U.S. Department of Agriculture, Agricultural Research Service, University of Kentucky, Lexingon, Kentucky 40546. ABSTRACT. The effect of inorganic phosphate ...
Received for publication April 6, 1989 and in revised form July 5, 1989
Plant Physiol. (1989) 91, 469-472 0032-0889/89/91/0469/04/$01 .00/0
Communication
Species and Environmental Variations in the Effect of Inorganic Phosphate on Sucrose-Phosphate Synthase
Activity'
Reliability of Assays Based Upon UDP Formation Steven J. Crafts-Brandner* and Michael E. Salvucci U.S. Department of Agriculture, Agricultural Research Service, University of Kentucky, Lexingon, Kentucky 40546 the authors indicated that, in spinach and barley, the use of the UDP method for determining SPS activity was preferable to the method based on sucrose measurement. In current investigations of the effect of phosphate nutrition on carbohydrate partitioning in tobacco, we have observed that SPS was not necessarily inhibited by Pi. Furthermore, the Pi effect and the apparent SPS activities per se were markedly affected by the assay method used. These observations prompted us to investigate the effect of Pi on SPS activity of various plant species and to evaluate the conventional assay methods based on either substrate dependent UDP or sucrose formation.
ABSTRACT The effect of inorganic phosphate (Pi) on sucrose-phosphate synthase (SPS) activity was determined for the enzyme from five plant species (Nicotiana tabacum L., Spinacia oleracea L., Triticum aestivum L., Zea mays L., Glycine max L.) using two assay methods. The assay method based on determination of uridine diphosphate glucose- (UDPG) and fructose-6-phosphate-dependent sucrose formation was linear up to 15 minutes for all species tested. When assayed in this way, the effect of Pi at levels of 5 or 10 millimolar in the assay was variable, ranging from 0 to 35% inhibition of SPS activity. The assay method based on substrate dependent UDP formation was linear for some, but not for all of the species tested. Deviations from linearity were caused by loss of UDP from the assay medium. In some species, the extent of UDP loss was influenced by the level of Pi in the assay medium and, for at least one species (tobacco), it was influenced by the environment in which the plants were grown. The results indicated that (a) the role of Pi as an effector of SPS may vary depending on the species, and (b) the UDP assay method should be used with caution for assays of crude or desalted extracts, particularly when evaluating the effect of Pi on SPS activity.
MATERIALS AND METHODS
Plant Material, Cultural Conditions, and Sampling
Tobacco (Nicotiana tabacum L. cv KY 14) was grown in the field (6) or the greenhouse (3) as previously described. Field-grown plants were sampled in August 1989; greenhousegrown plants were sampled in February and May 1989. At flowering, the apex of the plant including the top three to four small leaves was removed, a common cultural practice called topping. Subsequent axillary bud growth was removed two times per week. Tobacco plants that were subjected to this treatment are referred to as topped plants; these plants were sampled 15 to 20 d after applying the treatment. Nontopped tobacco plants of cultivar KY 14 were grown in the greenhouse as previously described (3) and sampled 14 weeks after planting. Spinach (Spinacia oleracea L. cv Melody) was grown in the field and sampled 75 d after planting. Wheat (Triticum aestivum L. cv Neeley) was grown in a growth chamber in a soil-perlite (3:1) mixture. The growth chamber day:night temperatures were 28 and 1 8°C, respectively, and PPFD was 700 M4mol m-2 s-'. The wheat plants were sampled 2 to 3 weeks after planting. Maize (Zea mays L. cv B73 x Mo17) was grown in the field and sampled 4 weeks after planting. Soybean (Glycine max L. Merr. cv McCall) plants were grown in the greenhouse in soil. Plants were sampled at growth stage R5 (7) (beginning seed development). All plants were supplied with adequate levels of mineral nutrients and, in addition, field-grown plants were irrigated as necessary to prevent mois-
Many studies have documented the importance of SPS2 in the regulation of sucrose synthesis in leaves (1, 4, 5, 8-10). Regulation of SPS has been ascribed, in part, to the effects of certain metabolites, particularly G6P, an activator of SPS activity, and Pi which acts as a partial competitive inhibitor of the substrates F6P and UDPG (1, 4, 5, 8, 10). Stitt et al. (10) have recently proposed that SPS may exist in kinetically different forms in the light and dark, citing as evidence marked changes in the sensitivity of SPS to Pi. These authors worked primarily with spinach, although they did note a less marked inhibition of barley SPS by Pi. In addition, ' Jointly supported by the U.S. Department of Agriculture, Agricultural Research Service and the Kentucky Agricultural Experiment Station, Lexington (paper No. 89-3-64). 2 Abbreviations: SPS, sucrose-phosphate synthase; F6P, fructose-6phosphate; G6P, glucose-6-phosphate; UDPG, uridine 5'-diphosphate glucose.
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ture stress. In all cases, leaves used for enzyme assays were sampled in the light from the upper third of the plants. Fieldand greenhouse-grown plants were sampled on clear sunny days between 1000 and 1300 h. Plants grown in the growth chamber were sampled 2 to 3 h after the beginning of the light cycle. Following sampling, leaves were immediately chilled to ice temperatures and maintained at 4°C for 10 to 20 min prior to enzyme extraction. In some cases (field tobacco and greenhouse tobacco grown in the winter of 1989) leaf tissue was immediately frozen and stored at -80°C until enzyme assay.
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Sucrose-Phosphate Synthase Measurements Three g fresh weight ofleaf tissue were frozen and powdered in liquid N2 in a mortar and pestle and subsequently homogenized in 6 mL of buffer containing 50 mm Hepes-NaOH (pH 7.5), 5 mM MgCl2, 1 mM EDTA, 2.5 mM DTT, 1% (w/ v) casein, and 1% (w/v) soluble PVP. After extraction, the homogenate was centrifuged at 4°C for 2 min in a microfuge at 13,000g. Immediately following centrifugation, the supernatant (300 ,uL) was desalted at 4°C on a column (8 x 40 mm) of Sephadex G50-300. Desalted extracts were used for all SPS measurements. SPS was assayed at 30°C by the two-stage assay for UDP formation (10) or by substrate dependent formation of sucrose (4). Assays were initiated by the addition of extract to a reaction mix containing 50 mM Hepes-NaOH (pH 7.4), 15 mM MgCl2, 1 mM EDTA, 7.5 mM F6P, and 7.5 mM UDPG in addition to 37.5 mm G6P. Pi (NaH2PO4, pH 7.4) was included at levels of 0, 5, or 10 mm. Assay blanks which lacked F6P and G6P were run for each assay condition. To test for the disappearance of product from the assay medium, 155 nmol of UDP or sucrose was added to reaction mixtures lacking F6P and G6P. The values reported for SPS activity represent the mean of two independent assays and are corrected for dilution caused by desalting the extracts. Pi was determined in the desalted extracts of all plant species by the method of Chifflet et al. (2). RESULTS AND DISCUSSION SPS activity was determined at relatively high levels of MgCl2, F6P, and UDPG (5, 8, 9) and in the presence of G6P. G6P was present at five times the level of F6P, the ratio at which these metabolites are adjusted to the thermodynamic equilibrium of the phosphoglucose isomerase reaction (10). The use of desalted extracts in this study minimized the carryover of Pi and other metabolites from the extracts. For example, desalted extracts contained less than 0.4 ,umol Pi/ mL which amounts to a concentration of 0.12 mM Pi in the 0 Pi assay. The time course of SPS activity assayed by measuring sucrose or UDP formation is presented in Figure 1. For all species, the method based on sucrose formation was linear at least up to 15 min (Fig. IA). For spinach, maize, and greenhouse-grown, nontopped tobacco, the assay method based on UDP determination (Fig. 1B) agreed well with results from the sucrose assay. However, for wheat, soybean, topped tobacco (field- or greenhouse-grown) (data not shown), and
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Minutes Figure 1. Time course of SPS assays based on sucrose formation (A) or UDP formation (B) for five plant species. The source of enzyme for the tobacco results was greenhouse-grown, nontopped tobacco. Data points represent the mean ± SEM of two replications. = 0 mM Pi = 5 mM Pi =10 mM Pi
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Wheat
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Figure 2. Loss of UDP from simulated SPS assays lacking F6P and G6P. Assays were conducted with 0, 5, or 10 mm Pi in the assay medium. Bars represent the mean % (two replications) of UDP (155 nmol) remaining after the 15 min assay period.
field-grown, nontopped tobacco (data not shown), the UDP assay was not linear and the apparent SPS activities were much lower relative to the sucrose assay method. This result was due to a loss of UDP during the assay. A loss of UDP from the SPS assay was demonstrated directly by adding representative amounts of UDP to assays lacking F6P and G6P (Figs. 2 and 3, 0 mM Pi). For spinach and maize, nearly 100% of the UDP added to a simulated assay medium remained after 15 min, whereas for wheat and
SPECIES VARIATIONS IN SUCROSE-PHOSPHATE SYNTHASE control
100
= 5 mM Pi 1iJ10 mM Pi
(non-topped) 80 +
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Table I. Effect of Inorganic Phosphate in the Assay Medium on SPS Activity Determined by Sucrose Formation or on Apparent SPS Activity Determined by UDP Formation in Desalted Extracts of Several Plant Species Plant Material
SPS Activity Pi
(non -topped) (topped)
jF\1A \r
Field
Figure 3. Loss of UDP from simulated SPS assays lacking F6P and G6P for tobacco grown under differing environments and subjected to different source:sink manipulations. Bars represent the mean % (two replications) of UDP (155 nmol) remaining after the 15 min assay period.
soybean nearly all of the UDP was depleted from the assay medium (Fig. 2). In addition, for wheat, the inclusion of Pi in the assay medium inhibited the loss of UDP (Fig. 2). For tobacco, the loss of UDP from the assay medium was variable and depended on the environment in which the plants were grown (Fig. 3). Loss of UDP was less for greenhouse-grown compared with field-grown tobacco, particularly for nontopped, greenhouse-grown tobacco. For field-grown tobacco, nearly all of the UDP was depleted from the assay medium. Inclusion of Pi in the assay had the general effect of inhibiting the loss of UDP, but the effect was quite variable depending on the environment in which the tobacco plants were grown (Fig. 3). Sucrose depletion from assays lacking F6P and G6P was minimal during the 15 min assay time for all of the species tested (data not shown). From these results it can be concluded that caution should be used when assaying for SPS activity in leaf extracts by the UDP method. The loss of UDP from the assay is highly variable among species, and in addition, the results for tobacco indicated that within species variation exists depending on the environment in which the plants are grown. The effect of Pi on light-activated SPS activity depended on the source of the enzyme. The inhibition of SPS activity by Pi was greatest for spinach relative to the other species tested (Tables I and II). At 10 mM Pi, spinach SPS activity was inhibited 35 to 40%, with similar results for both assay methods. This level of inhibition was similar to that reported by Doehlert and Huber (4) for similar assay conditions. Pi inhibition of the other species tested, when assayed by the sucrose method, was much less than for spinach, ranging from 0 to 17% inhibition at 10 mm Pi. With the exception of spinach and maize, and to a lesser extent greenhouse-grown, nontopped tobacco, the Pi effect observed using the UDP assay was markedly different from results for the sucrose assay. In fact, in several cases, Pi caused an apparent stimulation of SPS activity when measured by the UDP assay. This apparent stimulation of SPS activity by Pi was due to a decreased loss of UDP from the assay medium when Pi was present (Figs. 2 and 3). The effect of Pi on
471
mM
Spinach
Sucrose assay UDP assay % of controla
5 10 5 10 5 10 5 10
81 65 90 88 102 87 94 93
78 60 Maize 88 81 Wheat 123 121 Soybean 104 106 a SPS activities of 0 mm Pi controls as umol product g fresh wt-r hW1 ± SEM were, for the UDP and sucrose assays, respectively, 95.4 ± 1.3 and 92.7 ± 6.2 for spinach; 123.9 ± 1.8 and 127.3 ± 1.7 for maize; 25.9 ± 0.2 and 58.0 ± 0.1 for wheat; 10.9 ± 0.5 and 80.1 + 0.8 for soybean. Table II. Effect of Pi in the Assay Medium on SPS Activity Determined by Sucrose Formation or on Apparent SPS Activity Determined by UDP Formation in Extracts of Tobacco Grown Under Different Environments and Subjected to Different Source:Sink Manipulations SPS Activity
Environment
A. Greenhouse-May
Source:Sink
Pi
Sucrose
StatusSurs assay
Nontopped
UDP
UD
assay
mM
% of controla
5 10 5 10 5 10 5 10 5 10
86 83 85 84 85 83
91 85 B. Greenhouse-May Topped 92 91 C. Greenhouse-Feb Topped 98 99 D. Field 97 145 Nontopped 88 153 E. Field 97 116 Topped 98 129 a SPS activities of 0 mm Pi controls as Amol product g fresh wt-1 hW1 ± SEM were, for the UDP and sucrose assays, respectively, 49.2 ± 0.9 and 61.0 ± 3.7 for A; 32.2 ± 0.9 and 44.9 1.1 for B; 10.2 + 0.4 and 16.4 ± 2.8 for C; 22.4 ± 2.3 and 133.4 + 0.4 for D; 15.7 + 1.3 and 73.6 ± 2.0 for E.
decreasing the loss of UDP from the assay medium was quite variable and, as indicated by the results for tobacco (Fig. 3), depended on the environment in which the plants were grown. The results of the present study indicated that the effect of Pi as an inhibitor of light activated SPS activity was variable, having a much greater effect on spinach than on the other four species tested. The Pi inhibition may change depending on substrate levels and light/dark transitions (10), and therefore our results only suggest that Pi is not a universal inhibitor of light activated SPS. The effect of Pi should, therefore, be evaluated on a species by species basis, preferably with purified SPS. The results also demonstrated that determination of SPS activity via UDP formation has several potential problems.
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The metabolism of UDP by the desalted extracts varied markedly among the species tested and, perhaps more importantly, the growing environment also affected the metabolism of UDP. In addition, the presence of Pi decreased the metabolism of UDP for some of the species tested and this effect was also influenced by the growing environment. Thus, the possibility arises that UDP metabolism may vary during a time course experiment or for cultivars within a given species.
4. 5.
6.
ACKNOWLEDGMENTS
7.
We thank T. G. Sutton for the expert technical assistance and an anonymous reviewer for suggesting that loss of UDP from SPS assays may be species specific.
8.
LITERATURE CITED 1. Amir J, Preiss J (1982) Kinetic characterization of spinach leaf sucrose-phosphate synthase. Plant Physiol 69: 1027-1030 2. Chifnet S, Torriglia A, Chiesa R, Tolosa S (1988) A method for the determination of inorganic phosphate in the presence of labile organic phosphate and high concentrations of protein: Application to lens ATPases. Anal Biochem 168: 1-4 3. Crafts-Brandner SJ, Leggett JE, Sutton JG, Sims JL (1987)
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Effect of root system genotype and nitrogen fertility on physiological differences between burley and flue-cured tobacco. I. Single leaf measurements. Crop Sci 27: 535-539 Doehlert DC, Huber SC (1983) Regulation ofspinach leaf sucrose phosphate synthase by glucose-6-phosphate, inorganic phosphate and pH. Plant Physiol 73: 989-994 Doehlert DC, Huber SC (1984) Phosphate inhibition of spinach leaf sucrose phosphate synthase as affected by glucose-6-phosphate and phosphoglucoisomerase. Plant Physiol 76: 250-253 Evanylo GK, Sims JL (1988) Nitrogen and potassium fertilization effects on yield and quality of burley tobacco. Soil Sci Soc Am J 51: 1536-1540 Fehr WR, Caviness CE (1977) Stages of soybean development. Spec Rep No 80, Coop Ext Serv, Agric and Home Econ Exp Stn, Iowa State Univ, Ames, IA Harbron S, Foyer C, Walker D (1981) The purification and properties of sucrose-phosphate synthase from spinach leaves: The involvement of this enzyme and fructose bisphosphatase in the regulation of sucrose biosynthesis. Arch Biochem Biophys 212: 237-246 Sicher RC, Kremer DF (1984) Changes of sucrose-phosphate synthase activity in barley primary leaves during light/dark
transitions. Plant Physiol 76: 910-912
10. Stitt M, Wilke I, Feil R, Heldt HW (1988) Coarse control of sucrose-phosphate synthase in leaves: Alterations of the kinetic properties in response to the rate of photosynthesis and the accumulation of sucrose. Planta 174: 217-230
