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.

RNA sequence analysis data of Peronospora destructor maintained on onions.

Peronospora destructor (Berk.) is an important biotrophic oomycete that causes downy mildew on onion (Allium Cepa L.) worldwide, especially in humid and temperate regions. The disease attacks bulb and seed production of onion, resulting in losses in yield and quality of bulbs. Epidemiological studies have increased our understanding and control of downy mildew on onion; however, little is known about the molecular aspects of P. destructor behavior during infection. Here, we isolated RNA from four samples of sporangia and sporangiophores of P. destructor, which were maintained by spore inoculation onto onions in a growth chamber. We then used an Ion PGM next generation sequencer to acquire and assemble the RNA sequences of P. destructor. By transcriptome shotgun assembly, we obtained 2335 contigs (N50, 884 nucleotides (nt); mean length, 881.6 nt). The data are accessible at NCBI (BioProject PRJNA391849). Our data resource will facilitate further studies of the molecular events during P. destructor infection.

Characterization of coherence-or-power selectable operation of an external-cavity semiconductor diode laser.

The properties of the coherence-or-power selectable operation of an external-cavity semiconductor diode laser through the control of intracavity polarization states have been characterized in detail. In our technique, a diffraction grating and a reflector functioned as a polarization-dependent output coupler, such that the portion of light fed back to the gain medium was readily controlled by rotating the intracavity polarization axis, which resulted in the selectable operation of either a high degree of coherence or a high power for the laser output. We could continuously sweep the correlation widths over a range of approximately one order of magnitude, as well as four-fold output powers by simply rotating the intracavity half-wave plate. We also demonstrated experiments on optical phase locking, using two independent coherence-or-power selectable lasers.

Effects of carbon sources and amines on induction of trichothecene production by Fusarium asiaticum in liquid culture.

Fusarium asiaticum infects cereal crops and produces trichothecenes such as deoxynivalenol and nivalenol. To determine the trichothecene induction mechanism, effects of carbon sources on the production of deoxynivalenol, nivalenol, 3-acetyl deoxynivalenol (3ADON), and 4-acetyl nivalenol (4ANIV) were examined in liquid cultures incubated with various strains. Sucrose supported significantly higher levels of acetylated trichothecene production in all strains than did the other carbon sources. Structural isomers of sucrose did not induce trichothecene production. The inducing effect of sucrose on trichothecene production was lost after the carbon source in the culture medium changed from sucrose to maltose in the process of incubation. Tri4 and Tri5 expressions were specifically up-regulated in the sucrose-containing medium and down-regulated with sucrose exhaustion. These findings suggest that F. asiaticum recognizes sucrose molecules and regulates Tri gene expression and trichothecene production. Moreover, an accelerating effect on trichothecene production by acidification of the culture medium containing specific amines during fungal incubation was exhibited only in the presence of sucrose in the medium. F. asiaticum induces trichothecene production in the presence of sucrose and accelerates the production when the medium containing specific amines is acidified during incubation.

Comparative study of transgenic Brachypodium distachyon expressing sucrose:fructan 6-fructosyltransferases from wheat and timothy grass with different enzymatic properties.

Fructans can act as cryoprotectants and contribute to freezing tolerance in plant species, such as in members of the grass subfamily Pooideae that includes Triticeae species and forage grasses. To elucidate the relationship of freezing tolerance, carbohydrate composition and degree of polymerization (DP) of fructans, we generated transgenic plants in the model grass species Brachypodium distachyon that expressed cDNAs for sucrose:fructan 6-fructosyltransferases (6-SFTs) with different enzymatic properties: one cDNA encoded PpFT1 from timothy grass (Phleum pratense), an enzyme that produces high-DP levans; a second cDNA encoded wft1 from wheat (Triticum aestivum), an enzyme that produces low-DP levans. Transgenic lines expressing PpFT1 and wft1 showed retarded growth; this effect was particularly notable in the PpFT1 transgenic lines. When grown at 22 °C, both types of transgenic line showed little or no accumulation of fructans. However, after a cold treatment, wft1 transgenic plants accumulated fructans with DP = 3-40, whereas PpFT1 transgenic plants accumulated fructans with higher DPs (20 to the separation limit). The different compositions of the accumulated fructans in the two types of transgenic line were correlated with the differences in the enzymatic properties of the overexpressed 6-SFTs. Transgenic lines expressing PpFT1 accumulated greater amounts of mono- and disaccharides than wild type and wft1 expressing lines. Examination of leaf blades showed that after cold acclimation, PpFT1 overexpression increased tolerance to freezing; by contrast, the freezing tolerance of the wft1 expressing lines was the same as that of wild type plants. These results provide new insights into the relationship of the composition of water-soluble carbohydrates and the DP of fructans to freezing tolerance in plants.

Reliability of a newly-developed immunochromatography diagnostic kit for pandemic influenza A/H1N1pdm virus: implications for drug administration.

BACKGROUND: For the diagnosis of seasonal influenza, clinicians rely on point-of-care testing (POCT) using commercially available kits developed against seasonal influenza viruses. However, POCT has not yet been established for the diagnosis of pandemic influenza A virus (H1N1pdm) infection due to the low sensitivity of the existing kits for H1N1pdm. METHODOLOGY/PRINCIPAL FINDINGS: An immunochromatography (IC) test kit was developed based on a monoclonal antibody against H1N1pdm, which does not cross-react with seasonal influenza A or B viruses. The efficacy of this kit (PDM-IC kit) for the diagnosis of H1N1pdm infection was compared with that of an existing kit for the detection of seasonal influenza viruses (SEA-IC kit). Nasal swabs (n = 542) were obtained from patients with flu-like syndrome at 13 clinics in Osaka, Japan during the winter of 2010/2011. Among the 542 samples, randomly selected 332 were further evaluated for viral presence by reverse transcriptase polymerase chain reaction (RT-PCR). The PDM-IC kit versus the SEA-IC kit showed higher sensitivity to and specificity for H1N1pdm, despite several inconsistencies between the two kits or between the kits and RT-PCR. Consequently, greater numbers of false-negative and false-positive cases were documented when the SEA-IC kit was employed. Significant correlation coefficients for sensitivity, specificity, and negative prediction values between the two kits were observed at individual clinics, indicating that the results could be affected by clinic-related techniques for sampling and kit handling. Importantly, many patients (especially influenza-negative cases) were prescribed anti-influenza drugs that were incongruous with their condition, largely due to physician preference for patient responses to questionnaires and patient symptomology, as opposed to actual viral presence. CONCLUSIONS/SIGNIFICANCE: Concomitant use of SEA-IC and PDM-IC kits increased the likelihood of correct influenza diagnosis. Increasing the credibility of POCT is anticipated to decrease the inappropriate dispensing of anti-influenza drugs, thereby minimizing the emergence of drug-resistant H1N1pdm strains.

Graminan breakdown by fructan exohydrolase induced in winter wheat inoculated with snow mold.

Fructan structures vary widely among plant species. Graminan-type fructans, extensions of sucrose through ß-(2,6)-linked fructosyl units with branches of ß-(2,1)-linked fructosyl units, accumulate in tissues of winter wheat (Triticum aestivum) during cold hardening and are metabolized under persistent snow cover. Snow molds such as Typhula ishikariensis and Microdochium nivale opportunistically infect wheat under snow cover. Snow mold-resistant wheat cultivars tend to heavily accumulate and slowly metabolize water-soluble carbohydrates including graminans in comparison with snow mold-susceptible cultivars. We observed time-dependent changes in the amounts of water-soluble carbohydrates in snow mold-inoculated wheat tissues, and accumulated fructan levels significantly decreased as a result of snow mold inoculation and incubation under snow cover, especially in a snow mold-susceptible wheat cultivar. Three candidates for fructan exohydrolase (FEH) cDNAs with high homology to cell wall invertases were isolated from wheat leaf tissues inoculated with snow mold and incubated under snow cover. The substrate specificity of enzymes encoded by the isolated clones was analyzed by recombinant proteins expressed in Pichia pastoris. The recombinant protein (Wfh-sm3m) encoded by one (Wfh-sm3) of the isolated clones preferentially degraded 6-kestotriose and possessed minor hydrolase activity to 1-kestotriose and 1,1-kestotetraose. Moreover, Wfh-sm3m hydrolyzed almost all graminans that accumulated in hardened wheat tissues. Wfh-sm3 transcripts increased in wheat leaf tissues inoculated with snow mold and incubated under snow cover. These results suggest that Wfh-sm3 encodes a 6-FEH with minor 1-FEH activity and is associated with degradation of fructans in wheat leaf tissues during inoculation and incubation under snow cover.

Changes in nitrogen assimilation, metabolism, and growth in transgenic rice plants expressing a fungal NADP(H)-dependent glutamate dehydrogenase (gdhA).

In plants, glutamine synthetase (GS) is the enzyme that is mainly responsible for the assimilation of ammonium. Conversely, in microorganisms such as bacteria and Ascomycota, NADP(H)-dependent glutamate dehydrogenase (GDH) and GS both have important roles in ammonium assimilation. Here, we report the changes in nitrogen assimilation, metabolism, growth, and grain yield of rice plants caused by an ectopic expression of NADP(H)-GDH (gdhA) from the fungus Aspergillus niger in the cytoplasm. An investigation of the kinetic properties of purified recombinant protein showed that the fungal gdhA had 5.4-10.2 times higher V(max) value and 15.9-43.1 times higher K(m) value for NH(4)(+), compared with corresponding values for rice cytosolic GS as reported in the literature. These results suggested that the introduction of fungal GDH into rice could modify its ammonium assimilation pathway. We therefore expressed gdhA in the cytoplasm of rice plants. NADP(H)-GDH activities in the gdhA-transgenic lines were markedly higher than those in a control line. Tracer experiments by feeding with (15)NH(4)(+) showed that the introduced gdhA, together with the endogenous GS, directly assimilated NH(4)(+) absorbed from the roots. Furthermore, in comparison with the control line, the transgenic lines showed an increase in dry weight and nitrogen content when sufficient nitrogen was present, but did not do so under low-nitrogen conditions. Under field condition, the transgenic line examined showed a significant increase in grain yield in comparison with the control line. These results suggest that the introduction of fungal gdhA into rice plants could lead to better growth and higher grain yield by enhancing the assimilation of ammonium.

Sclerotia of Typhula ishikariensis biotype B (Typhulaceae) from archaeological sites (4000 to 400 BP) in Hokkaido, northern Japan.

Despite their close association with human activities, plant pathogenic fungi have rarely been found in archaeological excavations. We report here that a fungus was closely associated with human activities even in prehistoric times. Sclerotium-like objects were found at historical sites (4000 to 400 BP) on the island of Hokkaido, northern Japan. They were spherical, 0.3-1.0 mm in diameter, and had a medulla and rind. Some had leaf fragments on the surface or a protuberance that resembled emerging sporocarp primordia. These traits indicated that they were sclerotia of the snow mold fungus, Typhula ishikariensis biotype B.

Creating S-type characteristics in the F-type enzyme fructan:fructan 1-fructosyltransferase of Triticum aestivum L.

Invertases cleave sucrose in glucose and fructose, using water as an acceptor. Fructosyltransferases catalyse the transfer of a fructosyl residue between sucrose and/or fructan molecules. Plant fructosyltransferases (FTs) evolved from vacuolar invertases by small mutational changes, leading to differences in substrate specificity. The S-type of enzymes (invertases, sucrose:sucrose 1-fructosyltransferases or 1-SSTs, and sucrose:fructan 6-fructosyltransferases or 6-SFTs) prefer sucrose as the donor substrate while F-type enzymes (fructan:fructan 1-fructosyltransferases or 1-FFTs and fructan:fructan 6(G)-fructosyltransferases or 6(G)-FFTs) preferentially use fructan as the donor substrate. Recently, a functional Asp/Arg or Asp/Lys couple in the Hypervariable Loop (HVL) was suggested to be essential to keep Asp in a favourable orientation for binding sucrose as the donor substrate in S-type enzymes. However, the F-type enzyme 1-FFT of Triticum aestivum (Ta1-FFT) also contains the Asp/Arg couple in the HVL, although it prefers fructan as the donor substrate. In this paper, mutagenesis studies on Ta1-FFT are presented. In Ta1-SST, Tyr282 (the Asp281 homologue) seems to be essential in creating a tight H-bond Network (HBN) in which the Arg-residue of the Asp/Arg couple is held in a fixed position. This tight HBN is disrupted in Ta1-FFT, leading to a more flexible Arg-residue and a dysfunctional Asp/Arg couple. A single D281Y mutation in Ta1-FFT restored the tight HBN and introduced typical S-type characteristics. Conclusively, in wheat FTs Asp281 (and its homologues) is involved in donor substrate specificity.

Cloning and functional analysis of a fructosyltransferase cDNA for synthesis of highly polymerized levans in timothy (Phleum pratense L.).

Variation in the structures of plant fructans and their degree of polymerization (DP) can be explained as the result of diverse combinations of fructosyltransferases (FTs) with different properties. Although FT genes have been isolated in a range of plant species, sucrose:fructan 6-fructosyltransferase (6-SFT) cDNAs have only been functionally characterized in a few species such as wheat. A novel FT cDNA possessing 6-SFT activity has been identified and characterized from the temperate forage grass, timothy (Phleum pratense L.). The cDNA of an FT homolog, PpFT1, was isolated from cold-acclimated timothy. A recombinant PpFT1 protein expressed in Pichia pastoris showed 6-SFT/sucrose:sucrose 1-fructosyltransferase (1-SST) activity and produced linear beta(2,6)-linked levans from sucrose with higher DPs than present in graminans formed in vitro by wheat 6-SFT (Wft1). PpFT1 and Wft1 showed remarkably different acceptor substrate specificities: PpFT1 had high affinity for 6-kestotriose to produce levans and low affinity for 1-kestotriose, whereas Wft1 preferentially used 1-kestotriose as an acceptor. The affinity of the PpFT1 recombinant enzyme for sucrose as a substrate was lower than that of the Wft1 recombinant enzyme. It is also confirmed that timothy seedlings had elevated levels of PpFT1 transcripts during the accumulation of fructans under high sucrose and cold conditions. Our results suggest that PpFT1 is a novel cDNA with unique enzymatic properties that differ from those of previously cloned plant 6-SFTs, and is involved in the synthesis of highly polymerized levans in timothy.

Particle inflow gun-mediated transformation of multiple-shoot clumps in rhodes grass (Chloris gayana).

Rhodes grass (Chloris gayana) is one of the most important warm-season forage grasses. It is cultivated in tropical and subtropical parts of the world and is mostly used for grazing and hay production. We have established a particle-bombardment transformation protocol for rhodes grass using multiple-shoot clumps (MSCs) as the target tissue. A vector pAHC25 containing a herbicide-resistance gene (bar) together with the beta-glucuronidase (GUS) gene was used in transformation experiments. The most efficient recovery of bialaphos-resistant tissue was achieved when the bombarded MSCs were first cultured for 15 d on bialaphos-free medium before being subjected to selection pressure. The resistant tissues regenerated transgenic plants that displayed GUS gene expression. Under optimized conditions, 251 target pieces yielded 46 transgenic plants from 4 independent transgenic lines.

Effects of different carbon sources on trichothecene production and Tri gene expression by Fusarium graminearum in liquid culture.

Fusarium head blight caused by Fusarium graminearum is a disease of cereal crops that not only reduces crop yield and quality but also results in contamination with trichothecenes such as nivalenol and deoxynivalenol (DON). To analyze the trichothecene induction mechanism, effects of 12 carbon sources on the production of DON and 3-acetyldexynivalenol (3ADON) were examined in liquid cultures incubated with nine strains of 3ADON-producing F. graminearum. Significantly high levels of trichothecene (DON and 3ADON) production by sucrose, 1-kestose and nystose were commonly observed among all of the strains tested. On the other hand, the levels of trichothecene biosynthesis induced by the other carbon sources were strain-specific. Tri4 and Tri5 expressions were up-regulated in the sucrose-containing medium but not in glucose. Trichothecene accumulation in the sucrose-containing medium was not repressed by the addition of glucose, indicating that trichothecene production was not regulated by carbon catabolite repression. These findings suggest that F. graminearum recognizes sucrose molecules, activates Tri gene expression and induces trichothecene biosynthesis.

Genetic engineering of rice capable of synthesizing fructans and enhancing chilling tolerance.

Fructans are water-soluble fructose oligomers and polymers that are based on sucrose, and have been implicated in protecting plants against water stress. Rice (Oryza sativa L.) is highly sensitive to chilling temperatures, and is not able to synthesize fructans. Two wheat fructan-synthesizing enzymes, sucrose:sucrose 1-fructosyltransferase, encoded by wft2, or sucrose:fructan 6-fructosyltransferase, encoded by wft1, were introduced into rice plants, and rice transformants that accumulate fructans were successfully obtained. The mature leaf blades of transgenic rice lines with wft2 or wft1 accumulated 16.2 mg g(-1) FW of oligo- and polysaccharides mainly composed of inulin oligomers of more than DP7, and 3.7 mg g(-1) FW of oligo- and polysaccharides, mainly composed of phlein oligomers of more than DP15, respectively. The transgenic rice seedlings with wft2 accumulated significantly higher concentrations of oligo- and polysaccharides than non-transgenic rice seedlings, and exhibited enhanced chilling tolerance. The oligo- and polysaccharide concentrations of seedlings expressing wft1 were obviously lower than those of lines expressing wft2, and no correlation between oligo- and polysaccharide concentrations and chilling tolerance was detected in wft1-expressing rice lines. The results suggest that transgenic rice lines expressing wheat-derived fructosyltransferase genes accumulated large amounts of fructans in mature leaf blades and exhibited enhanced chilling tolerance at the seedling stage. This is the first report owing that fructan accumulation enhanced tolerance to non-freezing low temperatures.

Purification, cloning and functional differences of a third fructan 1-exohydrolase (1-FEHw3) from wheat (Triticum aestivum).

A third fructan exohydrolase isoform (1-FEHw3) was purified from wheat stems by a combination of ammonium sulfate precipitation, ConA affinity and ion-exchange chromatography. Homogeneity of the preparation was indicated by the presence of a single band (70 kDa) after SDS-PAGE. The enzyme hydrolyzed mainly beta2-1 linkages in fructans and was inhibited by sucrose. A cDNA could be obtained after reverse transcriptase polymerase chain reaction (RT-PCR)-based strategies and screening of a cDNA library. Functionality tests of the cDNA performed after heterologous expression in the yeast Pichia pastoris showed that the encoded protein has essentially the same characteristics as the native enzyme. Homology with previously described 1-FEH isoforms from wheat was high (97% identity), and the enzyme showed minor differences to the previously published enzymes. The relative abundance of 1-FEH transcripts in different tissues was investigated by using quantitative RT-PCR.

Fructan 1-exohydrolase is associated with flower opening in Campanula rapunculoides.

Fructans, typically reserve carbohydrates, may also fulfil other more specific roles in plants. It has been convincingly demonstrated that fructan hydrolysis contributes to osmoregulation during flower opening in the monocot species Hemerocallis. We report that a massive breakdown of inulin-type fructans in the petals of Campanula rapunculoides L. (Campanulaceae), associated with flower opening, is accompanied by a strong increase in fructan 1-exohydrolase (1-FEH; EC 3.2.1.153) activity and a decrease in sucrose : sucrose 1-fructosyl transferase (1-SST; EC 2.4.1.99) activity. The data strongly suggest that the drastic change in the 1-FEH/1-SST activity ratio causes the degradation of inulin, contributing to the osmotic driving force involved in flower opening. All characterised plant FEHs are believed to be derived from tissues that store fructans as a reserve carbohydrate either temporarily (grasses and cereals) or over a longer term (dicot roots and tubers). Here, we focussed on a physiologically distinct tissue and used a reverse transcriptase-polymerase chain reaction based strategy to clone the 1-FEH cDNA from the Campanula petals. The translated cDNA sequence groups along with other dicot FEHs and heterologous expression revealed that the cDNA encodes a 1-FEH without invertase activity. 1-FEH expression analysis in petals correlates well with 1-FEH activity and inulin degradation patterns in vivo, suggesting that this enzyme fulfils an important role during flower opening.

Multicenter phase II study of amrubicin, 9-amino-anthracycline, in patients with advanced non-small-cell lung cancer (Study 1): West Japan Thoracic Oncology Group (WJTOG) trial.

PURPOSE: Amrubicin is a novel 9-aminoanthracycline. This multicenter phase II study was conducted to evaluate the efficacy and safety of amrubicin in patients with non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: Sixty-one previously untreated patients with stage III or IV NSCLC were entered this study. The patients were required to have cytologically or histologically proven measurable NSCLC, an Eastern Cooperative Oncology Group (ECOG) performance status of 0-2, and adequate organ function. Amrubicin was administered by daily intravenous injection at 45 mg/m2/day for 3 consecutive days every 3 weeks. At least 3 cycles of treatment were administered to each patient. RESULTS: All 61 patients registered in this trial were eligible and assessable for efficacy and toxicity. Of them, 17 patients achieved objective responses, consisting of one complete response and 16 partial responses, and the overall response rate was 27.9% (95% confidence interval [CI], 17.1% to 40.8%). The median survival time was 9.8 months (95% CI, 7.7 months to 14.9 months). The major toxicity was myelosuppression. The incidences of grade 3 or 4 toxicity were 72.1% for neutropenia, 52.5% for leukopenia, 23.0% for anemia, and 14.8% for thrombocytopenia. As noticeable toxic events, grade 3 hypotention and alkaline phosphatase elevation were transiently observed in one patient each. In addition, three patients who had had asymptomatic interstitial pneumonitis, identified by diagnostic imaging before treatment, aggravated after amrubicin treatment; two of them died. Other non-hematologic toxicities were relatively mild. CONCLUSION: Amrubicin was an active, well-tolerated agent in the treatment of NSCLC.

Molecular characterization of a cold-induced plasma membrane protein gene from wheat.

As a means to study the function of plasma membrane proteins during cold acclimation, we have isolated a cDNA clone for wpi6 which encodes a putative plasma membrane protein from cold-acclimated winter wheat. The wpi6 gene encodes a putative 5.9 kDa polypeptide with two predicted membrane-spanning domains, the sequence of which shows high sequence similarity with BLT101-family proteins from plants and yeast. Strong induction of wpi6 mRNA was observed during an early stage of cold acclimation in root and shoot tissues of both winter and spring wheat cultivars. In contrast to blt101 in barley, wpi6 mRNA was also induced by drought and salinity stresses, and exogenous application of ABA. Expression of wpi6 in a Deltapmp3 mutant of Saccharomyces cerevisiae, which is disturbed in plasma membrane potential due to the lack of a BLT101-family protein, partially complemented NaCl sensitivity of the mutant. Transient expression analysis of a WPI6::GFP fusion protein in onion epidermal cells revealed that WPI6 is localized in the plasma membrane. Taken together, these data suggested that WPI6 may have a protective role in maintaining plasma membrane function during cold acclimation in wheat.

Molecular cloning and functional analysis of a novel 6&1-FEH from wheat (Triticum aestivum L.) preferentially degrading small graminans like bifurcose.

Like barley and other cereals, wheat (Triticum aestivum L.) accumulates branched graminan-type fructans containing both beta-(2,1) and beta-(2,6) fructosyl linkages, mainly with a quite low degree of polymerization (DP). 1&6-kestotetraose (bifurcose) is the major fructan oligosaccharide accumulating in crown tissues and leaves of cereals exposed to chilling. The fructan exohydrolase (FEH) cDNAs 1-FEH w1 and w2 were previously cloned from wheat crowns sampled in mid-November. Here, we report the cloning and functional analysis of another FEH cDNA from a mid-November wheat crown cDNA library. The cDNA encodes a long open reading frame (ORF) of 595 amino acids. Like other FEHs, it has a low iso-electric point (5.2) and it groups together with cell-wall type invertases and not with vacuolar invertases. The deduced amino acid sequence shows 67% identity to wheat 1-FEH w1 and w2. Functional characterization of the recombinant proteins in Pichia pastoris demonstrated that the recombinant enzyme had FEH activity towards the pure compounds 1-kestose, 6-kestose, 1,1-nystose and 1,1,1-kestopentaose. However, when incubated with its putative natural substrates (a mixture of low DP graminans from wheat crowns), it was shown that 1&6-kestotetraose (bifurcose) was preferentially removed from the graminan mixture. High DP wheat graminan and bacterial levan were only poor substrates. No hydrolase activities could be detected towards sucrose and high DP inulin, convincingly demonstrating that the enzyme is not a classic invertase or 1-FEH. The enzyme was termed 6&1-FEH w1. Northern blot analyses showed that 6&1-FEH w1 was expressed in crown tissue from autumn through winter under snow, while the expression levels in leaves were minimal or not detectable. The results strongly suggest that this unique FEH might play an important role in the degradation of branched, low DP wheat graminan (like bifurcose) in wheat crowns in the high fructan content season.

Fructan:fructan 1-fructosyltransferase, a key enzyme for biosynthesis of graminan oligomers in hardened wheat.

Fructans play important roles not only as a carbon source for survival under persistent snow cover but also as agents that protect against various stresses in overwintering plants. Complex fructans having both beta-(2,1)- and beta-(2,6)-linked fructosyl units accumulate in wheat (Triticum aestivum L.) during cold hardening. We detected fructan: fructan 1-fructosyltransferase (1-FFT; EC 2.4.1.100) activity for catalyzing the formation and extension of beta-(2,1)-linked fructans in hardened wheat tissues, cloned cDNAs (wft3 and wft4) of 1-FFT, and analyzed the enzymatic properties of a wft3 recombinant protein (Wft3m) produced by yeast. Wft3m transferred beta-(2,1)-linked fructosyl units to phlein, an extension of sucrose through beta-(2,6)-linked fructosyl units, as well as to inulin, an extension of sucrose through beta-(2,1)-linked fructosyl units, but could not efficiently synthesize long inulin oligomers. Incubation of a mixture of Wft3m and another recombinant protein of wheat, sucrose:fructan 6-fructosyltransferase (6-SFT), with sucrose and 1-kestotriose produced fructans similar to those that accumulated in hardened wheat tissues. The results demonstrate that 1-FFT produces branches of beta-(2,1)-linked fructosyl units to phlein and graminan oligomers synthesized by 6-SFT and contributes to accumulation of fructans containing beta-(2,1)- and beta-(2,6)-linked fructosyl units. In combination with sucrose:sucrose 1-fructosyltransferase (1-SST; EC 2.4.1.99) and 6-SFT, 1-FFT is necessary for fructan synthesis in hardened wheat.