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

Varietal Differences in Perennial Ryegrass for Fructan Metabolism and Their Relationship to Grazing Tolerance

Perennial ryegrass (Lolium perenne L.) is the most important grass in Europe; it often is defoliated. The link between fructan metabolism and defoliation tolerance has been studied in 2 Lolium perenne varieties, Aurora (high sugar perennial) and Perma (low-to-normal sugar perennial) (Turner et al., 2002)

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. vulgare

Plants Modify Biological Processes to Ensure Survival following Carbon Depletion: A Lolium perenne Model

BACKGROUND: Plants, due to their immobility, have evolved mechanisms allowing them to adapt to multiple environmental and management conditions. Short-term undesirable conditions (e.g. moisture deficit, cold temperatures) generally reduce photosynthetic carbon supply while increasing soluble carbohydrate accumulation. It is not known, however, what strategies plants may use in the long-term to adapt to situations resulting in net carbon depletion (i.e. reduced photosynthetic carbon supply and carbohydrate accumulation). In addition, many transcriptomic experiments have typically been undertaken under laboratory conditions; therefore, long-term acclimation strategies that plants use in natural environments are not well understood. METHODOLOGY/PRINCIPAL FINDINGS: Perennial ryegrass (Lolium perenne L.) was used as a model plant to define whether plants adapt to repetitive carbon depletion and to further elucidate their long-term acclimation mechanisms. Transcriptome changes in both lamina and stubble tissues of field-grown plants with depleted carbon reserves were characterised using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The RT-qPCR data for select key genes indicated that plants reduced fructan degradation, and increased photosynthesis and fructan synthesis capacities following carbon depletion. This acclimatory response was not sufficient to prevent a reduction (P<0.001) in net biomass accumulation, but ensured that the plant survived. CONCLUSIONS: Adaptations of plants with depleted carbon reserves resulted in reduced post-defoliation carbon mobilization and earlier replenishment of carbon reserves, thereby ensuring survival and continued growth. These findings will help pave the way to improve plant biomass production, for either grazing livestock or biofuel purposes

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

Diplôme : Dr. d'UniversiteFructans are the main storage compound in perennial ryegrass (Lolium perenne L.). To get a better un-derstanding on fructan involvement in leaf growth and defoliation tolerance, and to explain fructan biosynthesis at molecular level, several fructan biosynthetic enzymes were characterized and their regulation was studied. We screened a cDNA library of L. perenne by using the onion (Allium cepa) fructan:fructan 6G-fructosyltransferase (6G-FFT) and the Poa ampla sucrose:fructan 6-fructosyltransferase (6 SFT) as probes. Full length Lp6G-FFT and Lp6-SXT clones were isolated with significant homologies to vacuolar type fructosyltransferases and invertases. The functionality of the cDNAs was tested by heterologous expression in Pichia pastoris and/or in Sf9 cells. The recombinant proteins demonstrated, both 6G-FFT and fructan:fructan 1-fructosyltransferase activities (1 FFT), and 6-SFT activity, respect-tively. 6-SFT activity was not identical to that in barley since it did not use 1-kestotriose as fructosyl acceptor but 6G-kestotriose. Other putative fructosyl acceptors are under investigation. The present results have led us to reconsider fructan synthesis pattern in L. perenne. Fructans might be produced by a three-enzyme system instead of a four as previously suggested (Pavis et al., 2001a): 1 SST, 6G-FFT/1-FFT and 6-SFT. Sucrose:sucrose 1 fructosyltransferase (1 SST) allows the produc-tion of 1-kestotriose, 6G-FFT the synthesis of inulins and inulin neoseries by using 1-kestotriose as fructosyl donor and sucrose as fructosyl acceptor and an original 6-SFT would allow the synthesis of levan neoseries by using sucrose as fructosyl donor and probably 6G-kestotriose as fructosyl acceptor. The activity of 1-SST and 6G-FFT was investigated with respect to developmental stage, tissue distribution and alterations in carbohydrate status. Lp1-SST, Lp6G-FFT and Lp6-SXT were predo-minantly expressed in the basal part of elongating leaves and leaf sheaths. Expression of genes declined along the leaf axis, in parallel with the spatial occurrence of fructan and fructosyltransferase activities. Surprisingly, Lp6G-FFT was highly expressed in photosynthetically active tissues where very low extractable fructosyltransferase activity and fructan amounts were detected, suggesting a post-transcriptional expression regulation. Lp1 SST and Lp6-SXT were also highly expressed in leaf blades of plants, induced to accumulate fructans, without concomitant increase in 1-SST activity or fructan levels in this tissue. Regulation of Lp1-SST, Lp6G-FFT and Lp6-SXT gene expression depends on the tissue according to its sink-source status. To get a better understanding of fructan metabolism regulation during regrowth of Lolium perenne and to evaluate the role of carbon assimilation of new leaf tissues in refoliation, two contrasting varieties for carbohydrate metabolism, Aurora and Perma, were subject to severe and frequent or infrequent defoliations prior to regrowth. Aurora, which had a higher content of fructans in leaf sheaths than Perma before defoliation, produced more leaf biomass within the 4 d following the first cut. At the end of regrowth, Aurora produced more leaf biomass than Perma. Photosynthetic parameters were barely affected by defoliation frequency or varieties and could not explain the differences. Persistence of grassland species is dependant on their ability to accumulate high levels of carbohydrates in their tiller base. 1-SST and 6G-FFT activities declined after defoliation. In leaf sheaths, transcript levels mostly followed the time-course of the activities while in elongating leaf bases, they did not decrease concomitantly. This might represent an advantage for plants that regrow after defoliation.Les fructanes sont les réserves carbonées majeures du ray-grass anglais (Lolium perenne L.). Pour mieux comprendre leur implication dans la croissance foliaire et dans la tolérance à la défoliation, plusieurs enzymes de synthèse des fructanes ont été caractérisées et leur régulation a été étudiée. Nous avons construit et criblé une banque d’ADNc de ray-grass avec une sonde correspondant à la fructane:fructane 6G-fructosyltransférase (6G-FFT) d’oignon (Allium cepa) et à la saccharose:fructane 6-fructosyltransférase (6-SFT) du pâturin (Poa ampla). Les clones isolés, Lp6G-FFT et Lp6-SXT, ont été exprimés dans Pichia pastoris et/ou les cellules d’insecte Sf9. Les protéines recombinantes obtenues présentent respectivement une activité 6G-FFT associée à une activité fructane:fructane 1 fructosyltransférase (1-FFT), et une activité 6-SFT. L’activité 6-SFT n’est pas identique à celle décrite chez l’orge dans la mesure où elle n’utilise pas le 1-kestotriose comme accepteur de résidu fructose, mais le 6G-kestotriose. D’autres accepteurs potentiels sont actuellement à l’étude. Les résultats obtenus nous conduisent à reconsidérer la voie de biosynthèse des fructanes chez le ray grass. Les fructanes y seraient produits grâce à trois enzymes plutôt que quatre, comme cela avait été suggéré par Pavis et al. (2001a) : 1-SST, 6G-FFT/1-FFT et 6-SFT. La saccharose:saccharose 1 fructosyltransférase (1-SST) produit le 1-kestotriose, la 6G-FFT synthétise les inulines et les fructanes de la néosérie inuline en utilisant le 1-kestotriose comme donneur de résidu fructose et le saccharose comme accepteur, et la 6-SFT produit les fructanes de la néosérie lévane en utilisant le saccharose comme donneur de résidu fructose et probablement le 6G-kestotriose comme accepteur. L’activité de la 1-SST et celle de la 6G-FFT ont été suivies en fonction du stade de développement, du tissu et du statut carboné. Lp1-SST, Lp6G-FFT et Lp6-SXT sont majoritairement exprimés dans la partie basale des feuilles en croissance et des gaines. Leur expression diminue le long de l’axe des feuilles, en même temps que les teneurs en fructanes et les activités fructosyltransférasiques. Curieusement, Lp6G-FFT est fortement exprimé dans les tissus photosynthétiquement actifs alors que l’activité correspondante et les teneurs en fructanes y sont à peine détectables, ce qui suggère une régulation post-transcriptionnelle. Lp1-SST et Lp6-SXT sont aussi fortement exprimés dans les limbes de plantes dont la synthèse des fructanes est activée, sans que l’activité 1-SST et les fructanes ne soient accumulés. La régulation de Lp1-SST, Lp6G-FFT et Lp6-SXT dépend donc du tissu, selon qu’il est puits ou source de carbone. Pour mieux comprendre la régulation du métabolisme des fructanes en réponse à la défoliation chez Lolium perenne, et pour évaluer le rôle de l’assimilation du carbone des nouveaux tissus foliaires dans la repousse, deux variétés contrastées du point de vue de leur teneur en sucres solubles, Aurora et Perma, ont été soumises à des coupes sévères et répétées. Aurora, qui avait accumulé plus de fructanes dans ses gaines foliaires que Perma, produit davantage de biomasse foliaire au cours de la première repousse. Au terme de la cinétique de repousse de 29 jours, Aurora produit davantage de biomasse que Perma. A priori, les paramètres photosynthétiques, pratiquement identiques chez les deux variétés, ne permettent pas d’expliquer les différences observées de production de biomasse foliaire. La persistance des espèces prairiales dépend de leur capacité à accumuler des teneurs élevées de sucres à la base de leurs talles. Les activités 1-SST et 6G-FFT diminuent après la coupe. Dans les gaines, les transcrits évoluent comme les activités tandis que dans les bases de feuilles en croissance, leurs teneurs ne diminuent pas conjointement à la chute des activités. Ceci peut représenter un avantage pour la plante qui repousse après la coupe

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

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

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

Producing lambs while limiting concentrates in various pedoclimatic contexts: which performances?

International audienc

Producing lambs while limiting concentrates in various pedoclimatic contexts: which performances?

3rd cycl