NO and ROS homeostasis in mitochondria: a central role for alternative oxidase
Provide feedback or get help You are viewing our new enhanced HTML article.
If you can't find a tool you're looking for, please click the link at the top of the page to "Go to old article view". Alternatively, view our Knowledge Base articles for additional help. Your feedback is important to us, so please let us know if you have comments or ideas for improvement.
Multiple roles of alternative oxidase
Alternative oxidase (AOX) is an integral part of the mitochondrial electron transport chain (ETC) of plants, fungi and many lower vertebrates (Van Aken et al., 2009). AOX is a homodimeric protein ubiquinol oxidase which directly accepts electrons from the ubiquinone pool and transfers them to oxygen. AOX bypasses energy production associated with the complexes III and IV and lowers ATP generation. Although this could be thought to be detrimental, in fact, by preventing the over-reduction of respiratory chain components, AOX may lessen the production of reactive oxygen species (ROS). Given that ROS are commonly produced under a range of stress conditions, it is unsurprising that AOX modulates the plant’s responses to cold, drought stress, ozone injury (Van Aken et al., 2009) and biotic stresses (Thomazella et al., 2012).
‘The study revealing that AOX can also suppress NO concentration offers a coherent and integrated picture of how plants deal with stress-perturbed metabolism.’
Reactive nitrogen species (RNS), most prominently, nitric oxide (NO) have well-established roles in stress tolerance and are often generated contemporaneously with ROS. As such the study by Cvetkovska & Vanlerberghe in this issue of New Phytologist (pp. 32–39), by revealing that AOX can also suppress NO concentration, offers a coherent and integrated picture of how plants deal with stress-perturbed metabolism. Previously, it was found that AOX may be directly involved in reduction of nitrite to NO but only during anoxic conditions (Gupta & Kaiser, 2010). Crucially, Cvetkovska & Vanlerberghe measured NO under normoxic conditions and revealed that AOX decreases NO production.
Nitric oxide in mitochondria: what we know so far
There are two NO production pathways that have been suggested to be present in mitochondria. One is an L-arginine oxidizing nitric oxide synthase (NOS; Guo & Crawford, 2005) and the other one is a nitrite–NO reductase linked to the ETC (Gupta & Kaiser, 2010). A NOS-like enzyme was named AtNOS1 but later studies showed that the AtNOS1 gene encoded a GTPase and was renamed AtNOA1 (nitric oxide associated 1; Moreau et al., 2008). The decreased NO level observed in the Atnoa1 mutant apparently arises from an over-production of ROS which scavenge NO. Evidence was provided suggesting that mitochondria lack a NOS-like enzyme (Gupta & Kaiser, 2010). Isolated barley mitochondria were unable to synthesize NO after supplying NOS substrates or cofactors such as L-arginine, NADPH, Ca2+, calmodulin, tetrohydrobiopterin (BH4), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) (Gupta & Kaiser, 2010). However, under low oxygen the mitochondrial ETC reduces nitrite to NO and this can contribute to ATP production under hypoxic conditions (Gupta & Igamberdiev, 2011). Pharmacological evidence suggested that complex III, cytochrome c oxidase (CytOX) and AOX are all involved in nitrite to NO reduction; however the mechanism is established only for cytochrome oxidase and the reaction is questioned for complex III and AOX (reviewed in Gupta & Igamberdiev, 2011).
AOX modulates NO levels in mitochondria
By using transgenic tobacco plants with different levels of AOX, Cvetkovska & Vanlerberghe investigated how AOX could play a role in modulating both ROS and RNS. The authors found that lack of AOX leads to elevated levels of superoxide and NO; thus, the AOX respiratory pathway lowers ROS and RNS by dampening electron flow from the ETC to oxygen or nitrite in the cytochrome pathway. This would indicate that AOX controls NO generation by directly influencing the rate of electron leakage to nitrite.
The physiological relevance of these observations is immediately apparent. Robson & Vanlerberghe (2002) found that knocking down of AOX increases the susceptibility of plants to programmed cell death (PCD). According to the observations of Cvetkovska & Vanlerberghe this would undoubtedly be due to both superoxide and NO production (another factor of PCD; Delledonne et al., 2001). AOX was increased in response to treatment by the proteinaceous bacterial elicitor, harpin, and this may be due to NO generation (Huang et al., 2002). Fu et al. (2010) found that induction of AOX by NO protects the plants from tobacco mosaic virus (TMV) infection. In this case AOX will function as an anti-oxidative and anti-nitrosative agent and may protect the plants from viral infection. It has been shown that actively respiring mitochondria are able to scavenge superoxide in a NO-dependent manner (de Oliveira et al., 2008). Such observations clearly imply the existence of a negative feedback loop where NO acts to suppress excess mitochondrial RNS and presumably ROS via increased AOX expression to modulate the elicitation of PCD. Failure of this homeostatic mechanism through prolonged exposure of NO to mitochondria causes mitochondrial dysfunction via nitrosylation of complex I and ultimately to cell death (Brown & Borutaite, 2002).
Possible mechanism of AOX-mediated ROS and RNS homeostasis
Several AOX-related homeostasis mechanisms are suggested in the literature. The mitochondrial ETC is one of the major sources for ROS. The ROS generated in mitochondria depend on membrane potential (Møller, 2001); the higher the membrane potential, the greater the ROS production. Zottini et al. (2002) showed that NO increases membrane potential. To counter this, AOX can modulate the membrane potential and reduce NO levels. This action can lower ROS production (Møller, 2001). This AOX homeostatic step may be triggered by NO inhibition of CytOX (Millar & Day, 1996). When CytOX is inhibited then AOX is induced, the latter not being inhibited by NO. By decreasing NO levels, AOX may relieve CytOX inhibition and balance electron flow to the cytochrome pathway. Since electron transport via the cytochrome pathway leads to full ATP production, lowering NO levels by AOX would contribute to energy efficiency.
NO is also known to inhibit several tricarboxylic acid (TCA) cycle enzymes, perhaps most importantly aconitase (EC 22.214.171.124). This enzyme catalyses the reversible isomerization of citrate to isocitrate so that inhibition of aconitase leads to increase in citrate levels, which can act as a ‘circuit breaker’ of the TCA cycle. As the TCA cycle, by generating reductant, provides electrons which are transferred through the ETC, aconitase inhibition will affect the synthesis of ATP. However, citrate is known to induce AOX not only under normoxic conditions but also under hypoxic conditions (Gupta et al., 2012). This will have a two-fold role to maintain electron flow through the ETC and to lower NO concentrations thus relieving aconitase inhibition.
An additional mechanism of scavenging is suggested by our own data (Gupta et al., 2005). We found that tobacco root mitochondria consume externally supplied NO at much higher levels in the presence of NADH. As AOX activity also requires reducing equivalents, AOX scavenging of NO might help in decreasing ROS production by preventing over-reduction of the ubiquinone pool.
The elegance of such homeostatic mechanisms naturally leads on to questions of how these are overcome to lead to PCD or necrotic cell death in response to stress. The easiest explanation is that the potency of NO and ROS generation from nonmitochondrial sources, nitrate reductase and NADPH oxidase, respectively (Gupta et al., 2011), could swamp any AOX-mediated homeostatic mechanism. Pathogen-elicited PCD could be augmented by NO-dependent nitrosylation of the P-protein of the glycine decarboxylase complex (GDC; Gupta, 2011). This reaction leads to inhibition of its activity and is lethal. It was shown that AOX is co-expressed with GDC and the decrease of GDC amount in mitochondria also results in very low AOX levels (Bykova et al., 2005). It is also worth remembering older data showing how salicylic acid (SA) induces AOX (e.g. Yip & Vanlerberghe, 2001). This is of relevance here as both NO and ROS also induce the synthesis of SA (Delledonne et al., 2001). At high concentrations (> 1 mM) SA blocks electron flow from NADH dehydrogenases to the ubiquinone pool (Norman et al., 2004). This would effectively nullify any homeostatic mechanisms mediated by AOX and disrupt ETC to elicit cell death. The importance of such an anti-homeostatic SA-mediated mechanism is augmented when it is remembered that SA plays a role in the responses to many abiotic as well as biotic stresses (Fragnière et al., 2011).
Taking all of the earlier points together, it can be seen that the paper of Cvetkovska & Vanlerberghe places the maintenance of mitochondrial ETC as a central event in plant stress tolerance. Alternating the efficiency of this homeostatic mechanism by targeting lines with high AOX expression or AOX alleles with higher enzymatic efficiency is even possible as a prospective genetic modification (GM) approach and appears to be an attractive target in developing crops with greater stress tolerance.