Persulfidome of Sweet Pepper Fruits during Ripening: The Case Study of Leucine Aminopeptidase That Is Positively Modulated by H2S

Protein persulfidation is a thiol-based oxidative posttranslational modification (oxiPTM) that involves the modification of susceptible cysteine thiol groups present in peptides and proteins through hydrogen sulfide (H2S), thus affecting their function. Using sweet pepper (Capsicum annuum L.) fruits as a model material at different stages of ripening (immature green and ripe red), endogenous persulfidated proteins (persulfidome) were labeled using the dimedone switch method and identified using liquid chromatography and mass spectrometry analysis (LC-MS/MS). A total of 891 persulfidated proteins were found in pepper fruits, either immature green or ripe red. Among these, 370 proteins were exclusively present in green pepper, 237 proteins were exclusively present in red pepper, and 284 proteins were shared between both stages of ripening. A comparative analysis of the pepper persulfidome with that described in Arabidopsis leaves allowed the identification of 25% of common proteins. Among these proteins, glutathione reductase (GR) and leucine aminopeptidase (LAP) were selected to evaluate the effect of persulfidation using an in vitro approach. GR activity was unaffected, whereas LAP activity increased by 3-fold after persulfidation. Furthermore, this effect was reverted through treatment with dithiothreitol (DTT). To our knowledge, this is the first persulfidome described in fruits, which opens new avenues to study H2S metabolism. Additionally, the results obtained lead us to hypothesize that LAP could be involved in glutathione (GSH) recycling in pepper fruits.


Introduction
Hydrogen sulfide (H 2 S) is a gasotransmitter generated in different subcellular compartments of plant and animal cells which has signaling functions under physiological and adverse environmental conditions [1][2][3][4][5][6][7][8][9].From a chemical point of view, H 2 S is a weak acid that in an aqueous solution can be dissociated into hydrosulfide (HS − ) and sulfide (S 2− ) anions.It is estimated that under physiological pH conditions, around 20% of H 2 S occurs in a non-dissociated form, and the rest is dissociated into HS − and H + , whereas the amount of S 2− is very low at physiological pH [10].H 2 S exerts its regulatory function throughout the generation of persulfides (RSSH) which involves their interaction with either oxidized thiol derivatives such as disulfides (RSSR ′ ) and sulfenic acid (RSOH) or oxidized sulfur derivatives such as polysulfides (HSnS − n ≥ 1) [11][12][13][14][15]. Thus, polysulfides can also react with glutathione disulfide (GSSG) to generate glutathione persulfide California-type sweet pepper (Capsicum annuum L., cv.Melchor) fruits were harvested between January and February (2021) from plants grown in plastic-covered greenhouses (Syngenta Seeds, Ltd., El Ejido, Almería, Spain).Fruits were selected and harvested without any external apparent injury at two developmental stages: green immature (G) and red ripe (R).

Dimedone Switch Method and Proteomics
Endogenous persulfidated proteins were labelled using the dimedone switch method previously described [38] with some modifications, and were identified using mass spectrometry analysis.Briefly, green and red pepper fruit samples were ground to a fine powder in liquid nitrogen, resuspended in cold lysis buffer (PBS, pH 7.4, 2% (w/v) SDS, and 1 mM EDTA), together with 5 mM 4-chloro-7-nitrobenzofurazan (Cl-NBF, Sigma-Aldrich, Madrid, Spain) and 1% (v/v) protease inhibitor, incubated at 37 • C for 30 min, and protected from the light.Then, a methanol/chloroform precipitation was performed, and protein-enriched pellets were washed with cold methanol and dried afterward.The dried proteins were dissolved into 50 mM PBS with 2% (w/v) SDS, incubated with 200 µM DCP-Bio1 (a biotinylated form of dimedone) (Kerafast, Inc., Vizcaya, Spain) at 37 • C for 1 h, precipitated, and finally dissolved in 1 × PBS with 2% (w/v) SDS and 1% protease inhibitor.Proteins were incubated with Sera-Mag Magnetic Streptavidin beads (Cytiva S.L., Hospitalet de Llobregat, Spain) at 4 • C overnight with agitation.The microtubes containing the magnetic beads were placed in front of a magnet and the beads were separated from the supernatant.Then, they were washed with PBS supplemented with 0.001% (v/v) Tween-20 only the first time and after with PBS 1× several times.After washing, the beads were recovered again and incubated with 2.25 M ammonium hydroxide overnight at 4 • C with agitation.The final supernatants containing the persulfidated proteins labelled with DCP-Bio1 were transferred to new microtubes, and the beads were discarded.
For mass spectrometry analysis, samples were digested with trypsin following a standard protocol by using reduction conditions with dithiothreitol (DTT) and the carbamidomethylation of the reduced cysteines with iodoacetamide.In total, 1 µg of proteins from both green and red pepper fruits was analyzed using liquid chromatography-mass spectrometry, using a gradient of 120 min in length.A 75 µm × 50 cm C18 column was used, at a flow rate of 250 nL/min, for the chromatography, and the analysis was carried out in a Thermo Orbitrap Exploris OE240 mass spectrometer working in DDA mode.The MS1 and MS2 spectra were used to launch searches against a Capsicum annuum L. database, downloaded from UniProtKB, consisting of 38,701 entries.Peaks v7 was used as the search engine, and only proteins identified with at least one unique peptide were shown, using the 1% value for FDR (False Discovery Rate) as a criterion.The following modifications (variables) of the peptides including oxidation were selected as possible (M): 15.99 Da; Carbamidomethylation: 57.02 Da; NBF: 163.00 Da (in Lys, Arg, and Cys); hydrolyzed DCP-Bio1: 168.08 Da (in Cys); and DCP-Bio1: 394.16 Da (in Cys).

Functional Analysis
The sets of proteins identified as potential persulfidation targets were used to perform different functional analyses.First, a functional enrichment analysis was performed in GO (Gene Ontology) terms, using the web tool g:Profiler (v2023) [36].Then, the Python package GO-Figure!(v3) [37] was used to generate a more summarized and comprehensive version of the results.
To compare the persulfidated proteins identified in pepper fruits and the persulfidome previously described in Arabidopsis thaliana [17], we used the BLASTP tool (v2.15.0) [42] using the Arabidopsis proteome (UniProt Proteome ID: UP000006548; accessed on 20 March 2024) as a database.

Fruit Extracts and Leucine Aminopeptidase (LAP) and Glutathione Reductase (GR) Activities
Fruit samples stored at −80 • C were homogenized in liquid nitrogen using an IKA A11 Extraction Mill.The pulverized plant material was weighed and 50 mM Tris-HCl buffer, pH 7.5, containing 0.1 mM EDTA, 1 mM MgCl 2 , 10% (v/v) glycerol, and 0.1% (v/v) Triton X-100, was added.The ratio of plant material to used buffer was 1:1.
For the spectrophotometric leucine aminopeptidase (LAP; EC 3.4.11.1) activity assay, L-leucine-p-nitroanilide (Leu-p-NA) was used as a substrate which, by the action of the LAP activity, generated L-Leu plus p-nitroaniline whose absorbance was measured at 410 nm.Briefly, the reaction mixture contained 50 mM K-phosphate buffer pH 7.5, 10 mM β-mercaptoethanol, and 1 mM Leu-p-NA plus fruit samples, and was incubated at 39 • C for 30 min.The reaction was stopped with 30% (v/v) acetic acid, centrifuged at 10,000× g for 10 min, and then the supernatant was measured a 410 nm [43].One unit of LAP activity is defined as the change in one unit of absorbance at 39 • C which corresponds to the production of 1 nmol of p-nitroaniline per min, using an extinction coefficient ε 410 of 10 4 M −1 cm −1 [44].For comparisons of the LAP activity among treatments, Student's t-test was used with a statistical significance of 5% (p ≤ 0.05).
Protein concentration in fruit extracts was determined using the Bio-Rad protein assay with bovine serum albumin as standard.

In Vitro Treatment of Pepper Samples
To investigate the potential modulation of the LAP activity in pepper fruits, two different in vitro assays were performed.(1) Samples from green pepper fruits were preincubated at 25 • C for 1 h at 25 • C and darkness with different concentrations of sodium hydrosulfide (NaHS), an H 2 S donor.Afterward, samples were further incubated for 1 h either in the absence or the presence of 50 mM dithiothreitol (DTT), as reductant agent.
(2) Samples were pre-incubated with either 5 mM NaHS or 5 mM S-nitrosoglutathione (GSNO), a nitric oxide (NO) donor.Then, further incubations for 1 h were carried out in the presence of 5 mM GSNO or 5 mM NaHS, respectively.On the other hand, in the analysis of the simultaneous effect of H 2 S and NO, the samples were incubated for 30 min at 25 • C with the first donor, and then the second donor was added to complete together the full hour of incubation.In all cases, the solutions were freshly prepared before use.Then, either the GR or LAP activity was determined as described above.

Results
Three biological replicates were analyzed for each ripening stage (unripe green and ripe red).Considering the biological replicates independently, we identified a total of 2143 unique proteins that could undergo persulfidation during sweet pepper fruit ripening.However, for subsequent analyses, we considered common persulfidated proteins in the replicates for each ripening stage.Accordingly, the number of endogenously persulfidated proteins identified in sweet pepper fruits was 891.Thus, out of them, 370 were exclusively present in green pepper and 237 were exclusively present in red pepper, whereas 284 proteins were common to both ripening stages (Figure 1a).Supplementary Tables S1 and S2 shows the list of all the proteins identified as likely to be endogenously persulfidated in immature green and ripe red pepper fruits.
A comparative analysis of the pepper persulfidome against that described for Arabidopsis leaves under physiological conditions [17] was achieved with the help of the BLASTP tool.This allowed us to find that 25% of Arabidopsis shared proteins with pepper fruits, although the restriction limits, established by the BlastP engine during the comparative analysis, could only evaluate 774 out of the 891 proteins identified in pepper fruits due to the redundancy in the identifiers between both species.Thus, comparing the persulfidated proteins reported in Arabidopsis leaves with those found in pepper fruits, 402 were shared proteins (Figure 1b) (see Supplementary Table S3).
To elucidate the potential functions of persulfidation during pepper fruit ripening, we performed a functional enrichment analysis based on Gene Ontology (GO) terms (p < 0.05) (Figure 2).In our analysis of the persulfidated proteins uniquely identified in green and red bell pepper fruits, distinct patterns emerged regarding their involvement in various biological processes.For green pepper fruits, the most enriched terms corresponded to essential processes such as the biosynthesis of nitrogen-containing organic compounds, photosynthesis, as well as protein transport and vesicle trafficking between the endoplasmic reticulum and the Golgi apparatus.Furthermore, the analysis revealed the predominant distribution of these proteins within the cytoplasm and chloroplasts.Conversely, in red pepper fruits, persulfidated proteins exhibited a different spectrum of biological functions.The protein groups with higher enriched values were primarily associated with the metabolism of purine-based molecules, encompassing ribonucleotides and deoxyribonucleotides, the biosynthesis of amide compounds, amino acids, and proteins, as well as participation in the cellular response to oxidative stress.This indicates a distinct metabolic and regulatory landscape in red pepper fruits, underscoring the diverse roles of persulfidation in modulating cellular processes at different stages of fruit development and ripening.To elucidate the potential functions of persulfidation during pepper fruit ripening, we performed a functional enrichment analysis based on Gene Ontology (GO) terms (p < 0.05) (Figure 2).In our analysis of the persulfidated proteins uniquely identified in green and red bell pepper fruits, distinct patterns emerged regarding their involvement in various biological processes.For green pepper fruits, the most enriched terms corresponded to essential processes such as the biosynthesis of nitrogen-containing organic compounds, photosynthesis, as well as protein transport and vesicle trafficking between the endoplasmic reticulum and the Golgi apparatus.Furthermore, the analysis revealed the predominant distribution of these proteins within the cytoplasm and chloroplasts.Conversely, in red pepper fruits, persulfidated proteins exhibited a different spectrum of biological functions.The protein groups with higher enriched values were primarily associated with the metabolism of purine-based molecules, encompassing ribonucleotides To provide a more comprehensive physiological context, we categorized the identified proteins using the Mapman nomenclature.This classification revealed that the persulfidated proteins span 29 categories, each covering various plant metabolic aspects.Notably, over 26% of the proteins (246) were associated with protein metabolism, encompassing processes such as biosynthesis (89), homeostasis (114), modifications (28), and translocations (18).Additionally, significant representation was observed in primary metabolic pathways, including photosynthesis (74), amino acid (56), carbohydrate (50), and lipid (41) metabolism.While exhibiting fewer proteins, categories such as redox homeostasis (35) and secondary metabolism ( 14) were noteworthy, constituting 3.7% and 1.8% of the persulfidated proteins, respectively.Figure 3 provides a schematic summary of these findings.As shown in the figure, the majority of the persulfidated proteins in immature green fruits are especially, as expected, grouped in the photosynthetic pathways.On the contrary, in red fruits, the number of proteins that are related to carbohydrate metabolism is higher, possibly explaining the higher sweetness of these fruits compared to the green ones.Also, the catabolism of proteins seems to be enhanced in ripe fruits as a consequence of degenerative processes that are associated with the latest stages of fruit development.Supplementary Table S4 displays the list of the proteins shown in the MapMan categories of Figure 3.
Antioxidants 2024, 13, x FOR PEER REVIEW 6 of 18 and deoxyribonucleotides, the biosynthesis of amide compounds, amino acids, and proteins, as well as participation in the cellular response to oxidative stress.This indicates a distinct metabolic and regulatory landscape in red pepper fruits, underscoring the diverse roles of persulfidation in modulating cellular processes at different stages of fruit development and ripening.number of proteins that are related to carbohydrate metabolism is higher, possibly explaining the higher sweetness of these fruits compared to the green ones.Also, the catabolism of proteins seems to be enhanced in ripe fruits as a consequence of degenerative processes that are associated with the latest stages of fruit development.Supplementary Table S4 displays the list of the proteins shown in the MapMan categories of Figure 3. Notwithstanding the persulfidome provides us with a list of proteins that are susceptible to be persulfidated, it is important to evaluate the effect of this PTM on the function of the identified protein.Thus, as part of the characterization of the persulfidome and to corroborate the effect of the persulfidation events in pepper fruit metabolism, two enzymes were selected: glutathione reductase (GR), an enzyme of the ascorbate- Notwithstanding the persulfidome provides us with a list of proteins that are susceptible to be persulfidated, it is important to evaluate the effect of this PTM on the function of the identified protein.Thus, as part of the characterization of the persulfidome and to corroborate the effect of the persulfidation events in pepper fruit metabolism, two enzymes were selected: glutathione reductase (GR), an enzyme of the ascorbate-glutathione cycle that depends on NADPH to regenerate reduced glutathione (GSH), and leucine aminopeptidase (LAP), an enzyme that has recently been identified in pepper fruits to be modulated by nitric oxide (NO), another signal molecule that mediates different PTMs such as nitration and S-nitrosation [31].Both enzymes were detected in green and red fruits and were assigned to chloroplasts, according to their deduced amino acid sequences.This analysis of the redox and the peptide/amino acid metabolisms from a persulfidation perspective may shed light on a potential link between both processes, not explored thus far, thus contributing to envisage glutathione as a relevant metabolite in the physiology of pepper fruits.In fact, as well as being involved in the antioxidant metabolism, glutathione could be persulfidated, but it could also be broken down by LAP activity.
Figure 4 shows that GR activity preincubated with different concentrations of NaHS, as an H 2 S donor, had no effect at any of the concentrations tested.
explored thus far, thus contributing to envisage glutathione as a relevant metabolite in the physiology of pepper fruits.In fact, as well as being involved in the antioxidant metabolism, glutathione could be persulfidated, but it could also be broken down by LAP activity.
Figure 4 shows that GR activity preincubated with different concentrations of NaHS, as an H2S donor, had no effect at any of the concentrations tested.On the other hand, Figure 5a displays the in vitro analysis of pepper LAP activity in similar conditions to those used in Figure 4.In this case, it is observed that the pretreatment of the pepper extracts with 1 mM NaHS provoked a slight increase in LAP activity, but this effect increased at higher concentrations (white bars), with LAP activity being 2.5 times higher with 5 mM NaHS.Considering that persulfidation is suggested to be a reversible process, control (C)-and NaHS-treated pepper samples were further incubated with dithiothreitol (DTT).Thus, in Figure 5b, it is observed that the treatment of the control fruit samples with DTT induced a slight increase in the enzyme activity.However, in the samples pretreated with NaHS, the subsequent treatment with DTT reversed its effect by around 50% (brown bars).This indicates that the persulfidation of LAP in sweet pepper fruits is a reversible process which seems to be driven by reducing compounds.On the other hand, Figure 5a displays the in vitro analysis of pepper LAP activity in similar conditions to those used in Figure 4.In this case, it is observed that the pretreatment of the pepper extracts with 1 mM NaHS provoked a slight increase in LAP activity, but this effect increased at higher concentrations (white bars), with LAP activity being 2.5 times higher with 5 mM NaHS.Considering that persulfidation is suggested to be a reversible process, control (C)-and NaHS-treated pepper samples were further incubated with dithiothreitol (DTT).Thus, in Figure 5b, it is observed that the treatment of the control fruit samples with DTT induced a slight increase in the enzyme activity.However, in the samples pretreated with NaHS, the subsequent treatment with DTT reversed its effect by around 50% (brown bars).This indicates that the persulfidation of LAP in sweet pepper fruits is a reversible process which seems to be driven by reducing compounds.
In a previous study using S-nitrosoglutathione (GSNO) and nitroso-cysteine (NO-Cys), it was found that these NO donors caused the downregulation of the pepper LAP activity.However, this inhibition was not due to the NO released from these compounds because when GSH and Cys were used as internal controls, the same downregulation of LAP activity was also observed, thus indicating that these effects were due to the reductants [31].To get deeper insights into the modulation of LAP activity, the pepper fruit samples were incubated with GSNO and NaHS that may compete for the same Cys residue as a target.Figure 6 shows that when samples were subjected to a dual incubation with NaHS and GSNO, regardless of whether the pepper samples were first incubated with NaHS or with GSNO, the final effect was an inhibition.This suggests that the effect of the reducing compounds prevails over the positive effect of the H 2 S. In a previous study using S-nitrosoglutathione (GSNO) and nitroso-cysteine (NO-Cys), it was found that these NO donors caused the downregulation of the pepper LAP activity.However, this inhibition was not due to the NO released from these compounds because when GSH and Cys were used as internal controls, the same downregulation of LAP activity was also observed, thus indicating that these effects were due to the reductants [31].To get deeper insights into the modulation of LAP activity, the pepper fruit samples were incubated with GSNO and NaHS that may compete for the same Cys residue as a target.Figure 6 shows that when samples were subjected to a dual incubation with NaHS and GSNO, regardless of whether the pepper samples were first incubated with NaHS or with GSNO, the final effect was an inhibition.This suggests that the effect of the reducing compounds prevails over the positive effect of the H2S.

Discussion
Hydrogen sulfide (H2S) has gained great relevance in plant metabolism since it exerts signaling functions in numerous physiological processes [46][47][48][49][50] and in response to biotic and abiotic stresses [8,33,51,52].In fact, H2S is exogenously applied to vegetables and fruits using donors such as NaHS since it exerts positive effects.For example, in spinach, H2S increases resistance to drought [53]; in lettuce, it increases shoot elongation [54]; in ginger, it alleviates the toxic effects of cadmium by increasing the activity of different antioxidant enzymes [55]; in banana, it reduces chilling injury during postharvest [56][57][58]; in strawberry, H2S prolongs the shelf-life and reduces the decay rate of harvested fruits [59,60]; in cold-stored peaches, H2S regards fruit softening and avoid flesh browning [61,62]; and in apples, it promotes resistance against Penicillium expansum [63].
One of the mechanisms by which H2S regulates protein functions is by an oxiPTM named persulfidation [64][65][66], and the bulk of the proteins that undergo persulfidation are designated as the persulfidome.Research on the persulfidome is more advanced in animal cells [67-72], while in plants, as mentioned previously, most studies have focused on Arabidopsis, and to our knowledge, the persulfidome of any fruit is still unknown, as are the ways in which it could be modulated by ripening events since most studies have focused thus far on the Arabidopsis model plant under physiological and stress conditions [8,17,32,[73][74][75].Although the Arabidopsis persulfidome has been investigated mainly in leaves [17], whose metabolism is far different from that of the pepper fruits, the comparative analysis indicated that only 402 (25%) of the total proteins identified were shared by both organs and species.Furthermore, from the total persulfidated protein detected in pepper fruits, it is remarkable that 394 and 248 proteins were exclusively

Discussion
Hydrogen sulfide (H 2 S) has gained great relevance in plant metabolism since it exerts signaling functions in numerous physiological processes [46][47][48][49][50] and in response to biotic and abiotic stresses [8,33,51,52].In fact, H 2 S is exogenously applied to vegetables and fruits using donors such as NaHS since it exerts positive effects.For example, in spinach, H 2 S increases resistance to drought [53]; in lettuce, it increases shoot elongation [54]; in ginger, it alleviates the toxic effects of cadmium by increasing the activity of different antioxidant enzymes [55]; in banana, it reduces chilling injury during postharvest [56][57][58]; in strawberry, H 2 S prolongs the shelf-life and reduces the decay rate of harvested fruits [59,60]; in coldstored peaches, H 2 S regards fruit softening and avoid flesh browning [61,62]; and in apples, it promotes resistance against Penicillium expansum [63].
One of the mechanisms by which H 2 S regulates protein functions is by an oxiPTM named persulfidation [64][65][66], and the bulk of the proteins that undergo persulfidation are designated as the persulfidome.Research on the persulfidome is more advanced in animal cells [67-72], while in plants, as mentioned previously, most studies have focused on Arabidopsis, and to our knowledge, the persulfidome of any fruit is still unknown, as are the ways in which it could be modulated by ripening events since most studies have focused thus far on the Arabidopsis model plant under physiological and stress conditions [8,17,32,[73][74][75].Although the Arabidopsis persulfidome has been investigated mainly in leaves [17], whose metabolism is far different from that of the pepper fruits, the comparative analysis indicated that only 402 (25%) of the total proteins identified were shared by both organs and species.Furthermore, from the total persulfidated protein detected in pepper fruits, it is remarkable that 394 and 248 proteins were exclusively present in green and red pepper fruits, respectively.These data reflect the great metabolic difference between both stages of ripening, a physiological process that is characterized by an active nitro-oxidative metabolism [28].
At present, the most plausible effect of H 2 S at the protein level is the promotion of persulfidation, but this event has been little investigated in fruits and other vegetables.Thus, exogenous H 2 S delayed the ripening process of tomato by persulfidation linked to several proteins, including WRKY6, WRKY71 transcription factor, and E3 ligase BRG3 [93,94], and promoted Arabidopsis flowering by the persulfidation of transcript factor AtU2AF65a [50]; in pear, the persulfidation of transcription factor MYB10 triggered a lower content of anthocyanin on the skin [95].However, to our knowledge, there is no information about any persulfidome analysis of fleshy fruits during their ripening process.Based on the early information obtained from the Arabidopsis persulfidome [17,35], in previous studies, we selected different pepper fruit proteins involved in the metabolism of reactive oxygen species (ROS) such as catalase, APX, peroxidase, and NADPH-generating enzymes, among others, to evaluate, using in vitro approaches, how they could be regulated by H 2 S, either positively or negatively [28,86].However, concluding data about the potential persulfidation of those proteins could not be drawn from those analyses.Therefore, the information reported here for pepper fruits provides a set of potential protein targets for persulfidation, in competition with other thiol-based oxiPTMs such as S-nitrosation, S-glutathionylation, and S-sulfenylation, among others, for susceptible cysteines [86,87,96].Furthermore, according to the experimental design, the common persulfidome from both green and red fruits would allow us to attribute certain roles to specific persulfidated proteins in the ripening process.
In the present study, among the pepper proteins that are endogenously persulfidated, we add new enzymes, such as glutathione reductase (GR), whose activity was unaffected by persulfidation, suggesting that the effect of persulfidation on GR could be linked to other aspects not related to its activity, such as protection against protein overoxidation.On the contrary, leucine aminopeptidase activity was upregulated by this oxiPTM, and this effect was reversed by the reducing agent dithiothreitol (DTT).
GR is a key enzyme in the ascorbate-glutathione cycle that uses NADPH to keep the cellular levels of reduced glutathione (GSH), a non-enzymatic antioxidant that participates in the homeostasis of the cellular redox state [97,98] and which can interact with NO through an S-nitrosation event to generate GSNO, a compound that is considered a reservoir of NO in the cell [99,100].Remarkably, a previous study carried out in pea leaves demonstrated that GR can undergo tyrosine nitration and S-nitrosation, but none of these NO-derived PTMs had an effect on its activity, thus suggesting that it could be a mechanism to preserve the cellular GSH content.Therefore, the fact that persulfidation does not affect GR activity would be in concordance with being a mechanism to preserve GSH levels in pepper fruits, whose content decreases during ripening [26].
Plant leucine aminopeptidase (LAP) is a metalloenzyme, often requiring metal ions such as zinc or manganese for its catalytic activity [101][102][103][104]. LAP is typically found in the cytoplasm and plastids but can also be localized in other cellular compartments including mitochondria and peroxisomes, depending on the plant species and specific physiological conditions [31,43,[105][106][107][108][109][110].It plays a significant role in plant development [111][112][113] and its response to environmental adverse conditions including abiotic (Gu et al. [114]; Gu et al. [115]) and biotic stresses [116,117].LAP typically exhibits broad substrate specificity but has a preference for peptides with a leucine residue at the N-terminus, and is involved in the degradation of peptides into amino acids, particularly by removing leucine residues from the amino terminus of polypeptides.This process is crucial for protein turnover and the recycling of amino acids within plant cells.
Recently, in pepper fruit, we have shown that LAP activity increased during ripening.The enzyme was also a target of NO-derived PTMs (tyrosine nitration and S-nitrosation), and S-cyanylation, with its activity being either negatively or positively regulated, respectively [31].This seems to be correlated with the protein degradation associated with the disassembly of some organelles, such as it occurs in chloroplasts that transformed into chromoplasts during pepper ripening.Furthermore, LAP has been suggested to function in the free amino acid regulation, and peptide and protein turnover.Thus, LAP activity has been correlated with GSH recycling during the ripening of durian (Durio zibethinus L.) fruits [118].This fruit is rich in different sulfur-containing compounds including the dipeptide γ-glutamylcysteine and the tripeptide glutathione (γ-glutamyl-cysteinylglycine) [119,120], and LAP was shown to have Cys-Gly dipeptidase activity in the γglutamyl cycle, which would allow GSH recycling in the fruit.In this sense, Cys is the precursor of sulfur-containing volatiles during durian ripening due to the catabolism of GSH.Likewise, it has been shown that the GSH pool can be mobilized by physiological thiols like Cys and Cys-Gly, which, under certain oxidative conditions and in the presence of certain metal ions, can promote the formation of ROS [121][122][123].This situation may occur in pepper fruits, thus contributing to the nitro-oxidative metabolism that they undergo during ripening [26], where the GSH could regenerate Cys and Cys-Gly through transthiolation reactions.Accordingly, taking into account this last view plus, the role of GSH in the ascorbate-glutathione cycle and the detoxification processes through glutathione-Stransferase (GST), an enzymatic system which is also influenced by the ripening process (Figure 3), GSH could be envisaged as a relevant player in the physiology of pepper fruit.Recently, reactive polysulfides have been detected and quantified in different plant species, including pepper fruits [15].These polysulfides include cellular low-molecular thiols to form, for example, glutathione persulfide (GSSH), which could be involved in a transpersulfidation process of protein Cys residues.This process could provide an additional regulation mechanism equivalent to that attributed to S-nitrosoglutathione (GSNO), which mediates trans-nitrosation processes [124,125].However, these mechanisms will need future experimental analyses, although one of the main challenges is the technological difficulty in determining these compounds in plant tissue.

Conclusions
To the best of our knowledge, the present study provides the first report of the persulfidome of pepper fruit at different ripening stages and provides a new tool to study the relevance of H 2 S metabolism in this process that can be extrapolated as a starting point to other types of fleshy fruits, both climacteric and non-climacteric.Furthermore, it was found that whereas GR activity, which is involved in the regeneration of GSH, was unaffected by H 2 S, LAP activity was upregulated.Based on previous studies where it has been reported that (i) LAP participates in the GSH recycling and the generation of sulfur-containing volatiles involved in the process of fruit ripening [126,127]; (ii) pepper fruits have sulfur-containing volatiles which contribute to their flavor [119,126]; (iii) LAP activity increases during pepper ripening but is downregulated by reductants such as GSH and Cys [31]; and (iv) there is a decline in GSH content during pepper ripening [26], it could be hypothesized that H 2 S is a new element in this network which regulates the GSH content and sulfur-containing volatiles through the upregulation of the LAP. Figure 7 provides a working model that summarizes these new elements and previous ones.
was found that whereas GR activity, which is involved in the regeneration of GSH, was unaffected by H2S, LAP activity was upregulated.Based on previous studies where it has been reported that (i) LAP participates in the GSH recycling and the generation of sulfur-containing volatiles involved in the process of fruit ripening [126,127]; (ii) pepper fruits have sulfur-containing volatiles which contribute to their flavor [119,126]; (iii) LAP activity increases during pepper ripening but is downregulated by reductants such as GSH and Cys [31]; and (iv) there is a decline in GSH content during pepper ripening [26], it could be hypothesized that H2S is a new element in this network which regulates the GSH content and sulfur-containing volatiles through the upregulation of the LAP. Figure 7 provides a working model that summarizes these new elements and previous ones.

Figure 1 .
Figure 1.(a) Pepper persulfidome at different ripening stages, immature green and ripe red.A total of 891 proteins were identified, of which 370 proteins were exclusively present in green pepper, 237 were exclusively present in red pepper, and 284 were detected at both ripening stages.(b) Venn diagram of the comparative analysis of the persulfidomes from Arabidopsis thaliana leaves under physiological conditions and both green and red pepper fruits.In this last analysis, 2,015 proteins from Arabidopsis and 814 from pepper fruits were considered.

Figure 1 .
Figure 1.(a) Pepper persulfidome at different ripening stages, immature green and ripe red.A total of 891 proteins were identified, of which 370 proteins were exclusively present in green pepper, 237 were exclusively present in red pepper, and 284 were detected at both ripening stages.(b) Venn diagram of the comparative analysis of the persulfidomes from Arabidopsis thaliana leaves under physiological conditions and both green and red pepper fruits.In this last analysis, 2015 proteins from Arabidopsis and 814 from pepper fruits were considered.

Figure 2 .
Figure2.Gene ontology (GO) functional classification obtained for persulfidated proteins of pepper fruits.The scatterplots show GO terms as circles arranged such that those that are most similar in semantic space X and Y are placed nearest to each other.In this case, those proteins that appear exclusively at each stage of ripening have been analyzed, either green fruits (left panels) or red fruits (right panels), and the most enriched terms are shown.The number that appears inside each circle indicates which category proteins belong to.The size of the circles is directly proportional to the number of proteins in each sub-category that fall under each main category according to our data.The distance between the circles indicates a direct relationship between the categories.The p-value indicates the statistical significance of the GO category enrichment in our results.

Figure 2 .
Figure2.Gene ontology (GO) functional classification obtained for persulfidated proteins of pepper fruits.The scatterplots show GO terms as circles arranged such that those that are most similar in semantic space X and Y are placed nearest to each other.In this case, those proteins that appear exclusively at each stage of ripening have been analyzed, either green fruits (left panels) or red fruits (right panels), and the most enriched terms are shown.The number that appears inside each circle indicates which category proteins belong to.The size of the circles is directly proportional to the number of proteins in each sub-category that fall under each main category according to our data.The distance between the circles indicates a direct relationship between the categories.The p-value indicates the statistical significance of the GO category enrichment in our results.

Figure 3 .
Figure 3. MapMan categories assigned to the sweet pepper fruit persulfidome.Qualitative analysis o the diverse physiological aspects in which the proteins identified as potential persulfidation targets are involved.Each box represents a protein, color-coded to denote its presence in specific ripening stages o the fruit.Proteins exclusively identified in immature fruits are depicted in green, those exclusive to ripe fruits are shown in red, and proteins shared in both ripening stages are depicted in white.

Figure 3 .
Figure 3. MapMan categories assigned to the sweet pepper fruit persulfidome.Qualitative analysis of the diverse physiological aspects in which the proteins identified as potential persulfidation targets are involved.Each box represents a protein, color-coded to denote its presence in specific ripening stages of the fruit.Proteins exclusively identified in immature fruits are depicted in green, those exclusive to ripe fruits are shown in red, and proteins shared in both ripening stages are depicted in white.

Figure 4 .
Figure 4. Effect of NaHS on pepper fruit glutathione reductase (GR) activity.Green pepper extracts were pre-treated in the absence or presence of 0.1-5.0 mM NaHS for 1 h at 25 °C (white bars).The green bar corresponds to the control sample not treated with NaHS.Data are shown as the means ± SEM.No statistically significant differences were observed at p < 0.05 in relation to the control value.

Figure 4 .
Figure 4. Effect of NaHS on pepper fruit glutathione reductase (GR) activity.Green pepper extracts were pre-treated in the absence or presence of 0.1-5.0 mM NaHS for 1 h at 25 • C (white bars).The green bar corresponds to the control sample not treated with NaHS.Data are shown as the means ± SEM.No statistically significant differences were observed at p < 0.05 in relation to the control value.

Figure 5 .
Figure 5. (a) Effect of NaHS on the LAP activity of sweet pepper fruits where the samples were pre-treated in the absence or presence of 0.1-5.0 mM NaHS for 1 h at 25 °C (white bars).The green bar corresponds to the control sample not treated with NaHS (b) Effect of 5 mM NaHS on LAP activity in the presence and absence of dithiothreitol (DTT).Green pepper extracts were pre-treated in the absence or presence of 5 mM NaHS for 1 h at 25 °C (white bars), and then further incubation with 5 mM DTT was performed for 30 min (brown bars).Data are shown as the means ± SEM.Asterisks indicate that differences in relation to corresponding control values were statistically significant at p < 0.05.

Figure 5 .
Figure 5. (a) Effect of NaHS on the LAP activity of sweet pepper fruits where the samples were pre-treated in the absence or presence of 0.1-5.0 mM NaHS for 1 h at 25 • C (white bars).The green bar corresponds to the control sample not treated with NaHS (b) Effect of 5 mM NaHS on LAP activity in the presence and absence of dithiothreitol (DTT).Green pepper extracts were pre-treated in the absence or presence of 5 mM NaHS for 1 h at 25 • C (white bars), and then further incubation with 5 mM DTT was performed for 30 min (brown bars).Data are shown as the means ± SEM.Asterisks indicate that differences in relation to corresponding control values were statistically significant at p < 0.05.

Figure 6 .
Figure 6.Effect of NaHS and S-nitrosoglutathione (GSNO) on the LAP activity of sweet pepper fruit.Green pepper extracts were only treated with either 5 mM NaHS or 5 mM GSNO for 1 h at 25 °C.Additionally, samples were treated with the two compounds: first adding GSNO for 30 min at 25 °C and further incubating with NaHS for another 30 min at the same temperature (column GSNO + NaHS), and vice versa (column NaHS + GSNO).The white bar corresponds to the control sample without any treatment.The light brown bar corresponds to the treatment with NaHS.The blue bar corresponds to the GSNO treatment.The bars with both colors correspond to a combined and sequenced treatment, with the lower color, either blue (GSNO) or light brown, being applied first.Data are shown as the means ± SEM.Different letters indicate significant differences (p < 0.05).

Figure 6 .
Figure 6.Effect of NaHS and S-nitrosoglutathione (GSNO) on the LAP activity of sweet pepper fruit.Green pepper extracts were only treated with either 5 mM NaHS or 5 mM GSNO for 1 h at 25 • C. Additionally, samples were treated with the two compounds: first adding GSNO for 30 min at 25 • C and further incubating with NaHS for another 30 min at the same temperature (column GSNO + NaHS), and vice versa (column NaHS + GSNO).The white bar corresponds to the control sample without any treatment.The light brown bar corresponds to the treatment with NaHS.The blue bar corresponds to the GSNO treatment.The bars with both colors correspond to a combined and sequenced treatment, with the lower color, either blue (GSNO) or light brown, being applied first.Data are shown as the means ± SEM.Different letters indicate significant differences (p < 0.05).

Figure 7 .
Figure 7. Simple working model of the potential function of the LAP from pepper fruits in the mechanism of the GSH recycling process through its Cys-Gly peptidase activity and generation of sulfur-containing volatiles.During pepper fruit ripening, LAP activity is positively regulated by H2S (persulfidation) and cyanide (S-cyanylation), but it is negatively regulated by tyrosine nitration and S-glutathionylation.Based on our previous model [31].