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Storage and Remobilization of Carbohydrates in Meadow Fescue (Festuca pratensis Huds.)
Storage and Remobilization of Carbohydrates in Meadow Fescue (Festuca pratensis Huds.) HANS PETER BUCHER, FELIX MXCHLER *).
and JOSEF
N6SBERGER
Institut fur Pflanzenwissenschaftcn, Eidgcnossische Technische Hochschule, UniversitatstraBe 2, CH-8092 Zurich, Switzerland
Received January 5,1987· Accepted February 2,1987
Summary Vegetative meadow fescue plants (Festuca pratensis Huds.) were steady state labelled with for one light period under standard conditions, as well as at decreased CO 2 partial pressure or at increased temperature. Storage and remobilization of labelled and unlabelled fruelao and starch in blades, swbbles, and roots was studied at the end of the light period and at the end of the subsequent dark period, respectively. The content of polysaccharides in the leaf blades was high under standard conditions. Polysaccharides were associated with high sucrose contents and showed low turnover activity. Diurnal fluctuation of carbohydrate content was due more [0 sucrose than to polysaccharides. However, polysaccharides were remobilized readily both at decreased CO 2 parcial pressure and at increased temperature. Remohilization of starch was inhibited at low temperature. Monosaccharides decreased during the light period and increa....ed during the dark period, due probahly to increa....ed remobilization of polysaccharides in the dark. The content of polysaccharides in the stubbles was lower than in the blades and associated with a low sucrose content. Polysaccharides showed higher mrnover activity than in the blades. Diurnal fluctuation of carbohydrate content was due more to polysaccharides than to sucrose; however, remohilization of polysaccharides at decreased CO 2 partial pressure or increased temperature occurred less readily than in the blades. Remobilization of starch was inhibited at low temperature. The monosaccharide content was not affected by the diurnal cycle, by decreased CO 2 partial pressure or by increased temperature. The content of polysaccharides and sucrose in the roots was low. Monosaccharides were the predominant carbohydrates. The monosaccharide content was not affected by the diurnal cycle or by decreased CO 2 panial pressure. However, some decrease in monosaccharides occurred at increased temperature. 14C0 2
Key words: Festuca pratensis, carbohydrates, !ructan, starch, remobilization, storage. Introduction In temperate C) grasses, most excessively produced carbohydrates are stored as fructans in the vacuoles of cells (Wagner et aI., 1983; Labhart et al., 1983; Pollock, 1986). However, starch can also be synthesized. The relative effects of photosynthate availability and photosynthate demand on fructan and starch metabolism in fructan storing grasses are not well understood. The diurnal cycle of storage and remobiliza-
tion in barley leaves was studied by Gordon et a1. (1980) and by Sicher et a1. (1984). In the present study, synthesis and degradation of fructans and starch in blades, stubbles,
*) To whom correspondence should be addressed. }. Plant PhyslOl. Vol. 130. pp. 101-109 (1987)
102
HANS PETER BucHEA, FELIX MxCHLll, and JOSEF N()S8£RCER
and roots of meadow fescue were investigated under standard conditions, as well as at decreased CO2 partial pressure and at increased temperature, respectively.
Materials and Methods Plants and growing conditions Vegetative plants of meadow fescue (Festuca pratens£s Huds., cv. Bundy) were grown from seed in pots filled with washed silica sand (Experiment I) or with perlite (Experiment II) and placed in a growth chamber (PGV-36, Conviron, Winnipeg, Canada). The light period was 16h with light being provided by a bank of fluorescent tubes and incandescent bulbs giving a photon irradiance of 4S0-500p.ffiol quanta m - 2s- 1 (400-700nm) at the soil surface. Half of this photon irradiance was made available during the first and the last hour of the light period. Plants were irrigated daily with nutrient solution (Hammer et aI., 1978). Day/night temperatures were either 15/10°C (Experiment I) or 16/11 °C (Experiment II). Relative humidity was 70/85°C (day/night). The CO2 partial pressure during growth ranged between 35 and 40Pa
P(CO,).
Experiment! Steady s
Extraction of carbohydrates After removing the ethanol extracts the plant pans were homogenized in a mortar. Water soluble carbohydrates were extracted by suspending the homogenate in 25cml water (60°C) and shaking periodically during 30 min. After centrifugation (45 min at 40.000 g) the pellet was resuspended in 10cml water (60°C), periodically shaken during 15min and centrifuged. The water extracts were combined with the ethanol extract and dried under reduced pressure (50°C). The residue was suspended in 0.5 N N.OH (5 em' ) and periodically shaken during 1 h at 60 °C. Then Scml of O.5N HCl were added and the suspension centrifuged for 45 min at 40,000 g. The supernatant was removed, the residue dried at 60 °C for 48 h and the dry weight determined. The dried ethanol-water extract was dissolved in 3 cm 3 water, mixed with 1 em 3 chloroform and centrifuged. The chloroform fraction was dried and the weight determined. The sum of dry weights of the chloroform extract and of the residue is termed .,residual dry weight» in the results section. The ethanol-water extract and the NaOH extract were used separately for the determination of carbohydrates.
J.
Plant Physiol. Vol. 130. pp. 101-109 (1987)
Carbohydrates in meadow fescue
103
A nalysis ofcarbohydrates The carbohydrates in the ethanol-water extract were separated by thin-Iayer-chromatography using silica gel foils (ready foils F 1500, Schleicher & Schull, Kassel, FRG). The foils were developed continuously for 18h with n-butanol/ethanol/water (52/32/16. v/v/v). Two aliquots per sample were chromatographed, one of which had been digested by amyloglucosidase (Aspergillus niger. Boehringer, Mannheim, FRG) prior to chromatography. Amyloglucosidase digestion occurred for 60 min at 50 °C, pH 4.6. The excess glucose produced by amyloglucosidase digestion was used as an estimate of the soluble starch content of the samples. After separation, carbohydrate fractions on the foils were localized by autoradiography (X-ray films Osray M3, Agfa-Gevaert, exposed for 20 days) and eluted in 4 em) water. The carbohydrate content in each fraction was determined by the anthrone method (Dimler et aI., 1952) with the reaction occurring for 15 min at 95°C. Fructose was used as standard. Radioactivity was determined by liquid scintillation couming. Carbohydrates in the NaOH extract were assumed to be starch (inel. hemicellulose) and the content and radioactivity determined without chromatography. Total starch was calculated from starch in the ethanol-water and the NaOH extract.
Experiment II Determination of water soluble carbohydrates with high degree 01 polymerization The 28-day-old plants were either transferred to 23/18 °C or left at 16/11 °C (day/night temperatures) in growth chambers and grown for an additional 10 days. Plants were harvested on different days between 10 and 11 a.m., dissected into leaf blades and stubbles, dried at 105°C for 1 h and then at 60°C for 47 h. The dried tissue was ground. Samples of 200 mg were extracted by shaking in 25 cm) water for 1 h at room temperature. Soluble proteins were precipitated by adding O.S em) of 10 % lead acetate. The solution was filtered through Schleicher & SchUll paper (No. 590). Water was added to the filtrate to 50cm 3. An aliquot (1.2cm 3) was layered OntO a 1.6 x l00cm chromatography column filled with Sephadex G-50 fine gel. The carbohydrates were eluted with 0.2N NaCl at a flow rate of 20cm 3 h - I • The volume of the gel (V(- V o) was 108 cm 3 and was sampled in 27 fractions. K:2Cr04 and dextrans of known molecular weights were used ro calibrate the column. The carbohydrate content of the fractions was determined using the anthrone method.
Results and Discussion Contents and turnover of carbohydrates under standard conditions Plants grown at 38 Pa P(CO,) and 15/10 °C day/night temperatures had highest carbohydrate content in the leaf blades. lower content in the stubbles, and lowest content in the roots (Table 1). Fructans were the predominant storage polysac-
Table 1: Carbohydrate content in leaf blades, stubbles, and roots at the beginning of the light period under standard conditions (38 Pa p{C02 ), 15/10 °C day/night temperatures). Single determinations are shown. Experiment 1.
Blades Stubbles Roots
Monosaccharides Sucrose J.Lg/mg residual dry weight
Fructan
Starch
98 127 137 154 128
655 555 374 375
138 195 62 90 14 21
141
151 120 42 27 24 13
42
26
j. Planl Pbysiol. Vol. 130. pp. 101-109 (1987)
104
HANS PETER BUCHER, FEUX MACHLER,
and JOSEF
N6SBERGER
Table 2: Percentage of 14C labelled carbohydrates in various carbohydrate pools after one day/ night cycle in the presence of 14C02 under standard conditions. Single determinations are shown. Experiment I. ------~--~~----------------~
Monosaccharides %
Blades Stubble, Roots
26 24 40 45 33 37
Sucrose %
32 33
50 52 19
25
Fructan %
8 9
15 20 19
33
Starch %
9 7
22
22 40 41
chari des and contributed 78, 83, and 66 % to the polysaccharide fraction in the leaf blades, stubbles, and roots, respectively. The polysaccharides were associated with much higher sucrose content in the leaf blades than in the stubbles. Low content of polysaccharides and of sucrose were found in the roots. In contrast, monosaccharide content was similar in all plant pans. Monosaccharides were the predominant carbohydrates in the roots. The apparent turnover rates of the pools of each class of carbohydrates were es-
timated as percentages of labelled carbohydrates after one day/night cycle in the pre.. ence of "CO, (Table2). Percentages in leaf blades were low for all carbohydrate classes suggesting low turnover rates. In contrast, turnover of the carbohydrate pools in the stubbles and roots was higher with the exception of root sucrose which turned over slowly, possibly due to sucrose accumulated in old tissues with little metabolic activity.
Storage and remobilization of carbohydrates during one day/night cycle The diurnal cycle of storage and remobilization of the various carbohydrate classes
in the leaf blades, stubbles, and roots was studied under standard conditions, at decreased CO 2 partial pressure which brought about decreased assimilate supply, and at increased temperature causing an increase in assimjlate demand.
a) Sucrose Sucrose in the leaf blades increased during the light period and decreased during the following dark period. This diurnal fluctuation was due to the newly fixed "C labelled component, whereas the unlabelled component decreased throughout the cyde. This diurnal fluctuation in the leaf blades was associated with a smaller fluctua-
tion in the stubbles. No fluctuation of sucrose occurred in the roots (Fig. 1). A decrease in CO 2 partial pressure and an increase in temperature both resulted in decreased sucrose content in leaf blades, stubbles, and roots. The results show that the content of sucrose in the various plant pans depends strongly on the supply of assimilates due to photosynthesis as well as on their consumption due to growth processes. Diurnal fluctuation of sucrose has also been found in leaves of barley {Gordon et aI., /. Plant PbysioL VoL 130. pp. 101-109 (1987)
Carbohydrates in meadow fescue
105
Sucrose
Monosaccharides
Stubbles
rs::a
C
B
A
e-
!
Roots
C
B
A
C
2
C?a-
Time
Fig. t: Content of unlabelled (unshaded) and labelled (shaded) sucrose and monosaccharides in leaf blades, stubbles, and roots per plant at the end of one light period with !iteady state 14C02 photosynthesis and at the end of the subsequent dark period, (A) under standard conditions (38 Pa P(CO,). IS/10°C day/night). (B) at decreased CO, panial pressure (20Pa P(CO,). 15/ 10 °C) and (C) at increased temperature (38 Pa P(C02), 23/18 Oe). Vertical bars indicate standard errors for labelled and unlabelled carbohydrate fractions. The unshaded and shaded areas under the time axis represent light and dark periods. Experiment I.
1982; Sic her et a!.. 1984; Farrar and Farrar. 1985) and of Poa species (Borland and Farrar, 1985), and appears to be a characteristic of fructan storing species. This is in contrast to species with preferential storage of starch, like sugar beet (Fondy and Geiger, 1982). soybean (Chatterton and Silvius. 1979) and white clover (Scheidegger and Nosberger, 1984), where leaf sucrose content is low and diurnal fluctuation of carbohydrate content is mainly due to fluctuation of starch.
b) Monosaccharides The fluctuation of monosaccharides in the leaf blades was in contrast to the fluctuation of sucrose in that the monosaccharides decreased during the light period and increased during the dark period. The increase of monosaccharides during the dark period was due to the unlabelled component, suggesti ng that it resulted from polysaccharide degradation. the synthesis of which had occurred during previous light periods. The monosaccharide content in the leaf blades and stubbles was not much affected by a decrease in assimilate supply due lO decreased CO, panial pressure and by an increase in assimi late demand due to increased temperature. However, the content in the roots was decreased at increased temperature. In the roots, monosaccharides
J
Plant Pbysiol. Vol. 130. pp. 101 - 109(1987)
106
HANs PETER BUCHEJt., FELIX MXCHUR, and JOSEF N6SBERGER
-
Nuclan
Starch BIodes
A
c
B
160
I 80
40
Stubbles
Stubbles A
B
c
mg
I
40
4O[ o rl-:=I"
Roots
Roots
t::--J Time
'ii
Fig.2, Content of unlabelled (unshaded) and labelled (shaded) fructan and starch in leaf blades, stubbles, and roots per plant at the end of one light period with steady state 14C02 photosynthesis and at the end of the subsequent dark period. For details see Fig. 1. Experiment I.
were the predominant carbohydrates and were obviously needed to satisfy the increased assimilate demand at increased temperature.
c) Fructan and starch Under standard conditions, fructan and starch in the leaf blades and stubbles increased during the light period and decreased during the dark period (Fig. 2). Fruetan
and starch in the leaf blades were associated with high sucrose content and showed sma11er changes than sucrose. In contrast, fmetan and starch in the stubbles were associated with low sucrose content and showed greater diurnal changes than sucrose.
f. Plant Plrysiol. Vol. 130. pp. 101-109(1981)
Carbohydrates in meadow fescue
100
3 "en ~
LEAF BlADES
107
STUBBlES
80 60 40 20
15
25
5
VI
15
25 VI
Fraction NUf'1'lber
Fig.3; Effect of an increase in temperature on the content of water soluble carbohydrates (WSC) with high degree of polymerization in the leaf blades and stubbles. The plants were analyzed when they were 28 days old (e). Some of the 28-ciay..ald plants were transferred from 16/ 11 °C to 23/18 °C day/night temperatures and analyzed after 4 (0) and 10 days (6). Some of the plants were kept at 16/11 °C for a further 10 days and then analyzed (.&). Experiment II.
Roots showed some diurnal fluctuation in starch but no fluctuation in fructan. Decreased assimilate supply due to decreased CO2 partial pressure and increased assimilate demand due to increased temperature both resulted in remobilization of fruetan in the leaf blades. The content decreased during the light period, indicating that products of recent photosynthesis were mostly exported and that degradation and export of photoassimilates from earlier photosynthesis occurred at the same time. The decrease during the dark period was more rapid than under standard conditions. Fructan in the stubbles increased during the light period and decreased during the dark period. The increase at low CO2 partial pressure was smaller and the increase at high temperature was greater than under standard conditions. Fructan synthesis in the stubbles was obviously a sink for the carbohydrates from the leaf blades; during the light period, sink activity increased as temperature was increased. Stubbles contain leaf sheaths of mature leaves as well as growing bases of young leaves. The respective sink activities of these stubble components have been discussed elsewhere (Bucher et aI., 1987). Fructan in the roots was degraded considerably at decreased CO 2 partial pressure but only slightly at increased temperature. Strong remobilization of starch in the leaf blades and some remobilization of starch in the stubbles occurred at increased temperature. In contrast, no considerable rernabilization of starch was induced by a decrease in CO2 partial pressure, probably due to the relatively low temperature; The starch content in the blades decreased slightly during the light period and remained constant during the dark period, whereas the starch content in the stubbles did not decrease at low CO 2 • Starch in the roots was af-
J. Plant Pry,iol. Vol. 130. pp. 101-109 (1987)
108
fuNS PEl"D
BucHE.ll, FElIX MXcH1..U , and JOSEF NOSBERGER
feeted neither by an increase in temperature nor by a decrease in CO2 partial pressure. The results suggest that starch remobilization was inhibited at low temperature whereas fructan remobilization was not. A similar difference between starch and fructan was found with regard to synthesis. Starch synthesis decreased more than fructan synthesis as temperature was decreased in barley leaves (Sicher and Kremer, 1986) and in meadow fescue (Bucher et aI., 1987). Fructan storage appears, therefore, to be bener adapted to cold temperature than starch storage and seems to be predominant in plants of cold climates. Nevertheless, some starch was remobilized in the leaf blades during the light period at decreased CO, partial pressure despite low temperature. It is possible that this remobilization of starch in the light was needed to maintain high carbon fluxes through photosynthetic carbon reduction and oxidation cycles despite decreased ambient CO 2 partial pressure. Such remobilization processes have been found in illuminated drought stressed plants (Becker et aI., 1986).
Storage and remobilization of carbobydrates (long term) The effect of a long term increase in temperature on the content of water soluble carbohydrates in leaf blades and stubbles was studied in an additional experiment (ExperimentII). Four-week-old plants were either kept at 16/11 °C (day/night) and analyzed after 10 days or transferred to 23/18 °c(day/night) and analyzed after 4 and 10 days, respectively (Fig.3). Leaf blades and stubbles showed increasing content of carbohydrates with a high degree of polymerization when plants were kept at 16/ 11 °C for 10 more days. A considerable decrease in fructan content occurred in leaf blades when plants were transferred to 23/ 18 In contrast, the carbohydrate content of the stubbles was maintained when plants were exposed to the higher temperature . Remobilization of carbohydrates in stubbles appeared to be compensated for by new carbohydrates imported from the leaf blades. Polysaccharide content in the stubbles was therefore little affected by increased temperature in the long term. The degree of polymerization of fructans stored in leaf blades and stubbles of meadow fescue was about 90 units. No storage of shorter chains appeared to occur. The content of fructans with a degree of polymerization of about 20 units was always very low. Shoner chains were eluted together with disaccharides and monosaccharides. There appeared to be no relation between their content and those of long chain fructans.
0c.
Conclusions The results showed different characteristics of storage and remobilization for the various carbohydrate classes in the leaf blades, stubbles, and roots of meadow fescue. In the leaf blades, short term storage of carbohydrates occurred as sucrose, long term storage as fructan and starch. Carbohydrate storage in the stubbles occurred as fructan and starch, but not as sucrose. Only little fructan, starch, and sucrose were found in the roots. An increase in the ratio of assimilate demand to assimilate supply resulted in a ready decrease in carbohydrate content in the blades. In contrast, carbohydrates in the stubbles did not decrease so readily despite a high turnover rate. Re-
J. Plant Pbysiol. Vol.
130. pp. 101-109(1987)
Carbohydrates in meadow fescue
109
mobilization of starch in blades and stubbles was inhibited at low temperature, in contrast to remobilization of fructan. The results suggest that storage of carbohydrates in the stubbles was a sink with respect to the leaf blades. Acknowledgements We thank Ms. A. Allenbach for growing the plants, Dr. H. Schnyder for helpful discussions and Ms. M. Schonberg for checking the English translation. This work was supported by the Swiss National Science Foundation.
References BECKER, T. W., M_ HOPPE, and H. P. FOCK: Evidence for the participation of dissimilatory processes in maintaining high carbon fluxes through the photosynthetic carbon reduction and oxidation cycles in water stressed Phaseolus leaves. Photosynthetica 20,153-157 (1986). BORLAND, A. M. and]. F. FARRAR: Diel patterns of carbohydrate metabolism in leaf blades and leaf sheaths of Poa annua Land Poa x jemtlandica (Almq.) Rieht., New Phytol. lOa, 519-S31 (1985). BUCHER, H. P., F. MACHLER, and J. N6SBERGER; Sink control of assimilate partitioning in meadow fescue (Festuca prater/sis Huds). J. Plant PhysioJ. 129,469-477 (1987). CHATIERTON, N. J. and J. E. SILVIUS: Photosynthate partitioning into starch in soybean leaves. I. Effects of photoperiod versus photosymhetic period duration. Plant Physio!. 64, 749-753 (1979). DIMLER, R. J., W. C. SCHAEfER, C. S. WISE, and C. E. RlST: Quantitative paper chromatography of D-glucose and its oligosaccharides. Anal. Chern. 24, 1411-1414 (1952). FAK.ltAR, S. C. and J. F. FARRAR: Carbon fluxes in leaf blades of barley. New Phytol. lOa, 271-283 (1985). fONDY, B. R. and D. R. GEIGER: Diurnal pattern of translocation and carbohydrate metabolism in source leaves of Beta vulgaris L. Plant Physiol. 70, 671-676 (1982). GORDON, A. J., G.]. A. RYLE, and G. WEBB: The relationship between sucrose and starch during «dark» export from leaves of uniculm barley. J. Exp. Bot_ 31,845-850 (1980). GORDON, A. J., G. J. A_ RvLF., D. F. MITCHELL, and C. E. POWELL: The dynamics of carbon supply from leaves of barley plams grown in long or short days. J. Exp. Bot. 33, 241- 250 (1982). H AMMlOR, P. A., T. W. TIBBITTS, R. W. LANGHANS, and J. C. McFARLANE: Base-line growth studies of ... grand rapids» lettuce in controlled environments. J. Amer. Soc. Hort. Sci. 103, 649-655 (1978). LABHART, CH., J. NOSBERGER, and C. J. NELSON: Photosynthesis and degree of polymerization of fruetan during reproductive growth of meadow fescue at two temperatures and two photon flux densities. J. Exp. Bot. 34, 1037 -1046 (1983). POLLOCK, C. J.: fructans and the metabolism of sucrose in vascular plants. New Phytol. 104, 1-24 (1986). SCHEIDEGGER, U. C. and J. N6sBERGER.: Influence of carbon dioxide concentration on growth, carbohydrate content, translocation and photosynthesis of white clover. Ann. Bot. 54, 735-742 (1984). SrcHF.R, R. C, D. F. KREMER, and W. G. HARRIS: Diurnal carbohydrate metabolism of barlt!y primary leaves. Plant Physio!. 76, 165-169 (1984). 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). WAGNER, W., F. KELLER, and A. WrEMKEN: Fructan metabolism in cereals: Induction in leaves and compartmentation in protoplasts and vacuoles. j. Plant Physiol. 112, 359-372 (1983).
J.
Plant Pbysiol. Vol. 130.
pp.
101-109 (1987)
and JOSEF
N6SBERGER
Institut fur Pflanzenwissenschaftcn, Eidgcnossische Technische Hochschule, UniversitatstraBe 2, CH-8092 Zurich, Switzerland
Received January 5,1987· Accepted February 2,1987
Summary Vegetative meadow fescue plants (Festuca pratensis Huds.) were steady state labelled with for one light period under standard conditions, as well as at decreased CO 2 partial pressure or at increased temperature. Storage and remobilization of labelled and unlabelled fruelao and starch in blades, swbbles, and roots was studied at the end of the light period and at the end of the subsequent dark period, respectively. The content of polysaccharides in the leaf blades was high under standard conditions. Polysaccharides were associated with high sucrose contents and showed low turnover activity. Diurnal fluctuation of carbohydrate content was due more [0 sucrose than to polysaccharides. However, polysaccharides were remobilized readily both at decreased CO 2 parcial pressure and at increased temperature. Remohilization of starch was inhibited at low temperature. Monosaccharides decreased during the light period and increa....ed during the dark period, due probahly to increa....ed remobilization of polysaccharides in the dark. The content of polysaccharides in the stubbles was lower than in the blades and associated with a low sucrose content. Polysaccharides showed higher mrnover activity than in the blades. Diurnal fluctuation of carbohydrate content was due more to polysaccharides than to sucrose; however, remohilization of polysaccharides at decreased CO 2 partial pressure or increased temperature occurred less readily than in the blades. Remobilization of starch was inhibited at low temperature. The monosaccharide content was not affected by the diurnal cycle, by decreased CO 2 partial pressure or by increased temperature. The content of polysaccharides and sucrose in the roots was low. Monosaccharides were the predominant carbohydrates. The monosaccharide content was not affected by the diurnal cycle or by decreased CO 2 panial pressure. However, some decrease in monosaccharides occurred at increased temperature. 14C0 2
Key words: Festuca pratensis, carbohydrates, !ructan, starch, remobilization, storage. Introduction In temperate C) grasses, most excessively produced carbohydrates are stored as fructans in the vacuoles of cells (Wagner et aI., 1983; Labhart et al., 1983; Pollock, 1986). However, starch can also be synthesized. The relative effects of photosynthate availability and photosynthate demand on fructan and starch metabolism in fructan storing grasses are not well understood. The diurnal cycle of storage and remobiliza-
tion in barley leaves was studied by Gordon et a1. (1980) and by Sicher et a1. (1984). In the present study, synthesis and degradation of fructans and starch in blades, stubbles,
*) To whom correspondence should be addressed. }. Plant PhyslOl. Vol. 130. pp. 101-109 (1987)
102
HANS PETER BucHEA, FELIX MxCHLll, and JOSEF N()S8£RCER
and roots of meadow fescue were investigated under standard conditions, as well as at decreased CO2 partial pressure and at increased temperature, respectively.
Materials and Methods Plants and growing conditions Vegetative plants of meadow fescue (Festuca pratens£s Huds., cv. Bundy) were grown from seed in pots filled with washed silica sand (Experiment I) or with perlite (Experiment II) and placed in a growth chamber (PGV-36, Conviron, Winnipeg, Canada). The light period was 16h with light being provided by a bank of fluorescent tubes and incandescent bulbs giving a photon irradiance of 4S0-500p.ffiol quanta m - 2s- 1 (400-700nm) at the soil surface. Half of this photon irradiance was made available during the first and the last hour of the light period. Plants were irrigated daily with nutrient solution (Hammer et aI., 1978). Day/night temperatures were either 15/10°C (Experiment I) or 16/11 °C (Experiment II). Relative humidity was 70/85°C (day/night). The CO2 partial pressure during growth ranged between 35 and 40Pa
P(CO,).
Experiment! Steady s
Extraction of carbohydrates After removing the ethanol extracts the plant pans were homogenized in a mortar. Water soluble carbohydrates were extracted by suspending the homogenate in 25cml water (60°C) and shaking periodically during 30 min. After centrifugation (45 min at 40.000 g) the pellet was resuspended in 10cml water (60°C), periodically shaken during 15min and centrifuged. The water extracts were combined with the ethanol extract and dried under reduced pressure (50°C). The residue was suspended in 0.5 N N.OH (5 em' ) and periodically shaken during 1 h at 60 °C. Then Scml of O.5N HCl were added and the suspension centrifuged for 45 min at 40,000 g. The supernatant was removed, the residue dried at 60 °C for 48 h and the dry weight determined. The dried ethanol-water extract was dissolved in 3 cm 3 water, mixed with 1 em 3 chloroform and centrifuged. The chloroform fraction was dried and the weight determined. The sum of dry weights of the chloroform extract and of the residue is termed .,residual dry weight» in the results section. The ethanol-water extract and the NaOH extract were used separately for the determination of carbohydrates.
J.
Plant Physiol. Vol. 130. pp. 101-109 (1987)
Carbohydrates in meadow fescue
103
A nalysis ofcarbohydrates The carbohydrates in the ethanol-water extract were separated by thin-Iayer-chromatography using silica gel foils (ready foils F 1500, Schleicher & Schull, Kassel, FRG). The foils were developed continuously for 18h with n-butanol/ethanol/water (52/32/16. v/v/v). Two aliquots per sample were chromatographed, one of which had been digested by amyloglucosidase (Aspergillus niger. Boehringer, Mannheim, FRG) prior to chromatography. Amyloglucosidase digestion occurred for 60 min at 50 °C, pH 4.6. The excess glucose produced by amyloglucosidase digestion was used as an estimate of the soluble starch content of the samples. After separation, carbohydrate fractions on the foils were localized by autoradiography (X-ray films Osray M3, Agfa-Gevaert, exposed for 20 days) and eluted in 4 em) water. The carbohydrate content in each fraction was determined by the anthrone method (Dimler et aI., 1952) with the reaction occurring for 15 min at 95°C. Fructose was used as standard. Radioactivity was determined by liquid scintillation couming. Carbohydrates in the NaOH extract were assumed to be starch (inel. hemicellulose) and the content and radioactivity determined without chromatography. Total starch was calculated from starch in the ethanol-water and the NaOH extract.
Experiment II Determination of water soluble carbohydrates with high degree 01 polymerization The 28-day-old plants were either transferred to 23/18 °C or left at 16/11 °C (day/night temperatures) in growth chambers and grown for an additional 10 days. Plants were harvested on different days between 10 and 11 a.m., dissected into leaf blades and stubbles, dried at 105°C for 1 h and then at 60°C for 47 h. The dried tissue was ground. Samples of 200 mg were extracted by shaking in 25 cm) water for 1 h at room temperature. Soluble proteins were precipitated by adding O.S em) of 10 % lead acetate. The solution was filtered through Schleicher & SchUll paper (No. 590). Water was added to the filtrate to 50cm 3. An aliquot (1.2cm 3) was layered OntO a 1.6 x l00cm chromatography column filled with Sephadex G-50 fine gel. The carbohydrates were eluted with 0.2N NaCl at a flow rate of 20cm 3 h - I • The volume of the gel (V(- V o) was 108 cm 3 and was sampled in 27 fractions. K:2Cr04 and dextrans of known molecular weights were used ro calibrate the column. The carbohydrate content of the fractions was determined using the anthrone method.
Results and Discussion Contents and turnover of carbohydrates under standard conditions Plants grown at 38 Pa P(CO,) and 15/10 °C day/night temperatures had highest carbohydrate content in the leaf blades. lower content in the stubbles, and lowest content in the roots (Table 1). Fructans were the predominant storage polysac-
Table 1: Carbohydrate content in leaf blades, stubbles, and roots at the beginning of the light period under standard conditions (38 Pa p{C02 ), 15/10 °C day/night temperatures). Single determinations are shown. Experiment 1.
Blades Stubbles Roots
Monosaccharides Sucrose J.Lg/mg residual dry weight
Fructan
Starch
98 127 137 154 128
655 555 374 375
138 195 62 90 14 21
141
151 120 42 27 24 13
42
26
j. Planl Pbysiol. Vol. 130. pp. 101-109 (1987)
104
HANS PETER BUCHER, FEUX MACHLER,
and JOSEF
N6SBERGER
Table 2: Percentage of 14C labelled carbohydrates in various carbohydrate pools after one day/ night cycle in the presence of 14C02 under standard conditions. Single determinations are shown. Experiment I. ------~--~~----------------~
Monosaccharides %
Blades Stubble, Roots
26 24 40 45 33 37
Sucrose %
32 33
50 52 19
25
Fructan %
8 9
15 20 19
33
Starch %
9 7
22
22 40 41
chari des and contributed 78, 83, and 66 % to the polysaccharide fraction in the leaf blades, stubbles, and roots, respectively. The polysaccharides were associated with much higher sucrose content in the leaf blades than in the stubbles. Low content of polysaccharides and of sucrose were found in the roots. In contrast, monosaccharide content was similar in all plant pans. Monosaccharides were the predominant carbohydrates in the roots. The apparent turnover rates of the pools of each class of carbohydrates were es-
timated as percentages of labelled carbohydrates after one day/night cycle in the pre.. ence of "CO, (Table2). Percentages in leaf blades were low for all carbohydrate classes suggesting low turnover rates. In contrast, turnover of the carbohydrate pools in the stubbles and roots was higher with the exception of root sucrose which turned over slowly, possibly due to sucrose accumulated in old tissues with little metabolic activity.
Storage and remobilization of carbohydrates during one day/night cycle The diurnal cycle of storage and remobilization of the various carbohydrate classes
in the leaf blades, stubbles, and roots was studied under standard conditions, at decreased CO 2 partial pressure which brought about decreased assimilate supply, and at increased temperature causing an increase in assimjlate demand.
a) Sucrose Sucrose in the leaf blades increased during the light period and decreased during the following dark period. This diurnal fluctuation was due to the newly fixed "C labelled component, whereas the unlabelled component decreased throughout the cyde. This diurnal fluctuation in the leaf blades was associated with a smaller fluctua-
tion in the stubbles. No fluctuation of sucrose occurred in the roots (Fig. 1). A decrease in CO 2 partial pressure and an increase in temperature both resulted in decreased sucrose content in leaf blades, stubbles, and roots. The results show that the content of sucrose in the various plant pans depends strongly on the supply of assimilates due to photosynthesis as well as on their consumption due to growth processes. Diurnal fluctuation of sucrose has also been found in leaves of barley {Gordon et aI., /. Plant PbysioL VoL 130. pp. 101-109 (1987)
Carbohydrates in meadow fescue
105
Sucrose
Monosaccharides
Stubbles
rs::a
C
B
A
e-
!
Roots
C
B
A
C
2
C?a-
Time
Fig. t: Content of unlabelled (unshaded) and labelled (shaded) sucrose and monosaccharides in leaf blades, stubbles, and roots per plant at the end of one light period with !iteady state 14C02 photosynthesis and at the end of the subsequent dark period, (A) under standard conditions (38 Pa P(CO,). IS/10°C day/night). (B) at decreased CO, panial pressure (20Pa P(CO,). 15/ 10 °C) and (C) at increased temperature (38 Pa P(C02), 23/18 Oe). Vertical bars indicate standard errors for labelled and unlabelled carbohydrate fractions. The unshaded and shaded areas under the time axis represent light and dark periods. Experiment I.
1982; Sic her et a!.. 1984; Farrar and Farrar. 1985) and of Poa species (Borland and Farrar, 1985), and appears to be a characteristic of fructan storing species. This is in contrast to species with preferential storage of starch, like sugar beet (Fondy and Geiger, 1982). soybean (Chatterton and Silvius. 1979) and white clover (Scheidegger and Nosberger, 1984), where leaf sucrose content is low and diurnal fluctuation of carbohydrate content is mainly due to fluctuation of starch.
b) Monosaccharides The fluctuation of monosaccharides in the leaf blades was in contrast to the fluctuation of sucrose in that the monosaccharides decreased during the light period and increased during the dark period. The increase of monosaccharides during the dark period was due to the unlabelled component, suggesti ng that it resulted from polysaccharide degradation. the synthesis of which had occurred during previous light periods. The monosaccharide content in the leaf blades and stubbles was not much affected by a decrease in assimilate supply due lO decreased CO, panial pressure and by an increase in assimi late demand due to increased temperature. However, the content in the roots was decreased at increased temperature. In the roots, monosaccharides
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Plant Pbysiol. Vol. 130. pp. 101 - 109(1987)
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HANs PETER BUCHEJt., FELIX MXCHUR, and JOSEF N6SBERGER
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Nuclan
Starch BIodes
A
c
B
160
I 80
40
Stubbles
Stubbles A
B
c
mg
I
40
4O[ o rl-:=I"
Roots
Roots
t::--J Time
'ii
Fig.2, Content of unlabelled (unshaded) and labelled (shaded) fructan and starch in leaf blades, stubbles, and roots per plant at the end of one light period with steady state 14C02 photosynthesis and at the end of the subsequent dark period. For details see Fig. 1. Experiment I.
were the predominant carbohydrates and were obviously needed to satisfy the increased assimilate demand at increased temperature.
c) Fructan and starch Under standard conditions, fructan and starch in the leaf blades and stubbles increased during the light period and decreased during the dark period (Fig. 2). Fruetan
and starch in the leaf blades were associated with high sucrose content and showed sma11er changes than sucrose. In contrast, fmetan and starch in the stubbles were associated with low sucrose content and showed greater diurnal changes than sucrose.
f. Plant Plrysiol. Vol. 130. pp. 101-109(1981)
Carbohydrates in meadow fescue
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3 "en ~
LEAF BlADES
107
STUBBlES
80 60 40 20
15
25
5
VI
15
25 VI
Fraction NUf'1'lber
Fig.3; Effect of an increase in temperature on the content of water soluble carbohydrates (WSC) with high degree of polymerization in the leaf blades and stubbles. The plants were analyzed when they were 28 days old (e). Some of the 28-ciay..ald plants were transferred from 16/ 11 °C to 23/18 °C day/night temperatures and analyzed after 4 (0) and 10 days (6). Some of the plants were kept at 16/11 °C for a further 10 days and then analyzed (.&). Experiment II.
Roots showed some diurnal fluctuation in starch but no fluctuation in fructan. Decreased assimilate supply due to decreased CO2 partial pressure and increased assimilate demand due to increased temperature both resulted in remobilization of fruetan in the leaf blades. The content decreased during the light period, indicating that products of recent photosynthesis were mostly exported and that degradation and export of photoassimilates from earlier photosynthesis occurred at the same time. The decrease during the dark period was more rapid than under standard conditions. Fructan in the stubbles increased during the light period and decreased during the dark period. The increase at low CO2 partial pressure was smaller and the increase at high temperature was greater than under standard conditions. Fructan synthesis in the stubbles was obviously a sink for the carbohydrates from the leaf blades; during the light period, sink activity increased as temperature was increased. Stubbles contain leaf sheaths of mature leaves as well as growing bases of young leaves. The respective sink activities of these stubble components have been discussed elsewhere (Bucher et aI., 1987). Fructan in the roots was degraded considerably at decreased CO 2 partial pressure but only slightly at increased temperature. Strong remobilization of starch in the leaf blades and some remobilization of starch in the stubbles occurred at increased temperature. In contrast, no considerable rernabilization of starch was induced by a decrease in CO2 partial pressure, probably due to the relatively low temperature; The starch content in the blades decreased slightly during the light period and remained constant during the dark period, whereas the starch content in the stubbles did not decrease at low CO 2 • Starch in the roots was af-
J. Plant Pry,iol. Vol. 130. pp. 101-109 (1987)
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fuNS PEl"D
BucHE.ll, FElIX MXcH1..U , and JOSEF NOSBERGER
feeted neither by an increase in temperature nor by a decrease in CO2 partial pressure. The results suggest that starch remobilization was inhibited at low temperature whereas fructan remobilization was not. A similar difference between starch and fructan was found with regard to synthesis. Starch synthesis decreased more than fructan synthesis as temperature was decreased in barley leaves (Sicher and Kremer, 1986) and in meadow fescue (Bucher et aI., 1987). Fructan storage appears, therefore, to be bener adapted to cold temperature than starch storage and seems to be predominant in plants of cold climates. Nevertheless, some starch was remobilized in the leaf blades during the light period at decreased CO, partial pressure despite low temperature. It is possible that this remobilization of starch in the light was needed to maintain high carbon fluxes through photosynthetic carbon reduction and oxidation cycles despite decreased ambient CO 2 partial pressure. Such remobilization processes have been found in illuminated drought stressed plants (Becker et aI., 1986).
Storage and remobilization of carbobydrates (long term) The effect of a long term increase in temperature on the content of water soluble carbohydrates in leaf blades and stubbles was studied in an additional experiment (ExperimentII). Four-week-old plants were either kept at 16/11 °C (day/night) and analyzed after 10 days or transferred to 23/18 °c(day/night) and analyzed after 4 and 10 days, respectively (Fig.3). Leaf blades and stubbles showed increasing content of carbohydrates with a high degree of polymerization when plants were kept at 16/ 11 °C for 10 more days. A considerable decrease in fructan content occurred in leaf blades when plants were transferred to 23/ 18 In contrast, the carbohydrate content of the stubbles was maintained when plants were exposed to the higher temperature . Remobilization of carbohydrates in stubbles appeared to be compensated for by new carbohydrates imported from the leaf blades. Polysaccharide content in the stubbles was therefore little affected by increased temperature in the long term. The degree of polymerization of fructans stored in leaf blades and stubbles of meadow fescue was about 90 units. No storage of shorter chains appeared to occur. The content of fructans with a degree of polymerization of about 20 units was always very low. Shoner chains were eluted together with disaccharides and monosaccharides. There appeared to be no relation between their content and those of long chain fructans.
0c.
Conclusions The results showed different characteristics of storage and remobilization for the various carbohydrate classes in the leaf blades, stubbles, and roots of meadow fescue. In the leaf blades, short term storage of carbohydrates occurred as sucrose, long term storage as fructan and starch. Carbohydrate storage in the stubbles occurred as fructan and starch, but not as sucrose. Only little fructan, starch, and sucrose were found in the roots. An increase in the ratio of assimilate demand to assimilate supply resulted in a ready decrease in carbohydrate content in the blades. In contrast, carbohydrates in the stubbles did not decrease so readily despite a high turnover rate. Re-
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Carbohydrates in meadow fescue
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mobilization of starch in blades and stubbles was inhibited at low temperature, in contrast to remobilization of fructan. The results suggest that storage of carbohydrates in the stubbles was a sink with respect to the leaf blades. Acknowledgements We thank Ms. A. Allenbach for growing the plants, Dr. H. Schnyder for helpful discussions and Ms. M. Schonberg for checking the English translation. This work was supported by the Swiss National Science Foundation.
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