Planta
Planta 144, 325 332 (1979)
9 by Springer-Verlag 1979
Kinetics of Mitochondrial Phosphate Transport and Rates of Respiration and Phosphorylation during Greening of Etiolated Arena Leaves R. Hampp Institut ftir Botanik, TechnischeUniversit/it M/inchen, Arcisstral3e21, D-8000 Mfinchen 2, Federal Republic of Germany
Abstract. Using the technique of silicone oil filtration of organelles and the inhibitor stop method, the kinetics of transport of inorganic phosphate across the inner mitochondrial membrane were tested in relation to different stages of greening (0 to 24 h) of etiolated laminae of A r e n a s a t i v a L., and compared to the rates of oxygen consumption and ATP formation. The results demonstrate that there is a pronounced increase in phosphate transport after 3 h of greening, reaching values for V .... (about 1 7 g m o l m g protein-1 h-1) that are three times as high as those measured with mitochondria from etiolated tissue. This is also mirrored by the rates of respiration and oxidative phosphorylation. After 24 h of light treatment (4 Klx), respiration and ATP formation, as well as V decreased again to levels below those of the etiolated stage. In contrast to V, there was no change in the affinity between inorganic phosphate and the binding sites of the transporting systems involved, as indicated by a rather constant Km (0.23 mM) for phosphate transport. Of the inhibitors of phosphate transport tested, mersalyl and methyl mercuric iodide were most efficient with identical characteristics of inhibition ; but compared to animal mitochondria, the concentrations needed to result in similar amounts of inhibition, were more than ten times higher. The results are discussed with respect to plastid development. Key words: A r e n a - - Greening - Mitochondria Phosphate transport - Respiration.
BSA=bovine serum albumine; CH3HgJ=methyl mercuric iodide; Cyt-cytochrome; HEPES=N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonicacid; MDH=malate dehydrogenase; NEM = N-ethylmaleimide Abbreviations.
Introduction The major sites of intracellular energy conservation in autotrophic cells of higher plants are chloroplasts and mitochondria. Because energy is not only consumed within these organelles, transfer of energy is a prerequisite. Such a transfer of energy should be especially active with mitochondria during greening of etiolated tissue that contains photosynthetically incompetent plastids. It has been demonstrated that respiration of etiolated leaves is enhanced upon illumination of intact plants (De Greef and Verbelen, 1977; Hampp and Wellburn, 1976a) and that this change in the respiratory activity of mitochondria is accompanied by a transient change in the permeability properties of the inner mitochondrial membrane to a range of compounds (Hampp and Wellburn, 1976a). It is also known that the inner membrane of both animal (Chappell, 1968) and plant mitochondria (Phillips and Williams, 1973; Wiskich, 1975) is a site of specific transport where exchange translocators are involved. Phosphate, for example, can enter mitochondria by one of two different transport mechanisms, which can be functionally separated by the use of inhibitors. It may be taken up in exchange for O H - ions or cotransported with protons, which cannot be distinguished experimentally (Papa et al., 1969; Chappell and Crofts, 1966; McGivan and Klingenberg, 1971) - a process sensitive to N E M (Meijer et al., 1970), or it can be exchanged for dicarboxylate anions. The latter transport is inhibited by substrate analogs such as n-butyl malonate (Robinson and Williams, 1970; Palmieri etal., 1971). Both processes are also specifically inhibited by sulfhydrylblocking reagents (e.g., mersalyl; Meijer et al., 1970).
0032-0935/79/0144/0325/$01.60
326 It is a s s u m e d t h a t t h e P i / O H - e x c h a n g e serves to n e u t r a l i z e the c h a r g e i m b a l a n c e o c c u r r i n g f r o m t h e u p t a k e o f A D P 3- a n d t h e r e l e a s e o f A T P 4 - d u r i n g o x i d a t i v e p h o s p h o r y l a t i o n ( M c G i v a n et al., 1971), while the Pi/dicarboxylate exchange allows the net u p t a k e o f a n i o n i c s u b s t a n c e s at t h e e x p e n s e o f the mitochondrial pH gradient (Klingenberg, 1970; M c G i v a n a n d K l i n g e n b e r g , 1971). T h e r e f o r e , t r a n s p o r t o f a n i o n s i n t o m i t o c h o n d r i a is d e p e n d e n t o n r e s p i r a t i o n - l i n k e d p r o t o n efflux, c o u p l e d to an Pi/ OH-exchange (Pi t r a n s l o c a t o r ) , f o l l o w e d b y s u b strate/Pi e x c h a n g e ( d i c a r b o x y l a t e t r a n s l o c a t o r ) . Pj t r a n s p o r t v i a t h e p h o s p h a t e t r a n s l o c a t o r o f anim a l m i t o c h o n d r i a s h o w s v e r y h i g h rates a n d s e e m s n o t to b e r a t e l i m i t i n g f o r A T P f o r m a t i o n . O n t h e o t h e r h a n d , D a y a n d H a n s o n (1977) s h o w e d f o r p l a n t m i t o c h o n d r i a t h a t the r a t e o f Pi t r a n s p o r t ( r e d u c e d b y i n h i b i t o r s a n d u n c o u p l e r s ) is closely r e l a t e d to the a m o u n t o f t h e s u b s t r a t e o x i d a t i o n : r a p i d o x i d a tion of malate and succinate required high transport activities o f b o t h t h e Pi a n d t h e d i c a r b o x y l a t e t r a n s l o cator. It c a n t h e r e f o r e be s u g g e s t e d t h a t p h y s i o l o g i c stages w i t h h i g h e n e r g y d e m a n d (e.g., g r e e n i n g o f e t i o l a t e d tissue) s h o u l d be r e f l e c t e d by i n c r e a s e d rates o f m i t o c h o n d r i a l r e s p i r a t i o n , a c c o m p a n i e d b y enh a n c e d rates o f Pi t r a n s p o r t a c r o s s t h e i n n e r m i t o c h o n d r i a l m e m b r a n e . T h u s it was the a i m o f t h e exp e r i m e n t s d e s c r i b e d in this p a p e r to i n v e s t i g a t e t h e k i n e t i c s o f P~ t r a n s p o r t o f m i t o c h o n d r i a d u r i n g differe n t stages o f g r e e n i n g o f e t i o l a t e d l e a f tissue, a n d to c o m p a r e it to the rates o f o x y g e n c o n s u m p t i o n and ATP formation under the same conditions.
Materials and Methods Isolation of Mitochondria Seedlings of Arena sativa L. (var. Arnold) were grown in moist peat at 25 ~ C for 7 days in the dark. If different times of greening were required, partial illumination in the later stages of growth was achieved by a halogen lamp (Osram, HQLS, 400 W) providing a radiant light flux of 4 Klx at seedling level. About 50 g of laminae (uppermost 5 cm) were homogenized in t00ml of chitIed isolation medium (0.3 M sorbitol; 0.001 M MgC12; 0.001 M NaNO3; 0.001 M EDTA; 0.2% (w/v) BSA, Sigma Type V; in 0.05 M HEPES, finally adjusted to pH 7.3 M - prior to adding the plant tissue, cysteine (0.05%, w/v) was stirred in) using a Starmix (Braun, Melsungen, W. Germany) in three bursts of 5 s duration each. The homogenate was filtered twice through four layers of nylon sheet (Nytal 25 T 1, mesh 35 gm, Henry Simon, Stockport, U.K.) and centrifuged for 7 min at 1,000 g. The resulting pellet (mainly plastids) was discarded and the supernatant was centrifuged (1) for 10min at 10,000g, (2) after resuspension of the pellet (50 ml of homogenation medium) for 10 rain at 8,000 g. Finally the pellet was resuspended in isolation medium to give a protein content of about 2 3 mg/ml.
R. Hampp : Mitochondrial Phosphate Transport and Respiration All steps were carried out at temperatures ranging from 0 4 ~ C, and in the case of etiolated tissue in complete darkness. The amount of protein was measured by the method of Lowry et at. (1951).
Enzyme Assays Cytochrome oxidase (EC 1.9.3.1) was determined by adding an appropriate aliquot of particle suspension (50-100 gl) to 1.5 ml of 20 gM reduced Cyt c in 50 mM HEPES (pH 7.3) and following the change in optical density at 550 nm. NADH:Cyt c reductase (EC 1.6.99.3) was assayed according to Lord et al. (1973); the medium contained buffer as above, plus 20 gM oxidized Cyt c, 80 gM NADH, and 1 mM KCN. The reaction was initiated by adding the particle suspension, and the reduction of Cyt c was followed at 550 nm. Antimycin A was added in 10 gl of ethanol to give a final concentration of 1 gM. Catalase (EC 1.11.1.6) was assayed by following the 02 production using a Clark-type oxygen electrode. The reaction mixture contained in 50 mM HEPES (pH 7.3) 1.2mM H20 2 in a total volume of 1.5 ml. Oxygen was removed from the assay mixture by bubbling O2-free N 2 through it prior to measurement. Ribulose-bisphosphate carboxylase (EC 4.1.1.39) was activated and measured according to the method of Lorimer et al. (1977), NADH: MDH (EC 1.1.1.37) as described by Bonner (1974).
Measurement of Kinetics of Phosphate Transport All measurements of phosphate transport were made using an "inhibitor stop" technique, in which transport is stopped by the rapid addition of a transport inhibitor, followed by vigorous stirring (Pfaff and Klingenberg, 1968). The incubation was carried out in isolation medium, which contained additionally oligomycin (5 Ixg/ml) to prevent the incorporation of Pi into ATP. The uptake was started by the addition of [32p]orthophosphate (1-2 ~tCi; final concentrations, see Results) and terminated after time t by rapid addition of mersalyl (5 mM; exceptions see legends to Figures). This compound was shown (Meijer et al., 1970) to inhibit Pi transport via the phosphate and the dicarboxylate translocator almost completely. With this "inhibitor stop" method, the shortest time resolved was 2 s. The rates of uptake were calculated from the initial and linear parts of the time course of uptake, limited to a few seconds. The mitochondria with the trapped phosphate were separated from the incubation medium by rapid centrifugation through a layer of silicone oil (AR 80, Wacker-Chemie, Munich, W.-Germany) into 50 gl of 10% (v/v) HCIO4 (Microfuge B 152, Beckman, 30 s). With the exception of time-dependent experiments, incubation and centrifugation were performed within t min. Control experiments were carried out, in which the inhibitor was added before the labeled phosphate to the incubation mixture containing the mitochondria. In these controls, the time of contact of the mitochondria with phosphate was the same as in the case when merslyt was used to stop the uptake. The radioactivity in the supernatants and in the pellets was measured in a scintillation counter against standards using preset counting characteristics and quench curves constructed specifically for these experiments. 3H20, [U-3H]inulin, and [U-14C]sorbitol were added in parallel experiments to determine the intermembrane and matrix spaces. For the operational definition of these spaces see Klingenberg and Pfaff (1966). The amount of Pi taken up into the matrix space was calculated as described previously (Hampp and Wellburn, 1976a). All incubations and eentrifugations were carried out between 0 and 4~ C.
R. Hampp: Mitochondrial Phosphate Transport and Respiration
Chromatographic Analysis of 32pi In order to verify that 32pi taken up by mitochondria that have been incubated at 0~ C for up to 10 min in the presence of oligomycin, was not metabolized, the perchloric acid extracts of the collected mitochondrial pellets were neutralized (KOH) and subjected to chromatographic analysis (Coty and Pedersen, 1974). No detection of changes in the amount of migration of inorganic phosphate was observed by comparing aliquots at the beginning and at the end of the incubation period.
Respiration of Mitochondria Recordings of 0 2 consumption were carried out using a Clark-type oxygen electrode. The reaction vessel contained an assay volume of 2 ml with additives as indicated in Fig. 4. The temperature was 20~ C.
327 Table 1. Levels of activity of enzymes in the mitochondrial fraction. The activities are average values (• SD, n = 5) for different stages of greening of etiolated tissue (0, 3, 24 h); they are expressed as specific activity and as percentage of the respective levels of activity within the total homogenate Enzyme
Specific activity nmol min-a mg protein-
Ribulose-bisphosphate carboxylase
1.9 (0h) 3.8 (24h)
Cyt c oxidase
56.0 _+7.0
30.0 _+5.0
2.5 • 1.5
0.8 • 0.4
Cyt c reductase (antimycin A insensitive) Catalase
15,000 +_5,000
Per cent of total activity
0.9+_0.4
7.0 • 5.0
A TP Measurements Mitochondria (about 2 mg protein) were incubated at 20~ in 2 ml of isolation medium (EDTA and NaNO3 omitted), which contained in addition 1.3 gmol NADH, 10 gmol Pi, and 170 nmol ADP. Samples of 400 gl were withdrawn from the reaction vessel before and 1-3 min after the addition of ADP. The samples were kept for 2 min in a boiling water bath and afterward cooled down on ice; the denaturated mitochondria were pelleted at 6000 g for 5 min. A 200-ml aliquot of the supernatant was assayed for ATP with the luciferin/luciferase system, by mixing it with 0.8 ml of a solution containing (mM): Na2HAsO4 10, EDTA 1, MgSO4 5, glycine buffer 20 (pit 7.8). The complete sample was injected into a glass vial, which was inserted into a Farrand fluorometer and contained 100 ~tl of luciferin/luciferase (Sigma, Munich, FRG). The resulting luminescense was recorded and calibrated by the immediate addition of a known quantity of ATP. To determine adenytate kinase blanks, the same procedure was followed, but initial additions were made of 20 gM rotenone, 3 ~tM antimycin A, and 20 gg oligomycin, and NADH was omitted.
Significance of the Results The different experiments (5 parallels each) were repeated at least four times (see Table 3). Each developmental sequence (~24 h of illumination) was investigated as one complex.
Chemica& [32p]orthophosphoric acid, (U-a4C]sorbitol, [U-all]inulin, and [3H]H20 were obtained from the Radiochemical Center (Amersham, U.K.); antimycin, oligomycin, mersalyl and luciferin/luciferase were from Sigma (Munich, FRG); CH3HgJ was from Koch Light Labs, (Colnbrook, Bucks., U.K.).
Results
Purity and Integrity o f the Mitochondrial Fraction T a b l e 1 gives the average levels of activity associated with m i t o c h o n d r i a that have been isolated f r o m etiolated leaves after different times of i l l u m i n a t i o n , of
r i b u l o s e - b i s p h o s p h a t e carboxylase, Cyt c oxidase, N A D H : C y t c reductase ( a n t i m y c i n A insensitive), a n d catalase, which are m a r k e r s for plastids, mitochondria, e n d o p l a s m i c reticulum, a n d peroxisomes (Lord et al., 1973; Tolbert, 1971; T o l b e r t et al., 1968) in specific activity a n d in per cent of the respective rates m e a s u r e d in the total h o m o g e n a t e . The results indicate only a low c o n t a m i n a t i o n of the m i t o c h o n drial fraction by other particulate fractions; m i t o c h o n d r i a that had been passed t h r o u g h a layer of silicone oil ( b o t t o m - m o s t layer of centrifuge tubes 0.6 M sorbitol instead of HC104) showed an additional decrease of c o n t a m i n a t i o n of u p to 80% compared to the same m i t o c h o n d r i a l fraction before b e i n g i n t r o d u c e d to silicone oil filtration. These results dem o n s t r a t e that the rates of t r a n s p o r t given in this paper refer only to m i t o c h o n d r i a a n d that there is n o c o n t r i b u t i o n of other organelles. Similar conclusions have been d r a w n f r o m electron m i c r o g r a p h s of the m i t o c h o n d r i a l pellet ( H a m p p a n d W e l l b u r n , 1976a). I n T a b l e 2 a r e l a t i o n s h i p between the sorbitol c o n c e n t r a t i o n of the m e d i u m a n d the a m o u n t of m i t o c h o n d r i a (expressed as s o r b i t o l - i m p e r m e a b l e organelle space) able to penetrate a silicone layer of a distinct density is given. The results show that with decreasing c o n c e n t r a t i o n of sorbitol the n u m b e r of b r o k e n m i t o c h o n d r i a increases as m e a s u r e d by the level of activity of m i t o c h o n d r i a l N A D H : M D H after the a d d i t i o n of N A D H a n d oxaloacetate ( N A D H is n o t able to penetrate the intact m i t o c h o n d r i a l envelope; see B o n n e r , 1974). I n parallel, the n u m b e r of m i t o c h o n d r i a able to p e r m e a t e the silicone layer decreases. This f i n d i n g indicates that p r i m a r i l y organelles that retained their m e m b r a n e s are included in the e s t i m a t i o n of rates of u p t a k e by m e a n s of filtering centrifugation.
328
R. Hampp: Mitochondrial Phosphate Transport and Respiration
Table 2. Integrity of the mitochondrial membranes as measured by the levels of activity of N A D H : malate dehydrogenase, and number of organelles (expressed as sorbitol-impermeable space in the pellet after silicone centrifugal filtration) able to penetrate a silicone layer, in relation to the molarity of sorbitol in the incubation medium. The percantages are means ( + S D , n = 6 ) for mitochondria isolated from 0- to 48-h illuminated leaf tissue. The specific activity given for M D H was measured with mitochondria from etiolated tissue (with development there are changes in specific activity). Before centrifugation, the sorbitol concentration each time was made up to 0.3 M Sorbitol (M)
0.3
0.25
0.20
0.15
0.10
0
Malate dehydrogenase (~tmol N A D H mg protein-~ rain-1) Integrity (%) Matrix space in the pellet (~tl)
0.68 76_+5 0.26
0.88 68_+6 0.25
1.10 57_+6 0.21
1.38 51_+7 0.12
1.65 43_+5 0.10
2.75 0 0
100
medium concentration; for both compounds the increase in inhibitory action is most pronounced for concentrations up to 100 laM, but exceeds 90% only with 5-mM solutions. The effect of NEM, however, is much less expressed and requires at least 1 min of preincubation of the mitochondria before the addition of labeled P~ to exert maximum inhibition. The increase in inhibition is highest with NEM concentrations up to 5 mM ; but even with 25 mM NEM inhibition is only about 70% of the rates measured with untreated controls.
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) inhibitor concentration [mM] Fig. 1. Transport of inorganic phosphate into the matrix space of mitochondria in relation to the stage of greening of etiolated tissue ((~24 h of illumination, 4 Klx). Inhibition by mersalyl, methyl mercuric iodide, and N-ethylmaleimide; inorganic phosphate (0.1 raM), mersalyl, and CH3HgJ were applied prior to the addition of mitochondria. In the experiments with NEM, organelles were preincubated with the inhibitor for 60 s (longer times did not result in higher inhibition) before the addition of Pi; 1 m M inhibitor concentration is equivalent to about 500nmol/mg protein; 100%=5.5 (0 h: e - - e ) , 16.6 (3 h: 9 3.9 (24 h: x - - x ) ~tmol mg protein 1 h i. Temperature 0 ~ C
Effect of Sulfhydryl-Blocking Reagents on Mitochondrial Phosphate Transport during Greening In order to study the sensitivity for inhibitors of transport of inorganic phosphate during greening, the effects of the sulfhydryl-blocking reagents mersalyl, CH3HgJ, and NEM were tested. Figure 1 shows the efficiency of the different compounds in reducing the rates of uptake of Pi (0.1 mM). Generally, the values indicate that mitochondria isolated from leaf tissue after different stages of greening (0, 3, 24 h) respond in a similar way. Mersalyl and CH3HgJ cause nearly identically decreasing rates of uptake with increasing
Kinetics of Transport of Phosphate during Greening The time course of the uptake of inorganic phosphate by mitochondria, isolated from greening leaf tissue, is illustrated in Fig. 2. Uptake is rapid with all stages tested and shows a linear rate only up to about 6 s. In comparison to 24 h light treatment, mitochondria of less greened tissue exhibit higher rates of transport and also higher levels of accumulation of inorganic phosphate within the matrix space. Interestingly, both transport rates and accumulation show an intensive transient increase during the early stages of greening with a maximum at about 3 h of illumination. Figure 3 gives a typical example for the changing rates of uptake of Pi in relation to the medium concentration (0.1-1 mM) and the stages of greening of etiolated tissue. The concentration dependence reveals hyperbolic saturation characteristics, indicating substrate saturation of transport for all stages tested. Additionally, the results show accumulation of Pi in the matrix space, especially with low external concentrations; this accumulation is most pronounced for mitochondria isolated from etiolated tissue that had been given 3 h light treatment (about 5 times the external concentration within 4 s). Transport in mitochondria from etiolated and 24 h illuminated leaves is comparable, but on a much lower level.
R. Hampp : Mitochondrial Phosphate Transport and Respiration I U rl
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0.8
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Fig. 2. Time course of uptake of inorganic phosphate into the matrix space of mitochondria in relation to the stage of greening of etiolated tissue. Transport was initiated by the addition of P~ and stopped by the addition of mersalyl (final concentration 5 raM) at the times given ; illumination : 9 - e (Oh), o - - o (3h), x - - x (24h). One ~tl sorbitol-impermeable space is equivalent to about 0.08 mg of protein. Temperature 0 ~ C
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A double reciprocal plot of the data given in Fig. 3a yields linear functions (Fig. 3b), which enable the determination of the substrate concentrations that cause half maximal rates of transport (Kin) and the maximum velocities of transport (V). The results of the experiment, illustrated in Fig. 3 (Expt. 1) and of others (Exp. 2 to 4; Table 3) confirm that V (incubation time: 4 s) is highest after 3 h of illumination with about three times the rates measured with organelles from etiolated and 24 h illuminated leaves. In contrast, there is no change in the affinity between Pi and the binding sites of the transporting systems involved. The average K m a s calculated from the values shown in Table 3 is 0.23 mM.
Respiration and Oxidative Phosphorylation during Greening Figure 4 shows two typical traces for the oxidation of L-malate by leaf mitochondria from oats, obtained after the additions indicated. The symbols along the traces refer to the rates given in Table 4 for mitochondria, isolated from etiolated leaves after different times of light treatment. In general, the oxidation of L-malate (in the absence of Pi) was low and unaffected by the addition of ADP (lower trace). The addition of Pi stimulated the rate of oxidation (d'). When ADP was added
.13 0
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[mM] Fig. 3a and b. Concentration dependence of Pi transport into the matrix space of mitochondria in relation to the stage of greening of etiolated tissue. Transport was initiated by the addition of P~ and stopped 4 s later by mersalyl (final concentration 5 mM). Mitochondria were then immediately separated from the incubation layer by silicone oil filtration; illumi:lation: e - - e (0 h), o - - 9 (3 h), • - - x (24 h); (b) Lineweaver-Burk plot of the values given in (a). One gl sorbitol-impermeable space is equivalent to about 0.08 mg of protein. Temperature 0 ~ C
in the presence of Pi, it induced a rapid state 3 rate (C, C') of malate oxidation, which showed the property of respiratory control. Addition of mersalyl (2.5 mM) before injecting Pi into the electrode vessel, completely abolished Pi-stimulated rates of respiration (d, d') by inhibiting the Pi transport (see Fig. 1). A comparison of the rates of oxygen consumption of different developmental stages (Table 4) shows en-
330
R. Hampp: Mitochondrial Phosphate Transport and Respiration
Table 3. Kinetic constants of transport of inorganic phosphate
Table 4. Rates of L-malate oxidation by mitochondria, isolated
into the sorbitol-impermeable space of isolated mitochondria after different times of greening of etiolated leaves; the rates are calculated from the linear range of time-dependent uptake (4 s). Temperature 0~ C
from etiolated Arena leaves after 0- to 24-h of greening, as measured by oxygen electrode tracing. The rates refer to the letters along the traces in Fig. 4. The values (KCN-insensitve rates - 4-8 nmol 02 mg protein-1 min l-subtracted) are means of at least three different experiments
Time of V(gmol mg protein- 1 h- 1) K~/(mM) illumination (h) Expt. : 1 2 3 4 1 2 3 0 3 24
4.7/ 7.1/ 5.3/ 7.5 16.8/18.8/18.0/16.0 3.0/ 4.0/ 4.7/ 4.3
4
0.23/0.20/0.23/0.20 0.21/0.23/0.23/0.25 0.20/0.25/0.19/0.28
MW
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Planta 144, 325 332 (1979)
9 by Springer-Verlag 1979
Kinetics of Mitochondrial Phosphate Transport and Rates of Respiration and Phosphorylation during Greening of Etiolated Arena Leaves R. Hampp Institut ftir Botanik, TechnischeUniversit/it M/inchen, Arcisstral3e21, D-8000 Mfinchen 2, Federal Republic of Germany
Abstract. Using the technique of silicone oil filtration of organelles and the inhibitor stop method, the kinetics of transport of inorganic phosphate across the inner mitochondrial membrane were tested in relation to different stages of greening (0 to 24 h) of etiolated laminae of A r e n a s a t i v a L., and compared to the rates of oxygen consumption and ATP formation. The results demonstrate that there is a pronounced increase in phosphate transport after 3 h of greening, reaching values for V .... (about 1 7 g m o l m g protein-1 h-1) that are three times as high as those measured with mitochondria from etiolated tissue. This is also mirrored by the rates of respiration and oxidative phosphorylation. After 24 h of light treatment (4 Klx), respiration and ATP formation, as well as V decreased again to levels below those of the etiolated stage. In contrast to V, there was no change in the affinity between inorganic phosphate and the binding sites of the transporting systems involved, as indicated by a rather constant Km (0.23 mM) for phosphate transport. Of the inhibitors of phosphate transport tested, mersalyl and methyl mercuric iodide were most efficient with identical characteristics of inhibition ; but compared to animal mitochondria, the concentrations needed to result in similar amounts of inhibition, were more than ten times higher. The results are discussed with respect to plastid development. Key words: A r e n a - - Greening - Mitochondria Phosphate transport - Respiration.
BSA=bovine serum albumine; CH3HgJ=methyl mercuric iodide; Cyt-cytochrome; HEPES=N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonicacid; MDH=malate dehydrogenase; NEM = N-ethylmaleimide Abbreviations.
Introduction The major sites of intracellular energy conservation in autotrophic cells of higher plants are chloroplasts and mitochondria. Because energy is not only consumed within these organelles, transfer of energy is a prerequisite. Such a transfer of energy should be especially active with mitochondria during greening of etiolated tissue that contains photosynthetically incompetent plastids. It has been demonstrated that respiration of etiolated leaves is enhanced upon illumination of intact plants (De Greef and Verbelen, 1977; Hampp and Wellburn, 1976a) and that this change in the respiratory activity of mitochondria is accompanied by a transient change in the permeability properties of the inner mitochondrial membrane to a range of compounds (Hampp and Wellburn, 1976a). It is also known that the inner membrane of both animal (Chappell, 1968) and plant mitochondria (Phillips and Williams, 1973; Wiskich, 1975) is a site of specific transport where exchange translocators are involved. Phosphate, for example, can enter mitochondria by one of two different transport mechanisms, which can be functionally separated by the use of inhibitors. It may be taken up in exchange for O H - ions or cotransported with protons, which cannot be distinguished experimentally (Papa et al., 1969; Chappell and Crofts, 1966; McGivan and Klingenberg, 1971) - a process sensitive to N E M (Meijer et al., 1970), or it can be exchanged for dicarboxylate anions. The latter transport is inhibited by substrate analogs such as n-butyl malonate (Robinson and Williams, 1970; Palmieri etal., 1971). Both processes are also specifically inhibited by sulfhydrylblocking reagents (e.g., mersalyl; Meijer et al., 1970).
0032-0935/79/0144/0325/$01.60
326 It is a s s u m e d t h a t t h e P i / O H - e x c h a n g e serves to n e u t r a l i z e the c h a r g e i m b a l a n c e o c c u r r i n g f r o m t h e u p t a k e o f A D P 3- a n d t h e r e l e a s e o f A T P 4 - d u r i n g o x i d a t i v e p h o s p h o r y l a t i o n ( M c G i v a n et al., 1971), while the Pi/dicarboxylate exchange allows the net u p t a k e o f a n i o n i c s u b s t a n c e s at t h e e x p e n s e o f the mitochondrial pH gradient (Klingenberg, 1970; M c G i v a n a n d K l i n g e n b e r g , 1971). T h e r e f o r e , t r a n s p o r t o f a n i o n s i n t o m i t o c h o n d r i a is d e p e n d e n t o n r e s p i r a t i o n - l i n k e d p r o t o n efflux, c o u p l e d to an Pi/ OH-exchange (Pi t r a n s l o c a t o r ) , f o l l o w e d b y s u b strate/Pi e x c h a n g e ( d i c a r b o x y l a t e t r a n s l o c a t o r ) . Pj t r a n s p o r t v i a t h e p h o s p h a t e t r a n s l o c a t o r o f anim a l m i t o c h o n d r i a s h o w s v e r y h i g h rates a n d s e e m s n o t to b e r a t e l i m i t i n g f o r A T P f o r m a t i o n . O n t h e o t h e r h a n d , D a y a n d H a n s o n (1977) s h o w e d f o r p l a n t m i t o c h o n d r i a t h a t the r a t e o f Pi t r a n s p o r t ( r e d u c e d b y i n h i b i t o r s a n d u n c o u p l e r s ) is closely r e l a t e d to the a m o u n t o f t h e s u b s t r a t e o x i d a t i o n : r a p i d o x i d a tion of malate and succinate required high transport activities o f b o t h t h e Pi a n d t h e d i c a r b o x y l a t e t r a n s l o cator. It c a n t h e r e f o r e be s u g g e s t e d t h a t p h y s i o l o g i c stages w i t h h i g h e n e r g y d e m a n d (e.g., g r e e n i n g o f e t i o l a t e d tissue) s h o u l d be r e f l e c t e d by i n c r e a s e d rates o f m i t o c h o n d r i a l r e s p i r a t i o n , a c c o m p a n i e d b y enh a n c e d rates o f Pi t r a n s p o r t a c r o s s t h e i n n e r m i t o c h o n d r i a l m e m b r a n e . T h u s it was the a i m o f t h e exp e r i m e n t s d e s c r i b e d in this p a p e r to i n v e s t i g a t e t h e k i n e t i c s o f P~ t r a n s p o r t o f m i t o c h o n d r i a d u r i n g differe n t stages o f g r e e n i n g o f e t i o l a t e d l e a f tissue, a n d to c o m p a r e it to the rates o f o x y g e n c o n s u m p t i o n and ATP formation under the same conditions.
Materials and Methods Isolation of Mitochondria Seedlings of Arena sativa L. (var. Arnold) were grown in moist peat at 25 ~ C for 7 days in the dark. If different times of greening were required, partial illumination in the later stages of growth was achieved by a halogen lamp (Osram, HQLS, 400 W) providing a radiant light flux of 4 Klx at seedling level. About 50 g of laminae (uppermost 5 cm) were homogenized in t00ml of chitIed isolation medium (0.3 M sorbitol; 0.001 M MgC12; 0.001 M NaNO3; 0.001 M EDTA; 0.2% (w/v) BSA, Sigma Type V; in 0.05 M HEPES, finally adjusted to pH 7.3 M - prior to adding the plant tissue, cysteine (0.05%, w/v) was stirred in) using a Starmix (Braun, Melsungen, W. Germany) in three bursts of 5 s duration each. The homogenate was filtered twice through four layers of nylon sheet (Nytal 25 T 1, mesh 35 gm, Henry Simon, Stockport, U.K.) and centrifuged for 7 min at 1,000 g. The resulting pellet (mainly plastids) was discarded and the supernatant was centrifuged (1) for 10min at 10,000g, (2) after resuspension of the pellet (50 ml of homogenation medium) for 10 rain at 8,000 g. Finally the pellet was resuspended in isolation medium to give a protein content of about 2 3 mg/ml.
R. Hampp : Mitochondrial Phosphate Transport and Respiration All steps were carried out at temperatures ranging from 0 4 ~ C, and in the case of etiolated tissue in complete darkness. The amount of protein was measured by the method of Lowry et at. (1951).
Enzyme Assays Cytochrome oxidase (EC 1.9.3.1) was determined by adding an appropriate aliquot of particle suspension (50-100 gl) to 1.5 ml of 20 gM reduced Cyt c in 50 mM HEPES (pH 7.3) and following the change in optical density at 550 nm. NADH:Cyt c reductase (EC 1.6.99.3) was assayed according to Lord et al. (1973); the medium contained buffer as above, plus 20 gM oxidized Cyt c, 80 gM NADH, and 1 mM KCN. The reaction was initiated by adding the particle suspension, and the reduction of Cyt c was followed at 550 nm. Antimycin A was added in 10 gl of ethanol to give a final concentration of 1 gM. Catalase (EC 1.11.1.6) was assayed by following the 02 production using a Clark-type oxygen electrode. The reaction mixture contained in 50 mM HEPES (pH 7.3) 1.2mM H20 2 in a total volume of 1.5 ml. Oxygen was removed from the assay mixture by bubbling O2-free N 2 through it prior to measurement. Ribulose-bisphosphate carboxylase (EC 4.1.1.39) was activated and measured according to the method of Lorimer et al. (1977), NADH: MDH (EC 1.1.1.37) as described by Bonner (1974).
Measurement of Kinetics of Phosphate Transport All measurements of phosphate transport were made using an "inhibitor stop" technique, in which transport is stopped by the rapid addition of a transport inhibitor, followed by vigorous stirring (Pfaff and Klingenberg, 1968). The incubation was carried out in isolation medium, which contained additionally oligomycin (5 Ixg/ml) to prevent the incorporation of Pi into ATP. The uptake was started by the addition of [32p]orthophosphate (1-2 ~tCi; final concentrations, see Results) and terminated after time t by rapid addition of mersalyl (5 mM; exceptions see legends to Figures). This compound was shown (Meijer et al., 1970) to inhibit Pi transport via the phosphate and the dicarboxylate translocator almost completely. With this "inhibitor stop" method, the shortest time resolved was 2 s. The rates of uptake were calculated from the initial and linear parts of the time course of uptake, limited to a few seconds. The mitochondria with the trapped phosphate were separated from the incubation medium by rapid centrifugation through a layer of silicone oil (AR 80, Wacker-Chemie, Munich, W.-Germany) into 50 gl of 10% (v/v) HCIO4 (Microfuge B 152, Beckman, 30 s). With the exception of time-dependent experiments, incubation and centrifugation were performed within t min. Control experiments were carried out, in which the inhibitor was added before the labeled phosphate to the incubation mixture containing the mitochondria. In these controls, the time of contact of the mitochondria with phosphate was the same as in the case when merslyt was used to stop the uptake. The radioactivity in the supernatants and in the pellets was measured in a scintillation counter against standards using preset counting characteristics and quench curves constructed specifically for these experiments. 3H20, [U-3H]inulin, and [U-14C]sorbitol were added in parallel experiments to determine the intermembrane and matrix spaces. For the operational definition of these spaces see Klingenberg and Pfaff (1966). The amount of Pi taken up into the matrix space was calculated as described previously (Hampp and Wellburn, 1976a). All incubations and eentrifugations were carried out between 0 and 4~ C.
R. Hampp: Mitochondrial Phosphate Transport and Respiration
Chromatographic Analysis of 32pi In order to verify that 32pi taken up by mitochondria that have been incubated at 0~ C for up to 10 min in the presence of oligomycin, was not metabolized, the perchloric acid extracts of the collected mitochondrial pellets were neutralized (KOH) and subjected to chromatographic analysis (Coty and Pedersen, 1974). No detection of changes in the amount of migration of inorganic phosphate was observed by comparing aliquots at the beginning and at the end of the incubation period.
Respiration of Mitochondria Recordings of 0 2 consumption were carried out using a Clark-type oxygen electrode. The reaction vessel contained an assay volume of 2 ml with additives as indicated in Fig. 4. The temperature was 20~ C.
327 Table 1. Levels of activity of enzymes in the mitochondrial fraction. The activities are average values (• SD, n = 5) for different stages of greening of etiolated tissue (0, 3, 24 h); they are expressed as specific activity and as percentage of the respective levels of activity within the total homogenate Enzyme
Specific activity nmol min-a mg protein-
Ribulose-bisphosphate carboxylase
1.9 (0h) 3.8 (24h)
Cyt c oxidase
56.0 _+7.0
30.0 _+5.0
2.5 • 1.5
0.8 • 0.4
Cyt c reductase (antimycin A insensitive) Catalase
15,000 +_5,000
Per cent of total activity
0.9+_0.4
7.0 • 5.0
A TP Measurements Mitochondria (about 2 mg protein) were incubated at 20~ in 2 ml of isolation medium (EDTA and NaNO3 omitted), which contained in addition 1.3 gmol NADH, 10 gmol Pi, and 170 nmol ADP. Samples of 400 gl were withdrawn from the reaction vessel before and 1-3 min after the addition of ADP. The samples were kept for 2 min in a boiling water bath and afterward cooled down on ice; the denaturated mitochondria were pelleted at 6000 g for 5 min. A 200-ml aliquot of the supernatant was assayed for ATP with the luciferin/luciferase system, by mixing it with 0.8 ml of a solution containing (mM): Na2HAsO4 10, EDTA 1, MgSO4 5, glycine buffer 20 (pit 7.8). The complete sample was injected into a glass vial, which was inserted into a Farrand fluorometer and contained 100 ~tl of luciferin/luciferase (Sigma, Munich, FRG). The resulting luminescense was recorded and calibrated by the immediate addition of a known quantity of ATP. To determine adenytate kinase blanks, the same procedure was followed, but initial additions were made of 20 gM rotenone, 3 ~tM antimycin A, and 20 gg oligomycin, and NADH was omitted.
Significance of the Results The different experiments (5 parallels each) were repeated at least four times (see Table 3). Each developmental sequence (~24 h of illumination) was investigated as one complex.
Chemica& [32p]orthophosphoric acid, (U-a4C]sorbitol, [U-all]inulin, and [3H]H20 were obtained from the Radiochemical Center (Amersham, U.K.); antimycin, oligomycin, mersalyl and luciferin/luciferase were from Sigma (Munich, FRG); CH3HgJ was from Koch Light Labs, (Colnbrook, Bucks., U.K.).
Results
Purity and Integrity o f the Mitochondrial Fraction T a b l e 1 gives the average levels of activity associated with m i t o c h o n d r i a that have been isolated f r o m etiolated leaves after different times of i l l u m i n a t i o n , of
r i b u l o s e - b i s p h o s p h a t e carboxylase, Cyt c oxidase, N A D H : C y t c reductase ( a n t i m y c i n A insensitive), a n d catalase, which are m a r k e r s for plastids, mitochondria, e n d o p l a s m i c reticulum, a n d peroxisomes (Lord et al., 1973; Tolbert, 1971; T o l b e r t et al., 1968) in specific activity a n d in per cent of the respective rates m e a s u r e d in the total h o m o g e n a t e . The results indicate only a low c o n t a m i n a t i o n of the m i t o c h o n drial fraction by other particulate fractions; m i t o c h o n d r i a that had been passed t h r o u g h a layer of silicone oil ( b o t t o m - m o s t layer of centrifuge tubes 0.6 M sorbitol instead of HC104) showed an additional decrease of c o n t a m i n a t i o n of u p to 80% compared to the same m i t o c h o n d r i a l fraction before b e i n g i n t r o d u c e d to silicone oil filtration. These results dem o n s t r a t e that the rates of t r a n s p o r t given in this paper refer only to m i t o c h o n d r i a a n d that there is n o c o n t r i b u t i o n of other organelles. Similar conclusions have been d r a w n f r o m electron m i c r o g r a p h s of the m i t o c h o n d r i a l pellet ( H a m p p a n d W e l l b u r n , 1976a). I n T a b l e 2 a r e l a t i o n s h i p between the sorbitol c o n c e n t r a t i o n of the m e d i u m a n d the a m o u n t of m i t o c h o n d r i a (expressed as s o r b i t o l - i m p e r m e a b l e organelle space) able to penetrate a silicone layer of a distinct density is given. The results show that with decreasing c o n c e n t r a t i o n of sorbitol the n u m b e r of b r o k e n m i t o c h o n d r i a increases as m e a s u r e d by the level of activity of m i t o c h o n d r i a l N A D H : M D H after the a d d i t i o n of N A D H a n d oxaloacetate ( N A D H is n o t able to penetrate the intact m i t o c h o n d r i a l envelope; see B o n n e r , 1974). I n parallel, the n u m b e r of m i t o c h o n d r i a able to p e r m e a t e the silicone layer decreases. This f i n d i n g indicates that p r i m a r i l y organelles that retained their m e m b r a n e s are included in the e s t i m a t i o n of rates of u p t a k e by m e a n s of filtering centrifugation.
328
R. Hampp: Mitochondrial Phosphate Transport and Respiration
Table 2. Integrity of the mitochondrial membranes as measured by the levels of activity of N A D H : malate dehydrogenase, and number of organelles (expressed as sorbitol-impermeable space in the pellet after silicone centrifugal filtration) able to penetrate a silicone layer, in relation to the molarity of sorbitol in the incubation medium. The percantages are means ( + S D , n = 6 ) for mitochondria isolated from 0- to 48-h illuminated leaf tissue. The specific activity given for M D H was measured with mitochondria from etiolated tissue (with development there are changes in specific activity). Before centrifugation, the sorbitol concentration each time was made up to 0.3 M Sorbitol (M)
0.3
0.25
0.20
0.15
0.10
0
Malate dehydrogenase (~tmol N A D H mg protein-~ rain-1) Integrity (%) Matrix space in the pellet (~tl)
0.68 76_+5 0.26
0.88 68_+6 0.25
1.10 57_+6 0.21
1.38 51_+7 0.12
1.65 43_+5 0.10
2.75 0 0
100
medium concentration; for both compounds the increase in inhibitory action is most pronounced for concentrations up to 100 laM, but exceeds 90% only with 5-mM solutions. The effect of NEM, however, is much less expressed and requires at least 1 min of preincubation of the mitochondria before the addition of labeled P~ to exert maximum inhibition. The increase in inhibition is highest with NEM concentrations up to 5 mM ; but even with 25 mM NEM inhibition is only about 70% of the rates measured with untreated controls.
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) inhibitor concentration [mM] Fig. 1. Transport of inorganic phosphate into the matrix space of mitochondria in relation to the stage of greening of etiolated tissue ((~24 h of illumination, 4 Klx). Inhibition by mersalyl, methyl mercuric iodide, and N-ethylmaleimide; inorganic phosphate (0.1 raM), mersalyl, and CH3HgJ were applied prior to the addition of mitochondria. In the experiments with NEM, organelles were preincubated with the inhibitor for 60 s (longer times did not result in higher inhibition) before the addition of Pi; 1 m M inhibitor concentration is equivalent to about 500nmol/mg protein; 100%=5.5 (0 h: e - - e ) , 16.6 (3 h: 9 3.9 (24 h: x - - x ) ~tmol mg protein 1 h i. Temperature 0 ~ C
Effect of Sulfhydryl-Blocking Reagents on Mitochondrial Phosphate Transport during Greening In order to study the sensitivity for inhibitors of transport of inorganic phosphate during greening, the effects of the sulfhydryl-blocking reagents mersalyl, CH3HgJ, and NEM were tested. Figure 1 shows the efficiency of the different compounds in reducing the rates of uptake of Pi (0.1 mM). Generally, the values indicate that mitochondria isolated from leaf tissue after different stages of greening (0, 3, 24 h) respond in a similar way. Mersalyl and CH3HgJ cause nearly identically decreasing rates of uptake with increasing
Kinetics of Transport of Phosphate during Greening The time course of the uptake of inorganic phosphate by mitochondria, isolated from greening leaf tissue, is illustrated in Fig. 2. Uptake is rapid with all stages tested and shows a linear rate only up to about 6 s. In comparison to 24 h light treatment, mitochondria of less greened tissue exhibit higher rates of transport and also higher levels of accumulation of inorganic phosphate within the matrix space. Interestingly, both transport rates and accumulation show an intensive transient increase during the early stages of greening with a maximum at about 3 h of illumination. Figure 3 gives a typical example for the changing rates of uptake of Pi in relation to the medium concentration (0.1-1 mM) and the stages of greening of etiolated tissue. The concentration dependence reveals hyperbolic saturation characteristics, indicating substrate saturation of transport for all stages tested. Additionally, the results show accumulation of Pi in the matrix space, especially with low external concentrations; this accumulation is most pronounced for mitochondria isolated from etiolated tissue that had been given 3 h light treatment (about 5 times the external concentration within 4 s). Transport in mitochondria from etiolated and 24 h illuminated leaves is comparable, but on a much lower level.
R. Hampp : Mitochondrial Phosphate Transport and Respiration I U rl
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Fig. 2. Time course of uptake of inorganic phosphate into the matrix space of mitochondria in relation to the stage of greening of etiolated tissue. Transport was initiated by the addition of P~ and stopped by the addition of mersalyl (final concentration 5 raM) at the times given ; illumination : 9 - e (Oh), o - - o (3h), x - - x (24h). One ~tl sorbitol-impermeable space is equivalent to about 0.08 mg of protein. Temperature 0 ~ C
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A double reciprocal plot of the data given in Fig. 3a yields linear functions (Fig. 3b), which enable the determination of the substrate concentrations that cause half maximal rates of transport (Kin) and the maximum velocities of transport (V). The results of the experiment, illustrated in Fig. 3 (Expt. 1) and of others (Exp. 2 to 4; Table 3) confirm that V (incubation time: 4 s) is highest after 3 h of illumination with about three times the rates measured with organelles from etiolated and 24 h illuminated leaves. In contrast, there is no change in the affinity between Pi and the binding sites of the transporting systems involved. The average K m a s calculated from the values shown in Table 3 is 0.23 mM.
Respiration and Oxidative Phosphorylation during Greening Figure 4 shows two typical traces for the oxidation of L-malate by leaf mitochondria from oats, obtained after the additions indicated. The symbols along the traces refer to the rates given in Table 4 for mitochondria, isolated from etiolated leaves after different times of light treatment. In general, the oxidation of L-malate (in the absence of Pi) was low and unaffected by the addition of ADP (lower trace). The addition of Pi stimulated the rate of oxidation (d'). When ADP was added
.13 0
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[mM] Fig. 3a and b. Concentration dependence of Pi transport into the matrix space of mitochondria in relation to the stage of greening of etiolated tissue. Transport was initiated by the addition of P~ and stopped 4 s later by mersalyl (final concentration 5 mM). Mitochondria were then immediately separated from the incubation layer by silicone oil filtration; illumi:lation: e - - e (0 h), o - - 9 (3 h), • - - x (24 h); (b) Lineweaver-Burk plot of the values given in (a). One gl sorbitol-impermeable space is equivalent to about 0.08 mg of protein. Temperature 0 ~ C
in the presence of Pi, it induced a rapid state 3 rate (C, C') of malate oxidation, which showed the property of respiratory control. Addition of mersalyl (2.5 mM) before injecting Pi into the electrode vessel, completely abolished Pi-stimulated rates of respiration (d, d') by inhibiting the Pi transport (see Fig. 1). A comparison of the rates of oxygen consumption of different developmental stages (Table 4) shows en-
330
R. Hampp: Mitochondrial Phosphate Transport and Respiration
Table 3. Kinetic constants of transport of inorganic phosphate
Table 4. Rates of L-malate oxidation by mitochondria, isolated
into the sorbitol-impermeable space of isolated mitochondria after different times of greening of etiolated leaves; the rates are calculated from the linear range of time-dependent uptake (4 s). Temperature 0~ C
from etiolated Arena leaves after 0- to 24-h of greening, as measured by oxygen electrode tracing. The rates refer to the letters along the traces in Fig. 4. The values (KCN-insensitve rates - 4-8 nmol 02 mg protein-1 min l-subtracted) are means of at least three different experiments
Time of V(gmol mg protein- 1 h- 1) K~/(mM) illumination (h) Expt. : 1 2 3 4 1 2 3 0 3 24
4.7/ 7.1/ 5.3/ 7.5 16.8/18.8/18.0/16.0 3.0/ 4.0/ 4.7/ 4.3
4
0.23/0.20/0.23/0.20 0.21/0.23/0.23/0.25 0.20/0.25/0.19/0.28
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