Suspension-cultured pear fruit cells produce low levels of ethylene during growth and division in auxin containing medium. When deprived of auxin, division ...
Plant Physiol. (1985) 79, 973-976 0032-0889/85/79/0973/04/$0 1.00/0
Ethylene Production by Suspension-Cultured Pear Fruit Cells as Related to Their Senescence Received for publication May 28, 1985
ROLF PUSCHMANN', DANGYANG KE2, AND ROGER ROMANI* Department of Pomology, University of California, Davis, California 95616 ABSTRACT Suspension-cultured pear fruit cells produce low levels of ethylene during growth and division in auxin containing medium. When deprived of auxin, division gradually ceases and ethylene production falls to barely discernible levels. However, notable ethylene production can now be induced by indoleacetic acid, CuCl2, or 1-aminocyclopropane--carboxylic acid. If the auxin-deprived cells are transferred to 'aging' medium that lacks auxin but contains 0.4 molar mannitol, inducible ethylene production increases several-fold reaching levels of 40 to 60 nanoliters/10 cells per hour. Maximum inducible ethylene productivity is attained at varying times (1-6 days) after transfer to aging medium and appears to be temporally related to cell survival, i.e. the time of subsequent cell death. It is argued that auxin depletion initiates senescence which, in turn, leads to a transient increase in inducible ethylene production and eventual death. The limitations and potentials of the suspension-cultured pear cells as a system for the study of cellular senescence are discussed.
Suspension-cultured pear fruit cells first deprived of auxin and then supplied with 0.4 M mannitol undergo an extended, quiescent, pre-death phase (2). During their quiescent phase the cells exhibit a transient increase in protein synthesis (1, 8), an increase in the leakage of serine (1), and an increase, albeit induced by cycloheximide, in the alternative electron transport pathway (14). These transitions are close counterparts to events in senescent plant tissues and imply that the cultured cells hold promise for the study of cellular senescence. Although large differences undoubtedly exist between senescent cells in situ and in vitro, the potential utility of the latter as a model system justifies further search for the appearance of altered physiological state(s) as the cultured cells approach death. In an earlier study (11) we examined the kinetic and other characteristics of ethylene production by cultured pear fruit cells in response to auxin, CuCl2, and ACC.3 We now report on the senescence-related aspects of these responses. Our use of the terms 'aging' and 'senescence' is consonant with the recent suggestions of the Postharvest Working Group of the American Society of Horticultural Science (16). A preliminary report of this work has appeared (10).
strain of 'Passe Crassane' pear (Pyrus communis L.) fruit cells were grown in complete medium at 25 to 27°C and then deprived of 2,4-D by incubation for 9 to 11 d in medium lacking 2,4-D. To achieve a quiescent or senescent-like state, the cells were then transferred to aging medium which consisted of one-fourth the concentration of nutrients found in the complete medium, no 2,4-D, but supplemented with 0.385 M mannitol and 0.015 M sucrose. To assess the kinetics of ethylene production in response to an inducer, 50 ml aliquots of cell suspension were placed in 125 ml Erlenmeyer flasks and ethylene evolution determined at frequent intervals after the addition of IAA, CuC12, or ACC. Such kinetic data are represented as line drawings. To assess changes in rates of inducible ethylene production with cell age, 50 ml representative samples of aging cells were taken at daily or other stated intervals and their maximum rate of ethylene evolution measured at a predetermined (see below) number of hours following the addition of IAA, CuC12, or ACC. These age-related data are represented as bar graphs. Dependent upon the experimental objective, different flask sizes were used as recipients for the aging cells. Unless otherwise noted, the initial cell density (approximately 106 cells/ml) and culture volume/flask volume were approximately the same in all experiments. Rotary shaker speeds were adjusted to suit flask size and thereby achieve "visually" similar aeration.
RESULTS Cell Age, Kinetics of Ethylene Production, and Sampling Technique. Prior work has shown that induced ethylene production by suspension-cultured pear fruit cells follows a distinct kinetic pattern as determined by the particular inducer, i.e. IAA, CUC12, or ACC (1 1). Of consequence to the present study is the observation that each kinetic pattern does not change appreciably with cell age (days in aging medium) or intensity of the response. For instance, unless the cells are too old to respond, maximum ethylene production is attained at or near 12 h after addition of IAA regardless of cell age (Fig. 1). Similarly, ethylene production maximizes 4 to 4.5 h after the addition of CuCl2 or well within 2 h after the addition of ACC (Table I) irrespective of cell age, culture volume, or the quantity of ethylene evolved. Accordingly, ethylene production as a function of cell age is hereafter based on the maximum rates of ethylene evolution measured 12 h after the addition of IAA, 4 to 5 h after the addition of CuCl2, or 1 to MATERIALS AND METHODS 2 h after the addition of ACC. The levels of IAA (50 or 100 Mm), The requisite materials and experimental techniques were CuC12 (100 Mm), and ACC (100 Mm) employed have been shown described in detail elsewhere (9, 11). In brief, an established ( 11) to result in maximum ethylene production without incurring a lag phase in the case of IAA, or immediate cell injury in the 'Permanent address: Departamento de Biologia Vegetal, Universidade case of CUC12. Federal de Vicosa 36570, Vicosa, Minas Gerais, Brazil. Rates of Inducible Ethylene Production as a Function of Culture Medium and Cell Age. For the purposes of this experiment 2Under sponsorship of the Ministry of Education of China. 3 Abbreviations: ACC, I-aminocycloproprane-l-carboxylic acid; EFE, cells were allowed to "age" in each of three media (growth, auxinethylene forming enzyme. deprived, aging) until cell death. Cells cultured in growth medium 973
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FIG. 1. Effects of cell age on the kinetics of ethylene production following induction with 50 ;sM IAA. Numbers in parentheses refer to cell age (d in aging medium) before the addition of IAA.
(Fig. 2A) increased in number and produced low levels of ethylene (2-3 nl/106 cells- h) with only a slight increment in response to auxin. After I week a portion of these cells was transferred to auxin-deprived media. The transferred cells exhibited some additional increase in number accompanied by very low ethylene evolution, but the cells now responded to added auxin with moderate amounts (5 to 7 nl/106 cells- h) of ethylene production (Fig. 2B). After 10 d of auxin deprivation a portion of the cells was transferred to aging medium where the cells remained alive but quiescent and with barely discernible ethylene evolution. However, their capacity to produce ethylene following induction by IAA increased markedly to a maximum rate of over 20 nl/ 106 cells h by the 3rd d after transfer (Fig. 2C). Similar transitions in the propensity ofthe pear cells to produce ethylene were observed with CuCl2 or ACC as inducing agents. For example, in a subsequent experiment (Fig. 3) where the cells were again held in growth media deprived of 2,4-D, the patterns of growth, death, and IAA inducible ethylene production resemble those in Figure 2B. Ethylene production induced by CuCl2 or ACC follows the same temporal pattern but is appreciably higher. Data from two experiments comparing IAA, CuCl2, and ACC inducible ethylene production by auxin deprived cells that were transferred to aging medium are shown in Figure 4. Once again, the response to CuCl2 and ACC followed the same pattern of age-dependence as the response to IAA. Moreover, a difference in protocol (legend, Fig. 4) which resulted in longer survival times, or delayed senescence, also delayed the attainment of maximum inducible ethylene production. This effect of physical parameters on cell survival has not been resolved; however, its occurrence implies that the increase in inducible ethylene production which develops during the quiescent phase is not likely the result of osmotic stress. If it were, inducibility should be manifested at a fixed time after exposure to mannitol. The data of Figure 4 also imply a 'connection' between the transient rise -
Plant Physiol. Vol. 79, 1985
in inducible ethylene production and subsequent cell death, i.e. cells with maximum inducible ethylene production by d 1 or 2 showed signs of incipient death by d 7 or 8, cells with a delayed inducible ethylene production (d 5 or 6) did not evidence a substantial death rate until about d 30. Ethylene Production and Cell Division in Response to IAA. Since auxin deprivation leads to both the cessation of cell division and the rise of inducible ethylene production it was important to discern if the latter phenomenon was linked with the capacity for division. Accordingly, representative portions of progressively older cells were transferred from aging to growth medium while other portions were tested for auxin-stimulated ethylene evolution. Inducible ethylene production peaked on d 3 and ceased by d 8; however, the capacity to divide was retained even by cells held for up to 15 d in aging medium (Fig. 5). A similar persistence in growth response to auxin has been reported by Balague et al. (1). There is clearly no readily apparent connection between the loss of inducible ethylene production and the capacity to divide. It may be noted, however, that both maximum inducible ethylene production and subsequent cell death occurred at times intermediate between the two cultures in Figure 4.
DISCUSSION Ethylene evolution has long been associated with the senescence of various determinant plant organs including fruits (15) and flowers (4). The question giving impetus to this work was whether or not the capacity of cultured pear cells to produce ethylene is a manifestation of their senescence. In the main, ethylene production by suspension-cultured plant cells has been associated with active cell division (3, 5-7). There have, however, been a few reports of ethylene production by nongrowing cultured cells. MacKenzie and Street (7) observed a 3-fold increase in ethylene production by sycamore cells about 24 h after the addition of 4.5 gM 2,4-D to cultures which had been initiated in 2,4-D-deprived medium 13 or 20 d earlier. The authors concluded that 2,4-D can stimulate ethylene production in the absence of cell growth. We also observed modest increases in ethylene production with the addition of auxin, CuC12, or ACC to pear fruit cells aged in auxin-deprived medium (Figs. 2B and 3). MacKenzie and Street (7) reported that the addition of an osmoticum (NaCl, sucrose, or mannitol) to stationary cultures of sycamore cells increased their ethylene evolution 2- to 3-fold in 12 to 24 h. In contrast, we found that the addition of mannitol to stationary or growing cultures of pear cells did not stimulate ethylene production in the absence of an inducer (data not shown). Moreover, if the osmotic or even potentially metabolic (12) effects of mannitol were causing the increase in inducible ethylene production, it should have been manifested at a relatively fixed interval rather than at variable times after exposure to mannitol (Fig. 4; Table II). LaRue and Gamborg (5), noting a sharp increase in rate of ethylene production by rose cells at or shortly after the cessation of culture growth, suggested that the enhanced production may have been the result of senescence. The rates ofethylene production by the rose cells (12 nmol/h -600 mg dry weight) were about
Table I. Time Required for Cultured Pear Cells to Reach Maximum Rates ofEthylene Production as Induced by 100 gM, CuCl2, or ACC, Irrespective of Cell Age and Intensity of the Response Time to Reach Maximum Rate of Cell Age Time Maximum Rates Inducer and in Aging Medium No. of Experiments Ethylene Production Mean Range h d nl.106 cells'- h' 3.8-4.5 4.3 7-55 1-6 CuC12 (n = 12) 0.5-2 10-66 1.2 1-6 ACC(n = 15)
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FIG. 4. Age-related changes in the production of ethylene following the addition of 50 ,M IAA, 100 M CuC12, or 100 Mm ACC to representative samples of progressively older cultured pear fruit cells suspended in aging medium. Shown are the maximum ethylene production rates measured 12 h after addition of IAA, 4 h after addition of CuCI2, and 1 h after addition of ACC to aliquots of progressively older cells. Two different experimental protocols are represented. Cells were either distributed from the onset into small recipients (50 ml of suspension in 125 ml Erlenmeyer flasks) for subsequent estimation of inducible ethylene production (open bars) and % live cells (0) or, 1.8 L cell suspensions were retained in 3 L Fernbach flasks and 50 ml aliquots taken at daily or longer intervals for ethylene (closed bars) and % live cells (0) measurements. Ethylene production in aging, control (non-IAA, CuC12, or ACC treated) cells never exceeded 7 and 2 nl ethylene/106 cells-h in the small and large suspension volumes, respectively.
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~~~~CELL AGE (DAYS IN MEDIUM LACKNG 2.4-0) FIG. 3. Age-related changes in cell number and vitality (upper graph) and in induced ethylene production (lower graph) by pear fruit cells held in growth medium lacking 2,4-D. Ethylene production rates were measured 2, 4, and 12 h after the addition of 100 uM ACC, CuC12, or IAA, respectively, to 50 ml aliquots of progressively older cells. -
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the same as those observed by the aging pear cells in response to IAA (106 cells yield approximately 80 mg dry wt). The analogy appears warranted and senescence may also be implicated where maximum inducible ethylene production during auxin deprivation is roughly coincident with the cessation of growth and onset of cell death (Figs. 2B and 3). However, senescence is more readily implicated where the presence of 0.4 M mannitol, following auxin-deprivation, exacerbates a prolonged quiescent phase and even higher inducible ethylene production (Figs. 2C and 4). Our data do not permit a clear delineation between the onset of senescence and the rise in inducible ethylene production. However, since the peak in ethylene production occurs at varying times after transfer to aging medium and exhibits some temporal relationship with eventual cell death, we favor the notion that the onset of senescence is a consequence of auxin deprivation
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cells remaining in the aging medium (0). Volumes in this experiment 500 ml cell suspension per I L flask. In the absence of added IAA the aging cells produced less than 2 nl ethylene/106 cells-h. were
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PUSCHMANN ET AL. Table II. Variability in Maximum CuCl2 Inducible Ethylene Production and in the Cell Age at which it Was Attained No. of Experiments
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Cell Age Time in Aging
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Range Mean nl- 106 cells~'-hh ' n = 8a 16-55 30 1-6 3.6 n= 18b 7-24 16 0.5-6 2 a Experiments conducted January 1980 to June 1982 all consisting of b Experiments 1500 ml cell suspensions aged in 3 L Fernbach flasks. conducted July 1982 to February 1984 with adjustments in some variables, e.g. cell density, extent of aeration, days in medium lacking, 2,4-D before transfer to aging medium.
and that the rise in inducible ethylene production is, in turn, an early manifestation of progressive senescence. Such an interpretation bears directly on the interconnection between ethylene and the senescence of fruit tissue where it can be argued (13) that the onset of senescence precedes autocatalytic ethylene production. Potentials and Limitations of the Cultured Cells. Given that the several physiological transitions observed in aging, cultured pear cells, i.e. rise in protein synthesis (1, 8), elaboration of the alternative pathway (14), respiratory increase and accelerated death in response to ethylene (C. J. Brady and R. J. Romani, unpublished data), bear a close resemblance to events which characterize senescence in the intact tissue, it is tempting to consider the inducible ethylene production reported here as another manifestation of cellular senescence. We hasten to acknowledge that unknowns affect the production of ethylene by the cultured cells. For instance, neither cultured 'Bartlett' pear cells nor another strain of 'Passe Crassane' could be induced to produce high levels of ethylene. Even with a responsive cell system the maximum level of inducible ethylene production and the number of days in aging medium before the maximum is reached varied from experiment to experiment (Table II). On the other hand, the kinetics of ethylene production in response to IAA, CuCl2, or ACC have remained unchanged. The same is true for the pattern of increase in inducible ethylene production as the cells are held in auxin-deprived medium. For instance, one notes obvious similarities in the data of Figures 2B and 3 even though the latter were obtained 3 years or approximately
Plant Physiol. Vol. 79, 1985
100 cell generations later. Accepting that analogies between the behavior of undifferentiated cultured cells and their counterparts in situ must be drawn with caution, we believe the cell suspensions described here to be useful in examining ethylene production and other senescence-related phenomena.4 Acknowledgments-We are grateful to Drs. Lishar Huang and Neil Hoffman for many helpful suggestions and to Betty Hess for expert technical assistance. LITERATURE CITED 1. BALAGUE C, A LATCHE, J FALLOT, JC PECH 1982 Some physiological changes occurring during the senescence of auxin-deprived pear cells in culture. Plant Physiol 69: 1339-1343 2. CODRON H, A LATCHE, JC PECH, B NEBIE, J FALLOT 1979 Control of quiescence and viability in auxin-deprived pear cells in batch and continuous culture. Plant Sci Lett 17: 29-35 3. GAMBORG OL, TAG LARUE 1968 Ethylene produced by plant cells in suspension cultures. Nature 220: 404-605 4. KENDE H, AD HANSON 1977 On the role of ethylene in aging. In PE Pillet, ed, Plant Growth and Regulation. Springer-Verlag, New York, pp 172-180 5. LARUE TAG, OL GAMBORG 1971 Ethylene production by plant cell cultures. Plant Physiol 48: 394-398 6. LIEBERMAN M, SY WANG, LD OWENS 1979 Ethylene production by callus and suspension cells from cortex tissue of post climacteric apples. Plant Physiol 63: 811-815 7. MACKENZIE IA, HE STREET 1970 Studies on growth in culture of plant cells. I. Production of ethylene by suspension cultures of Acer pseudoplatanus L. J Exp Bot 21: 824-834 8. PECH JC, RJ ROMANI 1979 Senescence of pear fruit cells cultured in a continuously renewed auxin-deprived medium. Plant Physiol 64: 814-817 9. PUSCHMANN R 1982 Physiological transitions in senescent cultured pear (Pyrus communis L.) fruit cells as indicated by their ability to synthesize ethylene. Ph.D. Thesis. University of California, Davis 10. PUSCHMANN R, R ROMANI 1981 Senescence of cultured pear fruit cells: Physiological transition revealed by the response to auxin, copper or 1aminocyclopropane-l-carboxylic acid. Plant Physiol 67: S-l 16 1 1. PUSCHMANN R, R ROMANI 1983 Ethylene production by suspension cultured pear fruit cells in response to auxin, stress or precursor. Plant Physiol 73: 1013-1019 12. Riov J, SF YANG 1982 Stimulation of ethylene production in citrus discs by mannitol. Plant Physiol 70: 142-146 13. ROMANI RJ 1984 Respiration, ethylene, senescence, and homeostasis in an integrated view of postharvest life. Can J Bot 62: 2950-2955 14. ROMANI RJ, TJ Bos, JC PECH 1981 Cycloheximide stimulation of cyanideresistant respiration in suspension cultures of senescent pear fruit cells. Plant Physiol 68: 823-826 15. SACHER JA 1973 Senescence and postharvest physiology. Annu Rev Plant Physiol 24: 197-224 16. WATADA AE, RC HERNER, AA KADER, RJ ROMANI, GL STABY 1984 Terminology for the description of developmental stages of horticultural crops. Hortic Sci 19: 20-21
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