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
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
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
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
Leaves play a central role in the adaptation of nitrogen and sulfur metabolism to ammonium nutrition in oilseed rape (Brassica napus)
(BioMed Central, 2017) Coleto, Inmaculada; Peña, Marlon de la; Rodríguez Escalante, Jon; Bejarano, Iraide; Glauser, Gaëtan; Aparicio Tejo, Pedro María; González Moro, María Begoña; Marino Bilbao, Daniel; Ciencias del Medio Natural; Natura Ingurunearen Zientziak
Background: The coordination between nitrogen (N) and sulfur (S) assimilation is required to suitably provide plants with organic compounds essential for their development and growth. The N source induces the adaptation of many metabolic processes in plants; however, there is scarce information about the influence that it may exert on the functioning of S metabolism. The aim of this work was to provide an overview of N and S metabolism in oilseed rape (Brassica napus) when exposed to different N sources. To do so, plants were grown in hydroponic conditions with nitrate or ammonium as N source at two concentrations (0.5 and 1 mM). Results: Metabolic changes mainly occurred in leaves, where ammonium caused the up-regulation of enzymes involved in the primary assimilation of N and a general increase in the concentration of N-compounds (NH4 +, amino acids and proteins). Similarly, the activity of key enzymes of primary S assimilation and the content of S-compounds (glutathione and glucosinolates) were also higher in leaves of ammonium-fed plants. Interestingly, sulfate level was lower in leaves of ammonium-fed plants, which was accompanied by the down-regulation of SULTR1 transporters gene expression. Conclusions: The results highlight the impact of the N source on different steps of N and S metabolism in oilseed rape, notably inducing N and S assimilation in leaves, and put forward the potential of N source management to modulate the synthesis of compounds with biotechnological interest, such as glucosinolates.
Open Access
Improving the short‑term efficiency of rock phosphate‑based fertilizers in pastures by using edaphic biostimulants
(Springer, 2016) Fernández, L.; Baigorri, R.; Urrutia Vera, Olaia; Erro, J.; Aparicio Tejo, Pedro María; Yvin, J. C.; García Mina, José M.; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua
Background: The use of reactive rock phosphate (RP) in acidic soils as a phosphate (P) source for pastures and crops presents attractive economic advantages with respect to soluble phosphate. However, some studies have demonstrated that the short-term (1-year) efficiency of RP, compared with that of water-soluble P, is relatively poor. This fact penalizes not only the yield and quality of the earlier harvests, but even the whole final yield when the crop is affected by some abiotic or biotic stress at the beginning of the cycle. In the present study, we investigated the ability of new edaphic biostimulants to increase the short-term efficiency of RP-based fertilizer as a P source for pastures cultivated in acid soils. To this end, we have granulated rock phosphate with two edaphic biostimulants: tryptophan and a heteromolecular organic complex formed by humic acid and tryptophan through iron bridges, and compared their short-term P fertilizer efficacy with that of single superphosphate and rock phosphate. Results: Soil incubation studies showed that the heteromolecular complex humic acid–tryptophan and Tryptophan were able to increase soil CO2 production compared with native soil, rock phosphate, and superphosphate. Likewise, the presence of humic acid–tryptophan complex and Trp significantly increases plant-available phosphate compared with rock phosphate, up to levels similar to those of superphosphate. Plant (ray grass)–soil–pot studies showed that rock phosphate/(humic acid–tryptophan) formulation yielded values for both ray grass dry matter production and shoot P concentration, clearly higher than those of rock phosphate and rock phosphate/tryptophan. In addition, the results associated with rock phosphate/(humic acid–tryptophan) were similar to those for superphosphate, after 3 months of cultivation. Conclusions: Taken together, these results showed the suitability of the use of specific humic acid-based edaphic biostimulants to improve the short-term effect of rock phosphate fertilizers as a phosphate source for pastures cultivated in acid soils.
Open Access
Overexpression of a pine Dof transcription factor in hybrid poplars: A comparative study in trees growing under controlled and natural conditions
(Public Library of Science, 2017) Rueda López, Marina; Pascual, María Belén; Pallero, Mercedes; Henao, Luisa María; Lasa Larrea, Berta; Jáuregui Mosquera, Iván; Aparicio Tejo, Pedro María; Cánovas, Francisco M.; Ávila, Concepción; Ciencias del Medio Natural; Natura Ingurunearen Zientziak
In this work, the role of the pine transcriptional regulator Dof 5 in carbon and nitrogen metabolism has been examined in poplar trees. The overexpression of the gene and potential effects on growth and biomass production were compared between trees growing in a growth chamber under controlled conditions and trees growing in a field trial during two growth seasons. Ten-week-old transgenic poplars exhibited higher growth than untransformed controls and exhibited enhanced capacity for inorganic nitrogen uptake in the form of nitrate. Furthermore, the transgenic trees accumulated significantly more carbohydrates such as glucose, fructose, sucrose and starch. Lignin content increased in the basal part of the stem likely due to the thicker stem of the transformed plants. The enhanced levels of lignin were correlated with higher expression of the PAL1 and GS1.3 genes, which encode key enzymes involved in the phenylalanine deamination required for lignin biosynthesis. However, the results in the field trial experiment diverged from those observed in the chamber system. The lines overexpressing PpDof5 showed attenuated growth during the two growing seasons and no modification of carbon or nitrogen metabolism. These results were not associated with a decrease in the expression of the transgene, but they can be ascribed to the nitrogen available in the field soil compared to that available for growth under controlled conditions. This work highlights the paramount importance of testing transgenic lines in field trials.
Open Access
Insights into the regulation of nitrogen fixation in pea nodules: lessons from drought, abscisic acid and increased photoassimilate availability
(EDP Sciences, 2001) González García, Esther; Gálvez, Loli; Royuela Hernando, Mercedes; Aparicio Tejo, Pedro María; Arrese-Igor Sánchez, César; Ciencias del Medio Natural; Natura Ingurunearen Zientziak
Nitrogen fixation in legume nodules has been shown to be very sensitive to drought and other environmental constraints. It has been widely assumed that this decline in nitrogen fixation was a consequence of an increase in the so-called oxygen diffusion barrier and a subsequent impairment to bacteroid respiration. However, it has been recently shown that nitrogen fixation is highly correlated with nodule sucrose synthase (SS) activity under drought and other environmental stresses. Whether this correlation reflects a causative relationship or not has not been proven yet. The evidence presented here suggests that SS controls nitrogen fixation under mild drought conditions. However, nitrogen fixation cannot be enhanced only by increasing glycolytic flux, as under these conditions nodules become oxygen limited. Abscisic acid also induces a decline in nitrogen fixation that is independent of SS. The overall results suggest the occurrence of a complex regulation of nodule nitrogen fixation involving, at least, both carbohydrate and oxygen fluxes within the nodule.
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
Unraveling the role of transient starch in the response of Arabidopsis to elevated CO2 under long-day conditions
(Elsevier, 2018) Jáuregui Mosquera, Iván; Pozueta Romero, Javier; Aparicio Tejo, Pedro María; Baroja Fernández, Edurne; Aranjuelo Michelena, Iker; Zientziak; Ciencias; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Previous studies on Arabidopsis under long-term exposure to elevated CO2 have been conducted using starch synthesis and breakdown mutants cultured under short day conditions. These studies showed that starch synthesis can ameliorate the photosynthetic reduction caused by soluble sugar-mediated feedback regulation. In this work we characterized the effect of long-term exposure to elevated CO2 (800 ppm) on growth, photosynthesis and content of primary photosynthates in long-day grown wild type plants as well as the near starch-less (aps1) and the starch-excess (gwd) mutants. Notably, elevated CO2 promoted growth of both wild type and aps1 plants but had no effect on gwd plants. Growth promotion by elevated CO2 was accompanied by an increased net photosynthesis in WT and aps1 plants. However, the plants with the highest starch content (wild type at elevated CO2, gwd at ambient CO2, and gwd at elevated CO2) were the ones that suffered decreased in in vivo maximum carboxylation rate of Rubisco, and therefore, photosynthetic down-regulation. Further, the photosynthetic rates of wild type at elevated CO2 and gwd at elevated CO2 were acclimated to elevated CO2. Notably, elevated CO2 promoted the accumulation of stress-responsive and senescence-associated amino acid markers in gwd plants. The results presented in this work provide evidence that under long-day conditions, temporary storage of overflow photosynthate as starch negatively affect Rubisco performance. These data are consistent with earlier hypothesis that photosynthetic acclimation can be caused by accelerated senescence and hindrance of CO2 diffusion to the stroma due to accumulation of large starch granules.