Antibody toolkit to investigate eEF1A methylation dynamics in mRNA translation elongation.

Protein synthesis is a fundamental step in gene expression, with modulation of mRNA translation at the elongation step emerging as an important regulatory node in shaping cellular proteomes. In this context, five distinct lysine methylation events on eukaryotic elongation factor 1A (eEF1A), a fundamental nonribosomal elongation factor, are proposed to influence mRNA translation elongation dynamics. However, a lack of affinity tools has hindered progress in fully understanding how eEF1A lysine methylation impacts protein synthesis. Here we develop and characterize a suite of selective antibodies to investigate eEF1A methylation and provide evidence that methylation levels decline in aged tissue. Determination of the methyl state and stoichiometry on eEF1A in various cell lines by mass spectrometry shows modest cell-to-cell variability. We also find by Western blot analysis that knockdown of individual eEF1A-specific lysine methyltransferases leads to depletion of the cognate lysine methylation event and indicates active crosstalk between different sites. Further, we find that the antibodies are specific in immunohistochemistry applications. Finally, application of the antibody toolkit suggests that several eEF1A methylation events decrease in aged muscle tissue. Together, our study provides a roadmap for leveraging methyl state and sequence-selective antibody reagents to accelerate discovery of eEF1A methylation-related functions and suggests a role for eEF1A methylation, via protein synthesis regulation, in aging biology.

Genetic heterogeneity of engineered Escherichia coli Nissle 1917 strains during scale-up simulation.

Advanced microbiome therapeutics have emerged as a powerful approach for the treatment of numerous diseases. While the genetic instability of genetically engineered microorganisms is a well-known challenge in the scale-up of biomanufacturing processes, it has not yet been investigated for advanced microbiome therapeutics. Here, the evolution of engineered Escherichia coli Nissle 1917 strains producing Interleukin 2 and Aldafermin were investigated in two strain backgrounds with and without the three error-prone DNA polymerases polB, dinB, and umuDC, which contribute to the mutation rate of the host strain. Whole genome short-read sequencing revealed the genetic instability of the pMUT-based production plasmid after serial passaging for approximately 150 generations using an automated platform for high-throughput microbial evolution in five independent lineages for six distinct strains. While a reduction of the number of mutations of 12%-43% could be observed after the deletion of the error-prone DNA polymerases, the interruption of production-relevant genes could not be prevented, highlighting the need for additional strategies to improve the stability of advanced microbiome therapeutics.

Homozygosity for disease-causing variants in AMT and GLDC in a patient with severe nonketotic hyperglycinemia.

Nonketotic hyperglycinemia (NKH) is a relatively well-characterized inborn error of metabolism that results in a combination of lethargy, hypotonia, seizures, developmental arrest, and, in severe cases, death early in life. Three genes encoding components of the glycine cleavage enzyme system-GLDC, AMT, and GCSH-are independently associated with NKH. We report on a patient with severe NKH in whom the homozygous pathogenic variant in AMT (NM_000481.3):c.602_603del (p.Lys201Thrfs*75) and the homozygous likely pathogenic variant in GLDC(NM_000170.2):c.2852C>A (p.Ser951Tyr) were both identified. Our patient demonstrates a novel combination of two homozygous disease-causing variants impacting the glycine cleavage pathway at two different components, and elicits management- and genetic counseling-related challenges for the family.

Progress report on new medications for seizures and epilepsy: A summary of the 17th Eilat Conference on New Antiepileptic Drugs and Devices (EILAT XVII). I. Drugs in preclinical and early clinical development.

For >30 years, the Eilat Conference on New Antiepileptic Drugs and Devices has provided a forum for the discussion of advances in the development of new therapies for seizures and epilepsy. The EILAT XVII conference took place in Madrid, Spain, on May 5-8, 2024. Participants included basic scientists and clinical investigators from industry and academia, other health care professionals, and representatives from lay organizations. We summarize in this article information on treatments in preclinical and in early clinical development discussed at the conference. These include AMT-260, a gene therapy designed to downregulate the expression of Glu2K subunits of kainate receptors, in development for the treatment of drug-resistant seizures associated with mesial temporal sclerosis; BHV-7000, a selective activator of heteromeric Kv7.2/7.3 potassium channels, in development for the treatment of focal epilepsy; ETX101, a recombinant adeno-associated virus serotype 9 designed to increase NaV1.1 channel density in inhibitory γ-aminobutyric acidergic (GABAergic) neurons, in development for the treatment of SCN1A-positive Dravet syndrome; GAO-3-02, a compound structurally related to synaptamide, which exerts antiseizure activity at least in part through an action on cannabinoid type 2 receptors; LRP-661, a structural analogue of cannabidiol, in development for the treatment of seizures associated with Lennox-Gastaut syndrome, Dravet syndrome, and tuberous sclerosis complex; OV329, a selective inactivator of GABA aminotransferase, in development for the treatment of drug-resistant seizures; PRAX-628, a functionally selective potent sodium channel modulator with preference for the hyperexcitable state of sodium channels, in development for the treatment of focal seizures; RAP-219, a selective negative allosteric modulator of transmembrane α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor regulatory protein γ-8, in development for the treatment of focal seizures; and rozanolixizumab, a humanized anti-neonatal Fc receptor monoclonal antibody, in development for the treatment of LGI1 autoimmune encephalitis. Treatments in more advanced development are summarized in Part II of this report.

Powering prescription: Mitochondria as "Living Drugs" - Definition, clinical applications, and industry advancements.

Mitochondria's role as engines and beacons of metabolism and determinants of cellular health is being redefined through their therapeutic application as "Living Drugs" (LDs). Artificial mitochondrial transfer/transplant (AMT/T), encompassing various techniques to modify, enrich, or restore mitochondria in cells and tissues, is revolutionizing acellular therapies and the future of medicine. This article proposes a necessary definition for LDs within the Advanced Therapeutic Medicinal Products (ATMPs) framework. While recognizing different types of LDs as ATMPs, such as mesenchymal stem cells (MSCs) and chimeric antigen receptor T (CAR T) cells, we focus on mitochondria due to their unique attributes that distinguish them from traditional cell therapies. These attributes include their inherent living nature, diverse sources, industry applicability, validation, customizability for therapeutic needs, and their capability to adapt and respond within recipient cells. We trace the journey from initial breakthroughs in AMT/T to the current state-of-the-art applications by emerging innovative companies, highlighting the need for manufacturing standards to navigate the transition of mitochondrial therapies from concept to clinical practice. By providing a comprehensive overview of the scientific, clinical, and commercial landscape of mitochondria as LDs, this article contributes to the essential dialogue among regulatory agencies, academia, and industry to shape their future in medicine.

Exposure to four typical heavy metals induced telomere shortening of peripheral blood mononuclear cells in relevant with declined urinary aMT6s in rats.

Environmental heavy metals pollution have seriously threatened the health of human beings. An increasing number of researches have demonstrated that environmental heavy metals can influence the telomere length of Peripheral Blood Mononuclear Cells (PBMCs), which implicate biological aging as well as predicts diseases. Our previous study has shown that methylmercury (MeHg)-induced telomere shortening in rat brain tissue was associated with urinary melatonin metabolite 6-sulfatoxymelatonin (aMT6s) levels. Here, we aimed to further elucidate the impact of 4 typical heavy metals (As, Hg, Cd and Pb) on telomere length of PBMCs and their association with urinary aMT6s in rats. In this study, eighty-eight male Sprague-Dawley rats were randomized grouped into eleven groups. Among them, forty 3-month-old (young) and forty 12-month-old (middle-aged) rats were divided into young or middle-aged control groups as well as typical heavy metals exposed groups, respectively. Eight 24-month-old rats (old) was divided into aging control group. The results showed that MeHg exposure in young rats while sodium arsenite (iAs), MeHg, cadmium chloride (CdCl2), lead acetate (PbAc) exposure in middle-aged rats for 3 months significantly reduced the levels of and urinary aMT6s, as well as telomere length of PBMCs. In addition, they also induced abnormalities in serum oxidative stress (SOD, MDA and GPx) and inflammatory (IL-1ß, IL-6 and TNF-α) indicators. Notably, there was a significant positive correlation between declined level of urinary aMT6s and the shortening of telomere length in PBMCs in rats exposed to 4 typical heavy metals. These results suggested that 4 typical heavy metals exposure could accelerate the reduction of telomere length of PBMCs partially by inducing oxidative stress and inflammatory in rats, while ageing may be an important synergistic factor. Urinary aMT6s detection may be a alternative method to reflect telomere toxic effects induced by heavy metal exposure.

Autobiographical memory following weight gain in adult patients with Anorexia Nervosa: A longitudinal study.

BACKGROUND: Patients with anorexia nervosa (AN) show overgeneralization of memory (OGM) when generating autobiographical episodes related to food and body shape. These memories are central for the construction of a coherent self-concept, interpersonal relationships, and problem-solving abilities. The current study aims to investigate changes in autobiographical memory following weight gain. METHODS: OGM was assessed with an adapted version of the Autobiographical Memory Test including food-, body-, depression-related, and neutral cues. N = 41 female patients with AN (28 restricting-, 13 binge-eating/purging-subtype; mean disease duration: 4.5 years; mean BMI: 14.5 kg/m2) and N = 27 healthy controls (HC) were included at baseline. After inpatient treatment (mean duration: 11 weeks), 24 patients with AN and 24 age-matched HC were reassessed. Group differences were assessed using independent samples t-tests for cross-sectional comparisons and repeated measures ANOVAs for longitudinal data. RESULTS: At baseline, patients with AN generated significantly fewer specific memories than HC, independent of word category (F(1.66) = 27.167, p < 0.001). During inpatient stay, the average weight gain of patients with AN was 3.1 body mass index points. At follow-up, patients with AN showed a significant improvement in the number of specific memories for both depression-related and neutral cues, but not for food- and body-related cues. CONCLUSIONS: Generalised OGM (i.e., independent of word category) in patients with AN before weight restoration may be a general incapacity to recall autobiographical memory. After weight gain, the previously well-studied pattern of eating disorder-related OGM emerges. The clinical relevance of the continuing disorder-related OGM in patients with AN after weight gain is discussed.

A natural language model to automate scoring of autobiographical memories.

Biases in the retrieval of personal, autobiographical memories are a core feature of multiple mental health disorders, and are associated with poor clinical prognosis. However, current assessments of memory bias are either reliant on human scoring, restricting their administration in clinical settings, or when computerized, are only able to identify one memory type. Here, we developed a natural language model able to classify text-based memories as one of five different autobiographical memory types (specific, categoric, extended, semantic associate, omission), allowing easy assessment of a wider range of memory biases, including reduced memory specificity and impaired memory flexibility. Our model was trained on 17,632 text-based, human-scored memories obtained from individuals with and without experience of memory bias and mental health challenges, which was then tested on a dataset of 5880 memories. We used 20-fold cross-validation setup, and the model was fine-tuned over BERT. Relative to benchmarking and an existing support vector model, our model achieved high accuracy (95.7%) and precision (91.0%). We provide an open-source version of the model which is able to be used without further coding, by those with no coding experience, to facilitate the assessment of autobiographical memory bias in clinical settings, and aid implementation of memory-based interventions within treatment services.

Analysis of the AMT gene family in chili pepper and the effects of arbuscular mycorrhizal colonization on the expression patterns of CaAMT2 genes.

BACKGROUND: Ammonium (NH4+) is a key nitrogen source supporting plant growth and development. Proteins in the ammonium transporter (AMT) family mediate the movement of NH4+ across the cell membrane. Although several studies have examined AMT genes in various plant species, few studies of the AMT gene family have been conducted in chili pepper. RESULTS: Here, a total of eight AMT genes were identified in chili pepper, and their exon/intron structures, phylogenetic relationships, and expression patterns in response to arbuscular mycorrhizal (AM) colonization were explored. Synteny analyses among chili pepper, tomato, eggplant, soybean, and Medicago revealed that the CaAMT2;1, CaAMT2.4, and CaAMT3;1 have undergone an expansion prior to the divergence of Solanaceae and Leguminosae. The expression of six AMT2 genes was either up-regulated or down-regulated in response to AM colonization. The expression of CaAMT2;1/2;2/2;3 and SlAMT2;1/2;2/2;3 was significantly up-regulated in AM fungi-inoculated roots. A 1,112-bp CaAMT2;1 promoter fragment and a 1,400-bp CaAMT2;2 promoter fragment drove the expression of the ß-glucuronidase gene in the cortex of AM roots. Evaluation of AM colonization under different NH4+ concentrations revealed that a sufficient, but not excessive, supply of NH4+ promotes the growth of chili pepper and the colonization of AM. Furthermore, we demonstrated that CaAMT2;2 overexpression could mediate NH4+ uptake in tomato plants. CONCLUSION: In sum, our results provide new insights into the evolutionary relationships and functional divergence of chili pepper AMT genes. We also identified putative AMT genes expressed in AM symbiotic roots.

Arsenic metabolism, N6AMT1 and AS3MT single nucleotide polymorphisms, and their interaction on gestational diabetes mellitus in Chinese pregnant women.

BACKGROUND: Single nucleotide polymorphisms (SNPs) in N6AMT1 and AS3MT are associated with arsenic (As) metabolism, and efficient As methylation capacity has been associated with diabetes. However, little is known about the gene-As interaction on gestational diabetes mellitus (GDM). OBJECTIVE: This study aimed to explore the individual and combined effects of N6AMT1 and AS3MT SNPs with As metabolism on GDM. METHODS: A cross-sectional study was performed among 385 Chinese pregnant women (86 GDM and 299 Non-GDM). Four SNPs in N6AMT1 (rs1997605 and rs1003671) and AS3MT (rs1046778 and rs11191453) were genotyped. Urinary inorganic arsenic (iAs), monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA) were determined, and the percentages of As species (iAs%, MMA%, and DMA%) were calculated to assess the efficiency of As metabolism. RESULTS: Pregnant women with N6AMT1 rs1997605 AA genotype had lower iAs% (B: 2.11; 95% CI: 4.08, -0.13) and MMA% (B: 0.21; 95% CI: 0.39, -0.04) than pregnant women with GG genotype. The AS3MT rs1046778 and rs11191453 C alleles were negatively associated with iAs% and MMA% but positively associated with DMA%. Higher urinary MMA% was significantly associated with a lower risk of GDM (OR: 0.54; 95% CI: 0.30, 0.97). The A allele in N6AMT1 rs1997605 (OR: 0.46; 95% CI: 0.26, 0.79) was associated with a decreased risk of GDM. The additive interactions between N6AMT1 rs1997605 GG genotypes and lower iAs% (AP: 0.50; 95% CI: 0.01, 0.99) or higher DMA% (AP: 0.52; 95% CI: 0.04, 0.99) were statistically significant. Similar additive interactions were also found between N6AMT1 rs1003671 GG genotypes and lower iAs% or higher DMA%. CONCLUSIONS: Genetic variants in N6AMT1 and efficient As metabolism (indicated by lower iAs% and higher DMA%) can interact to influence GDM occurrence synergistically in Chinese pregnant women.

DNA N6-methyladenine involvement and regulation of hepatocellular carcinoma development.

DNA N6-methyladenine (6 mA) is a new type of DNA methylation identified in various eukaryotic cells. However, its alteration and genomic distribution features in hepatocellular carcinoma (HCC) remain elusive. In this study, we found that N6AMT1 overexpression increased HCC cell viability, suppressed apoptosis, and enhanced migration and invasion, whereas ALKBH1 overexpression induced the opposite effects. Further, 23,779 gain-of-6 mA regions and 11,240 loss-of-6 mA regions were differentially identified in HCC tissues. The differential gain and loss of 6 mA regions were considerably enriched in intergenic regions. Moreover, 7% of the differential 6 mA modifications were associated with tumors, with 60 associated with oncogenes and 57 with tumor suppressor genes (TSGs), and 17 were common to oncogenes and TSGs. The candidate genes affected by 6 mA were filtered by gene ontology (GO) and RNA-seq. Using quantitative polymerase chain reaction (qPCR), BCL2 and PARTICL were found to be correlated with DNA 6 mA in certain HCC processes.

Genome-Wide Identification and Expression Analysis of the Ammonium Transporter Family Genes in Soybean.

Ammonium transporters (AMTs) are responsible for ammonium absorption and utilization in plants. As a high-nitrogen-demand crop and a legume, soybean can also obtain ammonium from symbiotic root nodules in which nitrogen-fixing rhizobia convert atmospheric nitrogen (N2) into ammonium. Although increasing evidence implicates vital roles of ammonium transport in soybean, no systematic analyses of AMTs in soybean (named GmAMTs) or functional analyses of GmAMTs are available. In this study, we aimed to identify all GmAMT family genes and gain a better understanding of the characteristics of GmAMT genes in soybean. Here, due to the improved genome assembly and annotation of soybean, we tried to generate a phylogenetic tree of 16 GmAMTs based on new information. Consistent with reported data, GmAMT family members can be divided into two subfamilies of GmAMT1 (6 genes) and GmAMT2 (10 genes). Interestingly, unlike Arabidopsis, which has only one AMT2, soybean has substantially increased the number of GmAMT2s, suggesting enhanced demand for ammonium transport. These genes were distributed on nine chromosomes, of which GmAMT1.3, GmAMT1.4, and GmAMT1.5 were three tandem repeat genes. The gene structures and conserved protein motifs of the GmAMT1 and GmAMT2 subfamilies were different. All the GmAMTs were membrane proteins with varying numbers of transmembrane domains ranging from 4 to 11. Promoter analysis found that these GmAMT genes have phytohormone-, circadian control-, and organ expression-related cis-elements in their promoters, and notably, there were nodulation-specific and nitrogen-responsive elements in the promoters of the GmAMT1 and GmAMT2 genes. Further expression data showed that these GmAMT family genes exhibited different spatiotemporal expression patterns across tissues and organs. In addition, GmAMT1.1, GmAMT1.2, GmAMT2.2, and GmAMT2.3 were responsive to nitrogen treatment, while GmAMT1.2, GmAMT1.3, GmAMT1.4, GmAMT1.5, GmAMT1.6, GmAMT2.1, GmAMT2.2, GmAMT2.3, GmAMT3.1, and GmAMT4.6 showed circadian rhythms in transcription. RT-qPCR validated the expression patterns of GmAMTs in response to different forms of nitrogen and exogenous ABA treatments. Gene expression analysis also confirmed that GmAMTs are regulated by key nodulation gene GmNINa, indicating a role of GmAMTs in symbiosis. Together, these data indicate that GmAMTs may differentially and/or redundantly regulate ammonium transport during plant development and in response to environmental factors. These findings provide a basis for future research on the functions of GmAMTs and the mechanisms through which GmAMTs regulate ammonium metabolism and nodulation in soybean.

OsAMT1;1 and OsAMT1;2 Coordinate Root Morphological and Physiological Responses to Ammonium for Efficient Nitrogen Foraging in Rice.

Optimal plant growth and development rely on morphological and physiological adaptions of the root system to forage heterogeneously distributed nitrogen (N) in soils. Rice grows mainly in the paddy soil where ammonium (NH4+) is present as the major N source. Although root NH4+ foraging behaviors are expected to be agronomically relevant, the underlying mechanism remains largely unknown. Here, we showed that NH4+ supply transiently enhanced the high-affinity NH4+ uptake and stimulated lateral root (LR) branching and elongation. These synergistic physiological and morphological responses were closely related to NH4+-induced expression of NH4+ transporters OsAMT1;1 and OsAMT1;2 in roots. The two independent double mutants (dko) defective in OsAMT1;1 and OsAMT1;2 failed to induce NH4+ uptake and stimulate LR formation, suggesting that OsAMT1s conferred the substrate-dependent root NH4+ foraging. In dko plants, NH4+ was unable to activate the expression of OsPIN2, and the OsPIN2 mutant (lra1) exhibited a strong reduction in NH4+-triggered LR branching, suggesting that the auxin pathway was likely involved in OsAMT1s-dependent LR branching. Importantly, OsAMT1s-dependent root NH4+ foraging behaviors facilitated rice growth and N acquisition under fluctuating NH4+ supply. These results revealed an essential role of OsAMT1s in synergizing root morphological and physiological processes, allowing for efficient root NH4+ foraging to optimize N capture under fluctuating N availabilities.

Ammonium transporter 1 increases rice resistance to sheath blight by promoting nitrogen assimilation and ethylene signalling.

Sheath blight (ShB) significantly threatens rice yield production. However, the underlying mechanism of ShB defence in rice remains largely unknown. Here, we identified a highly ShB-susceptible mutant Ds-m which contained a mutation at the ammonium transporter 1;1 (AMT1;1) D358 N. AMT1;1 D358 N interacts with AMT1;1, AMT1;2 and AMT1;3 to inhibit the ammonium transport activity. The AMT1 RNAi was more susceptible and similar to the AMT1;1 D358 N mutant; however, plants with higher NH4+ uptake activity were less susceptible to ShB. Glutamine synthetase 1;1 (GS1;1) mutant gs1;1 and overexpressors (GS1;1 OXs) were more and less susceptible to ShB respectively. Furthermore, AMT1;1 overexpressor (AMT1;1 OX)/gs1;1 and gs1;1 exhibited a similar response to ShB, suggesting that ammonium assimilation rather than accumulation controls the ShB defence. Genetic and physiological assays further demonstrated that plants with higher amino acid or chlorophyll content promoted rice resistance to ShB. Interestingly, the expression of ethylene-related genes was higher in AMT1;1 OX and lower in RNAi mutants than in wild-type. Also, ethylene signalling positively regulated rice resistance to ShB and NH4+ uptake, suggesting that ethylene signalling acts downstream of AMT and also NH4+ uptake is under feedback control. Taken together, our data demonstrated that the AMT1 promotes rice resistance to ShB via the regulation of diverse metabolic and signalling pathways.

Simple construction of a two-component fluorescent sensor for turn-on detection of Hg2+ in human serum.

The simply constructed fluorescent sensor with inexpensive reagents and low toxicity has attracted increasing attention contributing to its practical application. However, the common construction methods usually required a few building blocks and complex procedures, which is inconvenient for their further application. Herein, a simply constructed fluorescent Hg2+ sensor has been developed based on the intrinsic fluorescence quenching power of G-quadruplex. Two components, AGRO 100 and AMT, were used to construct the sensor. AMT was selected as the fluorescent probe because of its distinct merits. The free AMT emits strongly. However, the fluorescence of AMT could be quenched by G-quadruplex DNA. Additionally, AMT is less toxic and inexpensive. AGRO 100 acts as both the quencher and the capture sequence because it consists of G-rich sequences and T-T mismatched base pairs. The fluorescence of AMT could be quenched by the formed G-quadruplex structure of AGRO 100 in the presence of K+. In the presence of Hg2+, G-quadruplex structure of AGRO 100 was switched to hairpin DNA structure because T-T mismatched base pairs in AGRO 100 could specifically recognize and capture Hg2+ with high affinity. Thus, AMT was released and the fluorescence of AMT was recovered. The developed sensing system was successfully applied to detect Hg2+ in human serum with good recovery and reproducibility.

Stability-indicating liquid chromatography method development for assay and impurity profiling of amitriptyline hydrochloride in tablet dosage form and forced degradation study.

Amitriptyline hydrochloride is an antidepressant drug with sedative effects used to treat the symptoms of anxiety, agitation with depression and schizophrenia with depression. A reversed-phase high-performance liquid chromatography method was developed to separate and quantitatively determine the assay and four organic impurities of amitriptyline in tablet dosage form and bulk drugs using a C18 column in an isocratic elution mode with mobile phase consisting of a mixture of pH 7.5 phosphate buffer and methanol. The pH conditions used in the chromatographic separation are discussed. The stability-indicating characteristics of the proposed method were proved using stress testing [5 m HCl at 80°C/1 h, 5 m NaOH at 80°C/1 h, H2 O (v/w) at 80°C/1 h, 6% H2 O2 (v/v) at 25°C/1 h, dry heat at 105°C/24 h and UV-vis light/4 days] and validated for specificity, detection limit, quantitation limit, linearity, precision, accuracy and robustness. For amitriptyline and its four known organic impurities, the quantitation limits, linearity and recoveries were in the ranges 0.25-3.0 µg/ml (r2 > 0.999) and 87.9-107.6%, respectively. The mass (m/z) spectral data of amitriptyline hydrochloride and its impurity are discussed. The proposed LC method is also suitable for impurity profiling and assay determination of amitriptyline in bulk drugs and pharmaceutical formulations.

Genome-Wide Identification and Expression Analysis of AMT and NRT Gene Family in Pecan (Carya illinoinensis) Seedlings Revealed a Preference for NH4+-N.

Nitrogen (N) is a major limiting factor for plant growth and crop production. The use of N fertilizer in forestry production is increasing each year, but the loss is substantial. Mastering the regulatory mechanisms of N uptake and transport is a key way to improve plant nitrogen use efficiency (NUE). However, this has rarely been studied in pecans. In this study, 10 AMT and 69 NRT gene family members were identified and systematically analyzed from the whole pecan genome using a bioinformatics approach, and the expression patterns of AMT and NRT genes and the uptake characteristics of NH4+ and NO3- in pecan were analyzed by aeroponic cultivation at varying NH4+/NO3- ratios (0/0, 0/100,25/75, 50/50, 75/25,100/0 as CK, T1, T2, T3, T4, and T5). The results showed that gene duplication was the main reason for the amplification of the AMT and NRT gene families in pecan, both of which experienced purifying selection. Based on qRT-PCR results, CiAMTs were primarily expressed in roots, and CiNRTs were majorly expressed in leaves, which were consistent with the distribution of pecan NH4+ and NO3- concentrations in the organs. The expression levels of CiAMTs and CiNRTs were mainly significantly upregulated under N deficiency and T4 treatment. Meanwhile, T4 treatment significantly increased the NH4+, NO3-, and NO2- concentrations as well as the Vmax and Km values of NH4+ and NO3- in pecans, and Vmax/Km indicated that pecan seedlings preferred to absorb NH4+. In summary, considering the single N source of T5, we suggested that the NH4+/NO3- ratio of 75:25 was more beneficial to improve the NUE of pecan, thus increasing pecan yield, which provides a theoretical basis for promoting the scale development of pecan and provides a basis for further identification of the functions of AMT and NRT genes in the N uptake and transport process of pecan.

Genome-Wide Identification of AMT2-Type Ammonium Transporters Reveal That CsAMT2.2 and CsAMT2.3 Potentially Regulate NH4+ Absorption among Three Different Cultivars of Camellia sinensis.

Ammonium (NH4+), as a major inorganic source of nitrogen (N) for tea plant growth, is transported and distributed across membranes by the proteins of ammonium transporters (AMTs). However, the AMT2-type AMTs from tea plants remain poorly understood. In this study, five CsAMT2 subfamily genes were identified in tea plant genomes, and their full-length coding sequences (CDS) were isolated from roots. Then, a NH4+ uptake kinetic comparison of Fudingdabaicha (FD), Huangdan (HD), and Maoxie (MX) showed that FD was a high N efficiency (HNE) cultivar that had a wide range of adaptability to NH4+, HD was a high N efficiency under high N conditions (HNEH) cultivar, in which it was easy to obtain higher yield in a high N environment, and MX was a high N efficiency under low N conditions (HNEL) cultivar, which had a higher affinity for NH4+ than the other two. Tissue-specific expression analysis suggested that CsAMT2.2 and CsAMT2.3 were highly expressed in the roots, indicating that these two members may be unique in the CsAMT2 subfamily. This is further supported by our findings from the temporal expression profiles in the roots among these three different N adaptation cultivars. Expression levels of CsAMT2.2 and CsAMT2.3 in FD and HD were upregulated by a short time (2 h) under high NH4+ treatment, while under low NH4+ treatment, CsAMT2.2 and CsAMT2.3 were highly expressed at 0 h and 2 h in the HNEL-type cultivar-MX. Furthermore, the functional analysis illustrated that CsAMT2.2 and CsAMT2.3 could make a functional complementation of NH4+-defective mutant yeast cells at low NH4+ levels, and the transport efficiency of CsAMT2.3 was higher than that of CsAMT2.2. Thus, we concluded that CsAMT2.2 and CsAMT2.3 might play roles in controlling the NH4+ uptake from the soil to the roots. These results will further the understanding of the NH4+ signal networks of AMT2-type proteins in tea plants.

LjAMT2;2 Promotes Ammonium Nitrogen Transport during Arbuscular Mycorrhizal Fungi Symbiosis in Lotus japonicus.

Arbuscular mycorrhizal fungi (AMF) are important symbiotic microorganisms in soil that engage in symbiotic relationships with legumes, resulting in mycorrhizal symbiosis. Establishment of strong symbiotic relationships between AMF and legumes promotes the absorption of nitrogen by plants. Ammonium nitrogen can be directly utilised by plants following ammonium transport, but there are few reports on ammonium transporters (AMTs) promoting ammonium nitrogen transport during AM symbiosis. Lotus japonicus is a typical legume model plant that hosts AMF. In this study, we analysed the characteristics of the Lotus japonicus ammonium transporter LjAMT2;2, and found that it is a typical ammonium transporter with mycorrhizal-induced and ammonium nitrogen transport-related cis-acting elements in its promoter region. LjAMT2;2 facilitated ammonium transfer in yeast mutant supplement experiments. In the presence of different nitrogen concentrations, the LjAMT2;2 gene was significantly upregulated following inoculation with AMF, and induced by low nitrogen. Overexpression of LjAMT2;2 increased the absorption of ammonium nitrogen, resulting in doubling of nitrogen content in leaves and roots, thus alleviating nitrogen stress and promoting plant growth.

[HEMK-Like Methyltransferases in the Regulation of Cellular Processes].

Human translational methyltransferase (methylase) HEMK2, whose orthologues are found in many prokaryotes and eukaryotes, methylates such diverse substrates as glutamine and lysine residues in proteins, deoxyadenosine in DNA, and arsenicals. One of the important substrate of HEMK2 methylase is a glutamine residue in the GGQ ultra-conservative motif of the eukaryotic release factor 1 (eRF1). Release factor methylation by HEMK2 orthologs is conserved among eukaryotes, archaea, and bacteria, although bacterial release factors differ in sequence and structure from eukaryotic ones. In this review, we consider the features of human HEMK2 methylase and its orthologs as multifunctional enzymes that regulate cellular processes, in particular, protein biosynthesis.