Improved Nitrogen Assimilation Research Articles

Leaves and roots metabolomic signatures underlying rootstock-mediated water stress tolerance in grafted pepper plants

Grafting onto pepper rootstock NIBER® is an effective strategy to mitigate water stress effects on the grafted variety. In this work, we comparatively explored the metabolomic responses to water stress in the pepper variety “Maestral F1” (V) grafted onto NIBER® (V/N) and self-grafted (V/V) by untargeted metabolomics on leaves and roots. Leaf water status was also evaluated by relative water content (RWC) and gas exchange measurements. Under water stress, the V/N water use efficiency (WUE) and leaf RWC were higher than V/V, in agreement with major stomata closure and water retention in leaves. V/N showed a tolerance response, which was manifested in the untargeted metabolomic analysis. NIBER® modulated the grafted variety response to water stress as reflected in the differential metabolomic profiles in leaves and roots. The V/N-enriched metabolic pathways showed that the NIBER® response to water stress involved cutin and suberin biosynthesis, which act as protection layers, and jasmonic acid (JA) and jasmonates biosynthesis to favor signaling pathways. NIBER® did not induce flavonols and chlorophyll b synthesis, but likely promoted anthocyanins biosynthesis and maintained an undisturbed chlorophyll a:chlorophyll b ratio. Moreover, NIBER® increased vitamin B6, anthocyanins and stearic acid concentration in the variety leaves, whereas siroheme content rose in roots to improve nitrogen assimilation. Further studies are required to understand the contribution of secondary metabolites, such as phenylpropanoids, glycoalkaloids, and nitrogen-containing secondary metabolites, to NIBER® water stress tolerance.

Aromatic compound 2-acetyl-1-pyrroline coordinates nitrogen assimilation and methane mitigation in fragrant rice.

Rice paddy has been the main source of anthropogenic methane (CH4) emissions, with significant variations among rice varieties. 2-Acetyl-1-pyrroline (2-AP) is the key component of the pleasant aroma in fragrant rice. Here, we show that fragrant rice is metabolically active in nitrogen assimilation and exhibits high levels of 2-AP and that CH4 fluxes at the booting stage and cumulative emissions are 25.5% and 14.8% lower, respectively, in fragrant rice paddies compared with nonfragrant rice paddies. Three precursors involved in 2-AP synthesis-proline, glutamic acid, and ornithine-are identified as crucial nitrogen compounds that significantly promote CH4 oxidation in the rhizosphere. Augmenting 2-AP synthesis, either through foliar spraying or by utilizing CRISPR-Cas9 technology to generate knockout lines of BETAINE ALDEHYDE DEHYDROGENASE 2 gene, effectively enhances CH4 oxidation and reduces CH4 fluxes. Our findings reveal that the 2-AP metabolic pathway coordinates the carbon/nitrogen cycle to improve nitrogen assimilation along with high 2-AP levels and mitigate CH4 emissions in paddy ecosystems.

Dissecting Enhanced Carbohydrate and Pigment Productivity in Mutants of Nannochloropsis oculata Using Metabolomics and Lipidomics.

Random mutagenesis is an effective strategy for enhancing cellular traits. In this study, we used the mutagen ethyl methanesulfonate to create fast-growing Nannochloropsis oculata mutants. When cultivated in a photobioreactor with a diel cycle, two mutants exhibited 2.2-fold higher carbohydrate productivity and 3.5-4.0-fold higher pigment productivity than the wild type, while one of them also showed 2.5-fold higher lipid productivity. A comprehensive physiological, metabolomic, and lipidomic study showed that the mutants had high levels of glucose-, galactose-, and xylose-based carbohydrates. Their high growth rate was attributed to increased chlorophyll a content, improved nitrogen assimilation, storage, and recycling, and low monogalactosyldiacyl glycerol/digalactosyldiacyl glycerol ratio, which was responsible for higher biomass productivity. The investigation revealed upregulation of lipid precursors, shedding light on high lipid accumulation. The derived algae strains are capable of increasing the biosynthesis of value-added storage molecules without impairing growth, rendering them promising candidates for commercial development in future biorefineries.

Magnetic Treatment Improves the Seedling Growth, Nitrogen Metabolism, and Mineral Nutrient Contents in Populus × euramericana ‘Neva’ under Cadmium Stress

This pot experiment was carried out to investigate the mechanism underlying nutrient metabolism and seedling growth responses to magnetic treatment following exposure to cadmium (Cd) stress. A magnetic device of 300 Gs was applied during Cd(NO3)2 solution treatment at 0 and 100 mM·L−1. One-year-old seedlings of Populus × euramericana ‘Neva’ were treated with different Cd(NO3)2 solutions in the presence or absence of magnetic treatment for 30 days. Seedling growth and physiological–biochemical indexes were measured under Cd stress. The contents of ammonium (NH4+–N), nitrate (NO3––N), and total nitrogen (TN) in leaves, as well as NH4+–N and TN in roots, were increased by magnetic treatment combined with Cd stress, although the NO3––N content was decreased. The activities of nitrate reductase (NR), nitrite reductase (NiR), glutathione reductase (GR), and glutamate synthase (GOGAT) in leaves and the activities of NR, glutamine synthetase (GS), and GOGAT in roots were stimulated by magnetic treatment; conversely, the NiR activity in roots was inhibited by magnetic effects. Magnetic treatment improved the synthesis of cysteine (Cys) and glutamine (Gln) in leaves and reduced the contents of glutamic acid (Glu) and glycine (Gly), while the contents of Cys, Glu, Gln, and Gly were increased in roots. The contents of Ca, Mg, Fe, Mn, Zn, and Cu in leaves were increased by magnetic treatment under Cd stress, whereas the content of K was reduced. In roots, the contents of K, Ca, and Fe were increased by magnetic treatment under Cd stress, but the contents of Na, Mg, Mn, Zn, and Cu were decreased. Magnetization could regulate the uptake of mineral nutrients by roots and translocation from the roots to the aboveground parts by affecting root morphology. Magnetic treatment could also improve nitrogen assimilation and the synthesis of free amino acids by stimulating the activities of key enzymes.

Unraveling metabolic alterations in Chlorella vulgaris cultivated on renewable sugars using time resolved multi-omics

The inherent metabolic versatility of Chlorella vulgaris that enables it to metabolize both inorganic and organic carbon under various trophic modes of cultivation makes it a promising candidate for industrial applications. To shed light on the metabolic flexibility of this microalga, time resolved proteomic and metabolomic studies were conducted in three distinct trophic modes (autotrophic, heterotrophic, mixotrophic) at two growth stages (end of linear growth at 6 days and during nutrient deprivation at 10 days). Sweet sorghum bagasse (SSB) hydrolysate was supplied to the cultivation medium as a renewable source of organic carbon mainly in the form of glucose. Integrated multi-omics data showed improved nitrogen assimilation, re-allocation, and recycling and increased levels of photosystem II (PS II) proteins indicating effective cellular quenching of excess electrons during mixotrophy. As external addition of organic carbon (glucose) to the cultivation medium decreases the cell's dependence on photosynthesis, an upregulation in the mitochondrial electron transport chain was recorded that led to increased cellular energy generation and hence higher growth rates under mixotrophy. Moreover, upregulation of the lipid-packaging proteins caleosin and 14_3_3 domain-containing protein resulted in maximum expression during mixotrophy suggesting a strong correlation between lipid synthesis, stabilization, and assembly. Overall, cells cultivated under mixotrophy showed better nutrient stress tolerance and redox balancing leading to higher biomass and lipid production. The study offers a panoramic view of the microalga's metabolic flexibility and contributes to a deeper understanding of the altered biochemical pathways that can be exploited to enhance algal productivity and commercial potential.

Transcription Factor TaDof1 Improves Nitrogen and Carbon Assimilation Under Low-Nitrogen Conditions in Wheat

A single transcription factor is known to coordinate expression of a set of metabolites in a biochemical pathway; its use therefore can be a functional strategy in generating plants with desired traits. Triticum aestivum Dof1 (TaDof1) transcription factor is mainly associated with improved nitrogen assimilation in plants. In the current research, the transgenic wheat overexpressing TaDof1 transcription factor, under a constitutive promoter, was developed by Agrobacterium-mediated transformation. The two elite wheat cultivars (Galaxy and Faisalabad-2008) were selected for transformation study. The T0 plants were subjected to screening using selection medium containing herbicide BASTA. PCR results confirmed that only 8 out of 31 plants possessed the complete TaDof1 cassette. A transformation efficiency of 0.46% for Galaxy and 0.08% for Faisalaad-2008 was obtained. The quantitative RT-PCR was performed on T1 plants grown under nitrogen-limiting conditions. A substantial rise in the expression of citrate synthase (CS), isocitrate dehydrogenase (ICDH), phosphoenolpyruvate carboxylase (PEPC), and pyruvate kinase (PK) genes regulated by TaDof1 was observed after 4 weeks of nitrogen stress in T1 plants. The maximum fold increase of 464 was recorded for ICDH. Our findings indicate a cooperative modification of nitrogen and carbon metabolisms since they are intimately linked together. Overexpression of TaDof1 in wheat resulted in a significant increase in various agronomic traits. Furthermore, various physiological and biochemical markers (chlorophyll, protein, and soluble sugar contents) exhibited a profound change in TaDof1 transgenic plants in comparison with wild type plants. The results clearly depict the merits of employing transcription factors in engineering plant metabolisms.

Overexpression of an NADP(H)-dependent glutamate dehydrogenase gene, TrGDH, from Trichurus improves nitrogen assimilation, growth status and grain weight per plant in rice.

As glutamate dehydrogenases (GDHs) of microorganisms usually have higher affinity for NH4+ than do those of higher plants, it is expected that ectopic expression of these GDHs can improve nitrogen assimilation in higher plants. Here, a novel NADP(H)-GDH gene (TrGDH) was isolated from the fungus Trichurus and introduced into rice (Oryza sativa L.). Investigation of kinetic properties in vitro showed that, compared with the rice GDH (OsGDH4), TrGDH exhibited higher affinity for NH4+ (Km = 1.48 ± 0.11 mM). Measurements of the NH4+ assimilation rate demonstrated that the NADP(H)-GDH activities of TrGDH transgenic lines were significantly higher than those of the controls. Hydroponic experiments revealed that the fresh weight, dry weight and nitrogen content significantly increased in the TrGDH transgenic lines. Field trials further demonstrated that the number of effective panicles, 1,000-grain weight and grain weight per plant of the transgenic lines were significantly higher than those of the controls, especially under low-nitrogen levels. Moreover, glutelin and prolamine were found to be markedly increased in seeds from the transgenic rice plants. These results sufficiently confirm that overexpression of TrGDH in rice can improve the growth status and grain weight per plant by enhancing nitrogen assimilation. Thus, TrGDH is a promising candidate gene for maintaining yields in crop plants via genetic engineering.

Nitrogen Supply and Leaf Age Affect the Expression of TaGS1 or TaGS2 Driven by a Constitutive Promoter in Transgenic Tobacco.

Glutamine synthetase (GS) plays a key role in nitrogen metabolism. Here, two types of tobacco transformants, overexpressing Triticum aestivum GS1 (TaGS1) or GS2 (TaGS2), were analysed. Four independent transformed lines, GS1-TR1, GS1-TR2, GS2-TR1 and GS2-TR2, were used for the nitrogen treatment. Under nitrogen-sufficient conditions, the leaves of GS2-TR showed high accumulation of the TaGS2 transcript, while those of GS1-TR showed a low TaGS1 transcript levels. However, compared with nitrogen-sufficient conditions, the TaGS1 transcript level increased in the leaves under nitrogen starvation, but the TaGS2 transcript level decreased. In addition, the TaGS1 and TaGS2 transcript levels were highest in the middle leaves under nitrogen-sufficient and starvation conditions. These results show that nitrogen supply and leaf age regulate TaGS expression, even when they are driven by a super-promoter. Additionally, in regard to nitrogen metabolism level, the lower leaves of the GS1-TR exhibited lower NH4+ and higher amino acid contents, while the upper leaves exhibited higher amino acid, soluble protein and chlorophyll contents. The leaves of the GS2-TR exhibited lower NH4+ but higher amino acid, soluble protein and chlorophyll contents. Given the role that GS isoforms play in nitrogen metabolism, these data suggest that TaGS1 overexpression may improve nitrogen transport, and that TaGS2 overexpression may improve nitrogen assimilation under nitrogen stress.

Tomato ethylene sensitivity determines interaction with plant growth-promoting bacteria.

Plant growth-promoting bacteria (PGPB) are soil micro-organisms able to interact with plants and stimulate their growth, positively affecting plant physiology and development. Although ethylene plays a key role in plant growth, little is known about the involvement of ethylene sensitivity in bacterial inoculation effects on plant physiology. Thus, the present study was pursued to establish whether ethylene perception is critical for plant-bacteria interaction and growth induction by two different PGPB strains, and to assess the physiological effects of these strains in juvenile and mature tomato ( Solanum lycopersicum ) plants. An experiment was performed with the ethylene-insensitive tomato never ripe and its isogenic wild-type line in which these two strains were inoculated with either Bacillus megaterium or Enterobacter sp. C7. Plants were grown until juvenile and mature stages, when biomass, stomatal conductance, photosynthesis as well as nutritional, hormonal and metabolic statuses were analysed. Bacillus megaterium promoted growth only in mature wild type plants. However, Enterobacter C7 PGPB activity affected both wild-type and never ripe plants. Furthermore, PGPB inoculation affected physiological parameters and root metabolite levels in juvenile plants; meanwhile plant nutrition was highly dependent on ethylene sensitivity and was altered at the mature stage. Bacillus megaterium inoculation improved carbon assimilation in wild-type plants. However, insensitivity to ethylene compromised B. megaterium PGPB activity, affecting photosynthetic efficiency, plant nutrition and the root sugar content. Nevertheless, Enterobacter C7 inoculation modified the root amino acid content in addition to stomatal conductance and plant nutrition. Insensitivity to ethylene severely impaired B. megaterium interaction with tomato plants, resulting in physiological modifications and loss of PGPB activity. In contrast, Enterobacter C7 inoculation stimulated growth independently of ethylene perception and improved nitrogen assimilation in ethylene-insensitive plants. Thus, ethylene sensitivity is a determinant for B. megaterium , but is not involved in Enterobacter C7 PGPB activity.

Over-expression of a fungal NADP(H)-dependent glutamate dehydrogenase PcGDH improves nitrogen assimilation and growth quality in rice

Glutamate dehydrogenase (GDH) tends to have a lower affinity for ammonium than glutamine synthetase (GS) in higher plants. Consequently, nitrogen is mostly assimilated as ammonium by the GS/glutamate synthase pathway which requires 2-oxoglutarate (2-OG) as carbon skeletons. In contrast, the NADP(H)-dependent GDH in fungi has a higher affinity for ammonium than that in higher plants and plays a more significant part in ammonium assimilation. We isolated an NADP(H)-GDH gene (PcGDH) from the fungus Pleurotus cystidiosus and heterologously expressed it in rice (Oryza sativa L.). Alterations in nitrogen assimilation, growth, metabolism, and grain yield were observed in the transgenic plants. An investigation of the kinetic properties of the purified recombinant protein demonstrated that the amination activity (7.05 ± 0.78 μmoL min−1 mg soluble protein−1) of PcGDH was higher than the deamination activity (3.36 ± 0.42 μmoL min−1 mg soluble protein−1) and that the Km value for ammonium (Km = 3.73 ± 0.23 mM) was lower than that for the glutamate (Km = 15.97 ± 0.31 mM), indicating that the PcGDH tends to interconvert 2-OG and glutamate. Examination of the activity of NADP(H)-GDH in control and transgenic lines demonstrated that NADP(H)-GDH activity in the transgenic lines was markedly higher than that in the control lines; in particular, the amination activity was significantly higher than the deamination activity in shoots of the transgenic lines. The results of the hydroponics experiment revealed that shoot and root length, fresh weight, chlorophyll content, nitrogen content, and amino acid levels (glutamate, glutamine, and total amino acids) were elevated in transgenic lines in comparison with those of the control line under different nitrogen conditions at seedling stage. The 1,000-grain weight and the panicle number in transgenic lines were considerably augmented in the field condition, yet the filled grain rate dropped slightly and there was no apparent change in the grain yield. The levels of glutelin and prolamine in the transgenic seeds were considerably higher than those in control seeds. In conclusion, these results demonstrate that heterologous expression of P. cystidiosus GDH (PcGDH) could improve nitrogen assimilation and growth in rice.

Вплив метанолу на вміст NAD(P)H, вільних амінокислот і протеїну в клітинах Chlamydomonas reinhardtii

It is known that the addition of methanol to the culture medium stimulates the photosynthetic productivity of some species of microalgae, but its influence on the biochemical composition of the biomass has not been investigated. The aim of the present work is to determine the effect of methanol (50 mM) on the content of free amino acids, soluble proteins and reduced nicotinamide coenzyme NAD(P)H in C. reinhartdii cells. It is shown that in case of illumination of C. reinhardtii methanol raises intracellular content of NAD(P)H four times more efficiently than in the darkness. Total content of free amino acids is increased and their ratio is changed. The concentration of glutamic acid, glutamine, alanine, serine and tyrosine also increases. The concentration of valine and methionine is reduced. Growth of culture with methanol is followed by an increase in the content of intracellular protein by 30% after 20 hours of cultivation. The obtained data indicate that methanol stimulates growth of C. reinhardtii, not only as a result of additional carbon utilization, but also due to improved nitrogen assimilation and the impact on the energy metabolism of cells.

Lactating mice (Mus musculus) exhibit compensatory flexibility in gut morphology in response to reduced dietary protein

Protein affects key life-history traits, and deficiencies in this nutrient may have selected for the ability to invoke physiological or morphological mechanisms to aid nutrient assimilation. I examined the effect of dietary protein on gut characters in lactating mice (Mus musculus L., 1758) and predicted that mice, to improve assimilation efficiency, would increase the mass of the stomach and small intestine and (or) increase food retention in these organs. Mice were maintained on isocaloric diets differing in protein and carbohydrate content (P:C) during the reproductive period. The hypothesis that food would be preferentially retained was not supported. However, both the stomach and the small intestine responded to low P:C with increased mass, and the small intestine exhibited increased diameter. This study demonstrates that mammalian gut morphology of lactating mice can respond to nutrient availability under conditions of constant energy intake. Further study is needed to determine if gut flexibility in response to decreasing P:C levels results in improved nitrogen assimilation efficiency and if this response is a general strategy of mammals or is limited to those with particular breeding strategies.

A novel feeding method in commercial Baker’s yeast production

The aim of this investigation was to determine a better leavening ability and shelf life for the same biomass yield of final product. A commercial fed-batch bioreactor equipped with circulation loop was used to study the effect of carbon source, molasses, profile on dough-leavening ability, shelf life and biomass yield of Baker's yeast, Saccharomyces cerevisiae. A set of 32 commercial batches were performed to investigate the effect of sugar concentration and compare with 32 control experiments. Higher local sugar concentration in circulation loop resulted in a better leavening ability and shelf life for the same biomass yield of the final product. In addition, this method improved nitrogen assimilation which resulted in higher protein content. Increase in leavening ability and protein content could be a result of the higher levels of glycolytic enzymes. It was observed that this change resulted in considerable improvement in leavening ability and shelf life at a commercial scale. It must be emphasized that to improve product quality, it is not necessary to pursue classical mutagenesis and selection strategies. A high-quality product can be achieved only by optimizing the feeding profile and strategy.

Nitrogen Assimilatory Enzymes and Amino Acid Content in Inoculated Foliar Fertilized Pea Plants Grown at Reduced Molybdenum Concentration

ABSTRACT A possibility to improve nitrogen assimilation in nitrogen fixing Molybdenum (Mo) deficient pea plants was shown. The influence of foliar supplied nutrients in addition to root nutrition resulted in reducing the unfavorable effects of inorganic nitrogen on nodule function and Mo deficiency on the nitrogen assimilatory enzymes. Inoculated pea plants were grown on liquid nutrient solution both with and without Mo. The following variants were tested: Mo supplied plants with root nutrition (F1 + Mo); Mo supplied plants with root and foliar nutrition (F2 + Mo); Mo deficient plants with root nutrition (F1 − Mo); and Mo deficient plants with root and foliar nutrition (F2 − Mo). Foliar application of nutrients had a positive effect on the glutamine synthetase and glutamate synthase enzyme activities in the roots and nodules of Mo deficient plants. It was found that the foliar fertilization reduced the inhibitory effect of Mo shortage on the aspartate/asparagine content in the pea shoots.

Metabolic engineering with Dof1 transcription factor in plants: Improved nitrogen assimilation and growth under low-nitrogen conditions.

Utilization of transcription factors might be a powerful approach to modification of metabolism for a generation of crops having superior characteristics because a single transcription factor frequently regulates coordinated expression of a set of key genes for respective pathways. Here, we apply the plant-specific Dof1 transcription factor to improve nitrogen assimilation, the essential metabolism including the primary assimilation of ammonia to carbon skeletons to biosynthesize amino acids and other organic compounds involving nitrogen in plants. Expressing Dof1 induced the up-regulation of genes encoding enzymes for carbon skeleton production, a marked increase of amino acid contents, and a reduction of the glucose level in transgenic Arabidopsis. The results suggest cooperative modification of carbon and nitrogen metabolisms on the basis of their intimate link. Furthermore, elementary analysis revealed that the nitrogen content increased in the Dof1 transgenic plants (approximately 30%), indicating promotion of net nitrogen assimilation. Most significantly, the Dof1 transgenic plants exhibit improved growth under low-nitrogen conditions, an agronomically important trait. These results highlight the great utility of transcription factors in engineering metabolism in plants.