Does the alternative respiratory pathway offer protection against the adverse effects resulting from climate change?

Elevated greenhouse gases (GHGs) induce adverse conditions directly and indirectly, causing decreases in plant productivity. To deal with climate change effects, plants have developed various mechanisms including the fine-tuning of metabolism. Plant respiratory metabolism is highly flexible due to the presence of various alternative pathways. The mitochondrial alternative oxidase (AOX) respiratory pathway is responsive to these changes, and several lines of evidence suggest it plays a role in reducing excesses of reactive oxygen species (ROS) and reactive nitrogen species (RNS) while providing metabolic flexibility under stress. Here we discuss the importance of the AOX pathway in dealing with elevated carbon dioxide (CO₂), nitrogen oxides (NOx), ozone (O₃), and the main abiotic stresses induced by climate change.

Recent advances in our understanding concerning the in vivo regulation of alternative oxidase (AOX) and its structural properties suggest that novel AOXs with altered regulatory properties could be used in future gene editing strategies. We suggest that fine-tuning modulation of the regulatory properties of AOX and targeting its expression in different plant tissues could improve plant growth and productivity under climate change conditions promoted by elevated greenhouse gasses (GHGs). Moreover, we also emphasize the need for extensive study on the interactive effects of major global change factors on AOX respiration and the importance of studies differentiating between the roles of AOX in sink versus source tissues under field conditions in order to improve plant productivity in response to elevated GHGs.

Climate change is associated with an elevation of the greenhouse gases such as (CO₂), nitrogen oxides (NOx), ozone (O₃), and methane (CH₄), and with increased events of adverse conditions for plants including drought and high temperature stress as well as flooding (Min et al., 2011; Pall et al., 2011). Such abiotic stress conditions in combination with increasing biotic stresses are challenging plant and agricultural research to adopt new strategies for developing more climate-resilient crops with high yield and productivity in order to meet the enhanced global population food demand (Dhankher and Foyer, 2018). Considering that respiration and photosynthesis are the main components of plant carbon balance, alterations in respiration can potentially affect plant growth and productivity (Zhang et al., 2018; Amthor et al., 2019). In particular, the AOX pathway has been demonstrated to improve plant performance under different physiological conditions—mainly due to its roles both in providing metabolic flexibility and in lowering the level of mitochondrial reactive oxygen species (ROS) (Vanlerberghe, 2013; Selinski et al., 2018; Del-Saz et al., 2018a). As such, it probably functions to protect plants against the adverse effects of climate change (Fig. 1).

Fig. 1.

The role of the alternative oxidase (AOX) pathway in mitigating the effects of climate change and improving plant growth. Environmental, human, and microbial activities lead to increased greenhouse gases (GHGs). These GHGs can elevate ROS and RNS directly or directly via inducing various stresses. AOX can reduce excess ROS and RNS while maintaining energy and carbon balance to improve plant growth. In the inset, a schematic representation of the plant mitochondrial electron transport chain (mETC) is shown. The mETC contains the classical components involved in oxidative phosphorylation [I, II, III, IV (or cytochrome oxidase, COX), and V], which yields ATP. Complexes I, III, III, and IV are also sources of superoxide (O₂^–) and nitric oxide (NO), which can be transformed into other ROS and RNS. The AOX is inserted at the inner mitochondrial membrane (IMM) and diverts electrons from the ubiquinone (UQ) pool by reducing O₂ to H₂O without proton (H⁺) translocation into the intermembrane space (IMS). In this way, the AOX can stabilize the reduction level of the UQ pool and other mETC components, thus preventing the formation of O₂^– and NO. At the same time, the AOX activity renders respiration independent of adenylate control, thus allowing the reoxidation of matrix and extramitochondrial NAD(P)H under high-energy charge or COX restriction. Several physiological situations can require the action of AOX to maintain or enhance the activities of the TCA cycle and other cellular metabolic processes under energy and carbon imbalance. Yellow and red boxes indicate induced and reduced molecule levels or processes by the action of AOX, respectively.

Susceptibility of plants to various abiotic and biotic stresses can be aggravated by climate change-induced ROS including the superoxide anion (O₂^–), hydrogen peroxide (H₂O₂), and the hydroxyl radical (·OH^–) (Cassia et al., 2018). These ROS originate from various sources such as the mitochondria, chloroplast, peroxisome, and the plasma membrane NADPH oxidase (Mittler, 2017). Various stresses additionally induce nitric oxide (NO), which in turn reacts with ROS, leading to the production of reactive nitrogen species (RNS) such as peroxynitrite (ONOO^–), nitric dioxide (NO₂), nitrosyl anion (NO^–), dinitrogen trioxide (N₂O₃), dinitrogen tetroxide (N₂O₄), and nitrous acid (HNO₂). Low levels of these free radicals trigger important signals; however, if they are produced in higher levels, they can cause adverse effects such as damage to lipids, proteins, and DNA, and consequently impact on plant growth and development. Plants have accordingly evolved various machineries that can deal with elevated ROS, including ascorbate peroxidase (APX), catalase (CAT), superoxide dismutase (SOD), and other enzymes involved in the ascorbate–glutathione cycle (Choudhury et al., 2017). Among mitochondrial proteins, the AOX controls mitochondrial ROS production and plays a role in adaptive plasticity. Briefly, AOX protein is inserted in the inner membrane of plant mitochondria and branches the cytochrome c oxidase (COX) pathway at the level of the ubiquinone (UQ) pool bypassing two sites of proton translocation associated with ATP production (Fig. 1). In this manner, the AOX pathway can stabilize the UQ reduction level and prevent the production of excessive ROS. Furthermore, the activity of the AOX pathway renders respiration independent of adenylate control, thus allowing the continuation of respiratory metabolism, which is crucial for plants to cope with different stress conditions (Del-Saz et al., 2018a) such as those promoted by climate change.

The AOX pathway in modulating ROS and RNS induced by greenhouse gases

Different studies have reported that AOX expression is responsive to a variety of greenhouse gases such as O₃, NO, and CO₂. O₃ is an important component of the stratosphere. It aids in filtering dangerous UV. In contrast, in the troposphere, O₃ is deleterious to plant performance. O₃ in the stratosphere is made by reaction between NOx, CO₂, CH₄, and volatile organic compounds (VOCs) in the presence of sunlight (Hickman, 2010). Increased O₃ can cause adverse effects on plants. For instance, exposure of plants to O₃ causes massive changes in transcription, translation, and metabolism, resulting in decreases in plant productivity of up to 15% (Wilkinson et al., 2012). In addition, increased formation of numerous free radicals has been reported in plants after O₃ exposure (Fiscus et al., 2005). These radicals can disrupt various organelles, causing programmed cell death and reducing yield in various crops (Mills et al., 2011). AOX can protect plants against damage imposed by ozone.

Intriguingly, Ederli et al. (2006) reported that exposure of tobacco plants to 300 ppb O₃ strongly induced AOX expression. Moreover, Tosti et al. (2006) reported that O₃ exposure resulted in the induction of AOX protein promoted by a crosstalk between ethylene and NO signalling. Furthermore, induction of AOX occurs via the inhibition of the cytochrome c pathway by O₃, and at the same time the inhibition of the cytochrome pathway by O₃ leads to production of H₂O₂. Consequently, the H₂O₂ produced causes further induction of AOX1a via retrograde signalling (Tosti et al., 2006). While the extent of the effect of O₃ on in vivo AOX activity has not yet been determined, it follows that the overexpression of AOX in crop plants may offer plant resistance to O₃ injury by reducing the levels of ROS.

Excess NOx (such as NO and NO₂), which occur within the natural atmosphere, can be problematic since they are both components of GHGs as well as being inducers of other GHGs such as O₃ (Hickman et al., 2010). NO, a free radical signal molecule, is a component of NOx. Excess NO can cause tyrosine nitration of proteins, thus inhibiting their activities. Several free radicals and metabolites such as pyruvate and citrate are inducers of AOX (Vanlerberghe, 2013). Among them, NO is an inducer of the AOX at the transcript and protein level (Huang et al., 2002; Kumari et al., 2019). Treatment of cell suspensions with NO leads to an increased capacity of the AOX pathway, and inhibition of AOX leads to increased NO sensitivity to cell death, suggesting that NO is induced to protect cells from cell death (Kumari et al., 2019). Fu et al. (2010) has shown that AOX is important in the prevention of cell death induced by Tobacco mosaic virus. Since O₃ also causes cell death, the induced AOX can help in the protection from cell death (Overmeyer et al., 2005). The AOX pathway prevents excess ROS and NO production (Maxwell et al., 1999; Cvetkovska and Vanlerberghe, 2012; Alber et al., 2017; Vishwakarma et al., 2018). NO reacts with superoxide, leading to production of ONOO^– which can cause tyrosine nitration and reduces function of various enzymes. In this context, the capacity of AOX to control both NO and ROS production makes it a very powerful machinery for the protection of plants against these molecules. Recently, it was demonstrated that AOX not only scavenges NO under normoxia induced by flg22 (flagellin) but also generates NO under hypoxia (Vishwakarma et al., 2018). In contrast to normoxia, hypoxia-induced NO does not react with superoxide, but rather is scavenged by phytoglobin1 via metaphytoglobin reductase activity. The scavenged NO has a role in recycling nitrate, maintenance of the redox status, and operation of the phytoglobin–NO cycle to generate ATP (Vishwakarma et al., 2018). NOx emissions also contribute to increased temperature, which indirectly can increase flash floods with the consequent hypoxic atmosphere in soil. Hence AOX can play a role under flooding conditions to improve energy efficiency and survival.

Although CO₂ is important for photosynthesis, elevated CO₂ can have some negative impacts on plants. The study of Loladze (2014) based on >130 species and crop species found that elevated CO₂ can reduce mineral content on average by 8% and increases the ratio of soluble carbohydrates to proteins. Several important elements such as zinc and iron diminished in several food crops such as rice, wheat, and soybean in the presence of high CO₂ (Myers et al., 2014). Elevated CO₂ also induces ROS (Cheeseman, 2006) and, in order to detoxify ROS, plants also induce various antioxidants (AbdElgawad et al., 2016). Several reports suggest that AOX protein is highly responsive to elevated CO₂ (Yoshida and Noguchi, 2009; Dahal and Vanlerberghe, 2018a). The relationships between yield, ROS production, and mineral nutrition in AOX-modified plants under elevated CO₂ (eCO₂) remain to be investigated and could provide more insights into the protective role of AOX.

The AOX pathway in providing metabolic adaptations of plants to stresses aggravated by climate change

The AOX pathway provides flexibility in cellular energy and carbon metabolism under drought, elevated temperature, and CO₂ (Del-Saz et al., 2018a; Dahal and Vanlerberghe, 2018a, b), which represent the major abiotic stresses challenging current agricultural productivity with regard to climate change. The beneficial role of such metabolic flexibility probably compensates the theoretical negative effects of AOX in reducing ATP and reductant availability required for growth (Vanlerberghe, 2013). In this context, Dahal and Vanlerberghe (2018b) importantly reported that plant growth was higher in tobacco plants overexpressing AOX as compared with wild-type plants after prolonged water deficit. The beneficial effect on growth has been linked to the ability of AOX to maintain a higher respiration in light, which improves chloroplast energy balance and photosynthesis (Dahal and Vanlerberghe, 2018b). The role of the AOX in improving photosynthesis has also been reported in other species under different conditions (reviewed by Del-Saz et al., 2018a) and is probably among the main reasons explaining the beneficial role of AOX in plant growth and productivity. Nevertheless, there is also evidence suggesting that the in vivo AOX activity favours the synthesis of tricarboxylic acid (TCA)-derived metabolites with specific roles in protecting against high light (Florez-Sarasa et al., 2016) and salinity stress (Del-Saz et al., 2016).

Adjustments to the partitioning of electrons between AOX and COX pathways were associated with changes in tissue energy demands of plants exposed to long-term elevated CO₂ conditions (Gomez-Casanovas et al., 2007). In addition, changes in mitochondrial electron partitioning to AOX were related to the improvement of leaf carbon balance and respiratory efficiency under different CO₂ growth conditions (Gonzàlez-Meler et al., 2009). Recently, AOX overexpression has been shown to prevent both carbohydrate and energy imbalances in leaves of tobacco plants grown at elevated CO₂ (Dahal and Vanlerberghe, 2018a). All these studies suggest that increased AOX activity can be beneficial for plant growth under elevated CO₂ conditions.

Future perspectives

As discussed above, the use of AOX-transgenic plants has provided important insights into the role of AOX in photosynthetic tissues and growth. However, the effects of AOX genetic modification on root growth and metabolism under stress have received much less attention (Smith et al., 2009, Keunen et al., 2016). Importantly, AOX has a role in the synthesis of carboxylates in white lupin (Florez-Sarasa et al., 2014), tobacco (Del-Saz et al., 2017), and tomato (Del-Saz et al., 2018b). The root exudation of carboxylates improves phosphate acquisition, which benefits photosynthesis and plant growth (Pang et al., 2018). On the other hand, information about the impact of the AOX pathway on the growth of other sink and reproductive tissues, such as tubers and fruits, is limited (Xu et al., 2012; Zidenga et al., 2012) and represents an important area for future research. Given the evidence for tissue-specific roles for AOX, the use of more sophisticated genetic approaches specifically targeting sink and/or source tissues (Sonnewald and Fernie, 2018) will be required for disentangling the roles of AOX and its impact on plant growth and productivity. Genetic engineering of respiration involving spatio-temporal changes of the target genes has been proposed as a crucial strategy to improve crop productivity (Amthor et al., 2019). Particularly, fine-tuning alterations of AOX have been predicted to be among the most efficient strategies to achieve high biomass gains (Amthor et al., 2019). In this respect, recent in vitro (Selinski et al., 2018) and in vivo (Florez-Sarasa et al., 2019) evidence on the predominant role of the TCA cycle intermediates on AOX regulation, together with new structural insights on its active site (May et al., 2017), is paving the way to design new AOXs with altered and desirable regulatory properties. Finally, the interactive effects of major global change factors on AOX respiration remain to be determined. Some studies have highlighted the importance of (photo) respiratory metabolism under stress combination (Obata et al., 2015; El Aou-Ouad et al., 2018), although AOX was not investigated in these studies. Thus, the specific role of AOX under stress combination remains to be explored by means of genetic approaches and in vivo activity measurements. Given the evidence reported about the AOX involvement in plant tolerance to several individual biotic and abiotic stresses (reviewed in Vanlerberghe, 2013; Saha et al., 2016; Del-Saz et al., 2018a), we envisage that AOX will provide a beneficial role for plants under combined stress conditions induced by climate change.