Fructan Exohydrolase Research Articles

Loss of function of 1-FEH IIb has more impact on post-harvest inulin degradation in Cichorium intybus than copy number variation of its close paralog 1-FEH IIa.

Key Message: The loss of mini-exon 2 in the 1-FEH IIb glycosyl-hydrolase results in a putative non-functional allele. This loss of function has a strong impact on the susceptibility to post-harvest inulin depolymerization. Significant variation of copy number was identified in its close paralog 1-FEH IIa, but no quantitative effect of copy number on carbohydrates-related phenotypes was detected.Inulin polyfructan is the second most abundant storage carbohydrate in flowering plants. After harvest, it is depolymerized by fructan exohydrolases (FEHs) as an adaptive response to end-season cold temperatures. In chicory, the intensity of this depolymerization differs between cultivars but also between individuals within a cultivar. Regarding this phenotypic variability, we recently identified statistically significant associations between inulin degradation and genetic polymorphisms located in three FEHs. We present here new results of a systematic analysis of copy number variation (CNV) in five key members of the chicory (Cichorium intybus) GH32 multigenic family, including three FEH genes and the two inulin biosynthesis genes: 1-SST and 1-FFT. qPCR analysis identified a significant variability of relative copy number only in the 1-FEH IIa gene. However, this CNV had no quantitative effect. Instead, cloning of the full length gDNA of a close paralogous sequence (1-FEH IIb) identified a 1028 bp deletion in lines less susceptible to post-harvest inulin depolymerization. This region comprises a 9 bp mini-exon containing one of the three conserved residues of the active site. This results in a putative non-functional 1-FEH IIb allele and an observed lower inulin depolymerization. Extensive genotyping confirmed that the loss of mini-exon 2 in 1-FEH IIb and the previously identified 47 bp duplication located in the 3′UTR of 1-FEH IIa belong to a single haplotype, both being statistically associated with reduced susceptibility to post-harvest inulin depolymerization. Emergence of these haplotypes is discussed.

Fructan metabolism and changes in fructan composition during cold acclimation in perennial ryegrass.

Perennial ryegrass (Lolium perenne L.) produces high levels of fructans as a mixture of oligosaccharides and polysaccharides with different degrees of polymerization (DP). The present study describes the analysis of the compositional changes in the full spectrum of fructans, fructan distribution between above ground biomass (top) and the roots, and the transcription of candidate genes involved in fructan metabolism during cold acclimation in perennial ryegrass variety “Veyo” and ecotype “Falster” from distinct geographical origins. We observed changes in fructan composition and induction of low-DP fructans, especially DP = 4, in both the top and the roots of “Veyo” and “Falster” in response to low-temperature stress. The accumulation of DP > 50 fructans was only apparent in the top tissues where the Lp1-FFT expression is higher compared to the roots in both “Veyo” and “Falster.” Our results also show the accumulation and depolymerization of fructans with different DP, together with the induction of genes encoding fructosyltransferases and fructan exohydrolases in both “Veyo” and “Falster” during cold acclimation, supporting the hypothesis that fructan synthesis and depolymerization occurring simultaneously. The ecotype “Falster,” adapted to cold climates, increased total fructan content and produced more DP > 7 fructans in the roots than the variety “Veyo,” adapted to warmer climates. This indicates that high-DP fructan accumulation in roots may be an adaptive trait for plant recovery after abiotic stresses.

Endogenous hormone concentrations correlate with fructan metabolism throughout the phenological cycle in Chrysolaena obovata.

Chrysolaena obovata, an Asteraceae of the Brazilian Cerrado, presents seasonal growth, marked by senescence of aerial organs in winter and subsequent regrowth at the end of this season. The underground reserve organs, the rhizophores, accumulate inulin-type fructans, which are known to confer tolerance to drought and low temperature. Fructans and fructan-metabolizing enzymes show a characteristic spatial and temporal distribution in the rhizophores during the developmental cycle. Previous studies have shown correlations between abscisic acid (ABA) or indole acetic acid (IAA), fructans, dormancy and tolerance to drought and cold, but the signalling mechanism for the beginning of dormancy and sprouting in this species is still unknown. Adult plants were sampled from the field across phenological phases including dormancy, sprouting and vegetative growth. Endogenous concentrations of ABA and IAA were determined by GC-MS-SIM (gas chromatography-mass spectrometry-selected ion monitoring), and measurements were made of fructan content and composition, and enzyme activities. The relative expression of corresponding genes during dormancy and sprouting were also determined. Plants showed a high fructan 1-exohydrolase (EC 3.2.1.153) activity and expression during sprouting in proximal segments of the rhizophores, indicating mobilization of fructan reserves, when ABA concentrations were relatively low and precipitation and temperature were at their minimum values. Concomitantly, higher IAA concentrations were consistent with the role of this regulator in promoting cell elongation and plant growth. With high rates of precipitation and high temperatures in summer, the fructan-synthesizing enzyme sucrose:sucrose 1-fructosyltransferase (EC 2.4.1.99) showed higher activity and expression in distal segments of the rhizophores, which decreased over the course of the vegetative stage when ABA concentrations were higher, possibly signalling the entry into dormancy. The results show that fructan metabolism correlates well with endogenous hormone concentrations and environmental changes, suggesting that the co-ordinated action of carbohydrate metabolism and hormone synthesis enables C. obovata to survive unfavourable field conditions. Endogenous hormone concentrations seem to be related to regulation of fructan metabolism and to the transition between phenophases, signalling for energy storage, reserve mobilization and accumulation of oligosaccharides as osmolytes.

Fructan and hormone connections.

Plants rely on “reserve” (stored) carbon (C) for growth and survival when newly synthesized C becomes limited. Besides a classic yet recalcitrant C reserve starch, fructans, a class of sucrose-derived soluble fructosyl-oligosaccharides, represent a major store of C in many temperate plant species including the economically important Asteraceae and Poaceae families (Hendry, 1993). Dicots typically accumulate inulin-type fructans as long-term storage (underground organs) whilst grasses and cereals accumulate fructans as short-term reserves in above-ground parts (Pollock and Cairns, 1991; Van Laere and Van den Ende, 2002). Unlike chloroplast-based water-insoluble starch, fructans are semi-soluble, possess flexible structures (Phelps, 1965; Valluru and Van den Ende, 2008), can be synthesized at low temperatures (Pollock and Cairns, 1991), and are degraded by a single type of fructan hydrolases, fructan exohydrolases (FEHs). Unlike starch that store in plastids, fructans store in vacuoles, which is physically less stressful to the active constituents of, and allows more C synthesis by, the photosynthetic cell, which may be different in dicots where fructans do not typically accumulate in green parts.

Cloning and functional characterization of two abiotic stress-responsive Jerusalem artichoke (Helianthus tuberosus) fructan 1-exohydrolases (1-FEHs).

Two fructan hydrolases were previously reported to exist in Jerusalem artichoke (Helianthus tuberosus) and one native fructan-β-fructosidase (1-FEH) was purified to homogeneity by SDS-PAGE, but no corresponding cDNA was cloned. Here, we cloned two full-length 1-FEH cDNA sequences from Jerusalem artichoke, named Ht1-FEH I and Ht1-FEH II, which showed high levels of identity with chicory 1-FEH I and 1-FEH II. Functional characterization of the corresponding recombinant proteins in Pichia pastoris X-33 demonstrated that both Ht1-FEHs had high levels of hydrolase activity towards β(2,1)-linked fructans, but low or no activity towards β(2,6)-linked levan and sucrose. Like other plant FEHs, the activities of the recombinant Ht1-FEHs were greatly inhibited by sucrose. Real-time quantitative PCR analysis showed that Ht1-FEH I transcripts accumulated to high levels in the developing leaves and stems of artichoke, whereas the expression levels of Ht1-FEH II increased in tubers during tuber sprouting, which implies that the two Ht1-FEHs play different roles. The levels of both Ht1-FEH I and II transcript were significantly increased in the stems of NaCl-treated plants. NaCl treatment also induced transcription of both Ht1-FEHs in the tubers, while PEG treatments slightly inhibited the expression of Ht1-FEH II in tubers. Analysis of sugar-metabolizing enzyme activities and carbohydrate concentration via HPLC showed that the enzyme activities of 1-FEHs were increased but the fructose content was decreased under NaCl and PEG treatments. Given that FEH hydrolyzes fructan to yield Fru, we discuss possible explanations for the inconsistency between 1-FEH activity and fructan dynamics in artichokes subjected to abiotic stress.

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.

Plant maturity and nitrogen fertilization affected fructan metabolism in harvestable tissues of timothy (Phleum pratense L.)

Timothy (Phleum pratense L.) is an important grass forage used for pasture, hay, and silage in regions with cool and humid growth seasons. One of the factors affecting the nutritive value of this grass is the concentration of non-structural carbohydrates (NSC), mainly represented by fructans. NSC concentration depends on multiple factors, making it hardly predictable. To provide a better understanding of NSC metabolism in timothy, the effects of maturity stage and nitrogen (N) fertilization level on biomass, NSC and N-compound concentrations were investigated in the tissues used for forage (leaf blades and stems surrounded by leaf sheaths) of hydroponically grown plants. Moreover, activities and relative expression level of enzymes involved in fructan metabolism were measured in the same tissues. Forage biomass was not altered by the fertilization level but was strongly modified by the stage of development. It increased from vegetative to heading stages while leaf-to-stem biomass ratio decreased. Total NSC concentration, which was not altered by N fertilization level, increased between heading and anthesis due to an accumulation of fructans in leaf blades. Fructan metabolizing enzyme activities (fructosyltransferase—FT and fructan exohydrolase—FEH) were not or only slightly altered by both maturity stage and N fertilization level. Conversely, the relative transcript levels of genes coding for enzymes involved in fructan metabolism were modified by N supply (PpFT1 and Pp6-FEH1) or maturity stage (PpFT2). The relative transcript level of PpFT1 was the highest in low N plants while that of Pp6-FEH1 was the highest in high N plants. Morevoer, transcript level of PpFT1 was negatively correlated with nitrate concentration while that of PpFT2 was positively correlated with sucrose concentration. This distinct regulation of the two genes coding for 6-sucrose:fructan fructosyltransferase (6-SFT) may allow a fine adequation of C allocation towards fructan synthesis in response to carbon and N availability. Contrary to fructans, starch content increased in low N plants, suggesting different regulatory mechanisms and/or sensitivity of starch and fructan metabolism in relation to the N status.

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.

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

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

Transcript profiling of fructan biosynthetic pathway genes reveals association of a specific fructosyltransferase isoform with the high sugar trait in Lolium perenne

Lolium perenne cultivars with elevated levels of fructans in leaf blades (high sugar-content grasses) have been developed to improve animal nutrition and reduce adverse environmental impacts of pastoral agricultural systems. Expression of the high sugar trait can vary substantially depending on genotype×environment (G×E) interactions. We grew three potential high sugar-content and a control cultivar in three temperature regimes and quantified water soluble carbohydrates (WSCs) and the expression of all functionally characterised L. perenne fructan pathway genes in leaf tissues. We also analysed the distribution, expression and sequence variation of two specific isoforms of Lp6G-FFT (fructan: fructan 6G-fructosyltransferase). Our study confirmed a significant G×E interaction affecting the accumulation of fructans in the high sugar-content cultivar AberDart, which accumulated higher levels of high DP (degree of polymerisation) fructans in blades compared to the control cultivar only when grown at 20°C (day)/10°C (night) temperatures. The cultivar Expo on the other hand accumulated significantly higher levels of high DP fructans in blades independent of temperature. Fructan levels in pseudostems were higher than in blades, and they increased markedly with decreasing temperature, but there was no consistent effect of cultivar in this tissue. The expression of the high sugar trait was generally positively correlated with transcript levels of fructosyltransferases. Presence and expression of only one of the two known 6G-FFT isoforms was positively correlated with high fructan biosynthesis, while the second isoform was associated with low fructan concentrations and positively correlated with fructan exohydrolase gene expression. The presence of distinct 6G-FFT sequence variants appears to be associated with the capacity of high sugar-content grasses to accumulate higher fructan levels particularly at warmer temperatures. These findings might be exploited for the selection and breeding of 'warm-effective' high sugar-content grasses to overcome some of the limitations of current high sugar-content ryegrass cultivars.

Fructan Metabolism in Developing Wheat (Triticum aestivum L.) Kernels

Although fructans play a crucial role in wheat kernel development, their metabolism during kernel maturation is far from being understood. In this study, all major fructan-metabolizing enzymes together with fructan content, fructan degree of polymerization and the presence of fructan oligosaccharides were examined in developing wheat kernels (Triticum aestivum L. var. Homeros) from anthesis until maturity. Fructan accumulation occurred mainly in the first 2 weeks after anthesis, and a maximal fructan concentration of 2.5 ± 0.3 mg fructan per kernel was reached at 16 days after anthesis (DAA). Fructan synthesis was catalyzed by 1-SST (sucrose:sucrose 1-fructosyltransferase) and 6-SFT (sucrose:fructan 6-fructosyltransferase), and to a lesser extent by 1-FFT (fructan:fructan 1-fructosyltransferase). Despite the presence of 6G-kestotriose in wheat kernel extracts, the measured 6G-FFT (fructan:fructan 6G-fructosyltransferase) activity levels were low. During kernel filling, which lasted from 2 to 6 weeks after anthesis, kernel fructan content decreased from 2.5 ± 0.3 to 1.31 ± 0.12 mg fructan per kernel (42 DAA) and the average fructan degree of polymerization decreased from 7.3 ± 0.4 (14 DAA) to 4.4 ± 0.1 (42 DAA). FEH (fructan exohydrolase) reached maximal activity between 20 and 28 DAA. No fructan-metabolizing enzyme activities were registered during the final phase of kernel maturation, and fructan content and structure remained unchanged. This study provides insight into the complex metabolism of fructans during wheat kernel development and relates fructan turnover to the general phases of kernel development.

Mutations in chicory FEH genes are statistically associated with enhanced resistance to post-harvest inulin depolymerization.

Nucleotidic polymorphisms were identified in fructan exohydrolases genes which are statistically associated with enhanced susceptibility to post-harvest inulin depolymerization. Industrial chicory (Cichorium intybus L.) root is the main commercial source of inulin, a linear fructose polymer used as dietary fiber. Post-harvest, inulin is depolymerized into fructose which drastically increases processing cost. To identify genetic variations associated with enhanced susceptibility to post-harvest inulin depolymerization and related free sugars content increase, we used a candidate-gene approach focused on inulin and sucrose synthesis and degradation genes, all members of the family 32 of glycoside hydrolases (GH32). Polymorphism in these genes was first investigated by carrying out EcoTILLING on two groups of chicory breeding lines exhibiting contrasted response to post-harvest inulin depolymerization. This allowed the identification of polymorphisms significantly associated with depolymerization in three fructan exohydrolase genes (FEH). This association was confirmed on a wider panel of 116 unrelated families in which the FEH polymorphism explained 35% of the post-harvest variance for inulin content, 36% of variance for sucrose content, 18% for inulin degree of polymerization, 23% for free fructose content and 22% for free glucose content. These polymorphisms were associated with significant post-harvest changes of inulin content, inulin chain length and free sugars content.

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.

Water stress drastically reduces root growth and inulin yield in Cichorium intybus (var. sativum) independently of photosynthesis.

Root chicory (Cichorium intybus var. sativum) is a cash crop cultivated for inulin production in Western Europe. This plant can be exposed to severe water stress during the last 3 months of its 6-month growing period. The aim of this study was to quantify the effect of a progressive decline in water availability on plant growth, photosynthesis, and sugar metabolism and to determine its impact on inulin production. Water stress drastically decreased fresh and dry root weight, leaf number, total leaf area, and stomatal conductance. Stressed plants, however, increased their water-use efficiency and leaf soluble sugar concentration, decreased the shoot-to-root ratio and lowered their osmotic potential. Despite a decrease in photosynthetic pigments, the photosynthesis light phase remained unaffected under water stress. Water stress increased sucrose phosphate synthase activity in the leaves but not in the roots. Water stress inhibited sucrose:sucrose 1-fructosyltransferase and fructan:fructan 1 fructosyltransferase after 19 weeks of culture and slightly increased fructan 1-exohydrolase activity. The root inulin concentration, expressed on a dry-weight basis, and the mean degree of polymerization of the inulin chain remained unaffected by water stress. Root chicory displayed resistance to water stress, but that resistance was obtained at the expense of growth, which in turn led to a significant decrease in inulin production.

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.

Comparison of fructan dynamics in two wheat cultivars with different capacities of accumulation and remobilization under drought stress

Remobilization of stored carbohydrates in the stem of wheat plants is an important contributor to grain filling under drought stress (DS) conditions. A massive screening on Iranian wheat cultivars was performed based on stem dry weight changes under well-watered and DS conditions. Two cultivars, Shole and Crossed Falat Hamun (CFH), with different fructan accumulation and remobilization behavior were selected for further studies. Water-soluble carbohydrates (WSCs) and fructan metabolizing enzymes were studied both in the stem penultimate and in sucrose (Suc) treated, excised leaves. Under drought, CFH produced higher grain yields than Shole (412 vs 220 g m(-2)). Also, grain yield loss under drought was more limited in CFH than in Shole (17 vs 54%). Under drought, CFH accumulated more graminan-type fructo-oligosaccharides than Shole. After anthesis, fructan 6-exohydrolase (6-FEH; EC 3.2.1.154) activities increased more prominently than fructan 1-exohydrolase (EC 3.2.1.153) activities during carbon remobilization. Interestingly, CFH showed higher 6-FEH activities in the penultimate than Shole. The field experiment results suggest that the combined higher remobilization efficiency and high 6-FEH activities in stems of wheat could contribute to grain yield under terminal drought. Similar to the penultimate, fructan metabolism differed strongly in Suc-treated detached leaves of selected cultivars. This suggests that variation in the stem fructan among wheat cultivars grown in the field could be traced by leaf blade induction experiments.

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 β-(2,6)-linked fructosyl units with branches of β-(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.