Plant Cell Reports (1983) 2:82-84
Plant Cell Reports © Springer-Verlag 1983
Influence of Diamines and Polyamines on the Senescence of Plant Suspension Cultures M. J. Muhitch, L. A. Edwards and J. S. Fletcher Department of Botany and Microbiology, University of Oklahoma, Norman, OK 73019, USA Received November 1, 1982 / February 15, 1983
Abstract The diamines putrescine and cadaverine and the polyamines sDermine and spermidine inhibited the senescence of nonphotosynthetic cultures of Paul's Scarlet rose. Response was observed when the media of stationary phase cultures was adjusted to either 1 mM of cadaverine or putrescine; or 0.i ~M of either spermine or s~ermidine along with 2% sucrose in all cases. Senescence of the cultures was followed by microscopic examination of cell aliquots removed at i0 day intervals and treated with the vital stain, fluorescein diacetate. Introduction The diamines cadaverine and putrescine and the polyamines spermine and spermidine have been recognized for over 25 years as modulators of microbial and animal cell growth (Dykstra and Herbst 1965), and more recently they have been shown to influence the physiology of plant cells. During the senescence of intact or detached leaves, the endogenous levels of polyamines decreased (Altman and Bachrach 1981; KaurSawhney et al. 1982b). The addition of polyamines to isolated protoplasts (Altman et al. 1977; Galston et al. 1978), detached leaves (Altman and Bachrach 1981; Kaur-Sawhney and Galston 1979; Altman 1982a) and isolated leaf discs (Kaur-Sawhney and Galston 1979; Cohen et al. 1979; Popovic et al. 1979) dramatically decreased the rate of senescence in these tissues. Although it is clear that the addition of polyamines to isolated organs and tissues can prevent or delay senescence, the ability of these compounds to act as senescence antagonists in undifferentiated plant tissue culture cells has not been explored. We reDort here the results of studies designed to determine the effects of added polyamines on the senescence of nonphotosynthetic cultures of Paul's Scarlet rose. The effect of polyamines added during the stationary phase of growth was evaluated by comparing the cell viability of treated versus control cells. Cell viability was determined by microscopic examination of cells treated with the vital stain fluorescein diacetate. The use of a vital stain offers an alternate method to the conventional practices of following plant senescence by measuring chlorophyll or protein loss. Materials and Methods Suspension cultures of Rosa sp. cv. Paul's Scarlet
were grown in I~R media as previously described (Reider et al. 1982). Sucrose and amines were added to the cultures as filter sterilized solutions on day 14, the normal transfer age, when cell growth has stopped (Fletcher and Beevers 1969). Culture viability was measured using the vital stain fluorescein diacetate as described previously (Smith et al. 1982~ Because of the tendency for suspension cultures of Paul's Scarlet rose to grow as cell clumps, it was necessary to evaluate the staining of cell clumps rather than individual cells. The degree to which clumps of cells stained was judged and relative staining values were assigned. Values were assigned according to the percent of cells within a clump which stained. Assigned values were as follows: no stained cells - 0, less than 25% stained - i, 25 to 50% stained - 2, over 50% stained - 3. One hundred clumps ranging in size from 50 to 200 cells were removed from each flask and served as the basis for the analysis of staining. The relative values assigned to each clump were totaled and this additive value for i00 clumps taken from a sample culture was compared to additive values calculated for other cultures. This method for following senescence has been reported in an earlier study (Smith et al. 1982), and appears to be more reliable for following the senescence of cultured cells than the measurement of protein loss (Reider et al. 1982). Results and Discussion The viability of stationary phase rose cultures declined steadily between 30-60 days (Figures 1 and 2). The addition of sucrose on day 14 to give the medium a 2% content of sucrose caused senescence to proceed at a slower rate, but by the end of the observational period (60 days) the viability of the sucrose supplemented cultures did not differ from that of the control cultures. However, when either spermine or spermidine was added (final concentration 0.i to i0 DM) on day 14 with sucrose (Figure i) the life span of the cells was extended such that on day 60 the treated cultures retained 50% of the viability observed on day 14. In contrast to this almost all of the cells in the control cultures were dead on day 60. A similar result was observed when either cadaverine or putrescine was added, but the effective concentration (i mM) was 10,000-times greater than that required for spermine or spermidine. Minor effects were observed when the amine additions were not accompanied by sucrose additions (data not shown). It has also been observed that the addition of only sucrose to
83 some cultures was as effective as the sucrose plus amine treatment in extending the life-span of cultured cells (Table i). This varied response may be due to changes in the degree of cell clumping and clump size which fluctuates in successive subcultures of this cell line. Larger clumps may possess elevated levels of polyamines as has previously been shown for hormones (Szweykowska 1974; Kinnersley and Dougall 1980). If this relationship exists for polyamines, it follows that the extent to which a culture responds to polyamine addition would depend on the number of large clumps present in the culture. Additional research is necessary to resolve this issue, and thereby permit determination of the lower limit of response to polyamine additions. Microscopic examination of sucrose plus amine treated cultures revealed that the antisenescent action of these treatments was not due to a reinitiation of cell division, because cell size remained unchanged in contrast to the marked reduction in cell size which normally accompanies increased mitotic activity and cell division in rose cultures (]~hanty and Fletcher 1978). Furthermore we do not believe that the antiseneseent influence of polyamines on rose cultures is a nutritional effect, since in two previous studies with rose cells (Jordan and Fletcher 1980; Mohanty and Fletcher 1980) it has been shown that the amount of soluble nitrogen (NO 3- plus soluble amino acids) present in mature cultures (14 days old) was over 90 times greater than the nitrogen content of the polyamines provided in this study to 14 day old cells. The necessity of sucrose additions to make
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Plant Cell Reports © Springer-Verlag 1983
Influence of Diamines and Polyamines on the Senescence of Plant Suspension Cultures M. J. Muhitch, L. A. Edwards and J. S. Fletcher Department of Botany and Microbiology, University of Oklahoma, Norman, OK 73019, USA Received November 1, 1982 / February 15, 1983
Abstract The diamines putrescine and cadaverine and the polyamines sDermine and spermidine inhibited the senescence of nonphotosynthetic cultures of Paul's Scarlet rose. Response was observed when the media of stationary phase cultures was adjusted to either 1 mM of cadaverine or putrescine; or 0.i ~M of either spermine or s~ermidine along with 2% sucrose in all cases. Senescence of the cultures was followed by microscopic examination of cell aliquots removed at i0 day intervals and treated with the vital stain, fluorescein diacetate. Introduction The diamines cadaverine and putrescine and the polyamines spermine and spermidine have been recognized for over 25 years as modulators of microbial and animal cell growth (Dykstra and Herbst 1965), and more recently they have been shown to influence the physiology of plant cells. During the senescence of intact or detached leaves, the endogenous levels of polyamines decreased (Altman and Bachrach 1981; KaurSawhney et al. 1982b). The addition of polyamines to isolated protoplasts (Altman et al. 1977; Galston et al. 1978), detached leaves (Altman and Bachrach 1981; Kaur-Sawhney and Galston 1979; Altman 1982a) and isolated leaf discs (Kaur-Sawhney and Galston 1979; Cohen et al. 1979; Popovic et al. 1979) dramatically decreased the rate of senescence in these tissues. Although it is clear that the addition of polyamines to isolated organs and tissues can prevent or delay senescence, the ability of these compounds to act as senescence antagonists in undifferentiated plant tissue culture cells has not been explored. We reDort here the results of studies designed to determine the effects of added polyamines on the senescence of nonphotosynthetic cultures of Paul's Scarlet rose. The effect of polyamines added during the stationary phase of growth was evaluated by comparing the cell viability of treated versus control cells. Cell viability was determined by microscopic examination of cells treated with the vital stain fluorescein diacetate. The use of a vital stain offers an alternate method to the conventional practices of following plant senescence by measuring chlorophyll or protein loss. Materials and Methods Suspension cultures of Rosa sp. cv. Paul's Scarlet
were grown in I~R media as previously described (Reider et al. 1982). Sucrose and amines were added to the cultures as filter sterilized solutions on day 14, the normal transfer age, when cell growth has stopped (Fletcher and Beevers 1969). Culture viability was measured using the vital stain fluorescein diacetate as described previously (Smith et al. 1982~ Because of the tendency for suspension cultures of Paul's Scarlet rose to grow as cell clumps, it was necessary to evaluate the staining of cell clumps rather than individual cells. The degree to which clumps of cells stained was judged and relative staining values were assigned. Values were assigned according to the percent of cells within a clump which stained. Assigned values were as follows: no stained cells - 0, less than 25% stained - i, 25 to 50% stained - 2, over 50% stained - 3. One hundred clumps ranging in size from 50 to 200 cells were removed from each flask and served as the basis for the analysis of staining. The relative values assigned to each clump were totaled and this additive value for i00 clumps taken from a sample culture was compared to additive values calculated for other cultures. This method for following senescence has been reported in an earlier study (Smith et al. 1982), and appears to be more reliable for following the senescence of cultured cells than the measurement of protein loss (Reider et al. 1982). Results and Discussion The viability of stationary phase rose cultures declined steadily between 30-60 days (Figures 1 and 2). The addition of sucrose on day 14 to give the medium a 2% content of sucrose caused senescence to proceed at a slower rate, but by the end of the observational period (60 days) the viability of the sucrose supplemented cultures did not differ from that of the control cultures. However, when either spermine or spermidine was added (final concentration 0.i to i0 DM) on day 14 with sucrose (Figure i) the life span of the cells was extended such that on day 60 the treated cultures retained 50% of the viability observed on day 14. In contrast to this almost all of the cells in the control cultures were dead on day 60. A similar result was observed when either cadaverine or putrescine was added, but the effective concentration (i mM) was 10,000-times greater than that required for spermine or spermidine. Minor effects were observed when the amine additions were not accompanied by sucrose additions (data not shown). It has also been observed that the addition of only sucrose to
83 some cultures was as effective as the sucrose plus amine treatment in extending the life-span of cultured cells (Table i). This varied response may be due to changes in the degree of cell clumping and clump size which fluctuates in successive subcultures of this cell line. Larger clumps may possess elevated levels of polyamines as has previously been shown for hormones (Szweykowska 1974; Kinnersley and Dougall 1980). If this relationship exists for polyamines, it follows that the extent to which a culture responds to polyamine addition would depend on the number of large clumps present in the culture. Additional research is necessary to resolve this issue, and thereby permit determination of the lower limit of response to polyamine additions. Microscopic examination of sucrose plus amine treated cultures revealed that the antisenescent action of these treatments was not due to a reinitiation of cell division, because cell size remained unchanged in contrast to the marked reduction in cell size which normally accompanies increased mitotic activity and cell division in rose cultures (]~hanty and Fletcher 1978). Furthermore we do not believe that the antiseneseent influence of polyamines on rose cultures is a nutritional effect, since in two previous studies with rose cells (Jordan and Fletcher 1980; Mohanty and Fletcher 1980) it has been shown that the amount of soluble nitrogen (NO 3- plus soluble amino acids) present in mature cultures (14 days old) was over 90 times greater than the nitrogen content of the polyamines provided in this study to 14 day old cells. The necessity of sucrose additions to make
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