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Fructan Metabolism in Expanded Primary Leaves of Barley (Hordeum vulgare L. cv. Gerbel): Change upon Ageing and Spatial Organization along the Leaf Blade
J.PlantPhysiol. Vol. 134.pp. 237-242 {1989}
Fructan Metabolism in Expanded Primary Leaves of Barley (Hordeum vulgare L. cv. Gerbel): Change upon Ageing and Spatial Organization along the Leaf Blade WALTER WAGNER
and ANDRES WIEMKEN
Department of Botany, University of Basel, Hebelstr. 1,4056 Basel, Switzerland Received June 10, 1988 . Accepted October 30, 1988
Summary Fructan metabolism in fully expanded primary leaf blades of Hordeum vulgare was found to depend on leaf age and tissue differentiation along the blade. The activities of the key enzyme of fructan synthesis, sucrose sucrose fructosyltransferase (SST), and of acid invertase were high in young leaves but decreased early after expansion. However, the leaves retained the capability to rapidly form SST activity upon excision and illumination for a prolonged period of time. In leaves treated with benzyladenine SST and invertase activities decreased more slowly, SST inducibility was maintained longer, and more sucrose and much more fructan accumulated than in untreated leaves. When young leaves were excised and illuminated or fed with sucrose, the basal part accumulated more fructan and had a higher SST activity than the tip. When seedlings were exposed to low temperatures fructan was accumulated in the basal part of the leaves although the activity of SST remained low. Fructan exohydrolase (FEH) and acid invertase gradually decreased in activity from the base to the tip of the blade. Both activities decreased after excision followed by sucrose feeding of the leaves and after the cold treatment whereas after excision followed by illumination only FEH activity decreased. The results show that the tissue of the fully expanded primary leaf blade of barley is remarkably heterogeneous along its longitudinal axis and changes rapidly with blade age.
Key words: Hordeum vulgare, carbohydrate partitioning, cytokinin, /ructan, SST, /ructan exohydrolase, invertase, sucrose. Abbreviations: FEH, fructan exohydrolase; SST, sucrose sucrose fructosyltransferase; BA, benzyladenine; TLC, thin layer chromatography; dwt, dry weight; fwt, fresh weight.
Introduction Fructan (polyfructosylsucrose) is the main polysaccharide reserve in vegetative tissues of many grasses that thrive in temperate and cold regions of the world (Nelson and Spollen, 1987; Pollock, 1986; Pontis and Del Campillo, 1985; Meier and Reid, 1982). The blades of primary leaves of barley and wheat have proved to be ideal experimental systems for studying fructan metabolism: they rapidly synthesize enormous quantities of fructan (up to 70 % of the dry weight) upon treatments which produce an excess supply of carbohydrates. Incubation of excised leaves in the light or in © 1989 by Gustav Fischer Verlag, Stuttgart
sugar solutions or exposure of intact seedlings to cool temperatures are particularly efficient (Wagner et aI., 1983; Wagner et aI., 1986). The fructan itself as well as the key enzymes for fructan synthesis and degradation, i.e. sucrose sucrose fructosyltransferase (SST: EC 2.4.1.99) and fructan exohydrolase (FEH: EC 2.4.1. x), respectively, have been found to be located in the vacuoles of barley mesophyll cells (Wagner et aI., 1983; Wagner and Wiemken, 1986). As expected from this location, both enzymes have pH activity optima in the acid range (pH 5-6) and appear to be glycoproteins (Wagner and Wiemken, 1986).
238
WALTER WAGNER and ANDRES WIEMKEN
SST is of particular interest since it is rapidly regulated upon induction or suppression of fructan synthesis in barley leaf blades, changing more than 20 fold in activity within 24h (Wagner et aI., 1986). Using protoplasts prepared from barley leaves highly induced for SST as starting material, a preparation of partially purified SST essentially free of invertase has been obtained (Wagner and Wiemken, 1987). This demonstrates that SST activity is due to a specific enzyme independent of invertase, and not to the known transfructosylating capability of invertase (Allen and Bacon, 1956; Straathof et aI., 1986). Interestingly, the purified SST of barley produced the trisaccharide isokestose (synonymes: 1kestose or fJ(2-1}fructosyl sucrose) exclusively, whereas the main invertase in barley mesophyll protoplasts yielded kestose (synonymes: 6-kestose or fJ(2-6}fructosyl sucrose) as a byproduct. Isokestose appears to occur ubiquitously in fructan-accumulating plants; it has been found to be generally the first and main fructosylsucrose isomer formed from sucrose in cell free extracts of such plants as well as after labelling leaves with 14C0 2 (Pollock, 1986). Therefore it has been speculated that an isokestose-producing SST may be the enzyme responsible for diverting sucrose into fructan metabolism in all fructan accumulating plants (Wiemken et aI., 1986; Pollock, 1986). Since grass leaves grow and develop in a highly polar fashion (Schnyder and Nelson, 1987), we became interested to find out how fructan metabolism is organized spatially and temporally along the barley leaf blade. Here we show that fructan metabolism differs conspicuously along the fully expanded young blade and during ageing of the blade. In particular, the ability of the tissue to produce SST activity and to accumulate fructan upon induction was confined to the basal part of the blade and was lost upon ageing. In the design and interpretation of experiments with grass leaves one should be aware of the marked heterogeneity of tissue even in fully expanded leaf blades.
Materials and Methods Plant and growth conditions Primary leaf blades of barley (Hordeum vulgare L. cv. Gerbel) were used for the experiments at an age of 7 - 28 days. Seeds were soaked in slowly running tap water for 24 h and sown in pots of 12cm diameter in commercially available soil (100-150 seeds per pot). The plants were grown in a growth chamber with a day/night cycle of 14 h, 25 °C/I0 h 15°C and a constant relative humidity of 70 %. Light intensity (fluorescent and incandescent) was ca. 500/Lmol photons m - 2 S - 1 at plant height. The seedlings were watered daily with tap water; no fertilizer was used.
Benzyladenine treatment Plants were sprayed with a solution of 1O- 4 M BA, 0.2% (v/v) Tween 80 at the age of 7 days (primary leaf stage).
photons m- 2 s- 1 at 22°C (Wagner et aI., 1986). b) Leaf blades were stripped of their abaxial epidermis and incubated with the stripped side down floating on a solution of 100 mM sucrose in a petri dish at 27°C in the dark. c) After 8 days of growth under the growth conditions described above, intact plants were transferred to a cool temperature regime for 17 days. The day/night cycle was 12 h, 15°C 60% relative humidity/12h, 5°C 80% relative humidity, with a photon flux density of 500 /Lmol photons m - 2 S - I.
Extraction and quantification of soluble carbohydrates Leaf samples were taken 2 h after start of the light period unless otherwise indicated, and extracted twice with boiling 25 % ethanol for 5 min under reflux (2 x 25 mllg fwt). The two extracts were combined and dried under reduced pressure at 40°C. The residue was dissolved in water (1 ml per g initial fwt) and incubated for 5 min with Serdolit Blue anion exchanger (carbonate form) and Serdolit Red cation exchanger (0.1 g each, from Serva, Heidelberg, FRG). After centrifugation (2000 x g, 5 min), the supernatant (= leaf extract) was analysed by TLC on silicagel; spots corresponding to fructose, glucose, sucrose and fructan were scraped off the plate and quantified calorimetrically (Wagner et a!., 1983). Glucose was analysed directly in aliquots of the leaf extracts by use of the glucose-oxidase-test (Boehringer, Mannheim, FRG).
Determination of starch Leaf samples of 1 g fwt were homogenized in 80% (v/v) ethanol in a mortar with a pestle, and the homogenate was centrifuged for 10 min at 2000 x g. The sediment obtained was resuspended in fresh 80 % ethanol, and the suspension was centrifuged again as above. The resulting sediment was suspended in 1 ml 0.5 N NaOH and shaken at 60°C for 1 h. The suspension was then neutralized by adding 1 ml 0.5M HCI, and the pH was adjusted to 4.8 with 2ml 0.5 M acetate (NaOH) buffer. After centrifugation (5 min, 2000 x g) aliquots of the supernatant (500 /LI) were incubated with 20/LI amyloglucosidase from Aspergillus niger (5 mg/ml, Boehringer, Mannheim, FRG) for 90 min at 50°C. The amount of glucose released during incubation was analysed using the glucose-oxidase-test (Boehringer, Mannheim, FRG).
Extraction and assay of/ructan enzymes Leaf samples (1 g fwt) were homogenized in 2 ml 20 mM citratephosphate-buffer (MacIlvaine), pH 5.7, per g fwt at 4°C in a mortar. The homogenates were centrifuged (10 min, 2000 xg) and the supernatants were desalted by dialysis against 5 mM McIlvaine buffer pH 5.7, at 4 °C for 16 h. The activities of sucrose sucrose fructosyltransferase (SST), invertase and fructan exohydrolase (FEH) were determined as described (Wagner and Wiemken, 1986). The samples for invertase and SST assay were incubated at 30°C for 3 h, those for FEH for 5 h. One unit of SST or invertase was defined as the activity using one /Lmol of substrate (sucrose) per min for fructan synthesis (calculated from fructose incorporated in fructan of DP ~ 3) or for hydrolysis (calculated from the fructose released), respectively. The newly formed fructan of DP ~ 3 and the fructose released were separated from the substrate sucrose by TLC (as above) and quantified calorimetrically (Wagner et aI., 1983). One unit of FEH is defined as the activity releasing one /Lmol of fructose from barley fructan per min at pH 5.2 (Wagner et aI., 1983).
Induction offructan synthesis Three different treatments were used to induce fructan synthesis in primary leaf blades. These were: a) Excised blades standing in water were illuminated for 24 h with a light intensity of 500/Lmol
Determination of chlorophyll and dry weight Leaf blades, 0.5 g fwt, were homogenized in 5 ml 80 % (v/v) acetone in a glass tissue grinder. The homogenate was centrifuged at
Fructan metabolism in barley leaves 2000 x g for 10 min and the volume of the supernatant was adjusted to 25 ml with 80 % acetone. The chlorophyll content was calculated from the absorption at 645 and 663 nm according to Arnon (1949). For the determination of dry weight, leaf samples were dried at 80°C for 20 h.
Results
Differentiation of/ructan metabolism upon ageing offully expanded primary leaf blades of barley In 7 to 8 day old seedlings, the primary leaf blades were fully expanded and the sheath and the blade were clearly discernible. The chlorophyll content per g fwt increased until the age of 10 d (Fig. 1 A). Thereafter the content slowly decreased; after 28 d chlorophyll was fully degraded and senescence completed. The dwt: fwt-ratio slightly decreased until the blade was dehydrated at the very end of its lifetime (Fig. 1 A).
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The main soluble carbohydrate found in the leaf blades was sucrose (Fig. 1 B). Its level (approximately 3 mg/g fwt) remained more or less constant during the whole developmental period observed. Interestingly, with progressive senescence the fructan content increased and finally reached the same level as sucrose. Fructan synthesis has been previously found to be correlated with SST activity (Wagner et al., 1986); here, in the contrary, SST activity rapidly decreased at an early stage as did invertase (Fig. 1 C). Since cytokinins are known to delay senescence of leaves (Kende, 1971), their influence on the contents of carbohydrates was studied. As expected, the degradation of chlorophyll was largely inhibited by a single treatment of the leaves with benzyladenine at an age of 7 d (Fig. 1 D). The chlorophyll content even increased initially up to the age of 17 d. The contents of sucrose and, after a lag period, of fructan were much enhanced (Fig. 1 E). The dwt/fwt-ratio of the leaves increased in comparison with the control (Fig. 1 D, compared with Fig. 1 A), probably reflecting the accumulation of more soluble carbohydrates. The accumulation of fructan occurred despite of a decrease of SST activity (Fig. 1 F). This decrease as well as that of invertase activity was slowed by the benzyladenine treatment (Fig. 1 F, compared with Fig. 1 C). Overall, benzyladenine induced an accumulation of sucrose and fructan and kept the leaves in a more juvenile state. The inducibility of SST activity in excised leaf blades by continuous illumination (Wagner et al., 1986) was found to depend on the leaf age as well. In leaves from untreated seedlings, SST was strongly induced up to an age of 14 d but no longer at the age of 21 d (Fig. 2). In the leaves from benzyladenine-treated seedlings the SST remained inducible even at an age of 28 d.
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7 10 14 17 21 24 28 7 10 14 17 21 24 28 d Fig. 1: Developmental changes of carbohydrate relationships in primary leaf blades of barley. The period between 7 d (fully expanded blades) and 28 d (senescence of blades terminated) was studied. Leaf blades from untreated seedlings (A - C) were compared with leaf blades from seedlings treated with 10~4M benzyladenine at the age of 7 d (D - F). A and D: Chlorophyll content (.) and dwt/fwt-ratio (0). Band E: Soluble carbohydrates. C and F activities of SST (.), acid invertase (O).
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Fig. 2: Dependence of the SST inducibility on the age of the leaf blade of barley. SST activity was induced by 24 h continuous illumination of excised primary leaf blades harvested at the age indicated. SST activity is given before (hatched part of bars) and after illumination (total height of bars).
240
WALTER WAGNER
and
ANDRES WIEMKEN
Spatial differentiation offructan metabolism along the blade offully expanded primary leaves of barley Fructan synthesis was induced in blades of fully expanded primary leaves at the age of 8 days by means of three different treatments. Thereafter the blades were dissected transversally into ten segments of equal length from the base (site of ligule) to the tip for further measurements. Untreated leaves contained very little fructan (Fig. 3 A). The content of sucrose was small and increased from the base to the tip of the blade (Fig. 3 A). Excision and subsequent continuous illumination of the blades for 24 h led to a drastic increase of the content of nonstructural carbohydrates (Fig. 3 B). Whereas sucrose was distributed more or less uniformly throughout the blade, fructan was accumulated mainly in the basal part and starch and hexoses mainly towards the tip of the blade. Another treatment inducing fructan synthesis consisted in the incubation of excised leaf blades on a solution of 100 mM sucrose in the dark for 24 h. Since the abaxial epidermis was removed, the supply of sucrose to cells was equal over the whole leaf. Nevertheless, the same asymmetric accumulation of fructan was found as before (Fig. 3 C). Section one (Fig. 3 B, C) has a much depressed fructan content probably because it is affected by the excision of the leaves. Finally, when fructan synthesis was induced by transferring seedlings to a cool temperature regime, the gradient of the
fructan content from the base to the tip of the blade became even more pronounced (Fig. 3 D). Starch was virtually absent after this treatment. Irrespective of the method used to induce fructan synthesis, sucrose concentrations were in all cases near 10 mg per g fwt throughout the whole length of the blades. This seems to be a threshold concentration at which surplus sucrose is either exported or transformed to a polymer, mainly fructan or starch (e.g. Fig. 3 B, segments of the leaf tip). The distribution of SST, the key enzyme of fructan synthesis, was found to be remarkably similar to the distribution of fructan (Fig. 4 compared with Fig. 3). The activity was localized chiefly in the basal part of the blade (Figs. 4 A - C). SST activity was high after treatments in which fructan synthesis was rapidly induced (i.e. illumination, Fig. 4 B, and sugar feeding, Fig. 4 C) whereas the activity was comparatively low after the treatment which induced fructan synthesis slowly, i.e. the cool temperature regime (Fig. 4D), as well as in the untreated control (Fig. 4A). The activity of fructan exohydrolase (FEH) decreased upon all the three treatments used, especially in the basal part of the leaf blades, where activity was high in the untreated control (Figs. 4 A - D). A decrease of FEH activity might be a prerequisite for the accumulation of fructan, as is induction of SST and a sufficient supply of sucrose. The high activity of acid invertase in the basal part of the untreated leaf blades
Fig. 3: Spatial distribution of nonstructural carbohydrates along the longitudinal axis of primary leaf blades of barley. The effects of three different treatments inducing accumulation of fructan in the leaves were compared: A: control; untreated blade excised at day 8. B: blade excised at day 7 and continuously illuminated for 24 h. C: blade excised at day 7 and fed with 100 mM sucrose for 24 h. D: blade of a seedling transferred at day 8 to the cool temperature regime for 17 days.
Fig. 4: Spatial distribution of the activities of SST, FEH and acid invertase along the longitudinal axis of primary leaf blades of barley. The effects of three different treatments inducing accumulation of fructans in the leaves were compared: A, B, C, D: see Figure 3. (e) SST, (6) FEH, (0) acid invertase.
(Fig. 4 A) may be connected with their relative youth (8 d) (see Fig. 1 C). Invertase activity was little affected by illumination but was strongly reduced upon sucrose feeding. Since the latter treatment also efficiently induces SST it is of particular interest in view of attempts for purification of SST where invertases represent a great inconvenience.
Discussion
We have demonstrated a marked differentiation of fructan metabolism in the tissue of fully grown barley leaf blades. Previous studies have shown that such a differentiation exists between sheath and blade of fully expanded tall fescue leaves (Housley and Volenec, 1988) and during the growth phase of tall fescue leaves in the different zones of cell development (Schnyder and Nelson, 1987; Schnyder et ai., 1988). Clearly, in studies of the regulation of fructan metabolism in grass leaves, one has to be aware of the fact that even the fully expanded blade is a heterogeneous system where the different parts respond differently to environmental changes. Measurements on the whole leaf blade might therefore miss a point of interest. Since acid invertase is known to be associated with expanding leaves (Morris and Arthur, 1984; Schaffer et aI., 1987) and SST activity, with the expansion zone of grass leaves (Schnyder et ai., 1988), the marked decrease of these acti-
vities found at the beginning (Fig. 1 C) might be related to the end of the leaf growth phase. Cytokinin treated leaves had a high sucrose content and accumulated fructan (Fig. 1 E). In combination with other phytohormones, cytokinin has been found to initiate fructan synthesis also in explants of dormant tubers of Jerusalem artichokes fed with sugar (Pontis, 1966). Quite generally, tissues treated with cytokinin appear to have a better supply of assimilates (Hayes and Patrick, 1985; Kende, 1971). In this context it is interesting to note that in a fungus-plant interaction, where phytohormones such as cytokinin are thought to play a prominent role, fructan was accumulated at the site of infection in a fructan accumulating plant (Holligan et ai., 1973). The data show that SST activity is not correlated with the sucrose content of the leaves. Thus, if sucrose acts as an effector for induction of SST, this role could only be attributed to a portion of the total pool of sucrose present in the cells, most probably to the cytosolic, not to the vacuolar pool (Kaiser et ai., 1982; Wagner et ai., 1983; Farrar and Farrar, 1986; Boller and Wiemken, 1986). Remarkably, in the tip parts of excised and illuminated leaves, where fructan apparently could not be synthesized from a surplus of photosynthates, starch was found to accumulate (Fig. 3 B). This was not the case when assimilate (sucrose) was supplied from the outside of the cell (Fig. 3 C). In a cool temperature regime, no starch accumulated (Fig. 3 D), in accordance with the inverse temperature dependence
242
WALTER WAGNER and ANDRES WIEMKEN
of the synthesis of starch and fructan in barley leaves which has been reported previously (Sicher and Kremer, 1986).
References ALLEN, P. J. and J. S. D. BACON: Oligosaccharides formed from sucrose by fructose-transferring enzymes of higher plants. Biochern. J. 63, 200-206 (1956). ARNON, D. J.: Copper enzymes in isolated chloroplasts. Polyp henoloxidase in Beta vulgaris. Plant Physiol. 24, 1-15 (1949). BOLLER, T. and A. WIEMKEN: Dynamics of vacuolar compartmentation. In: Ann. Rev. Plant Physiol., Vol. 37, pp. 137 -164 (1986). FARRAR, S. C. and J. F. FARRAR: Compartmentation and fluxes of sucrose in intact leaf blades of barley. New Phytol. 103, 645-657 (1986). HAYES, P. M. and J. W. PATRICK: Photosynthate transport in stems of Phaseolus vulgaris L. treated with gibberellic acid, indole-3-acetic acid or kinetin. Planta 166, 371-379 (1985). HOLLIGAN, P. M., C. CHEN, and D. H. LEWIS: Changes in the carbohydrate composition of leaves of Tussilago farfara during infection by Puccinia poarum. New Phytol. 72, 947 -955 (1973). HOUSLEY, TH. L. andJ. J. VOLENEC: Fructan content and synthesis in leaf tissues of Festuca arundinacea. Plant Physiol. 86, 1247 -1251 (1988). KAiSER, G., E. MARTINOIA, and A. WIEMKEN: Rapid appearance of photosynthetic products in the vacuoles isolated from barley mesophyll protoplasts by a new fast method. Z. Pflanzenphysiol. 107, 103-113 (1982). KENDE, H.: The cytokinins. Int. Rev. of Cytol. 31,301-338 (1971). MEIER, H. and J. S. G. REID: Reserve polysaccharides other than starch in higher plants. In: LOEwus, F. A. and W. TANNER (eds.): Encyclopedia of Plant Physiol. 13A, 419-469 (1982). MORRIs, D. A. and E. D. ARTHUR: An association between acid invertase activity and cell growth during leaf expansion in Pha· seolus vulgaris L. J. Exp. Bot. 35, No. 158, 1369-1379 (1984). NELSON, C. J. and W. G. SPOLLEN: Fructans. Physiol. Plant. 71, 512-516 (1987).
POLLOCK, C. J.: Fructans and the metabolism of sucrose in vascular plants. New Phytol. 104, 1-24 (1986). PONTIS, H. G.: Observations on the de novo synthesis of fructosans in vivo. Arch. Biochem. Biophys. 116,416-424 (1966). PONTIS, H. G. and E. DELCAMPILLO: Fructans. In: DEY, P. M. and R. A. DIXON (eds.): Biochemistry of Storage Carbohydrates in Green Plants. Academic Press, pp. 205-227 (1985). SCHAFFER, A. A., O. SAGEE, E. E. GOLDSCHMIDT, and R. GOREN: Invertase and sucrose synthase activity, carbohydrate status and endogenous IAA levels during Citrus leaf development. Physiol. Plant. 69, 151-155 (1987). SCHNYDER, H. and C. J. NELSON: Growth rates and carbonhydrate fluxes within the elongation zone of tall fescue leaf blades. Plant Physiol. 85, 548-553 (1987). SCHNYDER, H., C. J. NELSON, and W. G. SPOLLEN: Diurnal growth of tall fescue leaf blades. Plant Physiol. 86, 1077 -1083 (1988). SICHER, R. C. and D. F. KREMER: Effects of temperature and irradiance on non-structural carbohydrate accumulation in barley primary leaves. Physiol. Plant. 66, 365-369 (1986). STRAATHOF, A. J. J., A. P. G. KIEBOOM, and H. VAN BEKKUM: Invertase-catalysed fructosyl transfer in concentrated solutions of sucrose. Carbohyd. Res. 146, 154-159 (1986). WAGNER, W., F. KELLER, and A. WIEMKEN: Fructan metabolism in cereals: Induction in leaves and compartmentation in protoplasts and vacuoles. Z. Pflanzenphysiol. 112, 359-372 (1983). WAGNER, W., A. WIEMKEN, and P. MATILE: Regulation of fructan metabolism in leaves of barley (Hordeum vulgare L. cv. Gerbel). Plant Physiol. 81, 444-447 (1986). WAGNER, W. and A. WIEMKEN: Properties and subcellular localization of fructan hydrolase in the leaves of barley (Hordeum vulgare L. cv. Gerbel). J. Plant Physiol. 123, 429-439 (1986). - - Enzymology of fructan synthesis in grasses. Plant Physiol. 85,706-710 (1987). WIEMKEN, A., M. FREHNER, F. KELLER, and W. WAGNER: Fructan metabolism, enzymology and compartmentation. In: Current Topics of Plant Biochemistry and Physiology, Vol. 5, pp. 17 -31, Univ. of Missouri, Columbia, MO (1986).
Fructan Metabolism in Expanded Primary Leaves of Barley (Hordeum vulgare L. cv. Gerbel): Change upon Ageing and Spatial Organization along the Leaf Blade WALTER WAGNER
and ANDRES WIEMKEN
Department of Botany, University of Basel, Hebelstr. 1,4056 Basel, Switzerland Received June 10, 1988 . Accepted October 30, 1988
Summary Fructan metabolism in fully expanded primary leaf blades of Hordeum vulgare was found to depend on leaf age and tissue differentiation along the blade. The activities of the key enzyme of fructan synthesis, sucrose sucrose fructosyltransferase (SST), and of acid invertase were high in young leaves but decreased early after expansion. However, the leaves retained the capability to rapidly form SST activity upon excision and illumination for a prolonged period of time. In leaves treated with benzyladenine SST and invertase activities decreased more slowly, SST inducibility was maintained longer, and more sucrose and much more fructan accumulated than in untreated leaves. When young leaves were excised and illuminated or fed with sucrose, the basal part accumulated more fructan and had a higher SST activity than the tip. When seedlings were exposed to low temperatures fructan was accumulated in the basal part of the leaves although the activity of SST remained low. Fructan exohydrolase (FEH) and acid invertase gradually decreased in activity from the base to the tip of the blade. Both activities decreased after excision followed by sucrose feeding of the leaves and after the cold treatment whereas after excision followed by illumination only FEH activity decreased. The results show that the tissue of the fully expanded primary leaf blade of barley is remarkably heterogeneous along its longitudinal axis and changes rapidly with blade age.
Key words: Hordeum vulgare, carbohydrate partitioning, cytokinin, /ructan, SST, /ructan exohydrolase, invertase, sucrose. Abbreviations: FEH, fructan exohydrolase; SST, sucrose sucrose fructosyltransferase; BA, benzyladenine; TLC, thin layer chromatography; dwt, dry weight; fwt, fresh weight.
Introduction Fructan (polyfructosylsucrose) is the main polysaccharide reserve in vegetative tissues of many grasses that thrive in temperate and cold regions of the world (Nelson and Spollen, 1987; Pollock, 1986; Pontis and Del Campillo, 1985; Meier and Reid, 1982). The blades of primary leaves of barley and wheat have proved to be ideal experimental systems for studying fructan metabolism: they rapidly synthesize enormous quantities of fructan (up to 70 % of the dry weight) upon treatments which produce an excess supply of carbohydrates. Incubation of excised leaves in the light or in © 1989 by Gustav Fischer Verlag, Stuttgart
sugar solutions or exposure of intact seedlings to cool temperatures are particularly efficient (Wagner et aI., 1983; Wagner et aI., 1986). The fructan itself as well as the key enzymes for fructan synthesis and degradation, i.e. sucrose sucrose fructosyltransferase (SST: EC 2.4.1.99) and fructan exohydrolase (FEH: EC 2.4.1. x), respectively, have been found to be located in the vacuoles of barley mesophyll cells (Wagner et aI., 1983; Wagner and Wiemken, 1986). As expected from this location, both enzymes have pH activity optima in the acid range (pH 5-6) and appear to be glycoproteins (Wagner and Wiemken, 1986).
238
WALTER WAGNER and ANDRES WIEMKEN
SST is of particular interest since it is rapidly regulated upon induction or suppression of fructan synthesis in barley leaf blades, changing more than 20 fold in activity within 24h (Wagner et aI., 1986). Using protoplasts prepared from barley leaves highly induced for SST as starting material, a preparation of partially purified SST essentially free of invertase has been obtained (Wagner and Wiemken, 1987). This demonstrates that SST activity is due to a specific enzyme independent of invertase, and not to the known transfructosylating capability of invertase (Allen and Bacon, 1956; Straathof et aI., 1986). Interestingly, the purified SST of barley produced the trisaccharide isokestose (synonymes: 1kestose or fJ(2-1}fructosyl sucrose) exclusively, whereas the main invertase in barley mesophyll protoplasts yielded kestose (synonymes: 6-kestose or fJ(2-6}fructosyl sucrose) as a byproduct. Isokestose appears to occur ubiquitously in fructan-accumulating plants; it has been found to be generally the first and main fructosylsucrose isomer formed from sucrose in cell free extracts of such plants as well as after labelling leaves with 14C0 2 (Pollock, 1986). Therefore it has been speculated that an isokestose-producing SST may be the enzyme responsible for diverting sucrose into fructan metabolism in all fructan accumulating plants (Wiemken et aI., 1986; Pollock, 1986). Since grass leaves grow and develop in a highly polar fashion (Schnyder and Nelson, 1987), we became interested to find out how fructan metabolism is organized spatially and temporally along the barley leaf blade. Here we show that fructan metabolism differs conspicuously along the fully expanded young blade and during ageing of the blade. In particular, the ability of the tissue to produce SST activity and to accumulate fructan upon induction was confined to the basal part of the blade and was lost upon ageing. In the design and interpretation of experiments with grass leaves one should be aware of the marked heterogeneity of tissue even in fully expanded leaf blades.
Materials and Methods Plant and growth conditions Primary leaf blades of barley (Hordeum vulgare L. cv. Gerbel) were used for the experiments at an age of 7 - 28 days. Seeds were soaked in slowly running tap water for 24 h and sown in pots of 12cm diameter in commercially available soil (100-150 seeds per pot). The plants were grown in a growth chamber with a day/night cycle of 14 h, 25 °C/I0 h 15°C and a constant relative humidity of 70 %. Light intensity (fluorescent and incandescent) was ca. 500/Lmol photons m - 2 S - 1 at plant height. The seedlings were watered daily with tap water; no fertilizer was used.
Benzyladenine treatment Plants were sprayed with a solution of 1O- 4 M BA, 0.2% (v/v) Tween 80 at the age of 7 days (primary leaf stage).
photons m- 2 s- 1 at 22°C (Wagner et aI., 1986). b) Leaf blades were stripped of their abaxial epidermis and incubated with the stripped side down floating on a solution of 100 mM sucrose in a petri dish at 27°C in the dark. c) After 8 days of growth under the growth conditions described above, intact plants were transferred to a cool temperature regime for 17 days. The day/night cycle was 12 h, 15°C 60% relative humidity/12h, 5°C 80% relative humidity, with a photon flux density of 500 /Lmol photons m - 2 S - I.
Extraction and quantification of soluble carbohydrates Leaf samples were taken 2 h after start of the light period unless otherwise indicated, and extracted twice with boiling 25 % ethanol for 5 min under reflux (2 x 25 mllg fwt). The two extracts were combined and dried under reduced pressure at 40°C. The residue was dissolved in water (1 ml per g initial fwt) and incubated for 5 min with Serdolit Blue anion exchanger (carbonate form) and Serdolit Red cation exchanger (0.1 g each, from Serva, Heidelberg, FRG). After centrifugation (2000 x g, 5 min), the supernatant (= leaf extract) was analysed by TLC on silicagel; spots corresponding to fructose, glucose, sucrose and fructan were scraped off the plate and quantified calorimetrically (Wagner et a!., 1983). Glucose was analysed directly in aliquots of the leaf extracts by use of the glucose-oxidase-test (Boehringer, Mannheim, FRG).
Determination of starch Leaf samples of 1 g fwt were homogenized in 80% (v/v) ethanol in a mortar with a pestle, and the homogenate was centrifuged for 10 min at 2000 x g. The sediment obtained was resuspended in fresh 80 % ethanol, and the suspension was centrifuged again as above. The resulting sediment was suspended in 1 ml 0.5 N NaOH and shaken at 60°C for 1 h. The suspension was then neutralized by adding 1 ml 0.5M HCI, and the pH was adjusted to 4.8 with 2ml 0.5 M acetate (NaOH) buffer. After centrifugation (5 min, 2000 x g) aliquots of the supernatant (500 /LI) were incubated with 20/LI amyloglucosidase from Aspergillus niger (5 mg/ml, Boehringer, Mannheim, FRG) for 90 min at 50°C. The amount of glucose released during incubation was analysed using the glucose-oxidase-test (Boehringer, Mannheim, FRG).
Extraction and assay of/ructan enzymes Leaf samples (1 g fwt) were homogenized in 2 ml 20 mM citratephosphate-buffer (MacIlvaine), pH 5.7, per g fwt at 4°C in a mortar. The homogenates were centrifuged (10 min, 2000 xg) and the supernatants were desalted by dialysis against 5 mM McIlvaine buffer pH 5.7, at 4 °C for 16 h. The activities of sucrose sucrose fructosyltransferase (SST), invertase and fructan exohydrolase (FEH) were determined as described (Wagner and Wiemken, 1986). The samples for invertase and SST assay were incubated at 30°C for 3 h, those for FEH for 5 h. One unit of SST or invertase was defined as the activity using one /Lmol of substrate (sucrose) per min for fructan synthesis (calculated from fructose incorporated in fructan of DP ~ 3) or for hydrolysis (calculated from the fructose released), respectively. The newly formed fructan of DP ~ 3 and the fructose released were separated from the substrate sucrose by TLC (as above) and quantified calorimetrically (Wagner et aI., 1983). One unit of FEH is defined as the activity releasing one /Lmol of fructose from barley fructan per min at pH 5.2 (Wagner et aI., 1983).
Induction offructan synthesis Three different treatments were used to induce fructan synthesis in primary leaf blades. These were: a) Excised blades standing in water were illuminated for 24 h with a light intensity of 500/Lmol
Determination of chlorophyll and dry weight Leaf blades, 0.5 g fwt, were homogenized in 5 ml 80 % (v/v) acetone in a glass tissue grinder. The homogenate was centrifuged at
Fructan metabolism in barley leaves 2000 x g for 10 min and the volume of the supernatant was adjusted to 25 ml with 80 % acetone. The chlorophyll content was calculated from the absorption at 645 and 663 nm according to Arnon (1949). For the determination of dry weight, leaf samples were dried at 80°C for 20 h.
Results
Differentiation of/ructan metabolism upon ageing offully expanded primary leaf blades of barley In 7 to 8 day old seedlings, the primary leaf blades were fully expanded and the sheath and the blade were clearly discernible. The chlorophyll content per g fwt increased until the age of 10 d (Fig. 1 A). Thereafter the content slowly decreased; after 28 d chlorophyll was fully degraded and senescence completed. The dwt: fwt-ratio slightly decreased until the blade was dehydrated at the very end of its lifetime (Fig. 1 A).
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The main soluble carbohydrate found in the leaf blades was sucrose (Fig. 1 B). Its level (approximately 3 mg/g fwt) remained more or less constant during the whole developmental period observed. Interestingly, with progressive senescence the fructan content increased and finally reached the same level as sucrose. Fructan synthesis has been previously found to be correlated with SST activity (Wagner et al., 1986); here, in the contrary, SST activity rapidly decreased at an early stage as did invertase (Fig. 1 C). Since cytokinins are known to delay senescence of leaves (Kende, 1971), their influence on the contents of carbohydrates was studied. As expected, the degradation of chlorophyll was largely inhibited by a single treatment of the leaves with benzyladenine at an age of 7 d (Fig. 1 D). The chlorophyll content even increased initially up to the age of 17 d. The contents of sucrose and, after a lag period, of fructan were much enhanced (Fig. 1 E). The dwt/fwt-ratio of the leaves increased in comparison with the control (Fig. 1 D, compared with Fig. 1 A), probably reflecting the accumulation of more soluble carbohydrates. The accumulation of fructan occurred despite of a decrease of SST activity (Fig. 1 F). This decrease as well as that of invertase activity was slowed by the benzyladenine treatment (Fig. 1 F, compared with Fig. 1 C). Overall, benzyladenine induced an accumulation of sucrose and fructan and kept the leaves in a more juvenile state. The inducibility of SST activity in excised leaf blades by continuous illumination (Wagner et al., 1986) was found to depend on the leaf age as well. In leaves from untreated seedlings, SST was strongly induced up to an age of 14 d but no longer at the age of 21 d (Fig. 2). In the leaves from benzyladenine-treated seedlings the SST remained inducible even at an age of 28 d.
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7 10 14 17 21 24 28 7 10 14 17 21 24 28 d Fig. 1: Developmental changes of carbohydrate relationships in primary leaf blades of barley. The period between 7 d (fully expanded blades) and 28 d (senescence of blades terminated) was studied. Leaf blades from untreated seedlings (A - C) were compared with leaf blades from seedlings treated with 10~4M benzyladenine at the age of 7 d (D - F). A and D: Chlorophyll content (.) and dwt/fwt-ratio (0). Band E: Soluble carbohydrates. C and F activities of SST (.), acid invertase (O).
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Fig. 2: Dependence of the SST inducibility on the age of the leaf blade of barley. SST activity was induced by 24 h continuous illumination of excised primary leaf blades harvested at the age indicated. SST activity is given before (hatched part of bars) and after illumination (total height of bars).
240
WALTER WAGNER
and
ANDRES WIEMKEN
Spatial differentiation offructan metabolism along the blade offully expanded primary leaves of barley Fructan synthesis was induced in blades of fully expanded primary leaves at the age of 8 days by means of three different treatments. Thereafter the blades were dissected transversally into ten segments of equal length from the base (site of ligule) to the tip for further measurements. Untreated leaves contained very little fructan (Fig. 3 A). The content of sucrose was small and increased from the base to the tip of the blade (Fig. 3 A). Excision and subsequent continuous illumination of the blades for 24 h led to a drastic increase of the content of nonstructural carbohydrates (Fig. 3 B). Whereas sucrose was distributed more or less uniformly throughout the blade, fructan was accumulated mainly in the basal part and starch and hexoses mainly towards the tip of the blade. Another treatment inducing fructan synthesis consisted in the incubation of excised leaf blades on a solution of 100 mM sucrose in the dark for 24 h. Since the abaxial epidermis was removed, the supply of sucrose to cells was equal over the whole leaf. Nevertheless, the same asymmetric accumulation of fructan was found as before (Fig. 3 C). Section one (Fig. 3 B, C) has a much depressed fructan content probably because it is affected by the excision of the leaves. Finally, when fructan synthesis was induced by transferring seedlings to a cool temperature regime, the gradient of the
fructan content from the base to the tip of the blade became even more pronounced (Fig. 3 D). Starch was virtually absent after this treatment. Irrespective of the method used to induce fructan synthesis, sucrose concentrations were in all cases near 10 mg per g fwt throughout the whole length of the blades. This seems to be a threshold concentration at which surplus sucrose is either exported or transformed to a polymer, mainly fructan or starch (e.g. Fig. 3 B, segments of the leaf tip). The distribution of SST, the key enzyme of fructan synthesis, was found to be remarkably similar to the distribution of fructan (Fig. 4 compared with Fig. 3). The activity was localized chiefly in the basal part of the blade (Figs. 4 A - C). SST activity was high after treatments in which fructan synthesis was rapidly induced (i.e. illumination, Fig. 4 B, and sugar feeding, Fig. 4 C) whereas the activity was comparatively low after the treatment which induced fructan synthesis slowly, i.e. the cool temperature regime (Fig. 4D), as well as in the untreated control (Fig. 4A). The activity of fructan exohydrolase (FEH) decreased upon all the three treatments used, especially in the basal part of the leaf blades, where activity was high in the untreated control (Figs. 4 A - D). A decrease of FEH activity might be a prerequisite for the accumulation of fructan, as is induction of SST and a sufficient supply of sucrose. The high activity of acid invertase in the basal part of the untreated leaf blades
Fig. 3: Spatial distribution of nonstructural carbohydrates along the longitudinal axis of primary leaf blades of barley. The effects of three different treatments inducing accumulation of fructan in the leaves were compared: A: control; untreated blade excised at day 8. B: blade excised at day 7 and continuously illuminated for 24 h. C: blade excised at day 7 and fed with 100 mM sucrose for 24 h. D: blade of a seedling transferred at day 8 to the cool temperature regime for 17 days.
Fig. 4: Spatial distribution of the activities of SST, FEH and acid invertase along the longitudinal axis of primary leaf blades of barley. The effects of three different treatments inducing accumulation of fructans in the leaves were compared: A, B, C, D: see Figure 3. (e) SST, (6) FEH, (0) acid invertase.
(Fig. 4 A) may be connected with their relative youth (8 d) (see Fig. 1 C). Invertase activity was little affected by illumination but was strongly reduced upon sucrose feeding. Since the latter treatment also efficiently induces SST it is of particular interest in view of attempts for purification of SST where invertases represent a great inconvenience.
Discussion
We have demonstrated a marked differentiation of fructan metabolism in the tissue of fully grown barley leaf blades. Previous studies have shown that such a differentiation exists between sheath and blade of fully expanded tall fescue leaves (Housley and Volenec, 1988) and during the growth phase of tall fescue leaves in the different zones of cell development (Schnyder and Nelson, 1987; Schnyder et ai., 1988). Clearly, in studies of the regulation of fructan metabolism in grass leaves, one has to be aware of the fact that even the fully expanded blade is a heterogeneous system where the different parts respond differently to environmental changes. Measurements on the whole leaf blade might therefore miss a point of interest. Since acid invertase is known to be associated with expanding leaves (Morris and Arthur, 1984; Schaffer et aI., 1987) and SST activity, with the expansion zone of grass leaves (Schnyder et ai., 1988), the marked decrease of these acti-
vities found at the beginning (Fig. 1 C) might be related to the end of the leaf growth phase. Cytokinin treated leaves had a high sucrose content and accumulated fructan (Fig. 1 E). In combination with other phytohormones, cytokinin has been found to initiate fructan synthesis also in explants of dormant tubers of Jerusalem artichokes fed with sugar (Pontis, 1966). Quite generally, tissues treated with cytokinin appear to have a better supply of assimilates (Hayes and Patrick, 1985; Kende, 1971). In this context it is interesting to note that in a fungus-plant interaction, where phytohormones such as cytokinin are thought to play a prominent role, fructan was accumulated at the site of infection in a fructan accumulating plant (Holligan et ai., 1973). The data show that SST activity is not correlated with the sucrose content of the leaves. Thus, if sucrose acts as an effector for induction of SST, this role could only be attributed to a portion of the total pool of sucrose present in the cells, most probably to the cytosolic, not to the vacuolar pool (Kaiser et ai., 1982; Wagner et ai., 1983; Farrar and Farrar, 1986; Boller and Wiemken, 1986). Remarkably, in the tip parts of excised and illuminated leaves, where fructan apparently could not be synthesized from a surplus of photosynthates, starch was found to accumulate (Fig. 3 B). This was not the case when assimilate (sucrose) was supplied from the outside of the cell (Fig. 3 C). In a cool temperature regime, no starch accumulated (Fig. 3 D), in accordance with the inverse temperature dependence
242
WALTER WAGNER and ANDRES WIEMKEN
of the synthesis of starch and fructan in barley leaves which has been reported previously (Sicher and Kremer, 1986).
References ALLEN, P. J. and J. S. D. BACON: Oligosaccharides formed from sucrose by fructose-transferring enzymes of higher plants. Biochern. J. 63, 200-206 (1956). ARNON, D. J.: Copper enzymes in isolated chloroplasts. Polyp henoloxidase in Beta vulgaris. Plant Physiol. 24, 1-15 (1949). BOLLER, T. and A. WIEMKEN: Dynamics of vacuolar compartmentation. In: Ann. Rev. Plant Physiol., Vol. 37, pp. 137 -164 (1986). FARRAR, S. C. and J. F. FARRAR: Compartmentation and fluxes of sucrose in intact leaf blades of barley. New Phytol. 103, 645-657 (1986). HAYES, P. M. and J. W. PATRICK: Photosynthate transport in stems of Phaseolus vulgaris L. treated with gibberellic acid, indole-3-acetic acid or kinetin. Planta 166, 371-379 (1985). HOLLIGAN, P. M., C. CHEN, and D. H. LEWIS: Changes in the carbohydrate composition of leaves of Tussilago farfara during infection by Puccinia poarum. New Phytol. 72, 947 -955 (1973). HOUSLEY, TH. L. andJ. J. VOLENEC: Fructan content and synthesis in leaf tissues of Festuca arundinacea. Plant Physiol. 86, 1247 -1251 (1988). KAiSER, G., E. MARTINOIA, and A. WIEMKEN: Rapid appearance of photosynthetic products in the vacuoles isolated from barley mesophyll protoplasts by a new fast method. Z. Pflanzenphysiol. 107, 103-113 (1982). KENDE, H.: The cytokinins. Int. Rev. of Cytol. 31,301-338 (1971). MEIER, H. and J. S. G. REID: Reserve polysaccharides other than starch in higher plants. In: LOEwus, F. A. and W. TANNER (eds.): Encyclopedia of Plant Physiol. 13A, 419-469 (1982). MORRIs, D. A. and E. D. ARTHUR: An association between acid invertase activity and cell growth during leaf expansion in Pha· seolus vulgaris L. J. Exp. Bot. 35, No. 158, 1369-1379 (1984). NELSON, C. J. and W. G. SPOLLEN: Fructans. Physiol. Plant. 71, 512-516 (1987).
POLLOCK, C. J.: Fructans and the metabolism of sucrose in vascular plants. New Phytol. 104, 1-24 (1986). PONTIS, H. G.: Observations on the de novo synthesis of fructosans in vivo. Arch. Biochem. Biophys. 116,416-424 (1966). PONTIS, H. G. and E. DELCAMPILLO: Fructans. In: DEY, P. M. and R. A. DIXON (eds.): Biochemistry of Storage Carbohydrates in Green Plants. Academic Press, pp. 205-227 (1985). SCHAFFER, A. A., O. SAGEE, E. E. GOLDSCHMIDT, and R. GOREN: Invertase and sucrose synthase activity, carbohydrate status and endogenous IAA levels during Citrus leaf development. Physiol. Plant. 69, 151-155 (1987). SCHNYDER, H. and C. J. NELSON: Growth rates and carbonhydrate fluxes within the elongation zone of tall fescue leaf blades. Plant Physiol. 85, 548-553 (1987). SCHNYDER, H., C. J. NELSON, and W. G. SPOLLEN: Diurnal growth of tall fescue leaf blades. Plant Physiol. 86, 1077 -1083 (1988). SICHER, R. C. and D. F. KREMER: Effects of temperature and irradiance on non-structural carbohydrate accumulation in barley primary leaves. Physiol. Plant. 66, 365-369 (1986). STRAATHOF, A. J. J., A. P. G. KIEBOOM, and H. VAN BEKKUM: Invertase-catalysed fructosyl transfer in concentrated solutions of sucrose. Carbohyd. Res. 146, 154-159 (1986). WAGNER, W., F. KELLER, and A. WIEMKEN: Fructan metabolism in cereals: Induction in leaves and compartmentation in protoplasts and vacuoles. Z. Pflanzenphysiol. 112, 359-372 (1983). WAGNER, W., A. WIEMKEN, and P. MATILE: Regulation of fructan metabolism in leaves of barley (Hordeum vulgare L. cv. Gerbel). Plant Physiol. 81, 444-447 (1986). WAGNER, W. and A. WIEMKEN: Properties and subcellular localization of fructan hydrolase in the leaves of barley (Hordeum vulgare L. cv. Gerbel). J. Plant Physiol. 123, 429-439 (1986). - - Enzymology of fructan synthesis in grasses. Plant Physiol. 85,706-710 (1987). WIEMKEN, A., M. FREHNER, F. KELLER, and W. WAGNER: Fructan metabolism, enzymology and compartmentation. In: Current Topics of Plant Biochemistry and Physiology, Vol. 5, pp. 17 -31, Univ. of Missouri, Columbia, MO (1986).