Direct Effects of Low Temperature upon Components of Fructan Metabolism in Leaves of Lolium temulentum L.

f PlantPhysiol. Vol. 134. pp. 203-208(1989)

Direct Effects of Low Temperature upon Components of F ructan Metabolism in Leaves of Lolium temulentum L.

c. J. POLLOCK, A. J. CAIRNS, B. E. COLLIS, and R. P. WALKER Plant and Cell Biology Department, AFRC Institute for Grassland and Animal Production, Welsh Plant Breeding Station, Plas Gogerddan, Aberystwyth, Wales, UK Received June 10, 1988 . Accepted October 30,1988

Summary Specific effects of temperature upon fructan metabolism were measured in intact leaves of Lolium temulentum L. held at 5 °C by comparing them with leaves where fructan accumulation at 20 °C had been induced by excision. Changes in fructose 2,6-bisphosphate contents differed markedly, with leaves at 5°C showing a sustained decrease and excised leaves a marked increase, even though both treatments caused increased rates of sucrose accumulation in the tissue. Activity of cytoplasmic fructose-1,6-bisphosphatase, which is inhibited by fructose-2,6-bisphosphate, remained constant for 8 h following excision but increased by 64 % in chilled leaves. Measurements of incorporation of 14C02 into oligosaccharides showed that chilled and excised leaves differed in the patterns of accumulation of radioactivity in trisaccharide isomers and the relative abundance of labelled tetra- and pentasaccharides, but not in the progressive movement of radioactivity from sucrose to high molecular weight fructan. Low incubation temperatures also affected the relative proportions of the different isomeric trisaccharides produced in vitro by crude SST and purified invertase preparations supplied with sucrose, mainly by increased accumulation of kestose. These results are discussed in relation to the effects of different environmental conditions during the induction of fructan synthesis in leaves of temperate gramineae.

Key words: Fructan, isokestose, kestose, Lolium temulentum L., low temperature, neokestose, oligosaccharide, sucrose, thin layer chromatography. Abbreviations: DP, degree of polymerisation; F-2,6-BP, fructose-2,6-bisphosphate; FBP'ase, fructose1,6-bisphosphatase; SST, sucrose-sucrose fructosyl transferase.

Introduction Accumulation of fructans as storage carbohydrates has been associated with the response of certain higher plant species to low temperatures (Eagles, 1967; Edelman and Jefford, 1968; Pontis and Del Campillo, 1985). Low temperatures are known to have specific, direct and variable effects upon isolated enzymes and whole metabolic processes associated with carbohydrate metabolism (ap Rees et aI., 1982; Pollock and Lloyd, 1987) and low temperature has been shown to cause depolymerisation of fructan in tubers of Helianthus tuberosus Gefford and Edelman, 1963). Despite these observations, there is good evidence to suggest that induc© 1989 by Gustav Fischer Verlag. Stuttgart

tion of fructan accumulation in leaves of temperate grasses is initiated, not by low temperature per se, but by the accumulation of sucrose (Wagner et aI., 1983; Wagner et aI., 1986; Cairns and Pollock, 1988 a). A number of environmental perturbations, including chilling, have been shown to alter the patterns of supply and demand to cause such accumulation and there are similarities in patterns of fructan synthesis observed following different treatments (Pollock, 1986). Sucrose is thought to provide both the free energy for fructan synthesis and the fructosyl residues required; in addition it is closely associated with regulation of the activity of the enzyme systems involved (Wagner et aI., 1986; Pollock and Chatterton, 1988).

204

C. J. POLLOCK, A. J. CAIRNS, B. E. COLLIS, and R. P. WALKER

In this publication, we describe the direct effects of cold upon patterns of fructan synthesis in leaves of Lolium temulen tum L. We measured the differences in sucrose metabolism which occurred when fructan synthesis was stimulated by chilling (Pollock, 1984) or by illumination of excised leaves (Wagner et aI., 1983; Housley and Pollock, 1985). Cytoplasmic regulation of sucrose synthesis was studied by measurement of the content of F-2,6-BP. Concentrations of this regulatory metabolite are known to be sensitive to changes in the balance between photosynthesis and export (Stitt, 1985) and both chilling and excision cause rapid rises in sucrose content (Pollock, 1986). Temperature effects on fructan synthesis were measured by comparing the patterns of incorporation of 14C02 into fructans following either chilling or excision. High resolution TLC was used to investigate incorporation into different isomeric forms of individual fructo-oligosaccharides (Cairns and Pollock, 1988 a). In addition, measurements were made of the effects of temperature upon products of in vitro fructosyl transferase activity.

Materials and Methods Plant material For metabolite studies, plants of Lolium temulentum L. (Ba 3081, summer annual) were grown from seed in controlled environment chambers at 20°C (8 h photoperiod, 350 liE m - 2S -1) (Pollock and Lloyd, 1987). Plants were used in the week following the appearence of ligules on the 4th leaves. Leaves were excised just prior to the beginning of the photoperiod by cutting under water. The bases were immersed in 10 mM CaCh for 10 min, transferred to water and illuminated under the conditions described above (Housley and Pollock, 1985). Whole seedlings were also transferred to 5°C just prior to the onset of the photoperiod and then maintained at 350 liE m - 2S - 1 under an 8 h photoperiod. Control leaves were maintained at 20°C throughout. These treatments were used to produce material for measurements of sucrose, F-2,6-BP and FBPase and for 14C02 incorporation studies. SST was prepared from leaves of glasshouse-grown material extracted 12 h after excision (Cairns, 1987). Invertase was prepared from leaves of unexcised glasshouse-grown plants of Lolium temulentum, and coleoptiles of Triticum aestivum L. (cv. Avalon) following germination of seeds on damp filter paper for 4 d in darkness.

F-2,6-BP measurements Excised leaves at 20°C, attached leaves from control plants at 20 °C and attached leaves from plants transferred to 5 °C were harvested at various times during the first photoperiod, frozen in liquid N2 and stored at - 80°C until required. Extraction and assay of F-2,6-BP and total chlorophyll were as described by Sicher et al. (1986). Sucrose was measured in the same extracts using the method of Jones et al. (1977). Recoveries were estimated by splitting a leaf longitudinally and freezing and powdering each half separately. F2,6-BP (10 nmol, Sigma, UK) was added to one preparation prior to extraction and both preparations were extracted and assayed separately.

FBP'ase measurements Leaves were harvested as described above and extracted by grinding in a buffer containing 150 mM tris HCI (pH 7.5),20 mM sodium

diethyl dithiocarbamate, 10 mM MgCb, 5 mM EDT A, 20 mM {3mercaptoethanol and 10 % v/v ethanediol. The extract was centrifuged for 5 min at 20,000 x g and desalted on a 50 x 10 mm column of Bio-Gel P6DG into a buffer containing 20 mM tris-HCI (pH 7.5), 10 mM dithiothreitol and 5 mM MgCh. Extracts were assayed spectrophotometrically at 340 nm by the procedure of Zimmermann et al. (1978) except that the concentration of fructose-1,6bisphosphate was 1 mM.

Radiotracer studies Radioactive CO 2 (3.7MBq per plant or excised leaf) was administered by enclosing the leaf or seedling in sealed transparent chambers contained in the appropriate controlled environment chamber (Cairns and Pollock, 1988 a). Excised leaves were labelled at 20°C for 1 h commencing 8 h after excision. Chilled plants were labelled for 3 h commencing 24 h after transfer to 5°C. Following the labelling period, leaves and plants were re-exposed to the normal atmosphere and harvested at various times. Excised leaves were illuminated continuously throughout the feed and chase period (Cairns and Pollock, 1988 a). Chilled seedlings were maintained under the conditions described above. Neutral, water-soluble carbohydrates were extracted, purified and separated by TLC. Radioactivity on the TLC plates was visualised by autroradiography, and relative activity of individual components was measured by scanning the autoradiograms densitometrically. Compounds were identified on the basis of their chromatographic mobilities relative to known markers (Cairns and Pollock, 1988a). Radioactive standards (sucrose, 6 and 60 KBq) were incorporated on each TLC plate to check for uniformity of response.

Measurement 0/ the trisaccharide products 0/ in vitro /ructosyl transferase activity Four different preparations exhibiting fructosyltransferase activity were utilised. A partially purified SST from L. temulentum (Cairns. 1987), an invertase purified from a commercial product (Sigma, UK) by ion exchange chromatography and gel filtration (Trimble and Maley, 1977), a partially purified invertase from wheat coleoptiles (Krishnan et aI., 1985) and a partially purified invertase from L. temulentum. The latter was prepared from mature leaves which had been ground in liquid N2 and extracted in a buffer containing 100 mM Na P0 4 (pH 7.0), 20 mM EDTA, 20 mM Na diethyldithiocarbamate, 5 mM dithiothreitol and 10 % v/v ethanediol. After filtration and centrifugation at 3000 x g the supernatant was brought to pH 5.0 with 1M HCI, left overnight at 5°C and clarified by centrifugation at 3000 x g. The supernatant was fractionated by addition of solid (NH4h 50 4. Material precipitating between 30 and 70 % saturation was retained and dialysed overnight against a buffer containing 100mM Na P0 4 (pH 6.5), 0.5MNaCI and the following at 1 mM; MnCh, CaCh, MgCh and dithiothreitol. The soluble dialysate was fractionated on a 10 x 200 mm column of Concanavalin-A Sepharose 4B (Sigma, UK) equilibrated in the same buffer. Material which bound to the column but which was subsequently released by elution at 1.5 cm 3 min - 1 with 250 mM a-methyl mannos ide in the above buffer was pooled, concentrated by ultrafiltration and stored at - 80°C until use. In all cases, fructosyl transfer was measured using sucrose both as acceptor and donor. Assay mixtures contained 600 mM sucrose, 50 mM Na acetate at pH 5.5 and enzyme (5 nkat) in a final volume of 0.2 cm 3 • Mixtures were incubated for 4 h (SST) or 20 h (invertase) at a range of temperatures, the reaction being stopped by heating in a boiling water bath for 5 min. Production of total trisaccharide was linear with respect to time within the duration of the incubation. Total trisaccharide products were separated and quantified by HPLC, as described by Cairns and Pollock (1988 b). Individual tri-

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Results and Discussion

Changes in regulation of sucrose synthesis Imposition of environmental changes which alter carbon export caused rapid changes in leaf sucrose content (Fig. 1 a). Accumulation was linear throughout the photoperiod, and rates observed were similar to those reported in previous studies (Pollock, 1984; Housley and Pollock, 1985). During the same period there were also marked changes in tissue contents of F-2,6-BP (Fig. 1 b). In control leaves, contents decreased following illumination, followed by a slow rise, similar to the patterns observed in barley leaves (Sicher et al., 1986). In contrast, chilled leaves showed a larger decrease and no subsequent increase, whereas the contents of excised leaves rose markedly over the first hour of illumination and more slowly thereafter. Both excision and chilling elicited increased rates of sucrose accumulation, despite the contrasting patterns of F-2,6-BP metabolism. Measurements of cytoplasmic FBP'ase, the enzyme whose activity is regulated by tissue F-2,6-BP concentration, showed that no major changes in maximum catalytic activity occurred over this period in control or excised leaves, although there was a slight increase in activity during chilling (Table 1). The observed rates were sufficient to catalyse the maximum rates of sucrose accumulation observed in the tissues. The purified cytoplasmic FBP'ase from L. temulentum leaves was, however, inhibited 95 % by 5 JLM F-2,6-BP, (B. E. Collis, unpublished observations) a similar concentration to that which inhibits the enzyme from spinach (Stitt, 1985). Two conclusions can be drawn. Firstly, changes in F-2,6-BP contents do not correlate with the enhanced linear rates of sucrose accumulation which occur immediately following chilling or excision. Secondly, chilling does not produce the

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Table 1: Activity of cytoplasmic fructose 1,6-bisphosphatase in extracts from illuminated leaves of Lalium temulentum following chilling or excision. Extracts were assayed at 1 mM and pH 7.5 substrate to minimise interference from chloroplast enzyme. Recovery of purified cytoplasmic enzyme added prior to extraction was 65 %. Data are the mean ±sd of triplicate determinations expressed as a percentage of zero-time values. Time following treatment (h)

o 0.5 1 4 8

Percentage zero time activity in Control

5 °C chilled

20° C excised

100±18 102± 6 122± 12 95±16 114± 9

100±21 112± 9 124± 5 153±11 164±19

100±27 125± 4 108 ± 16 121± 8 128±19

Zero time values were, respectively, 8.4, 6.1, and 9.3 nKat g-l fro wt. for control, 5° and 20°C treatments.

increase in F-2,6-BP content which is observed in other treatments where sucrose accumulation is induced (Stitt, 1985). Reduction in F-2,6-BP content would be expected to increase activity of cytoplasmic FBP'ase (Stitt, 1985), and thus stimulate sucrose synthesis, but it appears that other factors are important at low temperature. If factors such as substrate availability or the direct effects of temperature upon the enzymes of sucrose synthesis are significant, then perturbations in F-2,6-BP contents or changes in cytoplasmic FBP'ase activity may indicate the operation of homeostatic compensatory mechanisms which serve to maintain steady rates of sucrose synthesis following environmental perturbations. The utilisation by temperate Gramineae of the vacuole for carbohydrate storage will minimise the significance of feedback inhibition of sucrose synthesis, and observations reported here may reflect the consequences of this pattern of intracellular carbohydrate partitioning (Pollock and Chatterton, 1988). It is clear, however, that specific cytoplasmic responses to low temperature do occur in leaves of fructan accumulators, even though the final result of such changes is still an increased rate of sucrose accumulation.

C. J. POLLOCK, A. ]. CAIRNS, B. E. COLLIS, and R. P. WALKER

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Fig. 2: Accumulation of radioactivity in specific oligosaccharides following in incorporation of 14C02 by (a) chilled and (b) excised leaves of Lalium temulentum. Fructans were separated by TLC and the relative radioactivity in individual components was estimated densitometrically from autoradiograms. Values are means of duplicate determinations (A kestose, • isokestose, • neokestose, 0 DP4, 0 DP5).

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Effects 0/low temperature on the incorporation 0/HC02 into /ructans

Previous measurements on the patterns of incorporation of radioactivity into fructan following chilling or excision of L. temulentum have shown progressive appearance of label in fructans of increasing molecular size (Pollock, 1982; Housley and Pollock, 1985). The experimental techniques used did not, however, permit resolution of individual isomeric oligosaccharides. Such resolution is possible up to DP 5 using TLC followed by autoradiography (Figs. 2 and 3). Rates of fructan synthesis were lower at 5°C than at 20°C (Pollock, 1982; Housley and Pollock, 1985). The duration of the chase

Table 2: Changes in radioactivity present in sucrose and fructan (DP> 5) following administration of 14C02 to chilled or excised leaves of Lalium temulentum. Data are means of triplicate determinations expressed as percentage of total radioactivity present in neutral water-soluble carbohydrate at the end of the feeding period (time 0) . Percentage of original radioactivity at time 0 Time after feeding (h)

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Time after feeding (h)

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45 40 32 25 18 15

1 2 4 6 8 10

61 53 42 35 31 25

5 16 31 44 47 54

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periods were such that in both treatments approximately two thirds of the radioactivity present in sucrose at the end of the feeding period was transferred into fructan during the chase. Substantial differences in patterns of labelling were observed both in terms of the initial relative abundance of individual oligosaccharides and of the changes which occurred during the subsequent chase period. Initially, isokestose was the most extensively labelled trisaccharide in both treatments, and this remained so in extracts from excised leaves harvested up to 10 h following feeding, even though activity declined after 2 - 3 h. Radioactivity in neokestose remained at 5 -7 % of the total throughout the photoperiod, whereas radioactive kestose was present in small amounts only up to 8 h following feeding. In chilled leaves, neokestose was the most radioactive trisaccharide towards the end of the 88 h chase period, and initial amounts of radioactive kestose were substantially higher. Initially, chilled leaves also contained more radioactive tetrasaccharide and less radioactive pentasaccharide than excised leaves, although in both cases there was the expected decline in radioactivity in sucrose and accumulation in high DP fructan throughout the course of the experiment (Table 2).

Low temperature and fructan synthesis in Latium

These data are consistent with the suggestion that isokestose is the major primary trisaccharide intermediate, with the others being synthesised either directly or indirectly via slower reactions (Cairns and Pollock, 1988 a). They also suggest that metabolism of individual components of the oligosaccharide pool may be affected differently by environmental variables which cause fructan accumulation to be stimulated. The data may also explain differences in the literature concerning detailed components of the fructan pool from individual species (Pollock and Chatterton, 1988).

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Effects of temperature on the products offructosyl transfer in vitro

Differential accumulation of specific fructo-oligosaccharides in vivo could be due to altered rates of synthesis or of utilisation for further chain elongation. The effect of temperature on the enzymatic synthesis of different trisaccharides was measured using sucrose as donor and acceptor. The enzyme preparation was derived from excised leaves of L. temulentum and the results are shown in Figure 4. Overall rates of fructosyl transfer increased six-fold between 2°C and 30°C. In addition, however, there were marked effects of temperature upon the proportions of the different trisaccharides produced. At low temperatures, kestose was a major product, with the proportion of isokestose increasing as incubation temperature was raised. Neokestose remained a small but relatively constant proportion of the overall product mixture. This distribution contrasts with the contents observed in vivo, where kestose formed only a small proportion of the pool (Fig. 2). This suggests that, even though it is a significant product of enzymatic transfructosylation, kestose

Percentage total trisaccharide Neokestose Isokestose Kestose

207

Neokestose Isokestose Kestose

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may be utilised rapidly in leaves of L. temulentum for the synthesis of {3-2,6 linked high DP fructan (Cairns and Pollock, 1988 a). The SST preparation used in this study was not pure, so it is possible that it contained multiple fructosyl transferase activities with different temperature optima. Purified {3-fructofuranosidase (invertase) preparations are known to catalyse fructosyl transfer at high sucrose concentrations (Schaffler and Morel Du Boil, 1972). In this study, purified preparations from yeast, mature leaves of L. temulentum and coleoptiles of T. aestivum were used, the latter because of the high initial specific activity within the tissue (Krishnan et aI., 1985). These preparations also showed a pronounced effect of incubation temperature upon the proportion of trisaccharide products of transfructosylation (Table 3), with the proportion of kestose declining as the incubation temperature increased. This suggests that a direct effect of temperature on the mechanism of fructosyl transfer may be a general property of such enzymes. There was also substantial variation in the proportions of the three isomeric trisaccharides synthesised by enzyme preparations from different sources. Such variation has been described previously for invertase preparations from different microorganisms (Albon et aI., 1953; Bacon and Bell, 1953). The occurrence of direct effects of temperature during subsequent chain elongation by fructan-fructan fructosyl transferase is currently under investigation using purified oligosaccharides as donors and acceptors. In conclusion, we propose that low temperatures can stimulate fructan synthesis by increasing the accumulation of sucrose. Low temperatures also alter the patterns of cytoplasmic regulatory interactions, change the metabolic interrelationships between individual oligosaccharide components of the fructan pool and alter the catalytic specificity of some of the enzymes of fructan synthesis.

40

Acknowledgements INCUBATION TEMPERATURE (Cl

Fig. 4: Effect of incubation temperature upon the synthesis of trisaccharide isomers by a preparation of SST from excised leaves of Lolium temulentum. Trisaccharides were separated by TLC and relative abundance estimated densitrometrically (.A. kestose, • isokestose, • neokestose).

We thank Jenny Ashton and Margaret Mack for excellent technical and secretarial assistance together with Tom Housley (Purdue University) and Richard Sicher (USDA, Beltsville) for technical advice. This work was carried out as part of the AFRC Scientific Initiative scheme and during the tenure of NATO grant CRG 0706/87 (to CJP).

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C. J. POLLOCK, A. J. CAIRNS, B. E. COLLIS, and R. P . WALKER

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