Fructosyltransferase Genes Research Articles

Integrated Transcriptomics and Metabolomics Analysis of the Fructan Metabolism Response to Low-Temperature Stress in Garlic.

As the main reserve carbohydrate in garlic, fructan contributes to garlic's yield and quality formation. Numerous studies have shown that plant fructan metabolism induces a stress response to adverse environments. However, the transcriptional regulation mechanism of garlic fructan in low-temperature environments is still unknown. In this study, the fructan metabolism of garlic seedlings under low-temperature stress was revealed by transcriptome and metabolome approaches. With the extension of stress time, the number of differentially expressed genes and metabolites increased. Using weighted gene co-expression network analysis (WGCNA), three key enzyme genes related to fructan metabolism were screened (a total of 12 transcripts): sucrose: sucrose 1-fructosyltransferase (1-SST) gene; fructan: fructan 6G fructosyltransferase (6G-FFT) gene; and fructan 1-exohydrolase (1-FEH) gene. Finally, two hub genes were obtained, namely Cluster-4573.161559 (6G-FFT) and Cluster-4573.153574 (1-FEH). The correlation network and metabolic heat map analysis between fructan genes and carbohydrate metabolites indicate that the expression of key enzyme genes in fructan metabolism plays a positive promoting role in the fructan response to low temperatures in garlic. The number of genes associated with the key enzyme of fructan metabolism in trehalose 6-phosphate was the highest, and the accumulation of trehalose 6-phosphate content may mainly depend on the key enzyme genes of fructan metabolism rather than the enzyme genes in its own synthesis pathway. This study not only obtained the key genes of fructan metabolism in garlic seedlings responding to low temperatures but also preliminarily analyzed its regulatory mechanism, providing an important theoretical basis for further elucidating the cold resistance mechanism of garlic fructan metabolism.

The Disparity Observed Between Murine Short-term and Long-term Hematopoietic Stem Cells in Their Ability to Migrate and Engraft When Introduced IV is Due to Distinct Adhesion Molecule Expression

CD34 has traditionally been used as a marker to enrich human bone marrow stem cells for clinical bone marrow transplants for over five decades. However, several studies support that the most primitive hematopoietic stem cells (HSCs) responsible for long-term hematopoiesis-long-term HSCs (LT-HSCs) -are found within the CD34 negative (CD34 neg) fraction of bone marrow cells. LT-HSCs are challenging to study due to their scarcity and lack of specific markers to identify them. Once these stem cells enter the circulation, they must find their way, through multistep process controlled by a variety of cell surface adhesion molecules, into the bone marrow. In contrast to the CD34 pos stem cells, studies have suggested that LT-HSCs have trouble migrating and engrafting when introduced intravenously. In order to better understand why these deficiencies exist, we set out to fully elucidate the adhesion mechanisms used by various murine hematopoietic stem cell populations to migrate. To this end, we assayed the homing effectors that are either necessary in the first step, i.e., the cell surface adhesion molecule Sialyl Lewis X (sLe x) and its interaction with selectins-mainly E-selectin-on the bone marrow endothelium, or the second step, i.e. the chemokine receptor CXCR4 and its binding to the bone marrow stroma resident chemokine SDF-1α, of cell migration.Specifically focusing on murine populations of short term HSCs (ST-HSCs; Flk2 negCD34 pos) and LT-HSCs (Flk2 negCD34 neg), flow cytometric and gene expression analysis clearly illustrated distinctive expression patterns of homing effectors with the highest contrast observed in sLe x where Flk2 negCD34 neg HSCs exhibited low expression (< 10%) and Flk2 negCD34 pos HSCs exhibited much higher expression (> 60%) which correlated to higher binding to E-selectin. Of the common E-selectin ligands identified, both Flk2 negCD34 pos and Flk2 negCD34 neg HSCs expressed CD44 and PSGL-1 to equivalent levels while CD43 was expressed at significantly lower levels on Flk2 negCD34 neg HSCs. Moreover, our data indicates that the variation observed in sLe x expression/E-selectin binding correlates with differences in the expression of key glycosyltransferase genes involved in the creation of the sLe x epitope on these E-selectin ligands. mRNA expression analysis of glycosyltransferases revealed that, in general, sialyltransferase (ST3Gals) genes were expressed at higher levels in Flk2 negCD34 neg HSCs while only one fructosyltransferase (FT4) gene was expressed at higher levels in this HSC population. Thus, these data suggest that α2,3 sialylation is intact in Flk2 negCD34 neg HSCs but α1,3 fucosylation is lacking. The expression levels of homing molecules involved in the stages of cell migration after E-selectin binding were also assessed. No differences in the expression of the integrins CD49d, CD49e and CD29 were observed between Flk2 negCD34 neg and Flk2 negCD34 pos HSCs. Interestingly, higher levels of the chemokine receptor CXCR4 were measured on the surface of Flk2 negCD34 pos HSCs compared to Flk2 negCD34 neg HSCs. Likewise, this was reflected in a much lower ability of the Flk2 negCD34 neg HSCs to migrate towards the CXCR4 ligand, SDF-1, in a transwell assay.Based on these findings that show that both E-selectin binding and CXCR4 mediated migration are compromised in Flk2 negCD34 neg HSCs, we sought to enhance this LT-HSC populations ability to migrate using well-known treatments including recombinant human fucosyltransferase 6 (rhFVI) to create the glycans necessary for selectin binding, as well as the CD26 inhibitor (Diprotin A; Dip A) to enhance the binding of chemokines to their receptors.Interestingly, although both CD26 inhibition and cell surface fucosylation remarkably enhanced the expression of homing molecules in both HSC populations in vitro, only pretreatment of Flk2 negCD34 neg HSCs with Dip A significantly enhanced engraftment in vivo after transplantation into C57BL/6N mice.This study represents the first direct analysis of E-selectin ligand expression on murine LT-HSC populations and strongly support that by temporarily enhancing the expression of migration-associated molecules on the surface of these cells, prolonged and efficient HSC engraftment can be achieved. We hope our finding will potentially shed light on methods to optimally utilize these very valuable long-term HSCs in clinical bone marrow and cord blood transplants worldwide. DisclosuresNo relevant conflicts of interest to declare.

Genome-wide association study reveals the genetic complexity of fructan accumulation patterns in barley grain.

We profiled the grain oligosaccharide content of 154 two-row spring barley genotypes and quantified 27 compounds, mainly inulin- and neoseries-type fructans, showing differential abundance. Clustering revealed two profile groups where the 'high' set contained greater amounts of sugar monomers, sucrose, and overall fructans, but lower fructosylraffinose. A genome-wide association study (GWAS) identified a significant association for the variability of two fructan types: neoseries-DP7 and inulin-DP9, which showed increased strength when applying a novel compound ratio-GWAS approach. Gene models within this region included three known fructan biosynthesis genes (fructan:fructan 1-fructosyltransferase, sucrose:sucrose 1-fructosyltransferase, and sucrose:fructan 6-fructosyltransferase). Two other genes in this region, 6(G)-fructosyltransferase and vacuolar invertase1, have not previously been linked to fructan biosynthesis and showed expression patterns distinct from those of the other three genes, including exclusive expression of 6(G)-fructosyltransferase in outer grain tissues at the storage phase. From exome capture data, several single nucleotide polymorphisms related to inulin- and neoseries-type fructan variability were identified in fructan:fructan 1-fructosyltransferase and 6(G)-fructosyltransferase genes. Co-expression analyses uncovered potential regulators of fructan biosynthesis including transcription factors. Our results provide the first scientific evidence for the distinct biosynthesis of neoseries-type fructans during barley grain maturation and reveal novel gene candidates likely to be involved in the differential biosynthesis of various types of fructan in barley.

Decreased expression of fructosyltransferase genes in asparagus roots may contribute to efficient fructan degradation during asparagus spear harvesting

Asparagus (Asparagus officinalis L.) accumulates inulin and inulin neoseries-type fructans in root, which are synthesized by three fructosyltransferases—sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99), fructan:fructan 1-fructosyltransferase (1-FFT, EC 2.4.1.100), and fructan:fructan 6G-fructosyltransferase (6G-FFT, EC 2.4.1.243). Fructans in roots are considered as energy sources for emerging of spears, and it has been demonstrated that a gradual decrease in root fructan content occurs during the spear harvesting season (budding and shooting up period). However, the roles of certain three fructosyltransferases during the harvest season have not yet been elucidated. Here, we investigated the variation in enzymatic activities and gene expression levels of three fructosyltransferases and examined sugar contents in roots before and during the spear harvest period. Two cDNAs, aoft2 and aoft3, were isolated from the cDNA library of roots. The respective recombinant proteins (rAoFT2 and rAoFT3), produced by Pichia pastoris, were characterized: rAoFT2 showed 1-FFT activity (producing nystose from 1-kestose), whereas rAoFT3 showed 1-SST activity (producing 1-kestose from sucrose). These reaction profiles of recombinant proteins were similar to those of native enzymes purified previously. These results indicate that aoft2 and aoft3 encoding 1-FFT and 1-SST are involved in fructan synthesis in roots. A gradual downregulation of fructosyltransferase genes and activity of respective enzymes was observed in roots during the harvest period, which also coincided with the decrease in fructooligosaccharides and increase in fructose due to fructan exohydrolase activity. These findings suggest that downregulation of fructosyltransferases genes during harvest time may contribute to efficient degradation of fructan required for the emergence of spears.

Glycoside hydrolase family 68 gene of halophilic archaeon Halalkalicoccus jeotgali B3T codes for an inulosucrase enzyme

Fructose polysaccharides and oligosaccharides are gaining strong importance due to their beneficial effects on human health and their applications in the food, feed and pharmaceutical industries. Fructan synthesis has been characterised in a wide variety of bacteria and plants. Metagenomic DNA sequence data suggests that several archaeal strains also contain fructosyltransferase (ftf) genes in their genome sequences (www.cazy.org), and thus may be able to synthesise fructans. The archaeal ftf genes are predicted to encode glycoside hydrolase family 68 enzymes. These genes are of strong interest in view of their potential role in the synthesis of novel prebiotic compounds. In the present studies, we have expressed putative ftf gene of an archaeon Halalkalicoccus jeotgali B3T in E. coli. The recombinant enzyme produced water-insoluble polysaccharide using sucrose as a substrate. Analysis by 13C and 1H NMR spectroscopy revealed the polysaccharide to be an inulin-type fructan polymer, depicting that the GH68 gene of H. jeotgali B3T codes for an inulosucrase enzyme. This is the first report on heterologous expression of an archaeal inulosucrase gene, which also provides conclusive evidence of the functional identity of this gene.

Cloning, expression and characterization of a novel fructosyltransferase from Aspergillus niger and its application in the synthesis of fructooligosaccharides.

Fructosyltransferases have been used in the industrial production of fructooligosaccharides (FOS). However, it is still not possible to explain the difference in FOS production based on the variations observed in the FOS synthesizing enzymes of A. niger. In the present study, a novel fructosyltransferase (FT-A) from A. niger TCCC41686 with high FOS synthesis ability was characterized. The FT-A gene was obtained and expressed in Pichia pastoris. A homology model of FT-A showed that the changes of residues identified outside the conserved domains were found to have an effect on its characteristics and its FOS-synthesis capacity. The optimal activity of the recombinant FT-A was observed at 50 °C and pH 6.0, and it was stable below 50 °C and over the range of pH 3.0–11.0. The Km and Vmax values of FT-A were 151.13 g L−1 and 6.55 g L−1 min−1, respectively. The production of FOS using the recombinant FT-A remained above 60% during 50–80 min of synthesis based on sucrose as substrate. The novel fructosyltransferase (FT-A) investigated in this study can potentially be applied for the efficient industrial production of FOS. The results also provide more valuable information for explaining the relationship between the structure and function of the FT-A.

Optical and physical mapping with local finishing enables megabase-scale resolution of agronomically important regions in the wheat genome

BackgroundNumerous scaffold-level sequences for wheat are now being released and, in this context, we report on a strategy for improving the overall assembly to a level comparable to that of the human genome.ResultsUsing chromosome 7A of wheat as a model, sequence-finished megabase-scale sections of this chromosome were established by combining a new independent assembly using a bacterial artificial chromosome (BAC)-based physical map, BAC pool paired-end sequencing, chromosome-arm-specific mate-pair sequencing and Bionano optical mapping with the International Wheat Genome Sequencing Consortium RefSeq v1.0 sequence and its underlying raw data. The combined assembly results in 18 super-scaffolds across the chromosome. The value of finished genome regions is demonstrated for two approximately 2.5 Mb regions associated with yield and the grain quality phenotype of fructan carbohydrate grain levels. In addition, the 50 Mb centromere region analysis incorporates cytological data highlighting the importance of non-sequence data in the assembly of this complex genome region.ConclusionsSufficient genome sequence information is shown to now be available for the wheat community to produce sequence-finished releases of each chromosome of the reference genome. The high-level completion identified that an array of seven fructosyl transferase genes underpins grain quality and that yield attributes are affected by five F-box-only-protein-ubiquitin ligase domain and four root-specific lipid transfer domain genes. The completed sequence also includes the centromere.

Evaluation of endophyte toxin production and its interaction with transgenic perennial ryegrass (Lolium perenne L.) with altered expression of fructosyltransferases.

Alkaloid concentration of perennial ryegrass herbage is affected by endophyte strain and host plant genotype. However, previous studies suggest that associations between host and endophyte also depends on environmental conditions, especially those affecting nutrient reserves and that water-soluble carbohydrate (WSC) concentration of perennial ryegrass plants may influence grass-endophyte associations. In this study a single transgenic event, with altered expression of fructosyltransferase genes to produce high WSC and biomass, has been crossed into a range of cultivar backgrounds with varying Epichloë endophyte strains. The effect of the association between the transgenic trait and alkaloid production was assessed and compared with transgene free control populations. In the vast-majority of comparisons there was no significant difference between alkaloid concentrations of transgenic and non-transgenic plants within the same cultivar and endophyte backgrounds. There was no significant difference between GOI+ (gene of interest positive) and GOI- (gene of interest negative) populations in Janthritrem response. Peramine concentration was not different between GOI+ and GOI- for 10 of the 12 endophytes-cultivar combinations. Cultivar Trojan infected with NEA6 and Alto with SE (standard endophyte) exhibited higher peramine and lolitrem B (only for Alto SE) concentration, in the control GOI- compared with GOI+. Similarly, cultivar Trojan infected with NEA6 and Alto with NEA3 presented higher ergovaline concentration in GOI-. Differences in alkaloid concentration may be attributable to an indirect effect in the modulation of fungal biomass. These results conclude that the presence of this transgenic insertion, does not alter the risk (toxicity) of the endophyte-grass associations. Endophyte-host interactions are complex and further research into associations with high WSC plant should be performed in a case by case basis.

Chicory R2R3-MYB transcription factors CiMYB5 and CiMYB3 regulate fructan 1-exohydrolase expression in response to abiotic stress and hormonal cues.

In the biennial Cichorium intybus, inulin-type fructans accumulate in the taproot during the first year. Upon cold or drought exposure, fructans are degraded by fructan exohydrolases, affecting inulin yield and degree of polymerization. While stress-induced expression of 1-FEH genes has been thoroughly explored, the transcriptional network mediating these responses has remained unknown. In this study, several R2R3-MYB transcriptional regulators were analysed for their possible involvement in 1-FEH regulation via transient transactivation of 1-FEH target promoters and for in vivo co-expression with target genes under different stress and hormone treatments. CiMYB3 and CiMYB5 selectively enhanced promoter activities of 1-FEH1, 1-FEH2a, and 1-FEH2b genes, without affecting promoter activities of fructosyltransferase genes. Both factors recognized the MYB-core motifs (C/TNGTTA/G) that are abundantly present in 1-FEH promoters. In chicory hairy root cultures, CiMYB5 displayed co-expression with its target genes in response to different abiotic stress and phytohormone treatments, whereas correlations with CiMYB3 expression were less consistent. Oligofructan levels indicated that the metabolic response, while depending on the balance of the relative expression levels of fructan exohydrolases and fructosyltransferases, could be also affected by differential subcellular localization of different FEH isoforms. The results indicate that in chicory hairy root cultures CiMYB5 and CiMYB3 act as positive regulators of the fructan degradation pathway.

Expression and Activity Analysis of Fructosyltransferase from Aspergillus oryzae.

The fructosyltransferase gene was isolated and cloned from Aspergillus oryzae. The gene was 1368bp, which encoded a protein of 455 amino acids. To analyze the activity of the expressed fructosyltransferase, the pET32a-fructosyltransferase recombined plasmid was transformed into Escherichia coli BL21. The fructosyltransferase gene was successfully expressed by Isopropyl-β-d-thiogalactoside (IPTG) induction. The molecular weight of the expression protein was about 45kDa. The optimal conditions of protein expression were 25 °C, 0.1mM IPTG, and 8h of inducing time. The optimal concentration of urea dealing with inclusion body was 2.5M. The expressed protein exhibited a strong fructosyl transfer activity. These results showed that the expressed fructosyltransferas owned transferase activity, and could catalyze the synthesis of sucrose-6-acetate.

Cloning of 1– fructan: fructan fructosyltransferase gene and expression of recombinant 1– fructan: fructan fructosyltransferase in yeast

Fructosyltransferases (FTases) were important enzymes for fructo-oligosaccharides (FOS) synthesis. Recently, FOS was considered as a potential prebiotic in food industry. In particularly, FOS with short chains was used as a new alternative sweetener. The production of recombinant 1- fructan: fructan fructosyltransferase (1- FFT) in yeast system was conducted in this research. The 1- fructan: fructan fructosyltransferase gene (1- fft) cloned from 105 days old tuber of Kaentawan ( Helianthus tuberosus L.) was sub cloned to expression vector, pPICZαB by adding Pst I and Sac II sites. The cloned gene was successfully transformed to yeast Pichia pastoris X-33 by lithium chloride transformation method. The yeast transformant; P. pastoris X- 33 PF1 showed the ability to produce recombinant 1- FFT. The enzyme activity at 3.57 and 3.33 unit/L was determined in cell and culture medium, respectively. It was also found that FOS was synthesized when recombinant 1- FFT was incubated with 1- kestose and synthesized FOS as substrates. The synthesis of FOS was not detected when sucrose was used as substrate of recombinant 1- FFT.

Different photosynthetic acclimation mechanisms are activated under waterlogging in two contrasting Lolium perenne genotypes.

Increased precipitation and snowmelt during warmer winters may lead to low-temperature waterlogging of plants. Perennial ryegrass (Lolium perenne L.) is one of the most important cool-season grasses in agriculture. It is well adapted to cold climates, and may be considered as a model system for studying the mechanisms involved in cold acclimation. The aim of this study was to evaluate the effects of waterlogging on photosynthetic acclimation to cold in perennial ryegrass. Two L. perenne genotypes that differ in their responses to waterlogging in terms of freezing tolerance were compared. We evaluated the effects of waterlogging during cold acclimation on the water-soluble carbohydrate concentration, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity, photochemical efficiency of PSII, and transcript levels of the Rubisco activase (RcaA) and sucrose-sucrose fructosyltransferase (1-SST) genes. The genotype that did not accumulate water-soluble carbohydrates in the leaf under waterlogging showed a lower degree of feedback inhibition of photosynthesis under low temperature, and activated a photochemical mechanism of photosynthetic acclimation to cold. The other genotype accumulated water-soluble carbohydrates in the leaf during waterlogging, and activated a non-photochemical mechanism under cold conditions. Different photosynthetic acclimation systems to cold under waterlogging may be activated in these two contrasting L. perenne genotypes.

Does gibberellin biosynthesis play a critical role in the growth of Lolium perenne? Evidence from a transcriptional analysis of gibberellin and carbohydrate metabolic genes after defoliation.

Global meat and milk production depends to a large extent on grazed pastures, with Lolium perenne being the major forage grass in temperate regions. Defoliation and subsequent regrowth of leaf blades is a major and essential event with respect to L. perenne growth and productivity. Following defoliation, carbohydrates (mainly fructans and sucrose) have to be mobilized from heterotrophic tissues to provide energy and carbon for regrowth of photosynthetic tissues. This mobilization of reserve carbohydrates requires a substantial change in the expression of genes coding for enzymes involved in carbohydrate metabolism. Here we tested the hypothesis that gibberellins (GA) are at the core of the processes regulating the expression of these genes. Thus, we examined the transcript profiles of genes involved in carbohydrate and GA metabolic pathways across a time course regrowth experiment. Our results show that following defoliation, the immediate reduction of carbohydrate concentrations in growing tissues is associated with a concomitant increase in the expression of genes encoding carbohydrate mobilizing invertases, and was also associated with a strong decrease in the expression of fructan synthesizing fructosyltransferase genes. We also show that the decrease in fructan levels is preceded by increased expression of the GA activating gene GA3-oxidase and decreased expression of the GA inactivating gene GA2-oxidase in sheaths. GA3-oxidase expression was negatively, while GA2-oxidase positively linked to sucrose concentrations. This study provides indicative evidence that gibberellins might play a role in L. perenne regrowth following defoliation and we hypothesize that there is a link between gibberellin regulation and sugar metabolism in L. perenne.

Changes in Lolium perenne transcriptome during cold acclimation in two genotypes adapted to different climatic conditions.

BackgroundActivation of numerous protective mechanisms during cold acclimation is important for the acquisition of freezing tolerance in perennial ryegrass (Lolium perenne L.). To elucidate the molecular mechanisms of cold acclimation in two genotypes (‘Veyo’ and ‘Falster’) of perennial ryegrass from distinct geographical origins, we performed transcriptome profiling during cold acclimation using RNA-Seq.MethodsWe cold-acclimated plants from both genotypes in controlled conditions for a period of 17 days and isolated Total RNA at various time points for high throughput sequencing using Illumina technology. RNA-seq reads were aligned to genotype specific references to identify transcripts with significant changes in expression during cold acclimation.ResultsThe genes induced were involved in protective mechanisms such as cell response to abiotic stimulus, signal transduction, redox homeostasis, plasma membrane and cell wall modifications, and carbohydrate metabolism in both genotypes. ‘Falster’ genotype, adapted to cold climates, showed a stronger transcriptional differentiation during cold acclimation, and more differentially expressed transcripts related to stress, signal transduction, response to abiotic stimulus, and metabolic processes compared to ‘Veyo’. ‘Falster’ genotype also showed an induction of more transcripts with sequence homology to fructosyltransferase genes (FTs) and a higher fold induction of fructan in response to low-temperature stress. The circadian rhythm network was perturbed in the ‘Veyo’ genotype adapted to warmer climates.ConclusionIn this study, the differentially expressed genes during cold acclimation, potentially involved in numerous protective mechanisms, were identified in two genotypes of perennial ryegrass from distinct geographical origins. The observation that the circadian rhythm network was perturbed in ‘Veyo’ during cold acclimation may point to a low adaptability of ‘Veyo’ to low temperature stresses. This study also revealed the transcriptional mechanisms underlying carbon allocation towards fructan biosynthesis in perennial ryegrass.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0643-x) contains supplementary material, which is available to authorized users.

Anti-cariogenic effects of erythritol on growth and adhesion of Streptococcus mutans

Erythritol is one of the most widely used low calorie sugar substitutes and has known inhibitory effects on the growth of Streptococcus mutans. However, the mechanism underlying this inhibition is poorly understood. Expression profiles of the glucosyltransferase (GTF) and fructosyltransferase (FTF) genes in S. mutans were evaluated in the presence of erythritol and other sweeteners. Adhesion of S. mutans to different carbohydrates was also determined across a range of concentrations. Erythritol significantly (p<0.05) inhibited adherence of S. mutans under multiple conditions, compared with sucrose. Erythritol significantly (p<0.05) inhibited expressions of gtfB, gtfC, gtfD, and ftf in the presence of various carbohydrates compared with sucrose. These findings were consistent with an anti-cariogenic effect of erythritol on S. mutans, and suggested mechanisms by which erythritol inhibits formation of dental caries.

Fructan metabolism and expression of genes coding fructan metabolic enzymes during cold acclimation and overwintering in timothy (Phleum pratense)

Metabolism of fructans in temperate grasses dynamically fluctuates before and during winter and is involved in the overwintering activity of plants. We monitored three candidate factors that may be involved in seasonal fructan metabolism in timothy (Phleum pratense): transcription levels of two fructosyltransferase (PpFT1 and PpFT2) genes and one fructan exohydrolase (Pp6-FEH1) gene during fall and winter and under artificially cold conditions. Functional analysis using a recombinant enzyme for PpFT2, a novel fructosyltransferase cDNA, revealed that it encoded sucrose:fructan 6-fructosyltransferase, with enzymatic properties different from previously characterized PpFT1. PpFT1 transcripts decreased from September to December as the amount of fructans increased, whereas PpFT2 transcripts increased in timothy crowns. PpFT2 was transcriptionally more induced than PpFT1 in response to cold and sucrose in timothy seedlings. A rapid increase in Pp6-FEH1 transcripts and increased monosaccharide content were observed in timothy crowns when air temperature was continuously below 0°C and plants were not covered by snow. Transcriptional induction of Pp6-FEH1 by exposure to −3°C was also observed in seedlings. These findings suggest Pp6-FEH1 involvement in the second phase of hardening. PpFT1 and PpFT2 transcription levels decreased under snow cover, whereas Pp6-FEH1 transcription levels were constant, which corresponded with the fluctuation of fructosyltransferase and fructan exohydrolase activities. Inoculation with snow mold fungi (Typhula ishikariensis) increased Pp6-FEH1 transcription levels and accelerated hydrolysis of fructans. These results suggest that transcriptional regulation of genes coding fructan metabolizing enzymes is partially involved in the fluctuation of fructan metabolism during cold acclimation and overwintering.

Effects of Lactobacillus reuteri-derived biosurfactant on the gene expression profile of essential adhesion genes (gtfB, gtfC and ftf) of Streptococcus mutans.

Background:Streptococci are the main causative agents in plaque formation and mutans streptococci are the principle etiological agent of dental plaque and caries. The process of biofilm formation is a step-wise process, starting with adhesion of planktonic cells to the surfaces. It is now a well known fact that expression of glucosyltransferases (gtfs) and fructosyltransferase (ftf) genes play a critical role in the initial adhesion of Streptococcus mutans to the tooth surface, which results in the formation of dental plaques and consequently caries and other periodontal diseases.Materials and Methods:In the present study, we have determined the effect of biosurfactants purified from Lactobacillus reuteri (DSM20016) culture on gene expression profile of gftB/C and fft of S. mutans (ATCC35668) using quantitative real-time polymerase chain reaction.Results:The application of biosurfactant caused considerable down-regulation of the expression of all three genes under study. The reduction in gene expression was statistically very significant (P > 0.0001 for all three genes).Conclusions:Inhibition of these genes by the extracted L. reuteri biosurfactant shows the emergence of a powerful alternative to the presently practicing alternatives. In view of the importance of these gene products for S. mutans attachment to the tooth surface, which is the initial important step in biofilm production and dental caries, we believe that the biosurfactant prepared in this study could be considered as a step ahead in dental caries prevention.

Effects of biosurfactant produced by Lactobacillus casei on gtfB, gtfC, and ftf gene expression level in S. mutans by real-time RT-PCR.

Background:The Streptococci are the pioneer strains in plaque formation and Streptococcus mutans are the main etiological agent of dental plaque and caries. In general, biofilm formation is a step-wise process, which begins by adhesion of planktonic cells to the surfaces. Evidences show that expression of glucosyltransferase B and C (gtfB and gtfC) and fructosyltransferase (ftf) genes play critical role in initial adhesion of S. mutans to the tooth surface which results in formation of dental plaques and consequently caries and other periodontal disease.Materials and Methods:The aim of this study was to determine the effect of biosurfactants produced by a probiotic strain; Lactobacillus casei (ATCC39392) on gene expression profile of gftB/C and tft of S. mutans (ATCC35668) using quantitative real-time PCR.Results:The application of the prepared biosurfactant caused dramatic down regulation of all the three genes under study. The reduction in gene expression was statistically highly significant (for gtfB, P > 0.0002; for gtfC, P > 0.0063, and for ftf, P > 0.0057).Conclusion:Considerable downregulation of all three genes in the presence of the prepared biosurfactant comparing to untreated controls is indicative of successful inhibition of influential genes in bacterial adhesion phenomena. In view of the importance of glucosyltransferase gene products for S.mutans attachment to the tooth surface which is the initial important step in biofilm production and dental caries, further research in this field may lead to an applicable alternative for successful with least adverse side effects in dental caries prevention.

Modification of the total soluble sugar content of the C4 grass Paspalum notatum expressing the wheat‐derived sucrose:sucrose 1‐fructosyltransferase and sucrose:fructan 6‐fructosyltransferase genes

AbstractFructans are water‐soluble fructose oligomers and polymers that are accumulated in many plants, especially the C3 temperate grasses. Fructan accumulation enhances cold and drought tolerance in grasses, and the increased soluble carbohydrates improve forage quality, especially the nutritional value animals can gain from these grasses. The C4 grasses yield high biomass but their inability to accumulate fructans coupled with their low water‐soluble carbohydrate (WSC) content are limiting factors for the forage quality of these grasses. The aim of this study was to produce fructans and increase the total soluble sugar content by introducing and expressing fructan biosynthesis genes in a C4 grass, bahiagrass (Paspalum notatum Flügge). Using particle bombardment, we transformed bahiagrass with a wheat‐derived sucrose:sucrose 1‐fructosyltransferase (1‐SST) gene and a sucrose:fructan 6‐fructosyltransferase (6‐SFT) gene, both of which were regulated by the cauliflower mosaic virus (CaMV) 35S promoter. Transcripts of the 1‐SST and 6‐SFT genes were detected in six out of 12 transgenic plants carrying both genes. Transgenic bahiagrass plants expressing the 1‐SST and 6‐SFT genes accumulated 1‐kestose and nystose. Although further polymerization of fructose units by 6‐SFT activity was not detected in transgenic plants, the total soluble sugar content increased. These results suggest that transgenic bahiagrass plants expressing wheat fructosyltransferase genes created a new metabolic pathway for fructan biosynthesis, which is an important requirement for producing fructans in C4 forage grasses with the objective of increasing the total soluble sugar content of these plants. This is the first report of introducing plant‐derived fructosyltransferases with the goal of increasing fructan accumulation in C4 forage grasses.

Molecular and Functional Characterization of Novel Fructosyltransferases and Invertases from Agave tequilana

Fructans are the main storage polysaccharides found in Agave species. The synthesis of these complex carbohydrates relies on the activities of specific fructosyltransferase enzymes closely related to the hydrolytic invertases. Analysis of Agave tequilana transcriptome data led to the identification of ESTs encoding putative fructosyltransferases and invertases. Based on sequence alignments and structure/function relationships, two different genes were predicted to encode 1-SST and 6G-FFT type fructosyltransferases, in addition, 4 genes encoding putative cell wall invertases and 4 genes encoding putative vacuolar invertases were also identified. Probable functions for each gene, were assigned based on conserved amino acid sequences and confirmed for 2 fructosyltransferases and one invertase by analyzing the enzymatic activity of recombinant Agave protein s expressed and purified from Pichia pastoris. The genome organization of the fructosyltransferase/invertase genes, for which the corresponding cDNA contained the complete open reading frame, was found to be well conserved since all genes were shown to carry a 9 bp mini-exon and all showed a similar structure of 8 exons/7 introns with the exception of a cell wall invertase gene which has 7 exons and 6 introns. Fructosyltransferase genes were strongly expressed in the storage organs of the plants, especially in vegetative stages of development and to lower levels in photosynthetic tissues, in contrast to the invertase genes where higher levels of expression were observed in leaf tissues and in mature plants.