Plant Fructan Research Articles

Research overview on the genetic mechanism underlying the biosynthesis of polysaccharide in tuber plants.

Tuber plants are of great significance in the world as human food crops. Polysaccharides, important metabolites in tuber plants, also serve as a source of innovative drugs with significant pharmacological effects. These drugs are particularly known for their immunomodulation and antitumor properties. To fully exploit the potential value of tuber plant polysaccharides and establish a synthetic system for their targeted synthesis, it is crucial to dissect their metabolic processes and genetic regulatory mechanisms. In this article, we provide a comprehensive summary of the basic pathways involved in the synthesis of various types of tuber plant polysaccharides. We also outline the key research progress that has been made in this area in recent years. We classify the main types and functions of tuber plant polysaccharides and analyze the biosynthetic processes and genetic regulation mechanisms of key enzymes involved in the metabolic pathways of starch, cellulose, pectin, and fructan in tuber plants. We have identified hexokinase and glycosyltransferase as the key enzymes involved in the polysaccharide synthesis process. By elucidating the synthesis pathway of polysaccharides in tuber plants and understanding the underlying mechanism of action of key enzymes in the metabolic pathway, we can provide a theoretical framework for enhancing the yield of polysaccharides and other metabolites in plant culture cells. This will ultimately lead to increased production efficiency.

Generation and characterization of two new monoclonal antibodies produced by immunizing mice with plant fructans: New tools for immunolocalization of β-(2 → 1) and β-(2 → 6) fructans

Fructans are water-soluble polymers of fructose in which fructose units are linked by β-(2 → 1) and/or β-(2 → 6) linkages. In plants, they are synthesized in the vacuole but have also been reported in the apoplastic sap under abiotic stress suggesting that they are involved in plasmalemma protection and in plant-microbial interactions. However, the lack of fructan-specific antibodies currently prevents further study of their role and the associated mechanisms of action, which could be elucidated thanks to their immunolocalization. We report the production of two monoclonal antibodies (named BTM9H2 and BTM15A6) using mice immunization with antigenic compounds prepared from a mixture of plant inulins and levans conjugated to serum albumin. Their specificity towards fructans with β-(2 → 1) and/or β-(2 → 6) linkage has been demonstrated by immuno-dot blot tests on a wide range of carbohydrates. The two mAbs were used for immunocytolocalization of fructans by epifluorescence microscopy in various plant species. Fructan epitopes were specifically detected in fructan-accumulating plants, inside cells as well as on the surface of root tips, confirming both extracellular and intracellular localizations. The two mAbs provide new tools to identify the mechanism of extracellular fructan secretion and explore the roles of fructans in stress resistance and plant-microorganism interactions.

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

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

Different evolutionary pathways to generate plant fructan exohydrolases.

This article comments on:Oku S, Ueno K, Sawazaki Y, Maeda T, Jitsuyama Y, Suzuki T, Onodera S, Fujino K, Shimura H. 2022. Functional characterization and vacuolar localization of fructan exohydrolase derived from onion (Allium cepa). Journal of Experimental Botany 73,4908–4922.

Interaction between fructan metabolism and plant growth regulators.

The relationship of fructan to plant growth regulators is clearly more complicated than it looks and is likely related to differences between fructan molecules in size and structure as well as localization. Fructans are a complex group of carbohydrates composed mainly of fructose units linked to a sucrose molecule. Fructans are present in plants as heterogeneous mixtures with diverse molecular structures and mass, different polymerization degrees, and linkage types between fructosyl residues. Like sucrose, they are frequently stored in leaves and other organs, acting as carbohydrate reserves. Fructans are synthesized in the cell vacuole by fructosyltransferase enzymes and catabolized by fructan exohydrolase enzymes. Several publications have shown that fructan metabolism varies with the stage of plant development and in response to the environment. Recent studies have shown a correlation between plant growth regulators (PGR), fructan metabolism, and tolerance to drought and cold. PGR are compounds that profoundly influence the growth and differentiation of plant cells, tissues, and organs. They play a fundamental role in regulating plant responses to developmental and environmental signals. In this review, we summarize the most up-to-date knowledge on the metabolism of fructans and their crosstalk with PGR signaling pathways. We identify areas that require more research to complete our understanding of the role of fructans in plants.

High-Throughput Extraction and Enzymatic Determination of Sugars and Fructans in Fructan-Accumulating Plants.

Fructans are carbohydrates present in more than 15% of flowering plants. They represent the major pool of carbohydrates in some species, especially when facing cold or drought. However, the functions of fructans with high or low degrees of polymerization (DP), their diurnal use, and the regulation of their synthesis and degradation in response to stresses still remain unclear. Here we present an enzymatic protocol adapted to 96-well microplates that simultaneously allows the determination of fructans and glucose, fructose, and sucrose. Moreover, the protocol allows to estimate the average DP of the fructans in the samples. The protocol is based on the enzymatic degradation of fructans into glucose and fructose and their subsequent conversion into gluconate 6-phosphate concomitant with the formation of NADH in the presence of ATP.

Fructan Structure and Metabolism in Overwintering Plants.

In northern regions, annual and perennial overwintering plants such as wheat and temperate grasses accumulate fructan in vegetative tissues as an energy source. This is necessary for the survival of wintering tissues and degrading fructan for regeneration in spring. Other types of wintering plants, including chicory and asparagus, store fructan as a reserve carbohydrate in their roots during winter for shoot- and spear-sprouting in spring. In this review, fructan metabolism in plants during winter is discussed, with a focus on the fructan-degrading enzyme, fructan exohydrolase (FEH). Plant fructan synthase genes were isolated in the 2000s, and FEH genes have been isolated since the cloning of synthase genes. There are many types of FEH in plants with complex-structured fructan, and these FEHs control various kinds of fructan metabolism in growth and survival by different physiological responses. The results of recent studies on the fructan metabolism of plants in winter have shown that changes in fructan contents in wintering plants that are involved in freezing tolerance and snow mold resistance might be largely controlled by regulation of the expressions of genes for fructan synthesis, whereas fructan degradation by FEHs is related to constant energy consumption for survival during winter and rapid sugar supply for regeneration or sprouting of tissues in spring.

Fructans (agavins) from Agave angustifolia and Agave potatorum as fat replacement in yogurt: Effects on physicochemical, rheological, and sensory properties

In this work, agavins (agave plant fructans) potential as fat replacer in dairy products was evaluated. Yogurt non-fat and fat reduced samples were prepared, agavins from Agave angustifolia and A. potatorum species were added to fat reduced products to evaluate their effect on the physicochemical, rheological, and sensory properties in the final products. Significant differences in the viscosity, syneresis and water retention capacity were observed among samples. All yogurts exhibited characteristics typical of a weak viscoelastic gel (G'>G'') and strain-thinning behavior. Physicochemical and rheological differences were strongly related to the agavins amount in yogurt fat-reduced samples. Agavins did not impact sensory attributes such color, smell, and taste in fact; they improved mouth feel and texture. HPAEC-PAD profiles demonstrated that agavins remain unaffected along the yogurt elaboration process. FTIR spectra showed remarkable differences on all final products composition, been more evident on fatty acids and carbohydrates regions. FTIR-PCA models allowed samples classification among treatments and samples discrimination according to the agavins content. Agavins addition to dairy products has a great potential as functional ingredient in the manufacture of low-fat, prebiotics and symbiotic products. Finally, compiled information increases our knowledge on the broad technological functionality that agavins may have in other food industry niches.

Fructan and antioxidant metabolisms in plants of Lolium perenne under drought are modulated by exogenous nitric oxide

Drought is a major environmental factor that can trigger oxidative stress and affect plant growth and productivity. Previous studies have shown that exogenous nitric oxide (NO) can minimize oxidative stress-related damage through the modulation of antioxidant enzyme activity. Fructan accumulation also has an important role in drought tolerance, since these carbohydrates participate in osmoregulation, membrane protection and oxidant scavenging. Currently, there are few studies investigating NO-regulated fructan metabolism in response to abiotic stresses. In the present study, we sought to determine if treating plants of Lolium perenne with S-nitrosoglutathione (GSNO), a NO donor, improved drought tolerance. Two-month-old plants received water (control), GSNO and reduced glutathione (GSH) as foliar spray treatments and were then maintained under drought or well-watered conditions for 23 days. At the end of drought period, we evaluated growth, pigment content and antioxidant and fructan metabolisms. None of these conditions influenced dry mass accumulation, but the leaves of plants treated with GSNO exhibited a slight increase in pigment content under drought. GSNO treatment also induced 1-SST activity, which was associated with a 3-fold increase in fructan content. GSNO-treated plants presented higher GR activity and, consequently, increased GSH levels. L. perenne cv. AberAvon was relatively tolerant to the water stress condition employed herein, maintaining ROS homeostasis and mitigating oxidative stress, possibly due to fructan, ascorbate and glutathione pools.

Purification, characterization, and functional analysis of a novel 6G&1-FEH mainly hydrolyzing neokestose from asparagus.

Asparagus (Asparagus officinalis L.) accumulates inulin- and inulin neoseries-type fructans. Fructose released by the hydrolysis of fructans is an energy source for emerging asparagus spears. Plant fructans are hydrolyzed by fructan exohydrolases (FEHs), whose presence in asparagus has not yet been fully characterized. Here, we describe for the first time the purification and characterization of an FEH from asparagus, and the functional analysis of its gene. The purified enzyme was predicted to exist as a dimer (approximately 130 kDa) consisting of two polypeptides with a molecular mass of approximately 68 kDa. N-terminal sequences of the purified enzyme were matched with the amino acid sequences of aoeh4a and aoeh4b cDNAs isolated from asparagus (cv. Gijnlim and Taihouwase). Native enzymes obtained from asparagus roots and recombinant enzymes produced by Pichia pastoris showed fructan 1-exohydrolase (1-FEH) activity via the hydrolysis of inulin-type fructan. Unlike other 1-FEHs, these enzymes showed minimal hydrolysis of 1-kestose but efficiently hydrolyzed neokestose. Therefore, the enzyme was termed 6G&1-FEH. Gene expression studies in asparagus roots showed that aoeh4 increased during root storage at 2 °C and spear harvesting. These findings suggest that 6G&1-FEH may be involved in fructan hydrolysis in asparagus roots to provide an energy source for emerging asparagus spears.

Expression and purification of plant fructan exohydrolases and their potential applications in fructose production

Inulinases from microorganisms have been extensively studied for their role in the production of fructose from fructan. Fructan can also be hydrolyzed by plant fructan exohydrolases (FEHs), but these enzymes have not been used to produce fructose commercially. Two Ht1-FEHs (Ht1-FEH I and Ht1-FEH II) were recently characterized in Jerusalem artichoke. In this study, we cloned the third member of the Ht1-FEH family in Jerusalem artichoke (i.e., Ht1-FEH III). When heterologously expressed in Pichia pastoris X-33, Ht1-FEH III not only demonstrated hydrolysis activity towards β (2, 1)-linked fructans and β (2, 6)-linked levan, but also towards sucrose. To explore the potential industrial applications, we heterologously expressed and purified six plant 1-FEHs from two typical fructan plants (i.e., chicory and Jerusalem artichoke) and showed that chicory Ci1-FEH IIa had the highest hydrolysis capacity to fructan in vitro. Furthermore, we immobilized Ci1-FEH IIa on resin and optimized the immobilization conditions. We found that inulin-type fructan or the tuber extract from Jerusalem artichoke could be rapidly degraded into fructose and sucrose by immobilized Ci1-FEH IIa. The capacity of Ci1-FEH IIa to release fructose from fructans was comparable to that of some inulinases from microorganisms. Thus, plant FEHs have potential applications in fructose production.

High production of plant type levan in sugar beet transformed with timothy (Phleum pratense) 6-SFT genes

Levan, a type of fructan, is an oligomer or polymer with mainly a β(2,6)-linked fructose chain attached to sucrose. We introduced two timothy genes, PpFT1 and PpFT2, coding for two homologous sucrose:fructan 6-fructosyltransferases into sugar beet. Sugar beet produces a high concentration of sucrose, a starting substrate in fructan synthesis, in the root. Among transgenic T1 lines, we obtained sugar beet transformants that accumulated large amounts of β(2,6)-linked levans (about 20 to 75mgg−1 FW) in the roots. The transformed sugar beet plants possessing PpFT1 or PpFT2 produced linear levans with different degrees of polymerization (DP). Namely, the PpFT1 transformants accumulated mainly high DP levans including those with DP>40, while the PpFT2 transformants accumulated levans with DP between 3 and 40. Chromatograms showed that PpFT2 produces pure β(2,6)-linked linear levans compared with fructans synthesized by PpFT1. These levans belong to the high DP class of plant fructans, but have much shorter DP than that of levans generally produced by microorganisms.

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.

Multifunctional fructans and raffinose family oligosaccharides

Fructans and raffinose family oligosaccharides (RFOs) are the two most important classes of water-soluble carbohydrates in plants. Recent progress is summarized on their metabolism (and regulation) and on their functions in plants and in food (prebiotics, antioxidants). Interest has shifted from the classic inulin-type fructans to more complex fructans. Similarly, alternative RFOs were discovered next to the classic RFOs. Considerable progress has been made in the understanding of structure–function relationships among different kinds of plant fructan metabolizing enzymes. This helps to understand their evolution from (invertase) ancestors, and the evolution and role of so-called “defective invertases.” Both fructans and RFOs can act as reserve carbohydrates, membrane stabilizers and stress tolerance mediators. Fructan metabolism can also play a role in osmoregulation (e.g., flower opening) and source–sink relationships. Here, two novel emerging roles are highlighted. First, fructans and RFOs may contribute to overall cellular reactive oxygen species (ROS) homeostasis by specific ROS scavenging processes in the vicinity of organellar membranes (e.g., vacuole, chloroplasts). Second, it is hypothesized that small fructans and RFOs act as phloem-mobile signaling compounds under stress. It is speculated that such underlying antioxidant and oligosaccharide signaling mechanisms contribute to disease prevention in plants as well as in animals and in humans.

Crystal structure of 6‐SST/6‐SFT from Pachysandra terminalis, a plant fructan biosynthesizing enzyme in complex with its acceptor substrate 6‐kestose

Fructans play important roles as reserve carbohydrates and stress protectants in plants, and additionally serve as prebiotics with emerging antioxidant properties. Various fructan types are synthesized by an array of plant fructosyltransferases belonging to family 32 of the glycoside hydrolases (GH32), clustering together with GH68 in Clan-J. Here, the 3D structure of a plant fructosyltransferase from a native source, the Pachysandra terminalis 6-SST/6-SFT (Pt6-SST/6-SFT), is reported. In addition to its 1-SST (1-kestose-forming) and hydrolytic side activities, the enzyme uses sucrose to create graminan- and levan-type fructans, which are probably associated with cold tolerance in this species. Furthermore, a Pt6-SST/6-SFT complex with 6-kestose was generated, representing a genuine acceptor binding modus at the +1, +2 and +3 subsites in the active site. The enzyme shows a unique configuration in the vicinity of its active site, including a unique D/Q couple located at the +1 subsite that plays a dual role in donor and acceptor substrate binding. Furthermore, it shows a unique orientation of some hydrophobic residues, probably contributing to its specific functionality. A model is presented showing formation of a β(2-6) fructosyl linkage on 6-kestose to create 6,6-nystose, a mechanism that differs from the creation of a β(2-1) fructosyl linkage on sucrose to produce 1-kestose. The structures shed light on the evolution of plant fructosyltransferases from their vacuolar invertase ancestors, and contribute to further understanding of the complex structure-function relationships within plant GH32 members.

Microbacterium sp. A-210이 생성하는 Levan fructotransferase의 정제 및 생물학적 특성에 관한 연구

DFA (Difructose anhydride)는 특유의 구조적인 안정성 때문에 당뇨병 환자를 위한 당원으로써 적합하다는 연구가 보고 되어 있다. DFA에는 4가지 type이 있는데 inulin에 의한 DFA I DFA III DFAV가 있고 levan에 의한 DFA IV가 있는 것으로 알려져 있다. 특히 DFA IV는 당뇨병 환자를 위한 당원 뿐 만 아니라 rat을 이용한 연구에서 칼슘의 흡수를 도와 준다는 보고가 있었다. 이러한 DFAIV를 생성하는 데 쓰이는 Microbacterium sp. AL-210에서 유래한 LFTase (Levan fructotransferase)의 wild-type과 mutants (D63A, D195N, N85S)의 구조적 특성을 밝히기 위해 정제하였다. LFTase의 wild-type과 mutants들을 대량 발현시킨 후 흡착 크로마토그래피, 이온교환 크로마토그래피 그리고 젤 여과 크로마토그래피를 이용하여 고순도로 분리 정제하였으며 이를 SDS-PAGE를 통하여 확인하였다. 분리 정제된 단백질을 JNET 이차 구조 예측 프로그램, solubility 측정, CD (원 편광 이색성 분광편광계), fluorescence spectroscopy (형광분석법), DSC (시차주사열량계)를 이용하여 분석하였다. 또한 다중 정렬과 2차 구조 예측 프로그램을 이용하여 wild-type의 2차 구조를 분석하였다. Solubility 측정에서 가장 적합한 온도는 <TEX>$55^{\circ}C$</TEX>, 최상의 pH는 7.5로 나타났다. CD 분석에서 wild-type과 비교한 결과 다른 mutant에 비해 N85S의 <TEX>$\alpha$</TEX>-helix가 많이 감소한 것과 <TEX>$\beta$</TEX> strand와 random coil이 증가한 것을 확인하였다. 또한 DSC 분석을 통해 wild-type이 다른 mutants에 비해 안정적인 구조를 지닌 것을 확인하였다. 형광분석에서 N85S가 wild-type과 가장 유사하게 나타났으며 D63A와 D195N은 wild-type에 비해 높은 강도를 나타내었다. 또한 wild-type의 sequence를 Exo-inulinase from Aspegillus awamori, a plant fructan 1-exohydrolase from Cichorium intybus 그리고 invertase from Thermotogo maritime (Tm)의 sequence와 다중 정렬한 결과 Exo-inulinase와 높은 identity를 보였다. Difractose anhydrides (DFAs) is studied as a sweetener for diabetics because of its structural property. DFAs have four types: DFA I, III, IV (degradation of levan) and V (degradation of inulin). Especially, DFA IV has been shown to enhance the absorption of calcium in experiments using rats. Levan fructotransferase is an enzyme for producing di-d-fructose-2,6':6,2-dianhydride (DFA IV). To identify structural characterization, we purified wild-type and mutants (D63A, D195N and N85S) of levan fructotransferase (LFTase) from Microbacterium sp. AL-210. These proteins were purified to apparent homogeneity by Ni-NTA affinity column, Q-sepharose ion exchange and gel filtration chromatography and detected by SDS-PAGE. They were also analyzed by circular dichroism (CD) measurements, JNET secondary structure prediction, activity measurements at various temperatures, and pH analysis. The optimum pH for the enzyme-catalyzed reaction was pH 7.5 and optimum temperature was observed at <TEX>$55^{\circ}C$</TEX>. Along with wild-type LFTase, mutants were analyzed by CD measurement, fluorescence analysis and differential scanning calorimetry (DSC). N85S showed less <TEX>$\alpha$</TEX>-helix and more <TEX>$\beta$</TEX> strand than others. Also, N85S showed almost the same curve as wild-type in their steady-state fluorescence spectra, whereas mutant D63A and D195N showed higher intensity than wild-type. The amino acid sequence of wild-type LFTase was compared to the sequences of exo-inulinase from Aspergillus awamori, a plant fructan 1-exohydrolase from Cichorium intybus, and Thermotogo maritime (Tm) invertase and showed a high identity with Exo-inulinase from Aspergillus awamori.

Donor and acceptor substrate selectivity among plant glycoside hydrolase family 32 enzymes

Plant family 32 glycoside hydrolase enzymes include hydrolases (cell wall invertases, fructan exohydrolases, vacuolar invertases) and fructosyltransferases. These enzymes are very similar at the molecular and structural levels but are functionally different. Understanding the basis of the functional diversity in this family is a challenging task. By combining structural and site-directed mutagenesis data, Asp239 in AtcwINV1 was identified as an amino acid critical for binding and stabilizing sucrose. Plant fructan exohydrolases lack such an Asp239 equivalent. Substitution of Asp239 led to the loss of invertase activity, while its introduction in fructan exohydrolases increased invertase activity. Some fructan exohydrolases are inhibited by sucrose. The difference between the inhibitor (fructan exohydrolase) and the substrate (invertase) binding configurations of sucrose can be explained by the different orientation of Trp82. Furthermore, the evolutionary hydrolase/transferase transition could be mimicked and the difference between S-type fructosyltransferases (sucrose as donor) and F-type fructosyltransferases (fructan as donor) could be unravelled.

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 β(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.

Inhibitors of plant invertases do not affect the structurally related enzymes of fructan metabolism.

Plant fructan active enzymes (FAZYs), including the enzymes involved in inulin metabolism, namely sucrose:sucrose 1-fructosyltransferase (1-SST; EC 2.4.1.99), fructan:fructan 1-fructosyltransferase (1-FFT; EC 2.4.1.100) and fructan 1-exohydrolase (1-FEH; EC 3.2.1.153), are evolutionarily related to acid invertases (AIs), that is, plant cell wall invertase (CWI) and vacuolar invertase (VI). Acid invertases are post-translationally controlled by proteinaceous inhibitors. Whether FAZYs are subject to similar controls is not known. To probe their possible interactions with invertase inhibitors, we transiently expressed chicory (Cichorium intybus) FAZYs, as well as several previously characterized invertase inhibitors from nonfructan species, and the C. intybus cell wall/vacuolar inhibitor of fructosidase (CiC/VIF), a putative invertase inhibitor of a fructan-accumulating plant, in leaves of Nicotiana benthamiana. Leaf extracts containing recombinant, enzymatically active FAZYs were used to explore the interaction with invertase inhibitors. Neither heterologous inhibitors nor CiC/VIF affected FAZY activities. CiC/VIF was confirmed as an AI inhibitor with a stronger effect on CWI than on VI. Its expression in planta was developmentally regulated (high in taproots, and undetectable in leaves and flowers). In agreement with its target specificities, CiC/VIF was associated with the cell wall. It is concluded that subtle structural differences between AIs and FAZYs result in pronounced selectivity of inhibitor action.

Plant fructans in stress environments: emerging concepts and future prospects

Plants are sessile and sensitive organisms known to possess various regulatory mechanisms for defending themselves under stress environments. Fructans are fructose-based polymers synthesized from sucrose by fructosyltransferases (FTs). They have been increasingly recognized as protective agents against abiotic stresses. Using model membranes, numerous in vitro studies have demonstrated that fructans can stabilize membranes by direct H-bonding to the phosphate and choline groups of membrane lipids, resulting in a reduced water outflow from the dry membranes. Inulin-type fructans are flexible random-coiled structures that can adopt many conformations, allowing them to insert deeply within the membranes. The devitrification temperature (T(g)) can be adjusted by their varying molecular weights. In addition, above T(g) their low crystallization rates ensure prolonged membrane protection. Supporting, in vivo studies with transgenic plants expressing FTs showed fructan accumulation and an associated improvement in freezing and/or chilling tolerance. The water-soluble nature of fructans may allow their rapid adaptation as cryoprotectants in order to give optimal membrane protection. One of the emerging concepts for delivering vacuolar fructans to the extracellular space for protecting the plasma membrane is vesicle-mediated, tonoplast-derived exocytosis. It should, however, be noted that natural stress tolerance is a very complex process that cannot be explained by the action of a single molecule or mechanism.