[Show abstract][Hide abstract] ABSTRACT: Main conclusion
The first 6-fructan exohydrolase (6-FEH) cDNA from
was cloned and characterized. Following defoliation,
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.
[Show abstract][Hide abstract] ABSTRACT: Sucrose transport between source and sink tissues is supposed to be a key-step for an efficient regrowth of perennial rye-grass after defoliation and might be altered by light conditions. We assessed the effect of different light regimes (high vs low light applied before or after defoliation) on growth, fructans and sucrose mobilization, as well as on sucrose transporter expression during 14 days of regrowth. Our results reported that defoliation led to a mobilization of C reserves (first sucrose and then fructans), which was parallel to an induction of LpSUT1 sucrose transporter expression in source and sink tissues (i.e. leaf sheaths and elongating leaf bases, respectively) irrespective to light conditions. Light regime (high or low light) had little effects on regrowth and on C reserves mobilization during the first 48 h of regrowth after defoliation. Thereafter, low light conditions, delaying the recovery of photosynthetic capacities, had a negative effect on C reserves re-accumulation (especially sucrose). Surprisingly, high light did not enhance sucrose transporter expression. Indeed, while light conditions had no effect on LpSUT1 expression, LpSUT2 transcripts levels were enhanced for low light grown plants. These results indicate that two sucrose transporter currently identified in Lolium perenne L. are differentially regulated by light and sucrose.
[Show abstract][Hide abstract] ABSTRACT: This work assessed the central carbohydrate metabolism of actively photosynthesizing leaf blades of a C3 grass (Lolium perenne L.). The study used dynamic (13)C labelling of plants growing in continuous light with contrasting supplies of nitrogen ('low N' and 'high N') and mathematical analysis of the tracer data with a four-pool compartmental model to estimate rates of: (i) sucrose synthesis from current assimilation; (ii) sucrose export/use; (iii) sucrose hydrolysis (to glucose and fructose) and resynthesis; and (iv) fructan synthesis and sucrose resynthesis from fructan metabolism. The contents of sucrose, fructan, glucose, and fructose were almost constant in both treatments. Labelling demonstrated that all carbohydrate pools were turned over. This indicated a system in metabolic steady state with equal rates of synthesis and degradation/consumption of the individual pools. Fructan content was enhanced by nitrogen deficiency (55 and 26% of dry mass at low and high N, respectively). Sucrose content was lower in nitrogen-deficient leaves (2.7 versus 6.7%). Glucose and fructose contents were always low (<1.5%). Interconversions between sucrose, glucose, and fructose were rapid (with half-lives of individual pools ranging between 0.3 and 0.8 h). Futile cycling of sucrose through sucrose hydrolysis (67 and 56% of sucrose at low and high N, respectively) and fructan metabolism (19 and 20%, respectively) was substantial but seemed to have no detrimental effect on the relative growth rate and carbon-use efficiency of these plants. The main effect of nitrogen deficiency on carbohydrate metabolism was to increase the half-life of the fructan pool from 27 to 62 h and to effectively double its size.
[Show abstract][Hide abstract] ABSTRACT: Carbohydrate and amino acid composition of phloem sap was studied in the grass Lolium perenne L., before and after defoliation. Leaf exudate was collected in a 5 mmol·L1 EDTA solution from cut leaf blades or stubble, and phloem sap was obtained through excised aphid (Rhopalosiphum padi L.) stylets. Results indicate that leaf exudates obtained from leaves devoid of petiole might not be relevant predictors of carbohydrate content of pure phloem sap. Sucrose was the dominating carbohydrate, accounting for 93% of the total soluble sugars in the phloem sap. Myo-inositol, glucose, and fructose were present in trace amounts, while fructans, raffinose, and loliose have never been detected. Predominant amino acid in the phloem sap was glutamine followed by glutamate, aspartate, and serine. Phloem sap component concentration declined during the first hours following defoliation. Sucrose was the main sugar transported in the phloem sap of Lolium perenne, despite the fact that the product of fructan degradation was fructose and not sucrose. The results are discussed in relation with the physiological mechanisms that contribute to plant recovery after defoliation.Key words: fructan, sucrose, loliose, defoliation, phloem sap, amino acids.
Canadian Journal of Botany 02/2011; 82(11):1594-1601. DOI:10.1139/b04-117 · 1.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Defoliation 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 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The pathway of carbon phloem loading was examined in leaf tissues of the forage grass Lolium perenne. The effect of defoliation (leaf blade removal) on sucrose transport capacity was assessed in leaf sheaths as the major carbon
source for regrowth. The pathway of carbon transport was assessed via a combination of electron microscopy, plasmolysis experiments
and plasma membrane vesicles (PMVs) purified by aqueous two-phase partitioning from the microsomal fraction. Results support
an apoplastic phloem loading mechanism. Imposition of an artificial proton-motive force to PMVs from leaf sheaths energized
an active, transient and saturable uptake of sucrose (Suc). The affinity of Suc carriers for Suc was 580 μM in leaf sheaths
of undefoliated plants. Defoliation induced a decrease of Km followed by an increase of Vmax. A transporter was isolated from stubble (including leaf sheaths) cDNA libraries and functionally expressed in yeast. The
level of L.perenne SUcrose Transporter 1 (LpSUT1) expression increased in leaf sheaths in response to defoliation. Taken together, the results indicate that Suc transport
capacity increased in leaf sheaths of L. perenne in response to leaf blade removal. This increase might imply de novo synthesis of Suc transporters, including LpSUT1, and
may represent one of the mechanisms contributing to rapid refoliation.
[Show abstract][Hide abstract] ABSTRACT: Fructosyltransferases (FTs) synthesize fructans, fructose polymers accumulating in economically important cool-season grasses and cereals. FTs might be crucial for plant survival under stress conditions in species in which fructans represent the major form of reserve carbohydrate, such as perennial ryegrass (Lolium perenne). Two FT types can be distinguished: those using sucrose (S-type enzymes: sucrose:sucrose 1-fructosyltransferase [1-SST], sucrose:fructan 6-fructosyltransferase) and those using fructans (F-type enzymes: fructan:fructan 1-fructosyltransferase [1-FFT], fructan:fructan 6G-fructosyltransferase [6G-FFT]) as preferential donor substrate. Here, we report, to our knowledge for the first time, the transformation of an F-type enzyme (6G-FFT/1-FFT) into an S-type enzyme (1-SST) using perennial ryegrass 6G-FFT/1-FFT (Lp6G-FFT/1-FFT) and 1-SST (Lp1-SST) as model enzymes. This transformation was accomplished by mutating three amino acids (N340D, W343R, and S415N) in the vicinity of the active site of Lp6G-FFT/1-FFT. In addition, effects of each amino acid mutation alone or in combination have been studied. Our results strongly suggest that the amino acid at position 343 (tryptophan or arginine) can greatly determine the donor substrate characteristics by influencing the position of the amino acid at position 340. Moreover, the presence of arginine-343 negatively affects the formation of neofructan-type linkages. The results are compared with recent findings on donor substrate selectivity within the group of plant cell wall invertases and fructan exohydrolases. Taken together, these insights contribute to our knowledge of structure/function relationships within plant family 32 glycosyl hydrolases and open the way to the production of tailor-made fructans on a larger scale.
[Show abstract][Hide abstract] ABSTRACT: The objectives of this study were (i) to evaluate the relative involvement of fructans from leaf sheaths and from elongating leaf bases to regrowth after defoliation of Lolium perenne L. by following fructan exohydrolase (FEH), sucrose-sucrose fructosyl transferase (SST) and invertase (INV) activities and (ii) to examine whether gibberellins could be implicated in regulation of FEH activity. In stubble, 36 % of fructans are located in leaf bases and 64 % in leaf sheaths. During the first phase of regrowth, the depletion of carbohydrates was mainly due to the decline of fructans, which represented 76% and 50 % of the carbohydrates mobilized from leaf sheaths and elongating leaf bases respectively. Despite a decrease in protein content, FEH activity increased 2-5-fold in leaf sheaths and sixfold in elongating leaf bases, so that the fructan-hydrolysing activity was four times higher in growing leaves than in leaf sheaths, 2 d after defoliation. INV activity also increased, whereas the pattern of SST activity was inversely related to the variations of both hydrolysing activities. SST activity, which is highest in growing leaves, declined approximately threefold in leaf sheaths and elongating leaf bases at the beginning of regrowth.
The increase in activity of FEH in stubble was strongly inhibited by an inhibitor of the biosynthesis of gibberellin, uniconazole. FEH activity was decreased to c. 67 %, 45 % and 33 % of the level in nontreated plants 24, 48 and 72 h following defoliation, respectively. The inhibition could be overcome by a subsequent treatment with gibberellic acids (GAs). For the first time, data are provided to support the view that GAs might play a role in the regulation of FEH activity, and the implication of this result is discussed.
[Show abstract][Hide abstract] ABSTRACT: 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 188.8.131.52) 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 beta-(2,1) trimming enzyme acting during fructan synthesis in concert with fructan synthesis enzymes.
[Show abstract][Hide abstract] ABSTRACT: Fructans are the main storage compound in Lolium perenne. To account for the prevailing neokestose-based fructan synthesis in this species, a cDNA library of L. perenne was screened by using the onion (Allium cepa) fructan:fructan 6G-fructosyltransferase (6G-FFT) as a probe. A full length Lp6G-FFT clone was isolated with significant homologies to vacuolar type fructosyltransferases and invertases. The functionality of the cDNA was tested by heterologous expression in Pichia pastoris. The recombinant protein demonstrated both 6G-FFT and fructan:fructan 1-fructosyltransferase activities (1-FFT) with a maximum 6G-FFT/1-FFT ratio of two. The activity of 6G-FFT was investigated with respect to developmental stage, tissue distribution, and alterations in carbohydrate status expression and compared to sucrose:sucrose 1-fructosyltransferase (1-SST). Lp6G-FFT and Lp1-SST were predominantly expressed in the basal part of elongating leaves and leaf sheaths. Expression of both 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 regulation of expression. Lp6G-FFT gene expression increased only in elongating leaves following similar increases of sucrose content in blades, sheaths, and elongating leaf bases. Regulation of Lp6G-FFT gene expression depends on the tissue according to its sink-source status.
[Show abstract][Hide abstract] ABSTRACT: The pharmacological activity of serine protease inhibitors, potential drugs for the treatment of thrombosis, is often linked to the presence of amidine functions. With the aim of developing a suitable formulation for these compounds, inulin and inulin acetate associated or not with 1,12-dodecanedicarboxylic acid, were chosen to prepare microspheres. Using a coacervation method, these biocompatible polymers led to microspheres of about 0.5-5 microm. The encapsulation of a water-soluble model drug (E,E)-bis(amidinobenzylidene)cycloheptanone [(E,E)-BABCH] in these microspheres was studied. In this investigation, factorial designs were used to determine the joint influence of several variables (drug mass, speed and time of formulation stirring, centrifugation time) for an optimum encapsulation efficiency. Results revealed that encapsulation efficiency reached 65% whatever the nature of the biopolymer, by using a stirring time of 30 min, a high stirring speed and a centrifugation time of 15 min. (E,E)-BABCH release from microspheres was examined in an in vitro model. The profiles were characterized by three phases strongly dependent on the microspheres and the diacid association displayed a crucial role. With inulin and inulin acetate, the initial phase was a rapid 'drug burst'. Within the first 5 min, 58-62% of the drug were delivered. Microspheres of inulin acetate associated with 1,12-dodecanedicarboxylic acid, showed a slower release with only 32% of the drug delivered after 15 min. After a slow diffusion phase (33 h), an increasing rate until complete drug release was observed for 2.5 days.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to evaluate the putative role of the sucrosyl-galactosides, loliose [alpha-D-Gal (1,3) alpha-D-Glc (1,2) beta-D-Fru] and raffinose [alpha-D-Gal (1,6) alpha-D-Glc (1,2) beta-D-Fru], in drought tolerance of perennial ryegrass and to compare it with that of fructans. To that end, the loliose biosynthetic pathway was first established and shown to operate by a UDP-Gal: sucrose (Suc) 3-galactosyltransferase, tentatively termed loliose synthase. Drought stress increased neither the concentrations of loliose and raffinose nor the activities of loliose synthase and raffinose synthase (EC 184.108.40.206). Moreover, the concentrations of the raffinose precursors, myoinositol and galactinol, as well as the gene expressions of myoinositol 1-phosphate synthase (EC 220.127.116.11) and galactinol synthase (EC 18.104.22.168) were either decreased or unaffected by drought stress. Taken together, these data are not in favor of an obvious role of sucrosyl-galactosides in drought tolerance of perennial ryegrass at the vegetative stage. By contrast, drought stress caused fructans to accumulate in leaf tissues, mainly in leaf sheaths and elongating leaf bases. This increase was mainly due to the accumulation of long-chain fructans (degree of polymerization > 8) and was not accompanied by a Suc increase. Interestingly, Suc but not fructan concentrations greatly increased in drought-stressed roots. Putative roles of fructans and sucrosyl-galactosides are discussed in relation to the acquisition of stress tolerance.
[Show abstract][Hide abstract] ABSTRACT: The role of fructans from leaf sheaths for the refoliation of Lolium perenne after severe defoliation was assessed by following the fate of (13)C-fructose supplied to leaf sheaths at the time of defoliation. At the end of the 4 h labelling period on defoliated plants, 77% of the (13)C incorporated was still located in leaf sheaths. Only 4% and 0.9% were, respectively, allocated to stem and roots, while 18% was imported by the growing leaves where (13)C was allocated first to the proximal part of the leaf growth zone (0-10 mm). In all tissues, the most highly (13)C-labelled carbohydrates was not fructose but sucrose. In leaf sheaths, (13)C-loliose was produced. In the leaf growth zone (0-20 mm), fructans were simultanously synthesized from (13)C entering the leaves and degraded. The export of (13)C from leaf sheaths continued during the first day of regrowth but stopped afterwards. There was no net loss of C from (13)C-fructose over the first 2 d of regrowth. The role of fructans and loliose is discussed as well as the physiological mechanisms contributing to defoliation tolerance in L. perenne.
Journal of Experimental Botany 05/2003; 54(385):1231-43. · 5.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gibberellin (GA) levels in leaf sheaths and in elongating leaf bases of perennial ryegrass (Lolium perenne L. cv. Bravo) were monitored in undefoliated and defoliated plants. Nine C-13-hydroxylated GAs (GA8, GA97, GA29, GA1, GA20, GA44, GA19, GA17, GA53) and one C-13-non-hydroxylated GA (GA9) were identified by combined gas chromatography-mass spectrometry in leaf extracts. The total level of GA8, GA29, GA1, GA20, GA44, GA19 and GA53, determined by selected ion monitoring, was 7 times higher in elongating leaf bases than in mature leaf sheaths. In both leaf tissues, defoliation induced an increase in GA53 level, while GA20 and GA1 levels decreased, suggesting that the GA53→GA44, as well as GA19→GA20, conversions were slowed down. The roles of GA1 in the control of leaf elongation and fructan mobilization following defoliation are discussed.
[Show abstract][Hide abstract] ABSTRACT: summaryAccumulation of water-soluble carbohydrates was studied in leaf tissues of 8-wk-old plants of perennial ryegrass (Lolium perenne L. var. Bravo). The roots and leaf bases were cooled to low temperatures to reduce sink activity while source activity was enhanced by continuous illumination of the shoots. This resulted in accumulation of fructans, first in sheaths, subsequently in expanding leaves, and finally in blades. Fructan concentration increased from 6 to 23 mg g−1f. wt in sheaths, from 8 to 30 mg g−1f. wt in expanding leaves and from 6 to only 17 mg g−1f. wt in mature leaf blades. Increase in concentration of low-DP fructans preceded accumulation of high-DP fructans. Expanding leaves accumulated significantly more glucose and fructose than did mature leaf tissue. Leaf blades contained a higher concentration of sucrose than either leaf sheaths or expanding leaves. Expanding leaves exhibited the greatest activity of SST (0.77 nkat g−1f. wt), the mature leaf blades the least (0.09 nkat g−1f. wt), whilst that of mature leaf sheaths was intermediate (0.27 nkat g−1f. wt). In each leaf tissue, invertase activity was higher than SST activity. Under conditions of fructan accumulation, SST activity increased threefold in mature leaf sheaths and 1.2-fold in expanding leaves. In mature leaf blades, however, the activity remained constant at a low level. Invertase activity never increased. Inhibitors of protein synthesis (cycloheximide) and of RNA metabolism (actinomycin D) prevented any increase of SST activity in leaf sheaths. The results are discussed in relation to fructan metabolism in leaf tissues of Lolium perenne.
New Phytologist 10/1996; 134(2):205-214. DOI:10.1111/j.1469-8137.1996.tb04625.x · 7.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Glucose, fructose, sucrose and fructans were the main non-structural carbohydrates of perennial ryegrass. Fructans accumulated predominantly in stubble, i.e. in the basal part of the leaves. Regrowth after defoliation involved two different phases. The first one (0–6 days) was characterized by the mobilization of carbohydrates in both roots and stubble to sustain foliage development in the absence of current photosynthate. During the second phase (6–29 days) carbohydrate content rose to the values observed prior to cutting. Concomitant with a decrease in soluble protein and in non-structural carbohydrate contents, sucrose-sucrose-fructosyltransferase (SST) activity decreased during the first 6 days in roots and stubble whereas fructan exohydrolase (FEH) activity increased and invertase activity was high. Prior to the subsequent increase in carbohydrate content, sucrose synthase activity increased sharply while sucrose phosphate synthase showed only slight variation. During the second period of regrowth FEH declined and SST increased. In regrowing leaves, however, fructan accumulation was not paralleled by an increase in SST activity. Results are interpreted and discussed in terms of source-sink relationships and carbohydrate partitioning.