ABSTRACT. Pear fruit cells undergoing a period of senescence in auxin-deprived media develop a substantial cyanide resistant respiration in response to.
Plant Physiol. (1981) 68, 823-826
0032-0889/81/68/0823/04/$00.50/0
Cycloheximide Stimulation of Cyanide-Resistant Respiration in Suspension Cultures of Senescent Pear Fruit Cells Received for publication October 28, 1980 and in revised form April 6, 1981
ROGER J. ROMANI1, TIMOTHY J. BOS2, AND JEAN-CLAUDE PECH3 Department of Pomology, University of California, Davis, CA 95616 ABSTRACT Pear fruit cells undergoing a period of senescence in auxin-deprived media develop a substantial cyanide resistant respiration in response to the adtion of 0.7 to 3.5 micromolar cycloheximide. The inhibitor does not affect overall cellular repiratory activity and titrations with salicylhydroxamic acid reveal that only a minor portion, about 10%, of the alternate pathway is utilized by the cycloheximide-treated senescent cells. The alternate respiratory pathway appears to be of mitochondrial origin but is not induced by chloramphenicol
Codron et al. (3) found that auxin deprivation, combined with an increase in medium osmolality, causes cultured pear fruit cells to cease division and to enter a protracted senescent phase. We (12) have since used similar and modified suspension cultures to observe a transient increase in protein synthesis that precedes the onset of cell death. CH4 not only affected protein synthesis and the timing of cell death (12), it also appeared to induce the development of CRR. Since CRR has been implicated in the senescence (ripening) of fruit cells (17), we have sought to define the presence and extent of the alternate respiratory pathway in suspension cultures of auxin-deprived pear cells. MATERIALS AND METHODS A strain of cells first established from Passe Crassane pear fruit (Pyrus communis L.) in 1972 was employed in these experiments. The growth medium, 'aging' medium (one-quarter concentration of nutrients in growth medium plus 0.4 M mannitol), and conditions of 'batch' culture or culture with continuously renewed media were as previously described (12). The cytokinin autotrophic pear fruit cells were normally subcultured every week. To establish the senescent phase the cells were transferred and held for 9 to 12 days in auxin-deprived growth medium. The cells then were allowed to settle and were washed twice with 'aging' medium before incubation in the latter. As previously described (12), CH was added to the cell suspension after about 6 days of aging. In those experiments *here the medium was under continuous renewal CH was also added to the media reservoirs. Respiration of aliquots of cells withdrawn from culture flasks ' To whom
reprint requests should be addressed.
2Present address: Department of Microbiology and Immunology, University of California, Los Angeles, California, 90024. 'Present address: Laboratoire de Biologie Vegetale, ENSAT, 145 Ave de Muret, F31076, Toulouse, France. 4Abbreviations: CH, cycloheximide; CRR, cyanide-resistant respiration; SHAM, salicylhydroxamic acid. 823
was measured polarographically with an 02 electrode in a 1-ml respiratory chamber. As cells senesced and declined in respiratory activity it was often necessary to either increase their concentration 1-fold by allowing them to settle and then decanting a portion of the supernatant, or to increase the sensitivity (recorder span) of the 02 measuring system, or both. Percentage of live cells was estimated by their exclusion of Evans blue as observed microscop-
ically. Mitochondria were isolated by pressing the cells through a fine (250 mm pore size) mesh screen suspended just below the surface of 2 to 3 volumes of isolation medium. Other commonly used forms of cell disruption, e.g. shaking with glass beads or use of a polytron homogenizer, resulted in higher yields but less functional mitochondria. The isolation and wash media, procedures for differential centrifugation and assay medium with succinate as substrate, were similar to those employed in our laboratory for mitochondria from intact fruit (14). Protein was determined by a modified Lowry procedure (11). SHAM was dissolved in dimethylformamide to avoid a metabolic reponse to ethanol, the more commonly used solvent. There was no observed cellular response to dimethylformamide.
RESULTS It has been demonstrated that the respiratory activity of auxindeprived pear fruit cells undergoes a progressive decline arriving at a reasonably constant basal rate 8 to 10 days after transfer to 'aging' media (12). The same general pattern was observed in the present experiments. Moreover, within the limits of the methods employed, CH at the three concentrations used in these experiments did not affect the magnitude or the time course of cellular respiration (Fig. 1). The addition of 0.1 mm KCN to aliquots of cells withdrawn from the suspension at various times throughout the senescent phase resulted in a 90 to 100%1o inhibition of respiratory activity (Fig. 2). However, 1 day after the addition of CH, KCN inhibition was much less. By the 5th day KCN inhibited only 10-15% of cellular respiration signaling the substantial development of an alternate CN-resistant electron transport pathway. As in its inhibition of protein synthesis (12), the CH induction of CRR was transient with a reversion to predominantly KCN sensitive respiration as the cells continued to senesce and approach death. Cellular sensitivity to KCN and SHAM was estimated from 02 electrode traces as shown in the upper portion of Figure 3. It has been observed (16, 18), and it has been our experience with pear fruit cells as well (12), that cell strains undergo some variability during progressive subcultures and do not always respond in the same way to similar perturbations. We have assessed the CH effect in nine separate experiments with different sequential subcultures of the pear fruit cells over a period of 18 months. Three of the cultures were with continuous media renewal, and six were in batch culture. The amount of CRR present in the control cultures varied from 5 to 45% of the total respiratory
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ROMANI ET AL.
Plant Physiol. Vol. 68, 1981
CELLS
N, cn
B3614
A308 KCN
KCN
+CH
20 1A0 2 8
108
226 32
min
2
0
M/ TOS
A Respiratory rate of auxmndeprived, senescent, cultured pear 1.75 Respiratory rates of treatments, controls (0) and 0.7 3.5 CH added at (i), fall within the cross-hatched (Om ,UM and FIG.
\
B 268
233
DAYS
4KCN
\,KCN
1.
fruit cells.
4SHAM 29
4SHAM
2
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al
area.
T139 SHAM tN
OD-1o4\
+CH
5
0
100
.o 0
0
z
FIG. 3. 02 consumption by cells and mitochondria extracted from them. CH treated (A), controls (B), KCN (0.1 mM) and SHAM (0.2 mM) added at arrows. Respiratory rates are shown in nmol 02 min-' ml-' (about 1.4 x 106) live cells and nmol 02 min-' mg-' mitochondrial protein.
00
z
60-
I0/
Table
Development and Subsequent Decline of the Potentialfor CRR in Cultured Pear Fruit CRR as % of Normal Respirationa
I.
20 --
4
8
12
16
20
24
3.5 Am Cycloheximide
DAYS
Experiment FIG. 2. Percent inhibition of
cells jAm
SHAM
90
(O-----O0),
(DJ-{)
respiratory
cells to which 0.7 CH
was
added at
JAm
rate
by 0.1I mm~KCN.
No.
Control
Control 1.75
JAm
or
3.5
(i). Cells cultured with continuously
renewed medium containing designated concentrations of CH.
With media renewal
activity (Table I). However, in all instances the addition of CH resulted in a marked increase in the potential for CRR so that it equaled from 55 to 92% of the total respiratory activity. In one experiment (No. 4) the addition of KCN actually stimulated cellular respiratory activity, a response also observed with potato tissue slices (8). In all instances there was some, though highly variable, return to a normal respiratory pattern, i.e., a decline in CRR, with continued senescence leading to cell death. As Theologis and Laties (19) have observed in ripening avocado and banana fruit, the presence of CRR does not necessarily mean that the alternate pathway is being utilized. Using the procedure of Bahr and Bonner (1) which is premised on the use of SHAM as an inhibitor of CRR, we have estimated the fraction, (p), of the total alternate respiratory pathway in actual use. Data thus obtained with duplicate CH-treated cell cultures are shown in Figure 4, A and B and replotted in Figure 4, A' and B' to estimate p. Within the limits of accuracy of this method it can be estimated that only about 10%Yo (p = 0.1) of the alternate, CRR pathway was being utilized in the CH-treated cells. It is presumed that mitochondria are the centers of cellular respiratory activity and the sites of the alternate pathway. The data in Table II, derived from cellular and mitochondrial respiratory measurements as shown in Figure 3, demonstrate that enhancement of CRR does occur in the mitochondria of CHtreated cells. In comparable experiments the substitution of 10 mg/l chlorophenicol for the CH did not result in the development of CRR. Cultured pear cells and mitochondria extracted from them exhibit a residual respiratory activity insensitive to both KCN and
2 3 Without media renewal
1
9 5 25
Maximum
Subsequent
developmentb
declinec
86 92 75
75 15
65
45 140' 15 30 75 60 6 18 55 25 7 22 78 65 35 92 65 8 30 85 58 9 a Respiratory rate in presence of KCN/initial uninhibited respiratory rate less the residual rate in presence of both 0.1 mm KCN and 0.2 mM SHAM. Rates were determined from oxygen electrode traces as shown in Figure 3. b Value determined at point of maximum observed effect of cyclohex4 5
imide, generally 4 to 6 days after addition of the inhibitor. ' Subsequent decline in CRR and return to a normal respiration pattern was measured at, and circumscribed by, the onset of cell death. d In these cycloheximide-treated cells the addition of KCN actually stimulated respiration.
SHAM (Fig. 3), not unlike that observed in potato (8) and fruit (19) tissue slices and avocado mitochondria (13). Residual respiration averaging about 10%1o of total respiration was substracted from the total respiratory activity in determining the percentage of inhibition by KCN (Fig. 2), when estimating p (Fig. 4), and in estimating CRR as a percentage of normal respiration (Tables 1, II).
Plant Physiol. Vol. 68, 1981
CULTURED CELLS AND CN-RESISTANCE
A
w3 0. 304
A' .
10
15
cR
24)5 25. .
0
1
0
2
15
20
25
15
20
25
cn w
zr20
~30
0.5
1.0 1.5 mM SHAM
2.0
cr
5
10 g(i)
B
B
>-20 a.
30
30-
0
25
+ KCN
w
0.5
1.0 1.5 mM SHAM
2.0
5
10
g(i)
FIG. 4. Effects of increasing levels of SHAM (± KCN) on the respiratory rate of cultured pear fruit cells 2 days after treatment with 3.5 PAa
CH. A and B represent duplicate cultures. A' and B' represent a replotting of the data to calculate p. All respiratory rates are in nmol 02 consumed min-' ml-' (about 1.4 x 106) live cells. Table II. CRR in Cultured Pear Fruit Cells and Mitochondria Extracted from Them
Determination was made 4.5 days after the addition of 3.5 ,AM CH to the suspension culture CRR as % of Normal Respirationa Treatment
Cells
Mitochondriab 12 None 32±3 74 +CH 64±4 aRespiratory rate in presence of 0.1 mm KCN/initial uninhibited respiratory rate-less the residual rate in presence of both 0.1 mm KCN and 0.2 mM SHAM. Rates determined from oxygen-electrode traces as shown in Figure 3. b Average of 2 mitochondrial respiratory assays.
DISCUSSION The metabolic effects of CR are ambiguous. McDonald and Ellis (9) noted that in some plant tissues CH inhibits energy transduction as well as protein synthesis. McMahon (10) observed additional deleterious effects of CH on Chlamydomonas reinhardi. Cocucci and Marre (2) discussed these and other anomalous effects of CH but their observations lead to the suggestion that CH (18 to 54 Mm) depression of respiration in Rhodotorula gracilis cells resulted from inhibited protein synthesis and the consequent decreased demand for ATP and the increase in energy charge. It is questionable whether energetics play a dominant role in the CH effect on senescent pear fruit cells whose respiration is ahlady depressed and show no measurable response to CH. It is also questionable whether inhibition of protein synthesis plays a direct role in the development of CRR. Even at the highest concentration (3.5 ,um) of CH employed, inhibition of protein synthesis is transient and is followed by a recovery 4 to 6 days after addition of the inhibitor (12). A transient inhibition by CH of protein synthesis in cultured plant cells was also observed by Davies and Exworth (4). If there exists a relationship between protein synthesis and the development of CRR in cultured pear fruit cells, it is likely to be associated with the burst of protein synthesis that follows transient CH inhibition and which precedes the onset of cell death (12). Indeed, the stage of maximum CRR seen in Figure 2 (13th day) corresponds very closely to the stage of maximum recovery in protein synthesis following transient inhibition by CH in these same cells (Fig. 5 in reference 12).
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Hence, it seems that the CH induced development of the alternate pathway in pear fruit cells is triggered by some as yet undefined perturbation by CH and is coincident with the development of CH insensitive protein synthesis. Such an interpretation is consistent with the finding by Dizengremel and Lance (5) that the development of CRR in potato slices is dependent on protein synthesis. In both potato slices (5) and pear fruit cells chloramphenicol did not stimulate the development of CRR. This suggests that the critical perturbation in protein synthesis is extra-mitochondrial. Ziogas and Georgopoulas (20) did find that chloramphenicol induced the alternate pathway in the mitochondria of Ustilago maydis. However, this effect was observed in actively growing cultures where perturbations at any locus are likely to be greatly amplified. The experimental utility of fruit cell suspensions for the study of cellular senescence hinges on the existence of definitive senescent changes that, ideally, have counterparts in the cells of senescent intact fruit. Auxin deprivation as utilized in these experiments to induce cellular senescence is consonant with Frenkel's suggestion (6) that lowered auxin levels trigger the ripening of pear fruit. The rise in protein synthesis that precedes the death of cultured pear cells (12) also has a counterpart in the increase in protein synthesis accompanying the senescence (ripening) of pears (7) and many other fruits (15). CRR had been implicated (17) in the respiratory climacteric of senescent fruit tissues (17), but more recently Theologis and Laties (19) have shown that the potential for CRR, though present in ripening avocado and banana, is normally not operative, i.e., p = 0. In a somewhat analogous fashion the potential for CRR, though greatly increased by CH treatment, is minimally operative in senescent, cultured, pear fruit cells. Caution is called for in drawing analogies between events in dedifferentiated cultured cells and their counterparts in intact tissues. Nonetheless, it does appear that auxin-deprived cultured fruit cells may be a useful system for the study of some senescence associated metabolic phenomena. LITERATURE CITED 1. BAHR JT, WD BONNER JR 1973 Cyanide-insensitive respiration. I. The steadystates of skunk cabbage spadix and bean hypocotyl mitochondria. J Biol Chem 10: 3441-3445 2. Cocucci, MC, E MARRE 1973 The effects of cycloheximide on respiration, protein synthesis and adenine nucleotide levels in Rhodotorula gracilis. Plant Sci Lett 1: 293-301 3. CODRON H, A LATCHE, JC PECH, J FALLOT 1978 Mise au point d'un nouveau systeme d'etude de la senescence des cellules vegetables. CR Acad Sci, Paris 287: 21-24 4. DAVIES ME, CP EXWORTH 1973 Transient inhibition by cycloheximide of protein synthesis in cultured plant cell suspension: a dose response paradox. Biochem Biophys Res Commun 50: 1075-1080 5. DIZENGREMEL P, C LANCE 1976 Control of changes in mitochondrial activities during aging of potato slices. Plant Physiol 58: 147-151 6. FRENKEL C 1975 Role of oxidative metabolism in the regulation of fruit ripening. In Facteurs et Regulation de la Maturation des Fruits. Colloq Int Cent Nat Rech Sci, Paris pp 201-209 7. FRENKEL C, I KLEIN, D DILLEY 1968 Protein synthesis in relation to ripening of pome fruits. Plant Physiol 43: 1146-1153 8. JANES HW, SC WIEST 1980 Comparison between aging of slices and ethylene treatment of whole white potatoes. Plant Physiol 66: 171-174 9. MAcDONALD IR, RJ ELLIS 1969 Does cycloheximide inhibit protein synthesis specifically in plant tissues? Nature 222: 791-792 10. MCMAHON, D 1975 Cycloheximide is not a specific inhibitor of protein synthesis in vivo. Plant Physiol 55: 815-821 11. MILLER GL 1959 Protein determination for large numbers of samples. Anal Chem 31: 964 12. PECH JC, RJ ROMANI 1979 Senescence of pear fruit cells cultured in a continuously renewed, auxin-deprived medium. Plant Physiol 64: 814-817 13. ROMANI RJ 1978 Long-term maintenance of mitochondrial functions in vitro and the course of cyanide insensitive respiration. In G Ducet, C Lance, eds, Plant Mitochondria Elsevier, Amsterdam, pp 3-10 14. ROMANI RJ, S OZELKOK 1973 "Survival" of mitochondria in vitro. Physiological and energy parameters. Plant Physiol 51: 702-707 15. SACHER J 1973 Senescence and postharvest physiology. Annu Rev Plant Physiol 24: 197-224
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16. SKIRVIN RM 1978 Natural and induced variation in tissue culture. Euphytica 27: 241-266 17. SoLoMos T, GG LATIES 1974 Similarities between the actions of ethylene and cyanide in initiating the climacteric and ripening of avocados. Plant Physiol 54: 506-511 18. SUNDERLAND N 1977 Nuclear cytology. In HE Street, ed, Plant Tissue and Cell Culture. University of California, Press, Berkeley pp 177-205
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19. THEOLOGIs A, GG LATIES 1978 Respiratory contribution of the alternate path during various stages of ripening of avocado and banana fruits. Plant Physiol 62: 249-255 20. ZIOGAS BN, SG GEORGOPOULOS 1980 Chloramphenicol-induction of a second cyanide- and azide-insensitive mitochondrial pathway in Ustilago maydis. Biochim Biophys Acta 592: 223-234
