Open Access
High irradiance increases NH4+ tolerance in Pisum sativum: higher carbon and energy availability improve ion balance but not N assimilation
(Elsevier, 2011-03-02) Ariz Arnedo, Idoia; Artola Rezola, Ekhiñe; Asensio, Aarón C.; Cruchaga Moso, Saioa; Aparicio Tejo, Pedro María; Morán Juez, José Fernando; Ciencias del Medio Natural; Natura Ingurunearen Zientziak; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Institute for Multidisciplinary Research in Applied Biology - IMAB
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 2.5–10 mM 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.
Open Access
Depletion of the heaviest stable N isotope is associated with NH4+/NH3 toxicity in NH4+-fed plants
(BioMed Central, 2011) Ariz Arnedo, Idoia; Cruz, Cristina; Morán Juez, José Fernando; González Moro, María Begoña; García Olaverri, Carmen; González Murua, Carmen; Martins Loucao, María A.; Aparicio Tejo, Pedro María; Estatistika eta Ikerketa Operatiboa; Estadística e Investigación Operativa; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
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.
Open Access
Effect of N-(n-butyl) thiophosphoric triamide on urea metabolism and the assimilation of ammonium by Triticum aestivum L.
(Springer, 2010-08-25) Artola Rezola, Ekhiñe; Cruchaga Moso, Saioa; Ariz Arnedo, Idoia; Morán Juez, José Fernando; Garnica, María; Houdusse, Fabrice; García Mina, José M.; Irigoyen Iriarte, Ignacio; Lasa Larrea, Berta; Aparicio Tejo, Pedro María; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Producción Agraria; Nekazaritza Ekoizpena
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 is to perform an in-depth analysis of the effects that NBPT use has on plant growth and N metabolism. Wheat plants were cultivated in a greenhouse experiment lasting four 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 harvest. 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.
Open Access
Use of recombinant iron-superoxide dismutase as a marker of nitrative stress
(Elservier, 2008-04-20) Larrainzar Rodríguez, Estíbaliz; Urarte Rodríguez, Estíbaliz; Auzmendi, Iñigo; Ariz Arnedo, Idoia; Arrese-Igor Sánchez, César; González García, Esther; Morán Juez, José Fernando; Ciencias del Medio Natural; Natura Ingurunearen Zientziak; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua, 57/2007
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 O2- 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.
Open Access
The physiological implications of urease inhibitors on N metabolism during germination of Pisum sativum and Spinacea oleracea seeds
(Elsevier, 2012-03-08) Ariz Arnedo, Idoia; Cruchaga Moso, Saioa; Lasa Larrea, Berta; Morán Juez, José Fernando; Jáuregui Mosquera, Iván; Aparicio Tejo, Pedro María; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Ciencias del Medio Natural; Natura Ingurunearen Zientziak
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.
Open Access
Expression and localization of a Rhizobium-derived cambialistic superoxide dismutase in pea (Pisum sativum) nodules subjected to oxidative stress
(The American Phytopathological Society, 2011-09-07) Asensio, Aarón C.; Marino Bilbao, Daniel; James, Euan K.; Ariz Arnedo, Idoia; Arrese-Igor Sánchez, César; Aparicio Tejo, Pedro María; Arredondo-Peter, Raúl; Morán Juez, José Fernando; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Ciencias del Medio Natural; Natura Ingurunearen Zientziak
Two phylogenetically unrelated superoxide dismutase (SOD) families, i.e., CuZnSOD (copper and zinc SOD) and FeMn-CamSOD (iron, manganese, or cambialistic SOD), eliminate superoxide radicals in different locations within the plant cell. CuZnSOD are located within the cytosol and plastids, while the second family of SOD, which are considered to be of bacterial origin, are usually located within organelles, such as mitochondria. We have used the reactive oxygen species¿producer methylviologen (MV) to study SOD isozymes in the indeterminate nodules on pea (Pisum sativum). MV caused severe effects on nodule physiology and structure and also resulted in an increase in SOD activity. Purification and N-terminal analysis identified CamSOD from the Rhizobium leguminosarum endosymbiont as one of the most active SOD in response to the oxidative stress. Fractionation of cell extracts and immunogold labeling confirmed that the CamSOD was present in both the bacteroids and the cytosol (including the nuclei, plastids, and mitochondria) of the N-fixing cells, and also within the uninfected cortical and interstitial cells. These findings, together with previous reports of the occurrence of FeSOD in determinate nodules, indicate that FeMnCamSOD have specific functions in legumes, some of which may be related to signaling between plant and bacterial symbionts, but the occurrence of one or more particular isozymes depends upon the nodule type.
Open Access
Mechanisms of ammonium toxicity and the quest for tolerance
(Elsevier, 2016) Esteban Terradillos, Raquel; Ariz Arnedo, Idoia; Cruz, Cristina; Morán Juez, José Fernando; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Ammonium sensitivity of plants is a worldwide problem, constraining crop production. Prolonged application of ammonium as the sole nitrogen source may result in physiological and morphological disorders that lead to decreased plant growth and toxicity. The main causes of ammonium toxicity/tolerance described until now include high ammonium assimilation by plants and/or low sensitivity to external pH acidification. The various ammonium transport-related components, especially the non-electrogenic influx of NH3 (related to the depletion of 15N) and the electrogenic influx of NH4+, may contribute to ammonium accumulation, and therefore to NH3 toxicity. However, this accumulation may be influenced by increasing K+ concentration in the root medium. Recently, new insights have been provided by “omics” studies, leading to a suggested involvement of GDP mannose-pyrophosphorylase in the response pathways of NH4+ stress. In this review, we highlight the cross-talk signaling between nitrate, auxins and NO, and the importance of the connection of the plants’ urea cycle to metabolism of polyamines. Overall, the tolerance and amelioration of ammonium toxicity are outlined to improve the yield of ammonium-grown plants. This review identifies future directions of research, focusing on the putative importance of aquaporins in ammonium influx, and on genes involved in ammonium sensitivity and tolerance.
Open Access
A self-induction method to produce high quantities of recombinant functional flavo-leghemoglobin reductase
(Elsevier, 2008-01-29) Urarte Rodríguez, Estíbaliz; Auzmendi, Iñigo; Rol, Selene; Ariz Arnedo, Idoia; Aparicio Tejo, Pedro María; Arredondo-Peter, Raúl; Morán Juez, José Fernando; Institute for Multidisciplinary Research in Applied Biology - IMAB; Gobierno de Navarra / Nafarroako Gobernua
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+.
Open Access
High irradiance induces photoprotective mechanisms and a positive effect on NH4+ stress in Pisum sativum L.
(Elsevier, 2010-04-29) Ariz Arnedo, Idoia; Esteban Terradillos, Raquel; García Plazaola, José Ignacio; Becerril, José María; Aparicio Tejo, Pedro María; Morán Juez, José Fernando; Ciencias; Zientziak; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
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 10 mM) under high (750 μmol photons m−2 s−1) or low PPFD conditions (350 μmol photons m−2 s−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 2.5 mM 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 NH4+ are not directly related to the generation of photo-oxidative stress.
Open Access
Short term physiological implications of NBPT application on the N metabolism of Pisum sativum and Spinacea oleracea
(Elsevier, 2011-03-01) Cruchaga Moso, Saioa; Artola Rezola, Ekhiñe; Lasa Larrea, Berta; Ariz Arnedo, Idoia; Irigoyen Iriarte, Ignacio; Morán Juez, José Fernando; Aparicio Tejo, Pedro María; Ciencias del Medio Natural; Natura Ingurunearen Zientziak; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Producción Agraria; Nekazaritza Ekoizpena
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.