Altered Plant and Nodule Development and Protein S-Nitrosylation in Lotus japonicus Mutants Deficient in S-Nitrosoglutathione Reductases.

Nitric oxide (NO) is a crucial signaling molecule that conveys its bioactivity mainly through protein S-nitrosylation. This is a reversible post-translational modification (PTM) that may affect protein function. S-nitrosoglutathione (GSNO) is a cellular NO reservoir and NO donor in protein S-nitrosylation. The enzyme S-nitrosoglutathione reductase (GSNOR) degrades GSNO, thereby regulating indirectly signaling cascades associated with this PTM. Here, the two GSNORs of the legume Lotus japonicus, LjGSNOR1 and LjGSNOR2, have been functionally characterized. The LjGSNOR1 gene is very active in leaves and roots, whereas LjGSNOR2 is highly expressed in nodules. The enzyme activities are regulated in vitro by redox-based PTMs. Reducing conditions and hydrogen sulfide-mediated cysteine persulfidation induced both activities, whereas cysteine oxidation or glutathionylation inhibited them. Ljgsnor1 knockout mutants contained higher levels of S-nitrosothiols. Affinity chromatography and subsequent shotgun proteomics allowed us to identify 19 proteins that are differentially S-nitrosylated in the mutant and the wild-type. These include proteins involved in biotic stress, protein degradation, antioxidant protection and photosynthesis. We propose that, in the mutant plants, deregulated protein S-nitrosylation contributes to developmental alterations, such as growth inhibition, impaired nodulation and delayed flowering and fruiting. Our results highlight the importance of GSNOR function in legume biology.

Hypoxia-Responsive Class III Peroxidases in Maize Roots: Soluble and Membrane-Bound Isoenzymes.

Flooding induces low-oxygen environments (hypoxia or anoxia) that lead to energy disruption and an imbalance of reactive oxygen species (ROS) production and scavenging enzymes in plants. The influence of hypoxia on roots of hydroponically grown maize (Zea mays L.) plants was investigated. Gene expression (RNA Seq and RT-qPCR) and proteome (LC-MS/MS and 2D-PAGE) analyses were used to determine the alterations in soluble and membrane-bound class III peroxidases under hypoxia. Gel-free peroxidase analyses of plasma membrane-bound proteins showed an increased abundance of ZmPrx03, ZmPrx24, ZmPrx81, and ZmPr85 in stressed samples. Furthermore, RT-qPCR analyses of the corresponding peroxidase genes revealed an increased expression. These peroxidases could be separated with 2D-PAGE and identified by mass spectrometry. An increased abundance of ZmPrx03 and ZmPrx85 was determined. Further peroxidases were identified in detergent-insoluble membranes. Co-regulation with a respiratory burst oxidase homolog (Rboh) and key enzymes of the phenylpropanoid pathway indicates a function of the peroxidases in membrane protection, aerenchyma formation, and cell wall remodeling under hypoxia. This hypothesis was supported by the following: (i) an elevated level of hydrogen peroxide and aerenchyma formation; (ii) an increased guaiacol peroxidase activity in membrane fractions of stressed samples, whereas a decrease was observed in soluble fractions; and (iii) alterations in lignified cells, cellulose, and suberin in root cross-sections.

Sulfate is transported at significant rates through the symbiosome membrane and is crucial for nitrogenase biosynthesis.

Legume-rhizobia symbioses play a major role in food production for an ever growing human population. In this symbiosis, dinitrogen is reduced ("fixed") to ammonia by the rhizobial nitrogenase enzyme complex and is secreted to the plant host cells, whereas dicarboxylic acids derived from photosynthetically produced sucrose are transported into the symbiosomes and serve as respiratory substrates for the bacteroids. The symbiosome membrane contains high levels of SST1 protein, a sulfate transporter. Sulfate is an essential nutrient for all living organisms, but its importance for symbiotic nitrogen fixation and nodule metabolism has long been underestimated. Using chemical imaging, we demonstrate that the bacteroids take up 20-fold more sulfate than the nodule host cells. Furthermore, we show that nitrogenase biosynthesis relies on high levels of imported sulfate, making sulfur as essential as carbon for the regulation and functioning of symbiotic nitrogen fixation. Our findings thus establish the importance of sulfate and its active transport for the plant-microbe interaction that is most relevant for agriculture and soil fertility.

Drought and Recovery: Independently Regulated Processes Highlighting the Importance of Protein Turnover Dynamics and Translational Regulation in Medicago truncatula.

Climate change in conjunction with population growth necessitates a systems biology approach to characterize plant drought acclimation as well as a more thorough understanding of the molecular mechanisms of stress recovery. Plants are exposed to a continuously changing environment. Extremes such as several weeks of drought are followed by rain. This requires a molecular plasticity of the plant enabling drought acclimation and the necessity of deacclimation processes for recovery and continuous growth.During drought stress and subsequent recovery, the metabolome and proteome are regulated through a sequence of molecular processes including synthesis and degradation and molecular interaction networks are part of this regulatory process. In order to study this complex regulatory network, a comprehensive analysis is presented for the first time, investigating protein turnover and regulatory classes of proteins and metabolites during a stress recovery scenario in the model legume Medicago truncatula The data give novel insights into the molecular capacity and differential processes required for acclimation and deacclimation of severe drought stressed plants.Functional cluster and network analyses unraveled independent regulatory mechanisms for stress and recovery with different dynamic phases that during the course of recovery define the plants deacclimation from stress. The combination of relative abundance levels and turnover analysis revealed an early transition phase that seems key for recovery initiation through water resupply and is independent from renutrition. Thus, a first indication for a metabolite and protein-based load capacity was observed necessary for the recovery from drought, an important but thus far ignored possible feature toward tolerance. The data indicate that apart from the plants molecular stress response mechanisms, plasticity may be related to the nutritional status of the plant prior to stress initiation. A new perspective and possible new targets as well as metabolic mechanisms for future plant-bioengineering toward enhanced drought stress tolerance are presented.

Changes in plastid proteome and structure in arbuscular mycorrhizal roots display a nutrient starvation signature.

During arbuscular mycorrhizal symbiosis, arbuscule-containing root cortex cells display a proliferation of plastids, a feature usually ascribed to an increased plant anabolism despite the lack of studies focusing on purified root plastids. In this study, we investigated mycorrhiza-induced changes in plastidic pathways by performing a label-free comparative subcellular quantitative proteomic analysis targeted on plastid-enriched fractions isolated from Medicago truncatula roots, coupled to a cytological analysis of plastid structure. We identified 490 root plastid protein candidates, among which 79 changed in abundance upon mycorrhization, as inferred from spectral counting. According to cross-species sequence homology searches, the mycorrhiza-responsive proteome was enriched in proteins experimentally localized in thylakoids, whereas it was depleted of proteins ascribed predominantly to amyloplasts. Consistently, the analysis of plastid morphology using transmission electron microscopy indicated that starch depletion associated with the proliferation of membrane-free and tubular membrane-containing plastids was a feature specific to arbusculated cells. The loss of enzymes involved in carbon/nitrogen assimilation and provision of reducing power, coupled to macromolecule degradation events in the plastid-enriched fraction of mycorrhizal roots that paralleled lack of starch accumulation in arbusculated cells, lead us to propose that arbuscule functioning elicits a nutrient starvation and an oxidative stress signature that may prime arbuscule breakdown.

Label-free quantitative proteomic analysis of tolerance to drought in Pisum sativum.

Abiotic stresses caused by adverse environmental conditions are responsible for heavy economic losses on pea crop, being drought one of the most important abiotic constraints. Development of pea cultivars well adapted to dry conditions has been one of the major tasks in breeding programs. The increasing food requirements drive the necessity to broaden the molecular basis of tolerance to drought to develop pea cultivars well adapted to dry conditions. We have used a shotgun proteomic approach (nLC-MSMS) to study the tolerance to drought in three pea genotypes that were selected based on differences in the level of water deficit tolerance. Multivariate statistical analysis of data unraveled 367 significant differences of 700 identified when genotypes and/or treatment were compared. More than half of the significantly changed proteins belong to primary metabolism and protein regulation categories. We propose different mechanisms to cope drought in the genotypes studied. Maintenance of the primary metabolism and protein protection seems a strategy for drought tolerance. On the other hand susceptibility might be related to maintenance of the homeostatic equilibrium, a very energy consuming process. Data are available via ProteomeXchange with identifier PXD004587.

Leghemoglobin is nitrated in functional legume nodules in a tyrosine residue within the heme cavity by a nitrite/peroxide-dependent mechanism.

Protein tyrosine (Tyr) nitration is a post-translational modification yielding 3-nitrotyrosine (NO2 -Tyr). Formation of NO2 -Tyr is generally considered as a marker of nitro-oxidative stress and is involved in some human pathophysiological disorders, but has been poorly studied in plants. Leghemoglobin (Lb) is an abundant hemeprotein of legume nodules that plays an essential role as an O2 transporter. Liquid chromatography coupled to tandem mass spectrometry was used for a targeted search and quantification of NO2 -Tyr in Lb. For all Lbs examined, Tyr30, located in the distal heme pocket, is the major target of nitration. Lower amounts were found for NO2 -Tyr25 and NO2 -Tyr133. Nitrated Lb and other as yet unidentified nitrated proteins were also detected in nodules of plants not receiving NO3- and were found to decrease during senescence. This demonstrates formation of nitric oxide (˙NO) and NO2- by alternative means to nitrate reductase, probably via a ˙NO synthase-like enzyme, and strongly suggests that nitrated proteins perform biological functions and are not merely metabolic byproducts. In vitro assays with purified Lb revealed that Tyr nitration requires NO2- + H2 O2 and that peroxynitrite is not an efficient inducer of nitration, probably because Lb isomerizes it to NO3-. Nitrated Lb is formed via oxoferryl Lb, which generates nitrogen dioxide and tyrosyl radicals. This mechanism is distinctly different from that involved in heme nitration. Formation of NO2 -Tyr in Lb is a consequence of active metabolism in functional nodules, where Lb may act as a sink of toxic peroxynitrite and may play a protective role in the symbiosis.

Medicago truncatula and Glycine max: Different Drought Tolerance and Similar Local Response of the Root Nodule Proteome.

Legume crops present important agronomical and environmental advantages mainly due to their capacity to reduce atmospheric N2 to ammonium via symbiotic nitrogen fixation (SNF). This process is very sensitive to abiotic stresses such as drought, but the mechanism underlying this response is not fully understood. The goal of the current work is to compare the drought response of two legumes with high economic impact and research importance, Medicago truncatula and Glycine max, by characterizing their root nodule proteomes. Our results show that, although M. truncatula exhibits lower water potential values under drought conditions compared to G. max, SNF declined analogously in the two legumes. Both of their nodule proteomes are very similar, and comparable down-regulation responses in the diverse protein functional groups were identified (mainly proteins related to the metabolism of carbon, nitrogen, and sulfur). We suggest lipoxygenases and protein turnover as newly recognized players in SNF regulation. Partial drought conditions applied to a split-root system resulted in the local down-regulation of the entire proteome of drought-stressed nodules in both legumes. The high degree of similarity between both legume proteomes suggests that the vast amount of research conducted on M. truncatula could be applied to economically important legume crops, such as soybean.

Heat-Treatment-Responsive Proteins in Different Developmental Stages of Tomato Pollen Detected by Targeted Mass Accuracy Precursor Alignment (tMAPA).

Recently, we have developed a quantitative shotgun proteomics strategy called mass accuracy precursor alignment (MAPA). The MAPA algorithm uses high mass accuracy to bin mass-to-charge (m/z) ratios of precursor ions from LC-MS analyses, determines their intensities, and extracts a quantitative sample versus m/z ratio data alignment matrix from a multitude of samples. Here, we introduce a novel feature of this algorithm that allows the extraction and alignment of proteotypic peptide precursor ions or any other target peptide from complex shotgun proteomics data for accurate quantification of unique proteins. This strategy circumvents the problem of confusing the quantification of proteins due to indistinguishable protein isoforms by a typical shotgun proteomics approach. We applied this strategy to a comparison of control and heat-treated tomato pollen grains at two developmental stages, post-meiotic and mature. Pollen is a temperature-sensitive tissue involved in the reproductive cycle of plants and plays a major role in fruit setting and yield. By LC-MS-based shotgun proteomics, we identified more than 2000 proteins in total for all different tissues. By applying the targeted MAPA data-processing strategy, 51 unique proteins were identified as heat-treatment-responsive protein candidates. The potential function of the identified candidates in a specific developmental stage is discussed.

Bimodal dynamics of primary metabolism-related responses in tolerant potato-Potato virus Y interaction.

BACKGROUND: Potato virus Y (PVY) is a major pathogen that causes substantial economic losses in worldwide potato production. Different potato cultivars differ in resistance to PVY, from severe susceptibility, through tolerance, to complete resistance. The aim of this study was to better define the mechanisms underlying tolerant responses of potato to infection by the particularly aggressive PVY(NTN) strain. We focused on the dynamics of the primary metabolism-related processes during PVY(NTN) infection. RESULTS: A comprehensive analysis of the dynamic changes in primary metabolism was performed, which included whole transcriptome analysis, nontargeted proteomics, and photosynthetic activity measurements in potato cv. Désirée and its transgenic counterpart depleted for accumulation of salicylic acid (NahG-Désirée). Faster multiplication of virus occurred in the NahG-Désirée, with these plants developing strong disease symptoms. We show that while the dynamics of responses at the transcriptional level are extensive and bimodal, this is only partially translated to the protein level, and to the final functional outcome. Photosynthesis-related genes are transiently induced before viral multiplication is detected and it is down-regulated later on. This is reflected as a deficiency of the photosynthetic apparatus at the onset of viral multiplication only. Interestingly, specific and constant up-regulation of some RuBisCO transcripts was detected in Désirée plants, which might be important, as these proteins have been shown to interact with viral proteins. In SA-deficient and more sensitive NahG-Désirée plants, consistent down-regulation of photosynthesis-related genes was detected. A constant reduction in the photochemical efficiency from the onset of viral multiplication was identified; in nontransgenic plants this decrease was only transient. The transient reduction in net photosynthetic rate occurred in both genotypes with the same timing, and coincided with changes in stomatal conductivity. CONCLUSIONS: Down-regulation of photosynthesis-related gene expression and decreased photosynthetic activity is in line with other studies that have reported the effects of biotic stress on photosynthesis. Here, we additionally detected induction of light-reaction components in the early stages of PVY(NTN) infection of tolerant interaction. As some of these components have already been shown to interact with viral proteins, their overproduction might contribute to the absence of symptoms in cv. Désirée.

Differential CO2 effect on primary carbon metabolism of flag leaves in durum wheat (Triticum durum Desf.).

C sink/source balance and N assimilation have been identified as target processes conditioning crop responsiveness to elevated CO2 . However, little is known about phenology-driven modifications of C and N primary metabolism at elevated CO2 in cereals such as wheat. Here, we examined the differential effect of elevated CO2 at two development stages (onset of flowering, onset of grain filling) in durum wheat (Triticum durum, var. Sula) using physiological measurements (photosynthesis, isotopes), metabolomics, proteomics and (15) N labelling. Our results show that growth at elevated CO2 was accompanied by photosynthetic acclimation through a lower internal (mesophyll) conductance but no significant effect on Rubisco content, maximal carboxylation or electron transfer. Growth at elevated CO2 altered photosynthate export and tended to accelerate leaf N remobilization, which was visible for several proteins and amino acids, as well as lysine degradation metabolism. However, grain biomass produced at elevated CO2 was larger and less N rich, suggesting that nitrogen use efficiency rather than photosynthesis is an important target for improvement, even in good CO2 -responsive cultivars.

Drought stress provokes the down-regulation of methionine and ethylene biosynthesis pathways in Medicago truncatula roots and nodules.

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 water-deficit conditions. To better understand this involvement, the drought-induced changes in expression and content of enzymes involved in the biosynthesis of Met, S-adenosyl-L-methionine (SAM) and ethylene in M. truncatula root and nodules were analyzed using targeted approaches. Nitrogen-fixing 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 down-regulation of the Met and ethylene biosynthesis pathways in roots and nodules in response to water-deficit conditions.

Isolation of cellular membranes from lignin-producing tissues of Norway spruce and analysis of redox enzymes.

There are no earlier reports with successful isolation of plasma membranes from lignin-forming tissues of conifers. A method to isolate cellular membranes from extracellular lignin-producing tissue-cultured cells and developing xylem of Norway spruce was optimized. Modifications to the homogenization buffer were needed to obtain membranes from these phenolics-rich tissues. Membranes were separated by aqueous polymer two-phase partitioning. Chlorophyll a determination, marker enzyme assays and western blot analyses using antibodies for each membrane type showed that mitochondrial, chloroplastic and to a certain extent also ER and Golgi membranes were efficiently diminished from the upper phase, but tonoplast and plasma membranes distributed evenly between the upper and lower phases. Redox enzymes present in the partially purified membrane fractions were assayed in order to reveal the origin of H(2)O(2) needed for lignification. The membranes of spruce contained enzymes able to generate superoxide in the presence of NAD(P)H. Besides members of the flavodoxin and flavodoxin-like family proteins, cytochrome b5, cytochrome P450 and several stress responsive proteins were identified by nitroblue tetrazolium staining of isoelectric focusing gels and by mass spectrometry. Naphthoquinones juglone and menadione increased superoxide production in activity-stained gels. Some juglone-activated enzymes were preferentially using NADH. With NADH, menadione activated only some of the enzymes that juglone did, whereas with NADPH the activation patterns were identical. Duroquinone, a benzoquinone, did not affect superoxide production. Superoxide dismutase, ascorbate peroxidase, catalase and an acidic class III peroxidase isoenzyme were detected in partially purified spruce membranes. The possible locations and functions of these enzymes are discussed.

Systemic long-distance sulfur transport and its role in symbiotic root nodule protein turnover.

Sulfur is an essential nutrient for all plants, but also crucial for the nitrogen fixing symbiosis between legumes and rhizobia. Sulfur limitation can hamper nodule development and functioning. Until now, it remained unclear whether sulfate uptake into nodules is local or mainly systemic via the roots, and if long-distance transport from shoots to roots and into nodules occurs. Therefore, this work investigates the systemic regulation of sulfur transportation in the model legume Lotus japonicus by applying stable isotope labeling to a split-root system. Metabolite and protein extraction together with mass spectrometry analyses were conducted to determine the plants molecular phenotype and relative isotope protein abundances. Data show that treatments of varying sulfate concentrations including the absence of sulfate on one side of a nodulated root was not affecting nodule development as long as the other side of the root system was provided with sufficient sulfate. Concentrations of shoot metabolites did not indicate a significant stress response caused by a lack of sulfur. Further, we did not observe any quantitative changes in proteins involved in biological nitrogen fixation in response to the different sulfate treatments. Relative isotope abundance of 34S confirmed a long-distance transport of sulfur from one side of the roots to the other side and into the nodules. Altogether, these results provide evidence for a systemic long-distance transport of sulfur via the upper part of the plant to the nodules suggesting a demand driven sulfur distribution for the maintenance of symbiotic N-fixation.

Rhizobium symbiosis improves amino acid and secondary metabolite biosynthesis of tungsten-stressed soybean (Glycine max).

The industrially important transition metal tungsten (W) shares certain chemical properties with the essential plant micronutrient molybdenum and inhibits the activity of molybdoenzymes such as nitrate reductase, impacting plant growth. Furthermore, tungsten appears to interfere with metabolic processes on a much wider scale and to trigger common heavy metal stress response mechanisms. We have previously found evidence that the tungsten stress response of soybeans (Glycine max) grown with symbiotically associated N2-fixing rhizobia (Bradyrhizobium japonicum) differs from that observed in nitrogen-fertilized soy plants. This study aimed to investigate how association with symbiotic rhizobia affects the primary and secondary metabolite profiles of tungsten-stressed soybean and whether changes in metabolite composition enhance the plant's resilience to tungsten. This comprehensive metabolomic and proteomic study presents further evidence that the tungsten-stress response of soybean plants is shaped by associated rhizobia. Symbiotically grown plants (N fix) were able to significantly increase the synthesis of an array of protective compounds such as phenols, polyamines, gluconic acid, and amino acids such as proline. This resulted in a higher antioxidant capacity, reduced root-to-shoot translocation of tungsten, and, potentially, also enhanced resilience of N fix plants compared to non-symbiotic counterparts (N fed). Taken together, our study revealed a symbiosis-specific metabolic readjustment in tungsten-stressed soybean plants and contributed to a deeper understanding of the mechanisms involved in the rhizobium-induced systemic resistance in response to heavy metals.

Integrative transcript to proteome analysis of barley during Ramularia collo-cygni leaf spot development identified several proteins that are related to fungal recognition and infection responses.

Introduction: Ramularia leaf spot (RLS) disease is a growing threat to barley cultivation, but with no substantial resistance identified to date. Similarly, the understanding of the lifestyle of Ramularia collo-cygni (Rcc) and the prediction of RLS outbreak severity remain challenging, with Rcc displaying a rather untypical long endophytic phase and a sudden change to a necrotrophic lifestyle. The aim of this study was to provide further insights into the defense dynamics during the different stages of colonization and infection in barley in order to identify potential targets for resistance breeding. Methods: Utilizing the strength of proteomics in understanding plant-pathogen interactions, we performed an integrative analysis of a published transcriptome dataset with a parallel generated proteome dataset. Therefore, we included two spring barley cultivars with contrasting susceptibilities to Rcc and two fungal isolates causing different levels of RLS symptoms. Results: Interestingly, early responses in the pathogen recognition phase of the host were driven by strong responses differing between isolates. An important enzyme in this process is a xylanase inhibitor, which protected the plant from cell wall degradation by the fungal xylanase. At later time points, the differences were driven by cultivar-specific responses, affecting mostly features contributing to the pathogenesis- and senescence-related pathways or photosynthesis. Discussion: This supports the hypothesis of a hemibiotrophic lifestyle of Rcc, with slight differences in trophism of the two analyzed isolates. The integration of these data modalities highlights a strength of protein-level analysis in understanding plant-pathogen interactions and reveals new features involved in fungal recognition and susceptibility in barley cultivars.

A giant virus infecting the amoeboflagellate Naegleria.

Giant viruses (Nucleocytoviricota) are significant lethality agents of various eukaryotic hosts. Although metagenomics indicates their ubiquitous distribution, available giant virus isolates are restricted to a very small number of protist and algal hosts. Here we report on the first viral isolate that replicates in the amoeboflagellate Naegleria. This genus comprises the notorious human pathogen Naegleria fowleri, the causative agent of the rare but fatal primary amoebic meningoencephalitis. We have elucidated the structure and infection cycle of this giant virus, Catovirus naegleriensis (a.k.a. Naegleriavirus, NiV), and show its unique adaptations to its Naegleria host using fluorescence in situ hybridization, electron microscopy, genomics, and proteomics. Naegleriavirus is only the fourth isolate of the highly diverse subfamily Klosneuvirinae, and like its relatives the NiV genome contains a large number of translation genes, but lacks transfer RNAs (tRNAs). NiV has acquired genes from its Naegleria host, which code for heat shock proteins and apoptosis inhibiting factors, presumably for host interactions. Notably, NiV infection was lethal to all Naegleria species tested, including the human pathogen N. fowleri. This study expands our experimental framework for investigating giant viruses and may help to better understand the basic biology of the human pathogen N. fowleri.

Mitochondria are an early target of oxidative modifications in senescing legume nodules.

Legume nodule senescence is a poorly understood process involving a decrease in N(2) fixation and an increase in proteolytic activity. Some physiological changes during nodule aging have been reported, but scarce information is available at the subcellular level. Biochemical, immunological and proteomic approaches were used to provide insight into the effects of aging on the mitochondria and cytosol of nodule host cells. In the mitochondria, the oxidative modification of lipids and proteins was associated with a marked decline in glutathione, a reduced capacity to regenerate ascorbate, and upregulation of alternative oxidase and manganese superoxide dismutase. In the cytosol, there were consistent reductions in the protein concentrations of carbon metabolism enzymes, inhibition of protein synthesis and increase in serine proteinase activity, disorganization of cytoskeleton, and a sharp reduction of cytosolic proteins, but no detectable accumulation of oxidized molecules. We conclude that nodule mitochondria are an early target of oxidative modifications and a likely source of redox signals. Alternative oxidase and manganese superoxide dismutase may play important roles in controlling ROS concentrations and the redox state of mitochondria. The finding that specific methionine residues of a cytosolic glutamine synthetase isoform are sulfoxidized suggests a regulatory role of this enzyme in senescing nodules.

Local inhibition of nitrogen fixation and nodule metabolism in drought-stressed soybean.

Drought stress is a major factor limiting symbiotic nitrogen fixation (NF) in soybean crop production. However, the regulatory mechanisms involved in this inhibition are still controversial. Soybean plants were symbiotically grown in a split-root system (SRS), which allowed for half of the root system to be irrigated at field capacity while the other half remained water deprived. NF declined in the water-deprived root system while nitrogenase activity was maintained at control values in the well-watered half. Concomitantly, amino acids and ureides accumulated in the water-deprived belowground organs regardless of transpiration rates. Ureide accumulation was found to be related to the decline in their degradation activities rather than increased biosynthesis. Finally, proteomic analysis suggests that plant carbon metabolism, protein synthesis, amino acid metabolism, and cell growth are among the processes most altered in soybean nodules under drought stress. Results presented here support the hypothesis of a local regulation of NF taking place in soybean and downplay the role of ureides in the inhibition of NF.

Metabolomics in Plant Research-From Ecometabolomics to Metabolotyping.

In this Special Issue, a state-of-the-art review of the current knowledge of sample preparation and LC-MS techniques for the analyses of nucleosides and nucleotides in plants was published [...].