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
Both free indole-3-acetic acid and photosynthetic performance are important players in the response of Medicago truncatula to urea and ammonium nutrition under axenic conditions
(Frontiers Media, 2016) Esteban Terradillos, Raquel; Royo Castillejo, Beatriz; Urarte Rodríguez, Estíbaliz; Zamarreño, Ángel M.; García Mina, José M.; Morán Juez, José Fernando; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
We aimed to identify the early stress response and plant performance of Medicago truncatula growing in axenic medium with ammonium or urea as the sole source of nitrogen, with respect to nitrate-based nutrition. Biomass measurements, auxin content analyses, root system architecture (RSA) response analyses, and physiological parameters were determined. Both ammonium and ureic nutrition severely affected the RSA, resulting in changes in the main elongation rate, lateral root development, and insert position from the root base. The auxin content decreased in both urea- and ammonium-treated roots; however, only the ammonium-treated plants were affected at the shoot level. The analysis of chlorophyll a fluorescence transients showed that ammonium affected photosystem II, but urea did not impair photosynthetic activity. Superoxide dismutase isoenzymes in the plastids were moderately affected by urea and ammonium in the roots. Overall, our results showed that low N doses from different sources had no remarkable effects on M. truncatula, with the exception of the differential phenotypic root response. High doses of both ammonium and urea caused great changes in plant length, auxin contents and physiological measurements. Interesting correlations were found between the shoot auxin pool and both plant length and the “performance index” parameter, which is obtained from measurements of the kinetics of chlorophyll a fluorescence. Taken together, these data demonstrate that both the indole-3-acetic acid pool and performance index are important components of the response of M. truncatula under ammonium or urea as the sole N source.
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
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
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
Comparison of four functionalization methods of gold nanoparticles for enhancing the enzyme-linked immunosorbent assay (ELISA)
(Beilstein-Institut, 2017) Ciáurriz Gortari, Paula; Fernández Santos, Fátima; Tellechea Malda, Edurne; Morán Juez, José Fernando; Asensio, Aarón C.; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua, proyecto SABioD
The enzyme-linked immunosorbent assay (ELISA) technique is based on the specific recognition ability of the molecular structure of an antigen (epitope) by an antibody and is likely the most important diagnostic technique used today in bioscience. With this methodology, it is possible to diagnose illness, allergies, alimentary fraud, and even to detect small molecules such as toxins, pesticides, heavy metals, etc. For this reason, any procedures that improve the detection limit, sensitivity or reduce the analysis time could have an important impact in several fields. In this respect, many methods have been developed for improving the technique, ranging from fluorescence substrates to methods for increasing the number of enzyme molecules involved in the detection such as the biotin–streptavidin method. In this context, nanotechnology has offered a significant number of proposed solutions, mainly based on the functionalization of nanoparticles from gold to carbon which could be used as antibody carriers as well as reporter enzymes like peroxidase. However, few works have focused on the study of best practices for nanoparticle functionalization for ELISA enhancement. In this work, we use 20 nm gold nanoparticles (AuNPs) as a vehicle for secondary antibodies and peroxidase (HRP). The design of experiments technique (DOE) and four different methods for biomolecule loading were compared using a rabbit IgG/goat anti-rabbit IgG ELISA model (adsorption, directional, covalent and a combination thereof). As a result, AuNP probes prepared by direct adsorption were the most effective method. AuNPs probes were then used to detect gliadin, one of the main components of wheat gluten, the protein composite that causes celiac disease. With this optimized approach, our data showed a sensitivity increase of at least five times and a lower detection limit with respect to a standard ELISA of at least three times. Additionally, the assay time was remarkably decreased.
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.
Open Access
Drought tolerance response of high-yielding soybean varieties to mild drought: physiological and photochemical adjustments
(Wiley, 2019) Buezo Bravo, Javier; Sanz Sáez, Álvaro; Morán Juez, José Fernando; Soba Hidalgo, David; Aranjuelo Michelena, Iker; Esteban Terradillos, Raquel; Ciencias; Zientziak; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
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.
Open Access
Evaluation of the anti-nitrative effect of plant antioxidants using a cowpea Fe-superoxide dismutase as a target
(Elsevier, 2014) Urarte Rodríguez, Estíbaliz; Asensio, Aarón C.; Tellechea Malda, Edurne; Pires, Laura; Morán Juez, José Fernando; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa; Gobierno de Navarra / Nafarroako Gobernua
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 beenyet clarified in plants. The objective of this study was to establish new approaches that could help to understand ONOOreactivity 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 ONOOby 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 f lavonoid 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 ONOOhomeostasis in vivo.