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Wim Van Den Ende - One of the best experts on this subject based on the ideXlab platform.
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Discovery of Fructans in Archaea.
Carbohydrate Polymers, 2019Co-Authors: Onur Kırtel, Wim Van Den Ende, Eveline Lescrinier, Ebru Toksoy OnerAbstract:Abstract Fructans are fructose-based oligo- and polysaccharides derived from sucrose that occur in a plethora of Eubacteria and plants. While fructan-producing (fructanogenic) Eubacteria are abundant in hypersaline environments, fructan production by Archaea has never been reported before. Exopolysaccharides accumulated by various Archaea from the Halobacteria class (belonging to the genera of Halomicrobium, Haloferax and Natronococcus) originating from different locations on Earth were structurally characterized as either levans or inulins with varying branching degrees (10%–16%). Thus, we show for the first time in the literature that Fructans are produced in all three domains of life, including Archaea. This proof of concept will not only provide insight into Archaeal glycans and evolution but it may also open new frontiers for innovative strategies to overcome the ever-increasing threat of excessive salinization.
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The fructan syndrome: Evolutionary aspects and common themes among plants and microbes
Plant Cell & Environment, 2017Co-Authors: Maxime Versluys, Onur Kırtel, Ebru Toksoy Oner, Wim Van Den EndeAbstract:Fructans are multifunctional fructose-based water soluble carbohydrates found in all biological kingdoms but not in animals. Most research has focused on plant and microbial Fructans and has received a growing interest because of their practical applications. Nevertheless, the origin of fructan production, the so-called "fructan syndrome," is still unknown. Why Fructans only occur in a limited number of plant and microbial species remains unclear. In this review, we provide an overview of plant and microbial fructan research with a focus on Fructans as an adaptation to the environment and their role in (a)biotic stress tolerance. The taxonomical and biogeographical distribution of Fructans in both kingdoms is discussed and linked (where possible) to environmental factors. Overall, the fructan syndrome may be related to water scarcity and differences in physicochemical properties, for instance, water retaining characteristics, at least partially explain why different fructan types with different branching levels are found in different species. Although a close correlation between environmental stresses and fructan production is quite clear in plants, this link seems to be missing in microbes. We hypothesize that this can be at least partially explained by differential evolutionary timeframes for plants and microbes, combined with potential redundancy effects.
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Fructans as damps or mamps evolutionary prospects cross tolerance and multistress resistance potential
Frontiers in Plant Science, 2017Co-Authors: Maxime Versluys, łukasz Pawel Tarkowski, Wim Van Den EndeAbstract:This perspective paper proposes that endogenous apoplastic Fructans in fructan accumulating plants, released after stress-mediated cellular leakage, or increased by exogenous application, can act as damage-associated molecular patterns (DAMPs), priming plant innate immunity through ancient receptors and defense pathways that most probably evolved to react on microbial Fructans acting as microbe-associated molecular patterns (MAMPs). The proposed model is placed in an evolutionary perspective. How this type of DAMP signaling may contribute to cross-tolerance and multistress resistance effects in plants is discussed. Besides apoplastic ATP, NAD and Fructans, apoplastic polyamines, secondary metabolites and melatonin may be considered potential players in DAMP-mediated stress signaling. It is proposed that mixtures of DAMP priming formulations hold great promise as natural and sustainable alternatives for toxic agrochemicals.
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Building a fructan LC–MS2 library and its application to reveal the fine structure of cereal grain Fructans
Carbohydrate Polymers, 2017Co-Authors: Joran Verspreet, Rudy Vergauwen, Wim Van Den Ende, Anders Holmgaard Hansen, Scott James Harrison, Christophe M. CourtinAbstract:Abstract A liquid chromatography-mass spectrometry (LC–MS) library is presented containing the relative retention times of 28 fructan oligomers and MS 2 spectra of 18 of them. It includes the main representatives of all fructan classes occurring in nature and with a degree of polymerization between three and five. This library enables a rapid and unambiguous detection of these 18 fructan structures in any type of sample without the need for fructan purification or the synthesis of fructan standards. Its wide applicability is demonstrated by the analysis of Fructans in a set of cereal flour samples. Marked differences were observed in the types of Fructans present in oat, barley, rye, spelt and wheat flour. A putative link between the accumulation of certain fructan types and cereal phylogeny is described.
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Fructose and Fructans: Opposite Effects on Health?
Plant Foods for Human Nutrition, 2015Co-Authors: Francesca Di Bartolomeo, Wim Van Den EndeAbstract:Fructans are fructose-based oligo-and polysaccharides of natural origin. Fructan and fructose species are sometimes confused by the great public, although they clearly have different biochemical and physiological properties. This review discusses aspects of the use of fructose and Fructans in foods in the context of human health, with possible differential effects on cellular autophagy in cells of the human body. Although there are uncertainties on the daily levels of ingested fructose to be considered harmful to human health, there is an emerging consensus on the benefits of the use of Fructans in functional foods, sustaining health via direct immunomodulatory and antioxidant effects or through indirect, prebiotic mechanisms.
André Van Laere - One of the best experts on this subject based on the ideXlab platform.
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Cloning and characterization of a novel fructan 6-exohydrolase strongly inhibited by sucrose in Lolium perenne
Planta, 2014Co-Authors: Jeremy Lothier, André Van Laere, Wim Van Den Ende, Marie-pascale Prud’homme, Annette Morvan-bertrandAbstract:Main conclusion 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.
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Fructan synthesizing and degrading activities in chicory roots (Cichorium intybus L.) during field-growth, storage and forcing
Journal of Plant Physiology, 2012Co-Authors: Wim Van Den Ende, André Van LaereAbstract:Summary An investigation of SST (E.C. 2.4.1.99) and FFT (E.C. 2.4.1.100) activities in chicory roots ( Cichorium intybus L. var foliosum cv. Flash) showed that both enzymes also had a small but genuine s-fructosidase activity. The activity of fructan exohydrolase towards small and large Fructans was comparable provided that they are present in the same molar concentration. During field growth the activity of SST decreased continuously to essentially disappear in October. FFT activities on the contrary remained high and even increased slighdy. Fructan exohydrolase activity was detected throughout field-growth but in crude extracts a part of it is likely to be due to the s-fructosidase activity of FFT. When this was taken into account the genuine fructan exohydrolase activity increased rapidly after mid-October. During cold storage a further rapid increase was detected. Forcing of the roots resulted in a considerable decrease of inulinase activity although large amounts of Fructans are mobilized during that period.
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unexpected presence of graminan and levan type Fructans in the evergreen frost hardy eudicot pachysandra terminalis buxaceae purification cloning and functional analysis of a 6 sst 6 sft enzyme
Plant Physiology, 2011Co-Authors: Wim Van Den Ende, Rudy Vergauwen, Willem Lammens, Marlies Coopman, Stefan Clerens, Katrien Le Roy, André Van LaereAbstract:About 15% of flowering plants accumulate Fructans. Inulin-type Fructans with β(2,1) fructosyl linkages typically accumulate in the core eudicot families (e.g. Asteraceae), while levan-type Fructans with β(2,6) linkages and branched, graminan-type Fructans with mixed linkages predominate in monocot families. Here, we describe the unexpected finding that graminan- and levan-type Fructans, as typically occurring in wheat (Triticum aestivum) and barley (Hordeum vulgare), also accumulate in Pachysandra terminalis, an evergreen, frost-hardy basal eudicot species. Part of the complex graminan- and levan-type Fructans as accumulating in vivo can be produced in vitro by a sucrose:fructan 6-fructosyltransferase (6-SFT) enzyme with inherent sucrose:sucrose 1-fructosyltransferase (1-SST) and fructan 6-exohydrolase side activities. This enzyme produces a series of cereal-like graminan- and levan-type Fructans from sucrose as a single substrate. The 6-SST/6-SFT enzyme was fully purified by classic column chromatography. In-gel trypsin digestion led to reverse transcription-polymerase chain reaction-based cDNA cloning. The functionality of the 6-SST/6-SFT cDNA was demonstrated after heterologous expression in Pichia pastoris. Both the recombinant and native enzymes showed rather similar substrate specificity characteristics, including peculiar temperature-dependent inherent 1-SST and fructan 6-exohydrolase side activities. The finding that cereal-type Fructans accumulate in a basal eudicot species further confirms the polyphyletic origin of fructan biosynthesis in nature. Our data suggest that the fructan syndrome in P. terminalis can be considered as a recent evolutionary event. Putative connections between abiotic stress and Fructans are discussed.
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Towards a better understanding of the generation of fructan structure diversity in plants: molecular and functional characterization of a sucrose:fructan 6-fructosyltransferase (6-SFT) cDNA from perennial ryegrass (Lolium perenne)
Journal of Experimental Botany, 2010Co-Authors: Bertrand Lasseur, André Van Laere, Wim Van Den Ende, Andres Wiemken, Jeremy Lothier, Annette Morvan-bertrand, Marie-pascale Prud'hommeAbstract:The main storage compounds in Lolium perenne are Fructans with prevailing β(2–6) linkages. A cDNA library of L. perenne was screened using Poa secunda sucrose:fructan 6-fructosyltransferase (6-SFT) as a probe. A full-length Lp6-SFT clone was isolated as shown by heterologous expression in Pichia pastoris. High levels of Lp6-SFT transcription were found in the growth zone of elongating leaves and in mature leaf sheaths where Fructans are synthesized. Upon fructan synthesis induction, Lp6-SFT transcription was high in mature leaf blades but with no concomitant accumulation of Fructans. In vitro studies with the recombinant Lp6-SFT protein showed that both 1-kestotriose and 6G-kestotriose acted as fructosyl acceptors, producing 1- and 6-kestotetraose (bifurcose) and 6G,6-kestotetraose, respectively. Interestingly, bifurcose formation ceased and 6G,6-kestotetraose was formed instead, when recombinant fructan:fructan 6G-fructosyltransferase (6G-FFT) of L. perenne was introduced in the enzyme assay with sucrose and 1-kestotriose as substrates. The remarkable absence of bifurcose in L. perenne tissues might be explained by a higher affinity of 6G-FFT, as compared with 6-SFT, for 1-kestotriose, which is the first fructan formed. Surprisingly, recombinant 6-SFT from Hordeum vulgare, a plant devoid of Fructans with internal glucosyl residues, also produced 6G,6-kestotetraose from sucrose and 6G-kestotriose. In the presence of recombinant L. perenne 6G-FFT, it produced 6G,6-kestotetraose from 1-kestotriose and sucrose, like L. perenne 6-SFT. Thus, we demonstrate that the two 6-SFTs have close catalytic properties and that the distinct Fructans formed in L. perenne and H. vulgare can be explained by the presence of 6G-FFT activity in L. perenne and its absence in H. vulgare.
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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.
Journal of Experimental Botany, 2007Co-Authors: Jeremy Lothier, André Van Laere, Wim Van Den Ende, Marie-pascale Prud'homme, Katrien Le Roy, Bertrand Lasseur, Philippe Barre, Annette Morvan-bertrandAbstract:Fructans, which are b-(2,1) and/or b-(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 b-(2,1) trimming enzyme acting during fructan synthesis in concert with fructan synthesis enzymes.
Marie-pascale Prud'homme - One of the best experts on this subject based on the ideXlab platform.
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Exogenous Classic Phytohormones Have Limited Regulatory Effects on Fructan and Primary Carbohydrate Metabolism in Perennial Ryegrass (Lolium perenne L.)
Frontiers in Plant Science, 2016Co-Authors: Anna Gasperl, Marie-pascale Prud'homme, Annette Morvan-bertrand, Eric Van Der Graaff, Thomas RoitschAbstract:Fructans are polymers of fructose and one of the main constituents of water-soluble carbohydrates in forage grasses and cereal crops of temperate climates. Fructans are involved in cold and drought resistance, regrowth following defoliation and early spring growth, seed filling, have beneficial effects on human health and are used for industrial processes. Perennial ryegrass (Lolium perenne L.) serves as model species to study fructan metabolism. Fructan metabolism is under the control of both synthesis by fructosyltransferases (FTs) and breakdown through fructan exohydrolases (FEHs). The accumulation of Fructans can be triggered by high sucrose levels and abiotic stress conditions such as drought and cold stress. However, detailed studies on the mechanisms involved in the regulation of fructan metabolism are scarce. Since different phytohormones, especially abscisic acid (ABA), are known to play an important role in abiotic stress responses, the possible short term regulation of the enzymes involved in fructan metabolism by the five classical phytohormones was investigated. Therefore, the activities of enzymes involved in fructan synthesis and breakdown, the expression levels for the corresponding genes and levels for water-soluble carbohydrates were determined following pulse treatments with ABA, auxin (AUX), ethylene (ET), gibberellic acid (GA), or kinetin (KIN). The most pronounced fast effects were a transient increase of I- I activities by AUX, KIN, ABA, and ET, while minor effects were evident for 1-FEH activity with an increased activity in response to KIN and a decrease by GA. Fructan and sucrose levels were not affected. This observed discrepancy demonstrates the importance of determining enzyme activities to obtain insight into the physiological traits and ultimately the plant phenotype. The comparative analyses of activities for seven key enzymes of primary carbohydrate metabolism revealed no co-regulation between enzymes of the fructan and sucrose pool.
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Plant maturity and nitrogen fertilization affected fructan metabolism in harvestable tissues of timothy (Phleum pratense L.)
Journal of Plant Physiology, 2014Co-Authors: Marouf Ould Ahmed, Marie-pascale Prud'homme, A. Morvan Bertrand, Marie-laure Decau, Carole Lafreniere, Pascal DrouinAbstract: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.
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Towards a better understanding of the generation of fructan structure diversity in plants: molecular and functional characterization of a sucrose:fructan 6-fructosyltransferase (6-SFT) cDNA from perennial ryegrass (Lolium perenne)
Journal of Experimental Botany, 2010Co-Authors: Bertrand Lasseur, André Van Laere, Wim Van Den Ende, Andres Wiemken, Jeremy Lothier, Annette Morvan-bertrand, Marie-pascale Prud'hommeAbstract:The main storage compounds in Lolium perenne are Fructans with prevailing β(2–6) linkages. A cDNA library of L. perenne was screened using Poa secunda sucrose:fructan 6-fructosyltransferase (6-SFT) as a probe. A full-length Lp6-SFT clone was isolated as shown by heterologous expression in Pichia pastoris. High levels of Lp6-SFT transcription were found in the growth zone of elongating leaves and in mature leaf sheaths where Fructans are synthesized. Upon fructan synthesis induction, Lp6-SFT transcription was high in mature leaf blades but with no concomitant accumulation of Fructans. In vitro studies with the recombinant Lp6-SFT protein showed that both 1-kestotriose and 6G-kestotriose acted as fructosyl acceptors, producing 1- and 6-kestotetraose (bifurcose) and 6G,6-kestotetraose, respectively. Interestingly, bifurcose formation ceased and 6G,6-kestotetraose was formed instead, when recombinant fructan:fructan 6G-fructosyltransferase (6G-FFT) of L. perenne was introduced in the enzyme assay with sucrose and 1-kestotriose as substrates. The remarkable absence of bifurcose in L. perenne tissues might be explained by a higher affinity of 6G-FFT, as compared with 6-SFT, for 1-kestotriose, which is the first fructan formed. Surprisingly, recombinant 6-SFT from Hordeum vulgare, a plant devoid of Fructans with internal glucosyl residues, also produced 6G,6-kestotetraose from sucrose and 6G-kestotriose. In the presence of recombinant L. perenne 6G-FFT, it produced 6G,6-kestotetraose from 1-kestotriose and sucrose, like L. perenne 6-SFT. Thus, we demonstrate that the two 6-SFTs have close catalytic properties and that the distinct Fructans formed in L. perenne and H. vulgare can be explained by the presence of 6G-FFT activity in L. perenne and its absence in H. vulgare.
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Carbon metabolism in grass leaf meristems
2007Co-Authors: Marie-pascale Prud'homme, A. Morvan Bertrand, Véronique Amiard, Bertrand Lasseur, Jeremy Lothier, Nathalie Noiraud RomyAbstract:In Lolium perenne, fructan polymers represent the main storage carbohydrates. Even if leaf meristems, located in elongating leaf bases, act as strong sink for imported assimilates, they also synthesize Fructans in substantial amounts. Fructans are generally not evenly distributed. Their highest content is found in the growth zone (0-30 mm from the leaf base) and decreased strongly in the differentiation zone (30-60 mm). Lp1-SST, Lp6G-FFT/1-FFT and Lp6-SFT, encoding the three main fructan synthesizing activities in Lolium perenne, were also predominantly expressed in the growth zone. Their expression declined along the leaf axis, in parallel with the spatial occurrence of Fructans, sucrose and enzyme activities. As a response to defoliation, the decline in fructan content occurred not only in the differentiation zone, but also in the growth zone. Before defoliation, the activity of fructan exohydrolase (FEH) was maximal in the differentiation zone. After defoliation, it increased in all segments, but peaked in the growth zone. These data strongly indicate that Fructans stored in the leaf growth zone were hydrolyzed and recycled in that zone to sustain refoliation immediately after defoliation. Leaf sheaths represent the other source of Fructans. When the product of fructan degradation, fructose, was supplied as 13C-fructose to leaf sheaths at the time of defoliation, its fate showed that the relative supply of C to roots was transiently reduced for the benefit of the growth zone where 13C was allocated first to the proximal part (0-10 mm). This preferential allocation of C could be at least partly explained by a strong and specific increase of the SuSy activity in that zone after defoliation.The results will be discussed in relation to plant development and defoliation tolerance.
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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.
Journal of Experimental Botany, 2007Co-Authors: Jeremy Lothier, André Van Laere, Wim Van Den Ende, Marie-pascale Prud'homme, Katrien Le Roy, Bertrand Lasseur, Philippe Barre, Annette Morvan-bertrandAbstract:Fructans, which are b-(2,1) and/or b-(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 b-(2,1) trimming enzyme acting during fructan synthesis in concert with fructan synthesis enzymes.
Annette Morvan-bertrand - One of the best experts on this subject based on the ideXlab platform.
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Exogenous Classic Phytohormones Have Limited Regulatory Effects on Fructan and Primary Carbohydrate Metabolism in Perennial Ryegrass (Lolium perenne L.)
Frontiers in Plant Science, 2016Co-Authors: Anna Gasperl, Marie-pascale Prud'homme, Annette Morvan-bertrand, Eric Van Der Graaff, Thomas RoitschAbstract:Fructans are polymers of fructose and one of the main constituents of water-soluble carbohydrates in forage grasses and cereal crops of temperate climates. Fructans are involved in cold and drought resistance, regrowth following defoliation and early spring growth, seed filling, have beneficial effects on human health and are used for industrial processes. Perennial ryegrass (Lolium perenne L.) serves as model species to study fructan metabolism. Fructan metabolism is under the control of both synthesis by fructosyltransferases (FTs) and breakdown through fructan exohydrolases (FEHs). The accumulation of Fructans can be triggered by high sucrose levels and abiotic stress conditions such as drought and cold stress. However, detailed studies on the mechanisms involved in the regulation of fructan metabolism are scarce. Since different phytohormones, especially abscisic acid (ABA), are known to play an important role in abiotic stress responses, the possible short term regulation of the enzymes involved in fructan metabolism by the five classical phytohormones was investigated. Therefore, the activities of enzymes involved in fructan synthesis and breakdown, the expression levels for the corresponding genes and levels for water-soluble carbohydrates were determined following pulse treatments with ABA, auxin (AUX), ethylene (ET), gibberellic acid (GA), or kinetin (KIN). The most pronounced fast effects were a transient increase of I- I activities by AUX, KIN, ABA, and ET, while minor effects were evident for 1-FEH activity with an increased activity in response to KIN and a decrease by GA. Fructan and sucrose levels were not affected. This observed discrepancy demonstrates the importance of determining enzyme activities to obtain insight into the physiological traits and ultimately the plant phenotype. The comparative analyses of activities for seven key enzymes of primary carbohydrate metabolism revealed no co-regulation between enzymes of the fructan and sucrose pool.
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A Simple and Fast Kinetic Assay for the Determination of Fructan Exohydrolase Activity in Perennial Ryegrass (Lolium perenne L.)
Frontiers in Plant Science, 2015Co-Authors: Anna Gasperl, Annette Morvan-bertrand, Eric Van Der Graaff, Marie-pascale Prud’homme, Thomas RoitschAbstract:Despite the fact that Fructans are the main constituent of water-soluble carbohydrates in forage grasses and cereal crops of temperate climates, little knowledge is available on the regulation of the enzymes involved in fructan metabolism. The analysis of enzyme activities involved in this process has been hampered by the low affinity of the fructan enzymes for sucrose and Fructans used as fructosyl donor. Further, the analysis of fructan composition and enzyme activities is restricted to specialized labs with access to suited HPLG equipment and appropriate fructan standards. The degradation of fructan polymers with high degree of polymerization (DP) by fructan exohydrolases (FEHs) to fructosyloligomers is important to liberate energy in the form of fructan, but also under conditions where the generation of low DP polymers is required. Based on published protocols employing enzyme coupled endpoint reactions in single cuvettes, we developed a simple and fast kinetic 1-FEH assay. This assay can be performed in multi-well plate format using plate readers to determine the activity of 1-FEH against 1-kestotriose, resulting in a significant time reduction. Kinetic assays allow an optimal and more precise determination of enzyme activities compared to endpoint assays, and enable to check the quality of any reaction with respect to linearity of the assay. The enzyme coupled kinetic 1-FEH assay was validated in a case study showing the expected increase in 1-FEH activity during cold treatment. This assay is cost effective and could be performed by any lab with access to a plate reader suited for kinetic measurements and readings at 340 nm, and is highly suited to assess temporal changes and relative differences in 1-FEH activities. Thus, this enzyme coupled kinetic 1-FEH assay is of high importance both to the field of basic fructan research and plant breeding.
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Cloning and characterization of a novel fructan 6-exohydrolase strongly inhibited by sucrose in Lolium perenne
Planta, 2014Co-Authors: Jeremy Lothier, André Van Laere, Wim Van Den Ende, Marie-pascale Prud’homme, Annette Morvan-bertrandAbstract:Main conclusion 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.
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Towards a better understanding of the generation of fructan structure diversity in plants: molecular and functional characterization of a sucrose:fructan 6-fructosyltransferase (6-SFT) cDNA from perennial ryegrass (Lolium perenne)
Journal of Experimental Botany, 2010Co-Authors: Bertrand Lasseur, André Van Laere, Wim Van Den Ende, Andres Wiemken, Jeremy Lothier, Annette Morvan-bertrand, Marie-pascale Prud'hommeAbstract:The main storage compounds in Lolium perenne are Fructans with prevailing β(2–6) linkages. A cDNA library of L. perenne was screened using Poa secunda sucrose:fructan 6-fructosyltransferase (6-SFT) as a probe. A full-length Lp6-SFT clone was isolated as shown by heterologous expression in Pichia pastoris. High levels of Lp6-SFT transcription were found in the growth zone of elongating leaves and in mature leaf sheaths where Fructans are synthesized. Upon fructan synthesis induction, Lp6-SFT transcription was high in mature leaf blades but with no concomitant accumulation of Fructans. In vitro studies with the recombinant Lp6-SFT protein showed that both 1-kestotriose and 6G-kestotriose acted as fructosyl acceptors, producing 1- and 6-kestotetraose (bifurcose) and 6G,6-kestotetraose, respectively. Interestingly, bifurcose formation ceased and 6G,6-kestotetraose was formed instead, when recombinant fructan:fructan 6G-fructosyltransferase (6G-FFT) of L. perenne was introduced in the enzyme assay with sucrose and 1-kestotriose as substrates. The remarkable absence of bifurcose in L. perenne tissues might be explained by a higher affinity of 6G-FFT, as compared with 6-SFT, for 1-kestotriose, which is the first fructan formed. Surprisingly, recombinant 6-SFT from Hordeum vulgare, a plant devoid of Fructans with internal glucosyl residues, also produced 6G,6-kestotetraose from sucrose and 6G-kestotriose. In the presence of recombinant L. perenne 6G-FFT, it produced 6G,6-kestotetraose from 1-kestotriose and sucrose, like L. perenne 6-SFT. Thus, we demonstrate that the two 6-SFTs have close catalytic properties and that the distinct Fructans formed in L. perenne and H. vulgare can be explained by the presence of 6G-FFT activity in L. perenne and its absence in H. vulgare.
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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.
Journal of Experimental Botany, 2007Co-Authors: Jeremy Lothier, André Van Laere, Wim Van Den Ende, Marie-pascale Prud'homme, Katrien Le Roy, Bertrand Lasseur, Philippe Barre, Annette Morvan-bertrandAbstract:Fructans, which are b-(2,1) and/or b-(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 b-(2,1) trimming enzyme acting during fructan synthesis in concert with fructan synthesis enzymes.
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Cloning and characterization of a novel fructan 6-exohydrolase strongly inhibited by sucrose in Lolium perenne
Planta, 2014Co-Authors: Jeremy Lothier, André Van Laere, Wim Van Den Ende, Marie-pascale Prud’homme, Annette Morvan-bertrandAbstract:Main conclusion 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.
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Towards a better understanding of the generation of fructan structure diversity in plants: molecular and functional characterization of a sucrose:fructan 6-fructosyltransferase (6-SFT) cDNA from perennial ryegrass (Lolium perenne)
Journal of Experimental Botany, 2010Co-Authors: Bertrand Lasseur, André Van Laere, Wim Van Den Ende, Andres Wiemken, Jeremy Lothier, Annette Morvan-bertrand, Marie-pascale Prud'hommeAbstract:The main storage compounds in Lolium perenne are Fructans with prevailing β(2–6) linkages. A cDNA library of L. perenne was screened using Poa secunda sucrose:fructan 6-fructosyltransferase (6-SFT) as a probe. A full-length Lp6-SFT clone was isolated as shown by heterologous expression in Pichia pastoris. High levels of Lp6-SFT transcription were found in the growth zone of elongating leaves and in mature leaf sheaths where Fructans are synthesized. Upon fructan synthesis induction, Lp6-SFT transcription was high in mature leaf blades but with no concomitant accumulation of Fructans. In vitro studies with the recombinant Lp6-SFT protein showed that both 1-kestotriose and 6G-kestotriose acted as fructosyl acceptors, producing 1- and 6-kestotetraose (bifurcose) and 6G,6-kestotetraose, respectively. Interestingly, bifurcose formation ceased and 6G,6-kestotetraose was formed instead, when recombinant fructan:fructan 6G-fructosyltransferase (6G-FFT) of L. perenne was introduced in the enzyme assay with sucrose and 1-kestotriose as substrates. The remarkable absence of bifurcose in L. perenne tissues might be explained by a higher affinity of 6G-FFT, as compared with 6-SFT, for 1-kestotriose, which is the first fructan formed. Surprisingly, recombinant 6-SFT from Hordeum vulgare, a plant devoid of Fructans with internal glucosyl residues, also produced 6G,6-kestotetraose from sucrose and 6G-kestotriose. In the presence of recombinant L. perenne 6G-FFT, it produced 6G,6-kestotetraose from 1-kestotriose and sucrose, like L. perenne 6-SFT. Thus, we demonstrate that the two 6-SFTs have close catalytic properties and that the distinct Fructans formed in L. perenne and H. vulgare can be explained by the presence of 6G-FFT activity in L. perenne and its absence in H. vulgare.
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Carbon metabolism in grass leaf meristems
2007Co-Authors: Marie-pascale Prud'homme, A. Morvan Bertrand, Véronique Amiard, Bertrand Lasseur, Jeremy Lothier, Nathalie Noiraud RomyAbstract:In Lolium perenne, fructan polymers represent the main storage carbohydrates. Even if leaf meristems, located in elongating leaf bases, act as strong sink for imported assimilates, they also synthesize Fructans in substantial amounts. Fructans are generally not evenly distributed. Their highest content is found in the growth zone (0-30 mm from the leaf base) and decreased strongly in the differentiation zone (30-60 mm). Lp1-SST, Lp6G-FFT/1-FFT and Lp6-SFT, encoding the three main fructan synthesizing activities in Lolium perenne, were also predominantly expressed in the growth zone. Their expression declined along the leaf axis, in parallel with the spatial occurrence of Fructans, sucrose and enzyme activities. As a response to defoliation, the decline in fructan content occurred not only in the differentiation zone, but also in the growth zone. Before defoliation, the activity of fructan exohydrolase (FEH) was maximal in the differentiation zone. After defoliation, it increased in all segments, but peaked in the growth zone. These data strongly indicate that Fructans stored in the leaf growth zone were hydrolyzed and recycled in that zone to sustain refoliation immediately after defoliation. Leaf sheaths represent the other source of Fructans. When the product of fructan degradation, fructose, was supplied as 13C-fructose to leaf sheaths at the time of defoliation, its fate showed that the relative supply of C to roots was transiently reduced for the benefit of the growth zone where 13C was allocated first to the proximal part (0-10 mm). This preferential allocation of C could be at least partly explained by a strong and specific increase of the SuSy activity in that zone after defoliation.The results will be discussed in relation to plant development and defoliation tolerance.
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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.
Journal of Experimental Botany, 2007Co-Authors: Jeremy Lothier, André Van Laere, Wim Van Den Ende, Marie-pascale Prud'homme, Katrien Le Roy, Bertrand Lasseur, Philippe Barre, Annette Morvan-bertrandAbstract:Fructans, which are b-(2,1) and/or b-(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 b-(2,1) trimming enzyme acting during fructan synthesis in concert with fructan synthesis enzymes.
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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
Journal of Experimental Botany, 2007Co-Authors: Jeremy Lothier, André Van Laere, Wim Van Den Ende, Marie-pascale Prud'homme, Katrien Le Roy, Bertrand Lasseur, Philippe Barre, A. Morvan BertrandAbstract: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 Lpl-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 Lpl-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.
