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
Increased ascorbate biosynthesis does not improve nitrogen fixation nor alleviate the effect of drought stress in nodulated Medicago truncatula plants
(Frontiers Media, 2021) Cobos Porras, Inmaculada Libertad; Rubia Galiano, María Isabel; Huertas, Raúl; Kum, David; Dalton, David A.; Udvardi, Michael; Arrese-Igor Sánchez, César; Larrainzar Rodríguez, Estíbaliz; Institute for Multidisciplinary Research in Applied Biology - IMAB; Gobierno de Navarra / Nafarroako Gobernua, PC112-113 LEGUSI
Legume plants are able to establish nitrogen-fixing symbiotic relations with Rhizobium bacteria. This symbiosis is, however, affected by a number of abiotic constraints, particularly drought. One of the consequences of drought stress is the overproduction of reactive oxygen (ROS) and nitrogen species (RNS), leading to cellular damage and, ultimately, cell death. Ascorbic acid (AsA), also known as vitamin C, is one of the antioxidant compounds that plants synthesize to counteract this oxidative damage. One promising strategy for the improvement of plant growth and symbiotic performance under drought stress is the overproduction of AsA via the overexpression of enzymes in the Smirnoff-Wheeler biosynthesis pathway. In the current work, we generated Medicago truncatula plants with increased AsA biosynthesis by overexpressing MtVTC2, a gene coding for GDP-L-galactose phosphorylase. We characterized the growth and physiological responses of symbiotic plants both under well-watered conditions and during a progressive water deficit. Results show that increased AsA availability did not provide an advantage in terms of plant growth or symbiotic performance either under well-watered conditions or in response to drought.
Open Access
Physiological, hormonal and metabolic responses of two alfalfa cultivars with contrasting responses to drought
(MDPI, 2019) Soba Hidalgo, David; Zhou, Bangwei; Arrese-Igor Sánchez, César; Munné Bosch, Sergi; Aranjuelo Michelena, Iker; Zientziak; Institute for Multidisciplinary Research in Applied Biology - IMAB; Ciencias; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Alfalfa (Medicago sativa L.) is frequently constrained by environmental conditions such as drought. Within this context, it is crucial to identify the physiological and metabolic traits conferring a better performance under stressful conditions. In the current study, two alfalfa cultivars (San Isidro and Zhong Mu) with different physiological strategies were selected and subjected to water limitation conditions. Together with the physiological analyses, we proceeded to characterize the isotopic, hormone, and metabolic profiles of the different plants. According to physiological and isotopic data, Zhong Mu has a water-saver strategy, reducing water lost by closing its stomata but fixing less carbon by photosynthesis, and therefore limiting its growth under water-stressed conditions. In contrast, San Isidro has enhanced root growth to replace the water lost through transpiration due to its more open stomata, thus maintaining its biomass. Zhong Mu nodules were less able to maintain nodule N2 fixing activity (matching plant nitrogen (N) demand). Our data suggest that this cultivar-specific performance is linked to Asn accumulation and its consequent N-feedback nitrogenase inhibition. Additionally, we observed a hormonal reorchestration in both cultivars under drought. Therefore, our results showed an intra-specific response to drought at physiological and metabolic levels in the two alfalfa cultivars studied.
Open Access
Drought stress provokes the down-regulation of methionine and ethylene biosynthesis pathways in Medicago truncatula roots and nodules
(Wiley, 2014) Larrainzar Rodríguez, Estíbaliz; Molenaar, Johanna A.; Wienkoop, Stefanie; Gil Quintana, Erena; Alibert, Bénédicte; Limami, Anis M.; Arrese-Igor Sánchez, César; González García, Esther; Ciencias del Medio Natural; Natura Ingurunearen Zientziak; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 735/2008
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.
Open Access
Split‐root systems applied to the study of the legume‐rhizobial symbiosis: what have we learned?
(Wiley, 2014) Larrainzar Rodríguez, Estíbaliz; Gil Quintana, Erena; Arrese-Igor Sánchez, César; González García, Esther; Marino Bilbao, Daniel; Ciencias del Medio Natural; Natura Ingurunearen Zientziak
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.
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, 57/2007
Superoxide 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.
Open Access
Nodule carbohydrate catabolism is enhanced in the Medicago truncatula A17-Sinorhizobium medicae WSM419 symbiosis
(Frontiers Media, 2014) Larrainzar Rodríguez, Estíbaliz; Gil Quintana, Erena; Seminario Huárriz, Amaia; Arrese-Igor Sánchez, César; González García, Esther; Ciencias del Medio Natural; Natura Ingurunearen Zientziak; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
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.
Open Access
Additive effects of heatwave and water stresses on soybean seed yield is caused by impaired carbon assimilation at pod formation but not at flowering
(Elsevier, 2022) Soba Hidalgo, David; Arrese-Igor Sánchez, César; Aranjuelo Michelena, Iker; Institute for Multidisciplinary Research in Applied Biology - IMAB; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Heatwave (HW) combined with water stress (WS) are critical environmental factors negatively affecting crop development. This study aimed to quantify the individual and combined effects of HW and WS during early reproductive stages on leaf and nodule functioning and their relation with final soybean seed yield (SY). For this purpose, during flowering (R2) and pod formation (R4) soybean (Glycine max L. Merr.) plants were exposed to different temperature (ambient[25ºC] versus HW[40ºC]) and water availability (full capacity versus WS[20% field capacity]). HW, WS and their combined impact on yield depended on the phenological stage at which stress was applied being more affected at R4. For gas exchange, WS severely impaired photosynthetic machinery, especially when combined with HS. Impaired photoassimilate supply at flowering caused flower abortion and a significant reduction in final SY due to interacting stresses and WS. On the other hand, at pod formation (R4), decreased leaf performance caused additive effect on SY by decreasing pod setting and seed size with combined stresses. At the nodule level, WS (alone or in combination with HW) caused nodule impairment, which was reflected by lower leaf N. Such response was linked with a poor malate supply to bacteroids and feed-back inhibition caused by nitrogenous compounds accumulation. In summary, our study noted that soybean sensitivity to interacting heat and water stresses was highly conditioned by the phenological stage at which it occurs with, R4 stage being the critical moment. To our knowledge this is the first soybean work integrating combined stresses at early reproductive stages.
Open Access
Long-term mannitol-induced osmotic stress leads to stomatal closure, carbohydrate accumulation and changes in leaf elasticity in Phaselous vulgaris leaves
(Academic Journals, 2010) Sassi, Sameh; Aydi, Samir; Hessini, Kamel; González García, Esther; Arrese-Igor Sánchez, César; Abdelly, Chedly; Ciencias del Medio Natural; Natura Ingurunearen Zientziak
The effect of long-term osmotic stress was investigated in leaves of two common bean lines, with contrasting tolerance: Flamingo (tolerant) and coco blanc (sensitive). Water relations, organic solute, ion accumulation and amino acids content as well as osmotic adjustment (OA) were studied during an extended exposure to osmotic stress. Osmotic stress was applied by means of 50 mM mannitol for 15 days. At the end of the stress period, both osmotic potential at full turgor (psi(100)) and at turgor loss point (psi(0)) decreased significantly in stressed plants compared with the control. The decrease being greater in the sensitive line, showed a greater OA compared with flamingo. Sugars contents increased in stressed plants and seem to be the major components of osmotic adjustment in stressed common bean leaves. The increase was more marked in coco blanc. Osmotic stress tolerance could thus not be associated with higher OA. The possible role of decreased leaf cell elasticity (epsilon(max)) is discussed in relation to osmotic stress tolerance in this species.
Open Access
Physiological responses of legume nodules to drought
(Global Science Books, 2011) Arrese-Igor Sánchez, César; González García, Esther; Marino Bilbao, Daniel; Ladrera Fernández, Rubén; Larrainzar Rodríguez, Estíbaliz; Gil Quintana, Erena; Ciencias del Medio Natural; Natura Ingurunearen Zientziak
Legumes include important agricultural crops, as their high protein content is of primary importance for human food and animal feed. In addition, the ability of most of them to establish symbiotic relationships with soil bacteria allows them to obtain their N requirements from nitrogen fixation in nodules and, therefore, avoids the use of nitrogen fertilizers. Thus, legumes are also essential to improve the soil fertility and quality of agricultural lands and to reclaim eroded or barren areas, making them crucial for agricultural and environmental sustainability. However, legume nitrogen fixation in crop species is very sensitive to environmental constraints and drought, in particular. The present contribution reviews our current knowledge on the processes involved in this inhibition, with particular emphasis on oxygen, nitrogen and carbon physiology. Emerging aspects such as oxidative damage, C/N interactions and sulphur metabolism together with future prospects are also discussed.