Trehalose-6-phosphate Synthase Research Articles

Optimizing the leaf-to-bunch ratio in ‘Mazafati’ date trees by considering source-sink limitation and physiological-molecular responses

The balance between carbon production and consumption is regulated by source and sink, which can limit plant performance. When there is an imbalance between source and sink, the plant‘s performance is affected. Thus, source and sink are interdependent. We conducted a two-year study (220-221) to investigate how different leaf-to–bunch ratios (LBR) (7-12) affect source–sink balance and yield in ’Mazafati’ date trees. We also determined the optimal number of bunches per tree as the main carbon sinks competing with other organs. Trehalose is a non-reducing sugar that protects cellular structure and function under stress. We found that 9–10 LBR-trees had no biotic stress due to carbon deficiency or excess, as indicated by minimal expression of the trehalose-6-phosphate synthase (TPS) gene and synthesis of trehalose. This alleviated source-sink limitation and resulted in the highest yield, bunch weight, fruit weight and flavor, water use efficiency, and reduced abscised fruit. Hormones gibberellic acid (GA3) and abscisic acid (ABA), gene expression of sucrose phosphate synthase 1 (SPS1), enzyme activities of sucrose phosphate synthase (SPS) and sucrose synthase (SuSy) related to sucrose synthesis, reducing and non-reducing sugars, and starch increased with increasing LBR in date palm fruit. Based on our findings, we concluded that a LBR of 9–10 optimizes fruit quality in date palm.

α,β-trehalose, an intracellular substance in resting cyst of colpodid ciliates as a key to environmental tolerances

α,α-trehalose is a well-known sugar that plays a key role in establishing tolerance to environmental stresses in many organisms, except unicellular eukaryotes. However, almost nothing is known about α,β-trehalose, including their synthesis, function, and even presence in living organisms. In this study, we identified α,β-trehalose in the resting cyst, a dormancy cell form characterized by extreme tolerance to environmental stresses, of the ciliated protist Colpoda cucullus, using high-performance liquid chromatography (HPLC), and a proton nuclear magnetic resonance (1H NMR). Gene expression analysis revealed that the expression of trehalose-6-phosphate synthase (TPS), glycosyltransferase (GT), alpha-amylase (AMY), and trehalose transporter 1 (TRET1), were up-regulated in encystment, while the expression of α-glucosidase 2 (AG2) and trehalase (TREH) was up-regulated in excystment. These results suggest that α,β-trehalose is synthesized during encystment process, while and contributes to extreme tolerances to environmental stressors, stored carbohydrates, and energy reserve during resting cyst and/or during excystment.

Overexpression of oilseed rape trehalose-6-phosphate synthesis gene BnaC02.TPS8 confers sensitivity to low nitrogen and high sucrose-induced anthocyanin accumulation in Arabidopsis.

Overexpression of BnaC02.TPS8 increased low N and high sucrose-induced anthocyanin accumulation. Anthocyanin plays a crucial role in safeguarding photosynthetic tissues against high light, UV radiation, and oxidative stress. Their accumulation is triggered by low nitrogen (N) stress and elevated sucrose levels in Arabidopsis. Trehalose-6-phosphate (T6P) serves as a pivotal signaling molecule, sensing sucrose availability, and carbon (C) metabolism. However, the mechanisms governing the regulation of T6P synthase (TPS) genes responsible for anthocyanin accumulation under conditions of low N and high sucrose remain elusive. In a previous study, we demonstrated the positive impact of a cytoplasm-localized class II TPS protein 'BnaC02.TPS8' on photosynthesis and seed yield improvement in Brassica napus. The present research delves into the biological role of BnaC02.TPS8 in response to low N and high sucrose. Ectopic overexpression of BnaC02.TPS8 in Arabidopsis seedlings resulted in elevated shoot T6P levels under N-sufficient conditions, as well as an increased carbon-to-nitrogen (C/N) ratio, sucrose accumulation, and starch storage under low N conditions. Overexpression of BnaC02.TPS8 in Arabidopsis heightened sensitivity to low N stress and high sucrose levels, accompanied by increased anthocyanin accumulation and upregulation of genes involved in flavonoid biosynthesis and regulation. Metabolic profiling revealed increased levels of intermediate products of carbon metabolism, as well as anthocyanin and flavonoid derivatives in BnaC02.TPS8-overexpressing Arabidopsis plants under low N conditions. Furthermore, yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) analyses demonstrated that BnaC02.TPS8 interacts with both BnaC08.TPS9 and BnaA01.TPS10. These findings contribute to our understanding of how TPS8-mediated anthocyanin accumulation is modulated under low N and high sucrose conditions.

Arabidopsis class II TPS controls root development and confers salt stress tolerance through enhanced hydrophobic barrier deposition.

The mechanism of conferring salt tolerance by AtTPS9 involves enhanced deposition of suberin lamellae in the Arabidopsis root endodermis, resulting in reduction of Na+ transported to the leaves. Members of the class I trehalose-6-phosphate synthase (TPS) enzymes are known to play an important role in plant growth and development in Arabidopsis. However, class II TPSs and their functions in salinity stress tolerance are not well studied. We characterized the function of a class II TPS gene, AtTPS9, to understand its role in salt stress response and root development in Arabidopsis. The attps9 mutant exhibited significant reduction of soluble sugar levels in the leaves and formation of suberin lamellae (SL) in the endodermis of roots compared to the wild type (WT). The reduction in SL deposition (hydrophobic barriers) leads to increased apoplastic xylem loading, resulting in enhanced Na+ content in the plants, which explains salt sensitivity of the mutant plants. Conversely, AtTPS9 overexpression lines exhibited increased SL deposition in the root endodermis along with increased salt tolerance, showing that regulation of SL deposition is one of the mechanisms of action of AtTPS9 in conferring salt tolerance to Arabidopsis plants. Our data showed that besides salt tolerance, AtTPS9 also regulates seed germination and root development. qRT-PCR analyses showed significant downregulation of selected SNF1-RELATED PROTEIN KINASE2 genes (SnRK2s) and ABA-responsive genes in the mutant, suggesting that AtTPS9 may regulate the ABA-signaling intermediates as part of the mechanism conferring salinity tolerance.

Direct and indirect responses of the Arabidopsis transcriptome to an induced increase in trehalose 6-phosphate.

Trehalose 6-phosphate (Tre6P) is an essential signal metabolite that regulates the level of sucrose, linking growth and development to the metabolic status. We hypothesized that Tre6P plays a role in mediating the regulation of gene expression by sucrose. To test this, we performed transcriptomic profiling on Arabidopsis (Arabidopsis thaliana) plants that expressed a bacterial TREHALOSE 6-PHOSPHATE SYNTHASE (TPS) under the control of an ethanol-inducible promoter. Induction led to a 4-fold rise in Tre6P levels, a concomitant decrease in sucrose, significant changes (FDR ≤ 0.05) of over 13,000 transcripts, and two-fold or larger changes of over 5000 transcripts. Comparison with nine published responses to sugar availability allowed some of these changes to be linked to the rise in Tre6P, while others were probably due to lower sucrose or other indirect effects. Changes linked to Tre6P included repression of photosynthesis-related gene expression and induction of many growth-related processes including ribosome biogenesis. About 500 starvation-related genes are known to be induced by SUCROSE-NON-FERMENTING-1-RELATED KINASE 1 (SnRK1). They were largely repressed by Tre6P in a manner consistent with SnRK1 inhibition by Tre6P. SnRK1 also represses many genes that are involved in biosynthesis and growth. These responded to Tre6P in a more complex manner, pointing toward Tre6P interacting with other C-signaling pathways. Additionally, elevated Tre6P modified the expression of genes encoding regulatory subunits of the SnRK1 complex and TPS class II and FCS-LIKE ZINC FINGER proteins that are thought to modulate SnRK1 function and genes involved in circadian, TARGET OF RAPAMYCIN-, light, abscisic acid, and other hormone signaling.

Exogenous trehalose improves the survival of date palm suckers by enhancing thioredoxin-antioxidant systems activity

Trehalose, a disaccharide, regulates osmotic pressure, stabilizes proteins, and plays in the signaling of carbon allocation in response to abiotic stresses. This study was evaluated the effects of 100 mM pre-separation (7 days before de-suckering) and post-separation (immediately after planting) spraying of trehalose on the survival of ‘Mazafati’ date palm suckers from a molecular perspective. Our results showed a 42% increase in the survival rate of the suckers sprayed with trehalose. By triggering trehalose-phosphate synthase (TPS) gene expression, promoting endogenous trehalose accumulation in suckers sprayed with trehalose could be responsible for sufficient intracellular NADP+ supplying arising from enhancing NAD kinase (NADK) enzyme activity. In addition, higher 2-cysteine peroxiredoxin (2-Cys-Prx) gene expression and enzyme activity could be ascribed to triggering NADPH-dependent thioredoxin reductase C (NTRC) gene expression and enzyme activity, promoting thioredoxin (Trx) enzyme activity concomitant with triggering nucleoside diphosphate kinase (NDPK) gene expression. Lower H2O2 accumulation in suckers sprayed with trehalose could be responsible for protecting membrane integrity as shown by lower electrolyte leakage and malondialdehyde (MDA) accumulation. In addition to promoting antioxidant system activity, higher phenols and anthocyanins accumulation in date palm suckers sprayed with trehalose could be ascribed to enhancing shikimate-phenylpropanoid pathways activity by Trx system activity. The results indicate that the Trx system could be employed by endogenous trehalose accumulation for improving photosynthesis efficiency and enhancing the survival rate of the suckers.

Transcriptome analysis reveals regulatory mechanisms of different drought-tolerant Gleditsia sinensis seedlings under drought stress

BackgroundGleditsia sinensis is a significant tree species from both ecological and economic perspectives. However, its growth is hampered by temporary droughts during the seedling stage, thereby impeding the development of the G. sinensis industry. Drought stress and rehydration of semi-annual potted seedlings using an artificial simulated water control method. RNA sequencing (RNA-seq) analyses were conducted on leaves collected from highly resistant (HR) and highly susceptible (HS) seedling families at five different stages during the process of drought stress and rehydration to investigate their gene expression patterns.ResultsThe differentially expressed genes (DEGs) were predominantly enriched in pathways related to “chloroplast” (GO:0009507), “photosynthesis” (GO:0015979), “plant hormone signal transduction” (map04075), “flavonoid biosynthesis” (map00941), “stress response”, “response to reactive oxygen species (ROS)” (GO:0000302), “signal transduction” (GO:0007165) in G. sinensis HR and HS families exposed to mild and severe drought stress. Additionally, the pathways related to “plant hormone signal transduction” (map04075), and osmoregulation were also enriched. The difference in drought tolerance between the two families of G. sinensis may be associated with “transmembrane transporter activity” (GO:0022857), “stress response”, “hormones and signal transduction” (GO:0007165), “cutin, suberine and wax biosynthesis” (map00073), “ribosome” (map03010), “photosynthesis” (map00195), “sugar metabolism”, and others. An enrichment analysis of DEGs under severe drought stress suggests that the drought tolerance of both families may be related to “water-soluble vitamin metabolic process” (GO:0006767), “photosynthesis” (map00195), “plant hormone signal transduction” (map04075), “starch and sucrose metabolism” (map00500), and “galactose metabolism” (map00052). Osmoregulation-related genes such as delta-1-pyrroline-5-carboxylate synthase (P5CS), Amino acid permease (AAP), Amino acid permease 2 (AAP2) and Trehalose-phosphate synthase (TPS), as well as the antioxidant enzyme L-ascorbate peroxidase 6 (APX6), may be significant genes involved in drought tolerance in G. sinensis. Five genes were selected randomly to validate the RNA-seq results using quantitative real-time PCR (RT-qPCR) and they indicated that the transcriptome data were reliable.ConclusionsThe study presents information on the molecular regulation of the drought tolerance mechanism in G. sinensis and provides a reference for further research on the molecular mechanisms involved in drought tolerance breeding of G. sinensis.

Trehalose 6-phosphate synthase gene rdtps1 contributes to thermal acclimation in Rhyzopertha dominica

BackgroundThe lesser grain borer (Rhyzopertha dominica), a worldwide primary pest of stored grain, causes serious economic losses and threatens stored food safety. R. dominica can respond to changes in temperature, especially the adaptability to heat. In this study, transcriptome analysis of R. dominica exposed to different temperatures was performed to elucidate differences in gene expression and the underling molecular mechanism.ResultsIsoform-sequencing generated 17,721,200 raw reads and yielded 20,416 full-length transcripts. A total of 18,880 (92.48%) transcripts were annotated. We extracted RNA from R. dominica reared at 5 °C (cold stress), 15 °C (cold stress), 27 °C (ambient temperature) and 40 °C (heat stress) for RNA-seq. Compared to those of control insects reared at 27 °C, 119, 342, and 875 differentially expressed genes (DEGs) were identified at 5 °C, 15 °C, and 40 °C, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that pathways associated with “fatty acid metabolism”, “fatty acid biosynthesis”, “AMPK signaling pathway”, “neuroactive ligand receptor interaction”, and “longevity regulating pathway-multiple species” were significantly enriched. The functional annotation revealed that the genes encoding heat shock proteins (HSPs), fatty acid synthase (FAS), phospholipases (PLA), trehalose transporter (TPST), trehalose 6-phosphate synthase (TPS), and vitellogenin (Vg) were most likely involved in temperature regulation, which was also validated by RT-qPCR. Seven candidate genes (rdhsp1, rdfas1, rdpla1, rdtpst1, rdtps1, rdvg1, and rdP450) were silenced in the RNA interference (RNAi) assay. RNAi of each candidate gene suggested that inhibiting rdtps1 expression significantly decreased the trehalose level and survival rate of R. dominica at 40 °C.ConclusionsThese results indicated that trehalose contributes to the high temperature resistance of R. dominica. Our study elucidates the molecular mechanisms underlying heat tolerance and provides a potential target for the pest management in R. dominica.

Identification of Two Enzymes for Trehalose Synthesis and Their Potential Function in Growth and Development in Peanut (Arachis hypogaea)

Plant trehalose has been regarded to play a key role in various biological processes during the growth and development stages. Trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) are two important enzymes for the synthesis of plant trehalose. Up till now, the TPS and TPP gene families have been identified and characterized in numerous higher plant species, but are rarely recorded in peanuts (Arachis hypogaea). In this study, a comprehensive search was performed to identify all putative TPS and TPP proteins in the peanut genome using Arabidopsis TPS and TPP proteins as queries. We then analyzed the characteristics of TPS and TPP members, including physic-chemical parameters, subcellular localization, phylogeny relationships, gene duplication, and expression patterns by various computational tools. As a result, a total of 17 ArahyTPS and 15 ArahyTPP genes were identified and annotated in the peanut genome, which was expanded by segmental duplication events. Our Neighbor-Joining based phylogenetic tree indicated that the ArahyTPS and ArahyTPP proteins could be categorized into three and two major branches. Gene structures and protein features analysis exhibited that the ArahyTPS and ArahyTPP proteins shared high structural and functional similarities. Based on previous RNA-Seq datasets, a majority of the ArahyTPS and ArahyTPP genes were found to specifically express in at least one major organ/tissue during the growth and development. This work will not only lead to a solid foundation on reveal the potential roles of ArahyTPS and ArahyTPP gene families in peanuts but also provide evidence to related trehalose research in other higher plant species.

Genome-wide identification of TPS and TPP genes in cultivated peanut (Arachis hypogaea) and functional characterization of AhTPS9 in response to cold stress.

Trehalose is vital for plant metabolism, growth, and stress resilience, relying on Trehalose-6-phosphate synthase (TPS) and Trehalose-6-phosphate phosphatase (TPP) genes. Research on these genes in cultivated peanuts (Arachis hypogaea) is limited. This study employed bioinformatics to identify and analyze AhTPS and AhTPP genes in cultivated peanuts, with subsequent experimental validation of AhTPS9's role in cold tolerance. In the cultivated peanut genome, a total of 16 AhTPS and 17 AhTPP genes were identified. AhTPS and AhTPP genes were observed in phylogenetic analysis, closely related to wild diploid peanuts, respectively. The evolutionary patterns of AhTPS and AhTPP genes were predominantly characterized by gene segmental duplication events and robust purifying selection. A variety of hormone-responsive and stress-related cis-elements were unveiled in our analysis of cis-regulatory elements. Distinct expression patterns of AhTPS and AhTPP genes across different peanut tissues, developmental stages, and treatments were revealed, suggesting potential roles in growth, development, and stress responses. Under low-temperature stress, qPCR results showcased upregulation in AhTPS genes (AhTPS2-5, AhTPS9-12, AhTPS14, AhTPS15) and AhTPP genes (AhTPP1, AhTPP6, AhTPP11, AhTPP13). Furthermore, AhTPS9, exhibiting the most significant expression difference under cold stress, was obviously induced by cold stress in cultivated peanut, and AhTPS9-overexpression improved the cold tolerance of Arabidopsis by protect the photosynthetic system of plants, and regulates sugar-related metabolites and genes. This comprehensive study lays the groundwork for understanding the roles of AhTPS and AhTPP gene families in trehalose regulation within cultivated peanuts and provides valuable insights into the mechanisms related to cold stress tolerance.

Genome-Wide Identification and Expression Analysis of the Trehalose-6-phosphate Synthase and Trehalose-6-phosphate Phosphatase Gene Families in Rose (Rosa hybrida cv 'Carola') under Different Light Conditions.

Trehalose, trehalose-6-phosphate synthase (TPS),and trehalose-6-phosphatase (TPP) have been reported to play important roles in plant abiotic stress and growth development. However, their functions in the flowering process of Rosa hybrida have not been characterized. In this study we found that, under a short photoperiod or weak light intensity, the content of trehalose in the shoot apical meristem of Rosa hybrida cv 'Carola' significantly decreased, leading to delayed flowering time. A total of nine RhTPSs and seven RhTPPs genes were identified in the genome. Cis-element analysis suggested that RhTPS and RhTPP genes were involved in plant hormones and environmental stress responses. Transcriptome data analysis reveals significant differences in the expression levels of RhTPSs and RhTPPs family genes in different tissues and indicates that RhTPPF and RhTPPJ are potential key genes involved in rose flower bud development under different light environments. The results of quantitative real-time reverse transcription (qRT-PCR) further indicate that under short photoperiod and weak light intensity all RhTPP members were significantly down-regulated. Additionally, RhTPS1a, RhTPS10, and RhTPS11 were up-regulated under a short photoperiod and showed a negative correlation with flowering time and trehalose content decrease. Under weak light intensity, RhTPS11 was up-regulated and negatively regulated flowering, while RhTPS5, RhTPS6, RhTPS7b, RhTPS9, and RhTPS10 were down-regulated and positively regulated flowering. This work lays the foundation for revealing the functions of RhTPS and RhTPP gene families in the regulation of rose trehalose.

Genome-Wide Identification of Trehalose-6-phosphate Synthase (TPS) Gene Family Reveals the Potential Role in Carbohydrate Metabolism in Peach.

Trehalose-6-phosphate synthase (TPS) is essential for plant growth and development, linking trehalose-6-phosphate (T6P) to carbon metabolism. However, little is known about the TPS gene family in peaches and their potential roles in regulating carbohydrates in peach fruit. In this study, nine TPS genes were identified in the peach genome and named according to the homologous genes in Arabidopsis. Phylogenetic analysis showed that three subfamilies were identified, including TPSI, TPSII-1, and TPSII-2, which were also consistent with gene structure analysis. Considerable cis-elements were enriched in the promoters, including plant hormone-related elements. Tissue-specific analysis showed that these TPS genes were mainly expressed in leaves, stems, and fruit, showing different expression patterns for each gene. In addition, during fruit development, the content of trehalose-6-phosphate (T6P) was positively correlated with the expression of PpTPS7a and negatively with sucrose non-fermenting-1-related kinase 1 (SnRK1) activity. Transient overexpression and silencing of PpTPS7a in peach fruit validated its function in regulating T6P content and SnRK1 activity.

Dual-Specificity Inhibitor Targets Enzymes of the Trehalose Biosynthesis Pathway.

To reduce the risk of resistance development, a novel fungicide with dual specificity is demanded. Trehalose is absent in animals, and its synthases, trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP), are safe fungicide targets. Here, we report the discovery of a dual-specificity inhibitor of MoTps1 (Magnaporthe oryzae Tps1, TPS) and MoTps2 (M. oryzae Tps2, TPP). The inhibitor, named A1-4, was obtained from a virtual screening and subsequent surface plasmon resonance screening. In in vitro assays, A1-4 interacts with MoTps1 and MoTps2-TPP (MoTps2 TPP domain) and inhibits their enzyme activities. In biological activity assays, A1-4 not only inhibits the virulence of M. oryzae on host but also causes aggregation of conidia cytosol, which is a characteristic phenotype of MoTps2. Furthermore, hydrogen/deuterium exchange mass spectrometry assays support the notion that A1-4 binds to the substrate pockets of TPS and TPP. Collectively, A1-4 is a promising hit compound for the development of safe fungicide with dual-target specificity.

Trehalose-6-Phosphate Synthase Contributes to Rapid Cold Hardening in the Invasive Insect Lissorhoptrus oryzophilus (Coleoptera: Curculionidae) by Regulating Trehalose Metabolism.

Rapid cold hardening (RCH) is known to rapidly enhance the cold tolerance of insects. Trehalose has been demonstrated to be a cryoprotectant in Lissorhoptrus oryzophilus, an important invasive pest of rice in China. Trehalose synthesis mainly occurs through the Trehalose-6-phosphate synthase (TPS)/trehalose-6-phosphate phosphatase (TPP) pathway in insects. In this study, the TPS gene from L. oryzophilus (LoTPS) was cloned and characterized for the first time. Its expression and trehalose content changes elicited by RCH were investigated. Our results revealed that RCH not only increased the survival rate of adults but also upregulated the expression level of LoTPS and increased the trehalose content under low temperature. We hypothesized that upregulated LoTPS promoted trehalose synthesis and accumulation to protect adults from low-temperature damage. To further verify the function of the LoTPS gene, we employed RNA interference (RNAi) technology. Our findings showed that RCH efficiency disappeared and the survival rate did not increase when the adults were fed dsRNA of LoTPS. Additionally, inhibiting LoTPS expression resulted in no significant difference in trehalose content between the RCH and non-RCH treatments. Furthermore, the expression patterns of trehalose transporter (TRET) and trehalase (TRE) were also affected. Collectively, these results indicate the critical role of LoTPS in L. oryzophilus cold resistance after RCH induction. LoTPS can enhance survival ability by regulating trehalose metabolism. These findings contribute to further understanding the role of TPS in insect cold resistance and the invasiveness of L. oryzophilus. Moreover, RNAi of LoTPS opens up possibilities for novel control strategies against L. oryzophilus in the future.

A sweet story from Phytophthora-soybean interaction.

Phytopathogenic microbes obtain nutrients from host plants to support their growth and metabolism. A recent study by Zhu et al. revealed that the oomycete pathogen Phytophthora sojae upregulates the activity of soybean trehalose 6-phosphate synthase 6 (GmTPS6) and increases trehalose accumulation (through an effector PsAvh413) to promote nutritional gain.

Elucidation of cold adaptation in Glaciimonas sp. PAMC28666 with special focus on trehalose biosynthesis.

Glaciimonas sp. PAMC28666, an extremophilic bacterium thriving in Antarctic soil and belonging to the Oxalobacteraceae family, represents the only complete genome of its genus available in the NCBI database. Its genome measures 5.2 Mb and comprises 4,476 genes (4,350 protein-coding and 72 non-coding). Phylogenetic analysis shows the strain PAMC28666 in a unique branch within the genus Glaciimonas, closely related to Glaciimonas alpine Cr9-12, supported by robust bootstrap values. In addition, strain PAMC28666 showed 77.08 and 23.3% ANI and DDH, respectively, with Glaciimonas sp. PCH181.This study focuses on how polar strain PAMC28666 responds to freeze-thaw conditions, Experimental results revealed a notable survival rate of 47.28% when subjected to a temperature of 15°C for a period of 10 days. Notably, two genes known to be responsive to cold stress, Trehalose 6-phosphate synthase (otsA) and Trehalose 6-phosphate phosphatase (otsB), exhibited increased expression levels as the temperature shifted from 25°C to 15°C. The upregulation of otsAB and the consequent synthesis of trehalose play pivotal roles in enhancing the cold resistance of strain PAMC28666, offering valuable insights into the correlation between trehalose production and adaptation to cold stress. Furthermore, research into this neglected cold-adapted variation, like Glaciimonas sp. PAMC28666, has the potential to shed light on how trehalose is produced in cold-adapted environments Additionally, there is potential to extract trehalose compounds from this strain for diverse biotechnological applications, including food and cosmetics, with ongoing research exploring its unique properties.

VdTps2 modulates plant colonization and symptom development in Verticillium dahliae.

The trehalose biosynthesis pathway is a potential target for antifungal drugs development. Trehalose phosphate synthase/phosphatase (TPS/TPP) are widely conserved components of trehalose biosynthesis in fungi. However, the role of trehalose biosynthesis in the vascular plant pathogenic fungus Verticillium dahliae remains unclear. Here, we investigated the functions of the TPS complex, including VdTps1, VdTps2, and VdTps3 in V. dahliae. Unlike VdTps2, deletion of VdTps1 or VdTps3 did not alter any phenotypes compared with the wild-type strain. In contrast, the ΔVdTps2 strain showed severely depressed radial growth due to the abnormal swelling of the hyphal tips. Further, deletion of VdTps2 increased microsclerotia formation, melanin biosynthesis, and resistance to cell wall perturbation and high-temperature stress. Virulence assays and quantification of fungal biomass revealed that deletion of VdTps2 delayed disease symptom development as evident by the reduced virulence and decreased biomass of the ΔVdTps2 strain in plant stem tissue following inoculation. Additionally, increases in penetration peg formation observed in the ΔVdTps2 strain in the present of H2O2 suggested that VdTps2 suppresses initial colonization. Our results also revealed the role of VdTps2 as a regulator of autophagy. Together, these results indicate that VdTps2 contributes to plant colonization and disease development.

Catalytic Mechanism of Nonglycosidic C-N Bond Formation by the Pseudoglycosyltransferase Enzyme VldE.

The pseudoglycosyltransferase (PsGT) enzyme VldE is a homologue of the retaining glycosyltransferase (GT) trehalose 6-phosphate synthase (OtsA) that catalyzes a coupling reaction between two pseudo-sugar units, GDP-valienol and validamine 7-phosphate, to give a product with α,α-N-pseudo-glycosidic linkage. Despite its biological importance and unique catalytic function, the molecular bases for its substrate specificity and reaction mechanism are still obscure. Here, we report a comparative mechanistic study of VldE and OtsA using various engineered chimeric proteins and point mutants of the enzymes, X-ray crystallography, docking studies, and kinetic isotope effects. We found that the distinct substrate specificities between VldE and OtsA are most likely due to topological differences within the hot spot amino acid regions of their N-terminal domains. We also found that the Asp158 and His182 residues, which are in the active site, play a significant role in the PsGT function of VldE. They do not seem to be directly involved in the catalysis but may be important for substrate recognition or contribute to the overall architecture of the active site pocket. Moreover, results of the kinetic isotope effect experiments suggest that VldE catalyzes a C-N bond formation between GDP-valienol and validamine 7-phosphate via an SNi-like mechanism. The study provides new insights into the substrate specificity and catalytic mechanism of a member of the growing family of PsGT enzymes, which may be used as a basis for developing new PsGTs from GTs.

Biological Activity of Acinetobacter calcoaceticus IMV B-7241 Surfactants Synthesized in the Presence of Competitive Bacteria Bacillus subtilis BT-2

Currently, the effectiveness of technologies for microbial surfactants, which are characterized by a complex of practically valuable physicochemical and biological properties is lower than that of synthetic analogues. To reduce the cost of these products of microbial synthesis, industrial waste is used as substrates for their biosynthesis. In previous studies, it has been established that surfactants synthesized by Acinetobacter calcoaceticus IMV B-7241 on crude glycerol have lower antimicrobial activity compared to that obtained on purified glycerol. The main approaches to the regulation of the biological activity of microbial surfactants are their post-fermentation chemical modification, as well as the improvement of producer strains by methods of metabolic and genetic engineering. In recent years, numerous studies have appeared on the co-cultivation of producers of antimicrobial compounds with competitive microorganisms (biological inductors), in response to the presence of which the antimicrobial activity of the final product increases. Aim. To study the effect of live and inactivated cells of Bacillus subtilis BT-2, as well as the corresponding supernatant, on the antimicrobial and anti-adhesive activity and the ability to destroy biofilms of A. calcoaceticus IМV B-7241 surfactants synthesized in a medium with glycerol of different degrees of purification. Methods. The A. calcoaceticus IMV B-7241 strain was grown in a liquid mineral medium with purified and crude glycerol, into which live and inactivated B. subtilis BT-2 cells as well as the supernatant after growing the B. subtilis BT-2 strain (2.5—10%, v/v) were added. Surfactants were extracted from the supernatant of the culture liquid with Folch’s mixture. Anti-adhesive activity and the degree of destruction of biofilms were determined by the spectrophotometric method, and antimicrobial activity — by the indicator of the minimum inhibitory concentration. The activity of enzymes of surface-active aminolipids biosynthesis (NADP+-dependent glutamate dehydrogenase) and glycolipids (phosphoenolpyruvate (PEP)-carboxylase, PEP-synthetase, PEP-carboxykinas, trehalose-phosphate synthase) was analyzed in cell-free extracts obtained after сells sonication. Results. It was established that the introduction of inactivated B. subtilis BT-2 cells and supernatant into the medium with both substrates did not affect the indicators of the surfactant synthesis, while in the presence of live cells of the B. subtilis BT-2 strain in the medium with purified glycerol, a decrease in the concentration of the final product by 1.5 times, and in the culture medium with crude glycerol — an increase by1.4 times were observed compared to the indicators with no inductor. The study of the antimicrobial activity of surfactants showed that the most effective of the used inductors (live, inactivated cells, supernatant) were live cells of B. subtilis BT-2. The introduction of B. subtilis BT-2 strain live cells into the culture medium with both substrates was accompanied by the formation of surfactants, the minimum inhibitory concentrations of which in relation to bacterial (Bacillus subtilis BT-2, Staphylococcus aureus BMS-1, Proteus vulgaris PA-12, Enterobacter cloacae С-8) and yeast (Candida albicans D-6, Candida tropicalis PE-2) test-cultures were 3—23 times lower than established for those synthesized on the medium with no inductor. Anti-adhesive activity of surfactants obtained on purified and crude glycerol in the presence of all types of inductors was higher compared to those synthesized in the culture medium without inductors (cells adhesion of bacterial and yeast test-cultures on polyvinyl chloride was 13—70 and 33—96%, respectively). Introduction of live and inactivated B. subtilis BT-2 cells or the supernatant into A. calcoaceticus IMV B-7241 cultivation medium was accompanied by the synthesis of surfactants, in the presence of which the disruption of bacterial biofilms was on average 10-20% higher compared to using surfactants synthesized without an inductor. In the presence of B. subtilis BT-2 in the medium, in the cells of the A. calcoaceticus IMV B-7241 strain, the activity of NADP+-dependent glutamate dehydrogenase (a key enzyme of aminolipids biosynthesis) increased by 1.5—2 times, while the activity of biosynthesis of glycolipids enzymes remained practically at the same level as without an inductor. Such data indicate that the higher biological activity of surfactants obtained by A. calcoaceticus IMV B-7241 in the presence of biological inductors might be due to an increase in the content of aminolipids in their composition. Conclusions. This research has established the possibility of regulating the antimicrobial and anti-adhesive activity as well as the ability to disrupt biofilms of A. calcoaceticus IМV B-7241 surfactants by introducing competitive bacteria B. subtilis BT-2 into the culture medium. It is important that under such cultivation conditions, the antimicrobial activity of surfactants synthesized on toxic crude glycerol significantly increases.

Identification, Evolutionary Relationship Analysis of the Trehalose-6-Phosphate Synthase (TPS) Gene Family in Common Bean (Phaseolus vulgaris) and Their Expression in Response to Cold Stress

Identification, Evolutionary Relationship Analysis of the Trehalose-6-Phosphate Synthase (TPS) Gene Family in Common Bean (Phaseolus vulgaris) and Their Expression in Response to Cold Stress