Fructan Exohydrolase Activity Research Articles

A Fructan Exohydrolase from Maize Degrades Both Inulin and Levan and Co-Exists with 1-Kestotriose in Maize.

Enzymes with fructan exohydrolase (FEH) activity are present not only in fructan-synthesizing species but also in non-fructan plants. This has led to speculation about their functions in non-fructan species. Here, a cell wall invertase-related Zm-6&1-FEH2 with no “classical” invertase motif was identified in maize. Following heterologous expression in Pichia pastoris and in Nicotiana benthamiana leaves, the enzyme activity of recombinant Zm-6&1-FEH2 displays substrate specificity with respect to inulin and levan. Subcellular localization showed Zm-6&1-FEH2 exclusively localized in the apoplast, and its expression profile was strongly dependent on plant development and in response to drought and abscisic acid. Furthermore, formation of 1-kestotriose, an oligofructan, was detected in vivo and in vitro and could be hydrolyzed by Zm-6&1-FEH2. In summary, these results support that Zm-6&1-FEH2 enzyme from maize can degrade both inulin-type and levan-type fructans, and the implications of the co-existence of Zm-6&1-FEH2 and 1-kestotriose are discussed.

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.

A 6&1-FEH Encodes an Enzyme for Fructan Degradation and Interact with Invertase Inhibitor Protein in Maize (Zea mays L.).

About 15% of higher plants have acquired the ability to convert sucrose into fructans. Fructan degradation is catalyzed by fructan exohydrolases (FEHs), which are structurally related to cell wall invertases (CWI). However, the biological function(s) of FEH enzymes in non-fructan species have remained largely enigmatic. In the present study, one maize CWI-related enzyme named Zm-6&1-FEH1, displaying FEH activity, was explored with respect to its substrate specificities, its expression during plant development, and its possible interaction with CWI inhibitor protein. Following heterologous expression in Pichia pastoris and in N. benthamiana leaves, recombinant Zm-6&1-FEH1 revealed substrate specificities of levan and inulin, and also displayed partially invertase activity. Expression of Zm-6&1-FEH1 as monitored by qPCR was strongly dependent on plant development and was further modulated by abiotic stress. To explore whether maize FEH can interact with invertase inhibitor protein, Zm-6&1-FEH1 and maize invertase inhibitor Zm-INVINH1 were co-expressed in N. benthamiana leaves. Bimolecular fluorescence complementation (BiFC) analysis and in vitro enzyme inhibition assays indicated productive complex formation. In summary, the results provide support to the hypothesis that in non-fructan species FEH enzymes may modulate the regulation of CWIs.

Correlation between Fructan Exohydrolase Activity and the Quality of Helianthus tuberosus L. Tubers

Jerusalem artichoke tubers have diverse applications in the food industry as well as in biotechnology. Their suitability depends mostly on the inulin content. Seasonal fluctuations of fructan exohydrolase activity responsible for inulin degradation was investigated in the tubers of three Jerusalem artichoke cultivars. The changes of fructan exohydrolase activity positively correlated with the changes of the content of total and short fructooligosaccharides. Therefore, to extract inulin with higher degree of polymerization for biotechnological purposes, the tubers of Jerusalem artichoke should be uprooted in autumn before the level of fructan exohydrolase reaches its maximum. If short fructooligosaccharides are desirable, the tubers in late autumn or spring tubers overwintered in soil are suitable.

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.

Factors affecting fructosyltransferases and fructan exohydrolase activities in Agave tequilana Weber var. azul

There is great interest in the fructosyltransferases (FTFs) involved in fructan metabolism and agents affecting their activity. Agaves accumulate fructans, fructose polymers linked by glycosidic β(2–1) and β(2–6) bonds in linear or branched configurations. In plants, fructans provide protection under stress conditions. The sucrose:sucrose 1-fructosyltransferase (1-SST), fructan:fructan 1-fructosyltransferase (1-FFT), fructan:fructan 6G-fructosyltransferase (6G-FFT), and fructan exohydrolase (FEH) activities were analyzed in micropropagated Agave tequilana plants in the absence and presence of HgCl2, AgNO3, MgCl2, sodium deoxycholate (DNa), and sodium dodecyl sulfate (SDS). Kestose, nystose and neokestose were synthesized by the respective FTFs. HgCl2 and AgNO3 inhibited all FTFs, mainly up to 90 % in 1-SST and 1-FFT. DNa increased 1-SST (32 %) and 1-FFT (45 %) activities, and SDS increased 6G-FFT activity by 96 %. Finally, AgNO3 inhibited FEH activity by 78 %. Our results might be relevant on the regulation of FTFs in agave and other crops, for instance by the increment the fructans synthesis in stressed plants.

Determination of fructan exohydrolase activity in the crude extracts of plants

article i nfo Background: The roots of chicory and the tubers of Jerusalem artichoke are used for the production of inulin. However, a quality of tubers and roots, i.e. the content of inulin, monosaccarides and disaccharides, depends on the activity of enzymes implicated in the metabolism of inulin. The knowledge on the changes of activities of inulin synthesizing and degrading enzymes is limited during plant sprouting, growth and dormancy. It happens due to complicated measurements of the product of enzymatic reaction in the presence of crude plant extract. Fructan exohydrolase (β-D-fructan fructohydrolase, FEH, EC 3.2.1.80) is an enzyme responsible for the hydrolysis of fructans in plants. For fructose as the reaction product measurement, a high-performance liquid chromatography is usually used. The aim of the study was to choose a simple and suitable method for FEH activity determination and the measurement of fructose in the presence of plant extracts. Results: Two chemical methods, i.e. copper(II)-neocuproine and 3,5-dinitrosalicylic acid, and the enzymatic one based on the reactions catalyzed by hexokinase, phosphoglucose isomerase and glucose-6-phosphate dehydrogenase were used. Enzymatic method was found to be suitable for FEH activity determination in plant extracts, and on the contrary to chemical methods no interference effects of compounds from crude plant extracts were observed. Conclusion: Enzymatic method is applicable for the routine analysis and will allow performing the investigations without special equipment on the inulin degrading enzyme in biotechnologically important crops.

The Relationship Between Yield and Fructan Exo-Hydrolases Activity in Two Drought Resistant Wheat Cultivars Grown Under Different Fertilizer and Tillage Treatments

Using two drought resistant wheat (Triticum aestivum L.) cultivars, ‘Changwu134’ and ‘Changhan58,’ a field experiment was conducted in ChangWu Agro-ecological Experiment Station on China's Loess Plateau during 2008 and 2009 to compare the effects of different fertilizer and tillage treatments on the fructan contents as well as fructan exo-hydrolase (FEH) activity in relation to wheat yield. We found that ‘Changhan58’ had greater yield and fructan content in the penultimate internode and higher FEH activity than did ‘Changwu134.’ For ‘Changhan58.’ applying 195 kg·ha−1(120 +75) N plus 45000 kg·ha−1 of pig manure and 120 kg·ha−1 phosphate under conservation tillage produced the highest yield (6769 ka/ha), fructan content in penultimate internode, water use efficiency (WUE), as well as FEH activity among the fertilizer and tillage treatments. Therefore, routine soil management for wheat should focus on combined use of manures and inorganic fertilizer to enhance the amount and transportation efficiency of WSC and ultimately ensure greater yield.

Cloning and characterization of a novel fructan 6-exohydrolase strongly inhibited by sucrose in Lolium perenne.

The first 6-fructan exohydrolase (6-FEH) cDNA from Lolium perenne was cloned and characterized. Following defoliation, Lp6 - FEHa transcript level unexpectedly decreased together with an increase in total FEH activity. Lolium perenne is a major forage grass species that accumulates fructans, mainly composed of β(2,6)-linked fructose units. Fructans are mobilized through strongly increased activities of fructan exohydrolases (FEHs), sustaining regrowth following defoliation. To understand the complex regulation of fructan breakdown in defoliated grassland species, the objective was to clone and characterize new FEH genes in L. perenne. To find FEH genes related to refoliation, a defoliated tiller base cDNA library was screened. Characterization of the recombinant protein was performed in Pichia pastoris. In this report, the cloning and enzymatic characterization of the first 6-FEH from L. perenne is described. Following defoliation, during fructan breakdown, Lp6-FEHa transcript level unexpectedly decreased in elongating leaf bases (ELB) and in mature leaf sheaths (tiller base) in parallel to increased total FEH activities. In comparison, transcript levels of genes coding for fructosyltransferases (FTs) involved in fructan biosynthesis also decreased after defoliation but much faster than FEH transcript levels. Since Lp6-FEHa was strongly inhibited by sucrose, mechanisms modulating FEH activities are discussed. It is proposed that differences in the regulation of FEH activity among forage grasses influence their tolerance to defoliation.

ヤーコン塊根のフルクトオリゴ糖含量とフルクタン分解酵素に対する低温減圧貯蔵の影響

ヤーコンの塊根にはプレバイオティクス素材として注目されるフルクトオリゴ糖（FOS）が豊富に含まれているが，貯蔵中に内在性酵素によって分解され，その含量が著しく減少する．収穫後の塊根を長期に FOS を保持した状態で貯蔵することが可能になれば，健康維持・増進に向けた有効活用につながる．そこで，細胞の代謝活性を低下できる低温減圧条件に FOS の分解・減少を抑制する効果があるか調べた．ヤーコン塊根を 4°C下，常圧（1 気圧）もしくは減圧（0.7気圧）条件にて 1ヶ月間貯蔵したところ，常圧では FOS が顕著に減少したのに対し，減圧下では FOS の減少が抑制されていた．塊根内にて FOS 分解に寄与するフルクタン 1-加水分解酵素（1-FEH）およびスクロースを分解するインベルターゼ（INV）の活性についても調べたところ，いずれの酵素も常圧に比べて減圧では活性が低下しており，糖質代謝が抑制されて FOS が分解を受けずに保持されたことが明らかとなった．

Sink filling, inulin metabolizing enzymes and carbohydrate status in field grown chicory (Cichorium intybus L.)

Inulin is a fructose-based polymer that is isolated from chicory (Cichorium intybus L.) taproots. The degree of polymerization (DP) determines its application and hence the value of the crop. The DP is highly dependent on the field conditions and harvest time. Therefore, the present study was carried out with the objective to understand the regulation of inulin metabolism and the process that determines the chain length and inulin yield throughout the whole growing season. Metabolic aspects of inulin production and degradation in chicory were monitored in the field and under controlled conditions. The following characteristics were determined in taproots: concentrations of glucose, fructose and sucrose, the inulin mean polymer length (mDP), yield, gene expression and activity of enzymes involved in inulin metabolism. Inulin synthesis, catalyzed by sucrose:sucrose 1-fructosyltransferase (EC 2.4.1.99) (1-SST) and fructan:fructan 1-fructosyltransferase (EC 2.4.1.100) (1-FFT), started at the onset of taproot development. Inulin yield as a function of time followed a sigmoid curve reaching a maximum in November. Inulin reached a maximum mDP of about 15 in September, than gradually decreased. Based on the changes observed in the pattern of inulin accumulation, we defined three different phases in the growing season and analyzed product formation, enzyme activity and gene expression in these defined periods. The results were validated by performing experiments under controlled conditions in climate rooms. Our results show that the decrease in 1-SST that starts in June is not regulated by day length and temperature. From mid-September onwards, the mean degree of polymerization (mDP) decreased gradually although inulin yield still increased. The decrease in mDP combined with increased yield results from fructan exohydrolase activity, induced by low temperature, and the back transfer activity of 1-FFT. Overall, this study provides background information on how to improve inulin yield and quality in chicory.

Carbohydrates Accumulation and Remobilization in Wheat Plants as Influenced by Combined Waterlogging and Shading Stress During Grain Filling

AbstractWheat plants were subjected to combined waterlogging and shading stress (WS) at 0–7, 8–15, 16–23 and 24–31 days after anthesis (DAA), respectively. Compared to the non‐stressed plants, WS significantly decreased the final grain yield. Grain number was dramatically lowered by WS imposed at 0–7 DAA but hardly affected by other WS treatments; while the thousand‐kernel‐weight was unaffected by WS imposed at 0–7 DAA and lowered by other WS treatments. Photosynthate accumulation in the stem was decreased by WS imposed at 0–7 and 8–15 DAA, but was unaffected by WS imposed at later stages. Grain‐filling rate was decreased, although the apparent remobilization of carbohydrate reserves from stem to grain was stimulated under WS. The carbohydrate reserves stored in the lower stem internodes were activated earlier and remobilized much more than those in the upper internodes; however, the proportion of the apparent remobilized reserves among the different stem internodes was consistent for all treatments. WS decreased contents of fructans, fructose and sucrose in the stem, which coincided with increased activity of fructan exohydrolase and decreased activities of sucrose‐1‐fructosyltransferase and fructan‐1‐fructosyltransferse. The results indicate that post‐anthesis WS stimulated carbohydrate reserves remobilization by modifying the activities of the fructans‐catalysing enzymes in the stem.

ENYMOLOGIC CHARACTERIZATION OF GARLIC FRUCTAN EXOHYDROLASE

Fructans represent over 75% of the dry mass of garlic (Allium sativum L.). Understanding the enzymologic characteristics of garlic fructan exohydrolase (FEH) can provide theoretical basis on garlic germination inhibition and mass production of high-quality garlic fructans. In this study, we isolated and localized FEH by ammonium sulfate fractional precipitation, analyzed the fructan enzymolysis products by thin-layer chromatography (TLC) and ion chromatography, and determined FEH activity by measuring changes of reducing sugars via 3,5-dinitrosalicylic acid assay. We further characterized the hydrolysis site of FEH and effects of temperature, pH level, ionic strength and substrate concentration on its activity. Results showed that FEH was present in the 0–20% ammonium sulfate fraction. Analysis of the hydrolysis product by both TLC and ion chromatography suggested that FEH was an excision enzyme. The optimum conditions for FEH activity were 45C, pH 5.5, ionic strength of 1.5 M (sodium chloride) and substrate concentration of 4 mg/mL. PRACTICAL APPLICATIONS During garlic storage, quality degradation such as shrinkage, softening, drying and decreased crispiness often occurs. These deteriorations might be related to garlic fructan exohydrolase (FEH), because FEH could hydrolyze storage carbohydrates (fructan) to produce monosaccharide for energy metabolism. Thus, to keep the high quality of garlic during storage, the key is to control the activity of garlic FEH. We studied the activity of the enzyme. Results obtained from this study will provide foundation for FEH research and insights for manufacturing homogeneous-quality garlic fructan products.

Hexokinase-dependent sugar signaling represses fructan exohydrolase activity in Lolium perenne

Defoliation of perennial ryegrass (Lolium perenne L.) by grazing animals leads to fructan mobilisation via an increase of fructan exohydrolase (FEH) activity. To highlight the regulation of fructan metabolism in perennial ryegrass, the role of sugars as signalling molecules for regulation of FEH activity after defoliation was evaluated. We used an original approach in planta by spraying stubble of defoliated plants (sugar starved plants) during 24 h with metabolisable sugars (glucose, fructose, sucrose) and sugar analogues (3-O-methylglucose, mannose, lactulose, turanose, palatinose). Metabolisable sugar (glucose, fructose, sucrose) supply following defoliation led to the repression of FEH activity increase. The supply of mannose, which is phosphorylated by hexokinase but not further metabolisable, led to the same repressive effect, whereas 3-O-methylglucose, which is not a substrate for hexokinase, had no effect. These results indicate that hexoses could be sensed by hexokinase, triggering a chain of events leading to the repression of FEH activity. By contrast, it was not possible to determine the role of sucrose as a signal since the supply of sucrose analogues (lactulose, turanose and palatinose) enhanced internal hexose content.

Cloning and functional characterization of a fructan 1-exohydrolase (1-FEH) in the cold tolerant Patagonian species Bromus pictus

Fructans are fructose polymers synthesized in a wide range of species such as bacteria, fungi and plants. Fructans are synthesized by fructosyltransferases (FTs) and depolymerized by fructan exohydrolases (FEHs). Bromus pictus is a graminean decaploid species from the Patagonian region of Argentina, which accumulates large amounts of fructans even at temperate temperatures. The first gene isolated from B. pictus fructan metabolism was a putative sucrose:fructan 6-fructosyltransferase (6-SFT). Here, a complete cDNA of the first fructan exohydrolase (FEH) from B. pictus (Bp1-FEHa) was isolated using RT-PCR strategies. The Bp1-FEHa encoding gene is present as a single copy in B. pictus genome. Functional characterization in Pichia pastoris confirmed Bp1-FEHa is a fructan exohydrolase with predominant activity towards beta-(2-1) linkages. Its expression was analyzed in different leaf sections, showing the highest expression levels in the second section of the sheath and the tip of the blade. Bp1-FEHa expression was studied along with FEH and FT activities and fructan accumulation profile in response to chilling conditions during a 7-day time course experiment. Bp1-FEHa expression and FEH activity followed a similar pattern in response to low temperatures, especially in basal sections of the sheaths. In these sections the FEH and FT activities were particularly high and they were significantly correlated to fructan accumulation profile, along with cold treatment.

Freezing tolerance by vesicle-mediated fructan transport

Fructans are fructose-based polymers associated with freezing tolerance. They might act directly via membrane stabilization or indirectly by stimulating alternative cryoprotectants. Fructans and fructan biosynthetic enzymes, in general, are believed to be present in the vacuole. This paper draws particular attention to the surprising presence of fructans and fructan exohydrolase activity in the apoplast of cold-stressed plants. This observation raises questions concerning the origin of apoplastic fructans and suggests that fructans are transported to the apoplast by post-synthesis mechanisms, perhaps induced by cold. We propose a conceptual vesicle-mediated transport model for the movement of vacuolar fructans to the apoplast, where they could assist in stabilizing the plasma membrane.

Cloning, gene mapping, and functional analysis of a fructan 1-exohydrolase (1-FEH) from Lolium perenne implicated in fructan synthesis rather than in fructan mobilization

Fructans, which are beta-(2,1) and/or beta-(2,6) linked polymers of fructose, are important storage carbohydrates in many plants. They are mobilized via fructan exohydrolases (FEHs). The cloning, mapping, and functional analysis of the first 1-FEH (EC 3.2.1.153) from Lolium perenne L. var. Bravo is described here. By screening a perennial ryegrass cDNA library, a 1-FEH cDNA named Lp1-FEHa was cloned. The Lp1-FEHa deduced protein has a low iso-electric point (5.22) and it groups together with plant FEHs and cell-wall type invertases. The deduced amino acid sequence shows 75% identity to wheat 1-FEH w2. The Lp1-FEHa gene was mapped at a distal position on the linkage group 3 (LG3). Functional characterization of the recombinant protein in Pichia pastoris demonstrated that it had high FEH activity towards 1-kestotriose, 1,1-kestotetraose, and inulin, but low activity against 6-kestotriose and levan. Like other fructan-plant FEHs, no hydrolase activity could be detected towards sucrose, convincingly demonstrating that the enzyme is not a classic invertase. The expression pattern analysis of Lp1-FEHa revealed transcript accumulation in leaf tissues accumulating fructans while transcript level was low in the photosynthetic tissues. The high expression level of this 1-FEH in conditions of active fructan synthesis, together with its low expression level when fructan contents are low, suggest that it might play a role as a beta-(2,1) trimming enzyme acting during fructan synthesis in concert with fructan synthesis enzymes.

The rice genome encodes two vacuolar invertases with fructan exohydrolase activity but lacks the related fructan biosynthesis genes of the Pooideae

* Fructans are believed to contribute to cold and drought tolerance in several plant families (Poaceae, Asparagaceae and Asteraceae), but it is not clear why the ability to accumulate these polymers is found in some genera (e.g. Triticum) but not in others (e.g. Oryza). * As fructan biosynthesis enzymes (FBEs) evolved from vacuolar invertases (VINs), we searched the rice genome sequence for genes related to both FBE and VIN genes of wheat and other members of the Pooideae. We compared them at the levels of exon-intron structure, protein sequence, and the enzymatic properties of recombinant proteins after expression in the yeast Pichia pastoris. * We found that rice possesses two VIN genes (OsVIN1 and OsVIN2) and no FBE genes. FBE genes appear to have arisen in the Pooideae by a series of gene duplications from an ancestor of wheat TaVIN3. Recombinant TaVIN2, OsVIN1 and OsVIN2 behaved as invertases with no FBE activity, but possessed high fructan exohydrolase activity, especially OsVIN1. * The engineering of fructan accumulation into rice for greater stress tolerance could founder on endogenous exohydrolases, but the fact that OsVIN1 transcripts are absent from peduncles of well watered and drought-stressed plants removes one potential obstacle to this endeavour.

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 β-(2,1) and β-(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.

Arabidopsis AtcwINV3 and 6 are not invertases but are fructan exohydrolases (FEHs) with different substrate specificities

ABSTRACTThe genome of Arabidopsis thaliana contains six putative cell‐wall type invertase genes (AtcwINV1‐6). Heterologous expression of AtcwINV1, 3 and 6 cDNAs in Pichia pastoris revealed that the enzymes encoded by AtcwINV3 and 6 did not show invertase activity. Instead, AtcwINV3 is a 6‐FEH and AtcwINV6 is a fructan exohydrolase (FEH) that can degrade both inulin and levan‐type fructans. For AtcwINV6 it is proposed to use the term (6&1) FEH. In contrast, AtcwINV1 is a typical invertase. FEH activity was also detected in crude extracts of different parts of Arabidopsis. To verify that the FEH activity of AtcwINV3 and 6 were not artefacts of the heterologous expression system, the protein corresponding to AtcwINV3 was isolated from whole Arabidopsis plants and indeed showed only 6‐FEH activity and no invertase activity. Although no fructans can be detected in Arabidopsis plants, it is shown that kestoses (trimers) can be synthesized in crude leaf extracts. The putative physiological significance of FEH in so‐called non‐fructan plants is discussed.