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Soole, Kathleen L. and Sweetman, Crystal. (2021) Plant Mitochondria - Substrates, Inhibitors, Uncouplers. In: Jez
Joseph (eds.) Encyclopedia of Biological Chemistry, 3rd Edition. vol. 2, pp. 536–544. Oxford: Elsevier.
http://dx.doi.org/10.1016/B978-0-12-819460-7.00014-1

© 2021 Elsevier Inc. All rights reserved.

 Author's personal copy

Respiration | Plant Mitochondria – Substrates, Inhibitors, Uncouplers☆

Kathleen L Soole and Crystal Sweetman, Flinders University, Bedford Park, SA, Australia
r 2021 Elsevier Inc. All rights reserved.

This is an update of K.L. Soole, R.I. Menz, ATP in Plant Mitochondria: Substrates, Inhibitors, and Uncouplers, in Encyclopedia of Biological Chemistry
(Second Edition), Edited by: William J. Lennarz, M. Daniel Lane, Elsevier Inc., 2013, https://doi.org/10.1016/B978-0-12-378630-2.00135-3.

Glossary with no consequent proton translocation across the

Alternative oxidase A ubiquinol oxidase located in the
inner mitochondrial membrane, which accepts electrons
from reduced ubiquinone and reduces oxygen to water.
During this process, no protons are translocated across the
inner mitochondrial membrane.
Non-phosphorylating NAD(P)H dehydrogenase
(s) Enzymes which accept electrons from NADH or
NADPH and reduce ubiquinone, a mobile lipophilic
electron carrier in the inner mitochondrial membrane
membrane.
Non-phosphorylating pathway A route of electron
transfer with no concomitant translocation of protons
across the inner mitochondrial membrane, hence this route
of electron flow does not contribute to the proton motive
force. Proton motive force refers to the proton gradient that
is established across the inner mitochondrial membrane
during electron transfer through Complexes I, II, and IV.
Often referred to as DmH þ or pmf.

Introduction

Plants cannot rely on dietary sources of complex metabolites and must generate all organic matter from fundamental environ-
mental elements. Therefore, while the major function of mitochondria in plants, as in any eukaryote, is synthesis and distribution
of the cellular energy carrier adenosine triphosphate (ATP), it also must fulfill an anabolic role. Plant respiratory pathways are
highly complex, from expanded gene families and enzyme isoforms with diversified kinetic properties, to entirely unique pathways
that branch from and feed into the classical pathways. This enables metabolic flexibility, redistributing carbon flux and balancing
energy status according to ever-changing needs of the cell. This is particularly important in an organism that has limited physical
ability to avoid harmful environmental situations. It also provides convenient subject matter for exploring mechanisms to improve
tolerance of future plant generations to increasingly unfavorable environmental conditions.

ATP Synthesis in Plants

ATP synthesis occurs not only through respiration but also photosynthesis, and the two pathways are linked through sophisticated
inter-organellar communication (Noguchi and Yoshida, 2008; van Lis and Atteia, 2004; Suzuki et al., 2012; Raghavendra and
Padmasree, 2003; Nunes-Nesi et al., 2011). As respiration is not limited to daylight hours nor cell type, it is still arguably the main
pathway of ATP synthesis in plants (Gardestrom and Igamberdiev, 2016). Within respiration there is a low level of ATP synthesis
associated with glycolysis in the cytoplasm; however, the majority of ATP is synthesized via oxidative phosphorylation that occurs
within mitochondria, specifically via the operation of an electron transport chain (ETC) in the inner mitochondrial membrane.
The ETC utilises electrons packaged in NADH, NADPH and FADH2, which predominantly arise from the TCA cycle in
the mitochondrial matrix. In mammals, flux through the respiratory pathway is tightly regulated by the ATP/ADP ratio or
adenylate energy charge of the cell. In plants, the presence of a non-phosphorylating pathway in the mitochondrial ETC and an
uncoupler protein in the inner membrane overcomes this restriction by adenylate control, which may facilitate biosynthesis and
anabolic reactions unique to plants (Millar et al., 2011; van Dongen et al., 2011).

TCA Cycle in Plants

Sucrose and starch are converted to metabolites that feed into the early steps of glycolysis, which occurs in the cytoplasm. The end
products of glycolysis in plants can be either pyruvate or malate, the latter being formed by the action of PEP carboxylase and
malate dehydrogenase, which together bypass pyruvate kinase (Plaxton and Podesta, 2006). Once within the mitochondrial
matrix, pyruvate is metabolized by pyruvate dehydrogenase and the enzymes of the TCA cycle. Being a substrate for mitochondrial
malate dehydrogenase, malate can either feed into the TCA cycle or be metabolized to pyruvate via NAD-malic enzyme. There
must be a balance between these processes, since because of the poor forward equilibrium of malate dehydrogenase, the

☆
Change History: January 2020. Crystal Sweetman and Kathleen L. Soole updated the text to the entire article including a new abstract, added new references to
the reading list and updated Fig. 1 and Table 1.

536 Encyclopedia of Biological Chemistry, 3rd Edition, Volume 2 doi:10.1016/B978-0-12-819460-7.00014-1

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Respiration | Plant Mitochondria – Substrates, Inhibitors, Uncouplers 537

accumulation of oxaloacetate would prevent further malate metabolism. At a fundamental level, the TCA cycle of plants is
comprised of the same enzymes and intermediates as in animals (Beevers, 1961). The net result of these reactions is the production
of NADH and FADH2 (via succinate dehydrogenase). During a turn of the TCA cycle, 1 ATP is produced directly by substrate level
phosphorylation, not GTP as in mammalian mitochondria.
Despite the major role of the TCA cycle in primary respiration, it also functions as a source of carbon intermediates necessary for
synthesis of a large array of compounds (Plaxton and Podesta, 2006). Flux analyses highlight the modularity of the TCA cycle in
plants; not all reactions carry the same flux and this is also tissue- and condition-dependent. For example, the TCA cycle of
heterotrophic (non-photosynthetic) tissues may generally occur as a complete cycle, whereas the TCA cycle of photosynthetic tissues
in the light is fragmented. The latter typically operates as two separate branches, one from stored citrate to a-ketoglutarate and the
other from OAA to fumarate (i.e., reverse flux), due to light-inhibition of pyruvate dehydrogenase and low flux through succinyl CoA
ligase and succinate dehydrogenase (Araujo et al., 2012). In addition, there exist numerous bypasses such as; the GABA shunt, which
can bypass a-ketoglutarate dehydrogenase and succinyl-CoA ligase steps of the TCA cycle; the malate/OAA shuttle, which exports
malate to the cytosol in return for OAA; the glyoxylate cycle, which shares citrate with peroxisomes; and amino acid biosynthesis,
which utilises carbon skeletons from the TCA cycle (Sweetlove et al., 2010; Popova and de Carvalho, 1998; Selinski et al., 2018;
Michaeli and Fromm, 2015). There is some evidence for the existence of multi-enzyme complexes of the TCA cycle in plants, which
could facilitate substrate-channeling; improving reaction efficiency and also potentially regulating the path of intermediates through
the TCA cycle and/or bypasses in plants (Klodmann et al., 2011; Araujo et al., 2012; Zhang et al., 2017). Some enzymes of the TCA
cycle are also regulated by redox state and there is evidence that thioredoxin may be important in this (Daloso et al., 2015).

Mitochondrial Electron Transport Chain in Plants

Oxidative phosphorylation occurs via the interaction of large lipo-protein complexes of the ETC and smaller mobile electron
carriers found in the inner mitochondrial membrane (Fig. 1) (Joseph-Horne et al., 2001). NADH is oxidized by Complex I
(NADH: ubiquinone oxidoreductase), which donates its electrons to a mobile lipophilic electron carrier, ubiquinone. Complex II
(succinate dehydrogenase) also donates electrons to the ubiquinone pool. Reduced ubiquinone (or ubiquinol) is oxidized by

Fig. 1 A schematic representation of the electron transport chain of plant mitochondria. Classical phosphorylating complexes and uncoupling
protein (UCP) that are common to plant and animal mitochondria are given in white. Cytochrome c and ubiquinone are identified in the key.
Non-phosphorylating components of the plant mETC are given in green, including the alternative oxidase (AOX) and non-phosphorylating NAD(P)H
dehydrogenases (NDs). The latter are classified based on the seven A. thaliana proteins, which have mostly been functionally characterized. The
antimycin A-insensitive NAD(P)H reductase of the outer membrane is also given in green. Enzymes of other metabolic pathways that may
contribute electrons directly to the mETC are given in blue. L-GLDH, L-galactonolactone dehydrogenase, DHODH, dihydroorotate dehydrogenase,
G3PDH, glycerol 3-phosphate dehydrogenase, PDH, proline dehydrogenase, D-LDH, D-lactate dehydrogenase. Localization studies have not yet
confirmed whether D-LDH is integrated in the IM or floating in the IMS.
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538 Respiration | Plant Mitochondria – Substrates, Inhibitors, Uncouplers

Complex III (coenzyme Q: cytochrome c-oxidoreductase) via the Q-cycle and reduces the mobile peripheral protein cytochrome c.
By interacting with Complex IV (cytochrome oxidase), the electrons carried by cytochrome c are donated to the terminal electron
acceptor, oxygen. During these electron transfer events, protons are translocated from the matrix side of the inner membrane to the
inter-membrane space at Complexes I, III, and IV, thus establishing the proton motive force (pmf or DmH þ ). Complex II is unique
to the other complexes, in that it does not contribute to the pmf and it directly participates in both the TCA cycle and
mitochondrial ETC.
Plant Complexes I-IV are similar to those from animals, however there are some structural and functional differences. Briefly,
Complex I of plants lacks 4–5 subunits seen in animals but contains 8–9 unique subunits, including a carbonic anhydrase domain
thought to facilitate CO2 supply from the mitochondrion to chloroplast, for carbon fixation (Senkler et al., 2017). Complex II
contains four additional subunits of unknown function compared to the animal counterpart, although some of these may recover
functions lost from truncated versions of the other “core” subunits (Millar et al., 2004; Huang et al., 2019). Complex III of plants
contains no unique subunits but demonstrates additional proteolytic functionality, due to the presence of intact active sites that
have been lost in other eukaryotes (Schertl and Braun, 2014). Complex IV from plants is lacking at least 6 of the core subunits seen
in mammalian cells (most of which are also in yeast) and contains up to ten additional subunits of unknown function (Millar
et al., 2004; Mansilla et al., 2018). Like other eukaryotes, “supercomplexes” of the ETC exist in plants, most commonly a
combination of Complex I, III and IV, also referred to as a “respirasome”, which may streamline the electron transport process
(Schertl and Braun, 2014).
If ADP is bound to the FoF1-ATPase in the inner membrane, then protons pass through this complex (also known as Complex V)
and the energy within the pmf is used to synthesize ATP from ADP and inorganic phosphate. The ATPase is similar to those of other
eukaryotes and can likewise form oligomers that influence the configuration of the inner mitochondrial membrane (i.e., participate
in cristae formation; Schertl and Braun (2014)). Once ATP is synthesized, it is then exported out of the matrix in exchange for another
ADP via the adenine nucleotide translocase (ANT). Thus, the flow of electrons and hence oxygen consumption is tightly under
control of cellular ADP levels. This is called acceptor or adenylate control. In animals and other eukaryotes, there also exist “ATP
synthasomes”, which consist of ATP synthase complexed with ANT and a phosphate carrier (Seelert and Dencher, 2011). As yet there
are no reports of such a supercomplex in plants, however the plant ATP synthase does oligomerise into rows and this is associated
with curved cristae structures, as seen in other eukaryotes (Davies et al., 2011).
In plant mitochondria, additional protein complexes are found associated with the ETC. They are distinct from Complexes I–IV
in that they participate in the transfer of electrons from NADH to oxygen, but do not contribute to the pmf. These enzymes are the
alternative or non-phosphorylating NAD(P)H dehydrogenases (NDs), named as NDE and NDI in yeast, which donate electrons to
the ubiquinone pool and the alternative oxidase (AOX), which accepts electrons from reduced ubiquinone (Fig. 1). These
dehydrogenases have different nomenclature in plants (Finnegan et al., 2004). There is also an outer membrane activity; antimycin
A-insensitive NAD(P)H reductase, which is often over-looked (Day and Wiskich, 1975; Soole, 1989). Under particular in vitro
assay conditions this enzyme can donate electrons to cytochrome c in the inner membrane space, and thereby has the capacity to
donate electrons to Complex IV. If this occurs in vivo, it may provide another pathway for cytosolic NAD(P)H to contribute to ATP
production. However, recently, an NADH: cytochrome reductase activity on the outer membrane has been linked to lipid
metabolism in the model plant Arabidopsis thaliana (Duncan et al., 2011) and this may be its role in vivo.
Evidence for the NDs comes from the ability of isolated plant mitochondria to oxidize externally supplied NAD(P)H, which
cannot pass through the inner membrane. The external cytosolic-facing NAD(P)H dehydrogenase (NDE) feeds directly into the
ubiquinone pool, as external NAD(P)H oxidation has an ADP/O ratio of 1.5, equivalent to succinate (Fredlund et al., 1991).
Matrix NADH oxidation in plants has a component which is insensitive to inhibition by the Complex I inhibitor, rotenone. This
oxidation is catalyzed by NDI. The rotenone-insensitive alternative matrix NADH oxidation has an ADP/O ratio also similar to
succinate indicating that NDI also feeds electrons into the ubiquinone pool, bypassing proton pumping at Complex I. In
plants, some of the proteins that catalyse these activities have been functionally characterized and annotated as NDA1, NDA2 and
NDC1 for the NDI family and NDB for the equivalent NDE family (Finnegan et al., 2004; Michalecka et al., 2004; Rasmusson
et al., 2004). NDC1 is dual-located in the cell as it can also be found in the chloroplast and has been functionally linked to a step
in Vitamin K1 synthesis (Fatihi et al., 2015). Its role in mitochondria has not been determined and therefore its ability to donate
electrons to the mETC and ATP synthesis is not conclusive.
The alternative oxidase is a cyanide- and antimycin A-resistant ubiquinol oxidase that catalyses the reduction of oxygen to water
with electrons from ubiquinol and thus bypasses the proton pumping activities of Complexes III and IV (Vanlerberghe, 2013).
Thus in plants, the ATP: O2 ratios can vary in vivo between 5.0 and 1.75 during sucrose oxidation in tissues, depending on the
relative contributions of electron flow through the cytochrome pathway (phosphorylating) and the alternative pathway (non-
phosphorylating), which, in turn, depends on environmental conditions (Munns et al., 2020). From this it follows that when only
the cytochrome pathway of the mETC operates, the yield of ATP from the complete oxidation of one sucrose molecule through the
whole of respiration yields 60 ATP (and an ATP:O2 of 5). If electron flow is via AOX, then this drops to 21 ATP (and an ATP:O2 of
1.75), assuming that intra-mitochondrial NADH is oxidized only via Complex I rather than through the internal alternative NADH
dehydrogenases. If mitochondrial electron flow was to occur entirely through the NDs and AOX, there could be electron transport/
transfer with no ATP synthesis in plant mitochondria. Therefore, due to the bypasses that exist around key regulatory sites in
glycolysis, flux through plant respiratory pathways can be less restricted by adenylate control, which gives plants great metabolic
flexibility. This flexibility can theoretically allow the release of biosynthetic intermediates from the respiratory pathway, inde-
pendent of energy charge of the cell. This role of the non-phosphorylating pathway is hypothesized and has not yet been clearly
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Respiration | Plant Mitochondria – Substrates, Inhibitors, Uncouplers 539

demonstrated in vivo. Both AOX and NDs can be regulated post-translationally (Day et al., 1995; Geisler et al., 2007) and there is
currently very limited data on the actual in vivo activity in any tissue.

Substrates for ATP Synthesis

The plant mitochondrial ETC can oxidize matrix NADH and, to a lesser extent, NADPH. The major source of NADH is from
pyruvate dehydrogenase and citric acid cycle, and plants have the full complement of these enzymes in addition to other unique
enzymes. The presence of matrix pools of NADPH is indicative of the mitochondrion’s anabolic role. NADP-dependent enzymes
involved in folate and thymidylate synthesis such as NADP-dihydrofolate reductase and methylene tetrahydrofolate dehy-
drogenase have been found in plant mitochondria (Neuburger et al., 1996) and their continued operation requires turnover of the
NADPH generated, which can occur via either the NDAs or NDC (Moller and Rasmusson, 1998). Apart from NAD-dependent
isocitrate dehydrogenase, there is also an NADP-dependent form in the matrix, which may be important for nitrogen assimilation
(Chen and Gadal, 1990; Igamberdiev and Gardestrom, 2003). Additionally, via the action of a soluble transhydrogenase detected
in pea leaf matrix, the mitochondrial oxidation of any strictly NAD-linked substrates such as pyruvate would also be able to
produce NADPH (Moller and Rasmusson, 1998). Another unique enzyme found in plant mitochondria is glycine decarboxylase,
which generates NADH in the matrix, is part of the photorespiratory cycle and is important in photosynthetic metabolism (Douce
et al., 2001). It is accepted that the mitochondrial ETC operates in the light, and that ATP synthesis in the light is not exclusively
generated from photosynthesis.
Plant mitochondria also have the capacity to oxidize cytosolic NADH and NADPH directly due to the NDB family (Rasmusson
et al., 2004). Thus, ATP synthesis can also occur from the recycling of cytosolic NAD(P)H. It has also been reported that plants
contain the components required for the oxidation of glycolytic NADH via the glycerol-3-phosphate (G-3-P) shuttle. This involves
the combination of two G-3-P dehydrogenases, one cytosolic and the other a UQ-reducing G-3-P dehydrogenase on the outer face
of the inner membrane feeding electrons directly into the UQ pool (Shen et al., 2003) (Fig. 1). Thus, plants have both strategies to
recycle cytosolic NADH. Enzymes of other metabolic pathways can also donate electrons directly to ubiquinone (Schertl and
Braun, 2014). Proline dehydrogenase is the first step in proline degradation, which is an important metabolite in the plant stress
response. It is a Flavin-dependent enzyme bound to the inner side of the inner membrane and donates its electrons to the UQ
pool, thus potentially contributing to ATP synthesis. Dihydro-orotate dehydrogenase (DHODH) is an enzyme involved in the de
novo pyrimidine biosynthesis pathway. Very little is known about this enzyme in plants, but activity has been detected in isolated
mitochondria from pea leaves and tomatoes, and the enzyme from Arabidopsis thaliana shows distinct properties to animal
DHODH in terms of substrate specificity and inhibitors (Ullrich et al., 2002). Its contribution to ATP is unclear yet possible as it
feeds its electron into the UQ pool. Other metabolic pathways can also directly reduce cytochrome c (Schertl and Braun, 2014).
For example, L-galactono-1,4-lactone dehydrogenase, responsible for the final step of L-ascorbate biosynthesis can donate elec-
trons to cytochrome c and also facilitates assembly of Complex I in plants. Recently D-lactate oxidation via lactate dehydrogenase
was also postulated to deliver electrons directly to cytochrome c (Welchen et al., 2016). D-lactate is produced via the glyoxalase
system in the metabolism of methylglyoxal: a by-product of glycolysis that can be toxic if over-accumulated. In these instances
the plant mitochondrial electron transport chain might be considered a sink for cytosolic electrons.
g-aminobutyric acid (GABA) can also act as a substrate for respiration in plant mitochondria and accumulates during abiotic
and biotic stress exposure (Kinnersley and Turano, 2000). During oxidative stress GABA can occur as a result of polyamine
(putrescine and spermidine) degradation, and potentially through a non-enzymatic conversion from proline. However, the main
biosynthetic precursor is glutamate, which is converted to GABA via glutamate decarboxylase in the cytosol. In stressed plants
GABA can act as a signal, potentially transported across the plasma membrane by aluminum-activated malate transporters
(ALMT), but it can also be used as a substrate for TCA cycle anaplerosis (Michaeli and Fromm, 2015; Ramesh et al., 2015). Entry
of cytosolic GABA to the mitochondrion is via a GABA permease. GABA transaminase converts GABA, in conjunction with one
of a-ketoglutarate, pyruvate or glyoxylate (depending on the isoform and substrate availability), to succinate semialdehyde and
one of glutamate, alanine or glycine (respectively, based on substrate). Succinate semialdehyde dehydrogenase can then intro-
duce succinate to the TCA cycle. Alternatively, GABA can be synthesized in the matrix from TCA cycle intermediates via
a-ketoglutarate dehydrogenase and glutamate decarboxylase as part of the GABA shunt, which completes via GABA transaminase
and succinate semialdehyde dehydrogenase as above (Michaeli and Fromm, 2015). This bypasses two steps of the TCA cycle and
decreases the net output by one NADH and an ATP molecule, providing flexibility for mitochondrial redox state and energetics.

Inhibitors of ATP Synthesis

There are a multitude of inhibitors that act on specific components of the ETC and the use of many of these has been invaluable in
elucidating the composition of the respiratory chain in different species (Scheffler, 2001; Table 1). The two most common and
specific Complex I inhibitors are rotenone and piericidin A; both inhibitors block electron flow between the final iron-sulfur center
and ubiquinone. However, they display different inhibition kinetics and it is postulated, although contested, that the transfer of
electrons to quinone may involve more than one quinone-binding site, similar to those observed in Complex III (Miyoshi, 1998;
Murphy et al., 1995; Friedrich et al., 1994). Many other inhibitors of mammalian Complex I have been discovered, such as
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540 Respiration | Plant Mitochondria – Substrates, Inhibitors, Uncouplers

Table 1 Specific inhibitors of components of the mitochondrial electron transport chain

Respiratory protein Inhibitor Site/mode of action

Complex I Rotenone Blocks e flow from FeS

Piericidin A Cluster N2 to quinone
DPI
Complex II Malonate Competitive substrate
Thenoyltrifluoroacetone (TTFA) Quinone binding (P site)
Carboxin Quinone binding
Nitric oxide
Complex III Antimycin A Quinone binding (N site)
Myxothiazol Quinone binding (P site)
Stigmatellin Quinone binding (P site)
Complex IV Cyanide Competitive inhibitor of O2 binding site
Azide Competitive inhibitor of O2 binding site
Nitric oxide
Complex V Oligomycin Binds to OSCP-subunit
Venturicidin Proton translocation (c-subunit)
Aurovertin F1
Tri-n-butyltin F0
Adenine nucleotide translocator Carboxyatractyloside
NDE1 (external NAD(P)H) DPI
Calcium chelators
NDE2 (external NADH) Calcium chelators

NDI1 (internal NADH)

NDI2 (internal NAD(P)H) DPI
Calcium chelators
Alternative oxidase n-Propyl gallate
Octyl gallate
SHAM

Note: Data from Orme-Johnson, N., 2001. Appendix 5: Direct and indirect inhibitors of mitochondrial ATP synthesis. In: Pon, L.A., Schon, E.A. (Eds.), Methods in Cell Biology,
vol. 65. Elsevier Inc., pp. 483–495.

aurachin A and B, thiangazole, phenoxan, and aureothin, and many are likely to inhibit plant Complex I; however, their efficacy
and specificity has not been demonstrated. Relatively few inhibitors of plant Complex II have been described (Table 1), the most
widely used is the competitive substrate inhibitor malonate; however, several new Complex II inhibitors that are potential
fungicides or pesticides have been reported recently, and nitric oxide has also recently been reported to inhibit activity (Simonin
and Galina, 2013). The two most significant specific Complex III inhibitors are antimycin A and myxothiazol, which are specific to
the N-side and P-side quinone-binding sites, respectively, and have been important for developing models of the Q-cycle which is
involved in proton translocation by Complex III. Aurachin C and derivatives have also been shown to inhibit Complex III in plants
but these also inhibit AOX (Ebiloma et al., 2019). Complex IV is inhibited by a variety of competitive inhibitors of the oxygen-
binding site, such as cyanide and azide. The most interesting of these is the rapid and reversible inhibition by nitric oxide, which
also inhibits Complex II and may play an important role in regulation of oxidative phosphorylation (Gupta et al., 2018).
There are several chemical inhibitors of ATP synthase (Table 1), the most prominent being oligomycin, which is a specific
inhibitor of F-type ATP synthases found in a variety of organisms. Apart from the chemical inhibitors, there are also inhibitor
proteins, which play a role in regulating the activity in vivo. The most characterized is the mammalian inhibitor protein (IF1),
whose conformation and inhibitory activity changes in response to pH. Proteins with low homology to IF1 have been found in
plants, and although they can inhibit ATP synthase activity, they do not appear to be regulated by pH (Norling et al., 1990). More
recently, a class of phosphoserine/phosphothreonine-binding proteins, called the 14-3-30 s, have been identified in plant mito-
chondria and shown to regulate the ATP synthase activity (Aducci et al., 2002). ATP synthesis can also be inhibited by carbox-
yatractyloside, which blocks the adenine nucleotide translocator and thus discontinues ADP supply to the F1 region of ATP
synthase (Vignais et al., 1976).
There are several inhibitors of the alternative NAD(P)H dehydrogenases. While a few show differential selectivity between the
various alternative NAD(P)H dehydrogenases many of these such as platanetin, IACA (7-iodo-acridone-4-carboxylic acid),
dicumarol, hydroxyflavones, and sulphydryl reagents can inhibit all of the alternative NAD(P)H dehydrogenases (Roberts et al.,
1996; Finnegan et al., 2004). Interestingly, many of these were originally reported as specific inhibitors of particular alternative
NAD(P)H activities. The most effective selective inhibitors are calcium chelators such as EGTA, which are potent inhibitors of the
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Respiration | Plant Mitochondria – Substrates, Inhibitors, Uncouplers 541

external NADH and NADPH utilizing enzymes, some of which have a calcium requirement, and DPI (diphenyleneiodonium)
which is more effective at inhibiting NADPH utilizing enzymes compared to NADH utilizing enzymes (Table 1). Recently,
2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) has been shown to bind to the quinone binding-site and inhibit both yeast and
Plasmodium falciparum NDs, suggesting a conserved mechanism of quinone binding in this protein family (Petri et al., 2018). This
inhibitor has not yet been tested on plant NDs and it is not known if would discriminate between the different ND protein
families, NDA, NDB and NDC. SHAM (salicylhydroxamic acid) and n-propylgallate are the most prominent inhibitors of the
alternative oxidase, the latter being more specific as SHAM can also affect other enzymes of oxidative metabolism such as
peroxidases, lipoxygenase, and xanthine oxidases.
Because the non-phosphorylating pathway components are not found in higher animals, there is an opportunity to target
these proteins in antimicrobial strategies (Ebiloma et al., 2019). Indeed, inhibitors of alternative NADH dehydrogenases and
alternative oxidase are being developed as potential antimicrobials against harmful human parasites and pathogens that rely on
these proteins instead of Complex I or Complex IV for mitochondrial electron transport. Such organisms include (but are not
limited to); Plasmodium falciparum (malaria), Streptococcus agalactiae (newborn meningitis), Mycobacterium tuberculosis (tubercu-
losis), Toxoplasma gondii (toxoplasmosis) and Trypanosoma brucei (African sleeping sickness). The crystal structure of plant AOX
remains elusive, however the enzyme from T. brucei has recently been resolved in the presence and absence of bound inhibitors.
This has facilitated chemical synthesis of new potent inhibitors, such as derivatives of ascofuranone, which may also be useful as
inhibitors of plant AOX but are yet to be tested as such (Shiba et al., 2013). The addition of lipophilic groups has also enabled
better targeting of inhibitors to T. brucei mitochondria (Ebiloma et al., 2019).
On the other hand, these proteins are also being explored as candidates for gene therapy, specifically in conditions where
mitochondrial biochemistry is perturbed and over-accumulation of reactive oxygen species is an issue. An AOX gene from the
marine tunicate “sea squirt” Ciona intestinalis can be actively expressed in mammalian cells and in Drosophila melanogaster flies. In
D. melanogaster this confers some resistance to cyanide, reducing accumulation of reactive oxygen species and improving the health
of flies that contained a defective homolog of the human Parkinson’s Disease gene (Fernandez-Ayala et al., 2009). It is also able to
complement human cells deficient in Complex IV (Dassa et al., 2009). Similar recent work with the ND from C. intestinalis has also
shown some promise and may be useful for complementing Complex I deficiencies, as previously demonstrated with the NDI of
Saccharomyces cerevisiae (Sanz et al., 2010; Gospodaryov et al., 2014).

Uncouplers of ATP Synthesis

Chemical Uncouplers
Oxidative phosphorylation in plants is sensitive to chemical uncouplers such as dinitrophenol (DNP), carbonyl cyanide
p-(trifluoromethoxy)phenylhydrazone (FCCP) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), which dissipate the
DmH þ by carrying protons across the inner membrane, thus equilibrating the proton gradient. In heterotrophically grown
tobacco suspension cells, cellular respiration is increased by the addition of the uncoupler, FCCP, therefore under these conditions
respiration is substrate limited (Vanlerberghe, McIntosh, 1992). FCCP and CCCP can also depolarise other membranes but
recently new uncouplers such as (2-fluorophenyl)[6-[(2-fluorophenyl)amino](1,2,5-oxadiazolo[3,4-e]pyrazin-5-yl)]amine (BAM15)
have been identified that depolarise the mitochondrial membrane but not plasma membrane in animal cells, although this is yet to
be tested in plants (Kenwood et al., 2014).

Non-Phosphorylating Respiration
Plants have the capacity to uncouple respiration and ATP synthesis using naturally occurring enzymes such as the NDA, NDB,
NDC, and AOX. Therefore, the level of “coupled” respiration will be dependent on the level and activity of these alternative ETC
enzymes. The number and make-up of gene family members varies among plant species (Costa et al., 2014; Sweetman et al.,
2019a, Wanniarachchi et al., 2018), suggesting different requirements for non-phosphorylating respiration between plant systems.
Furthermore, expression patterns change locally and temporally, while post-translational regulation occurs at multiple levels,
highlighting the need for flexibility in plant mitochondrial respiration. An increasing body of research suggests coordinated
expression and functional cooperation of ND and AOX genes and proteins (Clifton et al., 2005; Sweetman et al., 2019b), while an
imbalance between the capacity for feeding electrons into, and taking electrons out of the ubiquinone pool, could disrupt cellular
metabolism, signaling and growth (Vanlerberghe, 2013).

Alternative NAD(P)H dehydrogenases

In plants, Complex I has a much better affinity for NADH than the matrix-facing NDs, represented by NDAs and NDC. This
suggests that the latter route of electron flow will only be active when the matrix concentration of NADH is high. This has been
clearly demonstrated in tissues like potato, which lose NAD from their mitochondrial matrix during long-term storage. In
NAD-depleted mitochondria, the rate of electron flow through NDI was markedly reduced, i.e., malate oxidation in isolated
mitochondria was totally sensitive to rotenone; however, this could be overcome if the mitochondria were reloaded with NAD
from the bathing media (Tobin et al., 1980). NAD enters via a specific transporter on the inner membrane. It is not clear if this level
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of regulation occurs in vivo. One of the NDA/NDC enzymes that uses NADPH as a substrate is calcium-dependent and could be
regulated via matrix calcium levels. One question is whether these alternative pathways operate as overflow for Complex I or
operate simultaneously during respiration in vivo. In a mutant where expression of NDA1 was eliminated, the total respiration was
reduced, which suggests that NDA1 contributes to respiration in presence of ADP along with Complex I (Moore et al., 2003).
NDB1 and NDB2 are dependent on calcium for maximal activity. It has been suggested that electron flow through these enzymes
can be regulated by alteration of the local calcium concentration, which can be facilitated by polyamines that occur naturally in
plant cells, e.g., spermine and spermidine. There is also evidence of pH-dependence, whereby acidification of the cytosol lowers
the concentration of Ca2 þ required to activate the external NAD(P)H dehydrogenases (Geisler et al., 2007). NDB activities are also
limited by availability of cytosolic NAD(P)H.

Alternative oxidase
For many years, it was thought that the AOX acted as an overflow, only being used when there was an excess of reduced
ubiquinone and the cytochrome pathway (via Complexes III and IV) was saturated. It is now known that this is not the case, and
that AOX and cytochrome pathway can operate simultaneously. This finding was made possible through the development of a
non-invasive oxygen isotope fractionation technique, which exploits differences between Complex IV and AOX for their capacity to
discriminate between 18O2 and 16O2. Work with this technique demonstrated that AOX can contribute up to 50% of mito-
chondrial respiratory activity, depending on the plant species and growth condition, and that the activity of AOX in vivo does not
always align with the maximum capacity for AOX measured in vitro (Del-Saz et al., 2018). Thus, the level of ATP synthesis will rely
on the regulation of AOX as mentioned previously. AOX can be regulated at both the transcriptional and post-translational level.
AOX exists as a monomer or dimer, with the monomeric form being most active. Further, AOX is activated by organic acids such as
pyruvate, although some isoforms respond differently to specific organic acids due to the presence or absence of key regulatory
cysteine residues and nearby amino acid residues. For example, in tomato one AOX isoform has the characteristic cysteine residue
and can be activated by pyruvate, while another has a serine substitution and is instead activated by succinate (Holtzapffel et al.,
2003). In A. thaliana, three isoforms that all harbor this cysteine residue (but with either one or two additional conserved cysteine
residues) are also activated by pyruvate but not succinate. In addition, all three are activated by glyoxylate, two can be activated by
a-ketoglutarate and one of these two can also be activated by oxaloacetate and these differences may be due to amino acid
composition near to the key cysteine residues (Selinski et al., 2018). Pyruvate is the end product of glycolysis, glyoxylate a by-
product of photorespiration and a-ketoglutarate and oxaloacetate are intermediates of the TCA cycle that are produced along with
NADH, thus this activation of AOX can act as a positive feed-forward mechanism of control when glycolytic flux is high and when
there may be a high NADH:NAD in the matrix. AOX is induced upon inhibition of Complexes III and/or IV and also under various
environmental stresses. It is thought that the expression of AOX is an attempt by the plant to have a flexible control of ATP
synthesis (Moore et al., 2002; Vanlerberghe, 2013). For example oxidation of matrix NADH via the non-phosphorylating pathway
may enable continued operation of the TCA cycle when ATP needs are low, by releasing feedback inhibition. This could also divert
respiratory intermediates to other metabolic pathways in a real-time response to environmental change, e.g., biosynthesis of osmo-
protectants during salinity stress, or organic acids as root exudates for improving nutrient uptake. It is also thought to be part of the
cell’s defense against oxidative stress, as increased flux through the ETC would prevent the accumulation of high-energy electrons
in the respiratory pathway, which could react with oxygen to form harmful, reactive oxygen species (Vanlerberghe, 2013).

Uncoupling protein
In addition to AOX, plants also have uncoupling protein (UCP), which mediates proton re-uptake across the inner membrane
(Fig. 1). This activity is activated by free fatty acids and inhibited by pyridine nucleotides. UCP is active during respiration in
the presence of excess ADP when the free fatty acid concentration is approximately 4 mM, and thus can divert energy from oxidative
phosphorylation, impacting on the capacity for ATP synthesis (Jarmuszkiewicz, 2001). There is a reciprocal regulation between
UCP and AOX as free fatty acids inhibit AOX activity (Sluse and Jarmuszkiewicz, 2000). However a precise understanding of the
integration and regulation of non-phosphorylating respiratory pathway, UCP and ATP synthase remains a major research
challenge.

Measuring the Effect of Non-Phosphorylating Respiration in Plants

Effects of the non-phosphorylating pathway on ATP synthesis and other aspects of cellular biochemistry have been difficult to test
in vivo using traditional techniques. Interdisciplinary studies incorporating genetics, cellular energetics, physiology, in vivo
respiratory measurements, enzyme assays and “omics” approaches in different species under different conditions, will facilitate the
understanding of how this pathway influences energy use, metabolism, signaling and the stress response. New tools such as genetic
biosensors are enabling in vivo, real-time, quantitative measurement of plant cellular pH, redox state, Ca2 þ , H2O2, ATP and other
cellular markers that will be key to furthering this research. Signal peptides enable targeting of the sensors to specific cell
compartments (Schwarzlander and Finkemeier, 2013). Findings from such biosensors are already shedding light on plant
mitochondrial biology; such as elegant cooperation of chloroplasts and mitochondria in the balance of excess reducing equiva-
lents and ATP synthesis (Voon et al., 2018), and existence of transient calcium fluxes across the inner mitochondrial membrane
that generate a ‘pulsing’ membrane potential that may periodically uncouple the ETC (Schwarzlander et al., 2012). Understanding
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Respiration | Plant Mitochondria – Substrates, Inhibitors, Uncouplers 543

situational changes in flux, and the regulatory mechanisms of non-phosphorylating respiration and other branch points of plant
mitochondrial metabolism will be key to developing new strategies for improving plant survival and food security under
increasingly difficult environmental conditions.

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