Cyanide stimulation of respiration of Acer pseudoplatanus cells in batch suspension culture and activation of the alternative pathway

Plant Science Letters, 19 (1980) 65--71

65

© Ekevier/N0rth-Holland Scientific Publidzers Ltd.

CYANIDE STIMULATION OF RESPIRATION OF ACER PSEUDOPLATANUS CELLS IN BATCH SUSPENSION CULTURE AND ACTIVATION OF THE ALTERNATIVE PATHWAY J.P. BLEIN I.N. R.L Malherbologie, B V 1540, 21034 Dijon, Cddex (France)

(Received December 2lit, 1979) (Revkion received February 20th, 1980) (Accepted April 3rd, 1980)

SUMMARY

Cyanide induces an immediate increase of the rate of oxygen uptake by Acer pseudoplatanus cells grown in batch culture. This increase is followed by a progressive decline. Salicylhydroxamic acid (SHAM) inhibits the oxygen uptake through the cyanide-insensitive alternate pathway. Antimycin A, oligomycin, and carbonyl-cyanide-p-trifluorometoxy-phenylhydrazone (FCCP), had the same effect as cyanide. The alternative pathway is induced by changes in the cytochromes pathway. When oxygen uptake, via the cytochrome pathway is reduced by a given amount, the uptake via the alternate pathway is increased by twice this amount. Several herbicides are found to strongly inhibit the alternate pathway. INTRODUC~ON

Cyanide resistant respiration is commonly found in plants and microorganisms [1]. Moreover, several authors have reported that cyanide can stimulate the respiration of plant tissues [2--5] or cells [6,7]. Recent data have been discussed in a review by Solomos [8]. In cyanide resistant respiration, none of the cytochromes is involved, the branch point being on the substmte side of the cytochrome b [9]. One phosphorylation site occurs between the branch point and oxygen [ 10]. The cyanide insensitive oxidase is specifically inhibited by hydroxamic acids [11]. In the present work, we show that cyanide induces a transient stimulation of the respiration of Acer pseudoplatanus cells. The relatives activities of the A b breoiations: Bidisin, methyl 2-chloro-3-( 4-chlorophenyl)propionate; DMSO, dimethyl-

sulfoxide; DNOC, 2-methyl-4-6-dinitrophenol; FCCP, Carbonyl-cyanide-p-trifluorometroxyphenyl hydrazone; Ioxynil, 4-hydroxy-3,5-diiodobenzonitrile; Neburon, N-(3,4dichlorophenyl)-N'-methyl-N'-n-butylurea; SHAM, salicylhydroxamic acid.

66

two pathways (via cytochrome or alternate oxidase) are measured in the presence or absence of cyanide. The effects of antimycin A, oligomycin and FCCP are also studied. MATERIALS AND METHODS Cells of Acer pseudoplatanus were kindly supplied by Prof. J. Guern, and were grown in batch suspension culture, in 800-ml conical flasks, in an orbital shaker (120 rev./min) at 25°C. The basic medium was prepared according to Lamport [12] and Lescure [13], with a few modifications (Table I). Oxygen uptake was measured with a Clark type oxygen electrode (yellow springs Inc. OH) in a magnetically stirred glass vessel (4 ml). The respiration medium was the culture medium. The concentration of oxygen in air saturated medium was taken as 240/~M. Aqueous cyanide was adjusted to pH 7.0 and SHAM was dissolved in dimethylsulfoxide (DMSO). Cultures were used during the exponential phase of growth, when the density reached 1.3 × 106 cells/ml (3 mg dry wt/ml). Measurements of oxygen uptake were carried out during the first 2 rain following the addition of cyanide to the oxygraph vessel. RESULTS AND DISCUSSION

Activation of the alternate pathway by cyanide In the absence of inhibitor, the respiration rate of Acer pseudoplatanus cells was 3.42 -+ 0.18 nmol O2/min • mg dry wt, in agreement with the data of Bligny [14]. The addition of cyanide, at a concentration of 7.5 × 10 -s M, induced a rapid increase of respiration (Fig. 1). This increase ranged from 75 to 100% according to the age of the cells (data not shown) in agreement with the results of Wilson [15]. SHAM (2.5 × 10 -3 M) added alone increased the respiration of the cells by about 10% (Fig. 1). When cyanide and SHAM were added simultaneously, only a very small residual respiration was observed (Figs. 1 and 2, and Table IID).

TABLE I BASIC MEDIUM CULTURE AND MODIFICATIONS

Potmmium nitrate di Sodium sulfate di Sodium hydrogen phosphate 12 hydrate Sodium dihydrogen phosphate Potamfium dihydrogen phosphate

Original medium (13)

Modified medium

2.8 × 10 -s M 0.5 x 10 -3 M

1.9 × 10 -~ M

0 2.5 x 10 -~ M 0

2.7 x 10 -~ M 0 3.7 x 10 -3 M

0

67

~

0

f

o"

!. i

I

I

I

OD K C N jlA so

40

Fig. I. Dependence of respiration rates of AeerpseudopI~anue cells on cyanide concentration in the pa~oneo (o, 2.5 raM) or abeene0 (e) of SHAM. These ol~ervafions show that Acer pseudopIatanus cells have a constituent alternative respiratory pathway. Normally, all the respiration proceeds through the c y t o c h r o m e s pathway, and the alternate pathway is n o t operative. However, the alternate p a t h w a y can be activated b y cyanide inhibition o f the c y t o c h r o m e oxidxse.

!

i

|

| I

I 1000

I

I

I

2000 9HAM

Fig. 2. Dependeneu of mspkation rat~ ofAcerpseudoplatanw eolk on hydroxamic acid coneontration in the pmNnee (s, 1.00 ~M) or absence (e) of cyanide.

68 TABLE II DEPENDENCE OF RESPIRATION RATES (nmoles OJmin • m g dry wt) OF ACER PSEUDOPLATANUS CELLS ON VARIOUS COMPOUNDS. SEQUENTIALLY ADDED TO THE OXYGRAPH VESSEL

A B C D

None

Antimyein A Oligomycin 5 .M 1.25 , g ~ ml-'

3.57 3.50 3.25 3.44

4.72 a

FCCP 25 , M

5.25 a 5.54 a

SHAM 2.5 mM

KCN 100 , M

0.48 b 1.75 b 4.49 b 0.55 b

6.27 a

aCompound added first, bCompound added second.

In the presence of SHAM, the cyanide-cytochrome oxidase affinity constant was 18.2 ~mol. If measurements were performed in the original medium of Lescure, the constant was 20.7 ,tool. However, the stimulation of the respiration by cyanide was 20-fold greater in the modified medium than in the original one. Consequently, all experiments reported here were performed in the modified medium.

Time course of alternate pathway activation by cyanide Figure 3 shows that the addition of cyanide rapidly stimulated the oxygen x

"~ +5©

~o

I

I 120

I

• 240 mlnu tes

Fig. 3. Time course of 02 uptake b y Acer pseudoplahmus ceils in presence of cyanide ( I 0 0 #M). Results expressed as pereentage of control. Cyanide was added to the culture flasks and aliquots were removed to determine the O: uptake by the ceils.

69

uptake of the cells. Then the intensity of the oxygen consumption continuously decreased and after 240 min, it had fallen to 50% of the control value.

These data can be interpreted as a sudden activation of the alternate pathway, followed by a progressive inhibition of the respiratory systems of the cell.

Regulation of the alternative pathway activity Antimycin A, an inhibitor of the cytochromes b, gave an increase in the respiration (Table IIA). Since this enhanced respiration was reduced to a small residual respiration by the addition of SHAM, we may conclude that, like cyanide, antimycin A activates the alternate pathway. This activation was also caused by oligomycin which inhibits oxidative phosphorylations and thus decreases the oxygen uptake via the cytochromes pathway in presence of SHAM (Table IIB). Oligomycin alone increases oxygen uptake by cells via the alternate pathway. FCCP, uncoupler of oxidative phosphorylations, increases oxygen uptake by cells via the cytochromes and alternate pathways (Table IIC). Though these compounds act on various sites of the respiratory chain, they share the property of modifying the oxydo-reduction state of the cytochromes chain. Thus the activation of the alternate pathway could be due to this modification. These conclusions agree with those of Bahr and Bonner [16] who showed that mitochondria in state 3 did n o t use the alternate oxidase although they did use it in state 4, or in state 3 in presence of inhibitory concentration of cyanide. As it is known that oligomycin and FCCP depress the ATP level in the cell, this point will deserve a further study in our conditions. In conclusion, the alternate pathway activity may be dependent on the state of the cytochromes pathway, and was not fully active in the absence of cyanide.

TABLE III INHIBITOR CONSTANTS OF SEVERAL HERBICIDES Data calculated from plots of the reciprocal of the respiration rate qainst herbicide concentration. Compounds

K i (.M)

Loxynil Neburon Bidisin SHAM DNOC

0.26 58 109 193 244

70

Relative activities of cytochromes and alternate pathway Oligomycin enhances the respiration rate (Table IIB) although, unlike the effect of cyanide and antimycin, the enhanced respiration was only partially inhibited by SHAM. In the experiment reported in Table IIB, comparison of this residual respiration with the control level showed that oligomycin reduces the flux through the cytochromes pathway to the equivalent of 1.75 nmol O2/min. The alternate pathway activity calculated from the difference between the oxygen uptakes in presence of oligomycin alone or plus SHAM, was 3.5 nmol O2/min. Thus 2 nmol were used through the alternate pathway, when 1 nmol was lost from the cytochromes pathway. From the data of Fig. 1, it can be seen that when the cytochromes pathway was fully inhibited by cyanide, the oxygen uptake via the alternate pathway was twice the control level. When the total oxygen uptake increased by 1 nmol O2/min, the cytochromes pathway activity decreased by 1 nmol O2/min, but this fact may be casual or depending on the particular conditions of the experiment. Rychter et al. [ 17] observed the same relationships between the two pathways in cyanide insensitive potatoe mitochondria where the alternate pathway is about 2/3 the total respiratory capacity. Thus double rate of the alternate pathway explains the cyanide stimulation of the cell respiration rate. Inhibitors of the alternate pathway During the course of a study on the mode of action of some herbicides, we found that some of t h e m were inhibitors of the alternate pathway. The inhibitor constants were determined (Table III). Methyl 2-chloro-3-(4-chlorophenyl)propionate (Bidisin), 2-methyl-4,6dinitrophenol (DNOC) and SHAM had similar inhibitor constants. N-(3,4dichlorophenyl).N'-methyl-N'-n-butylurea (Neburon) and especially 4-hydroxy-3,5-diiodobenzonitrile (Ioxynll), were more active. Dinitrophenol and FCCP did n o t inhibit the alternate pathway. CONCLUSION The alternative pathway may be activated by c y a n i d e ' i n Acer pseudoplatanus cells and we shall research it, in vitro, at the mitochondrial level. We hope that understanding the action of these different compounds will lead to a better knowledge of the alternate pathway. ACKNOWLEDGEMENTS

I am grateful to Mr. D. Clair and Mrs M.R. Allard for skillful technical assistance and I am indebted to Dr. R. Scalla, Pr. G. Ducet, J. Guern and S.B. Wilson for critical discussion.

71 REFERENCES

I 2 3 4 5 6 ? 8 9 10 11 12 13 14 15 16 17

M.F. Henry and E.J. Nyns, Sub. Cell. B/ochem., 4 (1975) 1. P.J. Anen and D.R. Godsrd, Am. J. Bot., 25 (1988) 615. D.P. Haekett, J. Exp., 8 (1957) 157. 8.H. Lips and J.B. Biale, Plant Physiol., 41 (1966) 797. I.R. MacDonald and P.C. Dekock, Physiol. Plant., 11 (1958) 464. T.K. Sharplem and R.A. Butow, J. Biol. Chem., 245 (1970) 58. R.T. Wedding, C.C. MacCready and J.L. Harvey, New Phytol., 72 (1973) 15. T. 8olomo~ Ann. Rev. Plant Physiol., 28 (1977) 279. D.S. Bendall and W.D. Bonnet Jr, Plant Physiol., 47 (1971) 236. S.B. Wikon, Bioehim. Biophys. Acta, 228 (1970) 383. G.R. Sehonbaum, W.D. Bonner Jr. and B.T. Storey, Plant Physiol., 47 (1971) 124. D.T.A. Lamport, Exp. CeIL Res., 33 (1964) 195. A.M. Leseure, Physiol. V~g., 4 (1964) 365. R. B l i p y and R. Douce, Physiol. V ~ . , 14 (1976) 499. S.B. Wilson, J. Exp. Bot., 22 (1971) 725. J.T. Bahr and W.D. Bonnet Jr., J. Biol. Chem., 248 (1973) 3441. A. Rychter, H.W. Jones and C. Frenkei, Plant Physiol., 68 (1979) 149.