Transcriptomic and metabolomic analysis reveals that symbiotic nitrogen fixation enhances drought resistance in common bean.

Common bean (Phaseolus vulgaris L.), one of the most important legume crops, uses atmospheric nitrogen through symbiosis with soil rhizobia, reducing the need for nitrogen fertilization. However, this legume is particularly sensitive to drought conditions, prevalent in arid regions where this crop is cultured. Therefore, studying the response to drought is important to sustain crop productivity. We have used integrated transcriptomic and metabolomic analysis to understand the molecular responses to water deficit in a marker-class common bean accession cultivated under N2 fixation or fertilized with nitrate (NO3-). RNA-seq revealed more transcriptional changes in the plants fertilized with NO3- than in the N2-fixing plants. However, changes in N2-fixing plants were more associated with drought tolerance than in those fertilized with NO3-. N2-fixing plants accumulated more ureides in response to drought, and GC/MS and LC/MS analysis of primary and secondary metabolite profiles revealed that N2-fixing plants also had higher levels of abscisic acid, proline, raffinose, amino acids, sphingolipids, and triacylglycerols than those fertilized with NO3-. Moreover, plants grown under nitrogen fixation recovered from drought better than plants fertilized with NO3-. Altogether we show that common bean plants grown under symbiotic nitrogen fixation were more protected against drought than the plants fertilized with nitrate.

A New Perspective on the Role of Glutamine Synthetase in Nitrogen Remobilization in Wheat (Triticum aestivum L.).

Glutamine synthetase (GS), a key enzyme in plant nitrogen metabolism, is closely related to nitrogen remobilization. However, how GS isoforms participate in nitrogen remobilization remains unclear. Here, the spatiotemporal expression of the TaGS gene family after anthesis was investigated, and the results showed that TaGS1;1 was mainly encoded by TaGS1;1-6A, while the other isozymes were mainly encoded by TaGS localized on the A and D subgenomes. TaGS1;2-4A/4D had the highest expression level, especially in rachis and peduncle. Furthermore, immunofluorescence showed TaGS1;2 was located in the phloem of rachis and peduncle. GUS (ß-glucuronidase) staining confirmed that ProTaGS1;2-4A/4D::GUS activity was mainly present in the vascular system of leaves, roots, and petal of Arabidopsis. Ureides, an important transport form of nitrogen, were mainly synthesized in flag leaves and transported to grains through the phloem of peduncle and rachis during grain filling. TaAAH, which encodes the enzyme that degrades ureides to release NH4+, had a higher expression in rachis and peduncle and was synchronized with the increase in NH4+ concentration in phloem, indicating that NH4+ in phloem is from ureide degradation. Taking the above into account, TaGS1;2, which is highly expressed in the phloem of peduncle and rachis, may participate in N remobilization by assimilating NH4+ released from ureide degradation.

Response of soybean to soil waterlogging associated with iron excess in the reproductive stage.

Soil waterlogging is a common problem in some agricultural areas, including regions under soybean (Glycine max) cultivation. In waterlogged soils, soil O2 depletion occurs due to aerobic microorganisms and plants, affecting the metabolic and physiological processes of plants after suffering anoxia in their root tissue. Another harmful factor in this situation is the exponential increase in the availability of iron (Fe) in the soil, which may result in absorption of excess Fe. The present study sought to evaluate the response mechanisms in soybean leaves 'Agroeste 3680' by physiological and biochemical analyses associating them with the development of pods in non-waterlogged and waterlogged soil, combined with one moderate and two toxic levels of Fe. Gas exchange was strongly affected by soil waterlogging. Excess Fe without soil waterlogging reduced photosynthetic pigments, and potentiated this reduction when associated with soil waterlogging. Starch and ureide accumulation in the first newly expanded trifoliate leaves proved to be response mechanisms induced by soil waterlogging and excess Fe, since plants cultivated under soil non-waterlogged soil at 25 mg dm-3 Fe showed lower contents when compared to stressed plants. Thus, starch and ureide accumulation could be considered efficient biomarkers of phytotoxicity caused by soil waterlogging and excess Fe in soybean plants. The reproductive development was abruptly interrupted by the imposition of stresses, leading to a loss of pod dry biomass, which was largely due to the substantial decrease in the net photosynthetic rate, as expressed by area (A), the blockage of carbohydrate transport to sink tissues and an increase of malondialdehyde (MDA). The negative effect on reproductive development was more pronounced under waterlogged soil.

Allantoin accumulation through overexpression of ureide permease1 improves rice growth under limited nitrogen conditions.

In legumes, nitrogen (N) can be stored as ureide allantoin and transported by ureide permease (UPS) from nodules to leaves where it is catabolized to release ammonium and assimilation to amino acids. In non-leguminous plants especially rice, information on its roles in N metabolism is scarce. Here, we show that OsUPS1 is localized in plasma membranes and are highly expressed in vascular tissues of rice. We further evaluated an activation tagging rice overexpressing OsUPS1 (OsUPS1OX ) under several N regimes. Under normal field conditions, panicles from OsUPS1OX plants (14 days after flowering (DAF)) showed significant allantoin accumulation. Under hydroponic system at the vegetative stage, plants were exposed to N-starvation and measured the ammonium in roots after resupplying with ammonium sulphate. OsUPS1OX plants displayed higher ammonium uptake in roots compared to wild type (WT). When grown under low-N soil supplemented with different N-concentrations, OsUPS1OX exhibited better growth at 50% N showing higher chlorophyll, tiller number and at least 20% increase in shoot and root biomass relative to WT. To further confirm the effects of regulating the expression of OsUPS1, we evaluated whole-body-overexpressing plants driven by the GOS2 promoter (OsUPS1GOS2 ) as well as silencing plants (OsUPS1RNAi ). We found significant accumulation of allantoin in leaves, stems and roots of OsUPS1GOS2 while in OsUPS1RNAi allantoin was significantly accumulated in roots. We propose that OsUPS1 is responsible for allantoin partitioning in rice and its overexpression can support plant growth through accumulation of allantoin in sink tissues which can be utilized when N is limiting.

Allantoin attenuates cadmium-induced toxicity in cucumber plants.

Concentration-dependent responses of cucumber plants to cadmium (Cd, 5-15 µM) and/or allantoin (Alla, 10-1000 µM) have been investigated to detect a possible protective role of Alla under Cd excess. After 14 days of exposure, Alla often considerably reversed Cd-induced inhibition of growth and reduction of the content of photosynthetic pigments. Higher Alla doses depleted the Cd amount in shoots, which could be related to citric acid (increase in the shoots but depletion in the roots in Cd+Alla treatments) rather than to phytochelatins (Alla had a negative impact on the phytochelatin accumulation). An increase in the Alla concentration suppressed Cd-induced spatial H2O2 appearance, which does not seem to be related to antioxidative enzymes (low impact of Alla on catalase, ascorbate peroxidase, and guaiacol peroxidase). On the contrary, shoot glutathione and mainly ascorbic acid accumulation strongly increased in Cd+Alla treatments, indicating their prominent role in Alla-induced amelioration of Cd-stimulated oxidative stress and growth retardation. Similarly, phenolic metabolites (total soluble phenols and flavonols) were slightly influenced by Alla and their antioxidative action was not expected. We conclude that Alla-mediated attenuation of Cd-induced toxicity relies on enhanced accumulation of glutathione and ascorbate in the shoot tissue mainly, rather than on elevated antioxidative enzyme activities.

Allantoin Increases Cadmium Tolerance in Arabidopsis via Activation of Antioxidant Mechanisms.

Plants apply various molecular, physiological and morphological strategies in response to undesirable environmental conditions. One of the possible responses which may contribute to surviving stressful conditions is the accumulation of ureides. Ureides are recognized as important nitrogen-rich compounds involved in recycling nitrogen in plants to support growth and reproduction. Amongst them, allantoin not only serves as a transportable nitrogen-rich compound, but has also been suggested to protect plants from abiotic stresses via minimizing oxidative damage. This work focuses on the effect of cadmium (Cd) on ureide metabolism in Arabidopsis, in order to clarify the potential role of allantoin in plant tolerance to heavy metals. In response to Cd treatment, allantoin levels increase in Arabidopsis thaliana, ecotype Col-0, due to reduced allantoinase (ALN) gene expression and enzyme activity. This coincides with increases in uricase (UO) transcripts. UO and ALN encode the enzymes for the production and degradation of allantoin, respectively. ALN-negative aln-3 Arabidopsis mutants with elevated allantoin levels demonstrate resistance to soil-applied CdCl2, up to 1,500 µM. Although aln-3 mutants take up and store more Cd within their leaf tissue, they contain less damaging superoxide radicals. The protective mechanism of aln-3 mutants appears to involve enhancing the activity of antioxidant enzymes such as superoxide dismutase and ascorbate peroxidase.

Redox markers for drought-induced nodule senescence, a process occurring after drought-induced senescence of the lowest leaves in soybean (Glycine max).

BACKGROUND AND AIMS: Water is an increasingly scarce resource that limits crop productivity in many parts of the world, and the frequency and severity of drought are predicted to increase as a result of climate change. Improving tolerance to drought stress is therefore important for maximizing future crop yields. The aim of this study was to compare the effects of drought on soybean (Glycine max) leaves and nodules in order to define phenotypic markers and changes in cellular redox state that characterize the stress response in different organs, and to characterize the relationships between leaf and nodule senescence during drought. METHODS: Leaf and crown nodule metabolite pools were measured together with leaf and soil water contents, and leaf chlorophyll, total protein contents and chlorophyll a fluorescence quenching parameters in nodulated soybeans that were grown under either well-watered conditions or deprived of water for up to 21 d. KEY RESULTS: Ureides, ascorbate, protein, chlorophyll and the ratios of variable chlorophyll a fluorescence (Fv') to maximal chlorophyll a fluorescence (Fm') fell to levels below detection in the oldest leaves after 21 d of drought. While these drought-induced responses were not observed in the youngest leaf ranks, the Fv'/Fm' ratios, pyridine nucleotide levels and the reduction state of the ascorbate pool were lower in all leaf ranks after 21 d of drought. In contrast to leaves, total nodule protein, pyridine nucleotides, ureides, ascorbate and glutathione contents increased as a result of the drought treatment. However, the nodule ascorbate pool was significantly less reduced as a result of drought. Higher levels of transcripts encoding two peroxiredoxins were detected in nodules exposed to drought stress but senescence-associated transcripts and other mRNAs encoding redox-related proteins were similar under both conditions. CONCLUSIONS: While the physiological impact of the drought was perceived throughout the shoot, stress-induced senescence occurred only in the oldest leaf ranks. At this stage, a number of drought-induced changes in nodule metabolites were observed but no metabolite or transcript markers of senescence could be detected. It is concluded that stress-induced senescence in the lowest leaf ranks precedes nodule senescence, suggesting that leaves of low photosynthetic capacity are sacrificed in favour of nodule nitrogen metabolism.

Allantoate Amidohydrolase OsAAH is Essential for Preharvest Sprouting Resistance in Rice.

Preharvest sprouting (PHS) is an undesirable trait that decreases yield and quality in rice production. Understanding the genes and regulatory mechanisms underlying PHS is of great significance for breeding PHS-resistant rice. In this study, we identified a mutant, preharvest sprouting 39 (phs39), that exhibited an obvious PHS phenotype in the field. MutMap+ analysis and transgenic experiments demonstrated that OsAAH, which encodes allantoate amidohydrolase, is the causal gene of phs39 and is essential for PHS resistance. OsAAH was highly expressed in roots and leaves at the heading stage and gradually increased and then weakly declined in the seed developmental stage. OsAAH protein was localized to the endoplasmic reticulum, with a function of hydrolyzing allantoate in vitro. Disruption of OsAAH increased the levels of ureides (allantoate and allantoin) and activated the tricarboxylic acid (TCA) cycle, and thus increased energy levels in developing seeds. Additionally, the disruption of OsAAH significantly increased asparagine, arginine, and lysine levels, decreased tryptophan levels, and decreased levels of indole-3-acetic acid (IAA) and abscisic acid (ABA). Our findings revealed that the OsAAH of ureide catabolism is involved in the regulation of rice PHS via energy and hormone metabolisms, which will help to facilitate the breeding of rice PHS-resistant varieties.

Endogenous ureides are employed as a carbon source in Arabidopsis plants exposed to carbon starvation conditions.

Ureides, the degraded products of purine catabolism in Arabidopsis, have been shown to act as antioxidant and nitrogen sources. Herein we elucidate purine degraded metabolites as a carbon source using the Arabidopsis Atxdh1, Ataln, and Ataah knockout (KO) mutants vis-à-vis wild-type (WT) plants. Plants were grown under short-day conditions on agar plates containing half-strength MS medium with or without 1% sucrose. Notably, the absence of sucrose led to diminished biomass accumulation in both shoot and root tissues of the Atxdh1, Ataln, and Ataah mutants, while no such effect was observed in WT plants. Moreover, the application of sucrose resulted in a reduction of purine degradation metabolite levels, specifically xanthine and allantoin, predominantly within the roots of WT plants. Remarkably, an increase in proteins associated with the purine degradation pathway was observed in WT plants in the presence of sucrose. Lower glyoxylate levels in the roots but not in the shoot of the Atxdh1 mutant in comparison to WT, were observed under sucrose limitation, and improved by sucrose application in root, indicating that purine degradation provided glyoxylate in the root. Furthermore, the deficit of purine-degraded metabolites in the roots of mutants subjected to carbon starvation was partially mitigated through allantoin application. Collectively, these findings signify that under conditions of sucrose limitation and short-day growth, purines are primarily remobilized within the root system to augment the availability of ureides, serving as an additional carbon (as well as nitrogen) source to support plant growth.

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.

Physiological and biochemical role of nickel in nodulation and biological nitrogen fixation in Vigna unguiculata L. Walp.

Studies on the role of nickel (Ni) in photosynthetic and antioxidant metabolism, as well as in flavonoid synthesis and biological fixation nitrogen in cowpea crop are scarce. The aim of this study was to elucidate the role of Ni in metabolism, photosynthesis and nodulation of cowpea plants. A completely randomized experiment was performed in greenhouse, with cowpea plants cultivated under 0, 0.5, 1, 2, or 3 mg kg-1 Ni, as Ni sulfate. In the study the following parameters were evaluated: activity of urease, nitrate reductase, superoxide dismutase, catalase and ascorbate peroxidase; concentration of urea, n-compounds, photosynthetic pigments, flavonoids, H2O2 and MDA; estimative of gas exchange, and biomass as plants, yield and weight of 100 seeds. At whole-plant level, Ni affected root biomass, number of seeds per pot, and yield, increasing it at 0.5 mg kg-1 and leading to inhibition at 2-3 mg kg-1 (e.g. number of seeds per pot and nodulation). The whole-plant level enhancement by 0.5 mg Ni kg-1 occurred along with increased photosynthetic pigments, photosynthesis, ureides, and catalase, and decreased hydrogen peroxide concentration. This study presents fundamental new insights regarding Ni effect on N metabolism, and nodulation that can be helpful to increase cowpea yield. Considering the increasing population and its demand for staple food, these results contribute to the enhancement of agricultural techniques that increase crop productivity and help to maintain human food security.

Could nitrogen compounds be indicators of tolerance to high doses of Cu and Fe in the cultivation of Leucaena leucocephala?

Nitrogen metabolism and the production of primary and secondary metabolites vary according to biotic and abiotic factors such as trace elements (TE) stress, and can, therefore, be considered biomarkers. The present study evaluated the effect of copper (Cu) and iron (Fe) TE, separately, on the metabolism of nitrogen compounds and biomass production, partitioned into shoot and roots of Leucaena leucocephala (Lam.) de Wit., and identified possible defense mechanisms linked to nitrogen metabolism. At 120 days of cultivation, the biomass production of L. leucocephala was higher when exposed to excess Fe than Cu. Nonetheless, the biomass gain (%) of plants exposed to Cu was higher, especially the biomass gains in roots. The tolerance and biomass production of L. leucocephala is related to the regulation of nitrogen metabolism and production of secondary metabolites. The biochemistry of plant metabolism against the excess of Cu and Fe TE manifested similarly, but with some specifics regarding the chemical nature of each metal. There was a reduction in the content of ureides and proteins and an increase in amino acids in the roots in relation to the increase in Cu and Fe concentrations. There was low accumulation of proline in the roots in treatments 400 and 500 mg/dm3 compared to the control for both TE. On the other hand, the total phenolic compounds in the roots increased. Our results indicate that the increased synthesis of amino acids and the accumulation of phenolic compounds is involved in the tolerance of L. leucocephala to Cu and Fe.

Exogenous Allantoin Confers Rapeseed (Brassica campestris) Tolerance to Simulated Drought by Improving Antioxidant Metabolism and Physiology.

Allantoin is an emerging plant metabolite, but its role in conferring drought-induced oxidative stress is still elusive. Therefore, an experiment was devised to explore the role of allantoin (0.5 and 1.0 mM; foliar spray) in rapeseed (Brassica campestris cv. BARI Sarisha-17) under drought. Seedlings at fifteen days of age were subjected to drought, maintaining soil moisture levels at 50% and 25% field capacities, while well-irrigated plants served as the control group. Drought-stressed plants exhibited increased levels of lipid peroxidation and hydrogen peroxide, electrolyte leakage, and impaired glyoxalase systems. Thus, the growth, biomass, and yield attributes of rapeseed were significantly impaired under drought. However, the allantoin-supplemented plants showed a notable increase in their contents of ascorbate and glutathione and decreased dehydroascorbate and glutathione disulfide contents under drought. Moreover, the activity of antioxidant enzymes such as ascorbate peroxidase, dehydroascorbate reductase, glutathione reductase, glutathione peroxidase, and catalase were accelerated with the allantoin spray and the glyoxalase system was also enhanced under drought. Moreover, the improvement in water balance with reduction in proline and potassium ion contents was also observed when allantoin was applied to the plants. Overall, the beneficial effects of allantoin supplementation resulted in the improved plant growth, biomass, and yield of rapeseed under drought conditions. These findings suggest that allantoin acts as an efficient metabolite in mitigating the oxidative stress caused by reactive oxygen species by enhancing antioxidant defense mechanisms and the glyoxalase system.

Allantoin overaccumulation enhances production of metabolites under excess of metals but is not tightly regulated by nitric oxide.

The aln-3 mutant overaccumulating allantoin and respective wild type (WT) strain of Arabidopsis thaliana were exposed to cadmium (Cd) or mercury (Hg) with or without nitric oxide (NO) donor (sodium nitroprusside, SNP) to study cross-talk, metabolic and oxidative changes between these nitrogen sources (organic vs. inorganic). The aln-3 accumulated over 10-fold more allantoin than WT with the effect of Cd and Hg differing in leaf and root tissue: aln-3 contained more ascorbic acid and phytochelatins when treated with Cd or Hg and more Cd in both organs. SNP depleted leaf Cd and root Hg accumulation in aln3 but had a positive impact on the amount of metabolites typically in WT plants, indicating potentially negative relation between allantoin and NO. In agreement, aln-3 roots showed lower NO signals in control or metal treatments, but higher ROS signal, and SNP had more pronounced impact in WT roots. Flavonol glycosides were more abundant in aln-3 and were affected more by metals than by SNP. Malate was the most affected Krebs acid with strong reaction to SNP and Hg treatment. Data indicate that allantoin overaccumulation influences the accumulation of specific metabolites but nitric oxide has a greater impact on the metabolite profile in WT.

Design and synthesis of novel ureido and thioureido conjugated hydrazone derivatives with potent anticancer activity.

BACKGROUND: Compounds possessing urea/thiourea moiety have a wide range of biological properties including anticancer activity. On the other hand, taking advantage of the low toxicity and structural diversity of hydrazone derivatives, they are presently being considered for designing chemical compounds with hydrazone moiety in the field of cancer treatment. With this in mind, a series of novel ureido/thioureido derivatives possessing a hydrazone moiety bearing nitro and chloro substituents (4a-4i) have been designed, synthesized, characterized and evaluated for their in vitro cytotoxic effect on HT-29 human colon carcinoma and HepG2 hepatocarcinoma cell lines. RESULTS: Two compounds (4c and 4e) having the chloro phenylurea group hybridized with phenyl hydrazone bearing nitro or chloro moieties demonstrated potent anticancer effect with the IC50 values between 2.2 and 4.8 µM at 72 h. The mechanism of action of compound 4c was revealed in hepatocellular carcinoma cells as an inducer of apoptosis in a caspase-independent pathway. CONCLUSION: Taken together, the current work presented compound 4c as a potential lead compound in developing future hepatocellular carcinoma chemotherapy drugs. METHODS: The compounds were synthesized and then characterized by physical and spectral data (FT-IR, 1H-NMR, 13C-NMR, Mass). The anticancer activity was assessed using MTT assay, flowcytometry, annexin-V, DAPI staining and Western blot analysis.

Selenium increases photosynthetic capacity, daidzein biosynthesis, nodulation and yield of peanuts plants (Arachis hypogaea L.).

This study aimed to investigate the roles of selenium (Se) application on the profile of photosynthetic pigments, oxidant metabolism, flavonoids biosynthesis, nodulation, and its relation to agronomic traits of peanut plants. Two independent experiments were carried out: one conducted in soil and the other in a nutrient solution. When the plants reached the V2 growth stage, five Se doses (0, 7.5, 15, 30, and 45 µg kg-1) and four Se concentrations (0, 5, 10, and 15 µmol L-1) were supplied as sodium selenate. The concentration of photosynthetic pigments, activity of antioxidant enzymes and the concentration of total sugars in peanut leaves increased in response to Se fertilization. In addition, Se improves nitrogen assimilation efficiency by increasing nitrate reductase activity which results in a higher concentration of ureides, amino acids and proteins. Se increases the synthesis of daidzein and genistein in the root, resulting in a greater number of nodules and concentration and transport of ureides to the leaves. Se-treated plants showed greater growth, biomass accumulation in shoots and roots, yield and Se concentration in leaves and grains. Our results contribute to food security and also to increase knowledge about the effects of Se on physiology, biochemistry and biological nitrogen fixation in legume plants.

Environmental Factors Associated With Nitrogen Fixation Prediction in Soybean.

Biological nitrogen (N)-fixation is the most important source of N for soybean [Glycine max (L.) Merr.], with considerable implications for sustainable intensification. Therefore, this study aimed to investigate the relevance of environmental factors driving N-fixation and to develop predictive models defining the role of N-fixation for improved productivity and increased seed protein concentration. Using the elastic net regularization of multiple linear regression, we analyzed 40 environmental factors related to weather, soil, and crop management. We selected the most important factors associated with the relative abundance of ureides (RAU) as an indicator of the fraction of N derived from N-fixation. The most relevant RAU predictors were N fertilization, atmospheric vapor pressure deficit (VPD) and precipitation during early reproductive growth (R1-R4 stages), sowing date, drought stress during seed filling (R5-R6), soil cation exchange capacity (CEC), and soil sulfate concentration before sowing. Soybean N-fixation ranged from 60 to 98% across locations and years (n = 95). The predictive model for RAU showed relative mean square error (RRMSE) of 4.5% and an R2 value of 0.69, estimated via cross-validation. In addition, we built similar predictive models of yield and seed protein to assess the association of RAU and these plant traits. The variable RAU was selected as a covariable for the models predicting yield and seed protein, but with a small magnitude relative to the sowing date for yield or soil sulfate for protein. The early-reproductive period VPD affected all independent variables, namely RAU, yield, and seed protein. The elastic net algorithm successfully depicted some otherwise challenging empirical relationships to assess with bivariate associations in observational data. This approach provides inference about environmental variables while predicting N-fixation. The outcomes of this study will provide a foundation for improving the understanding of N-fixation within the context of sustainable intensification of soybean production.

Nucleoside Metabolism Is Induced in Common Bean During Early Seedling Development.

Nucleoside hydrolases (NSH; nucleosidases) catalyze the cleavage of nucleosides into ribose and free nucleobases. These enzymes have been postulated as key elements controlling the ratio between nucleotide salvage and degradation. Moreover, they play a pivotal role in ureidic legumes by providing the substrate for the synthesis of ureides. Furthermore, nucleotide metabolism has a crucial role during germination and early seedling development, since the developing seedlings require high amount of nucleotide simultaneously to the mobilization of nutrient in cotyledons. In this study, we have cloned two nucleosidases genes from Phaseolus vulgaris, PvNSH1 and PvNSH2, expressed them as recombinant proteins, and characterized their catalytic activities. Both enzymes showed a broad range of substrate affinity; however, PvNSH1 exhibited the highest activity with uridine, followed by xanthosine, whereas PvNSH2 hydrolyses preferentially xanthosine and shows low activity with uridine. The study of the regulation of nucleosidases during germination and early postgerminative development indicated that nucleosidases are induced in cotyledons and embryonic axes just after the radicle emergence, coincident with the induction of nucleases activity and the synthesis of ureides in the embryonic axes, with no remarkable differences in the level of expression of both nucleosidase genes. In addition, nucleosides and nucleobase levels were determined as well in cotyledons and embryonic axes. Our results suggest that PvNSH1 and PvNSH2 play an important role in the mobilization of nutrients during this crucial stage of plant development.

Agronomic biofortification of cowpea with zinc: Variation in primary metabolism responses and grain nutritional quality among 29 diverse genotypes.

Dietary zinc (Zn) deficiency is widespread globally, and is particularly prevalent in low- and middle-income countries (LMICs). Cowpea (Vigna unguiculata (L.) Walp) is consumed widely in LMICs due to its high protein content, and has potential for use in agronomic biofortification strategies using Zn. This study aimed to evaluate the effect of Zn biofortification on grain nutritional quality of 29 cowpea genotypes. Zn application did not increase cowpea yield. In 11 genotypes sucrose concentration, in 12 genotypes total sugar concentration, and in 27 genotypes storage protein concentration increased in response to Zn supply. Fifteen genotypes had lower concentrations of amino acids under Zn application, which are likely to have been converted into storage proteins, mostly comprised of albumin. Phytic acid (PA) concentration and PA/Zn molar ratio were decreased under Zn application. Six genotypes increased shoot ureides concentration in response to Zn fertilization, indicating potential improvements to biological nitrogen fixation. This study provides valuable information on the potential for Zn application to increase cowpea grain nutritional quality by increasing Zn and soluble storage protein and decreasing PA concentration. These results might be useful for future breeding programs aiming to increase cowpea grain Zn concentrations through biofortification.

Determination of Ureides Content in Plant Tissues.

The ureides allantoin and allantoate are the main organic nitrogen compounds transported in several legumes, predominantly from N2 fixation. Moreover, recent studies point out a remarkable role for allantoin during several stress responses of plants other than legumes. The goal of this protocol is to determine ureides concentration in different plant tissues. Ureides are extracted from plant material by boiling it in phosphate buffer. The allantoin and allantoate present in the supernatants are subjected to alkaline-acidic hydrolysis to glyoxylate. The glyoxylate is converted into glycoxylic acid phenylhydrazone, that is then oxidized to red-colored 1,5-diphenylformazan. The absorbance of supernatants is measured using a spectrophotometer at 520 nm. Ureides concentration can be inferred by using a glyoxylate calibration curve. Ureide quantification of different tissues of Arabidopsis thaliana and soybean plants were carried out following this protocol.