Journal of Experimental Botany, Vol. 53, No. 374, pp. 1581-1591,
July 1, 2002
© 2002 Oxford University Press
Kinetic characteristics of chloroplast glucose transport
Received 23 November 2001; Accepted 13 March 2002
1 Department of Biology, University of Dayton, Dayton, OH 45469–2320, USA
1 To whom correspondence should be addressed. Fax: +1 937 775 3320. E-mail: jerome.servaites{at}wright.edu
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Influx of labelled D-glucose into isolated spinach (Spinacia oleracea L. cv. Melody hybrid) chloroplasts was initially rapid followed by a period of slower influx. The stroma glucose concentration attained equilibrium rapidly with low external glucose concentrations and the two were linearly proportional. The period of slower influx resulted from conversion of glucose to acidic products that remained trapped in the chloroplast. As the external glucose concentration increased, the stroma glucose concentration increased less and less, attaining a maximal concentration of 72 mol m–3. The maintenance of an equilibrium stroma glucose concentration lower than that in the external medium is evidence that plastid glucose efflux involves secondary active transport. The equilibrium stroma glucose concentration increased in response to light and protonophoric uncouplers. It is proposed that glucose efflux is coupled with a proton and the stroma glucose concentration equilibrates in response to the proton gradient across the membrane. To determine if glucose is a significant product of starch mobilization, chloroplasts were isolated from spinach leaves labelled with 14CO2 during the preceding light period. Chloroplasts degraded starch at the same rate as the intact leaf. Glucose, maltose, and isomaltose were the principal labelled products that appeared in the medium during starch mobilization. The glucose concentration in the chloroplast was 2 mol m–3, which is similar to the measured Km for zero trans efflux. The data support the role of the glucose translocator as an important component in the pathway for sucrose synthesis at night.
Key words: Key words: Glucose translocator, maltose translocator, starch mobilization, stroma space.
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Introduction |
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During the day as much as one-half of the carbon assimilated by photosynthesis is temporarily stored in the chloroplast as starch. At night, this transitory starch is degraded, the degradation products exported to the cytosol, converted to sucrose, and exported from the leaf (Geiger and Batey, 1967). The biochemical pathway for starch mobilization is one of the least known in plant carbon metabolism. Little is known of the identity of the enzymes that attack the starch grain and produce the efflux products, the membrane translocators that transport the products of starch degradation from the plastid, and the enzymes that convert the products to precursors for sucrose synthesis. Elucidation of the pathway using traditional biochemical techniques has proved difficult (Caspar et al., 1991). Plants contain a number of enzymes that degrade starch or products derived from starch. Many of these enzymes are present as multiple isoforms, some of which are located outside the plastid. Most starch-metabolizing enzymes in photosynthetic tissues appear to be constitutively expressed and specific mechanisms of regulation common in photosynthetic enzymes have not been found (Steup, 1988). Although biochemical studies do not give clear evidence of regulation, physiological measurements have shown that starch mobilization is a highly regulated process (Fondy et al., 1989; Servaites et al., 1989a, b).
To date, confusion exists regarding the exact identity of the starch degradation products exported from the chloroplast (Veramendi et al., 1999; Paul and Foyer, 2001). This knowledge is important, because it indicates the end-products of the starch degradation pathway in the chloroplast and the substrates for the sucrose synthesis pathway in the cytosol. In an elegant labelling study with deuterium-labelled water, Schleucher et al. (1998) showed that starch carbon in leaves of tomato and bean is converted to sucrose by a pathway involving phosphoglucoisomerase and not triose-P isomerase. They concluded that most of the starch carbon leaves the chloroplast at night as Glc, maltose, or maltodextrins.
Schäfer et al. (1977) demonstrated the presence of a Glc translocator on the chloroplast envelope and suggested that it could play a role in starch mobilization. Recently, the authors were able to obtain the complete DNA sequence for a putative plastid Glc translocator from spinach leaf (Weber et al., 2000). Similar sequences were found in cDNA libraries from leaves of potato, tobacco, maize, and Arabidopsis thaliana and from developing apricot fruit, indicating that this same Glc translocator is expressed in both sink and source tissues. This translocator is specific for Glc or derivatives of Glc at the C2 position (mannose and 2-deoxy-Glc) and does not transport maltose (Weber et al., 2000). Herold et al. (1981), Beck (1985), and Rost et al. (1996) demonstrated that maltose is taken up by spinach chloroplasts and postulated the presence of a maltose translocator. The maltose translocator is specific for maltose and does not transport Glc (Rost et al., 1996). Rost et al. (1996) showed that maltodextrins (Glc3–Glc7) do not penetrate the chloroplast envelope.
Sequence information reveals little about the operation of the plastid Glc translocator in vivo. For this reason, both Glc influx and efflux were examined in isolated chloroplasts to learn more of the role of this translocator in starch mobilization. Schäfer et al. (1977) described plastid Glc influx as carrier-mediated facilitated diffusion and similar in mechanism to erythrocyte Glc transport. However, a number of peculiarities were associated with plastid Glc influx (Schäfer et al., 1977; Rost et al., 1996). At equilibrium, the stroma Glc concentration was only about half of the external Glc concentration and decreased with increasing external Glc concentration. Here, it is shown that these peculiarities result from the fact that plastid Glc efflux is an active process coupled with the transport of a proton. Furthermore, it is shown that Glc, maltose, and isomaltose are the major products of starch degradation exported from isolated chloroplasts during starch mobilization.
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Chemicals
Radioisotopes were purchased from NEN Life Science (Boston, MA, USA). Other chemicals were purchased from Sigma Chemical Co. (St Louis, MO, USA). Sorbitol (S-7547) contained 0.5 mg Glc g–1 based on enzymatic analysis and was used without further purification. Enzymes were purchased from Boehringer-Mannheim (Indianapolis, Indiana, USA).
Plant material
Spinach (Spinacia oleracea L. cv. Melody hybrid) plants were grown in 4 dm3 containers containing a mixture of Metromix 360 (Scotts Sierra, Marysville, OH, USA): sand (1:1, v/v) in an environmentally-controlled cabinet having a 12 h photoperiod (25/20 °C day/night). A combination of sodium vapour and metal halide lamps provided stepped increases in photosynthetic photon flux (PPF) culminating in a maximum of 600 µmol m–2 s–1 during the middle 4 h of the light period. Plants were irrigated at 6 h intervals with a balanced nutrient solution.
Chloroplast isolation
For transport studies, spinach leaves from 6-week-old plants were harvested about 1 h before the beginning of the photoentrained light period. Chloroplasts were isolated and purified by centrifugation through 40% (v/v) Percoll according to Robinson (1983), then collected by centrifugation and resuspended in solution B (330 mol m–3 sorbitol, 50 mol m–3 HEPES-KOH, pH 7.6, 1 mol m–3 MgCl2, 1 mol m–3 MnCl2, 2 mol m–3 EDTA) twice to remove residual Percoll. Chloroplasts were stored at a concentration of 0.75 kg Chl m–3 on ice until use and were >90% intact as measured by oxygen electrode (Robinson, 1983). Osmotically-shocked chloroplasts were prepared according to Foyer and Lelandais (1996).
Transport assays
Transport assays with times 
5 s were measured using the single oil layer method (Heldt, 1980). Influx was initiated by addition of D-[3H]-Glc (0.3 Ci mol–1) and [14C]-sorbitol (1 Ci m–3) to chloroplasts or osmotically-shocked chloroplasts at 20 °C in room light and terminated by centrifugation of chloroplasts through a single layer of polyphenylmethylsiloxane (silicone oil, AR200, Fluka, Milwaukee, WI, USA) into a termination layer containing glycerol:CH3OH:H2O (2:1:1, by vol). The sorbitol and water-permeable spaces were measured using [14C]-sorbitol and 3H2O, respectively (Heldt, 1980).
Transport assays with times <1 s were measured using the double oil layer method of Gross et al. (1990) as modified by Weber et al. (2000). The following were added in succession to a microcentrifuge tube (0.4 cm3, polypropylene): 50 mm3 of glycerol:methanol:water (2:1:1, by vol), 50 mm3 of polyphenylmethylsiloxane (AR200), 100 mm3 of an assay layer (solution B containing 220 mol m–3 sorbitol and 110 mol m–3 sucrose), 50 mm3 of polyphenylmethylsiloxane (AR200), and 100 mm3 of chloroplast suspension in solution B containing 50 µg of chlorophyll. From extrapolation of time-course measurements of [14C]-P-glycerate influx at 4 °C using a combination of single oil and double oil layer tubes, the residence time for passage through the assay layer was found to be 0.85±0.11 s. Influx was measured by including the labelled transport substrates in the assay layer. Efflux was measured by equilibrating chloroplasts in the labelled transport substrates for 5 min at 20 °C. Preloaded chloroplasts and substrates were then added to tubes and assays initiated by centrifugation of chloroplasts through an assay layer devoid of label. The amount of label present in chloroplasts before efflux was determined simultaneously with the efflux assays using single oil tubes. Howitz and McCarty (1985) describe the details and rationale of the double-oil method for measuring influx and efflux.
In some experiments, chloroplasts were illuminated with 600 µmol m–2 s–1 provided by a metal halide lamp for 5 min before the initiation of assays, during the assay and centrifugation. The lid of the microcentrifuge was fitted with a piece of clear plastic to irradiate the chloroplasts during centrifugation.
After centrifugation, the microcentrifuge tubes were frozen and stored at –70 °C until analysis. In some experiments, the aqueous medium above the oil layer was removed and analysed separately. The lower part of the microcentrifuge tube containing the chloroplast pellet and a small part of the oil layer was removed with a tubing cutter and placed in 1.5 cm3 polypropylene microcentrifuge tube containing 0.45 cm3 of 80% (v/v) methanol and 0.45 cm3 of Tris HCl-washed CHCl3. The chloroplast pellet was dispersed, the tip discarded, and insoluble material removed by centrifugation for 60 s at 12 000 g. Water (0.45 cm3) was added and the sample was mixed by vortex. The two phases were separated by centrifugation. The bottom organic layer was removed and diluted to 5 cm3 with ethanol. A649 and A665 were measured and Chl concentration determined using the extinction coefficients for Chl of Wintermans and DeMots (1965). The aqueous phase was diluted to 1 cm3 with water. An aliquot was removed and radioactivity present was determined by scintillation counting.
Measurement of labelled product efflux from spinach chloroplasts
Two spinach leaves on a mature plant were steady-state labelled with an atmospheric concentration of 14CO2 (0.91 mCi mol–1) during a 12 h day period. The PPF regimen was sinusoidal, increasing gradually from 0 to 600 µmol m–2 s–1 during the first 6 h and then decreasing to 0 during the last 6 h (Servaites et al., 1989a). Thirty minutes before the start of the photoentrained night period, leaves were removed and chloroplasts were extracted under sterile conditions. Chloroplasts were resuspended in sterile solution B and incubated at room temperature with gentle shaking to keep the chloroplasts suspended. At various times, after the start of the photoentrained night period, aliquots were removed and chloroplasts were separated from the medium by centrifugation through silicone oil. After centrifugation, tubes were frozen and stored at –70 °C until analysis. Leaf discs were removed from a leaf adjacent to those used for isolation of chloroplasts. Discs were extracted in chloroform and methanol and separated into insoluble, aqueous, and organic fractions as described previously (Fondy et al., 1989).
Analysis of products
The methanol-insoluble fraction was treated with amyloglucosidase to degrade starch and the liberated Glc was measured using an enzymatic assay and scintillation counting (Fondy et al., 1989). Aqueous products were separated into basic, acidic, and neutral fractions by ion exchange chromatography using Sephadex A25 and C25 according to Redgwell (1980). Aliquots were removed and radioactivity determined by scintillation counting. The remaining neutral fraction was taken to dryness in a rotary evaporator. Glc, fructose, sucrose, and maltose were measured in succession using enzyme-coupled assays (Jones et al., 1977; Beutler, 1984). After successive conversion of hexoses, sucrose, and maltose to 6-P-gluconate, aliquots of the enzymatic reaction mixture were removed and separated into neutral and acidic fractions by Sephadex C25 ion-exchange chromatography. These fractions were acidified, dried, and radioactivity determined by scintillation counting.
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Time-course of Glc influx
Zero trans influx of 4 mol m–3 D-[3H]-Glc into chloroplasts was initially rapid followed by a period of slower influx (Fig. 1). Fractionation of the radioactivity present in the chloroplast pellet using ion-exchange chromatography and enzymatic analyses showed that D-Glc was the radioactive species taken up into chloroplasts following addition of the labelled substrate. With time, some of the Glc in the choroplast was converted to acidic products and contributed to the accumulation of label in the chloroplasts, explaining the period of slower influx. There was no incorporation of label into basic, lipid, or insoluble products, such as starch. The equilibrium Glc minus sorbitol space remained fairly constant with time and was about 0.64 of the sorbitol-impermeable water space.
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The stroma equilibrium Glc concentration as a function of external Glc concentration
The apparent incomplete equilibration of Glc between the sorbitol-impermeable water space and the external medium (Fig. 1) could result if part of the sorbitol-impermeable water space, for example, the thylakoid space, were permeable to water, but not to Glc. To investigate this possibility, the steady-state Glc, water, and sorbitol spaces in intact and osmotically-shocked chloroplasts were measured simultaneously at external Glc concentrations of 0.6–262 mol m–3 (Fig. 2). Measurements were made at 0.5, 2, 5, and 10 min after addition of Glc to obtain accurate estimates of the steady-state spaces. When Glc was added to the external medium, the sorbitol concentration was decreased to keep the concentration of osmoticum at 330 mol m–3. Because with time some of the labelled Glc in the chloroplast is converted to acidic products and accumulates (Fig. 1), Glc was measured using an enzymatic assay. In osmotically-shocked chloroplasts, the sorbitol and Glc spaces were similar, about 40 cm3 g–1 Chl, at all external Glc concentrations, but the water space was about 10 cm3 g–1 Chl higher (Fig. 2A; Table 1). These data indicate that following the osmotic shock treatment the chloroplast envelope was made permeable to sorbitol. The fact that the water and Glc spaces were slightly higher than these spaces measured in intact chloroplasts (Fig. 2B) indicates that the chloroplasts were not ruptured by the mild osmotic shock and remained intact during filtration through silicone oil. These data are interpreted to indicate that the thylakoid membrane is permeable to water, but impermeable to Glc and sorbitol. In intact chloroplasts, the water and sorbitol spaces were about 45 and 20 cm3 g–1 Chl, respectively, at all Glc concentrations (Fig. 2B). The Glc space was intermediate between the sorbitol and water spaces, about 37 cm3 g–1 Chl at low external Glc concentrations. By contrast to osmotically-shocked chloroplasts, the Glc space decreased to 23 cm3 g–1 Chl with increasing external Glc concentration (Fig. 2B, open circles), indicating that Glc was increasingly excluded from the stroma space. These data are interpreted to indicate that in intact chloroplasts the chloroplast envelope is impermeable to sorbitol, but permeable to Glc because of the presence of the Glc translocator. The thylakoid membrane is permeable to water, but impermeable to Glc. As mentioned previously, the Glc and water spaces for osmotically-shocked and intact chloroplasts, at low external Glc concentrations, were very similar. In fact, the ratios of the Glc space to the water space for osmotically-shocked and intact chloroplasts are nearly identical (
0.8, Table 1). These data indicate that low external Glc concentrations equilibrate with the stroma and the two are linearly proportional. This is what would be expected if the Glc translocator were catalysing facilitated transport. However, the decline in the Glc space with increasing Glc in the medium is not typical of facilitated transport systems and requires further explanation.
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The decrease in the ‘Glc space’ could have resulted from either a reduction of the stroma volume or a decrease in the amount of Glc present in the stroma space at equilibrium. Heldt and Sauer (1971) showed that the stroma volume of intact chloroplasts decreased upon increasing the osmolarity of the external medium. Decreasing the stroma volume also decreased the total chloroplast volume, but had little effect upon the volume of the thylakoid and intermembrane spaces. The data in Fig. 2 show that the water space, which is a measure of the total chloroplast volume, did not change with increasing external Glc concentration when the osmolarity of the external medium was kept constant. Hence, it is concluded that, with increasing external Glc concentration, the net entry of Glc into the chloroplast increased less and less. A plot of the stroma equilibrium Glc concentration ([Glc]in) versus the external Glc concentration ([Glc]out) (Fig. 3) shows that the two are linearly proportional at low Glc. As the external Glc concentration increased, the stroma equilibrium Glc concentration increasingly departed from linearity with the external Glc concentration. An Eadie–Hofstee plot of the equilibrium stroma Glc concentration versus the equilibrium Glc minus sorbitol space as a fraction of the sorbitol-impermeable water space (Fig. 3, insert) is linear and intercepts a maximal stroma Glc concentration of 72 mol m–3 at saturating external Glc.
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Further experiments were conducted to determine the effect of various protein modifying agents, uncouplers, and light on the ratio of the equilibrium stroma Glc concentration to that of the medium ([Glc]in/[Glc]out) (Table 2). p-Chloromercuribenzene sulphonate (pCMBS) is a strong inhibitor of Glc influx and completely inhibited influx at a concentration of 40 mmol m–3 (Weber et al., 2000). At 20 mmol m–3, it inhibited zero trans influx by 50%, but inhibited zero trans efflux by only 17% and reduced the equilibrium [Glc]in/[Glc]out ratio to 0.33 (Table 2). Diethylpyrocarbonate (DEPC) at 0.5 mol m–3 inhibited zero trans influx by only 15%, but inhibited zero trans efflux by 43% and increased the [Glc]in/[Glc]out ratio to 2. These data indicate that the equilibrium stroma Glc concentration changes in response to the relative rates of influx and efflux and at equilibrium attains a concentration at which the steady-state rates of influx and efflux are equal. Chloroplasts maintained in the light prior to addition of 20 mol m–3 Glc had a higher equilibrium stroma Glc concentration than that of the medium (Table 2). Addition of gramicidin or CCCP (carbonyl cyanide m-chlorophenylhydrazone) increased the stroma Glc concentration to that of the medium.
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Concentration response of zero trans and equilibrium exchange influx and efflux
Rates of zero trans (Fig. 4A) and equilibrium exchange (Fig. 4B) influx and efflux were measured between 2.5 and 100 mol m–3 external Glc concentrations using a fixed time period of 0.85 s. Flux rates were estimated from changes of 2–46% of the equilibrium Glc space. As a consequence, flux rates measured at low Glc concentration were underestimated by 16% because of non-linearity of the time-course. Near zero trans influx was measured by the passage of chloroplasts through an assay layer containing varying concentrations of [3H]-Glc and a fixed amount of [14C]-sorbitol. Usually, zero trans influx is measured by fixing the substrate concentration at the trans or stroma face at zero and varying the substrate concentration at the cis or external face. The stroma Glc concentration measured in chloroplasts extracted from leaves at the end of the night period and equilibrated in a Glc-free medium was about 0.6 mol m–3. Darkened chloroplasts contain a small amount of Glc because of the mobilization of starch (Stitt and Heldt, 1981). Hence, these measurements are approximations of zero trans influx. To measure zero trans efflux, chloroplasts were allowed to equilibrate with label for 5 min. Efflux was then measured in tubes containing an assay layer without added Glc. The assay layer had a measured residual Glc concentration of 70 mmol m–3. The efflux space was the difference between the equilibrium Glc minus sorbitol space measured using the single-oil method and the same space after efflux. Equilibrium exchange influx and efflux were measured using equilibration and assay layers having the same Glc concentrations. Approximations of Km and Vmax values (Table 3) were calculated from a least squares fit of Eadie–Hofstee plots of v versus s–1 (Fig. 4A, B).
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Is Glc a significant product of starch mobilization?
Two leaves on a spinach plant were subjected to steady-state labelling with an atmospheric concentration of 14CO2 during the preceding light period to label the starch pool uniformly. Thirty minutes before the start of the photoentrained night period, these leaves were removed and chloroplasts isolated. At various times, chloroplasts were separated from the external medium by centrifugation through a single layer of silicone oil, terminated in 25% (v/v) CH3OH, and immediately frozen. The concentrations of starch and soluble basic, acidic, and neutral products (Glc, fructose, sucrose, and maltose) were measured in the aqueous medium above the silicone oil layer (Fig. 5A) and in the chloroplast pellet (Fig. 5B) using enzyme coupled assays and radioactivity measurement. For comparison, these same metabolites were measured in leaf discs removed at various times during the day and night periods from an intact spinach leaf adjacent to those used for preparing chloroplasts (Fig. 6). Starch levels in isolated chloroplasts and in the intact leaf were similar on a Chl basis and decreased at the same rate during the first 4 h of the night period, about 4.3 milliatom C kg–1 Chl s–1 (Fig. 6). Efflux of labelled carbon from chloroplasts during the first 0.5 h of the night period was about 2.9 milliatom C kg–1 Chl s–1, about two-thirds of the rate of starch degradation, and decreased to 0.7 milliatom C kg–1 Chl s–1 after 4 h (Fig. 5A). The remainder of the starch that was degraded was assumed to be lost as respired CO2. Maltose or compounds that yielded Glc in the presence of
-glucosidase accounted for about 50% of the label appearing in the medium (Fig. 5A). It is inferred that about 50% of the maltose that accumulated in the medium was isomaltose, because the time-course for maltose hydrolysis by -glucosidase was initially rapid (minutes) followed by a much slower (hours) rate of hydrolysis. The hydrolysis of standard maltose (4-O--D-glucopyranosyl-D-Glc) was completed within 25 min. However, hydrolysis of standard isomaltose (6-O--D-glucopyranosyl-D-Glc) was much slower and required 4 h for completion. Glc accumulated in the medium and accounted for about 20% of the starch carbon (Fig. 5A), while chloroplast levels remained constant (Fig. 5B). Sucrose increased in the medium (Fig. 5A) and decreased in the chloroplast slowly with time (Fig. 5B). Fructose concentrations in the medium and in the chloroplast were constant (data not shown), about 0.44±0.09 and 0.05±0.01 milliatom C g–1 Chl, respectively. Acidic and basic compounds in the chloroplast were constant (Fig 5B), only small amounts accumulated in the medium and at 4.5 h were 2.2±0.1 and 0.9±0.07 milliatom C g–1 Chl, respectively (data not shown). In intact leaves, sucrose, and Glc levels decreased slowly during the night period (Fig. 6). The levels of maltose and isomaltose in the leaf and in the chloroplast pellet were less than the detectable limit of 50 microatom C g–1 Chl (data not shown).
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Direct measurement of the intermembrane, stroma, and thylakoid spaces
The biphasic nature of labelled Glc influx into isolated chloroplasts (Schäfer et al., 1977; Rost et al., 1996; Fig. 1), i.e. a period of rapid influx followed by a period of slower influx, can be explained by the conversion of some of the labelled Glc in the chloroplast to acidic products, hexose-P that do not penetrate the chloroplast envelope, become trapped in the chloroplast, and accumulate (Fig. 1). The amount of labelled Glc converted to acidic products would be small during short-term incubations (<1 s), but would significantly overestimate the equilibrium Glc concentration and could be mistaken for the rate of Glc influx during longer incubations. To avoid this problem, Glc was measured using an enzymatic assay to estimate the equilibrium Glc space.
Estimates of the Glc, water, and sorbitol spaces measured in osmotically-shocked and intact chloroplasts indicated that Glc, like sorbitol, does not penetrate the thylakoid membrane (Fig. 2; Table 1). However, unlike sorbitol, Glc enters the stroma rapidly via the Glc translocator and at low concentration is present in the stroma and the medium at the same concentration (Fig. 3). Simultaneous measurements of the sorbitol, Glc, and water spaces allowed for estimates of the intermembrane, stroma, and thylakoid spaces (Table 1). The sizes of the thylakoid and stroma spaces have hitherto not been measurable directly in intact chloroplasts (Heldt, 1980). A rough estimation of the size of the thylakoid space based upon planimetry of electron micrographs from isolated spinach chloroplasts yielded a mean value of 3.3 cm3 g–1 Chl or 12.5% of the sorbitol-impermeable water space (Heldt et al., 1973). However, measurements of the volume of isolated thylakoids and osmotically-shocked chloroplasts using 3H2O and [14C]-sorbitol gave estimates of 10 (Pick and McCarty, 1980; Table 1) and 6.5 cm3 g–1 Chl (Foyer and Lelandais, 1996), values similar to those measured here for intact chloroplasts, 7.8 cm3 g–1 Chl (Table 1). It is estimated that the thylakoid space is about 31% of the internal volume of chloroplasts used in this study.
The stroma equilibrium Glc concentration as a function of external Glc concentration
The stroma equilibrium Glc concentration and the external Glc concentration were linearly proportional at low Glc concentrations. However, with increasing external Glc concentration, the equilibrium stroma Glc concentration increased less and less and approached a constant limit, i.e. 72 mol m–3 (Fig. 3). Schäfer et al. (1977) observed this same phenomenon and suggested that increasing Glc concentrations in the stroma increasingly inhibited the rate of Glc influx. An inhibition of either influx or efflux by external Glc concentrations up to 100 mol m–3 (Fig. 4) was not observed. It appears more likely that a higher rate of efflux relative to influx accounts for the reduced [Glc]in/[Glc]out ratio with increasing external Glc concentration. The non-permeable sulphhydryl inhibitor, pCMBS, reduced the [Glc]in/[Glc]out ratio by inhibiting influx more than efflux (Table 2). The opposite was true for DEPC, a reagent that specifically modifies histidyl residues (Lundblad, 1995). The four measured kinetic constants for equilibrium exchange (Table 3) should predict the relationship between the equilibrium stroma and the external Glc concentrations. Due to the imprecision of the methods for measuring Glc flux in chloroplasts, the measured kinetic constants for equilibrium exchange influx and efflux were not significantly different (Table 3). However, using a modeling approach, it was shown that the observed relationship between the equilibrium stroma Glc concentration and the external Glc concentration (Fig. 3) can only occur when the Vmax for equilibrium exchange efflux is slightly higher than that for influx (Table 3).
At equilibrium,
vin=vout, then(1)
Solving for [Glc]in, gives:
If Kin=Kout and Vin=Vout, then [Glc]in and [Glc]out would be the same at all Glc concentrations (Fig. 7, line A). This condition is seen in most facilitated transport systems. If the Km for equilibrium exchange influx (Kin) were 2-fold higher than that for equilibrium exchange efflux (Kout), then [Glc]in for a given [Glc]out would be 2-fold lower, but remain linear (Fig. 7, line B). Conversely, a 2-fold lower Kin than Kout would give a similar straight line as well and [Glc]in would be 2-fold greater than [Glc]out. (Fig. 7, line C). A mere 5% difference between the Vmax for equilibrium exchange influx and efflux would produce lines D (Vin<Vout) and E (Vin>Vout) (Fig. 7). Clearly, the only line in Fig. 7 resembling the measured relationship shown in Fig. 3 is line D, where the Kin=Kout and Vin<Vout. The means of the measured Km values for equilibrium exchange influx and efflux were not very different, but the Vmax for efflux was slightly higher than for influx (Table 3). Substituting the means of the kinetic constants for equilibrium exchange influx and efflux (Table 3) and the external Glc concentration into equation 3 above gives calculated values for [Glc]in (Fig. 3, dashed line) that show a close fit with the measured values.
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The maintenance of an equilibrium stroma Glc concentration lower than the external Glc concentration is best explained if plastid Glc efflux involved secondary active transport. Usually, secondary active transport systems involve cotransport with another molecule, e.g. a H+. Chloroplasts suspended in a buffered osmoticum of pH 7.6 had a measured stroma pH of about 7 in the dark, which increased to about 8 in the light (Heldt, 1980). If Glc equilibrates across the chloroplast envelope in response to a pH gradient, then illuminated chloroplasts should have an equilibrium [Glc]in/[Glc]out ratio >1, while this ratio should be <1 in darkened chloroplasts. In the absence of a proton gradient the ratio should be one. It was observed that chloroplasts incubated in an external medium (pH 7.6) containing 20 mol m–3 Glc had an equilibrium [Glc]in/[Glc]out ratio of 0.78 in the dark and a ratio of 1.21 in the light (Table 2). Furthermore, addition of the protonophoric uncouplers, CCCP and gramicidin, to chloroplasts in the dark increased the [Glc]in/[Glc]out ratio to 1. These data are preliminary evidence that chloroplast Glc transport is coupled with the transport of a proton. The apparent contradiction between the fact that Glc freely permeates the chloroplast envelope and the fact that Glc can be used as an osmoticum for chloroplast suspensions (Walker, 1980; Weber et al., 2000) without significantly changing the sorbitol and water spaces (Schäfer et al., 1977; Fig. 2) is resolved, because the Glc concentration in the stroma is kept low by active efflux of Glc. Darkened chloroplasts suspended in 330 mol m–3 Glc would have an equilibrium stroma Glc concentration of only 47 mol m–3, which is apparently insufficient to cause lysis.
Further support for glucose-H+ cotransport comes from sequence comparisons. A basic residue (arginine or lysine) is conserved at the beginning of the sixth predicted membrane spanning region of all transporters that are known to catalyse transport by substrate-H+ symport, but not in transporters that have been confirmed to catalyse transport by facilitated diffusion (Griffith et al., 1992). An arginine residue is present in this exact location in sequences of all six plastid Glc translocators sequenced to date (Weber et al., 2000). All of the plant Glc translocators described to date are Glc-H+ symporters. Most of these symporters have Km’s in the mmol m–3 range (Büttner and Sauer, 2000); the exceptions are AtSTP3, a plasmalemma Glc translocator found exclusively in green leaves (Büttner et al., 2000), and the plastid Glc translocator (Table 3), which both have Km’s of about 2 mol m–3.
The observed decrease in the stroma Glc concentration with increasing Glc in the medium could also be explained by an increasing rate of Glc metabolism in the stroma that is faster than the rate of Glc entry into the chloroplast. The relationship between [Glc]in and [Glc]out (Fig. 3) resembles an enzyme-catalysed reaction with a Km of about 70 mol m–3. However, for metabolism to maintain the stroma Glc concentration at a level lower than that in the medium, the rate of Glc metabolism in the chloroplast would need to be greater than transport and increase with increasing Glc concentration up to 250 mol m–3 (Fig. 3). Kruger and ap Rees (1983) found that labelled maltose accumulated in the medium following the incubation of pea chloroplasts in labelled Glc. They suggested that Glc was converted to maltose via stroma maltose phosphorylase and Glc-1-P formed from starch degradation. Maltose was rapidly effluxed from the chloroplast into the medium, thereby preventing the osmotic potential from increasing and rupturing the chloroplasts. In influx experiments using labelled Glc (Fig. 1), the medium was not examined to determine if labelled maltose was accumulating. Pea chloroplast maltose phosphorylase has a Km of 2 mol m–3 (Kruger and ap Rees, 1983), however, the reported Vmax is about three orders of magnitude lower than that for Glc exchange (Table 3). Hence, it appears unlikely that Glc metabolism via maltose phosphorylase could significantly lower the stroma Glc concentration. Glc is phosphorylated in the stroma and could be converted to starch (Stitt and Heldt, 1981). Stitt and Heldt (1981) mentioned that the rate of Glc phosphorylation by spinach chloroplasts increased with increasing Glc concentration in the medium. It was also found that Glc was phosphorylated in the stroma (Fig. 1), but at an external Glc concentration of 4 mol m–3 the rate was about 400 times slower than that of transport.
Glc, maltose, and isomaltose are the principal products of starch mobilization exported from the chloroplast
Previous studies (Peavey et al., 1977; Stitt and Heldt, 1981) investigated the nature of the labelled products appearing during the degradation of labelled starch in isolated spinach chloroplasts and whether these products accumulated inside the chloroplasts or in the medium, however, the results were mixed. Peavey et al. (1977) found that most of the label appearing in the medium was in the form of P-glycerate, and triose-P, while maltose and Glc remained inside the chloroplasts. Stitt and Heldt (1981) reported that Glc, maltose, and most of the phosphorylated intermediates were found in the medium. This question was re-examined by labelling spinach leaves with 14CO2 during the day, chloroplasts were isolated from these leaves, and the concentration of labelled products in the chloroplasts was measured and in the external medium at various times during the photoentrained night period. To prevent hydrolysis of disaccharides, chloroplast metabolism was terminated with methanol rather than acid and the labelled products were fractionated using weakly acidic and basic ion-exchange materials. For comparison, starch and sugar levels were measured simultaneously in an intact leaf from the same plant. The rates of starch degradation in isolated chloroplasts and in the intact leaf were nearly the same (Fig. 6). Most of the labelled starch carbon accumulating in the medium was in the form of neutral sugars, principally Glc, maltose, and isomaltose (Fig. 5A). Small amounts of acidic and basic compounds accumulated in the medium, however, at 4.5 h after extraction, the concentration of these compounds were 130-fold and 500-fold higher, respectively, in the stroma than in the medium. Maltose and isomaltose were found exclusively in the medium and accumulated to a concentration of 0.5 mol m–3; there were no measurable amounts of maltose or isomaltose in the chloroplast pellet fraction. Rost et al. (1996) measured a relatively high Km, 28 mol m–3, for zero trans influx of maltose into spinach chloroplasts. However, this study’s data indicate that the Km for zero trans efflux of maltose must be in the mmol m–3 range. Furthermore, maltose efflux from chloroplasts occurred against a concentration gradient. This could be explained if efflux involved co-transport with a H+ as occurs in yeast (Jiang et al., 2000) and as shown here for plastid Glc transport. It would appear that the maltose translocator also transports isomaltose. Glc accumulated to a concentration of 0.4 mol m–3 in the medium. Hence, on a molar basis the rates of Glc and maltose efflux from the chloroplast were about the same. The stroma Glc concentration was constant at about 2 mol m–3 (Fig. 5B). Stitt and Heldt (1981) reported a similar stroma Glc concentration in spinach chloroplasts during starch mobilization. These data are consistent with this study’s finding that the Km for zero trans efflux is about 2 mol m–3 also (Table 3). As previously reported by Stitt and Heldt (1981), a substantial amount of sucrose was present in spinach chloroplasts after isolation. Sucrose was the predominant neutral sugar in the chloroplast and was present at a concentration of 8 mol m–3 at the start of the night period and decreased slowly with time (Fig. 5A). Because the sucrose was labelled, it must have originated from photosynthesis during the day. The expression of sucrose metabolizing enzymes in plastids (Gerrits et al., 2001) showed substantial sucrose flux into chloroplasts and amyloplasts. The mechanism by which sucrose enters plastids is unknown. Sucrose efflux from the chloroplast was slow and does not appear to be carrier-mediated (Fig. 5B). Gerhardt et al. (1987) measured a maximal sucrose concentration in the cytosol of darkened spinach leaves of about 10 mol m–3. Hence, sucrose in the cytosol may slowly equilibrate by diffusion with the stroma during the day and vice versa at night.
Glc, maltose, and isomaltose are effluxed from the chloroplasts in significant amounts, indicating that there must be enzymes in the cytosol that metabolize these molecules to precursors for sucrose synthesis. The fact that no measurable amounts of maltose or isomaltose are present in leaves during starch mobilization (Herold et al., 1981; Fig. 6) indicates that these molecules are rapidly metabolized in the cytosol. Maltose accumulated in protoplasts treated with mannose, a sugar that rapidly depletes the Pi pool (Herold et al., 1981), indicating that metabolism of maltose may be through maltose phosphorylase. Hexokinase 1 is present in the outer membrane of spinach chloroplasts and explains how Glc is efficiently phosphorylated after efflux from the chloroplast (Wiese et al., 1999). Antisense repression of hexokinase 1 resulted in an overaccumulation of starch in leaves of potato (Veramendi et al., 1999) indicating that this enzyme catalyses a requisite step in starch mobilization and a necessary step for the metabolism of both maltose and Glc to sucrose (Fig. 8).
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Two complementary sources of carbon provide for uninterrupted sucrose synthesis
Studies that have measured rates of photosynthesis, starch synthesis and degradation, and export simultaneously have shown that these processes are highly regulated in relation to one another. When the supply of triose-P for maintaining sucrose synthesis is reduced, either because of a reduced level of light (Fondy et al., 1989; Servaites et al., 1989a, b), CO2 (Fox and Geiger, 1986), or a reduction in the capacity to transport triose-P from the chloroplast (Häusler et al., 1998) starch is mobilized to supplement sucrose synthesis. Newly-fixed carbon and starch are complementary sources of carbon for sucrose synthesis, providing a nearly continuous supply of carbon for export to plant organs throughout the day and night. The pathway for sucrose synthesis in leaves can be considered as one pathway with two sources supplying carbon for sucrose synthesis independently of photosynthesis rate (Fig. 8). Triose-P derived from the C3 photosynthetic cycle and exiting the chloroplast through the triose-P translocator is the primary source of carbon for sucrose synthesis when photosynthesis is high, while maltose and Glc derived from starch and exiting by separate carriers are the main source of carbon for sucrose synthesis during periods of low photosynthesis. When carbon flux through the C3 cycle falls below a threshold, these two sources of carbon maintain export near the daytime rate. These different metabolites exit the chloroplast through separate translocators and are metabolized in the cytosol to hexose-P through different biochemical reactions (Fig. 8), thereby permitting separate regulation. The nature of the translocated species imparts specificity to the associated biochemical reactions inside and outside the chloroplast. Triose-P exiting via the triose-P translocator enters a biochemical pathway regulated by cytosolic fructose bisphosphatase. Fructose 2,6-bisphosphate is a regulatory metabolite that strongly inhibits cytosolic fructose bisphosphatase (Stitt, 1990). During the transition from light to dark, the fructose 2,6-bisphosphate concentration in leaves increases markedly (Servaites et al., 1989a, b) and the concentrations of triose-P and fructose 1,6-bisphosphate decrease to zero (Gerhardt et al., 1987), indicating that the synthesis of triose-P and its conversion to hexose-P in the cytosol have ceased. Glc and maltose derived from starch and allocated to sucrose synthesis exit via the Glc and maltose translocators and are converted directly to hexose-P by hexokinase and cytosolic maltose phosphorylase, thereby, bypassing the cytosolic fructose bisphosphatase step. Carbon from the two sources ultimately mixes in a common hexose-P pool. Sucrose synthesis, which uses hexose phosphates derived from either or both sources of carbon, is regulated by sucrose-P synthase. Thus, the contribution of each carbon source to sucrose synthesis is regulated separately from sucrose synthesis itself.
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This work was supported in part by grants to DRG from the Monsanto Company, and grants to JCS from the US Department of Agriculture, National Research Initiative Competitive Grants Program, 9501219, the Ohio Plant Biotechnology Consortium, and the University of Dayton Venture Funds Program. We thank Mark Fuchs for growing the plants and for conducting the steady-state labelling experiments.
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|---|
Beck E. 1985. The degradation of transitory starch in chloroplasts. In: Heath RL, Preiss J, eds. Regulation of carbon partitioning in photosynthetic tissue. Baltimore: Waverly Press, 27–44.
Beutler H-O. 1984. Maltose. In: Bergmeyer HU, ed. Methods of enzymatic analysis, 3rd edn, Vol. 6, 119–126.
Büttner M, Sauer N. 2000. Monosaccharide transporters in plants: structure, function, and physiology. Biochimica et Biophysica Acta 1465, 263–274.[Medline]
Büttner M, Truernit E, Baier K, Scholz-Starke J, Sontheim M, Lauterbach C, Huss VAR, Sauer N. 2000. AtSTP3, a green leaf-specific, low affinity monosaccharide-H+ symporter of Arabidopsis thaliana. Plant, Cell and Environment 23, 175–184.
Caspar T, Lin T-P, Kakefuda G, Benbow L, Preiss J, Somerville C. 1991. Mutants of Arabidopsis with altered regulation of starch degradation. Plant Physiology 95, 1181–1188.
Fondy BR, Geiger DR, Servaites JC. 1989. Photosynthesis, carbohydrate metabolism and export in Beta vulgaris L. and Phaseolus vulgaris L. during square and sinusoidal light regimes. Plant Physiology 89, 396–402.
Fox TC, Geiger DR. 1986. Osmotic response of sugar beet source leaves at CO2 compensation point. Plant Physiology 80, 239–241.
Foyer CH, Lelandais M. 1996. A comparison of the relative rates of transport of ascorbate and glucose across the thylakoid, chloroplast and plasmalemma membranes of pea leaf mesophyll cells. Journal of Plant Physiology 148, 391–398.
Geiger DR, Batey JW. 1967. Translocation of 14C sucrose in sugar beet during darkness. Plant Physiology 42, 1743–1748.
Gerhardt R, Stitt M, Heldt HW. 1987. Subcellular metabolite levels in spinach leaves. Plant Physiology 83, 399–407.
Gerrits N, Turk SCHJ, van Dun KPM, Hulleman SHD, Visser RGF, Weibeek PJ, Smeekens SCM. 2001. Sucrose metabolism in plastids. Plant Physiology 125, 926–934.
Griffith JK. Baker ME, Rouch DA, Page MGP, Skurray RA, Paulson IT, Chater KF, Baldwin SA, Henderson PJF. 1992. Membrane transport proteins: implications of sequence comparisons. Current Opinion in Cell Biology 4, 684–695.[Medline]
Gross A, Brückner G, Heldt HW, Flügge U-I. 1990. Comparison of the kinetic properties, inhibition and labelling of the phosphate translocators from maize and spinach mesophyll chloroplasts. Planta 180, 262–271.
Häusler RE, Schlieben NH, Schulz B, Flügge U-I. 1998. Compensation of decreased triose phosphate/phosphate translocator activity by accelerated starch turnover and glucose transport in transgenic tobacco. Planta 204, 366–376.[ISI][Medline]
Heldt HW. 1980. Measurement of metabolite movement across the envelope and of the pH in the stroma and the thylakoid space in intact chloroplasts. Methods in Enzymology 69, 604–613.
Heldt HW, Sauer F. 1971. The inner membrane of the chloroplast envelope as the site of specific metabolite transport. Biochimica et Biophysica Acta 234, 83–91.[Medline]
Heldt HW, Werdan K, Milovancev M, Geller G. 1973. Alkalization of the chloroplast stroma caused by light dependent proton flux into the thylakoid space. Biochimica et Biophysica Acta 314, 224–241.[Medline]
Herold A, Leegood RC, McNeil PH, Robinson SP. 1981. Accumulation of maltose during photosynthesis in protoplasts isolated from spinach leaves treated with mannose. Plant Physiology 67, 85–88.
Howitz KT, McCarty RE. 1985. Kinetic characteristics of the chloroplast envelope glycolate transporter. Biochemistry 24, 2645–2652.
Jiang H, Medintz I, Zhang B, Michels CA. 2000. Metabolic signals trigger glucose-induced inactivation of maltose permease in Saccharomyces. Journal of Bacteriology 182, 647–654.
Jones MGK, Outlaw Jr WH, Lowry OH. 1977. Enzymic assay of 10–7 to 10–14 moles of sucrose in plant tissues. Plant Physiology 60, 379–383.
Kruger NJ, ap Rees T. 1983. Maltose metabolism by pea chloroplasts. Planta 158, 179–184.
Lundblad RL. 1995. Techniques in protein modification. Boca Raton, FL: CRC Press.
Paul MJ, Foyer CH. 2001. Sink regulation of photosynthesis. Journal of Experimental Botany 52, 1383–1400.
Peavey DG, Steup M, Gibbs M. 1977. Characterization of starch breakdown in the intact spinach chloroplast. Plant Physiology 60, 305–308.
Pick U, McCarty RE. 1980. Measurement of membrane 
pH. Methods in Enzymology 69, 538–546.
Redgwell RJ. 1980. Fractionation of plant extracts using ion-exchange sephadex. Analytical Biochemistry 107, 44–50.[ISI][Medline]
Robinson SP. 1983. Isolation of intact chloroplasts with high CO2 fixation from sugar beet leaves containing calcium oxalate. Photosynthesis Research 4, 281–287.
Rost S, Frank C, Beck E. 1996. The chloroplast envelope is permeable for maltose, but not for maltodextrins. Biochimica et Biophysica Acta 1291, 221–227.[Medline]
Schäfer G, Heber U, Heldt HW. 1977. Glucose transport into spinach chloroplasts. Plant Physiology 60, 286–289.
Schleucher J, Vanderveer PJ, Sharkey TD. 1998. Export of carbon from chloroplasts at night. Plant Physiology 118, 1439–1445.
Servaites JC, Fondy BR, Li B, Geiger DR. 1989a. Sources of carbon for export from spinach leaves throughout the day. Plant Physiology 90, 1168–1174
Servaites JC, Geiger DR, Tucci MA, Fondy BR. 1989b. Leaf carbon metabolism and metabolite levels during a period of sinusoidal light. Plant Physiology 89, 403–408.
Steup M. 1988. Starch degradation. In: The biochemistry of plants, Vol. 14. New York: Academic Press, 255–296.
Stitt M. 1990. Fructose-2,6-bisphosphate as a regulatory metabolite in plants. Annual Review of Plant Physiology and Molecular Biology 41, 153–185.[ISI]
Stitt M, Heldt HW. 1981. Physiological rates of starch breakdown in isolated intact spinach chloroplasts. Plant Physiology 68, 755–761.
Veramendi J, Roessner U, Renz A, Willmitzer L, Trethewey RN. 1999. Antisense repression of hexokinase 1 leads to an overaccumulation of starch in leaves of transgenic potato plants, but not to significant changes in tuber carbohydrate metabolism. Plant Physiology 121, 123–133.
Walker DA. 1980. Preparation of higher plant chloroplasts. Methods in Enzymology 69, 94–104.
Weber A, Servaites JC, Geiger DR, Schmidt R, Kofler H, Hille D, Gröner D, Hebbecker U, Flügge U-I. 2000. Identification, purification, and molecular cloning of a putative plastidic glucose translocator. The Plant Cell 12, 787–802.
Wiese A, Gröner F, Sonnewald U, Deppner H, Lerchl J, Hebbecker U, Flügge U-I, Weber A. 1999. Spinach hexokinase I is located in the outer envelope membrane of plastids. FEBS Letters 461, 13–18.[ISI][Medline]
Wintermans JF, DeMots A. 1965. Spectrophotometric characteristics of chlorophyll a and b and their pheophytins in ethanol. Biochimica et Biophysica Acta 109, 448–453.[Medline]
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