Relation to Photosynthesis by Isolated Spinach Chloroplasts - NCBI

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with the inhibitor for 2 min prior to illumination. Measurements of 02 evolution were carried out with an 02 electrode (4) supplied by Hansatech Limited, Norfolk.

Plant Physiol. (1980) 65, 574-577 0032-0889/80/65/0574/04/$00.50/0

Balance between Metabolite Accumulation and Transport in Relation to Photosynthesis by Isolated Spinach Chloroplasts' Received for publication May 29, 1979 and in revised form October 29, 1979

U. INGO FLUGGE, MARTHA FREISL, AND HANS W. HELDT

Institutffur Physiologische Chemie und Physikalische Biochemie der Universitat Munchen, Munchen, Federal

Republic of Germany

ABSTRACT The relationship between the rate of orthophosphate (Pi) transport into the stroma and the rate of CO2 fixation by intact chloroplasts was investigated. High Pi concentrations in the medium lead to a depletion of stromal metabolites, due to excessive Pi transport into the stroma, resulting in the inhibition of CO2 fixation. This inhibitory effect of Pi is released by inhibitors of Pi transport, such as pyrophosphate, citrate or pyridoxal-5phosphate. The latter compound appeared to be specially valuable in inhibiting Pi transport without affecting stromal reactions. The Pi optima of CO2 fixation were studied when the rate of CO2 fixation or the rate of Pi transport was varied by the application of specific inhibitors. For optimad performance the rates of CO2 fixation and Pi transport have to be matched in such a way, that transport neither Ulmits nor exceeds CO2 fixation. This accounts for the large variation in the Pi optima for CO2 fixation by different chloroplast preparations.

Triose-P and PGA3 are the main products of CO2 fixation released by spinach chloroplasts (3, 7). The export of these substances from the chloroplasts is controlled by the phosphate translocator, a specific carrier located in the inner envelope membrane (5), facilitating a counter exchange. In this way, during CO2 fLxation the net uptake of Pi into the chloroplasts is counterbalanced by a release of triose-P and PGA. Since CO2 fixation is a cyclic process, five-sixths of the carboxylation products are required in the stroma to regenerate the CO2 acceptor, ribulose bisP. High external Pi exaggerates export of PGA and triose-P, prolonging induction (12) and any factor which interferes with the exchange process might be expected to spare Pi inhibition. The effect of compounds such as pyridoxal 5'-P are considered in this

1 mM MgC12, 1 mM MnCI2, 2 mM EDTA, and 0.04 mg/ml catalase from beef liver (Boehringer Mannheim). If not stated otherwise, the chloroplast concentration was 0.1 mg Chl/ml. Other additions are indicated in the legends. When pyridoxal 5'-P or D,L-glyceraldehyde was to be added, the chloroplasts were preincubated with the inhibitor for 2 min prior to illumination. Measurements of 02 evolution were carried out with an 02 electrode (4) supplied by Hansatech Limited, Norfolk. The light intensity (RG 630 cut-off filter, Schott Mainz, Germany) was about 60 w/m2. Illumination was continued during centrifugation. Phosphate-containing metabolites were labeled by including 32Pi in the reaction mixtures. The chlloroplasts were preincubated with this reaction mixture for 5 min in order to exchange the stromal phosphates, and for another 2 min with pyridoxal 5'-P if it was to be added. Photosynthesis was then started by illumination, and the reaction was terminated by centrifugation of the chloroplasts through a silicone fluid layer into 20 pl of 1O0lo HC104. Following centrifugation the upper 200 ul. of the supernatant solution was also acidified with 5 ,ul of 60o HC104. Extracts were prepared by centrifugation of the precipitated protein, and neutralization of the supernatant with KOH or K2CO3. For further details, see reference 7. Ion exchange chromatography on Bio-Rad AG I-X8 was performed as described elsewhere (7). The values for the metabolites in the stroma were corrected for the metabolites in the medium, which had been carried through the silicone layer with the chloroplasts.

RESULTS AND DISCUSSION Effect of Pyrophosphate and Citrate on CO2 Fixation. When chloroplasts are illuminated, CO2 fixation undergoes an induction phase before a final rate is reached (12). The length of this induction phase and also the final rate depend on the external Pi concentration. Although Pi is required for CO2 fixation, external concentrations above about 0.5 mm were found to increase the context. induction period and to decrease the final rate of CO2 fixation. Furthermore, it was found that the addition of PPi shortened the MATERIALS AND METHODS induction phase observed with high external Pi concentrations Spinach (Spinacia oleracea, var. U.S. hybrid 424, from Ferry- (12). Figure 1 is typical of this effect showing that the induction Morse Seed Co., Mountain View, Calif.) was grown in water phase observed in the presence of 1 mm Pi was considerably culture according to Lilley and Walker (8). For the preparation of shorter if 10 mM PPi was also present. chloroplasts see reference (7). Shortening of the induction phase by PPi can be explained by The incubation was normally carried out in a medium contain- an inhibition of Pi uptake into the stroma, since it has been shown ing 0.33 M sorbitol, 50 mm Hepes adjusted to pH 7.6 with NaOH, that the phosphate translocator, which has a Km for Pi of about 0.2 mm, is competitively inhibited by PPi with a Ki of 1.8 mm (5). 'This work has been supported by the Deutsche Forschungsgemein- The maximal velocity of PPi transport into the chloroplasts was found to be two orders of magnitude lower than that of Pi schaft. 2 Present address: Lehrstuhl fur Biochemie der Pflanze der Universitat transport. From these findings, the rate of the transport of Pi (V) into the Gottingen, Untere Karspule 2, 3400 Gottingen, Federal Republic of chloroplasts in the presence of PPi can be calculated according to Germany. 'Abbreviations: PGA: 3-phosphoglycerate; RPP pathway; reductive the following equation (7) when Vm.x is maximal velocity of Pi transport. pentose phosphate pathway. 574

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metabolites were built up and subsequently triose-P and PGA were transported to the surrounding medium (see also ref. 7). The addition of pyridoxal 5'-P enhanced the formation of carboxylation products in the stroma, but strongly inhibited their release to the surrounding medium (Fig. 3). As a result, the over-all reaction of CO2 fixation was largely decreased once the initial phase of CO2 fixation was complete. The pattern of stromal metabolites observed in the presence of pyridoxal 5'-P was similar to that observed when there was a Pi deficiency in the medium (6). It seems that pyridoxal 5'-P does not penetrate the chloroplast envelope and thus has no marked effect on stromal reactions. Figure 4 shows the effect of pyridoxal 5'-P on C02-dependent 02 evolution in the presence of three different external Pi concentrations. (The pyridoxal 5'-P concentration was lower than in the experiment of Fig. 3.) In the absence of pyridoxal 5'-P induction increased with increasing external Pi concentrations, as in earlier work (12). When pyridoxal 5'-P was added to a chloroplast suspension containing 0.1 mM Pi, 02 evolution was inhibited as it was in the experiment of Figure 3. With 2.5 mm Pi in the medium, however, the addition of pyridoxal 5'-P led to an increase of the final rate of 02 evolution and also to a marked shortening of the induction phase. Figure 5 shows the rate of 02 evolution as a Metabolites in the stromo control

5 10 TIME (min)

Metabolites

FIG. 1. CO2 fixation in the presence of I mM Pi and 5 mM ["C]NaHCO3; PPi or citrate was added before onset of illumination.

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RuB In the simplified case in which only Pi was present in the medium the addition of 10 mm PPi would reduce the rate of Pi 2 4 6 2 4ht TIME (min) TIME (min) Light uptake to 52% of the control value. This explanation of the effect of PPi can be tested by introducing another competitive inhibitor FIG. 2. Distribution of 32Pi-labeled metabolites during CO2 fixation in of the phosphate translocator, namely citrate, which has a Km of the presence of 0.5 mm Pi in the medium. Pyridoxal-5'-P = 0; 02 evolution 1.5 mm and a maximal velocity of virtually zero (5). 135 ,smol/mg Chl.h. FBP: fructose-1,6-bisphosphate; FBPase: fructoseAccording to equation 1, the transport of Pi (1 mnm external 1,6-bisphosphate I-phosphohydrolase. concentration) should be inhibited by 8 mi citrate to 53% of the control rate. As shown in the experiment of Figure 1, 8 mm citrate Metabolites in the stroma shortened induction to precisely the same extent as 10 mm PPi. Pyridoxal P present Inasmuch as these two substances are very different in respect to 200. their metabolism, our results clearly indicate that the shortening of the induction phase is indeed due to a partial inhibition of _7/ PGA phosphate transport. Effect of Pyridoxal 5'-P on CO2 Fixation. p-Chloromercuriphenyl sulfonate, an inhibitor of the phosphate translocator in E Moetabolites in medium chloroplasts (5), partially reverses the inhibition of CO2 fixation presence of 0.5 mM Pi in the medium.Pyridoxal the0 by Pi in pea chloroplasts (10). Similar results have also been obtained with spinach chloroplasts (Heldt, unpublished). In our hands, however, this inhibitor was not very specific. Apparently, C250 depending on the chloroplast preparation, traces of this -SH 0-t~~~~~~~~DAP reagent also reached the stroma, resulting in an inhibition of stromal reactions to various extents. DHAPFB Pyridoxal 5'-P, another inhibitor of the phosphate translocator (min) Light (5), was found to be a much more suitable agent for specific inhibition of metabolite transport in intact chloroplasts. The conFIG. 3. Distribution of Pi-labeled metabolites during CO2 fixation in trol experiment (Fig. 2) demonstrates the appearance of the phos- the presence of 0.5 mmi Pi in the medium. Pyridoxal-5'-P 1.7 MM; 02 phorylated products of CO2 fixation in the chloroplast stroma and evolution 28 ,umol/mg Chl -h. A: metabolites in the stroma; B: metabolites also in the suspension medium. During induction the stromal in the medium. PMP

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of pyridoxal 5'-P reduced the excessive Pi uptake, thus enabling the maintenance of sufficient stromal metabolite levels required for CO2 fixation. Effect of D,L-Glyceraldehyde on the Pi Optimum of CO2 Fixation. It should be also possible to disturb the balance between the rates of Pi transport and CO2 fixation by inhibiting the latter reaction. A suitable inhibitor of the reductive CO2 fixation cycle is D,L-glyceraldehyde. This inhibitor acts at two sites in the regeneration of the CO2 acceptor from triose-P (1, 11). The partial inhibition of CO2 fixation by D,L-glyceraldehyde resulted in a lengthening of induction (Fig. 6). The addition of the inhibitor shifted the Pi optimum of CO2 fixation toward lower concentrations (Fig. 7). Apparently, when the capacity for CO2 fixation was diminished, a lower rate of Pi uptake was sufficient to maintain optimal CO2 fixation. Effects of this sort may contribute to the observed variability of the Pi

100 pM 30

132 136 B A 104

2min

Light

Pi OIl mM

Light Pi O.5mM

Light Pi 2.5mM

FIG. 4. C02-dependent CO2 evolution with different concentration of Pi in the medium; 64 ,ug Chl/ml. A: control; B: + I mm pyridoxal-5'-P. Numbers are rates of 02 evolution in umol/mg Chl. h.

Table I. Effect of Pi and Pyridoxal S'-P on Metabolite Levels in Chloroplasts Experiment of Figure 4, values obtained 4 min after illumination. Stroma volume 25 ,ld/mg Chl, trace means < 50S,uM. Pi, mM 0.5 2.0 2.0 Pyridoxal 5'-P, mM 0 0 1.0 Metabolite Concentration in Stroma mM

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Triose-P PGA Hexose and heptose monoP Fructose bisP Sedoheptulose bisP Pentose monoP Ribulose bisP ATP/ADP

~0

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trace trace 0.10 trace trace trace 0.06 1.75

0.18 3.69 1.85 0.37 0.10 0.02 0.30 0.98

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FIG. 5. Dependence of the rate of 02-dependent 02 evolution on the Pi concentration in the medium. Inhibition of the phosphate translocator by 0.46 mm pyridoxal-5'-P.

function of external Pi concentration in the presence and absence of pyridoxal 5'-P. In the control experiment, the optimal Pi concentration was 0.2 mm. In another experiment (Fig. 7) it was 0.5 mm. The addition of pyridoxal 5'-P resulted in a shift of the Pi optimum to about 1 mm, and about 90%o of the maximal rate of 02 evolution was still observed with 3 mm Pi. Table I shows the measurement of stromal metabolites in the presence of high and low external Pi concentrations. The low rate of C02-dependent 02 evolution in the presence of a high Pi concentration (2.0 mM) is reflected by very low levels of the intermediates of CO2 fixation. When pyridoxal 5'-P was added to these chloroplasts, the stimulation of 02 evolution was accompanied by a considerable increase in the stromal metabolite levels. Apparently, in the experiment of Figure 5, with 0.2 mm external Pi, the rate of net Pi transport into the chloroplasts was optimal for a maximal rate of CO2 fixation. A decrease of this optimal rate by the addition of pyridoxal 5'-P lead to a situation where the uptake of Pi was limiting CO2 fixation. With high external Pi concentrations, the rate of Pi, uptake was apparently too high, resulting in a depletion of the stromal metabolites. The addition

02

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/

FIG. 6. C02-dependent 02 evolution; 43 ,ug Chl/ml. Numbers are rates of 02 evolution in ,umol/mg Chl . h.

METABOLITE TRANSPORT IN CHLOROPLASTS

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100pmoIControl 100 %- 120 PMO0°l°2h

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2 3 Pi (mM) FIG. 7. Dependence of the rate of CO2 dependent 02 evolution on the Pi concentration in the medium. Inhibition of CO2 fixation by I mM D,Lglyceraldehyde.

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portion of the maximal carrier activity. As the Km for Pi transport into the chloroplasts was found to be 0.15-0.50 mm (5), a change of the external Pi concentration from 0.2 to 1.0 mm is expected to increase the rate of Pi transport by a factor of about 2. Nevertheless, Pi transport into the chloroplasts is decreased when there is also PGA or triose-P present in the external space, since these compounds compete with Pi for transportation. This explains why the inhibition of CO2 fixation by high external Pi concentrations is released by the addition of dihydroxyacetone-P or PGA (12). Furthermore, since the net transport of Pi into the stroma results from the difference of the fluxes in both directions, it is decreased by increasing Pi concentrations in the stroma. Due to these properties of the phosphate translocator, unless the external Pi concentration is artificially high, the rate of transport can be adjusted to the rate of CO2 fixation enabling the buildup of CO2 fixation cycle intermediates during the induction phase. Acknowledgment-The authors are indebted to Professor David Walker, University of Sheffield, England, for stimulating discussions and his collaboration in preparing this manuscript. LITERATURE CITED

optima of CO2 fixation in different chloroplast suspensions. For example, some chloroplasts exhibiting a low Pi optimum may have a diminished capacity for CO2 fixation as a result of loss of stromal enzymes. CONCLUSION As shown before, optimal concentrations of stromal metabolites are required for a maximal rate of CO2 fixation. Due to the counter exchange catalyzed by the phosphate translocator the sum of Pi and phosphorylated compounds in the stroma is kept about constant. Thus, a high level of stromal Pi is accompanied by low levels of intermediates of the RPP pathway. In the steady-state the levels of these intermediates in the stroma is the resultant of the velocity of CO2 fixation and the velocity of metabolite transport across the envelope. For this reason, the net uptake of Pi into the stroma must match the net release of triose-P plus PGA. If net Pi uptake exceeds carboxylation, the CO2 fixation cycle is depleted of its intermediates leading to an inhibition of the over-all reaction. When Pi uptake is too slow, the chloroplasts are depleted of Pi, and CO2 fixation is inhibited as well (see Fig. 3A). It has been recently demonstrated that the pH- and Mg"k-dependent light activation of ribulose bisP carboxylase in the chloroplasts requires the presence of Pi in the stroma (6). This could explain why in the experiment of Fig. 3A, when the chloroplasts have run out of Pi, CO2 fixation is inhibited by about 80%o even though the ribulose bisP level in these inhibited chloroplasts was slightly higher than that in fully active chloroplasts (Fig. 2A). As has been discussed elsewhere in detail (7), the net uptake of Pi into the chloroplasts during CO2 fixation will be only a small

1. BAMBERGER ES, M AVRON 1975 Site of the action of inhibitors of carbon dioxide assimilation by whole lettuce chloroplasts. Plant Physiol 56: 481-485 2. BASSHAM JA 1979 The reductive pentose phosphate cycle and its regulation. In M Gibbs, E Latzko, eds, Encyclopedia of Plant Physiology, Vol 6. SpringerVerlag, Heidelberg, pp 9-30 3. BASSHAM JA, M KIRK, RG JENSEN 1968 Diffusion of labelled photosynthetic intermediate between isolated chloroplasts and suspending medium. Biochim Biophys Acta 153: 211-218 4. DELIEu T, DA WALKER 1972 An improved cathode for the measurement of photosynthetic oxygen evolution by isolated chloroplasts. New Phytol 71: 201225 5. FLIEGE R, UI FLUGGE, K WERDAN, HW HELDT 1978 Specific transport of inorganic phosphate, 3-phosphoglycerate and triosephosphates across the inner membrane of the envelope in spinach chloroplasts. Biochim Biophys Acta 502: 232-247 6. HELDT HW, CJ CHON, GH LORIMER 1978 Phosphate requirement for the light activation of ribulose 1,5-bisphosphate carboxylase in intact spinach chloroplast. FEBS Lett 92: 234-240 7. LILLEY RMcC, CJ CHON, A MOSBACH, HW HELDT 1977 The distribution of metabolites between spinach chloroplasts and medium during photosynthesis in vitro. Biochim Biophys Acta 460: 259-272 8. LILLEY, RMcC, DA WALKER 1974 The reduction of 3-phosphoglycerate by reconstituted chloroplasts and by chloroplast extracts. Biochim Biophys Acta 368: 269-278 9. PEDERSEN TA, M KIRK, JA BASSHAM 1966 Light-dark transients in levels of intermediate compounds during photosynthesis in air-adapted Chlorella Physiol Plant 19: 219-231 10. ROBINSON SP, JT WISKICH 1977 p-Chloromercuriphenyl sulfonic acid as a specific inhibitor of the phosphate transporter in isolated chloroplasts. FEBS Lett 78: 203-206 11. STOKEs DM, DA WALKER 1972 Inhibition by D,L-glyceraldehyde of carbon dioxide assimilation. Biochem J 175: 1147-1157 12. WALKER DA 1978 Photosynthetic induction and its relation to the transport phenomena and control mechanisms in chloroplasts. In J. Barber, ed, The Intact Chloroplasts. Elsevier, Amsterdam, pp 235-278

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