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Publication Open Access Ecophysiological roles of abaxial anthocyanins in a perennial understorey herb from temperate deciduous forests(Oxford University Press, 2015) Fernández Marín, Beatriz; Esteban Terradillos, Raquel; Míguez, Fátima; Artetxe, Unai; Castañeda Presa, Verónica; Pintó Marijuan, Marta; Becerril, José María; García Plazaola, José Ignacio; Natura Ingurunearen Zientziak; Ciencias del Medio Natural; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako InstitutuaAccumulation of abaxial anthocyanins is an intriguing leaf trait particularly common among deeply shaded understorey plants of tropical and temperate forests whose ecological significance is still not properly understood. To shed light on it, possible ecophysiological roles of abaxial anthocyanins were tested in the perennial understorey herb of temperate deciduous forests Saxifraga hirsuta, chosen as a model species due to the coexistence of green and anthocyanic leaves and the presence of an easily removable lower anthocyanic epidermis. Anthocyanins accumulated during autumn, which temporally matched the overstorey leaf fall. Patterns of development of abaxial anthocyanins and direct measurements of photochemical efficiency under monochromatic light were not consistent with a photoprotective hypothesis. Enhancement of light capture also seemed unlikely since the back-scattering of red light towards the lower mesophyll was negligible. Seed germination was similar under acyanic and anthocyanic leaves. A relevant consequence of abaxial anthocyanins was the dramatic reduction of light transmission through the leaf. The dark environment generated underneath the Saxifraga canopy was enhanced by the horizontal repositioning of leaves, which occurs in parallel with reddening. This might play a role in biotic interactions by inhibiting vital processes of competitors, which may be of especial importance in spring before the overstorey leaves sprout.Publication Open Access Effect of feeding regime on composting in bins(Taylor & Francis, 2017) Storino, Francesco; Menéndez Villanueva, Sergio; Muro Erreguerena, Julio; Aparicio Tejo, Pedro María; Irigoyen Iriarte, Ignacio; Natura Ingurunearen Zientziak; Nekazaritza Ekoizpena; Ciencias del Medio Natural; Producción Agraria; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako InstitutuaComposting in bins is one of the most practical home composting methods. There is currently a need for greater information to improve the management of the composting process and to create home composting programs, which ensure sustainable production of high quality compost. This study investigates how two aspects of the bin feeding regime—the feeding frequency and the amount of waste applied at each feed—influence the process's evolution and the quality of the compost. Compost bins were assayed after introducing the same amount of kitchen and garden waste according to three different frequencies: in a single batch, weekly, or every 3 weeks. A fourth treatment was applied to calculate the potential waste reduction achieved by the composting process, filling the bins to the brim on a weekly basis. Temperature, mass, and volume changes; the microbial diversity (by Biolog); and gas emissions (CO2, CH4, N2O, and NH3) were all determined during the process. At the end of the experiment, all of the composts were weighed and characterized. Results show that the main differences were very dependent on the quantity of waste provided. Large amounts of waste were added increasing the compost's temperature and maturity during the process, while slightly affecting the salinity and phytotoxicity of the final compost but without any clear effects on microbial diversity and gas emission. Therefore, from a technical point of view, the shared use of compost bins among several households (community composting) is preferable to individual usPublication Open Access Elevated CO2 improved the growth of a double nitrate reductase defective mutant of Arabidopsis thaliana: the importance of maintaining a high energy status(Elsevier, 2017) Jáuregui Mosquera, Iván; Aparicio Tejo, Pedro María; Baroja Fernández, Edurne; Ávila, Concepción; Aranjuelo Michelena, Iker; Natura Ingurunearen Zientziak; Ciencias del Medio Natural; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako InstitutuaImpairments in leaf nitrogen (N) assimilation in C3 plants have been identified as processes conditioning photosynthesis under elevated [CO2], especially when N is supplied as nitrate. Leaf N status is usually improved under ammonium nutrition and elevated [CO2]. However, ammonium fertilization is usually accompanied by the appearance of oxidative stress symptoms, which constrains plant development. To understand how the limitations of direct fertilization with ammonium (growth reduction attributed to ammonium toxicity) can be overcome, the effects of elevated [CO2] (800 ppm) exposure were studied in the Arabidopsis thaliana double nitrate reductase defective mutant, nia1-1/chl3-5 (which preferentially assimilates ammonium as its nitrogen source). Analysis of the physiology, metabolites and gene expression was carried out in roots and shoot organs. Our study clearly showed that elevated [CO2] improved the inhibited phenotype of the nitrate reductase double mutant. Both the photosynthetic rates and the leaf N content of the NR mutant under elevated CO2 were similar to wild type plants. The growth of the nitrate reductase mutant was linked to its ability to overcome ammonium-associated photoinhibition processes at 800 ppm [CO2]. More specifically: (i) the capacity of NR mutants to equilibrate energy availability, as reflected by the electron transport equilibrium reached (photosynthesis, photorespiration and respiration), (ii) as well as by the upregulation of genes involved in stress tolerance were identified as the processes involved in the improved performance of NR mutants.Publication 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 ZientziakTwo 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.Publication 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 - IMABThe 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.Publication Open Access Isotopic composition of maize as related to N-fertilization and irrigation in the Mediterranean region(Escola Superior de Agricultura "Luiz de Queiroz", Universidade de São Paulo, 2011) Lasa Larrea, Berta; Irañeta, Iosu; Muro Erreguerena, Julio; Irigoyen Iriarte, Ignacio; Aparicio Tejo, Pedro María; Natura Ingurunearen Zientziak; Nekazaritza Ekoizpena; Ciencias del Medio Natural; Producción Agraria; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua, 17/2004Nitrate leaching as a result of excessive application of N-fertilizers and water use is a major problem of vulnerable regions. The farming of maize requires high N fertilization and water inputs in Spain. Isotopic techniques may provide information on the processes involved in the N and C cycles in farmed areas. The aim of this work was studying the impact of sprinkler and furrow irrigation and N input on maize (Zea mays L.) yields, and whether isotopic composition can be used as indicator of best farming practices. Trials were set up in Tudela (Spain) with three rates of N fertilization (0, 240 and 320 kg urea-N ha–1) and two irrigation systems (furrow and sprinkler). Yield, nitrogen content, irrigation parameters, N fate and C and N isotope composition were determined. The rate of N fertilization required to obtain the same yield is considerably higher under furrow irrigation, since the crop has less N at its disposal in furrow irrigation as a result of higher loss of nitrogen by NO3 –-N leaching and denitrification. A lower δ13C in plants under furrow irrigation was recorded.The δ15N value of plant increased with the application rate of N under furrow irrigation.Publication 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 GobernuaThe 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.Publication Open Access Nodule performance within a changing environmental context(Elsevier, 2014) Aranjuelo Michelena, Iker; Arrese-Igor Sánchez, César; Molero, Gemma; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Ciencias del Medio Natural; Natura Ingurunearen ZientziakGlobal climate models predict that future environmental conditions will see alterations in temperature, water availability and CO2 concentration ([CO2]) in the atmosphere. Climate change will reinforce the need to develop highly productive crops. For this purpose it is essential to identify target traits conditioning plant performance in changing environments. N2 fixing plants represent the second major crop of agricultural importance worldwide. The current review provides a compilation of results from existing literature on the effects of several abiotic stress conditions on nodule performance and N2 fixation. The environmental factors analysed include water stress, salinity, temperature, and elevated [CO2]. Despite the large number of studies analysing [CO2] effects in plants, frequently they have been conducted under optimal growth conditions that are difficult to find in natural conditions where different stresses often occur simultaneously. This is why we have also included a section describing the current state of knowledge of interacting environmental conditions in nodule functioning. Regardless of the environmental factor considered, it is evident that some general patterns of nodule response are observed. Nodule carbohydrate and N compound availability, together with the presence of oxygen reactive species (ROS) have proven to be the key factors modulating N2 fixation at the physiological/biochemical levels. However, with the exception of water availability and [CO2], it should also be considered that nodule performance has not been characterised in detail under other limiting growth conditions. This highlights the necessity to conduct further studies considering these factors. Finally, we also observe that a better understanding of these metabolic effects of changing environment in nodule functioning would require an integrated and synergistic investigation based on widely used and novel protocols such as transcriptomics, proteomics, metabolomics and stable isotopes.Publication Open Access Pea plant responsiveness under elevated [CO2] is conditioned by the N source (N2 fixation versus NO3 fertilization)(Elsevier, 2013) Aranjuelo Michelena, Iker; Cabrerizo Geijo, Pablo María; Arrese-Igor Sánchez, César; Aparicio Tejo, Pedro María; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Ciencias del Medio Natural; Natura Ingurunearen ZientziakThe main goal of this study was to test the effect of [CO2] on C and N management in 2different plant organs (shoots, roots and nodules) and its implication in the 3responsiveness of exclusively N2-fixing and NO3--fed plants. For this purpose, 4exclusively N2-fixingand NO3--fed (10 mM) pea (Pisum sativumL.) plants were 5exposed to elevated [CO2] (1000 mol mol-1versus360 mol mol-1CO2). Gas 6exchange analyses, together with carbohydrate, nitrogen, total soluble proteins and 7amino acids were determined in leaves, roots and nodules. The data obtained revealed 8that although exposure to elevated [CO2] increased total dry mass (DM)in both N 9treatments, photosynthetic activity was down-regulated in NO3--fed plants, whereas N2-10fixing plants were capable of maintaining enhanced photosynthetic rates under elevated 11[CO2]. In the case of N2-fixing plants, the enhanced C sink strength of nodules enabled 12the avoidance of harmful leaf carbohydrate build up. On the other hand, in NO3--fed 13plants, elevated [CO2] caused a large increase in sucrose and starch. The increase in root 14DM did not contribute to stimulation ofC sinks in these plants. Although N2fixation 15matched plant N requirementswith the consequent increase in photosynthetic rates, in 16NO3--fed plants, exposure to elevated [CO2] negatively affected N assimilationwith the 17consequent photosynthetic down-regulation.Publication 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 ZientziakThe 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.Publication 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 EkoizpenaThe 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.Publication Open Access Unravelling the mechanisms that improve photosynthetic performance of N₂-fixing pea plants exposed to elevated [CO₂](Elsevier, 2014) Aranjuelo Michelena, Iker; Cabrerizo Geijo, Pablo María; Aparicio Tejo, Pedro María; Arrese-Igor Sánchez, César; Natura Ingurunearen Zientziak; Ciencias del Medio Natural; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako InstitutuaAlthough the predicted enhanced photosynthetic rates of plants exposed to elevated [CO₂] are expected to increase carbohydrate and plant growth, recent findings have shown a complex regulation of these processes. The aim of this study was to determine the effect of elevated [CO₂] on pathways leading to the main forms of leaf C storage (starch) and export (sucrose) and the implications of this increased [CO₂] on photosynthetic performance of exclusively N2 fixing plants. For this purpose, exclusively N2-fixing pea plants were exposed to elevated [CO₂] (1000 mol mol−1 versus 360 mol mol−1 CO₂). The data obtained highlighted that plants exposed to elevated [CO₂] were capable of maintaining hexose levels (involved in Rubisco down regulation) at control levels with the consequent avoidance of photosynthetic acclimation. More specifically, in plants exposed to elevated [CO₂] there was an increase in the activity of pathways involved in the main forms of leaf C storage (starch) and export (sucrose). Furthermore, the study highlighted that although starch content increased by up to 40% under elevated [CO₂], there was also an increase in the proteins and compounds involved in starch degradation. Such a finding, together with an increase in the activity of proteins involved in sucrose synthesis revealed that these plants up-regulated the sucrose synthesis pathway in order to meet the large nodule photoassimilate requirements. As a consequence, the study highlighted the relevance of controlling the activity of pathways that determine leaf cellular carbohydrate availability and how this is linked with C-demanding organs such as nodules.Publication 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, 57/2007Superoxide 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.Publication Open Access Yield and quality of sugar snap pea in the Ebro Valley: sowing date and seed density(Escola Superior de Agricultura "Luiz de Queiroz", Universidade de São Paulo, 2012) Azpilicueta Unanua, Miren; Irigoyen Iriarte, Ignacio; Lasa Larrea, Berta; Muro Erreguerena, Julio; Aparicio Tejo, Pedro María; Nekazaritza Ekoizpena; Natura Ingurunearen Zientziak; Producción Agraria; Ciencias del Medio Natural; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako InstitutuaSugar snap pea (Pisum sativum L. var. macrocarpon Ser.) is an edible-podded sweet pea that is being considered as a new totally mechanized crop to supply raw material to the agri-food industry of the Ebro Valley (Northern Spain). It is of great interest from an agronomic and commercial standpoint but neither its agronomic behaviour nor its adaptation to the area are known. The objective of this study was to determine the effect of sowing date and seed density on the growth and yield of the sugar snap pea at industrial scale. Six randomized blocks experiments with four replicates were conducted on irrigated land in Villafranca (Navarra, Spain) in 1998, 1999, and 2000. Three experiments for testing sowing dates (Mar., Apr., and May) and another three for seed densities (from 75 to 150 plants m–2) were performed. Phenological development, thermal integral and qualitative and quantitative yield controls were performed. Sugar snap pea required 960 ºC d–1 (Tb = 3 ºC) from sowing to harvest. The early sowings gave more biomass, but yield was similar. However, Harvest Index and crop morphology varied. The sowing densities had similar yields sowing that sugar snap pea has a bigger adaptation availability. Sugar snap peas can be satisfactorily cultivated at industrial scale in the zone with sowings between Mar. and May and with seeding densities between 75 and 150 plants m–2.