Plant Physiol.

(1 992) 100, 1846-1851

0032-0889/92/100/1 846/06/$01 .00/0

Received for publication June 23, 1992

Accepted August 20, 1992

Coordinate Regulation of Cytochrome and Alternative

Pathway Respiration in Tobacco1
Greg C. Vanlerberghe and Lee McIntosh*
Michigan State University/Department of Energy Plant Research Laboratory and Biochemistry Department,
Michigan State University, East Lansing, Michigan 48824
ABSTRACT

It seems imperative that plants be able to regulate in a

coordinate fashion the partitioning of electrons between the
CP and the AP to meet changing metabolic requirements.
Two important factors to be regulated will be, first, the
capacity of each pathway to transfer electrons, and second,
the degree to which that capacity is used (i.e. the degree of
engagement of the pathway). The degree of engagement of
the AP is dependent upon the redox poise of the ubiquinone
pool (9) and, to some extent, upon the substrates being
oxidized by the mitochondria (7).
Although many studies indicate that plants are capable of
altering the capacity of the AP (6, 14), little is known about
the mechanisms by which this occurs. Some studies show
that inhibition of the CP with respiratory inhibitors increases
AP respiration. For example, treatment of rice roots with low
concentrations of KCN or NaN3 for 6 h resulted in increased
CN-resistant respiration, although it was not shown whether
this CN-resistant respiration was sensitive to SHAM (11). In
pea roots, a CN-resistant and SHAM-sensitive component of
respiration was induced after several hours of KCN treatment
(28). From the above reports, it is not clear whether the KCN
treatment increased the capacity or simply the engagement
of the AP. Also, interpretation of the data is hampered
because KCN is known to inhibit many enzymes of metabolism (29). Nevertheless, inhibition of the CP by a specific
respiratory inhibitor may be a useful tool to study mechanisms of induction of AP capacity in plants.
AA is a specific inhibitor that blocks electron transfer
between Cyt b and cl in the CP (20, 23). In Euglena gracilis,
addition of AA induced CN-resistant respiration within 5 h
(2-4). A similar induction was seen in the yeast Hansenula
anomala (17) and led to the identification of a 36-kD mitochondrial protein thought to be responsible for CN-resistant
respiration in this organism (16) and the isolation of a cDNA
clone encoding this protein (21). In the fungus Neurospora
crassa, the ability of AA to induce AP respiration has been
exploited to select mutants defective in AP respiration (5).
AA has been extensively used as a respiratory inhibitor in
higher plants (24) but, to our knowledge, the ability of AA
to induce AP respiration in higher plants has not been
investigated. In this paper, we show that treatment of suspension cells of tobacco (Nicotiana tabacum) with AA increases AP capacity, and we identify mechanisms involved
in this process. We also show that CP and AP capacities can
be regulated in a coordinate fashion.

In suspension cells of NT1 tobacco (Nicotiana tabacum L. cv

bright yellow), inhibition of the cytochrome pathway of respiration
with antimycin A induced a large increase in the capacity of the
alternative pathway over a period of approximately 12 h, as confirmed in both whole cells and isolated mitochondria. The increase
in alternative pathway capacity required de novo RNA and protein
synthesis and correlated closely with the increase of a 35-kD
alternative oxidase protein. When the cytochrome pathway of
intact cells was inhibited by antimycin A, respiration proceeded
exclusively through the alternative pathway, reached rates significantly higher than before antimycin A addition, and was not
stimulated by p-trifluoromethoxycarbonylcyanide (FCCP). When
inhibition of the cytochrome pathway was relieved, alternative
pathway capacity and the level of the 35-kD alternative oxidase
protein declined. Respiration rate also declined and could once
again be stimulated by FCCP. These observations show that the
capacities of the mitochondrial electron transport pathways can be
regulated in a coordinate fashion.

Mitochondrial electron transport in plants may proceed

through either the CP2 or the AP (6, 12, 14). Transfer of
electrons from NADH through the CP is coupled at three
sites to the translocation of protons from the matrix to the
intermembrane space. Reentry of protons to the matrix
through complex V (mitochondrial ATP synthetase) results
in the production of ATP. In this case, carbon oxidation in
respiration is coupled to the production of ATP, and in many
instances respiration rate is tightly regulated by the availability of ADP (i.e. adenylate control) (8, 12). Transfer of
electrons through the AP bypasses two of the three sites of
proton translocation. Under these conditions, carbon oxidation is not as tightly coupled to the production of ATP, and
it is expected that respiration rate would not be as strictly
regulated by the availability of ADP (8, 12).
This work was supported in part by Department of Energy Grant
DE-FG02-90ER20021 (to L.M.) and National Science Foundation
Grant D.C.B. 890451B (to L.M.). G.C.V. acknowledges support from
a Natural Sciences and Engineering Research Council of Canada
Postdoctoral Fellowship.
'Abbreviations: CP, cytochrome pathway; AA, antimycin A; AO,
alternative oxidase; AP, alternative pathway; FCCP, p-trifluoromethoxycarbonylcyanide; mETC, mitochondrial electron transport
chain; SHAM, salicylhydroxamic acid.
1

1846

CYTOCHROME AND ALTERNATIVE PATHWAY RESPIRATION

1847

MATERIALS AND METHODS

Organism and Culture Conditions

Suspension cells of NT1 tobacco (Nicotiana tabacum L. cv
bright yellow) were grown in axenic batch culture under
heterotrophic conditions in the medium previously described
(15). This medium contains 3% (w/v) sucrose as a carbon
source. The cells were grown on a rotary shaker at 300C and
were subcultured every 7 d to a density of 4% packed cell
volume. For experiments, cells were used at approximately 3
d after subculture when culture density was approximately
15 to 20% packed cell volume. All experiments were performed two to four times and representative results are
shown.
Respiration

E
0
E

a
0

C,

Cells (approximately 15-40 mg fresh weight) in their culture medium were placed in a Rank Brothers 02 electrode
cuvette at 300C. Steady rates of respiratory 02 consumption
could be determined after about 2 to 5 min under these
conditions. Respiratory pathway capacities were then determined as previously described (27). Briefly, CP capacity was
taken to be that portion of the 02 consumption inhibited by
1 mM KCN in the presence of 2 mm SHAM, and AP capacity
was taken to be that portion of the 02 consumption inhibited
by 2 mm SHAM in the presence of 1 mm KCN. Residual
respiration (in the presence of KCN and SHAM) was subtracted from all measures of total respiration. This residual
component represented 6.4 2.6% (SD; n = 24) of total 02
consumption. The effect of FCCP (1 gM) on respiration rate
was determined in the absence of KCN and SHAM.

E
0

E0
a
.2

E
0

03

6'

Mitochondria
IL

Isolation of washed mitochondria and determination of CP

and AP capacities in the presence of 10 mm succinate with
500 ,M ADP were done as described (27). Addition of FCCP
(1 uM) to these washed mitochondria results in a 2- to 3-fold
increase in the rate of NADH oxidation (our unpublished
data). AO protein levels in mitochondria were determined
with a monoclonal antibody raised against the Sauromatum
guttatum AO (AOA, ref. 10) as described (27).

a.+
.2

Thie (h)

Inhibitors

Stocks of KCN and SHAM were prepared fresh weekly.

For respiration assays on whole cells, SHAM was dissolved
in 95% ethanol, whereas for isolated mitochondria, SHAM
was dissolved in DMSO. FCCP and AA were dissolved in
95% ethanol. AA was filter-sterilized before being added to
cells.

RESULTS

Respiratory Characteristics and Growth

Before AA addition, AP capacity was approximately 31%
of the total capacity (AP capacity plus CP capacity) of electron
transport in whole cells (Fig. 1A). Addition of AA immedi-

Figure 1. Respiratory characteristics of suspension cells of N. tabacum in response to the addition of 2 gM AA. AA was added
where indicated by the arrow (0 h). A, The capacities of the AP and
the CP (determined as described in "Materials and Methods"). Note
that for the CP, the apparent reduced capacity after addition of AA
is presumably due to the ability of AA to chemically block electron
flux through the pathway (see "Results" for further explanation). B,
Respiration rate of cells in the absence of any inhibitors other than
AA. C, Adenylate control of respiration (defined here as the respiration rate in the presence of FCCP divided by the respiration rate
in the absence of FCCP). KCN and SHAM were not present.

1848

VANLERBERGHE AND MCINTOSH

ately decreased CP capacity by 95%, and this 'apparent'

capacity remained low for the next 12 h (Fig. 1A). The term
'apparent' CP capacity is used here because AA addition
presumably is not affecting the in vivo capacity of the pathway (as measured before AA addition) but rather is chemically blocking electron flow through the pathway. During
the first 12 h in the presence of AA, AP capacity gradually
increased from 54 to 174 natoms 0.mg-1 fresh weight.h-1
(Fig. 1A). Between 12 and 48 h after AA addition, there was
a gradual recovery in apparent CP capacity accompanied by
a gradual loss of AP capacity so that at 48 h the capacities
were similar to before AA addition (Fig. 1A).
Before AA addition, the respiration rate in whole cells was
approximately 108 natoms 0 * mg-' fresh weight. h-1 (Fig. 1B)
and could be stimulated 2.5-fold by FCCP (Fig. 1C). Within
12 h after AA addition, there was a large increase in the
respiration rate (Fig. 1B), which paralleled the increase in AP
capacity over this time (Fig. 1A). The AP was completely
engaged over this period (Fig. 2B), indicating that respiration
was proceeding almost exclusively through the AP. During
this time, respiration rate was unaffected by the uncoupler
FCCP (Fig. 1C). Between 12 and 48 h, respiration rate gradFigure 2. A, Growth of suspension cells of N.
tabacum in response to the addition of 2 AM
AA. AA was added where indicated by the
arrow (O h). These data are from the same
experiment as in Figure 1. The inset figure
shows growth of the cells from the time of
subculture until 52 h after AA addition. It also
shows the growth of a control culture not given
AA. B, The left axis shows the degree of engagement of the AP in response to addition of
2 AM AA. AA was added where indicated by
the arrow (O h). The engagement of the AP (p)
is the fraction (0-1) of the AP capacity that is
engaged under the given conditions. It is defined as vaIt/VAIt where va,I is that portion of 02
consumption that is inhibited by SHAM in the
absence of KCN and Vait is the capacity of the
AP (18). The right axis shows the calculated
ATP production, an estimate based on the respiration rate and on the partitioning of electrons
between the CP and the AP. Data are presented
as a percent of the ATP production before AA
addition. To calculate ATP production, the P/
O ratio for endogenous NADH oxidation is
assumed to be 3 for the CP and 1 for the AP
(6). ATP production by the AP is then (V,It.p. 1)
and ATP production by the CP is (vt - [p- Va1t]) * 3
where vt is the respiration rate in the absence
of KCN and SHAM, p is the engagement of the
AP, and Vait is the capacity of the AP. The above
terminology (i.e. vt, valt, Va,t, p) has been described by M0ller et al. (18), but note that these
terms are somewhat redefined here in that they
may be measured in the presence of AA. All of
the above data were calculated from the results
presented in Figure 1.

Plant

Physiol. Vol. 100,

1992

ually declined to a level similar to before AA addition (Fig.

1B). This was accompanied by a progressive increase in the
ability of FCCP to stimulate respiration rate (Fig. 1C).
Addition of 2 uM AA to suspension cells of N. tabacum
resulted in an almost complete inhibition of growth for a
period of about 30 h, but then growth resumed at control
rates (Fig. 2A). Once growth had resumed, addition of a
further 2 yM AA again inhibited growth for a 24- to 30-h
period (data not shown), indicating that these cells were still
sensitive to AA.

Isolated Mitochondria
Mitochondria isolated from control cells (before AA was

added) had a low capacity to oxidize succinate through the

AP (Fig. 3). Mitochondria isolated from cells treated with AA
showed changes in AP and CP capacity similar to those seen
in whole cells. There was a progressive increase in AP capacity (from 5-44 natoms 0 * mg-' protein. min within the first
10 h) when the CP was inhibited by AA. However, by 48 h
after AA addition the apparent CP capacity was recovering
and the AP capacity was decreased (Fig. 3). For both whole

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100

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40

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20

Tkim (h)

CYTOCHROME AND ALTERNATIVE PATHWAY RESPIRATION

1 849

I). Both of these responses were prevented by either an

inhibitor of RNA synthesis (actinomycin D) or an inhibitor
of cytosolic protein synthesis (cycloheximide) (Table I). The
ability of these inhibitors to prevent the increase in AP
capacity normally seen after AA addition was confirmed in
isolated mitochondria (Fig. 4). These inhibitors prevented the
increase of the 35-kD AO protein normally seen after AA
addition (Fig. 4).
DISCUSSION

Figure 3. The amount of a 35-kD AO protein in isolated mitochondria. A western blot of total mitochondrial protein was probed with
a monoclonal antibody to the AO of S. guttatum. Lane 1 is total
mitochondrial protein from S. guttatum used as a control and shows
the polypeptide profile previously reported (10). All other lanes are
mitochondrial protein (75 Ag of protein/lane) from N. tabacum cells
treated with 2 MM AA for different periods of time. Numbers at the
bottom of the figure are the measured capacities of these mitochondria to oxidize succinate through the AP and the CP.

cells and isolated mitochondria, we found that the degree of

recovery of the CP by 48 h after AA addition varied from
one experiment to the next. In some experiments, it was equal
to or greater than the AP capacity by this time (Fig. 1A; data
not shown).

AO Protein

A monoclonal antibody to the AO of S. guttatum also

recognizes an AO protein in N. tabacum (27) and was used
to compare levels of AO protein in isolated mitochondria.
Treatment of N. tabacum cells with AA resulted in a progressive increase of AO protein in isolated mitochondria over the
first 10- to 24-h period (Fig. 3). However, by 48 h after AA
addition the level of AO protein had declined (Fig. 3). In all
of our experiments, there was a good correlation between the
level of this protein and the capacity of isolated mitochondria
to oxidize succinate through the AP (Fig. 3).
In addition to the above quantitative changes in AO protein, there were also striking qualitative changes. The major
immunoreactive protein in these suspension cells is 35 kD.
However, at 24 and 48 h after AA addition, at least one other
immunoreactive protein of a slightly higher molecular mass
(about 36 kD) is also visible on western blots (Fig. 3).
Effect of Inhibitors

An 8-h treatment with AA resulted in a large increase in

both respiration rate and AP capacity in whole cells (Table

The addition of AA to suspension cells of N. tabacum

reduced apparent CP capacity by more than 95%. This was
confirmed in whole cells (Fig. 1A) and in mitochondria isolated from AA-treated cells (Fig. 3) and is due to the ability
of AA to block electron transfer between Cyt b and cl in
complex III (Cyt c reductase) of the mETC (20, 23). It should
be noted that AA also inhibits cyclic electron transport in
chloroplasts by binding to a b-type Cyt (22), but this is not
an important consideration in these heterotrophic suspension
cells. Inhibition of the CP resulted in a progressive increase
in the capacity of the AP over a period of 8 to 12 h. This
progressive increase was seen both in whole cells (Fig. 1A)
and in mitochondria isolated from AA-treated cells (Fig. 3)
and correlated with a progressive increase in the amount of
a 35-kD AO protein in mitochondria (Fig. 3). The increase in
AO protein was prevented by cycloheximide and by actinomycin D (Fig. 4), indicating the probable importance of de
novo RNA synthesis and cytoplasmic protein synthesis for
this process. These inhibitors also prevented the increase in
AP capacity, as confirmed in both whole cells (Table I) and
isolated mitochondria (Fig. 4), emphasizing the role of this
AO protein in determining AP capacity. A close correlation
between AP capacity and this AO protein was also seen in
response to changes in growth temperature of these suspension cells (27). Together, these studies provide strong evidence that increased AP capacity is due, at least in part, to
de novo synthesis of the 35-kD AO protein.
Addition of the uncoupler FCCP to whole cells (before AA
was added) increased respiration 2.5-fold (Fig. 1C), providing
Table I. Effect of Different Inhibitors on Respiratory Characteristics
of Suspension Cells of N. tabacum
All treatments were for 8 h. Inhibitors were used at a concentration of 2 FM (AA), 50 Ag/mL (cycloheximide), and 100 Ag/mL
(actinomycin D). Addition of cycloheximide or actinomycin D alone
had no effect on the respiration rate or pathway capacities after 8
h (data not shown). Adenylate control is defined here as the ratio
of the respiration rate in the presence versus the absence of 1 MM
FCCP.
Treatment

No addition
Antimycin A
Antimycin A +

Respiration

AP

CP

Capacity
natoms O-mg-' fresh wt-h-'
96
61
90
2
115
121
1
51
48

Rate

Capacity

Adenylate
Control
ratio

2
1
1

cycloheximide
Antimycin A +
actinomycin D

60

57

VANLERBERGHE AND MCINTOSH

1 850

i.,
C.
,_

35 k4)

--

AP capacat'.

.tP capacaty
1natoms

t4.

mg 8roter -r

Figure 4. The amount of a 35-kD AO protein in isolated mitochondria from N. tabacum. A western blot of total mitochondrial protein
was probed with a monoclonal antibody to the AO of S. guttatum.
The mitochondria were isolated from cells treated with the inhibitors for 8 h. Inhibitors were used at a concentration of 2 pM (AA),
50 ,ug/mL (cycloheximide), and 100 ,ug/mL (actinomycin D). Numbers at the bottom of the figure are the measured capacities of
these mitochondria to oxidize succinate through the AP and the
CP. Addition of cycloheximide or actinomycin D alone had no
effect on these capacities or the level of the 35-kD protein after 8
h (data not shown).
strong evidence that respiration rate was limited by the
availability of ADP (i.e. adenylate control) (8, 12). This ADP
limitation may be at the level of oxidative phosphorylation
or at the level of substrate supply to the mitochondria (12).
Under these conditions, respiration proceeded at only 61%
of the total capacity (AP capacity plus CP capacity) of the
mETC (compare Fig. 1, A and B) and the AP was not engaged
(Fig. 2B). For the first 12 h after addition of AA, apparent CP
capacity was very low and, hence, the bulk of respiration
proceeded through the AP, which was now fully engaged
(Fig. 2B). Because AP respiration bypasses two of the three
sites of proton translocation in the mitochondria, it is expected that over this time period carbon oxidation would be
largely uncoupled from ATP production and that respiration
rate would no longer be under strict adenylate control. Supporting this were the observations that respiration rate now
equaled the capacity of the mETC (compare Fig. 1, A and B),

Plant

Physiol. Vol. 100,

1992

reached levels considerably higher than in the absence of AA

(Fig. 1B), and was unaffected by the addition of FCCP (Fig.
1C). An increased respiration rate and engagement of the AP
after inhibition of the CP has also been reported for potato
tuber callus after an osmotic shock (26) and for Avena fatua
seeds after treatment with NaN3 (25).
Changes in respiratory characteristics and AO protein between 12 and 48 h after AA addition show that CP and AP
capacities can be regulated in a coordinate fashion. Between
12 and 48 h, apparent CP capacity gradually recovered (Figs.
1A and 3). At present, we do not know how this recovery
takes place, but, as indicated in 'Results," recovered cells are
still sensitive to subsequent AA addition. One possible explanation, then, is that the cells are able to degrade AA over
time. The CP recovery was paralleled by a loss of AP capacity
(Figs. 1A and 3) and engagement (Fig. 2B). The loss of AP
capacity was accompanied by a loss of 35-kD AO protein
(Fig. 3). We observed that the loss of the AO protein was
accompanied by the appearance of one or more slightly
higher molecular mass immunoreactive proteins on western
blots. This may represent accumulation of a mitochondrial
precursor form of the 35-kD protein. In the thermogenic
plant S. guttatum, there are at least 3 AO proteins of similar
molecular mass that most likely represent posttranslational
modifications of a single nuclear gene product (10, 19). As
CP capacity recovered (Fig. 1A), there was an increase in
adenylate control (as shown by the stimulation of respiration
by FCCP; Fig. 1C) and a lowering of respiration rate back to
levels seen before AA addition (Fig. 1B). By 48 h, respiration
was once again proceeding at only 66% of the total capacity
of the mETC (compare Fig. 1, A and B) and the AP was not
engaged (Fig. 2B). The above observations show that the
plant is capable of gradual alteration of AP capacity in
response to gradual changes in the CP. An important mechanism involved in this coordinate regulation appears to be
the level of the 35-kD AO protein.
Up to 30 h after AA addition, estimated ATP production
based on the respiration rate and on the partitioning of
electrons between the CP and the AP does not exceed 69%
of that found before AA addition (Fig. 2B) and there is little
growth during this period (Fig. 2A). By 36 h (when the CP is
substantially recovered), ATP production is increased to near
control levels (Fig. 2B) and growth is also resumed at control
rates (Fig. 2A). The respiratory ATP produced in the absence
of growth may represent that required for maintenance of
existing biomass (maintenance respiration). The concepts of
growth and maintenance respiration are discussed elsewhere
(1, 13). Pea roots were also shown to remain viable but not
to elongate after induction of AP respiration in response to
inhibition of the CP by KCN (28). These observations and
ours suggest that the AP may provide the maintenance
respiration required for cells to survive during periods when
the CP is suppressed.
As ATP production recovers (between 30 and 36 h; Fig.
2b), the degree of engagement of the AP drops off dramatically (from p = 0.88 at 30 h to p = 0.48 at 36 h; Fig. 2b).
Therefore, it is clear that both the capacity and the degree of
engagement of AP respiration are regulated in response to
changes in the CP during this time period. Much additional
research is required to elucidate how AP capacity and en-

CYTOCHROME AND ALTERNATIVE PATHWAY RESPIRATION

gagement are regulated in response to the metabolic demands

placed upon mitochondrial electron transport.
CONCLUSION
In suspension cells of N. tabacum, inhibition of the CP by
AA induced a rapid increase in the capacity and engagement
of the AP. Increased AP capacity was due, at least in part, to
de novo synthesis of a 35-kD AO protein. When respiration
proceeded exclusively through the AP, there was a lack of
adenylate control of respiration, resulting in a high respiration
rate. As the CP gradually recovered, AP capacity was gradually reduced due to a loss of the 35-kD AO protein, and
there was a decreased engagement of the AP. These changes
resulted in an increased adenylate control of respiration, and
the respiration rate declined. These observations show that
the mitochondrial electron transport pathways can be regulated in a coordinate fashion. Both the capacity and the degree
of engagement of the AP are important components of this
coordinate regulation.

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

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