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

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

Impact of PGPR inoculation on root morphological traits and root exudation in rapeseed and camelina: interactions with heat stress.

Root exudation is involved in the recruitment of beneficial microorganisms by trophic relationships and/or signalling pathways. Among beneficial microorganisms, Plant Growth-Promoting Rhizobacteria (PGPR) are known to improve plant growth and stress resistance. These interactions are of particular importance for species that do not interact with mycorrhizal fungi, such as rapeseed (Brassica napus L.) and camelina (Camelina sativa (L.) Crantz). However, heat stress is known to have a quantitative and qualitative impact on root exudation and could affect the interactions between plants and PGPR. We aimed to analyse the effects of PGPR inoculation on root morphology and exudation in rapeseed and camelina at the reproductive stage. The modulation of the effects of these interactions under heat stress was also investigated. The plants were inoculated twice at the reproductive stage with two different Pseudomonas species and were exposed to heat stress after the second inoculation. In non-stressing conditions, after bacterial inoculation, rapeseed and camelina exhibited two contrasting behaviours in C root allocation. While rapeseed plants seemed to suffer from the interactions with the bacteria, camelina plants appeared to control the relationship with the PGPR by modifying the composition of their root exudates. Under heat stress, the plant-PGPR interaction was unbalanced for rapeseed, for which the C allocation strategy is mainly driven by the C cost from the bacteria. Alternatively, camelina plants prioritized C allocation for their own above-ground development. This work opens up new perspectives for understanding plant-PGPR interactions, especially in an abiotic stress context.

Fructan exohydrolases (FEHs) are upregulated by salicylic acid together with defense-related genes in non-fructan accumulating plants.

The identification of several fructan exohydrolases (FEHs, EC 3.2.1.80) in non-fructan accumulating plants raised the question of their roles. FEHs may be defense-related proteins involved in the interactions with fructan-accumulating microorganisms. Since known defense-related proteins are upregulated by defense-related phytohormones, we tested the hypothesis that FEHs of non-fructan accumulating plants are upregulated by salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) using the model plant Arabidopsis thaliana and the agronomically relevant and genetically related species Brassica napus. By sequence homologies with the two known FEH genes of A. thaliana, At6-FEH, and At6&1-FEH, the genes coding for the putative B. napus FEHs, Bn6-FEH and Bn6&1-FEH, were identified. Plants were treated at root level with SA, methyl jasmonate (MeJA) or 1-aminocyclopropane-1-carboxylic acid (ACC). The transcript levels of defense-related and FEH genes were measured after treatments. MeJA and ACC did not upregulate FEHs, while HEL (HEVEIN-LIKE PREPROTEIN) expression was enhanced by both phytohormones. In both species, the expression of AOS, encoding a JA biosynthesis enzyme, was enhanced by MeJA and that of the defensine PDF1.2 and the ET signaling transcription factor ERF1/2 by ACC. In contrast, SA not only increased the expression of genes encoding antimicrobial proteins (PR1 and HEL) and the defense-related transcription factor WRKY70 but also that of FEH genes, in particular 6&1-FEH genes. This result supports the putative role of FEHs as defense-related proteins. Genotypic variability of SA-mediated FEH regulation (transcript level and activities) was observed among five varieties of B. napus, suggesting different susceptibilities toward fructan-accumulating pathogens.

Repeated heat stress events during the reproductive phase impact the dynamic development of seeds in Brassica napus L.

Many studies pointed out the deleterious effects of high temperatures events during the crop reproductive phase on seed yield and quality. However, plant responses to repeated stressing events remain poorly understood, while the increased frequency of extreme abiotic constraints, such as spring and summer heat waves, has been proven as one feature of the on-going and future climate change. The responses of oilseed rape plants subjected to three heat stress sequences that differed in the intensity, the timing of application, the duration and the frequency of the high temperature events were investigated throughout the seed development and maturation phases under controlled conditions. Seed yield and components were measured in three different harvest dates. Biochemical and histological analyses of seeds were carried out in order to monitor the evolution of the main storage compounds (fatty acids, proteins, sugars) involved in seed nutritional quality. Although the effects of heat stress were not significant on total yield, differences in seed number and weight highlighted the strong compensation capacity in indeterminate growth species. Heat stress induced significant decreases and increases in seed oil and protein content respectively, to different extent according to the age of the pods. Soluble sugars concentrations were impacted by heat during seed development, but not when the seeds reached physiological maturity, thus indicating compensatory mechanisms that set up after the stress exposure. Our results led to conclude that the effects of repeated heat stresses on seed yield and quality were tightly related to (i) the optimal temperature of a given compound biosynthesis process, and (ii) the synchrony between the temperature event and the period of biosynthesis of the targeted storage compound. These results highlight the complexity to design thermo-sensitizing protocols to maintain or even improve the various seed quality related criteria, especially in species with indeterminate growth.

The resilience of perennial grasses under two climate scenarios is correlated with carbohydrate metabolism in meristems.

Extreme climatic events (ECEs) such as droughts and heat waves affect ecosystem functioning and species turnover. This study investigated the effect of elevated CO2 on species' resilience to ECEs. Monoliths of intact soil and their plant communities from an upland grassland were exposed to 2050 climate scenarios with or without an ECE under ambient (390 ppm) or elevated (520 ppm) CO2. Ecophysiological traits of two perennial grasses (Dactylis glomerata and Holcus lanatus) were measured before, during, and after ECE. At similar soil water content, leaf elongation was greater under elevated CO2 for both species. The resilience of D. glomerata increased under enhanced CO2 (+60%) whereas H. lanatus mostly died during ECE. D. glomerata accumulated 30% more fructans, which were more highly polymerized, and 4-fold less sucrose than H. lanatus. The fructan concentration in leaf meristems was significantly increased under elevated CO2. Their relative abundance changed during the ECE, resulting in a more polymerized assemblage in H. lanatus and a more depolymerized assemblage in D. glomerata. The ratio of low degree of polymerization fructans to sucrose in leaf meristems was the best predictor of resilience across species. This study underlines the role of carbohydrate metabolism and the species-dependent effect of elevated CO2 on the resilience of grasses to ECE.

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

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

Short-term effects of defoliation intensity on sugar remobilization and N fluxes in ryegrass.

In grassland plant communities, the ability of individual plants to regrow after defoliation is of crucial importance since it allows the restoration of active photosynthesis and plant growth. The aim of this study was to evaluate the effects of increasing defoliation intensity (0, 25, 65, 84, and 100% of removed leaf area) on sugar remobilization and N uptake, remobilization, and allocation in roots, adult leaves, and growing leaves of ryegrass over 2 days, using a 15N tracer technique. Increasing defoliation intensity decreased plant N uptake in a correlative way and increased plant N remobilization, but independently. The relative contribution of N stored before defoliation to leaf growth increased when defoliation intensity was severe. In most conditions, root N reserves also contributed to leaf regrowth, but much less than adult leaves and irrespective of defoliation intensity. A threshold of defoliation intensity (65% leaf area removal) was identified below which C (glucose, fructose, sucrose, fructans), and N (amino acids, soluble proteins) storage compounds were not recruited for regrowth. By contrast, nitrate content increased in elongating leaf bases above this threshold. Wounding associated with defoliation is thus not the predominant signal that triggers storage remobilization and controls the priority of resource allocation to leaf meristems. A framework integrating the sequential events leading to the refoliation of grasses is proposed on the basis of current knowledge and on the findings of the present work.

Asparagine and sugars are both required to sustain secondary axis elongation after bud outgrowth in Rosa hybrida.

Nitrogen is required for optimal plant growth, especially in young organs such as secondary axes (axes II) after axillary bud outgrowth. Several studies have shown an increase of nitrogen concentration in xylem sap concomitantly with bud outgrowth, but the relation between nitrogen, sugars and plant hormones in axis II still remains unclear. We investigated in Rosa hybrida the involvement of nitrogen nutrition in axis II elongation in relation with sugars and cytokinins using 15N-labeled nitrate and sugars, amino acids and cytokinin quantifications. Besides, we measured the effect of the exogenous supply of these compounds on axis II elongation using in vitro excised bud culture. We demonstrated that nitrogen in the axis II comes mainly from new root uptake after decapitation. Asparagine, which concentration increases in sap exudates and tissues during axis II elongation, was the sole amino acid able to sustain an efficient elongation in vitro when supplied in combination with sucrose.

Trace element bioavailability, yield and seed quality of rapeseed (Brassica napus L.) modulated by biochar incorporation into a contaminated technosol.

BACKGROUND AND AIMS: Rapeseed (Brassica napus L.) is a Cd/Zn-accumulator whereas soil conditioners such as biochars may immobilize trace elements. These potentially complementary soil remediation options were trialed, singly and in combination, in a pot experiment with a metal(loid)-contaminated technosol. METHODS: The technosol [total content in mg kg(-1) Zn 6089, Cd 9.4, Cu 110, and Pb 956] was either amended (2% w/w) or not with a poultry manure-derived biochar. Rapeseed was cultivated for both soil treatments during 24 weeks up to harvest under controlled conditions. RESULTS: Biochar incorporation into the technosol promoted the As, Cd, Cu, Mo, Ni, Pb and Zn solubility. It decreased foliar B, Cu and Mo concentrations, and Mo concentration in stems, pericarps and seeds. But, it did not impact neither the biomass of aerial rapeseed parts (except a decrease for seeds), nor their C (except a decrease for stems), seed fatty acid, seed starch and soluble sugar contents, and antioxidant capacity in both leaves and seeds. Biochar amendment increased the phytoextraction by aerial plant parts for K, P, and S, reduced it for N, Ca, B, Mo, Ni and Se, whereas it remained steady for Mg, Zn, Fe, Mn, Cu, Cd and Co. CONCLUSIONS: The biochar incorporation into this technosol did not promote Cd, Cu and Zn phytoextraction by rapeseed and its potential oilseed production, but increased the solubility of several metal(loid)s. Here Zn and Cd concentrations in the soil pore water were decreased by rapeseed, showing the feasibility to strip available soil Zn and Cd in combination with seed production.

What functional strategies drive drought survival and recovery of perennial species from upland grassland?

BACKGROUND AND AIMS: Extreme climatic events such as severe droughts are expected to increase with climate change and to limit grassland perennity. The present study aimed to characterize the adaptive responses by which temperate herbaceous grassland species resist, survive and recover from a severe drought and to explore the relationships between plant resource use and drought resistance strategies. METHODS: Monocultures of six native perennial species from upland grasslands and one Mediterranean drought-resistant cultivar were compared under semi-controlled and non-limiting rooting depth conditions. Above- and below-ground traits were measured under irrigation in spring and during drought in summer (50 d of withholding water) in order to characterize resource use and drought resistance strategies. Plants were then rehydrated and assessed for survival (after 15 d) and recovery (after 1 year). KEY RESULTS: Dehydration avoidance through water uptake was associated with species that had deep roots (>1·2 m) and high root mass (>4 kg m(-3)). Cell membrane stability ensuring dehydration tolerance of roots and meristems was positively correlated with fructan content and negatively correlated with sucrose content. Species that survived and recovered best combined high resource acquisition in spring (leaf elongation rate >9 mm d(-1) and rooting depth >1·2 m) with both high dehydration avoidance and tolerance strategies. CONCLUSIONS: Most of the native forage species, dominant in upland grassland, were able to survive and recover from extreme drought, but with various time lags. Overall the results suggest that the wide range of interspecific functional strategies for coping with drought may enhance the resilience of upland grassland plant communities under extreme drought events.

Exogenous Classic Phytohormones Have Limited Regulatory Effects on Fructan and Primary Carbohydrate Metabolism in Perennial Ryegrass (Lolium perenne 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 FT 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.

A Simple and Fast Kinetic Assay for the Determination of Fructan Exohydrolase Activity in Perennial Ryegrass (Lolium perenne L.).

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 HPLC 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.

Plant maturity and nitrogen fertilization affected fructan metabolism in harvestable tissues of timothy (Phleum pratense L.).

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.

Cloning and characterization of a novel fructan 6-exohydrolase strongly inhibited by sucrose in Lolium perenne.

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 ß(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.

Differential regulation of two sucrose transporters by defoliation and light conditions in perennial ryegrass.

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.

Fluxes in central carbohydrate metabolism of source leaves in a fructan-storing C3 grass: rapid turnover and futile cycling of sucrose in continuous light under contrasted nitrogen nutrition status.

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.

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 ß(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.

Activation of sucrose transport in defoliated Lolium perenne L.: an example of apoplastic phloem loading plasticity.

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 microM in leaf sheaths of undefoliated plants. Defoliation induced a decrease of K(m) followed by an increase of V(max). 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.

Water-deficit accumulates sugars by starch degradation--not by de novo synthesis--in white clover leaves (Trifolium repens).

Labeling 13CO2 in steady-state condition was used to estimate quantitative mobilization of recently fixed carbon or stored sugar during water-deficit in white clover (Trifolium repens L.). Water-deficient gradually decreased leaf-water parameters and total amount of recently fixed carbon. Amount of 13C incorporated into glucose, sucrose and soluble sugars fraction rapidly decreased after 3 days of water-deficit treatment. In contrast, the previously stored soluble sugars significantly increased after 5 days of water-deficit with a coincidence of significant decrease in starch concentration. A highly significant (P < or = 0.001) relationship between the decrease in leaf-water potential caused by water-deficit and the increase in ratio of soluble sugar/starch concentration was observed in water deficit-stressed plants. The data indicate that soluble carbohydrate accumulated by water-deficit treatment is mainly because of the hydrolysis of previously stored starch rather than to de novo synthesis.

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.

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 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.