Open Access
Plant ammonium sensitivity is associated with external pH adaptation, repertoire of nitrogen transporters, and nitrogen requirement
(Oxford University Press, 2024-03-11) Rivero Marcos, Mikel; Lasa Larrea, Berta; Neves, Tomé; Zamarreño, Ángel M.; García Mina, José M.; García Olaverri, Carmen; Aparicio Tejo, Pedro María; Cruz, Cristina; Ariz Arnedo, Idoia; Ciencias; Zientziak; Estadística, Informática y Matemáticas; Estatistika, Informatika eta Matematika; Institute for Multidisciplinary Research in Applied Biology - IMAB; Institute for Advanced Research in Business and Economics - INARBE; Universidad Publica de Navarra / Nafarroako Unibertsitate Publikoa; Gobierno de Navarra / Nafarroako Gobernua
Modern crops exhibit diverse sensitivities to ammonium as the primary nitrogen source, influenced by environmental factors such as external pH and nutrient availability. Despite its significance, there is currently no systematic classification of plant species based on their ammonium sensitivity. We conducted a meta-analysis of 50 plant species and present a new classification method based on the comparison of fresh biomass obtained under ammonium and nitrate nutrition. The classification uses the natural logarithm of the biomass ratio as the size effect indicator of ammonium sensitivity. This numerical parameter is associated with critical factors for nitrogen demand and form preference, such as Ellenberg indicators and the repertoire of nitrogen transporters for ammonium and nitrate uptake. Finally, a comparative analysis of the developmental and metabolic responses, including hormonal balance, is conducted in two species with divergent ammonium sensitivity values in the classification. Results indicate that nitrate has a key role in counteracting ammonium toxicity in species with a higher abundance of genes encoding NRT2-type proteins and fewer of those encoding the AMT2-type proteins. Additionally, the study demonstrates the reliability of the phytohormone balance and methylglyoxal content as indicators for anticipating ammonium toxicity. This study emphasizes the importance of ecophysiological requirements and the repertoire of nitrogen transporters in understanding plant sensitivity to ammonium, and enhances our knowledge of plant nitrogen nutrition.
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
Foliar application of urea to "Sauvignon Blanc" and "Merlot" vines: doses and time of application
(Springer Nature, 2012-02-19) Lasa Larrea, Berta; Menéndez Villanueva, Sergio; Sagastizabal, Kepa; Calleja Cervantes, María Eréndira; Irigoyen Iriarte, Ignacio; Muro Erreguerena, Julio; Aparicio Tejo, Pedro María; Ariz Arnedo, Idoia; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Producción Agraria; Nekazaritza Ekoizpena
A careful control of the N nutritional status of grapevines can have a determining effect on wine characteristics; therefore a suitable management of N fertilization might allow some wine parameters to be modified, thereby improving product quality. The aim of this study was to determine the effect of foliar application of urea at different doses and different times of the growing season on the parameters of Sauvignon Blanc and Merlot grape juice. The research described herein involved Sauvignon Blanc and Merlot grapevines (V. vinifera L.) at a commercial vineyard and was conducted over 2 years. In the first year, N treatment involved a foliar application at a dose of 10 kg N ha−1 during veraison, whereas in the second year it involved a foliar urea application at two doses (10 and 50 kg N ha−1) and at three different times—3 weeks before veraison, during veraison and 3 weeks after veraison. In this second year, the urea applied at a dose of 10 kg N ha−1 was isotopically labelled with 1% 15N. Chemical parameters, yeast assimilable N, amino acid content, amino acid profile and N isotopic composition were determined for all treatments. Grape and grape-juice parameters for Merlot were found to be more affected by N fertilization than for Sauvignon Blanc and were also more affected during the second year than during the first year, thus indicating that the climatic characteristics of each campaign could affect these parameters. The yeast assimilable N in grape juice was found to be higher for late applications of foliar urea, with application of the higher dose of urea during veraison increasing the amino acid and proline contents in both varieties. The isotopic analysis data showed that the urea applied to leaves was transferred to the berries, with the maximum translocation in Sauvignon Blanc occurring for the post-veraison treatment and in Merlot for the veraison treatment. We can therefore conclude that foliar application of urea could modify grape juice quality and could therefore be used as a tool for obtaining quality wines.
Open Access
Leaf δ15N as a physiological indicator of the responsiveness of N2-fixing alfalfa plants to elevated CO2, temperature and low water availability
(Frontiers Media, 2015) Ariz Arnedo, Idoia; Cruz, Cristina; Neves, Tomé; Irigoyen, Juan J.; García Olaverri, Carmen; Nogués, Salvador; Aparicio Tejo, Pedro María; Aranjuelo Michelena, Iker; Estatistika eta Ikerketa Operatiboa; Natura Ingurunearen Zientziak; Estadística e Investigación Operativa; Ciencias del Medio Natural; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua
The natural 15N/14N isotope composition (δ15N) of a tissue is a consequence of its N source and N physiological mechanisms in response to the environment. It could potentially be used as a tracer of N metabolism in plants under changing environmental conditions, where primary N metabolism may be complex, and losses and gains of N fluctuate over time. In order to test the utility of δ15N as an indicator of plant N status in N2-fixing plants grown under various environmental conditions, alfalfa (Medicago sativa L.) plants were subjected to distinct conditions of [CO2] (400 vs. 700 μmol mol−1), temperature (ambient vs. ambient +4°C) and water availability (fully watered vs. water deficiency—WD). As expected, increased [CO2] and temperature stimulated photosynthetic rates and plant growth, whereas these parameters were negatively affected by WD. The determination of δ15N in leaves, stems, roots, and nodules showed that leaves were the most representative organs of the plant response to increased [CO2] and WD. Depletion of heavier N isotopes in plants grown under higher [CO2] and WD conditions reflected decreased transpiration rates, but could also be related to a higher N demand in leaves, as suggested by the decreased leaf N and total soluble protein (TSP) contents detected at 700 μmol mol−1 [CO2] and WD conditions. In summary, leaf δ15N provides relevant information integrating parameters which condition plant responsiveness (e.g., photosynthesis, TSP, N demand, and water transpiration) to environmental conditions.
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
Quantitative proteomics reveals the importance of nitrogen source to control glucosinolate metabolism in Arabidopsis thaliana and Brassica oleracea
(Oxford University Press, 2016) Marino Bilbao, Daniel; Ariz Arnedo, Idoia; Lasa Larrea, Berta; Santamaría Martínez, Enrique; Aparicio Tejo, Pedro María; Ciencias del Medio Natural; Natura Ingurunearen Zientziak
Accessing different nitrogen (N) sources involves a profound adaptation of plant metabolism. In this study, a quantitative proteomic approach was used to further understand how the model plant Arabidopsis thaliana adjusts to different N sources when grown exclusively under nitrate or ammonium nutrition. Proteome data evidenced that glucosinolate metabolism was differentially regulated by the N source and that both TGG1 and TGG2 myrosinases were more abundant under ammonium nutrition, which is generally considered to be a stressful situation. Moreover, Arabidopsis plants displayed glucosinolate accumulation and induced myrosinase activity under ammonium nutrition. Interestingly, these results were also confirmed in the economically important crop broccoli (Brassica oleracea var. italica). Moreover, these metabolic changes were correlated in Arabidopsis with the differential expression of genes from the aliphatic glucosinolate metabolic pathway. This study underlines the importance of nitrogen nutrition and the potential of using ammonium as the N source in order to stimulate glucosinolate metabolism, which may have important applications not only in terms of reducing pesticide use, but also for increasing plants’ nutritional value.
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
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+.