Identification of Superoxide Dismutase (SOD) Isozymes in Plant Tissues.

Superoxide and hydrogen peroxide are reactive oxygen species (ROS) involved in the oxidation of multiple biological molecules and the signaling processes during plant growth and stress response. Thus, control of ROS is fundamental for cell survival and development, with superoxide dismutase (EC 1.15.1.1, SOD) being one of the main enzymes involved. Different isoforms of SOD catalyze the dismutation of superoxide (O2.-) to hydrogen peroxide (H2O2) and oxygen (O2), such as Mn-SODs, Cu,Zn-SODs, and Fe-SODs. Using non-denaturing polyacrylamide gel electrophoresis (PAGE) combined with a specific staining method for SOD activity, the protocol describes the identification of different SOD isozymes, based on their differential inhibition by KCN and H2O2, in different organs and plant species such as pea (Pisum sativum L.) leaves and pepper (Capsicum annuum L.) fruits.

The integration of TRX/GRX systems and phytohormonal signalling pathways in plant stress and development.

Plant acclimation to changing environmental conditions involves the interaction of different signalling molecules, including reactive oxygen species and hormones. Redox regulation exerted by thioredoxin (TRX) and glutaredoxin (GRX), two oxidoreductases, is emerging as a specific point of control mediating signal transduction pathways associated with plant growth and stress response. Phytohormones are messengers that coordinate plant cell activities to regulate growth, defence, and productivity, although their cross-talk with components of the redox system is less known. The present review focuses on our current knowledge of the interplay that occurs between TRX and GRX systems and phytohormonal signalling pathways in connection with the control of plant development and stress responses. Here, we consider the regulation that phytohormones exert on TRX and GRX systems, as well as the involvement of these redox proteins in the control of phytohormone-mediated signalling pathways.

Redox regulation, thioredoxins, and glutaredoxins in retrograde signalling and gene transcription.

Integration of reactive oxygen species (ROS)-mediated signal transduction pathways via redox sensors and the thiol-dependent signalling network is of increasing interest in cell biology for their implications in plant growth and productivity. Redox regulation is an important point of control in protein structure, interactions, cellular location, and function, with thioredoxins (TRXs) and glutaredoxins (GRXs) being key players in the maintenance of cellular redox homeostasis. The crosstalk between second messengers, ROS, thiol redox signalling, and redox homeostasis-related genes controls almost every aspect of plant development and stress response. We review the emerging roles of TRXs and GRXs in redox-regulated processes interacting with other cell signalling systems such as organellar retrograde communication and gene expression, especially in plants during their development and under stressful environments. This approach will cast light on the specific role of these proteins as redox signalling components, and their importance in different developmental processes during abiotic stress.

Thioredoxin TRXo1 is involved in ABA perception via PYR1 redox regulation.

Abscisic acid (ABA) plays a fundamental role in plant growth and development processes such as seed germination, stomatal response or adaptation to stress, amongst others. Increases in the endogenous ABA content is recognized by specific receptors of the PYR/PYL/RCAR family that are coupled to a phosphorylation cascade targeting transcription factors and ion channels. Just like other receptors of the family, nuclear receptor PYR1 binds ABA and inhibits the activity of type 2C phosphatases (PP2Cs), thus avoiding the phosphatase-exerted inhibition on SnRK2 kinases, positive regulators which phosphorylate targets and trigger ABA signalling. Thioredoxins (TRXs) are key components of cellular redox homeostasis that regulate specific target proteins through a thiol-disulfide exchange, playing an essential role in redox homeostasis, cell survival, and growth. In higher plants, TRXs have been found in almost all cellular compartments, although its presence and role in nucleus has been less studied. In this work, affinity chromatography, Dot-blot, co-immunoprecipitation, and bimolecular fluorescence complementation assays allowed us to identify PYR1 as a new TRXo1 target in the nucleus. Studies on recombinant HisAtPYR1 oxidation-reduction with wild type and site-specific mutagenized forms showed that the receptor underwent redox regulation involving changes in the oligomeric state in which Cys30 and Cys65 residues were implied. TRXo1 was able to reduce previously-oxidized inactive PYR1, thus recovering its capacity to inhibit HAB1 phosphatase. In vivo PYR1 oligomerization was dependent on the redox state, and a differential pattern was detected in KO and over-expressing Attrxo1 mutant plants grown in the presence of ABA compared to WT plants. Thus, our findings suggest the existence of a redox regulation of TRXo1 on PYR1 that may be relevant for ABA signalling and had not been described so far.

Autophagy Is Involved in the Viability of Overexpressing Thioredoxin o1 Tobacco BY-2 Cells under Oxidative Conditions.

Autophagy is an essential process for the degradation of non-useful components, although the mechanism involved in its regulation is less known in plants than in animal systems. Redox regulation of autophagy components is emerging as a possible key mechanism with thioredoxins (TRXs) proposed as involved candidates. In this work, using overexpressing PsTRXo1 tobacco cells (OEX), which present higher viability than non-overexpressing cells after H2O2 treatment, we examine the functional interaction of autophagy and PsTRXo1 in a collaborative response. OEX cells present higher gene expression of the ATG (Autophagy related) marker ATG4 and higher protein content of ATG4, ATG8, and lipidated ATG8 as well as higher ATG4 activity than control cells, supporting the involvement of autophagy in their response to H2O2. In this oxidative situation, autophagy occurs in OEX cells as is evident from an accumulation of autolysosomes and ATG8 immunolocalization when the E-64d autophagy inhibitor is used. Interestingly, cell viability decreases in the presence of the inhibitor, pointing to autophagy as being involved in cell survival. The in vitro interaction of ATG4 and PsTRXo1 proteins is confirmed by dot-blot and co-immunoprecipitation assays as well as the redox regulation of ATG4 activity by PsTRXo1. These findings extend the role of TRXs in mediating the redox regulation of the autophagy process in plant cells.

Reactive oxygen species homeostasis and circadian rhythms in plants.

Elucidation of the molecular mechanisms by which plants sense and respond to environmental stimuli that influence their growth and yield is a prerequisite for understanding the adaptation of plants to climate change. Plants are sessile organisms and one important factor for their successful acclimation is the temporal coordination of the 24 h daily cycles and the stress response. The crosstalk between second messengers, such as Ca2+, reactive oxygen species (ROS), and hormones is a fundamental aspect in plant adaptation and survival under environmental stresses. In this sense, the circadian clock, in conjunction with Ca2+- and hormone-signalling pathways, appears to act as an important mechanism controlling plant adaptation to stress. The relationship between the circadian clock and ROS-generating and ROS-scavenging mechanisms is still not fully understood, especially at the post-transcriptional level and in stress situations in which ROS levels increase and changes in cell redox state occur. In this review, we summarize the information regarding the relationship between the circadian clock and the ROS homeostasis network. We pay special attention not only to the transcriptional regulation of ROS-generating and ROS-scavenging enzymes, but also to the few studies that have been performed at the biochemical level and those conducted under stress conditions.

Decreased Levels of Thioredoxin o1 Influences Stomatal Development and Aperture but Not Photosynthesis under Non-Stress and Saline Conditions.

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.

Thioredoxin Network in Plant Mitochondria: Cysteine S-Posttranslational Modifications and Stress Conditions.

Plants are sessile organisms presenting different adaptation mechanisms that allow their survival under adverse situations. Among them, reactive oxygen and nitrogen species (ROS, RNS) and H2S are emerging as components not only of cell development and differentiation but of signaling pathways involved in the response to both biotic and abiotic attacks. The study of the posttranslational modifications (PTMs) of proteins produced by those signaling molecules is revealing a modulation on specific targets that are involved in many metabolic pathways in the different cell compartments. These modifications are able to translate the imbalance of the redox state caused by exposure to the stress situation in a cascade of responses that finally allow the plant to cope with the adverse condition. In this review we give a generalized vision of the production of ROS, RNS, and H2S in plant mitochondria. We focus on how the principal mitochondrial processes mainly the electron transport chain, the tricarboxylic acid cycle and photorespiration are affected by PTMs on cysteine residues that are produced by the previously mentioned signaling molecules in the respiratory organelle. These PTMs include S-oxidation, S-glutathionylation, S-nitrosation, and persulfidation under normal and stress conditions. We pay special attention to the mitochondrial Thioredoxin/Peroxiredoxin system in terms of its oxidation-reduction posttranslational targets and its response to environmental stress.

Is Autophagy Involved in Pepper Fruit Ripening?

Autophagy is a universal self-degradation process involved in the removal and recycling of cellular constituents and organelles; however, little is known about its possible role in fruit ripening, in which the oxidation of lipids and proteins and changes in the metabolism of different cellular organelles occur. In this work, we analyzed several markers of autophagy in two critical maturation stages of pepper (Capsicum annuum L.) fruits where variations due to ripening become clearly visible. Using two commercial varieties that ripen to yellow and red fruits respectively, we studied changes in the gene expression and protein content of several autophagy (ATG) components, ATG4 activity, as well as the autophagy receptor NBR1 and the proteases LON1 and LON2. Additionally, the presence of intravacuolar vesicles was analyzed by electron microscopy. Altogether, our data reveal that autophagy plays a role in the metabolic changes which occur during ripening in the two studied varieties, suggesting that this process may be critical to acquiring final optimal quality of pepper fruits.

Lack of mitochondrial thioredoxin o1 is compensated by antioxidant components under salinity in Arabidopsis thaliana plants.

In a changing environment, plants are able to acclimate to new conditions by regulating their metabolism through the antioxidant and redox systems involved in the stress response. Here, we studied a mitochondrial thioredoxin in wild-type (WT) Arabidopis thaliana and two Attrxo1 mutant lines grown in the absence or presence of 100 mM NaCl. Compared to WT plants, no evident phenotype was observed in the mutant plants under control condition, although they had higher number of stomata, loss of water, nitric oxide and carbonyl protein contents as well as higher activity of superoxide dismutase (SOD) and catalase enzymes than WT plants. Under salinity, the mutants presented lower water loss and higher stomatal closure, H2 O2 and lipid peroxidation levels accompanied by higher enzymatic activity of catalase and the different SOD isoenzymes compared to WT plants. These inductions may collaborate in the maintenance of plant integrity and growth observed under saline conditions, possibly as a way to compensate the lack of TRXo1. We discuss the potential of TRXo1 to influence the development of the whole plant under saline conditions, which have great value for the agronomy of plants growing under unfavorable environment.

An Extract from the Plant Deschampsia antarctica Protects Fibroblasts from Senescence Induced by Hydrogen Peroxide.

The Antarctic plant Deschampsia antarctica (DA) is able to survive in extreme conditions thanks to its special mechanism of protection against environmental aggressions. In this work, we investigated whether an aqueous extract of the plant (EDA) retains some of its defensive properties and is able to protect our skin against common external oxidants. We evaluated EDA over young human fibroblasts and exposed to H2O2, and we measured cell proliferation, viability, and senescence-associated ß-galactosidase (SA-ß-Gal). We also tested the expression of several senescence-associated proteins including sirtuin1, lamin A/C, the replicative protein PCNA, and the redox protein thioredoxin 2. We found that EDA promoted per se cell proliferation and viability and increased the expression of anti-senescence-related markers. Then, we selected a dose of H2O2 as an inductor of senescence in human fibroblasts, and we found that an EDA treatment 24 h prior H2O2 exposure increased fibroblast proliferation. EDA significantly inhibited the increase in SA-ß-Gal levels induced by H2O2 and promoted the expression of sirtuin 1 and lamin A/C proteins. Altogether, these results suggest that EDA protects human fibroblasts from cellular senescence induced by H2O2, pointing to this compound as a potential therapeutic agent to treat or prevent skin senescence.

Thioredoxin (Trxo1) interacts with proliferating cell nuclear antigen (PCNA) and its overexpression affects the growth of tobacco cell culture.

Thioredoxins (Trxs), key components of cellular redox regulation, act by controlling the redox status of many target proteins, and have been shown to play an essential role in cell survival and growth. The presence of a Trx system in the nucleus has received little attention in plants, and the nuclear targets of plant Trxs have not been conclusively identified. Thus, very little is known about the function of Trxs in this cellular compartment. Previously, we studied the intracellular localization of PsTrxo1 and confirmed its presence in mitochondria and, interestingly, in the nucleus under standard growth conditions. In investigating the nuclear function of PsTrxo1 we identified proliferating cellular nuclear antigen (PCNA) as a PsTrxo1 target by means of affinity chromatography techniques using purified nuclei from pea leaves. Such protein-protein interaction was corroborated by dot-blot and bimolecular fluorescence complementation (BiFC) assays, which showed that both proteins interact in the nucleus. Moreover, PsTrxo1 showed disulfide reductase activity on previously oxidized recombinant PCNA protein. In parallel, we studied the effects of PsTrxo1 overexpression on Tobacco Bright Yellow-2 (TBY-2) cell cultures. Microscopy and flow-cytometry analysis showed that PsTrxo1 overexpression increases the rate of cell proliferation in the transformed lines, with a higher percentage of the S phase of the cell cycle at the beginning of the cell culture (days 1 and 3) and at the G2/M phase after longer times of culture (day 9), coinciding with an upregulation of PCNA protein. Furthermore, in PsTrxo1 overexpressed cells there is a decrease in the total cellular glutathione content but maintained nuclear GSH accumulation, especially at the end of the culture, which is accompanied by a higher mitotic index, unlike non-overexpressing cells. These results suggest that Trxo1 is involved in the cell cycle progression of TBY-2 cultures, possibly through its link with cellular PCNA and glutathione.

Mitochondrial AtTrxo1 is transcriptionally regulated by AtbZIP9 and AtAZF2 and affects seed germination under saline conditions.

Mitochondrial thioredoxin-o (AtTrxo1) was characterized and its expression examined in different organs of Arabidopsis thaliana. AtTrxo1 transcript levels were particularly high in dry seeds and cotyledons where they reached a maximum 36 h after imbibition with water, coinciding with 50% germination. Expression was lower in seeds germinating in 100 mM NaCl. To gain insight into the transcriptional regulation of the AtTrxo1 gene, a phylogenomic analysis was coupled with the screening of an arrayed library of Arabidopsis transcription factors in yeast. The basic leucine zipper AtbZIP9 and the zinc finger protein AZF2 were identified as putative transcriptional regulators. Transcript regulation of AtbZIP9 and AtAFZ2 during germination was compatible with the proposed role in transcriptional regulation of AtTrxo1. Transient over-expression of AtbZIP9 and AtAZF2 in Nicotiana benthamiana leaves demonstrated an activation effect of AtbZIP9 and a repressor effect of AtAZF2 on AtTrxo1 promoter-driven reporter expression. Although moderate concentrations of salt delayed germination in Arabidopsis wild-type seeds, those of two different AtTrxo1 knock-out mutants germinated faster and accumulated higher H2O2 levels than the wild-type. All these data indicate that AtTrxo1 has a role in redox homeostasis during seed germination under salt conditions.

Glutathionylation of Pea Chloroplast 2-Cys Prx and Mitochondrial Prx IIF Affects Their Structure and Peroxidase Activity and Sulfiredoxin Deglutathionylates Only the 2-Cys Prx.

Together with thioredoxins (Trxs), plant peroxiredoxins (Prxs), and sulfiredoxins (Srxs) are involved in antioxidant defense and redox signaling, while their regulation by post-translational modifications (PTMs) is increasingly regarded as a key component for the transduction of the bioactivity of reactive oxygen and nitrogen species. Among these PTMs, S-glutathionylation is considered a protective mechanism against overoxidation, it also modulates protein activity and allows signaling. This study explores the glutathionylation of recombinant chloroplastic 2-Cys Prx and mitochondrial Prx IIF from Pisum sativum. Glutathionylation of the decameric form of 2-Cys Prx produced a change in the elution volume after FPLC chromatography and converted it to its dimeric glutathionylated form, while Prx IIF in its reduced dimeric form was glutathionylated without changing its oligomeric state. Mass spectrometry demonstrated that oxidized glutathione (GSSG) can glutathionylate resolving cysteine (Cys174), but not the peroxidatic equivalent (Cys52), in 2-Cys Prx. In contrast, GSSG was able to glutathionylate both peroxidatic (Cys59) and resolving (Cys84) cysteine in Prx IIF. Glutathionylation was seen to be dependent on the GSH/GSSG ratio, although the exact effect on the 2-Cys Prx and Prx IIF proteins differed. However, the glutathionylation provoked a similar decrease in the peroxidase activity of both peroxiredoxins. Despite growing evidence of the importance of post-translational modifications, little is known about the enzymatic systems that specifically regulate the reversal of this modification. In the present work, sulfiredoxin from P. sativum was seen to be able to deglutathionylate pea 2-Cys Prx but not pea Prx IIF. Redox changes during plant development and the response to stress influence glutathionylation/deglutathionylation processes, which may represent an important event through the modulation of peroxiredoxin and sulfiredoxin proteins.

CP-154,526 Modifies CREB Phosphorylation and Thioredoxin-1 Expression in the Dentate Gyrus following Morphine-Induced Conditioned Place Preference.

Corticotropin-releasing factor (CRF) acts as neuro-regulator of the behavioral and emotional integration of environmental and endogenous stimuli associated with drug dependence. Thioredoxin-1 (Trx-1) is a functional protein controlling the redox status of several proteins, which is involved in addictive processes. In the present study, we have evaluated the role of CRF1 receptor (CRF1R) in the rewarding properties of morphine by using the conditioned place preference (CPP) paradigm. We also investigate the effects of the CRF1R antagonist, CP-154,526, on the morphine CPP-induced activation of CRF neurons, CREB phosphorylation and Trx expression in paraventricular nucleus (PVN) and dentate gyrus (DG) of the mice brain. CP-154,526 abolished the acquisition of morphine CPP and the increase of CRF/pCREB positive neurons in PVN. Moreover, this CRF1R antagonist prevented morphine-induced CRF-immunoreactive fibers in DG, as well as the increase in pCREB expression in both the PVN and DG. In addition, morphine exposure induced an increase in Trx-1 expression in DG without any alterations in PVN. We also observed that the majority of pCREB positive neurons in DG co-expressed Trx-1, suggesting that Trx-1 could activate CREB in the DG, a brain region involved in memory consolidation. Altogether, these results support the idea that CRF1R antagonist blocked Trx-1 expression and pCREB/Trx-1 co-localization, indicating a critical role of CRF, through CRF1R, in molecular changes involved in morphine associated behaviors.

Experimental evidences of the NO action on a recombinant PrxII F from pea plant and its effect preventing the citrate synthase aggregation.

S-nitrosylation is emerging as a key post-translational protein modification for the transduction of NO as a signaling molecule in plants. This data article supports the research article entitled "Functional and structural changes in plant mitochondrial PrxII F caused by NO" [1]. To identify the Cys residues of the recombinant PrxII F modified after the treatment with S-nitrosylating agents we performed the LC ESI-QTOF tandem MS and MALDI peptide mass fingerprinting analysis. Change in A 650 nm was monitored to estimate the thermal aggregation of citrate synthase in the presence S-nitrosylated PrxII F. The effect of the temperature on the oligomerization pattern and aggregation of PrxII F was analysed by SDS-PAGE and changes in absorbance at 650 nm, respectively.

Physiology of pepper fruit and the metabolism of antioxidants: chloroplasts, mitochondria and peroxisomes.

BACKGROUND AND AIMS: Pepper (Capsicum annuum) contains high levels of antioxidants, such as vitamins A and C and flavonoids. However, information on the role of these beneficial compounds in the physiology of pepper fruit remains scarce. Recent studies have shown that antioxidants in ripe pepper fruit play a key role in responses to temperature changes, and the redox state at the time of harvest affects the nutritional value for human consumption. In this paper, the role of antioxidant metabolism of pepper fruit during ripening and in the response to low temperature is addressed, paying particular attention to ascorbate, NADPH and the superoxide dismutase enzymatic system. The participation of chloroplasts, mitochondria and peroxisomes in the ripening process is also investigated. SCOPE AND RESULTS: Important changes occur at a subcellular level during ripening of pepper fruit. Chloroplasts turn into chromoplasts, with drastic conversion of their metabolism, and the role of the ascorbate-glutathione cycle is essential. In mitochondria from red fruits, higher ascorbate peroxidase (APX) and Mn-SOD activities are involved in avoiding the accumulation of reactive oxygen species in these organelles during ripening. Peroxisomes, whose antioxidant capacity at fruit ripening is substantially affected, display an atypical metabolic pattern during this physiological stage. In spite of these differences observed in the antioxidative metabolism of mitochondria and peroxisomes, proteomic analysis of these organelles, carried out by 2-D electrophoresis and MALDI-TOF/TOF and provided here for the first time, reveals no changes between the antioxidant metabolism from immature (green) and ripe (red) fruits. CONCLUSIONS: Taken together, the results show that investigation of molecular and enzymatic antioxidants from cell compartments, especially chloroplasts, mitochondria and peroxisomes, is a useful tool to study the physiology of pepper fruit, particularly in the context of expanding their shelf-life after harvest and in maintaining their nutritional value.

Over-expression of Trxo1 increases the viability of tobacco BY-2 cells under H2O2 treatment.

BACKGROUND AND AIMS: Reactive oxygen species (ROS), especially hydrogen peroxide, play a critical role in the regulation of plant development and in the induction of plant defence responses during stress adaptation, as well as in plant cell death. The antioxidant system is responsible for controlling ROS levels in these processes but redox homeostasis is also a key factor in plant cell metabolism under normal and stress situations. Thioredoxins (Trxs) are ubiquitous small proteins found in different cell compartments, including mitochondria and nuclei (Trxo1), and are involved in the regulation of target proteins through reduction of disulphide bonds, although their role under oxidative stress has been less well studied. This study describes over-expression of a Trxo1 for the first time, using a cell-culture model subjected to an oxidative treatment provoked by H2O2. METHODS: Control and over-expressing PsTrxo1 tobacco (Nicotiana tabacum) BY-2 cells were treated with 35 mm H2O2 and the effects were analysed by studying the growth dynamics of the cultures together with oxidative stress parameters, as well as several components of the antioxidant systems involved in the metabolism of H2O2. Analysis of different hallmarks of programmed cell death was also carried out. KEY RESULTS: Over-expression of PsTrxo1 caused significant differences in the response of TBY-2 cells to high concentrations of H2O2, namely higher and maintained viability in over-expressing cells, whilst the control line presented a severe decrease in viability and marked indications of oxidative stress, with generalized cell death after 3 d of treatment. In over-expressing cells, an increase in catalase activity, decreases in H2O2 and nitric oxide contents and maintenance of the glutathione redox state were observed. CONCLUSIONS: A decreased content of endogenous H2O2 may be responsible in part for the delayed cell death found in over-expressing cells, in which changes in oxidative parameters and antioxidants were less extended after the oxidative treatment. It is concluded that PsTrxo1 transformation protects TBY-2 cells from exogenous H2O2, thus increasing their viability via a process in which not only antioxidants but also Trxo1 seem to be involved.

Proteomic identification of mitochondrial carbonylated proteins in two maturation stages of pepper fruits.

Pepper fruits in green and red maturation stages were selected to study the protein pattern modified by oxidation measuring carbonylated proteins in isolated mitochondria, together with the accumulation of superoxide radical and hydrogen peroxide in the fruits. MALDI-TOF/TOF analysis identified as carbonylated proteins in both green and red fruits, formate dehydrogenase, NAD-dependent isocitrate dehydrogenase, porin, and defensin, pointing to a common regulation by carbonylation of these proteins independently of the maturation stage. However, other proteins such as glycine dehydrogenase P subunit and phosphate transporter were identified as targets of carbonylation only in green fruits, whereas aconitase, ATPase ß subunit, prohibitin, orfB protein, and cytochrome C oxidase, were identified only in red fruits. In general, the results suggest that carbonylation of mitochondrial proteins is a PTM that drives the complex ripening process, probably establishing the accumulation and functionality of some mitochondrial proteins in the nonclimacteric pepper fruit.