Exogenous classic phytohormones have limited regulatory effects on fructan and primary carbohydrate metabolism in perennial ryegrass (<i>Lolium perenne</i> L.)

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

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)

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. vulgar

Towards better understanding of the generation of fructan structure diversity in plants: molecular and functional characterization of a sucrose: fructan 6-fructoyltranferase (6-SFT) cDNA from perennial ryegrass (<em>Lolium perenne</em>)

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Plant fructosyltransferases, diversity for which purposes ?

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Métabolisme des fructanes chez Lolium perenne L. (identification de deux gènes codant des fructane exohydrolases (FEHs) et étude de la régulation de l activité FEH par les sucres solubles)

CAEN-BU Sciences et STAPS (141182103) / SudocSudocFranceF

Transport du saccharose chez le Ray-grass anglais (Lolium perenne L.) (réponse à la défoliation et à l intensité lumineuse)

Nous avons cherché à déterminer la nature du transport de saccharose, isoler, caractériser et localiser un ou des transporteurs de saccharose (SUTs) et voir si ces SUTs répondent à la défoliation ou à une variation d intensité lumineuse avant et/ou après coupe chez le Ray-grass anglais. Le transport de saccharose est de nature apoplastique. Il fait intervenir une famille multigénique de SUTs, pour la première fois identifiés chez le Ray-grass (LpSUT1, LpSUT2). La caractérisation fonctionnelle de LpSUT2, qui possède une boucle cytoplasmique interne, est une première chez les Monocotylédones. LpSUT2 est inhibé par le fructose, résultat remarquable chez une plante à fructanes, suggérant qu il pourrait être le site de perception d un signal sucre. Le suivi de l expression ainsi que la localisation de ces transporteurs, principalement au niveau du mésophylle, suggèrent qu ils jouent un rôle clé dans la distribution des ressources C au sein de la plante entière en repousse. Le niveau d expression des transcrits LpSUT1 augmente dans les premières heures suivant la coupe et semble régulé par la teneur en saccharose. LpSUT1 serait impliqué dans le transport latéral de saccharose associé à la mise en réserve et/ou à la mobilisation des fructanes. Les transcrits LpSUT2 sont insensibles à la coupe, par contre ils sont modulés par l intensité lumineuse avant et/ou après coupe, ce qui semble aller de pair avec le rôle suggéré de LpSUT2. Etant donné que ni LpSUT1, ni LpSUT2 n est localisé dans les tissus du phloème, cela suggère l existence d autres SUTs. Les résultats acquis permettent de mieux comprendre la repousse d une Poacée prairiale pérenne accumulatrice de fructanes.We wanted to determine the nature of sucrose transport, isolate, characterize and localize one or more sucrose transporters (SUTs) and assess if these SUTs respond to defoliation or to a modulation of light intensity before and/or after defoliation in rye-grass. Sucrose transport is apoplastic. It depends on a multigenic family of SUTs that were identified for the first time in rye-grass (LpSUT1, LpSUT2). The functional characterization of LpSUT2, which possess a cytoplasmic inner loop, was also successfully realized for the first time in a Monocot species. LpSUT2 is inhibited by fructose, which is a remarkable result for a fructan-accumulating plant, thus suggesting that this SUT could be the perception site of a sugar signal. The expression and the localization of these SUTs, mainly in the mesophyll, suggest that they play a great role for the distribution of C resources within the regrowing plant. The LpSUT1transcript level increases in the few hours following defoliation and might be regulated by sucrose content. LpSUT1 could be implicated in the lateral sucrose transport associated to the storage and/or the mobilization of fructans. The LpSUT2 transcripts are not sensitive to defoliation, but they are surprisingly modulated by light intensity before and/or after defoliation, which could strikingly match their putative role of sugar sensors. Because neither LpSUT1 nor LpSUT2 are localized within phloem tissues, this suggest the existence of other SUTs. The present results allow to better understand regrowth mechanism within a perennial forage species accumulating fructans.CAEN-BU Sciences et STAPS (141182103) / SudocSudocFranceF

Le métabolisme des fructanes chez Lolium perenne L. (clonage et expression fonctionnelle de deux fructosyltransférases, implication dans la croissance et la tolérance à la défoliation)

CAEN-BU Sciences et STAPS (141182103) / SudocSudocFranceF

Implication des frutanes et du loliose dans la croissance de Lolium perenne L. après défoliation et dans la tolérance à la sécheresse (Identification des formes de transport du carbone)

CAEN-BU Sciences et STAPS (141182103) / SudocSudocFranceF

Métabolisme des fructanes au cours du développement et après récolte chez la fléole des prés (Phleum pratense L) (identification et analyse fonctionnelle de deux gènes codant des Fructanes Exo-Hydrolases (FEHs) à activité invertase)

La fléole des prés (Phleum pratense L.) est une Poacée fourragère pérenne caractéristique des régions tempérées et froides, utilisée pour l alimentation animale en pâturage ou sous forme de foin ou d ensilage. Elle accumule ses réserves glucidiques principalement sous forme de fructanes, polymères solubles de fructose, qui participent à la valeur nutritive du fourrage et au processus de fermentation lors de l ensilage. Les objectifs de cette thèse étaient i) d étudier le métabolisme des fructanes chez cette espèce pendant la croissance dans le but d évaluer le stade de fauche permettant d obtenir des teneurs maximales en fructanes, en prenant en compte le niveau de fertilisation azotée, ii) étudier le métabolisme des fructanes après la fauche, pendant le fanage et iii) d identifier et caractériser les enzymes de dégradation des fructanes (les fructane exohydrolases, FEHs). Les tissus récoltés lors de la fauche (sommet des parties aériennes) ont été analysés pour quatre stades de développement (stade végétatif, montaison, épiaison et anthèse) et deux niveaux de nutrition azotée (0,375 et 3,75 mM de NH4NO3), en conditions hydroponiques. Le métabolisme de ces sucres a également été suivi après fauche, pendant le fanage, pour les deux derniers stades. Nos résultats montrent que l accumulation des fructanes est maximale à l anthèse. La diminution des teneurs en NH4NO3 n a pas d effet sur l'accumulation des fructanes alors qu elle provoque une forte augmentation de la concentration en amidon dans les limbes. Cela suggère que les mécanismes d interactions entre le métabolisme de l amidon et de l azote d une part; et le métabolisme des fructanes et de l azote d autre part sont différents. Pendant le fanage (à la lumière ou à l obscurité, à 20 ou 15C), les teneurs en sucres solubles sont assez stables pendant les 24 premières heures; tandis que les teneurs en protéines et en amidon diminuent puis se stabilisent dès que la matière sèche dépasse le seuil des 40 %. L activité d initiation de la synthèse des fructanes (SST) décroit rapidement après la fauche alors que les activités FEH et invertase acide soluble restent élevées pendant les premières heures, indiquant le maintien du métabolisme cellulaire en début de fanage. Par ailleurs, une banque d ADNc des tissus aériens de fléole a été réalisée et quatre nouveaux gènes codant potentiellement des FEHs ont été identifiés. Deux de ces FEHs, ont ainsi pu être fonctionnellement caractérisées et exprimées dans Pichia Pastoris. Ces nouvelles enzymes (Pp 1&6FEH1 et PpFEH-INV) sont capables d hydrolyser les liens b(2-1) et b(2-6) des fructanes mais également le saccharose. Ces FEHs non spécifiques, possédant une activité invertase, pourraient être impliquées dans une régulation fine des teneurs en fructanes et en saccharose en contribuant à une meilleure balance des flux de carbone entre les tissus aériens photosynthétiquement actifs et les tissus puits des parties inférieures.Timothy (Phleum pratense L.) is an important grass forage used for pasture, hay, and silage in regions with cool and humid growth season. One of the factors affecting its nutritive value and the silage fermentation process is the concentration of nonstructural carbohydrates (NSC), which are mainly fructans, soluble polymers of fructose. The objectives of this thesis were i) to study fructan metabolism in timothy during the growth to assess the stage of development allowing to obtain maximal fructan contents, taking into account the level of nitrogen fertilization, ii) to study the metabolism of fructans after mowing, during wilting and iii) to identify and characterize plants enzymes involved in fructan breakdown (fructan exohydrolases, FEHs). Harvested tissues (shoot above 5cm) were analyzed at four stages of development (vegetative stage, stem elongation stage, heading and anthesis) and two levels of nitrogen fertilization (0.375 and 3.75 mm of NH4NO3), in hydroponic conditions. The metabolism of these sugars was also followed during wilting, for tissues harvested at heading and anthesis. Our results show that the accumulation of fructans was maximal at anthesis. The decrease of NH4NO3 concentration had no effect on fructan accumulation while it caused a strong increase of starch concentration in leaves. These results suggest that the mechanisms of interaction between starch and nitrogen metabolisms on one hand, and fructans and nitrogen metabolisms on the other hand are different. During wilting (under light or darkness, at 20 or 15C), the contents in soluble sugars were rather stable during the first 24 hours; whereas protein and starch concentrations decreased during the first hours and then remained stable as soon as the dry material content reached 40 %. The sucrose:sucrose fructosyltransferase (SST) activity, which allows the initiation of fructan synthesis, decreased quickly after mowing while FEH and soluble acid invertase activities remained high during the first hours, indicating the preservation of cellular metabolism at the beginning of wilting. Besides, a cDNA library using timothy s shoot above 10 cm was built and four new genes coding putatively for FEHs were identified. Two of these genes were expressed in Pichia pastoris to be functionally characterized. These new enzymes (Pp1&6FEH1 and PpFEH-INV) are capable to hydrolyze the b(2-1) and b(2-6) linkages in fructans and also to hydrolyze sucrose. These non specific FEHs, possessing an invertase activity, could be involved in fine regulation of fructan and sucrose contents by contributing to a better balance of carbon flows between the photosynthetically active shoot tissues and the sink tissues at the plant base.CAEN-BU Sciences et STAPS (141182103) / SudocSudocFranceF