Over the past century, research has significantly advanced our understanding of fruit respiration, from (eco)physiological processes to molecular mechanisms. This review focuses on the functional relevance and regulatory roles of mitochondrial alternative respiratory pathways (ARPs) during fruit growth and ripening. We revisit classical distinctions between climacteric and nonclimacteric fruits, considering recent insights into the alternative oxidase, uncoupling proteins, and type II NAD(P)H dehydrogenases (NDIIs). These components are increasingly recognized as central to maintaining metabolic flexibility, energy balance, and redox homeostasis, supporting both primary and secondary metabolism. We highlight how CO2 refixation and organic acid metabolism, often displaying C4/CAM-like features, impose specific demands on mitochondrial electron transport, and how spatial heterogeneity in metabolism and O2 availability across fruit tissues can shape respiratory activity. Interactions between fruit photosynthesis and respiration remain poorly understood, particularly under stress. The interplay between respiration, ethylene biosynthesis, and signaling is discussed, emphasizing feedback loops involving mitochondrial retrograde regulation and redox-sensitive control of ripening. Key knowledge gaps include in vivo flux analyses, tissue-resolved energy profiling, and functional characterization of underexplored ARP components. Finally, we outline postharvest and metabolic engineering strategies targeting ARPs as complementary to ethylene-centered approaches to improve fruit quality, stress resilience, and nutritional value.

Tomato (Solanum lycopersicum) is a climacteric fruit displaying a respiration peak at the onset of ripening accompanied by increased synthesis of ethylene and carotenoid pigments. Chromoplast and mitochondrial respiration participate at different stages of fruit ripening, but their in vivo regulation and function remain unclear. We determined the in vivo activities of the mitochondrial alternative oxidase (AOX) and cytochrome oxidase pathways and quantified the levels of respiratory- and ripening-related gene transcripts, primary metabolites and carotenoids in ripening tomato fruits with or without functional chromorespiration. Furthermore, we carried out physiological, molecular, and metabolic analyses of CRISPR-Cas9 mutants defective in AOX1a, the main AOX isoform upregulated during tomato fruit ripening. We confirmed that plastid terminal oxidase-dependent chromorespiration is only relevant at late stages of ripening and found that in vivo AOX activity significantly increased at the breaker stage, becoming the main contributor to climacteric respiration when ripening initiates. This activation did not correlate with gene expression but was likely due to increased levels of AOX activators such as pyruvate (a metabolic precursor of carotenoids), 2-oxoglutarate, and succinate. A strong alteration of ripening-related metabolites was observed in aox1a mutant fruits, highlighting a key role of the AOX pathway at the onset of ripening. Our data suggest that increased supply of tricarboxylic acid cycle intermediates at the climacteric stage allosterically enhances AOX activity, thus allowing the reoxidation of NAD(P)H to ensure carbon supply for triggering ethylene and carotenoid biosynthesis.

La respiración celular es un proceso complejo que otorga vitalidad a los organismos, y que está estrechamente relacionado con el metabolismo y el estrés oxidativo. Las plantas poseen una respiración alternativa que, a pesar de disminuir la eficiencia energética de la respiración, brinda flexibilidad metabólica para permitir a la planta responder a situaciones adversas. Esto arroja nuevas perspectivas en el estudio de un proceso clave para el beneficio de una productividad vegetal amenazada por el cambio climático.

The viticulture sector is currently threatened by climate change, impacting grape quality and yield through altered weather patterns and reduced soil health. The incorporation of cover crops can significantly bolster sustainability by enhancing soil moisture retention and structural integrity, both of which are essential for the enduring viability of vineyards in the long term. Cover cropping presents numerous advantages, such as the enhancement of soil health, mitigation of erosion, and facilitation of nutrient cycling; however, it may also pose certain short-term risks, including competition for vital resources like water. In spite of the progress made in comprehending the advantages of cover crops in vineyard settings, the intricate dynamics between plant-microbe interactions and the leaf-level metabolic responses of grapevines at the leaf level to drought conditions remain unexplored. This study examines the impact of water availability and green cover (comprising perennial ryegrass and Trifolium repens) on grapevine photosynthetic and metabolism efficiency, positing that crop cover fosters a microhabitat that bolsters microbial communities and drought resilience. Through comprehensive examinations of gas exchange, isotopic analysis, metabolomics, transcriptomics, and soil metagenomics, this study clarifies the relationships among irrigation methodologies, photosynthesis, and soil health, ultimately aiding in the fortification of agricultural resilience in the face of climate change. Our investigation demonstrates that the adoption of cover crops yields unexpected immediate benefits in bolstering drought resilience for vineyards. Despite an observed increase in overall evapotranspiration during drought conditions, the use of cover crops facilitated carbon accumulation and enhanced osmolyte-acting metabolites (including sugars and sugar alcohols) and abscisic acid (ABA) concentrations, alongside a comprehensive molecular adaptation to drought stress. Moreover, cover cropping was shown to promote the expression of defense-related pathways, while vineyards devoid of cover crops exhibited minimal transcriptional responses; certain taxa exhibited responses contingent upon the treatment, with Tistrellales and Gaiellales being linked to crop cover under favorable conditions, whereas Rhizoctonia demonstrated a strong association with rhizospheric soil during drought conditions when crop cover was present. Our study is the first to show that cover cropping can boost cash crop resilience to drought through intricate plant-soil-microbe interactions, providing benefits from the outset.

In contrast to its close homolog PLANT UNCOUPLING MITOCHONDRIAL PROTEIN 1 (UCP1), which is an abundant carrier protein in the mitochondria, UCP2 localizes to the Golgi.

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.

When growing in search for light, plants can experience continuous or occasional shading by other plants. Plant proximity causes a decrease in the ratio of R to far-red light (low R:FR) due to the preferential absorbance of R light and reflection of FR light by photosynthetic tissues of neighboring plants. This signal is often perceived before actual shading causes a reduction in photosynthetically active radiation (low PAR). Here, we investigated how several Brassicaceae species from different habitats respond to low R:FR and low PAR in terms of elongation, photosynthesis, and photoacclimation. Shade-tolerant plants such as hairy bittercress (Cardamine hirsuta) displayed a good adaptation to low PAR but a poor or null response to low R:FR exposure. In contrast, shade-avoider species, such as Arabidopsis (Arabidopsis thaliana), showed a weak photosynthetic performance under low PAR but they strongly elongated when exposed to low R:FR. These responses could be genetically uncoupled. Most interestingly, exposure to low R:FR of shade-avoider (but not shade-tolerant) plants improved their photoacclimation to low PAR by triggering changes in photosynthesis-related gene expression, pigment accumulation, and chloroplast ultrastructure. These results indicate that low R:FR signaling unleashes molecular, metabolic, and developmental responses that allow shade-avoider plants (including most crops) to adjust their photosynthetic capacity in anticipation of eventual shading by nearby plants.

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.

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.

In plants, there is a complex interaction between carbon (C) and nitrogen (N) metabolism, and its coordination is fundamental for plant growth and development. Here, we studied the influence of thioredoxin (Trx) m on C and N partitioning using tobacco plants overexpressing Trx m from the chloroplast genome. The transgenic plants showed altered metabolism of C (lower leaf starch and soluble sugar accumulation) and N (with higher amounts of amino acids and soluble protein), which pointed to an activation of N metabolism at the expense of carbohydrates. To further delineate the effect of Trx m overexpression, metabolomic and enzymatic analyses were performed on these plants. These results showed an up-regulation of the glutamine synthetase-glutamate synthase pathway; specifically tobacco plants overexpressing Trx m displayed increased activity and stability of glutamine synthetase. Moreover, higher photorespiration and nitrate accumulation were observed in these plants relative to untransformed control plants, indicating that overexpression of Trx m favors the photorespiratory N cycle rather than primary nitrate assimilation. Taken together, our results reveal the importance of Trx m as a molecular mediator of N metabolism in plant chloroplasts.

Salinity has a negative impact on plant growth, with photosynthesis being downregulated partially due to osmotic effect and enhanced cellular oxidation. Redox signaling contributes to the plant response playing thioredoxins (TRXs) a central role. In this work we explore the potential contribution of Arabidopsis TRXo1 to the photosynthetic response under salinity analyzing Arabidopsis wild-type (WT) and two Attrxo1 mutant lines in their growth under short photoperiod and higher light intensity than previous reported works. Stomatal development and apertures and the antioxidant, hormonal and metabolic acclimation are also analyzed. In control conditions mutant plants displayed less and larger developed stomata and higher pore size which could underlie their higher stomatal conductance, without being affected in other photosynthetic parameters. Under salinity, all genotypes displayed a general decrease in photosynthesis and the oxidative status in the Attrxo1 mutant lines was altered, with higher levels of H2O2 and NO but also higher ascorbate/glutathione (ASC/GSH) redox states than WT plants. Finally, sugar changes and increases in abscisic acid (ABA) and NO may be involved in the observed higher stomatal response of the TRXo1-altered plants. Therefore, the lack of AtTRXo1 affected stomata development and opening and the mutants modulate their antioxidant, metabolic and hormonal responses to optimize their adaptation to salinity.

Geranylgeranyl diphosphate (GGPP) produced by GGPP synthase (GGPPS) serves as a precursor for many plastidial isoprenoids, including carotenoids. Phytoene synthase (PSY) converts GGPP into phytoene, the first committed intermediate of the carotenoid pathway. Here we used biochemical, molecular, and genetic tools to characterise the plastidial members of the GGPPS family in tomato (Solanum lycopersicum) and their interaction with PSY isoforms. The three tomato GGPPS isoforms found to localise in plastids (SlG1, 2 and 3) exhibit similar kinetic parameters. Gene expression analyses showed a preferential association of individual GGPPS and PSY isoforms when carotenoid biosynthesis was induced during root mycorrhization, seedling de-etiolation and fruit ripening. SlG2, but not SlG3, physically interacts with PSY proteins. By contrast, CRISPR-Cas9 mutants defective in SlG3 showed a stronger impact on carotenoid levels and derived metabolic, physiological and developmental phenotypes compared with those impaired in SlG2. Double mutants defective in both genes could not be rescued. Our work demonstrates that the bulk of GGPP production in tomato chloroplasts and chromoplasts relies on two cooperating GGPPS paralogues, unlike other plant species such as Arabidopsis thaliana, rice or pepper, which produce their essential plastidial isoprenoids using a single GGPPS isoform.

Plastids, the defining organelles of plant cells, undergo physiological and morphological changes to fulfill distinct biological functions. In particular, the differentiation of chloroplasts into chromoplasts results in an enhanced storage capacity for carotenoids with industrial and nutritional value such as beta-carotene (provitamin A). Here, we show that synthetically inducing a burst in the production of phytoene, the first committed intermediate of the carotenoid pathway, elicits an artificial chloroplast-to-chromoplast differentiation in leaves. Phytoene overproduction initially interferes with photosynthesis, acting as a metabolic threshold switch mechanism that weakens chloroplast identity. In a second stage, phytoene conversion into downstream carotenoids is required for the differentiation of chromoplasts, a process that involves a concurrent reprogramming of nuclear gene expression and plastid morphology for improved carotenoid storage. We hence demonstrate that loss of photosynthetic competence and enhanced production of carotenoids are not just consequences but requirements for chloroplasts to differentiate into chromoplasts.

Trabajo presentado al XV Meeting of Plant Molecular Biology, celebrado online del 26 al 27 de noviembre de 2020.

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.

Elevated greenhouse gases (GHGs) induce adverse conditions directly and indirectly, causing decreases in plant productivity. To deal with climate change effects, plants have developed various mechanisms including the fine-tuning of metabolism. Plant respiratory metabolism is highly flexible due to the presence of various alternative pathways. The mitochondrial alternative oxidase (AOX) respiratory pathway is responsive to these changes, and several lines of evidence suggest it plays a role in reducing excesses of reactive oxygen species (ROS) and reactive nitrogen species (RNS) while providing metabolic flexibility under stress. Here we discuss the importance of the AOX pathway in dealing with elevated carbon dioxide (CO2), nitrogen oxides (NOx), ozone (O-3), and the main abiotic stresses induced by climate change.

Climate change forecasts suggest temperature increases and lower rainfall rates, both of which challenge viticulture, particularly in semi-arid areas where water availability is critical. In this scenario, the use of the genetic variability in grapevine varieties reported around the world represents an important strategy for the selection of climate-resilient cultivars. In this work, physiological and metabolomics analyses were conducted to compare the water deficit stress (WDS) responses of red and white, local and widespread grapevines cultivars. Leaf gas exchange, water use efficiency (WUE) and water relation parameters were determined in plants under well-watered and WDS conditions alongside assessment of the levels of foliar primary metabolites using gas-chromatography coupled to mass-spectrometry. Results denote that red and white local cultivars displayed more adapted physiological performance under WDS as compared to the widely-distributed ones. Multivariate analyses and specific changes in leaf primary metabolites indicate genotype-specific responses of local cultivars as compared to widespread ones. Differences in ascorbate-related and shikimate/phenylpropanoid metabolism could explain the better physiological performance under WDS in red local as compared to widespread cultivars. On the other hand, coordinated changes in respiratory- and stress-related sugars and amino acids could underlie the better WUE under WDS in the white local cultivar. All these results suggest several metabolic targets that could be useful as metabolic markers or for metabolic engineering in grapevine breeding programs to improve drought tolerance.

Aluminum (Al) is an important factor that limits plant growth under acidic soil conditions. However, several plant species developed distinct mechanisms that limit the damage caused by high Al concentrations. In highbush blueberry (Vaccinium corymbosum), the Al resistance mechanisms are not fully understood. This study was designed to evaluate the effect of Al toxicity on roots and leaves of highbush blueberry genotypes with contrasting Al resistance [Star (Al-sensitive) and Camellia and Cargo (Al-resistant)] and identify the main molecular and physiological strategies underpinning adaptive Al stress responses in nutrient solution. After 48 h of Al treatment, the reduced form of ascorbate (ASC) was higher in roots, but unchanged in leaves of Cargo and Camellia genotypes compared to the control. We also observed decreased root exudation of oxalate in the Al-treated sensitive cultivar Star throughout the treatment period. However, in the resistant cultivar (Camellia), the exudation of oxalate increased 2.4- and 2.8-fold at 24 and 48 h, respectively. Al treatment differentially affected the enzyme activity and gene expression of the tricarboxylic acid (TCA) cycle enzymes. NAD-dependent malate dehydrogenase (NAD-MDH) expression in roots of cultivar Cargo was reduced at 24 h and increased at 48 h, whereas in leaves the expression was higher at 24 h and decreased at 48 h compared to the control. Citrate synthase (CS) activity in Al-resistant Cargo roots diminished at 24 h, increasing afterwards, without variation in the CS gene expression, compared with the initial time point (t = 0). In Al-resistant Camellia roots, the gene expression and the activity of CS decreased during Al exposure. NADP-dependent malate dehydrogenase (NADP-MDH) activity showed increased activity and gene expression at 24 h, in the leaves of cultivar Cargo, whereas in roots the gene expression decreased, but the activation state of NADP-MDH increased. The expression of genes encoding TCA cycle enzymes did not differ significantly in the Al-sensitive cultivar Star during Al exposure. In conclusion, the exudation of organic acid anions, particularly oxalate, plays an important role in Al resistance of highbush blueberry genotypes whilst elevated levels of ASC in roots, also contribute to the Al-resistance mechanisms exhibited by genotypes Camellia and Cargo.

Plant cells suffer alterations of their redox state and increase mitochondria] ROS generation during salinity. To avoid this, they activate several mitochondrial antioxidant and redox systems including the alternative oxidase (AOX), superoxide dismutase (SOD) and the ascorbate-glutathione (ASC-GSH) cycle components in a coordinated manner. The redox-sensitive mitochondrial thioredoxin (Trx) system may be responsible for this coordination through the redox regulation of target proteins. On top of this, metabolic perturbations induced by salinity may lead to alterations of the redox state of the Trx system. In order to explore the association between redox and metabolic changes occurring in mitochondria under saline conditions, we analyzed the salt-stress responses of mitochondrial antioxidant systems and metabolism in wild type (WT) and two knock-out (KO) AtTrxo1 lines. The activities of Mn-SOD and components of the ASC-GSH cycle were determined in isolated mitochondria, together with an evaluation of the AOX redox state, the oxidative stress, and catalase activity. Moreover, the in vivo activities of cytochrome (COX) and alternative mitochondrial respiratory pathways and primary metabolites profile were determined. Our results show that the lack of Trxo1 neither resulted in oxidative stress at the mitochondrial level nor in an upregulation of the antioxidant enzymes under salinity, although glutathione reductase (GR) maintained its high constitutive level as observed in control conditions. Moreover, the AOX was found invariably in its reduced monomeric state and displayed a reduction of its in vivo activity in all genotypes after the salt treatment, probably due to the mild severity of the treatment. Interestingly, trxo1 mutants displayed altered patterns in AOX isoforms and in the activities of the ASC-GSH cycle components and the electron partitioning to the AOX pathway indicating a reorganization of the different antioxidant systems. Furthermore, decreases on glucose and fructose levels in both trxo1 mutants coincided with an increased respiration through the COX pathway under control conditions. All these changes collaborate to maintain a low oxidative stress and the energy demand in both, control and salinity conditions and reflect the acclimation of all the genotypes to the applied stress.