BUSCADOR RECOLECTA

Drought stress is a major factor limiting symbiotic nitrogen fixation (NF) in soybean crop production. However, the regulatory mechanisms involved in this inhibition are still controversial. Soybean plants were symbiotically grown in a split-root system (SRS), which allowed for half of the root system to be irrigated at field capacity while the other half remained water deprived. NF declined in the water-deprived root system while nitrogenase activity was maintained at control values in the well-watered half. Concomitantly, amino acids and ureides accumulated in the water-deprived belowground organs regardless of transpiration rates. Ureide accumulation was found to be related to the decline in
their degradation activities rather than increased biosynthesis. Finally, proteomic analysis suggests that plant carbon metabolism, protein synthesis, amino acid metabolism, and cell growth are among the processes most altered in soybean nodules under drought stress. Results presented here support the hypothesis of a local regulation of NF taking place in soybean and downplay the role of ureides in the inhibition of NF, This work was financed by the Spanish Ministry of Economy and Competitiveness (AGL 2011–23738 and AGL 2011-30386-C02-01). EG-Q and AS are holders of PhD fellowships from the Public University of Navarre (735/2008 and 134/2012). EL is a recipient of a Marie Curie International Outgoing Fellowship for Career Development (PIOF-GA-2009–253141).

Drought stress is a major factor limiting nitrogen fixation (NF) in crop production. However, the regulatory mechanism involved and the origin of the inhibition, whether local or systemic, is still controversial and so far scarcely studied in temperate forage legumes. Medicago truncatula plants were symbiotically grown with a split-root system and exposed to gradual water deprivation. Physiological parameters, NF activity, and amino acid content were measured. The partial drought treatment inhibited NF in the nodules directly exposed to drought stress. Concomitantly, in the droughted below-ground organs, amino acids accumulated prior to any drop in evapotranspiration (ET). It is concluded that drought exerts a local inhibition of NF and drives an overall accumulation of amino acids in diverse plant organs which is independent of the decrease in ET. The general increase in the majority of single amino acids in the
whole plant questions the commonly accepted concept of a single amino acid acting as an N-feedback signal., This work was financed by the Spanish Ministry of Economy and Competitiveness (AGL 2011–23738 and AGL 2011-30386-C02-01). EG-Q is a holder of a PhD fellowship from the Public University of Navarre (735/2008). EL is a recipient of a Marie Curie International Outgoing Fellowship for Career Development (PIOF-GA-2009–253141).

The symbiotic association between Medicago truncatula and Sinorhizobium meliloti is a well-established model system in the legume–Rhizobium community. Despite its wide use, the symbiotic efficiency of this model has been recently questioned and an alternative microsymbiont, S. medicae, has been proposed. However, little is known about the physiological mechanisms behind the higher symbiotic efficiency of S. medicae WSM419. In the present study, we inoculated M. truncatula Jemalong A17 with either S. medicae WSM419 or S. meliloti 2011 and compared plant growth, photosynthesis, N2-fixation rates, and plant nodule carbon and nitrogen metabolic activities in the two systems. M. truncatula plants in symbiosis with S. medicae showed increased biomass and photosynthesis rates per plant. Plants grown in symbiosis with S. medicae WSM419 also showed higher N2-fixation rates, which were correlated with a larger nodule biomass, while nodule number was similar in both systems. In terms of plant nodule metabolism, M. truncatula–S. medicae WSM419 nodules showed increased sucrose-catabolic activity, mostly associated with sucrose synthase, accompanied by a reduced starch content, whereas nitrogen-assimilation activities were comparable to those measured in nodules infected with S. meliloti 2011. Taken together, these results suggest that S. medicae WSM419 is able to enhance plant carbon catabolism in M. truncatula nodules, which allows for the maintaining of high symbiotic N2-fixation rates, better growth and improved general plant performance., This
work has been partially funded by the Spanish National Research
and Development Programmes (AGL2011-23738 and AGL2011-
30386-C02-01). Estíbaliz Larrainzar and Erena Gil-Quintana are
funded by the European FP7-PEOPLE program (253141). Amaia
Seminario is funded by a predoctoral fellowship from the Public
University of Navarre.

Impairments 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., This work has been funded by the Spanish National Research and Development Programme (AGL2009-13339-C02-02, AGL2011-30386-C02-02 and AGL2012-37815-C05-05). Ivan Jauregui was the holder of a FPI fellowship from the Spanish Ministry of Economy and Competitiveness.

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., This work was supported by the Spanish Economy and Competiveness ministry (AGL-2012-37815-CO5-05, AGL2011-30386-C02-02 and Ramón y Cajal research grant) and by the Portuguese FCT (PTDC/BIA-ECS/122214/2010). IA was supported by a postdoctoral Fellowship from the Government of Navarra (Anabasid outgoing Programme) and by a postdoctoral Fellowship from the Portuguese FCT (SFRH/BPD/90436/2012).

Incluye 3 ficheros de datos, Symbiotic nitrogen fixation is one of the first physiological
processes inhibited in legume plants under water-deficit conditions.
Despite the progress made in the last decades, the
molecular mechanisms behind this regulation are not fully
understood yet. Recent proteomic work carried out in the
model legume Medicago truncatula provided the first indications
of a possible involvement of nodule methionine (Met)
biosynthesis and related pathways in response to waterdeficit
conditions. To better understand this involvement, the
drought-induced changes in expression and content of
enzymes involved in the biosynthesis of Met, S-adenosyl-Lmethionine
(SAM) and ethylene in M. truncatula root and
nodules were analyzed using targeted approaches. Nitrogenfixing
plants were subjected to a progressive water deficit and
a subsequent recovery period. Besides the physiological characterization
of the plants,the content of total sulphur,sulphate
and main S-containing metabolites was measured. Results
presented here show that S availability is not a limiting factor
in the drought-induced decline of nitrogen fixation rates in
M. truncatula plants and provide evidences for a downregulation
of the Met and ethylene biosynthesis pathways in
roots and nodules in response to water-deficit conditions., This work was supported by the Spanish Ministry of Economy and Competitiveness (AGL 2011–23738 and AGL 2011–30386-C02-01).E.L. is a recipient of a Marie Curie International Outgoing Fellowship for Career Development (PIOF-GA-2009–253141). E.G-Q. received a PhD fellowship from the Public University of Navarre (735/2008).

La actual población mundial junto con las predicciones de un mayor crecimiento sugieren que es necesario incrementar el rendimiento de los cultivos a nivel mundial. Las leguminosas son el segundo cultivo más importante para alimentación después de los cereales, y gracias a su capacidad de establecer una relación simbiótica con bacterias del suelo, se reduce el impacto del uso de fertilizantes nitrogenados sobre el medio ambiente. Esta simbiosis da lugar al proceso conocido como fijación biológica de nitrógeno (FBN), que consiste en la reducción de nitrógeno molecular a amonio, a partir del cual, las plantas sintetizan compuestos orgánicos nitrogenados esenciales para su nutrición. Desafortunadamente, la FBN es un proceso muy sensible a estreses bióticos y abióticos tales como salinidad, sequía o limitación de nutrientes, entre otros. El objetivo general de este trabajo es ampliar los conocimientos sobre la regulación de la FBN y los mecanismos fisiológicos y bioquímicos que activan las plantas en respuesta a estreses abióticos.
Para contrarrestar los efectos negativos de estreses osmóticos, las plantas y las bacterias son capaces de sintetizar compuestos osmoprotectores para mantener la viabilidad de las células, por ejemplo, el aminoácido prolina. El primer paso clave para entender las múltiples funciones de esta molécula bajo situaciones de estrés osmótico es una monitorización del uso de prolina a tiempo real. En el capítulo uno nuestros resultados mostraron que, en bacteroides, la acumulación de prolina no ocurre durante la fase de estrés, si no durante la recuperación, una vez las condiciones óptimas para el crecimiento de la planta se han reestablecido.
En el capítulo dos, se llevó a cabo un estudio proteómico y metabólico dirigido para ampliar el conocimiento sobre el metabolismo de aminoácidos en nódulos de guisante. En el modelo clásico de intercambio de nutrientes entre simbiontes, la planta suministra energía en forma de dicarboxilatos a los bacteroides fijadores de nitrógeno a cambio de amonio. Sin embargo, este modelo clásico fue cuestionado por la observación de que las mutaciones en los transportadores de aminoácidos ABC, AapJQMP and BraDEFGC, en Rhizobium leguminosarum dieron lugar a síntomas de falta de nitrógeno en plantas tanto de guisante como de alubia. Se encontró que era esencial la absorción de aminoácidos de cadena ramificada (AACRs) para una efectiva FBN, al menos en especies de R. leguminosarum. Otro enfoque experimental para comprender mejor el papel del metabolismo de los aminoácidos en los nódulos es la aplicación de compuestos que inhiben la biosíntesis de AACRs en las células de las plantas tales como los herbicidas del grupo B. Estos enfoques nos permitieron verificar como la inhibición del transporte de AACRs entre simbiontes tuvo un mayor efecto en el metabolismo nodular que la inhibición de la biosíntesis de AACRs. De hecho, la biosíntesis de AACRs fue también inhibida debido a la doble mutación de aap/bra. En el capítulo dos, también evaluamos el efecto del estrés hídrico sobre el proteoma nodular, ya que entre las estrategias que usan las plantas en respuesta a estreses abióticos hay varias relacionadas con el metabolismo de aminoácidos. Este estudio destaca la relevancia de aminoácidos poco abundantes, como metionina, aminoácidos aromáticos o el ácido γ-aminobutírico, en la respuesta al estrés hídrico.
Finalmente, hasta ahora no se ha intentado llevar a cabo un enfoque integral en el que se analicen los posibles cambios causados por sequía en la distribución de carbono (C) y, además, se analice el efecto sobre el consumo o la acumulación de metabolitos en todos los órganos de la planta. Con este propósito, en el capítulo tres, se analizó el efecto de la sequía tanto en la distribución de [U-13C]-sacarosa como en el contenido ureidos, ácidos orgánicos y carbohidratos. Descubrimos que la sequía disminuyó el transporte de 13C a los tejidos sumidero y cambió la prioridad en la distribución de C entre los órganos sumideros., The current world population together with the predictions of further growth suggest that it is necessary to increase crop yields worldwide. Legumes are the second most important food crop after cereals, and thanks to their ability to establish a symbiotic relationship with soil bacteria, the impact of the use of nitrogen fertilizers on the environment is reduced. This symbiosis gives rise to the process known as biological nitrogen fixation (BNF), which consists in the reduction of molecular nitrogen to ammonium, from which plants synthesize organic nitrogenous products essential for their nutrition. Unfortunately, BNF is a very sensitive process to biotic and abiotic stresses such as salinity, drought, or nutrient limitation, among others. The general aim of this work was to gain further insights in the regulation of BNF and the physiological and biochemical mechanisms that plant activate in response to abiotic stresses.
In order to counteract the negative effects of osmotic stresses, plant and bacteria are able to synthesise osmoprotectant compounds to maintain cell viability, e.g. the amino acid proline. A real-time monitoring of proline utilisation in both plant and bacterial systems is a first key step towards understanding the multiple roles of this molecule under osmotic stress situations. Our results in chapter one showed that, in bacteroids, proline accumulation does not occur during the stress phase, but during recovery, once optimal plant growth conditions are re-established.
In chapter two, a proteomic and metabolic study was performed to gain further insights about amino acid metabolism in pea nodules. In the classical model of nutrient exchange between symbionts, plant supplies energy in the form of dicarboxylates to the N2-fixing bacteroids in exchange for ammonium. However, this classic model was challenged upon the observation that mutations in the general ABC amino-acid transporters AapJQMP and BraDEFGC in Rhizobium leguminosarum resulted in N starvation symptoms in both pea and bean plants. The uptake of branched-chain amino acids (BCAAs) from the plant by the bacteroid was found to be essential for an effective BNF at least in R. leguminosarum species. Another experimental approach to further understand the role of amino acid metabolism in nodules is the application of compounds that inhibit the biosynthesis of BCAAs in plant cells such as group B herbicides. These approaches allowed us to verify how the blockage of BCAA transport between symbionts had a greater effect on nodule metabolism than the inhibition of BCAA biosynthesis. In fact, BCAA biosynthesis was also inhibited due to the aap/bra double mutation. In chapter two, we also evaluate the effect of water deficit on nodule proteome, since among the strategies that plants use in response to abiotic stresses there are several related to amino acid metabolism. This study highlights the relevance of low abundant amino acids, such as methionine, aromatic amino acids or γ-aminobutyric acid, in the response to water deficit.
Finally, until now no attempt has been made to carry out an integral approach in which possible changes caused by drought in carbon (C) allocation, and in addition, the effect on the consumption or accumulation of metabolites in all plant organs be analysed. For this purpose, in chapter three, the effect of drought on both the [U-13C]-sucrose distribution and ureides, organic acids and carbohydrates content were analysed. We found that drought decreased 13C transport to sink tissues and changed the priority of C allocation between sink organs., This work was funded by Ministerio de Economía y Competitividad, AGL2011-30386-C02-01 and AGL2014-56561-P. María Isabel Rubia Galiano has been holder of a PhD fellowship Formación de personal investigador from the Ministerio de Economía y Competitividad and she has received two mobility grants from the same institution., Programa de Doctorado en Agrobiología Ambiental (RD 99/2011), Ingurumen Agrobiologiako Doktoretza Programa (ED 99/2011)

Split-root system (SRS) approaches allow the differential treatment of separate and independent root systems, while sharing a common aerial part. As such, SRS is a useful tool for the discrimination of systemic (shoot origin) versus local (root/nodule origin) regulation mechanisms. This type of approach is particularly useful when studying the complex regulatory mechanisms governing the symbiosis established between legumes and Rhizobium bacteria. The current work provides an overview of the main insights gained from the application of SRS approaches to understand how nodule number (nodulation autoregulation) and nitrogen fixation are controlled both under non-stressful conditions and in response to a variety of stresses. Nodule number appears to be mainly controlled at the systemic level through a signal which is produced by nodule/root tissue, translocated to the shoot, and transmitted back to the root system, involving shoot Leu-rich repeat receptor-like kinases. In contrast, both local and systemic mechanisms have been shown to operate for the regulation of nitrogenase activity in nodules. Under drought and heavy metal stress, the regulation is mostly local, whereas the application of exogenous nitrogen seems to exert a regulation of nitrogen fixation both at the local and systemic levels., This work has been partially funded by the Spanish National Research and Development Program (AGL2011‐30386‐CO2‐1 and AGL2011‐23738). E. L. is a recipient of the Marie Curie International Outgoing Fellowships for Career Development (FP7‐PEOPLE).

Drought is considered the more harmful abiotic stress resulting in crops yield loss. Legumes in symbiosis with rhizobia are able to fix atmospheric nitrogen. Biological nitrogen fixation (SNF) is a very sensitive process to drought and limits legumes agricultural productivity. Several factors are known to regulate SNF including oxygen availability to bacteroids, carbon and nitrogen metabolisms; but the signalling pathways leading to SNF inhibition are largely unknown. In this work, we have performed a proteomic approach of pea plants grown in split-root-system where one half of the root was well-irrigated and the other was subjected to drought. Water stress locally provoked nodule water potential decrease that led to SNF local inhibition. The proteomic approach revealed 11 and 7 nodule proteins regulated by drought encoded by P. sativum and R. leguminosarum genomes respectively. Among these 18 proteins, three proteins related to flavonoid metabolism, two to sulphur metabolism and three RNA-binding proteins were identified. These proteins could be molecular targets for future studies focused on the improvement of legumes tolerance to drought. Moreover, this work also provides new hints for the deciphering of SNF regulation machinery in nodules., This work has been partially fundedby the Spanish National Research and Development Programme (AGL2011-30386-CO2-1 and AGL2011-23738).

Global 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., This work has been funded by the Spanish National Research and Development Programme-European Regional Development Fund ERDF (AGL2011-30386-C02-01 and AGL2011-30386-C02-02). IA was the recipient of a Ramon y Cajal research grant (Ministerio de Economia y Competitividad).

Although 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., This work has been funded by the Spanish National Research and Development Programme (AGL2011-30386-CO2-1 and AGL2011-30386-CO2-2). Iker Aranjuelo was the recipient of a Ramón y Cajal
research grant (Ministerio de Economía y Competitividad).

The 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., This work has been funded by the Spanish National Research and Development Programme (AGL2011-30386-CO2-1 and AGL2011-30386-CO2-2). Iker Aranjuelo was the recipient of a Ramón y Cajal research grant (Ministerio de Economía y Competitividad).