- Email: [email protected]
The Influence of Altered Gravity on Carbohydrate Metabolism in Excised Wheat Leaves
J. Plant Physiol. Vol. 144. pp. 696-699 (1994)
The Influence of Altered Gravity on Carbohydrate Metabolism in Excised Wheat Leaves 1 DAVID
M.
OBENLAND 2 and CHRISTOPHER
S. BROWN
Plant Space Biology Laboratory, The Bionetics Corporation, Mail Code BIO-3, Kennedy Space Center, Florida 32899, USA Received February 1, 1994 . Accepted April 15, 1994
Summary
We developed a system to study the influence of altered gravity on carbohydrate metabolism in excised wheat leaves by means of clinorotation. The use of excised leaves in our clinostat studies offered a number of advantages over the use of whole plants, most important of which were minimization of exogenous mechanical stress and a greater amount of carbohydrate accumulation during the time of treatment. We found that horizontal clinorotation of excised wheat leaves resulted in significant reductions in the accumulation of fructose, sucrose, starch and fructan relative to control, vertically clinorotated leaves. Photosynthesis, dark respiration and the extractable activities of ADP glucose pyrophosphorylase (EC 2.7.7.27), sucrose phosphate synthase (EC 2.4.4.14), sucrose sucrose fructosyltransferase (EC 2.4.1.99), and fructan hydrolase (EC 3.2.1.80) were unchanged due to altered gravity treatment. Key words: Triticum aestivum, carbohydrate, clinostat, dark respiration, enzyme, photosynthesis. Abbreviations: AGP = ADP glucose pyrophosphorylase; SPS = sucrose phosphate synthase; SST = sucrose sucrose fructosyltransferase; FH = fructan hydrolase.
Introduction
One consistent observation regarding the metabolism of plants grown in the micro gravity of space is that they accumulate less starch than ground-based controls (Abilov et aI., 1986; Johnson and Tibbitts, 1968; Moore et aI., 1987). In accordance with this result, we found that germinating, dark-grown soybean cotyledons have reduced starch content when subjected to altered gravity as imposed by clinorotation (Brown and Piastuch). In the current study we utilize an excised leaf system to study the effect of altered gravity on carbohydrate metabolism in light-grown wheat leaves. This system offers us five significant advantages over whole-plant systems: (i) exogenous mechanical stresses on plant tissues 1 Work done by D.M.O. while on a National Research Council Fellowship at the Kennedy Space Center, FL, U.S.A. 2 Present address and correspondence: Horticultural Crops Research Laboratory, USDA/ ARS, 2021 S. Peach Avenue, Fresno, CA, 93727, U.S.A.
© 1994 by Gustav Fischer Vedag, Stuttgart
due to clinorotation are greatly minimized due to the fact that the wheat leaves are fixed to the clinostat and little or no extraneous movement occurs; (ii) an excised, illuminated leaf accumulates large quantities of carbohydrates (Wagner et aI., 1986), making it possible to quantify fructan, a very important storage carbohydrate in the grasses that would normally be present in only very small amounts in attached, primary leaves; (iii) the accumulation of carbohydrate that occurs is known in the case of sucrose (Salerno et aI., 1989) and fructan (Obenland et aI., 1991) to depend on a rapid de novo synthesis of the biosynthetic enzyme systems for the particular carbohydrate, making it a good system to observe the effect of altered gravity on protein synthesis; (iv) it is very easy to introduce compounds via the transpiration stream into an excised leaf to aid in metabolic studies; and (v) we have found the set-up of the experiment to be easier and the time needed to complete the experiment less than for whole plants. In this paper we present a clinorotation experiment using excised wheat leaves. We show sucrose, starch and fructan to
The influence of altered gravity on Carbohydrate Metabolism
697
Small Test Tube Excised Leaf
Elastic Support
Fig. 1: Schematic diagram of clinostat used to impose omnilateral gravity stimulation on excised wheat leaves.
be significantly reduced in response to altered gravity treatment. Several possible mechanisms for the changes are discussed. Materials and Methods
Clinostat treatments Wheat (Triticum aestivum cv. Yecora rojo) plants were grown for 7 d in a growth chamber under a regime of 16 h light (325 f.1mol m- 2 S-I) and 8 h dark at a temperature of 25°C. Primary leaves were excised from the plants and the cut ends inserted into microcentrifuge tubes capped with parafilm and filled with distilled water. The microcentrifuge tubes were then mounted on a clinostat with the distal portion of the leaf being gently secured by elastic bands (Fig. 1). The altered gravity treatment consisted of a clinostat oriented horizontally (H) and rotating at 1 rpm. A vertically oriented clinostat (V) rotating at 1 rpm was used as a control. As a control for the effect of rotation alone, leaves were mounted on the vertical clinostat and not rotated. This treatment was designated as the stationary control (S). Fluorescent light banks were arranged directly above the vertical clinostats and in front of the horizontal clinostats so as to be perpendicular to the axis of rotation in each case, and to illuminate the leaves of both orientations with light coming directly parallel to the leaf surfaces. The light intensity as measured at the tops of the microcentrifuge tubes and directly facing the light banks was 325 f.1mol m -2 S-1 for both orientations. The duration of the gravity treatments was 24 h, during which time the excised leaves were exposed to continuous light. The temperature was maintained at 20°C throughout the 24-h period. At the end of the treatment the leaves were either frozen in liquid N 2 and stored at -80°C for subsequent carbohydrate or enzyme assay or were immediately used for determination of photosynthetic or respiratory rates.
Carbohydrate Analysis The tissue was extracted 3 times in boiling 80 % ethanol and 2 times in boiling water. Starch was quantified in the insoluble pellet as in Brown et aI., (1985). The 80 % ethanol and water extracts
CLiNOSTAT
were pooled and quantified enzymatically for sucrose, fructose, and glucose using a hexokinase (EC 2.7.1.1), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) linked assay Gones et al., 1977) and a glucose assay kit (Sigma Chemical Company, St. Louis, MO). Fructan was quantified in the 80 % ethanol and water extracts using the method of Wagner et al. ( 1983).
Photosynthesis and Dark Respiration Measurements At the end of the 24-hour treatment, excised leaves were removed from the clinostat or stationary control and placed in the leaf chamber of an LI-6200 portable photosynthesis system (Lambda instruments Inc., Lincoln, NE) in order to measure photosynthetic rate. These measurements were conducted under ambient CO 2 conditions. Following clinostat treatment, 4-mm leaf punches were taken from the leaves and used for oxygen uptake measurements to estimate dark respiration. The measurements were performed in distilled water using a YSI oxygen electrode (Yellow Springs Instruments, Yellow Springs, OH).
Enzyme Assays Enzyme extracts were prepared by grinding the leaf tissue using a mortar and pestle in 3 volumes of ice-cold buffer consisting of 50 mM HEPES-NaOH (PH 7.5), 5 mM MgCb, 2.5 mM DTT, 0.5 % BSA and 2 % PEG (average MW = 10,000). The homogenate was centrifuged at 14,000 X g for 20 minutes and the resulting supernatant desalted by passage through a column of Sephadex G-25 (Sigma Chemical, St. Louis, MO). Desalted extracts were used for all of the enzyme assays. The standard assay for AGP was composed of 50 mM HEPESNaOH (PH 7.5), 5 mM MgCb, 1 mM ADP-Glc, 1 mM NADP, 1 mM NaPPi, 0.5 mM 3-PGA, 2 U mL -1 glucose-6-phosphate dehydrogenase, 2 U mL -1 phosphoglucomuutase (EC 5.4.2.2), and 2 U mL -1 phosphogluconate dehydrogenase (EC 1.1.1.44). Addition of enzyme extract initiated the assay, the rate being determined by continuously monitoring absorbance at 340 nm. The same assay was also run in the absence of 3-PGA. SPS activity was assayed either under «limiting» or under «Vmax» conditions as described by Weiner et al. ( 1992). The limiting assay contained 50mM HEPES-
698
DAVID M. OBENLAND and CHRISTOPHER S. BROWN
NaOH (PH 7.5), 30mM MgCI2, 6mM Fru-6-P, 10mM UDP-Glc, 24mM Glc-6-P, 20mM Pi, 5mM DTT. The reaction was initiated by addition of enzyme extract. The total volume of the assay was 70 ilL. The Vmax assay was the same as the limiting assay except that Fru-6-P was increased to 20 mM, Glc-6-P increased to 80 mM, and that Pi was ommitted. After a reaction time of 30 to 60 min, the reaction was terminated by addition of 70 ilL of 1 N NaOH, followed by a 10-min incubation in a boiling water bath. After cooling, 250 ilL of 0.1 % resorcinol (in 95 % ethanol) and 750 ilL of 30 % HCI were added. The mixture was then heated at 80°C for 8 min, the absorbance measured at 520 nm and the amount of sucrose present in the sample determined by comparison to a sucrose standard curve. The SST assay contained 50 mM MES (pH 5.7), 100 mM sucrose, 0.1 % BSA, 10 mM pyridoxal-HCI (inhibitor of invertase) and the reaction was initiated with the enzyme extract. After a sufficient length of time the reaction was terminated by heating the reaction mixture for 5 min in a boiling water bath. Following 10 min of centrifugation at top speed in a microcentrifuge, the supernatant was assayed for glucose and fructose. The amount of fructose detected was used to correct the glucose concentration for the amount of glucose given off by the activity of invertase in the enzyme extract. The glucose remaining was considered to be due to the activity of SST. To assay for FH activity, enzyme extract was combined with a reaction mixture consisting of 50 mM McIvaine buffer (pH 5.2) and 1 mg of wheat fructan (prepared as in Wagner and Wiemken, 1986). The reaction was allowed to proceed at 34°C for 1 h after which the reaction was stopped by heating in a boiling water bath for 5 min. After 10 min of centrifugation in a microcentrifuge, the supernatant was assayed for fructose using the same methods as above. One unit of activity was defined for the AGP, SPS, and FH assays as being the amount able to produce 1 Ilmol of product per minute. The SST calculations were the same but on a substrate utilized basis.
Results and Discussion
The amount of sucrose, starch, and fructan was significantly reduced in the H treatment relative to the V treat-
~ ~
'"E
30
..c ~
~ 'OIl
20
..:.<
~...
-0
;>,
..c 0
-£:
10
'"
()
OIl
o
gf ucose
fructose
sucrose
starch
fructan
Fig. 2: Carbohydrate concentrations in 7-day-old excised wheat leaves exposed to constant light and subjected to either horizontal or vertical clinorotation or that remained stationary. All treatments were for a period of 24 h. Data represent the mean of 6 replications, each replication consisting of a separate extraction with 3 leaves per extraction. Results from this experiment were representative of those from other similar experiments. Bars designate standard error. Least significant differences at the 5 % level of significance were: fructose = 1.16, fructan = 2.53, glucose = 0.65, starch = 1.37, sucrose = 3.88.
Table 1: Effect of 24h of horizontal and vertical clinorotation and stationary treatment on photosynthesis, respiration, and the activities of enzymes of carbohydrate metabolism in excised wheat leaves. Treatment*
Parameter Measured
H
v
Photosynthesis ("mol C02 m- 2 s- l ) Dark Respiration ("mol C02 m- 2 s- l ) Enzyme Activity (Units kg-I fresh mass) (I)AGP +3·PGA -3·PGA
6.45 (0.35) a**
7.03 (0.12) ab
7.92(0.44)b
3.80(0.14)a
3.64 (0.12) a
(2)SPS limiting
vm'"
(3) SST
3.93 (0.12) a
s
1639.6(131.4) a 220.1 (14.1) a
1563.9 (30.7). 226.9(18.7).
1412.1 (163.9). 215.2 (9.2) a
80.1(7.7). 342.1 (28.2) a
61.6(7.4) a 315.0(18.5).
64.6(10.3). 334.8 (21.3) a
244.4(4.3).
238.7 (16.2).
338.5 (10.2) b
(4)FH 102.8 (3.1) a 98.6(4.9)ab 87.4(4.9)b * H = horizontal dinorotation, V = vertical c1inorotation, S = stationary.
**
Values ,represent the means of 4-6 replications with standard errors in parentheses.
Each replication was a measurement from a separate batch of three leaves. Means
within a row that are followed by the same letter are not statistically different at the 5 % level of significance.
ment by 26,22, and 41 %, respectively (Fig. 2). Of the hexoses, fructose was reduced by 11 % while the amount of glucose in both H and V treated tissues was the same (Fig. 2). Tests confirmed that these differences in carbohydrate amounts were not due to an increase in sucrose exudation from the cut end of the leaf from the H treatment (data not shown). With the exception of glucose, carbohydrates amounts for V and S were not statistically different, indicating that the effect of simply rotating the leaves in the vertical orientation had little effect on the amount of carbohydrate accumulated (Fig. 2). The lower starch concentration that was observed in this study is consistent with reductions that have been reported for space-grown plants (Moore et aI., 1990; Volkmann et al., 1986; Johnson and Tibbitts, 1968) and plants grown under altered gravity conditions on a clinostat Gohnson and Tibbitts, 1968; Brown and Piastuch). The effect of altered gravity on the accumulation of fructan has never been reported in the literature. Results from our laboratory indicate that there are no changes in sucrose, glucose, and fructose between H and V treatments in darkgrown soybean cotyledons (Brown and Piastuch). In comparison with the results in this study, it raises the possibility that different species and tissues may respond in a differing manner to altered gravity. A decrease in photosynthesis or an increase in respiration due to clinorotation conceivably could have caused a reduction in carbohydrate accumulation in the excised wheat leaves. Rates determined in clinorotated tissue, however, indicated that neither photosynthesis nor dark respiration were significantly altered by H in comparison to V (Table 1). Rates for the V and S treatments also were not statistically different from each other. A previous study in our lab (Brown and Piastuch) showed that alterations in starch accumulation in clinostat-treated or centrifuged soybean cotyledons was strongly correlated with changes in AGP activity. To test if clinorotation was also affecting carbohydrate metabolizing enzyme activities in ex-
The influence of altered gravity on Carbohydrate Metabolism
cised wheat leaves, activities of four key enzymes involved in the synthesis or degradation of the carbohydrates were measured (Table 1). AGP, the rate limiting enzyme of starch biosynthesis (Beck and Ziegler 1989), was assayed both in the presence and absence of 3-PGA, a positive effector of the enzyme (Table 1), in order to determine if changes in response to the effector might be involved in inhibiting AGP activity. No significant differences, however, were found between H and V treatments with or without 3-PGA (Table 1). SPS, an important enzyme in determining sucrose biosynthetic rates (Huber and Huber, 1992), was determined with both a «limiting» assay and a "Vmax » assay. The use of two different assay systems for SPS, which differed by concentrations of positive and negative effectors, was done for the same reason as was described for AGP. For both limiting and Vmax assays the rates were somewhat higher for the H treatment, though none of the differences were statistically significant (Table 1). Finally, neither SST nor FH, key enzymes of fructan biosynthesis and degradation, respectively, (Pollock and Chatterton, 1988), were changed in activity by altered gravity treatment (Table 1). Although our data cannot conclusively rule out that the in vivo activities of any of the four enzymes are affected by clinorotation, it is apparent that the extractable in vitro activity was not influenced by clinorotation to any great degree. The effect of rotation itself also did not appear to have a very dramatic influence on the enzyme activities measured. Of the four enzymes assayed, only in the case of SST was V statistically different than S (Table 1), and the greater activity of SST in S than in V did not lead to a greater accumulation of fructan in S. We have demonstrated the use of a new method that uses excised wheat leaves to study the effect of altered gravity on plant metabolism using the clinostat. The benefits of this system may be used to enhance future clinostat studies, especially in the area of carbohydrate metabolism in green leaf tissue. The results from this study gave evidence that in primary leaves of wheat carbohydrate accumulation is inhib-
699
ited in response to altered gravity. The causal mechanism for this reduction remains under investigation. References
ABILOV, Z. K., U. K. ALEKPEROV, A. L. MASHINSKIY, s. 1. FADEYEVA, and A. A. ALIYEV: USSR Space Life Sciences Digest 8, 15 -16 (1986). BECK, E. and P. ZIEGLER: In: BRIGGS, W. R. (ed.): Annual Review of Plant Physiology and Plant Molecular Biology. Annual Reviews, Inc., Palo Alto pp. 95-117 (1989). BROWN, C. s. and W. C. PIASTUCH: Plant Cell and Environ. (in press). BROWN, C. 5., E. YOUNG, and D. M. PHARR: J. Amer. Soc. of Hort. Sci. 110, 701-705 (1985). HUBER, S. C. andJ. L. HUBER: Plant Physiol. 99, 1275-1278 (1992). JOHNSON, s. P. and T. W. TIBBITTS: BioScience 18, 655-661 (1968). JONES, M. G. K., W. H. OUTLAW Jr., and o. H. LOWRY: Plant Physiol. 60, 379-383 (1977). MOORE, R.: Aim. Bot. 65, 213-216 (1990). MOORE, R., W. M. FONDREN, E. C. KOON, and c.-L. WANG: Amer. J. Bot. 74,216-221 (1987). OBENLAND, D. M., U. SIMMEN, T. BOLLER, and A. WIEMKEN: Plant Physiol. 97, 811- 813 (1991). POLLOCK, C. J. and N. J. CHATTERTON: In: PREISS, J. (ed.): The Biochemistry of Plants, Vol. 14, pp. 109-140. Academic Press, Inc., London (1988). SALERNO, G. L., J. L. lANIRO, J. A. TOGNETTI, M. D. CRESPI, and H. G. PONTIS: J. Plant Physiol. 134,214-217 (1989). VOLKMANN, D., H. M. BEHRENS, and A. SIEVERS: Naturwissenschaften 73, 438-441 (1986). WAGNER, W., F. KELLER, and A. WIEMKEN: Z. Pflanzenphysiol. 112, 359-372 (1983). WAGNER, W. and A. WIEMKEN: J. Plant Physiol. 123, 429-439 (1986). WAGNER, W., A. WIEMKEN, and P. MATILE: Plant Physiol. 81, 444447 (1986). WEINER, H., R. W. McMICHAEL Jr., and S. HUBER: Plant Physiol. 99, 1435-1442 (1992).
The Influence of Altered Gravity on Carbohydrate Metabolism in Excised Wheat Leaves 1 DAVID
M.
OBENLAND 2 and CHRISTOPHER
S. BROWN
Plant Space Biology Laboratory, The Bionetics Corporation, Mail Code BIO-3, Kennedy Space Center, Florida 32899, USA Received February 1, 1994 . Accepted April 15, 1994
Summary
We developed a system to study the influence of altered gravity on carbohydrate metabolism in excised wheat leaves by means of clinorotation. The use of excised leaves in our clinostat studies offered a number of advantages over the use of whole plants, most important of which were minimization of exogenous mechanical stress and a greater amount of carbohydrate accumulation during the time of treatment. We found that horizontal clinorotation of excised wheat leaves resulted in significant reductions in the accumulation of fructose, sucrose, starch and fructan relative to control, vertically clinorotated leaves. Photosynthesis, dark respiration and the extractable activities of ADP glucose pyrophosphorylase (EC 2.7.7.27), sucrose phosphate synthase (EC 2.4.4.14), sucrose sucrose fructosyltransferase (EC 2.4.1.99), and fructan hydrolase (EC 3.2.1.80) were unchanged due to altered gravity treatment. Key words: Triticum aestivum, carbohydrate, clinostat, dark respiration, enzyme, photosynthesis. Abbreviations: AGP = ADP glucose pyrophosphorylase; SPS = sucrose phosphate synthase; SST = sucrose sucrose fructosyltransferase; FH = fructan hydrolase.
Introduction
One consistent observation regarding the metabolism of plants grown in the micro gravity of space is that they accumulate less starch than ground-based controls (Abilov et aI., 1986; Johnson and Tibbitts, 1968; Moore et aI., 1987). In accordance with this result, we found that germinating, dark-grown soybean cotyledons have reduced starch content when subjected to altered gravity as imposed by clinorotation (Brown and Piastuch). In the current study we utilize an excised leaf system to study the effect of altered gravity on carbohydrate metabolism in light-grown wheat leaves. This system offers us five significant advantages over whole-plant systems: (i) exogenous mechanical stresses on plant tissues 1 Work done by D.M.O. while on a National Research Council Fellowship at the Kennedy Space Center, FL, U.S.A. 2 Present address and correspondence: Horticultural Crops Research Laboratory, USDA/ ARS, 2021 S. Peach Avenue, Fresno, CA, 93727, U.S.A.
© 1994 by Gustav Fischer Vedag, Stuttgart
due to clinorotation are greatly minimized due to the fact that the wheat leaves are fixed to the clinostat and little or no extraneous movement occurs; (ii) an excised, illuminated leaf accumulates large quantities of carbohydrates (Wagner et aI., 1986), making it possible to quantify fructan, a very important storage carbohydrate in the grasses that would normally be present in only very small amounts in attached, primary leaves; (iii) the accumulation of carbohydrate that occurs is known in the case of sucrose (Salerno et aI., 1989) and fructan (Obenland et aI., 1991) to depend on a rapid de novo synthesis of the biosynthetic enzyme systems for the particular carbohydrate, making it a good system to observe the effect of altered gravity on protein synthesis; (iv) it is very easy to introduce compounds via the transpiration stream into an excised leaf to aid in metabolic studies; and (v) we have found the set-up of the experiment to be easier and the time needed to complete the experiment less than for whole plants. In this paper we present a clinorotation experiment using excised wheat leaves. We show sucrose, starch and fructan to
The influence of altered gravity on Carbohydrate Metabolism
697
Small Test Tube Excised Leaf
Elastic Support
Fig. 1: Schematic diagram of clinostat used to impose omnilateral gravity stimulation on excised wheat leaves.
be significantly reduced in response to altered gravity treatment. Several possible mechanisms for the changes are discussed. Materials and Methods
Clinostat treatments Wheat (Triticum aestivum cv. Yecora rojo) plants were grown for 7 d in a growth chamber under a regime of 16 h light (325 f.1mol m- 2 S-I) and 8 h dark at a temperature of 25°C. Primary leaves were excised from the plants and the cut ends inserted into microcentrifuge tubes capped with parafilm and filled with distilled water. The microcentrifuge tubes were then mounted on a clinostat with the distal portion of the leaf being gently secured by elastic bands (Fig. 1). The altered gravity treatment consisted of a clinostat oriented horizontally (H) and rotating at 1 rpm. A vertically oriented clinostat (V) rotating at 1 rpm was used as a control. As a control for the effect of rotation alone, leaves were mounted on the vertical clinostat and not rotated. This treatment was designated as the stationary control (S). Fluorescent light banks were arranged directly above the vertical clinostats and in front of the horizontal clinostats so as to be perpendicular to the axis of rotation in each case, and to illuminate the leaves of both orientations with light coming directly parallel to the leaf surfaces. The light intensity as measured at the tops of the microcentrifuge tubes and directly facing the light banks was 325 f.1mol m -2 S-1 for both orientations. The duration of the gravity treatments was 24 h, during which time the excised leaves were exposed to continuous light. The temperature was maintained at 20°C throughout the 24-h period. At the end of the treatment the leaves were either frozen in liquid N 2 and stored at -80°C for subsequent carbohydrate or enzyme assay or were immediately used for determination of photosynthetic or respiratory rates.
Carbohydrate Analysis The tissue was extracted 3 times in boiling 80 % ethanol and 2 times in boiling water. Starch was quantified in the insoluble pellet as in Brown et aI., (1985). The 80 % ethanol and water extracts
CLiNOSTAT
were pooled and quantified enzymatically for sucrose, fructose, and glucose using a hexokinase (EC 2.7.1.1), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) linked assay Gones et al., 1977) and a glucose assay kit (Sigma Chemical Company, St. Louis, MO). Fructan was quantified in the 80 % ethanol and water extracts using the method of Wagner et al. ( 1983).
Photosynthesis and Dark Respiration Measurements At the end of the 24-hour treatment, excised leaves were removed from the clinostat or stationary control and placed in the leaf chamber of an LI-6200 portable photosynthesis system (Lambda instruments Inc., Lincoln, NE) in order to measure photosynthetic rate. These measurements were conducted under ambient CO 2 conditions. Following clinostat treatment, 4-mm leaf punches were taken from the leaves and used for oxygen uptake measurements to estimate dark respiration. The measurements were performed in distilled water using a YSI oxygen electrode (Yellow Springs Instruments, Yellow Springs, OH).
Enzyme Assays Enzyme extracts were prepared by grinding the leaf tissue using a mortar and pestle in 3 volumes of ice-cold buffer consisting of 50 mM HEPES-NaOH (PH 7.5), 5 mM MgCb, 2.5 mM DTT, 0.5 % BSA and 2 % PEG (average MW = 10,000). The homogenate was centrifuged at 14,000 X g for 20 minutes and the resulting supernatant desalted by passage through a column of Sephadex G-25 (Sigma Chemical, St. Louis, MO). Desalted extracts were used for all of the enzyme assays. The standard assay for AGP was composed of 50 mM HEPESNaOH (PH 7.5), 5 mM MgCb, 1 mM ADP-Glc, 1 mM NADP, 1 mM NaPPi, 0.5 mM 3-PGA, 2 U mL -1 glucose-6-phosphate dehydrogenase, 2 U mL -1 phosphoglucomuutase (EC 5.4.2.2), and 2 U mL -1 phosphogluconate dehydrogenase (EC 1.1.1.44). Addition of enzyme extract initiated the assay, the rate being determined by continuously monitoring absorbance at 340 nm. The same assay was also run in the absence of 3-PGA. SPS activity was assayed either under «limiting» or under «Vmax» conditions as described by Weiner et al. ( 1992). The limiting assay contained 50mM HEPES-
698
DAVID M. OBENLAND and CHRISTOPHER S. BROWN
NaOH (PH 7.5), 30mM MgCI2, 6mM Fru-6-P, 10mM UDP-Glc, 24mM Glc-6-P, 20mM Pi, 5mM DTT. The reaction was initiated by addition of enzyme extract. The total volume of the assay was 70 ilL. The Vmax assay was the same as the limiting assay except that Fru-6-P was increased to 20 mM, Glc-6-P increased to 80 mM, and that Pi was ommitted. After a reaction time of 30 to 60 min, the reaction was terminated by addition of 70 ilL of 1 N NaOH, followed by a 10-min incubation in a boiling water bath. After cooling, 250 ilL of 0.1 % resorcinol (in 95 % ethanol) and 750 ilL of 30 % HCI were added. The mixture was then heated at 80°C for 8 min, the absorbance measured at 520 nm and the amount of sucrose present in the sample determined by comparison to a sucrose standard curve. The SST assay contained 50 mM MES (pH 5.7), 100 mM sucrose, 0.1 % BSA, 10 mM pyridoxal-HCI (inhibitor of invertase) and the reaction was initiated with the enzyme extract. After a sufficient length of time the reaction was terminated by heating the reaction mixture for 5 min in a boiling water bath. Following 10 min of centrifugation at top speed in a microcentrifuge, the supernatant was assayed for glucose and fructose. The amount of fructose detected was used to correct the glucose concentration for the amount of glucose given off by the activity of invertase in the enzyme extract. The glucose remaining was considered to be due to the activity of SST. To assay for FH activity, enzyme extract was combined with a reaction mixture consisting of 50 mM McIvaine buffer (pH 5.2) and 1 mg of wheat fructan (prepared as in Wagner and Wiemken, 1986). The reaction was allowed to proceed at 34°C for 1 h after which the reaction was stopped by heating in a boiling water bath for 5 min. After 10 min of centrifugation in a microcentrifuge, the supernatant was assayed for fructose using the same methods as above. One unit of activity was defined for the AGP, SPS, and FH assays as being the amount able to produce 1 Ilmol of product per minute. The SST calculations were the same but on a substrate utilized basis.
Results and Discussion
The amount of sucrose, starch, and fructan was significantly reduced in the H treatment relative to the V treat-
~ ~
'"E
30
..c ~
~ 'OIl
20
..:.<
~...
-0
;>,
..c 0
-£:
10
'"
()
OIl
o
gf ucose
fructose
sucrose
starch
fructan
Fig. 2: Carbohydrate concentrations in 7-day-old excised wheat leaves exposed to constant light and subjected to either horizontal or vertical clinorotation or that remained stationary. All treatments were for a period of 24 h. Data represent the mean of 6 replications, each replication consisting of a separate extraction with 3 leaves per extraction. Results from this experiment were representative of those from other similar experiments. Bars designate standard error. Least significant differences at the 5 % level of significance were: fructose = 1.16, fructan = 2.53, glucose = 0.65, starch = 1.37, sucrose = 3.88.
Table 1: Effect of 24h of horizontal and vertical clinorotation and stationary treatment on photosynthesis, respiration, and the activities of enzymes of carbohydrate metabolism in excised wheat leaves. Treatment*
Parameter Measured
H
v
Photosynthesis ("mol C02 m- 2 s- l ) Dark Respiration ("mol C02 m- 2 s- l ) Enzyme Activity (Units kg-I fresh mass) (I)AGP +3·PGA -3·PGA
6.45 (0.35) a**
7.03 (0.12) ab
7.92(0.44)b
3.80(0.14)a
3.64 (0.12) a
(2)SPS limiting
vm'"
(3) SST
3.93 (0.12) a
s
1639.6(131.4) a 220.1 (14.1) a
1563.9 (30.7). 226.9(18.7).
1412.1 (163.9). 215.2 (9.2) a
80.1(7.7). 342.1 (28.2) a
61.6(7.4) a 315.0(18.5).
64.6(10.3). 334.8 (21.3) a
244.4(4.3).
238.7 (16.2).
338.5 (10.2) b
(4)FH 102.8 (3.1) a 98.6(4.9)ab 87.4(4.9)b * H = horizontal dinorotation, V = vertical c1inorotation, S = stationary.
**
Values ,represent the means of 4-6 replications with standard errors in parentheses.
Each replication was a measurement from a separate batch of three leaves. Means
within a row that are followed by the same letter are not statistically different at the 5 % level of significance.
ment by 26,22, and 41 %, respectively (Fig. 2). Of the hexoses, fructose was reduced by 11 % while the amount of glucose in both H and V treated tissues was the same (Fig. 2). Tests confirmed that these differences in carbohydrate amounts were not due to an increase in sucrose exudation from the cut end of the leaf from the H treatment (data not shown). With the exception of glucose, carbohydrates amounts for V and S were not statistically different, indicating that the effect of simply rotating the leaves in the vertical orientation had little effect on the amount of carbohydrate accumulated (Fig. 2). The lower starch concentration that was observed in this study is consistent with reductions that have been reported for space-grown plants (Moore et aI., 1990; Volkmann et al., 1986; Johnson and Tibbitts, 1968) and plants grown under altered gravity conditions on a clinostat Gohnson and Tibbitts, 1968; Brown and Piastuch). The effect of altered gravity on the accumulation of fructan has never been reported in the literature. Results from our laboratory indicate that there are no changes in sucrose, glucose, and fructose between H and V treatments in darkgrown soybean cotyledons (Brown and Piastuch). In comparison with the results in this study, it raises the possibility that different species and tissues may respond in a differing manner to altered gravity. A decrease in photosynthesis or an increase in respiration due to clinorotation conceivably could have caused a reduction in carbohydrate accumulation in the excised wheat leaves. Rates determined in clinorotated tissue, however, indicated that neither photosynthesis nor dark respiration were significantly altered by H in comparison to V (Table 1). Rates for the V and S treatments also were not statistically different from each other. A previous study in our lab (Brown and Piastuch) showed that alterations in starch accumulation in clinostat-treated or centrifuged soybean cotyledons was strongly correlated with changes in AGP activity. To test if clinorotation was also affecting carbohydrate metabolizing enzyme activities in ex-
The influence of altered gravity on Carbohydrate Metabolism
cised wheat leaves, activities of four key enzymes involved in the synthesis or degradation of the carbohydrates were measured (Table 1). AGP, the rate limiting enzyme of starch biosynthesis (Beck and Ziegler 1989), was assayed both in the presence and absence of 3-PGA, a positive effector of the enzyme (Table 1), in order to determine if changes in response to the effector might be involved in inhibiting AGP activity. No significant differences, however, were found between H and V treatments with or without 3-PGA (Table 1). SPS, an important enzyme in determining sucrose biosynthetic rates (Huber and Huber, 1992), was determined with both a «limiting» assay and a "Vmax » assay. The use of two different assay systems for SPS, which differed by concentrations of positive and negative effectors, was done for the same reason as was described for AGP. For both limiting and Vmax assays the rates were somewhat higher for the H treatment, though none of the differences were statistically significant (Table 1). Finally, neither SST nor FH, key enzymes of fructan biosynthesis and degradation, respectively, (Pollock and Chatterton, 1988), were changed in activity by altered gravity treatment (Table 1). Although our data cannot conclusively rule out that the in vivo activities of any of the four enzymes are affected by clinorotation, it is apparent that the extractable in vitro activity was not influenced by clinorotation to any great degree. The effect of rotation itself also did not appear to have a very dramatic influence on the enzyme activities measured. Of the four enzymes assayed, only in the case of SST was V statistically different than S (Table 1), and the greater activity of SST in S than in V did not lead to a greater accumulation of fructan in S. We have demonstrated the use of a new method that uses excised wheat leaves to study the effect of altered gravity on plant metabolism using the clinostat. The benefits of this system may be used to enhance future clinostat studies, especially in the area of carbohydrate metabolism in green leaf tissue. The results from this study gave evidence that in primary leaves of wheat carbohydrate accumulation is inhib-
699
ited in response to altered gravity. The causal mechanism for this reduction remains under investigation. References
ABILOV, Z. K., U. K. ALEKPEROV, A. L. MASHINSKIY, s. 1. FADEYEVA, and A. A. ALIYEV: USSR Space Life Sciences Digest 8, 15 -16 (1986). BECK, E. and P. ZIEGLER: In: BRIGGS, W. R. (ed.): Annual Review of Plant Physiology and Plant Molecular Biology. Annual Reviews, Inc., Palo Alto pp. 95-117 (1989). BROWN, C. s. and W. C. PIASTUCH: Plant Cell and Environ. (in press). BROWN, C. 5., E. YOUNG, and D. M. PHARR: J. Amer. Soc. of Hort. Sci. 110, 701-705 (1985). HUBER, S. C. andJ. L. HUBER: Plant Physiol. 99, 1275-1278 (1992). JOHNSON, s. P. and T. W. TIBBITTS: BioScience 18, 655-661 (1968). JONES, M. G. K., W. H. OUTLAW Jr., and o. H. LOWRY: Plant Physiol. 60, 379-383 (1977). MOORE, R.: Aim. Bot. 65, 213-216 (1990). MOORE, R., W. M. FONDREN, E. C. KOON, and c.-L. WANG: Amer. J. Bot. 74,216-221 (1987). OBENLAND, D. M., U. SIMMEN, T. BOLLER, and A. WIEMKEN: Plant Physiol. 97, 811- 813 (1991). POLLOCK, C. J. and N. J. CHATTERTON: In: PREISS, J. (ed.): The Biochemistry of Plants, Vol. 14, pp. 109-140. Academic Press, Inc., London (1988). SALERNO, G. L., J. L. lANIRO, J. A. TOGNETTI, M. D. CRESPI, and H. G. PONTIS: J. Plant Physiol. 134,214-217 (1989). VOLKMANN, D., H. M. BEHRENS, and A. SIEVERS: Naturwissenschaften 73, 438-441 (1986). WAGNER, W., F. KELLER, and A. WIEMKEN: Z. Pflanzenphysiol. 112, 359-372 (1983). WAGNER, W. and A. WIEMKEN: J. Plant Physiol. 123, 429-439 (1986). WAGNER, W., A. WIEMKEN, and P. MATILE: Plant Physiol. 81, 444447 (1986). WEINER, H., R. W. McMICHAEL Jr., and S. HUBER: Plant Physiol. 99, 1435-1442 (1992).