Plant Physiol. (1 995) 109: 11 3-1 21
lnduction of Hexose-Phosphate Translocator Activity in Spinach ChIoropIasts’ W. Paul Quick*, Renate Scheibe, and H. Ekkehard Neuhaus Department of Animal and Plant Sciences, University of Sheffield, P.O. Box 601, Sheffield SI O 2UQ, United Kingdom (W.P.Q.); and Pflanzenphysiologie, Universitat Osnabrück, D-49069 Osnabrück, Cermany (R.S., H.E.N.) tein is not generally used for the import of carbohydrates into nonphotosynthetic plastids for conversion to starch. It is now known that most heterotrophic plastids do not possess a stromal Fru-l,6-bisphosphatase necessary for the conversion of triose phosphates to hexose phosphates, the precursor required for starch synthesis (Entwistle and ap Rees, 1990; Kossmann et al., 1992; Neuhaus et al., 1993b), and there is considerable evidence to suggest that most heterotrophic plastids import hexose phosphate as the major precursor for starch synthesis (Tyson and ap Rees, 1988; Hill and Smith, 1991; Neuhaus et al., 1993a, 1993b). The uptake of Glc-6-P is mediated by a specific hexose-phosphate translocator, which is also strictly coupled to the simultaneous uptake of Pi (Borchert et al., 1989; Mohlmann et al., 1995). The situation is more complex in tissues that not only import carbohydrates but also carry out primary CO, fixation, e.g. the green tissues of developing fruits. Recently, experiments have demonstrated that chloroplasts isolated from the fruits of green pepper (Capsicum annuum L.) also possess a highly active hexose-phosphate translocator protein (Batz et al., 1995). The possibility arises that the type of translocator found in any tissue may change to fulfill the metabolic demands placed upon that tissue. The presence of a protein catalyzing the uptake of Glc-6-P into plastids may therefore reflect the physiological state of a tissue (e.g. source or sink for carbohydrates) rather than the specific tissue type. This hypothesis is reinforced by the recent finding that the amount of mRNA encoding the triosephosphate translocator protein is strongly decreased after feeding Suc to tobacco seedlings (Knight and Gray, 1994). To test the hypothesis that the presence of a hexosephosphate translocator in plastids reflects the physiological state of a tissue, we fed Glc via the transpiration stream to mature spinach leaves to induce a switch between source and sink metabolism. This system has already been well described (Krapp et al., 1991, 1993), and it has been clearly demonstrated that Glc feeding leads to a large accumulation of starch and a corresponding decrease of the activities of enzymes involved in the process of CO, fixation (Krapp et al., 1991, 1993). In this paper, we characterize the trans-
Many environmental and experimental conditions lead t o accumulation of carbohydrates in photosynthetic tissues. This situation i s typically associated with major changes in the mRNA and protein complement of the cell, including metabolic repression of photosynthetic gene expression, which can be induced by feeding carbohydrates directly t o leaves. I n this study we examined the carbohydrate transport properties of chloroplasts isolated from spinach (Spinacia oleracea L.) leaves fed with glucose for severa1 days. lhese chloroplasts contain large quantities of starch, can perform photosynthetic 3-phosphoglycerate reduction, and surprisingly also have the ability t o perform starch synthesis from exogenous glucose-6phosphate (Clc-6-P) both in the light and in darkness, similarly to heterotrophic plastids. Clucose-1 -phosphate does not act as an exogenous precursor for starch synthesis. Light, ATP, and 3-phosphoglyceric acid stimulate Clc-6-P-dependent starch synthesis. Short-term uptake experiments indicate that a nove1 Clc-6-P-translocator capacity i s present in the envelope membrane, exhibiting an apparent K, of 0.54 mM and a V,,, of 2.9 pmol Clc-6-P mg-’ chlorophyll h-’. Similar results were obtained with chloroplasts isolated from glucose-fed potato leaves and from water-stressed spinach leaves. l h e generally held view that sugar phosphates transported by chloroplasts are confined to triose phosphates i s not supported by these results. A physiological role for a Clc-6-P translocator in green plastids i s presented with reference t o the source/ sink function of the leaf.
Plant plastids contribute significantly to the carbon metabolism of a cell, particularly in the synthesis and degradation of storage carbohydrates. In terms of carbon metabolism plastids can be divided into two basic types: (a) plastids that export carbohydrates derived either from recently fixed carbon or from the degradation of storage reserves and (b) plastids that import carbohydrates. Typically, carbohydrate-exporting plastids are the chloroplasts localized in leaf tissues. The release of newly synthesized carbohydrate occurs by export of triose phosphates catalyzed by the triose-phosphate translocator (Fliege et al., 1978) and is strictly coupled to the simultaneous uptake of Pi (Fliigge and Heldt, 1984). However, this transport prol This work was financially supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 171, C16 (H.E.N., R.S.) and by a Biotechnology and Biological Sciences Research Council trave1 award to W.P.Q. * Corresponding author; e-mail
[email protected]; fax 44-114-2760159.
Abbreviations: DIDS, 4,4’-diisothiocyano-2,2’-stilbenedisulfonate; NADP-GAPDH, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase; PGA, 3-phosphoglycerate. 113
Quick et al.
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port properties of chloroplasts purified from Glc-fed leaves.
MATERIALS A N D METHODS Leaf Material
Spinach (Spinacia oleracea L.) was grown hydroponically in a growth chamber maintained at 18”C, with a 10-h photoperiod and an irradiance of 250 pmol m-’ s-’. Mature, fully expanded leaves were harvested from 6-weekold plants just prior to the start of the photoperiod. The petioles of harvested leaves were recut under water prior to their incubation in either 50 mM Glc or water. The incubation was carried out in the same growth chamber but at an irradiance of 50 pmol m-’s-’. This light intensity was sufficient to maintain water-incubated leaves in a healthy condition for several days but does not lead to an accumulation of carbohydrates. Potato (Solanum tuberosum L.) plants were grown in a greenhouse at 25°C and a 16-h photoperiod was maintained by supplementary illumination (250 pmol m-’ s-I). Fully expanded leaves were harvested and incubated in 50 mM Glc as described for spinach. Water-stressed spinach leaf material was obtained by floating 1-cm2 discs on 500 mM sorbitol. Discs were then incubated for several days in the growth conditions described above.
lsolation of Chloroplasts
A11 steps of the isolation were carried out at O to 4°C. De-ribbed spinach leaves (15 g fresh weight) were cut into small (5 mm2) pieces and homogenized for three times for 3 s in the 1-dm3 beaker of a Waring Blendor in 120 cm3 of buffer A containing 330 mM mannitol, 30 mM Hepes (pH 7.8, NaOH), 2 mM EDTA, and 0.5% acetone-washed BSA. The homogenate was filtered through three layers of muslin and one layer of Miracloth (Calbiochem),and the filtrate was centrifuged at 2,OOOg in a Sorvall SS34 rotor for 2 min. The supernatant was discarded, and the pellet was resuspended in 1 to 2 cm3 of buffer A. To this chloroplast suspension, 100 (1111, of buffer A containing 35% Perco11 (Pharmacia) were added, mixed, and divided between four 25-cm3 tubes. These were spun at 50,OOOg for 25 min in an ultracentrifuge (Kontron, Munich, Germany; equipped with a Sorvall T 1250 rotor). The chloroplast band, which appeared close to the bottom of the tube, was carefully removed from each tube with a Pasteur pipette; the fractions were pooled and diluted with 50 cm3 of buffer A. This suspension was divided between two tubes and centrifuged at 3,500g for 90 s. The resulting pellet was resuspended in 1cm3 of buffer A minus EDTA and stored on ice. Chloroplasts were used within 2 h of preparation. Chl was determined according to the method of Arnon (1949), and protein was determined according to the method of Bradford (1976) using Coomassie brilliant blue (Serva, Heidelberg, Germany) with BSA as a standard and correcting for BSA present in the medium.
Plant Physiol. Vol. 109, 1995
Chloroplast lntactness and Purity
Chloroplast purity was estimated by measuring markerenzyme activities specific for cellular compartments in both crude extracts and purified chloroplasts. NADP-GAPDH (EC 1.2.1.13) was assayed as a chloroplast marker according to the method of Holtum and Winter (1982); UDP-Glcpyrophosphorylase (EC 2.7.7.9)was assayed as a cytosolic marker according to the method of Bergmeyer (1974); and citrate synthase (EC 4.1.3.7) and a-mannosidase (EC 3.2.1.24) were assayed as mitochondrial and vacuolar marker enzymes, respectively, according to the method of Stitt et al. (1989). Intactness of the chloroplasts was estimated by measuring the latent activity of the chloroplastic marker enzyme NADP-GAPDH in an intact sample (addition of 330 mM sorbitol to the measuring buffer) and in a lysed sample in which the plastids had been disintegrated by ultrasonication (three times for 3 s) prior to measurement (Entwistle and ap Rees, 1988). Chloroplast intactness was also determined by measuring the light-induced rate of ferricyanidedependent O, evolution for intact and osmotically lysed chloroplasts according to the method of Lilley et al. (1975). Photosynthesis Measurements
The rate of photosynthetic evolution was measured using an O, electrode (Hansatech-Bachofer,Reutlingen, Germany). Chloroplasts, equivalent to 50 pg of Chl, were incubated in 1 cmP3of buffer containing 50 mM Hepes (pH 7.6, KOH), 1 mM MgCl,, 1mM MnCl,, 2 mM EDTA, 330 mM sorbitol, 10 mM NaHCO,, 0.1 mM KH,PO,, and catalase (1000 units cm-”) at 20°C and illuminated with white light at an irradiance of 500 pmol m-’ s-’. After an initial lag period (1-3 min), rates of O2 evolution were generally linear for at least 20 min. Photosynthetic rates were determined in the presence or absence of PGA (0.5 mM). Chloroplasts isolated from Glc-fed leaves often exhibited much higher rates of O, evolution in the presence of PGA. This stimulation was not generally observed in control chloroplasts (see later). Starch Content
The starch content of chloroplasts was assayed as described by Batz et al. (1992); Chloroplasts, equivalent to 50 pg of protein, were added to 500 p L of 50 mM sodiumacetate buffer (pH 4.7) and autoclaved for 3 h. After the sample was cooled, starch degradation was started by the addition of 3 units of both a-amylase and amyloglucosidase and incubated at 37°C for 3 h. The reaction was stopped by incubation at 95°C for 3 min. Glc was subsequently assayed by the addition of 100 FL of the supernatant to a cuvette containing 100 mM Hepes (pH 7.8, KOH), 5 mM MgCl,, 1 mM NADP+, 1 mM ATP, Glc-6-P dehydrogenase (1 unit cm-,) and hexokinase (1 unit cm-”) and following the change in A,,,. Clc-6-P Uptake
The rate of Glc-6-P uptake was measured using the silicon oil centrifugation technique previously described by
Hexose-Phosphate Translocator in Spinach Chloroplasts Heldt and Sauer (1971). Eppendorf reaction tubes (400 pL) were filled with three layers of liquid. The lower layer was 50 pL of 800 mM SUC,the middle layer was 70 pL of silicon oil AR 200 (generous gift of Wacker-Chemie, Munich, Germany), and the upper layer was 100 p L of buffer B containing 15 mM Hepes (pH 7.2, NaOH), 300 mM sorbitol, 1 mM EDTA, 2 mM MgCl,, and various concentrations of [‘4C]Glc-6-P (specific activity, 10-30 MBq/ mmol). Chloroplasts were preloaded with phosphate by addition of KHJ’O, to a final concentration of 10 mM and incubation on ice for 30 min. Just prior to the experiment the chloroplasts were pelleted by centrifugation at 4°C for 1 min at 3000g. The chloroplast pellet was resuspended in buffer B to a final concentration of 1mg Chl ~ m - The ~ . reaction was started by adding 50 pL of the chloroplast suspension to the top layer of the Eppendorf tube with gentle mixing. Incubation was carried out at room temperature (20°C) for 15 s and was terminated by centrifugation for 40 s in a centrifuge equipped with a horizontal rotor (Beckman Microfuge E). Transport rates were corrected for radioactivity trapped in the sorbitol-permeable space. Sorbitol-Permeable Volume
Chloroplasts were incubated as described above but with [14C]sorbitoland 3H,0 to allow calculation of the sorbitolpermeable space as described by Heldt and Sauer (1971). Starch Synthesis from I4C-Labeled Substrates
Incorporation of I4C-labeled substrates into starch was carried out in buffer B according to the method of Batz et al. (1994). Starch synthesis was initiated by adding 150 pL of the chloroplast suspension (diluted with medium B to a concentration of 200 pg Chl ~ m - to ~ )an equal volume of medium B containing l4C-labeled substrates and effectors at double concentration. Incubation was carried out at room temperature for 20 min in Eppendorf reaction vessels (1.5 cm3 volume) that were either maintained in the dark or illuminated with a cold tungsten light source providing an irradiance of 150 pmol m-’ s-’ as measured inside the reaction vessel. Starch synthesis was terminated by incubating the reaction mixture at 95°C for 3 min and subsequent centrifugation of the samples at 16,0008 for 5 min. The pellets were washed twice with 1 cm3 of water and in a final wash with 1 cm3 of 90% ethanol. The washed pellet was resuspended in 100 pL of 100 mM sodium acetate (pH
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4.7) and subjected to amylolytic starch digestion as described above. After a further centrifugation step the resultant supernatant was measured for radioactivity using a Tricarb 2500 scintillation counter (Packard, Darmstadt, Germany). RESULTS High-Starch-Containing Chloroplasts That Are lntact and Free of Contaminants
Glc feeding to leaves led to a large increase in starch content (Krapp et al., 1991, 1993). Isolation of chloroplasts from material with a high starch content by traditional techniques leads to considerable disruption of the envelope membrane and hence reduced yield, activity, and intactness of chloroplast preparations (Walker, 1992). Therefore, we developed a protocol for chloroplast isolation based on a Perco11 density gradient centrifugation procedure that has been described for nonphotosynthetic plastids (Neuhaus et al., 1993b). This protocol produces chloroplasts with a high starch content that are devoid of most other cellular components and that exhibit a high degree of intactness (Table I). Contamination with other cellular components was extremely low; a minor cytosolic contamination (0.2-0.3%) was estimated from the activity of the UDP-Glc pyrophosphorylase, known to be a highly active cytosolic enzyme. Chloroplast intactness was typically greater than 90% as calculated by measuring the latent activity of the stromal marker enzyme NADP-GAPDH in an intact and a lysed sample. This high degree of intactness was confirmed using the rate of ferricyanide-dependent O, evolution (data not shown). The starch content of chloroplasts isolated from Glc-fed leaves was approximately 20 times higher than that of the control plastids isolated from either water-fed leaves or freshly harvested material, demonstrating that the Glcfeeding treatment induced a substantial accumulation of starch. We routinely used detached leaves incubated in water as a control for Glc-feeding experiments. However, in the measurements described here, no differences were observed between detached leaves fed water for up to 8 d and freshly detached material. Incubation of leaves with 50 mM sorbitol resulted in chloroplasts with identical rates of photosynthesis and Glc-6-P-dependent starch synthesis as controls (data not shown), suggesting that the effects observed with 50 mM Glc were not due to osmotic stress.
Table 1. Characterization o f purity, intactness, and starch content o f chloroplasts isolated from mature spinach and potato leaves and from detached leaves fed either water o r 50 mM Glc for 7 d lntactness was determined using latency experiments. See “Materials and Methods” for further details. n.d., Nondetectable. Origin of Plastids
Contamination CVtosol
Mitochondria
Vacuole
%
Spinach, undetached leaves Spinach, water-fed leaves Spinach, Glc-fed leaves Potato, undetached leaves Potato. Clc-fed leaves
0.21 0.27 0.1 9 0.23 0.20
n.d. n.d. n.d. n.d. n.d.
lntactness %
n.d. n.d. n.d. n.d. n.d.
92 91 94 92 92
Starch Content pmol Glc mg-
Chl
0.8 1.5 19.3 Not determined Not determined
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Plant Physiol. Vol. 109, 1995
Photosynthetic Characteristics
Chloroplasts isolated from control leaves according to our protocol were intact and able to perform C0,-dependent O, evolution at high rates (50 pmol mg-I Chl h-l, Fig. 1). Chloroplasts isolated from Glc-fed leaves showed a progressive decline in the rate of C0,-dependent O, evolution with increased times of incubation in Glc. Interestingly, the decline in O, evolution observed in Glc-fed chloroplasts could be prevented by the addition of externa1 PGA (Fig. 1). 1 7 -
Hexose-Phosphate-Dependent Starch Synthesis
c
O
The major question we wished to address was whether the activity of a hexose-phosphate translocator could be induced by feeding carbohydrates to green leaves. In fact, chloroplasts isolated from Glc-fed leaves are able to perform Glc-6-P-dependent starch synthesis (Fig. 2). The rate of Glc-6-P-dependent starch synthesis was increased after 2 d of incubation in 50 mM Glc, and after 7 d of incubation the rate was 15 times higher than in the control chloroplasts purified at the beginning of the experiment (Fig. 2). In contrast, chloroplasts purified from spinach leaves incubated for several days in water did not show any stimulation of the rate of Glc-6-P-dependent starch synthesis, indicating that the effect was Glc specific. Physiological intactness of the plastids was required for Glc-6-P incorporation into starch, which was almost completely abolished when the plastids were lysed prior to the assay (Table 11). Glc feeding not only induced the ability to synthesize starch from Glc-6-P but also led to a decrease in the rate of C0,-dependent starch synthesis (Table 111). The rate of
30 20
200
-
4
6
E
n
n
o '
O
'
I
"
4
2
"
"
6
8
T i m e of I n c u b a t i o n (days)
Figure 2. The rate of Clc-6-P-dependent starch synthesis by spinach or 50 mM Glc chloroplasts isolated from leaves fed either water (O) (O)for varying times. Substrates and effectors were supplied at the following concentrations: Glc-6-P, 5 mM; PGA, 0.5 mM; ATP, 2.0 0.1 mM; and NaHCO,, 5.0 mM. mM; KH,PO,,
Glc-6-P-dependent starch synthesis in chloroplasts isolated from Glc-fed leaves was comparable to the rate of C0,dependent starch synthesis in control chloroplasts (Table 111). To demonstrate whether Glc feeding could induce the ability to use Glc-6-P as a precursor for starch synthesis in other plant species, we repeated the experiments with potato leaves. Potato chloroplasts performed Glc-6-P-dependent starch synthesis when they were purified from leaf tissues incubated with Glc (Table IV). After 4 d of incubation in 50 mM Glc, the rate of Glc-6-P-dependent starch synthesis was 5 times higher than at the beginning of the experiment. In potato, the incorporation of Glc-6-P was strictly light dependent, unlike in spinach, in which light had only a stimulatory effect (see below). Chloroplasts isolated from fruits of green pepper also showed high rates of Glc-6-P-dependent starch synthesis (Batz et al., 1995). However, this rate was strongly enhanced in the presence of light, ATP, and PGA. Chloroplasts isolated from Glc-fed spinach leaves had character-
-
10 -
I
01
O
2
4
6
a
T i m e of Incubation (days)
Figure 1. Rate of O, evolution by spinach chloroplasts isolated from leaves fed Glc or water for several days. The light-saturated rate of photosynthetic O, evolution, measured in a liquid-phase O, electrode, is shown for chloroplasts isolated from spinach leaves fed or 50 mM Clc ( 0 )for varying times. The assay buffer either water (O) and catalase (1 O00 units contained 5 mM NaHCO,, 0.1 mM KH,PO,, ~ m - as ~ effectors. ) Data are also shown for chloroplasts isolated from Glc-fed leaves with 1 mM PGA included in the assay medium (V).
Table II. The Clc-6-P-dependent rate of starch synthesis in chloroplasts that were either intact or lysed by sonication prior to assay Glc-6-P was 5 mM, and 0.5 mM PGA, 2.0 mM ATP, 0.1 mM KH2P0,, and 5.0 mM NaHCO, were present i n addition. Chloroplasts were isolated either from fresh control spinach leaves or from leaves that had been incubated for 7 d with either water or 50 mM Glc. Data are means t SE. Origin of Plastids
Rate of Clc-6-P-Dependent Starch Synthesis lntact plastids nmol
Control leaves H,O-fed leaves Glc-fed leaves
Lysed plastids
C, units mg-' Chl h-'
122 t 23 188 ? 7 1 3 5 7 ? 16
22 2 7 37 ? 3 73 ? 8
Hexose-Phosphate Translocator in Spinach Chloroplasts
Table 111. Comparison of the rates of starch synthesis from 74C02 fixed during photosynthesis or from exogenous [74CIGlc-6-P in chloroplasts isolated from spinach leaves incubated for 7 d with either water or 50 mM Glc Substrates and effectors were supplied at the following concentrations: Glc-6-P, 5 mM; PGA, 0.5 mM; ATP, 2.0 mM; KH,P04, 0.1 mM;
and NaHCO,, 5.0
mM. lncubations were carried out for 20 min at room temperature at an irradiance of 150 pmol m-’ s-’. Rates of photosynthesis were not measured simultaneously but were typically 50 pmol mg-’ Chl h-’. Data are means 2 SE.
Origin of Plastids
Rate of C0,-Dependent Starch Synthesis
Rate of Clc-6-P-Dependent Starch Synthesis
nmol C, units mg-
H,O-fed leaves Glc-fed leaves
’ Chl h- ‘
622 t 130
129 t 17
162 t 1 3
896 2 191
istics similar to those isolated from green pepper fruits. Illumination led to a 35% increase in the rate of Glc-6-Pdependent starch synthesis (Table V). The omission of PGA from the incubation medium reduced the rate of starch synthesis by 55% in the light and by 84% in the dark. The absence of ATP had no significant effect in the light, but in the dark, the Glc-6-P-dependent starch synthesis was virtually abolished. Furthermore, the incorporation of Glc-6-P into starch in chloroplasts isolated from Glc-fed spinach leaves appeared to be specific, since addition of Glc-1-P did not lead to a significant rate of starch synthesis (Table V). To analyze whether Glc-6-P-dependent starch synthesis occurred within a physiological range of Glc-6-P concentrations, we measured the effect of Glc-6-P concentration upon the rate of starch synthesis. Increasing concentrations of Glc-6-P increased the rate of starch synthesis reaching saturation at below 7 mM, both in the dark and in the light (Fig. 3). It has already been established that spinach chloroplasts do not normally take up Glc-6-P at appreciable rates (Fliege et al., 1978). Kinetics of Glc-6-P Uptake
Our data indicated that chloroplasts purified from Glcfed spinach leaves are able to incorporate Glc-6-P into starch; therefore we examined the biochemical features of the uptake mechanism. We used rapid silicon oil centrifugation to measure the rate of Glc-6-P uptake across the chloroplast envelope. Chloroplasts purified from Glc-fed leaves are able to take up external Glc-6-P in a concentration-dependent manner exhibiting typical Michaelis-Menten kinetics (Fig. 4). An Eadie-Hofstee transformation of the data obtained from the Glc treatment yields an estimated V,,, and K, for the uptake mechanism of 2.9 pmol Glc-6-P mg-’ Chl h-1 and 0.54 mM, respectively (Fig. 4, inset). The nonlinearity observed with this transformation means that our determination of V,,, and K , are at best only estimates but could reflect the nonspecific uptake of Glc-6-P that occurs at high concentrations, as shown by control chloroplasts. Chloroplasts isolated from control leaves did not take up Glc-6-l‘ at significant rates and did not reach saturation at the concentration range used here (Fig. 4). DIDS, a noncompetitive inhibitor of the chloroplast
117
envelope triose-phosphate translocator (Rumpo and Edwards, 1985), has been demonstrated to also inhibit the Glc-6-P translocator of cauliflower-bud amyloplasts (Batz et al., 1993, 1994). The effect of DIDS on the rate of incorporation of Glc-6-P into starch in chloroplasts isolated from Glc-fed leaves is shown in Figure 5 . The absolute rates of Glc-6-P incorporation were lower in this experiment because the concentration of substrate was reduced to 1 mM. Glc-6-P-dependent starch synthesis is inhibited by DIDS at concentrations similar to those reported previously for triose phosphate and Glc-6-P uptake into chloroplasts or amyloplasts (Rumpo and Edwards, 1985; Batz et al., 1993). Glc-6-P-Dependent Starch Synthesis Can Be lnduced by Water Stress
As a first attempt to identify whether a hexose-phosphate translocator activity could be induced by other treatments, leaf discs of spinach were artificially water stressed by incubation of leaf discs in 500 mM sorbitol and then by isolation of chloroplasts after various times of incubation. Sorbitol applied in this concentration induces symptoms of water stress in spinach leaves (Quick et al., 1989). Water stress led to the induction of Glc-6-P-dependent starch synthesis (Fig. 6), and the rate of induction was very similar to that induced by Glc feeding (Fig. 2). DISC USSI ON lnduction of Clc-6-P-Dependent Starch Synthesis
In this paper we have demonstrated that chloroplasts isolated from leaves can possess a hexose-phosphate translocator and that this transport function can be induced by feeding the carbohydrate Glc (Fig. 4). Chloroplasts isolated from Glc-fed leaves retain the photosynthetic capacity to reduce PGA (Fig. 1) and show light-stimulated rates of Glc-6-P-dependent starch synthesis (Figs. 3 and 6). This is more evident in the absence of the externally applied effectors PGA and ATP (Table V). The data presented indicate that the translocator involved is specific for Glc-6-P, because rates of Glc-1-P incorporation into starch are very low, despite the presence of external PGA and ATP (Table V). These results are similar to those from other studies characterizing the precursor dependency of starch syntheTable IV. The rate of Glc-6-P-dependent starch synthesis in chlo-
roplasts isolated either from freshly detached potato leaves or from leaves that were fed 50 mM Glc for 4 d The actinic light intensity was 150 pmol m-’ s-’, and samples were incubated for 20 min at room temperature. Substrates and effectors were supplied at the following concentrations: Glc-6-P, 5 mM; PGA, 0.5 mM; ATP, 2.0 mM; KH,PO,, 0.1 mM; and NaHCO,, 5.0 mM. Data are means ? SE. Rate of Starch Synthesis lncubation Time Lieht
d
O 4
Dark
nmol C, units mg-’ Chl h-’
56.9 ? 2.9 254.4 ? 11.9
38.0 f 5.2 36.3 t 2.0
Quick et al.
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Plant Physiol. Vol. 109, 1995
Table V. Substrate and effector dependence of hexose-phosphate-dependent starch synthesis performed by chloroplasts isolated from spinach leaves incubated with Glc for 7 d Glc-6-P and Glc-1-P were added at 5.0 mM, and the effector concentrations of PGA and ATP were 0.5 mM and 2.0 mM, respectively. KH,PO, (0.1 mM) and NaHCO, (5.0 mM) were also included in all samples. Data are means 2 SE. Rate of Starch Synthesis Precursor Effectorb) Dark Light CIC-6-P Glc-6-P GIc-6-P Glc-1 -P
ATP, PGA ATP PGA ATP, PGA
sis in heterotrophic plastids (Hill and Smith, 1991; Neuhaus et al., 1993a, 1993b; Batz et al., 1994) and pepper-fruit chloroplasts (Batz et al., 1995). Also, previous studies of the metabolism of radiolabeled Glc suggested that unicellular algae (Raven, 1976; Marker and Whittingham, 1966) and tobacco leaf discs (Maclachlan and Porter, 1959) had the capacity to incorporate supplied hexose sugars directly into starch. The apparent K , for Glc-6-P uptake measured for Glcinduced chloroplasts is very low (0.54 mM, Fig. 5) and comparable to values associated with the transport of triose phosphates in noninduced spinach chloroplasts (Fliege et al., 1978). Previous studies have shown that transport of Glc-6-P into noninduced chloroplasts is slow and is largely insensitive to competitive inhibition by phosphate (Fliege et al., 1978). Also, the Ki of Glc-6-P for the uptake of phosphate by the triose-phosphate translocator was very high (40 mM), suggesting that these two compounds were again noncompetitive and therefore not transported on the same transport protein (Fliege et al., 1978). Indeed, uptake of Glc-6-P was more sensitive to inhibition by D-G~c, sug-
400
I
526 232 497 13
nmol C, units mg-’ Chl h-’ ? 52 389 2 6 ? 37 62 2 5
25
?
8?6 1421
2 3
gesting that the low rates of uptake observed were catalyzed by the Glc transporter. Our data indicate that Glc-6-P uptake, induced by the Glc treatment, is due to the production of a nove1 translocator protein in photosynthetic chloroplasts exhibiting some structural similarities to the one recently identified in cauliflower amyloplasts (Batz et al., 1993). The rate of Glc-6-P-dependent starch synthesis in the dark was also dependent on the presence of the externa1 effectors ATP and PGA (Table V). A supply of ATP is directly required to sustain starch synthesis from Glc-6-P, and import is mediated by a specific ATP/ ADP transloca-
4.0
3.0 2.5
~
2.0
-
1.5
-
1 1 .o .o ’ .
0.5 0.0
0.0
t f n
0.5
1 .o
1.5
2.0
2.5
Glc6P (mM)
G l c 6 P (mM)
Figure 3. Substrate concentration dependence of the rate of Glc-6P-dependent starch synthesis for chloroplasts isolated from spinach leaves fed with 50 mM Glc for 7 d. Data are shown for chloroplasts incubated both in the light (O) and in the dark (O).Effectors were supplied at the following concentrations: PGA, 0.5 mM; ATP, 2.0 mM; KH,PO,, 0.1 mM; and NaHCO,, 5.0 mM.
Figure 4. Effect of increasing substrate concentrations on the rate of Glc-6-P uptake into spinach chloroplasts. Data are shown for chloroplasts isolated from spinach leaves fed 50 mM Glc for 7 d (0)or from freshly harvested leaves (O). Chloroplasts were preloaded with 10 mM KH,PO, prior to assay as described in the “Materials and Methods.” The chloroplast volume as calculated from the water (3H,0) permeable space was 16 p L and 17.3 p L mg-’ Chl for chloroplasts isolated from control and Glc fed leaves, respectively. Correction was made for the [’4C]sorbitol-permeable space, which was calculated as 6.6 p L and 6.8 p L mg-’ Chl for the control and Glc treatments, respectively. The inset graph shows the same data but as an Eadie-Hofstee transformation. The estimated apparent K,,. and V, for the rate of Glc-6-P uptake were 0.54 mM and 2.9 p m o l mg-1 Chl h-’, respectively.
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Hexose-Phosphate Translocator in Spinach Chloroplasts
00
70 60
50 40 30
20
10 O O
10
20
30
DIDS
40
50
60
(,UM)
Figure 5. lnhibition of the rate of Clc-6-P-dependentstarch synthesis by DIDS. The sensitivity of the light-saturated rate of Clc-6-P-dependent starch synthesis to inhibition by DIDS is shown for chloroplasts isolated from spinach leaves fed 50 mM Glc for 4 d. Substrates and effectors were supplied at the following concentrations: Glc-6-P, 1 mM; PCA, 0.5 mM; ATP, 2.0 mM; KH,PO,, 0.1 mM; and NaHCO,,
5.0 mM.
tor. The requirement for PGA is less obvious but could be due to the allosteric activation of ADP-Glc pyrophosphorylase by an increasing internal PGA/Pi ratio (Preiss, 1991). The requirement for PGA and ATP is reduced in the presente of light; the absence of exogenous PGA resulted in a 56% inhibition of Glc-6-P-dependent starch synthesis compared to 84% in the dark. Photosynthesis may provide, to some extent, either an internal source of PGA or the required change in the PGA/Pi ratio necessary for the activation of ADP-Glc pyrophosphorylase. It is interesting that PGA is also required to induce maximum rates of photosynthetic O, evolution in Glc-induced chloroplasts (Fig. 1). This could indicate that CO, fixation by the Calvin cycle is impaired, while electron transport capacity is retained; the provision of an exogenous supply of PGA would facilitate electron transport by acting as an acceptor for both ATP and NADPH. This notion is supported by the work of Krapp et al. (1991), who have shown that Calvin cycle activity declines faster than electron transport after Glc feeding to intact spinach leaves. Omission of ATP in the light had very little effect on the rate of Glc-6-P-dependent starch synthesis, demonstrating that electron transport was capable of supplying the required ATP. Also, the ATP/ ADP ratio has been shown to be high in Glc-fed leaves in both the light and the dark (Krapp et al., 1991).
hexose-phosphate translocator could afford two major possibilities for the chloroplast. First, when cytosolic carbohydrate availability is high, starch synthesis is made possible during dark periods when the stromal Fru-bisphosphatase is normally inactive. This allows carbohydrate to be accumulated as starch throughout the diurna1 cycle, either from sugars arising from primary CO, fixation during the day or as a result of carbohydrate import. Alternatively, the dark synthesis of starch may provide a mechanism to inhibit/impair starch degradation during the night. This could be regulated by the concentration of hexose phosphates in the cytosol. The simultaneous synthesis and degradation of starch has already been observed in isolated chloroplasts undergoing active photosynthesis (Stitt and Heldt, 1981).In that study, the investigators were able to show that the rate of starch degradation under these conditions was controlled by the rate of carbon remova1 from the chloroplast (Stitt and Heldt, 1981), which is normally controlled by cytosolic factors in the intact cell. This would suggest that the rate of starch degradation is controlled by the concentration of hexose phosphates inside the plastid. Regulation of this type would be precluded in darkened chloroplasts that do not possess a hexose-phosphate translocator. Chloroplasts localized in source leaves may not possess a Glc-6-P-translocator, since they have to mobilize transitory starch during the night to maintain carbon export from the leaf. Starch degradation products would be converted in the cytosol to glycolytic intermediates, and these could easily be used for starch biosynthesis if a hexose-phosphate translocator were present. Second, when carbohydrate availability is low, hexose phosphates can act as a source of energy for the chloroplast (generated through the oxidative pentose-phosphate path-
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