodules., This work was financed by Acciones Hispano-Britá́nicas HB91-218 and HB92-185, and grant PB95-0091 from the Dirección General de Enseñanza Superior (Ministry of Education and Culture, Spain).

24 Pags. The definitive version, with Figs. and Tabls., is available at: http://www.sciencedirect.com/science/journal/08915849, 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., This work was supported by grants from Dirección General de Investigación Científica y Tecnológica (PB92-0058) to M.B., Acciones Integradas Hispano-Británicas (HB91-218 and HB92-185) to M.B. and R.J. G., and U.S. Department of Agriculture (93-0318) to R.V.K.

22 Pags. The definitive version, with Figs. and Tabls., is available at: http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1365-3040, Iron (Fe) deficiency in pea leaves caused a large decrease (44–62&) in chlorophyll a, chlorophyll b and carotenoids, and smaller decreases in soluble protein (18&) and net photosynthesis (28&). Catalase, non-specific peroxidase and ascorbate peroxidase activities declined by 51& in young Fe-deficient leaves, whereas monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase activities remained unaffected. Ascorbate peroxidase activity was highly correlated (r2= 0. 99, P < 0. 001) with the Fe content of leaves, which allows its use as an indicator of the Fe nutritional status of the plant. Fe deficiency resulted in an increase of CuZn-superoxide dismutase but not of Mn-superoxide dismutase. The content of ascorbate decreased by only 24& and those of reduced and oxidized glutathione and vitamin E did not vary. The low-molecular-mass fraction of Fe-sufficient leaves contained 30–65 μg (g dry weight)−1 Mn. This concentration was 15–60 times greater than that of Fe and Cu in the same fraction, and was further enhanced (1. 5- to 2. 5-fold) by Fe deficiency without causing Mn toxicity. The concentration of catalytic Fe, that is, of Fe active for free radical generation, was virtually zero and that of catalytic Cu did not change with severe Fe deficiency. Because catalytic metals mediate lipid and protein oxidation in vivo, the above findings would explain why oxidatively damaged lipids and proteins do not accumulate in Fe-deficient leaves., I.I.O. and J.F.M. were the recipients of predoctoral fellowships from the Comunidades Autónomas del País Vasco and Aragón, respectively, and Y. Gogorcena was the recipient of a postdoctoral contract from the Mininsterio de Educación y Ciencia. This work was supported by grant PB92-0058 from the Dirección General de Investigación Científica y Técnica to M.B. and grant 93-0318 from the U.S. Department of Agriculture to R.V.K.

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.

Cowpea (Vigna unguiculata) nodules contain three leghemoglobins (LbI, LbII and LbIII) that are encoded by at least two genes. We have cloned and sequenced the gene that encodes for LbII (lbII), the most abundant Lb in cowpea nodules, using total DNA as the template for PCR. Primers were designed using the sequence of the soybean lbc gene. The lbII gene is 679 bp in length and codes for a predicted protein of 145 amino acids. Using sequences of the cowpea lbII gene for the synthesis of primers and total nodule RNA as the template, we cloned a cDNA for LbII into a constitutive expression vector (pEMBL19+) and then expressed it in Escherichia coli. Recombinant LbII (rLbII) and native LbII (nLbII) from cowpea nodules were purified to homogeneity using standard techniques. Properties of rLbII were compared with nLbII by partially sequencing the proteins and by sodium dodecyl sulfate- and isoelectric focusing polyacrylamide gel electrophoresis, western-blot analysis using anti-soybean Lba antibodies, tryptic and chymotryptic mapping, and spectrophotometric techniques. The data showed that the structural and spectral characteristics of rLbII and nLbII were similar. The rLbII was reversibly oxygenated/deoxygenated, showing that it is a functional hemoglobin.