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Chenopodium quinoa Willd. is a native species that originated in the High Andes plateau (Altiplano) and its cultivation spread out to the south of Chile. Because of the different edaphoclimatic characteristics of both regions, soils from Altiplano accumulated higher levels of nitrate (NO3-) than in the south of Chile, where soils favor ammonium (NH4 +) accumulation. To elucidate whether C. quinoa ecotypes differ in several physiological and biochemical parameters related to their capacity to assimilate NO3- and NH4 +, juvenile plants of Socaire (from Altiplano) and Faro (from Lowland/South of Chile) were grown under different sources of N (NO3- or NH4 +). Measurements of photosynthesis and foliar oxygen-isotope fractionation were carried out, together with biochemical analyses, as proxies for the analysis of plant performance or sensitivity to NH4 +. Overall, while NH4 + reduced the growth of Socaire, it induced higher biomass productivity and increased protein synthesis, oxygen consumption, and cytochrome oxidase activity in Faro. We discussed that ATP yield from respiration in Faro could promote protein production from assimilated NH4 + to benefit its growth. The characterization of this differential sensitivity of both quinoa ecotypes for NH4 + contributes to a better understanding of nutritional aspects driving plant primary productivity.

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In plants salt and water stress result in an induction of respiration and accumulation of stress-related metabolites (SRMs) with osmoregulation and osmoprotection functions that benefit photosynthesis. The synthesis of SRMs may depend on an active respiratory metabolism, which can be restricted under stress by the inhibition of the cytochrome oxidase pathway (COP), thus causing an increase in the reduction level of the ubiquinone pool. However, the activity of the alternative oxidase pathway (AOP) is thought to prevent this from occurring while at the same time, dissipates excess of reducing power from the chloroplast and thereby improves photosynthetic performance. The present research is based on the hypothesis that the accumulation of SRMs under osmotic stress will be affected by changes in folial AOP activity. To test this, the oxygen isotope-fractionation technique was used to study the in vivo respiratory activities of COP and AOP in leaves of wild-type Arabidopsis thaliana plants and of aox1a mutants under sudden acute stress conditions induced by mannitol and salt treatments. Levels of leaf primary metabolites and transcripts of respiratory-related proteins were also determined in parallel to photosynthetic analyses. The lack of in vivo AOP response in the aox1a mutants coincided with a lower leaf relative water content 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.

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Projected rises in atmospheric CO2 concentration and minimum night-time temperatures may have important effects on plant carbon metabolism altering the carbon balance of the only two vascular plant species in the Antarctic Peninsula. We assessed the effect of nocturnal warming (8/5 °C vs. 8/8 °C day/night) and CO2 concentrations (400 ppm and 750 ppm) on gas exchange, non-structural carbohydrates, two respiratory-related enzymes, and mitochondrial size and number in two species of vascular plants. In Colobanthus quitensis, light-saturated photosynthesis measured at 400 ppm was reduced when plants were grown in the elevated CO2 or in the nocturnal warming treatments. Growth in elevated CO2 reduced stomatal conductance but nocturnal warming did not. The short-term sensitivity of respiration, relative protein abundance, and mitochondrial traits were not responsive to either treatment in this species. Moreover, some acclimation to nocturnal warming at ambient CO2 was observed. Altogether, these responses in C. quitensis led to an increase in the respiration-assimilation ratio in plants grown in elevated CO2. The response of Deschampsia antarctica to the experimental treatments was quite distinct. Photosynthesis was not affected by either treatment; however, respiration acclimated to temperature in the elevated CO2 treatment. The observed short-term changes in thermal sensitivity indicate type I acclimation of respiration. Growth in elevated CO2 and nocturnal warming resulted in a reduction in mitochondrial numbers and an increase in mitochondrial size in D. antarctica. Overall, our results suggest that with climate change D. antarctica could be more successful than C. quitensis, due to its ability to make metabolic adjustments to maintain its carbon balance.

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"Memory imprint" refers to the process when prior exposure to stress prepares the plant for subsequent stress episodes. Seed priming is a strategy to change the performance of seedlings to cope with stress; however, mechanisms associated with the metabolic response are fragmentary. Salinity is one of the major abiotic stresses that affect crop production in arid and semiarid areas. Chenopodium quinoa Willd. (Amaranthaceae) is a promising crop to sustain food security and possesses a wide genetic diversity of salinity tolerance. To elucidate if the metabolic memory induced by seed halo-priming (HP) differs among contrasting saline tolerance plants, seeds of two ecotypes of Quinoa (Socaire from Atacama Salar, and BO78 from Chilean Coastal/lowlands) were treated with a saline solution and then germinated and grown under different saline conditions. The seed HP showed a more positive impact on the sensitive ecotype during germination and promoted changes in the metabolomic profile in both ecotypes, including a reduction in carbohydrates (starch) and organic acids (citric and succinic acid), and an increase in antioxidants (ascorbic acid and α-tocopherol) and related metabolites. These changes were linked to a further reduced level of oxidative markers (methionine sulfoxide and malondialdehyde), allowing improvements in the energy use in photosystem II under saline conditions in the salt-sensitive ecotype. In view of these results, we conclude that seed HP prompts a "metabolic imprint" related to ROS scavenger at the thylakoid level, improving further the physiological performance of the most sensitive ecotype.

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The alternative oxidase pathway (AOP) is associated with excess energy dissipation in leaves of terrestrial plants. To address whether this association is less important in palustrine plants, we compared the role of AOP in balancing energy and carbon metabolism in palustrine and terrestrial environments by identifying metabolic relationships between primary carbon metabolites and AOP in each habitat. We measured oxygen isotope discrimination during respiration, gas exchange, and metabolite profiles in aerial leaves of ten fern and angiosperm species belonging to five families organized as pairs of palustrine and terrestrial species. We performed a partial least square model combined with variable importance for projection to reveal relationships between the electron partitioning to the AOP (τa) and metabolite levels. Terrestrial plants showed higher values of net photosynthesis (AN) and τa, together with stronger metabolic relationships between τa and sugars, important for water conservation. Palustrine plants showed relationships between τa and metabolites related to the shikimate pathway and the GABA shunt, to be important for heterophylly. Excess energy dissipation via AOX is less crucial in palustrine environments than on land. The basis of this difference resides in the contrasting photosynthetic performance observed in each environment, thus reinforcing the importance of AOP for photosynthesis.

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Plant respiration provides metabolic flexibility under changing environmental conditions by modulating the activity of the nonphosphorylating alternative pathways from the mitochondrial electron transport chain, which bypass the main energy-producing components of the cytochrome oxidase pathway (COP). While adjustments in leaf primary metabolism induced by changes in day length are well studied, possible differences in the in vivo contribution of the COP and the alternative oxidase pathway (AOP) between different photoperiods remain unknown. In our study, in vivo electron partitioning between AOP and COP and expression analysis of respiratory components, photosynthesis, and the levels of primary metabolites were studied in leaves of wild-type (WT) plants and cytochrome c (CYTc) mutants, with reduced levels of COP components, under short- and long-day photoperiods. Our results clearly show that differences in AOP and COP in vivo activities between WT and cytc mutants depend on the photoperiod likely due to energy and stress signaling constraints. Parallel responses observed between in vivo respiratory activities, TCA cycle intermediates, amino acids, and stress signaling metabolites indicate the coordination of different pathways of primary metabolism to support growth adaptation under different photoperiods.

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The interaction of the alternative oxidase (AOX) pathway with nutrient metabolism is important for understanding how respiration modulates ATP synthesis and carbon economy in plants under nutrient deficiency. Although AOX activity reduces the energy yield of respiration, this enzymatic activity is upregulated under stress conditions to maintain the functioning of primary metabolism. The in vivo metabolic regulation of AOX activity by phosphorus (P) and nitrogen (N) and during plant symbioses with Arbuscular mycorrhizal fungi (AMF) and Rhizobium bacteria is still not fully understood. We highlight several findings and open questions concerning the in vivo regulation of AOX activity and its impact on plant metabolism during P deficiency and symbiosis with AMF. We also highlight the need for the identification of which metabolic regulatory factors of AOX activity are related to N availability and nitrogen-fixing legume-rhizobia symbiosis in order to improve our understanding of N assimilation and biological nitrogen fixation.

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Phytopathogen infection alters primary metabolism status and plant development. The alternative oxidase (AOX) has been hypothesized to increase under pathogen attack preventing reductions, thus optimizing photosynthesis and growth. In this study, two genotypes of Medicago truncatula, one relatively resistant (Jemalong A17) and one susceptible (TN1.11), were infected with Fusarium oxysporum and Rhizoctonia solani. The in vivo foliar respiratory activities of the cytochrome oxidase pathway (COP) and the alternative oxidase pathway (AOP) were measured using the oxygen isotope fractionation. Gas exchange and photosynthesis-related parameters were measured and calculated together with antioxidant enzymes activities and organic acids contents. Our results show that the in vivo activity of AOX (valt ) plays a role under fungal infection. When infected with R. solani, the increase of valt in A17 was concomitant to an increase in net assimilation, in mesophyll conductance, to an improvement in the maximum velocity of Rubisco carboxylation and to unchanged malate content. However, under F. oxysporum infection, the induced valt was accompanied by an enhancement in the antioxidant enzymes, superoxide dismutase (SOD; EC1.15.1.1), catalase (CAT; EC1.11.1.6) and guaiacol peroxidase (GPX; EC1.11.1.7), activities and to an unchanged tricarboxylic acid cycle intermediates. These results provide new insight into the role of the in vivo activity of AOX in coordinating primary metabolism interactions that, partly, modulate the relative resistance of M. truncatula to diseases caused by soil-borne pathogenic fungi.

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The species Deschampsia antarctica (DA) is one of the only two native vascular species that live in Antarctica. We performed ecophysiological, biochemical, and metabolomic studies to investigate the responses of DA to low temperature. In parallel, we assessed the responses in a non-Antarctic reference species (Triticum aestivum [TA]) from the same family (Poaceae). At low temperature (4°C), both species showed lower photosynthetic rates (reductions were 70% and 80% for DA and TA, respectively) and symptoms of oxidative stress but opposite responses of antioxidant enzymes (peroxidases and catalase). We employed fused least absolute shrinkage and selection operator statistical modelling to associate the species-dependent physiological and antioxidant responses to primary metabolism. Model results for DA indicated associations with osmoprotection, cell wall remodelling, membrane stabilization, and antioxidant secondary metabolism (synthesis of flavonols and phenylpropanoids), coordinated with nutrient mobilization from source to sink tissues (confirmed by elemental analysis), which were not observed in TA. The metabolic behaviour of DA, with significant changes in particular metabolites, was compared with a newly compiled multispecies dataset showing a general accumulation of metabolites in response to low temperatures. Altogether, the responses displayed by DA suggest a compromise between catabolism and maintenance of leaf functionality.

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Understanding the strategies employed by plant species that live in extreme environments offers the possibility to discover stress tolerance mechanisms. We studied the physiological, antioxidant and metabolic responses to three temperature conditions (4, 15, and 23°C) of Colobanthus quitensis (CQ), one of the only two native vascular species in Antarctica. We also employed Dianthus chinensis (DC), to assess the effects of the treatments in a non-Antarctic species from the same family. Using fused LASSO modelling, we associated physiological and biochemical antioxidant responses with primary metabolism. This approach allowed us to highlight the metabolic pathways driving the response specific to CQ. Low temperature imposed dramatic reductions in photosynthesis (up to 88%) but not in respiration (sustaining rates of 3.0-4.2 µmol CO2  m-2  s-1 ) in CQ, and no change in the physiological stress parameters was found. Its notable antioxidant capacity and mitochondrial cytochrome respiratory activity (20 and two times higher than DC, respectively), which ensure ATP production even at low temperature, was significantly associated with sulphur-containing metabolites and polyamines. Our findings potentially open new biotechnological opportunities regarding the role of antioxidant compounds and respiratory mechanisms associated with sulphur metabolism in stress tolerance strategies to low temperature.

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The present study aims to unravel ecophysiological mechanisms underlying plant-microbe interactions under natural abiotic stress conditions, specifically heavy metal pollution. Effect of plant growth promoting rhizobacteria (PGPR) bioaugmentation on Spartina maritima in vivo root respiration and oxidative stress was investigated. This autochthonous plant is a heavy metal hyperaccumulator cordgrass growing in one of the most polluted estuaries in the world. The association with native PGPR is being studied with a view to their biotechnological potential in environmental decontamination. As a novelty, the oxygen-isotope fractionation technique was used to study the in vivo activities of cytochrome oxidase (COX) and alternative oxidase (AOX) pathways. Inoculated plants showed decreased antioxidant enzymatic activities and in vivo root respiration rates. The reduction in respiratory carbon consumption and the stress alleviation may explain the increments observed in S. maritima root biomass and metal rhizoaccumulation after inoculation. For the first time, plant carbon balance and PGPR are interrelated to explain the effect of rhizobacteria under abiotic stress.

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Plants exhibit respiratory bypasses (e.g., the alternative oxidase [AOX]) and increase the synthesis of carboxylates in their organs (leaves and roots) in response to phosphorus (P) deficiency, which increases P uptake capacity. They also show differential expression of high-affinity inorganic phosphorus (Pi) transporters, thus avoiding P toxicity at a high P availability. The association between AOX and carboxylate synthesis was tested in Solanum lycopersicum plants grown at different soil P availability, by using plants grown under P-sufficient and P-limiting conditions and by applying a short-term (24 hr) P-sufficient pulse to plants grown under P limitation. Tests were also performed with plants colonized with arbuscular mycorrhizal fungi, which increased plant P concentration under reduced P availability. The in vivo activities of AOX and cytochrome oxidase were measured together with the concentration of carboxylates and the P concentration in plant organs. Gene transcription of Pi transporters (LePT1 and LePT2) was also studied. A coordinated response between plant P concentration with these traits was observed, indicating that a sufficient P availability in soil led to a suppression of both AOX activity and synthesis of citrate and a downregulation of the transcription of genes encoding high-affinity Pi transporters, presumably to avoid P toxicity.

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Despite intense research on the in vitro characterization of regulatory factors modulating the alternative oxidase (AOX) pathway, the regulation of its activity in vivo is still not fully understood. Advances concerning in vivo regulation of AOX based on the oxygen-isotope fractionation technique are reviewed, and regulatory factors that merit future research are highlighted. In addition, we review and discuss the main biological functions assigned to the plant AOX, and suggest future experiments involving in vivo activity measurements to test different hypothesized physiological roles.

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Plant respiration is characterized by the existence of the alternative oxidase pathway (AOP) that competes with cytochrome oxidase pathway (COP) for the electrons of the ubiquinone pool of the mitochondrial electron transport chain, thus reducing ATP synthesis. The oxygen (O2) isotope fractionation technique is the only available to determine the electron partitioning between the two pathways and their in vivo activities in plant tissues. In this chapter, the basis of the O2 isotope fractionation technique and its derived calculations are carefully explained together with a detailed description of the dual-inlet isotope ratio mass spectrometry (DI-IRMS) system and the protocol developed at the University of Balearic Islands. The key advantages of the DI-IRMS over other systems are highlighted as well as the potential problems of this technique. Among these problems, those associated with leakage, diffusion, and inhibitor treatments are noted and solutions to prevent, detect, and repair these problems are detailed.

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Growth and maintenance partly depend on both respiration and ATP production during oxidative phosphorylation in leaves. Under stress, ATP is needed to maintain the accumulated biomass. ATP production mostly proceeds from the cytochrome oxidase pathway (COP), while respiration via the alternative oxidase pathway (AOP) may decrease the production of ATP per oxygen consumed, especially under phosphorus (P) limitation and salinity conditions. Symbiosis with arbuscular mycorrhizal (AM) fungi is reputed by their positive effect on plant growth under stress at mature stages of colonization; however, fungal colonization may decrease plant growth at early stages. Thus, the present research is based on the hypothesis that AM fungus colonization will increase both foliar respiration and ATP production at mature stages of plant growth while decreasing them both at early stages. We used the oxygen-isotope-fractionation technique to study the in vivo respiratory activities and ATP production of the COP and AOP in AM and non-AM (NM) tobacco plants grown under P-limiting and saline conditions in sand at different growth stages (14, 28 and 49days). Our results suggest that AM symbiosis represents an ATP cost detrimental for shoot growth at early stages, whilst it represents a benefit on ATP allowing for faster rates of growth at mature stages, even under salinity conditions.

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The effect of arbuscular mycorrhiza (AM) symbiosis on plant growth is associated with the balance between costs and benefits. A feedback regulation loop has been described in which the higher carbohydrate cost to plants for AM symbiosis is compensated by increases in their photosynthetic rates. Nevertheless, plant carbon balance depends both on photosynthetic carbon uptake and respiratory carbon consumption. The hypothesis behind this research was that the role of respiration in plant growth under AM symbiosis may be as important as that of photosynthesis. This hypothesis was tested in Arundo donax L. plantlets inoculated with Rhizophagus irregularis and Funneliformis mosseae. We tested the effects of AM inoculation on both photosynthetic capacity and in vivo leaf and root respiration. Additionally, analyses of the primary metabolism and ion content were performed in both leaves and roots. AM inoculation increased photosynthesis through increased CO2 diffusion and electron transport in the chloroplast. Moreover, respiration decreased only in AM roots via the cytochrome oxidase pathway (COP) as measured by the oxygen isotope technique. This decline in the COP can be related to the reduced respiratory metabolism and substrates (sugars and tricarboxylic acid cycle intermediates) observed in roots.

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The mitochondrial alternative oxidase pathway (AOP) has been suggested to act as a sink for excess reducing power generated in the chloroplast under high-light (HL) stress and thus may reduce photoinhibition. The aim of this study was to compare different species to investigate the in vivo regulation and role of AOP under HL stress. The in vivo activities of AOP (νalt ) and the cytochrome oxidase pathway, chlorophyll fluorescence, metabolite profiles, alternative oxidase (AOX) capacity and protein amount were determined in leaves of five C3 species under growth light and after HL treatment. Differences in respiration and metabolite levels were observed among species under growth light conditions. The HL response of νalt was highly species dependent, correlated with the AOP capacity and independent of AOX protein content. Nevertheless, significant correlations were observed between νalt , levels of key metabolites and photosynthetic parameters. The results show that the species-specific response of νalt is caused by the differential post-translational regulation of AOX. Significant correlations between respiration, metabolites and photosynthetic performance across species suggest that AOP may permit stress-related amino acid synthesis, whilst maintaining photosynthetic activity under HL stress.

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Salt respiration is defined as the increase of respiration under early salt stress. However, the response of respiration varies depending on the degree of salt tolerance and salt stress. It has been hypothesized that the activity of the alternative pathway may increase preventing over-reduction of the ubiquinone pool in response to salinity, which in turn can increase respiration. Three genotypes of Medicago truncatula are reputed as differently responsive to salinity: TN1.11, A17 and TN6.18. We used the oxygen-isotope fractionation technique to study the in vivo respiratory activities of the cytochrome oxidase pathway (COP) and the alternative oxidase pathway (AOP) in leaves and roots of these genotypes treated with severe salt stress (300 mM) during 1 and 3 days. In parallel, AOX capacity, gas exchange measurements, relative water content and metabolomics were determined in control and treated plants. Our study shows for first time that salt respiration is induced by the triggered AOP in response to salinity. Moreover, this phenomenon coincides with increased levels of metabolites such as amino and organic acids, and is shown to be related with higher photosynthetic rate and water content in TN6.18.

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