The ripening of fleshy fruits is a highly coordinated developmental process that integrates physiological, biochemical, and structural modifications that determine organoleptic quality and post-harvest stability. It involves a progressive accumulation of sugars, which modifies taste and osmotic balance, decline in organic acids that affects flavour and acidity, and profound changes in hormonal signalling pathways that regulate the transition from growth to maturation. Additional features include the synthesis of volatile and aromatic compounds that define fruit aroma, the breakdown of chlorophyll, and cell wall remodelling, which confers texture and softness (Giovannoni et al., 2017). Together, these events confer the specific quality traits that distinguish ripe fruits, but they also expose tissues to greater metabolic demands and vulnerability to oxidative stress.

Reactive oxygen species (ROS) are continuously produced as by-products of aerobic metabolism, making their presence unavoidable during fruit development. Mitochondria are major contributors, as electron leakage from complexes I and III of the respiratory chain leads to superoxide formation. However, other compartments, including chloroplasts, the apoplast and the endoplasmic reticulum, are also important sources of ROS in developing plant tissues (Møller, 2001; Neill, 2002; Veal et al., 2007; Birk et al., 2013). In addition to organellar metabolism, a range of enzymes catalyse ROS production: NADPH oxidases at the plasma membrane, cell wall peroxidases, xanthine oxidase and amine oxidase, among others (Corpas et al., 2001; Bolwell, 2002; Torres et al., 2002; Bindschedler et al., 2006; Suzuki et al., 2011). The resulting ROS species include singlet oxygen (¹O₂), superoxide anion (O₂•^–), hydroxyl radicals (•OH), and hydrogen peroxide (H₂O₂). Each of these has distinct chemical properties: hydroxyl radicals are highly reactive and short-lived, causing immediate oxidative damage, whereas H₂O₂ is more stable, freely diffusible, and capable of acting as a versatile signalling intermediate. This duality illustrates the paradoxical role of ROS in plant physiology: moderate accumulation functions as an essential signal in development and adaptive responses, whereas excessive ROS generation promotes oxidation of proteins, lipids, and nucleic acids, leading to structural and functional impairment (Imlay, 2003; Ogilby, 2010).

To mitigate this intrinsic risk, plants employ a highly sophisticated antioxidant defence network that ensures redox homeostasis. This network integrates small-molecule antioxidants, such as ascorbate, glutathione, polyphenols and carotenoids, with an extensive set of antioxidant enzymes. Key enzymatic components include superoxide dismutase (SOD), which catalyses the dismutation of superoxide to H₂O₂ and O₂; catalase (CAT), which detoxifies H₂O₂ at high concentrations; ascorbate peroxidase (APX), with high affinity for H₂O₂ under basal conditions; and glutathione peroxidase (GPX), glutathione reductase (GR) and peroxiredoxins (Prx), which contribute to finely tuned ROS scavenging throughout different cellular compartments. The operation of this system depends on a continuous supply of reducing power, especially NADPH. The regeneration of glutathione and thioredoxin pools, which are essential for GPX and Prx activity, relies on NADPH-dependent enzymes such as glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH) and isocitrate dehydrogenase (ICDH) (Foyer et al., 1997; Jimenez et al., 1997; Corpas et al., 1998; Corpas et al., 1999; Arnér & Holmgren, 2000; Rhee & Woo, 2011; Halliwell & Gutteridge, 2015). Thus, the integration of ROS generation and detoxification constitute a dynamic equilibrium, critical for cell survival and developmental regulation.

Berry ripening represents a particularly relevant developmental context in which redox equilibrium must be preserved. Enhanced metabolic activity, including intensified respiration, sugar accumulation and hormonal reprogramming, inevitably enhances ROS generation during the transition from growth to maturation (Zeng et al., 2010). Controlled ROS accumulation has been associated with the onset of berry ripening, indicating that oxidative events may participate in developmental regulation rather than merely reflect metabolic disorder (Pilati et al., 2014). Nonetheless, despite elevated ROS contents during post-veraison, many fleshy fruits retain metabolic coordination and tissue functionality, avoiding premature cell death (Chan et al., 2016). This observation suggests that antioxidant defences are not only active but dynamically modulated throughout ripening to ensure viability. Understanding how berries orchestrate this balance between oxidative metabolism and antioxidant capacity is therefore essential in order to unravel the molecular bases of development and quality maintenance.

Grape berries constitute a suitable system for addressing this question. As non-climacteric fruits, grape berries ripen through finely coordinated interactions between sugar accumulation, hormonal regulation, and redox-related signalling rather than through a primarily ethylene-driven programme (Giovannoni et al., 2017; Lu et al., 2024; Pilati et al., 2014). Previous studies have provided valuable insights into transcriptional regulation and enzymatic activities associated with antioxidant responses in grapevine. For example, alterations in catalase or superoxide dismutase activity have been reported in association with developmental stages or stress exposure (Wang et al., 2019). However, although controlled ROS accumulation has been described at the onset of ripening, these approaches have generally focused on skin tissues, individual enzymes, or transcript-level responses (Pilati et al., 2014). They therefore provide only an incomplete framework for understanding the coordinated redox adjustments occurring in the mesocarp, where tissue functionality and fruit texture must be preserved throughout maturation, particularly because transcript abundance does not necessarily correspond to protein accumulation or catalytic activity. A more comprehensive interpretation requires the integration of proteomic datasets with enzymatic activity profiles, capturing both abundance and functional capacity of redox-related pathways.

In this study, we employed quantitative proteomic profiling combined with enzymatic assays to characterise the dynamics of redox metabolism during the ripening of Vitis labrusca × Vitis vinifera cv. Niagara Rosada. This cultivar is an economically important hybrid widely cultivated in Brazil, providing a relevant model for understanding the ripening physiology of table grapes grown in warm regions. By examining three key developmental stages, we aimed to identify proteins and enzymatic responses directly or indirectly associated with ROS metabolism. We hypothesised that stage-specific regulation of antioxidant enzymes and NADPH-generating pathways contributes to the control of oxidative processes in the mesocarp, supporting tissue functionality despite enhanced ROS-associated metabolism during ripening.

This study aimed to characterise redox metabolism during the ripening of cv. Niagara Rosada berries through the integration of proteomic profiling and enzymatic assays. Pi and lipid peroxidation profiles suggested moderate membrane-associated oxidative changes during ripening, without evidence of extensive oxidative deterioration. Antioxidant enzyme activities were stage-dependent, where CAT followed a biphasic profile, SOD increased towards maturity, PX declined progressively, and APX, GR, and GPX exhibited distinct temporal patterns. Proteomic analysis supported these patterns, identifying 956 differentially accumulated proteins, with a predominance of antioxidant isoforms and NADPH-generating dehydrogenases at specific developmental stages. Overall, Niagara Rosada berries exhibited coordinated redox adjustment during ripening, associating antioxidant enzyme activity with proteomic reprogramming.

Grape berry development involved coordinated morphological and physiological transitions that define the onset and progression of ripening. Berry diameter and fresh mass increased progressively, reflecting active cell expansion and the accumulation of soluble compounds, primarily sugars and water (Figure 1A-C). These physical changes occurred concomitantly with a decline in titratable acidity and an increase in pH (Figure 1D-E), consistent with the established shift from organic acid accumulation to hexose accumulation during grape berry ripening (Lu et al., 2024).

In our proteomic dataset, several proteins associated with oxidative responses and cellular oxidant detoxification accumulated progressively during ripening. This indicates an active remodelling of redox metabolism during berry development, enhancing the capacity to maintain ROS within physiological limits. Such regulation is essential, because ROS function not only as oxidants but also as signalling molecules that mediate developmental transitions (Møller, 2001; Neill, 2002; Imlay, 2003; Pilati et al., 2014).

The abundance patterns of PX and AO in the mesocarp are consistent with their possible contribution to H₂O₂ formation at early and late ripening stages (Figure 3B-C). This points to an active and developmentally regulated redox flux rather than passive oxidative accumulation. AO, localised in mitochondria, contributes to controlled H₂O₂ formation through alternative respiratory routes that modulate the cellular redox environment, as reported in ROS-mediated mitochondrial signalling (Møller & McPherson, 1998; Belenghi, 2004; Cona et al., 2005). PX, although commonly associated with H₂O₂ removal, may operate reversibly under specific redox conditions, sustaining ROS turnover in metabolically active tissues. Comparable enzyme behaviour has been recorded in grapevine buds, where PX activity increases alongside H₂O₂ during deep endodormancy (Pérez et al., 2021). Overall, these responses maintain redox homeostasis during ripening while allowing H₂O₂ to operate within its signalling range.

Although ROS-related enzymatic activity changed during ripening, Pi and MDA profiles did not support extensive oxidative deterioration of the mesocarp. Pi increased from EL 33 to EL 35 and EL 39, whereas no further increase occurred between EL 35 and EL 39 (Table 1). MDA also increased during ripening, with higher values at EL 39 than at EL 33. These responses indicate moderate membrane-associated oxidative changes during berry development rather than progressive cellular collapse. This interpretation is consistent with previous evidence that vitality loss in Niagara Rosada mesocarp occurs independently of extensive cell death (Monteiro et al., 2025). Similar regulation has been described in grape berries, where oxidative changes may accompany ripening while tissue functionality is retained during development (Caravia et al., 2015; Krasnow et al., 2008).

Our enzyme profiles identified CAT as the early, high-capacity peroxide buffer, with maximal abundance at initial stages (Figure 2D-E) and a biphasic activity course (lower mid-development, recovery at maturity; Figure 2A). This pattern is compatible with post-translational control to handle episodic H₂O₂ rises (Van Breusegem et al., 2001; Gupta, 2010). APX, with high affinity for H₂O₂, accumulated at EL 33 (Figure 2B) while keeping activity near-constant through maturation (Figure 2F), thereby securing baseline scavenging by maintaining peroxide within its functional range (De Gara, 2004; Dąbrowska et al., 2007). In contrast, SOD gains both abundance and activity at EL 39 (Figure 2C-G), contributing to the peroxide pool late in ripening and supporting signalling-dependent adjustments linked to maturation (Mittler, 2002; Bhattacharjee, 2010). These profiles outline a division of functionality: CAT covers high-load detoxification, APX provides fine control, and SOD participates in late-stage superoxide conversion, contributing to the redox set-point compatible with mesocarp functionality from early growth to maturity. Adjustments in oxidative metabolism have also been reported in other fruits. In peach, for instance, CAT and GPX activities rise during late ripening in response to temperature shifts (Huan et al., 2016), while in grape, declining internal O₂ concentrations drive metabolic transitions that alter mitochondrial respiration (Romieu et al., 1992; Xiao et al., 2018a; Xiao et al., 2018b). These converging patterns emphasise the conserved nature of oxidative control mechanisms that sustain metabolic flexibility in fleshy fruits.

NADPH-producing dehydrogenases displayed predominantly early-stage profiles during Niagara Rosada berry development. G6PDH activity was highest at EL 33 and declined markedly towards EL 39–EL 41, while the corresponding G6PDH protein also prevailed at the early stage (Figure 5A-B). Similarly, ICDH isoforms were more abundant at EL 33 and decreased towards EL 39 (Figure 7C-D). These profiles suggest that NADPH-generating capacity through G6PDH- and ICDH-linked routes was more prominent during early mesocarp development, when biosynthetic activity and basal antioxidant buffering are expected to impose a high demand for reducing power. This interpretation is consistent with the recognised role of NADPH-generating dehydrogenases, including G6PDH, 6PGDH, and ICDH, in sustaining antioxidant systems and cellular redox homeostasis in plants (Corpas & Barroso, 2014; Mhamdi et al., 2010). By contrast, the two 6PGDH isoforms remained unchanged during the analysed stages (Figure 5D-E), indicating a comparatively stable proteomic contribution of this oxidative pentose phosphate pathway component. Therefore, rather than reflecting a generalised late increase in NADPH-producing enzymes, the data point to a developmental redistribution of redox support, with early NADPH-generating capacity preceding the late activation of glutathione-dependent antioxidant components. This redistribution is represented schematically by the early profiles of G6PDH and ICDH and the comparatively stable 6PGDH isoforms in the integrated redox model (Figure 8B-C).

GR abundance and activity followed distinct temporal profiles, indicating post-transcriptional or post-translational regulation of this glutathione-dependent component (Figure 4A-C). GR protein reached its highest abundance at EL 35, whereas enzymatic activity became detectable only at EL 39–EL 41. This late catalytic activation is consistent with an increased requirement for glutathione recycling as ROS-associated metabolism intensifies during the final stages of development (Dumanović et al., 2021; Foyer et al., 1997). In contrast, TrxR abundance remained steady throughout ripening (Figure 7A), indicating a constitutive function in sustaining thiol-disulphide equilibrium under stable metabolic conditions (Sevilla et al., 2015). The GPX and Prx (Figure 7B) pattern refined this balance as GPX isoforms (Figure 4D-E) became more abundant at later stages, whereas Prx predominates in early development. Despite more abundant, GPX catalytic activity decreased during maturation (Figure 4B). This regulation between GPX and Prx supports a shift from peroxide-processing routes as the berry matures, reinforcing the flexibility of the antioxidant network in meeting distinct metabolic demands throughout ripening (Figure 8A).

The data delineate a stage-dependent modulation of ROS generation and detoxification that maintains a controlled oxidative environment throughout ripening. Coordination among AO, PX, NADPH-generating dehydrogenases and antioxidant enzymes sustains redox balance, limiting oxidative injury while preserving the signalling capacity of H₂O₂ (Foyer & Noctor, 2011; Neill, 2002). Such regulation supports the interpretation that oxidative metabolism contributes to developmental progression rather than stress-related disruption. Within this controlled environment, redox homeostasis emerges as a key determinant of mesocarp functionality, consistent with the maintenance of tissue viability reported for ripening in grape berries (Caravia et al., 2015; Krasnow et al., 2008; Monteiro et al., 2025). This integrated organisation is summarised in Figure 8.

The integrated scheme summarises the main redox routes detected in Niagara Rosada mesocarp and highlights the temporal redistribution of antioxidant and NADPH-linked components during ripening (Figure 8). The enzymatic and proteomic profiles converge on a model in which early development is characterised by higher abundance or activity of selected peroxide-removing and NADPH-generating components, whereas late ripening involves increased SOD abundance/activity and the late activation of glutathione-dependent recycling. This organisation suggests that ROS metabolism is not uniformly intensified during ripening, but redistributed among enzymatic routes that combine peroxide detoxification, reducing-power supply and redox signalling.

For Niagara Rosada berries, softening is more consistent with regulated cell wall metabolism than with oxidative damage, in agreement with previous evidence that mesocarp vitality loss occurs independently of cell death (Monteiro et al., 2025). In the present study, the increase in MDA suggests ripening-associated lipid peroxidation, but not necessarily uncontrolled oxidative deterioration. Proteins involved in polysaccharide modification, such as pectin methylesterase, β-galactosidase, and expansins, may therefore operate within a controlled redox framework, enabling wall relaxation without generalised cellular collapse (Giovannoni et al., 2017; Lijavetzky et al., 2012). The persistence of aquaporins and membrane-associated proteins supports gradual turgor adjustment instead of structural failure (Caravia et al., 2015; Krasnow et al., 2008). Controlled ROS activity may further mediate local wall extensibility through peroxidase-driven crosslinking, associating redox signals with mechanical transitions in the mesocarp (Bolwell, 2002; De Gara, 2004).

The present findings indicate a developmental strategy in which oxidative control and structural remodelling are coordinated to sustain mesocarp functionality during ripening. This integrated regulation enables Niagara Rosada berries to attain full maturity while maintaining tissue organisation and mechanical stability, traits of great relevance to fruit quality and post-harvest performance.

Luan C. Corrêa is grateful for the scholarship by the Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Eduardo Monteiro is grateful for the scholarship funding provided by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Rodrigo R.R.F. Lucas is grateful for the scholarship by the Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF).