全 文 ：植物学通报 2004, 21 (3): 296~305
Chinese Bulletin of Botany
①Foundation items: Supported by the National Natural Science Foundation of China(No.30170081)and by the
Chengdu Science ＆ Technology Foundation.
②Author for correspondence. E-mail: honghui968@sina.com
Received: 2003-03-18 Accepted: 2003-04-01 Managing editor: SUN Dong-Hua
不同低温胁迫对烟草愈伤组织抗氰交替途径
诱导和交替氧化酶表达影响的比较①
晏婴才 林宏辉② 梁厚果 张年辉
（四川大学生命科学学院 成都 610064）
摘要 比较了不同低温（14℃和4℃）胁迫对烟草（Nicotiana rustica L.）愈伤组织抗氰交替途径诱导
和交替氧化酶表达的影响。 结果显示，不同低温胁迫处理能显著诱导烟草愈伤组织交替途径容量和实
际运行的增加，且都呈现出基本相同的变化模式：在胁迫的初期（1～3 d）持续增加，在3 d时达到最
高，而后下降到一个相对恒定的水平。但交替途径容量增加的幅度与温度下降的程度密切相关，而交替
途径实际运行量的诱导程度在不同低温胁迫下的差异却很小。 表明交替途径容量和实际运行对低温胁
迫的响应是不同的。免疫印迹分析结果表明：低温胁迫明显诱导了交替氧化酶总蛋白的增加，且其随低
温胁迫进程的变化与交替途径容量的变化基本一致；而对交替氧化酶单体与二聚体在低温胁迫下的含量
变化检测结果则显示，烟草愈伤组织中交替氧化酶主要以二聚体形式存在，且这一存在形式并不随低温
胁迫程度的加深而发生改变。两种形式的交替氧化酶蛋白含量都能被低温胁迫诱导增加，但其单体水平
在两种不同的低温胁迫下并无明显差别，而4℃低温胁迫诱导的二聚体交替氧化酶蛋白含量明显高于14
℃。表明不同程度低温对抗氰交替途径发生的不同影响主要是由于对交替氧化酶蛋白二聚体形式的不同
诱导程度所致；而高活性的交替氧化酶单体形式则不因低温胁迫程度的加重而被明显诱导升高，使得抗
氰交替途径的运行程度在两种不同的低温胁迫处理条件下无显著差异。
关键词 烟草愈伤组织, 低温胁迫, 抗氰交替途径, 交替氧化酶表达
Comparison of the Effects of Different Low Temperature
Stresses on the Induction of the Cyanide-Resistant
Alternative Pathway and the Expression of
Alternative Oxidase in Tobacco Callus
YAN Ying-Cai LIN Hong-Hui② LIANG Hou-Guo ZHANG Nian-Hui
(College of Life Sciences, Sichuan University, Chengdu 610064)
Abstract Effects of different low temperature stresses (14℃ and 4℃) on cyanide-resistant alterna-
tive pathway development and operation, as well as on alternative oxidase expression in plant calli of
tobacco (Nicotiana rustica L.) were compared. The results showed that both alternative pathway
研 究 论 文
2972004 晏婴才等：不同低温胁迫对烟草愈伤组织抗氰交替途径诱导和交替氧化酶表达影响的比较
capacity (Valt) and its actual operation activity (rValt) were dramatically induced under the two
different low temperature stresses, and the change patterns of Valt and rValt were similar: both of
them were induced at the early stage of stress treatment, the highest was found on 3 d and then
declined to a relatively constant level. The increase of Valt was in relation to the temperature decreased,
while no significant difference of rValt induced between 14℃ and 4℃. This suggests that responses
of Valt and rValt to different temperature stress are different. The immunoblot results indicated that
low temperature stress remarkably induced the content of total alternative oxidase (AOX) protein,
and the changing mode throughout the stress was similar to Valt. The results of variation in AOX
monomer and homodimer contents under stress treatment showed that AOX is largely located in
mitochondria membrane of tobacco callus as the oxidized form (homodimer), and the existing state
could not be changed by low temperature stress. The amount of monomer and homodimer both
induced by low temperature stress, but no appreciable differences in the content of monomer was
observed between two different low temperatures, and the level of homodimer induced under 4℃ was
higher than that under 14℃. Our results suggest that, effects of low temperature on the development
of cyanide-resistant alternative pathway are mainly due to the differently induced content of AOX
homodimer under different low temperatures. The content of highly active AOX monomer form is not
obviously induced by aggravating low temperature stress, thus the operation degree of cyanide-
resistant alternative pathway shows no notable difference between two kinds of low temperatures
tested.
Key words Tobacco callus, Low temperature stresses, Cyanide-resistant alternative pathway, Ex
pression of AOX
The higher plant mitochondria electron transport chain contains, in addition to the cytochrome
chain which terminates with cytochrome oxidase, an alternative pathway that terminates with an alter-
native oxidase(AOX)(Siedow and Umbach, 1995; Vanlerberghe and McIntosh, 1997). Because of the
special heat-instability, AOX is very difficult to be eluted from the inner membrane of mitochondria and
maintained the activity(Liang and Liang,1998). Until 1987, AOX was successfully purified from mito-
chondria of Sauromatum guttatum Schott (voodoo lily) and the polyclonal antibodies raised to AOX
were prepared(Elthon and McIntosh,1987). Two years later, monoclonal antibodies against AOX were
produced and designated as AOA, AOU and AOL(Elthon et al.,1989). With the polyclonal and mono-
clonal antibodies against AOX, the studies on cyanide-resistant respiration were carried out in-depth
at molecular level to elucidate the mechanisms of its development and operation. In addition, the anti-
synthetic polypeptide antibodies against AOX using synthetic polypeptide were prepared in our lab
and used to study the relationship between cyanide-resistant respiration and the expression of AOX
in mung bean(Li et al., 2000) and to confirm its detecting effects on AOX expression in tobacco callus
as compared with AOX monoclonal antibody(Yan et al.,2002).
The reported studies showed cyanide-resistant respiration exists in a great variety of plant species
(Moore and Siedow, 1991), and its development and operation are, in addition related with the internal
development and physiological status of the plant(Liang and Liang,1997), affected by many external
298 21(3)
factors such as environmental stresses those can influence the ability of cyanide-resistant respiration
or change the degree of its engagement (Jolivet et al., 1990; Rycher et al., 1992; Zhang et al., 2001).
Low temperature is a general environmental stress, and its effects on cyanide-resistant respiration
have been investigated with different plant tissues or organs(Stewart et al., 1990a; 1990b; Vanlerberghe
and McIntosh,1997; Meler et al., 1999). The results indicated that Valt is higher in plants grown at low
temperature (10-15℃) compared to those controls grown at near optimal temperature (25-30℃), so is
the AOX protein level. However, in most of these studies, only one kind low temperature was applied,
and merely examined the response of alternative pathway respiration to short-term low temperature
stress. Moreover, some studies showed AOX located in inner membrane of mitochondria with two
forms: oxidized homodimer and reduced monomer(Umbach and Siedow, 1993), and the activity of
monomer is 4-5 folds higher than that of homodimer(Moore et al., 1995). However, only the total AOX
protein content induced by low temperature was investigated in most of the previous studies, the
relative changes of AOX homodimer and monomer were not concerned. We have found the cyanide-
resistant respiration existed obviously in tobacco callus(Liang et al., 1984), and was significantly
enhanced when treated with 4℃ low temperature(Zhou et al., 2000). In the present study, tobacco
callus were used to measure the dynamic changes of the cyanide-resistant respiration throughout the
low temperature stresses of 14℃ and 4℃. The changes of the total AOX content and the relative
amount of AOX homodimer and monomer were detected in the stress course. Our objective is to better
compare the effects of different low temperature stresses on cyanide-resistant respiration and the
relationship between the ability of cyanide-resistant respiration and the expression of AOX under low
temperature stresses.
1 Materials and Methods
1.1 Plant material and low temperature treatment
Tobacco (Nicotiana rustica L.) callus was dedifferentiated from seedling leaves on a basal
Murashige and Skoog medium supplemented with 2, 4-D(2 mg.L-1), 6-BA(0.5 mg.L-1), 3%(W/V)
sucrose and 0.8%(W/V) agar(pH 5.8). The calli were maintained in a growth chamber at 25℃ and
illuminated with a 12 h photoperiod at light intensity of 100 mmol.m-2.s-1, and subcultured once a
month. When grown to the 15-day stage, tobacco calli were treated with low temperatures of 14℃ or
4℃ under the same photoperiod and light intensity. Control samples were maintained at 25℃, and all
samples were grown for 1, 3, 5 and 7 d under the above light and temperature conditions. At different
stages of low temperature stress, the respiration parameters were determined and the mitochondria
were isolated in stressed or non-stressed tobacco callus.
1.2 Determination of tobacco callus respiration
The respiratory rates of tobacco callus were measured and calculated according to the equations
described by Liang and Lü(1984) with Clark-type oxygen electrode at 25℃ in a dark room. The respi-
ratory parameters were determined by Bahr and Bonner’s hydroxamic acid titration method(Bahr and
Bonner,1973), the data were calculated with the equation modified by Theologis and Laties(1978):
2992004 晏婴才等：不同低温胁迫对烟草愈伤组织抗氰交替途径诱导和交替氧化酶表达影响的比较
Vt=rValt +r Vcyt + Vres
Where, Vt is the total respiratory rate, r Vcyt is the cytochrome-mediated respiration, Vres is the
residual O2 uptake which is not inhibited by KCN plus SHAM, and r presents the fraction of the
alternative pathway which is operating, and Valt is the maximal capacity of the alternative pathway.
Accordingly, rValt represents the extent of operation activity of the alternative pathway in the ab-
sence of inhibitor. Optimal concentrations of inhibitors used with tobacco calluses were: KCN
1 mmol.L-1; SHAM 1 mmol.L-1. The respiration rate was calculated as mLO2.g-1 FW.h-1. The data
presented were the means of at least four independent measurements.
1.3 Isolation and purification of tobacco callus mitochondria
Mitochondria were isolated and purified from tobacco callus suffered from different degree of low
temperatures based on the procedure of previous study(Wen and Liang,1993). Purified mitochondria
were resuspended in washing medium devoid of bovine serum albumin and stored at -20℃. Protein
concentration was measured according to the method of Bradford (1976).
1.4 SDS-PAGE and immunoblotting analysis of mitochondrial protein
Mitochondria proteins were separated by reducing and nonreducing SDS-PAGE according to the
methods described by Umbach and Siedow(1993) and Vanlerberghe et al. (1998). For reducing SDS-
PAGE, mitochondria (50 mg of protein) were dissolved in equal volume sample buffer (10% [V/V] b
mercaptoethanol, 20%[V/V] glycerol, 4%[W/V] SDS, 0.005%[W/V] bromophenol blue and
50 mmol.L-1 Tris, pH 6.8 ) and incubated in boiling water bath for 3 min. Nonreducing SDS-PAGE
analysis was performed without b-mercaptoethanol in sample buffer. SDS-PAGE analysis was per-
formed according to the standard method of Laemmli (1970). A 5% (W/V) polyacrylamide stacking gel
and 13% polyacrylamide gradient resolving gel were used. The resolved proteins were then electro-
transferred to a nitrocellulose membrane and probed with a 1:100 dilution of a monoclonal antibody
(AOA) raised against the Sauromatum guttatum AOX by the standard method of Sambrook et al.
(1989). The secondary antibody was goat-anti-mouse IgG conjugated with alkaline phosphatase (diluted
1:500). The color was developed by reaction with color buffer supplemented with r-nitro blue tetrazo-
lium chloride and 5-bromo-4-chloro-indolyl phosphate.
2 Results
2.1 Effects of different low temperature stresses on capacity of alternative pathway in tobacco callus
The capacity of alternative pathway(Valt) refers to its maximal engagement when cytochrome
pathway (CP) is completely inhibited. It represents the maximal extent to which alternative pathway
(AP) is engaged in vivo(Day et al., 1995). AP operation is dependent upon the degree of its development
(McIntosh, 1994). For this reason, we first examined the effects of low temperature (14℃ and 4℃)
stresses on AP capacity. The results in Fig.1 indicate that, low temperature stress could dramatically
induce the increase in Valt during tissue recovering. However, the changing models of Valt under
different low temperatures were not so consistent. When the calli were treated with 4℃ for 1 d, Valt
gave rise to 137.1 mLO2.g-1 FW.h-1 from 83.6 mLO2.g-1 FW.h-1 in control. By contrast, Valt at 14℃
300 21(3)
engagement when there is no inhibitor on cytochrome pathway, it represents the actual demanded
extent of AP for plant tissue in specific physiological conditions(Day et al., 1995). Though the opera-
tion of AP is dependent on its development, the developed alternative pathway respiration is not
necessarily engaged totally. So, we also determined changes in rValt at different low temperatures. As
shown in Fig. 2, there was a rapid increase in rValt in the first three days of low temperature treatment.
On 3 d of the stress, rValt increased to 128.3 mLO2.g-1 FW.h-1 from 80.1 mLO2.g-1 FW.h-1 at 14℃
treatment and from 88.1 mLO2.g-1 FW.h-1 to 134.1 mLO2.g-1 FW.h-1 under 4℃, increasing by 60.2％
and 52.2％, respectively. As compared with the control, rValt increased by 87.8％ under 14℃ and
○. Control; ■. Treated with 14℃; ▲. Treated with 4℃
Fig.1 Effects of different low temperature stresses on
capacity of AP in tobacco callus
went up to 93.1 mLO2.g-1 FW.h-1. It increased
by 64.0％ and 11.4％ of control respectively.
Following the time of stress, Valt remained rais-
ing until it reached the highest level observed
on 3 d of 166.4 mLO2.g-1 FW.h-1 under 4℃ and
137.8 mLO2.g-1 FW.h-1 under 14℃, increas-
ing by 69.6％ and 40.5％, respectively, in com-
parison to control. Thereafter, both of the
Valt under 14℃ and 4℃ declined slightly, and
then kept on relatively stable level which is
still significantly higher than the value of
control.
2.2 Effects of different low temperature
stresses on activity of AP in tobacco callus
The activity of AP (rValt) is the actual
○. Control; ■. Treated with 14℃; ▲. Treated with 4℃
Fig.2 Effects of different low temperature stresses on AP
activity in tobacco callus
96.3％ under 4℃ on 3 d of the low tempera-
ture stress. Unlike Valt, rValt decreased soon
after it reached its peak, and then maintained
at about the same level of each. So, the in-
duced pattern of rValt under different low
temperatures was nearly the same, only for
the extent induced under 4℃ is higher than
that induced under 14℃.
2.3 Effects of different low temperature
stresses on total AOX protein in tobacco cal-
lus
The changes in alternative pathway ca-
pacity in plant tissues were dependent upon
the expression of AOX protein, and their
changes were usually in parallel (McIntosh,
3012004 晏婴才等：不同低温胁迫对烟草愈伤组织抗氰交替途径诱导和交替氧化酶表达影响的比较
1994; Day et al.,1995). In order to analyze effects of different low temperature stresses on AOX protein
expression, mitochondria were isolated from tobacco calli treated with low temperatures for 1, 3, 5 and
7 d and the controls grown for the same time. After separated by reducing SDS-PAGE, mitochondria
proteins were probed with the monoclonal antibody of AOA. As shown in Fig.3A and Fig.3B, the
change pattern of total AOX protein content under the two low temperatures were the same: there was
an increase at the early stage of stress, maximal AOX amount was induced on 3 d, after then (on 5 d and
7 d), it showed no appreciable variation that was still higher than that in control. The changes of the
total AOX protein content under different low temperature stress treatment showed excellent correla-
tion with measurement of Valt (Fig.1). The results of Fig.3 also indicate the level of the total AOX
protein at each stress treatment time under 4℃ was higher than that under 14℃. This is also in good
coincidence with our results of higher Valt induced under 4℃.
2.4 Effects of different low temperature stresses on AOX homodimer and monomer in tobacco callus
To further examine changes in AOX homodimer and monomer under low temperature stresses,
mitochondrial protein was separated by nonreducing SDS-PAGE and analyzed with immunoblotting.
As shown in Fig. 4A and Fig. 4B, in tobacco callus grown at either low or normal temperature, the level
of 70 kD AOX protein, i.e., the oxidized form, was higher than that of 35 kD protein of AOX monomer
form. As far as change mode of each AOX form under low temperature stresses was concerted, 70 kD
protein was markedly induced during the early three days. Afterwards, no obvious changes could be
seen. To the contrary, though maximal levels of 35 kD protein were induced on 3 d, its expression
declined slightly following the stress. However, if the comparison was made between total AOX
protein content at the same time of different temperature stress treatment the same amount of 35 kD
protein was found under 14℃ and 4℃. So, the changes of 35 kD AOX protein level under different
temperatures were generally in accordance to rValt at 14℃ and 4℃.
Fig. 3 Immunoblots of mitochondrial protein separated by reducing SDS-PAGE
Mitochondria were isolated from tobacco callus treated with different low temperature stresses for 1, 3, 5 and
7 d. 1. Control; 2. Treated with 14℃; 3. Treated with 4℃. Equal amounts (50 mg) of mitochondrial protein were
loaded in each lane and blotted with the AOA monoclonal antibody
302 21(3)
3 Discussion
Low temperature stress can significantly enhance the ability of the cyanide-resistant respiration
in plants. Our results also showed that low temperature stresses markedly induced an increase in the
capacity and activity of alternative pathway in tobacco callus. We further compared the dynamic
changes of Valt and rValt under different temperature stresses. The results showed that their change
patterns were nearly the same: they increased at the early stage of low temperature treatment, reached
its maximal level on 3 d and began to decline, then maintained at a stable level. This indicates that the
induction of alternative pathway has an adaptation process. Though Valt was induced at 14℃ and 4℃,
and the induced extent at 4℃ was higher than that at 14℃, the actual operation(rValt) under the two
temperatures had no significant difference, indicating that Valt is inclined to be influenced by low
temperatures, and the responses of alternative pathway development and operation to different low
temperature are different.
AOX activity can be rapidly regulated by means of interconversion of its homodimer and monomer,
and plant is capable of fast adapting to the changes of environmental conditions by this way(Rhoads
et al., 1998). Furthermore, the results of some previous works also indicated AP capacity(Valt) and the
level of AOX protein are in parallel (McIntosh, 1994; Day et al., 1995; Vanlerberghe et al., 1995). Here,
we show that changes in total AOX protein content (Fig. 3) are in relation to Valt throughout the stress.
Moreover, the analysis of the changes in relative extent of AOX homodimer and monomer demon-
strated that, though the difference of AOX homodimer content under different temperatures was
remarkable, there was no significant difference in AOX monomer content. Because the activity of
monomer was 4-5 folds higher than that of homodimer(Moore et al., 1995), and rValt was mainly
decided by the content of the monomer, the measurement of little difference in rValt at the two tempera-
tures may be a result of nearly the same extent of AOX monomer induced. However, the increase in Valt
induced by low temperatures mainly depends upon the increase in total AOX protein, especially upon
Fig. 4 Immunoblots of mitochondrial protein separated by nonreducing SDS-PAGE
Mitochondria were isolated from tobacco callus treated with different low temperature stresses for 1, 3, 5 and
7 d. 1. Control; 2. Treated with 14℃; 3. Treated with 4℃. Equal amounts (50 mg ) of mitochondrial protein were
loaded in each lane and blotted with the AOA monoclonal antibody
3032004 晏婴才等：不同低温胁迫对烟草愈伤组织抗氰交替途径诱导和交替氧化酶表达影响的比较
the increase in AOX homodimer. So, the results of our study not only find distinct effects of different
low temperatures on plant alternative pathway development and operation, in addition, we further
elucidate the mechanisms of the differences at AOX protein expression level.
The operation of alternative pathway could result in heat evolution(Kapulnik et al., 1992; Wen
and Liang, 1994; Vanlerberghe and McIntosh, 1997). Accordingly, plants can be classified into typical
thermogenic plants and atypical thermogenic plants. For the AOX existing states in the mitochondria
of the two kinds plants, Kinden and Akerlund implied that AOX exists with generally high-activity
monomer in typical thermogenic plant mitochondria and with low-activity homodimer in atypical ther-
mogenic plant(Lidén and Akerlund,1993). In our study, we found that, AOX in tobacco callus mito-
chondria was largely existed in its oxidized state (homodimer), and low temperature stress could not
modify the status of AOX protein in mitochondria. It provided good direct evidence for the implication
of Kinden and Akerlund that there was different AOX protein existing states in the two kinds of plants.
Acknowledgement We thank Dr Thomas E. Elthon (University of Nebraska) for the generous gift
of the OA monoclonal antibody.
References
Bahr J T, Bonner W D Jr, 1973. Cyanide-insensitive respiration. 1. The steady states of Skunk cabbage spadix and
bean hypocotyl mitochondria. J Biol Chem, 248: 3441~3445
Bradford M M, 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing
the principle of protein-dye binding. Anal Biochem, 72: 248~254
Day D A, Whelan J, Millar A H, Siedow J N, Wiskich J T, 1995. Regulation of alternative oxidase in plants and fungi.
Aust J Plant Physiol, 22: 497~509
Elthon T E, McIntosh L, 1987. Identification of the alternative terminal oxidase of higher plant mitochondria. Proc
Natl Acad Sci USA, 84: 8399~8403
Elthon T E, Nickels R L, McIntosh L, 1989. Monoclonal antibodies to the alternative oxidase of higher plant
mitochondria: dependence on tricarboxylic acid cycle-mediated redox regulation and pyruvate activation. Plant
Physiol, 89: 1311~1317
Jolivet Y, Pireaux J C, Dizengremel P, 1990. Changes in properties of barley leaf mitochondria isolated from NaCl-
treated plants. Plant Physiol, 94: 641~646
Kapulnik Y, Yalpani N, Raskin I, 1992. Salicylic acid induces cyanide-resistant respiration in tobacco cell-suspension
cultures. Plant Physiol, 100:1921~1926
Laemmli U K, 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature,
227: 680~685
Li C J(李驰峻), Liang H G（梁厚果）, Du L F（杜林方）, Wang R（王锐）, 2000. The relationship between
the activity of cyanide-resistant respiration and the expression of alternative oxidase in different organs of
mung bean seedlings. Acta Bot Sin（植物学报）, 42: 646~648 (in Chinese)
Liang W S(梁五生), Liang H G(梁厚果), 1997. Progress of the study on alternative oxidase. Chin Bull Bot(植物学
304 21(3)
通报), 14(2): 9~16（in Chinese）
Liang H G（梁厚果），Liu L H(刘良寰)，Lü T H(吕太和)，Pu T L(浦铜良), 1984. Studies on the CN-
resistant respiration in callus of Nicotiana rustica, Gansu yellow flower. Acta Bot Sin（植物学报）, 26：
499~505 ( in Chinese)
Liang H G, Lü Z S, 1984. A comparative study of CN-resistant respiration in different cultures of tobacco callus. Plant
Physiol, 75: 876~878
Liang Z(梁峥)，Liang H G(梁厚果), 1998. The respiratory metabolism of plants. In: Yu S W(余叔文)，Tang Z C
(汤章城) eds. Plant Physiology and Molecular Biology. 2nd ed. Beijing: Science Press, 344~365 ( in Chinese)
Lidén A C, Akerlund H E, 1993. Induction and activation of the alternative oxidase of potato tuber mitochondria.
Physiol Plant, 86: 134~141
McIntosh L, 1994. Molecular biology of the alternative oxidase. Plant Physiol, 105: 781~786
Meler M A G, Carbo M R, Giles L, Siedow J N , 1999. The effect of growth and measurement temperature on the
activity of the alternative respiratory pathway. Plant Physiol, 120: 765~772
Moore A L, Siedow J N, 1991. The regulation and nature of the cyanide-resistant alternative oxidase of plant
mitochondria. Biochim Biophys Acta, 1059: 121~140
Moore A L, Umbach A L, Siedow J N, 1995. Structure-function relationships of the alternative oxidase of plant
mitochondria: a model of the active site. J Bioenerg Biomembr, 27: 367~377
Rhoads D M, Umbach A L, Sweet C R, Lennon A M, Rauch G S, Siedow J N, 1998. Regulation of the cyanide-resistant
alternative oxidase of plant mitochondria . J Biol Chem, 273: 30750~30756
Rychter A M, Chauveau M, Bomsel J L, Lance C, 1992. The effect of phosphate deficiency on mitochondrial activity
and adenylate in bean roots. Physiol Plant, 84: 80~86
Sambrook J, Fritsch E F, Maniatis T, 1989. Molecular clone: a laboratory manual 2nd ed. New York: Cold Spring
Harbor Laboratory Press
Siedow J N, Umbach A L, 1995. Plant mitochondrial electron transfer and molecular biology. Plant Cell, 7: 821~831
Stewart C K, Martin B A, Reding L, Cerwick S, 1990a. Respiration and alternative oxidase in corn seedling tissues
during germination at different temperatures. Plant Physiol, 92: 755~760
Stewart C K, Martin B A, Reding L, Cerwick S, 1990b. Seedling growth, mitochondrial characteristics, and alternative
respiratory capacity of corn genotypes differing in cold tolerance. Plant Physiol, 92:761~766
Theologis A, Laties G G, 1978. Relative contribution of cytochrome-mediated and cyanide-resistant electron trans-
port in fresh and aged potato slices. Plant Physiol, 62: 232~237
Umbach A L, Siedow J N, 1993. Covalent and noncovalent dimmers of the cyanide-resistant alternative oxidase
protein in higher plant mitochondria and their relationship to enzyme activity. Plant Physiol, 103: 845~854
Vanlerberghe G C, Day D A, Wiskich J T, Vanlerberghe A E, McIntosh L, 1995. Alternative oxidase activity in tobacco
leaf mitochondria. Plant Physiol, 109: 353~361
Vanlerberghe G C, McIntosh L, 1997. Alternative oxidase: from gene to function. Annu Rev Plant Phys Plant Mol
Biol, 48: 703~734
Vanlerberghe G C, McIntosh L, Yip J Y H, 1998. Molecular localization of a redox-modulated process regulating plant
mitochondrial electron transport. Plant Cell, 10: 1551~1560
3052004 晏婴才等：不同低温胁迫对烟草愈伤组织抗氰交替途径诱导和交替氧化酶表达影响的比较
Wen J Q, Liang H G, 1994. Comparison of the effects of salicylic acid on the alternative pathway in slices of dormant
and dormancy-breaking potato tubers. Plant Sci, 102:127~131
Wen J Q, Liang H G, 1993. Studies on energy status and mitochondria respiration during growth and senescence of
mung bean cotyledons. Physiol Plant, 89: 805~810
Yan Y C(晏婴才), Lin H H(林宏辉), Liang H G（梁厚果）, Du L F（杜林方）, 2002. A comparative study on
detection of the expression of alternative oxidase in tobacco callus with the monoclonal antibody against
alternative oxidase and antibody against-synthetic polypeptide antibody. Acta Bot Sin（植物学报）, 44:
1255~1257 (in Chinese)
Zhang N H(张年辉)，Wei Z Q(韦振泉)，He J X(何军贤)，Liang H G(梁厚果), 2001. Responses of the cyanide-
resistant respiration to mild water stress in wheat leaves. Acta Bot Boreal-Occident Sin (西北植物学报)， 21：
21~25 ( in Chinese)
Zhou G K（周功克）, Li H Y（李红玉）, Wen J Q（文江祁）, Kong Y Z（孔英珍）, Liang H G（梁厚果）,
2000. The cyanide-resistant respiration in callus of Nicotiana rustica cv. Gansu yellow flower under low
temperature. Acta Bot Sin（植物学报）, 42: 679~683 (in Chinese)