Planta (Berl.)131, 2 1 - 26 (1976)
P I ~ ) H ~ 9 by Springer-Verlag 1976
Changes in the Permeability of the Inner Mitochondrial Membrane Associated with Plastid Development R. Hampp* and A.R. Wellburn Department of Biological Sciences, University of Lancaster, Lancaster LA1 4YQ, U.K.
Summary. Mitochondria isolated from greening etiolated laminae of Arena sativa L. show changes in the permeability of their inner membranes during chloroplast development similar to those described earlier for plastids. Oxalo-acetate, succinate and c~keto-glutarate permeate most readily inner membranes of mitochondria isolated from laminae given 2 h illumination whilst glutamate and glycine show later and more general penetration into the matrix spaces of mitochondria from greening tissue. Aminolevulinic acid (ALA) by contrast does not readily enter Arena mitochondria especially those isolated from laminae illuminated for longer than 2 h.
Introduction
Cellular systems show a high degree of structural complexity and the regulation of metabolic processes is of central importance in maintaining this organisation. This is partly achieved by separated compartments surrounded by selectively permeable membranes such as those of the mitochondria and plastids. The inner envelope membrane of chloroplasts has been shown to be a site of specific metabolite transport from the cytosol into the stroma of the plastid (Heldt and Sauer, 1971; Gimmler et al., 1974) and corresponding observations have already been made with mitochondria (see Klingenberg and Pfaff, 1966). Using the technique of silicone oil centrifugal filtration (Klingenberg and Pfaff, 1967) it was demonstrated that there is a change in permeability of the transport limiting membrane of the envelope of plastids Abbreviations: ALA = amino-levulinic acid; HEPES = N-2-hydroxyethyl-piperazine-N'-2-ethane-sulphonic acid * Permanent address: Institut ftir Botanik, Technische Universit,it, Arcisstr. 21, D-8000 Mfinchen 2, Federal Republic of Germany
(Hampp and Wellburn, 1976) during development. Oxaloacetate and succinate for example, showed a pronounced increase in uptake after 1 to 2 h of greening whilst other compounds showed characteristic permeability changes over longer periods of plastid development. Most of the metabolites examined were precursors for the synthesis of components of membranes and pigments, an extensive process in the early stages of chloroplast morphogenesis. As long as the plastids have not reached that developmental stage when the ability to fix CO2 is aquired such precursors in the main are probably imported from outside the developing plastid, in some cases having originated in the mitochondria. Consequently the permeability of the inner mitochondrial membranes during the period of the development of plastids were investigated using slightly modified techniques to those used earlier (Hampp and Schmidt, 1976; Hampp and Wellburn, 1976). The results indicate that mitochondria isolated from etiolated Arena laminae given the same light treatment as was used for the plastid investigations show similar changes in the permeability of the inner mitochondrial membranes to those that occur in the inner envelope membranes of corresponding etioplasts or 1-24 h etio-chloroplasts.
Materials and Methods Preparation of Mitochondria from Greening Arena Laminae
Seedlings of Arena sativa L. (cv. Mostyn) were grown in gravel trays for ten days at 22~ in moist peat in the dark. Some of the trays were partially illuminated (6 klux; 1, 2, 4, 8 or 24 h) during the later stages of growth. Mitochondria were isolated from batches of these different laminae using a modified method based upon that of Bonner (1967) allowing when necessary the simultaneous isolation of plastids from the same initial homogenate. About 100 g of laminae were homogeuised in 170 ml of chilled isolation medium (0.45 M sorbitol; 0.001 M MgCI2; 0.001 M KH2PO4; 0.001 M NaNO3; 0.2% (w/v) bovine serum albumin,
22
R. Hampp and A.R. Wellburn: Permeability of the Mitochondrial Membrane
Sigma Type V; all in 0.05 M HEPES, finally adjusted to pH 7.3 with KOH) using a Townson and Mercer top-drive homogeniser in 3 bursts of 7s duration. The homogenate was filtered twice through 4 layers of nylon sheet (Nytal 25T1, mesh 35 gm, Hemy Simon, Stockport) and centrifuged in 4 cooled polypropylene tubes (50 ml) for 7 min at 1000 x g in a swing-out bench centrifuge kept at 4 ~ After centrifugation the pellets were suitable for a subsequent plastid isolation but for mitochondrial preparations the combined supematants were diluted with a half volume of 3 mM EDTA (i.e. sorhitol concentration reduced to 0.3 M). At the point of mixing powdered cysteine was added so as to give a final concentration of 0.05% (w/v), followed by centrifugation at 10,000 x g for I5 rain at 2~ using an MSE 18 ultracentrifuge and an 8 x 50 ml fixed anne rotor. The crude mitochondrial pellets were resuspended in a total of 30 ml of isolation medium B (2 volumes of isolation medium A plus 1 volume of 3 mM EDTA, but without added cysteine). Two turns of a loosely fitting TenBroeck homogeniser was used to assist the resuspension and a further centrifugation carried out at 6,000 x g for 15 min at 2~ The pellet was resuspended as before in 10 ml of isolation medium B and used for 02 electrode, volume and permeability studies or prepared for electron microscopy.
G.F.R.) and 100 I~1 mitochondrial suspensions incorporating aliquots (5%) of the labelled solutions (final concentration 5 raM), were accelerated to full speed for 10 s and back to rest using a Beckman Microfuge B after a minimum of 1 min incubation at 0~ The tubes were then frozen in liquid N2 and the tips (bases) cut off with cable cutters and dropped into scintillation vials containing 0.5 ml water. After vibration to dislodge and disperse the pellets, 10 ml of Bray's solution (Bray, 1960) was added to each vial and the amount of radioactivity in each pellet determined by counting in a Packard 3375 liquid scintillation counter against standards using pre-set counting characteristics for 3H and 14C and quench curves constructed specifically for these experiments. At least 8 parallel treatments were carried out, each having a standard deviation of counting of 1.5%. From the mean of these a calculation of the matrix space concentration, relative to the incubation medium concentration of 5 mM, is possible after determining the total volume by the uptake of tritiated water. The amount of unspecific permeation into the inner membrane space was corrected for by the uptake of [U-14C]sucrose and the amount of medium adhering to the outer mitochoudrial surfaces by the use of dextran (M.W. 40x 103 daltons, 2 mgm1-1, see Heldt and Sauer, 1971). The protein content of mitochondrial suspensions was determined by the method of Lowry et al. (1951).
Measurement of 02 Uptake by Arena Mitochondria
Results Oxygen consumption of mitochondrial preparations was measured in a stoppered chamber of total volume I ml with a side entry pore occupied by a Clark type Oz electrode and stirred by means of a Teflon coated magnetic "flea" at the base. Rates of oxidation of succinate (8 pmoles) and ADP (50 nmoles) were recorded on a scale established between that of air saturated water and that of zero-free oxygen water achieved after adding sodium dithionite crystals.
Electron Microscopy of Mitochondrial Suspensions An equal volume of 5% (v/v) glutaraldehyde in isolation medium B was added to mitochondrial suspensions in 2 ml polyethylene centrifuge tubes (Beckman) and left for 1 h at 0% The tubes were centrifuged for 30 s using a Beckman Microfuge B and the pellets washed with isolation medium B and left overnight in 1% (w/v) OsO4 in isolation medium B still at 0 ~ They were dehydrated through a graded ethanol series made up in isolation medium of decreasing sorbitol, bovine serum albumin and buffer concentration, allowed to rise to room temperature and finally dried in propylene oxide. The pellets were embedded in Epon and sectioned on a Reichert OMU2 ultramicrotome. Sections were double stained in uranyl acetate and lead citrate (Reynolds, 1963) and examined in a GEC-AEI EM801 electron microscope.
Measurement of the Matrix Space Concentrations of Plant Mitochondria Labelled compounds were obtained from the Radiochemical Centre with the exception of oxalo-acetate which was prepared from malate using the method of Kirk and Leech (1972), All compounds were dissolved in isolation medium B and corrected with unlabelled compounds so as to give a concentration of 100 mM and a specific activity of 0.3 mCi mM -1, readjusting the pH where necessary with KOH. The modified silicone oil centrifugal filtration was based on that described by Klingenberg and Pfaff (1967). Polyethylene centrifuge tubes (250 gl, Beckman), containing 50 gl 10% (v/v) HC104 (lower-most) plus 50 gl silicone oil (Types AR50 and AR150 in a ratio 2: 1, both from Wacker Chemic, Burghausen,
Many different methods of preparation of plant mitoc h o n d r i a w e r e initially t e s t e d b u t f i n a l l y the m o d i f i e d f o r m o f the p r o c e d u r e o f B o n n e r (1967) d e s c r i b e d here was adopted. Those preparations which advocate d the use o f d e n s i t y g r a d i e n t c e n t r i f u g a t i o n s u c h as t h a t o f D o u c e et aI. (I972) w e r e r e j e c t e d o n the grounds of yield and osmotic problems after removal o f the c u s h i o n m e d i u m . R o u t i n e m o n i t o r i n g o f p r e p a rations by electron microscopy was adopted and a r e p r e s e n t a t i v e field o f s u c h a p r e p a r a t i o n is s h o w n in F i g u r e 1. L i t t l e p l a s t i d c o n t a m i n a t i o n was enc o u n t e r e d e v e n in p r e p a r a t i o n s f r o m e t i o l a t e d tissue a n d the m i t o c h o n d r i a w e r e w e l l p r e s e r v e d w i t h little evidence of osmotic damage. The main contaminant a p p e a r e d to be p e r o x i z o m a l m a t e r i a l . T h e s i l i c o n e oil c e n t r i f u g a l f i l t r a t i o n t e c h n i q u e p r o v e d to be a u s e f u l d i a g n o s t i c test o f o r g a n e l l e i n t a c t n e s s . F u r t h e r m o r e b y a d j u s t i n g the r a t i o s o f the s i l i c o n e oils a n y p r e p a r a t i o n c o u l d be q u i c k l y c h e c k e d f o r the p r e s e n c e o r a b s e n c e o f i n t a c t plastids. F i g u r e 2 s h o w s the t r i t i a t e d w a t e r p e r m e a b l e s p a c e o f m i t o c h o n d r i a e x p r e s s e d o n p r o t e i n basis in r e l a t i o n to t h e l i g h t t r e a t m e n t o f the l e a f tissue. W h i l e the r a t i o o f v o l u m e to p r o t e i n is r e l a t i v e l y c o n s t a n t f o r 0, 2, 4, 8 a n d 2 4 h o f l i g h t t r e a t m e n t , b e t w e e n 0 a n d 2 h t h e r e is a m a r k e d i n c r e a s e in the m i t o c h o n d r i a l v o l u m e . T h e o s c i l l a t i o n o f the v o l u m e s u g g e s t e d b e t w e e n 2 a n d 8 h i l l u m i n a t i o n h a s n o t b e e n statistic a l l y s u b s t a n t i a t e d . It m a y b e seen in F i g u r e 3 t h a t the r e s p i r a t o r y a c t i v i t i e s o f the s a m e m i t o c h o n d r i a , u s e d for the volume determination, mirror these changes in v o l u m e . M a x i m u m u p t a k e o f 0 2 in t h e p r e s e n c e o f s u c c i n a t e a n d s u c c i n a t e p l u s A D P o c c u r s in mito-
R. Hampp and A.R. Wellburn: Permeability of the Mitochondrial Membrane
23
Fig. 1. Electron micrograph of a typical mitochondrial suspension. In this case, the preparation was isolated from etiolated Arena laminae given 8 h illumination, x 20,000
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Hours of iLLumination of laminae
Fig. 2. Changes of the tritiated water permeable volume of isolated Arena mitochondria with respect to the illumination of etiolated laminae from which they were isolated
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Hours of illumination of laminae
Fig. 3. Consumption of oxygen in the presence of succinate and succinate ptus ADP by isolated A~'ena mitochondria with respect to the illumination of etiolated laminae from which they were isolated
24
R. Hampp and A.R. Wellburn: Permeabilityof the MitochondrialMembrane
361
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Fig. 4. Penetration of 14C-labelled glutamate, e-ketoglutarate and 6-aminolaevulinate into the matrix space of mitochondria in relation to the illumination of etiolated Arena laminae (0 ~ C, 1 min; dashed line: external concentration of the respective compound)
Fig. 5. Penetration of 14C-labelled succinate, oxaloacetate and glycine into the matrix space of mitochondria in relation to the illumination of etiolated Arena laminae (0 ~ C, 1 min; dashed line: external concentration of the respective compound)
chondria isolated from 1 h illuminated laminae although the proportional increase on addition of ADP (state 4 to state 3) is much the same throughout indicating the maintenance of respiratory control. In a mitochondrial suspension three different spaces have been defined (see Klingenberg and Pfaff; 1966) : the extra-mitochondrial space (suspension medium), the cristae space and the matrix space. These spaces are separated by two membranes, the outer and the inner mitochondrial membranes. Only the inner membrane contains characteristic particles or spheres which face the matrix space (Parsons, 1963) and functions as a metabolic barrier between the cristae space and the tightly enclosed matrix space. In contrast the outer membrane is unspecifically permeable to metabolites of low molecular weight. Measurement of the penetration of labelled metabolites into the matrix space is therefore only possible when experimental values are corrected for unspecific penetration into the cristae space and the amounts of label adhering to the outer membrane (see Methods). In these experiments the matrix space was found to be about 40% of the total mitochondrial volume. This is in accordance with the results of other authors using mitochondria isolated in isotonic conditions (Malamed and Recknagel, 1959; Klingenberg and Pfaff, 1966). Figures 4 and 5 show the matrix concentrations of different compounds in mitochondria isolated from Arena laminae after 0-24 h of greening. The time
of incubation was 1 min and the concentration of the respective compound 5 mM (dashed horizontal lines). All the compounds tested showed an increase in intra-mitochondrial concentration up to 4 h of greening. For some compounds there is even an accumulation in the matrix space relative to that of the extra-mitochondrial space. This uptake is pronounced for both succinate and oxalo-acetate. Although the maximum uptake of succinate is limited to a period of 1 h of greening, the permeability for oxalo-acetate remains at a high value for almost all the stages of light treatment and, in this respect, is similar to that of glutamate. This increase in permeability (penetrability) during greening is not so pronounced for c~-keto-glutarate and glycine; nevertheless these compounds show distinct maxima for the matrix space concentrations after 2 and 4 h illumination respectively. No accumulation in the matrix space of ALA could be shown for any of the stages of greening, although those mitochondria isolated from etiolated tissue or from 1 h illuminated laminae showed a higher internal concentration than those from seedlings given longer illumination. In general the penetrability of the inner mitochondrial membranes of mitochondria from etiolated tissue is similar to that of 24 h illuminated mitochondria. The observed changes in the permeability of the inner mitochondrial membrane during development are reflected in the changes of the volume to protein ratio and also in the changes of the respiratory activ-
R. Hampp and A.R. Wellburn: Permeability of the Mitochondrial Membrane
ities of mitochondria during the different stages of greening. Discussion
These results identify a change in the permeability of the inner mitochondrial membrane during the greening of etiolated A r e n a laminae. This change in permeability is very similar to that of the inner envelope membrane of etioplasts and etio-chloroplasts isolated at the same stages of greening (Hampp and Schmidt, 1976; Hampp and Wellburn, 1976). For both organelles, oxalo-acetate and succinate show a maximum in internal concentration after 1 h of light treatment, with the internal concentrations markedly higher than those of the external medium. The maxima in permeability of each with respect to ~-ketoglutarate are also in accordance with each other after 2 h illumination. However, there are different uptake characteristics with respect to glycine and glutamate and the permeability for ALA is low for both transport limiting membranes throughout development. The importance of oxalo-acetate for the synthesis of aspartate in plastids has been discussed elsewhere (Hampp and Wellburn, 1976). Kirk and Leech (1972) have demonstrated that the transport of oxalo-acetate from the cytoplasm into the plastid is a pre-requisite for aspartate formation. Our results point towards the possibility that the mitochondrion is the site of oxaloacetate formation. There is ample evidence that the complete pathway of porphyrin synthesis from ALA to the chlorophylls exists in chloroplasts (see Rebeiz and Castelfranco, 1973). In contrast the origin of ALA in higher plants is unknown and a subject of much investigation. More recent papers (Beale and Castelfranco, 1974; Beale et al., 1975; Wellburn, 1975) indicate that e-keto-glutarate, glutamate or glutamine may be possible precursors for ALA synthesis but no convincing evidence is available to suggest that all the enzymes necessary to synthesise these compounds exist in plastids. The implication of this is that the metabolites required for ALA synthesis are supplied from a source outside the plastid. Mitochondria possess the ability so synthesize porphyrins and to provide the necessary precursors. They could also supply compounds for similar processes in plastids. Our results support this possibility. Succinate and, to a lesser extent, c~-keto-glutarate and glutamate show permeability of the inner mitochondrial membrane at the appropriate time when plastidic requirements for ALA are highest. The belief that the major portion of the ALA is translocated from mitochondria to plastids by simple permeation is not supported by our results.
25
A number of studies have established that mitochondria undergo volume changes which depend on the anions composition of both extra-mitochondrial and intra-mitochondrial spaces (Lynn and Brown, 1965). The mitochondrial swelling or contraction is believed to be, in part, the result of osmotic adjustments to the movement of anions (see Miller et al., 1975). Certain anions, such as phosphate and acetate cause a substrate dependent swelling of mitochondria which is thought to reflect ion transport and accumulation. Active swelling in the presence of a number of oxidizable and non-oxidizable organic acids has also been demonstrated (Lee and Wilson, 1972). Since active swelling is correlated with active ion accumulation, the observed increase in volume of the tritiated water permeable space of mitochondria isolated from 1 h light treated tissue could be linked to the enhanced synthetic activities within these organelles. This high metabolic activity, which is also shown by the increased respiratory activity, is followed by an increase in membrane permeability starting between 1 and 2 h of illumination. As a result of the higher permeability the osmotic pressure inside the mitochondria decreases accounting for the decrease in volume down to that shown by mitochondria isolated from etiolated tissue. All these results substantiate the importance of mitochondria in supporting the development of plastids during the greening of etiolated A r e n a laminae and imply changes in the nature of the inner mitochondrial membrane during this maturation. The nature of the control of these apparently co-ordinated changes is immediately questioned. Perhaps one should look to phytochrome as a modifier of envelope permeabilities or seek alternative or additional mechanisms to understand the nature of permeability control especially when co-ordinated changes in different organelles are involved. What is certain is that mitochondria isolated from etiolated tissue differ from those of 1 h illuminated laminae, and so on, especially in respect to the permeabilities of their inner membranes. Although no structural differences were suggested from the electron microscope study of the different preparations, the respiratory behaviour suggests that this may not be the only difference. It may well be that in the future one might refer to (1 h) etio-mitochondria (i.e. mitochondria isolated from etiolated plant tissue given illumination of 1 h ) j u s t like the present use of the accepted term etio-chloroplasts, originally suggested by Egnfius et al. (1972); if only to save on descriptive text. We thank the Deutsche Forschungsgemeinschaftand the Royal Society for the award of a European Exchange Fellowship to one of us (R.H.) and Mr. T. Travis for technical assistance.
26
R. Hampp and A.R. Wellburn: Permeability of the Mitochondrial Membrane
References Beale, S.I., Castelfranco, P.A. : The biosynthesis of 6-aminolevulinic acid in higher plants. II. Formation of l~C-6-aminolevulinic acid form labelled precursors in greening plant tissues. Plant Physiol. (Baltimore) 53, 297-303 (1974) Beale, S.I., Gough, S.P., Granick, S. : Biosynthesis of 3-aminolevutinic acid from the intact carbon skeleton of glutamic acid in greening barley. Proc. Nat. Acad. Sci. (U.S.A.) 72, 2719-2723 (1975) Bonner, W.D.: A general method for the preparation of plant mitochondria. In: Methods in enzymology. Vol. X. pp 126-133. Eds: Colowick, S.P., Kaplan, N.L New York: Academic Press 1967 Bray, G.A.: A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Analyt. Biochem. 1, 279-285 (1960) Douce, R., Christensen, E.L., Bonnet, W.D. : Preparation of intact plant mitochondria. Biochim. biophys. Acta (Amst.) 275, 148 160 (1972) Egnhus, H., Reftel, S., Selldhn, G. : The appearance and development of photosynthetic activity in etiolated barley leaves and isolated etiochloroplasts. Plant Physiol. 27, 48-55 (1972) Gimmler, A., Sch~ifer, G., Kraminer, H., Heber, U. : Amino acid permeability of the chloroplast envelope as measured by light scattering, volumetry and amino acid uptake. Planta (Berl.) 120, 47-61 (1974) Hampp, R., Schmidt, H.W. : Changes in envelope permeability during chloroplast development. Planta (Berl.) 129, 69-73 (1976) Hampp, R., Wellburn, A.R.: Early changes in the envelope permeability of developing chloroplasts. J. exp. Bot. (in press) Heldt, A.W., Saner, F.: The inner membrane of the chloroplast envelope as the site of specific metabolite transport. Biochim. biophys. Acta (Amst.) 234, 83 91 (1971) Kirk, P.R., Leech, R.M.: Amino acid biosynthesis by isolated chloroplasts during photosynthesis. Plant Physiol. (Baltimore) 50, 228-234 (1972)
Klingenberg, M., Pfaff, E. : Structural and functional compartmentation in mitochondria. In: Regulation of metabolic processes in mitochondria, Eds: Tager, J.M., Papa, S., Quagliariello, E., Slater, E.C.: BBA Library Vol. 7, pp 180-201, Amsterdam: Elsevier (1966) Klingenberg, M., Pfaff, E.: Means of terminating reactions, in: Methods in enzymology. Vol. X, pp 680~84. Eds. : Colowick, S.P., Kaplan, N.I. New York: Academic Press 1967 Lee, D.C., Wilson, R.H.: Swelling in bean shoot mitochondria induced by a series of potassium salts of organic anions. Physiol. Plant. 27, 195-201 (197_9) Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. : Protein measurement with the FoIin phenol reagent. J. biol. Chem. 193, 265-275 (1951) Lynn, W.S., Brown, R.H. : Role of anions in mitochondrial swelling and contraction. Biochim. biophys. Acta (Amst.) 110, 445-458 (1965) Malamed, S., Recknagel, R.O. : The osmotic behaviour of the sucrose-inaccessible space of mitochondrial pellets from rat liver. J. Biol. Chem. 234, 3027 3030 (1959) Miller, J.E., Koeppe, D.E., Miller, R.J. : Effects of anions on swelling, respiration, and phosphorylation of isolated corn mitochondria. Physiol. Plant. 34, 153-156 (1975) Parsons, D.F. : Mitochondrial structure: two types of subunits on negatively stained mitochondrial membranes. Science 140, 985987 (1963) Rebeiz, C.A., Castelfranco, P.A.: Protochlorophyll and chlorophyll biosynthesis in cell-free systems from higher plants. Ann. Rev. Plant Physiol. 24, 129-172 (1973) Reynolds, E.S. : The use of lead citrate at high pH as an electronopaque stain in electron microscopy. J. Cell Biol. 17, 208-212 (1963) Wellburn, A.R.: 6-aminolaevulinic acid formation in greening Arena laminae. Phytochemistry 14, 699-701 (1975)
Received 27 February ; accepted 12 March 1976
P I ~ ) H ~ 9 by Springer-Verlag 1976
Changes in the Permeability of the Inner Mitochondrial Membrane Associated with Plastid Development R. Hampp* and A.R. Wellburn Department of Biological Sciences, University of Lancaster, Lancaster LA1 4YQ, U.K.
Summary. Mitochondria isolated from greening etiolated laminae of Arena sativa L. show changes in the permeability of their inner membranes during chloroplast development similar to those described earlier for plastids. Oxalo-acetate, succinate and c~keto-glutarate permeate most readily inner membranes of mitochondria isolated from laminae given 2 h illumination whilst glutamate and glycine show later and more general penetration into the matrix spaces of mitochondria from greening tissue. Aminolevulinic acid (ALA) by contrast does not readily enter Arena mitochondria especially those isolated from laminae illuminated for longer than 2 h.
Introduction
Cellular systems show a high degree of structural complexity and the regulation of metabolic processes is of central importance in maintaining this organisation. This is partly achieved by separated compartments surrounded by selectively permeable membranes such as those of the mitochondria and plastids. The inner envelope membrane of chloroplasts has been shown to be a site of specific metabolite transport from the cytosol into the stroma of the plastid (Heldt and Sauer, 1971; Gimmler et al., 1974) and corresponding observations have already been made with mitochondria (see Klingenberg and Pfaff, 1966). Using the technique of silicone oil centrifugal filtration (Klingenberg and Pfaff, 1967) it was demonstrated that there is a change in permeability of the transport limiting membrane of the envelope of plastids Abbreviations: ALA = amino-levulinic acid; HEPES = N-2-hydroxyethyl-piperazine-N'-2-ethane-sulphonic acid * Permanent address: Institut ftir Botanik, Technische Universit,it, Arcisstr. 21, D-8000 Mfinchen 2, Federal Republic of Germany
(Hampp and Wellburn, 1976) during development. Oxaloacetate and succinate for example, showed a pronounced increase in uptake after 1 to 2 h of greening whilst other compounds showed characteristic permeability changes over longer periods of plastid development. Most of the metabolites examined were precursors for the synthesis of components of membranes and pigments, an extensive process in the early stages of chloroplast morphogenesis. As long as the plastids have not reached that developmental stage when the ability to fix CO2 is aquired such precursors in the main are probably imported from outside the developing plastid, in some cases having originated in the mitochondria. Consequently the permeability of the inner mitochondrial membranes during the period of the development of plastids were investigated using slightly modified techniques to those used earlier (Hampp and Schmidt, 1976; Hampp and Wellburn, 1976). The results indicate that mitochondria isolated from etiolated Arena laminae given the same light treatment as was used for the plastid investigations show similar changes in the permeability of the inner mitochondrial membranes to those that occur in the inner envelope membranes of corresponding etioplasts or 1-24 h etio-chloroplasts.
Materials and Methods Preparation of Mitochondria from Greening Arena Laminae
Seedlings of Arena sativa L. (cv. Mostyn) were grown in gravel trays for ten days at 22~ in moist peat in the dark. Some of the trays were partially illuminated (6 klux; 1, 2, 4, 8 or 24 h) during the later stages of growth. Mitochondria were isolated from batches of these different laminae using a modified method based upon that of Bonner (1967) allowing when necessary the simultaneous isolation of plastids from the same initial homogenate. About 100 g of laminae were homogeuised in 170 ml of chilled isolation medium (0.45 M sorbitol; 0.001 M MgCI2; 0.001 M KH2PO4; 0.001 M NaNO3; 0.2% (w/v) bovine serum albumin,
22
R. Hampp and A.R. Wellburn: Permeability of the Mitochondrial Membrane
Sigma Type V; all in 0.05 M HEPES, finally adjusted to pH 7.3 with KOH) using a Townson and Mercer top-drive homogeniser in 3 bursts of 7s duration. The homogenate was filtered twice through 4 layers of nylon sheet (Nytal 25T1, mesh 35 gm, Hemy Simon, Stockport) and centrifuged in 4 cooled polypropylene tubes (50 ml) for 7 min at 1000 x g in a swing-out bench centrifuge kept at 4 ~ After centrifugation the pellets were suitable for a subsequent plastid isolation but for mitochondrial preparations the combined supematants were diluted with a half volume of 3 mM EDTA (i.e. sorhitol concentration reduced to 0.3 M). At the point of mixing powdered cysteine was added so as to give a final concentration of 0.05% (w/v), followed by centrifugation at 10,000 x g for I5 rain at 2~ using an MSE 18 ultracentrifuge and an 8 x 50 ml fixed anne rotor. The crude mitochondrial pellets were resuspended in a total of 30 ml of isolation medium B (2 volumes of isolation medium A plus 1 volume of 3 mM EDTA, but without added cysteine). Two turns of a loosely fitting TenBroeck homogeniser was used to assist the resuspension and a further centrifugation carried out at 6,000 x g for 15 min at 2~ The pellet was resuspended as before in 10 ml of isolation medium B and used for 02 electrode, volume and permeability studies or prepared for electron microscopy.
G.F.R.) and 100 I~1 mitochondrial suspensions incorporating aliquots (5%) of the labelled solutions (final concentration 5 raM), were accelerated to full speed for 10 s and back to rest using a Beckman Microfuge B after a minimum of 1 min incubation at 0~ The tubes were then frozen in liquid N2 and the tips (bases) cut off with cable cutters and dropped into scintillation vials containing 0.5 ml water. After vibration to dislodge and disperse the pellets, 10 ml of Bray's solution (Bray, 1960) was added to each vial and the amount of radioactivity in each pellet determined by counting in a Packard 3375 liquid scintillation counter against standards using pre-set counting characteristics for 3H and 14C and quench curves constructed specifically for these experiments. At least 8 parallel treatments were carried out, each having a standard deviation of counting of 1.5%. From the mean of these a calculation of the matrix space concentration, relative to the incubation medium concentration of 5 mM, is possible after determining the total volume by the uptake of tritiated water. The amount of unspecific permeation into the inner membrane space was corrected for by the uptake of [U-14C]sucrose and the amount of medium adhering to the outer mitochoudrial surfaces by the use of dextran (M.W. 40x 103 daltons, 2 mgm1-1, see Heldt and Sauer, 1971). The protein content of mitochondrial suspensions was determined by the method of Lowry et al. (1951).
Measurement of 02 Uptake by Arena Mitochondria
Results Oxygen consumption of mitochondrial preparations was measured in a stoppered chamber of total volume I ml with a side entry pore occupied by a Clark type Oz electrode and stirred by means of a Teflon coated magnetic "flea" at the base. Rates of oxidation of succinate (8 pmoles) and ADP (50 nmoles) were recorded on a scale established between that of air saturated water and that of zero-free oxygen water achieved after adding sodium dithionite crystals.
Electron Microscopy of Mitochondrial Suspensions An equal volume of 5% (v/v) glutaraldehyde in isolation medium B was added to mitochondrial suspensions in 2 ml polyethylene centrifuge tubes (Beckman) and left for 1 h at 0% The tubes were centrifuged for 30 s using a Beckman Microfuge B and the pellets washed with isolation medium B and left overnight in 1% (w/v) OsO4 in isolation medium B still at 0 ~ They were dehydrated through a graded ethanol series made up in isolation medium of decreasing sorbitol, bovine serum albumin and buffer concentration, allowed to rise to room temperature and finally dried in propylene oxide. The pellets were embedded in Epon and sectioned on a Reichert OMU2 ultramicrotome. Sections were double stained in uranyl acetate and lead citrate (Reynolds, 1963) and examined in a GEC-AEI EM801 electron microscope.
Measurement of the Matrix Space Concentrations of Plant Mitochondria Labelled compounds were obtained from the Radiochemical Centre with the exception of oxalo-acetate which was prepared from malate using the method of Kirk and Leech (1972), All compounds were dissolved in isolation medium B and corrected with unlabelled compounds so as to give a concentration of 100 mM and a specific activity of 0.3 mCi mM -1, readjusting the pH where necessary with KOH. The modified silicone oil centrifugal filtration was based on that described by Klingenberg and Pfaff (1967). Polyethylene centrifuge tubes (250 gl, Beckman), containing 50 gl 10% (v/v) HC104 (lower-most) plus 50 gl silicone oil (Types AR50 and AR150 in a ratio 2: 1, both from Wacker Chemic, Burghausen,
Many different methods of preparation of plant mitoc h o n d r i a w e r e initially t e s t e d b u t f i n a l l y the m o d i f i e d f o r m o f the p r o c e d u r e o f B o n n e r (1967) d e s c r i b e d here was adopted. Those preparations which advocate d the use o f d e n s i t y g r a d i e n t c e n t r i f u g a t i o n s u c h as t h a t o f D o u c e et aI. (I972) w e r e r e j e c t e d o n the grounds of yield and osmotic problems after removal o f the c u s h i o n m e d i u m . R o u t i n e m o n i t o r i n g o f p r e p a rations by electron microscopy was adopted and a r e p r e s e n t a t i v e field o f s u c h a p r e p a r a t i o n is s h o w n in F i g u r e 1. L i t t l e p l a s t i d c o n t a m i n a t i o n was enc o u n t e r e d e v e n in p r e p a r a t i o n s f r o m e t i o l a t e d tissue a n d the m i t o c h o n d r i a w e r e w e l l p r e s e r v e d w i t h little evidence of osmotic damage. The main contaminant a p p e a r e d to be p e r o x i z o m a l m a t e r i a l . T h e s i l i c o n e oil c e n t r i f u g a l f i l t r a t i o n t e c h n i q u e p r o v e d to be a u s e f u l d i a g n o s t i c test o f o r g a n e l l e i n t a c t n e s s . F u r t h e r m o r e b y a d j u s t i n g the r a t i o s o f the s i l i c o n e oils a n y p r e p a r a t i o n c o u l d be q u i c k l y c h e c k e d f o r the p r e s e n c e o r a b s e n c e o f i n t a c t plastids. F i g u r e 2 s h o w s the t r i t i a t e d w a t e r p e r m e a b l e s p a c e o f m i t o c h o n d r i a e x p r e s s e d o n p r o t e i n basis in r e l a t i o n to t h e l i g h t t r e a t m e n t o f the l e a f tissue. W h i l e the r a t i o o f v o l u m e to p r o t e i n is r e l a t i v e l y c o n s t a n t f o r 0, 2, 4, 8 a n d 2 4 h o f l i g h t t r e a t m e n t , b e t w e e n 0 a n d 2 h t h e r e is a m a r k e d i n c r e a s e in the m i t o c h o n d r i a l v o l u m e . T h e o s c i l l a t i o n o f the v o l u m e s u g g e s t e d b e t w e e n 2 a n d 8 h i l l u m i n a t i o n h a s n o t b e e n statistic a l l y s u b s t a n t i a t e d . It m a y b e seen in F i g u r e 3 t h a t the r e s p i r a t o r y a c t i v i t i e s o f the s a m e m i t o c h o n d r i a , u s e d for the volume determination, mirror these changes in v o l u m e . M a x i m u m u p t a k e o f 0 2 in t h e p r e s e n c e o f s u c c i n a t e a n d s u c c i n a t e p l u s A D P o c c u r s in mito-
R. Hampp and A.R. Wellburn: Permeability of the Mitochondrial Membrane
23
Fig. 1. Electron micrograph of a typical mitochondrial suspension. In this case, the preparation was isolated from etiolated Arena laminae given 8 h illumination, x 20,000
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Fig. 2. Changes of the tritiated water permeable volume of isolated Arena mitochondria with respect to the illumination of etiolated laminae from which they were isolated
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Hours of illumination of laminae
Fig. 3. Consumption of oxygen in the presence of succinate and succinate ptus ADP by isolated A~'ena mitochondria with respect to the illumination of etiolated laminae from which they were isolated
24
R. Hampp and A.R. Wellburn: Permeabilityof the MitochondrialMembrane
361
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Fig. 4. Penetration of 14C-labelled glutamate, e-ketoglutarate and 6-aminolaevulinate into the matrix space of mitochondria in relation to the illumination of etiolated Arena laminae (0 ~ C, 1 min; dashed line: external concentration of the respective compound)
Fig. 5. Penetration of 14C-labelled succinate, oxaloacetate and glycine into the matrix space of mitochondria in relation to the illumination of etiolated Arena laminae (0 ~ C, 1 min; dashed line: external concentration of the respective compound)
chondria isolated from 1 h illuminated laminae although the proportional increase on addition of ADP (state 4 to state 3) is much the same throughout indicating the maintenance of respiratory control. In a mitochondrial suspension three different spaces have been defined (see Klingenberg and Pfaff; 1966) : the extra-mitochondrial space (suspension medium), the cristae space and the matrix space. These spaces are separated by two membranes, the outer and the inner mitochondrial membranes. Only the inner membrane contains characteristic particles or spheres which face the matrix space (Parsons, 1963) and functions as a metabolic barrier between the cristae space and the tightly enclosed matrix space. In contrast the outer membrane is unspecifically permeable to metabolites of low molecular weight. Measurement of the penetration of labelled metabolites into the matrix space is therefore only possible when experimental values are corrected for unspecific penetration into the cristae space and the amounts of label adhering to the outer membrane (see Methods). In these experiments the matrix space was found to be about 40% of the total mitochondrial volume. This is in accordance with the results of other authors using mitochondria isolated in isotonic conditions (Malamed and Recknagel, 1959; Klingenberg and Pfaff, 1966). Figures 4 and 5 show the matrix concentrations of different compounds in mitochondria isolated from Arena laminae after 0-24 h of greening. The time
of incubation was 1 min and the concentration of the respective compound 5 mM (dashed horizontal lines). All the compounds tested showed an increase in intra-mitochondrial concentration up to 4 h of greening. For some compounds there is even an accumulation in the matrix space relative to that of the extra-mitochondrial space. This uptake is pronounced for both succinate and oxalo-acetate. Although the maximum uptake of succinate is limited to a period of 1 h of greening, the permeability for oxalo-acetate remains at a high value for almost all the stages of light treatment and, in this respect, is similar to that of glutamate. This increase in permeability (penetrability) during greening is not so pronounced for c~-keto-glutarate and glycine; nevertheless these compounds show distinct maxima for the matrix space concentrations after 2 and 4 h illumination respectively. No accumulation in the matrix space of ALA could be shown for any of the stages of greening, although those mitochondria isolated from etiolated tissue or from 1 h illuminated laminae showed a higher internal concentration than those from seedlings given longer illumination. In general the penetrability of the inner mitochondrial membranes of mitochondria from etiolated tissue is similar to that of 24 h illuminated mitochondria. The observed changes in the permeability of the inner mitochondrial membrane during development are reflected in the changes of the volume to protein ratio and also in the changes of the respiratory activ-
R. Hampp and A.R. Wellburn: Permeability of the Mitochondrial Membrane
ities of mitochondria during the different stages of greening. Discussion
These results identify a change in the permeability of the inner mitochondrial membrane during the greening of etiolated A r e n a laminae. This change in permeability is very similar to that of the inner envelope membrane of etioplasts and etio-chloroplasts isolated at the same stages of greening (Hampp and Schmidt, 1976; Hampp and Wellburn, 1976). For both organelles, oxalo-acetate and succinate show a maximum in internal concentration after 1 h of light treatment, with the internal concentrations markedly higher than those of the external medium. The maxima in permeability of each with respect to ~-ketoglutarate are also in accordance with each other after 2 h illumination. However, there are different uptake characteristics with respect to glycine and glutamate and the permeability for ALA is low for both transport limiting membranes throughout development. The importance of oxalo-acetate for the synthesis of aspartate in plastids has been discussed elsewhere (Hampp and Wellburn, 1976). Kirk and Leech (1972) have demonstrated that the transport of oxalo-acetate from the cytoplasm into the plastid is a pre-requisite for aspartate formation. Our results point towards the possibility that the mitochondrion is the site of oxaloacetate formation. There is ample evidence that the complete pathway of porphyrin synthesis from ALA to the chlorophylls exists in chloroplasts (see Rebeiz and Castelfranco, 1973). In contrast the origin of ALA in higher plants is unknown and a subject of much investigation. More recent papers (Beale and Castelfranco, 1974; Beale et al., 1975; Wellburn, 1975) indicate that e-keto-glutarate, glutamate or glutamine may be possible precursors for ALA synthesis but no convincing evidence is available to suggest that all the enzymes necessary to synthesise these compounds exist in plastids. The implication of this is that the metabolites required for ALA synthesis are supplied from a source outside the plastid. Mitochondria possess the ability so synthesize porphyrins and to provide the necessary precursors. They could also supply compounds for similar processes in plastids. Our results support this possibility. Succinate and, to a lesser extent, c~-keto-glutarate and glutamate show permeability of the inner mitochondrial membrane at the appropriate time when plastidic requirements for ALA are highest. The belief that the major portion of the ALA is translocated from mitochondria to plastids by simple permeation is not supported by our results.
25
A number of studies have established that mitochondria undergo volume changes which depend on the anions composition of both extra-mitochondrial and intra-mitochondrial spaces (Lynn and Brown, 1965). The mitochondrial swelling or contraction is believed to be, in part, the result of osmotic adjustments to the movement of anions (see Miller et al., 1975). Certain anions, such as phosphate and acetate cause a substrate dependent swelling of mitochondria which is thought to reflect ion transport and accumulation. Active swelling in the presence of a number of oxidizable and non-oxidizable organic acids has also been demonstrated (Lee and Wilson, 1972). Since active swelling is correlated with active ion accumulation, the observed increase in volume of the tritiated water permeable space of mitochondria isolated from 1 h light treated tissue could be linked to the enhanced synthetic activities within these organelles. This high metabolic activity, which is also shown by the increased respiratory activity, is followed by an increase in membrane permeability starting between 1 and 2 h of illumination. As a result of the higher permeability the osmotic pressure inside the mitochondria decreases accounting for the decrease in volume down to that shown by mitochondria isolated from etiolated tissue. All these results substantiate the importance of mitochondria in supporting the development of plastids during the greening of etiolated A r e n a laminae and imply changes in the nature of the inner mitochondrial membrane during this maturation. The nature of the control of these apparently co-ordinated changes is immediately questioned. Perhaps one should look to phytochrome as a modifier of envelope permeabilities or seek alternative or additional mechanisms to understand the nature of permeability control especially when co-ordinated changes in different organelles are involved. What is certain is that mitochondria isolated from etiolated tissue differ from those of 1 h illuminated laminae, and so on, especially in respect to the permeabilities of their inner membranes. Although no structural differences were suggested from the electron microscope study of the different preparations, the respiratory behaviour suggests that this may not be the only difference. It may well be that in the future one might refer to (1 h) etio-mitochondria (i.e. mitochondria isolated from etiolated plant tissue given illumination of 1 h ) j u s t like the present use of the accepted term etio-chloroplasts, originally suggested by Egnfius et al. (1972); if only to save on descriptive text. We thank the Deutsche Forschungsgemeinschaftand the Royal Society for the award of a European Exchange Fellowship to one of us (R.H.) and Mr. T. Travis for technical assistance.
26
R. Hampp and A.R. Wellburn: Permeability of the Mitochondrial Membrane
References Beale, S.I., Castelfranco, P.A. : The biosynthesis of 6-aminolevulinic acid in higher plants. II. Formation of l~C-6-aminolevulinic acid form labelled precursors in greening plant tissues. Plant Physiol. (Baltimore) 53, 297-303 (1974) Beale, S.I., Gough, S.P., Granick, S. : Biosynthesis of 3-aminolevutinic acid from the intact carbon skeleton of glutamic acid in greening barley. Proc. Nat. Acad. Sci. (U.S.A.) 72, 2719-2723 (1975) Bonner, W.D.: A general method for the preparation of plant mitochondria. In: Methods in enzymology. Vol. X. pp 126-133. Eds: Colowick, S.P., Kaplan, N.L New York: Academic Press 1967 Bray, G.A.: A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Analyt. Biochem. 1, 279-285 (1960) Douce, R., Christensen, E.L., Bonnet, W.D. : Preparation of intact plant mitochondria. Biochim. biophys. Acta (Amst.) 275, 148 160 (1972) Egnhus, H., Reftel, S., Selldhn, G. : The appearance and development of photosynthetic activity in etiolated barley leaves and isolated etiochloroplasts. Plant Physiol. 27, 48-55 (1972) Gimmler, A., Sch~ifer, G., Kraminer, H., Heber, U. : Amino acid permeability of the chloroplast envelope as measured by light scattering, volumetry and amino acid uptake. Planta (Berl.) 120, 47-61 (1974) Hampp, R., Schmidt, H.W. : Changes in envelope permeability during chloroplast development. Planta (Berl.) 129, 69-73 (1976) Hampp, R., Wellburn, A.R.: Early changes in the envelope permeability of developing chloroplasts. J. exp. Bot. (in press) Heldt, A.W., Saner, F.: The inner membrane of the chloroplast envelope as the site of specific metabolite transport. Biochim. biophys. Acta (Amst.) 234, 83 91 (1971) Kirk, P.R., Leech, R.M.: Amino acid biosynthesis by isolated chloroplasts during photosynthesis. Plant Physiol. (Baltimore) 50, 228-234 (1972)
Klingenberg, M., Pfaff, E. : Structural and functional compartmentation in mitochondria. In: Regulation of metabolic processes in mitochondria, Eds: Tager, J.M., Papa, S., Quagliariello, E., Slater, E.C.: BBA Library Vol. 7, pp 180-201, Amsterdam: Elsevier (1966) Klingenberg, M., Pfaff, E.: Means of terminating reactions, in: Methods in enzymology. Vol. X, pp 680~84. Eds. : Colowick, S.P., Kaplan, N.I. New York: Academic Press 1967 Lee, D.C., Wilson, R.H.: Swelling in bean shoot mitochondria induced by a series of potassium salts of organic anions. Physiol. Plant. 27, 195-201 (197_9) Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. : Protein measurement with the FoIin phenol reagent. J. biol. Chem. 193, 265-275 (1951) Lynn, W.S., Brown, R.H. : Role of anions in mitochondrial swelling and contraction. Biochim. biophys. Acta (Amst.) 110, 445-458 (1965) Malamed, S., Recknagel, R.O. : The osmotic behaviour of the sucrose-inaccessible space of mitochondrial pellets from rat liver. J. Biol. Chem. 234, 3027 3030 (1959) Miller, J.E., Koeppe, D.E., Miller, R.J. : Effects of anions on swelling, respiration, and phosphorylation of isolated corn mitochondria. Physiol. Plant. 34, 153-156 (1975) Parsons, D.F. : Mitochondrial structure: two types of subunits on negatively stained mitochondrial membranes. Science 140, 985987 (1963) Rebeiz, C.A., Castelfranco, P.A.: Protochlorophyll and chlorophyll biosynthesis in cell-free systems from higher plants. Ann. Rev. Plant Physiol. 24, 129-172 (1973) Reynolds, E.S. : The use of lead citrate at high pH as an electronopaque stain in electron microscopy. J. Cell Biol. 17, 208-212 (1963) Wellburn, A.R.: 6-aminolaevulinic acid formation in greening Arena laminae. Phytochemistry 14, 699-701 (1975)
Received 27 February ; accepted 12 March 1976
