Enlisting wild grass genes to combat nitrification in wheat farming: A nature-based solution.

Active nitrifiers and rapid nitrification are major contributing factors to nitrogen losses in global wheat production. Suppressing nitrifier activity is an effective strategy to limit N losses from agriculture. Production and release of nitrification inhibitors from plant roots is termed "biological nitrification inhibition" (BNI). Here, we report the discovery of a chromosome region that controls BNI production in "wheat grass" Leymus racemosus (Lam.) Tzvelev, located on the short arm of the "Lr#3Nsb" (Lr#n), which can be transferred to wheat as T3BL.3NsbS (denoted Lr#n-SA), where 3BS arm of chromosome 3B of wheat was replaced by 3NsbS of L. racemosus We successfully introduced T3BL.3NsbS into the wheat cultivar "Chinese Spring" (CS-Lr#n-SA, referred to as "BNI-CS"), which resulted in the doubling of its BNI capacity. T3BL.3NsbS from BNI-CS was then transferred to several elite high-yielding hexaploid wheat cultivars, leading to near doubling of BNI production in "BNI-MUNAL" and "BNI-ROELFS." Laboratory incubation studies with root-zone soil from field-grown BNI-MUNAL confirmed BNI trait expression, evident from suppression of soil nitrifier activity, reduced nitrification potential, and N2O emissions. Changes in N metabolism included reductions in both leaf nitrate, nitrate reductase activity, and enhanced glutamine synthetase activity, indicating a shift toward ammonium nutrition. Nitrogen uptake from soil organic matter mineralization improved under low N conditions. Biomass production, grain yields, and N uptake were significantly higher in BNI-MUNAL across N treatments. Grain protein levels and breadmaking attributes were not negatively impacted. Wide use of BNI functions in wheat breeding may combat nitrification in high N input-intensive farming but also can improve adaptation to low N input marginal areas.

Drug discovery-based approach identifies new nitrification inhibitors.

Nitrogen (N) fertilization is crucial to sustain global food security, but fertilizer N production is energy-demanding and subsequent environmental N losses contribute to biodiversity loss and climate change. N losses can be mitigated be interfering with microbial nitrification, and therefore the use of nitrification inhibitors in enhanced efficiency fertilizers (EEFs) is an important N management strategy to increase N use efficiency and reduce N pollution. However, currently applied nitrification inhibitors have limitations and do not target all nitrifying microorganisms. Here, to identify broad-spectrum nitrification inhibitors, we adopted a drug discovery-based approach and screened 45,400 small molecules on different groups of nitrifying microorganisms. Although a high number of potential nitrification inhibitors were identified, none of them targeted all nitrifier groups. Moreover, a high number of new nitrification inhibitors were shown to be highly effective in culture but did not reduce ammonia consumption in soil. One archaea-targeting inhibitor was not only effective in soil, but even reduced - when co-applied with a bacteria-targeting inhibitor - ammonium consumption and greenhouse gas emissions beyond what is achieved with currently applied nitrification inhibitors. This advocates for combining different types of nitrification inhibitors in EEFs to optimize N management practices and make agriculture more sustainable.

Evaluation of a crop rotation with biological inhibition potential to avoid N2O emissions in comparison with synthetic nitrification inhibition.

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.

Mitigation of yield-scaled nitrous oxide emissions and global warming potential in an oilseed rape crop through N source management.

Enhanced-efficiency nitrogen (N) fertilizers, such as those containing nitrification or urease inhibitors, can mitigate the carbon (C) footprint linked to the production of bioenergy crops through a reduction in direct nitrous oxide (N2O) emissions and indirect N2O losses. These indirect emissions are derived from ammonia (NH3) volatilization, which also have important environmental and health implications. The evaluation of the global warming potential (GWP) of different N sources using site-specific data of yield and direct and indirect emissions is needed for oilseed rape under rainfed semi-arid conditions, especially when meteorological variability is taken into account. Using urea as a N source, the N2O mitigation efficacy of the urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT) alone or combined with the nitrification inhibitor 2-(3,4-dimethyl-1H-pyrazol-1-yl) succinic acid isomeric mixture (DMPSA) was evaluated under field conditions in a rainfed oilseed rape (Brassica napus L.) crop. Two additional N sources from calcium ammonium nitrate (CAN), with and without DMPSA, were included. The GWP of the treatments was estimated considering the emissions from inputs, operations and other direct and indirect emissions of greenhouse gases (GHGs), such as methane (CH4) and the volatilization of NH3. We also measured the abundance of key genes involved in nitrification and denitrification to improve the understanding of N2O emissions on a biochemical basis under the conditions of our study. The results show that due to the intense rainfall after fertilization and a rewetting event, N2O losses from fertilizers without inhibitors were greater than those previously reported under Mediterranean conditions, while NH3 losses were low and not affected by the urease inhibitor. The cumulative N2O emissions (which were greatly influenced by a rewetting peak three months after fertilization) from the urea fertilization were significantly higher than those from CAN. The presence of NBPT significantly reduced N2O losses by an average of 71%, with respect to urea. The use of DMPSA with CAN resulted in an abatement of N2O emissions (by 57%) and a significant increase in oil yield in comparison with CAN alone. All inhibitor-based treatments were effective in abating N2O emissions during the rewetting peak. The abundances of the nitrifier and denitrifier communities, especially ammonia-oxidizing bacteria (AOB), significantly decreased relative to the urea or CAN treatments as inhibitors were applied. Under the conditions of our study, the sustainability of a bioenergy crop such as oilseed rape can be improved by using inhibitors because they mitigated N2O emissions and/or enhanced the oil yield.

C and N metabolism in barley leaves and peduncles modulates responsiveness to changing CO2.

Balancing of leaf carbohydrates is a key process for maximising crop performance in elevated CO2 environments. With the aim of testing the role of the carbon sink-source relationship under different CO2 conditions, we performed two experiments with two barley genotypes (Harrington and RCSL-89) exposed to changing CO2. In Experiment 1, the genotypes were exposed to 400 and 700 ppm CO2. Elevated CO2 induced photosynthetic acclimation in Harrington that was linked with the depletion of Rubisco protein. In contrast, a higher peduncle carbohydrate-storage capacity in RSCL-89 was associated with a better balance of leaf carbohydrates that could help to maximize the photosynthetic capacity under elevated CO2. In Experiment 2, plants that were grown at 400 ppm or 700 ppm CO2 for 5 weeks were switched to 700 ppm or 400 ppm CO2, respectively. Raising CO2 to 700 ppm increased photosynthetic rates with a reduction in leaf carbohydrate content and an improvement in N assimilation. The increase in nitrate content was associated with up-regulation of genes of protein transcripts of photosynthesis and N assimilation that favoured plant performance under elevated CO2. Finally, decreasing the CO2 from 700 ppm to 400 ppm revealed that both stomatal closure and inhibited expression of light-harvesting proteins negatively affected photosynthetic performance and plant growth.

Elevated CO2 Induces Root Defensive Mechanisms in Tomato Plants When Dealing with Ammonium Toxicity.

An adequate carbon supply is fundamental for plants to thrive under ammonium stress. In this work, we studied the mechanisms involved in tomato (Solanum lycopersicum L.) response to ammonium toxicity when grown under ambient or elevated CO2 conditions (400 or 800 p.p.m. CO2). Tomato roots were observed to be the primary organ dealing with ammonium nutrition. We therefore analyzed nitrogen (N) and carbon (C) metabolism in the roots, integrating the physiological response with transcriptomic regulation. Elevated levels of CO2 preferentially stimulated root growth despite the high ammonium content. The induction of anaplerotic enzymes from the tricarboxylic acid (TCA) cycle led to enhanced amino acid synthesis under ammonium nutrition. Furthermore, the root transcriptional response to ammonium toxicity was improved by CO2-enriched conditions, leading to higher expression of stress-related genes, as well as enhanced modulation of genes related to signaling, transcription, transport and hormone metabolism. Tomato roots exposed to ammonium stress also showed a defense-like transcriptional response according to the modulation of genes related to detoxification and secondary metabolism, involving principally terpenoid and phenolic compounds. These results indicate that increasing C supply allowed the co-ordinated regulation of root defense mechanisms when dealing with ammonium toxicity.

Quantitative proteomics reveals the importance of nitrogen source to control glucosinolate metabolism in Arabidopsis thaliana and Brassica oleracea.

Accessing different nitrogen (N) sources involves a profound adaptation of plant metabolism. In this study, a quantitative proteomic approach was used to further understand how the model plant Arabidopsis thaliana adjusts to different N sources when grown exclusively under nitrate or ammonium nutrition. Proteome data evidenced that glucosinolate metabolism was differentially regulated by the N source and that both TGG1 and TGG2 myrosinases were more abundant under ammonium nutrition, which is generally considered to be a stressful situation. Moreover, Arabidopsis plants displayed glucosinolate accumulation and induced myrosinase activity under ammonium nutrition. Interestingly, these results were also confirmed in the economically important crop broccoli (Brassica oleracea var. italica). Moreover, these metabolic changes were correlated in Arabidopsis with the differential expression of genes from the aliphatic glucosinolate metabolic pathway. This study underlines the importance of nitrogen nutrition and the potential of using ammonium as the N source in order to stimulate glucosinolate metabolism, which may have important applications not only in terms of reducing pesticide use, but also for increasing plants' nutritional value.

Exploring ammonium tolerance in a large panel of Arabidopsis thaliana natural accessions.

Plants are dependent on exogenous nitrogen (N) supply. Ammonium (NH4(+)), together with nitrate (NO3(-)), is one of the main nitrogenous compounds available in the soil. Paradoxically, although NH4 (+) assimilation requires less energy than that of NO3(-), many plants display toxicity symptoms when grown with NH4(+) as the sole N source. However, in addition to species-specific ammonium toxicity, intraspecific variability has also been shown. Thus, the aim of this work was to study the intraspecific ammonium tolerance in a large panel of Arabidopsis thaliana natural accessions. Plants were grown with either 1mM NO3(-) or NH4(+) as the N source, and several parameters related to ammonium tolerance and assimilation were determined. Overall, high variability was observed in A. thaliana shoot growth under both forms of N nutrition. From the parameters determined, tissue ammonium content was the one with the highest impact on shoot biomass, and interestingly this was also the case when N was supplied as NO3(-). Enzymes of nitrogen assimilation did not have an impact on A. thaliana biomass variation, but the N source affected their activity. Glutamate dehydrogenase (GDH) aminating activity was, in general, higher in NH4 (+)-fed plants. In contrast, GDH deaminating activity was higher in NO3(-)-fed plants, suggesting a differential role for this enzyme as a function of the N form supplied. Overall, NH4 (+) accumulation seems to be an important player in Arabidopsis natural variability in ammonium tolerance rather than the cell NH4(+) assimilation capacity.

Spatial and temporal dynamics of the colonization of Pinus radiata by Fusarium circinatum, of conidiophora development in the pith and of traumatic resin duct formation.

· Fusarium circinatum causes pitch canker disease in a wide range of pine trees, including Pinus radiata, with devastating economic consequences. · To assess the spatial and temporal dynamics of growth of this pathogen in radiata pine, we examined the process of infection using both real-time PCR to quantify fungal biomass inside the plant host, and confocal microscopy using a green fluorescent protein (GFP)-tagged strain of F. circinatum. · Pathogen growth exhibited three distinct phases: an initial exponential increase in fungal biomass, concomitant with pathogen colonization of the cortex and phloem; a slowdown in fungal growth coincident with sporulating hyphae deep within the host; and stabilization of the fungal biomass when the first wilting symptoms appeared. The number of resin ducts in the xylem was found to increase in response to infection and the fungus grew inside both constitutive and traumatic resin ducts. · These results indicate that conidiation may contribute to the spatial or temporal dissemination of the pathogen. Moreover, the present findings raise the intriguing possibility that the generation of traumatic resin ducts may be of more benefit to the fungus than to the plant.

Physiological aspects underlying the improved outplanting performance of Pinus pinaster Ait. seedlings associated with ectomycorrhizal inoculation.

Mycorrhizal inoculation of conifer roots is a key strategy to optimize establishment and performance of forest tree species under both natural and cultivated conditions and also to mitigate transplantation shock. However, despite being a common practice, inoculation in outdoor nursery conditions has been poorly studied. Here, we have evaluated effectiveness of four fungal species (Lactarius deliciosus, Lactarius quieticolor, Pisolithus arhizus, and Suillus luteus) in the production of mycorrhizal Pinus pinaster seedlings in an outdoor commercial nursery and their ability to improve seedling physiology and field performance. All inoculated seedlings showed a significant increase in growth at the end of the nursery stage and these differences remained after 3 years of growth in the field. Differences observed in the content of malondialdehyde, total chlorophyll, carotenoids, anthocyanins, and phenolic compounds from needles of mycorrhizal and control seedlings may reflect a different sensitivity to photo-oxidative damage. We conclude that ectomycorrhizal inoculation improves adaptability to changeable growing conditions of an outdoor nursery and produces a higher quality nursery stock, thereby enhancing seedling performance after planting.

Ammonium as sole N source improves grain quality in wheat.

BACKGROUND: The skilful handling of N fertilizer, including N source type and its timing, is necessary to obtain maximum profitability in wheat crops in terms of production and quality. Studies on grain yield and quality with ammonium as sole N source have not yet been conducted. The aim of this study was to evaluate the effect of N source management (nitrate vs. ammonium), and splitting it into two or three amendments during the wheat life cycle, on grain yield and quality under irrigated conditions. RESULTS: This experiment demonstrates that Cezanne wheat plants growing with ammonium as exclusive N source are able to achieve the same yield as plants growing with nitrate and that individual wheat plants grown in irrigated pots can efficiently use late N applied in GS37. Ammonium nutrition increased both types of grain reserve proteins (gliadins and glutenins) and also increased the ratio gli/glu with respect to nitrate nutrition. The splitting of the N rate enhanced the ammonium effect on grain protein composition. CONCLUSIONS: The application of ammonium N source, especially when split into three amendments, has an analogous effect on grain protein content and composition to applications at a higher N rate, leading to higher N use efficiency.

Dimethylpyrazole-based nitrification inhibitors have a dual role in N2O emissions mitigation in forage systems under Atlantic climate conditions.

Nitrogen fertilization is the most important factor increasing nitrous oxide (N2O) emissions from agriculture, which is a powerful greenhouse gas. These emissions are mainly produced by the soil microbial processes of nitrification and denitrification, and the application of nitrification inhibitors (NIs) together with an ammonium-based fertilizer has been proved as an efficient way to decrease them. In this work the NIs dimethylpyrazole phosphate (DMPP) and dimethylpyrazole succinic acid (DMPSA) were evaluated in a temperate grassland under environmental changing field conditions in terms of their efficiency reducing N2O emissions and their effect on the amount of nitrifying and denitrifying bacterial populations responsible of these emissions. The stimulation of nitrifying bacteria induced by the application of ammonium sulphate as fertilizer was efficiently avoided by the application of both DMPP and DMPSA whatever the soil water content. The denitrifying bacteria population capable of reducing N2O up to N2 was also enhanced by both NIs provided that sufficiently high soil water conditions and low nitrate content were occurring. Therefore, both NIs showed the capacity to promote the denitrification process up to N2 as a mechanism to mitigate N2O emissions. DMPSA proved to be a promising NI, since it showed a more significant effect than DMPP in decreasing N2O emissions and increasing ryegrass yield.

Biological nitrification inhibitor-trait enhances nitrogen uptake by suppressing nitrifier activity and improves ammonium assimilation in two elite wheat varieties.

Synthetic nitrification inhibitors (SNI) and biological nitrification inhibitors (BNI) are promising tools to limit nitrogen (N) pollution derived from agriculture. Modern wheat cultivars lack sufficient capacity to exude BNIs, but, fortunately, the chromosome region (Lr#n-SA) controlling BNI production in Leymus racemosus, a wild relative of wheat, was introduced into two elite wheat cultivars, ROELFS and MUNAL. Using BNI-isogenic-lines could become a cost-effective, farmer-friendly, and globally scalable technology that incentivizes more sustainable and environmentally friendly agronomic practices. We studied how BNI-trait improves N-uptake, and N-use, both with ammonium and nitrate fertilization, analysing representative indicators of soil nitrification inhibition, and plant metabolism. Synthesizing BNI molecules did not mean a metabolic cost since Control and BNI-isogenic-lines from ROELFS and MUNAL presented similar agronomic performance and plant development. In the soil, ROELFS-BNI and MUNAL-BNI plants decreased ammonia-oxidizing bacteria (AOB) abundance by 60% and 45% respectively, delaying ammonium oxidation without reducing the total abundance of bacteria or archaea. Interestingly, BNI-trait presented a synergistic effect with SNIs since made it also possible to decrease the AOA abundance. ROELFS-BNI and MUNAL-BNI plants showed a reduced leaf nitrate reductase (NR) activity as a consequence of lower soil NO 3 - formation and a higher amino acid content compared to BNI-trait lacking lines, indicating that the transfer of Lr#-SA was able to induce a higher capacity to assimilate ammonium. Moreover, the impact of the BNI-trait in wheat cultivars was also noticeable for nitrate fertilization, with improved N absorption, and therefore, reducing soil nitrate content.

Depletion of the heaviest stable N isotope is associated with NH4+/NH3 toxicity in NH4+-fed plants.

BACKGROUND: In plants, nitrate (NO3-) nutrition gives rise to a natural N isotopic signature (δ15N), which correlates with the δ15N of the N source. However, little is known about the relationship between the δ15N of the N source and the 14N/15N fractionation in plants under ammonium (NH4+) nutrition. When NH4+ is the major N source, the two forms, NH4+ and NH3, are present in the nutrient solution. There is a 1.025 thermodynamic isotope effect between NH3 (g) and NH4+ (aq) which drives to a different δ15N. Nine plant species with different NH4+-sensitivities were cultured hydroponically with NO3- or NH4+ as the sole N sources, and plant growth and δ15N were determined. Short-term NH4+/NH3 uptake experiments at pH 6.0 and 9.0 (which favours NH3 form) were carried out in order to support and substantiate our hypothesis. N source fractionation throughout the whole plant was interpreted on the basis of the relative transport of NH4+ and NH3. RESULTS: Several NO3--fed plants were consistently enriched in 15N, whereas plants under NH4+ nutrition were depleted of 15N. It was shown that more sensitive plants to NH4+ toxicity were the most depleted in 15N. In parallel, N-deficient pea and spinach plants fed with 15NH4+ showed an increased level of NH3 uptake at alkaline pH that was related to the 15N depletion of the plant. Tolerant to NH4+ pea plants or sensitive spinach plants showed similar trend on 15N depletion while slight differences in the time kinetics were observed during the initial stages. The use of RbNO3 as control discarded that the differences observed arise from pH detrimental effects. CONCLUSIONS: This article proposes that the negative values of δ15N in NH4+-fed plants are originated from NH3 uptake by plants. Moreover, this depletion of the heavier N isotope is proportional to the NH4+/NH3 toxicity in plants species. Therefore, we hypothesise that the low affinity transport system for NH4+ may have two components: one that transports N in the molecular form and is associated with fractionation and another that transports N in the ionic form and is not associated with fractionation.

Mechanism of action of nitrification inhibitors based on dimethylpyrazole: A matter of chelation.

In agriculture, the applied nitrogen (N) can be lost in the environment in different forms because of microbial transformations. It is of special concern the nitrate (NO3-) leaching and the nitrous oxide (N2O) emissions, due to their negative environmental impacts. Nitrification inhibitors (NIs) based on dimethylpyrazole (DMP) are applied worldwide in order to reduce N losses. These compounds delay ammonium (NH4+) oxidation by inhibiting ammonia-oxidizing bacteria (AOB) growth. However, their mechanism of action has not been demonstrated, which represent an important lack of knowledge to use them correctly. In this work, through chemical and biological analysis, we unveil the mechanism of action of the commonly applied 3,4-dimethyl-1H-pyrazole dihydrogen phosphate (DMPP) and the new DMP-based NI, 2-(3,4-dimethyl-1H-pyrazol-1-yl)-succinic acid (DMPSA). Our results show that DMP and DMPSA form complexes with copper (Cu2+) cations, an indispensable cofactor in the nitrification pathway. Three coordination compounds namely [Cu(DMP)4Cl2] (CuDMP1), [Cu(DMP)4SO4]n (CuDMP2) and [Cu(DMPSA)2]·H2O (CuDMPSA) have been synthesized and chemical and structurally characterized. The CuDMPSA complex is more stable than those containing DMP ligands; however, both NIs show the same nitrification inhibition efficiency in soils with different Cu contents, suggesting that the active specie in both cases is DMP. Our soil experiment reveals that the usual application dose is enough to inhibit nitrification within the range of Cu and Zn contents present in agricultural soils, although their effects vary depending on the content of these elements. As a result of AOB inhibition by these NIs, N2O-reducing bacteria seem to be beneficed in Cu-limited soils due to a reduction in the competence. This opens up the possibility to induce N2O reduction to N2 through Cu fertilization. On the other hand, when fertilizing with micronutrients such as Cu and Zn, the use of NIs could be beneficial to counteract the increase of nitrification derived from their application.

Impact of dimethylpyrazole-based nitrification inhibitors on soil-borne bacteria.

Nitrogen (N) input from fertilizers modifies the properties of agricultural soils as well as bacterial community diversity, composition and relationships. This can lead to negative impacts such as the deterioration of system multifunctionality, whose maintenance is critical to normal nutrient cycling. Synthetic nitrification inhibitors (NIs) can be combined with fertilizers to improve the efficiency of N use by reducing N losses. However, analysis of their effects on non-target bacteria are scarce. This study aimed to analyze the effect of applying the NIs DMPP and DMPSA on the whole bacterial community. Through 16S rRNA amplicon sequencing we determined the differences between samples in terms of microbial diversity, composition and co-occurrence networks. The application of DMPP and DMPSA exerted little impact on the abundance of the dominant phyla. Nevertheless, several significant shifts were detected in bacterial diversity, co-occurrence networks, and the abundance of particular taxa, where soil water content played a key role. For instance, the application of NIs intensified the negative impact of N fertilization on bacterial diversity under high water-filled pore spaces (WFPS) (>64%), reducing community diversity, whereas alpha-diversity was not affected at low WFPS (<55%). Interestingly, despite NIs are known to inhibit ammonia monooxygenase (AMO) enzyme, both NIs almost exclusively inhibited Nitrosomonas genera among AMO holding nitrifiers. Thus, Nitrosomonas showed abundance reductions of up to 47% (DMPP) and 66% (DMPSA). Nonetheless, non-target bacterial abundances also shifted with NI application. Notably, DMPSA application partially alleviated the negative effect of fertilization on soil multifunctionality. A remarkable increase in populations related to system multifunctionality, such as Armatimonadetes (up to +21%), Cyanobacteria (up to +30%) and Fibrobacteres (up to +25%) was observed when DMPSA was applied. NI application substantially influenced microbial associations by decreasing the complexity of co-occurrence networks, decreasing the total edges and node connectivity, and increasing path distances.

Short-Term Exposure to High Atmospheric Vapor Pressure Deficit (VPD) Severely Impacts Durum Wheat Carbon and Nitrogen Metabolism in the Absence of Edaphic Water Stress.

Low atmospheric relative humidity (RH) accompanied by elevated air temperature and decreased precipitation are environmental challenges that wheat production will face in future decades. These changes to the atmosphere are causing increases in air vapor pressure deficit (VPD) and low soil water availability during certain periods of the wheat-growing season. The main objective of this study was to analyze the physiological, metabolic, and transcriptional response of carbon (C) and nitrogen (N) metabolism of wheat (Triticum durum cv. Sula) to increases in VPD and soil water stress conditions, either alone or in combination. Plants were first grown in well-watered conditions and near-ambient temperature and RH in temperature-gradient greenhouses until anthesis, and they were then subjected to two different water regimes well-watered (WW) and water-stressed (WS), i.e., watered at 50% of the control for one week, followed by two VPD levels (low, 1.01/0.36 KPa and high, 2.27/0.62 KPa; day/night) for five additional days. Both VPD and soil water content had an important impact on water status and the plant physiological apparatus. While high VPD and water stress-induced stomatal closure affected photosynthetic rates, in the case of plants watered at 50%, high VPD also caused a direct impairment of the RuBisCO large subunit, RuBisCO activase and the electron transport rate. Regarding N metabolism, the gene expression, nitrite reductase (NIR) and transport levels detected in young leaves, as well as determinations of the δ15N and amino acid profiles (arginine, leucine, tryptophan, aspartic acid, and serine) indicated activation of N metabolism and final transport of nitrate to leaves and photosynthesizing cells. On the other hand, under low VPD conditions, a positive effect was only observed on gene expression related to the final step of nitrate supply to photosynthesizing cells, whereas the amount of 15N supplied to the roots that reached the leaves decreased. Such an effect would suggest an impaired N remobilization from other organs to young leaves under water stress conditions and low VPD.

Response of Wheat Storage Proteins and Breadmaking Quality to Dimethylpyrazole-Based Nitrification Inhibitors under Different Nitrogen Fertilization Splitting Strategies.

Improving fertilizer nitrogen (N) use efficiency is essential to increase crop productivity and avoid environmental damage. This study was conducted during four crop cycles of winter wheat under humid Mediterranean conditions (Araba, northern Spain). The effects of N-fertilization splitting and the application of the nitrification inhibitors (NIs) 3,4-dimethylpyrazole phosphate (DMPP) and 2-(3,4-dimethyl-1H-pyrazol-1-yl) succinic acid isomeric mixture (DMPSA) as strategies to improve grain quality were examined. The hypothesis of this study was to test if the partial ammonium nutrition and the reduction of fertilizer losses presumably induced by the application of NIs can modify the grain gliadin and glutenin protein contents and the breadmaking quality (dough rheological properties). Among both NIs assayed, only DMPP showed a slight effect of decreasing the omega gliadin fraction, following splitting either two or three times, although this effect was dependent on the year and was not reflected in terms of dough extensibility. The slight decreases observed in grain quality in terms of dough strength and glutenin content induced by DMPP suggest that DMPSA is more promising in terms of maintaining grain quality. Nonetheless, these poor effects exerted by NI application on grain quality parameters did not lead to changes in the quality parameters defining the flour aptitudes for breadmaking.

Involvement of the metabolically active bacteria in the organic matter degradation during olive mill waste composting.

RNA-based high-throughput sequencing is a valuable tool in the discernment of the implication of metabolically active bacteria during composting. In this study, "alperujo" composting was used as microbial model for the elucidation of structure-function relationships with physicochemical transformation of the organic matter. DNA and RNA, subsequently retrotranscribed into cDNA, were isolated at the mesophilic, thermophilic and maturation phases. 16S rRNA gene was amplified by quantitative PCR (qPCR) and Illumina MiSeq platform to assess bacterial abundance and diversity, respectively. The results showed that the abundance of active bacteria assessed by qPCR was maximum at thermophilic phase, which confirm it as the most active stage of the process. Concerning diversity, Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria were the main phyla presented in composts. Concomitantly, three different behaviours were observed for bacterial dynamics: some genera decreased during the whole process meanwhile others proliferated only at thermophilic or maturation phase. Statistical correlation between physicochemical transformations of the organic matter and bacterial diversity revealed bacterial specialisation. This result indicated that specific groups of bacteria were only involved in the organic matter degradation during bio-oxidative phase or humification at maturation. Metabolic functions predictions confirmed that active bacteria were mainly involved in carbon (C) and nitrogen (N) cycles transformations, and pathogen reduction.

Nitrogen Assimilation in the Highly Salt- and Boron-Tolerant Ecotype Zea mays L. Amylacea.

The Lluta Valley in Northern Chile is an important agricultural area affected by both salinity and boron (B) toxicity. Zea mays L. amylacea, an ecotype arisen because of the seed selection practiced in this valley, shows a high tolerance to salt and B levels. In the present study the interaction between B and salt was studied after 20 days of treatment at low (100 mM) and high salinity (430 mM NaCl), assessing changes in nitrogen metabolites and in the activity of key nitrogen-assimilating enzymes. Under non-saline conditions, the presence of excessive B favored higher nitrate and ammonium mobilization to leaves, increasing nitrate reductase (NR) activity but not glutamine synthetase (GS). Thus, the increment of nitrogen use efficiency by B application would contribute partially to maintain the biomass production in this ecotype. Positive relationships between NR activity, nitrate, and stomatal conductance were observed in leaves. The increment of major amino acids alanine and serine would indicate a photoprotective role of photorespiration under low-salinity conditions, thus the inhibition of nitrogen assimilation pathway (NR and GS activities) occurred only at high salinity. The role of cytosolic GS regarding the proline accumulation is discussed.