Download The `Uniqueness` of Plant Mitochondria

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The ‘Uniqueness’ of Plant Mitochondria
J. M. PALMER
Department of Botany, Imperial College of Science and Technology,
Prince Consort Road, London S W7 2BB, U.K.
The accepted biochemical view of the mammalian mitochondrion is that it functions
as the powerhouse of the cell. It is equipped with a linear respiratory chain that is
tightly coupled to synthesis of ATP, which is the intermediate needed to run the energy
economy of the cell. It is not thought to have any other major metabolic role in the cell.
For many years plant mitochondria were thought to be very similar to mammalian
mitochondria. However, it is becoming increasingly apparent that mitochondria isolated from plants and some micro-organisms have a considerably more complex
respiratory chain, which may enable them to fulfil a more complex metabolic function in the cell. It is these differences that form the basis of the ‘uniqueness’ that is the
subject of the present review. Here I shall limit my discussion to what I consider are the
major differences. These are (a) the characteristics of the cyanide-resistant oxidase,
which is not uniquely associated with plant mitochondria and is frequently encountered
in microbial mitochondria; ( b ) the organization of the NADH dehydrogenase system,
which is a less-well-documented area; nevertheless it raises important issues concerning
the regulation of electron flux along branched pathways and the metabolic significance
of the mitochondrion in the cell; and finally (c) the nature of the enzymes responsible
for the oxidation of malate, which differ from those present in mammalian mitochondria
by enabling malate oxidation to proceed in the presence of oxaloacetate. This complex
system for the oxidation of malate makes it possible to postulate that the tricarboxylic
acid cycle in plant mitochondria can oxidize to completion any intermediate of the cycle
without the necessity to supply pyruvate from glycolysis. Thus the tricarboxylic acid
cycle in plants is a more versatile system than is its mammalian counterpart.
Cyanide-resistant respiration
Progress in elucidating the molecular basis of cyanide-resistant respiration in plant
mitochondria has been frustratingly slow. The alternative oxidase is found in different
amounts in many different types of plant mitochondria. Mitochondria isolated from
storage tubers of Solanum tuberosum (potatoes) or Helianthus tuberosus (Jerusalem
artichokes) are very sensitive to inhibition by cyanide, which is rather surprising, since
oxygen uptake in the intact tubers is stimulated by gaseous cyanide. The fate of the
cyanide-resistant oxygen uptake on isolation of the mitochondria is not known; it is
possible that it may not have been originally associated with the mitochondria or that
the resistant oxidase may have been inactivated during the isolation of the organelles.
It has been suggested that the loss of the cyanide-resistant oxidase is caused by the
destruction of phospholipids caused by the release of phospholipases during the
isolation procedure (Solomos, 1977). Mitochondria isolated from tubers of Ipomoea
batatas (sweet potatoes) or Manihot esculenta (cassava) have about equal concentrations of both the cyanide-sensitive and -resistant oxidases, whereas mitochondria
isolated from the mature spadix of Arum maculatum (cuckoo pint) have ten times more
cyanide-resistant oxidase than cytochrome oxidase. The spadix of the Arum is a very
interesting tissue; its physiological role is to become thermogenic for a limited period
during the life of the flower; the heat releases amines that attract flies involved in the
pollination process. Mitochondria isolated from this tissue are capable of very high
rates of oxygen uptake, in the order of 4000nmol/min per mg of protein, most of
which is mediated by the cyanide-resistant oxidase.
Little is definitely known about the characteristics of this alternative cyanide-resistant
oxidase. Many theories have been proposed and none has stood the test of time. It
was first thought that the oxidase had a high K,,, for oxygen, and on this basis it was
suggested that the oxidase may be a flavoprotein (James & Beevers, 1950). The K,,,
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measurements were subsequently shown to be incorrect, owing to problems of oxygen
accessibility to the oxidase, and the idea that the oxidase was a flavoprotein has dropped
from favour. However, it has been frequently observed that mitochondria that contain
significant amounts of the alternative oxidase also contain significantly higher amounts
of flavoprotein; it is also clear that the induction of cyanide resistance in microorganisms alters the spectral behaviour of the flavoproteins (Lloyd, 1974) ; and finally,
kinetic measurements show that there is a pool of flavoprotein that could act as the
oxidase (Erecinska & Storey, 1970). A second theory was that the autoxidation
of cytochrome b could mediate the oxidase; initial kinetic experiments showed that,
in the presence of cyanide, there was a species of cytochrome 6 that could fulfil this
role. However, when antimycin A was added, it prevented the redox changes associated with cytochrome b without having any inhibitory effect on the cyanide-resistant
oxidase (Bendall & Bonner, 1971). In recent years the most significant observation
relating to the operation of the alternative oxidase has been that it can be specifically
inhibited by substituted benzhydroxamic acids (Schonbaum et al., 1971). Since these
compounds can chelate iron, much effort has been expended to prove that the oxidase
is an iron-sulphur protein. This theory has not met with any success, and direct examination of Arum mitochondria with e.p.r. techniques has failed to show any unique ironsulphur centre that could be a candidate for the oxidase (Cammack & Palmer, 1977).
These e.p.r. studies showed, however, that Arum mitochondria did differ from animal
mitochondria by having very weak e.p.r. signals that could be attributed to centres
N1-4 of the NADH dehydrogenase and very strong signals associated with centres
S1-3 of the succinic dehydrogenase. Therefore at present we have no definitive evidence
concerning the nature of the oxidase. Even simple facts such as the nature of the products
formed when the oxidase interacts with oxygen remain to be clarified, although recent
studies appear to confirm that water is the first detectable product (Huq & Palmer,
1978a); this information is significant when speculating on the nature of the oxidase.
Some progress has been made concerning the relationship between the alternative
oxidase and the conventional respiratory chain. Kinetic measurements (Storey, 1976)
show that at least a portion of the ubiquinone pool acts as if it were mediating electron
flux to the alternative oxidase. Rich & Moore (1976) have produced evidence that
an e.p.r. signal at g = 1.98, resulting from spin-spin interaction between two ubisemiquinone radicals, is closely associated with the alternative oxidase, and have
proposed that the cyanide-resistant oxidase acts by oxidizing the ubiquinol to the
ubisemiquinone. More recently it has been possible to extract the quinones from
freeze-dried mitochondria and demonstrate that cyanide-resistant oxidation ceases
until ubiquinone is re-incorporated (Huq & Palmer, 19786). The elucidation of the
mechanism by which the reduced quinones are oxidized remains a prize to be won.
Recently a quinol oxidase with the correct properties and specific activities to be the
alternative oxidase has been solubilized from Arum mitochondria in my laboratory
(Huq & Palmer, 1978c) and by Rich & Bonner (1978). We have partially purified and
characterized our preparation and find it contains a fluorescent compound, that is
probably a flavoprotein, and significant quantities of copper. It is not clear whether
these components are involved in mediating electron flux between the quinol and oxygen.
It is noteworthy that quinols can naturally interact with molecular oxygen, and it is
possible that the oxidase could be a protein with no redox component that provides a
favourable environment to enhance the rate of autoxidation. However, it seems likely
that the nature of the oxidase will be resolved in the near future.
From the discussion so far it may be concluded that the alternative oxidase may
simply operate as a branch from the conventional respiratory chain at the level of
ubiquinone. However, data are accumulating to suggest that the system may not be
this simple. In the aroid spadices, which use the cyanide-resistant oxidase to produce
heat, all substrates appear to have equal access to the oxidase. However, in mitochondria from non-thermogenic tissue, such as Triticum vulgare (wheat) roots, Ipomoea
batatas and Munihot esculenta, it appears that, whereas electrons from endogenous
NADH can have access to the cyanide-resistant oxidase, electrons from exogenous
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NADH do not have access to this oxidase (Huq & Palmer, 19786). It is thought that
electrons from endogenous and exogenous NADHJoin the main chain at the level of the
quinone. If there is a single homogeneous pool of quinone receiving electrons from the
various dehydrogenases and distributing them to the different oxidases, it is difficult to
see, if using the foregoing model, how electrons emanating from exogenous NADH can
be excluded from the cyanide-resistant oxidase. In order to achieve this discrimination, more than one functional pool of quinone must be present. Experimental evidence
is now available to suggest that the quinone associated with the cyanide-resistant
oxidase is more readily extracted with pentane than is the quinone associated with the
cytochrome c oxidase (Huq & Palmer, 19786). This would be consistent with the
existence of two functional pools of ubiquinone. If this should prove to be correct, then
the alternative oxidase would branch from the conventional respiratory chain before
the quinone pool. There is evidence to support the speculation that the two respiratory
systems do not necessarily branch from each other but represent two parallel chains
which can in vitro, under appropriate conditions of phosphate potential and inhibitors,
communicate with each other. There is little evidence to show that, in mitochondria from
higher plants, electrons must traverse the first site of oxidative phosphorylation before
becoming available to the cyanide-resistant oxidase. Lips & Biale (1 966) observed that
amytal, normally considered to be an inhibitor of the NADH dehydrogenase, failed
to inhibit respiration in Persea gratissima (avocado) tissue and proposed that two
parallel pathways of electron transport existed ;one was resistant to amytal, antimycin A
and cyanide and was unable to support oxidative phosphorylation; the other chain had
conventional sensitivity to inhibitors and was coupled to the synthesis of three molecules
of ATP. Treatments that induce cyanide-resistant oxidation also increase the activity of
piericidin-resistant NADH dehydrogenase (Cammack & Palmer, 1973). If the two
oxidases represent the terminal components of two parallel chains rather than the end
of two branches with a common origin, then it becomes necessary to re-examine the
mechanism by which electron flux to the two oxidase systems is regulated. The study by
Bahr & Bonner (1973) represents the only viable model for regulation and visualizes
that the alternative electron-transport sequence branches from, and is in rapid equilibrium
with, the conventional electron-transport chains as shown in Scheme 1. In this scheme
the alternative electron-transport sequence branches from the conventional respiratory
chain at the unidentified component B, and the first component of the alternative
sequence, represented by A, has a standard redox potential approximately 50 mV
more negative than that of component B. Under normal State-3 conditions, component
B would be partially reduced and capable of acting as a donor to the cytochrome
system, whereas component A, in equilibrium with B, would be much less reduced and
consequently would fail to act as an efficient donor to the alternative oxidase. According
to the scheme proposed by Bahr & Bonner (1973) the alternative oxidase only works
when the cytochrome c oxidase is saturated, which only occurs under State-4 conditions
or in the presence of inhibitors of the conventional oxidase. Under these conditions
compound B becomes very reduced and consequently compound A becomes more
Succinate
--f
Fp
-B
A
c--,
cyt
CI
alt oxidase
t
,
-
cyt c
c+
cyt u
C,
cyt u3 -----+ o2
O2
Scheme 1. Possible relationship between the conventional and alternative oxidases in
plant mitochondria
The horizontal axis represents an approximate measure of standard redox potential,
hence A is more reducing than B. Abbreviations used: Fp, flavoprotein; cyt, cytochrome;
alt, alternative.
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Step (I)
NADH --+
F p + Fe-S -+ Q
+ cyt b
-+
cyt c t ---+ cyt c -+ cyt u -+ cyt u3 -+
O2
I \
ll
/I 1I
i l
Step(2)
NADH + F p
____+
Q
+ alt oxidase
’
0 2
Scheme 2. Possible organization of electron transport as two parallel steps
Step (1) is stimulated by AMP, and step ( 2 ) inhibited by AMP (Sotthibandhu &Palmer,
1975). Q , quinone; other abbreviations as in Scheme 1.
reduced and can then donate electrons to the cyanide-resistant oxidase. There is some
doubt as to whether the energy charge becomes high enough in intact plant cells to
impose State-4 control, and the concentration of cyanide in higher-plant cells, even
in tissues containing cyanogenic glucosides, is not thought to be sufficient to inhibit
cytochrome oxidase. Therefore, it is not certain whether the control mechanisms
described by Bahr & Bonner (1973) can play a role in uiuo. If the respiratory system is
organized as two parallel chains, as shown in Scheme 2, then the control system outlined
by Bahr & Bonner (1973) would not apply. It is, however, possible to speculate that
factors such as AMP, which regulate electron flux through the different dehydrogenases
(Palmer & Coleman, 1974; see also below), may influence which oxidase is used.
NA DH dehydrogenase system
Plant mitochondria also differ from their mammalian counterpart in having a more
complex NADH dehydrogenase system. These differences are illustrated by the observations that (a) plant mitochondria can oxidize exogenous NADH and (b) the
oxidation of endogenous NADH is only partially inhibited by rotenone or piericidin
(Palmer & Coleman, 1974).
Intact mammalian mitochondria cannot oxidize exogenous NADH because the
NADH molecule cannot traverse the inner membrane. This compartmentation allows
the nicotinamide nucleotide in the cytosol and mitochondria1 matrix to adopt different
redox poises. The ability of plant mitochondria to oxidize exogenous NADH was
originally thought to be due to the NADH entering the matrix space and being oxidized
by the internal dehydrogenase. This apparent ‘leakiness’ was thought to be due to
damage induced by the rigorous grinding techniques used in isolating the organelles.
However, evidence suggests that plant mitochondria are relatively tough structures
capable of withstanding far greater osmotic stress than are their mammalian counterparts. The osmotic stability may be related to the large amounts of polyuronides
associated with the outer membrane (Mannella & Bonner, 1975). It is now generally
agreed that exogenous and endogenous NADH are oxidized by different NADH
dehydrogenases and that the enzyme responsible for the oxidation of exogenous NADH
may have its active site located on the outer surface of the inner membrane (Palmer &
Coleman, 1974). However, the existence of an NADH dehydrogenase associated
with the outer membrane has hampered research into the precise location of the external
dehydrogenase associated with the inner membrane. A similar NADH dehydrogenase
is known to exist in mitochondria from the fungus Neurospora crassa (Von Jajow &
Klingenberg, 1970). The external dehydrogenase feeds electrons into ubiquinone
without involving the piericidin A-sensitive site of the respiratory chain or the first site
of oxidative phosphorylation. The metabolic significance of this external dehydrogenase
capable of oxidizing cytosolic NADH is not known. At first sight it appears to be an
undesirable feature, and it seems reasonable to expect that the electron flux through
this dehydrogenase would be closely regulated; little evidence is available on how this
may be achieved. The oxidation of external NADH appears to be dependent on the
presence of bivalent cations, particularly calcium, and is inhibited by bivalent-cation
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chelators such as EGTA and citrate (Palmer & Coleman, 1974). The mechanism by
which calcium stimulates the electron flux remains a mystery. It is therefore possible,
but not certain, that the distribution of bivalent cations in the cell may be involved
in the regulation of dehydrogenase activity. Results have been obtained (R. C. Cowley
& J. M. Palmer, unpublished work) that show that the oxidation of succinate takes
precedence over the oxidation of exogenous NADH. Thus the addition of succinate
to mitochondria oxidizing exogenous NADH severely curtails the NADH oxidase.
This is clearly a potential control mechanism, although the molecular basis of the
interaction between internal and external dehydrogenases remains unsolved.
The NADH dehydrogenase responsible for the oxidation of endogenous NADH
appears to be more complex than its enzymic counterpart in mammalian mitochondria.
The most obvious manifestation of this phenomenon is the observation that inhibitors
such as rotenone or piericidin A, which inhibit the oxidation of endogenous NADH
in animal mitochondria by interacting with the iron-sulphur centres of the dehydrogenase, only cause a partial inhibition in the plant system. By measuring the efficiency
of ATP synthesis it is apparent that the piericidin-resistant pathway is not coupled
to the first site of ATP synthesis, whereas the piericidin-sensitive component does
result in ATP synthesis in this region of the chain. The components involved in catalysing this inhibitor-resistant non-phosphorylating oxidation of endogenous NADH are
not yet known. It seems clear, however, that some system must exist to regulate the
flux of electrons through the two internal NADH dehydrogenase systems. Early observations seem to suggest that under normal State-3 respiratory conditions the nonphosphorylating dehydrogenase plays only a small role, since the oxidation of NAD+linked substrates can be shown to be coupled to ATP synthesis at three sites. However,
as soon as piericidin A is added, only two sites of ATP remain functional. The electron
flux through the two internal NADH dehydrogenases appears to be under the control
of the adenine nucleotides in the cytosol. This conclusion was reached when investigating why plant mitochondria did not oxidize NAD+-linked substrates rapidly in the
presence of weak acid uncouplers (Sotthibandhu & Palmer, 1975). The results clearly
showed that adenine nucleotides, and particularly AMP, were necessary in order to
obtain maximum rates of oxidation in the presence of uncoupling agents. AMP stimulated electron flux from NADH to cytochrome b via a piericidin A-sensitive pathway.
It was also apparent that the AMP did not have to enter the mitochondrion in order
to bring about the stimulation. This appears to be a potential control mechanism,
whereby an increase in AMP in the cytosol results in the stimulation of the internal
NADH dehydrogenase coupled to ATP synthesis. The mechanism by which external
AMP stimulates the oxidation of internal NADH is at present speculative, but presumably it occurs as the result of AMP binding to the outer loop of the proton-pumping
NADH dehydrogenase.
The physiological significance of the multiple systems for oxidizing NADH is not
understood. It has already been suggested in the present review that the piericidin
A-sensitive phosphorylating dehydrogenase may be associated with the cyanidesensitive oxidase, whereas the non-phosphorylating dehydrogenase, which works best
in the absence of AMP, is associated with the cyanide-resistant oxidase providing a
totally non-phosphorylating pathway for the oxidation of NADH when the energy
charge is high. It must be emphasized that this is speculation, and further experimental
justification is necessary.
Oxidation of malate
There is evidence that the different NADH dehydrogenases described above may
be closely associated with the NAD+-linked dehydrogenases responsible for the oxidation of malate. Malate oxidation in plant mitochondria is a much more complex
process than in mammalian mitochondria, in which oxidation is achieved by a single
enzyme, malate dehydrogenase, and continual oxidation of malate depends on the
removal of oxaloacetate, which is a product of the reaction. It has long been observed
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25 1
that plant mitochondria were capable of oxidizing malate without the need to remove
oxaloacetate, indicating that plant mitochondria either have a system for removing
the oxaloacetate or do not produce oxaloacetate as a product of malate oxidation.
It is now clear that both mechanisms are involved, and malate is oxidized by the malate
dehydrogenase and an NAD+-linked ‘malic’ enzyme to produce oxaloacetate and
pyruvate respectively, which then give rise to citrate. The ability to produce pyruvate
from malate in the matrix of the mitochondrion makes it possible to completely oxidize
any tricarboxylic acid-cycle acid without the necessity to supply acetyl-CoA to complete
the cycle (Palmer, 1976). This scheme has many attractions, since large quantities
of organic acids are known to be stored in plant tissue. It also has the disadvantage
that it could allow the oxidation of the catalytic pool of acids necessary for the operation
of the tricarboxylic acid cycle, and would also keep the concentration of oxaloacetate
very low. The concentration of this acid is believed to be an important factor in integrating the activity of catabolic and anabolic metabolism. Factors that determine the
relative contribution of the malate dehydrogenase and ‘malic’ enzyme to the overall
rate of malate oxidation are not fully understood. There is, however, evidence to suggest
that although both enzymes are located inside the inner membrane of the mitochondrion, they are spatially separate from each other (Brunton & Palmer, 1973). This
can be shown by using exogenous oxaloacetate to oxidize endogenous NADH. Plant
mitochondria are readily permeable to added oxaloacetate, which, if added at a suitable
concentration, will enter the matrix and cause the malate dehydrogenase to run in
reverse; this reaction oxidizes any NADH being produced more readily than does the
respiratory chain. Hence when oxaloacetate is added to mitochondria oxidizing NAD+linked substrates, there is an immediate and essentially complete inhibition of oxygen
uptake until all the oxaloacetate has been removed. This inhibition was observed
with all substrates except malate, which continued to supply NADH to the respiratory
chain in the presence of oxaloacetate, clearly indicating spatial separation of the malate
dehydrogenase and ‘malic’ enzyme. The evidence (Brunton & Palmer, 1973) clearly
suggests that the NADH produced by malate dehydrogenase and ‘malic’ enzyme
appears to be oxidized by different NADH dehydrogenase systems. The NADH produced by the ‘malic’ enzyme appears to be oxidized by the NADH dehydrogenase
closely associated with the piericidin A-sensitive component and coupled phosphorylation, whereas the NADH from the malate dehydrogenase appears more readily
accessible to the piericidin A-resistant, non-phosphorylating dehydrogenase. This
possible association of the malate oxidoreductases with different NADH dehydrogenases
provides a potential physiological role for the multiple dehydrogenase systems and
a regulating mechanism for malate oxidation via the two enzymes involved.
The physiological importance of the complex respiratory chain and tricarboxylic
acid cycle known to exist in plant mitochondria can only be the subject of speculation.
It seems clear, however, that plant mitochondria have a complex respiratory chain
that can by-pass all the sites of ATP synthesis and a tricarboxylic acid cycle that can
oxidize to completion a variety of compounds other than pyruvate. The combination
of these two abilities would result in a system that could metabolize carbon compounds
through the mitochondria1 enzyme system much more rapidly than the turnover of
the ADP/ATP pool. This is clearly important in thermogenesis, but it could also be
significant in photosynthetic tissue, where photosynthesis maintains a high energy
charge, or in tissue in which carbon metabolism is very rapid, particularly when one
form is being converted into another, when substrate-level phosphorylation may
maintain a high energy charge.
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Brunton, C . J. & Palmer, J. M. (1973) Eur. J . Biochem. 39,283-291
Cammack, R. & Palmer, J. M. (1973) Ann. N . Y.Acad. Sci. 222,16-23
Cammack, R. & Palmer, J. M. (1977) Biochem. J . 166,347-355
Erecinska, M. & Storey, B. T. (1970) Plant Physiol. 46, 618-624
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Huq, S. &Palmer, J. M. (1978~)Plant Sci. Lett. 11, 351-358
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James, W. 0. & Beevers, H. (1950) New Phytol. 49,353-374
Lips, S . H. & Riale, J. B. (1966) Plant Physiol. 41,797-802
Lloyd, D. (1974) The Mitochondria of Microorganisms pp. 137-158, Academic Press, London
Mannella, C. A. & Bonner, W. D. (1975) Biochim. Biophys. Acta 413,213-225
Palmer, J. M. (1976) Annu. Rev. Plant Physiol. 27, 133-157
Palmer, J. M. & Coleman, J. 0. D. (1974) Horizons Biochem. Biophys. 1,220-260
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1979