Fructan 6-fructosyltransferase Research Articles

Modified fructan accumulation through overexpression of wheat fructan biosynthesis pathway fusion genes Ta1SST:Ta6SFT

BackgroundFructans are water-soluble carbohydrates that accumulate in wheat and are thought to contribute to a pool of stored carbon reserves used in grain filling and tolerance to abiotic stress.ResultsIn this study, transgenic wheat plants were engineered to overexpress a fusion of two fructan biosynthesis pathway genes, wheat sucrose: sucrose 1-fructosyltransferase (Ta1SST) and wheat sucrose: fructan 6-fructosyltransferase (Ta6SFT), regulated by a wheat ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (TaRbcS) gene promoter. We have shown that T4 generation transgene-homozygous single-copy events accumulated more fructan polymers in leaf, stem and grain when compared in the same tissues from transgene null lines. Under water-deficit (WD) conditions, transgenic wheat plants showed an increased accumulation of fructan polymers with a high degree of polymerisation (DP) when compared to non-transgenic plants. In wheat grain of a transgenic event, increased deposition of particular fructan polymers such as, DP4 was observed.ConclusionsThis study demonstrated that the tissue-regulated expression of a gene fusion between Ta1SST and Ta6SFT resulted in modified fructan accumulation in transgenic wheat plants and was influenced by water-deficit stress conditions.

Single and co-inoculum of endophytic bacteria promote growth and yield of Jerusalem artichoke through upregulation of plant genes under drought stress.

Helianthus tuberosus L. (Jerusalem artichoke) produce inulin, a type of fructan, which possesses several biotechnology applications, e.g., sugar syrup, prebiotics, fiber in diabetic food, enabling blood sugar and cholesterol reduction. Drought reduces inulin accumulation in the tubers of Jerusalem artichoke as the plants protect themselves from this stress by induction of stress gene responses, effecting growth reduction. Endophytic bacteria are promising candidates to promote plant growth and yield particularly under abiotic stress. Therefore, three endophytic bacteria with plant growth promoting properties were examined for their ability to improve Jerusalem artichoke growth and yield under both well-watered and drought conditions when inoculated individually or in combinations in pot experiments with 2 factorial random complete block design. The interactions of the endophytic bacteria and plant host determined the differential gene expression in response to drought as revealed by quantitative polymerase chain reaction. Single inoculum of the endophytic bacteria increased the height, weight, root traits, and harvest index of Jerusalem artichoke compared to co-inocula under both well-watered and drought conditions. However, the co-inocula of Rossellomorea aquimaris strain 3.13 and Bacillus velezensis strain 5.18 proved to be a synergistic combination leading to high inulin accumulation; while the co-inocula of B. velezensis strain 5.18 and Micrococcus luteus strain 4.43 were not beneficial when used in combination. The genes, dehydrin like protein and ethylene responsive element binding factor, were upregulated in the plants inoculated with single inoculum and co-inocula of all endophytic bacteria during drought stress. Moreover, the gene expression of indole-3-acetic acid (IAA) amido synthetase were up-regulated in Jerusalem artichoke inoculated with M. luteus strain 4.43 during drought stress. The fructan:fructan 1-fructosyltransferase (1-FFT) was also stimulated by the endophytic bacteria particularly in drought condition; the results of this study could explain the relationship between endophytic bacteria and plant host for growth and yield promotion under well-watered and drought conditions.

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.

Comparison of carbohydrate partitioning and expression patterns of some genes involved in carbohydrate biosynthesis pathways in annual and biennial species of Cichorium spp.

Variation in metabolism and partitioning of carbohydrates, particularly fructans, between annual and perennial Cichorium species remains a challenging topic. To address this problem, an annual (endive, Cichorium endive L. var. Crispum; Asteraceae) and a biennial species (chicory, Cichorium intybus L. var. Witloof; Asteraceae) were compared with in terms of variability in carbohydrate accumulation and expression patterns of fructan-active enzyme genes, as well as sucrose metabolism at various growth and developmental stages. In general, constituents such as 1-kestose, nystose, and inulin were detected only in the root of chicory and were not present in any of the endive tissues. For both species, flower tissue contained maximum levels of both fructose and glucose, while for sucrose, more fluctuations were observed. On the other hand, all the genes under study exhibited variation, not only between the two species but also among different tissues at different sampling times. In endive root compared to endive leaf, the expression of cell wall invertase genes and sucrose accumulation decreased simultaneously, indicating the limited capacity of its roots to absorb sucrose, a precursor to inulin production. In addition, low expression of fructan: fructan fructosyltransferase in endive root compared to chicory root confirmed the inability of endive to inulin synthesis. Overall, annual and biennial species were different in the production of inulin, transport, remobilization, and unloading of sucrose.

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.

Fructans Are Differentially Distributed in Root Tissues of Asparagus.

Inulin- and neoseries-type fructans [fructooligosaccharides (FOS) and fructopolysaccharides] accumulate in storage roots of asparagus (Asparagus officinalis L.), which continue to grow throughout the lifespan of this perennial plant. However, little is known about the storage of fructans at the spatial level in planta, and the degree of control by the plant is largely uncertain. We have utilized mass spectrometry imaging (MSI) to resolve FOS distribution patterns in asparagus roots (inner, middle, and outer tissues). Fructan and proteome profiling were further applied to validate the differential abundance of various fructan structures and to correlate observed tissue-specific metabolite patterns with the abundance of related fructan biosynthesis enzymes. Our data revealed an increased abundance of FOS with higher degree of polymerization (DP > 5) and of fructopolysaccharides (DP11 to DP17) towards the inner root tissues. Three isoforms of fructan:fructan 6G-fructosyltransferase (6G-FFT), forming 6G-kestose with a β (2–6) linkage using sucrose as receptor and 1-kestose as donor, were similarly detected in all three root tissues. In contrast, one ß-fructofuranosidase, which likely exhibits fructan:fructan 1-fructosyltransferase (1-FFT) activity, showed very high abundance in the inner tissues and lower levels in the outer tissues. We concluded a tight induction of the biosynthesis of fructans with DP > 5, following a gradient from the outer root cortex to the inner vascular tissues, which also correlates with high levels of sucrose metabolism in inner tissues, observed in our study.

Abscisic acid associated with key enzymes and genes involving in dynamic flux of water soluble carbohydrates in wheat peduncle under terminal drought stress

Remobilization of stem water soluble carbohydrates (WSC) can supply crucial carbon resources for grain filling under drought stress, while the regulatory metabolism associated with abscisic acid (ABA) is still limited. Two cultivars, LJ196 (drought-tolerant) and XD18 (drought-prone), were pot-grown under well-watered (WW) and drought-stressed (DS) conditions. Concentrations of WSC components and ABA, and fructan metabolizing enzymes and genes were investigated in peduncle after anthesis. When compared with those under the WW, LJ196 remained higher grain yield and grain-filling rate than XD18 under the DS. During the early period of grain filling (0–14 DAA), DS increased concentrations of total WSC and its components, but thereafter substantially reduced them. The gene expression levels and enzymatic activities of fructan 1-exohydrolases (1-FEH) and fructan 6-exohydrolases (6-FEH) showed similar trends, whereas those of fructan: fructan 1-fructosyltransferase (1-FFT), and sucrose: fructan 6-fructosyltransferase (6-SFT) were depressed and declined over the period of examination. LJ196 still showed higher levels of ABA and fructan metabolizing. The ABA concentration under the DS was positively and significantly correlated with total WSC and fructan concentration, and expression levels of these enzymes and genes as well, with more prominently with those of 6-FEH. Presumably, ABA could enhance fructan hydrolysis by strongly up-regulating the gene expression and enzymatic activity of 6-FEH to accelerate WSC remobilization. However, stem WSC induced by DS could be not fully remobilized to grains, due to its weaker correlation with grain-filling rate and finally indicating lower grain yield. The findings would provide useful information for wheat production under water-deficit environments.

Whole-Transcriptome Analysis Unveils the Synchronized Activities of Genes for Fructans in Developing Tubers of the Jerusalem Artichoke.

Helianthus tuberosus L., known as the Jerusalem artichoke, is a hexaploid plant species, adapted to low-nutrient soils, that accumulates high levels of inulin in its tubers. Inulin is a fructose-based polysaccharide used either as dietary fiber or for the production of bioethanol. Key enzymes involved in inulin biosynthesis are well known. However, the gene networks underpinning tuber development and inulin accumulation in H. tuberous remain elusive. To fill this gap, we selected 6,365 expressed sequence tags (ESTs) from an H. tuberosus library to set up a microarray platform and record their expression across three tuber developmental stages, when rhizomes start enlarging (T0), at maximum tuber elongation rate (T3), and at tuber physiological maturity (Tm), in “VR” and “K8-HS142”clones. The former was selected as an early tuberizing and the latter as a late-tuberizing clone. We quantified inulin and starch levels, and qRT-PCR confirmed the expression of critical genes accounting for inulin biosynthesis. The microarray analysis revealed that the differences in morphological and physiological traits between tubers of the two clones are genetically determined since T0 and that is relatively low the number of differentially expressed ESTs across the stages shared between the clones (93). The expression of ESTs for sucrose:sucrose 1-fructosyltransferase (1-SST) and fructan:fructan 1-fructosyltransferase (1-FFT), the two critical genes for fructans polymerization, resulted to be temporarily synchronized and mirror the progress of inulin accumulation and stretching. The expression of ESTs for starch biosynthesis was insignificant throughout the developmental stages of the clones in line with the negligible level of starch into their mature tubers, where inulin was the dominant polysaccharide. Overall, our study disclosed candidate genes underpinning the development and storage of carbohydrates in the tubers of two H. tuberosus clones. A model according to which the steady-state levels of 1-SST and 1-FFT transcripts are developmentally controlled and might represent a limiting factor for inulin accumulation has been provided. Our finding may have significant repercussions for breeding clones with improved levels of inulin for food and chemical industry.

Expression of Jerusalem artichoke (Helianthus tuberosus L.) fructosyltransferases, and high fructan accumulation in potato tubers

Fructans are polymers of fructose that are present as storage carbohydrates in various plants. Jerusalem artichoke (Helianthus tuberosus L.) contains a high amount of inulin. Two enzymes are involved in inulin biosynthesis. The sucrose:sucrose 1-fructosyltransferase (1-SST) enzyme mainly catalyzes the synthesis of 1-kestose from sucrose. In the next step, fructan:fructan 1-fructosyltransferase (1-FFT) catalyzes the synthesis of inulin from 1-kestose. In this study, the Ht1-SST and Ht1-FFT genes were isolated from Jerusalem artichoke and expressed in potato (Solanum tuberosum L.), either separately or together, via Agrobacterium-mediated transformation. Transgenic potato tubers overexpressing Ht1-SST accumulated 1-kestose to a high level (up to 3.36 mg/g), while tubers overexpressing both Ht1-SST and Ht1-FFT accumulated up to 3.14 mg/g short-chain inulin-type fructans, with the degree of polymerization (DP) ranging from 3 to 5, excluding high DP inulins. Transgenic potato plants accumulated fructo-oligosaccharides to a high level, following the fructan biosynthetic pathway of Jerusalem artichoke, and therefore present a high potential for the mass production of inulin through established potato breeding and cultivation methods.

Label-free quantitative proteomic analysis determines changes in amino acid and carbohydrate metabolism in three cultivars of Jerusalem artichoke tubers

Jerusalem artichoke (JA) tubers are an important bio-economy developing crop because of its invaluable bioproducts in both food and biofuel aspects. However, the molecular mechanism of its tuberization, and the differences among different cultivars have been little studied to date. Therefore, here we selected PJA, DJA, and HJA cultivars of JA tubers, showing variations in their tuber epidermal pigmentation, underground tuberization, and inulin content. A comparative proteome analysis led to the identification of 402 proteins in the tubers of which 114 were significantly modulated among different cultivars. Gene Ontology (GO) analysis showed proteins related to the biosynthesis of amino acids and carbohydrate metabolism were differentially modulated in the tubers of three cultivars. Results from the inulin content measurement and proteome analysis suggest that Sucrose:sucrose 1-fructosyltransferase (1-SST) prioritizes inulin biosynthesis rather than rate-limiting enzyme fructan:fructan 1-fructosyltransferases (1-FFT). Furthermore, we confirmed the relationship between transcript-protein expression levels was in discord within inulin biosynthesis enzymes 1-SST and 1-FFT with the terms in previous RT-qPCR results using the same tubers. Our data represent the first report of comparative tuber proteome profiling of different JA and provide the metabolic and molecular basis for understanding carbohydrate metabolism in the tuber tissue.

Review and in silico analysis of fermentation, bioenergy, fiber, and biopolymer genes of biotechnological interest in Agave L. for genetic improvement and biocatalysis.

Several of the over 200 known species of Agave L. are currently used for production of distilled beverages and biopolymers. The plants live in a wide range of stressful environments as a result of their resistance to abiotic stress (drought, salinity, and extreme temperature) and pathogens, which gives the genus potential for germplasm conservation and biotechnological applications that may minimize economic losses as a result of the global climate change. However, the limited knowledge in the genus of genome structure and organization hampers development of potential improved biotechnological applications by means of genetic manipulation and biocatalysis. We reviewed Agave and plant sequences in the GenBank NCBI database for identifying genes with biotechnological potential for fermentation, bioenergy, fiber improvement, and in vivo plant biopolymer production. Three-dimensional modeling of enzyme structures in plant accessions revealed structural differences in sucrose 1-fructosyltransferase, fructan 1-fructosyltransferase, fructan exohydrolase (1-FEH), cellulose synthase (CES), and glucanases (EGases) with possible effects in fructan, sugar, and biopolymer production. Although the coding genes of FEH and enzymes involved in biopolymer production (CES, sucrose synthase, and EGases) remain unidentified in Agave L., our results could aid isolation of such genes in Agave. By comparing nucleotide and amino acid sequences in accessions of Agave and other plants, knowledge may be gained about transcriptional regulation and enzymatic activity factors. Future study is needed of biotechnological application of Agave genes for crop breeding aided by genetic engineering and biocatalysis. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1314-1334, 2018.

Accumulation of water-soluble carbohydrates and gene expression in wheat stems correlates with drought resistance

In order to understand the effects of sugar metabolism on drought resistance in wheat, two wheat cultivars with different levels of drought resistance were used in this study. We investigated the accumulation pattern of water-soluble carbohydrates (WSC) and expression profiles of twelve fructan metabolism-related genes in peduncle (PED), penultimate (PEN), and lower internode (LOW) stem tissues under drought stress. LH7, a higher drought-resistance cultivar, contained a higher stem dry weight and higher content of WSC in PED, PEN, and LOW tissues, while XN979, a lower drought-resistance cultivar, contained lower values. The tissues from LOW internodes had the highest WSC content, while PED had the lowest. The mRNA levels of genes encoding fructan synthesis-related enzymes, sucrose: sucrose 1-fructosyltransferase (1-SST), sucrose: fructan 6-fructosyltransferase (6-SFT), and fructan: fructan 1- fructosyltransferase (1-FFT) showed higher expression levels at early time points following stress, whilst the genes encoding degradation-related enzymes, fructan exohydrolases (1-FEH), and invertase (INV), showed higher expression at a later time point. Compared with XN979, LH7 showed higher expression levels of genes encoding fructan synthesis-related enzymes at all growth stages, whilst the expression of 1-FEH-W3, 6-FEH, and INV3 were higher at a later stage; these expression levels would benefit fructan accumulation and remobilization at early and later stages, respectively. Drought stress induced most of fructan metabolism related genes expression level decreasing in LH7 PED, but enhancing in LH7 LOW part at early time points following stress. The results confirm that there are complex, coordinated expression patterns of fructan synthesis- and degradation-related genes in stems under drought stress. In summary, 1–SST-A2, 6-SFT, 1-FFT-A, 1-FEH-W3, 6-FEH, and INV3 play important roles in fructan accumulation. In addition, higher expression of genes related to fructan synthesis and degradation occurs during early and later stages of drought stress, respectively, enhancing the drought resistance of wheat cultivar LH7.

Characterization of Fructan Metabolism During Jerusalem Artichoke (Helianthus tuberosus L.) Germination.

The inulin-type fructans in Jerusalem artichoke (Helianthus tuberosus L.) tubers exhibit different degrees of polymerization and are critical for germination. We aimed to characterize the sugar metabolism dynamics in the tubers without bud eyes or shoots (T) and BE/S of indoor- and field-grown Jerusalem artichokes during germination. Ht1-FEH II and Ht1-FEH III (1-fructan exohydrolases II and III, inulin-degrading enzymes) expression increased 5 days after planting indoors, whereas Ht1-FEH II expression increased 72 days after planting in the field in T and BE/S. Ht1-SST (sucrose:sucrose 1-fructosyl transferase, inulin synthesis initiator), and Ht1-FFT (fructan:fructan 1-fructosyl transferase, inulin elongator) expression generally decreased in indoor-grown T. The enzyme activities of 1-FEH and 1-FFT were unchanged during germination in both indoor- and field-grown T and BE/S, whereas 1-SST activity decreased in indoor-grown T, while 1-FEH and 1-FFT activities increased as a function of germination time in BE/S of both indoor- and field-grown tubers. The total soluble sugar content gradually decreased in T after germination indoors or in the field, while at the end of germination, the sucrose and fructan contents decreased, and fructose content increased in the field. The enzyme activities of soluble vacuolar (VI) or neutral invertase (NI) did not change significantly, except at the late germination stage. Sucrose synthase (SS) and sucrose-phosphate synthase (SPS) activities were not significantly changed in T and BE/S in indoor-grown artichokes, while SS activity gradually increased, and SPS activity gradually decreased in field-grown artichokes, alongside sucrose degradation. Compared to T, BE/S generally had higher enzyme activities of 1-FEH and 1-FFT, promoting inulin hydrolysis. This work shows that the process of tuber germination is similar indoors and in the field, and germination studies can therefore be conducted in either environment.

Fructan dynamics and antioxidant capacity of 4-day-old seedlings of wheat (Triticum aestivum) cultivars during drought stress and recovery.

One of the inevitable consequences of drought stress is enhanced production of reactive oxygen species (ROS). Fructan might function as effective candidate for capturing ROS in a wide range of stresses. Herein, 4-day-old seedlings of drought-tolerant and -sensitive wheat cultivars were exposed to drought stress for 7 days by water cessation, followed by further 7 days re-watering. The content, metabolism, related enzymes activity, degree of polymerisation (DP) and antioxidant capacity of fructan were compared in the two cultivars. High resolution HPAEC-PAD analysis of fructan showed an increase in the activities of fructan: fructan 1-fructosyltransferase (1-FFT) in the tolerant cultivar and sucrose: sucrose 1-fructosyltransferase (1-SST) and 1-FFT in the sensitive cultivar under drought condition. The activity of fructan exohydrolase (FEH) did not show any significant change in tolerant cultivar, but decreased in a sensitive one. In comparison with the sensitive cultivar, the tolerant one accumulated fructan (0.9% of dry matter) with higher degree of polymerisation (10.67±1.1), accompanied by increased OH radical scavenging activity, during drought condition. In regard to the fact that OH radical is the most prevalent ROS in damaging membrane lipids, the results suggest that fructans play a crucial role in the tolerance of wheat seedlings against drought stress.

Stem carbohydrate dynamics and expression of genes involved in fructan accumulation and remobilization during grain growth in wheat (Triticum aestivum L.) genotypes with contrasting tolerance to water stress.

The genetic and physiological mechanisms underlying the relationship between water-soluble carbohydrates (WSC) and water stress tolerance are scarcely known. This study aimed to evaluate the main WSC in stems, and the expression of genes involved in fructan metabolism in wheat genotypes growing in a glasshouse with water stress (WS; 50% field capacity from heading) and full irrigation (FI; 100% field capacity). Eight wheat genotypes (five tolerant and three susceptible to water stress) were evaluated initially (experiment 1) and the two most contrasting genotypes in terms of WSC accumulation were evaluated in a subsequent experiment (experiment 2). Maximum accumulation of WSC occurred 10–20 days after anthesis. Under WS, the stress-tolerant genotype exhibited higher concentrations of WSC, glucose, fructose and fructan in the stems, compared to FI. In addition, the stress-tolerant genotype exhibited higher up-regulation of the fructan 1-fructosyltransferase B (1-FFTB) and fructan 1-exohydrolase w2 (1-FEHw2) genes, whereas the susceptible cultivar presented an up-regulation of the fructan 6-fructosyltransferase (6-SFT) and fructan 1-exohydrolase w3 (1-FEHw3) genes. Our results indicated clear differences in the pattern of WSC accumulation and the expression of genes regulating fructan metabolism between the tolerant and susceptible genotypes under WS.

CiMYB17, a stress-induced chicory R2R3-MYB transcription factor, activates promoters of genes involved in fructan synthesis and degradation.

In Cichorium intybus, inulin metabolism is mediated by fructan-active enzymes (FAZYs): sucrose:sucrose 1-fructosyltransferase (1-SST), fructan:fructan 1-fructosyltransferase (1-FFT), and fructan 1-exohydrolases 1, 2a and 2b (1-FEH1, -2a and -2b), respectively. While these enzymes have been rigorously characterized, the transcriptional network orchestrating their development- and stress-related expression has remained largely unknown. Here, the possible role of R2R3-MYB transcription factors in FAZY regulation was explored via bioinformatic identification of R2R3-MYBs (using an RNA sequencing (RNAseq) database), studies of co-expression of these factors with target genes, invivo transient transactivation assays of FAZY target promoters (dual luciferase assay), and a yeast one-hybrid assay investigating the specificity of the binding of these factors to cis-elements. The chicory MYB transcription factor CiMYB17 specifically activated promoters of 1-SST and 1-FFT by binding to the consensus DNA-motif DTTHGGT. Unexpectedly, CiMYB17 also activated promoters of fructan exohydrolase genes. The stimulatory effect on promoter activities of sucrose transporter and cell wall invertase genes points to a general role in regulating the source-sink relationship. Co-induction of CiMYB17 with 1-SST and 1-FFT (and, less consistently, with 1-FEH1/2) in nitrogen-starved or abscisic acid (ABA)-treated chicory seedlings and in salt-stressed chicory hairy roots supports a role in stress-induced fructan metabolism, including de novo fructan synthesis and trimming of pre-existing fructans, whereas the reduced expression of CiMYB17 in developing taproots excludes a role in fructan accumulation under normal growth conditions.

Single-nucleotide polymorphisms, mapping and association analysis of 1-FFT-A1 gene in wheat

Fructans are major nonstructural carbohydrates in wheat (Triticum aestivum L.). Fructan 1-fructosyltransferase (1-FFT) is the key enzyme in fructan biosynthesis. In the present study, 96 sequence variants were detected in the 1-FFT-A1 gene among 26 wheat accessions including UR208, and 15 of them result in amino acid substitutions, forming four haplotypes. Two markers M39 and M2164 were developed based on the InDel21-39 and SNP-2164 polymorphisms to distinguish the three haplotypes in the 1-FFT-A1. 1-FFT-A1 was located on chromosome 4A using marker M2164 and was flanked by markers Xcwm27 and 6-SFT-A1. By association analysis using a natural wheat population consisted of 154 accessions, the results showed that the two markers were significantly associated with water-soluble carbohydrate (WSC) content in the lower internode stem and total stem at the early and middle grain filling stages, 1000-grain weight (TGW) at different grain filling stages and peduncle length (PLE). Comparison of the effects of three haplotypes on agronomic traits indicated that TGW, PLE and total number of spikelets per spike (TNSS) were significantly influenced by haplotypes. HapIII showed a significant positive effect on TGW, PLE and TNSS.

オオムギのフルクタン合成系酵素sucrose:fructan 6-fructosyltransferase(6-SFT)遺伝子の品種間変異

オオムギのフルクタン合成系酵素sucrose:fructan 6-fructosyltransferase(6-SFT)遺伝子の品種間変異

Fructan:fructan 1-fructosyltransferase and inulin hydrolase activities relating to inulin and soluble sugars in Jerusalem artichoke (Helianthus tuberosus Linn.) tubers during storage.

Influences of harvest time and storage conditions on activities of fructan:fructan1-fructosyltransferase (1-FFT) and inulin hydrolase (InH) in relation to inulin and soluble sugars of Jerusalem artichoke (Helianthus tuberosus L.) tubers were investigated. Maturity affected 1-FFT-activity, inulin contents, and inulin profiles of the tubers harvested between 30 and 70days after flowering (DAF). Decreases in 1-FFT activity, high molecular weight inulin, and inulin content were observed in late-harvested tubers. The tubers harvested at 50 DAF had the highest inulin content (734.9±20.5gkg-1 DW) with a high degree of polymerization (28% of DP >30). During storage of the tubers, increases in InH activity (reached its peak at 15days of storage) and gradual decreases in 1-FFT activity took placed. These changes were associated with inulin depolymerization, causing decreases in inulin content and increases in soluble sugars. As well, decreasing storage temperatures would retain high inulin content and keep low soluble sugars; and freezing at -18°C would best retard 1-FFT, InH, and inulin changes.

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.