Response of mitochondrial alternative oxidase (AOX) to light signals

Abstract

Mitochondrial alternative oxidase (AOX), the unique respiratory terminal oxidase in plants, catalyzes the energy wasteful cyanide (CN)-resistant respiration and plays a role in optimizing photosynthesis. Recent studies from our group indicated that AOX plays a crucial role in chloroplast protection under extreme environments, such as high light (HL). Genetic data suggest that AOX is upregulated by light that was mediated by photoreceptors (phytochromes, phototropins and cryptochromes), and it also might have a particular role in relieving the overreduction of chloroplasts. Physiological analyses further suggest that AOX is essential for the dark-to-light transition, especially in de-etiolation course. In this mini-review, we highlight recent progresses in understanding the beneficial interaction between photosynthesis and mitochondria metabolism and discuss the possible role and mechanism of AOX in dissipation of excess reduced equivalents for chloroplasts under high light condition.

Introduction

For the last two decades, metabolic interaction between photosynthesizing chloroplasts and oxidative-respiring mitochondria has been intensively studied.1,2 In illuminated leaves, intracellular metabolism is dynamically modulated depending on environmental changes. Previous studies showed that excess light energy is harmful for plants and leads to disruption of the photosynthetic apparatus, especially the accumulation lots of reducing equivalents by the photochemical reaction in the thylakoid membrane of chloroplast under high light (HL) conditions.3–6 Depending on the developmental stage and/or environment, reducing equivalents generated in the form of NADPH are often in excess in the chloroplast stroma.7 The accumulation of reducing equivalents in the stroma causes over-reduction of the photosynthetic electron transport chain and accelerates generation of ROS. Under such conditions, the function of chloroplasts and mitochondria is closely coordinated. Recently, metabolic interaction between chloroplasts and mitochondria has been intensively studied.1 It has been suggested that AOX plays an essential role in preventing chloroplast over-reduction through the efficient dissipation of excess cellular reducing equivalents.8,9 These results may suggest that AOX would function as a sink for excess reducing equivalents generated in the chloroplasts, and this role of AOX has attracted much attention in terms of its interaction with photosynthesis. However, evidence for the role of AOX as a system for dissipation of excess reducing equivalents is still weak. Here, we reviewed and discussed the recent advances about the possible relationships between photosynthesis and mitochondrial metabolism and the light signaling involved in it.

AOX Gene Families and Physiological Functions in Plants

Mitochondria in higher plants have two ubiquinol-oxidizing pathways in the respiratory chain. One is the cytochrome pathway (CP), which is common in the respiratory chain in all aerobic organisms, and the other is the cyanide-insensitive alternative oxidase (AOX).9,10 The AOX branches from the cytochrome pathway at the level of the ubiquinone pool and couples the oxidation of ubiquinol to the four-electron reduction of oxygen to water. In angiosperms, two types of AOX genes exist: AOX1 genes have been found in all angiosperms examined to date, while AOX2 genes appear to be limited to dicot speces.11,12 In Arabidopsis, there are five AOX genes: AOX1a, AOX1b, AOX1c, AOX1d and AOX2.13,14

Accumulating evidence suggests that AOX may play a significant role in cell adaptation under different types of stress. Changes in AOX gene expression and AOX protein activity are induced in response to diverse biotic and abiotic stresses.15,16 As was suggested a long time ago and as has been shown by the results of many studies, AOX reduces the effects of oxidative stress by preventing accumulation of ROS in mitochondria. Moreover, ROS was regarded as a signaling molecule that can induce or upregulate AOX expression. However, there are multiple signaling pathways leading to AOX induction that can be either ROS-dependent or -independent.17 Therefore, what the true factors are that affect AOX expression is largely dependent upon a given set of metabolic conditions. Recently, many works have demonstrated that in plants AOX can be used as a functional marker for efficient cell reprogramming under stress.18–20

The Possible Role of AOX in Balancing Photosynthesis and Respiration Metabolism Under High Light (HL)

Light is a key regulator of gene expression in plants, altering the transcription of thousands of genes through direct (photo-receptor-mediated) or indirect (photosynthetic product-mediated) pathways.21 However, the vast majority of detailed studies on light regulation have focused on photosynthesis-associated nuclear genes, and little is known about the effects of light on mitochondria and the respiratory chain. Recently, it has been demonstrated that the type II NAD(P)H dehydrogenase genes (nda1 and nadc1) are regulated by light in potato (Solanum tuberosum) and Arabidopsis22,23 and that the alternative oxidase genes are also light induced, not only monocots but also in dicots.24–27 The mode of signaling underlying light-regulated changes in mitochondria is unclear as of yet: how does the light signal reach the mitochondrion and how does the light signal modulate mitochondrial functions? Previous studies demonstrated that at least three pathways mediated the light-related cross-signaling between chloroplast and mitochondria (Fig. 1).

Under excess light conditions, AOX might serve to regenerate oxidised reductants (NAD⁺ and NADP⁺) and thereby prevent the inhibition of carbon metabolism under conditions of cytochrome (cyt) impairment.9 Recently, many works have demonstrated that in plants AOX can be used to remove excess reducing power from the chloroplast.25–27 To prove this, AOX antisense and overexpressor lines were used to demonstrate that these reducing equivalents are transported from the chloroplast to the mitochondria where AOX serves to dissipate the excess reducing equivalents. Moreover, the oxaloacetate/malate shuttle was regarded as the most likely pathway for transporting those reducing equivalents from chloroplast to mitochondria, where they are taken up by the electron transport chain (ETC) and dissipated via mitochondrial non-phosphorylating pathways (including AOX).2 Thus, AOX appears to contribute to the protection against photo-inhibition in plants.

In addition, controlling reactive oxygen species (ROS) generation may be another explanation for the role of AOX in alleviating photo-oxidative damage. As we all know, photosynthetic electron transport is known to be one of the main sources of ROS production in the photosynthesizing cell in light, and ROS generated in chloroplasts are involved in the cell metabolism and its regulation.28,29 Under light condition, ROS production increases with increasing light intensity.30,31 Earlier studies suggested that ROS can act as signal molecules in the cell and function as environmental sensors. Excess ROS imposes photo-oxidative damage to the photosynthetic apparatus.2 AOX is thought to function as an antioxidant system and to suppress the generation of ROS.9,32 Thus, AOX would be essential for maintaining the photosynthetic electron transport chain in a more oxidized state, especially under stressful conditions. However, this idea may not necessarily be true in illuminated leaves, because they contain chloroplasts that are more susceptible to light energy and are the major sites of ROS generation in the light.33 In the chloroplasts, ROS production could be avoided by several systems dissipating excess light energy, such as thermal dissipation of light energy by the conformational changes in photosystem II (PSII) and by the xanthophyll cycle, which is induced by PSI cyclic electron flow.34 Therefore, it appears that upregulation of AOX expression and capacity under HL condition was affected directly or indirectly by ROS (Fig. 1).

In our study, more ROS and reducing equivalents accumulated in the Arabidopsis aox1a mutant than in wild type after HL treatment. In addition, all enzymes (NADP-MDH, NAD-MDH, citrate synthase (CS), NADP-ICDH and NAD-ME) involved in the malate/oxaloacetate shuttle were increased by the HL treatment and were always higher in the aox1a mutant after the HL treatment. Interestingly, ROS accumulation was so little in the aox1a mutant compared to the control that the role of AOX in relieving the over-reduction of chloroplasts may be more important than ROS scavenging in seedlings grown under HL conditions.27

In addition to the indirect pathways of light-regulation of mitochondria described above, light could directly influence the respiratory electron transport chain via photoreceptor-mediated transcriptional control.21,35 Escobar et al.21 demonstrated that alternative oxidase (AOX), uncoupling protein (UCP) and type II NAD(P)H dehydrogenase gene families were directly regulated by light in etiolated Arabidopsis seedlings. Feng et al.26 showed that AOX pathway activation in the initial greening stage appears to be independent of photosynthetic metabolism and light would be a direct signal to induce the AOX pathway. In our work, we found that photoreceptors mediated light-induced AOX gene family expression in Arabidopsis. When Arabidopsis seedlings were illuminated with red (R) or blue (B) light, AOX1a expression could be doubled; while a clear decrease in transcript abundance was apparent in the phyA or phyB mutant and no increase could be found for the phyA,phyB double mutant, indicating that photoreceptors are a central component of the light signaling transduction pathway to AOX genes.27 In addition, analysis of the light-responsive motifs in AOX1a promoter further improved the critical functions of photoreceptors in light signaling to AOX.27

Previous work demonstrated that AOX plays an important role in dark-to-light transition, especially in the course of de-etiolation. Several studies revealed that the AOX pathway was involved in chloroplast carbon assimilation.36,37 Chlorophyll accumulation and CO₂ fixation rate induced by light were largely delayed by SHAM (an AOX inhibitor) pretreatment and in the Ataox1a mutant,26,27 which is likely to be related to the inactivion or downregulation, respectively, of alternative pathways. These results imply that the AOX pathway may play a key role in chloroplast functions and that there might be a retrograde signaling from mitochondria to chloroplasts (Fig. 1).

Perspectives

Several studies on the modulation of photosynthesis and respiration have been made using light as one of the regulatory tools.8 However, limited data are available, and the beneficial interaction between photosynthesis and mitochondria metabolism is still unclear. Under high light conditions, reductants are exported from the chloroplasts to the cytosol, probably via the NADP-MDH and Mal-OAA shuttle; and the respiratory chain is thought to dissipate excess reductants produced in the chloroplasts and AOX is thought to play an important role in relieving these reducing equivalents. Therefore, more research is needed to identify transporters functioning in the shuttle machinery from chloroplast to mitochondria in detail. Since ROS produced in the chloroplasts under excess light could induce AOX expression and capacity, further investigations are needed to examine the signal transduction pathways between ROS and AOX. Many more experiments are needed to characterize the nature and the role of the alternative pathway in the initial greening process of etiolated plants. What is the retrograde communication between chloroplast to mitochondria, and which process of mitochondrial retrograde regulation (MRR) occurs during the dark-to-light transition? Consequently, more attention should be paid to the effects of MRR on chlorophyll and chloroplast biosynthesis.

It is worth noting that mitochondrial uncoupling proteins (UCP) also play an important role in maintaining photosynthesis redox status.38 Consequently, AOX probably works in conjunction with UCP under extreme environments, such as high light. Thus, further studies are needed to clarify the role and relationships of the two energy-dissipating respiratory proteins. Moreover, transgenic plants and novel experimental techniques are also needed to clarify the interaction between chloroplasts and mitochondria in the future.

Figures and Tables

Figure 1 Putative signaling pathways from light to organelles. Under excess light conditions, reducing equivalents (such as NADPH) generated in chloroplast which can be transported to mitochondria by Malate/OAA shuttle; Excess reducing equivalents can be dissipated by non-phosphorylating pathways, including NDin and AOX. Photoreceptors also mediate light-induced expression of NAD(P)H and AOX. In addition, alternative pathway may play an important role in the initial greening process of etiolated plants through mitochondrial MRR pathways. AOX, alternative oxidase; OAA, oxaloacetate; CI, Complex I; CII, Complex II; CIII, Complex III; CIV, Complex IV; Cyt c, cytochrome c oxidase; MRR, mitochondria retrograde regulation; NDex, external type II NAD(P)H dehydrogenase; NDin, internal type II NAD(P)H dehydrogenase; ROS, reactive oxygen species; TFs, target factors; UQ, ubiquinone.

Acknowledgements

This work was supported by the National Key Basic Research ‘973’ Program of China (2009CB118500), National Nature Science Foundation of China (30970214, 30800071, 31070210 and 910170004) and the Sichuan Nature Science Foundation (2010JQ0080).