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.

Molecular and physiological mechanisms of tea (Camellia sinensis (L.) O. Kuntze) leaf and root in response to nitrogen deficiency.

BACKGROUND: As an economically important crop, tea is strongly nitrogen (N)-dependent. However, the physiological and molecular mechanisms underlying the response of N deficiency in tea are not fully understood. Tea cultivar "Chunlv2" [Camellia sinensis (L.) O. Kuntze] were cultured with a nutrient solution with 0 mM [N-deficiency] or 3 mM (Control) NH4NO3 in 6 L pottery pots containing clean river sands. RESULTS: N deficiency significantly decreased N content, dry weight, chlorophyll (Chl) content, L-theanine and the activities of N metabolism-related enzymes, but increased the content of total flavonoids and polyphenols in tea leaves. N deficiency delayed the sprouting time of tea buds. By using the RNA-seq technique and subsequent bioinformatics analysis, 3050 up-regulated and 2688 down-regulated differentially expressed genes (DEGs) were isolated in tea leaves in response to N deficiency. However, only 1025 genes were up-regulated and 744 down-regulated in roots. Gene ontology (GO) term enrichment analysis showed that 205 DEGs in tea leaves were enriched in seven GO terms and 152 DEGs in tea roots were enriched in 11 GO items based on P < 0.05. In tea leaves, most GO-enriched DEGs were involved in chlorophyll a/b binding activities, photosynthetic performance, and transport activities. But most of the DEGs in tea roots were involved in the metabolism of carbohydrates and plant hormones with regard to the GO terms of biological processes. N deficiency significantly increased the expression level of phosphate transporter genes, which indicated that N deficiency might impair phosphorus metabolism in tea leaves. Furthermore, some DEGs, such as probable anion transporter 3 and high-affinity nitrate transporter 2.7, might be of great potential in improving the tolerance of N deficiency in tea plants and further study could work on this area in the future. CONCLUSIONS: Our results indicated N deficiency inhibited the growth of tea plant, which might be due to altered N metabolism and expression levels of DEGs involved in the photosynthetic performance, transport activity and oxidation-reduction processes.

Exogenous Melatonin Modulates Physiological Response to Nitrogen and Improves Yield in Nitrogen-Deficient Soybean (Glycine max L. Merr.).

Melatonin (MT) is a key plant growth regulator. To investigate its effect at different growth stages on the yield of soybean under nitrogen deficiency, 100 µM MT was applied to soybean supplemented with zero nitrogen (0N), low nitrogen (LN), and control nitrogen (CK) levels, during the plant vegetative growth (V3) and filling (R5) stages. This study revealed that the application of MT mainly enhanced the nitrogen fixation of plants by increasing the root nodule number and provided more substrates for glutamine synthetase (GS) under 0N supply. However, under the LN supply, more ammonium was assimilated through the direct promotion of nitrate reductase (NR) activity by MT. MT enhanced the activity of ammonium-assimilation-related enzymes, such as GOGAT and GDH, and the expression of their coding genes, promoted the synthesis of chlorophyll and amino acids, and increased the photosynthetic capacity under nitrogen deficiency. Exogenous MT directly upregulated the expression of genes involved in the photosynthetic system and stimulated dry-matter accumulation. Thus, MT alleviated the inhibitory effect of nitrogen deficiency on soybean yield. This mitigation effect was better when MT was applied at the V3 stage, and the seed weight per plant increased by 16.69 and 12.20% at 0N and LN levels, respectively. The results of this study provide a new theoretical basis to apply MT in agriculture to improve the resilience of soybean plants to low nitrogen availability.

The GC-TOF/MS-based Metabolomic analysis reveals altered metabolic profiles in nitrogen-deficient leaves and roots of tea plants (Camellia sinensis).

BACKGROUND: Nitrogen (N) fertilizer is commonly considered as one of the most important limiting factors in the agricultural production. As a result, a large amount of N fertilizer is used to improve the yield in modern tea production. Unfortunately, the large amount of N fertilizer input has led to increased plant nitrogen-tolerance and decreased amplitude of yield improvement, which results in significant N loss, energy waste and environment pollution. However, the effects of N-deficiency on the metabolic profiles of tea leaves and roots are not well understood. RESULTS: In this study, seedlings of Camellia sinensis (L.) O. Kuntze Chunlv 2 were treated with 3 mM NH4NO3 (Control) or without NH4NO3 (N-deficiency) for 4 months by sandy culture. The results suggested that N-deficiency induced tea leaf chlorosis, impaired biomass accumulation, decreased the leaf chlorophyll content and N absorption when they were compared to the Control samples. The untargeted metabolomics based on GC-TOF/MS approach revealed a discrimination of the metabolic profiles between N-deficient tea leaves and roots. The identification and classification of the altered metabolites indicated that N deficiency upregulated the relative abundances of most phenylpropanoids and organic acids, while downregulated the relative abundances of most amino acids in tea leaves. Differentially, N-deficiency induced the accumulation of most carbohydrates, organic acids and amino acids in tea roots. The potential biomarkers screened in N-deficient leaves compared to Control implied that N deficiency might reduce the tea quality. Unlike the N-deficient leaves, the potential biomarkers in N-deficient roots indicated an improved stress response might occur in tea roots. CONCLUSIONS: The results demonstrated N deficiency had different effects on the primary and secondary metabolism in tea leaves and roots. The findings of this study will facilitate a comprehensive understanding of the N-deficient tea plants and provide a valuable reference for the optimized N nutrient management and the sustainable development in the tea plantations.

Impacts of Nitrogen Deficiency on Wheat (Triticum aestivum L.) Grain During the Medium Filling Stage: Transcriptomic and Metabolomic Comparisons.

Nitrogen (N) supplementation is essential to the yield and quality of bread wheat (Triticum aestivum L.). The impact of N-deficiency on wheat at the seedling stage has been previously reported, but the impact of distinct N regimes applied at the seedling stage with continuous application on filling and maturing wheat grains is lesser known, despite the filling stage being critical for final grain yield and flour quality. Here, we compared phenotype characteristics such as grain yield, grain protein and sugar quality, plant growth, leaf photosynthesis of wheat under N-deficient and N-sufficient conditions imposed prior to sowing (120 kg/hm2) and in the jointing stage (120 kg/hm2), and then evaluated the effects of this continued stress through RNA-seq and GC-MS metabolomics profiling of grain at the mid-filling stage. The results showed that except for an increase in grain size and weight, and in the content of total sugar, starch, and fiber in bran fraction and white flour, the other metrics were all decreased under N-deficiency conditions. A total of 761 differentially expressed genes (DEGs) and 77 differentially accumulated metabolites (DAMs) were identified. Under N-deficiency, 51 down-regulated DEGs were involved in the process of impeding chlorophyll synthesis, chloroplast development, light harvesting, and electron transfer functions of photosystem, which resulted in the SPAD and Pn value decreased by 32 and 15.2% compared with N-sufficiency, inhibited photosynthesis. Twenty-four DEGs implicated the inhibition of amino acids synthesis and protein transport, in agreement with a 17-42% reduction in ornithine, cysteine, aspartate, and tyrosine from metabolome, and an 18.6% reduction in grain protein content. However, 14 DEGs were implicated in promoting sugar accumulation in the cell wall and another six DEGs also enhanced cell wall synthesis, which significantly increased fiber content in the endosperm and likely contributed to increasing the thousands-grain weight (TGW). Moreover, RNA-seq profiling suggested that wheat grain can improve the capacity of DNA repair, iron uptake, disease and abiotic stress resistance, and oxidative stress scavenging through increasing the content levels of anthocyanin, flavonoid, GABA, galactose, and glucose under N-deficiency condition. This study identified candidate genes and metabolites related to low N adaption and tolerance that may provide new insights into a comprehensive understanding of the genotype-specific differences in performance under N-deficiency conditions.

Nitrogen-Regulated Theanine and Flavonoid Biosynthesis in Tea Plant Roots: Protein-Level Regulation Revealed by Multiomics Analyses.

Theanine and flavonoids (especially proanthocyanidins) are the most important and abundant secondary metabolites synthesized in the roots of tea plants. Nitrogen promotes theanine and represses flavonoid biosynthesis in tea plant roots, but the underlying mechanism is still elusive. Here, we analyzed theanine and flavonoid metabolism in tea plant roots under nitrogen deficiency and explored the regulatory mechanism using proteome and ubiquitylome profiling together with transcriptome data. Differentially expressed proteins responsive to nitrogen deficiency were identified and found to be enriched in flavonoid, nitrogen, and amino acid metabolism pathways. The proteins responding to nitrogen deficiency at the transcriptional level, translational level, and both transcriptional and translational levels were classified. Nitrogen-deficiency-responsive and ubiquitinated proteins were further identified. Our results showed that most genes encoding enzymes in the theanine synthesis pathway, such as CsAlaDC, CsGDH, and CsGOGATs, were repressed by nitrogen deficiency at transcriptional and/or protein level(s). While a large number of enzymes in flavonoid metabolism were upregulated at the transcriptional and/or translational level(s). Importantly, the ubiquitylomic analysis identified important proteins, especially the hub enzymes in theanine and flavonoid biosynthesis, such as CsAlaDC, CsTSI, CsGS, CsPAL, and CsCHS, modified by ubiquitination. This study provided novel insights into the regulation of theanine and flavonoid biosynthesis and will contribute to future studies on the post-translational regulation of secondary metabolism in tea plants.

Nitrogen, phosphorus and high CO2 modulate photosynthesis, biomass and lipid production in the green alga Chlorella vulgaris.

Climate change could impact nutrient bioavailability in aquatic environment. To understand the interaction of nutrient bioavailability and elevated CO2, Chlorella vulgaris cells were grown in ambient air or 5% CO2 in different concentrations of nitrogen and phosphorus in a photobioreactor. The chlorophyll content, photosynthesis and respiration rates increased in 5% CO2 to support higher biomass production. The nutrient limitation in the growth media resulted in reduced photosynthetic rates of the algal cells and their PSI, PSII, and whole chain electron transport rates and biomass production. Conversely, their lipid content increased partly due to upregulation of expression of several lipid biosynthesis genes. The order of downregulation of photosynthesis and upregulation in lipid production due to nutrient limitation was in the order of N > P. The N-50 and 5% CO2 culture had only 10% reduction in biomass and 32% increase in lipids having 85% saturated fat required for efficient biofuel production. This growth condition is ideal for generation of biodiesel required to reduce the consumption of fossil fuel and combat global warming.

Cytosolic ascorbate peroxidase 1 modulates ascorbic acid metabolism through cooperating with nitrogen regulatory protein P-II in tea plant under nitrogen deficiency stress.

Nitrogen (N) element is essential nutrient, and affect metabolism of secondary metabolites in higher plants. Ascorbate peroxidase (APX) plays an important role in ascorbic acid (AsA) metabolism of tea plant. However, the roles of cytosolic ascorbate peroxidase 1 (CsAPX1) in AsA metabolism under N deficiency stress in tea plant remains unclear in detail. In this work, nitrogen regulatory protein P-II (CsGLB1) and CsAPX1 were identified by isobaric tags for relative and absolute quantitation (iTRAQ) from tea plant. The cell growth rates in transgenic Escherichia coli overexpressing CsAPX1 and CsGLB1 were higher than empty vector under N sufficiency condition. Phenotype of shoots and roots, AsA accumulation, and expression levels of AtAPX1 and AtGLB1 genes were changed in transgenic Arabidopsis hosting CsAPX1 under N deficiency stress. These findings suggested that cytosolic CsAPX1 acted a regulator in AsA accumulation through cooperating with GLB1 under N deficiency stress in tea plant.

High fatty acid productivity from Scenedesmus obliquus in heterotrophic cultivation with glucose and soybean processing wastewater via nitrogen and phosphorus regulation.

In this study, the effects of nitrogen and phosphorus supply on biodiesel production from Scenedesmus obliquus with glucose as the carbon source were investigated. It was found that sufficient phosphorus could further improve biodiesel production under nitrogen starvation. S. obliquus was cultivated in soybean processing wastewater. The removal efficiencies of carbon oxygen demand (COD), total nitrogen (TN), and total phosphorus (TP) after 8-day cultivation were 72%, 95%, and 54%, respectively. Moreover, the fatty acid productivity after eight-day cultivation reached as high as 99.3 mg·L-1·d-1, which was 1.15 times higher than the highest efficiency using a glucose culture. This result was due to two naturally-formed stages occurring with sufficient phosphorus: nitrogen sufficiency stage for biomass and nitrogen starvation stage for lipid accumulation. It verified the conclusion of the roles of nitrogen and phosphorus obtained in the glucose culture and provided an economic and environmentally friendly choice for biodiesel production with efficient soybean wastewater treatment.

Untargeted liquid chromatography coupled with mass spectrometry reveals metabolic changes in nitrogen-deficient Isatis indigotica Fortune.

Isatis indigotica Fortune is a popular herb in traditional Chinese medicine, and various types of metabolites are the basis for its pharmacological efficacy. The biosynthesis and accumulation of these metabolites are closely linked to nitrogen availability; the benefits of low nitrogen application on the environment and herb quality are increasingly prominent. To analyze metabolic changes in the leaves and roots of I.indigotica in nitrogen deficiency conditions, and to identify the pathways and metabolites induced by low nitrogen availability, we used untargeted liquid chromatography coupled with mass spectrometry (UHPLC-TripleTOF) to obtain metabolomics profiling of I.indigotica under two N-deficiency treatments (0 kg/hm2; 337.5 kg/hm2) and normal nitrogen treatment (675 kg/hm2). A total of 447 metabolites were annotated. Principal component analysis separated the three nitrogen treatments. A greater diversity of metabolites was observed in roots than in leaves under N-deficiency treatments, suggesting that roots have a more important function in low N tolerance. Differential metabolites were mainly enriched in purine metabolism, phenylpropanoid biosynthesis, the shikimate pathway, tryptophan metabolism, and flavonoid biosynthesis that notably induced only in leaves in low nitrogen stress. Moderate N-deficiency benefits carbohydrate accumulation, whereas accumulation of most amino acids decreases. Uniquely, L-tryptophan was maintained at a high concentration in N-deficiency conditions. Low nitrogen stress induced the accumulation of some specialized metabolites (matairesinol, dictamnine, 5-hydroxyindoleacetate (serotonin) in roots and vitexin, xanthohumol, sinapyl alcohol in leaves). N-deficiency also increased the accumulation of adenosine and quality indicators of I.indigotica (indirubin-indigo, epigoitrin and anthranilic acid) in a certain degree. Our findings showed that nitrogen deficiency modified roots and leaves conditions of I.indigotica, affecting both the primary and secondary metabolism. Moderate nitrogen reduction was beneficial to the accumulation of active ingredients. Our methods and analysis are expected to provide an insight regarding the diversity of metabolites and regulation of their synthesis in low nitrogen application, and better investigate the nitrogen deficiency effect on I.indigotica.

An enzymatic mechanism for balancing the stoichiometry of nitrogen and phosphorus in a shallow Chinese eutrophic lake.

The over-enrichment of lake waters with nitrogen (N) and phosphorus (P) has become a serious environmental problem, but modes of change in stoichiometry and enzymatic regeneration along trophic gradients are largely unknown. Seasonal variations in the kinetics of extracellular aminopeptidase (LAP) and alkaline phosphatase (AP), together with the composition of phytoplankton and concentrations of N and P, were examined from Jun 2013 to September 2014 in a Chinese shallow lake in which two basins had contrasting trophic states. The turbid basin had a significantly higher concentration of chlorophyll a and lower ratios of N to P. In parallel, the turnover time of organic N mediated by LAP (LAPT) was significantly shorter, and its maximum velocity (Vmax) was significantly higher compared to those in the clear basin. Considering data from both basins, there were linear decreases in N/P and the ratios between dissolved inorganic N and total N with an increasing trophic state index, coupled with a significantly positive relationship between N/P and LAPT. Additionally, with decreasing TN/TP, the Michaelis constant (Km) of the AP increased linearly, reducing the efficiency of P regeneration. In contrast, the Km value of LAP decreased, and Vmax increased, which enhanced N mineralization by simultaneously increasing the reaction velocity and improving the affinity for substrate. Additionally, the Km value of LAP was significantly related to that of AP and the ammonium concentration. Thus, substrate affinity acted as a key factor modifying the pathways of enzymatic degradation of organic N and P according to their stoichiometry in the water column. Phytoplankton composition was directly linked to LAPT. Overall, this study seemed to be the first to connect a stoichiometric shift of N and P with kinetics of extracellular enzymes responsible for their regeneration along trophic gradients, presenting an additional pathway to overcome nitrogen deficiency in eutrophic lakes.

iTRAQ-based quantitative proteomic analysis gives insight into sexually different metabolic processes of poplars under nitrogen and phosphorus deficiencies.

Male and female poplars (Populus cathayana Rehd.) respond differently to nitrogen (N) and phosphorus (P) deficiencies. In this study, an iTRAQ-based quantitative proteomic analysis was performed. N and P deficiencies caused 189 and 144 proteins to change in abundance in males and 244 and 464 in females, respectively. Compared to N- and P-deficient males, both N- and P-deficient females showed a wider range of changes in proteins that are involved in amino acid, carbohydrate and protein metabolism, and the sexual differences were significant. When comparing the effects of N- and P-deficiencies, N-deficient females expressed more changes in proteins that are involved in stress responses and gene expression regulation, while P-deficient females showed more changes in proteins that are involved in energy and lipid metabolism, stress responses and gene expression regulation. The quantitative RT-PCR analysis of stress-related proteins showed that males have a better expression correlation between mRNA and protein levels than do females. This study shows that P. cathayana females are more sensitive and have more rapid metabolic mechanisms when responding to N and P deficiencies than do males, and P deficiency has a wider range of effects on females than does N deficiency.

Saline wastewater treatment by Chlorella vulgaris with simultaneous algal lipid accumulation triggered by nitrate deficiency.

Chlorella vulgaris, a marine microalgae strain adaptable to 0-50 g L(-1) of salinity, was selected for studying the coupling system of saline wastewater treatment and lipid accumulation. The effect of total nitrogen (T N) concentration was investigated on algal growth, nutrients removal as well as lipid accumulation. The removal efficiencies of TN and total phosphorus (TP) were found to be 92.2-96.6% and over 99%, respectively, after a batch cultivation of 20 days. To illustrate the response of lipid accumulation to nutrients removal, C. vulgaris was further cultivated in the recycling experiment of tidal saline water within the photobioreactor. The lipid accumulation was triggered upon the almost depletion of nitrate (<5 mg L(-1)), till the final highest lipid content of 40%. The nitrogen conversion in the sequence of nitrate, nitrite, and then to ammonium in the effluents was finally integrated with previous discussions on metabolic pathways of algal cell under nitrogen deficiency.

Influence of the N:P supply ratio on biomass productivity and time-resolved changes in elemental and bulk biochemical composition of Nannochloropsis sp.

This work reports for the first time the detailed impacts of dual nitrogen (N) and phosphorus (P) stress on growth dynamics and biochemical composition in the Eustigmatophyte Nannochloropsis sp. P-stress concurrent with N-stress had subtle effects on culture bulk biochemical composition, but negatively influenced biomass productivity. However, the N:P supply ratio can be raised to at least 32:1 without compromising productivity (yielding a maximum lipid content of 52% of dry weight and volumetric lipid concentration of 233 mg L(-1)). The maximum biomass and lipid yields per unit of cell-P were 1.2 kg DW (gP)(-1) and 0.54 kg lipid (gP)(-1). The P concentration of many common media is thus in surplus for optimal Nannochloropsis sp. biomass and lipid production, offering potential for significant savings in P usage and improving the sustainability of algal cultivation.

Induction of phenolic metabolites and physiological changes in chamomile plants in relation to nitrogen nutrition.

Alternative tools, such as the manipulation of mineral nutrition, may affect secondary metabolite production and thus the nutritional value of food/medicinal plants. We studied the impact of nitrogen (N) nutrition (nitrate/NO3(-) or ammonium/NH4(+) nitrogen) and subsequent nitrogen deficit on phenolic metabolites and physiology in Matricaria chamomilla plants. NH4(+)-fed plants revealed a strong induction of selected phenolic metabolites but, at the same time, growth, Fv/Fm, tissue water content and soluble protein depletion occurred in comparison with NO3(-)-fed ones. On the other hand, NO3(-)-deficient plants also revealed an increase in phenolic metabolites but growth depression was not observed after the given exposure period. Free amino acids were more accumulated in NH4(+)-fed shoots (strong increase in arginine and proline mainly), while the pattern of roots' accumulation was independent of N form. Among phenolic acids, NH4(+) strongly elevated mainly the accumulation of chlorogenic acid. Within flavonoids, flavonols decreased while flavones strongly increased in response to N deficiency. Coumarin-related metabolites revealed a similar increase in herniarin glucosidic precursor in response to N deficiency, while herniarin was more accumulated in NO3(-)- and umbelliferone in NH4(+)-cultured plants. These data indicate a negative impact of NH4(+) as the only source of N on physiology, but also a higher stimulation of some valuable phenols. Nitrogen-induced changes in comparison with other food/crop plants are discussed.

Effect of phosphorus on biodiesel production from Scenedesmus obliquus under nitrogen-deficiency stress.

In order to study the effect of phosphorus on biodiesel production from Scenedesmus obliquus especially under nitrogen deficiency conditions, six types of media with combinations of nitrogen repletion/depletion and phosphorus repletion/limitation/depletion were investigated in this study. It was found that nitrogen starvation compared to nitrogen repletion enhanced biodiesel productivity. Moreover, biodiesel productivity was further strengthened by varying the supply level of phosphorus from depletion, limitation, through to repletion. The maximum FAMEs productivity of 24.2 mg/L/day was obtained in nitrogen depletion with phosphorus repletion, which was two times higher than that in nutrient complete medium. More phosphorus was accumulated in cells under the nitrogen starvation with sufficient phosphorus condition, but no polyphosphate was formed. This study indicated that nitrogen starvation plus sufficient P supply might be the real "lipid trigger". Furthermore, results of the current study suggest a potential application for utilizing microalgae to combine phosphorus removal from wastewater with biodiesel production.