Environmental stresses cause large economic losses in agriculture every year, constraining crop yield and production. Owing to the consequences of the climate change, different combinations of abiotic stress conditions are severely impacting on plants in the natural field (Mittler, 2006; Suzuki et al., 2014; Zandalinas et al., 2017a). Although research on plants is traditionally based on the study of the responses to single abiotic factors, further effort has been made over the last years to analyze plant responses to different combined stresses, either abiotic or biotic (Suzuki et al., 2014; Zandalinas et al., 2017a). Particularly, drought and heat are considered one of the most frequent abiotic stress combinations that drastically affect global agricultural systems [International Panel of Climate Change (IPCC, 2014)].

Reactive oxygen species are normally produced as a result of aerobic metabolism. However, metabolic imbalances produced by changes in environmental conditions promote the over-accumulation of ROS (Suzuki et al., 2012). In general, abiotic stresses that limit CO₂ availability due to stomatal closure enhance the accumulation of ROS. Interestingly, while ROS, such as H₂O₂, are considered important signal transduction molecules (Baxter et al., 2014; Mittler, 2016), they are also toxic, causing extensive cellular damage and inhibition of photosynthesis (Choudhury et al., 2016). To prevent damage, ROS accumulation is mitigated by the antioxidant machinery including ROS-scavenging enzymes and increased levels of antioxidants such as AsA and GSH, components of the so-called Halliwell-Asada cycle (Mittler et al., 2004). One of the key enzymes of the antioxidant defense system is the SOD, which constitutes the first level of defense against superoxide radicals. SOD-catalyzed O2•- dismutation renders H₂O₂ as a reaction product, which in turn is removed by APX and CAT activities (Mittler et al., 2004). APX reduces H₂O₂ using AsA as the electron donor and the balance between GSH and GSSG is critical for maintaining a favorable redox status for the detoxification of H₂O₂. In addition, GR, the rate-limiting enzyme of AsA–GSH cycle, keeps the GSH/GSSG ratio favorable for AsA reduction (Foyer and Noctor, 2005).

Several studies have reported that the ability of plants to balance ROS production and scavenging is associated to a higher tolerance to different environmental stresses (Hernandez et al., 2000; Lin et al., 2004; Arbona et al., 2008; Martinez et al., 2016). The accumulation of high amount of ROS-response transcripts in plants subjected to different combinations of stress factors, reflects the relevance of ROS as an important component of acclimation pathways during combined stresses (reviewed in Suzuki et al., 2014). For example, it has been suggested the key role of cytosolic APX1 for the acclimation of plants to a combination of drought and heat (Koussevitzky et al., 2008). In that work, Arabidopsis mutants deficient in this enzyme (apx1), were found to be highly sensitive to this stress combination. Furthermore, ROS–ABA interactions are very important for plant acclimation to stress combination. In this way, previous reports have shown that mutants impaired in the function of the ABA and ROS-regulated protein phosphates 2C (PP2Cs) (abi-1) were sensitive to the combined impact of drought and heat, as well as salinity and heat (Suzuki et al., 2016; Zandalinas et al., 2016a). Furthermore, several studies have reported that the expression of different ROS-scavenging enzymes and the accumulation of different antioxidants exhibit a unique mechanism of response during stress combination that is different than that found in response to each individual stress (Rizhsky et al., 2002, 2004; Srivastava et al., 2012; Prasch and Sonnewald, 2013; Rivero et al., 2013; Pandey et al., 2015; Jin et al., 2016).

We recently demonstrated the different ability of two citrus genotypes, Carrizo citrange and Cleopatra mandarin, to tolerate drought and heat applied alone or in combination. Therefore, physiological responses in terms of gas exchange parameters and chlorophyll fluorescence, evidenced the higher susceptibility of Cleopatra mandarin to combined drought and heat conditions (Zandalinas et al., 2016b). Moreover, metabolite profiling of leaves of both citrus genotypes in response to combined drought and heat revealed that the accumulation of secondary metabolites with antioxidant function is associated to sensitivity to this stress combination (Zandalinas et al., 2017b). Therefore, the higher sensitivity of Cleopatra plants required a higher accumulation of protective metabolites oriented to mitigate the damaging effects of stress, including flavonols, flavones, and limonoids (Zandalinas et al., 2017b). However, the role of the antioxidant defense involving ROS-scavenging enzymes in the tolerance of citrus plants to combined drought and heat is currently unknown. Previous reports have associated the ability to modulate the antioxidant system with the tolerance of citrus plants to waterlogging (Arbona et al., 2008; Hossain et al., 2009), salinity (Arbona et al., 2003), or WS (Wu et al., 2006). In general, these investigations concluded that coordinated antioxidant activity associated to increased activities of SOD and CAT, along with a modulation of the AsA–GSH cycle, allowed citrus plants to reduce stress-induced oxidative damage.

The aim of the present work was to determine the importance of the modulation of the antioxidant system in citrus tolerance to the combination of drought and high temperatures. To achieve this, oxidative metabolism and related antioxidants were studied in two citrus genotypes (Carrizo citrange and Cleopatra mandarin) with different ability to cope with this combined stresses (Zandalinas et al., 2016b).

Abiotic stresses including high temperatures, drought or different combinations of environmental challenges, induce metabolic imbalances that can cause an oxidative stress in plant cells. This effect results in the generation and accumulation of ROS, promoting oxidation of cellular components, hindering metabolic activities and affecting organelle integrity (Suzuki et al., 2012). In citrus plants, it has been proposed that environmental cues such as waterlogging, Cu toxicity, salinity or drought induce oxidative damage (Arbona et al., 2003, 2008; Wu et al., 2006; Hossain et al., 2009; Hippler et al., 2016), highlighting the importance of modulating the antioxidant system efficiently to cope with these abiotic stresses. In the present work, the antioxidant machinery of two citrus genotypes, Carrizo citrange and Cleopatra mandarin, with contrasting ability to tolerate the combination of drought and heat (Zandalinas et al., 2016b) was investigated to correlate differences in the modulation of the antioxidant system with tolerance to this stress combination. In this sense, Cleopatra constitutes a genotype more sensitive than Carrizo to drought combined with heat according to data presented in Figure 1 and also reported in Zandalinas et al. (2016b). Therefore, the percentage of damaged leaves in response to heat or a combination of drought and heat was significantly higher in Cleopatra than in Carrizo (Figure 1), demonstrating the higher ability of Carrizo to deal with stresses that involve high temperatures. Moreover, a positive correlation between stress sensitivity and proline accumulation was observed. Hence, Cleopatra accumulated higher amounts of proline in leaves and roots in response to the combination of drought and heat compared to Carrizo. Additionally, both citrus genotypes accumulated higher levels of this metabolite in response to combined stress factors, a more damaging situation with respect to individual stresses. This result matches other works in which a higher stress pressure exerts a major proline accumulation (Claussen, 2005; Kaur and Asthir, 2015) due to its protective roles, including maintenance of redox balance and radical scavenging, maintenance of protein structure and contribution to reduce cell membrane damage (Shao et al., 2008; Szabados and Savouré, 2010).

In addition, oxidative damage (estimated by MDA accumulation) was also higher in leaves and roots of Cleopatra in response to combined stresses (Figure 3), suggesting that the extent of oxidative damage is directly linked to susceptibility of citrus plants to the combination of WS+HS. The increased SOD and CAT activities of Carrizo in both basal and stress conditions compared to Cleopatra (Figures 4A, 5A) could be related to an active and efficient antioxidant response that might be involved in maintaining a lower MDA content (and oxidative stress, Gill and Tuteja, 2010) especially under the combination of drought and heat, and therefore helping citrus plants to cope with the combined stresses. On the contrary, whereas SOD activity of Cleopatra leaves increased in response to stress imposition, CAT activity did not change in response to individual stresses and even decreased below control levels under WS+HS conditions. In addition to CAT, APX removes H₂O₂ and this reaction has been previously reported to be a crucial process for the tolerance of plants to combined drought and heat (Koussevitzky et al., 2008). In our work, APX activity was significantly induced by HS and WS+HS in Carrizo leaves (Figure 6A), suggesting an efficient H₂O₂ scavenging ability under these stress conditions. However, in Cleopatra, only HS significantly induced an increased APX activity (Figure 6A). However, under high temperatures, this enzyme activity could be insufficient to scavenge the excess of H₂O₂ when CAT activity is not activated (Figure 5A), rendering an increased oxidative damage. Additionally, APX dismutase H₂O₂ using AsA as the electron donor (Foyer and Noctor, 2011). Both citrus genotypes showed increases in leaf AsA and tAsA contents in response to WS+HS, suggesting that the accumulation of AsA could be related with a stronger stress pressure. In addition, Cleopatra showed higher tAsA and DHA levels as well as a lower AsA/DHA ratio with respect to Carrizo during this stress combination (Table 2), which are according to the lower APX activity observed during WS+HS in this citrus genotype (Figure 6A).

Accurate modulation of the GSH cycle is involved in maintaining a favorable GSH/GSSG ratio required for cellular redox regulation. In this way, GR activity could effectively recycle GSH at the expense of NADPH (Foyer and Noctor, 2011). The pattern observed for GR activity in Figure 7 indicates that Carrizo plants, despite increasing tGSH, GSH, and GSSG levels in response to HS and WS+HS (Table 3), preserved the GR activity as well as the GSH/GSSG ratio around control values, probably as a result of a lower incidence of the oxidative damage. In contrast, in Cleopatra leaves, the reduction in GSH/GSSG ratio with respect to control values, especially under WS+HS (Table 3), suggests an impairment of GSH recycling. This result points to a better ROS non-enzymatic detoxification system and to an efficient GSH recycling in Carrizo plants compared to Cleopatra, with no apparent NADPH limitation. In this sense, it has been previously reported that maintenance of a more GSSG status could be a consequence of an enhanced ROS accumulation (Foyer and Noctor, 2011). Our results are in accordance with this statement since MDA specially accumulated in Cleopatra in response to WS+HS (Figure 3). Furthermore, the activation of GR activity observed in Cleopatra under this stress combination (Figure 7A) might be insufficient to keep a proper GSH/GSSH ratio, leading to a lower ability for ROS detoxification (Arbona et al., 2008) and, as a result, to a higher sensitivity of this citrus genotype to WS+HS. These results also demonstrated the previous hypothesis, suggesting that a deficient antioxidant system in Cleopatra plants under the combination of drought and heat would lead to an enhanced activation of secondary metabolites with antioxidant properties including flavonols, flavones and limonoids to supplement the antioxidant deficiency and mitigate the damaging effects of stress (Zandalinas et al., 2017b). However, all these metabolic strategies, including proline accumulation (Figure 2), do not seem to be effective as Cleopatra mandarin suffered important damage under WS+HS conditions.

According to our data, the combination of drought and heat negatively impacted both citrus genotypes (Figure 1) but the effective activation of the antioxidant machinery was associated to the ability to tolerate this stress combination. As a result, in Cleopatra plants in response to WS+HS, the increment of SOD activity (Figure 4A) along with the decline in CAT activity (Figure 5A) and the lack of APX activity increase (Figure 6A), compared to control values could be partially responsible of its increased oxidative damage and sensitivity to the combination of drought and high temperatures. In contrast, the ability of Carrizo plants to efficiently activate antioxidant enzymes involved in ROS detoxification along with preserving a favorable GSH/GSSG ratio would be partially related to genotype tolerance to combined stresses. This work provides physiological basis for directing future genetic programs to improve the antioxidant system of Cleopatra mandarin, a genotype that has been very useful as a rootstock for plants cultivated under conditions of water scarcity. However, its future use can be seriously compromised in a scenario of climatic change due to the high sensibility to combined conditions of heat and drought.

SZ and DB performed the research. AG-C and VA supervised the project and provided funding. SZ, DB, and AG wrote the manuscript and prepared figures. SZ, DB, VA, and AG-C revised the final version. All authors have read and approved the final version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.