ORIGINAL RESEARCH published: 17 May 2022 doi: 10.3389/fpls.2022.833113 The Lack of Alternative Oxidase 1a Restricts in vivo Respiratory Activity and Stress-Related Metabolism for Edited by: Leaf Osmoprotection and Redox Miquel A. Gonzalez-Meler, University of Illinois at Chicago, Balancing Under Sudden Acute United States Reviewed by: Water and Salt Stress in Arabidopsis Brendan Michael O’Leary, Agriculture and Agri-Food Canada thaliana (AAFC), Canada Dinakar Challabathula, Néstor F. Del-Saz 1* † , Ariadna Iglesias-Sanchez 2† , David Alonso-Forn 3 , Central University of Tamil Nadu, India Miguel López-Gómez 4 , Francisco Palma 4 , María José Clemente-Moreno 5 , *Correspondence: Alisdair R. Fernie 6 , Miquel Ribas-Carbo 5 and Igor Florez-Sarasa 2,7* Nestor F. Del-Saz 1 Laboratorio de Fisiología Vegetal, Universidad de Concepción, Concepción, Chile, 2 Centre for Research in Agricultural nesfernandez@udec.cl Genomics CSIC-IRTA-UAB-UB, Barcelona, Spain, 3 Unidad de Recursos Forestales, Centro de Investigación y Tecnología Igor Florez-Sarasa Agroalimentaria de Aragón, Zaragoza, Spain, 4 Department of Plant Physiology, University of Granada, Granada, Spain, igor.florez@cragenomica.es 5 Grup de Recerca en Biologia de les Plantes en Condicions Mediterranies, Departament de Biologia, Universitat de les Illes † These authors have contributed Balears, Palma, Spain, 6 Max Planck Institute of Molecular Plant Physiology, Potsdam, Germany, 7 Institut de Recerca i equally to this work Tecnología Agroalimentàries (IRTA), Edifici CRAG, Barcelona, Spain Specialty section: This article was submitted to In plants salt and water stress result in an induction of respiration and accumulation of Plant Physiology, stress-related metabolites (SRMs) with osmoregulation and osmoprotection functions a section of the journal Frontiers in Plant Science that benefit photosynthesis. The synthesis of SRMs may depend on an active respiratory Received: 10 December 2021 metabolism, which can be restricted under stress by the inhibition of the cytochrome Accepted: 19 April 2022 oxidase pathway (COP), thus causing an increase in the reduction level of the ubiquinone Published: 17 May 2022 pool. However, the activity of the alternative oxidase pathway (AOP) is thought to Citation: prevent this from occurring while at the same time, dissipates excess of reducing Del-Saz NF, Iglesias-Sanchez A, Alonso-Forn D, López-Gómez M, power from the chloroplast and thereby improves photosynthetic performance. The Palma F, Clemente-Moreno MJ, present research is based on the hypothesis that the accumulation of SRMs under Fernie AR, Ribas-Carbo M and Florez-Sarasa I (2022) The Lack osmotic stress will be affected by changes in folial AOP activity. To test this, the of Alternative Oxidase 1a Restricts oxygen isotope-fractionation technique was used to study the in vivo respiratory in vivo Respiratory Activity activities of COP and AOP in leaves of wild-type Arabidopsis thaliana plants and of and Stress-Related Metabolism for Leaf Osmoprotection and Redox aox1a mutants under sudden acute stress conditions induced by mannitol and salt Balancing Under Sudden Acute Water treatments. Levels of leaf primary metabolites and transcripts of respiratory-related and Salt Stress in Arabidopsis thaliana. Front. Plant Sci. 13:833113. proteins were also determined in parallel to photosynthetic analyses. The lack of in vivo doi: 10.3389/fpls.2022.833113 AOP response in the aox1a mutants coincided with a lower leaf relative water content Frontiers in Plant Science | www.frontiersin.org 1 May 2022 | Volume 13 | Article 833113 Del-Saz et al. Alternative Oxidase Role in Osmoprotection and a decreased accumulation of crucial osmoregulators. Additionally, levels of oxidative stress-related metabolites and transcripts encoding alternative respiratory components were increased. Coordinated changes in metabolite levels, respiratory activities and photosynthetic performance highlight the contribution of the AOP in providing flexibility to carbon metabolism for the accumulation of SRMs. Keywords: water stress, salinity, alternative oxidase, oxygen-isotope fractionation, primary metabolism, photosynthesis, Arabidopsis thaliana INTRODUCTION show the highest expression levels, being expressed in most tissues and induced by stress treatments in several species Plants display several mechanisms for drought and salinity (Clifton et al., 2006; Vanlerberghe et al., 2020). Over the last tolerance, however, these come with high energy and two decades, reverse genetics has been used to decrease AOX1 carbon demands that plant metabolism meets by adjusting gene expression in order to test hypotheses regarding the photosynthesis and respiration (Munns et al., 2020; Vanlerberghe role of the AOP. Following the suppression of AOX1, the et al., 2020). An important tolerance mechanism to drought amount of the AOX protein has been found to be markedly and salinity resides in the accumulation of metabolites that decreased concomitantly with a decrease in the AOP capacity play multiple protective roles, including primary metabolites (the maximum possible flux of electrons to AOX; Guy and such as organic acids, sugars and polyols (Sanchez et al., 2008; Vanlerberghe, 2005; Umbach et al., 2005; Watanabe et al., 2010; Fàbregas and Fernie, 2019), as well as polyamines (Alcázar et al., Florez-Sarasa et al., 2011; Xu et al., 2012; Vishwakarma et al., 2020). These stress-related metabolites (SRMs) from primary 2015; Zhang et al., 2016; Garmash et al., 2020). In parallel, many metabolism, protect against osmotic and oxidative stress, studies have observed an increase in AOX protein or capacity serve as energy sources and/or are involved in stress-signalling under stress in wild-type plants (reviewed in Vanlerberghe, 2013; pathways (Slama et al., 2015; Pires et al., 2016; Fàbregas and and in Vanlerberghe et al., 2020). In fact, a regulated overcapacity Fernie, 2019). At the same time, the synthesis and degradation of AOP is thought to be required for ensuring an adequate activity of several SRMs involve metabolic pathways that are tightly under conditions inducing metabolic fluctuations (Rasmusson linked to respiratory metabolism (Pires et al., 2016; Batista-Silva et al., 2009; Florez-Sarasa et al., 2011). By contrast to AOP et al., 2019; Bandehagh and Taylor, 2020). Thus, SRMs or their capacity, in vivo AOP activity can be directly determined in the carbon precursors [i.e., glycolytic or tricarboxylic acid (TCA) absence of added inhibitors by measuring the differential oxygen cycle intermediates] are tightly associated to the generation isotope discrimination during respiration (Day et al., 1996; Del- of reducing equivalents (NADH and FADH2 ), from which Saz et al., 2017). While there is substantial evidence on the oxidation is coupled to the mitochondrial electron transport molecular mechanisms regulating the AOX gene expression in chain (mETC). A distinctive feature of the plant mETC is response to drought and salinity stresses (Vanlerberghe, 2013; the presence of two terminal oxidases. In addition to the Dahal and Vanlerberghe, 2017; Zhu et al., 2018; Gong et al., cyanide sensitive cytochrome oxidase (COX), the presence 2020), only some studies have addressed when and to which of a cyanide-resistant alternative oxidase (AOX) that couples extent the in vivo AOP activity is effectively responding. The the oxidation of ubiquinol with the reduction of O2 to H2 O, in vivo AOP activity was reported to increase in response to severe confers to mETC the capacity to sustain O2 consumption drought and salt stress (Ribas-Carbo et al., 2005; Del-Saz et al., under COX restriction (Del-Saz et al., 2018; Vanlerberghe 2016), while unaltered activities were reported under milder or et al., 2020). Electron transport via the AOX pathway (AOP) longer drought/salinity periods (Galle et al., 2010; Martí et al., is not coupled with energy conservation (Del-Saz et al., 2018; 2011). These different responses denote, as previously highlighted Vanlerberghe et al., 2020). Nonetheless, the AOP provides (Munns et al., 2020), that in vivo AOP activity determinations metabolic flexibility that helps plants acclimate to stress by in combination with detailed metabolic and molecular analyses allowing the oxidation of reducing equivalents coupled to remain crucial for understanding the key changes in energy TCA cycle and/or cytosolic reactions under conditions where and carbon metabolism conferring drought (Vanlerberghe et al., the COX pathway (COP) is restricted (Del-Saz et al., 2018). 2020) and salt (Munns et al., 2020) tolerance. In fact, the accumulation of SRMs has been reported to In order to gain insight into the role of the AOP during coincide with higher respiratory rates (Mattoo et al., 2006; stress-related metabolism, wild-type Arabidopsis thaliana plants Del-Saz et al., 2016; Pires et al., 2016; Bandehagh and Taylor, and aox1a T-DNA insertional mutants, from which information 2020), indicating an active mitochondrial metabolism under regarding in vivo activity is still lacking, were treated with stress. Nevertheless, direct evidence about the influence of high concentrations (1 day, 300 mM) of mannitol and salt the AOP activity in modulating the synthesis of stress-related (NaCl), known to greatly induce the accumulation of several metabolites remains scarce. SRMs (Del-Saz et al., 2016; Darko et al., 2019). The in vivo The AOX protein is encoded by a multigene family which activities of COP and AOP in leaves were measured using the exhibits tissue-specific expression (Saisho et al., 1997; Clifton oxygen-isotope discrimination technique and in parallel, leaf et al., 2006; Selinski et al., 2018a). AOX1 isoforms generally levels of primary metabolites and respiratory-related transcripts Frontiers in Plant Science | www.frontiersin.org 2 May 2022 | Volume 13 | Article 833113 Del-Saz et al. Alternative Oxidase Role in Osmoprotection were also determined. In addition, leaf relative water content as Leaf Water Status well as photosynthetic performance, determined by chlorophyll Leaf relative water content (RWC) was determined as follows: fluorescence analysis in the dark and in the light, was also studied. We hypothesized that the lack of AOX1a will be associated with RWC (%) = (FW − DW)/(TW − DW) × 100 lower leaf rates of AOP respiration and less accumulation of SRMs under severe water and salt stress, with consequences for where fresh (FW), turgid (TW) and dry (DW) weight of leaves plant performance under stress. were measured. FW was determined immediately after sampling; TW was obtained after incubating leaf discs in distilled water for 48 h in the dark at 4 ◦ C (to minimize respiration losses) and DW MATERIALS AND METHODS was determined after drying for at least 2 days in an oven at 60– 70◦ C. Plant Material and Growth Conditions Seeds of wild-type (WT) A. thaliana Columbia 0 (Col 0) Photosynthetic Measurements and aox1a T-DNA insertion line (SALK_084897) were sown Chlorophyll fluorescence measurements were carried out on in pre-wetted pots with a mixture of peat-perlite (3:1). The rosette leaves using a MAXI-PAM fluorometer (Heinz Walz T-DNA insertion within the AtAOX1a gene was confirmed by GmbH) as previously described (Morelli et al., 2021). Briefly, genomic PCR with T-DNA-specific primers and seeds from a for every measurement six rosette leaves from each plant were homozygous line were used for the experiments. Pots were placed considered. The actual quantum efficiency of the photosystem II in plastic trays used for sub-irrigation. Plants were grown in (PSII)-driven electron transport (8PSII) was determined under a growth chamber for 32–35 days at 12 h/12 h photoperiod, different light intensities (in µmol photons m−2 s−1 of photon 150 µmol quanta m−2 s−1 light intensity, 25◦ C/20◦ C day/night flux density, PPFD): 800, 700, 600, 500, 400, 300, 200, 150, 100, 75, temperature and relative humidity above 50%. Plants were 50, 25 and 0. The chloroplast electron transport rate (ETR) was watered twice a week with half-strength Hoagland’s solution calculated as the product of 8PSII x PPFD x 0.84 x 0.5, where 0.84 (Epstein et al., 1972). On days 32–35, plants were divided in and 0.5 were assumed values for the leaf light absorptance and three groups, one was watered normally (control plants) while the fraction of absorbed quanta available for PSII. From the light the other two were irrigated with watering solution containing response curves, the associated parameters ETRm (maximum 300 mM of sodium chloride (NaCl-treated plants) or 300 mM electron transport rate) and alpha (photosynthetic rate in light mannitol (Mannitol-treated plants). The analyses of control and limited region of the light curve) were characterized by fitting treated plants were performed one day after the irrigation. iteratively the model of the rETR versus E curves using MS Excel All the experiments were repeated at least twice, and data is Solver (Platt and Gallegos, 1980). The fit was very good in all the presented as a pool of at least two individual experiments (i.e., cases (r > 0.98). After the light curves, the maximum quantum each consisting of two to four biological replicates/plants per efficiency of PSII, Fv /Fm , was calculated as (Fm – Fo )/Fm , where genotype and treatment), since similar results were obtained in Fm and Fo are the maximum and the minimum fluorescence of each individual experiment. dark-adapted samples, respectively. For dark acclimation, plants were incubated for at least 30 min in darkness to allow the full Respiration and Oxygen-Isotope relaxation of photosystems. Fractionation Measurements Leaves were placed in the dark for 30 min to avoid light- Metabolite Profiling enhanced dark respiration. The capacity of the alternative Metabolite extractions were performed as described previously pathway (Valt ) was determined with an oxygen electrode as (Lisec et al., 2006) using approximately 50 mg of leaf tissue, previously described (Del-Saz et al., 2016), and results were previously frozen-powdered. Derivatization and GC–TOF–MS expressed in nmol O2 g−1 dry weight s−1 as the mean ± SE of analyses were carried out as described previously (Lisec et al., five or six biological replicates. Respiration and oxygen isotope 2006). Metabolites were identified manually by TagFinder fractionation measurements were performed as previously software (Luedemann et al., 2012) using the reference library described (Del-Saz et al., 2016). Respiratory partitioning between mass spectra and retention indices housed in the Golm the two respiratory pathways was calculated from the oxygen Metabolome Database;1 (Kopka et al., 2005). The parameters isotope fractionation by the alternative oxidase (1a ) and the used for the peak annotation of the 46 metabolites can be found cytochrome oxidase (1c ). The 1a values, determined in the in Supplementary Table 1, which follows previously reported presence of 10 mM of KCN, were not significantly different recommendations (Fernie et al., 2011). between the genotypes and thus, the 1a mean value of To process the GC-TOF-MS results, the intensity of a selected 30.8 ± 0.445h (six replicates) was used for all the partitioning unique ion shown in Supplementary Table 1 was normalized calculations. The 1c , determined in presence of 25 mM of to that of ribitol which was added to each sample as an salicylhydroxamic acid (SHAM), was 20.5h (four replicates). internal standard, as well as to the fresh weight of the materials Dry weights were obtained after drying leaves for 2 days at 60– used for metabolite extraction. Fresh weight was then corrected 70◦ C. Results were expressed in nmol O2 g−1 dry weight s−1 as the mean ± SE of six or eight biological replicates. 1 http://gmd.mpimp-golm.mpg.de/ Frontiers in Plant Science | www.frontiersin.org 3 May 2022 | Volume 13 | Article 833113 Del-Saz et al. Alternative Oxidase Role in Osmoprotection FIGURE 1 | Alternative oxidase (AOX) pathway capacity (Valt ) and expression analysis. (A) Valt determined (see section “Materials and Methods”) in leaves of wild-type Col 0 (WT) and aox1a Arabidopsis thaliana plants untreated with NaCl or Mannitol (Control), and after 1 day of severe NaCl or Mannitol treatments (300 mM). Values are means ± SE of 5–6 biological replicates. Significant differences (P < 0.05) are denoted by different letters. (B) Western blot analysis of AOX and porin (VDAC) protein levels from total leaf protein extracts (see section “Material and Methods” for details). The intensities of the signals from AOX were normalized to those from porin (VDAC) and are expressed relative to WT levels under control conditions. The normalized intensity values shown are averages of three independent blots. (C) Transcript levels of genes encoding AOX isoforms determined by qPCR analyses (see Materials and Methods for details) under control and stress conditions. Data are shown as fold-changes relative to control conditions (i.e., all control values were set to 1, which is denoted by the dashed line). Primers used and gene information can be found in Supplementary Table 4. Values are means ± SE of 5–6 replicates and asterisks denote significant differences (P < 0.05) to the control condition for each gene expression analysis. Letters denote significant differences (P < 0.05) among genotypes and stress conditions for each gene expression analysis. n.d. (not detected). by dry weight/fresh weight ratios used to determine the leaf 20 mg of frozen leaf powder resuspended in 100 µl SDS RWC (see Section “Leaf Water Staus”). Thereafter, data were sample buffer [2% (w/v) SDS, 62.5mM Tris–HCl (pH 6.8), normalized to the mean value of Col-0 wild-type (WT) plants 10% (v/v) glycerol and 0.007% (w/v) bromophenol blue], under control conditions (i.e., the value of all metabolites for WT 50 mM DTT and protease inhibitor cocktail (Roche Basel, at control conditions was set to 1). This normalization allowed Switzerland). Samples were incubated 30 min at 4◦ C to the comparison of the relative metabolite levels among genotypes allow full reduction of the AOX protein and then boiled and treatments (Supplementary Table 2). In addition, leaf (95◦ C) for 5 min. The resuspended sample was centrifuged absolute metabolite levels (µmol gDW− 1 ) were also determined at 14,000 rpm for 10 min. After loading 25 µl on a 12% for all the metabolites detected and present in the authentic SDS-PAGE gel, proteins were transferred to a 0.45 µm standards mixture (Supplementary Table 3). The experimental Nitrocellulose membrane (Armersham Protan) using a semi-dry samples and standards mixtures containing 0, 100, 200, 500, Trans-Blot TurboTM Transfer System of Bio-Rad. Membranes R and 1,000 ng of each compound were analysed in the same were incubated overnight at 4◦ C with diluted 1:500 polyclonal GC- MS runs, and the absolute metabolite amounts in the anti-AOX, AOX1 and 2 (AS04054, Agrisera, Sweden) or samples were calculated by extrapolation from standard curves diluted 1:5,000 anti-Porin, voltage-dependent anion-selective using the peak intensity of a representative mass fragment. channel protein 1–5 (AS07212, Agrisera, Sweden). Incubation Values presented for both relative and absolute levels are with 1:20,000 horseradish peroxidase secondary antibodies means ± SE of four to six replicates corresponding to several fully (Cytiva) during 1h at room temperature. The detection of expanded rosette leaves. immunoreactive bands was performed using ECL SuperSignalTM West Femto Maximum Sensitivity Substrate (Thermo Fisher). Western Blot Analysis Chemiluminescent signals were collected by IQ 800 Armersham Protein extraction and western blot analysis was performed (Cytiva). The protein band quantifications were performed as previously described (Florez-Sarasa et al., 2016) with some with ImageJ analysis software according to the manufacturer’s modifications. Total plant protein extracts were obtained from instructions. The obtained band intensities for AOX were Frontiers in Plant Science | www.frontiersin.org 4 May 2022 | Volume 13 | Article 833113 Del-Saz et al. Alternative Oxidase Role in Osmoprotection corrected for their corresponding porin band intensities and RESULTS then normalized to the levels of the WT plants under control conditions (i.e., WT levels were set to 1). Three different The Alternative Oxidase Capacity and immunoblot experiments per protein were performed and Expression in aox1a and Wild-Type the image shown in Figure 1B belong to one of the three membranes obtained. Samples used in each of the three western Plants Under Control and Stress blot experiments were a mixture of aliquots belonging to two Conditions biological replicates from the same six biological replicates The capacity of the alternative oxidase pathway (Valt ), measured used for the gene expression analysis. The relative values as the KCN-resistant respiration, was determined in both presented in Figure 1B are means of the three western genotypes under control and stress treatments (Figure 1). blot experiments. As expected, Valt was significantly (P < 0.05) lower in aox1a mutants than in WT plants (5.9 and 19.1 nmol O2 g−1 DW s−1 , respectively) under control conditions. Gene Expression Analysis Under stress, Valt increased in WT plants after salt treatment, RNA was isolated from lyophilized leaves by using Maxwell R while mannitol-treated plants displayed an intermediate Valt RSC Plant RNA Kit (Promega Biotech Ibérica, Madrid, Spain) (Figure 1A). On the other hand, Valt remained similar in aox1a and automated system Maxwell RSC Instrument (Promega R mutants under control and stress conditions, and it was much Biotech Ibérica, Madrid, Spain) according to the manufacturer’s lower as compared to WT plants under stress conditions. Then, instructions, and was quantified using a NanoDrop 1000 the AOX protein levels were determined in leaf crude extracts spectrophotometer (Thermo Scientific,2 ). Afterward cDNA after immunoblotting analyses and quantified after normalization synthesis was performed following the recommendations of the to mitochondrial porin levels (Figure 1B). In the line with Transcriptor First Strand cDNA Synthesis Kit (Roche Basel, respiratory capacity, the AOX protein levels were similarly Switzerland). Relative mRNA abundance was evaluated by increased after both stress treatments in WT plants. We did quantitative PCR using LightCycler 480 SYBR Green I Master not detect any signal of AOX protein in the aox1a mutants as Mix (Roche Basel, Switzerland) on a LightCycler 480 real- previously reported (Giraud et al., 2008; Strodtkötter et al., 2009). time PCR system (Roche Basel, Switzerland). Primers used Thereafter, the expression profiles of the five genes encoding and the related information are detailed in the Supplementary the AOX protein in Arabidopsis were determined by qPCR Table 4. Two technical replicates of each biological replicate analyses in both genotypes under control and stress conditions were performed, and the mean values were used for further (Figure 1C). Transcript levels of the AOX1a gene were not calculations. Arabidopsis AtUbqC (At5g25760) was used as detected in the aox1a mutant, thus confirming the expected effect a reference gene to correct for differences in the total of the aox1a mutation, while the expression of this gene was amount of transcripts and the 2−11Ct method (Livak and induced in WT plants under both stress conditions (Figure 1C), Schmittgen, 2001) was used to calculate the fold-change of particularly under salinity (7.2-fold increase). The transcript gene expression. Finally, data were normalized to the mean levels of AOX1b and AOX2 were below the level of detection in value of plants under control conditions in each genotype all control and most of the stress-treated samples. The transcript (i.e., the level of all transcripts for WT and aox1a mutants at levels of AOX1c were generally not responsive to stress, except control was set to 1). for the slight significant (P < 0.05) increase in WT plants after mannitol treatment. On the other hand, the levels of AOX1d Statistical Analyses transcripts were highly responsive to both stress conditions, For the statistical analyses in Figures 1–4 and 6, a one- particularly in the aox1a mutants, which display a much higher way ANOVA with a level of significance of P < 0.05 was response than in WT plants. performed with SPSS statistical software package, version 25 (IBM Corp., 2016, Armonk, New York, NY, USA), and Leaf Phenotype and Water Status in Duncan’s posthoc test was used to determine statistically aox1a and Wild-Type Plants Under significant differences. In addition, Student’s t tests were used for the statistical analyses in Figures 1B, 5, 6, Supplementary Control and Stress Conditions Figure 1, Supplementary Tables 2,3 in order to determine Wild-type (WT) and aox1a mutant plants did not show visual significant (P < 0.05) differences between genotypes under differences under control conditions, as previously observed each experimental condition (Figure 5, Supplementary in several other studies (Figure 2A). Acute salt and mannitol Figure 1, Supplementary Tables 2,3), or to determine treatments caused minor leaf wilting in both WT and aox1a significant (P < 0.05) differences between control and stress mutant plants after one day of stress (Figure 2A). Leaf relative treatments in each genotype (Figures 1B, 6, Supplementary water content (RWC) was determined as an indicator of water Tables 2,3). status in both genotypes after salt and mannitol treatments (Figure 2B). Salinity provoked a reduction of leaf RWC in both genotypes, while a significant (P < 0.05) reduction of leaf RWC after mannitol treatment was only observed in the 2 http://www.nanodrop.com/ aox1a mutants. Under both stress conditions, the leaf RWC Frontiers in Plant Science | www.frontiersin.org 5 May 2022 | Volume 13 | Article 833113 Del-Saz et al. Alternative Oxidase Role in Osmoprotection FIGURE 2 | Visual phenotype and relative water content. (A) Photographs representative of WT and aox1a plants untreated with NaCl or Mannitol (Control), and after 1 day of severe NaCl or Mannitol treatments (300 mM). (B) Leaf relative water content (RWC) in leaves of WT and aox1a plants under control and stress conditions. Values are means ± SE of 10–12 biological replicates. Significant differences (P < 0.05) are denoted by different letters. was significantly (P < 0.05) lower in the aox1a mutants as Photosynthetic Parameters in aox1a and compared to WT plants. Wild-Type Plants Under Control and Stress Conditions Respiration and Electron Partitioning to The chloroplast electron transport rate (ETR) was determined at the Alternative Oxidase Pathway in different light intensities in aox1a and WT plants under control aox1a and Wild-Type Plants Under and stress conditions (Figure 4A). While similar light-response Control and Stress Conditions curves were obtained in both genotypes under control conditions, Both total respiration and the electron partitioning between some differences were observed under stress (Figure 4A). Light the cytochrome oxidase pathway (COP) and the AOP were curves were modelled to obtain photosynthetic parameters (see determined by using the oxygen isotope discrimination technique section “Materials and Methods” for details) such as alpha, (Figure 3). Total oxygen uptake (Vt ) and electron partitioning which indicates the photosynthetic rate in the light-limited region to the alternative pathway (τa ) were similar in WT and aox1a of the light curve, and the maximum electron transport rate mutants under control conditions. Salt and mannitol treatments (ETRmax ; Figure 4B). Both parameters were similar among significantly (P < 0.05) increased respiration only in WT genotypes under control conditions. Regarding alpha, only aox1a plants from 23.9 to 36.6 and 32.9 nmol O2 g−1 DW s−1 , mutants displayed a significant (P < 0.05) reduction after respectively. Similarly, τa significantly increased after salt and salt treatment as compared to control conditions, and this mannitol treatments in WT plants from 0.17 to 0.33 and 0.28, parameter became significantly (P < 0.05) lower in aox1a as respectively. On the other hand, no significant (P < 0.05) compared to WT under salinity. On the other hand, the ETRmax change in τa was observed in aox1a mutants after salt and was clearly reduced under both stress conditions and in both mannitol treatments. The in vivo AOP activity (ν alt ) was genotypes but notably, this parameter was significantly lower in similar in both lines under control conditions and increased the aox1a mutants as compared to WT plants under mannitol significantly in WT plants after salt (1.94-fold) and mannitol stress. In addition, the maximum efficiency of PSII (Fv /Fm ) was (1.27-fold) treatments, whilst it was unaltered in aox1a mutants. determined by measuring chlorophyll fluorescence after dark The activity of the COP (ν cyt ) was similar without significant adaptation, in order to evaluate the level of photoinhibitory stress (P < 0.05) differences between control and treated plants after salt and mannitol treatments (Figure 4B). The Fv /Fm was in both genotypes. also reduced in both genotypes under both stress conditions, Frontiers in Plant Science | www.frontiersin.org 6 May 2022 | Volume 13 | Article 833113 Del-Saz et al. Alternative Oxidase Role in Osmoprotection FIGURE 3 | Respiration and electron partitioning between the cytochrome and the alternative oxidase pathways (COP and AOP). Total respiration (Vt ), FIGURE 4 | Photosynthetic activity and capacity under different light electron partitioning to the AOP (τa ), COP activity (vcyt ) and AOP activity (valt ) intensities. (A) Light curves showing light dependent response (see Materials in leaves of Col 0 (WT) and aox1a A. thaliana plants untreated with NaCl or and Methods for details) of the chloroplast electron transport rate (ETR) in Mannitol (Control), and after 1 day of severe NaCl or Mannitol treatments leaves of wild-type Col 0 (WT) and aox1a A. thaliana plants untreated with (300 mM). Values are means ± SE of 6–8 biological replicates. Significant NaCl or Mannitol (Control), and after 1 day of severe NaCl or Mannitol differences (P < 0.05) are denoted by different letters. (Continued) Frontiers in Plant Science | www.frontiersin.org 7 May 2022 | Volume 13 | Article 833113 Del-Saz et al. Alternative Oxidase Role in Osmoprotection FIGURE 4 | treatments (300 mM). Values are means ± SE of 5–6 biological replicates. (B) Maximum electron transport rate (ETRmax ) and photosynthetic rate in light limited region (alpha) were derived from light curves (see Materials and Methods for details) performed under control and stress conditions. The maximum quantum efficiency of PSII (Fv/Fm) was determined after 30 min in darkness (see Materials and Methods for details) under control and stress conditions. Values are means ± SE of 5–6 biological replicates. Significant differences (P < 0.05) are denoted by different letters. and salinity was the condition inducing higher photoinhibition in both genotypes (Figure 4B). Notably, the aox1a mutants displayed significantly lower Fv /Fm after both stress treatments as compared to WT plants (Figure 4B). Overall, these results indicate a similar photosynthetic performance among genotypes under control conditions, however, the aox1a mutants displayed higher photoinhibition and lower photosynthetic activity under stress conditions. Metabolite Profiling in aox1a and Wild-Type Plants Under Control and Stress Conditions In order to further investigate the metabolic changes underlying the different physiological responses observed in the aox1a mutants, gas chromatography-time of flight-mass spectrometry (GC–TOF–MS) metabolite profiling analysis was performed on rosette leaves from plants grown under control and stress conditions (Figure 5 and Supplementary Table 2). A total of 45 metabolites were identified using gas chromatography-mass spectrometry (GC-MS), including several amino acids, organic acids, sugars and sugar alcohols (Supplementary Tables 1,2; Figure 5). Relative metabolite levels were normalized to the mean levels of WT plants under control conditions (Supplementary Table 1 and Figure 5). Under control conditions, the aox1a FIGURE 5 | Metabolite profiles showing relative changes in primary mutants displayed higher levels of putrescine (2.31-fold), glycine metabolism. Heat map showing the relative levels of the GC-MS-analysed (1.37-fold), erythritol (2.59-fold) and maltose (1.69-fold) as well metabolites in leaves of wild-type Col 0 (WT) and aox1a A. thaliana plants as of the TCA cycle intermediates, citrate (1.62-fold), fumarate untreated with NaCl or Mannitol (Control), and after 1 day of severe NaCl or (1.30-fold) and malate (1.55-fold). Under stress, leaves of WT Mannitol treatments (300 mM). Metabolites were clustered per class into amino acids, organic acids, sugars and sugar alcohols, and other metabolites. plants displayed significant increases in 31 metabolites after Relative metabolite levels were normalized to the mean level of the WT plants salt and mannitol treatments (Figure 5 and Supplementary under control conditions and fold-change values were log2 transformed (i.e., Table 2). While many of the metabolites increasing after mannitol the level of all metabolites of WT plants under control conditions is 0). In this treatment were the same as those increasing under salinity, heat map, red and blue colours represent log2 fold-increased and -decreased metabolites, respectively. Gray colours indicate the metabolites not detected there were several exceptions: alanine, glutamate, ornithine, in some experimental conditions. Values are means ± SE of 4–6 replicates citrate, glycerol and urea, which did not significantly (P < 0.05) and asterisks denote significant differences (P < 0.05) to the WT plants in increase after mannitol treatment; and glutamine, fumarate, each experimental condition. The statistical differences between control and erythritol, galactinol, xylose and trehalose, which increased stress treatments in each genotype are presented in Supplementary Table 2. only after mannitol treatment (Figure 5 and Supplementary Table 2). On the other hand, only glycerate and pyruvate displayed decreased levels after both stress treatments, and dehydroascorbate was reduced only after salt treatment (Figure 5 significantly (P < 0.05) lower in aox1a mutants than in WT plants and Supplementary Table 2). after salt treatment. Regarding mannitol stress, only aspartate Metabolite levels in the aox1a mutants displayed similar and glycerate levels were significantly (P < 0.05) higher in aox1a patterns after both stress treatments as compared with WT plants mutants than in WT plants. On the other hand, fumarate, citrate, (Figure 5). Nevertheless, the levels of asparagine, glycine, proline, fructose and glucose were significantly (P < 0.05) lower in aox1a malate and spermidine, although following similar stress-induced mutants than in WT plants after mannitol treatment. patterns in both genotypes, were significantly (P < 0.05) higher in Additionally, absolute levels of metabolites were determined aox1a mutants than in WT plants under salinity (Figure 5). On to evaluate the physiological relevance for the accumulation the other hand, aspartate, dehydroascorbate and fructose were of the stress-related metabolites with protective functions. Frontiers in Plant Science | www.frontiersin.org 8 May 2022 | Volume 13 | Article 833113 Del-Saz et al. Alternative Oxidase Role in Osmoprotection The absolute levels of 37 metabolites, present in our standard mixture, were determined for all conditions in both genotypes (Supplementary Figure 2 and Supplementary Table 3). Metabolites in order of absolute abundance under salinity conditions are shown in Supplementary Figure 3. Among the metabolites observed as significantly different between WT or aox1a mutants under salinity (Figure 5 and Supplementary Table 2), glycine and proline were above 2 µmol gDW−1 in either WT or aox1a mutants, while malate, fructose and asparagine displayed lower amounts in the range of 0.3–0.8 µmol gDW−1 (Supplementary Figure 2 and Supplementary Table 3). With respect to mannitol treatment, glucose was by far the most abundant metabolite (56.5 and 34.1 µmol gDW−1 in WT and aox1a mutant, respectively) that was significantly lower in the aox1a mutants, followed FIGURE 6 | Gene expression analyses of alternative respiratory components. by fumarate (10.4 and 7.6 µmol gDW−1 ) and fructose Transcript levels of genes encoding uncoupling proteins and alternative (4.1 and 2.0 µmol gDW−1 ; Supplementary Figure 2 and NAD(P)H dehydrogenases were determined by qPCR analyses (see Materials Supplementary Table 3). and Methods for details) in leaves of wild-type Col 0 (WT) and aox1a A. thaliana plants untreated with NaCl or Mannitol (Control), and after 1 day of severe NaCl or Mannitol treatments (300 mM). Data is shown as fold-changes Gene Expression of Other Alternative relative to control conditions (i.e., all control values were set to 1, which is denoted by the dashed line). Primers used and gene information can be found Respiratory Components in aox1a and in Supplementary Table 4. Values are means ± SE of 5–6 replicates and Wild-Type Plants Under Control and asterisks denote significant differences (P < 0.05) to the control condition for each gene expression analysis. Letters denote significant differences Stress Conditions (P < 0.05) among genotypes and stress conditions for each gene expression The relative levels of transcripts encoding mitochondrial analysis. proteins of the alternative respiratory pathways, other than AOX, were determined by qPCR in both WT and aox1a mutants (Supplementary Figure 1 and Figure 6). The aim of these gene expression analysis was to evaluate possible 2019) and water stress (Guy and Vanlerberghe, 2005; Ribas- compensations from other alternative respiratory pathways Carbo et al., 2005; Galle et al., 2010; Sanhueza et al., 2013) are for the lack of the AOX1a gene expression as well as the variable, however, a common pattern emerge with the electron degree of mitochondrial stress perturbation. Regarding genes partitioning to AOX usually increasing, either by a decrease in encoding alternative NAD(P)H dehydrogenases, transcript levels COP activity, increase in AOP activity or both (Del-Saz et al., of NDB3 were under the limit of detection while NDB2 2018). Notably, a specific increase in the in vivo AOP activity expression displayed a higher stress-response in the aox1a (i.e., in the absence of COP changes), was observed in leaves mutants as compared to WT (Figure 6), a similar trend of Medicago truncatula genotypes after a sudden severe salt as the observed for the AOX1d (Figure 1C). On the other stress (Del-Saz et al., 2016). Such a particular AOP activation hand, genes encoding NDA1 and NDA2 proteins were not in vivo is in line with its proposed role in providing metabolic responsive to stress conditions. Finally, the transcript levels of flexibility to carbon and energy metabolism under short-term genes encoding uncoupling proteins (UCPs) were also evaluated environmental perturbations (Del-Saz et al., 2018), as well as its (Supplementary Figure 1 and Figure 6); UCP2 transcript levels role as a first line of defence against oxidative stress (Møller, were under the limit of detection while UCP1 transcripts 2001). Following previous evidence, we have here investigated did not display any significant change under stress in both the role of the AOP under sudden acute salt stress in more genotypes (Figure 6). detail. Our hypothesis was that in vivo AOP response in aox1a mutants will be curtailed under acute stress, which would impair stress-related metabolism as compared to WT plants. DISCUSSION In addition, we also applied a high mannitol concentration to both genotypes for exploring whether similar effects on the Responses of plant respiration to abiotic stresses are largely respiration and stress-related metabolism were observed after variable (Lambers and Oliveira, 2019; O’Leary et al., 2019). This an acute osmotic stress in the absence of ionic toxicity. We are variability can be partly explained by plants’ ability to adjust aware that osmotic stress conditions applied here were not fully respiratory energy and carbon demands through changes in the controlled by our experimental approach with plants grown in partitioning of electrons between the cytochrome and alternative soil, however, our previous observations (Del-Saz et al., 2016) oxidase pathways (COP and AOP; Del-Saz et al., 2018; Lambers and those presented here show and track typical physiological and Oliveira, 2019; O’Leary et al., 2019), without necessarily (Figures 2,4) and molecular (Figure 5) responses to salt and involve a major change in total respiration rate. In particular, water stress, thus allowing the establishment of relationships the responses of the in vivo AOP and COP activities to salt between the degree of water and (photo)oxidative stress with the (Martí et al., 2011; Del-Saz et al., 2016; Sánchez-Guerrero et al., metabolic changes studied. Frontiers in Plant Science | www.frontiersin.org 9 May 2022 | Volume 13 | Article 833113 Del-Saz et al. Alternative Oxidase Role in Osmoprotection FIGURE 7 | Schematic overview illustrating the impact of in vivo AOP restriction on leaf cell physiology and metabolism in aox1a plants after (A) salt and (B) mannitol treatments. Blue and red boxes indicate significant (P < 0.05) reductions and increases, respectively, in physiological indicators and metabolite levels in aox1a mutants as compared to WT plants. After both stress treatments, the lack of in vivo AOP response was associated to reduced sugars (glucose, fructose or both) accumulation in aox1a mutants, which can be partially explained by their lower photosynthetic activity. Reduced accumulation of sugars in aox1a mutants could affect their osmotic adjustment as reflected by their lower relative water content (RWC). The reduction of fumarate and citrate accumulation in aox1a plants, the former likely contributing the decreased osmotic adjustment, may be explained by a TCA activity impairment triggered by the lack of response on the in vivo AOP activity, which allows matrix NADH re-oxidation without energy demand (i.e., ATP for growth) constrains. This limitation in NADH reoxidation can also explain the accumulation of photorespiratory cycle intermediates glycine (A) and glycerate (B). In addition, ion toxicity effects imposed by salinity could be aggravated by the lack of AOP response and, according to its role in avoiding ROS formation, thus trigger additional oxidative stress that was reflected in the alteration of other metabolic pathways involved in redox balancing and ROS detoxification (A), as denoted by the accumulation of malate, dehydroascorbate, proline, polyamines (spermidine) and asparagine. Image created with BioRender (https://biorender.com). The Lack of AOX1a Does Not Impair or enrichment in mitochondrial membranes are required for the Alternative Oxidase Pathway in vivo detection of low abundance AOX proteins (Oh et al., 2022). Indeed, AOX1D protein was detected in enriched mitochondrial Activity Under Control Conditions but membrane samples from aox1a mutants after proline incubation Restricted Its Response to Stress (Oh et al., 2022). Furthermore, evidence for the involvement As expected, the capacity of the AOP was significantly lower in AOX capacity of isoforms other than AOX1a has recently in the aox1a mutant than in WT plants (Figure 1A), although been provided (Oh et al., 2022). To this respect, while single it was not completely abolished in agreement with previous aox1a and aox1d mutants displayed lower AOX capacity as observations (Watanabe et al., 2010; Vishwakarma et al., 2015; compared to WT plants, the reduction in AOX capacity was Zhang et al., 2016). In the present study, the oxygen isotope clearly more pronounced in double mutants aox1a-aox1d (Oh discrimination in the presence of KCN (1a of approx. 31h) et al., 2022). While the relationship between protein levels provides additional evidence supporting that remaining O2 and cyanide-resistant respiration may not be linear (Gonzalez- consumption after KCN treatment was mainly due to leaf Meler et al., 1999) our and previous results (Oh et al., 2022) AOX (maximum) activity. Indeed, aox1a mutants displayed suggest that both AOX1a and AOX1d proteins can be responsible similar total respiration, electron partitioning and in vivo AOP for AOX capacity in Arabidopsis plants. Notably, transcript activity as WT plants under control conditions (Figure 3). levels of other AOX isoforms or alternative components of These results are very similar to those reported in AtAOX1a the mitochondrial respiratory chain in aox1a mutants were not anti-sense plants, previously obtained at the laboratory of the higher than in WT plants (Supplementary Figure 1). Therefore, honourable Prof. James Siedow, grown under different light our and previous results in transcript and protein amounts intensities (Florez-Sarasa et al., 2011). Our previous (Figure 1 suggest that in vivo AOP activity under non-stress conditions from Florez-Sarasa et al., 2011) and current data (compare could be achieved without any additional induction of other Valt at Figure 1A with ν alt at Figure 3) show that AOX is AOX isoforms (i.e., AOX1c or AOX1d proteins). However, almost fully engaged (ν alt to Valt ratio close to 1) in aox1a post-translational activation of the remaining AOX isoforms in impaired plants. Therefore, results presented here confirm that aox1a mutants could also contribute to the observed in vivo AOX1a isoform is not essential for maintaining leaf in vivo AOP activity. To this respect, while moderate increases in AOX activity under non-stress conditions. On the other hand, the levels of some TCA cycle intermediates (citrate, malate AOX protein signal could not be detected in the aox1a mutants and fumarate) were observed in aox1a mutants under control under control conditions (Figure 1B) as in previous reports conditions (Figure 5), none of them have been shown to act as (Giraud et al., 2008; Strodtkötter et al., 2009; Oh et al., 2022). allosteric activators of any AOX isoform (Selinski et al., 2018b). However, it is likely that samples after mitochondrial isolation However, analysis of AOX allosteric activators at the subcellular Frontiers in Plant Science | www.frontiersin.org 10 May 2022 | Volume 13 | Article 833113 Del-Saz et al. Alternative Oxidase Role in Osmoprotection (mitochondrial) level would be required to attribute their effects conditions when plant growth declines before photosynthesis, on AOX activation in vivo. which can result in a relatively favourable carbon status allowing Both stress treatments, significantly increased transcript levels the use of photosynthates for osmolyte production at low of AOX1a and AOX1d in WT plants (Figure 1B), thus both cost (Hummel et al., 2010). In our short-term stress study, isoforms could potentially contribute to the observed increase in photosynthesis was not strongly affected, particularly at growth AOP capacity (Figure 1A) and in vivo activity (Figure 3). On light intensity (Figure 4), which suggests minor limitations on the other hand, the absence of AOX1a expression in the aox1a C availability. In agreement, a large accumulation of sugars, mutants coincided with a much higher induction of the AOX1d sugar alcohols and amino acids was observed in both genotypes transcript levels (approx. 20 times higher) under both stress after stress treatments (Figure 5). Previous reports suggest that conditions, as compared to WT plants (Figure 1B). However, this accumulation of sugars in leaves of Arabidopsis and other response was not coupled to a significant (P < 0.05) increase species after mannitol and salt treatments have a prominent in the AOX capacity or protein level under our experimental role on osmotic adjustment (Thalmann and Santelia, 2017; conditions (Figure 1). Moreover, the lack of in vivo AOP activity Gurrieri et al., 2020). On the other hand, the accumulation response observed in aox1a mutant leaves (Figure 3) clearly of amino acids (i.e., proline) is a well-known mechanism suggests that the absence of AOX1a was not compensated by contributing to osmoprotection and osmoregulation in different other AOX isoforms (i.e., AOX1d) under stress conditions. species (Slama et al., 2015). The relatively low concentrations These results are in line with previous observations suggesting of amino acids as typically observed in Arabidopsis leaves no functional compensation for the lack of AOX1a under suggest their roles as osmoprotectants of subcellular structures, stress via high induction of AOX1d after respiratory inhibitor in scavenging reactive oxygen species or counteracting redox (Strodtkötter et al., 2009) and metal stress treatments (Keunen imbalances, rather than contributing to osmotic adjustment et al., 2015). In here, the aox1a mutants did not show respiratory (Shabala and Shabala, 2011; Slama et al., 2015; Gurrieri et al., response to acute salt and water stress, since AOP was the main 2020). Our results are in line with these observations since the contributor to this respiratory response in our experimental levels of sucrose, glucose and fructose were not only highly conditions (Figure 3). This AOP-specific response to acute accumulated, but also reached the highest absolute amounts, salt stress was previously observed in a Medicago truncatula as compared to highly accumulated amino acids such as sensitive genotype (Del-Saz et al., 2016), which fits with the proline and glycine (Supplementary Figure 2). Importantly, the Arabidopsis behaviour, as a salt-sensitive species (Kazachkova accumulation of glucose and fructose in the aox1a mutants was et al., 2018). In addition, we here show that mannitol treatment lower as compared with WT plants (Figure 5), and together with induces a similar in vivo respiratory response as salt stress their lower relative water content (RWC), suggests a decreased treatment (Figure 3), which suggests that the osmotic stress osmotic adjustment in the aox1a mutants (Figure 7). In this line, generated after both treatments is likely the main responsible the aox1a mutants after mannitol treatment also displayed lower for the observed in vivo AOP specific response. In fact, the levels of fumarate, which was among the top three most abundant lack of in vivo AOP response in aox1a mutants coincided metabolites under stress (Supplementary Figure 2), and this with a more pronounced reduction in leaf RWC under both metabolite was previously suggested to have a high contribution stress conditions, as compared to WT plants (Figure 2), which to osmotic adjustment in Arabidopsis under severe drought could be related to a reduced osmotic adjustment. Osmotic (Hummel et al., 2010). The lower photosynthetic performance adjustment is an essential trait that plants use to maintain of aox1a mutants could partly explain their lower sugar levels, turgor by balancing the osmotic pressure imposed during salt which in turn could slow down the carbon flow through glycolysis or water stresses (Munns and Gilliham, 2015). While osmotic and consequently the TCA cycle activity for the fumarate adjustment using inorganic ions is energetically “cheaper” than production (Figure 7). Nevertheless, fumarate can also be using organic solutes, salt-sensitive species usually rely in ion produced from malate which levels could be altered by the redox exclusion (particularly in leaves) and accumulation of organic imbalances in aox1a plants. Furthermore, the accumulation of solutes (Munns and Gilliham, 2015). In the present study, osmolytes is thought to be the result of the coordinated regulation salt and mannitol treatments induced a large accumulation of of biosynthetic and catabolic pathways (Slama et al., 2015), many several stress-related metabolites (SRMs; Figure 5), many of of which are tightly linked to respiratory pathways (Bandehagh them being well-known for their roles in osmoregulation and and Taylor, 2020). Therefore, we cannot discard that the lower osmoprotection (Slama et al., 2015). accumulation of fumarate and citrate observed in aox1a mutants after mannitol treatment could be partly due to a reduced TCA The Lack in vivo Alternative Oxidase cycle activity as a direct consequence of a reduction in matrix Pathway Response Under Stress Altered NADH reoxidation by the AOP. Future experiments determining carbon fluxes (i.e., with 13 C labelled substrates under control and the Accumulation of Stress-Related osmotic stress conditions) could unveil the precise nature of the Metabolities Involved in Leaf observed altered accumulation of TCA cycle intermediates. Osmoregulation and Redox Balance Impairment of photosynthetic performance and higher Osmotic stress has immediate effects on plant growth, and photoinhibition have been observed in other aox1a suppressed the accumulation of osmolytes is part of the typical plant plants under drought (Giraud et al., 2008; Dahal et al., 2014, responses involved in osmotic adjustment (Munns and Gilliham, 2017; Dahal and Vanlerberghe, 2017, 2018) high light (Florez- 2015). The accumulation of osmolytes is favoured under Sarasa et al., 2011) and temperature (Watanabe et al., 2010; Frontiers in Plant Science | www.frontiersin.org 11 May 2022 | Volume 13 | Article 833113 Del-Saz et al. Alternative Oxidase Role in Osmoprotection Li et al., 2020) stress, which have been attributed to the role our results provide evidence on the contribution of the AOP of the AOP in providing flexibility to carbon and energy in providing flexibility to carbon metabolism for biosynthesis, metabolism in photosynthetic tissues, particularly under stress in addition to its role in cell redox balancing and preventing (Del-Saz et al., 2018; Vanlerberghe et al., 2020). In our study, mitochondrial ROS formation (Smith et al., 2009; Vanlerberghe while both stress conditions induced photoinhibition in both et al., 2020). Metabolites including TCA cycle intermediates genotypes (Figure 3), the decrease in Fv/Fm was higher (malate, citrate and fumarate), proline and hexoses, which under salinity as compared to mannitol stress (Figure 3), thus were altered by the absence of in vivo AOP activity in this suggesting that ion toxicity probably led to higher oxidative study, are similar to those typically altered when comparing stress at the chloroplast level. In line with a higher oxidative salt-sensitive species A. thaliana with its relatives exhibiting stress after salt treatment, as compared to mannitol treatment, salt-tolerance (Kazachkova et al., 2018). Therefore, our results both genotypes displayed higher levels of different metabolites provide important additional evidence reinforcing previous with known functions on protecting against oxidative stress suggestions for AOX manipulation as a reasonable strategy for themselves (i.e., dehydroascorbate, proline, asparagine; Figure 5 improving salt-tolerance in salt-sensitive crops (Smith et al., and Supplementary Table 2) or as precursors of oxidative stress- 2009), notwithstanding other important strategies involving salt- related specialized metabolites (i.e., phenylalanine and tyrosine tolerant crops (Ventura et al., 2015; Fernie and Yan, 2019). for phenylpropanoids synthesis; Figure 5 and Supplementary Whether a higher AOP capacity or activity is primed for a Table 2), as well as higher levels of transcripts encoding better performance in case of salinity exposure remains to be oxidative stress-responsive genes (AOX1a and NDB2; Figure 6). investigated in a wider range of salt-tolerant crops. In addition, In agreement, the AOP activity in WT plants was significantly the relevance of AOP and its coordinated regulation with other higher after salt than after mannitol stress (Figure 3). alternative respiratory pathways under longer exposure to salt Importantly, the aox1a mutants, lacking stress response of and/or water stress remains to be investigated in depth. the in vivo AOP activity, displayed higher levels of proline, asparagine, spemidine, and dehydroascorbate when compared to WT plants (Figures 5, 7). Increased levels of all these metabolites DATA AVAILABILITY STATEMENT indicate a higher induction of metabolic pathways involved in The original contributions presented in the study are included redox balancing and avoidance of excessive ROS accumulation in the article/Supplementary Material, further inquiries can be under stress (Szabados and Savouré, 2010; Maaroufi-Dguimi directed to the corresponding authors. et al., 2011; Alcázar et al., 2020; Foyer et al., 2020). Moreover, higher glycine levels in aox1a mutants under salinity denotes a restriction in photorespiration due to reduction of glycine AUTHOR CONTRIBUTIONS decarboxylation (Figure 7), as has been previously reported in aox1a mutants (Strodtkötter et al., 2009; Zhang et al., ND-S, MR-C, and IF-S conceived and designed the idea of this 2017; Li et al., 2020). Finally, higher malate levels in aox1a experiment. ND-S, AI-S, and DA-F carried out oxygen electrode mutants could indicate an impairment in the reductant export and mass spectrometry measurements of respiration as well from the chloroplast via the malate valve (Figure 7), as as leaf relative water content determinations. AI-S performed previously suggested (Yoshida et al., 2007; Dinakar et al., 2010; photosynthetic and gene expression analyses. ML-G, FP, and Vishwakarma et al., 2015). MC-M performed samples preparation, metabolite extractions and analyses. IF-S and AF performed the metabolite data analysis. IF-S and ND-S performed the statistical analysis. ND-S and IF-S CONCLUSION wrote the first draft of the manuscript with subsequent inputs from all co-authors. All authors have read and agreed to the Mitochondrial function has been suggested to be essential published version of the manuscript. for drought and salinity tolerance in plants as it can have roles in signal transduction, ion exclusion and homeostasis, ROS detoxification, the generation of ATP, and carbon balance FUNDING (Bandehagh and Taylor, 2020; Munns et al., 2020; Vanlerberghe This work was financed by the MICINN–project CTM2014- et al., 2020). However, information about the in vivo AOP and 53902-C2-1-P and FONDECYT No. 1191118 from National COP activity and their metabolic roles under water and salt Agency for Research and Development (ANID). AI-S was stress remain scarce. The different metabolic responses in aox1a supported by a predoctoral fellowship from MICINN (PRE2018- mutants lacking in vivo AOP response under stress (Figure 7) 083610). IF-S has received funding the “Ramon y Cajal” reinforce the relevant roles of the AOX previously suggested contract RYC2019-027244-I/AEI/10.13039/501100011033 and in redox balancing, avoidance of excessive ROS accumulation the European Social Fund. and photosynthetic performance. Furthermore, we here provide new evidence on the in vivo regulation and metabolic role of the AOP in the accumulation of stress-related metabolites ACKNOWLEDGMENTS associated to osmoregulation. While further experiments would be required to unravel the precise direction of subcellular carbon We would like to thank Biel Martorell, Yunuen Avalos-Padilla fluxes and its direct relationship with the in vivo AOP response, and Javier Hidalgo for their technical help as well as Miquel Frontiers in Plant Science | www.frontiersin.org 12 May 2022 | Volume 13 | Article 833113 Del-Saz et al. Alternative Oxidase Role in Osmoprotection Truyols and collaborators of the UIB Experimental Field and described in Figure 5A, 6. Data is shown as fold-changes relative to WT (i.e., all Greenhouses which are supported by the UIBGrant 15/2015. WT values were set to 1, which is denoted by the dashed line). Primers used and gene information can be found in Supplementary Table 4. Values are means ± SE of 5–6 replicates and asterisks denote significant differences (P < 0.05) to the WT for each gene expression analysis. n.d. (not detected). SUPPLEMENTARY MATERIAL Supplementary Figure 2 | Metabolite profiles showing absolute changes in primary metabolism. Coloured table showing the absolute levels (µmol gDW−1 ) of The Supplementary Material for this article can be found the GC-MS-analysed metabolites in leaves of wild-type Col 0 (WT) and aox1a online at: https://www.frontiersin.org/articles/10.3389/fpls.2022. A. thaliana plants untreated with NaCl or Mannitol (Control), and after 1 day of 833113/full#supplementary-material severe NaCl or Mannitol treatments (300 mM). Metabolites are shown in order of absolute abundance under 300 mM NaCl conditions. In this table, the intensity of Supplementary Figure 1 | Gene expression analyses of alternative respiratory the red colour indicates the absolute level of the metabolite as well as the green components under control conditions. Transcript levels of genes encoding bars inside the cells. Values are means of 4–6 replicates and the statistical alternative oxidases, uncoupling proteins and alternative NAD(P)H differences to the WT plants in each experimental condition as well as between dehydrogenases were determined by qPCR analyses (see Materials and Methods control and stress treatments in each genotype are presented in for details) in leaves of wild-type Col 0 (WT) and aox1a A. thaliana plants as Supplementary Table 3. REFERENCES Dinakar, C., Raghavendra, A. S., and Padmasree, K. (2010). Importance of AOX pathway in optimizing photosynthesis under high light stress: role of pyruvate Alcázar, R., Bueno, M., and Tiburcio, A. F. (2020). Polyamines: small amines and malate in activating AOX. Physiol. Plant. 139, 13–26. doi: 10.1111/j.1399- with large effects on plant abiotic stress tolerance. Cells 9:2373. doi: 10.3390/ 3054.2010.01346.x cells9112373 Epstein, E. H. Jr., Levin, D. L., Croft, J. D. Jr., and Lutzner, M. A. (1972). MYCOSIS Bandehagh, A., and Taylor, N. L. 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Plant Cell Physiol. 51, 810–822. doi: 10.1093/pcp/ pcq033 Publisher’s Note: All claims expressed in this article are solely those of the authors Xu, F., Yuan, S., Zhang, D. W., Lv, X., and Lin, H. H. (2012). The and do not necessarily represent those of their affiliated organizations, or those of role of alternative oxidase in tomato fruit ripening and its regulatory the publisher, the editors and the reviewers. Any product that may be evaluated in interaction with ethylene. J. Exp. Bot. 63, 5705–5716. doi: 10.1093/jxb/er this article, or claim that may be made by its manufacturer, is not guaranteed or s226 endorsed by the publisher. Yoshida, K., Terashima, I., and Noguchi, K. (2007). Up-regulation of mitochondrial alternative oxidase concomitant with chloroplast over- Copyright © 2022 Del-Saz, Iglesias-Sanchez, Alonso-Forn, López-Gómez, Palma, reduction by excess light. Plant Cell Physiol. 48, 606–614. doi: 10.1093/pcp/p Clemente-Moreno, Fernie, Ribas-Carbo and Florez-Sarasa. This is an open-access cm033 article distributed under the terms of the Creative Commons Attribution License Zhang, D. W., Yuan, S., Xu, F., Zhu, F., Yuan, M., Ye, H. X., et al. (2016). Light (CC BY). The use, distribution or reproduction in other forums is permitted, provided intensity affects chlorophyll synthesis during greening process by metabolite the original author(s) and the copyright owner(s) are credited and that the original signal from mitochondrial alternative oxidase in Arabidopsis. Plant Cell publication in this journal is cited, in accordance with accepted academic practice. No Environ. 39, 12–25. doi: 10.1111/pce.12438 use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Plant Science | www.frontiersin.org 15 May 2022 | Volume 13 | Article 833113