Jan 23, 1984 - MARK R. SCHMITT2, WILLIAM D. HITZ, WILLY LIN*, AND ROBERT T. GIAQUINTA. Central Research and Development Department, ...
Plant Physiol. (1984) 75, 941-946 0032-0889/84/75/0941 /06/$0 1.00/0
Sugar Transport into Protoplasts Isolated from Developing Soybean Cotyledons' II. SUCROSE TRANSPORT KINETICS, SELECTIVITY, AND MODELING STUDIES Received for publication January 23, 1984 and in revised form April 18, 1984
MARK R. SCHMITT2, WILLIAM D. HITZ, WILLY LIN*, AND ROBERT T. GIAQUINTA Central Research and Development Department, Experimental Station, E. L du Pont de Nemours and
Company, Wilmington, Delaware 19898 ABSTRACT
The effects of metabolic inhibitors, pH, and temperature on the kinetics of sucrose uptake protoplasts isolated from developing soybean Glycine max L. cv Wye cotyledons were studied. Strcturl requirements for substrate recognition by the sucrose carrier were examined by observing the effects of potential alternate substrates for the saturable component on sucrose uptake. Uptake by the three components (saturable, sulfhydryl reagent-sensitive nonsaturable, and diffusive) was calculated over a range of sucrose concentrations. The saturable component dominated uptake at external sucrose concentrations below 12 millimolar and was approximately equal to the nonsaturable and diffusive components at 44 and 22 millimolar external sucrose, respectively. The three uptake components showed different temperature sensitivities. Increasing external pH decreased both the linear component and the V.,., calculated for the saturable component. Conversely, increasing pH increased the calculated K. (sucrose) for the saturable component. Sucrose uptake by the saturable component was insensitive to several mono- and divalent cations. Competition for uptake of 0.5 millimolar sucrose by several sugars suggested that the 6-D-fructofuranoside bond and molecular size of sucrose were particularly important in sugar recognition by the saturable component carrier.
Understanding the cellular mechanisms associated with assimilate unloading and utilization in agronomically important sinks is central to identifying yield-limiting target sites for chemical or genetic manipulation. In the preceding paper (10), we have shown that sucrose uptake into protoplasts isolated from the developing soybean cotyledons follows biphasic kinetics consisting of a saturable component selective for sucrose and a linear uptake component evident at higher substrate levels. At low external concentrations of sucrose, uptake into protoplasts was markedly sensitive to pH, temperature, metabolic inhibitors, and the membrane-modifying sulfhydryl reagent PCMBS3. Sucrose uptake into protoplasts was also 'Contribution No. 3441 from the Central Research and Development Department, Experimental Station, E. I. du Pont de Nemours, and Co., Wilmington, DE 19898. 2 Visiting scientist. 3Abbreviations: PCMBS, p-chloromercuribenzene sulfonate; TBAH, tetrabutylammonium hydroxide; FCCP, (p-trifluoromethoxy)carbonyl cyanide; E., apparent Arrhenius activation energy. 941
stimulated by fusicoccin, a potent promotor of active H+/K' exchange. In this paper, we present a detailed characterization of sugar uptake into soybean cotyledon protoplasts. Specifically, we focus on the proton, sulfhydryl reagent, and temperature sensitivities of sucrose uptake kinetics; we consider the operation of multiple sucrose uptake mechanisms; and we examine the sugar recognition characteristics of the saturable uptake component.
MATERIALS AND METHODS Protoplasts were isolated from developing 'Wye' soybean (Glycine max L.) embryos essentiallv as described previously (10). Sugar uptake into the protoplasts was also according to Lin et al. (10) except as noted. Tetrabutylammonium hydroxide was obtained from Aldrich and other chemicals from Sigma. For studying the effects of mono- and divalent cations on sugar uptake, purified protoplasts were washed twice in 0.5 M sorbitol, 25 mm Mes-TBAH (pH 6.0) prior to addition to the uptake solution containing sorbitol, Mes-TBAH (pH 6.0), 0.5 mm sucrose, and the appropriate cation. In experiments where pH was varied, 25 mm Hepes-25 mM citrate-KOH was substituted for the standard Mes buffer. The temperature response of sucrose uptake into cotyledon protoplasts was determined using an aluminum thermal gradient block (3). The apparatus consisted of a solid block of aluminum 15.2 cm wide x 10.2 cm high x 61 cm long equipped to allow temperature-regulated fluids to be circulated through channels in each end of the block. By equilibrating the ends of the block at different temperatures, a linear temperature gradient was established along the length of the block. Evenly spaced holes drilled in the top of the block allowed test tubes containing the uptake solutions to be equilibrated simultaneously at a number of temperatures. At appropriate intervals after addition of protoplasts to the uptake solution, aliquots were taken and uptake measured using the silicone oil techniques described above. Effects of PCMBS treatment were studied by providing the indicated concentration of the sulfhydryl reagent throughout the 20-min uptake period (9). Competition by other sugars for sites on sucrose carriers was measured by providing 50 to 100 mm of potential competitors and 0.5 mm radiolabeled sucrose. Total osmotic concentration was maintained at 0.6 M in all assays by reducing the sorbitol in the assay by an amount appropriate to compensate for the added competing sugar. Inulobiose was isolated by the partial hydrolysis of inulin from Dahlia tubers (Sigma) at pH 2 and 65°C for 10 min. The ethanolsoluble fraction from the dried hydrolysate was first fractionated by silica gel chromatography (eluting solvent acetone: l-bu-
SCHMITT ET AL.
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tion dependence of sugar uptake in soybean cotyledon protoplasts can be described by a two-component (saturable and linear) model. PCMBS at 500 Mm in the uptake assay medium inhibits both components of uptake (Fig. 2A). No saturable response is observed in the presence of 500 MM PCMBS. Instead, sucrose uptake in the presence of high PCMBS is linearly dependent on external sucrose concentration. The slope of the linear response in the presence of this level of PCMBS is significantly less than the slope of the linear component of uptake seen in uninhibited protoplasts. In parallel experiments, Maynard and Lucas (13) reported that high (2 mM) PCMBS treatment reduced the linear and essentially eliminated the saturable component of sucrose uptake into Beta leaf discs. In our hands, high PCMBS reduced the sucrose uptake rate to that of L-glucose which is thought to be due to diffusion (11, 16). PCMBS does not appear to affect the permeability of the plasmalemma per se since PCMBS (500 ,uM) did not significantly affect the uptake of either L-glucose or 3-0-methyl glucose over a range (0.5-30 mM) of sugar concenRESULTS AND DISCUSSION trations (data not shown). Low concentrations of PCMBS effect a qualitatively different PCMBS Sensitivity. The relatively nonpermeant sulfhydrylmodifying reagent PCMBS markedly inhibits sucrose uptake into response to increasing concentrations of sucrose than do PCMBS soybean cotyledon protoplasts (10). The extent of inhibition of levels. Figure 2B shows that 10 uM PCMBS selectivity inhibits sugar uptake is dependent upon both the concentration of su- the saturable components of sucrose uptake by soybean cotylecrose present during assay and also the concentration of sulfhy- don protoplasts while leaving the linear component of uptake dryl reagent used (Fig. 1, A and B). At low (0.5 mM) sucrose unaffected. Energy Dependence. We have shown previously (10) that concentrations, uptake is inhibited 90% by 50 MM PCMBS. The apparent Io of PCMBS at 0.5 mm sucrose was between 5 and 20 sucrose uptake from 0.5 mm solution is strongly energy dependent, being inhibited approximately 80% by 15 ,uM FCCP. Sucrose Mm. At higher (50 mM) sucrose concentrations, inhibition by high PCMBS concentrations (200-500 uM) only reached approximately 40% of total uptake. The PCMBS concentration depen300 B -100 AA dence of uptake inhibition at 50 mM sucrose was more compli250 cated than that observed for 0.5 mm sucrose. That the shapes of * 80 A 200 A~~ the inhibition curves for 0.5 and 50 mm sucrose differ qualitatively suggests that more than one component of uptake at 50 }60 150 mM sucrose was affected by PCMBS and that the components 100 were differentially sensitive to PCMBS treatment. 50 In the previous paper (10), we have shown that the concentraE5 20
tanol:water; 14:2:1 v/v) to give a low mol wt fraction. This fraction was treated overnight (25°C, pH 6.5) with excess aglucosidase to remove sucrose, then separated into monosaccharide, disaccharide, and larger carbohydrate'classes by gel exclusion chromatography using Bio-Gel P-2 (2 x 29 cm) eluted with water at 0.8 ml/min. The disaccharide fraction yielded only fructose upon hydrolysis by invertase. Methyl-fl-D-fructofuranoside was synthesized by acidification of an anhydrous methanolic solution of D-fructose with H2SO4. The reaction was stopped after 30 min by addition of 10 N NaOH. The reaction mixture was reduced to a syrup under vacuum at 35C and fractionated by silica chromatography as described above. Eluant fractions containing methyl-p-D-fructofuranoside were pooled and reduced to a thick syrup under vacuum. The '3C-NMR spectra of the resulting product corresponded with published spectra (15).
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FIG. 2. Sucrose uptake kinetics in developing soybean cotyledon protoplasts. A, Uninhibited sucrose uptake (A) and PCMBS (500 gM inhibited sucrose uptake (0) are compared to uptake of L-glucose (0). B, Uninhibited sucrose uptake (A) and sucrose uptake inhibited by 500 #M PCMBS (0) are compared to sucrose uptake in the presence of 10 uM PCMBS (0). C, Uninhibited sucrose uptake (A) and 15 Mm FCCPinhibited sucrose uptake (0) are compared to uptake of L-glucose (0). D, Kinetic model of sucrose uptake data from A above. Total uptake ) and two linear ( ) is separated into a saturable component ( components. Diffusion ( ) is estimated by entry of L-glucose. The PCMBS-sensitive linear component is estimated by the difference between the total linear uptake (- -) and diffusive uptake. -
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SUGAR TRANSPORT IN SOYBEAN COTYLEDON PROTOPLASTS II
uptake kinetics in the presence of FCCP show that the inhibition observed is due to an inhibition of the saturable component of uptake (Fig. 2C) and not of the linear component. In the presence of 20 AM FCCP, we observe no saturable component but rather a linear dependence on sucrose concentration, the slope of the uptake response curve in the preesnce of FCCP being equivalent to the slope of the total linear component in the absence of FCCP. The uptake rate of sucrose in the presence of FCCP is greater than that expected for diffusion alone as estimated by the rate of entry of L-glucose (Fig. 2C). Modeling of Uptake Components. The data presented above suggest to us that a refinement of the two-component model for sucrose uptake previously presented (9, 16) may be in order. Previous workers have treated the linear component of uptake as if it was composed of a single mechanism. However, our observations of the fractional sensitivity of uptake to PCMBS and FCCP lead us to divide the total linear uptake component further into two separate linear components. The sum of these two linear components (diffusive and PCMBS-sensitive nonsaturable) combine to produce the total linear component. This model is illustrated in Figure 2D where total sucrose uptake ( ) is divided into a saturable, energy-dependent component .r-r), and the energy-independent total linear component (-- -). The total linear component is further divided into the diffusive component (-) and the PCMBS-sensitive linear component, illustrated in Figure 2D as the difference between the total linear component and the diffusive component. This model can predict the proportional contribution of the three components (saturable, PCMBS-sensitive nonsaturable, and diffusive) to total sucrose uptake into soybean cotyledon protoplasts at sucrose concentrations up to 50 mm (Fig. 3). The saturable component dominates uptake as external sucrose concentrations approach zero, contributing almost 80% of total uptake at 0.5 mM sucrose. At increasing sucrose concentrations, the proportion of total uptake attributable to the saturable component decreases to a point equaling uptake by the total linear component (nonsaturable and diffusion) at between 10 and 12 mm sucrose. We calculate that uptake by the saturable component equals diffusional uptake and PCMBS-sensitive nonsaturable uptake at about 22 and 44 mM sucrose, respectively. Hence, at purported apoplastic concentrations of sucrose in the seed (1, 8), all three components of uptake may contribute significantly to total sucrose uptake. Extrapolation of proportional contribution to uptake in protoplasts to conditions in situ must be made with caution due to uncertainties as to whether bulk apoplastic sucrose concentrations estimated in intact seeds in fact correctly reflect sucrose concentrations immediately external to the plasmalemma. Nonetheless, it is clear that a number of mechanisms
involved in sucrose acquisition by cotyledonary cells may contribute significantly to total uptake. pH Response. Previously (10), we have shown that uptake at 0.5 mM sucrose was strongly inhibited by alkaline external pH. Data show that pH markedly alters the kinetic responses of the protoplasts to external sucrose from 0.25 to 10 mm (Fig. 4). A summary of the effects of pH on kinetic constants for sucrose uptake is shown in Figure 5. With external pH increasing from 4.0 to 7.5, both Vmax and Km values were altered, with the maximal velocity of the saturable component decreasing with increasing pH and the Km values increasing with pH. A similar response ofKm (sucrose) to pH has been reported by Thorne (17) who found a 3-fold increase in Km from pH 6.0 to 8.0, compared to the 2-fold increase we see between pH 6.0 and 7.5. However, Thorne ( 16) also reported that the Vm,, of the saturable component of sucrose uptake did not change between pH 6.0 and 8.0. In work with Ricinus cotyledons, Komor (5) reported uptake of sucrose at approximately its Km was not affected by pH between 4.5 and 7.0. In contrast, we see the Vma,x of the saturable component doubling between pH 7.5 and 6.0 with a further doubling between 6.0 and 4.0. One possible explanation of the differences in the results of these two studies could be related to the buffering capacity of the cell wall present in whole embryos. The plasma membranes of protoplasts are directly exposed to the pH of the suspending medium and so may be expected to respond more directly to external pH. Using Lineweaver-Burk and Eadie-Hofstee data transformations of our Vmaxc versus pH data (not shown), we calculate a Vmax (H+) of 0.08 to 0.1 1 Mm (r2 = 0.71-0.80). However, note that a change of greater than three orders of magnitude (pH 4.07.5) in proton concentration resulted in only an approximately 4-fold change in maximal velocity. It may be that the H+ binding site on the transport protein is somewhat shielded from changes in external proton concentration. We also consistently noted a decrease in the permeability coefficient (first order constant for total linear uptake) with increasing pH (data not shown) similar to that reported by Maynard and Lucas (12) for phloem loading in sugar beet leaves. We have not separated the effects of pH on the nonsaturable and diffusive components of total linear uptake at this time. A striking feature of the pH effects on Km and Vm< shown in 120 100 1-
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Plant Physiol. Vol. 75, 1984
SCHMITT ET AL. A
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FIG. 5. Dependence ofkinetic parameters ofthe saturable component on external pH. A, Variations in Vm as a function of pH in two experiments (0, A) are expressed as a per cent of the V,",= determined at ph 6.0. B, Variations in Km (sucrose) as a function of pH were measured in twelve experiments.
Figure 5 is the change in both kinetic parameters around pH 6.0, the pH thought to be present in the apoplastic space in vivo (4). Although little or no information is available on the constancy of cotyledon apoplastic pH or on processes which might regulate apoplastic pH, the observed pH effects on the kinetic constants for the saturable component suggest that a relatively small change in apoplastic pH from 6.0 can markedly affect the rate of uptake. It is interesting to note that the responses to pH of Km and Vm., resemble titration curves with pK values around 6.0. The imidazolium group of histidine is singular among protein amino acids with a side chain pK value around 6.0. This group is also commonly involved in H+ catalysis (7, 14) by a number of enzymes. It is intriguing to speculate that a histidyl residue or residues might be involved in sucrose/H+ cotransport. Temperature Response. Rates of sucrose uptake at 0.5 and 50 mm respond to temperature (Fig. 6) with optima at around 40°C. Sucrose accumulation from 0.5 and 50 mm solutions was linear with time at assay temperatures of 13 to 16 and 25 to 27°C over the 20-min assay period (data not shown). Calculations of apparent activation energies and Qio values from Arrhenius plots of the data (Fig. 6, insets) show substantial differences in the temperature responses (Table I). Qio values (1 5-25°C) for uptake at 0.5 and 50 mm sucrose were calculated to be 4.23 and 2.02, respectively, with corresponding apparent E. values of 24.2 and 1 1.9 kcal/mol. Thorne (16) had previously shown a temperature
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FIG. 6. Effects of temperature on sucrose uptake. Sucrose uptake from 50 (A) or 0.5 (B) mm sucrose solutions. Inserts show Arrhenius activation energy plots. Table
Sucrose Uptake Response to Temperatures between 15 and 25°C Tissue Ea Substrate Concn. Qio kcal/mol Sucrose 0.5 mm 24.2 Cotyledon protoplasts 4.23 Sucrose + 0.5 mM PCMBS 20 uM 15.1 2.43 Sucrose 50 mM 11.9 2.02 Sucrose + 50 mM PCMBS 20uM 2.81 1.18 0.5 mM 0.47 1.03 L-glucose I.
Intact embryoa Sucrose 9.39 I mm from J. H. Thorne (personal communication).
1.94
' Data
optimum of 35C for uptake of 1 mm sucrose into intact cotyledons. From these and other data (Thorne, personal communication), we calculate a Qi0 of 1.94 and Ea of 9.4 kcal/mol for uptake into whole embryos at 1 mm external sucrose. Reasons for the lower calculated Qlo and Ea values in intact tissue are unclear but may be related to the greater role of bulk diffusion in whole embryos compared to isolated protoplasts. From the measured Qlo values for uptake of 0.5 and 50 mm sucrose and the calculated proportional contribution of the three components to total uptake at these concentrations (Fig. 3), we can calculate Qlo values for the saturable and PCMBS-sensitive linear components of uptake. The Qlo for the diffusive component was
SUGAR TRANSPORT IN SOYBEAN COTYLEDON PROTOPLASTS II assumed to be 1 from the temperature response of L-glucose uptake shown in Table I. The Qio value for the saturable component is 5.1 compared to 1.2 for the PCMBS-sensitive nonsaturable component. Using these estimates of the Qio values for the three components of uptake, we can predict Qio values under conditions in which the proportional contributions of the components have been altered. Low (10 ,gM) PCMBS concentrations inhibit the saturable component of uptake, but not either linear component (Fig. 2B). Assuming an 89% inhibition of the saturable component at 20 gM PCMBS (Figs. 1 and 3), we can recalculate the expected contributions at the two sucrose concentrations in the presence of 20 Mm PCMBS (Table I). Using the predicted contributions in the presence of low PCMBS, and the estimated Qlo values of the three components, we predict Q,o values for uptake from 0.5 and 50 mM sucrose (containing 20 ,uM PCMBS) to be 2.24 and 1.21, respectively. The observed Qio values under these conditions (2.43 for 0.5 mM and 1.18 for 50 mM) agree well with the predicted values. The slight underestimate of the Q,o at low sucrose and low PCMBS could result from an overestimate of the degree of inhibition achieved at 20 ,Mm PCMBS. While this does not conclusively establish the validity of the modeled response, the agreement between predicted and observed responses increases our confidence in the model. Sugar Recognition Characteristics. To examine the sucrose carrier oligosaccharide recognition characteristics, we tested several sugars as alternate-substrate inhibitors of ['4C]sucrose uptake. Uptake rates of 0.5 mM ['4C]sucrose in the presence of alternate substrates are shown in Table II. The sucrose carrier did not recognize the a-glucopyranosides trehalose, palatinose, or turanose. The glycosidic bond in trehalose is similar to that in sucrose inasmuch as both are dihemiacetals; also, both turanose and palatinose are fructosecontaining a-glycopyranosides. Maltose was recognized by the soybean cotyledon sucrose carrier as has been observed for sucrose carriers in sugar beet leaves ( 13). However, the recognition was low with the apparent Ki about 30 times the Km for sucrose uptake. Since no other f,-fructofuranoside disaccharides were commercially available, inulobiose was isolated from a partial hydrolysate of inulin. Inulobiose was an effective competitor of sucrose for uptake by the carrier, exhibiting an apparent Ki of about twice the Km for sucrose uptake. Methyl-,3-fructofuranoside was also recognized by the carrier. But, like maltose, recognition was weak compared to sucrose. Trisaccharides which contain a sucrose moiety were not recognized by the carrier, as shown by the inability of raffinose and melezitose to compete with sucrose uptake. Some general characteristics of substrate recognition by the sucrose carrier can be derived from the above competition studies. First, the rather strong recognition of inulobiose in addition to the very poor recognition of palatinose and turanose indicate that the recognition site relies most strongly on the fructose portion of the sucrose for binding. In particular, the 13-fructofuranoside bond and its neighboring groups on fructose may particTable II. Competition of Several Glucosides and Fructosides with 0.5 mMf[ 4C]Sucrose Uptake into Soybean Cotyledon Protoplasts Concn. Competitor Uptake Rate mM % control Cellobiose 100 82 Palatinose 100 86 100 89 Raffinose 84 50 Melezitose 54 50 Maltose Inulobiose 22 50 50 58 Methyl-B-D-fructofuranoside
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ipate in binding to the carrier. Second, the poor inhibition by methyl-f3-D-fructofuranoside and the trisaccharides tested indicates that, while the exact stereoconfiguration of glucose moiety may not be required for binding, the sugar must be near a disaccharide in size. Sucrose uptake is thought to occur via a proton cotransport mechanism (4). Other cations appear to have no influence on uptake since no significant differences in uptake rate among saltfree and salt-added (1 or 10 mM K+, Na+, Li', Ca2+, or Mg2+) treatments were found (data not shown). This extends the findings of Lichtner and Spanswick (9) who reported that uptake of 10 mm sucrose into intact cotyledons was insensitive to Na+ and K. CONCLUSIONS Sucrose uptake by protoplasts isolated from developing soybean cotyledons appears to result from simultaneous operation of at least three distinct mechanisms. The first, observable at low (
