Multisubstrate specificity shaped the complex evolution of the aminotransferase family across the tree of life.

Aminotransferases (ATs) are an ancient enzyme family that play central roles in core nitrogen metabolism, essential to all organisms. However, many of the AT enzyme functions remain poorly defined, limiting our fundamental understanding of the nitrogen metabolic networks that exist in different organisms. Here, we traced the deep evolutionary history of the AT family by analyzing AT enzymes from 90 species spanning the tree of life (ToL). We found that each organism has maintained a relatively small and constant number of ATs. Mapping the distribution of ATs across the ToL uncovered that many essential AT reactions are carried out by taxon-specific AT enzymes due to wide-spread nonorthologous gene displacements. This complex evolutionary history explains the difficulty of homology-based AT functional prediction. Biochemical characterization of diverse aromatic ATs further revealed their broad substrate specificity, unlike other core metabolic enzymes that evolved to catalyze specific reactions today. Interestingly, however, we found that these AT enzymes that diverged over billion years share common signatures of multisubstrate specificity by employing different nonconserved active site residues. These findings illustrate that AT family enzymes had leveraged their inherent substrate promiscuity to maintain a small yet distinct set of multifunctional AT enzymes in different taxa. This evolutionary history of versatile ATs likely contributed to the establishment of robust and diverse nitrogen metabolic networks that exist throughout the ToL. The study provides a critical foundation to systematically determine diverse AT functions and underlying nitrogen metabolic networks across the ToL.

Light recovery after maize harvesting promotes soybean flowering in a maize-soybean relay strip intercropping system.

Moving from sole cropping to intercropping is a transformative change in agriculture, contributing to yield. Soybeans adapt to light conditions in intercropping by adjusting the onset of reproduction and the inflorescence architecture to optimize reproductive success. Maize-soybean strip intercropping (MS), maize-soybean relay strip intercropping (IS), and sole soybean (SS) systems are typical soybean planting systems with significant differences in light environments during growth periods. To elucidate the effect of changes in the light environment on soybean flowering processes and provide a theoretical basis for selecting suitable varieties in various planting systems to improve yields, field experiments combining planting systems (IS, MS, and SS) and soybean varieties (GQ8, GX7, ND25, and NN996) were conducted in 2021 and 2022. Results showed that growth recovery in the IS resulted in a balance in the expression of TERMINAL FLOWER 1 (TFL1) and FLOWERING LOCUS T (FT) in the meristematic tissues of soybeans, which promoted the formation of new branches or flowers. IS prolonged the flowering time (2-7 days) and increased the number of forming flowers compared with SS (93.0 and 169%) and MS (67.3 and 103.3%) at the later soybean flowering stage. The higher carbon and nitrogen content in the middle and bottom canopies of soybean contributed to decreased flower abscission by 26.7 and 30.2%, respectively, compared with SS. Canopy light environment recovery promoted branch and flower formation and transformation of flowers into pods with lower flower-pod abscission, which contributed to elevating soybean yields in late-maturing and multibranching varieties (ND25) in IS.

Integrated morphological, physiological and transcriptomic analyses reveal the responses of Toona sinensis seedlings to low-nitrogen stress.

Nitrogen is one of the most essential elements for plant growth and development. In this study, the growth, physiology, and transcriptome of Toona sinensis (A. Juss) Roem seedlings were compared between low-nitrogen (LN) and normal-nitrogen (NN) conditions. These results indicate that LN stress adversely influences T. sinensis seedling growth. The activities of key enzymes related to nitrogen assimilation and phytohormone contents were altered by LN stress. A total of 2828 differentially expressed genes (DEGs) in roots and 1547 in leaves were identified between the LN and NN treatments. A differential enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways indicated that nitrogen and sugar metabolism, flavonoid biosynthesis, plant hormone signal transduction, and ABC transporters, were strongly affected by LN stress. In summary, this research provides information for further understanding the response of T. sinensis to LN stress.

Arabidopsis thaliana argininosuccinate lyase structure uncovers the role of serine as the catalytic base.

Arginine is an important amino acid in plants, as it not only plays a structural role and serves as nitrogen storage but is also a precursor for various molecules, including polyamines and proline. Arginine is produced by argininosuccinate lyase (ASL) which catalyzes the cleavage of argininosuccinate to arginine and fumarate. ASL belongs to the fumarate lyase family and while many members of this family were well-characterized, little is known about plant ASLs. Here we present the first crystal structures of ASL from the model plant, Arabidopsis thaliana (AtASL). One of the structures represents the unliganded form of the AtASL homotetramer. The other structure, obtained from a crystal soaked in argininosuccinate, accommodates the substrate or the reaction products in one of four active sites of the AtASL tetramer. Each active site is located at the interface of three neighboring protomers. The AtASL structure with ligands allowed us to analyze the enzyme-substrate and the enzyme-product interactions in detail. Furthermore, based on our analyses, we describe residues of AtASL crucial for catalysis. The structure of AtASL gives the rationale for the open-to-close transition of the GSS mobile loop and indicates the importance of serine 333 from this loop for the enzymatic action of the enzyme. Finally, we supplemented the structural data with the identification of sequence motifs characteristic for ASLs.

Pine has two glutamine synthetase paralogs, GS1b.1 and GS1b.2, exhibiting distinct biochemical properties.

The enzyme glutamine synthetase (EC 6.3.1.2) is mainly responsible for the incorporation of inorganic nitrogen into organic molecules in plants. In the present work, a pine (Pinus pinaster) GS1 (PpGS1b.2) gene was identified, showing a high sequence identity with the GS1b.1 gene previously characterized in conifers. Phylogenetic analysis revealed that the presence of PpGS1b.2 is restricted to the genera Pinus and Picea and is not found in other conifers. Gene expression data suggest a putative role of PpGS1b.2 in plant development, similar to other GS1b genes from angiosperms, suggesting evolutionary convergence. The characterization of GS1b.1 and GS1b.2 at the structural, physicochemical, and kinetic levels has shown differences even though they have high sequence homology. GS1b.2 had a lower optimum pH (6 vs. 6.5) and was less thermally stable than GS1b.1. GS1b.2 exhibited positive cooperativity for glutamate and substrate inhibition for ammonium. However, GS1b.1 exhibited substrate inhibition behavior for glutamate and ATP. Alterations in the kinetic characteristics produced by site-directed mutagenesis carried out in this work strongly suggest an implication of amino acids at positions 264 and 267 in the active center of pine GS1b.1 and GS1b.2 being involved in affinity toward ammonium. Therefore, the amino acid differences between GS1b.1 and GS1b.2 would support the functioning of both enzymes to meet distinct plant needs.

Prominent transcriptomic changes in Mycobacterium intracellulare under acidic and oxidative stress.

BACKGROUND: Mycobacterium avium complex (MAC), including Mycobacterium intracellulare is a member of slow-growing mycobacteria and contributes to a substantial proportion of nontuberculous mycobacterial lung disease in humans affecting immunocompromised and elderly populations. Adaptation of pathogens in hostile environments is crucial in establishing infection and persistence within the host. However, the sophisticated cellular and molecular mechanisms of stress response in M. intracellulare still need to be fully explored. We aimed to elucidate the transcriptional response of M. intracellulare under acidic and oxidative stress conditions. RESULTS: At the transcriptome level, 80 genes were shown [FC] ≥ 2.0 and p < 0.05 under oxidative stress with 10 mM hydrogen peroxide. Specifically, 77 genes were upregulated, while 3 genes were downregulated. In functional analysis, oxidative stress conditions activate DNA replication, nucleotide excision repair, mismatch repair, homologous recombination, and tuberculosis pathways. Additionally, our results demonstrate that DNA replication and repair system genes, such as dnaB, dinG, urvB, uvrD2, and recA, are indispensable for resistance to oxidative stress. On the contrary, 878 genes were shown [FC] ≥ 2.0 and p < 0.05 under acidic stress with pH 4.5. Among these genes, 339 were upregulated, while 539 were downregulated. Functional analysis highlighted nitrogen and sulfur metabolism pathways as the primary responses to acidic stress. Our findings provide evidence of the critical role played by nitrogen and sulfur metabolism genes in the response to acidic stress, including narGHIJ, nirBD, narU, narK3, cysND, cysC, cysH, ferredoxin 1 and 2, and formate dehydrogenase. CONCLUSION: Our results suggest the activation of several pathways potentially critical for the survival of M. intracellulare under a hostile microenvironment within the host. This study indicates the importance of stress responses in M. intracellulare infection and identifies promising therapeutic targets.

A comprehensive assessment of photosynthetic acclimation to shade in C4 grass (Cynodon dactylon (L.) Pers.).

BACKGROUND: Light deficit in shaded environment critically impacts the growth and development of turf plants. Despite this fact, past research has predominantly concentrated on shade avoidance rather than shade tolerance. To address this, our study examined the photosynthetic adjustments of Bermudagrass when exposed to varying intensities of shade to gain an integrative understanding of the shade response of C4 turfgrass. RESULTS: We observed alterations in photosynthetic pigment-proteins, electron transport and its associated carbon and nitrogen assimilation, along with ROS-scavenging enzyme activity in shaded conditions. Mild shade enriched Chl b and LHC transcripts, while severe shade promoted Chl a, carotenoids and photosynthetic electron transfer beyond QA- (ET0/RC, φE0, Ψ0). The study also highlighted differential effects of shade on leaf and root components. For example, Soluble sugar content varied between leaves and roots as shade diminished SPS, SUT1 but upregulated BAM. Furthermore, we observed that shading decreased the transcriptional level of genes involving in nitrogen assimilation (e.g. NR) and SOD, POD, CAT enzyme activities in leaves, even though it increased in roots. CONCLUSIONS: As shade intensity increased, considerable changes were noted in light energy conversion and photosynthetic metabolism processes along the electron transport chain axis. Our study thus provides valuable theoretical groundwork for understanding how C4 grass acclimates to shade tolerance.

Metabolomics analysis of different diameter classes of Taxus chinensis reveals that the resource allocation is related to carbon and nitrogen metabolism.

Taxus chinensis (Taxus cuspidata Sieb. et Zucc.) is a traditional medicinal plant known for its anticancer substance paclitaxel, and its growth age is also an important factor affecting its medicinal value. However, how age affects the physiological and metabolic characteristics and active substances of T. chinensis is still unclear. In this study, carbon and nitrogen accumulation, contents of active substances and changes in primary metabolites in barks and annual leaves of T. chinensis of different diameter classes were investigated by using diameter classes instead of age. The results showed that leaves and barks of small diameter class (D1) had higher content of non-structural carbohydrates and C, which were effective in enhancing defense capacity, while N content was higher in medium (D2) and large diameter classes (D3). Active substances such as paclitaxel, baccatin III and cephalomannine also accumulated significantly in barks of large diameter classes. Moreover, 21 and 25 differential metabolites were identified in leaves and barks of different diameter classes, respectively. The differential metabolites were enhanced the TCA cycle and amino acid biosynthesis, accumulate metabolites such as organic acids, and promote the synthesis and accumulation of active substances such as paclitaxel in the medium and large diameter classes. These results revealed the carbon and nitrogen allocation mechanism of different diameter classes of T. chinensis, and its relationship with medicinal components, providing a guidance for the harvesting and utilization of wild T. chinensis.

Effects of cadmium stress on the growth and physiological characteristics of sweet potato.

This study evaluated the responses of sweet potatoes to Cadmium (Cd) stress through pot experiments to theoretically substantiate their comprehensive applications in Cd-polluted agricultural land. The experiments included a CK treatment and three Cd stress treatments with 3, 30, and 150 mg/kg concentrations, respectively. We analyzed specified indicators of sweet potato at different growth periods, such as the individual plant growth, photosynthesis, antioxidant capacity, and carbohydrate Cd accumulation distribution. On this basis, the characteristics of the plant carbon metabolism in response to Cd stress throughout the growth cycle were explored. The results showed that T2 and T3 treatments inhibited the vine growth, leaf area expansion, stem diameter elongation, and tuberous root growth of sweet potato; notably, T3 treatment significantly increased the number of sweet potato branches. Under Cd stress, the synthesis of chlorophyll in sweet potato was significantly suppressed, and the Rubisco activity experienced significant reductions. With the increasing Cd concentration, the function of PS II was also affected. The soluble sugar content underwent no significant change in low Cd concentration treatments. In contrast, it decreased significantly under high Cd concentrations. Additionally, the tuberous root starch content decreased significantly with the increase in Cd concentration. Throughout the plant growth, the activity levels of catalase, peroxidase, and superoxide dismutase increased significantly in T2 and T3 treatments. By comparison, the superoxide dismutase activity in T1 treatment was significantly lower than that of CK. With the increasing application of Cd, its accumulation accordingly increased in various sweet potato organs. The the highest bioconcentration factor was detected in absorbing roots, while the tuberous roots had a lower bioconcentration factor and Cd accumulation. Moreover, the transfer factor from stem to petiole was the highest of the potato organs. These results demonstrated that sweet potatoes had a high Cd tolerance and a restoration potential for Cd-contaminated farmland.

RNA-seq reveals the gene expression in patterns in Populus × euramericana 'Neva' plantation under different precision water and fertilizer-intensive management.

BACKGROUND: Populus spp. is a crucial fast-growing and productive tree species extensively cultivated in the mid-latitude plains of the world. However, the impact of intensive cultivation management on gene expression in plantation remains largely unexplored. RESULTS: Precision water and fertilizer-intensive management substantially increased key enzyme activities of nitrogen transport, assimilation, and photosynthesis (1.12-2.63 times than CK) in Populus × euramericana 'Neva' plantation. Meanwhile, this management approach had a significant regulatory effect on the gene expression of poplar plantations. 1554 differential expression genes (DEGs)were identified in drip irrigation (ND) compared with conventional irrigation. Relative to ND, 2761-4116 DEGs, predominantly up-regulated, were identified under three drip fertilization combinations, among which 202 DEGs were mainly regulated by fertilization. Moreover, drip irrigation reduced the expression of cell wall synthesis-related genes to reduce unnecessary water transport. Precision drip and fertilizer-intensive management promotes the synergistic regulation of carbon and nitrogen metabolism and up-regulates the expression of major genes in nitrogen transport and assimilation processes (5 DEGs), photosynthesis (15 DEGs), and plant hormone signal transduction (11 DEGs). The incorporation of trace elements further enhanced the up-regulation of secondary metabolic process genes. In addition, the co-expression network identified nine hub genes regulated by precision water and fertilizer-intensive management, suggesting a pivotal role in regulating the growth of poplar. CONCLUSION: Precision water and fertilizer-intensive management demonstrated the ability to regulate the expression of key genes and transcription factor genes involved in carbon and nitrogen metabolism pathways, plant hormone signal transduction, and enhance the activity of key enzymes involved in related processes. This regulation facilitated nitrogen absorption and utilization, and photosynthetic abilities such as light capture, light transport, and electron transport, which faintly synergistically regulate the growth of poplar plantations. These results provide a reference for proposing highly efficient precision intensive management to optimize the expression of target genes.

Identification of candidate genes and residues for improving nitrogen use efficiency in the N-sensitive medicinal plant Panax notoginseng.

BACKGROUND: Nitrogen (N) metabolism-related key genes and conserved amino acid sites in key enzymes play a crucial role in improving N use efficiency (NUE) under N stress. However, it is not clearly known about the molecular mechanism of N deficiency-induced improvement of NUE in the N-sensitive rhizomatous medicinal plant Panax notoginseng (Burk.) F. H. Chen. To explore the potential regulatory mechanism, the transcriptome and proteome were analyzed and the three-dimensional (3D) information and molecular docking models of key genes were compared in the roots of P. notoginseng grown under N regimes. RESULTS: Total N uptake and the proportion of N distribution to roots were significantly reduced, but the NUE, N use efficiency in biomass production (NUEb), the recovery of N fertilizer (RNF) and the proportion of N distribution to shoot were increased in the N0-treated (without N addition) plants. The expression of N uptake- and transport-related genes NPF1.2, NRT2.4, NPF8.1, NPF4.6, AVP, proteins AMT and NRT2 were obviously up-regulated in the N0-grown plants. Meanwhile, the expression of CIPK23, PLC2, NLP6, TCP20, and BT1 related to the nitrate signal-sensing and transduction were up-regulated under the N0 condition. Glutamine synthetase (GS) activity was decreased in the N-deficient plants, while the activity of glutamate dehydrogenase (GDH) increased. The expression of genes GS1-1 and GDH1, and proteins GDH1 and GDH2 were up-regulated in the N0-grown plants, there was a significantly positive correlation between the expression of protein GDH1 and of gene GDH1. Glu192, Glu199 and Glu400 in PnGS1 and PnGDH1were the key amino acid residues that affect the NUE and lead to the differences in GDH enzyme activity. The 3D structure, docking model, and residues of Solanum tuberosum and P. notoginseng was similar. CONCLUSIONS: N deficiency might promote the expression of key genes for N uptake (genes NPF8.1, NPF4.6, AMT, AVP and NRT2), transport (NPF1.2 and NRT2.4), assimilation (proteins GS1 and GDH1), signaling and transduction (genes CIPK23, PLC2, NLP6, TCP20, and BT1) to enhance NUE in the rhizomatous species. N deficiency might induce Glu192, Glu199 and Glu400 to improve the biological activity of GS1 and GDH, this has been hypothesized to be the main reason for the enhanced ability of N assimilation in N-deficient rhizomatous species. The key genes and residues involved in improving NUE provide excellent candidates for the breeding of medicinal plants.

Auxin synthesis promotes N metabolism and optimizes root structure enhancing N acquirement in maize (Zea mays L.).

MAIN CONCLUSION: Foliar NAA increases photosynthate supplied by enhancing photosynthesis, to strengthen root activity and provide a large sink for root carbohydrate accumulation, which is beneficial to acquire more nitrogen. The improvement of grain yield is an effective component in the food security. Auxin acts as a well-known plant hormone, plays an important role in maize growth and nutrient uptake. In this study, with maize variety Zhengdan 958 (ZD958) as material, the effects of auxin on nitrogen (N) uptake and assimilation of seedling maize were studied by hydroponic experiments. With water as the control, naphthalene acetic acid (NAA, 0.1 mmol/L) and aminoethoxyvinylglycine (AVG, 0.1 mmol/L, an auxin synthesis inhibitor) were used for foliar spraying. The results showed that NAA significantly improved photosynthetic rate and plant biomass by 58.6% and 91.7%, respectively, while the effect of AVG was opposite to that of NAA. At the same time, key enzymes activities related N assimilation in NAA leaves were significantly increased, and the activities of nitrate reductase (NR), glutamine synthetase (GS) and glutamate synthase (GOGAT) were increased by 32.3%, 22.9%, and 16.2% in new leaves. Furthermore, NAA treatment promoted underground growth. When compared with control, total root length, root surface area, root tip number, branch number and root activity were significantly increased by 37.8%, 22.2%, 35.1%, 28.8% and 21.2%. Root growth is beneficial to N capture in maize. Ultimately, the total N accumulation of NAA treatment was significantly increased by 74.5%, as compared to the control. In conclusion, NAA foliar spraying increased endogenous IAA content, and enhanced the activity of N assimilation-related enzymes and photosynthesis rate, in order to build a large sink for carbohydrate accumulation. In addition, NAA strengthened root activity and regulated root morphology and architecture, which facilitated further N uptake and plant growth.

Engineering Halomonas bluephagenesis for Synthesis of Polyhydroxybutyrate (PHB) in the Presence of High Nitrogen Containing Media.

The trade-offs exist between microbial growth and bioproduct synthesis including intracellular polyester polyhydroxybutyrate (PHB). Under nitrogen limitation, more carbon flux is directed to PHB synthesis while growth is inhibited with diminishing overall carbon utilization, similar to the suboptimal carbon utilization during glycolysis-derived pyruvate decarboxylation. This study reconfigured the central carbon network of Halomonas bluphagenesis to improve PHB yield theoretically and practically. It was found that the downregulation of glutamine synthetase (GS) activity led to a synchronous improvement on PHB accumulation and cell growth under nitrogen non-limitation condition, increasing the PHB yield from glucose (g/g) to 85% of theoretical yield, PHB titer from 7.6 g/L to 12.9 g/L, and from 51 g/L to 65 g/L when grown in shake flasks containing a rich N-source, and grown in a fed-batch cultivation conducted in a 7-L bioreactor also containing a rich N-source, respectively. Results offer better metabolic balance between glucose conversion efficiency and microbial growth for economic PHB production.

Unraveling the genetic potential of nitrous oxide reduction in wastewater treatment: insights from metagenome-assembled genomes.

This study explores the genetic landscape of nitrous oxide (N2O) reduction in wastewater treatment plants (WWTPs) by profiling 1,083 high-quality metagenome-assembled genomes (HQ MAGs) from 23 Danish full-scale WWTPs. The focus is on the distribution and diversity of nitrous oxide reductase (nosZ) genes and their association with other nitrogen metabolism pathways. A custom pipeline for clade-specific nosZ gene identification with higher sensitivity revealed 503 nosZ sequences in 489 of these HQ MAGs, outperforming existing Kyoto Encyclopedia of Genes and Genomes (KEGG) module-based methods. Notably, 48.7% of the total 1,083 HQ MAGs harbored nosZ genes, with clade II being predominant, accounting for 93.7% of these genes. Taxonomic profiling highlighted the prevalence of nosZ-containing taxa within Bacteroidota and Pseudomonadota. Chloroflexota exhibited unexpected affiliations with both the sec and tat secretory pathways, and all were found to contain the accessory nosB gene, underscoring the importance of investigating the secretory pathway. The majority of non-denitrifying N2O reducers were found within Bacteroidota and Chloroflexota. Additionally, HQ MAGs with genes for dissimilatory nitrate reduction to ammonium and assimilatory nitrate reduction frequently co-occurred with the nosZ gene. Traditional primers targeting nosZ often focus on short-length amplicons. Therefore, we introduced custom-designed primer sets targeting near-full-length nosZ sequences. These new primers demonstrate efficacy in capturing diverse and well-characterized sequences, providing a valuable tool with higher resolution for future research. In conclusion, this comprehensive analysis enhances our understanding of N2O-reducing organisms in WWTPs, highlighting their potential as N2O sinks with the potential for optimizing wastewater treatment processes and mitigating greenhouse gas emissions. IMPORTANCE: This study provides critical insights into the genetic diversity of nitrous oxide reductase (nosZ) genes and the microorganisms harboring them in wastewater treatment plants (WWTPs) by exploring 1,083 high-quality metagenome-assembled genomes (MAGs) from 23 Danish full-scale WWTPs. Despite the pivotal role of nosZ-containing organisms, their diversity remains largely unexplored in WWTPs. Our custom pipeline for detecting nosZ provides near-full-length genes with detailed information on secretory pathways and accessory nos genes. Using these genes as templates, we developed taxonomically diverse clade-specific primers that generate nosZ amplicons for phylogenetic annotation and gene-to-MAG linkage. This approach improves detection and expands the discovery of novel sequences, highlighting the prevalence of non-denitrifying N2O reducers and their potential as N2O sinks. These findings have the potential to optimize nitrogen removal processes and mitigate greenhouse gas emissions from WWTPs by fully harnessing the capabilities of the microbial communities.

One-shot 13 C15 N-metabolic flux analysis for simultaneous quantification of carbon and nitrogen flux.

Metabolic flux is the final output of cellular regulation and has been extensively studied for carbon but much less is known about nitrogen, which is another important building block for living organisms. For the tuberculosis pathogen, this is particularly important in informing the development of effective drugs targeting the pathogen's metabolism. Here we performed 13 C15 N dual isotopic labeling of Mycobacterium bovis BCG steady state cultures, quantified intracellular carbon and nitrogen fluxes and inferred reaction bidirectionalities. This was achieved by model scope extension and refinement, implemented in a multi-atom transition model, within the statistical framework of Bayesian model averaging (BMA). Using BMA-based 13 C15 N-metabolic flux analysis, we jointly resolve carbon and nitrogen fluxes quantitatively. We provide the first nitrogen flux distributions for amino acid and nucleotide biosynthesis in mycobacteria and establish glutamate as the central node for nitrogen metabolism. We improved resolution of the notoriously elusive anaplerotic node in central carbon metabolism and revealed possible operation modes. Our study provides a powerful and statistically rigorous platform to simultaneously infer carbon and nitrogen metabolism in any biological system.

One-shot 13C15N-metabolic flux analysis for simultaneous quantification of carbon and nitrogen flux.

Metabolic flux is the final output of cellular regulation and has been extensively studied for carbon but much less is known about nitrogen, which is another important building block for living organisms. For the tuberculosis pathogen, this is particularly important in informing the development of effective drugs targeting the pathogen's metabolism. Here we performed 13C15N dual isotopic labeling of Mycobacterium bovis BCG steady state cultures, quantified intracellular carbon and nitrogen fluxes and inferred reaction bidirectionalities. This was achieved by model scope extension and refinement, implemented in a multi-atom transition model, within the statistical framework of Bayesian model averaging (BMA). Using BMA-based 13C15N-metabolic flux analysis, we jointly resolve carbon and nitrogen fluxes quantitatively. We provide the first nitrogen flux distributions for amino acid and nucleotide biosynthesis in mycobacteria and establish glutamate as the central node for nitrogen metabolism. We improved resolution of the notoriously elusive anaplerotic node in central carbon metabolism and revealed possible operation modes. Our study provides a powerful and statistically rigorous platform to simultaneously infer carbon and nitrogen metabolism in any biological system.

Non-foliar photosynthesis and nitrogen assimilation influence grain yield in durum wheat regardless of water conditions.

There is a need to generate improved crop varieties adapted to the ongoing changes in the climate. We studied durum wheat canopy and central metabolism of six different photosynthetic organs in two yield-contrasting varieties. The aim was to understand the mechanisms associated with the water stress response and yield performance. Water stress strongly reduced grain yield, plant biomass, and leaf photosynthesis, and down-regulated C/N-metabolism genes and key protein levels, which occurred mainly in leaf blades. By contrast, higher yield was associated with high ear dry weight and lower biomass and ears per area, highlighting the advantage of reduced tillering and the consequent improvement in sink strength, which promoted C/N metabolism at the whole plant level. An improved C metabolism in blades and ear bracts and N assimilation in all photosynthetic organs facilitated C/N remobilization to the grain and promoted yield. Therefore, we propose that further yield gains in Mediterranean conditions could be achieved by considering the source-sink dynamics and the contribution of non-foliar organs, and particularly N assimilation and remobilization during the late growth stages. We highlight the power of linking phenotyping with plant metabolism to identify novel traits at the whole plant level to support breeding programmes.

Overexpressing GLUTAMINE SYNTHETASE 1;2 maintains carbon and nitrogen balance under high-ammonium conditions and results in increased tolerance to ammonium toxicity in hybrid poplar.

The glutamine synthetase/glutamic acid synthetase (GS/GOGAT) cycle plays important roles in N metabolism, growth, development, and stress resistance in plants. Excess ammonium (NH4+) restricts growth, but GS can help to alleviate its toxicity. In this study, the 84K model clone of hybrid poplar (Populus alba × P. tremula var. glandulosa), which has reduced biomass accumulation and leaf chlorosis under high-NH4+ stress, showed less severe symptoms in transgenic lines overexpressing GLUTAMINE SYNTHETASE 1;2 (GS1;2-OE), and more severe symptoms in RNAi lines (GS1;2-RNAi). Compared with the wild type, the GS1;2-OE lines had increased GS and GOGAT activities and higher contents of free amino acids, soluble proteins, total N, and chlorophyll under high-NH4+ stress, whilst the antioxidant and NH4+ assimilation capacities of the GS1;2-RNAi lines were decreased. The total C content and C/N ratio in roots and leaves of the overexpression lines were higher under stress, and there were increased contents of various amino acids and sugar alcohols, and reduced contents of carbohydrates in the roots. Under high-NH4+ stress, genes related to amino acid biosynthesis, sucrose and starch degradation, galactose metabolism, and the antioxidant system were significantly up-regulated in the roots of the overexpression lines. Thus, overexpression of GS1;2 affected the carbon and amino acid metabolism pathways under high-NH4+ stress to help maintain the balance between C and N metabolism and alleviate the symptoms of toxicity. Modification of the GS/GOGAT cycle by genetic engineering is therefore a potential strategy for improving the NH4+ tolerance of cultivated trees.

Effect of nitrogen reduction by chemical fertilization with green manure (Vicia sativa L.) on soil microbial community, nitrogen metabolism and and yield of Uncaria rhynchophylla by metagenomics.

Uncaria rhynchophylla is an important herbal medicine, and the predominant issues affecting its cultivation include a single method of fertilizer application and inappropriate chemical fertilizer application. To reduce the use of inorganic nitrogen fertilization and increase the yield of Uncaria rhynchophylla, field experiments in 2020-2021 were conducted. The experimental treatments included the following categories: S1, no fertilization; S2, application of chemical NPK fertilizer; and S3-S6, application of chemical fertilizers and green manures, featuring nitrogen fertilizers reductions of 0%, 15%, 30%, and 45%, respectively. The results showed that a moderate application of nitrogen fertilizer when combined with green manure, can help alleviate soil acidification and increase urease activity. Specifically, the treatment with green manure provided in a 14.71-66.67% increase in urease activity compared to S2. Metagenomics sequencing results showed a decrease in diversity in S3, S4, S5, and S6 compared to S2, but the application of chemical fertilizer with green manure promoted an increase in the relative abundance of Acidobacteria and Chloroflexi. In addition, the nitrification pathway displayed a progressive augmentation in tandem with the reduction in nitrogen fertilizer and application of green manure, reaching its zenith at S5. Conversely, other nitrogen metabolism pathways showed a decline in correlation with diminishing nitrogen fertilizer dosages. The rest of the treatments showed an increase in yield in comparison to S1, S5 showing significant differences (p < 0.05). In summary, although S2 demonstrate the ability to enhance soil microbial diversity, it is important to consider the long-term ecological impacts, and S5 may be a better choice.

The role of NtrC in the adaptation of Herbaspirillum seropedicae SmR1 to nitrogen limitation and to nitrate.

The RNA-Seq profiling of Herbaspirillum seropedicae SmR1 wild-type and ntrC mutant was performed under aerobic and three nitrogen conditions (ammonium limitation, ammonium shock, and nitrate shock) to identify the major metabolic pathways modulated by these nitrogen sources and those dependent on NtrC. Under ammonium limitation, H. seropedicae scavenges nitrogen compounds by activating transporter systems and metabolic pathways to utilize different nitrogen sources and by increasing proteolysis, along with genes involved in carbon storage, cell protection, and redox balance, while downregulating those involved in energy metabolism and protein synthesis. Growth on nitrate depends on the narKnirBDHsero_2899nasA operon responding to nitrate and NtrC. Ammonium shock resulted in a higher number of genes differently expressed when compared to nitrate. Our results showed that NtrC activates a network of transcriptional regulators to prepare the cell for nitrogen starvation, and also synchronizes nitrogen metabolism with carbon and redox balance pathways.