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Effects of Chilling and Temperature Preconditioning on the Activity of Polyamine Biosynthetic Enzymes in Zucchini Squash
J. Plant Physiol. Vol. 136. pp. 115 -119 (1990)
Effects of Chilling and Temperature Preconditioning on the Activity of Polyamine Biosynthetic Enzymes in Zucchini Squash G. F.
KRAMER
and C. Y.
WANG'~
Horticultural Crops Quality Lab., Agricultural Research Service, U.S. Dept. of Agriculture, Beltsville, MD 20705, USA Received August 14, 1989 . Accepted December 1, 1989
Summary Polyamine biosynthesis during chilling stress in zucchini squash (Cucurbita pepo L., cv. «Ambassador») was measured by determining the activities of SAMDC, ADC, and ODe. Stress induced increases in Put were correlated with increased ODC activity. Decreases in the levels of Spd and Spn during chilling may be related to decreased SAMDC activity. Temperature preconditioning adapts squash to subsequent chilling stress and results in increased Spn and Spd levels in response to chilling. The concomitant increase in these two polyamines is correlated with elevated SAMDC activity. These data support the view that the mechanism by which temperature preconditioning inhibits chilling injury involves the induction of polyamine biosynthesis.
Key words: Cucurbita pepo, ADC, chilling injury, ODC, polyamines, preconditioning, putrescine, SAMDC, spermidine, spermine, zucchini squash. Abbreviations: ADC = arginine decarboxylase (EC 4.1.1.49); AP = aminopropyl; DAP = diaminopropane; DAO = diamino oxidase; FW = fresh weight; HPLC = high pressure liquid chromatography; ODC = ornithine decarboxylase (EC 4.1.1.17); PAO = polyamine oxidase; Put = putrescine; SAM = Sadenosylmethionine; SAMDC = SAM decarboxylase (EC 4.1.1.50); Spd = spermidine; Spn = spermine. Introduction Polyamines can act to inhibit a variety of senescence-related processes in plant tissues (GaIston and Kaur-Sawhney, 1987). These antisenescent properties include the inhibition of the activity of degradative enzymes such as RNase, protease (Galston and Kaur-Sawhney, 1987), and polygalacturonase (Kramer et al., 1989); the stabilization of membrane structure (Roberts et al., 1986); antioxidant activity (Drolet et al., 1986); and the inhibition of lipid peroxidation (Kitada et al., 1979). Put levels increase in plant tissues in response to a number of stresses, including acid treatment (Young and Galston, 1983), osmotic shock (Flores and Galston, 1982), and water stress (Wang and Steffens, 1985). Chilling injury in a variety of fruit also results in significant increases in Put
*
Corresponding author.
© 1990 by Gustav Fischer Verlag, Stuttgart
levels (McDonald and Kushad, 1986; Wang and Ji, 1989). Our results indicate that polyamines may be involved in protecting plant tissues from chilling injury. Treatment of squash with Spn after harvest inhibited injury during subsequent chilling stress (Kramer and Wang, 1989). Also, the reduction of chilling injury in squash and senescence in Chinese cabbage and apples by controlled atmosphere storage is coupled with increased polyamine levels (Kramer et al., 1989; Wang, 1988; Wang and Ji, 1989). These results are consistent with the suggestion that polyamines increase cell viability during chilling by retarding membrane senescence (Guye et aI., 1986). Put, a precursor to the synthesis of both Spd and Spn (Fig. 1), can be synthesized via two separate pathways in plant tissues: from arginine via ADC and from ornithine via ODe (Smith, 1985). The relative activity of these pathways can vary depending on species, tissue, and growth and devel-
116
G. F.
KRAMER
and C. Y. WANG
=1-:
l
ARmIlNE-
l!QII\TI1IE:---- ror - - - - -
SAMIlC
~lsFO
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PM
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N113
H:02 Pyrrol~
+
: AP-pyrrol~
PM
+
SIt{-1- - - - - Il1\P
+
H202
Fig. 1: Polyamine metabolic pathways in plant tissues. opmental stage (Smith, 1985). Stress-induced Put accumulation is a result of increased ADC activity (Flores and Galston, 1982; Smith, 1985; Young and Galston, 1983; Wang and Steffens, 1985). Chilling injury symptoms develop rapidly in zucchini squash during storage at 2.5 °C (Hardenburg et aI., 1986; Kramer and Wang, 1989). This chilling stress is correlated with a > 5-fold increase in Put and a 2- to 4-fold decrease in Spd and Spn (Kramer and Wang, 1989). Preconditioning squash at 10°C for 2 days prior to storage at 2.5 °C significantly delays the development of chilling injury. This adaptive response to chilling is correlated with increased levels of Spd and Spn (Kramer and Wang, 1989). The present study was undertaken to determine the mechanism by which the levels of polyamines are regulated during chilling stress and preconditioning treatment.
Materials and Methods Zucchini squash (Cucurbita pepo L., cv. «Ambassadon» used for this study were freshly harvested from a local farm near Beltsville, MD. Samples were selected for their uniformity of size (16-22cm in length) and randomly divided into two lots. The first group (control) was placed in storage at 2.5 DC. The second lot was preconditioned at 10°C for the first 2 days of storage and then moved to 2.5 °C for the remainder of the study. Samples were taken on various days throughout the storage period. Three squash were chosen at random from each group being sampled. Two 2.0-g samples of skin were removed from each fruit and immediately frozen. Samples were stored at - 80°C prior to analysis.
Enzyme assays ADC, ODC, and SAMDC were determined by the method of Kaur-Sawhney et al. (1982). Skin samples (2.0 g) were homogenized in 15mL of chilled 100mM phosphate pH7.6 (for ADC and SAMDC) or 100 mM Tris-CI pH 8.0 (for ODC). Crude extracts were obtained by centrifugation of the homogenates at 47,000 xg for 20 min. The assays were carried out in 12 x 75-mm polystyrene tubes (InterLab) sealed with snap caps. A 6-mm filter paper (Whatman GF/B) disc, saturated with 50 JLL 2 N KOH, was transfixed onto a 22-gauge needle that was inserted through the tube cap. Reaction mixtures contained 100 JLL crude extract plus 10 JLL 5 mM pyridoxal phosphate (ICN) and 10 JLL of 20 JLCi/mL L-[U- 14C]arginine (270 mCil mmol, ICN) diluted with unlabelled L-arginine (Sigma) to give a final concentration of 9 mM for ADC, 10 JLL 5 mM pyridoxal
phosphate plus 10JLL of 20JLCi/mL DL-e 4C]ornithine (50mCil mmol, ICN) diluted with cold L-ornithine (Sigma) to give a final concentration of 66mM for ODC, and 10JLL O.lmM pyridoxal phosphate plus 10 JLL of 20 JLCi/mL SAM-[14C]carboxyl (59 mCil mmol, Amersham) diluted with cold SAM (Sigma) to give a final concentration of 2.7 mM for SAMDC. These mixtures were incubated 30 min (SAMDC) or 60 min (ODC, ADC) at 37°C. The reactions were stopped by the addition of 0.2 mL 10 % trichloroacetic acid (Sigma) through the needle. The incubation was continued for an additional 45 min. The filter paper discs were removed and dried at 70°C for 30 min. The liberated CO 2 was determined by placing the discs in 3 mL ScintiLene (Fisher) and counting with a Beckman LS 6800 scintillation counter. Each time point was the average of the three individual fruits sampled. Data presented are the average of 2 to 4 separate experiments.
Polyamine oxidase and dzamzne oxzdase assays Polyamine-dependent H 20 Z formation was determined by measuring guaiacol (Sigma) oxidation with the method of Kaur-Sawhney et al. (1981). More sensitive assays (Zaitsu and Ohkura, 1980) involved substitution of guaiacol with 3-(p-hydroxyphenyl)propionic acid (Sigma) and measuring fluorescence (excitation wavelength = 320 nm, emission wavelength = 404 nm). Activity was determined in crude extracts prepared by homogenization of skin tissue in 100 mM phosphate pH 6.5 followed by centrifugation at 17,000 g for 10 min and in cell wall fractions prepared by the method of Torrigiani et al. (1988).
Protein determination Protein concentration in the crude extract was determined with the BCA Protein Assay Reagent (Pierce) using the standard room temperature protocol.
Polyamine analysis Free polyamines were analyzed as dansylated derivatives vIa HPLC as described previously (Kramer and Wang, 1989).
Figure preparation The figures were prepared using SigmaPlot software Oandel Scientific). The traces presented are computer generated regression lines.
Results
Put biosynthesis The activities of both ADC and ODC in squash skin extracts were determined as the skin is the tissue most sensitive to chilling and has been monitored in earlier studies. Chilling stress induced a 2- to 3-fold increase by day 5 and a 4- to 6-fold increase in ODC activity by day 12 (Fig. 2), which correlates well with the 2- to 3-fold increase by day 5 (Table 1) and the 5- to 6-fold increase in Put by day 12 observed under these conditions (Kramer and Wang, 1989). The ODC activity in the preconditioned fruit also increased during chilling (Fig. 2). This activity may initially (days 4 - 9) increase more rapidly than the control activity but appears to be somewhat lower than the control at later times (after day 12). The ADC activity remained unchanged in both groups during chilling (data not shown).
Effects of Chilling on Polyamine Biosynthetic Enzymes Table 1: Induction of polyamines and polyamine biosynthetic enzymes by chilling stress in preconditioned squash. Day of Maximal Induction SAMDC (nmollmg· hr) Spd + Spn (nmollg) ODC (nmollmg· hr) Protein (mg/mL) Put (nmoll g)*
6 7 5 6 6
Treatment
no PC
+PC
significance, P
0.664 398 1.85 1.61 1135
2.06 940 3.16 1.87 1138
<0.005 <0.005 <0.005 0.0125 n.s.
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* Initial Put levels were 333 (nmollg).
117
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T
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Fig.3: Effect of chilling on SAMDC activity. Open circles (0) control group (r = 0.873). Closed triangles (....) - preconditioned group (r = 0.866). Error bars represent ±SE.
-+--'
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2.0
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Duration of Chilling (Days) Fig.2: Effect of chilling on the activity of ODC in squash skin. Open circles (0) - control fruit that had been held at 2.5°C throughout the experiment (r = 0.949). Closed triangles (....) - preconditioned fruit, held at 10 °C for days 0 - 2 prior to transfer to 2.5 °C (r = 0.869). Error bars represent ±SE.
SAMDC activity The product of SAMDC, decarboxylated SAM, serves as an aminopropyl donor in the synthesis of both Spd and Spn (Fig. 1). The activity of this enzyme decreased during chilling stress in zucchini squash (Fig. 3). In the preconditioned fruit, SAMDC activity began increasing on day 2 after transfer to the chilling temperature (Fig. 3). After day 6, the enzyme activity decreased but remained elevated relative to the nontreated fruit. These patterns of activity are closely correlated with the observed levels of Spd and Spn, the ultimate products of SAMDC action. Spd + Spn decreased in the nontreated fruit but increased in the preconditioned fruit after transfer to chilling stress (Fig. 4). After day 7, the levels decreased but remained elevated relative to the control. The elevation of SAMDC could thus account for the observed increases in Spd and Spn in the preconditioned fruit.
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Fig. 4: Effect of chilling on the levels of Spd + Spn. Open circles (0) - control group (r = 0.911). Closed triangles (....) - preconditioned group (r = 0.895). Error bars represent ±SE.
Polyamine degradative pathways Put can be degraded via DAO and Spd and Spn via PAO (Smith, 1985; Fig. 1), both of which are assayed by detection
118
G. F. KRAMER and C. Y. WANG
of polyamine-dependent H 20 2 formation. Using guaiacol oxidation to detect H 20 2 (Kaur-Sawhney et al., 1981), there was no detectable activity of DAO or PAO in the cytoplasmic or cell wall fractions of squash skin homogenates (data not shown). Using a more sensitive fluorogenic substrate, hydroxyphenyl propionic acid (Zaitsu and Ohkura, 1980), the activities of both enzymes remained below the limit of detection (data not shown). These results may indicate that degradative enzymes do not playa major role in the regulation of polyamine levels in squash. Discussion
Plants respond to a variety of stresses by inducing the synthesis of Put (Smith, 1985). In all cases reported, this increase in Put results from induction of ADC activity (Smith, 1985). Chilling stress in zucchini squash results in 5- to 6-fold increase in Put concomitant with a 4- to 7-fold increase in ODC activity. The stress-induced Put thus appears to originate from the ODC pathway. The temperature preconditioning of squash prior to chilling induces an adaptive response to this stress (Kramer and Wang, 1989). Fruit that are preconditioned accumulate Spn and Spd after transfer to chilling temperatures; whereas in nontreated fruit, Spd and Spn decline. The pattern of accumulation of both Spd and Spn in preconditioned fruit is the same: an increase for 5 days after initiation of chilling followed by declining levels that remain higher than those in the nontreated fruit. This result may imply coordinated regulation of Spd and Spn, which could be explained by an effect on SAMDC, the product of which is used to synthesize both Spd and Spn. The pattern of SAMDC activity correlated closely with the levels of Spd and Spn. SAMDC declined in nontreated fruit after transfer to chilling conditions as did Spd + Spn. In contrast, SAMDC increased in preconditioned fruit for four days after transfer to chilling temperatures along with Spd + Spn. Thus it appears that Chilling-induced increases in Spd + Spn in preconditioned fruit may involve increased SAMDC activity. In preconditioned fruit, SAMDC activity and maximal Spd and Spn levels are induced four to five days after transfer to chilling temperature. At this time, the activity of ODC and the concentration of protein are also higher in preconditioned squash than in the control (Table 1). This early increase in ODC may explain how Put levels are induced to the same extent in the preconditioned as in the control fruit, but Spd and Spn production show a differential response, increasing in preconditioned fruit and decreasing in the control. Further metabolism of PUT (i.e., conjugation or conversion to alkaloids) could also be involved. Increased protein levels in the preconditioned fruit may indicate that either protein synthesis was induced or that protease levels were reduced as a result of preconditioning. Determination of protease indicated that the levels of this enzyme were unaffected by chilling or preconditioning in squash (unpublished data). Many questions remain to be answered concerning the mechanism of adaptation to chilling in squash. The metabolic changes during preconditioning that allow the induction
of polyamine biosynthetic enzyme activity in response to chilling are unknown. Also, it is unclear to what extent polyamines are involved in this response. We have shown that preconditioning is correlated with increases in Spd and Spn and that direct treatment of squash with Spn inhibits the development of chilling injury (Kramer and Wang, 1989), but these results do not rule out the possibility of factors other than polyamines being involved in this adaptive response. The general increase in protein levels (almost 20 %) resulting from preconditioning may suggest the involvement of other enzymes as the polyamine biosynthetic enzymes are probably not abundant enough to account for this increase. The elucidation of the mechanism of this adaptive response may suggest possible approaches to developing more chilling-resistant crops in the future. Acknowledgements We would like to thank Hilarine Repace for technical assistance.
References DROLET, G., E. B. DUMBROFF, R. L. LEGGE, and]. E. THOMPSON: Radical scavenging properties of polyamines. Phytochemistry
25, 367 -371 (1986). FLORES, H. E. and A. W. GALSTON: Polyamines and plant stress: activation of putrescine biosynthesis by osmotic shock. Science
217, 1259-1260 (1982). GALSTON, A. W. and R. KAUR-SAWHNEY: Polyamines and senescence in plants. In: THOMSON, W., E. NOTHNAGEL, and R. HUFFAKER (eds.): Plant Senescence: Its Biochemistry and Physiology, pp. 167 -171. American Society of Plant Physiologists, Rockville
(1987). GUYE, M. G., L. VIGH, and]. M. WILSON: Polyamine titre in relation to chill-sensitivity in Phaseolus sp. J. Exp. Bot. 37, 1036-1043
(1986).
HARDENBURG, R. E., A. E. WATADA, and C. Y. WANG: The commercial storage of fruits, vegetables, and florist and nursery stocks. u.S. Dept. Agric., Agriculture Handbook No. 66 (1986). KAUR-SAWHNEY, R., H. E. FLORES, and A. W. GALSTON: Polyamine oxidase in oat leaves: a cell wall-localized enzyme. Plant Physiol.
68,494-498 (1981).
KAUR-SAWHNEY, R., L.-M. SHIH, H. E. FLORES, and A. W. GALSTON: Relation of polyamine synthesis and titer to aging and senescence in oat leaves. Plant Physiol. 69, 405-410 (1982). KITADA, M., K. IGARASHI, S. HIROSE, and H. KITAGAWA: Inhibition by polyamines of lipid peroxide formation in rat liver microsomes. Biochem. Biophys. Res. Commun. 87, 388-394 (1979). KRAMER, G. F. and C. Y. WANG: Correlation of reduced chilling injury with increased spermine and spermidine levels in zucchini squash. Physiol. Plant. 76, 479-484 (1989). KRAMER, G. F., C. Y. WANG, and W. S. CONWAY: Correlation of reduced softening and increased polyamine levels during low-oxygen storage of McIntosh apples. ]. Amer. Soc. Hort. Sci. 114,
942-946 (1989). McDONALD, R. E. and M. M. KUSHAD: Accumulation of putrescine during chilling injury of fruits. Plant Physiol. 82, 324-326
(1986).
ROBERTS, D. R., E. B. DUMBROFF, and J. E. THOMPSON: Exogenous polyamines alter membrane fluidity - a basis for potential misinterpretation of their physiological role. Planta 167, 395-401
(1986).
Effects of Chilling on Polyamine Biosynthetic Enzymes SMITH, T. A.: Polyamines. Ann. Rev. Plant Physio!. 36, 117 -143 (1985). TORRlGIANI, P., V. SCOCCIANTI, and N. BAGNI: Polyamine oxidase activity and polyamine content in maize during seed germination. Physio!. Plant. 74, 427-432 (1988). WANG, C. Y.: Changes in polyamine content in chinese cabbage during storage in air or low oxygen atmosphere. ]. Food Quality 11,289-302 (1988). WANG, C. Y. and Z. L.JI: Effect oflow oxygen storage on chillinginjury and polyamines in zucchini squash. Sci. Hortic. 39, 1-7 (1989).
119
WANG, S. Y. and G. L. STEFFENS: Effect of paclobutrazol on water stress-induced ethylene biosynthesis and polyamine accumulation in apple seedling leaves. Phytochemistry 24, 2185-2190 (1985). YOUNG, N. D. and A. W. GALSTON: Putrescine and acid stress. Plant Physio!. 71,767-777(1983). ZAITSU, K. and Y. OHKURA: New flu orogenic substrates for horseradish peroxidase: rapid and sensitive assays for hydrogen peroxide and the peroxidase. Ana!. Biochem. 109,109-113 (1980) .
Effects of Chilling and Temperature Preconditioning on the Activity of Polyamine Biosynthetic Enzymes in Zucchini Squash G. F.
KRAMER
and C. Y.
WANG'~
Horticultural Crops Quality Lab., Agricultural Research Service, U.S. Dept. of Agriculture, Beltsville, MD 20705, USA Received August 14, 1989 . Accepted December 1, 1989
Summary Polyamine biosynthesis during chilling stress in zucchini squash (Cucurbita pepo L., cv. «Ambassador») was measured by determining the activities of SAMDC, ADC, and ODe. Stress induced increases in Put were correlated with increased ODC activity. Decreases in the levels of Spd and Spn during chilling may be related to decreased SAMDC activity. Temperature preconditioning adapts squash to subsequent chilling stress and results in increased Spn and Spd levels in response to chilling. The concomitant increase in these two polyamines is correlated with elevated SAMDC activity. These data support the view that the mechanism by which temperature preconditioning inhibits chilling injury involves the induction of polyamine biosynthesis.
Key words: Cucurbita pepo, ADC, chilling injury, ODC, polyamines, preconditioning, putrescine, SAMDC, spermidine, spermine, zucchini squash. Abbreviations: ADC = arginine decarboxylase (EC 4.1.1.49); AP = aminopropyl; DAP = diaminopropane; DAO = diamino oxidase; FW = fresh weight; HPLC = high pressure liquid chromatography; ODC = ornithine decarboxylase (EC 4.1.1.17); PAO = polyamine oxidase; Put = putrescine; SAM = Sadenosylmethionine; SAMDC = SAM decarboxylase (EC 4.1.1.50); Spd = spermidine; Spn = spermine. Introduction Polyamines can act to inhibit a variety of senescence-related processes in plant tissues (GaIston and Kaur-Sawhney, 1987). These antisenescent properties include the inhibition of the activity of degradative enzymes such as RNase, protease (Galston and Kaur-Sawhney, 1987), and polygalacturonase (Kramer et al., 1989); the stabilization of membrane structure (Roberts et al., 1986); antioxidant activity (Drolet et al., 1986); and the inhibition of lipid peroxidation (Kitada et al., 1979). Put levels increase in plant tissues in response to a number of stresses, including acid treatment (Young and Galston, 1983), osmotic shock (Flores and Galston, 1982), and water stress (Wang and Steffens, 1985). Chilling injury in a variety of fruit also results in significant increases in Put
*
Corresponding author.
© 1990 by Gustav Fischer Verlag, Stuttgart
levels (McDonald and Kushad, 1986; Wang and Ji, 1989). Our results indicate that polyamines may be involved in protecting plant tissues from chilling injury. Treatment of squash with Spn after harvest inhibited injury during subsequent chilling stress (Kramer and Wang, 1989). Also, the reduction of chilling injury in squash and senescence in Chinese cabbage and apples by controlled atmosphere storage is coupled with increased polyamine levels (Kramer et al., 1989; Wang, 1988; Wang and Ji, 1989). These results are consistent with the suggestion that polyamines increase cell viability during chilling by retarding membrane senescence (Guye et aI., 1986). Put, a precursor to the synthesis of both Spd and Spn (Fig. 1), can be synthesized via two separate pathways in plant tissues: from arginine via ADC and from ornithine via ODe (Smith, 1985). The relative activity of these pathways can vary depending on species, tissue, and growth and devel-
116
G. F.
KRAMER
and C. Y. WANG
=1-:
l
ARmIlNE-
l!QII\TI1IE:---- ror - - - - -
SAMIlC
~lsFO
Synthase
PM
S1'!\M~I~~-- decartlaxylated S1tIt- ~SFOlsm Synthase
~l~
N113
H:02 Pyrrol~
+
: AP-pyrrol~
PM
+
SIt{-1- - - - - Il1\P
+
H202
Fig. 1: Polyamine metabolic pathways in plant tissues. opmental stage (Smith, 1985). Stress-induced Put accumulation is a result of increased ADC activity (Flores and Galston, 1982; Smith, 1985; Young and Galston, 1983; Wang and Steffens, 1985). Chilling injury symptoms develop rapidly in zucchini squash during storage at 2.5 °C (Hardenburg et aI., 1986; Kramer and Wang, 1989). This chilling stress is correlated with a > 5-fold increase in Put and a 2- to 4-fold decrease in Spd and Spn (Kramer and Wang, 1989). Preconditioning squash at 10°C for 2 days prior to storage at 2.5 °C significantly delays the development of chilling injury. This adaptive response to chilling is correlated with increased levels of Spd and Spn (Kramer and Wang, 1989). The present study was undertaken to determine the mechanism by which the levels of polyamines are regulated during chilling stress and preconditioning treatment.
Materials and Methods Zucchini squash (Cucurbita pepo L., cv. «Ambassadon» used for this study were freshly harvested from a local farm near Beltsville, MD. Samples were selected for their uniformity of size (16-22cm in length) and randomly divided into two lots. The first group (control) was placed in storage at 2.5 DC. The second lot was preconditioned at 10°C for the first 2 days of storage and then moved to 2.5 °C for the remainder of the study. Samples were taken on various days throughout the storage period. Three squash were chosen at random from each group being sampled. Two 2.0-g samples of skin were removed from each fruit and immediately frozen. Samples were stored at - 80°C prior to analysis.
Enzyme assays ADC, ODC, and SAMDC were determined by the method of Kaur-Sawhney et al. (1982). Skin samples (2.0 g) were homogenized in 15mL of chilled 100mM phosphate pH7.6 (for ADC and SAMDC) or 100 mM Tris-CI pH 8.0 (for ODC). Crude extracts were obtained by centrifugation of the homogenates at 47,000 xg for 20 min. The assays were carried out in 12 x 75-mm polystyrene tubes (InterLab) sealed with snap caps. A 6-mm filter paper (Whatman GF/B) disc, saturated with 50 JLL 2 N KOH, was transfixed onto a 22-gauge needle that was inserted through the tube cap. Reaction mixtures contained 100 JLL crude extract plus 10 JLL 5 mM pyridoxal phosphate (ICN) and 10 JLL of 20 JLCi/mL L-[U- 14C]arginine (270 mCil mmol, ICN) diluted with unlabelled L-arginine (Sigma) to give a final concentration of 9 mM for ADC, 10 JLL 5 mM pyridoxal
phosphate plus 10JLL of 20JLCi/mL DL-e 4C]ornithine (50mCil mmol, ICN) diluted with cold L-ornithine (Sigma) to give a final concentration of 66mM for ODC, and 10JLL O.lmM pyridoxal phosphate plus 10 JLL of 20 JLCi/mL SAM-[14C]carboxyl (59 mCil mmol, Amersham) diluted with cold SAM (Sigma) to give a final concentration of 2.7 mM for SAMDC. These mixtures were incubated 30 min (SAMDC) or 60 min (ODC, ADC) at 37°C. The reactions were stopped by the addition of 0.2 mL 10 % trichloroacetic acid (Sigma) through the needle. The incubation was continued for an additional 45 min. The filter paper discs were removed and dried at 70°C for 30 min. The liberated CO 2 was determined by placing the discs in 3 mL ScintiLene (Fisher) and counting with a Beckman LS 6800 scintillation counter. Each time point was the average of the three individual fruits sampled. Data presented are the average of 2 to 4 separate experiments.
Polyamine oxidase and dzamzne oxzdase assays Polyamine-dependent H 20 Z formation was determined by measuring guaiacol (Sigma) oxidation with the method of Kaur-Sawhney et al. (1981). More sensitive assays (Zaitsu and Ohkura, 1980) involved substitution of guaiacol with 3-(p-hydroxyphenyl)propionic acid (Sigma) and measuring fluorescence (excitation wavelength = 320 nm, emission wavelength = 404 nm). Activity was determined in crude extracts prepared by homogenization of skin tissue in 100 mM phosphate pH 6.5 followed by centrifugation at 17,000 g for 10 min and in cell wall fractions prepared by the method of Torrigiani et al. (1988).
Protein determination Protein concentration in the crude extract was determined with the BCA Protein Assay Reagent (Pierce) using the standard room temperature protocol.
Polyamine analysis Free polyamines were analyzed as dansylated derivatives vIa HPLC as described previously (Kramer and Wang, 1989).
Figure preparation The figures were prepared using SigmaPlot software Oandel Scientific). The traces presented are computer generated regression lines.
Results
Put biosynthesis The activities of both ADC and ODC in squash skin extracts were determined as the skin is the tissue most sensitive to chilling and has been monitored in earlier studies. Chilling stress induced a 2- to 3-fold increase by day 5 and a 4- to 6-fold increase in ODC activity by day 12 (Fig. 2), which correlates well with the 2- to 3-fold increase by day 5 (Table 1) and the 5- to 6-fold increase in Put by day 12 observed under these conditions (Kramer and Wang, 1989). The ODC activity in the preconditioned fruit also increased during chilling (Fig. 2). This activity may initially (days 4 - 9) increase more rapidly than the control activity but appears to be somewhat lower than the control at later times (after day 12). The ADC activity remained unchanged in both groups during chilling (data not shown).
Effects of Chilling on Polyamine Biosynthetic Enzymes Table 1: Induction of polyamines and polyamine biosynthetic enzymes by chilling stress in preconditioned squash. Day of Maximal Induction SAMDC (nmollmg· hr) Spd + Spn (nmollg) ODC (nmollmg· hr) Protein (mg/mL) Put (nmoll g)*
6 7 5 6 6
Treatment
no PC
+PC
significance, P
0.664 398 1.85 1.61 1135
2.06 940 3.16 1.87 1138
<0.005 <0.005 <0.005 0.0125 n.s.
2.50 +--t---+---t---t--t----t----t---t------r
~ L
I
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O"l
1
E
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o
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Preconditioned
o 1.50
* Initial Put levels were 333 (nmollg).
117
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T ...T .L 1
...T T 0 1 1
Fig.3: Effect of chilling on SAMDC activity. Open circles (0) control group (r = 0.873). Closed triangles (....) - preconditioned group (r = 0.866). Error bars represent ±SE.
-+--'
()
«
2.0
0
1.0
u 0
0
1000
...T 1
900
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Duration of Chilling (Days) Fig.2: Effect of chilling on the activity of ODC in squash skin. Open circles (0) - control fruit that had been held at 2.5°C throughout the experiment (r = 0.949). Closed triangles (....) - preconditioned fruit, held at 10 °C for days 0 - 2 prior to transfer to 2.5 °C (r = 0.869). Error bars represent ±SE.
SAMDC activity The product of SAMDC, decarboxylated SAM, serves as an aminopropyl donor in the synthesis of both Spd and Spn (Fig. 1). The activity of this enzyme decreased during chilling stress in zucchini squash (Fig. 3). In the preconditioned fruit, SAMDC activity began increasing on day 2 after transfer to the chilling temperature (Fig. 3). After day 6, the enzyme activity decreased but remained elevated relative to the nontreated fruit. These patterns of activity are closely correlated with the observed levels of Spd and Spn, the ultimate products of SAMDC action. Spd + Spn decreased in the nontreated fruit but increased in the preconditioned fruit after transfer to chilling stress (Fig. 4). After day 7, the levels decreased but remained elevated relative to the control. The elevation of SAMDC could thus account for the observed increases in Spd and Spn in the preconditioned fruit.
~
3: 800
Preconditioned
LL
/
O"l
700
...
o 600
E
c '--" 500
,----,
z
0..- 400 IJJ
+ 0
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0
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T
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.L
0..IJJ 200
~
1
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T
T
0
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Q
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1
0
L-.J
0
100 O+---t---r--+--+--~--t---r--+---+
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2
4
6
8
10 12 14 16 18
Duration of Chilling (Days)
Fig. 4: Effect of chilling on the levels of Spd + Spn. Open circles (0) - control group (r = 0.911). Closed triangles (....) - preconditioned group (r = 0.895). Error bars represent ±SE.
Polyamine degradative pathways Put can be degraded via DAO and Spd and Spn via PAO (Smith, 1985; Fig. 1), both of which are assayed by detection
118
G. F. KRAMER and C. Y. WANG
of polyamine-dependent H 20 2 formation. Using guaiacol oxidation to detect H 20 2 (Kaur-Sawhney et al., 1981), there was no detectable activity of DAO or PAO in the cytoplasmic or cell wall fractions of squash skin homogenates (data not shown). Using a more sensitive fluorogenic substrate, hydroxyphenyl propionic acid (Zaitsu and Ohkura, 1980), the activities of both enzymes remained below the limit of detection (data not shown). These results may indicate that degradative enzymes do not playa major role in the regulation of polyamine levels in squash. Discussion
Plants respond to a variety of stresses by inducing the synthesis of Put (Smith, 1985). In all cases reported, this increase in Put results from induction of ADC activity (Smith, 1985). Chilling stress in zucchini squash results in 5- to 6-fold increase in Put concomitant with a 4- to 7-fold increase in ODC activity. The stress-induced Put thus appears to originate from the ODC pathway. The temperature preconditioning of squash prior to chilling induces an adaptive response to this stress (Kramer and Wang, 1989). Fruit that are preconditioned accumulate Spn and Spd after transfer to chilling temperatures; whereas in nontreated fruit, Spd and Spn decline. The pattern of accumulation of both Spd and Spn in preconditioned fruit is the same: an increase for 5 days after initiation of chilling followed by declining levels that remain higher than those in the nontreated fruit. This result may imply coordinated regulation of Spd and Spn, which could be explained by an effect on SAMDC, the product of which is used to synthesize both Spd and Spn. The pattern of SAMDC activity correlated closely with the levels of Spd and Spn. SAMDC declined in nontreated fruit after transfer to chilling conditions as did Spd + Spn. In contrast, SAMDC increased in preconditioned fruit for four days after transfer to chilling temperatures along with Spd + Spn. Thus it appears that Chilling-induced increases in Spd + Spn in preconditioned fruit may involve increased SAMDC activity. In preconditioned fruit, SAMDC activity and maximal Spd and Spn levels are induced four to five days after transfer to chilling temperature. At this time, the activity of ODC and the concentration of protein are also higher in preconditioned squash than in the control (Table 1). This early increase in ODC may explain how Put levels are induced to the same extent in the preconditioned as in the control fruit, but Spd and Spn production show a differential response, increasing in preconditioned fruit and decreasing in the control. Further metabolism of PUT (i.e., conjugation or conversion to alkaloids) could also be involved. Increased protein levels in the preconditioned fruit may indicate that either protein synthesis was induced or that protease levels were reduced as a result of preconditioning. Determination of protease indicated that the levels of this enzyme were unaffected by chilling or preconditioning in squash (unpublished data). Many questions remain to be answered concerning the mechanism of adaptation to chilling in squash. The metabolic changes during preconditioning that allow the induction
of polyamine biosynthetic enzyme activity in response to chilling are unknown. Also, it is unclear to what extent polyamines are involved in this response. We have shown that preconditioning is correlated with increases in Spd and Spn and that direct treatment of squash with Spn inhibits the development of chilling injury (Kramer and Wang, 1989), but these results do not rule out the possibility of factors other than polyamines being involved in this adaptive response. The general increase in protein levels (almost 20 %) resulting from preconditioning may suggest the involvement of other enzymes as the polyamine biosynthetic enzymes are probably not abundant enough to account for this increase. The elucidation of the mechanism of this adaptive response may suggest possible approaches to developing more chilling-resistant crops in the future. Acknowledgements We would like to thank Hilarine Repace for technical assistance.
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HARDENBURG, R. E., A. E. WATADA, and C. Y. WANG: The commercial storage of fruits, vegetables, and florist and nursery stocks. u.S. Dept. Agric., Agriculture Handbook No. 66 (1986). KAUR-SAWHNEY, R., H. E. FLORES, and A. W. GALSTON: Polyamine oxidase in oat leaves: a cell wall-localized enzyme. Plant Physiol.
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KAUR-SAWHNEY, R., L.-M. SHIH, H. E. FLORES, and A. W. GALSTON: Relation of polyamine synthesis and titer to aging and senescence in oat leaves. Plant Physiol. 69, 405-410 (1982). KITADA, M., K. IGARASHI, S. HIROSE, and H. KITAGAWA: Inhibition by polyamines of lipid peroxide formation in rat liver microsomes. Biochem. Biophys. Res. Commun. 87, 388-394 (1979). KRAMER, G. F. and C. Y. WANG: Correlation of reduced chilling injury with increased spermine and spermidine levels in zucchini squash. Physiol. Plant. 76, 479-484 (1989). KRAMER, G. F., C. Y. WANG, and W. S. CONWAY: Correlation of reduced softening and increased polyamine levels during low-oxygen storage of McIntosh apples. ]. Amer. Soc. Hort. Sci. 114,
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WANG, S. Y. and G. L. STEFFENS: Effect of paclobutrazol on water stress-induced ethylene biosynthesis and polyamine accumulation in apple seedling leaves. Phytochemistry 24, 2185-2190 (1985). YOUNG, N. D. and A. W. GALSTON: Putrescine and acid stress. Plant Physio!. 71,767-777(1983). ZAITSU, K. and Y. OHKURA: New flu orogenic substrates for horseradish peroxidase: rapid and sensitive assays for hydrogen peroxide and the peroxidase. Ana!. Biochem. 109,109-113 (1980) .