TPS1 Gene Research Articles

Impact of tps1 Deletion and Overexpression on Terpene Metabolites in Trichoderma atroviride.

Terpenoids are structurally diverse natural products that have been widely used in the pharmaceutical, food, and cosmetic industries. Research has shown that fungi produce a variety of terpenoids, yet fungal terpene synthases remain not thoroughly explored. In this study, the tps1 gene, a crucial component of the terpene synthetic pathway, was isolated from Trichoderma atroviride HB20111 through genome mining. The function of this gene in the terpene synthetic pathway was investigated by constructing tps1-gene-deletion- and overexpression-engineered strains and evaluating the expression differences in the tps1 gene at the transcript level. HS-SPME-GC-MS analysis revealed significant variations in terpene metabolites among wild-type, tps1-deleted (Δtps1), and tps1-overexpressed (Otps1) strains; for instance, most sesquiterpene volatile organic compounds (VOCs) were notably reduced or absent in the Δtps1 strain, while nerolidol, β-acorenol, and guaiene were particularly produced by the Otps1 strain. However, both the Δtps1 and Otps1 strains produced new terpene metabolites compared to the wild-type, which indicated that the tps1 gene played an important role in terpene synthesis but was not the only gene involved in T. atroviride HB20111. The TPS1 protein encoded by the tps1 gene could function as a sesquiterpene cyclase through biological information and evolutionary tree analysis. Additionally, fungal inhibition assay and wheat growth promotion assay results suggested that the deletion or overexpression of the tps1 gene had a minimal impact on fungal inhibitory activity, plant growth promotion, and development, as well as stress response. This implies that these activities of T. atroviride HB20111 might result from a combination of multiple metabolites rather than being solely dependent on one specific metabolite. This study offers theoretical guidance for future investigations into the mechanism of terpenoid synthesis and serves as a foundation for related studies on terpenoid metabolic pathways in fungi.

Growth enhancement, metabolic profile improvement, and DXR and TPS2 gene expression changes in Thymus vulgaris L. by cyanobacterial inoculation.

In recent decades cyanobacterial species have attracted research attention as potential sources of new biostimulants. In this study, the biostimulant effects of five cyanobacterial suspensions on the growth and essential oil composition of Thymus vulgaris L. were evaluated. The expression of key genes involved in the biosynthesis of thymol and carvacrol, such as DXR and TPS2, were investigated. A pot culture experiment revealed that cyanobacterial application significantly improved T. vulgaris L. growth indices, including plant height, dry and fresh weight, leaf and flower number, leaf area, and photosynthetic pigment content. Total phenol and flavonoid content in inoculated plants also showed a significant increase compared with the control. Anabaena torulosa ISB213 inoculation significantly increased root and shoot biomass by about 65.38% and 92.98% compared with the control, respectively. Nostoc calcicola ISB215 inoculation resulted in the highest amount of essential oil accumulation (18.08 ± 0.62) in T. vulgaris leaves, by about 72.19% compared with the control (10.5 ± 0.50%). Interestingly, the amount of limonene in the Nostoc ellipsosporum ISB217 treatment (1.67%) increased significantly compared with the control and other treatments. The highest expression rates of DXR and TPS2 genes were observed in the treatment of N. ellipsosporum ISB217, with 5.92-fold and 5.22-fold increases over the control, respectively. This research revealed the potential of the cyanobacteria that were studied as promising biostimulants to increase the production of biomass and secondary metabolites of T. vulgaris L., which could be a suitable alternative to chemical fertilizers. © 2024 Society of Chemical Industry.

Investigating the transcriptomic variances in two phases Ecytonucleospora hepatopenaei (EHP) in Litopenaeus vannamei

This study explores the transcriptomic differences in two distinct phases of Ecytonucleospora hepatopenaei (EHP) in Litopenaeus vannamei, a crucial aspect in shrimp health management. We employed high-throughput sequencing to categorize samples into two phases, 'Phase A' and 'Phase B', defined by the differential expression of PTP2 and TPS1 genes. Our analysis identified 2057 genes, with 78 exhibiting significant variances, including 62 upregulated and 16 downregulated genes. Enrichment analyses via GO and KEGG pathways highlighted these genes' roles in cellular metabolism, signal transduction, and immune responses. Notably, genes like IQGAP2, Rhob, Pim1, and PCM1 emerged as potentially crucial in EHP's infection process and lifecycle. We hypothesize that these genes may influence trehalose metabolism and glucose provision, impacting the biological activities within EHP during different phases. Interestingly, a lower transcript count in 'Phase A' EHP suggests a reduction in biological activities, likely preparing for host cell invasion. This research provides a foundational understanding of EHP infection mechanisms, offering vital insights for future studies and therapeutic interventions.

Wolbachia Effect on Drosophila melanogaster Lipid and Carbohydrate Metabolism.

The effect of maternally inherited endosymbiotic bacteria Wolbachia on triglyceride and carbohydrate metabolism, starvation resistance and feeding behavior of Drosophila melanogaster females was studied. Eight D. melanogaster lines of the same nuclear background were investigated; one had no infection and served as the control, and seven others were infected with different Wolbachia strains pertaining to wMel and wMelCS groups of genotypes. Most of the infected lines had a higher overall lipid content and triglyceride level than the control line and their expression of the bmm gene regulating triglyceride catabolism was reduced. The glucose content was higher in the infected lines compared to that in the control, while their trehalose levels were similar. It was also found that the Wolbachia infection reduced the level of tps1 gene expression (coding for enzyme for trehalose synthesis from glucose) and had no effect on treh gene expression (coding for trehalose degradation enzyme). The infected lines exhibited lower appetite but higher survival under starvation compared to the control. The data obtained may indicate that Wolbachia foster their hosts' energy exchange through increasing its lipid storage and glucose content to ensure the host's competitive advantage over uninfected individuals. The scheme of carbohydrate and lipid metabolism regulation under Wolbachia's influence was suggested.

A secondary function of trehalose-6-phosphate synthase is required for resistance to oxidative and desiccation stress in Fusarium verticillioides

The disaccharide trehalose has long been recognized for its role as a stress solute, but in recent years some of the protective effects previously ascribed to trehalose have been suggested to arise from a function of the trehalose biosynthesis enzyme trehalose-6-phosphate (T6P) synthase that is distinct from its catalytic activity. In this study, we use the maize pathogenic fungus Fusarium verticillioides as a model to explore the relative contributions of trehalose itself and a putative secondary function of T6P synthase in protection against stress as well as to understand why, as shown in a previous study, deletion of the TPS1 gene coding for T6P synthase reduces pathogenicity against maize. We report that a TPS1-deletion mutant of F. verticillioides is compromised in its ability to withstand exposure to oxidative stress meant to simulate the oxidative burst phase of maize defense and experiences more ROS-induced lipid damage than the wild-type strain. Eliminating T6P synthase expression also reduces resistance to desiccation, but not resistance to phenolic acids. Expression of catalytically-inactive T6P synthase in the TPS1-deletion mutant leads to a partial rescue of the oxidative and desiccation stress-sensitive phenotypes, suggesting the importance of a T6P synthase function that is independent of its role in trehalose synthesis.

Silencing CaTPS1 Increases the Sensitivity to Low Temperature and Salt Stresses in Pepper

Trehalose, as a non-reductive disaccharide, plays a vital role in plant growth and development and resistance to abiotic stress. Trehalose-6-phosphate synthase (TPS) is a key enzyme in the synthesis mechanism of trehalose and TPS1 genes play a crucial role in the response to abiotic stress in plants. However, it has rarely been reported that CaTPS1 responds to cold and salt stresses in pepper. To verify the function of CaTPS1 in response to cold and salt stresses, CaTPS1 was silenced by virus-induced gene silencing (VIGS). Subsequently, the expressions of CaTPS1, plant morphology and some physiological indexes were analyzed after cold and salt stresses in pepper. The results showed that the expression of CaTPS1 was significantly lower in CaTPS1-silenced (pTRV2-CaTPS1) plant than that in the non-VIGS (CK) and negative control (PTRV2-00) plants. The parameters of response to cold and salt stresses have changed accordingly. The chlorophyll content decreased, while the trehalose content, peroxidase (POD) activity, catalase (CAT) activity and ascorbate peroxidase (APX) activity increased in all treatments. However, these parameters of response to cold and salt stresses were significantly lower in pTRV2-CaTPS1 plant than in CK and PTRV2-00 plants. This study suggested that CaTPS1 was involved in the response to cold and salt stresses in pepper.

Disruption of GRR1 in Saccharomyces cerevisiae rescues tps1Δ growth on fermentable carbon sources.

In Saccharomyces cerevisiae , trehalose-6-phosphate synthase (Tps1) catalyzes the formation of trehalose-6-phophate in trehalose synthesis. Deletion of the TPS1 gene is associated with phenotypes including inability to grow on fermentable carbon sources, survive at elevated temperatures, or sporulate. To further understand these pleiotropic phenotypes, we conducted a genetic suppressor screen and identified a novel suppressor, grr1 Δ, able to restore tps1 Δ growth on rapidly fermentable sugars. However, disruption of GRR1 did not rescue tps1 Δ thermosensitivity. These results support the model that trehalose metabolism has important roles in regulating glucose sensing and signaling in addition to regulating stress resistance.

THE SAGA COMPLEX IS ESSENTIAL FOR THE REGULATION OF GENES INVOLVED IN YEAST TREHALOSE METABOLISM

Saccharomyces cerevisiae accumulates trehalose as a stress metabolite in adverse environmental conditions. The trehalose synthesis and breakdown are important for the regulation of trehalose levels within the yeast cell. Therefore, TPS1 and NTH1 gene expressions are tightly regulated during transcription and also translation. Since both genes contain Stress Response Elements (STRE) in the promoter regions, they are co-activated under stress conditions. However, the presence of similar regulatory elements in the promoter of both genes shows that these genes undergo a different regulation at the transcriptional level. In our study, the role of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex in the transcriptional regulation of TPS1 and NTH1 genes was determined in nutrient-poor environment. For that purpose, the wild type and Δada1 mutant yeast cells, where Ada1p is a member of the SAGA complex, were grown in normal and nitrogen starvation conditions. In addition, trehalose level was detected enzymatically in both wild type and mutant yeast cells. In silico promoter analysis of TPS1 and NTH1 promoters revealed that the STRE sequences required for binding of Msn2/4 transcription factors are closed by nucleosomes at the NTH1 promoter, but open at the TPS1 promoter. In the absence of Ada1p, stress-induced promoter activation in the TPS1 gene was observed, while NTH1 gene expression was not activated. According to these results, the nucleosomes spanning the STRE sequences could not be mobilized in the absence of Ada1 protein, and therefore the Msn2/4 transcription factors cannot bind to the promoter and activate the NTH1 gene expression under stress conditions. It was also observed that in the absence of Ada1p, trehalose accumulation was reduced regardless of stress conditions.

Agrobacterium-mediated transformation of spring camelina with yeast genes of trehalose biosynthesis

Aim. The aim of the study was the obtaining of Camelina sativa (L.) Crantz lines with yeast genes of trehalose synthesis TPS1 and TPS2 to increase their resistance to drought. Methods. Seeds of C. sativa genotype FEORZhYaF-1 were used for in vitro culture establishment. For this hypocotyl segments and shoot meristems of 5-days-old camelina seedlings were cultivated on three different nutrient media for regeneration supplemented with various hormone combinations. Vector constructions pGWB2-TPS1 and pGWB2-TPS2 with TPS1 and TPS2 genes have been used for genetic transformation. Results. The highest efficiency of plant regeneration from hypocotyl explants was found on medium supplemented with 1 mg/l BAP and 0.1 mg/l NAA, and from meristem explants – on medium with 1.5 mg/l BAP and 0.5 mg/l NAA. Agrobacterium-mediated transformation was conducted out, and camelina lines were picked up on corresponding medium with selective concentration of hygromycin. Transgenic nature of obtained plants was confirmed by PCR-analysis. Conclusions. The efficiency of in vitro plant regeneration of C. sativa genotype FEORZhYaF-1 has been investigated. Two types of explants and two vector constructions pGWB2-TPS1 and pGWB2-TPS2 with TPS1 and TPS2 yeast trehalose synthesis genes have been used for obtaining of transgenic camelina lines.

Characterizing phenotypic diversity of trehalose biosynthesis mutants in multiple wild strains of Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, trehalose-6-phospahte synthase (Tps1) and trehalose-6-phosphate phosphatase (Tps2) are the main proteins catalyzing intracellular trehalose production. In addition to Tps1 and Tps2, 2 putative regulatory proteins with less clearly defined roles also appear to be involved with trehalose production, Tps3 and Tsl1. While this pathway has been extensively studied in laboratory strains of S. cerevisiae, we sought to examine the phenotypic consequences of disrupting these genes in wild strains. Here we deleted the TPS1, TPS2, TPS3, and TSL1 genes in 4 wild strains and 1 laboratory strain for comparison. Although some tested phenotypes were not shared between all strains, deletion of TPS1 abolished intracellular trehalose, caused inability to grow on fermentable carbon sources and resulted in severe sporulation deficiency for all 5 strains. After examining tps1 mutant strains expressing catalytically inactive variants of Tps1, our results indicate that Tps1, independent of trehalose production, is a key component for yeast survival in response to heat stress, for regulating sporulation, and growth on fermentable sugars. All tps2Δ mutants exhibited growth impairment on nonfermentable carbon sources, whereas variations were observed in trehalose synthesis, thermosensitivity and sporulation efficiency. tps3Δ and tsl1Δ mutants exhibited mild or no phenotypic disparity from their isogenic wild type although double mutants tps3Δ tsl1Δ decreased the amount of intracellular trehalose production in all 5 strains by 17-45%. Altogether, we evaluated, confirmed, and expanded the phenotypic characteristics associated trehalose biosynthesis mutants. We also identified natural phenotypic variants in multiple strains that could be used to genetically dissect the basis of these traits and then develop mechanistic models connecting trehalose metabolism to diverse cellular processes.

Stimulation of Secondary Metabolites and γ-Terpinene Synthase by Silver Nanoparticles in Callus Cultures of Carum carvi.

Biotechnology and nanotechnology are important tools for understanding biochemical pathways. They can be used efficiently for stimulating and increasing the production of secondary metabolites in medicinal plants. The present study aimed to identify the γ-terpinene synthase gene (CcTPS2) as an effective contributor to the biosynthetic pathway of monoterpenes. The effects of silver nanoparticles (AgNPs; 50 and 100mg l- 1) and time (24 and 48h) were examined on secondary metabolites in cell suspension cultures of Carum carvi. This involved the identification, isolation, and sequencing of a partial sequence in the CcTPS2 gene of C. carvi. The genomic sequence of CcTPS2 comprised 292bp which were organized into two exons (110 and 82bp) and one intron (100bp), while the cDNA was 192bp. In the scale of nucleotides, the CcTPS2 gene showed 96% similarity with the TPS2 gene of Oliveria decumbens. We generated sequence data of the CcTPS2 gene for the first time in this species, thereby enabling further developments in understanding the molecular mechanisms responsible for terpene biosynthesis and other chemical derivatives in C. carvi. The results of GC/MS and GC/FID showed that AgNPs strongly affected the secondary metabolites in cell suspension cultures of C. carvi. According to the results, the AgNPs (50mg l- 1) increased p-cymene and carvone contents in comparison with the control. The exposure of plants to 100mg l- 1 AgNPs induced the production of thymol and carvacrol. The results of real-time PCR revealed that the exposure of plants to 100mg l- 1 AgNPs caused a significant upregulation of CcTPS2 expression for 24h. These cell suspension cultures were elicited by AgNPs, the application of which proved as an effective method to improve the production of secondary metabolites in vitro.

Transcriptomic Analysis of a Susceptible African Maize Line to Fusarium verticillioides Infection.

Maize (Zea mays L.) is a staple crop providing food security to millions of people in sub Saharan Africa. Fusarium verticillioides, an important fungal pathogen, infects maize causing ‘Fusarium Ear Rot’ disease, which decreases maize kernel yield and the quality of the crop harvested. Currently, no African maize line is completely resistant to infection by F. verticillioides. This study investigated an African maize line, Zea mays CML144, infected with F. verticillioides. Analysis of morphological characteristics showed significant differences between mock-infected and infected plants. RNA-sequencing (RNA-seq) was conducted on plants 14 days post-inoculation to identify differentially expressed genes (DEGs) involved in F. verticillioides infection. Analysis of RNA-seq data revealed DEGs that were both significantly up- and down-regulated in the infected samples compared to the mock-infected control. The maize TPS1 and cytochrome P450 genes were up-regulated, suggesting that kauralexins were involved in the CML144 defense response. This was substantiated by kauralexin analyses, which showed that kauralexins, belonging to class A and B, accumulated in infected maize tissue. Gene ontology terms relating to response to stimulus, chemical stimulus and carbohydrate metabolic processes were enriched, and the genes belonging to these GO-terms were down-regulated. Quantitative real-time PCR was performed on selected DEGs and measurement of phytoalexin accumulation validated the RNA-seq data and GO-analysis results. A comparison of DEGs from this study to DEGs found in F. verticillioides (ITEM 1744) infected susceptible (CO354) and resistant (CO441) maize genotypes in a previous study, matched 18 DEGs with 17 up-regulated and one down-regulated, respectively. This is the first transcriptomic study on the African maize line, CML144, in response to F. verticillioides infection.

The Genome-Wide Mutation Shows the Importance of Cell Wall Integrity in Growth of the Psychrophilic Yeast Metschnikowia australis W7-5 at Different Temperatures.

In this study, it was found that a Cre/loxP system could be successfully used as a tool for editing the genome of the psychrophilic yeast Metschnikowia australis W7-5 isolated from Antarctica. The deletion and over-expression of the TPS1 gene for trehalose biosynthesis, the GSY gene for glycogen biosynthesis, and the GPD1 and GPP genes for glycerol biosynthesis had no influence on cell growth of the mutants and transformants compared to cell growth of their wild-type strain M. australis W7-5, indicating that trehalose, glycogen, and glycerol had no function in growth of the psychrophilic yeast at different temperatures. However, removal of the SLT2 gene encoding the mitogen-activated protein kinase in the cell wall integrity (CWI) signaling pathway and the SWI4 and SWI6 genes encoding the transcriptional activators Swi4/6 had the crucial influence on cell growth of the psychrophilic yeast at the low temperature, especially at 25 °C and expression of the genes related to cell wall and lipid biosynthesis. Therefore, the cell wall could play an important role in growth of the psychrophilic yeast at different temperatures and biosynthesis of cell wall was actively regulated by the CWI signaling pathway. This was the first time to show that the genome of the psychrophilic yeast was successfully edited and the molecular evidences were obtained to elucidate mechanisms of low temperature growth of the psychrophilic yeast from Antarctica.

Obtaining Wheat (Triticum aestivum L.) Lines with Yeast Genes for Trehalose Biosynthesis

Yeast (Saccharomyces cerevisiae) genes for trehalose biosynthesis (TPS1 and TPS2) were transferred into genomes of several common wheat cultivars using two methods of Agrobacterium-mediated transformation (in vitro and in planta) to enhance drought tolerance. For this purpose, vectors pBract214-TPS1 and pBract214-TPS2 were constructed using the Gateway-cloning technique. Both the vectors contained TPS1 and TPS2 genes under control of the constitutive maize ubiquitin promoter (PUbi) and selectable marker hygromycin-phosphotransferase (hpt) gene. Three- to five-day-old calluses obtained from immature wheat embryos were used as explants for the transformation in vitro. Selection of transgenic plants was carried out on nutrient medium supplemented with 30 mg/L hygromycin (as a selectable agent). Seeds of wheat (transgenic generation T1) were obtained by the in planta method of transformation. Integration and the presence of yeast genes in wheat genomic DNA isolated from transgenic plants were confirmed by PCR analysis using primers specific to TPS1 and TPS2 genes.

Deletion of the trehalose tps1 gene in Kluyveromyces lactis does not impair growth in glucose.

Trehalose is a non-reducing disaccharide composed of two α-glucose molecules and synthesized by an enzyme complex containing four subunits TPS1 (EC 2.4.1.15), TPS2 (EC 3.1.3.12), TPS3 and TSL1. First reports about trehalose classified this sugar as an energy reserve compound like glycogen. However, lately, trehalose is known to assist yeast cells during heat, osmotic and starvation stresses. In Saccharomyces cerevisiae, the deletion of the tps1 encoding gene eliminated the yeast ability to grow on glucose as the sole carbon source. Kluyveromyces lactis is a yeast present in various dairy products and is currently utilized for the synthesis of more than 40 industrial heterologous products. In this study, the deletion of the tps1 gene in K. lactis showed that unlike S. cerevisiae, tps1 gene disruption does not cause growth failure in glucose, galactose, or fructose. The µMAX rate values of K. lactis tps1Δ strains were equal than the non-disrupted strains, showing that the gene deletion does not affect the yeast growth. After gene disruption, the absence of trehalose into the metabolism of K. lactis was also confirmed.

MYB21 interacts with MYC2 to control the expression of terpene synthase genes in flowers of Freesia hybrida and Arabidopsis thaliana.

Previously, linalool was found to be the most abundant component among the cocktail of volatiles released from flowers of Freesia hybrida. Linalool formation is catalysed by monoterpene synthase TPS1. However, the regulatory network developmentally modulating the expression of the TPS1 gene in Freesia hybrida remains unexplored. In this study, three regulatory genes, FhMYB21L1, FhMYB21L2, and FhMYC2, were screened from 52 candidates. Two MYB transcription factor genes were synchronously expressed with FhTPS1 and could activate its expression significantly when overexpressed, and the binding of FhMYB21L2 to the MYBCORE sites in the FhTPS1 promoter was further confirmed, indicating a direct role in activation. FhMYC2 showed an inverse expression pattern compared with FhTPS1; its expression led to a decreased binding of FhMYB21 to the FhTPS1 promoter to reduce its activation capacity when co-expressed, suggesting a role for an MYB-bHLH complex in the regulation of the FhTPS1 gene. In Arabidopsis, both MYB21 and MYC2 regulators were shown to activate the expression of sesquiterpene synthase genes, and the regulatory roles of AtMYB21 and AtMYC2 in the expression of the linalool synthase gene were also confirmed, implying conserved functions of the MYB-bHLH complex in these two evolutionarily divergent plants. Moreover, the expression ratio between MYB21 and MYC2 orthologues might be a determinant factor in floral linalool emission.

Genetic transformation of Nicotiana tabacum with the yeast TPS1 and TPS2 genes involved in trehalose synthesis

Aim. The aim of the study was the creation of vector constructions with yeast genes of trehalose synthesis TPS1 and TPS2 and their using for Agrobacterium-mediated transformation of N. tabacum. Methods. Strain of Agrobacterium tumefaciens GV3101 carrying vector constructions — pGWВ2-TPS1 and pGWВ2-TPS2 with TPS1 and TPS2 target genes respectively under 35S promoter of cauliflower mosaic virus and selectable hpt gene of hygromycin phosphotransferase has been used for plants transformation. N. tabacum leaf explants were used for Agrobacterium-mediated transformation. The medium with the addition of hygromycin was applied to transgenic lines selection. Results. The created vector constructions pGWВ2-TPS1 and pGWВ2-TPS2 has been used in genetic N. tabacum transformation. Target genes TPS1 and TPS2 were integrated applied of Agrobacterium-mediated transformation and transgenic lines of N. tabacum were selected with addition at 25 mg/L hygromycin into the medium. Molecular analysis confirmed the transgenic nature of selected lines. Conclusions. Sensitivity of selected lines for sugars content into the medium was established for shoots and roots formation of tobacco plants. Conditions for increase of transformation frequency and rooting of transgenic lines of plant after integrated of target TPS1 and TPS2 genes were investigated. Keywords: trehalose, yeast genes TPS1, TPS2, genetic transformation, Nicotiana tabacum.

Insoluble solids at high concentrations repress yeast\u2019s response against stress and increase intracellular ROS levels

Lignocellulosic ethanol production requires high substrate concentrations for its cost-competitiveness. This implies the presence of high concentrations of insoluble solids (IS) at the initial stages of the process, which may limit the fermentation performance of the corresponding microorganism. The presence of 40–60% IS (w/w) resulted in lower glucose consumption rates and reduced ethanol volumetric productivities of Saccharomyces cerevisiae F12. Yeast cells exposed to IS exhibited a wrinkled cell surface and a reduced mean cell size due to cavity formation. In addition, the intracellular levels of reactive oxygen species (ROS) increased up to 40%. These ROS levels increased up to 70% when both lignocellulose-derived inhibitors and IS were simultaneously present. The general stress response mechanisms (e.g. DDR2, TPS1 or ZWF1 genes, trehalose and glycogen biosynthesis, and DNA repair mechanisms) were found repressed, and ROS formation could not be counteracted by the induction of the genes involved in repairing the oxidative damage such as glutathione, thioredoxin and methionine scavenging systems (e.g. CTA1, GRX4, MXR1, and TSA1; and the repression of cell cycle progression, CLN3). Overall, these results clearly show the role of IS as an important microbial stress factor that affect yeast cells at physical, physiological, and molecular levels.

Knockdown of glycogen phosphorylase and glycogen synthase influences expression of chitin synthesis genes of rice brown planthopper Nilaparvata lugens

Abstract Glycogen synthase (GS) and glycogen phosphorylase (GP) are two key enzymes in the glycogen synthesis pathway, which catalyze trehalose and glucose transformation in insects. GS and GP can be regulated by trehalose metabolism, which plays an important role in insect growth. However, it is not known whether these genes can be targeted for pest control through regulation of chitin metabolism. We studied the function of Nilaparvata lugens GS and GP (NLGS and NLGP, respectively) using RNA interference, and reported that trehalose and the chitin biosynthesis pathways are regulated by GP and GS, especially TPS3, TRE1-1, and G6PI1, which decreased following knockdown of these two genes. The expression levels of TPS1, TPS2, and several chitin synthesis pathway family genes were significantly increased following dsNlGP injection. Additionally, despite there being no apparent change to the chitin content, an abnormal molting phenotype and wing deformity appeared, and close to 25% insects died. These results demonstrate that silencing of NLGP or NLGS can lead to molting deformities and an elevated mortality rate through the regulation of chitin pathway genes and chitinase genes. NLGP may play a key role in chitin synthesis due to the number of genes regulated, and higher deformity and mortality rates resulting from its knockdown.

Overexpression of TPS2 gene to increase thymol content using Agrobacterium tumefaciens-mediated transformation in Trachyspermum ammi (Qom ecotype)

Trachyspermum ammi is a well-known pharmaceutical plant in Iran and has many medicinal properties including anti-digestive, anti-spasm, antimicrobial, antiviral, and relieving rheumatic pains. This plant is important for its essential oil and its main constituent; i.e., thymol. Several properties of this plant are related to this valuable constituent. This research was conducted to increase thymol content of this plant by genetic transformation using Agrobacterium tumefaciens. To enhance thymol production, we used TPS2 gene (Terpene Synthase 2), which is a key enzyme involved in thymol synthesis pathway. In this research, an effective genetic transformation system was developed for Trachyspermum ammi with Agrobacterium tumefaciens. To overexpress TPS2 gene in Trachyspermum ammi, a gene construct called pBI121-TP harboring the neomycin phosphotransferase (nptII) gene as a plant-selected marker was designed. This work was performed using three strains of Agrobacterium tumefaciens (LBA4404, GV3101, and EHA101), hypocotyl explants, and factors including inoculation time (5, 10, 15, and 20 min) and co-cultivation time (1–3 days). Polymerase chain reaction (PCR) and Southern blot analysis were performed to confirm the integration of TPS2 and nptII transgenes into the plant genome. Based on these findings, the EHA101 strain in 5-min inoculation and 1-day co- cultivation showed a higher efficiency compared to two other strains. Transformation efficiency varied between 6.12 and 18.36% based on the Agrobacterium strains for transformation. The qPCR results demonstrated that the TPS2 gene was more expressed in the transgenic plants than non-transgenic plants. HPLC analysis showed that the aerial part of transgenic plants has the greater thymol compared to the non-transgenic plants. To the best of our knowledge, this study demonstrates, for the first time, a suitable and efficient method for overexpression TPS2 gene to enhance thymol by Agrobacterium-mediated transformation method in Trachyspermum ammi.