BUSCADOR RECOLECTA

In plants, symbiotic hemoglobins act as carriers and buffers of O2 in nodules, whereas nonsymbiotic hemoglobins or phytoglobins (Glbs) are ubiquitous in tissues and may perform multiple, but still poorly defined, functions related to O2 and/or nitric oxide (NO). Here, we have identified a Glb gene of the model legume Medicago truncatula with unique properties. The gene, designated MtGlb1-2, generates four alternative splice forms encoding Glbs with one or two heme domains and 215–351 amino acid residues. This is more than double the size of any hemoglobin from plants or other organisms described so far. A combination of molecular, cellular, biochemical, and biophysical methods was used to characterize these novel proteins. RNA-sequencing showed that the four splice variants are expressed in plant tissues. MtGlb1-2 is transcriptionally activated by hypoxia and its expression is further enhanced by an NO source. The gene is preferentially expressed in the meristems and vascular bundles of roots and nodules. Two of the proteins, bearing one or two hemes, were characterized using mutants in the distal histidines of the hemes. The Glbs are extremely reactive toward the physiological ligands O2, NO, and nitrite. They show very high O2 affinities, NO dioxygenase activity (in the presence of O2), and nitrite reductase (NiR) activity (in the absence of O2) compared with the hemoglobins from vertebrates and other plants. We propose that these Glbs act as either NO scavengers or NO producers depending on the O2 tension in the plant tissue, being involved in the fast and fine tuning of NO concentration in the cytosol in response to sudden changes in O2 availability.

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. © 2021 The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology.

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.

Background and aims: Sorghum (Sorghum bicolor) is able to exude allelochemicals with biological nitrifcation inhibition (BNI) capacity. Therefore, sorghum might be an option as cover crop since its BNI ability may reduce N pollution in the following crop due to a decreased nitrifcation. However, BNI exudation is related to the physiological state and development of the plant, so abiotic stresses such as drought might modify the rate of BNI exudation. Hence, the objective was to determine the efect of drought stress on sorghum plants’ BNI release. Methods: The residual efects of sorghum crops over ammonia-oxidizing bacteria (AOB) were monitored in a 3-year feld experiment. In a controlled-conditions experiment, sorghum plants were grown under Watered (60% WFPS) or Moderate drought (30% WFPS) conditions, and fertilized with ammonium sulphate (A), ammonium sulphate+DMPP (A+D), or potassium nitrate (KNO3 −). Soil mineral N was determined, and AOB populations were quantifed. Additionally, plant biomass, isotopic discrimination of N and C, and photosynthetic parameters were measured in sorghum plants. Results: In the driest year, sorghum was able to reduce the AOB relative abundance by 50% at feld conditions. In the plant-soil microcosm, drought stress reduced leaf photosynthetic parameters, which had an impact on plant biomass. Under these conditions, sorghum plants exposed to Moderate drought reduced the AOB abundance of A treatment by 25% compared to Watered treatment. Conclusion: The release of BNI by sorghum under limited water conditions might ensure high soil NH4 +-N pool for crop uptake due to a reduction of nitrifying microorganisms., Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This project was funded by the Spanish Government (RTI2018-094623-B-C21 and C22 MCIU/AEI/FEDER, UE) and by the Basque Government (IT-932-16; IT-1560-22). Dr Adrián Bozal-Leorri held a grant from the Basque Government (PRE-2020-2-0142). Dr. Fernando Torralbo was supported by the European Union – Next Generation EU program. Authors thank the Jesus de Gangoiti Barrera Foundation for the financial support.

In plants, symbiotic hemoglobins act as carriers and buffers of O2 in nodules, whereas nonsymbiotic hemoglobins or phytoglobins (Glbs) are ubiquitous in tissues and may perform multiple, but still poorly defined, functions related to O2 and/or nitric oxide (NO). Here, we have identified a Glb gene of the model legume Medicago truncatula with unique properties. The gene, designated MtGlb1-2, generates four alternative splice forms encoding Glbs with one or two heme domains and 215–351 amino acid residues. This is more than double the size of any hemoglobin from plants or other organisms described so far. A combination of molecular, cellular, biochemical, and biophysical methods was used to characterize these novel proteins. RNA-sequencing showed that the four splice variants are expressed in plant tissues. MtGlb1-2 is transcriptionally activated by hypoxia and its expression is further enhanced by an NO source. The gene is preferentially expressed in the meristems and vascular bundles of roots and nodules. Two of the proteins, bearing one or two hemes, were characterized using mutants in the distal histidines of the hemes. The Glbs are extremely reactive toward the physiological ligands O2, NO, and nitrite. They show very high O2 affinities, NO dioxygenase activity (in the presence of O2), and nitrite reductase (NiR) activity (in the absence of O2) compared with the hemoglobins from vertebrates and other plants. We propose that these Glbs act as either NO scavengers or NO producers depending on the O2 tension in the plant tissue, being involved in the fast and fine tuning of NO concentration in the cytosol in response to sudden changes in O2 availability., IV was a Formación de Personal Investigador fellow (BES-2015-073059) and EL is a Ramón y Cajal fellow (RYC2018-023867-I), both from the Spanish State Research Agency-Ministry of Economy, Industry and Competitiveness (MINECO). LM was the recipient of a fellowship partially funded by MIUR-Italy ('Progetto Dipartimenti di Eccellenza 2018-2022' allocated to Department of Chemistry 'Ugo Schiff'). This work was supported by grants AGL2017-85775-R and RTI2018-094623-B-C22 from MINECO, co-funded by the European Regional Development Fund, and by Government of Aragón (group A09_17R).

Background: The increasing demand for food production has led to a tenfold increase in nitrogen (N) fertilizer use since the Green Revolution. Nowadays, agricultural soils have been turned into high-nitrifying environments that increase N pollution. To decrease N losses, synthetic nitrification inhibitors (SNIs) such as 3,4-dimethylpyrazole phosphate (DMPP) have been developed. However, SNIs are not widely adopted by farmers due to their biologically limited stability and soil mobility. On the other hand, allelopathic substances from root exudates from crops such as sorghum are known for their activity as biological nitrification inhibitors (BNIs). These substances are released directly into the rhizosphere. Nevertheless, BNI exudation could be modified or even suppressed if crop development is affected. In this work, we compare the performance of biological (sorghum crop) and synthetic (DMPP) nitrification inhibitors in field conditions. Results: Sorghum crop BNIs and DMPP prevented an increase in the abundance of ammonia-oxidizing bacteria (AOB) without affecting the total bacterial abundance. Both nitrification inhibitors maintained similar soil NH4+ content, but at 30 days post-fertilization (DPF), the sorghum BNIs resulted in higher soil NO3− content than DMPP. Even so, these inhibitors managed to reduce 64% and 96%, respectively, of the NO3−-N/NH4+-N ratio compared to the control treatment. Similar to soil mineral N, there were no differences in leaf δ15N values between the two nitrification inhibitors, yet at 30 DPF, δ15N values from sorghum BNI were more positive than those of DMPP. N2O emissions from DMPP-treated soil were low throughout the experiment. Nevertheless, while sorghum BNIs also maintained low N2O emissions, they were associated with a substantial N2O emission peak at 3 DPF that lasted until 7 DPF. Conclusions: Our results indicate that while sorghum root exudates can reduce nitrification in field soil, even at the same efficiency as DMPP for a certain amount of time, they are not able to prevent the N pollution derived from N fertilization as DMPP does during the entire experiment., This project was funded by the Spanish Government (RTI2018-094623-B-C22 MCIU/AEI/FEDER, UE) and by the Basque Government (IT-932-16). Adrián Bozal-Leorri holds a Grant from the Basque Government (PRE-2020-2-0142). Mario Corrochano-Monsalve holds a Grant from the Ministry of Economy and Business of the Spanish Government (BES-2016-076725).

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., This work had been funded by the Spanish Ministry of Science and Innovation-European Regional Development Fund (grant RTI2018-094623-B-C22) and the Government of Navarra (project PC112-113 LEGUSI). EL was a Ramón y Cajal fellow (RYC2018-023867-I) and LC-P and MR were Formación de Personal Investigador fellows from the Spanish Ministry of Economy and Competitiveness (BES-2015-074411 and BES-2012-059972, respectively).

En un mundo que requiere urgentemente una agricultura más sostenible, los cultivos de leguminosas representan una importante alternativa. A nivel agroecológico, la característica más interesante de esta familia de plantas es su capacidad para llevar a cabo la fijación biológica de nitrógeno atmosférico (FBN), reduciendo los insumos de fertilizantes nitrogenados con una baja huella de carbono, lo que disminuye la contaminación ambiental y enriquece el suelo para los siguientes cultivos. Sin embargo, para optimizar el potencial de las leguminosas en un escenario donde los efectos del cambio climático conducirán a un aumento del impacto de las sequías, es preciso dilucidar los mecanismos fisiológicos y las vías de señalización molecular responsables de una mayor tolerancia de estos cultivos a esta situación de estrés. La disminución de la FBN provocada por la sequía es consecuencia de una serie de mecanismos fisiológicos entre los que destacan la disminución de la disponibilidad de sustratos carbonados para los bacteroides, la inhibición de la actividad nitrogenasa debido a la acumulación de compuestos nitrogenados, el incremento de la resistencia a la difusión de oxígeno y el estado redox. Con este fin de profundizar en estos factores, en este trabajo se han evaluado diferentes mecanismos implicados en la regulación del proceso de FBN bajo estrés hídrico., Ministerio de Economía y Competitividad AGL2014-56561-P, RTI2018-094623-B-C22 y sus correspondientes fondos FEDER. La autora ha contado con una ayuda del programa de Formación de Personal Investigador (FPI) del Ministerio de Economía y Competitividad, Programa de Doctorado en Agrobiología Ambiental (RD 99/2011), Ingurumen Agrobiologiako Doktoretza Programa (ED 99/2011)

Agriculture has increased the release of reactive nitrogen to the environment due to
crops’ low nitrogen-use efficiency (NUE) after the application of nitrogen-fertilisers. Practices like the use of stabilized-fertilisers with nitrification inhibitors such as DMPP (3,4-
dimethylpyrazole phosphate) have been adopted to reduce nitrogen losses. Otherwise, cover
crops can be used in crop-rotation-strategies to reduce soil nitrogen pollution and benefit
the following culture. Sorghum (Sorghum bicolor) could be a good candidate as it is drought
tolerant and its culture can reduce nitrogen losses derived from nitrification because it exudates biological nitrification inhibitors (BNIs). This work aimed to evaluate the effect of
fallow-wheat and sorghum cover crop-wheat rotations on N2O emissions and the grain
yield of winter wheat crop. In addition, the suitability of DMPP addition was also analyzed.
The use of sorghum as a cover crop might not be a suitable option to mitigate nitrogen
losses in the subsequent crop. Although sorghum–wheat rotation was able to reduce 22%
the abundance of amoA, it presented an increment of 77% in cumulative N2O emissions
compared to fallow–wheat rotation, which was probably related to a greater abundance of
heterotrophic-denitrification genes. On the other hand,the application of DMPP avoided the
growth of ammonia-oxidizing bacteria and maintained the N2O emissions at the levels of
unfertilized-soils in both rotations. As a conclusion, the use of DMPP would be recommendable regardless of the rotation since it maintains NH4
+ in the soil for longer and mitigates
the impact of the crop residues on nitrogen soil dynamics, This work was supported by the Spanish Government (RTI2018-094623-B-C21 and C22 MCIU/AEI/FEDER, UE) and the Basque Government (IT-932-16). Dr. Adrián Bozal-Leorri held a grant from the Basque Government (PRE-2020-2-0142). Dr. Mario Corrochano-Monsalve held a grant from the Ministry of Economy and Business of the Spanish Government (BES-2016-076725).

The use of nitrification inhibitors is an interesting tool to achieve a higher N efficiency in plants while decreasing the environmental impact of N fertilization. However, an integrated evaluation of the efficiency of nitrification inhibitors over time, understood as the period in which the nitrifying activity is inhibited or slows down, is necessary to assess whether their use is ecofriendly and sustainable.

To test the direct efficiency of 3,4-dimethyl-1H-pyrazole dihydrogen phosphate (DMPP) on nitrification, a study has been carried out in two cultures enriched with ammonia-oxidizing bacteria (AOB) obtained from a soil with continuous N fertilization (80 kg N ha−1 year−1 as NH4NO3) and from soil without N fertilization. In addition, Cu has been evaluated as a cofactor of ammonia monoxygenase, a key enzyme in the nitrifying activity of AOBs. On the other hand, the stability of DMP has been studied both in the cultivation system enriched in AOBs and in soil to assess the efficiency of the inhibitor due to its persistence over time.

Our work reveals that nitrification rates observed in cultures enriched in AOBs from genus Nitrosospira isolated from soils with continuous N fertilization were not higher than those of cultures without N fertilization. In AOB cultures, DMPP was a very efficient inhibitor of nitrification (> 50 % inhibition of integrated AMO activity), mainly due to the stability of DMP (3,4-dimethyl-1 H-pyrazole) in the cultures. However, DMP stability was significantly lower under soil conditions (> 90 % of DMP was degraded in the first 30 days of incubation). Other metals are suggested as cofactors of the enzyme ammonia monooxygenase alternatively to Cu., This work was supported by Spanish Government-Ministry of Science, Innovation and Universities (RTI2018-094623-B-C22), Spain. MCIN/AEI/10.130039/501100011033/FEDER, EU. J.M.R. was supported by a doctoral fellowship from Coordination for the Improvement of Higher Education Personnel (CAPES), of Brazilian Government (1I11903/13-9), Brazil. Open access funding provided by the Public University of Navarra.

In plants, symbiotic hemoglobins act as carriers and buffers of O2 in nodules, whereas nonsymbiotic hemoglobins or phytoglobins (Glbs) are ubiquitous in tissues and may perform multiple, but still poorly defined, functions related to O2 and/or nitric oxide (NO). Here, we have identified a Glb gene of the model legume Medicago truncatula with unique properties. The gene, designated MtGlb1-2, generates four alternative splice forms encoding Glbs with one or two heme domains and 215–351 amino acid residues. This is more than double the size of any hemoglobin from plants or other organisms described so far. A combination of molecular, cellular, biochemical, and biophysical methods was used to characterize these novel proteins. RNA-sequencing showed that the four splice variants are expressed in plant tissues. MtGlb1-2 is transcriptionally activated by hypoxia and its expression is further enhanced by an NO source. The gene is preferentially expressed in the meristems and vascular bundles of roots and nodules. Two of the proteins, bearing one or two hemes, were characterized using mutants in the distal histidines of the hemes. The Glbs are extremely reactive toward the physiological ligands O2, NO, and nitrite. They show very high O2 affinities, NO dioxygenase activity (in the presence of O2), and nitrite reductase (NiR) activity (in the absence of O2) compared with the hemoglobins from vertebrates and other plants. We propose that these Glbs act as either NO scavengers or NO producers depending on the O2 tension in the plant tissue, being involved in the fast and fine tuning of NO concentration in the cytosol in response to sudden changes in O2 availability., IV was a Formación de Personal Investigador fellow (BES-
2015-073059) and EL is a Ramón y Cajal fellow (RYC2018-
023867-I), both from the Spanish State Research Agency-Ministry of Economy, Industry and Competitiveness (MINECO). LM was the recipient of a fellowship partially funded by MIURItaly (“Progetto Dipartimenti di Eccellenza 2018-2022” allocated to Department of Chemistry “Ugo Schiff ”). This work was supported by grants AGL2017-85775-R and RTI2018-094623-B-C22 from MINECO, co-funded by the European Regional Development Fund, and by Government of Aragón (group A09_17R)., Peer reviewed

Atmospheric carbon dioxide (CO) is increasing, and this affects plant photosynthesis and biomass production. Under elevated CO conditions (eCO), plants need to cope with an unbalanced carbon-to-nitrogen ratio (C/N) due to a limited C sink strength and/or the reported constrains in leaf N. Here, we present a physiological and metabolic analysis of ammonium (NH)-tolerant pea plants (Pisum sativum L., cv. snap pea) grown hydroponically with moderate or high NH concentrations (2.5 or 10 mM), and under two atmospheric CO concentrations (400 and 800 ppm). We found that the photosynthetic efficiency of the NH tolerant pea plants remain intact under eCO thanks to the capacity of the plants to maintain the foliar N status (N content and total soluble proteins), and the higher C-skeleton requirements for NH assimilation. The capacity of pea plants grown at 800 ppm to promote the C allocation into mobile pools of sugar (mainly sucrose and glucose) instead of starch contributed to balancing plant C/N. Our results also support previous observations: plants exposed to eCO and NH nutrition can increase of stomatal conductance. Considering the C and N source-sink balance of our plants, we call for exploring a novel trait, combining NH tolerant plants with a proper NH nutrition management, as a way for a better exploitation of eCO in C3 crops., Ivan Jauregui acknowledge financial support of the Belgian FNRS (Fonds de la Recherché Scientifique) grant number 1.B.216.20F.

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, RTI2018–094623-B-C22., Peer reviewed