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1 Pag., Symbio􀆟c hemoglobins provide O2 to N2-fixing bacteria within legume nodules. However, the func􀆟ons of nonsymbio􀆟c hemoglobins or phytoglobins (Glbs) are less defined. Three Glb classes can be dis􀆟nguished based on phylogene􀆟c and biochemical analyses and may coexist in plant 􀆟ssues: class 1 Glbs have extreme O2 affinity and are induced by hypoxia; class 2 Glbs have moderate O2 affinity and are precursors of leghemoglobins; and class 3 have low O2 affinity and high sequence homology with bacterial truncated hemoglobins. Immunogold labeling combined with confocal microscopy of Glbs tagged with GFP at the C-terminus was used to determine the subcellular localiza􀆟ons of Glbs in the model plants Arabidopsis and Lotus japonicus. To this end, we used overexpressing and knockout or silenced lines of Arabidopsis, performed quan􀆟ta􀆟ve immunolabeling, and monitored the GFP-tagged proteins in leaf cells and protoplasts., This work was funded by grant AGL2017-85775-R from the Spanish Ministry of Economy, Industry and Competitiveness/European Regional Development Fund.

1 Pag., Nitric oxide (NO) is a gaseous free radical and signaling molecule with multiple functions in plants, including the onset of the legume-rhizobia symbiosis and the development and senescence of root nodules (Hichri et al., 2016). Some of these functions may rest on NO acting as intermediate in the signaling cascades of most phytohormones (Hill, 2012). To gain insight into the functions of NO in nodules, it is therefore crucial to localize the sites of NO production and accumulation at the tissue, cell, and organelle levels. For this purpose, we have employed two different techniques: electron paramagnetic resonance (EPR) spectroscopy and the specific fluorescent dye 4,5-diaminofluorescein diacetate (DAF-2 DA). The EPR detects the highly stable nitrosyl-leghemoglobin (Lb2+NO) complex, while the DAF-2 DA probe detects free NO. Also, we have used Bradyrhizobium diazoefficiens mutant strains lacking the enzymes of the denitrification pathway: nitrate reductase (Nap), nitrite reductase (NirK), or nitric oxide reductase (Nor) (Meakin et al., 2007)., Funded by grants AGL2014-53717-R and AGL2017-85775-R to M.B. and grant CTQ2015-64486-R to J.I.M. from the Ministry of Economy and Competitiveness - European Regional Development Fund.

1 Pag., Reactive oxygen (ROS; superoxide radicals and H2O2) and nitrogen (RNS; NO and nitrosothiols) species are of paramount importance in biology. In plants, ROS and RNS are generated in most cellular compartments. At the light microscopy level, the production of superoxide radicals, H2O2 and NO may be detected based on their reactions with nitroblue tetrazolium, diaminobenzidine and 4,5-diaminofluorescein diacetate, respectively. Additional techniques are the HyPer bioprobe for H2O2 and electron paramagnetic resonance (EPR) spectroscopy for free radicals. However, strict controls must be used to ensure specificity, discard interferences and minimize artifacts during tissue extraction. In the rhizobia-legume symbiosis, all three reactive species were detected in the infection threads and in mature and senescent nodules. Leghemoglobins (Lbs) are heme proteins found at millimolar concentrations in the cytoplasm of infected cells, where they transport and deliver O2 to the symbiosomes. We have used two experimental approaches involving Lbs to localize ROS and RNS in nodules and identify potential pitfalls of the methodology.

Resumen del trabajo presentado a la XIV Reunión Nacional del Metabolismo del Nitrógeno, celebrada en Segovia del 16 al 18 de mayo de 2018., El óxido nítrico (NO) es una molécula señal que desempeña múltiples funciones en las plantas, incluida la formación y desarrollo de los nódulos de leguminosas. Considerando la especial importancia biológica y reactividad del NO como radical libre gaseoso, hemos examinado la producción de NO en nódulos de leguminosas utilizando dos técnicas muy diferentes con fines comparativos: espectroscopia de resonancia paramagnética electrónica (EPR) y una sonda fluorescente específica (DAF-2 DA). La primera detecta la nitrosil-leghemoglobina (LbN0) y la segunda el NO libre. El EPR de nódulos intactos reveló la ausencia de LbNO en nódulos de judía o soja tratados o no con nitrato; por el contrario, la LbNO se acumuló tras el tratamiento con nitrato en nódulos de soja que eran deficientes en nitrito reductasa o NO reductasa, o que habían estado expuestos a temperatura ambiente durante 30-60 min desde la cosecha. Por consiguiente, concluimos que: (a) los bacteroides son una fuente principal de NO; (b) las enzimas desnitrificantes intervienen en la homeostasis de NO en los nódulos; (e) la producción de LbNO no es responsable de la inhibición de la fijación de nitrógeno por el nitrato; y (d) la LbNO se genera como artefacto en extractos de nódulos o en nódulos intactos que no han sido procesados inmediatamente después de su cosechado. El uso de la sonda fluorescente nos permitió detectar NO en células infectadas de nódulos de judía y soja y en el parénquima de nódulos de soja. En el parénquima nodular se ha localizado la barrera a la difusión de oxígeno, por lo que el NO podría intervenir en este mecanismo de control de la concentración de oxígeno. La señal de· NO disminuyó ligeramente tras la incubación con inhibidores de la nitrato reductasa pero no de la NO sintasa, lo que evidencia una contribución poco relevante de la nitrato reductasa y apoya la existencia en los nódulos de vías independientes del nitrato y de la arginina para la producción de NO. En conjunto, nuestros datos indican que los métodos de EPR y fluorescencia son complementarios para obtener conclusiones fiables sobre la producción de NO en los tejidos vegetales. El EPR es capaz de detectar NO in vivo pero solamente en la zona infectada del nódulo, donde se encuentra confinada la leghemoglobina, mientras que la sonda fluorescente permite localizar las células y tejidos de los nódulos donde se puede producir NO., Este trabajo ha sido financiado por los proyectos AGL2014-53717-R (M. B.) y CTQ2015-B4486-R (J.I.M.) del MINECO-FEDER.

Resumen del trabajo presentado al 7th Plant Nitric Oxide International Meeting (PNO), celebrado en Niza (Francia) del 24 al 26 de octubre de 2018., Nitric oxide (NO) is a gaseous free radical and signaling molecule with multiple functions in plants, including the onset of the Iegume-rhizobia symbiosis and the development and senescence of root nodules. Sorne of these functions may rest on NO acting as intermediate in the signaling cascades of most phytohormones. To gain insight into the functions of NO in nodules, it is therefore crucial to localize the sites of NO production and accumulation at the tissue, cell, and organelle levels. For this purpose, we have employed two different techniques: electron paramagnetic resonance (EPR) spectroscopy and the specific fluorescent dye 4,5- diaminofluorescein diacetate (DAF-2 DA). The EPR detects the highly stable nitrosylleghemoglobin (Lb2+NO) complex, while the DAF-2 DA probe detects free NO. Also, we have used Bradyrhizobium diazoefficiens mutant strains lacking the enzymes of the denitrification pathway: nitrate reductase (Nap ), ni tri te reductase (NirK), or nitric oxide reductase (Nor). EPR of intact nodules revealed that Lb2+No was absent from bean or soybean nodules regardless of nitrate supply. In contrast, NO accumulated in soybean nodules treated with nitrate that were defective in bacteria! NirK or Nor, as well as in nodules that had been exposed to ambient temperature for one hour after their harvest from the roots. These observations indicate that: (i) the bacteroids are a major source of NO in nodules treated with nitrate; (ii) the denitrification enzymes NirK and Nor are required for NO homeostasis; and (iii) Lb2+NO is not responsible for the inhibition of nitrogen fixation by nitrate. Further, we noted that Lb2+NO is artifactually generated in nodule extracts or in intact nodules that were not analyzed immediately after detachment. The fluorescent probe detected NO formation in bean and soybean nodule infected cells and in soybean nodule parenchyma. The NO signal was slightly decreased by inhibitors of nitrate reductase but not of nitric oxide synthase, which could indicate a minor contribution of plant nitrate reductase and supports the existence of nitrateand arginine-independent pathways for NO production. Collectively, our data indicate that EPR and fluorometric methods are complementary to draw reliable conclusions about NO production in plants., This work was supported by grants AGL2014-53717-R (M.B.) and CTQ2015-64486-R (J.I.M.) from Ministerio de Economía y Competitividad-Fondo Europeo de Desarrollo Regional.

[ES] Se estudia la exportación de sales y sedimentos en una cuenca de regadío de ambiente semiárido en Bardenas (Zaragoza). Los resultados muestran un incremento de sedimentos en el agua de salida de cuenca respecto a la de entrada, correspondiendo mayoritariamente a sales disueltas y muy poco a material en suspensión. Durante el periodo riego la concentración de sales es más baja que en los días sin riego, debido a una mayor dilución, si bien las pérdidas totales son superiores en fase de riego en relación con un caudal de retorno mucho más elevado., [EN] In this work, we analyse the salt and sediment load from an irrigation area located in a semi-arid environment in the Ebro Valley (Bardenas, Zaragoza). The results show higher solute and sediment load in the waters exiting the irrigation area compared to the inputs waters. Changes in the suspended load are very small, and most of the mercase corresponds to soluble salts. During the period of irrigation, solute concentration is lower than during the non-irrigation periods because of higher dilution. However, the total load output during the irrigation periods is greater because the irrigation flows and returns are also higher., This study has been supported by: Water resources management in a changing envirornnent: the impact of sediment on sustainabilitv” (WARMICE, ENV 4-CT98-0789), (Comisión Europea, Dirección General XII) La identificación de fuentes de sedimento y áreas generadoras de escorrentía en relación con los cambios de uso del suelo” CICYT (REN2000-1709-C04-O1/GLO). La recuperación del espacio agrícola como estrategia de gestión integrada del territorio en áreas de montaña: El ejemplo de los Altos Valles del Aragón y del Gállego” DGA(P049/2000). Los retornos salinos del sistema de riego Bardenas 1 y su contribución a la salirilzación de las aguas superficiales” CICYT (Nat 90-1077 - C02-02). “Impacto ambiental del regadío de Bardenas 1. Fuentes de contaminación y transporte de nutrientes en los efluentes de riego CICYT (HID. 96-1306).

40 Pags., 4 Tabls., 9 Figs. The definitive version is available at: www3.interscience.wiley.com, •Aluminum (Al) toxicity is a major limiting factor of crop production on acid soils, but the implication of oxidative stress in this process is controversial. A multidisciplinary approach was used here to address this question in the forage legume Lotus corniculatus.•Plants were treated with low Al concentrations in hydroponic culture, and physiological and biochemical parameters, together with semiquantitative metabolic and proteomic profiles, were determined.•The exposure of plants to 10 μM Al inhibited root and leaf growth, but had no effect on the production of reactive oxygen species or lipid peroxides. By contrast, exposure to 20 μM Al elicited the production of superoxide radicals, peroxide and malondialdehyde. In response to Al, there was a progressive replacement of the superoxide dismutase isoforms in the cytosol, a loss of ascorbate and consistent changes in amino acids, sugars and associated enzymes.•We conclude that oxidative stress is not a causative factor of Al toxicity. The increased contents in roots of two powerful Al chelators, malic and 2-isopropylmalic acids, together with the induction of an Al-activated malate transporter gene, strongly suggest that both organic acids are implicated in Al detoxification. The effects of Al on key proteins involved in cytoskeleton dynamics, protein turnover, transport, methylation reactions, redox control and stress responses underscore a metabolic dysfunction, which affects multiple cellular compartments, particularly in plants exposed to 20 μM Al., Joaquín Navascués was the recipient of a postdoctoral contract (JAE-CSIC program). This work was funded by the European Commission (FP6-2003-INCO-DEV2- 517617), the Ministry of Science and Innovation-Fondo Europeo de Desarrollo Regional (AGL2008-01298 and AGL2010-16515), and Gobierno de Aragón-Fondo Social Europeo (group A53).

1 copia .pdf (a3) de póster original presentado por los autores. 4 Figs., The thiol tripeptide GSH (yGIu-Cys-GIy) is a major antioxidant and redox buffer in plants, where it also performs critical functions in ccli cycle regulation, development, sulfur transport and storage, stress response, and heavy metal detoxification. In legumes, hGSH (yGlu-Cys-bAla) may partially or completely replace GSH with presumably the same functions. The synthesis of GSH is accomplished in two sequential reactions catalyzed by yECS and GSHS, whereas the synthesis of hGSH shares the same first enzyme and then requires a specific hGSHS. A better understanding of the regulation of GSH and hGSH biosynthesis in legumes during the stress response requires a precise determination of the subcellular localization of the enzymes and a quantitative expression analysis of the genes involved. For this purpose, two types of experiments were performed. First, these proteins were immunolocalized in legumes using electron microscopy. Second, the expression pattern of the three genes was determined in the model legume Lotus japonicus following treatment with several hormones that are crucial for plant development and stress signaling., This work was funded by grant AGL2008-01298 from MICINN-FEDER.

Beca de apoyo a la investigación de Ibercaja ; Beca para estancia en el extranjero de la European Science Foundation (ESF).

ISHS Acta Horticulturae 448: III International Symposium on Mineral Nutrition of Deciduous Fruit Trees. 5 Pag., 2 Tabl., 1 Fig. The definitive version is available at: http://www.actahort.org/, The performance of three different rootstock (Adara (Prunus cerasifera L.), SL 64 (Prunus mahaleb), and Colt (Prunus avium x Prunus pseudocerasus)) as nutrient suppliers has been studied for sweet cherry cultivars (Prunus avium L.). The results obtained suggested that, depending on the rootstock, the trees are submitted to different degrees of stress. According to the data obtained, it could be expected a different photosynthetic pattern of these trees for the same sweet cherry cultivar. Fluorescence measurements as well as photosynthetic pigment composition has been assessed to know the photosynthetic capacity of the trees. The highest Fv/Fm ratio was found in leaves from trees grafted over the rootstock Adara. An increase of the zeaxanthin (Z) content could be observed in the less adequate rootstock variety.

14 Pags.- 7 Figs.- 1 Tabl. © The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (cc by 4.0), Legumes express two major types of hemoglobins, namely symbiotic (leghemoglobins) and non-symbiotic (phytoglobins), with the latter being categorized into three classes according to phylogeny and biochemistry. Using knockout mutants, we show that all three phytoglobin classes are required for optimal vegetative and reproductive development of Lotus japonicus. The mutants of two class 1 phytoglobins showed different phenotypes: Ljglb1-1 plants were smaller and had relatively more pods, whereas Ljglb1-2 plants had no distinctive vegetative phenotype and produced relatively fewer pods. Non-nodulated plants lacking LjGlb2-1 showed delayed growth and alterations in the leaf metabolome linked to amino acid processing, fermentative and respiratory pathways, and hormonal balance. The leaves of mutant plants accumulated salicylic acid and contained relatively less methyl jasmonic acid, suggesting crosstalk between LjGlb2-1 and the signaling pathways of both hormones. Based on the expression of LjGlb2-1 in leaves, the alterations of flowering and fruiting of nodulated Ljglb2-1 plants, the developmental and biochemical phenotypes of the mutant fed on ammonium nitrate, and the heme coordination and reactivity of the protein toward nitric oxide, we conclude that LjGlb2-1 is not a leghemoglobin but an unusual class 2 phytoglobin. For comparison, we have also characterized a close relative of LjGlb2-1 in Medicago truncatula, MtLb3, and conclude that this is an atypical leghemoglobin., This work was supported by grants AGL2017-85775-R, PID2020-113985GB-I00, and RTI2018-094623-B-C22 from the Agencia Estatal de Investigación (AEI) and by Gobierno de Aragón (group A09_20R). IV was a ‘Formación de Personal Investigador’ fellow (BES-2015-073059) and EL is a ‘Ramón y Cajal’ fellow (RYC2018-023867-I), both funded by AEI.

Nitric oxide (NO) is a signaling molecule with multiple functions in plants. Given its critical importance and reactivity as a gaseous free radical, we have examined NO production in legume nodules using electron paramagnetic resonance (EPR) spectroscopy and the specific fluorescent dye 4,5-diaminofluorescein diacetate. Also, in this context, we critically assess previous and current views of NO production and detection in nodules. EPR of intact nodules revealed that nitrosyl-leghemoglobin (Lb2+NO) was absent from bean or soybean nodules regardless of nitrate supply, but accumulated in soybean nodules treated with nitrate that were defective in nitrite or nitric oxide reductases or that were exposed to ambient temperature. Consequently, bacteroids are a major source of NO, denitrification enzymes are required for NO homeostasis, and Lb2+NO is not responsible for the inhibition of nitrogen fixation by nitrate. Further, we noted that Lb2+NO is artifactually generated in nodule extracts or in intact nodules not analyzed immediately after detachment. The fluorescent probe detected NO formation in bean and soybean nodule infected cells and in soybean nodule parenchyma. The NO signal was slightly decreased by inhibitors of nitrate reductase but not by those of nitric oxide synthase, which could indicate a minor contribution of plant nitrate reductase and supports the existence of nitrate- and arginine-independent pathways for NO production. Together, our data indicate that EPR and fluorometric methods are complementary to draw reliable conclusions about NO production in plants., This work was supported by grants AGL2014-53717-R (to MB), CTQ2015-64486-R (to JIM), and AGL2013-45087-R (to MJD) from the Ministry of Economy and Competitiveness. These grants were co-funded by Fondos Europeos de Desarrollo Regional (FEDER).