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Early Biochemical Changes during Acclimation of Poplar to Low Temperature
J. Plant Physiol.
Vol. 147. pp. 247-250 (1995)
Early Biochemical Changes during Acclimation of Poplar to Low Temperature L. JOUVE*, J.
G. FOUCHE, AND
T. GASPAR
Laboratoire d'Hormonologie Fondamentale et Appliquee, Institut de Botanique B22, Universite de Liege-Sart Tilman, B-4000 Liege (Belgium) Received April 4, 1995 . Accepted June 28,1995
Summary
Microcuttings of Populus tremula were cultured in vitro for 0 to 15 days at 10°C and compared with cultures kept at 23 °C. The changes in the levels of polyamines were followed in relation to frost resistance (LTso). At 23°C there was no variation in the level of putrescine; the level of spermidine decreased in the first 5 days and remained relatively stable afterwards. Treatment at 10°C induced a sharp temporary peaking of putrescine level after 1 day, followed by a transitory decrease. A second peak, higher in duration and intensity, was observed after the 4th day. After a transitory decrease the level of spermidine increased to twice the standard level after 6 days storage. An increase in the freezing tolerance of poplar shoots was achieved when cultures were exposed to 10°C for at least 3 days; however, 3 to 15 days pretreatment at 10°C increased the freezing tolerance.
Key words: Populus tremula L., acclimation, freezing resistance, polyamine, putrescine, spermidine. Abbreviations list: LTso - lethal temperature 50 %; 2-iP - 2-isopentenyl-adenine. Introduction
The injury suffered by many crop species, which results when they are exposed to temperatures between O°C and about 15°C, is now commonly referred to as chilling injury (Lyons, 1973). Generally, for plants adapted to a cool climate, the exposure to low temperature induces biochemical and physiological changes that allow them to withstand this stress (Levitt, 1980; Graham and Patterson, 1982). The resulting adjustment, known as the acclimation process, increases resistance to further freezing temperatures. Elevation of endogenous polyamine levels in response to stress conditions has been well documented. For example 502 fumigation (Priebe et al., 1978), water stress (Kandpal and Rao, 1985), acidification (Shen et al., 1994), salinity and osmotic stress (Smith, 1985) have been reported to increase polyamine levels, particularly putrescine, in plants. Accumulation of polyamines during cold exposure was also reported (Guye et al., 1986; Wang, 1987; Nadeau et al., 1987; K:ushad .. Correspondence. © 1995 by Gustav Fischer Verlag, Stuttgart
and Yelenosky, 1987). Guye et al. (1986) have suggested that the relative changes rather than the absolute levels of putrescine are more important in predicting responses of Phaseolus species to chilling temperature. Other reports have indicated that spermidine accumulation contributed significantly towards maintaining cell membrane integrity during freezing stress (Smith, 1971, 1982; Guye et al., 1986). In this paper, we report on the modification of polyamine levels in poplar shoots during acclimation to cold. The results are related to survival and growth rates of poplar shoots in the experimental conditions and to frost resistance during the acclimation process.
Materials and Methods
Plant material In vitro stock proliferating cultures of Populus tremula L. were used as the source of material. The explants used were orthotropic axes three nodes long (±30 mm long; ±6Ieaves), without roots.
248
L.JOUVE,J. G. FOUCHE, and T. GASPAR
Methods Before assays, the proliferating clusters were subcultured every 4 weeks on Murashige and Skoog (1962) medium supplemented with 0.8 mg' L -I 2-iP. Isolated shoots were transferred to Murashige and Skoog (1962) medium without growth regulator. Cultures were placed at 23 and 10°C with a photoperiod of 16h under a photon flow of 55~mol'm-2's-1 (Sylvania Gro-Lux fluorescent lamps) at the culture level inside the jar plus lid. Frost resistance was tested after 0, 1, 3, 6 and 15 days pretreatment at 10°C. Three jars (13 plants per jar) were used for each freezing temperature assay (0, - 2, - 5, - 8, -10, -12, -15 and - 17 0C). Samples were cooled at the rate of 6 °C.h -I to 0 °C and held 120 min for medium supercooling. Then samples were frozen to each desired temperature at the rate of 6°C·h-1 for 15 min. Thereafter, jars were placed at 23°C for 12 h and microcuttings were transferred onto fresh Murashige and Skoog medium supplemented with 0.8mg·L -I 2-iP. Survival was estimated after 2 weeks culture. The temperature that resulted at 50 % survival was graphically determined and defined as freezing tolerance (LT so). Biochemical changes were measured after several low temperature treatment durations: 0, 1, 2, 3, 4, 6, 8, 11 and 15 days. At the end of each time period, the survival rate was estimated as a function of the total number of remaining microcuttings. At each sampling date, several jars were randomly chosen in each condition. For polyamine and enzyme assays plant material was frozen in liquid nitrogen and stored at - 80°C for ulterior measurements. Weights measured during sampling were used for growth rate determination. Free polyamine extraction, separation, identification and measurement by direct dansylation and HPLC were done as described by Walter and Geuns (1987). Results were expressed in ~M polyamine per g of fresh weight. All results are the means of measurements from at least three separate experiments.
120 110 100
00 90
.§,
~
80
~
70
iOl)
60 50 40
0
2
4
6
8
10
12
14
Time (days)
Fig. 1: Evolution of fresh weight of microcuttings of poplar during storage at different temperatures (23°C 0, 10°C.).
100
90 80
70
~
60
>
.~
50
=' tn
40
<;i
Results
Survival and growth rate No significant variation concerning survival was measured during the course of the experiment. After 15 days at 10 and 23°C survival was 100 %. Low temperature had a significant effect on growth (Fig. 1): culture at 10°C stopped growth, and 23 °C permitted two-fold multiplication after 15 days.
Cold acclimation The capacity of poplar shoots for cold acclimation was assessed by determining the freezing tolerance of non-acclimated plants (microcuttings grown at 23°C) and those that were exposed to cold hardening treatment (Fig. 2). The LT 50 of non-acclimated plants was - 8.6 DC. When shoots were transferred to 10°C for 3 and 6 days the freezing tolerance increased to -10.7 and -lOA DC, respectively. When they were transferred to 10°C for 15 days the freezing tolerance was more increased (-1204 0C). Nevertheless, the LT50 of plants cold treated during 1 day at 10°C was worse (- 6.9 0C) than that of the non-acclimated ones.
Polyamine Variations in endogenous levels of putrescine and spermidine are shown in Fig. 3. The endogenous spermine level is
30 20 10
o L....t~_II!I:..........L..............L.............l-.........L-..---"-.............l-.o-LJ -18
-16
-14
-12
-10
-8
-6
-4
-2
0
Temperature (OC)
Fig. 2: Freezing tolerance of microcuttings of poplar cultured at 23°C (0) and pretreated during 1 day (.), 3 days (_), 6 days (0) and 15 days (A) at 10°C, as expressed in % survival.
not represented because there was no variation between 10 and 23 °C treatments. Spermine content remained at a relatively low level (0.12±0.05IJ.M 'g-I fresh weight) during the experiment. At 23°C no variation in the level of putrescine occurred; the level of spermidine decreased in the first 5 days and stayed relatively stable afterwards. Culture at 10°C induced a sharp temporary peaking of the putrescine level af· ter 1 day followed by a transitory decrease. A second peak, higher in duration and intensity, was observed between the 4tli and 15th days. After a transitory decrease, the level of spermidine increased to twice the standard level after 6 days and three times after 15 days.
Poplar acclimation: biochemical changes 1.6
1986; Kushad and Yelenosky, 1987) and in plant cold acclimation (Nadeau et al., 1987). We have also seen that storage at 10°C induced a sharp temporary peaking of the putrescine level after 1 day, followed by a rapid decrease. This may be the first part of the reaction against cold stress. Putrescine may be quickly produced for membrane stabilisation or osmoregulation (Kushad and Yelenosky, 1987).
1.4
1.:2
~
-;'
249
1.0
bO
~
::t.
Cold acclimation andfreezing resistance
0.8
0.6
:2
4
6
8
10
1:2
14
Time (days) Fig. 3: Evolution of the endogenous concentration of putrescine (0 and .) and spermidine (0 and .) of microcuttings of poplar during storage at different temperatures (23°C 0 and 0; 10°C. and .), as expressed in IJ.M 'g-l Fresh Weight. Disscusslon
Changes linked to cold treatment The results indicated that growth of poplar microcuttings
in vitro was almost completely stopped by the low temper-
ature as already seen by Hausman et al. (1994) for another poplar species. Similar results were observed from cold conservation of different crop species (Sagisaka, 1985; Borkowska, 1990; Orlikowska, 1992). The present results suggest that a temperature of 10°C is sufficient to stop growth of poplar microcuttings without killing or damaging them. The polyamine concentration varied differently during conservation, depending on the culture temperature and duration. We observed a peak in the putrescine level from the 4th to the 15th day of cold storage. Guye et al. (1986) reported that, in Phaseolus sp., an increase in polyamine level, particularly putrescine, occurred in hardened plants. This was also reported when wheat or alfalfa were exposed to a cold hardening temperature (Nadeau et al., 1987). An increase of spermidine (after 6 days) has been shown when poplar shoots were stored at 10°C. This is in accordance with the results of Wang (1987), which indicated that spermidine content in cucumber exposed to chilling (5°C) was higher in chilled plants than in non-chilled plants. A high level of spermidine was maintained from 1- 3 weeks when Citrus was exposed to low temperature (Kushad and Yelenosky, 1987). These results seem to show that polyamine metabolism is involved in the reaction against cold treatments. First, the putrescine level increases (from the 4th to the 8th day) and afterwards decreases. This decrease seems to be related to the increase (from the 6th to the 15th day) of spermidine. This evolution during cold exposure is in accordance with an activation of the polyamine biosynthetic pathway (Guye et al., 1986; ~mith, 1985). That increase in polyamine level may play an Important role in cell protection (Smith, 1985; Guye et al.,
An increase in the freezing tolerance of poplar shoots was achieved when cultures were exposed to 10°C for at least 3 days. The acquisition of freezing tolerance via cold acclimation generally requires exposure to low non-freezing exposure (Sakai and Yoshida, 1968; Weiser, 1970; Fennell and Li, 1985). Moreover, we have seen that from 3 to 15 days pretreatment the freezing tolerance was increased. This is in accordance with the observed increase at a gradual rate of the freezing resistance to maximum hardiness (Sakai and Yoshida, 1968; Steponkus and Lanphear, 1968; Fennell and Li, 1985). Maximum freezing tolerance is reached in 1- 3 weeks of low temperature exposure. Exposure of plants to cold results in a progressive adjustment of growth and metabolism to low temperature conditions and in an increased resistance to freezing temperatures. We have seen first a decrease in the resistance to freezing temperatures (when plants were pretreated 1 day at 10°C), then an increase up to a maximum of cold hardiness after 15 days culture at 10 dc. In the first stage (1 day) low temperature may stress plants before the acclimation reaction. After 3 days of low temperature culture the resistance to freezing temperatures may be increased by a second reaction consisting of a sharp temporary peaking in putrescine level. A prolonged stage at 10 °C (15 days) induced a higher freezing resistance; this may by in accordance with changes in the second phase response of putrescine and spermidine metabolism (Guye et al., 1986; Kushad and Yelenosky, 1987; Nadeau et al., 1987; Wang, 1987). This work will be continued to elucidate early changes in other metabolisms (protein biosynthesis, scavenging enzyme involvement, ... ) and growth regulators such as abscisic acid. Acknowledgement
This research was carried out in the frame-work of the EC AIR contract l-CT92-0349 on poplar.
References
Influence of low temperature storage on regenerative capacity of sour cherry cultures. Fruit Science Reports 17,
BORKOWSKA, B.:
1-7 (1990).
FENNELL, A. and P. H. LI: Rapid cold acclimation and deacclimation in winter spinach. Acta Hortic. 168, 179-183 (1985). GRAHAM, D. and B. D. PATIERSON: Responses of plants to low, nonfreezing temperatures: proteins, metabolism and acclimation. Annu. Rev. Plant Physiol. 33,347 -372 (1982).
250
L. JOUVE, J G. FOUCHE, and T. GASPAR
GUYE, M. G., L. VIGH, and J M. Wn.soN: Polyamine titre in relation to chill-sensitivity in Phaseolus sp. J. Exp. Bot. 31, 10361043 (1986). HAUSMAN, J F., O. NEYs, C. KEvus, and T. GASPAR: Effect of in vitro storage at 4 °C on survival and proliferation of poplar shoots. Plant Cell Tiss. Org. Cult. 38, 65-67 (1994). KAN!>PAL, R. P. and N. A. RAo: Changes in levels ofpolyamines in ragi seedlings during water stress. Biochem. Int. 11, 365 - 370 (1985). KUSHAD, N. M. and G. YELENOSI.Y: Evaluation of polyamine and proline levels during low temperature acclimation of Citrus. Plant Physiol. 84, 692-695 (1987). LEvrrr, J: Responses of Plants to Environmental Stresses. Vol. 1. Chilling, Freezing and High Temperature Stresses. 497p. New York Academic. 2nd ed. (1980). LYONS, J M.: Chilling injury in plants. Annu. Rev. Plant Physiol. 24,445-466 (1973). MU1lASHlGE, T. and F. SI.OOG: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473-497 (1962). NADEAU, T., S. DELANEY, and L. CHOUINARD: Effects of cold hardening of the regulation of polyamine levels in wheat (Triticum aes· tivum L.) and alfalfa (Medicago sativa L.). Plant Physiol. 84, 7377 (1987). OIUll.OWSI.A, T.: Effect of in vitro storage at 4°C on survival and proliferation of two apples rootstocks. Plant Cell Tissue Organ Culture 31,1-7 (1992).
PIUEBE, A., H. K.i.EIN, and A. J. JAGER: Role of polyamines in SOT polluted pea plants. J Exp. Bot. 29, 1045 -1050 (1978). SAGISAI.A, S.: Injuries of cold acclimatised poplar twigs resulting from enzyme inactivation and substrate depression during frozen storage at ambient temperatures for a long period. Plant Cell Physiol. 26, 1135-1145 (1985). SAI.AI, A. and S. YOSHIDA: The role of sugars and related compounds in variation of freezing resistance. Cryobiology 5, 160-174 (1968). SHEN, H. J., Y. F. XIE, and R. T. LI: Effects of acid stress on polyamine levels, ion efflux, protective enzymes and macromolecular synthesis in cereal leaves. Plant Growth Reg. 14, 1-5 (1994). SMITH, T. A.: The occurrence, metabolism and function of amines in plants. Biol. Rev. 46, 201-241 (1971). - Polyamines. Annu. Rev. Plant Physiol. 36, 117 -143 (1985). STEPONl.US, P. L. and F. O. lANPHEAll: The relationship of carbohydrates to cold acclimation of Hedera helix L. cv. Thorndale. Physiol. Plant. 21,777 -791 (1968). WALTER, H. J P. and J M. GEUNS: High speed HPLC analysis of polyamines in plant tissues. Plant Physiol. 83, 232 - 234 (1987). WANG, C. Y.: Changes of polyamines and ethylene in cucumber seedlings in response to chilling stress. Physiol. Plant. 69, 253257 (1987). WEISER, C. J.: Cold resistance and injury in woody plants. Science 169, 1269-1278 (1970).
Vol. 147. pp. 247-250 (1995)
Early Biochemical Changes during Acclimation of Poplar to Low Temperature L. JOUVE*, J.
G. FOUCHE, AND
T. GASPAR
Laboratoire d'Hormonologie Fondamentale et Appliquee, Institut de Botanique B22, Universite de Liege-Sart Tilman, B-4000 Liege (Belgium) Received April 4, 1995 . Accepted June 28,1995
Summary
Microcuttings of Populus tremula were cultured in vitro for 0 to 15 days at 10°C and compared with cultures kept at 23 °C. The changes in the levels of polyamines were followed in relation to frost resistance (LTso). At 23°C there was no variation in the level of putrescine; the level of spermidine decreased in the first 5 days and remained relatively stable afterwards. Treatment at 10°C induced a sharp temporary peaking of putrescine level after 1 day, followed by a transitory decrease. A second peak, higher in duration and intensity, was observed after the 4th day. After a transitory decrease the level of spermidine increased to twice the standard level after 6 days storage. An increase in the freezing tolerance of poplar shoots was achieved when cultures were exposed to 10°C for at least 3 days; however, 3 to 15 days pretreatment at 10°C increased the freezing tolerance.
Key words: Populus tremula L., acclimation, freezing resistance, polyamine, putrescine, spermidine. Abbreviations list: LTso - lethal temperature 50 %; 2-iP - 2-isopentenyl-adenine. Introduction
The injury suffered by many crop species, which results when they are exposed to temperatures between O°C and about 15°C, is now commonly referred to as chilling injury (Lyons, 1973). Generally, for plants adapted to a cool climate, the exposure to low temperature induces biochemical and physiological changes that allow them to withstand this stress (Levitt, 1980; Graham and Patterson, 1982). The resulting adjustment, known as the acclimation process, increases resistance to further freezing temperatures. Elevation of endogenous polyamine levels in response to stress conditions has been well documented. For example 502 fumigation (Priebe et al., 1978), water stress (Kandpal and Rao, 1985), acidification (Shen et al., 1994), salinity and osmotic stress (Smith, 1985) have been reported to increase polyamine levels, particularly putrescine, in plants. Accumulation of polyamines during cold exposure was also reported (Guye et al., 1986; Wang, 1987; Nadeau et al., 1987; K:ushad .. Correspondence. © 1995 by Gustav Fischer Verlag, Stuttgart
and Yelenosky, 1987). Guye et al. (1986) have suggested that the relative changes rather than the absolute levels of putrescine are more important in predicting responses of Phaseolus species to chilling temperature. Other reports have indicated that spermidine accumulation contributed significantly towards maintaining cell membrane integrity during freezing stress (Smith, 1971, 1982; Guye et al., 1986). In this paper, we report on the modification of polyamine levels in poplar shoots during acclimation to cold. The results are related to survival and growth rates of poplar shoots in the experimental conditions and to frost resistance during the acclimation process.
Materials and Methods
Plant material In vitro stock proliferating cultures of Populus tremula L. were used as the source of material. The explants used were orthotropic axes three nodes long (±30 mm long; ±6Ieaves), without roots.
248
L.JOUVE,J. G. FOUCHE, and T. GASPAR
Methods Before assays, the proliferating clusters were subcultured every 4 weeks on Murashige and Skoog (1962) medium supplemented with 0.8 mg' L -I 2-iP. Isolated shoots were transferred to Murashige and Skoog (1962) medium without growth regulator. Cultures were placed at 23 and 10°C with a photoperiod of 16h under a photon flow of 55~mol'm-2's-1 (Sylvania Gro-Lux fluorescent lamps) at the culture level inside the jar plus lid. Frost resistance was tested after 0, 1, 3, 6 and 15 days pretreatment at 10°C. Three jars (13 plants per jar) were used for each freezing temperature assay (0, - 2, - 5, - 8, -10, -12, -15 and - 17 0C). Samples were cooled at the rate of 6 °C.h -I to 0 °C and held 120 min for medium supercooling. Then samples were frozen to each desired temperature at the rate of 6°C·h-1 for 15 min. Thereafter, jars were placed at 23°C for 12 h and microcuttings were transferred onto fresh Murashige and Skoog medium supplemented with 0.8mg·L -I 2-iP. Survival was estimated after 2 weeks culture. The temperature that resulted at 50 % survival was graphically determined and defined as freezing tolerance (LT so). Biochemical changes were measured after several low temperature treatment durations: 0, 1, 2, 3, 4, 6, 8, 11 and 15 days. At the end of each time period, the survival rate was estimated as a function of the total number of remaining microcuttings. At each sampling date, several jars were randomly chosen in each condition. For polyamine and enzyme assays plant material was frozen in liquid nitrogen and stored at - 80°C for ulterior measurements. Weights measured during sampling were used for growth rate determination. Free polyamine extraction, separation, identification and measurement by direct dansylation and HPLC were done as described by Walter and Geuns (1987). Results were expressed in ~M polyamine per g of fresh weight. All results are the means of measurements from at least three separate experiments.
120 110 100
00 90
.§,
~
80
~
70
iOl)
60 50 40
0
2
4
6
8
10
12
14
Time (days)
Fig. 1: Evolution of fresh weight of microcuttings of poplar during storage at different temperatures (23°C 0, 10°C.).
100
90 80
70
~
60
>
.~
50
=' tn
40
<;i
Results
Survival and growth rate No significant variation concerning survival was measured during the course of the experiment. After 15 days at 10 and 23°C survival was 100 %. Low temperature had a significant effect on growth (Fig. 1): culture at 10°C stopped growth, and 23 °C permitted two-fold multiplication after 15 days.
Cold acclimation The capacity of poplar shoots for cold acclimation was assessed by determining the freezing tolerance of non-acclimated plants (microcuttings grown at 23°C) and those that were exposed to cold hardening treatment (Fig. 2). The LT 50 of non-acclimated plants was - 8.6 DC. When shoots were transferred to 10°C for 3 and 6 days the freezing tolerance increased to -10.7 and -lOA DC, respectively. When they were transferred to 10°C for 15 days the freezing tolerance was more increased (-1204 0C). Nevertheless, the LT50 of plants cold treated during 1 day at 10°C was worse (- 6.9 0C) than that of the non-acclimated ones.
Polyamine Variations in endogenous levels of putrescine and spermidine are shown in Fig. 3. The endogenous spermine level is
30 20 10
o L....t~_II!I:..........L..............L.............l-.........L-..---"-.............l-.o-LJ -18
-16
-14
-12
-10
-8
-6
-4
-2
0
Temperature (OC)
Fig. 2: Freezing tolerance of microcuttings of poplar cultured at 23°C (0) and pretreated during 1 day (.), 3 days (_), 6 days (0) and 15 days (A) at 10°C, as expressed in % survival.
not represented because there was no variation between 10 and 23 °C treatments. Spermine content remained at a relatively low level (0.12±0.05IJ.M 'g-I fresh weight) during the experiment. At 23°C no variation in the level of putrescine occurred; the level of spermidine decreased in the first 5 days and stayed relatively stable afterwards. Culture at 10°C induced a sharp temporary peaking of the putrescine level af· ter 1 day followed by a transitory decrease. A second peak, higher in duration and intensity, was observed between the 4tli and 15th days. After a transitory decrease, the level of spermidine increased to twice the standard level after 6 days and three times after 15 days.
Poplar acclimation: biochemical changes 1.6
1986; Kushad and Yelenosky, 1987) and in plant cold acclimation (Nadeau et al., 1987). We have also seen that storage at 10°C induced a sharp temporary peaking of the putrescine level after 1 day, followed by a rapid decrease. This may be the first part of the reaction against cold stress. Putrescine may be quickly produced for membrane stabilisation or osmoregulation (Kushad and Yelenosky, 1987).
1.4
1.:2
~
-;'
249
1.0
bO
~
::t.
Cold acclimation andfreezing resistance
0.8
0.6
:2
4
6
8
10
1:2
14
Time (days) Fig. 3: Evolution of the endogenous concentration of putrescine (0 and .) and spermidine (0 and .) of microcuttings of poplar during storage at different temperatures (23°C 0 and 0; 10°C. and .), as expressed in IJ.M 'g-l Fresh Weight. Disscusslon
Changes linked to cold treatment The results indicated that growth of poplar microcuttings
in vitro was almost completely stopped by the low temper-
ature as already seen by Hausman et al. (1994) for another poplar species. Similar results were observed from cold conservation of different crop species (Sagisaka, 1985; Borkowska, 1990; Orlikowska, 1992). The present results suggest that a temperature of 10°C is sufficient to stop growth of poplar microcuttings without killing or damaging them. The polyamine concentration varied differently during conservation, depending on the culture temperature and duration. We observed a peak in the putrescine level from the 4th to the 15th day of cold storage. Guye et al. (1986) reported that, in Phaseolus sp., an increase in polyamine level, particularly putrescine, occurred in hardened plants. This was also reported when wheat or alfalfa were exposed to a cold hardening temperature (Nadeau et al., 1987). An increase of spermidine (after 6 days) has been shown when poplar shoots were stored at 10°C. This is in accordance with the results of Wang (1987), which indicated that spermidine content in cucumber exposed to chilling (5°C) was higher in chilled plants than in non-chilled plants. A high level of spermidine was maintained from 1- 3 weeks when Citrus was exposed to low temperature (Kushad and Yelenosky, 1987). These results seem to show that polyamine metabolism is involved in the reaction against cold treatments. First, the putrescine level increases (from the 4th to the 8th day) and afterwards decreases. This decrease seems to be related to the increase (from the 6th to the 15th day) of spermidine. This evolution during cold exposure is in accordance with an activation of the polyamine biosynthetic pathway (Guye et al., 1986; ~mith, 1985). That increase in polyamine level may play an Important role in cell protection (Smith, 1985; Guye et al.,
An increase in the freezing tolerance of poplar shoots was achieved when cultures were exposed to 10°C for at least 3 days. The acquisition of freezing tolerance via cold acclimation generally requires exposure to low non-freezing exposure (Sakai and Yoshida, 1968; Weiser, 1970; Fennell and Li, 1985). Moreover, we have seen that from 3 to 15 days pretreatment the freezing tolerance was increased. This is in accordance with the observed increase at a gradual rate of the freezing resistance to maximum hardiness (Sakai and Yoshida, 1968; Steponkus and Lanphear, 1968; Fennell and Li, 1985). Maximum freezing tolerance is reached in 1- 3 weeks of low temperature exposure. Exposure of plants to cold results in a progressive adjustment of growth and metabolism to low temperature conditions and in an increased resistance to freezing temperatures. We have seen first a decrease in the resistance to freezing temperatures (when plants were pretreated 1 day at 10°C), then an increase up to a maximum of cold hardiness after 15 days culture at 10 dc. In the first stage (1 day) low temperature may stress plants before the acclimation reaction. After 3 days of low temperature culture the resistance to freezing temperatures may be increased by a second reaction consisting of a sharp temporary peaking in putrescine level. A prolonged stage at 10 °C (15 days) induced a higher freezing resistance; this may by in accordance with changes in the second phase response of putrescine and spermidine metabolism (Guye et al., 1986; Kushad and Yelenosky, 1987; Nadeau et al., 1987; Wang, 1987). This work will be continued to elucidate early changes in other metabolisms (protein biosynthesis, scavenging enzyme involvement, ... ) and growth regulators such as abscisic acid. Acknowledgement
This research was carried out in the frame-work of the EC AIR contract l-CT92-0349 on poplar.
References
Influence of low temperature storage on regenerative capacity of sour cherry cultures. Fruit Science Reports 17,
BORKOWSKA, B.:
1-7 (1990).
FENNELL, A. and P. H. LI: Rapid cold acclimation and deacclimation in winter spinach. Acta Hortic. 168, 179-183 (1985). GRAHAM, D. and B. D. PATIERSON: Responses of plants to low, nonfreezing temperatures: proteins, metabolism and acclimation. Annu. Rev. Plant Physiol. 33,347 -372 (1982).
250
L. JOUVE, J G. FOUCHE, and T. GASPAR
GUYE, M. G., L. VIGH, and J M. Wn.soN: Polyamine titre in relation to chill-sensitivity in Phaseolus sp. J. Exp. Bot. 31, 10361043 (1986). HAUSMAN, J F., O. NEYs, C. KEvus, and T. GASPAR: Effect of in vitro storage at 4 °C on survival and proliferation of poplar shoots. Plant Cell Tiss. Org. Cult. 38, 65-67 (1994). KAN!>PAL, R. P. and N. A. RAo: Changes in levels ofpolyamines in ragi seedlings during water stress. Biochem. Int. 11, 365 - 370 (1985). KUSHAD, N. M. and G. YELENOSI.Y: Evaluation of polyamine and proline levels during low temperature acclimation of Citrus. Plant Physiol. 84, 692-695 (1987). LEvrrr, J: Responses of Plants to Environmental Stresses. Vol. 1. Chilling, Freezing and High Temperature Stresses. 497p. New York Academic. 2nd ed. (1980). LYONS, J M.: Chilling injury in plants. Annu. Rev. Plant Physiol. 24,445-466 (1973). MU1lASHlGE, T. and F. SI.OOG: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473-497 (1962). NADEAU, T., S. DELANEY, and L. CHOUINARD: Effects of cold hardening of the regulation of polyamine levels in wheat (Triticum aes· tivum L.) and alfalfa (Medicago sativa L.). Plant Physiol. 84, 7377 (1987). OIUll.OWSI.A, T.: Effect of in vitro storage at 4°C on survival and proliferation of two apples rootstocks. Plant Cell Tissue Organ Culture 31,1-7 (1992).
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