Alternative oxidase and plant stress tolerance

ABSTRACT

Alternative oxidase (AOX) is one of the terminal oxidases of the plant mitochondrial electron transport chain. AOX acts as a means to relax the highly coupled and tensed electron transport process in mitochondria thus providing and maintaining the much needed metabolic homeostasis by directly reducing oxygen to water. In the process AOX also act as facilitator for signaling molecules conveying the metabolic status of mitochondria to the nucleus and thus able to influence nuclear gene expression. Since AOX indirectly, is able to control the synthesis of important signaling molecules like hydrogen peroxide, superoxide, nitric oxide, thus it is also helping in stress signaling. AOX mediated signaling and metabolic activities are very much important for plant stress response. This include both biotic (fungal, bacterial, viral, etc.) and abiotic (drought, salinity, cold, heavy metal, etc.) stresses. The review provides a gist of regulation and functioning of AOX.

Plants being sessile, are exposed to various environmental stressors, viz, drought, salinity, metal toxicity, low or high temperature, pathogen attack, nutrient deficiency, hypoxia etc; which aims to hamper their lifestyle and lifespan to a great extent. So, it has developed certain inbuilt mechanism for perception of minute changes in the environment and responders which facilitates, either tolerance or avoidance responses to alleviate the stress. This review rotates round one of these molecules which helps in stress perception and mediates a retrograde signaling pathway to architect a tolerance/avoidance response.

What is Alternative Oxidase?

The discovery of alternative oxidase (AOX) was at the beginning of the 20^th century from thermogenic plants during anthesis. AOXs are interfacial membrane bound, cyanide insensitive, metallo-protein involved in mitochondrial redox reactions. AOX branches off from the cytochrome pathway of mitochondria at the level of Ubiquinone (UQ) and is responsible for coupling, oxidation of ubiquinol, to 4 electron reduction of oxygen to water.¹ As AOX bypasses Complex III and IV of the cytochrome pathway, it dramatically reduces ATP generation and the energy thus released is dissipated as heat.² It helps in maintaining metabolic homeostasis and signaling dynamics in mitochondria. Stressors effect plant growth resulting in misbalance of energy demands and production. The ability of plants to maintain the delicate balance of energy production and utilization is fundamentally important for their survival. Presence of AOX forms the striking functional difference between mitochondria of higher plants (as well as some fungi and protists) and animals, i.e., presence of two terminal oxidases, AOX and cytochrome oxidase. AOX is encoded by two nuclear gene subfamily, AOX1 and AOX2, where dicotyledons possesses both gene ubfamilies, while monocots have only AOX1 genes.³ AOX is responsible for thermogenesis (attract insects for pollination) and stress tolerance. In response to stress AOX mediates a retrograde signaling pathway which in turn regulates gene expression both transcriptionally and post transcriptionally. This review is aimed to primarily focus on AOX functioning and regulation.

Mechanism of AOX in plants

AOX acts by introducing a branch into ETC at the ubiquinone pool preventing excessive reduction of the downstream complexes (cytochrome pathway), in case of any dysfunction, thus cutting down the single electron leakage from any of the ETC complexes to O₂ or nitrite. Hence preventing excessive mitochondrial ROS, RNS (reactive nitrogen species, designate NO and other NO-related molecules) production by maintaining carbon pool homeostasis, ¹⁹ redox state homeostasis in nitrogen assimilation process.^20,21 and energy state homeostasis during mineral ion uptake.²² AOX provides the plant respiratory system with, in built flexibility regarding coupling among carbon metabolic pathways, ETC activity and ATP turnover.

Regulation of AOX

Genetic control of AOX respiration

As described earlier AOX proteins are encoded by two subfamilies of genes AOX1 and AOX2. This AOX family of genes are induced to transcribe, by various stimulants. One of the important stimulants is the dysfunction in mitochondrial electron transport chain complexes I, III and IV.^4,5 Thus any component leading to cytochrome pathway complex dysfunction will lead to induction of AOX. Besides that accumulation of Tricarboxylic acid (TCA) cycle intermediate citrate is an important signal controlling expression as was observed in tobacco cells.⁶ But these signaling pathways are independent of each other since inhibition of cytochrome pathway doesn't increase citrate and vice versa. Another important reason for AOX activation is to balance momentum of chloroplasts and mitochondria in presence of light. Since both systems produce a large amount of energy, it is of utmost necessity to maintain coordination between the activities of chloroplasts and mitochondria in the light.⁷ The expression of AOXs also is known to be induced by a wide range of biotic and abiotic stresses.^8,3 The induced expression of AOX genes under stress conditions is associated with stress-dependent reactive oxygen species (ROS) production. So not surprising that ROS also induces AOX expression.^6,9 On the other hand plant hormone salicylic acid concentration also shows dramatic effect toward AOX expression as low concentration induces AOX whereas higher concentration do not.^10,11

Biochemical control of AOX activity

Expression of the AOX genes and protein formation do not determine the activity of the enzyme. AOX are inner mitochondrial membrane protein existing as a homodimer, regulated by 2 step biochemical regulation. The linkage between the monomers, determines the activity of the enzyme. When the 2 monomers are non-covalently linked it is in reduced active form whereas when covalently linked, it is in its oxidised inactive form.¹² In wheat, for instance, drought stress was associated with an increased conversion of AOX protein from its inactive (oxidized) to active (reduced) form¹³ Once in reduced active form second regulatory mechanism comes into action wherein the α-keto acids especially pyruvate come into play.^14,15 The exact mechanism on how pyruvate control AOX has not been elucidated yet. Two conserved cysteine (Cys_I and Cys_II) are present in AOX and interactions with these residues have been found to modulate the enzyme activity. Pyruvate stimulation occurs only at Cys_I, whereas glyoxylate alters AOX activity by interacting with Cys_I and Cys_II.^16,17 AOX proteins with different regulatory properties have also been reported, which depend on whether a cys or ser is present in the conserved position. In a case of serine substitution for cysteine as observed in rice and tomato AOX is activated by succinate.¹⁸

AOX and retrograde signaling

The signaling from organelles controlling nuclear gene expression is called retrograde signaling (opposite of anterograde signaling). Mitochondria has been increasingly regarded as a signaling organelle able to relay their metabolic status to the nucleus thus having an influence in latter's gene expression but it is still less unravelled and less understood in comparison to chloroplastic retrograde signaling network.²³ ROS and RNS molecules act as mediators to the process with AOX playing the role of an important linker.²⁴ AOX is playing a link between metabolic activities (mitochondria) and signaling (nucleus); and mediates in the creation of a retrograde signaling network. AOX activity reduces the generation of O²⁻ which further reduces the conversion of O²⁻ to other ROS species, whereas AOX knockdown heightens the activity of ROS scavenging enzyme throughout the cell.²⁵ In other words, mechanisms that control mitochondrial ROS generation such as AOX is actually playing an important role on how cell manages the ROS load and maintain its homeostasis (Fig. 1). Similar, is the control of AOX toward RNS signaling and in turn RNS, like, NO controls glycine decarboxylase (GDC) which is responsible for conversion of glycine to serine, an important step in the photo- respiratory cycle.³ROS and RNS plays a major role in developmental as well as abiotic and biotic stress signaling in plants attributed to the regulatory mechanisms controlling their production.²⁶

Figure 1.

Schematic representation for AOX induction under stress and response of the plant Electron Transport Chain (ETC). Under unstressed condition NADH oxidation by complex I is coupled to proton transport from matrix to inter-membrane space (IMS), whereas oxidation of FADH₂ and NAD(P)H doesn't lead to such fate. Similar is the electron flow from ubiquinol to complex III and then to complex IV which is additionally associated with reduction of O₂ to H₂O. Proton transport across the membrane generates a proton motive force which is dissipated by complex V to produce ATP. But under stress due ETC complex dysfunction, citrate accumulation there is electron leakage from complexes as ROS and RNS (shown in dotted lines) which induces AOX. AOX puts a branch in ETC after ubiquinol pool and directly reduces O₂ to H₂O with production of heat. I, II, III, IV, V: Complex 1–5; IMS: Inter Mitochondrial Space; IMM: Inner Mitochondrial Membrane.

Abiotic stress

AOX has important roles to play in countering abiotic stress in plants ranging from drought, light, and salinity to heavy metal stress. High expression of AOX induces enhanced salt tolerance capability in Medicago truncatula through regulation of ROS and protection of photosystem.²⁷ It has been reported that, for several species of monocot and dicot leaves, drought stress can lead to an increase in the transcription of gene(s) encoding AOX, the amount of AOX protein, or the maximum capacity of the AOX pathway to consume electrons.⁷ AOX controls respiration, photosynthesis and chlorophyll synthesis during drought to maintain an overall homeostasis and enhance plant lifespan.⁷ AOX has also been reported to be responsible for development of cold resistance in winter wheat seedlings, whereas at temperatures below zero AOX activity slightly decreases.²⁸ Phosphorous deficiency leads to shortage of adenylates and Pi, which are substrates for oxidative phosphorylation and hence AOX is induced to maintain electron flow.³ Aluminum stress causes ETC dysfunction which also leads to higher AOX expression to maintain cellular homeostasis²⁹ (Fig. 1). AOX1 was found to modulate oxidative challenge due to cadmium exposure.³⁰ Since AOX is regulating ROS synthesis it is actually regulating all ROS signaling pathways responsible for stress responses in a cell.

Biotic stress

Though much work has not been done on the role of AOX in mediating plants' response to attack by fungal, bacterial and viral pathogens, still a vague impression can be made. Plants responds to biotic stress by eliciting a salicylic acid mediated hypersensitive reaction to limit pathogen spread and in the process AOX expression is enhanced. Salicylic acid has been reported to inhibit mitochondrial ETC in tobacco cell cultures and this inhibition might be inducing AOX expression.³¹ AOX enhanced expression causes lesions which causes programmed cell death.³²

AOX in intra-organelle cross-talk

Mitochondria acts as key component of all bioenergetics of a cell, being sensors of environmental changes and metabolic reactions on stress perception. Mitochondrial AOX and UCPs (Uncoupling proteins, responsible for dissipating proton gradient) provide plants metabolic flexibility and take part in intra-organelle cross-talk, mediating level of energetic molecules (NAD(P)H and ATP/ADP) and messenger molecules such as ROS and calcium. Roles and importance of AOX and UCP in stress signaling were recently reviewed.¹⁸

Conclusions

AOX in last decade has developed as an important player in plant stress response linking metabolic status to that of signaling and thus regulating nuclear gene expression. Though much work has been done a lot is yet to do to unravel the whole mechanism of retrograde signaling and its control. The story of AOX reveals how important is to maintain the homeostasis of mitochondrial energetics and function for plant survival.

Disclosure of potential conflicts of interest

Authors declare no conflict of interest.