Synthetase Gene Research Articles

Pan-genome wide identification and analysis of the SAMS gene family in sunflowers (Helianthus annuus L.) revealed their intraspecies diversity and potential roles in abiotic stress tolerance

IntroductionS-adenosylmethionine (SAM), a key molecule in plant biology, plays an essential role in stress response and growth regulation. Despite its importance, the SAM synthetase (SAMS) gene family in sunflowers (Helianthus annuus L.) remains poorly understood.MethodsIn this study, the SAMS genes were identified from the sunflower genome. Subsequently, the protein properties, gene structure, chromosomal location, cis-acting elements, collinearity, and phylogeny of the SAMS gene family were analyzed by bioinformatic methods. Finally, the expression patterns of SAMS genes in different tissues, under different hormonal treatment and abiotic stress were analyzed based on transcriptome data and qRT-PCR.ResultsThis study identified 58 SAMS genes across nine cultivated sunflower species, which were phylogenetically classified into seven distinct subgroups. Physicochemical properties and gene structure analysis showed that the SAMS genes are tightly conserved between cultivars. Collinearity analysis revealed segmental duplications as the primary driver of gene family expansion. The codon usage bias analysis suggested that natural selection substantially shapes the codon usage patterns of sunflower SAMS genes, with a bias for G/C-ending high-frequency codons, particularly encoding glycine, leucine, and arginine. Analysis of the cis-regulatory elements in promoter regions, implied their potential roles in stress responsiveness. Differential expression patterns for HanSAMS genes were observed in different tissues as well as under hormone treatment or abiotic stress conditions by analyzing RNA-seq data from previous studies and qRT-PCR data in our current study. The majority of genes demonstrated a robust response to BRA and IAA treatments in leaf tissues, with no significant expression change observed in roots, suggesting the response of HanSAMS genes to hormones is tissue-specific. Expression analyses under abiotic stresses demonstrated diverse expression profiles of HanSAMS genes, with HanSAMS5 showing significant upregulation in response to both drought and salt stresses.DiscussionThis comprehensive genomic and expression analysis provides valuable insights into the SAMS gene family in sunflowers, laying a robust foundation for future functional studies and applications in crop improvement for stress resilience.

Targeting glutamine synthetase with AS1411-modified exosome-liposome hybrid nanoparticles for inhibition of choroidal neovascularization.

Choroidal neovascularization (CNV) is a leading cause of visual impairment in wet age-related macular degeneration (wAMD). Recent investigations have validated the potential of reducing glutamine synthetase (GS) to inhibit neovascularization formation, offering prospects for treating various neovascularization-related diseases. In this study, we devised a CRISPR/Cas9 delivery system employing the nucleic acid aptamer AS1411 as a targeting moiety and exosome-liposome hybrid nanoparticles as carriers (CAELN). Exploiting the binding affinity between AS1411 and nucleolin on endothelial cell surfaces, the delivery system was engineered to specifically target the glutamine synthetase gene (GLUL), thereby attenuating GS levels and continuously suppressing CNV. CAELN exhibited spherical and uniform dispersion. In vitro cellular investigations demonstrated gene editing efficiencies of CAELN ranging from 42.05 to 55.02% and its capacity to inhibit neovascularization in HUVEC cells. Moreover, in vivo pharmacodynamic studies conducted in CNV rabbits revealed efficacy of CAELN in restoring the thickness of intra- and extranuclear tissues. The findings suggest that GS is a novel target for the inhibition of pathological CNV, while the development of AS1411-modified exosome-liposome hybrid nanoparticles represents a novel delivery method for the treatment of neovascular-related diseases.

Genome editing of an oxalyl-CoA synthetase gene in Lathyrus sativus reveals its role in oxalate metabolism.

Established an Agrobacterium-mediated hairy root transformation system for gene function analysis in Lathyrus sativus. Arabidopsis mutant complementation and genome editing in Lathyrus confirmed role of LsOCS in the oxalate metabolism. Grass pea (Lathyrus sativus) is a resilient legume cultivated for its protein-rich seeds and fodder. However, the presence of a naturally occurring neurotoxin, β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP), which causes neurolathyrism, limits its extensive cultivation. This paper reports the in-planta characterization of oxalyl-CoA synthetase (OCS), an enzyme involved in oxalate metabolism and important in the oxalylating step leading to β-ODAP production in Lathyrus. For this, we used complementation experiments in an Arabidopsis OCS mutant. The LsOCS-complemented lines showed oxalate content similar to wild-type levels, and the analysis of seeds by field emission scanning electron microscope (FESEM) showed that the LsOCS-complemented lines were rescued from seed-coat defects found in the mutant seeds. We used genome editing of LsOCS in Lathyrus hairy roots to further characterize LsOCS function. The mutations in LsOCS resulted in the accumulation of oxalate in the hairy roots of Lathyrus, as observed in Arabidopsis mutants, but did not affect the ODAP levels. The hairy root genome editing system could serve as a rapid tool for functional studies of Lathyrus genes and optimizing the agronomic traits.

Pleiotropic effects of Ebony on pigmentation and development in the Asian multi-coloured ladybird beetle, Harmonia axyridis (Coleoptera: Coccinellidae).

Melanin plays a pivotal role in insect body pigmentation, significantly contributing to their adaptation to diverse biotic and abiotic environmental challenges. Several genes involved in insect melanin synthesis showed pleiotropic effects on insect development and reproduction. Among these, the N-β-alanyl dopamine synthetase gene (Ebony) is integral to the pigmentation process. However, the full spectrum of its pleiotropic impacts is not yet thoroughly understood. In this study, we identified and characterised the HaEbony gene in the Asian multi-coloured ladybird beetle (Harmonia axyridis) and found that HaEbony gene is a conserved gene within the Coleoptera order. We aimed to further explore the multiple roles of HaEbony in the physiology and behaviour in H. axyridis. The CRISPR/Cas9 system was applied to generate multiple HaEbony knockout allele (HaEbony+/-), showing nucleotide deletion in the G0 and G1 generations. Remarkably, the resultant HaEbony+/- mutants consistently displayed darker pigmentation than their wild-type counterparts across larval, pupal and adult stages. Furthermore, these HaEbony+/- individuals (G0) demonstrated an enhanced predatory efficiency, evidenced by a higher number of aphids consumed compared to the wild type. A significant finding was the reduced egg hatchability in both G0 and G1 generations of the HaEbony+/- group, highlighting a potential reproductive fitness cost associated with HaEbony deficiency. In conclusion, our study not only sheds light on the multifaceted roles of HaEbony in H. axyridis but also highlights the potential of employing CRISPR/Cas9-targeted modifications of the Ebony gene. Such genetic interventions could enhance the environmental adaptability and predatory efficacy of ladybirds, presenting a novel strategy in biological control application.

Overlapping Transcriptome Alterations Reveal the Mechanism of Interaction between Selenium and Zinc and Their Common Effect on Essential Nutrient Metabolism in Mung Bean (Vigna radiata L.).

Selenium (Se) and zinc (Zn) deficiencies have become serious global food security and public health problems. Biofortification through foliar fertilizer is a nonspecific, low-tech, and cost-effective strategy. Se and Zn have overlapping physiological roles and interacting relationships in plants. Mung bean is superior for Se enrichment and an excellent Zn carrier. However, the molecular mechanism underlying the interaction between Se and Zn in the mung bean remains unclear. Herein, Se and Zn accumulation, antioxidant activities, physiological determination, and transcriptomic analysis were performed under both Se and Zn treatments. Common essential roles of Se and Zn in mung bean were reflected by the comprehensively altered ten physiological indexes under both Se2 (24 g·ha-1) and Zn1 (1.2 kg·ha-1) treatments. Overlapping transcriptome changes and common DEGs in two compared groups revealed that the upregulated expression of sulfate transporters (SULTRs), phosphate transporters (PHTs), and Zinc-regulated/Iron-regulated-like protein (ZIP) family genes under Se and Zn treatments directly promoted both Se and Zn intakes. Furthermore, the altered Se/Sulfur, nitrogen, and carbohydrate metabolisms are closely interlinked with the uptake and assimilation of Se and Zn via the 20 key genes that we filtered through the protein-protein interaction (PPI) network analysis. Further analysis indicated that l-methionine γ-lyase (E 4.4.1.11) genes may play an important role in the transamination of selenomethionine and its derivatives; glutamine synthetase (GS), nitrate reductase (NR), and starch synthase (SS) genes may regulate the nitrogen assimilation and carbohydrate metabolism, which provide more carriers for Se and Zn; glutathione peroxidase (GPx), glutamate-cysteine ligase catalytic subunit (GCLC), and serine acetyltransferase (SAT) genes may accelerate the GSH-GSSH cycle and promote Se and Zn storages. This study provides new molecular insights into the comprehensive improvement of the nutritional quality of mung beans in Se and Zn biofortification productions.

Screening of Sugarcane Commercial GMP Varieties Tolerant to Drought Stress Based on Molecular Detection of P5CS Gene

Sugarcane (Saccharum officinarum L.) requires sufficient a water supply to produce optimal productivity. The selection of the superior varieties of sugarcane that are tolerant to drought can be done by molecular detection of the presence of the pyrroline-5-carboxylate synthetase (P5CS) gene. The research is aimed at molecular detection of P5CS gene in a commercial varieties at PT Gunung Madu Plantations (GMP), Indonesia that are potentially drought resistant. Similar research has never been conducted at PT GMP, as evidenced by the absence of official publications. The varieties used are GMP 3, GMP 5, RGM 06-654, PS 864, RGM 08-1026, PSJT 941, RGM 01-1834, GP11, RGM 07-099, and RGM 02-108. The research phases include DNA isolation, quantitative and qualitative DNA amplification, and PCR product visualization. Molecular data analysis is based on DNA band scoring results in the form of binary data which is then used to calculate the level of polimorphism information content (PIC) on the primary data used. The results of the study showed that nine out of ten varieties had P5CS-specific band sizes of ±167 bp, namely GMP 3, GMP 5, RGM 06-654, PS 864, PSJT 941, RGM 01-1834, GP 11, RGM 07-099, and RGM 02-108. A total of four of the nine varieties showed P5CS gene band thicknesses of RGM06-654, PSJT 941, PS864, and RGM 01-1834 which indicates the selected varieties most resistant to drought. The result of the PIC calculation has a value of > 0.25 which indicates the primary P5CS used is quite informative. The molecular detection was continued on one selected variety is PSJT 941, which showed that the relationship analysis of P5CS gene shows close affinity with the isolates from China and Bogor.

Sink-source driven metabolic acclimation of winter oilseed rape leaves (Brassica napus L.) to drought

The crop cycle of winter oilseed rape (WOSR) incorporates source-to-sink remobilisation during the vegetative stage as a principal factor influencing the ultimate seed yield. These processes are supported by the coordinated activity of the plant’s central metabolism. However, climate change-induced drought will affect the metabolic acclimation of WOSR sink/source relationships at this vegetative stage, with consequences that remain to be determined. In this study, we subjected WOSR to severe soil dehydration for 18 days and analysed the physiological and metabolic acclimation of sink and source leaves along the kinetics in combination with measurements of enzymatic activities and transcript levels. Overall, the acclimation of WOSR to drought led to subtle regulations of central metabolism in relation to leaf growth and Pro-induced osmotic adjustment. Notably, sink leaves drastically reduced their growth and transiently accumulated starch. Subsequent starch degradation correlated with the induction of beta-amylases, sucrose transporters, pyrroline-5-carboxylate synthases and proline accumulation. The functioning of the tricarboxylic acid cycle was also altered in sink leaves, as evidenced by variations in citrate, malate and associated enzymatic activities. The metabolic origin of Pro in sink leaves is discussed in relation to Pro accumulation in source leaves and the up-regulation of amino acid permease 1 and glutamine synthetase genes.

Application of CRISPR/Cas9 Genome Editing to Improve Recombinant Protein Production in CHO Cells.

Genome editing has become an important aspect of Chinese hamster ovary (CHO) cell line engineering for improving the production of recombinant protein therapeutics. Currently, the engineering focus is directed toward expanding product diversity while controlling and improving product quality and yields. In this chapter, we present our protocol for using the genome editing tool Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) to knock out engineering target genes in CHO cells. As an example, we describe how to knock out the glutamine synthetase (GS) gene, which increases the selection efficiency of the GS-mediated gene amplification system.

Phylogenomic analysis of glutamine synthetase gene family in Helianthus annuus L.

Glutamine synthetase is one of the predominant enzymes in nitrogen metabolism. In developing leaves, glutamine is mainly produced in chloroplasts by the activity of the GS2 isoenzyme. It catalyzes glutamine synthesis from glutamate and ammonia in an ATP-dependent reaction. The genes encoding glutamine synthetase play a crucial role in ammonia and glutamate detoxification, acid-base homeostasis, cell signaling, and cell proliferation. The gene family responsible for producing glutamine synthetase has been previously documented in model plants like Arabidopsis. Nevertheless, there has been no exploration into its existence and attributes in oilseed crops like sunflower (Helianthus annuus L.). This study thoroughly analyzes, gene structure, conserved motifs, chromosomal location, phylogenetic relationships, and expression patterns to identify the glutamine synthetase genes in H. annuus. Our findings unveiled 19 genes encoding glutamine synthetase within the H. annuus genome. These genes were distributed across 11 chromosomes of H. annuus. Furthermore, we examined the expression patterns of all the HaGS genes using RNA-seq datasets, specifically focusing on their response to biotic and abiotic stress conditions. Under biotic stress, H. annuus expresses genes for mycorrhizal fungi named Rhizoglomus irregulare at four days post inflorescence (dpi) and 16 dpi. Under abiotic stress, the effect of drought and hormones was investigated. In drought, one gene, HaGS6D, showed the highest expression in the leaf. Meanwhile, in roots, gene HaGS7B showed the highest expression. Under hormonal stress, the effects of auxin, brassinosteroid, and cytokinin were studied on the leaf. For auxin, the gene HaGS7C showed the highest expression. For brassinosteroid, the gene HaGS7D showed the highest expression; for cytokinin, the gene HaGS6E showed the highest expression. Thus, these findings can significantly contribute to our understanding of the arrangement of glutamine synthetase genes in H. annuus and offer valuable insights for developing of drought-resistant cultivars of this species.

Overexpression of bacterial γ-glutamylcysteine synthetase increases toxic metal(loid)s tolerance and accumulation in Crambe abyssinica.

Transgenic Crambe abyssinica lines overexpressing γ-ECS significantly enhance tolerance to and accumulation of toxic metal(loid)s, improving phytoremediation potential and offering an effective solution for contaminated soil management. Phytoremediation is an attractive environmental-friendly technology to remove metal(loid)s from contaminated soils and water. However, tolerance to toxic metals in plants is a critical limiting factor. Transgenic Crambe abyssinica lines were developed that overexpress the bacterial γ-glutamylcysteine synthetase (γ-ECS) gene to increase the levels of non-protein thiol peptides such as γ-glutamylcysteine (γ-EC), glutathione (GSH), and phytochelatins (PCs) that mediate metal(loid)s detoxification. The present study investigated the effect of γ-ECS overexpression on the tolerance to and accumulation of toxic As, Cd, Pb, Hg, and Cr supplied individually or as a mixture of metals. Compared to wild-type plants, γ-ECS transgenics (γ-ECS1-8 and γ-ECS16-5) exhibited a significantly higher capacity to tolerate and accumulate these elements in aboveground tissues, i.e., 76-154% As, 200-254% Cd, 37-48% Hg, 26-69% Pb, and 39-46% Cr, when supplied individually. This is attributable to enhanced production of GSH (82-159% and 75-87%) and PC2 (27-33% and 37-65%) as compared to WT plants under AsV and Cd exposure, respectively. The levels of Cys and γ-EC were also increased by 56-67% and 450-794% in the overexpression lines compared to WT plants under non-stress conditions, respectively. This likely enhanced the metabolic pathway associated with GSH biosynthesis, leading to the ultimate synthesis of PCs, which detoxify toxic metal(loid)s through chelation. These findings demonstrate that γ-ECS overexpressing Crambe lines can be used for the enhanced phytoremediation of toxic metals and metalloids from contaminated soils.

Evaluation of the Role of Tanshinone I in an In Vitro System of Charcot-Marie-Tooth Disease Type 2N.

Charcot-Marie-Tooth disease type 2N (CMT2N) is an inherited nerve disorder caused by mutations in the alanyl-tRNA synthetase (AlaRS) gene, resulting in muscle weakness and sensory issues. Currently, there is no cure for CMT2N. Here, we found that all five AlaRS mutations in the aminoacylation domain can interact with neuropilin-1 (Nrp1), which is consistent with our previous findings. Interestingly, three of these mutations did not affect alanine activation activity. We then performed a high-throughput screen of 2000 small molecules targeting the prevalent R329H mutant. Using thermal stability assays (TSA), biolayer interferometry (BLI), ATP consumption, and proteolysis assays, we identified Tanshinone I as a compound that binds to and modifies the conformation of the R329H mutant and other CMT-related AlaRS mutants interacting with Nrp1. Molecular docking and dynamic simulation studies further clarified Tanshinone I's binding mode, indicating its potential against various AlaRS mutants. Furthermore, co-immunoprecipitation (Co-IP) and pull-down assays showed that Tanshinone I significantly reduces the binding of AlaRS mutants to Nrp1. Collectively, these findings suggest that Tanshinone I, by altering the conformation of mutant proteins, disrupts the pathological interaction between AlaRS CMT mutants and Nrp1, potentially restoring normal Nrp1 function. This makes Tanshinone I a promising therapeutic candidate for CMT2N.

Chitosan nanoparticles (CSNPs) conferred salinity tolerance in maize by upregulating E3 ubiquitin-protein ligase, P5CS1, HKT1, NHX1, and PMP3 genes.

This study explored the transcriptional behaviors of several candidate genes in response to the application of CSNPs (50 and 100 mgl-1) in maize seedlings grown under two salinity levels (NaCl of 0.07 and 0.14 gkg-1soil). Employing CSNPs at both concentrations mitigated the inhibitory role of salinity on the leaf and root fresh weights. The application of CSNPs enhanced the transcription of the E3 ubiquitin-protein ligase gene by an average of threefold, contrasted with the salinity controls. The Δ1-pyrroline-5-carboxylate synthetase (P5CS1) gene was upregulated in response to both individual and mixed treatments of CSNPs and salinity. The transcription of the high-affinity K+ transporter (HKT1) gene displayed an upward trend in response to the CSNPs and salinity treatments. The Na+/H+ exchangers (NHX1) gene exhibited a similar trend to that of the HKT1 gene. The utilization of CSNPs was accompanied by an upregulation in the plasma membrane proteolipid 3 (PMP3) gene, contrasted with the salinity controls. The phenylalanine ammonia-lyase (PAL) activity displayed an upward trend in response to the foliar application of CSNPs. The CSNPs at the 100 mgl-1 concentration were more capable of inducing the ascorbate peroxidase enzyme under both salinity conditions than the 50 mgl-1 dose. The simultaneous exposure of maize seedlings to CSNPs and salinity resulted in the drastic upregulation of the catalase activities. This study provides novel insights into the major mechanisms underlying the stress-mitigating effects of CSNPs, thereby providing a suitable platform for their application in sustainable agricultural practices.

Advantages of compost tea: Promotion of nitrogen influx into the fruit and improvement of fruit nitrogen metabolism in tomato

The use of compost tea is important to improve food safety. However, the effect of compost tea on N uptake and partitioning in tomato is unclear. In this study, we measured temporal and spatial changes in nitrogen content, enzyme activities, and expression levels of nitrogen transporters genes in different organs of tomato treated with five nutrient solutions. The results showed that the expression levels of ammonium transporter protein genes (AMT1s) increased and that of a nitrogen transporters gene (NRT2.1) decreased under treatment with compost tea, which promoted NH4+ transport to the fruit and reduced nutrient wastage compared with the response to chemical fertilizers. In addition, the lowermost leaves on the stem showed reduced nitrate content, faster metabolism, and decreased chlorophyll a content, but fruit yield was not increased, in compost tea-treated plants. These changes were dependent on the expression level of the glutamine synthetase gene (GS1.1), which was increased in leaves and decreased in fruit. Compost tea influenced the expression of critical genes in the fruits and leaves, and improved the competitiveness of sexual reproductive growth as a sink for nitrogen. However, the benefits of compost tea were reduced when it was mixed chemical fertilizers. This research establishes a theoretical framework for optimization of organic vegetable cultivation and promoting the widespread production of organic crops.

Pioneering precision in markerless strain development for Synechococcus sp. PCC 7002

Marine cyanobacteria such as Picosynechococcus sp. (formerly called Synechococcus sp.) PCC 7002 are promising chassis for photosynthetic production of commodity chemicals with low environmental burdens. Genetic engineering of cyanobacteria conventionally employs antibiotic resistance markers. However, limited availability of antibiotic-resistant markers is a problem for highly multigenic strain engineering. Although several markerless genetic manipulation methods have been developed for PCC 7002, they often lack versatility due to the requirement of gene disruption in the host strain. To achieve markerless transformation in Synechococcus sp. with no requirements for the host strain, this study developed a method in which temporarily introduces a mutated phenylalanyl-tRNA synthetase gene (pheS) into the genome for counter selection. Amino acid substitutions in the PheS that cause high susceptibility of PCC 7002 to the phenylalanine analog p-chlorophenylalanine were examined, and the combination of T261A and A303G was determined as the most suitable mutation. The mutated PheS-based selection was utilized for the markerless knockout of the nblA gene in PCC 7002. In addition, the genetic construct containing the lldD and lldP genes from Escherichia coli was introduced into the ldhA gene site using the counter selection strategy, resulting in a markerless recombinant strain. The repeatability of this method was demonstrated by the double markerless knockin recombinant strain, suggesting it will be a powerful tool for multigenic strain engineering of cyanobacteria.

Exploiting phenotypic heterogeneity to improve production of glutathione by yeast

BackgroundGene expression noise (variation in gene expression among individual cells of a genetically uniform cell population) can result in heterogenous metabolite production by industrial microorganisms, with cultures containing both low- and high-producing cells. The presence of low-producing individuals may be a factor limiting the potential for high yields. This study tested the hypothesis that low-producing variants in yeast cell populations can be continuously counter-selected, to increase net production of glutathione (GSH) as an exemplar product.ResultsA counter-selection system was engineered in Saccharomyces cerevisiae based on the known feedback inhibition of gamma-glutamylcysteine synthetase (GSH1) gene expression, which is rate limiting for GSH synthesis: the GSH1 ORF and the counter-selectable marker GAP1 were expressed under control of the TEF1 and GSH-regulated GSH1 promoters, respectively. An 18% increase in the mean cellular GSH level was achieved in cultures of the engineered strain supplemented with D-histidine to counter-select cells with high GAP1 expression (i.e. low GSH-producing cells). The phenotype was non-heritable and did not arise from a generic response to D-histidine, unlike that with certain other test-constructs prepared with alternative markers.ConclusionsThe results corroborate that the system developed here improves GSH production by targeting low-producing cells. This supports the potential for exploiting end-product/promoter interactions to enrich high-producing cells in phenotypically heterogeneous populations, in order to improve metabolite production by yeast.

Transcriptomic and Proteomic Insights into Host Immune Responses in Pediatric Severe Malarial Anemia: Dysregulation in HSP60-70-TLR2/4 Signaling and Altered Glutamine Metabolism.

Severe malarial anemia (SMA, Hb < 6.0 g/dL) is a leading cause of childhood morbidity and mortality in holoendemic Plasmodium falciparum transmission zones. This study explored the entire expressed human transcriptome in whole blood from 66 Kenyan children with non-SMA (Hb ≥ 6.0 g/dL, n = 41) and SMA (n = 25), focusing on host immune response networks. RNA-seq analysis revealed 6862 differentially expressed genes, with equally distributed up-and down-regulated genes, indicating a complex host immune response. Deconvolution analyses uncovered leukocytic immune profiles indicative of a diminished antigenic response, reduced immune priming, and polarization toward cellular repair in SMA. Weighted gene co-expression network analysis revealed that immune-regulated processes are central molecular distinctions between non-SMA and SMA. A top dysregulated immune response signaling network in SMA was the HSP60-HSP70-TLR2/4 signaling pathway, indicating altered pathogen recognition, innate immune activation, stress responses, and antigen recognition. Validation with high-throughput gene expression from a separate cohort of Kenyan children (n = 50) with varying severities of malarial anemia (n = 38 non-SMA and n = 12 SMA) confirmed the RNA-seq findings. Proteomic analyses in 35 children with matched transcript and protein abundance (n = 19 non-SMA and n = 16 SMA) confirmed dysregulation in the HSP60-HSP70-TLR2/4 signaling pathway. Additionally, glutamine transporter and glutamine synthetase genes were differentially expressed, indicating altered glutamine metabolism in SMA. This comprehensive analysis underscores complex immune dysregulation and novel pathogenic features in SMA.

Characterization of a novel heterozygous variant in the histidyl-tRNA synthetase gene associated with Charcot-Marie-Tooth disease type 2W.

Heterozygous pathogenic variants in the histidyl-tRNA synthetase (HARS) gene are associated with Charcot-Marie-Tooth (CMT) type 2W disease, classified as an axonal peripheral neuropathy. To date, at least 60 variants causing CMT symptoms have been identified in seven different aminoacyl-tRNA synthetases, with eight being found in the catalytic domain of HARS. The genetic data clearly show a causative role of aminoacyl-tRNA synthetases in CMT; however, the cellular mechanisms leading to pathology can vary widely and are unknown in the case of most identified variants. Here we describe a novel HARS variant, c.412T>C; p.Y138H, identified through a CMT gene panel in a patient with peripheral neuropathy. To determine the effect of p.Y138H we employed a humanized HARS yeast model and recombinant protein biochemistry, which identified a deficiency in protein dimerization and a growth defect which shows mild but significant improvement with histidine supplementation. This raises the potential for a clinical trial of histidine.

Overexpression of the pullulan synthetase gene enhanced pullulan production and its molecular weight by a mutant of Aureobasidium melanogenum P16

The pullulan synthetase gene (PUL1), involved in pullulan biosynthesis in Aureobasidium species, remains poorly understood. The open reading frame (ORF) of the PUL1 gene from the high pullulan-producing yeast Aureobasidium melanogenum P16 strain was cloned and characterized. The ORF of the PUL1 gene was determined to be 592 bp in length, encoding 178 amino acid residues. It was observed that an intron of 55 bp disrupted the gene. The promoter of the PUL1 gene contained a CAAT box, a TATA box, and a 5’-HGATAR-3′ sequence. The deduced protein possessed a signal peptide comprising 18 amino acids and harbored five potential N-glycosylation sites. Following the disruption of the PUL1 gene in strain P16, the disruptant DP108 yielded 34.7 ± 0.3 g/L of pullulan from sucrose, significantly lower than the production by its wild-type strain P16. This discrepancy underscores the close association between the PUL1 gene and pullulan biosynthesis. The majority of the fused Gfp-Pul1 proteins were found to be localized in the cell membrane and on the surface of vacuoles within the yeast-like fungal cells, indicating that pullulan biosynthesis occurred at these subcellular sites. Following the overexpression of the PUL1 gene, strain G14 produced >72.0 g/L of pullulan from sucrose, surpassing the production of its wild-type counterpart strain P16, which yielded 65.5 g/L of pullulan under the identical conditions. This outcome demonstrated that the overexpression of the PUL1 gene significantly enhanced pullulan production. The apparent molecular mass of the purified pullulan increased to 4.4 × 105 Da. As an auxiliary protein, Pul1 was predicted to bind to AmAgs2, the key enzyme in pullulan biosynthesis, facilitating enhanced pullulan production.

An evaluation of cyanobacterial occurrence and bloom development in Adirondack lakes

Cyanobacterial harmful algal blooms (cyanoHABs) have occurred in many low nutrient (oligotrophic) lakes in the northeastern United States. The Adirondack Park in New York is a large, mountainous region with many low nutrient lakes. There is a gap in understanding regarding whether cyanoHAB reporting data are truly reflective of the susceptibility of lakes to develop bloom conditions. We evaluated lakes with and without documented cyanoHABs for cyanotoxin synthetase gene quantification, phytoplankton community composition, and akinete abundance to identify conditions associated with the observation of cyanoHABs. We analyzed (1) contributions of cyanobacteria to the overall phytoplankton community, (2) differences in cyanobacterial communities and the presence of cyanotoxin synthetase genes, and (3) lake physical and geomorphological attributes as drivers of differences in cyanobacteria occurrence. Two sample types (water and sediment) were collected from 2 sample locations (nearshore and open water) in 5 lakes in 2021. We found cyanobacteria in all lakes and sample locations. Phytoplankton biovolume and cyanotoxin synthetase genes differed among lakes and by cyanoHAB history. Samples from lakes with documented blooms were associated with marginally higher total phosphorus. Nonmetric multidimensional scaling was used to identify which environmental factors influenced community structure. Our study demonstrates the importance of multifaceted approaches to detect cyanobacteria that may only be apparent during ephemeral bloom events and the similarities among lakes with and without a history of bloom reports. This work contributes to a better understanding of cyanoHAB occurrence in Adirondack lakes, and of conditions that may cause low nutrient lakes to be susceptible to cyanoHABs.

Exploration of Biomass-related Genes in the FOX-superroot Lines of Lotus corniculatus and its Impact on Agronomic Traits

Background: Identifying biomass-related genes to meet the increasing demand for food supply is important approach for agricultural and scientific efforts. In this study, we employed the Full-length cDNA Over-eXpressing gene hunting system (FOX hunting system) to identify biomass-related genes through ectopic expression of Arabidopsis thaliana full-length cDNA in bird’s-foot trefoil (Lotus corniculatus). Methods: A total of 66 FOX superroot lines (FSLs) from Lotus corniculatus cv. Viking were transferred in vermiculite after two weeks in half-strength Murashige and Skoog (MS) medium. These FSLs were then grown in a growth chamber for four weeks, during which their plant length and root length were measured. High biomass lines were selected from this group. FSL #83 and #121 were then grown for an additional four months in a greenhouse until they reached maturity, at which point they were evaluated for their agronomic traits. Result: Out of the 66 analyzed FSLs, 15 displayed superior growth characteristics in plant length and total root length. Arabidopsis cDNA, categorized into genes related to various functions such as membrane transport, photosystems, phytohormone synthesis, amino acid synthesis and unknown genes, was introduced into these lines. Two lines were grown to maturity in the greenhouse and their agronomic traits were analyzed in detail. FSL #83 expressed brassinosteroid biosynthetic pathway gene (CPD) led to significant differences in internode length, number of stems, number of flowers and above-ground dry matter weight compared to non-transgenic Super-growing roots (SR). On the other hand, FSL #121 expressed Asparaginyl-tRNA synthetase (SYNC1) gene exhibited increases in plant length and the number of stem nodes.