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Role of Abscisic Acid, Ethylene and Polyamines in Flooding-Promoted Senescence of Tobacco Leaves
J Plant Physiol. Vol. 143. pp. 102-105 (1994)
Role of Abscisic Acid, Ethylene and Polyamines in FloodingPromoted Senescence of Tobacco Leaves WEEI PIRNG HuRNG, Huu SHENG LuR, CHERNG-KANG LIAo,
and CHING HuEI KAo
Department of Agronomy, National Taiwan University, Taipei, Taiwan, Republic of China Received December 19, 1992 · Accepted August 3, 1993
Summary
The role of abscisic acid (ABA), ethylene and polyamines in leaf senescence of tobacco exposed to flooding was investigated. Senescence of tobacco leaves was followed by measuring the decrease of protein. Flooding significantly promoted senescence of tobacco leaves and resulted in a higher ABA content than the controls throughout the entire 4-day experimental duration. Ethylene production in floodingtreated leaves was much higher than that in control leaves during the first day of the experiment. Putrescine and spermidine, but not spermine, were present in tobacco leaves throughout the experimental duration. Flooded leaves had higher levels of putrescine and spermidine than untreated leaves. It is concluded that ethylene, but neither ABA nor polyamines, is possibly involved in the regulation of flooding-promoted senescence of tobacco leaves.
Key words: Abscisic acid, ethylene, lea/senescence, Nicotiana tabacum, polyamines. Abbreviations: ABA = abscisic acid; BSA = bovine serum albumin; ELISA = enzyme-linked immunosorbent assay; HPLC = high performance chromatography; PVP = polyvinylpyrrolidone. Introduction
Both ABA and ethylene are considered to be the endogenous promoters of leaf senescence (Thimann, 1980). Polyamines (putrescine, spermidine and spermine), recently recognized as a new class of plant growth substances, are present in all plant leaves examined to date. It has been postulated that polyamines are antisenescent factor of leaf senescence (Galston and Sawhney, 1990). There are many reports of leaf senescence being promoted in flooded plants (Drew and Sisworo, 1977; Jackson, 1979; Kawase, 1974; Kramer, 1951; Trought and Drew, 1980; Wenkert et al., 1981). The concentrations of ABA and the production of ehtylene increase markedly in response to flooding (Bradford and Yang, 1980; Hiron and Wright, 1975; Jackson and Campbell, 1976; Kawase, 1974; Wadman-van Schravendijk and van Andel, 1985; Zhang and Davies, 1987). Kawase (1974) reported that an increase in ethylene production was largely responsible for flooding-induced damage including leaf senescence of sunflower. However, Reid and Bradford (1984) have shown that ethylene was not the primary factor of leaf senescence © 1994 by Gustav Fischer Verlag, Stuttgart
in flooded sunflower. No direct evidence has been documented to support a role for ABA or polyamines in regulating flooding-promoted leaf senescence. In the present investigation, we examined the role of ABA, ethylene, and polyamines in flooding-promoted senescence of tobacco leaves.
Materials and Methods
Plant material and cultural conditions
Seeds of Nicotiana tabacum (cv. Speight G-70, provided by Speight Seed Farm, U.S.A.) were sown in plastic trays containing vermiculite and calcined clay (1: 1). Seedlings were grown in a greenhouse with natural light at 30 °C day/25 °C night. At 4 weeks after sowing, seedlings were transplanted to pots (0.02 m) containing sandy loam. Each pot contained a seedling and received 15 g compound fertilizer (N-K20-P20s, 7-21-21). Pots were placed in the same greenhouse as described above. Flooding treatment started at 40 d after transplanting when the plants had 7 or 8 leaves. Tobacco plants were flooded by maintaining the water level at 1 em above the soil surface. Unflooded control
Abscisic acid, ethylene, polyamines and leaf senescence plants were allowed to receive the optimum amount of water by watering twice a day. At the times indicated, the lowest leaves were collected for analysis of protein, ABA, polyamines, and ethylene. Protein determination Leaf samples were homogenized in 25 mM sodium phosphate buffer (pH 7.5) in ice bath with a mortar and pestle. The extracts were centrifuged at 17,000 x g for 20 min, and the supernatants were used for determination of protein by the method c1f Lowry et al. (1951). ABA determination For ABA extraction, leaf samples were homogenized with a mortar and pestle in extraction solution (80% methanol containing 100mg/mL butylated hydroxytoluene). Extracts were filtered through Whatman No. 1 filter paper and rinsed twice with extraction solution. Filtrates were reduced to dryness in vacuo at 30 °C. Samples were resuspended in 100% methanol. A solution of 500 mM (~)2 HP04 was then added, and the samples were allowed to stand for 10 min at 4 °C until ammonium salts formed. Pigments and phenolics in the ammonium salt solution were removed by passing through a PVP column (Mousdale and Knee, 1979). The combined PVP column-filtered solutions were adjusted to pH 3.0 with acetic acid. The acidified solution was eluted through a C 18 cartridge to remove polar compounds. ABA trapped in the C18 cartridge was then eluted with 65% ethanol (pH 3.2). The ABA solution was reduced to dryness in vacuo, resuspended in Tris-buffered saline (50 mM Tris HCl, 10 mM NaCl, 1mM MgCh, 15mM NaN3, pH 7.5), and then subjected to HPLC. Samples were injected into a Waters M-6 UK Universal Liquid Chromatograph. They were eluted through a 250 x 4-mm C 18 reverse-phase column (5 11m particle size) at a flow rate of 1 mL/ min with 40% methanol in 0.02% acetic acid. ABA was collected with a fraction collector. The ABA fractions were then dried in vacuo. Samples were resuspended in Tris-buffered saline and stored at -20 °C for ELISA analysis. ABA was quantitated by indirect ELISA according to WalkerSimmons (1987) and Norman et al. (1988). ABA-4' -BSA conjugate was prepared according to Weiler (1980) and used to determine ABA. ABA levels are expressed as J.1mol/kg fresh mass.
were obtained each time. The data reported here are from a single experiment. Results Senescence of tobacco leaves was followed by measuring the decrease of protein. Fig. 1 shows the time courses of protein content in leaves of tobacco plants treated with or without flooding. The decrease of protein in control leaves was evident at 2 d after the start of experiment. Flooding significantly promoted the decrease in protein content. Fig. 2 shows the time courses of changes of ABA in flooding-treated and control leaves. ABA content in control leaves increased at 2 d after the start of experiment, reached a maximum at d 3, and subsequently declined. Flooding treatment resulted in a higher ABA content than the controls
0-0control
9
•-•flooded
.
3
0
DAYS
2
FLOODED
4
Fig. 1: Effects of flooding on protein content of tobacco leaves. Vertical bars represent standard errors (n = 4).
0-0control
Polyamine determination Leaf tissues were homogenized in 5% perchloric acid. Polyamine levels were determined using HPLC after benzoylation as described previously (Chen and K.ao, 1991). Levels of polyamines are expressed as Jlmollkg fresh mass.
103
•-•flooded 4
1
.s:. 3
Ethylene determination Leaf discs were floated abaxially down under cool-white fluorescent light for about 3 h in open Petri dishes containing water. This preincubation allowed wound ethylene to subside. Leaf discs were then transfered to flasks sealed with serum caps. After 3 h of incubation in the dark at 27 °C, a 1-mL gas sample was withdrawn from the head space of the flasks. Ethylene was then assayed as described previously (K.ao and Yang, 1983). Experimental design For protein, ABA, and ethylene determinations, each treatment contained four replicates, whereas for polyamine determination, each treatment was repeated three times. All experiments described were repeated at least twice. Similar results and identical trends
I
C( - .
lllj
c:c......_
~ 2
3
0 0
DAYS
2
FLOODED
4
Fig. 2: Effects of flooding on ABA content of tobacco leaves. Vertical bars represent standard errors (n = 4).
104
WEEI PIRNG HUBNG, Huu SHENG Lm, CHERNG-lUNG LIAo, and CHING Hum KAo 0-0control
16
• - • flooded
----0
4
0 0
-
····-·------..--.....4
2
DAYS FLOODED
Fig. 3: Effects of flooding on ethylene production of tobacco leaves. Vertical bars represent standard errors (n • 4). throughhout the entire 4-d experimental duration. ABA content in flooded leaves was 20% and 4-fold higher at d 1 and d3, respectively, than the control leaves. Effects of flooding on ethylene production of tobacco leaves are presented in Fig. 3. Ethylene production in leaves of the controls increased significantly during the first day of the experiment and decreased subsequently. Flooding treatment resulted in a higher ethylene production than the controls during the first 2 d of the experiment. Putrescine and spermidine were present in tobacco leaves throughout the experimental duration (Fig. 4). However, spermine was not detectable in tobacco leaves. Putrescine content in control leaves decreased at 1 d after the start of the experiment, reached a minimum at d3, and subsequently increased (Fig. 4). Flooding treatment resulted in a higher putrescine content than the controls during the first 3 d of the experiment. Spermidine content in control leaves remained unchanged during the first 2 d of the experiment, decreased at d3 and subsequently increased (Fig. 4). Flooding treatment resulted in a higher spermidine content than the control at d2 and 3.
is to correlated changes in endogenous hormone content with senescence. Our results show that ethylene production in flooding-treated leaves is much higher than that in control leaves during the first day of the experiment, whereas ABA content in flooding-treated leaves is slightly (about 20%) higher than that in control leaves during the first day of the experiment. A significant rise of ABA in flooded-treated leaves appears to be associated with the rapid phase of protein breakdown. We conclude that flooding-promoted leaf senescence is triggered by an early increase in ethylene. The increase of ABA in flooded-treated leaves is probably caused by earlier events taking place in the course of leaf senescence. It has long been recognized that cytokinins play an important role in regulating leaf senescence (Thimann, 1980). Thus, the possibility that cytokinins may be involved in regulating flooding-promoted senescence of tobacco leaves can not be excluded. It has been shown that exogenous application of polymines retard senescence of detached leaves (Altman, 1982; Kaur-Sawhney and Galston, 1979; Shih et al., 1982). If endogenous polyamines play a role in regulating flooding-promoted senescence of tobacco leaves, lower content of polyamines in flooding-treated leaves would be expected. However, this was not observed. It is concluded that endogenous polyamines are unlikely to be the primary factor responsible for flooding-promoted senescence of tobacco leaves. The present investigation provides support to the suggestion of Birecka et al. (1984) that endogenous polyamines may have
90
70
I'E
-' .c
.... ~ 0
E
:a.
Discussion
In the present work, protein content of the leaves of control (unflooded) plants decreased substantially throughout the experiment period. The effects of flooding on the reported changes of the leaves therefore may only apply to leaves that have already started the senescence process. The present investigation shows that flooding promotes leaf senescence of tobacco leaves. This result is in agreement with earlier reports of other investigators (Drew and Sisworo, 1977; Jackson, 1979; Kawase, 1974; Kramer, 1951; Trought and Drew, 1980; Wenkert et al., 1981). In the study of hormonal control of leaf senescence, one approach
0-0control •-•flooded
50
30
Putrescine
60
40
20
I ...... _
0 0
2
_.___. . . .__,
DAYS FLOODED
4
Fig. 4: Effects of flooding on the contents of putrescine and spermidine of tobacco leaves. Vertical bars represent standard errors (n- 3).
Abscisic acid, ethylene, polyamines and leaf senescence
effects different from the possibly nonspecific effects frequently reported for exogenous polyamines. Exposure of plants to a variety of stresses often results in a higher polyamine content (Flores, 1990). The higher polyamines induced by flooding has not previously been demonstrated. In the present investigation, we provide the evidence that flooding results in a higher polyamine content. There is considerable evidence in the literature to support a role for putrescine in the tolerance of plants to stresses, but there is also accumulating evidence associating higher putrescine level with the development of the symptoms of stress (Flores, 1990). The physiological role of putrescine in tobacco leaves under flooding stress remains to be investigated. It has been suggested that ethylene and polyamine formation impose competitive demand on S-adenosylmethionine and that allocation of it to either pathway may constitute a point of regulation in ethylene and polyamine biosynthesis (Miyazaki and Yang, 1987). The present investigation shows that the pattern of ethylene production in either control or flooding-treated leaves was not inversely correlated with that of spermidine, suggesting that the two pathways (i.e. spermidine and ethylene biosynthesis in tobacco leaves) do not actively compete for the substrate, S-adenosylmethionine. Similar conclusion has been reached in studies of rice leaves and avocado fruits (Chen and Kao, 1992; Kushad et al., 1988). Acknowledgement
This work was supported by the National Science Council of the Republic of China (NSC 282410).
References ALTMAN, A.: Retardation of radish leaf senescence by polyamines. Physiol. Plant. 54, 189-193 (1982). BIRECXA, H., T. S. DINOLFO, W. B. MARTIN, and M. W. F&oucH: Polyamines and leaf senescence in pyrrolizidine alkaloid-bearing Heliotropium plants. Phytochemistry 23,991-994 (1984). BRADFORD, K. J. and S. F. YANG: Xylem transport of 1-aminocyclopropane-1-carboxylic acid, an ethylene precursor, in waterlogging tomato plants. Plant Physiol. 65, 322-326 (1980). CHEN, C. T. and C. H. KAo: Senescence of rice leaves XXX. Levels of endogenous polyamines and dark-induced senescence of rice leaves. Plant Cell Physiol. 32, 935-941 (1991). DREw, M. C. and E. J. SISWORO: Early effects of flooding on nitrogen deficiency and leaf chlorosis in barley. New Phytol. 79, 567-571 (1977). FLORES, H. E.: Polyamines and plant stress. In: Ar.scHER, R. G. and J. R. CUMMING (eds.): Stress Responses in Plants: Adaptation and Acclimation Mechanisms, pp. 217-239. Willey-Liss Inc., New York. GALSTON, A. W. and R. K. SAWHNEY: Polyamines in plant physiology. Plant Physiol. 94, 406-410 (1990).
105
HmoN, R. W. P. and S. T. C. WRIGHT: The role of endogenous abscisic acid in the response of plants to stress. J. Exp. Bot. 24, 769-781 (1973). jACKSON, M. B.: Rapid injury to peas by soil waterlogging. J. Sci. Food Agric. 30, 143 -152 (1979). jACKSON, M. B. and D. J. CAMPBELL: Waterlogging and petiole epinasty in tomato :the role of ethylene and low oxygen. New Phytol. 76, 21-29 (1976). KAo, C. H. and S. F. YANG: Role of ethylene in the senescence of detached rice leaves. Plant Physiol. 73, 881-885 (1983). KAUll-SAWHNEY, R. and A. W. GALSTON: Interaction of polyamines and light on biochemical process involved in senescence. Plant Cell Environ. 2, 189-196 (1979). KAwASE, M.: Role of ethylene in induction of flooding damage in sunflower. Physiol. Plant. 31, 29-38 (1974). KllAME:&, P. J.: Causes of injury of plants resulting from flooding of the soil. Plant Physiol. 26, 722-736 (1951). KusHAD, M. M., G. YELENOSKY, and R. KNIGHT: Interrelationship of polyamine and ethylene biosynthesis during avocado fruit development and ripening. Plant Physiol. 87, 463-467 (1988). LoWRY, 0. H., N.J. RossENBROUGH, A. L. FAllll, and R. J. RANDALL: Protein measurement with the Folin phenol reagent. J. Bioi. Chern. 193, 265-275 (1951). MIYAZAKI,}. H. and S. F. YANG: The methionine salvage pathway in relation to polyamine biosynthesis. Physiol. Plant. 69, 366-370 (1987). MousoALE, D. M. A. and M. KNEE: Poly-N-vinylpyrrolidone column chromatography of plant hormones with methanol as eluent. J. Chromatogr. 177, 398-400 (1979). REID, D. M. and K. J. BRADFORD: Effects of flooding on hormone relations. In: KozLOwsKI, T. T. (ed.): Flooding and Plant Growth, pp. 195-219. Academic Press Inc., New York (1984). SHIH, L. N., R. KAUll-SAWHNEY, J. FUHllEll, S. SAMANT, and A. W. GALSTON: Effects of exogenous 1,3-diaminopropane and spermidine on senescence of oat leaves I. Inhibition of protease activity, ethylene production, and chlorophyll loss as related to polyamine content. Plant Physiol. 70, 1592-1596 (1982). THIMANN, K. V.: The senescence of leaves. In: THIMANN, K. V. (ed.): Senescence in Plants, pp. 85-115. CRC Press Inc., Boca Raton, Florida (1980). TROUGHT, M. C. T. and M. C. DREW: The development of waterlogging damage in wheat seedlings (Triticum aestivum L.). I. shoot and root growth in relation to changes in the concentration of dissolved gases and solutes in the soil solution. Plant Soil 54, 77-94 (1980). WADMAN-VAN ScHRAVENDIJK, W. and 0. M. VAN ANDm.: Interdependence of growth, water relations and abscisic acid level in Phaseolus vulgaris during waterlogging. Physiol. Plant. 63, 215220. WALKER-SIMMONS, M.: ABA levels and sensitivity in developing embryos of sprouting and susceptible cultivars. Plant Physiol. 84, 61-66 (1987). WENKERT, W., N. R. FAUSEY, and H. D. WATTERS: Flooding responses in Zea mays L. Plant Soil62, 351-366 (1981). WEILER, E. W.: Radioimmunoassays for the differential and direct analysis of free and conjugated abscisic acid in plant extracts. Planta 148, 262-272. ZHANG, J. and W. J. DAVIES: ABA in roots and leaves of flooded pea plants. J. Exp. Bot. 38, 649-659 (1987).
Role of Abscisic Acid, Ethylene and Polyamines in FloodingPromoted Senescence of Tobacco Leaves WEEI PIRNG HuRNG, Huu SHENG LuR, CHERNG-KANG LIAo,
and CHING HuEI KAo
Department of Agronomy, National Taiwan University, Taipei, Taiwan, Republic of China Received December 19, 1992 · Accepted August 3, 1993
Summary
The role of abscisic acid (ABA), ethylene and polyamines in leaf senescence of tobacco exposed to flooding was investigated. Senescence of tobacco leaves was followed by measuring the decrease of protein. Flooding significantly promoted senescence of tobacco leaves and resulted in a higher ABA content than the controls throughout the entire 4-day experimental duration. Ethylene production in floodingtreated leaves was much higher than that in control leaves during the first day of the experiment. Putrescine and spermidine, but not spermine, were present in tobacco leaves throughout the experimental duration. Flooded leaves had higher levels of putrescine and spermidine than untreated leaves. It is concluded that ethylene, but neither ABA nor polyamines, is possibly involved in the regulation of flooding-promoted senescence of tobacco leaves.
Key words: Abscisic acid, ethylene, lea/senescence, Nicotiana tabacum, polyamines. Abbreviations: ABA = abscisic acid; BSA = bovine serum albumin; ELISA = enzyme-linked immunosorbent assay; HPLC = high performance chromatography; PVP = polyvinylpyrrolidone. Introduction
Both ABA and ethylene are considered to be the endogenous promoters of leaf senescence (Thimann, 1980). Polyamines (putrescine, spermidine and spermine), recently recognized as a new class of plant growth substances, are present in all plant leaves examined to date. It has been postulated that polyamines are antisenescent factor of leaf senescence (Galston and Sawhney, 1990). There are many reports of leaf senescence being promoted in flooded plants (Drew and Sisworo, 1977; Jackson, 1979; Kawase, 1974; Kramer, 1951; Trought and Drew, 1980; Wenkert et al., 1981). The concentrations of ABA and the production of ehtylene increase markedly in response to flooding (Bradford and Yang, 1980; Hiron and Wright, 1975; Jackson and Campbell, 1976; Kawase, 1974; Wadman-van Schravendijk and van Andel, 1985; Zhang and Davies, 1987). Kawase (1974) reported that an increase in ethylene production was largely responsible for flooding-induced damage including leaf senescence of sunflower. However, Reid and Bradford (1984) have shown that ethylene was not the primary factor of leaf senescence © 1994 by Gustav Fischer Verlag, Stuttgart
in flooded sunflower. No direct evidence has been documented to support a role for ABA or polyamines in regulating flooding-promoted leaf senescence. In the present investigation, we examined the role of ABA, ethylene, and polyamines in flooding-promoted senescence of tobacco leaves.
Materials and Methods
Plant material and cultural conditions
Seeds of Nicotiana tabacum (cv. Speight G-70, provided by Speight Seed Farm, U.S.A.) were sown in plastic trays containing vermiculite and calcined clay (1: 1). Seedlings were grown in a greenhouse with natural light at 30 °C day/25 °C night. At 4 weeks after sowing, seedlings were transplanted to pots (0.02 m) containing sandy loam. Each pot contained a seedling and received 15 g compound fertilizer (N-K20-P20s, 7-21-21). Pots were placed in the same greenhouse as described above. Flooding treatment started at 40 d after transplanting when the plants had 7 or 8 leaves. Tobacco plants were flooded by maintaining the water level at 1 em above the soil surface. Unflooded control
Abscisic acid, ethylene, polyamines and leaf senescence plants were allowed to receive the optimum amount of water by watering twice a day. At the times indicated, the lowest leaves were collected for analysis of protein, ABA, polyamines, and ethylene. Protein determination Leaf samples were homogenized in 25 mM sodium phosphate buffer (pH 7.5) in ice bath with a mortar and pestle. The extracts were centrifuged at 17,000 x g for 20 min, and the supernatants were used for determination of protein by the method c1f Lowry et al. (1951). ABA determination For ABA extraction, leaf samples were homogenized with a mortar and pestle in extraction solution (80% methanol containing 100mg/mL butylated hydroxytoluene). Extracts were filtered through Whatman No. 1 filter paper and rinsed twice with extraction solution. Filtrates were reduced to dryness in vacuo at 30 °C. Samples were resuspended in 100% methanol. A solution of 500 mM (~)2 HP04 was then added, and the samples were allowed to stand for 10 min at 4 °C until ammonium salts formed. Pigments and phenolics in the ammonium salt solution were removed by passing through a PVP column (Mousdale and Knee, 1979). The combined PVP column-filtered solutions were adjusted to pH 3.0 with acetic acid. The acidified solution was eluted through a C 18 cartridge to remove polar compounds. ABA trapped in the C18 cartridge was then eluted with 65% ethanol (pH 3.2). The ABA solution was reduced to dryness in vacuo, resuspended in Tris-buffered saline (50 mM Tris HCl, 10 mM NaCl, 1mM MgCh, 15mM NaN3, pH 7.5), and then subjected to HPLC. Samples were injected into a Waters M-6 UK Universal Liquid Chromatograph. They were eluted through a 250 x 4-mm C 18 reverse-phase column (5 11m particle size) at a flow rate of 1 mL/ min with 40% methanol in 0.02% acetic acid. ABA was collected with a fraction collector. The ABA fractions were then dried in vacuo. Samples were resuspended in Tris-buffered saline and stored at -20 °C for ELISA analysis. ABA was quantitated by indirect ELISA according to WalkerSimmons (1987) and Norman et al. (1988). ABA-4' -BSA conjugate was prepared according to Weiler (1980) and used to determine ABA. ABA levels are expressed as J.1mol/kg fresh mass.
were obtained each time. The data reported here are from a single experiment. Results Senescence of tobacco leaves was followed by measuring the decrease of protein. Fig. 1 shows the time courses of protein content in leaves of tobacco plants treated with or without flooding. The decrease of protein in control leaves was evident at 2 d after the start of experiment. Flooding significantly promoted the decrease in protein content. Fig. 2 shows the time courses of changes of ABA in flooding-treated and control leaves. ABA content in control leaves increased at 2 d after the start of experiment, reached a maximum at d 3, and subsequently declined. Flooding treatment resulted in a higher ABA content than the controls
0-0control
9
•-•flooded
.
3
0
DAYS
2
FLOODED
4
Fig. 1: Effects of flooding on protein content of tobacco leaves. Vertical bars represent standard errors (n = 4).
0-0control
Polyamine determination Leaf tissues were homogenized in 5% perchloric acid. Polyamine levels were determined using HPLC after benzoylation as described previously (Chen and K.ao, 1991). Levels of polyamines are expressed as Jlmollkg fresh mass.
103
•-•flooded 4
1
.s:. 3
Ethylene determination Leaf discs were floated abaxially down under cool-white fluorescent light for about 3 h in open Petri dishes containing water. This preincubation allowed wound ethylene to subside. Leaf discs were then transfered to flasks sealed with serum caps. After 3 h of incubation in the dark at 27 °C, a 1-mL gas sample was withdrawn from the head space of the flasks. Ethylene was then assayed as described previously (K.ao and Yang, 1983). Experimental design For protein, ABA, and ethylene determinations, each treatment contained four replicates, whereas for polyamine determination, each treatment was repeated three times. All experiments described were repeated at least twice. Similar results and identical trends
I
C( - .
lllj
c:c......_
~ 2
3
0 0
DAYS
2
FLOODED
4
Fig. 2: Effects of flooding on ABA content of tobacco leaves. Vertical bars represent standard errors (n = 4).
104
WEEI PIRNG HUBNG, Huu SHENG Lm, CHERNG-lUNG LIAo, and CHING Hum KAo 0-0control
16
• - • flooded
----0
4
0 0
-
····-·------..--.....4
2
DAYS FLOODED
Fig. 3: Effects of flooding on ethylene production of tobacco leaves. Vertical bars represent standard errors (n • 4). throughhout the entire 4-d experimental duration. ABA content in flooded leaves was 20% and 4-fold higher at d 1 and d3, respectively, than the control leaves. Effects of flooding on ethylene production of tobacco leaves are presented in Fig. 3. Ethylene production in leaves of the controls increased significantly during the first day of the experiment and decreased subsequently. Flooding treatment resulted in a higher ethylene production than the controls during the first 2 d of the experiment. Putrescine and spermidine were present in tobacco leaves throughout the experimental duration (Fig. 4). However, spermine was not detectable in tobacco leaves. Putrescine content in control leaves decreased at 1 d after the start of the experiment, reached a minimum at d3, and subsequently increased (Fig. 4). Flooding treatment resulted in a higher putrescine content than the controls during the first 3 d of the experiment. Spermidine content in control leaves remained unchanged during the first 2 d of the experiment, decreased at d3 and subsequently increased (Fig. 4). Flooding treatment resulted in a higher spermidine content than the control at d2 and 3.
is to correlated changes in endogenous hormone content with senescence. Our results show that ethylene production in flooding-treated leaves is much higher than that in control leaves during the first day of the experiment, whereas ABA content in flooding-treated leaves is slightly (about 20%) higher than that in control leaves during the first day of the experiment. A significant rise of ABA in flooded-treated leaves appears to be associated with the rapid phase of protein breakdown. We conclude that flooding-promoted leaf senescence is triggered by an early increase in ethylene. The increase of ABA in flooded-treated leaves is probably caused by earlier events taking place in the course of leaf senescence. It has long been recognized that cytokinins play an important role in regulating leaf senescence (Thimann, 1980). Thus, the possibility that cytokinins may be involved in regulating flooding-promoted senescence of tobacco leaves can not be excluded. It has been shown that exogenous application of polymines retard senescence of detached leaves (Altman, 1982; Kaur-Sawhney and Galston, 1979; Shih et al., 1982). If endogenous polyamines play a role in regulating flooding-promoted senescence of tobacco leaves, lower content of polyamines in flooding-treated leaves would be expected. However, this was not observed. It is concluded that endogenous polyamines are unlikely to be the primary factor responsible for flooding-promoted senescence of tobacco leaves. The present investigation provides support to the suggestion of Birecka et al. (1984) that endogenous polyamines may have
90
70
I'E
-' .c
.... ~ 0
E
:a.
Discussion
In the present work, protein content of the leaves of control (unflooded) plants decreased substantially throughout the experiment period. The effects of flooding on the reported changes of the leaves therefore may only apply to leaves that have already started the senescence process. The present investigation shows that flooding promotes leaf senescence of tobacco leaves. This result is in agreement with earlier reports of other investigators (Drew and Sisworo, 1977; Jackson, 1979; Kawase, 1974; Kramer, 1951; Trought and Drew, 1980; Wenkert et al., 1981). In the study of hormonal control of leaf senescence, one approach
0-0control •-•flooded
50
30
Putrescine
60
40
20
I ...... _
0 0
2
_.___. . . .__,
DAYS FLOODED
4
Fig. 4: Effects of flooding on the contents of putrescine and spermidine of tobacco leaves. Vertical bars represent standard errors (n- 3).
Abscisic acid, ethylene, polyamines and leaf senescence
effects different from the possibly nonspecific effects frequently reported for exogenous polyamines. Exposure of plants to a variety of stresses often results in a higher polyamine content (Flores, 1990). The higher polyamines induced by flooding has not previously been demonstrated. In the present investigation, we provide the evidence that flooding results in a higher polyamine content. There is considerable evidence in the literature to support a role for putrescine in the tolerance of plants to stresses, but there is also accumulating evidence associating higher putrescine level with the development of the symptoms of stress (Flores, 1990). The physiological role of putrescine in tobacco leaves under flooding stress remains to be investigated. It has been suggested that ethylene and polyamine formation impose competitive demand on S-adenosylmethionine and that allocation of it to either pathway may constitute a point of regulation in ethylene and polyamine biosynthesis (Miyazaki and Yang, 1987). The present investigation shows that the pattern of ethylene production in either control or flooding-treated leaves was not inversely correlated with that of spermidine, suggesting that the two pathways (i.e. spermidine and ethylene biosynthesis in tobacco leaves) do not actively compete for the substrate, S-adenosylmethionine. Similar conclusion has been reached in studies of rice leaves and avocado fruits (Chen and Kao, 1992; Kushad et al., 1988). Acknowledgement
This work was supported by the National Science Council of the Republic of China (NSC 282410).
References ALTMAN, A.: Retardation of radish leaf senescence by polyamines. Physiol. Plant. 54, 189-193 (1982). BIRECXA, H., T. S. DINOLFO, W. B. MARTIN, and M. W. F&oucH: Polyamines and leaf senescence in pyrrolizidine alkaloid-bearing Heliotropium plants. Phytochemistry 23,991-994 (1984). BRADFORD, K. J. and S. F. YANG: Xylem transport of 1-aminocyclopropane-1-carboxylic acid, an ethylene precursor, in waterlogging tomato plants. Plant Physiol. 65, 322-326 (1980). CHEN, C. T. and C. H. KAo: Senescence of rice leaves XXX. Levels of endogenous polyamines and dark-induced senescence of rice leaves. Plant Cell Physiol. 32, 935-941 (1991). DREw, M. C. and E. J. SISWORO: Early effects of flooding on nitrogen deficiency and leaf chlorosis in barley. New Phytol. 79, 567-571 (1977). FLORES, H. E.: Polyamines and plant stress. In: Ar.scHER, R. G. and J. R. CUMMING (eds.): Stress Responses in Plants: Adaptation and Acclimation Mechanisms, pp. 217-239. Willey-Liss Inc., New York. GALSTON, A. W. and R. K. SAWHNEY: Polyamines in plant physiology. Plant Physiol. 94, 406-410 (1990).
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