YkuR functions as a protein deacetylase in Streptococcus mutans.

Protein acetylation is a common and reversible posttranslational modification tightly governed by protein acetyltransferases and deacetylases crucial for various biological processes in both eukaryotes and prokaryotes. Although recent studies have characterized many acetyltransferases in diverse bacterial species, only a few protein deacetylases have been identified in prokaryotes, perhaps in part due to their limited sequence homology. In this study, we identified YkuR, encoded by smu_318, as a unique protein deacetylase in Streptococcus mutans. Through protein acetylome analysis, we demonstrated that the deletion of ykuR significantly upregulated protein acetylation levels, affecting key enzymes in translation processes and metabolic pathways, including starch and sucrose metabolism, glycolysis/gluconeogenesis, and biofilm formation. In particular, YkuR modulated extracellular polysaccharide synthesis and biofilm formation through the direct deacetylation of glucosyltransferases (Gtfs) in the presence of NAD+. Intriguingly, YkuR can be acetylated in a nonenzymatic manner, which then negatively regulated its deacetylase activity, suggesting the presence of a self-regulatory mechanism. Moreover, in vivo studies further demonstrated that the deletion of ykuR attenuated the cariogenicity of S. mutans in the rat caries model, substantiating its involvement in the pathogenesis of dental caries. Therefore, our study revealed a unique regulatory mechanism mediated by YkuR through protein deacetylation that regulates the physiology and pathogenicity of S. mutans.

Cellulose synthase-like OsCSLD4: a key regulator of agronomic traits, disease resistance, and metabolic indices in rice.

KEY MESSAGE: Cellulose synthase-like OsCSLD4 plays a pivotal role in regulating diverse agronomic traits, enhancing resistance against bacterial leaf blight, and modulating metabolite indices based on the multi-omics analysis in rice. To delve deeper into this complex network between agronomic traits and metabolites in rice, we have compiled a dataset encompassing genome, phenome, and metabolome, including 524 diverse accessions, 11 agronomic traits, and 841 metabolites, enabling us to pinpoint eight hotspots through GWAS. We later discovered four distinct metabolite categories, encompassing 15 metabolites that are concurrently present on the QTL qC12.1, associated with leaf angle of flag and spikelet length, and finally focused the cellulose synthase-like OsCSLD4, which was pinpointed through a rigorous process encompassing sequence variation, haplotype, ATAC, and differential expression across diverse tissues. Compared to the wild type, csld4 exhibited significant reductions in the plant height, flag leaf length, leaf width, spikelet length, 1000-grain weight, grain width, grain thickness, fertility, yield per plant, and bacterial blight resistance. However, there were significant increase in tiller numbers, degree of leaf rolling, flowering period, growth period, grain length, and empty kernel rate. Furthermore, the content of four polyphenol metabolites, excluding metabolite N-feruloyltyramine (mr1268), notably rose, whereas the levels of the other three polyphenol metabolites, smiglaside C (mr1498), 4-coumaric acid (mr1622), and smiglaside A (mr1925) decreased significantly in mutant csld4. The content of amino acid L-tyramine (mr1446) exhibited a notable increase, whereas the alkaloid trigonelline (mr1188) displayed a substantial decrease among the mutants. This study offered a comprehensive multi-omics perspective to analyze the genetic mechanism of OsCSLD4, and breeders can potentially enhance rice's yield, bacterial leaf blight resistance, and metabolite content, leading to more sustainable and profitable rice production.

Structural basis for synthase activation and cellulose modification in the E. coli Type II Bcs secretion system.

Bacterial cellulosic polymers constitute a prevalent class of biofilm matrix exopolysaccharides that are synthesized by several types of bacterial cellulose secretion (Bcs) systems, which include conserved cyclic diguanylate (c-di-GMP)-dependent cellulose synthase modules together with diverse accessory subunits. In E. coli, the biogenesis of phosphoethanolamine (pEtN)-modified cellulose relies on the BcsRQABEFG macrocomplex, encompassing inner-membrane and cytosolic subunits, and an outer membrane porin, BcsC. Here, we use cryogenic electron microscopy to shed light on the molecular mechanisms of BcsA-dependent recruitment and stabilization of a trimeric BcsG pEtN-transferase for polymer modification, and a dimeric BcsF-dependent recruitment of an otherwise cytosolic BcsE2R2Q2 regulatory complex. We further demonstrate that BcsE, a secondary c-di-GMP sensor, can remain dinucleotide-bound and retain the essential-for-secretion BcsRQ partners onto the synthase even in the absence of direct c-di-GMP-synthase complexation, likely lowering the threshold for c-di-GMP-dependent synthase activation. Such activation-by-proxy mechanism could allow Bcs secretion system activity even in the absence of substantial intracellular c-di-GMP increase, and is reminiscent of other widespread synthase-dependent polysaccharide secretion systems where dinucleotide sensing and/or synthase stabilization are carried out by key co-polymerase subunits.

Postnatal Wing Morph of Pea Aphids Regulates Hemolymph Trehalose Levels.

Trehalose, a nonreducing disaccharide composed of two glucose molecules, functions as a critical energy source in various insect tissues and organs and is the predominant sugar component of the hemolymph. The pea aphid, Acyrthosiphon pisum, exhibits higher hemolymph trehalose levels than other insects. However, the dynamics of hemolymph trehalose levels throughout its life stages remain unclear owing to the challenges associated with obtaining hemolymph from these small insects. Therefore, this study was conducted to quantify hemolymph trehalose levels in A. pisum using a fluorescent trehalose sensor (Tre-C04), which enhances green fluorescent protein fluorescence through the binding of trehalose to a ligand-binding protein fused to the fluorophore. Trehalose levels were successfully quantified in minimal hemolymph samples from individual aphids, with measurements spanning from the first nymphal stage to the adult stage in both the winged and wingless forms of A. pisum. Hemolymph trehalose levels remained relatively stable throughout the life cycle but exhibited a gradual increase with each developmental stage. Notably, adult winged aphids exhibited significantly higher hemolymph trehalose levels than wingless aphids. Given that wing morph determination occurs early in the nymphal stage, these findings suggest that hemolymph trehalose levels are regulated post-wing morph development. Further investigation of the expression of genes associated with trehalose metabolism revealed that trehalose phosphate synthase 2 levels were downregulated in early-stage wingless adults, whereas insulin-related peptide 5 levels were upregulated in wingless aphids. These findings indicate that A. pisum synthesizes trehalose during the winged adult stage to serve as an energy source for flight.

Evolutionary dynamics in gut-colonizing Candida glabrata during caspofungin therapy: Emergence of clinically important mutations in sphingolipid biosynthesis.

Invasive fungal infections are associated with high mortality, which is exacerbated by the limited antifungal drug armamentarium and increasing antifungal drug resistance. Echinocandins are a frontline antifungal drug class targeting ß-glucan synthase (GS), a fungal cell wall biosynthetic enzyme. Echinocandin resistance is generally low but increasing in species like Candida glabrata, an opportunistic yeast pathogen colonizing human mucosal surfaces. Mutations in GS-encoding genes (FKS1 and FKS2 in C. glabrata) are strongly associated with clinical echinocandin failure, but epidemiological studies show that other, as yet unidentified factors also influence echinocandin susceptibility. Furthermore, although the gut is known to be an important reservoir for emergence of drug-resistant strains, the evolution of resistance is not well understood. Here, we studied the evolutionary dynamics of C. glabrata colonizing the gut of immunocompetent mice during treatment with caspofungin, a widely-used echinocandin. Whole genome and amplicon sequencing revealed rapid genetic diversification of this C. glabrata population during treatment and the emergence of both drug target (FKS2) and non-drug target mutations, the latter predominantly in the FEN1 gene encoding a fatty acid elongase functioning in sphingolipid biosynthesis. The fen1 mutants displayed high fitness in the gut specifically during caspofungin treatment and contained high levels of phytosphingosine, whereas genetic depletion of phytosphingosine by deletion of YPC1 gene hypersensitized the wild type strain to caspofungin and was epistatic to fen1Δ. Furthermore, high resolution imaging and mass spectrometry showed that reduced caspofungin susceptibility in fen1Δ cells was associated with reduced caspofungin binding to the plasma membrane. Finally, we identified several different fen1 mutations in clinical C. glabrata isolates, which phenocopied the fen1Δ mutant, causing reduced caspofungin susceptibility. These studies reveal new genetic and molecular determinants of clinical caspofungin susceptibility and illuminate the dynamic evolution of drug target and non-drug target mutations reducing echinocandin efficacy in patients colonized with C. glabrata.

Insights into phosphoethanolamine cellulose synthesis and secretion across the Gram-negative cell envelope.

Phosphoethanolamine (pEtN) cellulose is a naturally occurring modified cellulose produced by several Enterobacteriaceae. The minimal components of the E. coli cellulose synthase complex include the catalytically active BcsA enzyme, a hexameric semicircle of the periplasmic BcsB protein, and the outer membrane (OM)-integrated BcsC subunit containing periplasmic tetratricopeptide repeats (TPR). Additional subunits include BcsG, a membrane-anchored periplasmic pEtN transferase associated with BcsA, and BcsZ, a periplasmic cellulase of unknown biological function. While cellulose synthesis and translocation by BcsA are well described, little is known about its pEtN modification and translocation across the cell envelope. We show that the N-terminal cytosolic domain of BcsA positions three BcsG copies near the nascent cellulose polymer. Further, the semicircle's terminal BcsB subunit tethers the N-terminus of a single BcsC protein in a trans-envelope secretion system. BcsC's TPR motifs bind a putative cello-oligosaccharide near the entrance to its OM pore. Additionally, we show that only the hydrolytic activity of BcsZ but not the subunit itself is necessary for cellulose secretion, suggesting a secretion mechanism based on enzymatic removal of translocation incompetent cellulose. Lastly, protein engineering introduces cellulose pEtN modification in orthogonal cellulose biosynthetic systems. These findings advance our understanding of pEtN cellulose modification and secretion.

Development of a Chitin-Based Purification System Utilizing Chitin-Binding Domain and Tobacco Etch Virus Protease Cleavage for Efficient Recombinant Protein Recovery.

This study aims to develop an efficient chitin-based purification system, leveraging a novel design where the target proteins, superfolding green fluorescent protein (sfGFP) and Thermus antranikianii trehalose synthase (TaTS), fused with a chitin-binding domain (ChBD) from Bacillus circulans WL-12 chitinase A1 and a tobacco etch virus protease (TEVp) cleavage site. This configuration allows for the effective immobilization of the target proteins on chitin beads, facilitating the removal of endogenous proteins. A mutant TEVp, H-TEVS219V-ChBD, fused with the His-tag and ChBD, is employed to cleave the target proteins from the chitin beads specifically. Subsequently, fresh chitin beads are added for adsorption to remove H-TEVS219V-ChBD in the solution, thereby significantly improving the purity of the target protein. Our results confirm that this system can efficiently and specifically purify and recover sfGFP and TaTS, achieving electrophoretic-grade purity exceeding 90%. This system holds significant potential for industrial production and other applications.

UDP-glucose ceramide glucosyltransferase specifically upregulated in plasmacytoid dendritic cells regulates type I interferon production upon CpG stimulation.

Plasmacytoid dendritic cells (pDCs) are a distinct subset of DCs involved in immune regulation and antiviral immune responses. Recent studies have elucidated the metabolic profile of pDCs and reported that perturbations in amino acid metabolism can modulate their immune functions. Glycolipid metabolism is suggested to be highly active in pDCs; however, its significance remains unclear. In this study, bulk RNA-sequencing analysis confirmed the known pDC-marker expressions, including interleukin (IL)-3R (CD123), BDCA-2 (CD303), BDCA-4 (CD304), and toll-like receptor 9, compared with that of myeloid DCs (mDCs). Among the differentially expressed genes, UDP-glucose-ceramide glucosyltransferase (UGCG) expression was significantly upregulated in pDCs than in mDCs. Moreover, pDC-specific UGCG expression was observed at both the mRNA and protein levels in pDCs and pDC-like cell lines, including CAL-1 and PMDC05 cell lines. Pharmacological or clustered regularly interspaced palindromic repeat (CRISPR)/CRISPR-associated protein 9-mediated genetic inhibition of UGCG did not affect the pDC phenotype as evidenced by the persistent expression of IL-3R and BDCA-2 in pDC-like cell lines. However, UGCG knockout resulted in reduced type I interferon production in pDCs upon CpG activation. In addition, UGCG-knockout pDC-like cell lines exhibited reduced transduction by vesicular stomatitis virus-G pseudo-typed lentiviral vectors, suggesting that low UGCG expression hinders infectivity. Collectively, our findings suggest that pDC-specific UGCG expression is critical for cytokine production and antiviral immune responses in pDCs.

Effect of delignification on the adsorption of loofah sponge-based immobilized metal affinity chromatography adsorbent for His-tagged trehalose synthase.

The effect of delignification on the adsorption capacity of loofah sponge-based immobilized metal affinity chromatography adsorbents was investigated with recombinant His-tagged trehalose synthase as the model protein. Pretreatments with [EMIM][Ac] ionic liquid at 80 °C for 5 h and with sodium chlorite/acetic acid at 80 °C for 2 h were found effective for the removal of lignin, leading to a loss in biomass of 15.7% and 25.2%, respectively. Upon delignification, the metal chelating capacities of the loofah sponge-based adsorbents prepared with 5-h ionic liquid pretreatment (712 ± 82 µmole Cu(II)/g) and with 2-h sodium chlorite/acetic acid pretreatment (1012 ± 18 µmole Cu(II)/g) were 38% and 97% higher than that of the control (514 ± 55 µmole Cu(II)/g), adsorbent prepared with untreated loofah sponge, respectively. Results of protein adsorption study indicated that the Co(II)-loaded adsorbent prepared with 2-h sodium chlorite/acetic acid pretreatment exhibited the highest adsorption capacity and selectivity for the recombinant His-tagged trehalose synthase, giving a purification product with a specific activity of 7.62 U/mg protein. The predicted maximum adsorption capacity of the delignified loofah sponge-based adsorbent, 2.04 ± 0.14 mg/g, was 73% higher than that of the control.

Advancements in the Heterologous Expression of Sucrose Phosphorylase and Its Molecular Modification for the Synthesis of Glycosylated Products.

Sucrose phosphorylase (SPase), a member of the glycoside hydrolase GH13 family, possesses the ability to catalyze the hydrolysis of sucrose to generate α-glucose-1-phosphate and can also glycosylate diverse substrates, showcasing a wide substrate specificity. This enzyme has found extensive utility in the fields of food, medicine, and cosmetics, and has garnered significant attention as a focal point of research in transglycosylation enzymes. Nevertheless, SPase encounters numerous obstacles in industrial settings, including low enzyme yield, inadequate thermal stability, mixed regioselectivity, and limited transglycosylation activity. In-depth exploration of efficient expression strategies and molecular modifications based on the crystal structure and functional information of SPase is now a critical research priority. This paper systematically reviews the source microorganisms, crystal structure, and catalytic mechanism of SPase, summarizes diverse heterologous expression systems based on expression hosts and vectors, and examines the application and molecular modification progress of SPase in synthesizing typical glycosylated products. Additionally, it anticipates the broad application prospects of SPase in industrial production and related research fields, laying the groundwork for its engineering modification and industrial application.

PagTPS1 and PagTPS10, the trehalose-6-phosphate synthase genes, increase trehalose content and enhance drought tolerance.

Trehalose-6-phosphate synthase (TPS) genes play an active role in the trehalose metabolism pathway that regulates the responses of plants to diverse stresses. However, the functional identification, comparison, and conservatism of TPS genes in the responses of woody plants, especially poplars, to drought stress remain unclear. Here, the trehalose content of 84K (Populus alba × P. glandulosa) poplars was down-regulated and PagTPS and PagTPP genes had diverse response patterns under drought stress. Physicochemical properties, expression patterns, and functions of PagTPS1 and PagTPS10, two class I members of TPS gene family, were identified and compared. Transgenic 84K poplars overexpressing PagTPS1 and PagTPS10 had significantly higher trehalose content with approximately 138% and 123%, respectively, and stronger drought tolerance compared to WT. PagTPS1 and PagTPS10 promoted the expression of TPPA genes and drought-responsive genes. Accordingly, poplars inhibiting PagTPS1 and PagTPS10 expression via RNA interference had lower trehalose content and drought tolerance. Simultaneously, overexpressing PagTPS1 and PagTPS10 improved the trehalose content and drought tolerance of Arabidopsis. Overall, we proposed a model of the effects of PagTPS1 and PagTPS10 as conservative regulators on the responses of plants to drought, which would provide new insights into the functional explorations of TPS genes in plants.

[Research progress in bacterial cellulose synthase subunit diversity and fiber structure formation].

Bacterial cellulose (BC) is the glucose polymer produced by bacterial metabolism. The bacterial cellulose synthase (BCS) is the key enzyme for catalyzing the formation of BC. The cooperation between different submits of BCS is necessary for the intracellular formation and extracellular secretion of BC. This review summarized the BC-producing strains and the differences of BCS among different strains. Furthermore, we detailed the BC synthesis mechanism, the interactions between BCS subunits, and the relationship between the structural characteristics of strains and the formation of highly ordered fiber structures. A comprehensive insight into the mechanism of BC synthesis and secretion will supply more strategies for optimizing the BC synthesis via methods of synthetic biology.

[Efficient synthesis of salidroside from tyrosol based on UDPG recycling system].

Salidroside is a functional ingredient with wide applications in food and pharmaceutical fields. It is conventionally produced by extraction from plants, the application of which is limited by the scarcity of raw materials and cumbersome process. This study achieved the efficient production of salidroside by biosynthesis with tyrosol as the substrate. While utilizing glycosyltransferases for tyrosol glycosylation, we introduced sucrose synthase to construct the uridine diphosphate glucose (UDPG) recycling system. The glycosyltransferase UGT33 and sucrose synthase AtSUS were screened out by comparison, and the recombinant strain Escherichia coli BL21/pETDuet-AtSUS-UGT33 was constructed. The copy number of the gene was optimized and the optimal copy number ratio of glycosyltransferase to sucrose synthase was determined to be 3:1. The whole-cell transformation conditions (temperature, pH, inoculum amount, substrate concentration, and concentrations of metal ions) of the recombinant strain were optimized, and the highest yield of salidroside reached 8.17 g/L after fermentation under the optimal conditions in a 5 L fermenter for 24 h. This study provides a reference for the efficient production of salidroside by microorganisms.

Insights into the interaction between UGGT, the gatekeeper of folding in the ER, and its partner, the selenoprotein SEP15.

The enzyme UDP-glucose: glycoprotein glucosyltransferase (UGGT) is the gatekeeper of protein folding within the endoplasmic reticulum (ER). One-third of the human proteome traverses the ER where folding and maturation are facilitated by a complex protein homeostasis network. Both glycan modifications and disulfide bonds are of key importance in the maturation of these ER proteins. The actions of UGGT are intimately linked to the glycan code for folding and maturation of secretory proteins in the ER. UGGT selectively glucosylates the N-linked glycan of misfolded proteins so that they can reenter the lectin-folding chaperone cycle and be retained within the ER for further attempts at folding. An intriguing aspect of UGGT function is its interaction with its poorly understood cochaperone, the 15 kDa selenoprotein known as SELENOF or SEP15. This small protein contains a rare selenocysteine residue proposed to act as an oxidoreductase toward UGGT substrates. AlphaFold2 predictions of the UGGT1/SEP15 complex provide insight into this complex at a structural level. The predicted UGGT1/SEP15 interaction interface was validated by mutagenesis and coimmunoprecipitation experiments. These results serve as a springboard for models of the integrated action of UGGT1 and SEP15.

Glucosylceramide synthase modulation ameliorates murine renal pathologies and promotes macrophage effector function in vitro.

While significant advances have been made in understanding renal pathophysiology, less is known about the role of glycosphingolipid (GSL) metabolism in driving organ dysfunction. Here, we used a small molecule inhibitor of glucosylceramide synthase to modulate GSL levels in three mouse models of distinct renal pathologies: Alport syndrome (Col4a3 KO), polycystic kidney disease (Nek8jck), and steroid-resistant nephrotic syndrome (Nphs2 cKO). At the tissue level, we identified a core immune-enriched transcriptional signature that was shared across models and enriched in human polycystic kidney disease. Single nuclei analysis identified robust transcriptional changes across multiple kidney cell types, including epithelial and immune lineages. To further explore the role of GSL modulation in macrophage biology, we performed in vitro studies with homeostatic and inflammatory bone marrow-derived macrophages. Cumulatively, this study provides a comprehensive overview of renal dysfunction and the effect of GSL modulation on kidney-derived cells in the setting of renal dysfunction.

Trehalose mediates salinity-stress tolerance in natural populations of a freshwater crustacean.

Salinization poses an increasing problem worldwide, threatening freshwater organisms and raising questions about their ability to adapt. We explored the mechanisms enabling a planktonic crustacean to tolerate elevated salinity. By gradually raising water salinity in clonal cultures from 185 Daphnia magna populations, we showed that salt tolerance strongly correlates with native habitat salinity, indicating local adaptation. A genome-wide association study (GWAS) further revealed a major effect of the Alpha,alpha-trehalose-phosphate synthase (TPS) gene, suggesting that trehalose production facilitates salinity tolerance. Salinity-tolerant animals showed a positive correlation between water salinity and trehalose concentrations, while intolerant animals failed to produce trehalose. Animals with a non-functional TPS gene, generated through CRISPR-Cas9, supported the trehalose role in salinity stress. Our study highlights how a keystone freshwater animal adapts to salinity stress using an evolutionary mechanism known in bacteria, plants, and arthropods.

Kinetics of Secoisolariciresinol Glucosyltransferase LuUGT74S1 and Its Mutants.

The lignan secoisolariciresinol (SECO) diglucoside (SDG) is a phytoestrogen with diverse effects. LuUGT74S1 glucosylates SECO to SDG, whereby only small amounts of the monoglucoside SMG are formed intermediately, which exhibit increased activity. To identify critical amino acids that are important for enzymatic activity and the SMG/SDG ratio, 3D structural modeling and docking, as well as site-directed mutation studies, were performed. Enzyme assays with ten mutants revealed that four of them had identical kinetic data to LuUGT74S1, while three showed reduced and one increased catalytic efficiency kcat/Km. S82F and E189L substitutions resulted in the complete absence of activity. A17 and Q136 are crucial for the conversion of SMG to SDG as A17S and Q136F mutants exhibited the highest SMG/SDG ratios of 0.7 and 0.4. Kinetic analyses show that diglucosylation is an essentially irreversible reaction, while monoglycosylation is kinetically favored. The results lay the foundation for the biotechnological production of SMG.

Ceramide metabolism alterations contribute to Tumor Necrosis Factor-induced melanoma dedifferentiation and predict resistance to immune checkpoint inhibitors in advanced melanoma patients.

Introduction: Advanced cutaneous melanoma is a skin cancer characterized by a poor prognosis and high metastatic potential. During metastatic spread, melanoma cells often undergo dedifferentiation toward an invasive phenotype, resulting in reduced expression of microphthalmia-associated transcription factor (MITF)-dependent melanoma antigens and facilitating immune escape. Tumor Necrosis Factor (TNF) is known to be a key factor in melanoma dedifferentiation. Interestingly, accumulating evidence suggests that TNF may play a role in melanoma progression and resistance to immunotherapies. Additionally, TNF has been identified as a potent regulator of sphingolipid metabolism, which could contribute to melanoma aggressiveness and the process of melanoma dedifferentiation. Methods: We conducted RNA sequencing and mass spectrometry analyses to investigate TNF-induced dedifferentiation in two melanoma cell lines. In vitro experiments were performed to manipulate sphingolipid metabolism using genetic or pharmacologic alterations in combination with TNF treatment, aiming to elucidate the potential involvement of this metabolism in TNF-induced dedifferentiation. Lastly, to evaluate the clinical significance of our findings, we performed unsupervised analysis of plasma sphingolipid levels in 48 patients receiving treatment with immune checkpoint inhibitors, either alone or in combination with anti-TNF therapy. Results: Herein, we demonstrate that TNF-induced melanoma cell dedifferentiation is associated with a global modulation of sphingolipid metabolism. Specifically, TNF decreases the expression and activity of acid ceramidase (AC), encoded by the ASAH1 gene, while increasing the expression of glucosylceramide synthase (GCS), encoded by the UGCG gene. Remarkably, knockdown of AC alone via RNA interference is enough to induce melanoma cell dedifferentiation. Furthermore, treatment with Eliglustat, a GCS inhibitor, inhibits TNF-induced melanoma cell dedifferentiation. Lastly, analysis of plasma samples from patients treated with immune checkpoint inhibitors, with or without anti-TNF therapy, revealed significant predictive sphingolipids. Notably, the top 8 predictive sphingolipids, including glycosphingolipids, were associated with a poor response to immunotherapy. Discussion: Our study highlights that ceramide metabolism alterations are causally involved in TNF-induced melanoma cell dedifferentiation and suggests that the evolution of specific ceramide metabolites in plasma may be considered as predictive biomarkers of resistance to immunotherapy.

Genome-Wide Identification and Analysis of SUS and AGPase Family Members in Sweet Potato: Response to Excessive Nitrogen Stress during Storage Root Formation.

(1) The development of sweet potato storage roots is impacted by nitrogen (N) levels, with excessive nitrogen often impeding development. Starch synthesis enzymes such as sucrose synthase (SUS) and ADP-glucose pyrophosphorylase (AGPase) are pivotal in this context. Although the effects of excessive nitrogen on the formation of sweet potato storage roots are well documented, the specific responses of IbSUSs and IbAGPases have not been extensively reported on. (2) Pot experiments were conducted using the sweet potato cultivar "Pushu 32" at moderate (MN, 120 kg N ha-1) and excessive nitrogen levels (EN, 240 kg N ha-1). (3) Nine IbSUS and nine IbAGPase genes were categorized into three and two distinct subgroups based on phylogenetic analysis. Excessive nitrogen significantly (p < 0.05) suppressed the expression of IbAGPL1, IbAGPL2, IbAGPL4, IbAGPL5, IbAGPL6, IbAGPS1, and IbAGPS2 in fibrous roots and IbSUS2, IbSUS6, IbSUS7, IbSUS8, IbSUS9, IbAGPL2, and IbAGPL4 in storage roots, and then significantly (p < 0.05) decreased the SUS and AGPase activities and starch content of fibrous root and storage root, ultimately reducing the storage root formation of sweet potato. Excessive nitrogen extremely significantly (p < 0.01) enhanced the expression of IbAGPL3, which was strongly negatively correlated with the number and weight of storage roots per plant. (4) IbAGPL3 may be a key gene in the response to excessive nitrogen stress and modifying starch synthesis in sweet potato.

Melatonin mitigates drought stress by increasing sucrose synthesis and suppressing abscisic acid biosynthesis in tomato seedlings.

The increasing prevalence of drought events poses a major challenge for upcoming crop production. Melatonin is a tiny indolic tonic substance with fascinating regulatory functions in plants. While plants can respond in several ways to alleviate drought stress, the processes underpinning stress sensing and signaling are poorly understood. Hereafter, the objectives of this investigation were to explore the putative functions of melatonin in the regulation of sugar metabolism and abscisic acid biosynthesis in drought-stressed tomato seedlings. Melatonin (100 µM) and/or water were foliar sprayed, followed by the plants being imposed to drought stress for 14 days. Drought stress significantly decreased biomass accumulation, inhibited photosynthetic activity, and stimulated senescence-associated gene 12 (SAG12) expression. Melatonin treatment effectively reversed drought-induced growth retardation as evidenced by increased leaf pigment and water balance and restricted abscisic acid (ABA) accumulation. Sugar accumulation, particularly sucrose content, was higher in drought-imposed seedlings, possibly owing to higher transcription levels of sucrose non-fermenting 1-related protein kinase 2 (SnKR2.2) and ABA-responsive element binding factors 2 (AREB2). Melatonin addition further uplifted the sucrose content, which coincided with increased activity of sucrose synthase (SS, 130%), sucrose phosphate synthase (SPS, 137%), starch degradation encoding enzyme ß-amylase (BAM, 40%) and α-amylase (AMY, 59%) activity and upregulated their encoding BAM1(10.3 folds) and AMY3 (8.1 folds) genes expression at day 14 relative to the control. Under water deficit conditions, melatonin supplementation decreased the ABA content (24%) and its biosynthesis gene expressions. Additionally, sugar transporter subfamily genes SUT1 and SUT4 expression were upregulated by the addition of melatonin. Collectively, our findings illustrate that melatonin enhances drought tolerance in tomato seedlings by stimulating sugar metabolism and negatively regulating ABA synthesis.