Nitrogen Acquisition Strategies Mediated by Insect Symbionts: A Review of Their Mechanisms, Methodologies, and Case Studies

Nitrogen is usually a restrictive nutrient that affects the growth and development of insects, especially of those living in low nitrogen nutrient niches. In response to the low nitrogen stress, insects have gradually developed symbiont-based stress response strategies—biological nitrogen fixation and nitrogenous waste recycling—to optimize dietary nitrogen intake. Based on the above two patterns, atmospheric nitrogen or nitrogenous waste (e.g., uric acid, urea) is converted into ammonia, which in turn is incorporated into the organism via the glutamine synthetase and glutamate synthase pathways. This review summarized the reaction mechanisms, conventional research methods and the various applications of biological nitrogen fixation and nitrogenous waste recycling strategies. Further, we compared the bio-reaction characteristics and conditions of two strategies, then proposed a model for nitrogen provisioning based on different strategies.

A Mathematical Model for Characterizing the Biomass and the Physiological/Biochemical Indicators of Salvia miltiorrhiza Based on Growth-Defense Tradeoff

Carbon(C) and nitrogen(N) metabolisms are important for plant growth and defense, and enzymes play a major role in these two metabolisms. Current studies show that the enzymes of N Metabolism, C Metabolism, and defense are correlated with biomass. Then, we conducted this research under the assumption that enzymes could characterize the relationship based on growth-defense tradeoff, and some of the enzymes could be used to represent the plant growth. From the mechanism model, we picked out 18 physiological/biochemical indicators and obtained the data from 24 tissue culture seedlings of Salvia miltiorrhiza (S.miltiorrhiza) which were grafted with 11 endophytic fungi. Then, the relationship between the biomass and the physiological/biochemical indicators was investigated by using statistical analysis, such as correlation analysis, variable screening, and regression analysis. The results showed that many physiological/biochemical indicators, especially enzyme activities, were related to biomass accumulation. Through a rigorous logical reasoning process, we established a mathematical model of the biomass and 6 key physiological/biochemical indicators, including glutamine synthetase (GS), glutamate synthase (GLS), glutamate dehydrogenase (GDH), peroxidase (POD), catalase (CAT), and soluble protein from Cobb-Douglas production function. This model had high prediction accuracy, and it could simplify the measurement of biomass. During the artificial cultivation of S.miltiorrhiza, we can monitor the biomass accumulation by scaling the key physiological/biochemical indicators in the leaves. Interestingly, the coefficients of Lasso regression during our analysis were consistent with the mechanism of growth-defense tradeoff. Perhaps, the key physiological/biochemical indicators obtained in the statistical analysis are related to the indicators affecting biomass accumulation in practice.

Comparative analysis of Lysine and Arginine biosynthesis pathway in Deinococcus genomes

The members of the Deinococcaceae family have the ability to survive extreme environmental conditions. Deinococcus species have a complex cell envelope composed of L-ornithine containing peptidoglycan. Anabolism of L-ornithine is intrinsically linked to L-lysine and L-arginine biosynthetic pathways. To understand these two pathways, we analyzed the L-lysine and L-arginine pathways using 23 Deinococcus genomes, including D. indicus. We used BLAST-P based ortholog identification using D. radiodurans genes as the query. We identified some BLAST-P hits that shared the same functional annotation. We analyzed three (class I aminotransferase, acetyl-lysine deacetylase, and acetyl glutamate/acetyl aminoadipate kinase) from L-lysine biosynthesis pathway and three (bifunctional ornithine acetyltransferase or N-acetyl glutamate synthase protein, nitric oxide synthase-like protein, and Acetyl-lysine deacetylase) from L-arginine biosynthesis pathway. Two proteins showed certain structural variations. Specifically, [LysW]-lysine hydrolase protein sequence and structure level changes indicated changes in oligomeric conformation, which could likely be a result of divergent evolution. And, bifunctional ornithine acetyltransferase or N-acetyl glutamate synthase had its active site pocket positions shifted at the structural level and we hypothesize that it may not perform at the optimal level. Thus, we were able to compare and contrast different Deinococcus species indicating some genes occurring because of divergent evolution.

Effects of Se Application on Polyamines and Carbon–Nitrogen Metabolism of Pepper Plants Suffering from Cd Toxicity

Previous studies have shown that the application of selenium (Se) can efficiently mitigate the toxic effects of cadmium (Cd) on various crops. The objective of the present work is to decipher the mechanisms responsible for the efficiency of Se against the effects of Cd in pepper plants, with respect to the carbon and nitrogen metabolism. The following were analyzed: the concentrations of anions related with this metabolism, such as nitrates, nitrites, and ammonium, the activities of different enzymes such as nitrate reductase, nitrite reductase, and glutamate synthase, polyamines in their different forms, organic acid salts, amino acids, and sugars in the leaf and root tissues of the pepper plants grown in a hydroponics system. Four different treatments were applied: plants without Cd or Se applied (−Cd/−Se); plants grown with Cd added to the nutrient solution (NS) but without Se (+Cd/−Se); plants grown with Cd in the NS, and with the foliar application of Se (+CD/+SeF); and lastly, plants grown with Cd in the NS, and with Se applied to the root (+Cd/+SeR). The metabolites and enzymes related with carbon and nitrogen metabolism were analyzed 15 days after the application. The results showed the superiority of the +Cd/+SeR treatment with respect to the +Cd/+SeF treatment, as shown by an increase in the conjugated polyamines, the decrease in glutamate and phenylalanine, and the increase of malate and chlorogenic acid. The results indicated that SeR decreased the accumulation and toxicity of Se as polyamine homeostasis improved, defense mechanisms such as the phenylpropanoid increased, and the entry of Cd into the plants was blocked.

A glutamate synthase mutant of Bradyrhizobium sp. strain ORS285 is unable to induce nodules on Nod factor-independent Aeschynomene species

The Bradyrhizobium sp. strain ORS285 is able to establish a nitrogen-fixing symbiosis with both Nod factor (NF) dependent and NF-independent Aeschynomene species. Here, we have studied the growth characteristics and symbiotic interaction of a glutamate synthase (GOGAT; gltD::Tn5) mutant of Bradyrhizobium ORS285. We show that the ORS285 gltD::Tn5 mutant is unable to use ammonium, nitrate and many amino acids as nitrogen source for growth and is unable to fix nitrogen under free-living conditions. Moreover, on several nitrogen sources, the growth rate of the gltB::Tn5 mutant was faster and/or the production of the carotenoid spirilloxanthin was much higher as compared to the wild-type strain. The absence of GOGAT activity has a drastic impact on the symbiotic interaction with NF-independent Aeschynomene species. With these species, inoculation with the ORS285 gltD::Tn5 mutant does not result in the formation of nodules. In contrast, the ORS285 gltD::Tn5 mutant is capable to induce nodules on NF-dependent Aeschynomene species, but these nodules were ineffective for nitrogen fixation. Interestingly, in NF-dependent and NF-independent Aeschynomene species inoculation with the ORS285 gltD::Tn5 mutant results in browning of the plant tissue at the site of the infection suggesting that the mutant bacteria induce plant defence responses.

Regulation of Nitrate (NO3) Transporters and Glutamate Synthase-Encoding Genes under Drought Stress in Arabidopsis: The Regulatory Role of AtbZIP62 Transcription Factor

Nitrogen (N) is an essential macronutrient, which contributes substantially to the growth and development of plants. In the soil, nitrate (NO3) is the predominant form of N available to the plant and its acquisition by the plant involves several NO3 transporters; however, the mechanism underlying their involvement in the adaptive response under abiotic stress is poorly understood. Initially, we performed an in silico analysis to identify potential binding sites for the basic leucine zipper 62 transcription factor (AtbZIP62 TF) in the promoter of the target genes, and constructed their protein–protein interaction networks. Rather than AtbZIP62, results revealed the presence of cis-regulatory elements specific to two other bZIP TFs, AtbZIP18 and 69. A recent report showed that AtbZIP62 TF negatively regulated AtbZIP18 and AtbZIP69. Therefore, we investigated the transcriptional regulation of AtNPF6.2/NRT1.4 (low-affinity NO3 transporter), AtNPF6.3/NRT1.1 (dual-affinity NO3 transporter), AtNRT2.1 and AtNRT2.2 (high-affinity NO3 transporters), and AtGLU1 and AtGLU2 (both encoding glutamate synthase) in response to drought stress in Col-0. From the perspective of exploring the transcriptional interplay of the target genes with AtbZIP62 TF, we measured their expression by qPCR in the atbzip62 (lacking the AtbZIP62 gene) under the same conditions. Our recent study revealed that AtbZIP62 TF positively regulates the expression of AtPYD1 (Pyrimidine 1, a key gene of the de novo pyrimidine biosynthesis pathway know to share a common substrate with the N metabolic pathway). For this reason, we included the atpyd1-2 mutant in the study. Our findings revealed that the expression of AtNPF6.2/NRT1.4, AtNPF6.3/NRT1.1 and AtNRT2.2 was similarly regulated in atzbip62 and atpyd1-2 but differentially regulated between the mutant lines and Col-0. Meanwhile, the expression pattern of AtNRT2.1 in atbzip62 was similar to that observed in Col-0 but was suppressed in atpyd1-2. The breakthrough is that AtNRT2.2 had the highest expression level in Col-0, while being suppressed in atbzip62 and atpyd1-2. Furthermore, the transcript accumulation of AtGLU1 and AtGLU2 showed differential regulation patterns between Col-0 and atbzip62, and atpyd1-2. Therefore, results suggest that of all tested NO3 transporters, AtNRT2.2 is thought to play a preponderant role in contributing to NO3 transport events under the regulatory influence of AtbZIP62 TF in response to drought stress.

Transcriptomic and Biochemical Analysis Reveal Integrative Pathways Between Carbon and Nitrogen Metabolism in Guzmania monostachia (Bromeliaceae) Under Drought

Most epiphytes are found in low-nutrient environments with an intermittent water supply. To deal with water limitation, many bromeliads perform crassulacean acid metabolism (CAM), such as Guzmania monostachia, which shifts from C3 to CAM and can recycle CO2 from the respiration while stomata remain closed during daytime and nighttime (CAM-idling mode). Since the absorbing leaf trichomes can be in contact with organic (urea) and inorganic nutrients (NO3−, NH4+) and the urea hydrolysis releases NH4+ and CO2, we hypothesized that urea can integrate the N and C metabolism during periods of severe drought. Under this condition, NH4+ can be assimilated into amino acids through glutamine synthetase (GS), while the CO2 can be pre-fixated by phosphoenolpyruvate carboxylase (PEPC). In this context, we evaluated the foliar transcriptome of G. monostachia to compare the relative gene expression of some genes involved with CAM and the N metabolism when bromeliads were submitted to 7days of drought. We also conducted a controlled experiment with an extended water deficit period (21days) in which bromeliads were cultivated in different N sources (urea, NH4+, and NO3−). Our transcriptome results demonstrated an increment in the expression of genes related to CAM, particularly those involved in the carboxylation metabolism (PEPC1, PPCK, and NAD-MDH), the movement of malate through vacuolar membrane (ALMT9), and the decarboxylation process (PEPCK). Urea stimulated the expression of PEPC1 and ALMT9, while Urease transcripts increased under water deficit. Under this same condition, GS1 gene expression increased, indicating that the NH4+ from urea hydrolysis can be assimilated in the cytosol. We suggest that the link between C and N metabolism occurred through the supply of carbon skeleton (2-oxoglutarate, 2-OG) by the cytosolic isocitrate dehydrogenase since the number of NADP-ICDH transcripts was also higher under drought conditions. These findings indicate that while urea hydrolysis provides NH4+ that can be consumed by glutamine synthetase-cytosolic/glutamate synthase (GS1/GOGAT) cycle, the CO2 can be used by CAM, maintaining photosynthetic efficiency even when most stomata remain closed 24h (CAM-idling) as in the case of a severe water deficit condition. Thus, we suggest that urea could be used by G. monostachia as a strategy to increase its survival under drought, integrating N and C metabolism.

Drought, Salinity, and Low Nitrogen Differentially Affect the Growth and Nitrogen Metabolism of Sophora japonica (L.) in a Semi-Hydroponic Phenotyping Platform

Abiotic stresses, such as salinity, drought, and nutrient deficiency adversely affect nitrogen (N) uptake and assimilation in plants. However, the regulation of N metabolism and N pathway genes in Sophora japonica under abiotic stresses is unclear. Sophora japonica seedlings were subjected to drought (5% polyethylene glycol 6,000), salinity (75mM NaCl), or low N (0.01mM NH4NO3) for 3weeks in a semi-hydroponic phenotyping platform. Salinity and low N negatively affected plant growth, while drought promoted root growth and inhibited aboveground growth. The NH4+/NO3− ratio increased under all three treatments with the exception of a reduction in leaves under salinity. Drought significantly increased leaf NO2− concentrations. Nitrate reductase (NR) activity was unaltered or increased under stresses with the exception of a reduction in leaves under salinity. Drought enhanced ammonium assimilation with increased glutamate synthase (GOGAT) activity, although glutamine synthetase (GS) activity remained unchanged, whereas salinity and low N inhibited ammonium assimilation with decreased GS activity under salt stress and decreased GOGAT activity under low N treatment. Glutamate dehydrogenase (GDH) activity also changed dramatically under different stresses. Additionally, expression changes of genes involved in N reduction and assimilation were generally consistent with related enzyme activities. In roots, ammonium transporters, especially SjAMT1.1 and SjAMT2.1a, showed higher transcription under all three stresses; however, most nitrate transporters (NRTs) were upregulated under salinity but unchanged under drought. SjNRT2.4, SjNRT2.5, and SjNRT3.1 were highly induced by low N. These results indicate that N uptake and metabolism processes respond differently to drought, salinity, and low N conditions in S. japonica seedlings, possibly playing key roles in plant resistance to environmental stress.

RNA-Seq analysis and transcriptome assembly of Salicornia neei reveals a powerful system for ammonium detoxification

ABSTRACTBackgroundSalicornia neei is a halophyte plant that has been proposed for phytoremediation of saline wastewater generated by land-based aquaculture, which usually contains elevated concentrations of ammonium resulting from protein metabolism. To identify the molecular mechanisms related to ammonium response through of analysis results in silico and the Michaelis–Menten ammonium removal biokinetics and the transcriptome of S. neei in response to growth in saline water containing 3 mM ammonium.ResultsThe parameters for ammonium uptake by S. neei root cuttings were estimated: 1) maximum uptake rate Imax = 7.07 ± 0.27 mM N g−1 fresh weight h−1; and 2) half-saturation constant Km = 0.85 ± 0.12 mM N L−1. Further, a total of 45,327 genes were annotated, which represents 51.2% of the contig predicted from de novo assembly. A total of 9,140 genes were differentially expressed in response to ammonium in saline water, but only 7,396 could be annotated against functional databases. According to the GO enrichment and as well as KEGG pathway analyses showed these upregulated genes were involved in pr cellular anatomical entity, cellular process, and metabolic process, including biological KEGG pathways linked to biosynthesis amino acid biosynthesis, nitrogen metabolism and autophagy and other. In addiction, a set of 72 genes were directly involved in ammonium metabolism, including glutamine synthetase 1 (GLN1), glutamate synthase 1 (GLT1), and ferredoxin-dependent glutamate synthase chloroplastic (Fd-GOGAT).ConclusionOur results support the hypothesis that an ammonium detoxification system mediated by glutamine and glutamate synthase was activated in S. neei when exposed to ammonium and saline water. These results provide novel insight into understanding the molecular mechanisms of ammonium nutrition and aid for investigating the response of halophyte plants to saline wastewater from land-based aquaculture