全 文 ：The Changes of G6PDHase , ATPase and Protein during Low
Temperature-induced Freezing Tolerance of P .suaveollens
Lin Shanzhi , Zhang Zhiyi.College of Life Science
and Bio technology , Beijing Forest ry University , Bei-
jing 100083 , P.R.China
Received October 17 ,2000
Lin Shanzhi Zhang Zhiyi
ABSTRACT The effect of cold acclimation on the f reezing tolerance of P.suaveollens seedlings and the
changes of G6PDHase , ATPase , and protein in branches of P.suaveol lens seedling s during cold acclima-
tio n w ere studied.In addition , the seedling s are pretreated w ith protein synthesis inhibitor (cyclohex-
imide)befo re cold acclimat ion in order to examine whether cold acclimat ion-induced proteins play a role in
the development of f reezing tolerance.The results show that f reezing tolerance of P .suaveol lens
seedlings could be obviously induced by cold acclimation , this cold acclimation w ill require experiencing
tw o stages in o rder to acquire higher f reezing tolerance.Although the first stage of cold acclimation , in-
volving a temperature of-10℃, has few effects on the increase in f reezing tolerance , it might provide the
basis fo r acclimation at -20℃ of the second stage and the acquisition of f reezing tolerance.Cold acclima-
tio n distinct ly increased no t only the act ivit ies of G6PDHase and ATPase , and the content of protein in
branches of seedling s but also the f reezing tolerance of seedlings.After 2 days of deacclimation , the f reez-
ing tolerance of seedlings decrease to the level of nonacclimat ion , but the activities of G6PDHase and A T-
Pase , and the content of protein in branches of seedlings still maintained a lit tle higher than those in
branches of nonacclimated seedlings.Pro tein synthesis and the acquisi tion of freezing tolerance are inhibit-
ed by cycloheximide.Further analy sis found that low temperature-induced changes in the activities of
G6PDHase and ATPase , and the content of protein directly correlated w ith the development of f reezing
tolerance.
KEYWORDS Populus suaveol lens , cold acclimation , f reezing tolerance , survival rates , pro tein
1 Introduction
Freezing temperature constitutes one of the
most important environmental constrains limit-
ing the productivity and distribution of plants.
Many studies have shown that higher plants ex-
hibit an increase in freezing tolerance when ex-
posed to low temperature.This process , termed
cold acclimation , involves a series of physiologi-
cal and metabolic changes , including alterations
in the concentration of carbohydrates , soluble
sugars , proteins and free amino acids , changes
in isozyme activities as w ell as in membrane
composition and it has been suggested that these
changes occuring during cold acclimation have
more or less relation to the increase in freezing
tolerance (Guy 1990 , Hughes et al .1990 ,
Thomashow 1990).Until recently , the mecha-
nism of cold acclimation for increasing freezing
tolerance has not been investigated clearly.Al-
though various biochemical responses of plants to
low temperature have been w idely documented
and review ed , little is known about the relation
among the changes of energy , the contents of
protein and freezing tolerance , especially in
woody plants.
In order to further investigate the molecular
mechanism of cold acclimation and freezing tol-
erance , the changes of the activities of
G6PDHase and ATPase , the content of protein ,
and the freezing tolerance of Populus suaveol-
lens seedlings during cold acclimation at -10℃
17
Forest ry Studies in China 2(2):17 ～ 26
and-20℃ are examined , and the possible rela-
tions betw een the change of protein content and
the increase of freezing tolerance at energy-
metabolic angle are studied.These may be use-
ful in making scientific researches on the freez-
ing tolerance of woody plants.
2 Metarials and methods
2.1 Plant material and experimental condi-
tions
Poplar(P .suaveollens)plants were estab-
lished f rom dormant branch cuttings collected
from Heilongjiang Province in China on March
2000.The cuttings w ere rooted and grown in the
g reenhouse in the pots containing a 2∶1(v/ v)
mixture of soil and sand.After the seedlings
were grown for 5 weeks in the g reenhouse , they
were placed outdoor for 3 weeks.Part of eight-
week old seedlings that were maintained at 20℃
w ith an 8h photoperiod for 14 days was referred
to as nonacclimated seedlings , the others were
further divided into tw o groups.One group of
seedlings held at -10℃w ith an 8h photoperiod
for 6 days were referred to as preacclimated
seedlings , part of the preacclimated seedlings
further transferred to -20℃ w ith an 8h pho-
toperiod for 6 days were referred to as cold-accli-
mated seedlings.The other group of seedlings
first pretreated w ith cycloheximide(CH ) at
20℃ for 6 days were transferred to the condi-
tions of cold acclimation and these seedlings were
referred to as inhibitor-pretreated seedlings.Fi-
nally , part of cold-acclimated seedlings trans-
ferred to the conditions of nonacclimation for 2
days were referred to as deacclimated seedlings.
Branches were randomly harvested from nonac-
climated , preacclimated , cold-acclimated , and
inhibitor-pretreated seedlings , the bark tissue
from the collected branches were removed and
immediately f rozen on dry ice until used for pro-
tein , ATPase and G6PDHase ex traction.
2.2 Evaluation of freezing tolerance and sur-
vival ratio of seedlings
Survival ratio w as examined f rom nonaccli-
mated , preacclimated , cold-acclimated , and in-
hibitor-pret reated seedlings.Some of the above-
mentioned seedlings were cooled gradually to
-15℃, -20℃, -23℃, -25℃, -27℃,
-30℃, -32℃, and -35℃, respectively , in
a programmed freezer.The chamber was cooled
at a constant rate of 0.5℃·min-1 and then held
for 12h at each temperature.These seedlings
were transferred to 20℃ and then thawed for 2
days.The standard of seedlings survival rates de-
pends upon whether these seedlings regrow nor-
mally and the degree of freezing injury of bark
tissue and buds at -35℃ for 12h.Freezing tol-
erance was determined on the basis of the tem-
perature at which 100% of seedlings survived
and the survival rates of seedlings at -35℃ for
12h.
2.3 Protein extraction and measurement
Protein from bark of branches was obtained in
borate buffer (50mM sodium brote , 50mM
ascorbic acid , 1mM phenylmethylsulfonyl fluo-
ride , 1%-mercaptoethanol , 0.5%sodium do-
decyl sulfate , pH9.0)at 4℃as described earlier
(Coleman et al.1991 , GU Ruisheng et al.
1999).The homogeneous liquid was extracted
at 4℃ for 2h , and then centrifuged at 4℃with
12 000g for 30min.The resulting supernatant
w as collected.2.5 volumes of acetone pretreated
in -20℃was added to the supernatant and held
at -20℃ for 1h.The precipitate w as collected
by centrifugation at 12 000g for 20min , and
then dried at -20℃ for 20min.The protein pel-
let was resuspended in Tris-Hcl buffer[ 12.5%
(v/v)0.5M Tris-Hcl , pH6.8 , 5%(v/v)β-
mercaptoethanol , 10%(v/v)glycerol , 72.5%
(v/v)deionized water] and the supernatant w as
collected at 4℃ w ith 12 000g for 10min.The
18 Forest ry Studies in China ,Vol.2 ,No.2 ,2000
resulting supernatant w as used as the souble pro-
tein and the protein concentrations were assayed
by using the Bradford(1979)dye-binding tech-
nique with bovine serum albumin(BSA) as a
standard.
2.4 G6PDHase extraction and analysis
G6PDHase was extracted f rom bark of
branches and analyzed using the technique de-
scribed by Li Jinshu(1985).
2.5 ATPase extraction and analysis
ATPase was ex tracted from bark of branches
and analyzed using the method described by Ao-
yan Guangcha(1985).
3 Results
3.1 Effects of low temperature on the survival
rates and freezing tolerance of seedlings
From Table 1 , the preacclimated and cold ac-
climated seedlings had an increase in the survival
rate and freezing tolerance compared to nonaccli-
mated seedlings and then decreased to the level
of nonacclimated seedlings after 2 days of deac-
climation.The survival rates of seedlings at
-35℃ for 12h increased from 0% in nonaccli-
mated seedlings to 30% in preacclimated
seedlings , and to 100% in cold-acclimated
seedlings.The temperature at which 100% of
seedlings survived are -15℃ in nonacclimated
and deacclimated seedlings , -20℃ in preaccli-
mated seedlings and -35℃ in cold-acclimated
seedlings.
In order to demonstrate in detail the effect of
low temperature on the development of freezing
tolerance of seedlings , we determined the kinetic
changes of freezing tolerance at -20℃ for 1 ～ 6
days and at 20℃ for 2 days (Figure 1).At 1
day after transferring seedlings from -10℃ to
-20℃, freezing tolerance of seedlings started
to increase distinctly from -20℃ to -23℃ and
continued to g radually increase during a period of
2 to 5 days to reach a maximum level(-35℃)
on the 6th day.After 2 days of deacclimation ,
the acquired freezing tolerance of seedlings dur-
ing acclimation of -20℃ decreased rapidly to
the level of nonacclimated seedlings(-15℃).
TABLE 1 The changes of the survival rates and freezing
tolerance of seedlings under dif ferent conditions
T reatment
The survival rates
of seedings at
-35℃ f or 12h / %
T he temperatu re at
which 100% of see-
dlings survived / ℃
Nonacclimated 0 -15
Preacclimated 30 -20
Cold-acclimated 100 -35
Deacclimated 0 -15
FIGURE 1 The effect of cold acclimation of
-35℃ on the freezing tolerance of seedlings
Preacclimated conditions w ere used as zero
time point
3.2 Effects of low temperature on the protein
content in branches of seedlings
Table 2 show s that the protein content in-
creased f rom 0.451mg·g-1 FW in branches of
nonacclimated seedlings to 0.554mg-1·g FW in
branches of preacclimated seedlings , and to
0.937 mg·g-1 FW in branches of cold-acclimat-
ed seedlings.That is to say , the protein content
has increased by 30% in branches of preaccli-
mated seedlings and by 120% in branches of
cold-acclimated seedlings as compared with that
of branches of nonacclimated seedlings.Af ter 2
days of deacclimation at 20℃, the protein con-
tent decreased from 0.937 mg · g-1 FW in
branches of cold-acclimated seedlings to 0.466
mg · g-1 FW in branches of deacclimated
seedlings , but the level of protein was still high-
er than that of nonacclimated seedlings.
To examine the changes of protein in branches
19Lin Shanzhi et al .
during cold acclimation , we determined the ki-
netics of protein content in branches during cold
acclimation at -20℃(Figure 2).The protein
content in branches increased from 0.554 mg·
g-1 FW to 0.653 mg·g-1FW as the 1day hold-
ing temperature decreased from -10℃ to -
20℃, and further increased gradually during a
period of 2 to 5 days to reach the highest amount
(0.937 mg·g-1 FW)on the 6th day.
TABLE 2 The changes of the content of protein and the
activities of G6PDHase and ATPase in branches under
different conditions
Treatment
Protein
content /
mg·g-1 FW
G6PDHase
activity /
n(enzyme unit)·
g-1 FW
ATPase
activity /
Umpi·g-1
apoenzyme·h-1
Nonacclimated 0.426 47.2 33.7
Preacclimated 0.553 67.4 44.8
Cold-acclimated 0.937 134.9 78.1
Deaccl imated 0.466 58.3 39.6
FIGURE 2 The effect of cold accl imation of
-20℃on protein content of seedlings
Preacclimated conditions w ere used as
zero time point
3.3 Effects of low temperature on the activi-
ties of G6PDHase and ATPase in branch-
es of seedlings
The branches of seedlings exhibited a signifi-
cant increase in the activities of G6PDHase and
ATPase after 6 days of preacclimation or cold ac-
climation compared to nonacclimated ones , re-
spectively , preacclimated branches showed a rel-
ative lesser.2 days of deacclimation at 20℃ re-
sulted in a rapid decrease in the activities of
G6PDHase and ATPase.Comparatively speak-
ing , the decrease in G6PDHase activity was
more marked , but they still maintained a higher
content than those of nonacclimated branches(
Table2).
The activities of G6PDHase and ATPase in
branches were daily on the increase during cold
acclimation at -20℃ and reached the maximum
levels of about 134.9 n·g-1FW and 78.1 Umpi
·g-1 apoenzyme· h-1 on the 6th day , respec-
tively .Comparing the G6PDHase with ATPase ,
there was obvious enhancement in the
G6PDHase activity during cold acclimation.For
example , the G6PDHase activity had been en-
hanced by 110% in branches of cold-acclimated
seedlings , whereas , the ATPase activity in
branches of the cold-acclimated seedlings had
been enhanced only by 70%(Table 2 and Fig-
ure 2).
3.4 Effect of cycloheximide(CH)pretreat-
ment on the protein content and freezing
tolerance
P.suaveollens seedlings pretreated w ith
20ppm cycloheximide(CH )during nonacclim-
ation for 6 days were transferred to -10℃ for 6
days and further transferred to -20℃ for 6
days , the changes of the protein content and
freezing tolerance of seedlings were showed in
Table 3 .After CH pretreatment , the protein
TABLE 3 The effect of cyclocheximide(CH)pretreatment
on the protein content and freezing tolerance
Treatment
Protein content
/mg·g-1FW
The temperature at
which 100% of see-
dl ings survived/ ℃
Nonacclimated 0.426 -15
Cold-acclimated 0.937 -35
CH-pretreated 0.426 -15
content and freezing tolerance decreased to the
levels of nonacclimation , in other w ords , the
temperature at which 100% seedlings survived
decreased f rom -35℃ to -10℃, at the same
time , the content of protein in branches had de-
creased about 55%.The results indicated that
the protein synthesis and the acquisition of freez-
ing tolerance could be inhibited by CH.
20 Forest ry Studies in China ,Vol.2 ,No.2 ,2000
FIGURE 3 The effect of cold accl imation of
-20℃ on the protein content of seedlings
Preacclimated content and freezing tolerance
4 Dicussion
4.1 Relationship between cold-acclimation
and freezing tolerance
Many plants have an ability to cold-acclimate
and to develop freezing tolerance , as exposure to
low temperature is a major trigger for the induc-
tion of freezing tolerance (Levitt 1980).The
acclimation response is a complex phenomenon
and freezing tolerance in plants is considered to
be a quantitative genetic trait , mainly influenced
by the genotype of the plant and the acclimating
temperature used(Hughes et al .1990 , Dunn et
al.1994).Although it has been reported that
cold-acclimation induced an increase in freezing
tolerance in many plant species(Levit t 1980 ,
Guy 1990 , Thomashow 1990), not all plants
are able to acclimate for f reezing tolerance and
not all temperatures are suitable for the induc-
tion of f reezing tolerance.For example , a low
temperature of 6℃ to 2℃ that can cold-accli-
mate genetically competent plants (e.g., cere-
als)will damage a chill-sensitive species(e.g.,
maize)(Hughes et al.1996).Studies by Shen
Zhenyan et al.(1983)had shown that in order
to acquire higher freezing tolerance , the tomoto
seedlings should be first transferred to a cooler
w ith a temperature of 5 ～ 10℃ for 5 days and
then further t ransferred to -2℃ for 5 days.
Our results clearly demonstrated that exposure
of the nonacclimated seedlings to low tempera-
ture of -10℃ or exposure of the preacclimated
seedlings to -20℃resulted in an increase in the
survival rates and freezing tolerance of seedlings ,
but the seedlings of acclimation at - 10℃
showed a relative lower(Table 1).Moreover ,
freezing tolerance of seedlings had been en-
hanced gradually during cold-acclimation at -
20℃ and reached the maximum level on the 6th
day (Figure 1).In addition , our previous stud-
ies found that the seedlings started to exhibit
damage when they were directly exposed to -
20℃ for 4h;if the seedlings that have been
preacclimated at -10℃ for 6 days were trans-
ferred to -20℃ for 6 days , they have acquired
the highest freezing tolerance (-35℃).These
facts indicated that the induction and the devel-
opmental process of f reezing tolerance in P.
suaveollens seedlings require a certain tempera-
ture and duration.The acquisition of the highest
freezing tolerance induced by adaptive cold accli-
mation will require two stage experiences.Al-
though the first stage of cold acclimation , in-
volving a temperature of -10℃ with 6days ,
had few effect on the increase of freezing toler-
ance , it might provide the basis for acclimation
at -20℃of the second stage and the acquisition
of freezing tolerance.A shift to low er tempera-
ture(from -10℃ to -20℃)had much effect
on the acquisition of freezing tolerance.There-
fore , to choose positive temperature which can
effectively acclimate but will not damage plant is
significant for the development of freezing toler-
ance.
4.2 Relationship between low temperature-in-
duced changes in the content of protein
and freezing tolerance
It has been reported that an increase of pro-
tein content in many plants induced by cold ac-
climation was directly correlated w ith the freez-
ing tolerance , the increase of protein content
21Lin Shanzhi et al .
during cold acclimation was accompanied by the
enhancement of freezing tolerance(Guy 1987 ,
1990 , Kazuoka et al .1992 , G raham et al.
1982 , Pan Jie et al.1994 , Lin Shanzhi 1997).
Moreover , the increase of protein content during
cold acclimation may play a specific role in con-
ferring freezing tolerance in plants(Guy et al.
1989 , 1990 , Perras et al .1989 , Arora et al.
1994).But few er investigator pointed out that
the amount of soluble protein has not essentially
increased during cold acclimation;the increase
of freezing tolerance may not be due to the in-
crease in soluble protein(Levit t 1980 , Uemura
et al .1996).It w as known from our experi-
ments that the amount of protein in branches
g radually increased during clod acclimation at
-20℃ and decreased after 2 days of deacclima-
tion at 20℃, but still maintained a relative high-
er compared to nonacclimated branches(Figure
2).The branches of cold-acclimatied seedlings
contained the highest amount of protein (
0.937mg·g-1 FW )and was the most freezing
tolerance(-35℃);the branches of nonaccli-
mated seedlings was the least freezing tolerance
(-15℃)and contained the low er amount of
protein(0.462mg·g-1FW)(Table 1 , Table 2
).Further analysis found that an increase in
protein content during clod acclimation at
-20℃was obviously paralleled by the acquisi-
tion of freezing tolerance(Figure 1 , Figure 2).
For example , the rise in protein amount to a
maximum on day6 concided w ith the highest
freezing tolerance on the 6th day.These results
showed that the protein was accumulated in re-
sponse to low temperature.An increase in pro-
tein content during cold acclimation may associ-
ate w ith the establishment of freezing tolerance ,
and may play a specific role in the cold-acclima-
tion process and in the regrow th of the cold-ac-
climated seedlings after deacclimation(Neven et
al.1993).
To examine whether the increase in protein
during cold acclimation at -20℃was correlated
with the acquisition of freezing tolerance , we
determined the changes of protein content and
freezing tolerance in the cycloheximide (CH)-
pretreated seedlings during clod acclimation at
-20℃(Table 3 ).After CH-pretreatment ,
the temperature at which 100% seedlings sur-
vived decreased from -35℃ to -10℃.At the
same time , the content of protein in branches
decreased by 55%.The fact that the decrease of
freezing tolerance was accompanied by the de-
crease of protein content indicates that the de-
crease of freezing tolerance may due to the inhi-
bition of protein synthesis resulting in a decrease
of protein content.An increase in the content of
cold acclimation-induced protein was directly
correlated w ith the acquisition of freezing toler-
ance.
It is , therefore , of interest to considering how
low-temperature-induced proteins might con-
tribute to the establishment of freezing tolerance
in plants.On the basis of recent studies , the
characteristics of the role of proteins can be sum-
marized as follow s:the direct affection on ext ra-
cellular ice formation;the indirect alteration in
the percentage of w ater frozen at a given tem-
perature by restricting the rate of grow th of ex-
tracellular ice crystals;the depression in the
freezing point of the body fluid and the stabiliza-
tion in cell membranes against either rupture or
loss of its semipermeablity(Duman et al .1991 ,
Kazuoka et al .1992 , Boothe et al.1995 ,
Hughes et al .1996).
4.3 Role of NADPH2 and ATP in freezing tol-
erance
ATPase in the cell membranes is thought as a
functional protein and plays a critical role in so-
lute transport , energy metabolism and so on
(Mito et al.1996).Early studies of changes in
ATPase with cold acclimation reported increase
22 Forest ry Studies in China ,Vol.2 ,No.2 ,2000
in the activity of A TPase that paralleled the ac-
quisition of freezing tolerance and a decline in
the activity of ATPase as freezing tolerance was
lost (Jian Lingchen et al.1983 , Zhao et al.
1993 , Salzman et al .1993 , Lin Shanzhi 1997).
Studies by Dai Jinpin et al(1991)has shown
that the plasma membrane ATPase in cucumber
seedlings has an acquisition of f reezing tolerance
during cold aaclimation at 5℃.Recent reports
have implicated cold liability of endomembrane
ATPase as a possible cause of cold-induced injury
in chilling sensitive plants , that is to say , AT-
Pase in cell membranes may be the primary sites
of freezing injury(Steponkus 1984 , Yoshida et
al.1989 , Jian Lingchen 1992 , Boothe et al.
1995 , Lin Shanzhi 1997).We observed that an
increase in ATPase activity induced by cold ac-
climation was paralleled not only by the increase
in protein content but also by the acquisition of
freezing tolerance(Figure 1 , Figure 2 and Fig-
ure 3).It appears that ATPase may play an im-
portant role in the induction of freezing toler-
ance , presumable at least in part by energizing
the synthesis of protein associated w ith freezing
tolerance.
Sagisaka (1985 ) first reported that the
G6PDHase activity in poplar twigs exhibited a
marked increase in fall and winter follow ed by a
g radual decrease in spring , and suggested that
an increase in G6PDHase activity was required
for the tolerant to freezing.As reported previ-
ously (Jian Lingchen 1990 , Liu Hong xian et
al.1991)and confirmed in this paper (Figure
3), the activity of G6PDHase in branches grad-
ually increased during cold acclimation at -20℃
and decreased af ter 2days of deacclimation at
20℃.Moreover , this increasing trend was par-
alleled by both the increase of the protein con-
tent and the acquisition of freezing tolerance
(Figure 1 , Figure 2 and Figure 3).The analy-
sis of the changes of the G6PDHase activity and
the ATPase activity found that the increasing
trend of the G6PDHase activity and the ATPase
activity w as very similar(Figure 3).These re-
sults indicated that increase in the G6PDHase
activity and the ATPase activity induced by cold
acclimationan was directly correlated w ith both
the increase of the protein content and the acqui-
sition of freezing tolerance.It is because that
G6PDHase is a key regulatory enzyme of pentose
phosphate cycle , cold acclimation-induced in-
crease in G6PDHase activity accomplanied by
the increase of ATPase activity will accelerate
pentose phosphate cycle , by which many inter-
mediates for use in protein synthesis and
NADPH2 are produced , NADPH2 can be used as
a specific elect ron donor for many biosynthesis
reaction and also be oxidated to ATP by bio-oxi-
dation-systems , ATP can be hydrolyzed to ADP
or AMP by ATPase and then generate many en-
ergy for biosynthesis.Therefore , an alteration
in metabolic pathway as the result of an increase
in G6PDHase activity induced by cold acclima-
tion was closely associated with the energy dissi-
pation as the result of an increase in A TPase ac-
tivity.The higher NADPH2 levels and increas-
ing A TP supply are essential for the synthesis of
protein and the induction of freezing tolerance.
5 Conclusion
This study indicates that freezing tolerance of
P.suaveollens seedlings can be effectively in-
duced by cold acclimation that w ill require expe-
riencing tw o stages in order to acquire the high-
est freezing tolerance.Although the first stage
of cold acclimation , involving a temperature of
-10℃, has few effects on the increase of freez-
ing tolerance , it may provide the basis for accli-
mation at -20℃of the second stage and the ac-
quisition of freezing tolerance.The fact that the
increase in activities of G6PDHase and ATPase
23Lin Shanzhi et al .
as a result of adaptive cold acclimation parallel
not only to the increase of protein content but al-
so to the enhancement of f reezing tolerance
shows that there is a close correlation among
them.An marked increase of G6PDHase and
ATPase induced by adaptive cold acclimation
may be a form of energy generation , and may be
a adaptive reaction to low temperature environ-
ment.Moreover , they may provide the energy
for the synthesis of protein associated w ith the
development of freezing tolerance , and for the
activation in enzymes correlated with the mem-
brane stability.So this may be just the cause of
cold acclimation-induced increase in freezing tol-
erance of P .suaveollens seedlings.
To further elucidate the mechanism of the ac-
quisition of freezing tolerance induced by cold ac-
climation in P .suaveollens seedlings , it is nec-
essary to isolate cold acclimation-induced specific
protein that may be involved in the development
of freezing tolerance , to determine the nu-
cleotide sequence of this protein and to identify
the function of this protein from its deduced
amino acid sequences.
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25Lin Shanzhi et al .
甜杨抗冻性低温诱导过程中 G6PDHase ,ATPase及蛋白质的变化
林善枝 张志毅
(北京林业大学生命科学和生物技术学院 ,北京 100083)
摘要 首次研究了低温锻炼对甜杨(Populus suaveot lens)幼苗抗冻性的效应 , 并对低温锻炼中的
G6PDHase ,ATPase及蛋白质的动态变化过程进行了测定.另外 ,在低温锻炼前用蛋白质合成抑制剂
环己亚胺对甜杨幼苗进行预处理.试验结果表明 ,适当的低温锻炼可明显提高甜杨幼苗的抗冻性 ,但
整个低温锻炼过程必须分两个阶段进行 ,第一阶段在-10℃的温度下锻炼 6 天 ,虽对甜杨幼苗抗冻性
的提高效应不明显 ,但它是不可缺少的 ,它可为第二阶段的-20℃锻炼的进行及抗冻性的完全发育提
供基础.伴随着幼苗枝条中蛋白质含量及 G6PDHase和 ATPase 活性提高的同时 ,也明显提高了幼苗
的抗冻性;脱锻炼 2天后 ,幼苗抗冻性下降到未锻炼水平 ,而枝条中蛋白质含量及 G6PDHase 和 A T-
Pase活性虽有下降但略高于未锻炼.蛋白质合成抑制剂环己亚胺预处理则明显降低了幼苗抗冻性和
蛋白质含量.进一步分析发现 ,低温锻炼中的 G6PDHase 和 ATPase活性及蛋白质含量的变化与幼苗
抗冻性的提高密切相关.
关键词 甜杨 ,低温锻炼 ,抗冻性 ,蛋白质
26 Forest ry Studies in China ,Vol.2 ,No.2 ,2000