Tissue-Specific Metabolic Changes During Postharvest Storage of Friariello Napoletano

Abstract

Brassica rapa L. subsp. sylvestris L. Janch. var. esculenta Hort., commonly known as Friariello Napoletano, is a traditional Italian landrace valued for its distinctive flavor, nutritional richness, and cultural relevance in Mediterranean cuisine. The present study investigates the biochemical changes during postharvest storage at two temperatures (4 °C and 10 °C) for 2 and 20 days in its inflorescences and leaves. The experiment aimed to evaluate the evolution of primary and secondary metabolites, with a focus on pigments, amino acids, antioxidants, and glucosinolates. Significant degradation of chlorophylls was observed, particularly in leaves, with reductions of over 90% after 20 days at both temperatures. Conversely, α-tocopherol content increased significantly, especially in inflorescences, indicating an antioxidant response to storage stress. Amino acid analysis revealed a sharp decline in glutamate (up to 79%) and glutamine (up to 83%) in leaves, while proline levels increased across both tissues, reflecting an osmoprotective response. Essential amino acids (EAAs) showed variable responses, with certain EAAs, such as histidine and phenylalanine, accumulating under specific storage conditions. Soluble sugars, starch, and glucosinolates also decreased significantly, with soluble sugars dropping by 87% in inflorescences and 90% in leaves after 20 days at 10 °C. Pathway analysis revealed distinct tissue-specific metabolic responses, with inflorescences exhibiting more stable antioxidant levels and greater resilience to oxidative stress compared to leaves. These findings provide insights into the metabolic adjustments during postharvest senescence and may support future strategies aimed at preserving shelf life and nutritional quality of this traditional Mediterranean vegetable.

1. Introduction

Brassica rapa L. subsp. sylvestris (L.) encompasses a group of leafy vegetables traditionally cultivated in the Mediterranean basin, commonly known in Italy as cime di rapa, friariello, broccoletti, or rapini. Among these, the Friariello Napoletano (FN) represents a distinctive and culturally iconic ecotype, appreciated for its aromatic taste and nutritional richness [1,2,3]. FN has been described since the late 19th century as a regrowth crop obtained from the basal shoots after the main stem is cut, a practice well documented by De Rosa [4] and later reaffirmed by Angelini [5] and Tesi [6]. These local landraces have historically been referred to as Brassica campestris but are now classified as Brassica rapa L. subsp. sylvestris (L.) Janch. var. esculenta Hort., as confirmed in “The Brassica rapa Genome” book [7]. Its cultivation spans over 3500 hectares both in the Campania and Apulia regions, according to national statistics [8], and the vegetable is recognized as a traditional regional product due to its role in historic and contemporary local cuisine [9]. The edible portion, consisting of tender leaves and immature inflorescences, is appreciated for its aromatic, slightly pungent flavor and moderate bitterness. This sensory profile is attributed mainly to its high content of glucosinolates and related sulfur-containing compounds, which are also responsible for its health-promoting properties [1,10,11].

From a nutritional point of view, FN is particularly rich in bioactive compounds, including glucosinolates, vitamin C, phenolics, carotenoids, and essential amino acids—in particular, branched-chain amino acids [12]. These phytochemicals are known to exert antioxidant, anti-inflammatory, and even anticancer effects, as extensively documented in Brassica vegetables such as broccoli, kale, and turnip greens [11,13]. Glucosinolates, in particular, are hydrolyzed upon tissue damage to isothiocyanates and other biologically active metabolites that contribute to chemopreventive mechanisms [14]. Ascorbic acid (vitamin C) not only enhances the nutritional value of the product but also stabilizes other sensitive molecules and plays a crucial role in delaying oxidative damage in postharvest tissues [15].

Despite its health benefits and growing popularity beyond Italy, including in North America, Argentina, and Australia where it is marketed as broccoli rabe, rapini, or spring broccoli [16], FN remains largely under-investigated from both physiological and postharvest perspectives. Furthermore, due to the absence of registered cultivars and reliance on landraces selected by farmers through empirical methods, the species displays wide morphological and genetic variability, with potential implications for its response to storage and handling conditions [10,11].

Postharvest deterioration is a critical bottleneck for FN. As with other leafy vegetables, the plant tissues remain metabolically active after harvest, leading to rapid senescence [17]. Key processes responsible for quality loss include elevated respiration rates, ethylene production, oxidative stress, pigment degradation, and dehydration [13,18,19]. Ethylene, a key ripening hormone even in non-climacteric crops, accelerates chlorophyll breakdown and initiates a cascade of biochemical events such as peroxidase activation, lipid peroxidation, and ascorbate depletion. Respiration rates tend to spike immediately after harvest and remain high, leading to the consumption of sugars, organic acids, and antioxidants, thus depleting the energy reserves and nutritional quality of the product [9,17]. Visual indicators of senescence in FN include yellowing of the inflorescences due to chlorophyll loss, wilting caused by water deficit and cell-wall degradation, and browning of cut surfaces through enzymatic oxidation of phenolic compounds [20,21]. These phenomena are intensified under suboptimal storage temperatures and relative humidity, as well as through mechanical injuries during harvesting and handling [22]. In addition, the reduction in key metabolites such as vitamin C, glucosinolates, and chlorophyll not only compromises sensory and nutritional attributes but also undermines the potential nutraceutical role of the vegetable [13].

A wide array of technological solutions has been proposed for extending the shelf life of Brassica vegetables, including modified atmosphere packaging, pre-treatment with calcium chloride or antioxidants, and the application of edible coatings [19,20,23,24]. Among these, temperature remains the most critical and practical parameter to manage. Several studies suggest that storage at 0–4 °C significantly delays senescence, inhibits chlorophyllase and peroxidase activities, and preserves firmness and nutritional quality [20,25]. However, temperature regimes above 5 °C are often encountered in commercial distribution chains, especially in the early phases of postharvest management, which may compromise the benefits of cold storage [21,26]. Although the effects of postharvest temperature on broccoli and related species have been studied, little is known about how storage temperature and duration affect the metabolic profile of B. rapa subsp. sylvestris. A better understanding of the physiological and biochemical changes during storage is needed to support the development of optimized protocols for handling and conservation.

To address this gap, the present study analyzed the evolution of key primary and secondary metabolites in FN leaves and inflorescences during storage at two temperature regimes (4 °C and 10 °C) over two time intervals (2 and 20 days). Inflorescences and leaves of Friariello Napoletano differ in morphology, function, and metabolic activity, which may influence their postharvest behavior and senescence patterns. For clarity, the term “tissue” is used throughout this study to indicate metabolically distinct plant organs, namely inflorescences and leaves. By monitoring a comprehensive panel of biochemical parameters, including pigments, sugars, amino acids, glucosinolates, organic acids, and antioxidants, this work aims to provide mechanistic insights into the senescence process and to support future efforts aimed at preserving shelf life, nutritional integrity, and enhancing the marketability of this high-value vegetable.

2. Materials and Methods

2.1. Plant Material and Experimental Design

The experiment was performed on the local ecotype ”Sessantino Riccia di San Marzano” of Brassica rapa L. subsp. sylvestris L. Janch. var. esculenta Hort. Seeds were provided by Agrisementi Lebbioli Srl (San Tammaro, Caserta, Italy) and sown in a conventional open-field farm located in Capua (41°06′42.7″ N, 14°14′45.5″ E; 36 m a.s.l.), in the province of Caserta, Campania Region (Southern Italy). Plants were grown under conventional agronomic conditions and harvested 60 days after sowing on 20 November 2023. The experimental field was located in the Volturno River plain in an area characterized by deep, fertile silty clay loam soils of fluvio-alluvial origin. According to the USDA Soil Taxonomy, the soil was classified as a Typic Haplustept, a moderately developed Inceptisol with good drainage and medium structural development. The pH was slightly alkaline (ranging from 7.8 to 8.0), and the soil showed moderate organic matter content (~2%), with adequate cation exchange capacity to support intensive horticultural production. This area was designated as a Nitrate Vulnerable Zone (NVZ) under the European Union Nitrates Directive (91/676/EEC); consequently, nitrogen fertilization was carefully managed in compliance with environmental legislation without exceeding the regulatory threshold of 170 kg N ha⁻¹ year⁻¹, and was adjusted according to crop requirements and baseline soil fertility in order to minimize the risk of nitrate leaching into groundwater.

At commercial maturity, a large, homogeneous population of plants was harvested allowing for the setup of three biological replicates per postharvest condition, each consisting of pooled tissue from six individual plants. The outer young but fully developed leaves were separated from the edible inflorescences and processed as distinct sample types. A portion of the material was immediately frozen in liquid nitrogen and stored at −80 °C to serve as the control. The rest of the plant material was stored under controlled postharvest conditions at 4 °C and 10 °C, and sampled at 2 and 20 days after harvest. The experiment followed a completely randomized design. Treatments included two storage temperatures (4 °C and 10 °C) and two storage durations (2 and 20 days), resulting in four postharvest conditions, plus a freshly harvested control. Each treatment was applied to two tissue types (inflorescences and leaves) for a total of ten experimental groups. The term tissue was used consistently to refer to plant organs (inflorescences vs. leaves), as clarified throughout the manuscript. The selected storage temperatures (4 °C and 10 °C) reflect commonly encountered postharvest conditions for fresh vegetables. A setting of 4 °C corresponds to standard refrigeration in domestic and retail storage [27]. Conversely, 10 °C approximates the temperature typically measured in vegetable drawers of household refrigerators and commercial storage rooms with less precise climate control [28]. This value is also considered the upper optimal limit for maintaining quality in climacteric vegetables such as tomatoes [29], and is broadly applied to leafy greens to avoid chilling injury or accelerated senescence [30]. In Campania, Friariello Napoletano is traditionally cultivated in succession to industrial tomato on the same fields as a winter crop. As a result, the same postharvest infrastructure—originally designed for tomatoes—is frequently reused for Friariello, making 10 °C a practical and operationally relevant storage condition. After postharvest treatment, all samples were finely ground in liquid nitrogen using a pre-chilled ceramic mortar and pestle, aliquoted into pre-cooled 1.5 mL Eppendorf tubes, and stored at −80 °C for subsequent biochemical analyses.

2.2. Reagents and Laboratory Materials

All biochemical analyses were carried out using certified analytical-grade reagents and authenticated reference standards. High-purity amino acid standards, 2-mercaptoethanol, tetrahydrofuran, sodium acetate, sodium borate, and ninhydrin—all suitable for HPLC applications—were sourced from Merck KGaA (Darmstadt, Germany). Additional reagents included Trizma^® base, multielement anion and cation standards, bovine serum albumin (BSA), HEPES, adenosine 5′-triphosphate (ATP), disodium salt hydrate, dithiothreitol, potassium hydroxide hydrate, and glucose. Enzymes used in the assays—such as α-amylase, amyloglucosidase, hexokinase, phosphoglucose isomerase, invertase, and glucose-6-phosphate dehydrogenase (from Leuconostoc mesenteroides)—as well as β-nicotinamide adenine dinucleotide hydrate, glacial acetic acid, gallic acid, and Folin–Ciocalteu reagent were also obtained from Merck KGaA (Darmstadt, Germany). The Bradford reagent for protein quantification was purchased from Bio-Rad Laboratories (Hercules, CA, USA). HPLC-grade methanol and ethanol were supplied by VWR International (Radnor, PA, USA). Ultrapure water used in all preparations was generated using a Milli-Q Gradient A10 purification system (Millipore, Billerica, MA, USA).

2.3. Soluble Sugars and Starch

Soluble sugars were extracted from 50 mg of powdered tissue using 250 µL of 80% ethanol (v/v), followed by incubation at 80 °C for 20 min in a ThermoMixer (Eppendorf, Hamburg, Germany). Samples were centrifuged at 15,000× g for 10 min at 4 °C. The procedure was repeated with 250 µL of 50% ethanol and then with 150 µL of 80% ethanol. Supernatants were pooled for quantification of glucose, fructose, and sucrose. The remaining pellet was used for starch analysis: it was solubilized in 100 µL of 0.1 M KOH, heated at 90 °C for 2 h, neutralized with glacial acetic acid (pH 4.5), and incubated for 20 h at 37 °C in a buffer (50 mM sodium acetate, pH 4.5) containing α-amylase (2 U mL⁻¹) and amyloglucosidase (20 U mL⁻¹). After centrifugation, glucose released from starch was quantified by enzymatic assays based on pyridine nucleotide reduction [31]. Absorbance was recorded at 340 nm using a Synergy HT spectrophotometer (BioTEK Instruments, Winooski, VT, USA). Sugar and starch contents are expressed in g per kg of fresh weight (g kg⁻¹ FW).

2.4. Pigments, α-Tocopherol, Ascorbate, and Glucosinolates Determination

Tocopherols were quantified by HPLC as described by Annunziata et al. [17] and expressed as mg kg⁻¹ FW. Ascorbate (reduced and total) and chlorophylls were extracted in 0.2 N HCl, neutralized, and quantified spectrophotometrically at 265 nm and 666, 655, and 470 nm, respectively, according to Annunziata et al. [17]. Ascorbate is expressed as µmol kg⁻¹ FW, while chlorophylls and carotenoids are expressed as g kg⁻¹ FW and mg kg⁻¹ FW, respectively. Glucosinolates were extracted in 70% methanol, sonicated, centrifuged, and analyzed by HPLC with UV detection at 227 nm using desulfo-sinigrin as an external standard [32]. Results are expressed as g kg⁻¹ FW.

2.5. Soluble Proteins, Organic Acids, and Free Amino Acids

Proteins were extracted from 20 mg of lyophilized tissue using Tris–HCl buffer (200 mM, pH 7.5) with 500 mM MgCl₂, stored at 4 °C for 24 h, and centrifuged (15,000× g, 5 min). The protein content was determined using the Bio-Rad assay and a BSA standard curve (595 nm) [33] and expressed as g kg⁻¹ FW. Free amino acids were extracted in 60% ethanol, derivatized, and quantified by HPLC after OPA derivatization, while proline was measured calorimetrically using a ninhydrin-based assay modified by [34], and the results are expressed as mmol kg⁻¹ FW [35]. Organic acids and anions were measured by ion chromatography as in Woodrow et al. [35] and results are expressed as mmol kg⁻¹ FW.

2.6. Statistical Analysis and Data Visualization

Each biochemical parameter was analyzed using three independent biological replicates per condition, and each biological replicate consisted of a pooled sample of five leaves or five inflorescences collected from five different plants to minimize biological variability. Data were subjected to a two-way analysis of variance (ANOVA) using SPSS software version 25 (IBM Corp., Armonk, NY, USA), with tissue type (inflorescences vs. leaves) and postharvest treatment (defined by temperature and storage duration) as the main factors. When significant effects were detected, treatment means were compared using Tukey’s HSD post hoc test at a significance level of p < 0.05—significant differences are indicated by different lowercase letters. Results are expressed as mean ± standard deviation (n = 3). Interaction terms (Tissue × treatment and Inflorescence × Leaves) were also tested to assess both tissue-specific responses and overall differences between organs across treatments. Significance is indicated by asterisks (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001), and non-significance by ns. Changes in metabolite levels were also visualized through pathway maps based on log_1.5-transformed fold changes (treatment vs. control), and represented by a false-color scale, with red indicating increased and blue decreased values, as described in [36].

3. Results

3.1. Responses of Pigments, α-Tocopherol, and Ascorbate to Different Storage Conditions

At harvest, FN leaves and inflorescences exhibited clearly distinct biochemical profiles. Cold storage significantly influenced pigment and antioxidant composition in FN leaves and inflorescences (Figure 1, Table S1). In particular, leaves had higher average concentrations of total chlorophyll (Figure 1A) and carotenoids (Figure 1B) compared to inflorescences. Total chlorophyll content in leaves was 2.11 g kg⁻¹ FW, nearly five times higher than in inflorescences (0.43 g kg⁻¹ FW) (Table S1). A similar trend was observed for carotenoids, with leaves showing a value of 0.80 g kg⁻¹ FW compared to 0.32 in inflorescences. Compared with the control, storage at 4 °C caused a marked reduction in the total chlorophyll in leaves—by 95% after 2 days and 96% after 20 days (Figure 1A). At 10 °C, the reduction was 62% and 66%, respectively. In contrast, inflorescences showed no significant variation in total chlorophyll content under any storage condition. Chlorophyll a decreased in leaves under all treatments, while in inflorescences it remained unchanged (Table S1). Chlorophyll b showed a 44% decrease in inflorescences at 4 °C but increased by 67% at 10 °C after 2 days. In leaves, Chl b was strongly affected by storage, with reductions of 94% and 96% at 4 °C, and 53% and 55% at 10 °C (Table S1). The Chl a/Chl b ratio was not significantly affected, although it tended to decrease in leaves under stress conditions (Figure 1C).

Carotenoids in leaves were also highly sensitive to storage. Compared with the control, concentrations declined by 97% and 94% at 4 °C, and by 65% and 68% at 10 °C after 2 and 20 days, respectively (Figure 1B). In inflorescences, the carotenoid levels decreased by 37.5% at 10 °C, while no significant changes were observed at 4 °C.

α-Tocopherol responded differently depending on the tissue. In inflorescences, all storage treatments led to a significant increase in α-tocopherol content, with an average rise of 342% compared to the control (Figure 1D). In leaves, the increase was limited to 20 days after harvest, with values rising by 70% at 4 °C and 255% at 10 °C (Table S1).

Total ascorbate content was higher in leaves than in inflorescences under control conditions (80.85 vs. 29.31 µmol g⁻¹ FW) (Table S1). However, storage induced a sharp decline in both tissues. In inflorescences, ascorbate levels decreased by 49% at 4 °C and 88% at 10 °C after 20 days (Figure 1E). In leaves, the reduction reached 62% at 4 °C and 90% at 10 °C. The DHA/ASC ratio did not change significantly, although the highest value was observed in inflorescences stored at 10 °C for 20 days (Figure 1F).

3.2. Responses of Soluble Sugars, Starch, and Glucosinolates to Different Postharvest Conditions

Soluble sugars, starch, and glucosinolates were significantly affected by postharvest conditions, with distinct tissue- and time-dependent patterns (Figure 2, Table S1). In particular, soluble sugar concentrations declined sharply in both tissues. Compared with the control, values decreased by 87% in inflorescences and 90% in leaves after 20 days at 10 °C (Figure 2A). A consistent reduction was observed under all cold storage treatments, although the decrease was more pronounced at higher temperatures and longer durations.

According to Supplementary Table S2, glucose and fructose had high baseline concentrations in both tissues, and both were reduced after storage. The effect was more marked after 20 days at 4 °C and 10 °C. The glucose/fructose ratio increased significantly in response to storage, particularly in inflorescences, where values more than doubled compared to the control (Figure 2B). In leaves, the increase was less marked and became evident only after 20 days at 10 °C.

Glucosinolates also declined progressively during storage. In both tissues, the strongest reduction occurred after 20 days at 10 °C, with a 75% decrease relative to the control (Figure 2D). Despite different initial concentrations, inflorescences and leaves showed a similar downward trend under prolonged cold exposure, indicating a shared sensitivity of glucosinolates to temperature- and time-dependent degradation.

3.3. Responses of Organic Acids and Inorganic Anions to Different Postharvest Conditions

Storage conditions induced tissue-specific changes in the accumulation of organic acids and inorganic anions (Figure 3, Table S2). Significant Tissue × treatment interactions were observed for acetate, pyruvate, citrate, and malate (p ≤ 0.01). Leaves generally showed higher concentrations of organic acids, while inflorescences appeared more sensitive to cold storage.

Acetate increased in both tissues following storage. In inflorescences, the rise was particularly marked at 10 °C after 20 days, with values exceeding the control by 418% (Figure 3A). Pyruvate concentrations also increased with storage, especially in inflorescences, where levels rose by 233% at 4 °C and 200% at 10 °C after 20 days. In leaves, the most pronounced increase (+500%) was observed at 4 °C after 2 days (Figure 3B).

Citrate levels (Figure 3C) did not vary significantly across treatments, although inflorescences showed a significant increase (+65%) after 20 days at 10 °C. Malate concentrations were consistently higher in leaves than in inflorescences and showed a tendency to increase after long-term storage at 10 °C (Figure 3D).

Among inorganic anions, fluoride, phosphate, and sulfate responded significantly to storage (p ≤ 0.01), while chloride and oxaloacetate remained unchanged (ns) (Table S2). Nitrate exhibited a moderate but significant Tissue × treatment interaction (p ≤ 0.05), with the highest concentrations observed in leaves at 10 °C after 20 days. However, high variability among replicates limited the statistical resolution of pairwise post hoc comparisons. Although Tukey’s HSD test did not always detect significant differences between treatments within each tissue, the overall ANOVA confirmed biologically meaningful and tissue-dependent shifts in nitrate levels.

3.4. Protein and Amino Acid Response to Different Storage Conditions

Soluble protein content decreased in both tissues under all storage conditions compared with freshly harvested samples (Figure 4A), although the reduction was not statistically significant in the two tissues compared to respective controls. By contrast, free amino acid profiles were more strongly affected by temperature and duration, showing highly significant Tissue × treatment interactions for most metabolites (Figure 4B; Table S3). Compared with the control, total amino acid content in inflorescences decreased by 36% at 2 postharvest days (PHD) at 4 °C and by 53% at 2 PHD at 10 °C. In leaves, total amino acids were reduced by approximately 61% under all storage conditions (Figure 4B). Several amino acids, including alanine, arginine, asparagine, glutamine, glutamate, proline, phenylalanine, and threonine, declined sharply in leaves, while inflorescences maintained more stable levels (Figure 4B; Table S3). Glutamate was particularly sensitive: in leaves, concentrations dropped by 79% under all postharvest conditions compared to the control (Figure 4C). Glutamine was also depleted, most severely after 2 days at 10 °C (−83%) (Figure 4D). Interestingly, proline levels increased in both tissues across all treatments, suggesting a possible osmoprotective role in response to storage-induced stress (Figure 4E).

Some amino acids accumulated under specific conditions. For example, histidine peaked in inflorescences at 20 PHD at 4 °C (Table S3), while phenylalanine showed maximum accumulation at 20 PHD at 10 °C (Figure 4G). Branched-chain amino acids (BCAAs), calculated as the sum of valine, leucine, and isoleucine, decreased markedly in both tissues after 2 PHD at 4 °C but partially recovered in leaves at later stages (Table S3).

Essential amino acids (EAAs), calculated as the sum of histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine, showed distinct trends. In inflorescences, they reached their highest values at 20 PHD at 4 °C, while in leaves, levels remained lower and less responsive. Total amino acids followed a similarly divergent pattern, with peaks in inflorescences at 2 PHD at 4 °C and 20 PHD at 10 °C from 1.5- to 2.5-fold higher than control levels.

3.5. Heatmap Pathway Analysis

The tissue-specific metabolic responses to cold storage are summarized in the pathway heatmap (Figure 5 for 4 °C) and Figure S1 (for 10 °C). The visualizations integrate the relative changes in pigments, sugars, antioxidants, amino acids, and carboxylic acids, expressed as fold changes compared to freshly harvested controls. At 4 °C, most metabolic alterations appeared moderate and variable, with some amino acids and antioxidants decreasing primarily in leaves. In contrast, the map corresponding to 10 °C (Figure S1) reveals more extensive and intense shifts across several metabolite classes, especially in leaf tissues. Changes in carboxylic acids such as acetate and pyruvate were consistently observed in both tissues under both storage temperatures. These diagrams provide a comparative overview of postharvest metabolic trends and complement the individual results detailed in Figure 1, Figure 2, Figure 3 and Figure 4 and Tables S1–S3.

4. Discussion

The postharvest physiology of leafy and inflorescent tissues of Friariello Napoletano revealed complex metabolic responses to cold storage, indicating both tissue-specific sensitivity and general biochemical trends. Metabolic adjustments involved pigment degradation, antioxidant dynamics, nitrogen mobilization, and organic acid accumulation, collectively evidencing orchestrated senescence-like changes under low temperature.

A consistent finding was the strong degradation of chlorophylls and carotenoids in leaves, with total chlorophyll a content dropping by over 90% at 4 °C. Interestingly, this degradation was more severe at 4 °C than at 10 °C, suggesting that low-temperature stress may accelerate senescence in leaves. Although FN is a non-climacteric vegetable, basal levels of ethylene are still physiologically relevant in Brassicaceae, including broccoli and cabbage, where ethylene has been shown to accelerate chlorophyll degradation and senescence [37]. These findings suggest a cold-sensitive degradation process, likely involving chlorophyll catabolic enzymes such as chlorophyllase and pheophorbide a oxygenase, initiating pigment breakdown prior to the disassembly of the photosynthetic machinery [38], and support the hypothesis that 10 °C, although warmer, may impose less stress and allow for better pigment retention. In parallel, the α-tocopherol content increased, especially in inflorescences, suggesting a compensatory antioxidant response as chlorophyll-derived phytol is recycled into tocopherol biosynthesis [17]. Redox metabolism was further altered during storage. The progressive depletion of total ascorbate and the shift in the DHA/ASC ratio indicate a compromised antioxidant buffering capacity and the onset of oxidative stress, as supported by Smirnoff [39], who emphasized the relevance of the ascorbate redox state as a marker of oxidative imbalance in plant tissues [40]. These redox perturbations are characteristic of early postharvest senescence and have been associated with increased ROS generation under cold exposure [41,42].

Amino acid profiles showed strong tissue × treatment interactions. Leaves showed a relevant reduction in glutamate, glutamine, and alanine, key nitrogen-rich compounds known to be remobilized during senescence [43]. In contrast, proline and γ-aminobutyric acid (GABA) accumulated significantly in both tissues [44,45]. These metabolites are recognized for their multifunctional role in abiotic stress adaptation, acting as osmoprotectants, ROS scavengers, and in the case of GABA, linking stress signaling with respiratory metabolism via the GABA shunt [46,47].

The accumulation of carboxylic acids such as acetate and pyruvate, particularly in inflorescences, further suggests a shift toward enhanced mitochondrial activity and catabolic energy production. These changes are indicative of a metabolic transition supporting cell maintenance and nutrient recycling under stress [41,48].

The increase in nitrate observed at 20 days and 10 °C may be primarily attributed to microbial nitrification of ammonia released during amino acid deamination in senescing tissues. Although classical autotrophic nitrifiers such as Nitrosomonas and Nitrobacter were not detected in microbial studies on Brassica rapa during storage [49], Proteobacteria, comprising over 75% of the microbiota of Brassicaceae, may include heterotrophic taxa capable of oxidizing ammonia to nitrate. This process, typically soil-based, can occur in stored plant material when residual nitrifying bacteria, oxygen, and suitable pH are present. Recent studies have demonstrated that nitrifying bacteria can also reside on tree leaves, where they convert atmospheric ammonia into nitrates, suggesting that similar nitrification may occur in postharvest tissues [50]. Alternatively, under sustained oxidative stress, non-enzymatic oxidation of ammonia to nitrate—though less efficient—might also contribute, especially in aging tissues where ROS accumulate. These transformations, whether microbial or chemical, are consistent with the nitrogen remobilization processes that characterize early senescence phases [51].

Essential amino acids (EAAs) and branched-chain amino acids (BCAAs) displayed relative accumulation under specific conditions, particularly in inflorescences, suggesting a buffering role for nitrogen and energy metabolism. Their contribution to osmotic adjustment and mitochondrial electron transport has been previously described in other species under abiotic stress [35,52]. Recent integrative reviews have highlighted how postharvest stress triggers profound metabolic rearrangements involving redox balance, membrane integrity, and nitrogen remobilization pathways [53]. These changes are mediated by a coordinated modulation of antioxidants, amino acids, and storage compounds, as confirmed by earlier studies on oxidative stress [42], senescence [38,54], and osmolyte dynamics under abiotic stress [35,36,52].

This study provides a detailed overview of tissue-specific metabolic responses in Friariello Napoletano during cold storage. Although only two storage temperatures and time points were investigated, the results reveal consistent differences between leaves and inflorescences in terms of pigment degradation, antioxidant profiles, amino acid turnover, and ion homeostasis. The data suggest that inflorescences may retain greater metabolic stability and buffering capacity, while leaves are more prone to senescence-related changes. These findings offer a valuable framework for improving the postharvest handling of this traditional leafy vegetable and may support future efforts to preserve its nutritional and functional quality under realistic storage conditions.

5. Conclusions

This work provides a detailed assessment of the tissue-specific metabolic responses of Friariello Napoletano during postharvest storage under cold conditions at 4 °C and 10 °C. Both temperatures, although commonly applied to extend shelf life, induced clear biochemical alterations. Leaves showed rapid pigment degradation (with chlorophyll losses exceeding 90%), depletion of antioxidants, and significant amino acid catabolism, indicating a higher sensitivity to cold stress. Inflorescences, in contrast, retained greater metabolic stability and may contribute to nitrogen buffering through sustained amino acid pools. The accumulation of stress-related metabolites and alterations in ion profiles suggest the activation of defense and adaptive responses. Notably, the observed rise in nitrate at later stages raises the hypothesis of post-storage nitrification processes. Taken together, these results provide a useful perspective on the tissue-specific postharvest behavior of this traditional vegetable and may support the development of more effective storage practices.