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Differential effects of digitonin on mitochondria isolated from male-sterile and non male-sterile cytoplasms of corn
Plant Science Letters, 9 (1977) 17--21 O Elsevier/North-Holland Scientific Publishers, Ltd.
17
D I F F E R E N T I A L EFFECTS OF DIGITONIN ON MITOCHONDRIA ISOLATED FROM MALE-STERILE AND NON MALE-STERILE CYTOPLASMS OF CORN
PETER GREGORY, VERNON KRAUS
E. G R A C E N , O L E N C. Y O D E R and N A N C Y
A. STEIN-
Department of Plant Breeding and Biometry and Department of Plant Pathology, CorneU University, Ithaca, N e w York 14853 (U.S.A.)
(Received June 24th, 1976) (Revision received August 28th, 1976)
(Accepted November 18th, 1976)
SUMMARY Mitochondria were isolated from corn roots containing T or C (male-sterile) cytoplasms and from N (non male-sterile) cytoplasm. The mitochondrial preparations (T, C and N mitochondria respectively) were treated with various concentrations of digitonin and assayed for respiratory activity. The T mitochondria were markedly more digitonin-sensitive than C or N mitochondria, as shown by the inhibition of malate and succinate respiration and uncoupling of oxidative phosphorylation. There were also differences between C and N mitochondria with respect to digitonin-sensitivity. These data strongly suggest that T, C and N mitochondria differ from each other with respect to biochemical structure. The chemical, genetic and agricultural signifance of these data is discussed.
INTRODUCTION There is little or no evidence for the existence of mitochondrial m u t a n t s in higher plants except for that derived from studies on Southern Corn Leaf Blight disease. This disease is caused by the fungus Itelminthosporium maydis Race T which produces host specific toxic chemicals (HmT toxin). Susceptibility to H. maydis Race T is cytoplasmically inherited [6,7 ]. Corn lines containing Texas male-sterile (T) cytoplasm are severely damaged by H. maydis Race T and HmT toxin, whereas other cytoplasms, including other malesterile types such as C cytoplasm and the non male-sterile N cytoplasm, are resistant to both the fungus and the toxin. There is convincing biochemical and ultrastructural evidence that the susceptibility of corn to H. maydis Race T is determined by sensitivity of mitochondria to HmT toxin and t h a t resisPaper No. 670 in the Plant Breec]ing Series. Abbreviations: BSA, bovine serum albumin; DTT dithiothreitol; RCR, respiratory control ratios.
18 tance is caused by insensitivity of mitochondria to HmT toxin [1,2,4,10,13]. It seems possible t h a t a m u t a t i o n in mitochondrial DNA controls the differential responses to the toxin. Several groups have attempted to identify differences between T and N mitochondria by studying chemical composition. Pring [ 14] has reported that mitochondria of T cytoplasm contain a full complement of cytochromes a+a3, b and c as detected by difference spectra at 25°C. There was evidence [14] that T cytoplasm contains 7--12% more c y t o c h r o m e b than does N cytoplasm. Shah and Levings [15] have reported that the b u o y a n t densities and base compositions of mitochondrial DNA were the same for T and N cytoplasm. However, Levings and Pring [8] extracted mitochondrial DNA from normal and T cytoplasm maize and digested the DNA with restriction endonucleases. Electrophoresis of the resulting fragments revealed distinctions between the two cytoplasmic types. Gengenbach et al. [3 ] have shown that while both T and N mitochondria respond to valinomycin, gramicidin and decenyl-succinic acid, there were some small quantitative differences in their sensitivities to these substances. These studies have not demonstrated any major biochemical difference between T and N mitochondria. To date, reaction to HmT toxin is the only major difference between these mitochondria. However, the data presented here show that T, N and also C mitochondria differ markedly in their sensitivities to digitonin. Our finding suggests that the three types of mitochondria differ with respect to membrane structure. This m a y have agricultural as well as genetic and chemical significance. METHODS Mitochondria were isolated from the roots of 3-day-old etiolated corn seedlings by modifications of a m e t h o d reported previously [11]. Modifications of this m e t h o d are indicated in the legend to Fig. 1. The corn lines (Zea mays L.) were: W64A T (male sterile, HmT toxin-susceptible), W64A C (male sterile, HmT to~xin-resistant) and W64A N (male fertile, HmT toxin-resistant). Respiratory assays, using either malate or succinate as substrates, were performed with the addition of mitochondria to a buffered KC1 medium in an oxygen electrode (for details see legend to Fig. 1}. Approx. 0.5 mg mitochondrial protein was used in each assay. Digitonin concentrations up to 2 mg per mg mitochondrial protein were used. Because digitonin action is time-dependent, as well as concentration-dependent, care was taken to measure respiratory activity (State 4) after a 10 minute incubation of the mitochondria with digitonin. RCR indicating the degree of coupling between mitochondrial electron transport and oxidative phosphorylation were determined by measuring the ratio of the respiration rate in the presence of added ADP and after exhaustion of ADP. The mitochondrial preparations used in these experiments were well coupled and also showed good purity and membrane integrity as judged by electron-
19
40
"E -"
Malate
._~ "~ c 0 co 0
2o
lo
~ E ._o
,~~.,.. .
"I
•~. ~ (~ ~ CC N
,
A,A
Succinate
~ l~ 30 I ~
o W64A-N • W64A -T "W64A-C ~Uncoupled
(3 20
*
,ot 0
2
4
.6
.8
1.0
1.2
1.4
1.6
1.8
20
Digitonin Concentration (mg digitonin/mg mitochondrial protein ) Fig. 1. T h e e f f e c t o f d i g i t o n i n o n r e s p i r a t i o n a n d c o u p l i n g in m i t o c h o n d r i a f r o m c o r n seedlings c o n t a i n i n g T, N or C c y t o p l a s m . I s o l a t i o n a n d final s u s p e n s i o n o f t h e m i t o c h o n dria f r o m r o o t s f r o m e t i o l a t e d seedlings were p e r f o r m e d b y m o d i f i c a t i o n s o f a p r e v i o u s m e t h o d [11 ] : a p p r o x . 5 g (fresh w e i g h t ) o f c o r n r o o t s were g r o u n d in a m o r t a r w i t h 50 ml o f a s o l u t i o n ( S o l u t i o n A) c o n t a i n i n g 0.4 M sucrose, 0.03 M H e p e s b u f f e r ( p H 7.4), 50 m M KH:PO4, 5 m M E D T A , 1 m g B S A / m l a n d 1 m M DTT. S u i t a b l e a l i q u o t s o f D T T a n d B S A s o l u t i o n s were s t o r e d at --20°C. T h e a l i q u o t s were t h a w e d a n d a d d e d t o S o l u t i o n A i m m e d i a t e l y p r i o r t o grinding. T h e h o m o g e n a t e was s t r a i n e d t h r o u g h 4 layers o f cheesec l o t h a n d c e n t r i f u g e d a t 28 0 0 0 g for 5 min. T h e pellet was r e s u s p e n d e d , using a sable artist b r u s h , in 20 m l o f S o l u t i o n A a n d c e n t r i f u g e d at 1 5 0 0 g for 10 min. T h e s u p e r n a t a n t was t r a n s f e r r e d t o a c l e a n c e n t r i f u g e t u b e , u n d e r l a y e r e d w i t h 10 ml o f a s o l u t i o n c o n t a i n i n g 0.6 M s u c r o s e a n d 0 . 0 2 M H e p e s b u f f e r pH 7.4 a n d c e n t r i f u g e d at 18 0 0 0 g for 18 min. T h e m i t o c h o n d r i a l pellet was r e s u s p e n d e d , w i t h t h e b r u s h , in I ml o f a s o l u t i o n c o n t a i n i n g 0.4 M s u c r o s e a n d 0 . 0 2 M H e p e s b u f f e r pH 7.4. T h e p r e c e d i n g o p e r a t i o n s were carried o u t o n ice at or n e a r 0°C. M e a s u r e m e n t o f t h e rate of 0 2 u p t a k e was m a d e in a n o x y g e n e l e c t r o d e ( Y e l l o w Springs I n s t r u m e n t Co.) in a 3 ml r e a c t i o n m i x t u r e (22°C) c o n t a i n i n g 0.4 M KCI, 0.04 M H e p e s b u f f e r ( p H 7.4), 8 m M KH2PO 4 a n d 2 m g B S A / m l , a p p r o x . 0 . 5 m g m i t o c h o n drial p r o t e i n a n d t h e a p p r o p r i a t e c o n c e n t r a t i o n o f d i g i t o n i n (see text). O x i d a t i o n was i n i t i a t e d b y t h e a d d i t i o n o f e i t h e r 3 0 p m o l e s m a l a t e plus 3 0 p m o l e s p y r u v a t e o r 30 pmoles succinate. U n c o u p l i n g o f m i t o c h o n d r i a ( w h e r e t h e R C R = I ) is d e n o t e d b y a n asterisk.
20
micrographic data (York, unpublished data). Protein measurements were made by the m e t h o d of Lowry et al. [9]. Digitonin was purchased from the Sigma Chemical Company, St. Louis, Mo. Each graph shown in Fig. 1 represents one experiment and is representative of at least two other separate experiments with respect to respiration rates and values. RESULTS
The effects of digitonin on malate and succinate respiration in T, N and C mitochondria are shown in Fig. 1. Digitonin action on T mitochondria resulted in complete inhibition of malate respiration (Fig. 1A) and strong inhibition of succinate respiration (Fig. 1B). The effect of digitonin on N mitochondria was markedly different from that on T mitochondria: malate respiration was uninhibited in N and C cytoplasm (Fig. 1A) and maximal inhibition of succinate respiration in N mitochondria required a 4-fold higher digitonin concentration than that needed for T mitochondria (Fig. 1B). The C mitochondria differed from T and N mitochondria in their response to digitonin (Fig. 1). The effects of digitonin on uncoupling of oxidative phosphorylation in T, N and C mitochondria were determined by measurements of RCR values (Fig. 1). The digitonin concentrations at which the various mitochondria were completely uncoupled (RCR=I) are designated by asterisks in Fig. 1. In the absence of digitonin treatment the T, N and C mitochondria were well coupled and typical respiratory ratios were 2.7 and 2.1 for malate and succinate respiration respectively. It may be seen in Fig. 1 that the coupling of oxidative phosphorylation to either malate or succinate respiration was more digitoninsensitive in T than in N or C mitochondria, and that the N and C mitochondria were uncoupled at similar concentrations of digitonin. DISCUSSION
Our data show t h a t T and N mitochondria differ markedly in their response to low concentrations of digitonin. This suggests that these mitochondria differ structurally. Digitonin has long been used in the disruption and fractionation of biological membranes. At high concentrations, digitonin exhibits detergent action and can disrupt many kinds of membranes. At low concentrations, digitonin is selective for steroid-rich membranes. Evidence for this is the finding of Schnaitman et al. [16] that the outer membrane of rat liver mitochondria is more digitonin-sensitive than the inner membrane. These authors suggested [16] that as digitonin is k n o w n to combine with cholesterol (and certain other steroids) on a 1:1 basis, it is possible that the outer membranes were higher in cholesterol than the inner membrane. Subsequent studies with Guinea pig liver mitochondria showed t h a t the steroid c o n t e n t of the outer mitochondrial membrane was six times more concentrated, on a protein basis, than the inner membrane [ 12]. Thus the differential effects of digitonin on the T and
21
N mitochondria possibly reflect differences in membrane steroid composition. This is especially feasible because the digitonin concentrations used in our experiments were similar to those used by Schnaitman et al. [ 1 6 ] . It is w o r t h y of note that the difference in response of C and N mitochondria to digitonin (Fig. 1) may well reflect structural (possibly steroidal) differences b e t w e e n them. Such differences could prove important to corn breeders who are increasing their use of C cytoplasm. The effects of digitonin on mitochondria were inhibition of malate and succinate oxidation and uncoupling of oxidative phosphorylation (Fig. 1). These effects are similar to those of HmT toxin on T mitochondria [2,4,10,13] These similarities between the activities of digitonin and H m T toxin may have significance with respect to the mechanism of toxin action. Our finding that T, C and N mitochondria differ in their sensitivities to digitonin supports the concept that mitochondrial mutations exist in higher plants. As digitonin is selective for steroid-rich membranes, the difference between T, C and N mitochondria may be phenotypically expressed in the steroid fraction. We intend to analyze the steroids of T, C and N mitochondria. These proposed analyses might or might n o t have direct bearing on the mechanism of Southern Corn Leaf Blight disease and the nature of cytoplasmic male-sterility in corn. However, such studies should provide valuable insight into the genetic and biochemical bases of cytoplasmic mutation. ACKNOWLEDGEMENTS
We wish to thank Drs. Efraim Racker, Andre T. Jagendorf, Hans D. van Etten and Elizabeth D. Earle of Cornell University for reviewing the first draft of the manuscript and for making helpful suggestions. The w o r k was supported in part by grant No. 75002 from the Rockefeller Foundation. REFERENCES 1 H.C. Aldrich, V.E. Gracen, D.W. York, E.D. Earle and O.C. Yoder, Tissue and Cell (In Press). 2 C.J. Arntzen, M.F. Haugh and S. Bobick, Plant Physiol., 52 (1973) 569. 3 B.G. Gengenbach, D.E. Koeppe and R.J. Miller, Physiol. Plant., 29 (1973) 103. 4 B.G. Gengenbach, R.J. Miller, D.E. Koeppe and C.J. Arntzen, Can. J. Bot., 51 (1973) 2119. 5 V.E. Gracen, C.O. Grogan and M.J. Forster, Can. J. Bot., 50 (1972) 2167. 6 A.L. Hooker, D.R. Smith, S.M. Lira and J.B. Beckett, Plant Dis. Rep., 54 (1970) 708. 7 A.L. Hooker, D.R. Smith, S.M. Lim and M.D. Musson, Plant Dis. Rep., 54 (1970) 1109. 8 C.S. Levings, III and D.R. Pring, Science, 193 (1976) 158. 9 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193"(1951) 265. 10 R.J. Miller and D.E. Koeppe, Science 173 (1971) 67. 11 R.J. Miller, S.W. Dumford, D.E. Koeppe and J.B. Hanson, Plant Physiol., 45 (1970) 649. 12 D.F. Parsons and Y. Yano, Biophys. Acta, 135 (1967) 362. 13 P.A~ Peterson, R.B. Flavell and D.H.P. Barratt, Theor. Appl. Genet., 45 (1975) 309. 14 D.R. Pring, Plant Physiol., 55 (1975) 203. 15 D.M. Shah and C.S. Levings, III, Crop Science, 14 (1974) 852. 16 C. Schnaitman, V.G. Erwin and J.W. Greenawalt, J. Cell Biol., 32 (1967) 719.
17
D I F F E R E N T I A L EFFECTS OF DIGITONIN ON MITOCHONDRIA ISOLATED FROM MALE-STERILE AND NON MALE-STERILE CYTOPLASMS OF CORN
PETER GREGORY, VERNON KRAUS
E. G R A C E N , O L E N C. Y O D E R and N A N C Y
A. STEIN-
Department of Plant Breeding and Biometry and Department of Plant Pathology, CorneU University, Ithaca, N e w York 14853 (U.S.A.)
(Received June 24th, 1976) (Revision received August 28th, 1976)
(Accepted November 18th, 1976)
SUMMARY Mitochondria were isolated from corn roots containing T or C (male-sterile) cytoplasms and from N (non male-sterile) cytoplasm. The mitochondrial preparations (T, C and N mitochondria respectively) were treated with various concentrations of digitonin and assayed for respiratory activity. The T mitochondria were markedly more digitonin-sensitive than C or N mitochondria, as shown by the inhibition of malate and succinate respiration and uncoupling of oxidative phosphorylation. There were also differences between C and N mitochondria with respect to digitonin-sensitivity. These data strongly suggest that T, C and N mitochondria differ from each other with respect to biochemical structure. The chemical, genetic and agricultural signifance of these data is discussed.
INTRODUCTION There is little or no evidence for the existence of mitochondrial m u t a n t s in higher plants except for that derived from studies on Southern Corn Leaf Blight disease. This disease is caused by the fungus Itelminthosporium maydis Race T which produces host specific toxic chemicals (HmT toxin). Susceptibility to H. maydis Race T is cytoplasmically inherited [6,7 ]. Corn lines containing Texas male-sterile (T) cytoplasm are severely damaged by H. maydis Race T and HmT toxin, whereas other cytoplasms, including other malesterile types such as C cytoplasm and the non male-sterile N cytoplasm, are resistant to both the fungus and the toxin. There is convincing biochemical and ultrastructural evidence that the susceptibility of corn to H. maydis Race T is determined by sensitivity of mitochondria to HmT toxin and t h a t resisPaper No. 670 in the Plant Breec]ing Series. Abbreviations: BSA, bovine serum albumin; DTT dithiothreitol; RCR, respiratory control ratios.
18 tance is caused by insensitivity of mitochondria to HmT toxin [1,2,4,10,13]. It seems possible t h a t a m u t a t i o n in mitochondrial DNA controls the differential responses to the toxin. Several groups have attempted to identify differences between T and N mitochondria by studying chemical composition. Pring [ 14] has reported that mitochondria of T cytoplasm contain a full complement of cytochromes a+a3, b and c as detected by difference spectra at 25°C. There was evidence [14] that T cytoplasm contains 7--12% more c y t o c h r o m e b than does N cytoplasm. Shah and Levings [15] have reported that the b u o y a n t densities and base compositions of mitochondrial DNA were the same for T and N cytoplasm. However, Levings and Pring [8] extracted mitochondrial DNA from normal and T cytoplasm maize and digested the DNA with restriction endonucleases. Electrophoresis of the resulting fragments revealed distinctions between the two cytoplasmic types. Gengenbach et al. [3 ] have shown that while both T and N mitochondria respond to valinomycin, gramicidin and decenyl-succinic acid, there were some small quantitative differences in their sensitivities to these substances. These studies have not demonstrated any major biochemical difference between T and N mitochondria. To date, reaction to HmT toxin is the only major difference between these mitochondria. However, the data presented here show that T, N and also C mitochondria differ markedly in their sensitivities to digitonin. Our finding suggests that the three types of mitochondria differ with respect to membrane structure. This m a y have agricultural as well as genetic and chemical significance. METHODS Mitochondria were isolated from the roots of 3-day-old etiolated corn seedlings by modifications of a m e t h o d reported previously [11]. Modifications of this m e t h o d are indicated in the legend to Fig. 1. The corn lines (Zea mays L.) were: W64A T (male sterile, HmT toxin-susceptible), W64A C (male sterile, HmT to~xin-resistant) and W64A N (male fertile, HmT toxin-resistant). Respiratory assays, using either malate or succinate as substrates, were performed with the addition of mitochondria to a buffered KC1 medium in an oxygen electrode (for details see legend to Fig. 1}. Approx. 0.5 mg mitochondrial protein was used in each assay. Digitonin concentrations up to 2 mg per mg mitochondrial protein were used. Because digitonin action is time-dependent, as well as concentration-dependent, care was taken to measure respiratory activity (State 4) after a 10 minute incubation of the mitochondria with digitonin. RCR indicating the degree of coupling between mitochondrial electron transport and oxidative phosphorylation were determined by measuring the ratio of the respiration rate in the presence of added ADP and after exhaustion of ADP. The mitochondrial preparations used in these experiments were well coupled and also showed good purity and membrane integrity as judged by electron-
19
40
"E -"
Malate
._~ "~ c 0 co 0
2o
lo
~ E ._o
,~~.,.. .
"I
•~. ~ (~ ~ CC N
,
A,A
Succinate
~ l~ 30 I ~
o W64A-N • W64A -T "W64A-C ~Uncoupled
(3 20
*
,ot 0
2
4
.6
.8
1.0
1.2
1.4
1.6
1.8
20
Digitonin Concentration (mg digitonin/mg mitochondrial protein ) Fig. 1. T h e e f f e c t o f d i g i t o n i n o n r e s p i r a t i o n a n d c o u p l i n g in m i t o c h o n d r i a f r o m c o r n seedlings c o n t a i n i n g T, N or C c y t o p l a s m . I s o l a t i o n a n d final s u s p e n s i o n o f t h e m i t o c h o n dria f r o m r o o t s f r o m e t i o l a t e d seedlings were p e r f o r m e d b y m o d i f i c a t i o n s o f a p r e v i o u s m e t h o d [11 ] : a p p r o x . 5 g (fresh w e i g h t ) o f c o r n r o o t s were g r o u n d in a m o r t a r w i t h 50 ml o f a s o l u t i o n ( S o l u t i o n A) c o n t a i n i n g 0.4 M sucrose, 0.03 M H e p e s b u f f e r ( p H 7.4), 50 m M KH:PO4, 5 m M E D T A , 1 m g B S A / m l a n d 1 m M DTT. S u i t a b l e a l i q u o t s o f D T T a n d B S A s o l u t i o n s were s t o r e d at --20°C. T h e a l i q u o t s were t h a w e d a n d a d d e d t o S o l u t i o n A i m m e d i a t e l y p r i o r t o grinding. T h e h o m o g e n a t e was s t r a i n e d t h r o u g h 4 layers o f cheesec l o t h a n d c e n t r i f u g e d a t 28 0 0 0 g for 5 min. T h e pellet was r e s u s p e n d e d , using a sable artist b r u s h , in 20 m l o f S o l u t i o n A a n d c e n t r i f u g e d at 1 5 0 0 g for 10 min. T h e s u p e r n a t a n t was t r a n s f e r r e d t o a c l e a n c e n t r i f u g e t u b e , u n d e r l a y e r e d w i t h 10 ml o f a s o l u t i o n c o n t a i n i n g 0.6 M s u c r o s e a n d 0 . 0 2 M H e p e s b u f f e r pH 7.4 a n d c e n t r i f u g e d at 18 0 0 0 g for 18 min. T h e m i t o c h o n d r i a l pellet was r e s u s p e n d e d , w i t h t h e b r u s h , in I ml o f a s o l u t i o n c o n t a i n i n g 0.4 M s u c r o s e a n d 0 . 0 2 M H e p e s b u f f e r pH 7.4. T h e p r e c e d i n g o p e r a t i o n s were carried o u t o n ice at or n e a r 0°C. M e a s u r e m e n t o f t h e rate of 0 2 u p t a k e was m a d e in a n o x y g e n e l e c t r o d e ( Y e l l o w Springs I n s t r u m e n t Co.) in a 3 ml r e a c t i o n m i x t u r e (22°C) c o n t a i n i n g 0.4 M KCI, 0.04 M H e p e s b u f f e r ( p H 7.4), 8 m M KH2PO 4 a n d 2 m g B S A / m l , a p p r o x . 0 . 5 m g m i t o c h o n drial p r o t e i n a n d t h e a p p r o p r i a t e c o n c e n t r a t i o n o f d i g i t o n i n (see text). O x i d a t i o n was i n i t i a t e d b y t h e a d d i t i o n o f e i t h e r 3 0 p m o l e s m a l a t e plus 3 0 p m o l e s p y r u v a t e o r 30 pmoles succinate. U n c o u p l i n g o f m i t o c h o n d r i a ( w h e r e t h e R C R = I ) is d e n o t e d b y a n asterisk.
20
micrographic data (York, unpublished data). Protein measurements were made by the m e t h o d of Lowry et al. [9]. Digitonin was purchased from the Sigma Chemical Company, St. Louis, Mo. Each graph shown in Fig. 1 represents one experiment and is representative of at least two other separate experiments with respect to respiration rates and values. RESULTS
The effects of digitonin on malate and succinate respiration in T, N and C mitochondria are shown in Fig. 1. Digitonin action on T mitochondria resulted in complete inhibition of malate respiration (Fig. 1A) and strong inhibition of succinate respiration (Fig. 1B). The effect of digitonin on N mitochondria was markedly different from that on T mitochondria: malate respiration was uninhibited in N and C cytoplasm (Fig. 1A) and maximal inhibition of succinate respiration in N mitochondria required a 4-fold higher digitonin concentration than that needed for T mitochondria (Fig. 1B). The C mitochondria differed from T and N mitochondria in their response to digitonin (Fig. 1). The effects of digitonin on uncoupling of oxidative phosphorylation in T, N and C mitochondria were determined by measurements of RCR values (Fig. 1). The digitonin concentrations at which the various mitochondria were completely uncoupled (RCR=I) are designated by asterisks in Fig. 1. In the absence of digitonin treatment the T, N and C mitochondria were well coupled and typical respiratory ratios were 2.7 and 2.1 for malate and succinate respiration respectively. It may be seen in Fig. 1 that the coupling of oxidative phosphorylation to either malate or succinate respiration was more digitoninsensitive in T than in N or C mitochondria, and that the N and C mitochondria were uncoupled at similar concentrations of digitonin. DISCUSSION
Our data show t h a t T and N mitochondria differ markedly in their response to low concentrations of digitonin. This suggests that these mitochondria differ structurally. Digitonin has long been used in the disruption and fractionation of biological membranes. At high concentrations, digitonin exhibits detergent action and can disrupt many kinds of membranes. At low concentrations, digitonin is selective for steroid-rich membranes. Evidence for this is the finding of Schnaitman et al. [16] that the outer membrane of rat liver mitochondria is more digitonin-sensitive than the inner membrane. These authors suggested [16] that as digitonin is k n o w n to combine with cholesterol (and certain other steroids) on a 1:1 basis, it is possible that the outer membranes were higher in cholesterol than the inner membrane. Subsequent studies with Guinea pig liver mitochondria showed t h a t the steroid c o n t e n t of the outer mitochondrial membrane was six times more concentrated, on a protein basis, than the inner membrane [ 12]. Thus the differential effects of digitonin on the T and
21
N mitochondria possibly reflect differences in membrane steroid composition. This is especially feasible because the digitonin concentrations used in our experiments were similar to those used by Schnaitman et al. [ 1 6 ] . It is w o r t h y of note that the difference in response of C and N mitochondria to digitonin (Fig. 1) may well reflect structural (possibly steroidal) differences b e t w e e n them. Such differences could prove important to corn breeders who are increasing their use of C cytoplasm. The effects of digitonin on mitochondria were inhibition of malate and succinate oxidation and uncoupling of oxidative phosphorylation (Fig. 1). These effects are similar to those of HmT toxin on T mitochondria [2,4,10,13] These similarities between the activities of digitonin and H m T toxin may have significance with respect to the mechanism of toxin action. Our finding that T, C and N mitochondria differ in their sensitivities to digitonin supports the concept that mitochondrial mutations exist in higher plants. As digitonin is selective for steroid-rich membranes, the difference between T, C and N mitochondria may be phenotypically expressed in the steroid fraction. We intend to analyze the steroids of T, C and N mitochondria. These proposed analyses might or might n o t have direct bearing on the mechanism of Southern Corn Leaf Blight disease and the nature of cytoplasmic male-sterility in corn. However, such studies should provide valuable insight into the genetic and biochemical bases of cytoplasmic mutation. ACKNOWLEDGEMENTS
We wish to thank Drs. Efraim Racker, Andre T. Jagendorf, Hans D. van Etten and Elizabeth D. Earle of Cornell University for reviewing the first draft of the manuscript and for making helpful suggestions. The w o r k was supported in part by grant No. 75002 from the Rockefeller Foundation. REFERENCES 1 H.C. Aldrich, V.E. Gracen, D.W. York, E.D. Earle and O.C. Yoder, Tissue and Cell (In Press). 2 C.J. Arntzen, M.F. Haugh and S. Bobick, Plant Physiol., 52 (1973) 569. 3 B.G. Gengenbach, D.E. Koeppe and R.J. Miller, Physiol. Plant., 29 (1973) 103. 4 B.G. Gengenbach, R.J. Miller, D.E. Koeppe and C.J. Arntzen, Can. J. Bot., 51 (1973) 2119. 5 V.E. Gracen, C.O. Grogan and M.J. Forster, Can. J. Bot., 50 (1972) 2167. 6 A.L. Hooker, D.R. Smith, S.M. Lira and J.B. Beckett, Plant Dis. Rep., 54 (1970) 708. 7 A.L. Hooker, D.R. Smith, S.M. Lim and M.D. Musson, Plant Dis. Rep., 54 (1970) 1109. 8 C.S. Levings, III and D.R. Pring, Science, 193 (1976) 158. 9 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193"(1951) 265. 10 R.J. Miller and D.E. Koeppe, Science 173 (1971) 67. 11 R.J. Miller, S.W. Dumford, D.E. Koeppe and J.B. Hanson, Plant Physiol., 45 (1970) 649. 12 D.F. Parsons and Y. Yano, Biophys. Acta, 135 (1967) 362. 13 P.A~ Peterson, R.B. Flavell and D.H.P. Barratt, Theor. Appl. Genet., 45 (1975) 309. 14 D.R. Pring, Plant Physiol., 55 (1975) 203. 15 D.M. Shah and C.S. Levings, III, Crop Science, 14 (1974) 852. 16 C. Schnaitman, V.G. Erwin and J.W. Greenawalt, J. Cell Biol., 32 (1967) 719.