A good model would have an R² value of more than 0.6, Q² value of more than 0.6, and a low value of RMSEE and RMSECV [36]. We constructed 3 OPLS models from metabolites annotated in flesh, peel and crown. The constructed model of flesh and peel showed R² value of more than 0.6, Q² value of more than 0.6, and a low value of RMSEE and RMSECV, whereas crown model showed R² value of 0.896, Q² value of 0.432, RMSEE value of 0.509, and RMSECV value of 1.066. These results indicated that only peel and flesh model meet the thresholds for a valid model with a good fit. The low Q² value in crown model showed that the samples from crown part cannot be used to predict ripening stages in pineapple. This is in line with the results obtained from PCA.

Contributing metabolites related to ripening stages could be obtained from the variable importance in the projection (VIP) scores. Based on these scores, the five highest VIP metabolites in the flesh part are melezitose, inositol, xylonic acid, gluconic acid, and raffinose; while for peel part are inositol, mannose, galactose, sucrose, and aspartic acid. Figure 6 shows the dynamic of these VIP metabolites relative intensity (normalized with ribitol) along the ripening process of pineapple. These metabolites were shown to be increased or decreased during the pineapple ripening process. From these VIP metabolites in flesh parts, the raffinose level was in agreement with the previous report that showed a decreased level during ripening process [38]. In addition to the previously reported metabolites that correlate with the ripening process, this study also reports the dynamics of inositol, melezitose, xylonic acid, and gluconic acid in the flesh part during ripening process. Inositol or commonly known as myo-inositol was known to regulate osmotic pressure in blueberry fruit thus maintaining turgor and fruit firmness between the firm cultivar and soft cultivar [19]. In addition to that, inositol might be oxidized to D-glucuronic acid known as a major precursor of the cell wall in Arabidopsis [39]. Therefore, the presence of inositol might also relate to the cell wall in fruit. Melezitose is known to play a role in osmoregulation system [40]. However, comparing the relative intensity trend with inositol, the mechanism underlying these two metabolites might be different to regulate the osmoregulation system during the pineapple ripening process. Even though melezitose relative intensity was considered low compared to other sugar, it was reported in the previous study that it could attract ants in honeydew fruit [41]. Therefore, the accumulation of melezitose in the latter stage of ripening might reflect the attractancy of the fruit in the fully ripe stage.

Xylonic acid relative intensity was shown to be increased during the ripening process (Figure 6a). The increase of this organic acid might be related to the reactive oxygen species (ROS). During fruit ripening, oxidative stress was increased and might result in some changes in fruit, such as changes in skin color or fruit softening. Due to the presence of ROS, fruit antioxidants might act to balance the reduction–oxidation homeostasis [42]. One of the most known fruit antioxidants is ascorbic acid. The previous report stated that xylonic acid is a product of ascorbic acid degradation, thus explaining the increase of xylonic acid during the ripening process [43]. During the pineapple ripening process, raffinose was found to be decreased along with the progression of ripening (Figure 6). In agreement with this result, previous report showed that the raffinose level also decreases in Japanese plum non-climacteric cultivar during its ripening [38]. They reported that the raffinose level in non-climacteric fruit might be related to its ability to alleviate the oxidative process during fruit ripening [38]. Therefore, not only xylonic acid but the level of raffinose might also be related to the reduction–oxidation process along the ripening process. The gluconic acid concentration was shown to be increased during the late-ripening process in pineapple (Figure 6a). This increase might be triggered by the increase of carbon molecules availability during the later stage of pineapple ripening [44]. In addition, gluconic acid intensity increase might also cause by the effect of cell wall degradation, change in cuticle composition and pH of host cells that allow the transition of fungi into their aggressive colonization [44]. The presence of gluconic acid might indicate an infection that could secrets gluconic acid and acidify the pH in fruit such as in apple and mango [45,46].

Metabolite that shows the highest VIP score in peel after inositol is mannose. Mannose is known as a component of the plant cell wall, specifically hemicellulose [47]. The previous report mentioned that the concentration of mannose was decreased during fruit development, hence support our findings
shown in Figure 6b [48]. Similar to mannose, decrease level of aspartic acid in the pineapple ripening process were also reported during ripening of banana [49]. Aspartic acid was known to be a source for umami taste along with glutamic acid [50]. Its level was known to be varied among the fruits. In banana and *Vasconcellea quercifolia*, the level of aspartic acid was found to be decreased along with the ripening progress, while mature or ripe tomato contained more aspartic acid that brings out the umami taste [49,51,52]. Other than the source of umami taste, the level of aspartic acid might also connect with the free auxin level in fruits that affect its ripening [53]. Aspartic acid was found to be conjugated with indole acetic acid (IAA) and lead to degradation of the IAA hormones [53].

Other than the previously reported metabolites, this study found the change of sucrose and galactose level in the peel part might correlate with the pineapple ripening process. This study showed the sucrose level in pineapple peel part was increased during ripening (Figure 6b) while previous report mentioned the increase of sucrose during ripening process in pineapple flesh [21]. Sucrose was known to become a source of sweet taste in food and commonly used as a standard solution for sweetness [54]. However, sucrose in the peel part might not directly affect the sweetness in the flesh. It is reported that sucrose in the peel is lower if compared with the flesh part [55]. Sucrose not only contributes to sweetness, but it also plays a role to regulate fruit development and ripening in strawberry fruit, a non-climacteric fruit [56]. Sucrose accumulation might induce the expression level of the key enzymes in abscisic acid (ABA) hormones pathway, hence promote ripening process in non-climacteric fruit via ABA hormones [57]. Galactose in Figure 6b was shown a decreasing trend along the ripening stages. This might be explained by the relation of galactose with the plant cell wall. The previous report showed that galactose is the major non-cellulosic sugar in the cell wall and significantly decreased during fruit ripening [58,59]. All these metabolites with high VIP score consist of sugars (melezitose, inositol, raffinose, mannose, galactose, and sucrose) and organic acids (xylonic acid, gluconic acid, and aspartic acid).

During ripening process, many biological processes occur, such as cell wall loosening, texture changes, flavor development, chlorophyll degradation, and pigment accumulation [60]. Changes in melezitose and inositol level might indicate the texture changes during pineapple ripening process. Melezitose and inositol are known to regulate fruit firmness that affects the texture or hardness of fruit [19]. In addition to that, inositol, galactose, and mannose levels might be related with cell wall loosening during ripening. Inositol were known to be precursor of D-glucuronic acid of plant cell wall, galactose is a major non-cellulosic sugar in plant cell wall, and mannose are component of hemicellulose in plant cell wall. Therefore, the decrease of these three metabolites might correlate with loosening of cell wall that usually accompanied with decrease level of firmness and increase of gluconic acid [19,44]. Reactive oxygen species also play a role in ripening process to regulate programed cell death and cell aging [61]. This report in line with our results that show decrease level of xylonic acid and increase level of raffinose during ripening process. Both metabolites were known to respond to reactive oxygen species as discussed previously. These biological processes that affected by the ripening process are product of biochemical changes that mediated by plant hormone. Abscisic acid (ABA) and auxin were known to be affecting the ripening process in non-climacteric fruit [60]. Changes in sucrose and aspartic acid during ripening process might affected abscisic acid and auxin, respectively. Therefore, it might modulate ripening process in pineapple fruit.

This study showed the significance of sample preparation to gain more metabolite coverage that is useful for further analysis. Based on metabolites data acquired from GC-MS analysis, flesh and peel data could show clustering separation between C0-C2 stages and C3-C4 stages using principal component analysis (PCA), while the crown part does not show correlation with the ripening process. Orthogonal projection to latent structures (OPLS) regression analysis reveals metabolites that have possible relations to the pineapple ripening process in flesh and peel parts. In the flesh part, melezitose, inositol, xylonic acid, gluconic acid, and raffinose were found to be the five highest important metabolites, while for the peel part are inositol, mannose, galactose, sucrose, aspartic acid. These metabolites were known to be involved during plant cell wall metabolism and osmoregulation system thus affecting the firmness and
shelf life of pineapple, in addition to the redox defense system and non-climacteric ripening hormones. For future applications, these VIP metabolites could be added exogenously to regulate specific effects, for example, the addition of polyamine and ascorbic acid to regulate the shelf life of fruit, as shown in the previous reports [62,63]. In addition, influencing the level of the metabolites through post-harvest treatment was also feasible to be conducted, such as regulating inositol, galactose, and raffinose by cold or heat treatment [64–66]. It must be noted that this study only limited to the “Queen” cultivar. Future study using other widely known cultivars, such as “Smooth Cayenne”, is still needed to enrich the information regarding pineapple ripening process. Regardless, this study might become a basis for resolving the post-harvest issue in the pineapple industry by controlling important metabolites influenced in the ripening process.

# 4. Materials and Methods

## 4.1. Plant Materials

Pineapple (*Ananas comosus*) fruit from Indonesia corresponding to 5 different ripening stages were used in this study (Figure 1). To set the same harvest time at the end of April 2019, ethephon treatment was used to induce fruit development around November to December 2018. Cultivars of pineapple used in this study to represent the important cultivar from the pineapple Industry is cv. Mahkota Bogor 'Queen'. Three samples (biological replicates) from different plants were collected from each ripening stage for Queen cultivars from the cultivation period of November 2018—April 2019 in Center for Tropical Horticulture Studies, Bogor Agricultural University (CENTROHS, Bogor Agriculture University), Bogor, Indonesia (minimum temperature 21 °C and maximum temperature 35 °C). Ripening stage determination was conducted with the help of a trained panelist according to peel color changes [5].

## 4.2. Optimization of Sample Preparation

### 4.2.1. Comparison between Food Processor and Freeze-Drying Methods

Pineapple fruit was cut into half then the flesh was diced using a stainless-steel knife. Diced flesh with 1 × 1 cm size from one half of fruit was subjected to freeze dry-extraction method using VD-800F Freeze dryer (Taitec, Saitama, Japan). This flesh was put into a Pyrex tube and closed with holed parafilm before being quenched with liquid nitrogen and lyophilized. Diced flesh with 1 × 1 cm size from the other half of the fruit was homogenized using hand immersion-blender WSB-33XJ (Waring Commercial, Pennsylvania, United States of America) before extraction. Ten milligrams of both samples were subjected to extraction following the method described previously prior to GC-MS analysis [66].

### 4.2.2. Comparison of Crown, Flesh, and Peel of Pineapple Fruit

The pineapple was cut into three different parts, crown, flesh, and peel. Crown part was analyzed by cutting the leaves into a 1x1 cm size before quenching by liquid nitrogen and freeze-dried. Peel part was analyzed by scraping the peel into a 1x1 cm size using a stainless-steel knife before quenching and freeze-drying. Flesh part was analyzed according to above description. Lyophilized sample was homogenized using Multi-beads shocker (Yasui Kikai, Osaka, Japan). Ten milligrams of the homogenized sample of each part of pineapple was subjected to extraction and GC-MS analysis [66].

## 4.3. Sample Preparation and Extraction of Pineapple from Different Ripening Stages

Pineapple fruit of different ripening stages collected in Indonesia was divided into three parts: Crown, flesh, and peel. Each part was cut into small pieces and placed into a Pyrex tube covered with holed parafilm. Samples were quenched by immersing the Pyrex tubes in liquid nitrogen prior to lyophilization using the VD-800R Freeze dryer (Taitec, Saitama, Japan). Freeze-dried pineapple
samples were transported from Indonesia to Japan within a day. The samples were homogenized and ground into a fine powder using Multi-beads shocker. This extraction method was conducted based on the method described in our previous study [66]. Pineapple samples (10 mg), blank samples and quality control (QC) samples were extracted together and lyophilized in a single day. QC samples were prepared by collecting small aliquots of each sample obtained in this study.

All samples were extracted using the mixture of methanol (Wako Chemical, Osaka, Japan), chloroform (Kishida Chemical Co. Ltd, Osaka, Japan), ultrapure water (Wako Chemical, Osaka, Japan); in the ratio of 2.5/1/1 (v/v/v) containing 100 µg/mL ribitol as an internal standard. The mixture was incubated at 37 °C, 1200 rpm for 30 min followed by centrifugation for 3 min at 40 °C. Six hundred microliters of supernatant was transferred to a new 1.5 mL microtube and 300 µL water was added into the mixture. The sample mixture was centrifuged for 3 min at 40 °C and 400 µL of supernatant was transferred to a new microtube and closed with a holed cap. The solvent from the sample mixture was evaporated for 1 h at room temperature followed by lyophilization overnight. All samples were analyzed in triplicates (n = 3). One hundred microliters of methoxyamine hydrochloride (20 mg/mL in pyridine) was added into lyophilized samples and incubated in thermomixer for 90 min at 30 °C. Subsequently, 50 µL N-Methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA) (GL Sciences) was added to the samples and incubated for 30 min at 37 °C.

## 4.4. GC-MS Analysis

GC-Q/MS analysis was performed on a GC-MS QP2010 Ultra (Shimadzu, Kyoto, Japan) equipped with an InertCap 5 MS/NP column (GL Sciences). Tuning and calibration of the mass spectrometer were done prior to analysis. One microliter of the derivatized sample was injected in split mode, 25:1 (v/v), with an injection temperature of 230 °C. The carrier gas (He) flow was 1.12 mL/min with a linear velocity of 39 cm/s. The column temperature was held at 80 °C for 2 min, increased by 15 °C/min to 330 °C, and then held for 6 min. The transfer line and ion source temperatures were 250 and 200 °C, respectively. Ions were generated by electron ionization (EI) at 0.94 kV. Spectra were recorded at 10,000 u/s (check value) over the mass range m/z 85–500. A standard alkane mixture (C8–C40) was injected prior to analysis for peak identification.

## 4.5. GC-MS Data Analysis

The raw data obtained from the analysis was converted to the AIA file using GCMS solution software package (Shimadzu, Kyoto, Japan). Peak alignment, peak filtering and annotation was conducted by MS-DIAL ver. 4.00 using GCMS-5MP Library (Riken, Kanagawa, Japan). Peak confirmation of important metabolites, namely inositol, mannose, galactose, melezitose were conducted by co-injection with authentic standard (Wako Pure Chemical Industries Ltd., Osaka, Japan; Sigma-Aldrich Japan Ltd., Tokyo, Japan; Alfa Aesar Ltd., Heysham, UK).

## 4.6. Statistical Analysis

Annotated metabolites from GC-MS analysis were pre-treated by normalizing each metabolite peak height to internal standard (ribitol). Normalized data were scaled by autoscaling and without transformation subjected to PCA (Principal Component Analysis) using SIMCA-P+ version 13 (Umetrics, Umea, Sweden). Principal component analysis is an unsupervised analysis that is useful as a dimension-reduction tool in order to easily observe trends, clusters and outliers [25]. Other than PCA, a projections to latent structures (PLS) regression model that is constructed from the maximal correlation of explanatory variable (x-variable) with response variable (y-variable) offers ranking of metabolites correlation with a certain quantitative phenotype [67,68]. In particular, orthogonal projections to latent structures (OPLS) regression model is very useful for reducing many variables to limited latent variables [26]. Parts that show ripening trends in PCA were subjected to OPLS (Orthogonal Projections to Latent Structures) analyses using SIMCA-P+ version 13. From OPLS analyses, variable importance in projection (VIP) were calculated for each metabolite. The top five highest VIP score's metabolites in
each pineapple part were statistically analyzed by analysis of variance (ANOVA) with Tukey's post hoc test performed using JASP Version 0.11.1 (JASP Team, Amsterdam, Netherlands). The statistical analysis was conducted to evaluate the differences among mean values of VIP metabolites obtained from all ripening stages. Differences were considered significant if *p* < 0.05.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2218-1989/10/4/134/s1, Table S1: Complete list of annotated metabolites with RSD < 20% for sample preparation analysis, Table S2: Complete list of annotated metabolites with RSD < 20% for different part analysis, Table S3: Complete list of annotated metabolites with RSD < 20% for ripening process analysis, Table S4: metabolites with VIP score (more than 1) and its coefficient, Figure S1: Visual experimental design on pineapple ripening study, Figure S2: Venn diagram of annotated metabolites in crown, flesh, and peel part on different parts analysis, Figure S3: Venn diagram of annotated metabolites in crown, flesh, and peel part on pineapple ripening analysis.

**Author Contributions:** M.M.M.I. and S.P.P. designed the experiments; M.M.M.I. performed the experiments, analyzed the data, and wrote the manuscript; S.R. advised sample treatment design and aided in data interpretation; S.P.P. analyzed the data and wrote the manuscript. E.F. conceived the study and participated in its design and coordination. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors thank Baesuni and Rizal from the Center for Tropical Horticultural Studies for their help during sample collection. The authors also thank Fenny Martha Dwivany and Intan Taufik from School of Life Sciences and Technology, Institut Teknologi Bandung for assistance in sample preparation and freeze-drying. This study represents a portion of a dissertation submitted by MM. Malikul Ikram to Osaka University in partial fulfillment of requirements for his Ph.D.

**Conflicts of Interest:** The authors declare no conflict of interest.

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Article

# Cross-Species Comparison of Fruit-Metabolomics to Elucidate Metabolic Regulation of Fruit Polyphenolics Among Solanaceous Crops

Carla Lenore F. Calumpang, Tomoki Saigo, Mutsumi Watanabe and Takayuki Tohge *

Graduate School of Science and Technology, Nara Institute of Science and Technology (NAIST), Ikoma, Nara 630-0192, Japan; calumpang.carla_lenore.bw2@bs.naist.jp (C.L.F.C.); saigo.tomoki.sn6@bs.naist.jp (T.S.); mutsumi@bs.naist.jp (M.W.)

* Correspondence: tohge@bs.naist.jp; Tel.: +81-743-72-5480

Received: 20 March 2020; Accepted: 14 May 2020; Published: 19 May 2020

**Abstract:** Many solanaceous crops are an important part of the human daily diet. Fruit polyphenolics are plant specialized metabolites that are recognized for their human health benefits and their defensive role against plant abiotic and biotic stressors. Flavonoids and chlorogenates are the major polyphenolic compounds found in solanaceous fruits that vary in quantity, physiological function, and structural diversity among and within plant species. Despite their biological significance, the elucidation of metabolic shifts of polyphenols during fruit ripening in different fruit tissues, has not yet been well-characterized in solanaceous crops, especially at a cross-species and cross-cultivar level. Here, we performed a cross-species comparison of fruit-metabolomics to elucidate the metabolic regulation of fruit polyphenolics from three representative crops of Solanaceae (tomato, eggplant, and pepper), and a cross-cultivar comparison among different pepper cultivars (*Capsicum annuum* cv.) using liquid chromatography-mass spectrometry (LC-MS). We observed a metabolic trade-off between hydroxycinnamates and flavonoids in pungent pepper and anthocyanin-type pepper cultivars and identified metabolic signatures of fruit polyphenolics in each species from each different tissue-type and fruit ripening stage. Our results provide additional information for metabolomics-assisted crop improvement of solanaceous fruits towards their improved nutritive properties and enhanced stress tolerance.

**Keywords:** polyphenolics; solanaceous crops; *capsicum annuum*; pepper; tomato; eggplant; fruit ripening; metabolomics; tissue-specificity; flavonoid

## 1. Introduction

Solanaceae (nightshade family) is an agronomically- and botanically-diverse plant taxonomic group, with members ranging from vegetable crops through medicinal plants to ornamentals. Few representative crops of economic importance include tomato (*Solanum lycopersicum*), eggplant (*Solanum melongena*), and pepper (*Capsicum annuum*). With available genome sequences [1,2] and genetic resources from different tomato varieties and natural mutants [3], tomato has become the first model crop for fleshy fruit ripening, fruit pigmentation, specialized (secondary) metabolism, and plant defense [4–9]. Subsequently, metabolomic data of specialized metabolites from tomato fruits [4,10–12] and a tomato metabolite database [5,13–15] have been published and developed via a mass-spectrometry (MS)-based metabolomic analysis for fruit-omics approach. The metabolomics approach, focusing on specialized metabolism, is also being used on other solanaceous plants, including tobacco (*Nicotiana spp.*) [16,17], potato (*Solanum tuberosum*) [18–20], petunia (*Petunia spp.*) [21], and *Atropa* spp. [22]. However, developing similar resources for related crops is still a goal of the scientific community [23].
In the point of fruit-omics, pepper (Capsicum spp.) is increasingly gaining recognition as an excellent model plant for solanaceous fruit-omics [8,12,24,25]. Pepper (Capsicum spp.) has a world yield of 184,742 hg/ha and a world production of almost 40 million tons in 2018 [26]. Consumed either raw, cooked, as a spice, or food colorant [27], pepper have known human health benefits, such as weight reduction [28], pain relief [29], and cancer prevention [30]. Peppers can also be used as sprays for crowd control and personal defense since some cultivars cause skin irritation [31,32]. Capsaicinoids (e.g., capsaicin, 8-methyl-N-vanillyl-6-nonenamide) have been the main focus of metabolic diversity studies in pepper due to their dramatic bioactivities; however, pepper polyphenols are less highlighted, since fruit metabolomics focusing on polyphenols have already been well-investigated in tomato species. Furthermore, tissue-specific patterns of polyphenol distribution in pepper fruit have not been well-characterized during fruit ripening, specifically in the peel and pericarp. There is still a need to investigate metabolic shifts of polyphenols among different fruit tissues during different stages of ripening in pepper through fruit-omics analysis, because of the fact that significant metabolic shift of polyphenolic compounds during fruit ripening is observed in different fruit tissues, such as in the case of tomato species [10,33–35]. Furthermore, capsaicinoids share the same biosynthetic precursors, e.g., phenylalanine and *p*-coumarate, which can cause differences in metabolic shifts of polyphenols in pepper versus tomato. The pungent properties in pepper are due to the bioactivity of capsaicinoids, an exclusive trait amongst capsicum species and is not exhibited by other solanaceous crops. Pepper also has a variety of fruit colors among cultivars during fruit ripening, which mainly indicate variation in fruit carotenoids and chlorophylls, but also in fruit polyphenolics, in the case of purple pepper cultivars. While total amount of flavonoid aglycones have been compared between pungent and non-pungent pepper cultivars [36], further metabolomic analysis of fruit polyphenolics in terms of their tissue specificity and metabolic shift during fruit ripening should be highlighted.

On the other hand, fruit-metabolomics on eggplant polyphenolics is still in progress with only a few studies having been performed. The metabolomic analysis of different *Solanum* species, including five eggplant (*S. melongena*) accessions, three accessions from an eggplant wild ancestor (*Solanum insanum*), and two from scarlet eggplant (*Solanum aethiopicum*), through a LC-MS based strategy revealed metabolic diversity of anthocyanins, chlorogenic acid derivatives, flavonoids, triterpenoid alkaloids and triterpenoids, and novel biosynthetic frameworks [37]. In the untargeted metabolomics of the fruit of twenty-one eggplant (*S. melongena*) accessions using GC-MS and LC-MS, some accession-specific specialized metabolites were putatively identified [38]. Moreover, the metabolic quantitative trait locus (mQTL) from eggplant fruit was investigated for identification of a genomic region of productivity for chlorogenic acid and two anthocyanin pigments [39].

Plant polyphenols are a large group of plant specialized metabolites, which can be subdivided into several large sub-groups of major phyto-antioxidants, for example flavonoids and hydroxycinnamates [40,41]. In solanaceous plants, flavonoids involved in the stress resistance against abiotic and biotic stressors, such as pathogen infection [42,43], ultraviolet light (UV) [44,45], nitrogen deficiency [46], and cold temperature [47]. Flavonoids are also involved in plant reproduction, such as attracting insect pollinators and seed dispersers [48]. Pigment flavonoids, such as anthocyanins, upon absorbing visible (VIS) light contribute to the red and purple pigmentation of solanaceous fruits [8]. Intake of flavonoids and hydroxycinnamates also reduce the risk of human disease due to their anti-cancer and antioxidant activities based on in vitro assays [49–51]. Chlorogenates have recognized human health benefits, such as antioxidant, antiviral, hepatoprotective, and anti-hypoglycemic properties [52]. With their beneficial roles in human health, tissue-specific accumulation patterns of polyphenols in pepper fruit during ripening can be focused. Since polyphenols contribute a role in plant stress response, there is a propensity for such compounds to accumulate more in the fruit peel than in the pericarp, which has already been observed in tomato fruit [10,53–56].

To develop a baseline on the polyphenolic compounds already detected in our solanaceous crops of study, a phytochemical survey of polyphenolics reported in the fruits of tomato, eggplant, and pepper was investigated via a phytochemical database KNApSAcK (http://www.knapsackfamily.
com/KNApSAcK_Family) and literature search of fruit metabolomic analyses conducted on these species [4,10,25,57–59] (Figure 1A, Table 1). In tomato species, major polyphenolic compounds include derivatives of hydroxycinnamates, flavonols, and anthocyanins [5,10,13–15,57]. For eggplant, its fruit peel is rich in anthocyanins, mainly delphinidin derivatives [60], while the pericarp consists mainly of chlorogenates (CGAs) [61]. Flavonol glycosides have also been detected in the pericarp of eggplant fruit [62]. Polyphenolic compounds detected from pepper are much more diverse, with flavonoids being the main compound group and flavones and flavonols the major flavonoid subfamilies detected. Chlorogenates have also been identified from pepper fruit [59]. Based on the data for all three solanaceous crops of study, common polyphenolic compounds present in their fruit include hydroxycinnamates (specifically chlorogenates and their positional-isomers), chalconoids/stilbenoids, flavanones, and flavonol derivatives, such as rutin (quercetin-3-O-rutinoside) and kaempferol-3-O-glucosides (Figure 1A, Table 1). A survey of representative polyphenolic compounds have been reported in the metabolic analysis of the solanaceous fruits, a metabolic framework of major polyphenolics among three solanaceous species is illustrated (Figure 1B).

<figure><img src="image_82.png" /><figcaption>**Figure 1.** Major polyphenolic compounds reported in mature fruit of solanaceous crops. (A) chemical structure of major polyphenolic specialized metabolites found in solanaceous crops. Chlorogenates (CGAs), naringenins, and flavonols are shown. (B) the biosynthetic framework of polyphenolic compounds in solanaceous crops. Biosynthetic pathways were constructed by an online database search and literature review of major polyphenolic compounds in solanaceous crops. Color: Blue, hydroxycinnamates; black, stilbenoids/flavanones and chalcones; purple, anthocyanins; and green, flavonols/flavones; orange, amino acid. Yellow color inside of circle indicates major accumulation form in the plant. Abbreviations: Phe, phenylalanine; cou, coumaroyl; caf, caffeoyl; fer, feruloyl; sin, sinapoyl; glyc, glycoside; deriv, derivative. K, kaempferol; Q, quercetin; Narichal, naringenin chalcone; CGA, chlorogenate; DiCGA, dicaffeoyl-chlorogenate; Glc, glucose; Rha, rhamnose; Api, apiose; and hex, hexose.</figcaption></figure>

Peppers consist of approximately 35 species [63] with five of them domesticated independently (C. annuum, C. chinense, C. frutescens, C. baccatum, and C. pubescens). C. annuum is the largest and most widely-cultivated species having both spicy (chili) and sweet varieties with different kinds of pigmentation in their fruits [8,64]. However, due to hybridization, pepper currently has around 50,000 varieties, providing a wide range of chemical variability within the same species exhibiting similar physical characteristics [12]. Despite such physical similarity within species, such genetic diversity still provides a large pool of chemical variability within species in terms of tissue-specificity, developmental ripening stage, and cultivar. Furthermore, the process of fruit ripening involves a tight metabolic regulation in conjunction with developmental stage [65], involving biochemical reactions resulting in changes in fruit flavor, texture, aroma, hardness, nutrient composition, and color [25,66].
Since some polyphenolic compounds are involved with flavor and fruit color, changes in polyphenolic content can occur during fruit ripening [15,67]. Accumulation patterns of major flavonoid glycosides in terms of subspecies and cultivar-specificity were evaluated from ripe fruits across thirty-two pepper accessions, including *C. annuum*, *C. chinense*, *C. frutescens*, and *C. baccatum* [25]. As one of the results given by this research, main pepper flavonoid decorations were observed as a metabolic polymorphism within these pepper species. Previously, primary metabolites (sugars, amino acids, and organic acids) were compared between tomato (*S. lycopersicum*) and pepper (*Capsicum chilense*) during fruit ripening [66,68] but not specialized metabolites, specifically polyphenols. Since some metabolomic studies of primary metabolites have been conducted for pepper fruit, such fruit-metabolomics studies should be expanded to include tissue-specificity of polyphenol accumulation in pepper peel versus pericarp during different stages of fruit ripening among various pepper cultivars. Furthermore, after metabolic polymorphisms of polyphenolic profiling were characterized through metabolic profiling with acid hydrolysis [49], a complete representation of the polyphenolic profile for this pepper variety can additionally be focused with metabolic profiling of the individual derivative forms. Meanwhile in hot pepper (*Capsicum annuum* "CM334") fruit, only the pericarp was analyzed for polyphenolic accumulation patterns during ripening [69]. Additionally, the identification of polyphenolic compounds specific for pepper against other solanaceous crops has not yet been performed, nor have the patterns of accumulation in relation with fruit tissue type been reported in some pepper species. Therefore, polyphenolic fruit-omics data developed in tomato research can possibly be used for the extension to other solanaceous crops, such as pepper species and cultivars through other fruit-omics approaches.

<table><caption>**Table 1.** Major polyphenolic compound groups reported in the solanaceous fruits.</caption><thead><tr><th>Compound Family</th><th>Compound Group</th><th>Tomato<br>(S. Lycopersicum)</th><th>Eggplant<br>(S. Melongena)</th><th>Pepper<br>(C. Annuum)</th></tr></thead><tbody><tr><td rowspan="6">Hydroxycinnamates</td><td>CGAs</td><td>√</td><td>√</td><td>√</td></tr><tr><td>di-CGAs</td><td>√</td><td>√</td><td></td></tr><tr><td>tri-CGA</td><td>√</td><td></td><td></td></tr><tr><td>p-coumaroyl-glycosides</td><td>√</td><td></td><td>√</td></tr><tr><td>caffeoyl-glycosides</td><td>√</td><td>√</td><td>√</td></tr><tr><td>feruloyl-glycosides</td><td>√</td><td>√</td><td>√</td></tr><tr><td>Chalconoids/stilbenoids</td><td>sinapoyl-glycosides</td><td>√</td><td>√</td><td>√</td></tr><tr><td rowspan="3">Flavonoids<br>flavonols</td><td>phloretin deriv.</td><td>√</td><td></td><td>√</td></tr><tr><td>naringenin chalcone deriv.</td><td>√</td><td>√</td><td></td></tr><tr><td>naringenin deriv.</td><td>√</td><td>√</td><td></td></tr><tr><td rowspan="5">anthocyanins</td><td>kaempferol deriv.</td><td>√</td><td>√</td><td>√</td></tr><tr><td>quercetin deriv.</td><td>√</td><td>√</td><td>√</td></tr><tr><td>isorhamnetin deriv.</td><td>√</td><td></td><td>√</td></tr><tr><td>myricetin deriv.</td><td>√</td><td>√</td><td></td></tr><tr><td>delphinidin deriv.</td><td>√</td><td>√</td><td>√</td></tr><tr><td rowspan="3">flavones</td><td>petunidin deriv.</td><td>√</td><td>√</td><td>√</td></tr><tr><td>malvidin deriv.</td><td>√</td><td></td><td></td></tr><tr><td>apigenin deriv.</td><td></td><td></td><td>√</td></tr><tr><td></td><td>luteolin deriv.</td><td></td><td></td><td>√</td></tr></tbody></table>

Here, we performed a cross-species comparison of representative phenolic compounds among three solanaceous crops (tomato, eggplant, and pepper) during fruit ripening, additionally including different pepper cultivars to address metabolic regulation of fruit polyphenolic metabolism among pepper varieties exhibiting differences in pigmentation and pungency. We focused on differences in the metabolic accumulation pattern between tissue types (peel and pericarp) and three fruit ripening stages (immature, green/purple mature, and final mature) to develop a better understanding of metabolic trade-off in fruit polyphenolics. Such information can allow breeders to optimize the biosynthesis
of health-related polyphenolic compounds in pepper during their developmental stage of harvest. Metabolic signatures provided here, provide significant information for future functional genomics approach as well as for the metabolomics-assisted crop breeding of solanaceous fruits towards their nutritive improvement and stress tolerance enhancements.

## 2. Results

### 2.1. Metabolite Profiling of Major Polyphenolic Compounds in Fruit Tissues Among Solanaceous Crops

Metabolomic profiling of representative polyphenolic compounds from the fruit extracts of two tissue types (peel and pericarp) was conducted on three major solanaceous crops through liquid-chromatography-mass spectrometry (LC-MS) (Supplemental Table S1). Tomato, eggplant, and pepper were cultivated in the field and harvested for fruit-metabolomics analysis. Jalapeño pepper, a variety of chili pepper producing capsaicinoid compounds [70], was chosen for our cross-species analysis in order to understand tissue-specific metabolic trade-offs during metabolic shifts in fruit polyphenolics. Due to available studies on tissue-specific accumulation patterns of polyphenolic compounds in tomato fruit, tomato was used for the reference extracts for the peak annotation and analysis of metabolic change during fruit ripening. In our analysis, thirty-nine polyphenolic compounds were detected and annotated (Figure 2A; $log_2$FC (mature/immature), up, > 2.0; down, < 0.5), including eggplant and pepper-specific flavonoid-derivatives. An upregulation of 19 polyphenolic compounds in the peel (4 chalcones, 9 flavonols, and 6 hydroxycinnamates) and 14 polyphenols in the pericarp (4 chalcones, 5 flavonols, and 5 hydroxycinnamates), were observed (Figure 2B) in tomato. Additionally, 8 polyphenolic compounds had higher upregulation in the peel than in the pericarp for tomato, which include one hydroxycinnamate (cou_hex-II, coumaroyl-hexoside II) and 7 flavonols (Q3G2''A6''R7G, quercetin-3-O-(2''-O-apiosyl-6''-O-rhamnosyl)glucoside-7-O-glucoside; Q3G2''A6''R, quercetin-3-O-(2''-O-apiosyl-6''-O-rhamnosyl)glucoside; K3G2''A6''R, kaempferol-3-O-(2''-O-apiosyl-6''-O-rhamnosyl) glucoside; rutin; K3G6''R, kaempferol-3-O-(6''-O-rhamnosyl)glucoside; Q3G, quercetin-3-O-glucoside; Q3R, quercetin-3-O-rhamnoside; and K3R, kaempferol-3-O-rhamnoside), as reported by Mintz-Oron et al. 2008 [10] in metabolomic analysis of tomato cv. Ailsa Craig. As reported in the other reports [53–56], the most abundant flavonol-glycoside, rutin, had more elevated levels in the peel than the pericarp (Figure 2A).

A cross-species comparison of the accumulation patterns of representative polyphenolic compounds between two tissue types from the fruits of three solanaceous crops was conducted. Two hydroxycinnamates (cou_hex-II; caf_hex_III, caffeoyl-hexoside III) were upregulated in the fruit peel among all three crops, while none were commonly upregulated in the pericarp. Between tomato cv. M82 and Jalapeño pepper, six polyphenols were commonly upregulated among their fruit peel while none were upregulated in the pericarp. Out of the six polyphenols upregulated in the fruit peel of both tomato cv. M82 and jalapeño pepper, two were hydroxycinnamates (caff_hex_I and caf_hex-II), two were flavonols (Q3G6''R7G; K3G6''R), and two were chalcones (narichal, naringenin chalcone; narichal_hex_I, naringenin chalcone-hexoside I). Meanwhile, a comparison between jalapeño pepper and eggplant indicated an upregulation of three hydroxycinnamates (cou_hex_III; feru_hex_I, feruloyl-hexoside I; feru_hex_II) in both crops for their fruit peel, while only feru_hex_I was upregulated in the pericarp for both. Species-specific polyphenols were also upregulated in jalapeño pepper, wherein coumaroyl-hexoside I (cou_hex_I) was specific for pepper in both the fruit peel and pericarp. Two other hydroxycinnamates were specifically upregulated only in pepper, with 5-chlorogenate (5CGA) upregulated in the peel and feru_hex_II in the pericarp. Jalapeño pepper showed a greater number of downregulated polyphenols in the peel (14) and pericarp (26), with an especially significant reduction of flavonol-derivatives in both peel and pericarp at fruit mature stage. Our cross-species comparison of fruit-omics with tissue specificity, indicated that pepper polyphenolic metabolism clearly has an opposite metabolic shift between hydroxycinnamate and flavonoid biosynthesis during fruit ripening in both peel and pericarp (Figure 2).
<figure><img src="image_83.png" /><figcaption>**Figure 2.** Metabolite profiling of major polyphenolic compounds between fruit tissues among three major solanaceous crops. (A) metabolic shift during fruit ripening (log<sub>2</sub>FC(mature/immature)) in peel and pericarp of tomato, eggplant and pepper fruits is presented. (B) venn's diagrams show conserved metabolic changes within and between fruit tissues among three solanaceous crops. Metabolites which are commonly up- (>2.0) or down-regulated (<0.5) are shown. MeV software (http://www.mev.tm4.org/) was used for data visualization. Abbreviations: cou, coumaroyl; caf, caffeoyl; fer, feruloyl; CGA, chlorogenate; DiCGA, dicaffeoyl-chlorogenate; K, kaempferol; Q, quercetin; Glc/G, glucose; Rha/R, rhamnose; Api/A, apiose; hex, hexose; Phi, phloretin; narichal, naringenin-chalcone; flv, putative flavonol-derivatives.</figcaption></figure>

## 2.2. Metabolic Shifts of Polyphenolics Different Pepper Cultivars During Fruit Ripening

The metabolic analysis of polyphenolic compounds in pepper fruits was extended from the pungent jalapeño pepper to include other sweet pepper cultivars. Due to the high variability among pepper cultivars in terms of color, shape, and pungency, six pepper cultivars of different visible phenotypes caused by differences in pigment composition (elphinidinnone, carotenoid-type, and anthocyanin-type) were selected. Six different cultivars of pepper (C. annuum) including four sweet cultivars (green paprika, green pepper, yellow paprika, and red paprika), a pungent cultivar (jalapeño pepper), and an anthocyanin-type pepper (purple pepper) were selected for comparison during three fruit ripening stages (immature, green mature, and red or final mature) (Figure 3). In terms of color, five cultivars had red-colored final mature fruits while yellow paprika was yellow at final maturity. During the immature and green mature stages of ripening, purple pepper was purple-colored while the other five cultivars were colored green. In terms of pungency, jalapeño pepper was the only pungent cultivar and the other cultivars were non-pungent or sweet. In terms of fruit shape, purple and jalapeño peppers were pointed while the other cultivars were bell-shaped. Purple pepper differs from other sweet pepper cultivars since most sweet cultivars are bell-shaped [71].

Upon comparing the polyphenol distribution patterns between fruit peel and pericarp, no clear common pattern was observed among the six pepper cultivars, however they are slightly separated in each tissues and cultivars (Figure 4A). All anthocyanins which are involved in pigmentation, specifically purple to black pigmentation in pepper tissues [72], were only detected in purple pepper and not in other pepper cultivars, since purple pepper is the only cultivar exhibiting a developmental stage with purple color fruit (Figure 4B and Supplemental Table S1).
<figure><img src="image_84.png" /><figcaption>**Figure 3.** Pepper cultivars used in this study. Fruits from jalapeño pepper cv. "Jalapeño", red paprika pepper cv. "Flupy Red EX", purple pepper cv. "Nara Murasaki", green pepper cv. "Miogi", yellow paprika cv. "Sonia Gold", and green paprika pepper cv. "New Ace", were cultivated for our analysis.</figcaption></figure>

In the pungent cultivar (jalapeño pepper), most polyphenols were upregulated in the peel during both green mature (14 polyphenols) and final mature (14 polyphenols) stages. Meanwhile, most polyphenols were upregulated in the pericarp during its immature stage (24 polyphenols). More hydroxycinnamates were upregulated in the peel (9) than in the pericarp (4) during its red mature stage. An elevation of eight hydroxycinnamates, namely coumaroyl-hexosides (cou_hex_I, II, III), caffeoyl-hexosides (caff_hex_I, II, III), and feruloyl-hexosides (feru_hex_I and II), as well as a reduction of almost all flavonoids except rutin and putative flavonoid one (flv_1), were suggested as candidate fruit ripening markers in the peel in pungent jalapeño pepper. However, these metabolic shifts were not observed in the other pepper cultivars except purple pepper. For the non-pungent cultivar (green pepper), more hydroxycinnamates were upregulated in the pericarp (11) than in the peel (1) at its red mature stage. Reduction of chlorogenates (3CGA, 4CGA, and 5CGA), were observed in the peel, but CGAs were elevated in the pericarp. Di-caffeoyl-chlorogenate_I (DiCGA_I) is the product of both 3CGA and 4CGA while DiCGA_II is the product of both 3CGA and 5CGA. The upregulation of both DiCGA_I and DiCGA_II suggested to explain the absence of 3CGA from the pericarp. In the sweet green paprika pepper, flavonoids were upregulated in the pericarp during its red mature stage. Furthermore, more hydroxycinnamates were upregulated in the pericarp (11) than in the peel (1) at its red mature stage. Eleven candidate ripening markers were identified for green paprika pepper, which include: Three chlorogenates (3CGA, 4CGA, and 5CGA), three di-chlorogenates (DiCGA_I, II, III), two coumaroyl-hexosides (cou_hex II, III), one caffeoyl-hexoside (caff_hex_III), and two feruloyl-hexosides (feru_hex I, II). In red paprika pepper, most polyphenols were upregulated in both peel (21 polyphenols) and pericarp (22 polyphenols) during its immature stage. Only two hydroxycinnamates were upregulated its red mature stage in the peel (cou_hex_I and cou_hex_III) while one hydroxycinnamate accumulated more at its red mature stage in the pericarp (cou_hex_I). In yellow paprika pepper, only two hydroxycinnamates (3CGA and DiCGA_III) were upregulated in the peel, while three hydroxycinnamates (DiCGA_III, fer_hex_I and fer_hex_II) were upregulated in the pericarp at its red mature stage. Flavonoid mono- and di-glycosides were upregulated during the course of ripening for both tissues, specifically K3G6''R in the peel and K3R in the pericarp. In purple pepper, more hydroxycinnamates were upregulated in the peel (7) than in the pericarp (3) in its red mature stage. Finally, one chlorogenate (3CGA), two di-chlorogenates (DiCGA_I, III), one
coumaroyl-hexoside (cou_hex_I), two caffeoyl-hexosides (caff_hex I, II), and one feruloyl-hexoside (fer_hex_II), were selected as ripening marker for polyphenolics of purple pepper (Figure 4B). K3G6''R, Q3R, rutin, and Q3G2''A6''R were upregulated in both tissues during immature stage in purple pepper. Finally, the metabolic shifts observed in the cross-species comparison was the opposing direction of metabolic shift between hydroxycinnamate and flavonoid biosynthesis during fruit ripening in both the pungent cultivar (jalapeño pepper) and anthocyanin-producing purple pepper, but not in other carotenoid-type pepper cultivars.

<figure><img src="image_85.png" /><figcaption>**Figure 4.** Metabolic shifts of polyphenolics in different fruit tissue among six pepper cultivars during fruit ripening. (A) principal component analysis (PCA) of polyphenolics in different pepper cultivars during fruit ripening. The plots were applied for the 39 metabolites with the average values from 3 biological replications. PCA was conducted by the MultiExperiment Viewer [73]. Principal component (PC) triangles and circles indicate peel and pericarp, respectively. Coefficient correlation was estimated by person correlation method using MeV software. (B) heatmap visualization of metabolite data is normalized and scaled by log<sub>2</sub>FC (mean/average_mean) for each metabolite. Abbreviations: cou, coumaroyl; caf, caffeoyl; fer, feruloyl; CGA, chlorogenate; DiCGA, dicaffeoyl-chlorogenate; Glc/G, glucose; Rha/R, rhamnose; Api/A, apiose; hex, hexose; Phi, phloretin; flv, putative flavonol-derivatives; Narichal, naringenin chalcone; Im, immature stage; Gm(Pm), green mature (or purple mature stage); and Rm(Ym), red mature (or yellow mature stage).</figcaption></figure>
### 3. Discussion

Polyphenols are primarily involved in physiological response against abiotic and biotic stressors [42, 44,46,47] and occasionally in plant reproduction [48]. Due to the phytochemical functions of these compounds, polyphenolic which are accumulating on the surface of the fruit, have been focused. Tomato polyphenols including flavonoids and hydroxycinnamates indeed were identified at higher levels in the fruit peel than in the pericarp [10]. Because of the recognized human health benefits of these polyphenolic compounds, their accumulation patterns during different stages of ripening in economically-important crops would be important to both farmers and consumers. Comparison of representative polyphenolic compounds that accumulate in the peel and pericarp during three different stages of fruit ripening, particularly polyphenols that are specifically up-regulated only in jalapeño pepper. These polyphenolic compounds are also upregulated in the other sweet pepper cultivars, but at different stages of fruit ripening (Figure 4).

Comparison of individual polyphenolic compounds suggest possible metabolic markers that are specifically upregulated during fruit ripening. In the pungent pepper cultivar (jalapeño pepper), we observed an elevation of three coumaroyl-hexosides, three caffeoyl-hexosides, and two feruloyl-hexosides, as well as reduction of almost all flavonoids in the peel at the final mature stage (Figure 4B). However, these metabolic shifts were not observed in the other pepper cultivars except purple pepper, which happens to be non-pungent but is the only cultivar in our study that has purple pigmentation before reaching its final maturity stage. In the previous study of hot and semi-hot pepper cultivars (C. annuum vs. Cyklon, Bronowicka Ostra, Tajfun, and Tornado), individual hydroxycinnamate content increased in the pericarp from green mature to red mature stage of development in all cultivars [74]. Additionally, in the analysis of a sweet bell pepper cultivar (C. annuum L. cv. Vergasa) exhibited a decrease in total hydroxycinnamate content in the pericarp from their immature green to green mature stage and then slightly increasing to immature red and red mature stages [75]. Taking into both results and our result in this study, results from the previous studies are consistent with our results such that hydroxycinnamate content increased in pungent cultivars from green to red stages of fruit ripening while hydroxycinnamate content decreased in sweet cultivars from immature to mature stages. Interestingly, purple pepper which is an anthocyanin-producing type of pepper cultivar showed similar metabolic changes with the pungent pepper cultivar, wherein the accumulation of hydroxycinnamates is inversely related with that of flavonoids. In both cultivars, hydroxycinnamates were generally upregulated while flavonoids were downregulated in the peel during their red mature stage. The pungent nature of jalapeño pepper due to its production of capsaicinoids could explain its downregulation of flavonoids during its red mature stage, given that both compounds groups share the same biosynthetic precursor.

Previously, in the metabolic analysis of fruit pericarp of the pungent hot pepper (Capsicum annuum "CM334") during six stages of fruit ripening, the flavonoid, Q3R, had high levels during earlier stages (16 and 25 DPA) and then gradually decreased until their last stage (48 DPA) [69]. In our study, Q3R in the pericarp of the hot cultivar (jalapeño pepper) was upregulated during its immature stage and then downregulated at its green and red mature stages, which are consistent with the decrease in Q3R observed from the previous study. Total flavonoid content, total O-glycosylflavone content and total C-glycosylflavone content in the pericarp of the sweet cultivar (C. annuum L. cv. Vergasa) decreased during four stages of ripening. Q3R decreased in the pericarp during the four stages of ripening [75], while in our study, Q3R was upregulated in the pericarp during immature stage and then downregulated in the middle stage (green or purple mature) and final mature (red or yellow mature) stages in the sweet cultivars (red paprika, yellow paprika), a non-pungent cultivar (purple pepper), and a pungent cultivar (jalapeño pepper). However, in another sweet cultivar (green paprika), Q3R was downregulated in immature and green mature stages and upregulated during red mature stage. In another non-pungent cultivar (green pepper), Q3R was upregulated during immature and red mature stages and downregulated in green mature stage of ripening. Variability in Q3R regulation among the six pepper cultivars is consistent with the subspecies or genotype-specific accumulation
pattern of flavonoid glycosides among 32 pepper accessions from a previous study, wherein specific accessions contained higher levels of flavones, flavanones, and flavonol glycosides [25].

In our results, flavonol mono-glycosides (Q3G, Q3R, and K3R) were generally decreased during ripening in the peel for almost all pepper cultivars. This metabolic shift pattern was observed inversely in the level of naringenin chalcones which increased in the peels during fruit ripening. In spite of the metabolic changes of flavonol mono-glycosides in peel, these flavonols were increased in green paprika pericarp during fruit ripening which also the same for the naringenin chalcones, including Phi35diGlc. Rutin and flavonol-tri-glycosides showed a slight shift in decrease at late stages among all pepper cultivars for both peel and pericarp during ripening. In green pepper, flavonol di- and tri-glycosides decreased in the peel during ripening, were increased in the pericarp during fruit development. The flavonol-tetra-glycosides, Q3G2'''A6''R7G which was detected in tomato fruits, was not detected in all pepper species in both tissue types. In red paprika pepper, flavonol di- and tri-glycosides decreased in the peel during fruit ripening, while in the pericarp, flavonol di-glycosides also decreased. There were nine putative flavonoids identified during analysis with most of them decreasing in the peel and pericarp of three cultivars (red paprika, jalapeño, and purple peppers). For green pepper, most of the putative flavonoids decreased in the fruit peel during ripening but increased in the pericarp. No obvious pattern among the nine flavonoids were observed for green and yellow paprika peppers. Flavonol mono-glycosides (Q3G, Q3R, and K3R) decreased in the peel during fruit development for most pepper cultivars, except in green paprika pepper which exhibited highest accumulation during its green mature stage. Meanwhile, in pepper pericarp, flavonol mono-glycosides decreased during maturity only for both red paprika and jalapeño peppers, while no concrete pattern was present for the other pepper cultivars.

Anthocyanin derivatives (anthocyanin_I, delphinidin-3-O-(-feruloyl) rutinoside; anthocyanin_II, delphinidin-3-O-(-p-coumaroyl) rutinoside-5-O-glucoside) were upregulated in purple pepper during either the purple immature or purple mature stages in both fruit tissues (Figure 4). Anthocyanin II (delphinidin-3-O-(-p-coumaroyl)rutinoside-5-O-glucoside) was also previously detected in immature purple pepper whole fruit [76] and peel [77]. Anthocyanin I being one of the major anthocyanins identified from eggplant fruit peel [78]. Anthocyanins I, II, and III were also upregulated in eggplant peel in our study (Figure 2). Anthocyanins are involved in pigmentation, specifically purple to black pigmentation in pepper fruit tissues [72] and in the purple pigmentation in the peel of eggplant fruit [78]. Metabolic changes of these anthocyanins are slightly different in terms of ripening stage in peel and pericarp. Anthocyanins I and II were upregulated in the peel during immature stage in purple pepper while they were upregulated in the pericarp during purple mature stage. Purple pepper is the only cultivar that was unable to detect more than one of the putative flavonoid compounds and the only cultivar that detected anthocyanins, indicating a possible inverse relationship between the two compound groups.

Most naringenin chalcones and Phi35diGlc (phloretin-3',5'-di-C-glucoside) were upregulated in the fruit peel in sweet and non-pungent cultivars during their final mature stage (red or yellow mature). Most naringenin chalcones and Phi35diGlc were upregulated in the peel of pungent jalapeño pepper during green mature stage. Due to the pungent nature of jalapeño pepper, differences in metabolic regulation with sweet cultivars could account for the down-regulation of most naringenin chalcones during red mature stage in jalapeño pepper. The pattern of accumulation is different in the fruit pericarp, with all three naringenin chalcones being upregulated at final maturity only for green pepper and green paprika peppers. Only naringenin-chalcone-hexose and phloretin dihexoside have been previously detected in non-pungent pepper (C. annuum) cultivars [79]. Accumulation of naringenin chalcones in the fruit peel is related with their functions of attracting seed dispersers, moderating damage against UV-light [55], and providing a structural role in the cuticle by controlling water movement [80]. Concentration of naringenin chalcone and its derivatives might have important roles after fruit maturation, particularly in the non-pungent and sweet cultivars where naringenin chalcones are upregulated in the peel during final mature stage of ripening.
Environmental stress, such as high temperature and UV-light, can increase reactive oxygen
species (ROS) concentration resulting in fruit oxidative damage. Specialized metabolites can act as
antioxidants protecting the fruit against photooxidative damage [81,82]. Polyphenols, such as flavonoids
and hydroxycinnamates, act as antioxidants [40,41]. In solanaceous crop tomato (*S. lycopersicum*),
total antioxidant activity in ripe fruit was in agreement with their total polyphenol content which
corroborates that polyphenols can act as antioxidants [83]. In the perennial shrub *Ribes stenocarpsum*
Maxim, polyphenol content was significantly higher in immature than in mature fruits, with antioxidant
activities consequently being higher in immature fruits [84]. In Alphonso mango (*Mangifera indica*) fruit,
a positive correlation between phenolic and antioxidant content in healthy tissues was present during
the course of fruit ripening [85]. Ripening in sweet pepper (*C. annuum*) is associated with oxidative
stress due to an increase in lipid peroxidation [86,87] and decrease in antioxidant enzymes during its
red ripe stage [86]. In our study, polyphenols were generally upregulated during red mature stage in
the pericarp of sweet green paprika (*C. annuum*) and non-pungent green pepper (*C. annuum*), which is
in agreement that fruit ripening in sweet pepper is related with oxidative stress since polyphenols
can act as antioxidants. In tomato, total phenolics and free radical scavenging activity increased
during ripening for all cultivars under study [88]. Decrease in antioxidant enzyme activity during fruit
ripening involved the enzymes catalase [89] and ascorbate peroxidase [86] which are not relevant with
polyphenol biosynthesis.

Comparing the number of polyphenolic compounds that accumulated more in the peel against the pericarp showed difference between fruit tissue type for the six pepper cultivars. However, not in all cases were the polyphenolic compounds more abundant during the same stage of development between tissue types. In peel, the compounds upregulated were 5CGA and fer_hex-II. 5CGA only increased during fruit ripening for the pungent cultivar and decreased for the sweet cultivars. Fer_hex_I and fer_hex_II increased remarkably in the peel of the pungent cultivar (jalapeño pepper) although some increase was observed in purple pepper as well. In the fruit pericarp, fer_hex_I increased noticeably in the pungent jalapeño pepper; however, there was also some increase in yellow and green paprika peppers. To determine whether such accumulation patterns are specific to pungent cultivars or whether this accumulation is only cultivar-specific, accumulation patterns for these polyphenolic compounds need to be conducted among pungent pepper cultivars during fruit ripening. We also observed that similar metabolic shift of pepper specific flavonoid derivatives between green pepper and green paprika, but these metabolic changes were negatively correlated to the metabolic shift in the jalapeño pepper which is the capsaicinoids producing-type of cultivar. This result provides a metabolic trade-off of fruit polyphenolics metabolism in capsaicinoids-producing cultivars, since polyphenolics and capsaicinoids share the biosynthetic precursors. In contrast to this point, jalapeño pepper showed clear elevation of hydroxycinnamates, such as caffeoyl-hexosides, in both peel and pericarp. However, these metabolic changes were also observed in the peel of green pepper and green paprika, but not in the other red, yellow, and purple peppers. Taking into account both metabolic changes of polyphenolic subgroups, changes of metabolic flux in polyphenolics are specific metabolic regulation in each types of peppers with different tissue specific manners. Finally, our results may have been convoluted by the absence of any pattern among polyphenols in terms of maturity stage and cultivar type, however integration of metabolomics data with previous studies [24] will provide other novel insights to understand this convoluted metabolic regulations. Importantly, naringenin chalcone which is one of the major ripening marker metabolites in tomato fruits, was conserved among almost all pepper species. Metabolomics analysis presented here suggested metabolic shift including convoluted metabolic trade-off in the solanaceous crops and provided hints for metabolomics-assisted crop improvement for the polyphenolic metabolism in the solanaceous fruits for the improvement of nutritive properties and enhanced stress tolerance.
# 4. Materials and Methods

## 4.1. Plant Material and Sampling

Fruits from tomato (S. lycopersicum cv. "M82") (TGRC, Tomato Genetics Resource Center, Davis, CA, USA), eggplant cv. "Ryoma" (S. melongena) (Takii, Japan), and different pepper (C. annuum) cultivars (green paprika cv. "New Ace" (Takii, Japan), red paprika cv. "Flupy Red EX" (e-taneya, Japan), purple pepper cv. "Nara Murasaki" (Tsurushin, Japan), green pepper cv. "Miogi" (e-taneya, Japan), yellow paprika cv. "Sonia Gold" (Sakata-no-Tane, Japan), and jalapeño pepper cv. "Jalapeño" (Marche, Japan)) were grown from May–December 2019 in standard soil under open field (longitude, 34.734433; latitude 135.736754) conditions. Three developmental stages were defined for analysis from fruit peel and flesh. Immature fruit, mid-stage, and final mature stages were selected and defined based on the height of each fruit and the color of peel and seeds. Immature fruit are half the height of mature fruit and green in color. In the case of purple pepper and eggplant, the immature fruit color was purple. Green mature fruit had approximately the same height as the final mature stage but still green in color. Purple pepper was also purple during its green mature stage. The final mature stage is when peel color has completely changed from green to its ripe fruit color (red, yellow). Mature eggplant fruit were distinguished through their dark brown seed color. Three independent biological replicates per plant tissue during each ripening stage were harvested and used for metabolomic analysis. After tissue separation, fruit peel and pericarp were grounded into powder using liquid nitrogen and stored at –80 °C for further analysis.

## 4.2. Sample Extraction and LC-MS Analysis

Metabolite extraction was performed as described previously [90,91]. Fifty milligrams of powdered frozen tissue sample were aliquoted and weighed in a 2.0 mL centrifuge tube. Two hundred fifty microliters of extraction solvent (80% methanol in LC-MS grade water with 5 µg/mL isovitexin standard) were added per tissue sample in liquid nitrogen and a Zirconia bead. All frozen tissue samples were ground into powder using Mixer Mill TissueLyser II (Qiagen, Hilden, Germany) for 3 min at 25l/s at room temperature, and centrifuged at 15,366× g at 4 °C for 10 min. After centrifugation, to take an additional cleaning step of extracts to exclude plant tissues, two hundred microliters of supernatant per sample were transferred into a 1.5 mL centrifuge tube then all 1.5 mL tubes were centrifuged at 15,366× g at 4 °C for 10 min. One hundred microliters supernatant per sample were transferred into LC-MS vials and stored at 4 °C in the dark until analysis. For detection of polyphenolic metabolites, LC-electrospray ionization (ESI)-MS was used. Nanoflow-HPLC "Paradigm MS4 system" (Michrom BioResources, Inc., Auburn, CA, USA), equipped with a Luna C18 column (150 by 2.00 mm i.d. 3 micron particle size, Phenomenex, Torrance, CA, USA) operated at a temperature of 25 °C was used. The mobile phases consisted of 0.1% formic acid in water (Solvent A) and 0.1% formic acid in acetonitrile (Solvent B). The flow rate of the mobile phase was 200 µL/min, and 10 µL sample were loaded per injection. The following gradient profile was applied: The concentration of mobile phase A was 100% at 0 min, 93% at 1 min, decreased to 80% at 8 min, 60% at 17 min, 15% at 21 min, and 0% at 25 min and 28 min for column wash, then increased to 100% at 28.01 min and 31 min for the equilibration of the column in the gradient description. The LC was connected to an MS TSQ Vantage (Thermo Fisher Scientific, San Jose, CA, USA). The spectra were recorded using full scan mode, covering a mass range from m/z 200–1500 by both positive and negative ion detection. The transfer capillary temperature was set to 350 °C. The spray voltage was fixed at 3.00 kV. Peak identification of major polyphenolic compounds (rutin, quercetin-3-O-rutinoside; Q3G, quercetin-3-O-glucoside; and 3CGA, chlorogenate) was performed using standard compounds. Peak annotation of major polyphenolic compounds in Solanaceae species was performed via combination approach of co-elution profile of common tomato fruit extracts and phytochemical database (KomicMarket [14] and MotoDB [13,92]) as well as metabolites table in the literatures [5,15].
## 4.3. Data Analysis

Molecular masses, retention time, and associated peak intensities were extracted from the raw files using the Xcalibur software version 4.1.31.9 (Thermo Fisher Scientific, San Jose, CA, USA). Compounds were identified and putatively identified by comparing with corresponding retention time (minutes) and molecular weight with those provided by tomato cv. M82 and eggplant. Previous information on polyphenolic compounds identified from different pepper species and varieties from published journal articles were also used for cross-referencing. Peak picking in the Xcalibur software was performed with the parameter of RT tolerance window (20 s), base window 80, area noise factor 5.0, peak noise factor 10, and "nearest RT". MeV software version 4.9.0 (http://www.mev.tm4.org/, Dana Farber Cancer Institute, Boston, MA, USA) was used for data visualization and PCA analysis. The plots were applied for the 39 metabolites with the average values from 3 biological replications. Heatmap visualization of metabolite data is normalized and scaled by $log_2FC$ (mean/average_mean) for each metabolite. Coefficient correlation was estimated by person correlation method using MeV software.

## 5. Conclusions

With several fruit-metabolomics studies on the specialized metabolism of tomato recently available, such an approach could, therefore, be extended to generate information on other members from Solanaceae. Comparing the number of polyphenolic compounds that accumulated more in the peel against the pericarp showed differences between fruit tissue type in the six pepper cultivars. However, not in all cases were the polyphenolic compounds more abundant during the same stage of development between tissue types. We also observed that a similar metabolic shift of pepper-specific flavonoid derivatives between green pepper and green paprika cultivars, but these metabolic changes were negatively correlated to the metabolic shift in jalapeño pepper, which biosynthesizes capsaicinoids. This result exhibits a metabolic trade-off in fruit polyphenolics metabolism in the capsaicinoid-producing cultivar, since polyphenolics and capsaicinoids share the biosynthetic precursors. In support of this point, hydroxycinnamates in the pungent jalapeño and anthocyanin-producing purple pepper cultivars were clearly elevated in both peel and pericarp during red mature stage. However, flavonoids from both cultivars were downregulated during red mature stage suggesting a metabolic trade-off between both compound groups during fruit development. Taking into account both metabolic changes of polyphenolic subgroups, changes of metabolic flux in polyphenolics are specifically regulated in each pepper cultivar with different tissue specific manners. Finally, our results may have been convoluted by the absence of any accumulation patterns of polyphenol in terms of ripening stage and cultivar type, however integration of metabolomics data with previous studies [24] will provide other novel insights to understand these convoluted metabolic regulations. Importantly, naringenin chalcone which is one of the major ripening marker metabolites in tomato fruits, was conserved among almost all pepper cultivars. Metabolomic analysis presented here suggested metabolic shift including convoluted metabolic trade-off in three solanaceous crops and provided hints for metabolomics-assisted crop improvement of polyphenolic metabolism in three solanaceous crops towards their improved nutritive properties and enhanced stress tolerance.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2218-1989/10/5/209/s1, Figure S1: LC-MS TIC (total ion chromatograms) of fruit extracts of tomato, eggplant and pepper; Supplemental Table S1: Metabolite table of peaks detected in this study using LC-MS.

**Author Contributions:** Conceptualization, M.W. and T.T.; software, C.L.F.C. and T.T.; validation, C.L.F.C., T.S. and T.T.; formal analysis, C.L.F.C.; project administration, M.W. and T.T.; supervision, M.W. and T.T.; investigation, C.L.F.C.; writing—original draft preparation, C.L.F.C. and T.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Japan Society for the Promotion of Science (JSPS) Scientific Research B (19H03249) and C (19K06723).
**Acknowledgments:** C.L.F.C.: T.S., M.W. and T.T gratefully acknowledge partial support by NAIST. We are grateful Jay Camisora Delfin who helped for growing tomato plants. Research activity of M.W and T.T. were additionally supported by JSPS KAKENHI Grant-in-Aid for Scientific Research B (19H03249) and C (19K06723).

**Conflicts of Interest:** The authors declare no conflict of interest.

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© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Review

# Metabolite Changes during Postharvest Storage: Effects on Fruit Quality Traits

Delphine M. Pott, José G. Vallarino *,† and Sonia Osorio *

Departamento de Biología Molecular y Bioquímica, Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora", Universidad de Málaga-Consejo Superior de Investigaciones Científicas, Campus de Teatinos, 29071 Málaga, Spain; dpott@uma.es

* Correspondence: vallarino@uma.es (J.G.V.); sosorio@uma.es (S.O.); Tel.: +34-952134271 (J.G.V. & S.O.)

† Current address: Max-Planck-Institut für Molekulare Pflanzenphysiologie, 14476 Potsdam-Golm, Germany.

Received: 14 April 2020; Accepted: 6 May 2020; Published: 8 May 2020

**Abstract:** Metabolic changes occurring in ripe or senescent fruits during postharvest storage lead to a general deterioration in quality attributes, including decreased flavor and 'off-aroma' compound generation. As a consequence, measures to reduce economic losses have to be taken by the fruit industry and have mostly consisted of storage at cold temperatures and the use of controlled atmospheres or ripening inhibitors. However, the biochemical pathways and molecular mechanisms underlying fruit senescence in commercial storage conditions are still poorly understood. In this sense, metabolomic platforms, enabling the profiling of key metabolites responsible for organoleptic and health-promoting traits, such as volatiles, sugars, acids, polyphenols and carotenoids, can be a powerful tool for further understanding the biochemical basis of postharvest physiology and have the potential to play a critical role in the identification of the pathways affected by fruit senescence. Here, we provide an overview of the metabolic changes during postharvest storage, with special attention to key metabolites related to fruit quality. The potential use of metabolomic approaches to yield metabolic markers useful for chemical phenotyping or even storage and marketing decisions is highlighted.

**Keywords:** fruit; postharvest; metabolomics; quality traits; stress; biomarkers

## 1. Introduction

Fruit growth, ripening and senescence are complex processes, controlled by multiple developmental and environmental signals, and their molecular mechanisms remain unclear [1]. Fruits undergo important metabolic changes during ripening, including chlorophyll breakdown, anthocyanin or carotenoid pigment accumulation, cell wall degradation and the synthesis of low-weight metabolites (such as sugars, acids and volatiles), which function to increase their attractiveness to seed dispersers [2]. Once fruits are removed from the plant and until they reach consumers on the market, a period known as postharvest ripening or senescence occurs—the duration of which is variable (from days to weeks) and the effects of which mainly depend on fruit metabolism and ripening status at harvest. Indeed, climacteric fruits, such as tomatoes, kiwi or avocados, which exhibit a concomitant peak of ethylene production and a sudden rise in respiration at the onset of ripening [3], can ripen after harvest. In this sense, the control of ethylene production is fundamental to optimize the postharvest storage of these types of fruits [4]. On the other hand, non-climacteric fruits, e.g., strawberries and grapes, do not exhibit respiration and ethylene production peaks, and have to be harvested (almost) fully ripe. Postharvest storage initiates fruit senescence—the effects of which on biological processes are unavoidable and largely negative. Senescence leads to protein, lipid and nucleic acid degradation and cell dysfunction, disintegration and death [5]. Several factors influence and accelerate fruit senescence, with the most relevant being respiration, providing energy for maintaining metabolism, dehydration
and fungal activity [6]. Consequently, the degradative processes associated with postharvest senescence impact fruit quality traits, i.e., aspect, texture, taste, aroma and nutritional characteristics, leading to consumer rejection and important economic losses for the fruit industry.

Currently, depending on fruit crops, different postharvest strategies are commercially practiced in order to adapt ripening to industry needs, delay senescence, maintain fruit quality attributes and, thus, prolong shelf-life. In general, fruits are highly perishable at ambient temperature. Thus, refrigerated storage is the most common method used to delay ripening, fruit respiration, enzymatic activities, and the development of pathogen infections, and, therefore, extend fruit shelf-life [7]. However, cold storage can provoke the development of a physiological disorder called chilling injury (CI). Although CI symptoms are species dependent, CI includes internal and external browning, mealiness, flesh bleeding, pitting or the inability to soften. These physiological disorders tend to appear once the fruits are acquired by consumers, having a negative impact on palatability and acceptance [8,9]. To reduce CI symptoms and depending on the type of fruit, the industry combines low-temperature storage with some complementary strategies. For example, prior heat treatment to cold storage is widely used in several crops, including *Citrus* and loquat [10], while controlled atmosphere (CA, increased CO₂ and decreased O₂ levels) is commonly applied to apple, strawberry, peach and pear, among others [11]. Additionally, delayed cooling has been successfully applied in apple to reduce soft scald, a chilling-dependent physiological disorder [12]. While CA reduces fruit respiration, heat treatment has a protective effect by acting on membrane integrity and heat shock protein accumulation and by promoting antioxidant and sugar metabolism [13]. Further, it is known that heat treatment induces defense mechanisms and induces physiological changes that allow *Citrus* fruit to withstand stressful conditions during storage. For example, GC-MS analysis in heat-treated oranges during storage showed a higher concentration of sugars while no changes were observed in organic acid levels [14].

In the case of climacteric fruits, such as tomato or banana, the application of ethylene antagonist 1-methylcyclopropene (1-MCP) is commonly used to increase shelf-life [15]. However, the aforementioned strategies have different degrees of effectiveness at reducing CI and prolonging shelf-life, depending on fruit species and varieties. In addition, it must be highlighted that these postharvest techniques constitute abiotic stresses for the fruits, which have to adapt their metabolism to maintain homeostasis [16]. In particular, stress situations induce the synthesis of compounds involved in plant protection, and trigger the accumulation of compatible metabolites, reactive oxygen species (ROS)-scavenging enzymes and changes in carbon metabolism [17,18]. In this sense, metabolomic platforms, allowing the simultaneous detection and quantification of hundreds of metabolites, offer the possibility to improve our knowledge about the molecular mechanisms underlying fruit senescence under commercial storage conditions.

## 2. Metabolomic Platforms in Postharvest Studies

The plant metabolome comprises a wide range of small molecules, with a large variety in physico-chemical properties and extremely variable concentrations. Metabolomics is defined as the field of the research that generates a profile of small molecules in a biological system. Thus, it can directly reflect the outcome of complex networks of biochemical reactions and, therefore, provides essential information about the underlying biological status on the system in question.

For these reasons, successful analysis of the complex network of fruit metabolites requires highly sensitive and selective analytical techniques, with each displaying both advantages and limitations and showing differential coverage depending on the nature of the metabolite. In particular, mass spectrometry (MS) coupled with gas chromatography (GC), liquid chromatography (LC) and, to a lesser extent, capillary chromatography (CE) and nuclear magnetic resonance spectroscopy (NMR) have been the most extensively applied methodologies to study the plant metabolome, including its reconfiguration during fruit postharvest senescence [17,19,20].
GC-MS is the technique of choice for measuring small polar metabolites, which are thermally stable and can be made volatile through a derivatization approach [21]. The main advantages of GC-MS are its robustness and reproducibility, which have allowed the establishment of libraries and databases facilitating the identification of metabolites. As a result of its characteristics, GC-MS is mainly used in plant metabolomic studies to investigate central primary metabolism, which includes sugars, sugar alcohols, amino acids, organic acids and polyamines [17,19]. In addition, GC-MS can be coupled with headspace solid-phase microextraction (HS-SPME), which allows the detection of specific volatiles present in a sample [22]. Both volatile and primary metabolite changes occurring during fruit postharvest storage have been extensively studied, as they are key compounds of fruit taste and aroma.

To overcome the limitations of GC-MS, which is restricted to volatile and thermally stable molecules, LC-MS is broadly used to detect a wider range of metabolites. In particular, the enormous diversity of plant secondary metabolites, which includes tens of thousands of different compounds [23], is mainly studied using LC-MS due to its versatility. However, and as a consequence of LC-MS flexibility, metabolite identification remains difficult, as no universal mass spectral library has been created [24].

Another technique used to study plant metabolomics, although rather uncommon, is capillary electrophoresis (CE)-MS. This technique allows the detection of a wide range of highly polar or charged metabolites by separating them based on their mass-to-charge ratio [25]. In this sense, this method has been proposed as a valuable complementary approach for samples that cannot be readily resolved by the more established GC- and LC-MS platforms [26].

Even if NMR presents a low sensitivity compared to that of MS approaches, it provides a series of advantages over the previously mentioned approaches by providing structural information, involving non-destructive sample preparation and providing rapid metabolite screening [19,27]. Integrated NMR platforms, allowing the monitoring of changes in both primary and secondary metabolites, have been developed and can be useful to study metabolic shifts in senescent fruits during postharvest [28].

### 3. Primary Metabolic Pathways Affected by Postharvest Storage: Effects on Fruit Texture and Taste

Fruit organoleptic quality is a complex trait that is influenced by taste, aroma, color and texture. In particular, fruit acceptance by consumers is directly influenced by sugar and acid content and the ratios of both groups of primary metabolites [29,30]. Fruit respiration during postharvest storage directly affects primary metabolic pathways, such as glycolysis, starch metabolism, and the tricarboxylic acid cycle (TCA), which account for changes in sugar, amino and organic acid levels. Indeed, carbohydrates, organic acids, proteins and fats are the main respiratory substrates during fruit storage. Furthermore, they are involved in gluconeogenesis, a process which has been described to be upregulated during the postharvest in orange and apple fruits [31–33]. Thus, it contributes to fruit depletion and also to important changes in primary metabolite composition. In the next paragraphs, we described alterations in sugars, organic and amino acids as a consequence of postharvest storage.

#### 3.1. Sugars and Sugar Derivatives

Sugar content, which is commonly estimated by the soluble solid content (SSC), shows differential behavior during postharvest, depending mainly on the species and storage conditions. The SSC trend normally coincides with changes in the main sugar profiles present in ripe fruits, i.e., glucose, fructose and sucrose.

While main carbohydrates tend to decrease in some species, as profiled by GC-MS and NMR analysis in tomatoes kept at room temperature [34] or blackberries stored at 4 °C [35], in other fruits, such as bananas and kiwis, their level increases as a consequence of starch hydrolysis, which takes place during postharvest storage [36,37]. In turn, sucrose can be hydrolyzed, leading to a concomitant increase in hexoses, as monitored by GC-MS in Powell oranges stored at room temperature [33]. Interestingly, growing evidence seems to point to sugars playing a regulatory role in senescence
processes [38,39]. During fruit ripening and senescence, cross-talk between sugars and hormones involved in ripening and senescence processes, such as abscisic acid, ethylene and auxin, has been described [40–43], and sucrose degradation during postharvest storage can be crucial for inducing senescence [33,44]. Furthermore, sugar uptake during fruit ripening may affect postharvest water loss by interfering with cuticle development. Indeed, stable silencing of the cell wall invertase LIN5, a key determinant of SSC content, led to a diminished water loss rate and wrinkling in transgenic tomato fruits kept at room temperature for 12 days. Even though the complete molecular mechanism has not been described, it was clearly established that sugar entry during fruit development impacts the cell wall and cuticle structure, resulting in a radical effect on tomato senescence [45].

Apart from the most abundant sugars, i.e., sucrose, glucose and fructose, fruits also contain minor sugar and alcohol derivatives, such as sorbitol, galactinol, raffinose, *myo*-inositol and trehalose [46]. Even if those compounds may be at low concentrations, they seem to be crucial for fruit behavior during storage, as they can alleviate the negative effects of the abiotic stresses underlying postharvest conditions. Indeed, soluble sugars are important metabolites in ROS metabolism, being the primary carbon and energy source and contributing to the generation of reducing power generation via the oxidative pentose phosphate pathway [41,47,48]. Furthermore, they play key roles in osmoprotection and cell membrane stabilization [49–51]. As an example, important increases in raffinose and galactinol levels were measured by GC-MS in peaches after heat treatment (three days at 39 °C) followed by storage at 0 °C for two days and may confer improved tolerance to CI [52]. Moreover, comparing the levels of galactinol (detected by LC-MS/MS), raffinose, trehalose and *myo*-inositol (identified by NMR) in climacteric and non-climacteric plum varieties during postharvest storage at 20 °C and in presence of 1-MCP, propylene (ethylene analogue) or control air, Farcuh et al. [46] noticed that the levels were more enhanced in the latter variety. These data could explain the capacity of the non-climacteric variety to cope better with postharvest stress conditions, and the identified sugars could be used as biomarkers to evaluate fruit physiological status during storage (Table 1).

Softening during postharvest storage is a key physiological process leading to ripe fruit firmness; however, excessive loss of firmness as a consequence of overripening can prompt physical damage and pathogen attack, and consequently lead to an important decrease in fruit quality. Softening is the result of several factors, including cell wall disassembling metabolism. Metabolites originated from cell wall disassembly, mainly monosaccharides, can be monitored by primary metabolite profiling. Indeed, in pitaya fruit, the content of several monosaccharides, including xylose, galactose, arabinose, and mannonic acid and glucuronic acid, which originate from cell wall disassembly, was measured by GC-MS [53]. Interestingly, these metabolites were decreased after blue light treatment (2 h at 25 °C under blue light emitting diode) compared to control fruits kept in the dark, suggesting that this treatment has a significant effect in delaying cell wall degradation and postharvest decay of pitaya fruit [53]. Another study, using LC coupled with tandem MS (LC-MS/MS), detected an increase in glucuronic acid, a component of pectin, among the major elements of plant cell wall, in pears stored 18 days at room temperature [54]. Pectin de-polymerization and de-esterification were also evidenced by the detection of galacturonic acid by two-dimensional GC-MS (GC x GC-MS) in overripe kiwi fruits stored at 20 °C and treated for 24 h with 200 ppm ethylene. On the contrary, no increase in galactose was observed using GC-MS measurements, suggesting that this sugar is directly metabolized after its release from cell wall, or that it is liberated as different form [55]. Among the main symptoms of CI in peach is mealiness, which is the result of a cell wall metabolism disorder. Xylose, the central constituent of hemicellulose, among the key components of plant cell wall, was increased during cold storage in peach chilling-susceptible genotypes, but not in the varieties resistant to CI, confirming a link between cell wall disassembly and mealiness in sensitive cultivars [56].
<table><caption>**Table 1.** Primary metabolites (sugars, organic and amino acids) identified as putative biomarkers by metabolomic profiling studies to assess fruit quality changes during postharvest storage, d: day; RT: room temperature; HPLC: high-performance liquid chromatography; UHPLC-MS/MS: ultra-high-pressure liquid chromatography-tandem mass spectrometry; $^{1}$H-NMR: proton-NMR; CI: chilling injury; ROS: reactive oxygen species.</caption><thead><tr><th>Metabolite</th><th>Effect on Fruit</th><th>Postharvest Treatment</th><th>Behavior during Postharvest</th><th>Fruit Species</th><th>Metabolomic Platform</th><th>Reference</th></tr></thead><tbody><tr><td>raffinose, galactinol</td><td>Tolerance to CI</td><td>39 °C, 3 d + 0 °C, 2 d</td><td>Increase</td><td>peach</td><td>GC-MS</td><td>[52]</td></tr><tr><td>raffinose, galactinol, myo-inositol, trehalose</td><td>Enhanced capacity to cope with postharvest stress conditions</td><td>20 °C, 14 d</td><td>Increase</td><td>non-climacteric plum</td><td>NMR, UHPLC-MS/MS<sup>[6]</sup></td><td></td></tr><tr><td>malate</td><td>Decrease in water loss and in susceptibility to opportunistic fungal infections</td><td>RT, 20 d</td><td>Decrease</td><td>tomato</td><td>GC-MS</td><td>[57]</td></tr><tr><td>malate</td><td>Correlation with fruit firmness and shelf-life</td><td>25 °C until first symptoms of deterioration</td><td>Decrease</td><td>tomato</td><td>GC-MS, <sup>1</sup>H-NMR</td><td>[58]</td></tr><tr><td>mannose, citramalate, gluconate, keto-gulonate</td><td></td><td>18 °C, 10 d</td><td>Increase</td><td>tomato</td><td>GC-MS</td><td>[36,59]</td></tr><tr><td>dopamine</td><td>Conversion to salsolinol at late postharvest stages, decrease in fruit quality</td><td>25 °C until senescence</td><td>Decrease</td><td>banana</td><td><sup>1</sup>H-NMR</td><td>[36]</td></tr><tr><td>proline</td><td>Osmoprotection and ROS-scavenging functions</td><td>0 °C, 20 kPa CO<sub>2</sub>/20 kPa O<sub>2</sub>/60 kPa N<sub>2</sub>, 3 d + 0 °C, air, 30 d + 20 °C, 2 d</td><td>Increase</td><td>grape</td><td>LC-MS</td><td>[60]</td></tr><tr><td>proline</td><td>Osmoprotection and ROS-scavenging functions</td><td>1 mM GABA treatment, 20 min + 4 °C, 18 d (dark)</td><td>Increase</td><td>zucchini</td><td>HPLC</td><td>[61]</td></tr></tbody></table>
## 3.2. *Organic Acids*

Several organic acids are related to fruit postharvest metabolism. Surprisingly, particularly in tomato, the levels of malate, among the most abundant organic acids, impact fruit shelf-life (Table 1). The malate content, measured by GC-MS, decreases during the ripening and postharvest storage at room temperature of several tomato genotypes, including *delayed fruit deterioration, non-ripening* and *ripening inhibitor* mutants as well as genotypes that are commercially used because of their delayed maturation and senescence. Interestingly, it was also shown that malate levels were lower in mature tomato fruits that ripened on the vine than off the vine [34]. However, when the malate concentration in tomato fruits was manipulated by reducing the expression of two TCA cycle enzymes (*fumarase* and *malate dehydrogenase (MDH)*), a differential postharvest behavior was observed compared to that in wild-type fruits at room temperature. Interestingly, fruits of the MDH-deficient genotype showed higher malate content and poorer postharvest behavior than non-transformed fruits by losing more water and being more susceptible to opportunistic fungal infections and *Botrytis cinerea* spores. In contrast, the fumarase-deficient genotype, with a relatively low malate content, presented a decrease in water loss and in susceptibility to opportunistic fungi [57]. The mechanism underlying malate's role in postharvest responses could not be clearly explained; however, the authors suggested a role for SSC, which changes in the opposite manner in *MDH-* and *fumarase*-silenced lines, in osmotic potential and subsequent water loss during storage. Another study using recombinant inbred lines originating from the cultivated tomato *Solanum lycopersicum* and the wild-type species *Solanum pimpinellifolium* also pointed out the association between malate content, fruit firmness and shelf-life [58]. A comprehensive polar metabolite profiling was performed by GC-MS and NMR and a combination of neuronal clustering and network construction displayed a strong correlation between glycerate and malate content and postharvest, which also showed a negative correlation with fructose levels [58]. This association between metabolites and agronomic traits such as firmness and storage behavior suggested that malate could be a good biomarker to select genotypes with enhanced quality traits, such as improved postharvest life [58].

By performing a GC-MS metabolic characterization of *S. lycopersicum* cv. 'Plaisance' fruits during ripening and postharvest stages, Oms-Oliu et al. [59] showed that one sugar (mannose) and three organic acids (citramalate, gluconate and keto-gulonate) were strongly increased once the fruit was removed from the vine and that these compounds could be indicators of metabolic shifts during postharvest storage [59] (Table 1). As an example, the enhanced gluconate levels could be a consequence of tartarate biosynthesis from ascorbate degradation or energy balance changes during tomato storage [62,63]. Free mannose levels are generally low, as this monomer usually composes carbohydrate polymers. However, it can be found in a free form as a result of cell wall disassembly and hemicellulose breakdown during fruit senescence, as described in tomato, apple and pear [59,62,64].

Organic acids, particularly citric acid, accumulate at high levels in *Citrus* fruits, such as lemons, oranges, grapefruits or pummelos. A study on 'Hirado Buntan' pummelo focused on the relationship between organic acids, measured by high-performance capillary electrophoresis, and fruit senescence during postharvest storage at both ambient and cold temperatures. The authors observed a general decrease in malate, citrate, aconitate and fumarate during storage, accompanied by important fluctuations in their levels; this decrease was associated with a loss of fruit quality [31]. The combination of transcriptomic analysis paralleled the metabolomic data, suggesting that the *peroxisomal MDH*—the expression of which correlated with malate levels—is responsible for organic acid metabolism regulation during postharvest. This result indicated that the glyoxylate cycle, which occurs in peroxisomes and glyoxysomes, is central to organic acid regulation by supplying succinate for the TCA cycle [31]. Tang et al. [33] also observed a decrease in several organic acids analyzed by GC-MS, such as malate, citrate and α-ketoglutarate, during postharvest storage of 'Powell' oranges at room temperature. In this case, they suggested that malate could be used as a substrate for gluconeogenesis, being converted into phosphoenolpyruvate (PEP) by the action of two enzymes upregulated in oranges kept at room temperature: PEP carboxykinase and pyruvate orthophosphate dikinase (PPDK). Similarly,
an increased abundance of PPDK proteins associated with decreased malate content was observed in peaches subjected to heat treatment followed by storage at 20 °C [65]. Another study using GC-MS analysis in different varieties from the *Citrus* genus suggested that a conversion of organic acids to sugars during fruit postharvest senescence at ambient temperature occurs, as negative correlations were frequently observed between metabolites belonging to the two groups and that the SSC/titratable acidity ratio increased during storage [66]. The succinate content increased during pummelo postharvest storage, showing a positive correlation with GABA and glutamine [31]. In addition, GABA increased during the postharvest senescence of Powell oranges, matched by an upregulation of the genes involved in the GABA shunt [33]. In this sense, the GABA shunt was outlined as an important pathway for organic acid catabolism and for balancing organic acid and amino acid levels. Indeed, superfluous citrate can be converted into amino acids via the GABA shunt [33,67]. Moreover, Sun et al. [31] observed an increase in ROS during pummelo storage, which correlated with enhanced mitochondrial damage. Cross-talk between ROS and organic acids could occur during postharvest senescence, as TCA enzymes have been described to be very sensitive to inhibition by ROS [68,69], while organic acids could be involved in the direct ROS scavenging [70,71].

### 3.3. *Amino Acids*

Amino acid content is also affected to a large extent by postharvest storage, as these compounds are involved in several pathways induced during fruit ripening [47]. In particular, during senescence, amino acid catabolism can counteract the reduction in electron supply from the TCA cycle [72]. Ubiquitination of proteins controls their degradation to free amino acids, and upregulation of the ubiquitin pathway has been reported in stored peaches that were previously were heat treated [73]. Dopamine, a derivative of the aromatic amino acid tyrosine, has been proposed as a postharvest marker in banana fruit stored at 25 °C [36] (Table 1). Indeed, NMR-based metabolite profiling of the senescence of bananas stored at room temperature showed that dopamine levels were undetectable at the last postharvest stage. Concomitant with dopamine disappearance was the sudden appearance of salsolinol, which has been described to originate from dopamine and acetaldehyde, the latter formed from ethanol, which is also generated in the late postharvest stage [36,74]. The authors concluded that the conversion of dopamine to salsolinol led to a decrease in fruit quality, making bananas less fit for consumption [36].

Additionally, several amino acids play a key role in tolerance to abiotic stresses in fruits during postharvest senescence. Indeed, a GC-MS comparative study between pineapple varieties tolerant and susceptible to CI stored at 10 °C outlined that amino acid increases during chilling stress may be associated with a delay in symptom appearance, such as internal browning, by presumably contributing to the synthesis of enzymes involved in tissue repair and, in the case of cysteine, aspartate and valine, by acting as osmoprotectants [75]. Proline is a well-documented stress-related amino acid and among the main osmolytes that are accumulated during plant stresses, playing important membrane protection and ROS-scavenging functions [76,77] (Table 1). Grape storage in a CO₂-enriched atmosphere resulted in a threefold endogenous proline increase when compared to that in air-stored grapes [60], and proline accumulation is a common trend in postharvest fruits subjected to treatments to attenuate CI, such as zucchinis [61], mangoes [78], bananas [79], pears [80] and loquats [81]. However, the possible role of amino acids in counteracting CI seems to be species dependent, as GABA, aspartate, phenylalanine and proline increase in peach stored at 0 °C for 21 days was not associated with CI protection, since their levels, quantified by GC-MS, were enhanced in both resistant and susceptible genotypes [56].

A recent study in strawberry also outlined the possible role of amino acids in plant defense, as pathogen resistance mechanisms implicated this group of metabolites [82]. The increase in asparagine, aspartic acid, threonine, glutamic acid, glutamine, alanine and glycine in CO₂-treated strawberries compared to control fruits could, at least partially, explain the lower fungal decay observed in the first group [83].
# 4. Postharvest Impact on Secondary Metabolites

The two main families of secondary metabolites present in fruits are polyphenol and terpenoid compounds, responsible for their appealing color and also important for their organoleptic and nutritional characteristics [84]. Apart from their importance in the human diet, these molecules are involved in plant defense and responses against biotic and abiotic stresses. In particular, metabolomic approaches have helped in deciphering their role during fruit storage and how different postharvest strategies impact on them. Here, impact on polyphenols, including anthocyanins, and carotenoids during fruit shelf-life is discussed in the next paragraphs.

## 4.1. Polyphenol Compounds

Dynamic metabolite changes, profiled by high-performance LC-MS (HPLC-MS), were observed during grape postharvest ripening and dehydration, the metabolic responses being genotype dependent [85]. A particular feature was the cultivar-specific accumulation of stilbenes, a class of phenylpropanoid compounds, with antifungal activity. On the other hand, anthocyanins and other flavonoids, belonging to another phenylpropanoid class, were depleted along postharvest dehydration [85]. An untargeted HPLC-MS profiling during grape ripening and withering (postharvest drying), combined with transcriptomic and proteomic data integration, also correlated the presence of stress-related secondary compounds (stilbenes and acylated anthocyanins) with the postharvest phase. The synthesis of defense molecules could be a response to abiotic stress (dehydration) or biotic stress (eventual pathogen attack). In addition, three metabolites (two taxifolins and tetrahydroxyflavanone-O-deoxyhexoside), belonging to the flavonoid class, have been proposed as putative markers in order to assess berry fruit quality traits (Table 2) [86]. In grapes, the accumulation of different stilbenes during cold postharvest storage was monitored by UHPLC-MS/MS [87]. This increase was also observed when grape fruits were kept at high CO₂ [87]. In contrast, CA storage has been described to have negative effects on anthocyanin accumulation in strawberry fruits, compounds responsible for the color of the ripe fruit [88]. In this sense, postharvest cold storage is a mandatory strategy to enhance anthocyanin content in some fruits such as blood oranges, some varieties of plums and anthocyanin-rich tomatoes [89,90]. Interestingly, it has been described that tomato anthocyanin-rich lines are able to maintain fruit quality for longer during storage, mainly by reducing their susceptibility to *Botrytis cinerea* [91,92].

In mandarins, heat treatment previous to storage at 12-16 °C positively impacts polyphenol metabolism by increasing flavonoids and lignin content (flavonoids measured by HPLC-MS). The effect of this postharvest strategy can be seen as a modulation of fruit defense against biotic and abiotic stress during postharvest storage, by supplying chemical (flavonoids) and physical barriers against pathogen attack [93]. The relationship between polyphenol content and resistance to postharvest decay caused by *Penicillium expansum* has also been described in apple; indeed, resistant and susceptible apple genotypes could be discriminated based on polyphenol content, measured by UPLC-MS (Table 2) [94]. However, a general polyphenol increase during fruit shelf-life does not always occur, as described by untargeted UHPLC-MS in several mango varieties stored at room temperature during six days, in which it was found that only gallic acid and epicatechin content was enhanced after storage [95].
<table><caption>**Table 2.** Secondary metabolites (polyphenols and carotenoids) identified as putative biomarkers by metabolomic profiling studies to assess fruit quality changes during postharvest storage. d: day; w: weeks; UHPLC-HRAM MSn: ultra-high-performance liquid chromatography coupled to high resolution multiple-stage mass spectrometry.</caption><thead><tr><th>Metabolite</th><th>Effect on Fruit</th><th>Postharvest Treatment</th><th>Behavior during Postharvest</th><th>Fruit Species</th><th>Metabolomic Platform</th><th>Reference</th></tr></thead><tbody><tr><td>taxifolin deoxyhexoside, taxifolin hexoside tetrahydroxyflavanone-O-deoxyhexoside procyanidin B1, epi-catechin</td><td>Antifungal activity, withering stress responses</td><td>withering, 91 d</td><td>Increase</td><td>grape</td><td>Untargeted HPLC-MS</td><td>[86]</td></tr><tr><td>β-cryptoxanthin</td><td>Resistance to Penicillium expansum</td><td>2 °C storage</td><td>Increase</td><td>apple</td><td>UHPLC-HRAM MSn</td><td>[94]</td></tr><tr><td></td><td>Part of β, β-xanthophyll pool in mature oranges</td><td>12 °C up to 7 w</td><td>Increase</td><td>sweet orange</td><td>HPLC</td><td>[96]</td></tr></tbody></table>
## 4.2. *Carotenoids*

Carotenoids are an important class of terpenoids, responsible for the attractive color of many fruits and vegetables. While their low stability during postharvest storage, mainly due to a rapid turnover of β-carotene, has been described in many staple crops, postharvest accumulation in *Citrus* and tomato seems to be temperature dependent [96,97]. Carotenoid levels in grapefruit, determined by HPLC, stored at 2 and 12 °C established a link between carotenoid content and CI symptom suppression, suggesting that they play a role in preventing cold damage by protecting plastid structures [98,99]. Furthermore, the ratio between 9-Z-violaxanthin (yellow hues) and β-citraurin (orange-red pigments), responsible for the external orange fruit color, was lower in sweet oranges stored at 12 °C than at 2 °C, outlining that this important quality indicator is better maintained at moderate temperatures. Additionally, increased levels of β-cryptoxanthin in orange pulp stored at 12 °C should be pointed out, due to health-beneficial provitamin A activity (Table 2) [96]. Carotenoid content, measured by HPLC, was also drastically increased during postharvest storage of winter squash at 21 °C, even if no induction of the biosynthetic genes could be observed. Starch degradation during winter squash storage, with the concomitant release of soluble sugars which may act as substrates for terpenoid synthesis, and downregulation of genes involved in carotenoid turnover, could be the explanation of their enhanced content [100]. In other fruits, such as green pepper, carotenoid accumulation during postharvest storage has a negative impact on consumer acceptance. Pepper reddening depends on the metabolic dynamic of chlorophyll degradation and active synthesis of carotenoids, such as β-carotene and capsanthin, as depicted by HPLC-based profiling of these pigments [101]. Quantification of chlorophyll by spectrophotometry has also pointed out its breakdown as a deterioration factor occurring during pear or lime shelf-life [102,103]. In this sense, postharvest strategies, such as chlorine dioxide fumigation or hot water treatment, may be effective in downregulating genes involved in chlorophyll-degrading enzymes [101,102].

# 5. Volatile Profiles during Postharvest and Their Impact on Fruit Aroma

In fruits, there are three major classes of metabolites responsible for flavor: sugars, acids, and volatile. While fruit taste is mostly dependent on the ratio of sugars and acids, it is the volatiles that determine the unique flavor of fruits. Most volatiles present in mature fruits originate from terpenoid and phenylpropanoid pathways or are fatty and amino acid derivatives [104]. Volatile profiling is typically achieved by extracting them from the headspace (HS), i.e., the airspace around the fruit, and detecting them by GC-MS. Sampling from headspace is most often performed by the adsorption of the volatiles on a stationary phase coated on a fused silica fiber and is known as solid-phase microextraction (SPME) [104]. Another GC-MS-based strategy for volatile profiling is their collection from chopped fruits on a Super Q column, followed by elution with methylene chloride [105]. To overcome metabolite co-elution by one-dimensional GC, GC × GC-MS has been implemented to increase separation efficiency and volatile detection [106,107]. As not all volatiles impact fruit aroma, a complementary approach, known as GC-olfactometry, can be used to determine odor-active compounds [108]. During postharvest, it could be established that important shifts in fruit volatile profiles are normally observed and are often responsible for the decreased sensory acceptability after prolonged storage. For instance, general trends profiled by GC-olfactometry, describe a loss of 'green' or 'fresh' notes and a concomitant increase in 'fruity', 'overripe' or 'musty' aromas [109]. Changes in aroma are a consequence of metabolic pathways that are active during postharvest and, in turn, appear to be largely depend on the storage strategies used by industry. For example, among the symptoms related to CI is the negative impact perceived on aroma production, a phenomenon described in many species, such as strawberries [110], kiwifruit [111], tomatoes [112] and peaches [113]. Tomatoes stored at 5 °C for 7 days were significantly less palatable than fruits recently harvested, and this decrease in consumer acceptance, established by taste panels, was a consequence of changes in volatile emissions [105]. Furthermore, a higher increase in 'musty' and 'damp' aroma notes was observed in tomatoes stored
at 10 °C than in those stored at 12.5 °C, suggesting that the latter temperature storage was able to maintain better sensory attributes [114].

Fermentation metabolism and amino acid and fatty acid catabolism are of great importance regarding the production and accumulation of volatiles in harvested fruits. Indeed, the activation of amino acid and fatty acid degradation to generate TCA cycle acetyl-CoA precursors and thus maintain energy production leads to the accumulation of specific substrates for volatile formation. In mandarin, a combination of metabolomic and transcriptomic data outlined the upregulation of genes involved in branched-chain amino acid catabolism, fatty acid cleavage and ethanol fermentation, which suggested that central metabolism modifications are accountable for the increase in branched-chain esters ('fruity', 'overripe' aroma), fatty acid-derived volatiles ('musty' notes) and ethanol [115]. The activation of anaerobic fermentative metabolism due to postharvest abiotic stress is especially important in 'off-aroma' compound generation and has been described in fruits of several species, including strawberries [116–119], apples [120], mandarins [109] and peaches [121], among others. Indeed, the glycolysis end-product pyruvate can alternatively serve as a substrate for anaerobic respiration and ATP production under O₂-limiting conditions, which produces a shift from aerobic respiration to the fermentation pathway [16,120,122,123]. As a consequence, off-aroma volatiles, namely ethanol, acetaldehyde and ethyl acetate, accumulate, playing a key role in fruit quality decline [117,124] (Table 3).
<table><caption>**Table 3.** Volatile compounds identified as putative biomarkers to evaluate the effects of postharvest storage on fruit aroma. d: day; w: week; GC–O: gas chromatography–olfactometry; GLC–MS: capillary gas–liquid chromatography–mass spectrometry.</caption><thead><tr><th>Volatile</th><th>Effect on Fruit</th><th>Postharvest Treatment</th><th>Behavior during Postharvest</th><th>Fruit Species</th><th>Metabolomic Platform</th><th>Reference</th></tr></thead><tbody><tr><td>ethanol, ethyl acetate, acetaldehyde</td><td>'Off-aroma' generation, alcohol' aroma</td><td>3 °C, 3 ws supplemented with different CO₂ concentration</td><td>Increase</td><td>strawberry</td><td>HS-SPME-GC-MS</td><td>[117]</td></tr><tr><td>ethanol, ethyl acetate, acetaldehyde</td><td>'Off-aroma' generation, alcohol' aroma</td><td>5 °C, 6 ws + 20 °C, 1 w</td><td>Increase</td><td>mandarin</td><td>HS-SPME-GC-MS and GC-O</td><td>[109]</td></tr><tr><td>ethanol, ethyl acetate, acetaldehyde</td><td>'Off-aroma' generation, alcohol' aroma</td><td>2.5 °C, 7 d + 1 °C followed by two different low oxygen protocols up to 240 d</td><td>Increase</td><td>apple</td><td>HS-SPME-GC-MS</td><td>[111]</td></tr><tr><td>ethanol, ethyl acetate, acetaldehyde</td><td>'Off-aroma' generation, alcohol' aroma</td><td>0 °C, 6 w + 20 °C, 2 days supplemented with different CO₂ concentration</td><td>Increase</td><td>grape</td><td>HS-SPME-GC-MS</td><td>[87]</td></tr><tr><td>β-mycene</td><td>Decrease in aroma quality</td><td>2 °C or 12 °C, 7 w</td><td>Increase at 2 °C, decrease at 12 °C</td><td>grapefruit</td><td>HS-SPME-GC-MS</td><td>[98]</td></tr><tr><td>ketone rootkatone</td><td>Confers characteristic ripe aroma fragrance</td><td>12 °C, 7 w</td><td>Increase in 12 °C</td><td>grapefruit</td><td>HS-SPME-GC-MS</td><td>[98]</td></tr><tr><td>limonene</td><td>Cold-induced responses</td><td>2 °C, 7 w</td><td>Increase</td><td>grapefruit</td><td>HS-SPME-GC-MS</td><td>[98]</td></tr><tr><td>limonene</td><td>Cold-induced responses</td><td>1 °C, 7 w</td><td>Increase</td><td>lemon</td><td>HS-SPME-GC-MS</td><td>[125]</td></tr><tr><td>limonene</td><td>Cold-induced responses</td><td>5 °C, up to 6 w</td><td>Increase</td><td>mandarin</td><td>HS-SPME-GC-MS</td><td>[126]</td></tr><tr><td>limonene</td><td>Senescence predictor</td><td>Combination of treatments, including 15 °C, 7 d + 2 °C, 18 d, 13 °C, 17 d</td><td>Increase</td><td>grapefruit</td><td>GLC-MS</td><td>[127]</td></tr><tr><td>α-farnesene</td><td>Correlation with Cl symptom development in 0 °C storage</td><td>0 °C up to 12 w, with or without ethylene</td><td>Increase</td><td>lime, mandarin, grapefruit, orange</td><td>GC-MS</td><td>[128]</td></tr><tr><td>linalool</td><td>Key component of fruit aroma</td><td>10 °C, 10 d + 22 °C until fully ripe</td><td>Decrease in low-temperature storage</td><td>papaya</td><td>HS-SPME-GC-MS</td><td>[129]</td></tr><tr><td>linalool</td><td>Key component of fruit aroma</td><td>0, 2, 5, and 10 °C up to 3 months</td><td>Decrease in low-temperature storage</td><td>Muscat table grapes</td><td>HS-SPME-GC-MS</td><td>[130]</td></tr></tbody></table>
Fermentative metabolism activation and off-flavor compound formation are mainly associated with low oxygen concentration under CA storage [118,123,124,131]. However, the production of ethanol via fermentation may also be a consequence of a decline in cellular energy status [132]. As long as energy demand is maintained, fermentation can be endured; nevertheless, failure of cellular homeostasis, such as an imbalance in the pH or ROS production, will lead to storage-induced disorders, strongly affecting fruit quality [133]. Understanding how or when fermentation occurs can help to limit ethanol production. Metabolomic approaches using ¹H NMR and GC-MS profiling were used to assess metabolite gradients within the fruit, which may be related to in situ hypoxia in the central part of the ripening fruit [134,135]. CA is of special importance for the long-term storage of fruits such as apples, and could maintain a better aroma quality [136,137]. It appears that the low-oxygen pressure employed during CA affects volatile emissions in a genotype-dependent manner [11]. Indeed, a multiplatform metabolomic approach (proton-NMR, GC-MS and HS-SPME-GC-MS) comparing 'Red Delicious' and 'Granny Smith' apple varieties showed strong activation of fermentative metabolism in the former, with ethanol and acetaldehyde accumulation, while the latter dealt with hypoxia by a reconfiguration of nitrogen metabolism through the intensification of alanine levels to prevent excessive accumulation of pyruvate [11]. Low oxygen may induce changes in metabolite concentrations that reflect a decrease in biosynthetic process, inhibition of the TCA cycle, and activation of anaerobic metabolism, which means accumulation of sucrose and organic acids and diversion of pyruvate to ethanol and alanine [134]. Table grapes stored under elevated CO₂ concentrations (5 kPa O₂ and 15 kPa CO₂) showed an upregulation of genes involved in pyruvate synthesis (*pyruvate kinase*, *PEP carboxykinase* and *NADP-dependent malic acid enzyme*) and a concomitant increase in volatiles, detected by HS-SPME-GC-MS, derived from pyruvate degradation—some of which were suspected to generate 'off flavor'. Additionally, the increased expression of a specific *alcohol dehydrogenase* gene (*ADH*) under anaerobic atmospheric conditions enhanced the accumulation of off-aroma volatiles, including ethanol, acetaldehyde and ethyl acetate [87].

Metabolic reconfiguration during postharvest affects volatile patterns beyond the generation of off-aroma compounds, and changes occurring in most important volatile classes are described in the next sections.

## 5.1. Fatty and Amino Acid-Derived Volatiles

Fatty acid-derived volatiles, responsible for aldehyde, alcohol and ester accumulation, the last being the predominant class of aromatic compounds in fruits of several species, seem to be strongly impacted by low-temperature storage [110,138,139]. Free fatty acids such as linoleic acid and linolenic acid are reduced to aldehydes by the lipoxygenase pathway (LOX). Next, aldehydes are reduced to alcohols followed by alcohols to esters by ADH and alcohol acyltransferase (AAT), respectively (for a review, see [84]). Interestingly, correlations among LOX, ADH and AAT activities, gene expression and decreased volatile production under refrigerated postharvest conditions have been established in several fruit-bearing species [140,141]. In particular, a relationship between a reduction in ADH activity and decreased ester content, monitored by SPME-GC-MS technology, during pear cold storage has been established [138]. In tomatoes, ADH activity was diminished as a consequence of refrigerated conditions at both 10 and 12.5 °C, and storage was associated with an increase in the aldehyde/alcohol ratio at 10 °C [112,114]. Furthermore, a decrease in *ADH2*, *LoxC* and *AAT1* transcripts after 8 days of cold storage (5 °C) was associated with lower levels of C6 and C5 (fatty acid-derived) volatiles in chilled tomatoes [105]. Additionally, low temperature also seems to affect upstream lipid catabolism by downregulating the expression of several genes involved in the formation of the unsaturated free fatty acids linoleic acid and linolenic acid, limiting substrate availability for ester biosynthesis [142]. Furthermore, membrane damage during cold storage has also been suggested to impair ester synthesis, as a relatively high leakage rate, a commonly used marker for membrane permeability, was measured in pears stored for a long time under refrigerated conditions [138]. The impact of cold storage on the aromatic compound profile reaches further than that on the pattern of lipid-derived volatiles.
Branched-chain volatiles derived from the direct precursors of branched-chain amino acids, measured by GC, after methylene chloride extraction, were also shown to decrease during tomato cold storage and are correlated with a lower expression of two branched-chain aminotransferases (*BCAT1* and *BCAT7*) involved in the first step of the catabolism of these amino acids [105]. Additional treatments, such as hot air or UV-C, combined with cold storage could counteract the negative effect on ester biosynthesis by promoting the *LOX* pathway, as has been demonstrated in peaches [143]. Similarly, a pre-chilling heat treatment (52 °C, 5 min) has been shown to alleviate the depletion of important volatiles for tomato aroma quality during its postharvest storage; in this case, the volatiles include amino acid- and carotenoid-derived compounds profiled by HS-SPME–GC–MS [144]. Fatty acid-derived alcohols were also higher under elevated CO₂ concentrations compared to those of recently harvested grapes and cold-stored berries under atmospheric conditions due to the upregulation of the *LOX* pathway, together with *ADH* [87].

Low-oxygen storage has a broader impact on volatile content than ethanol and off-aroma compound generation, as demonstrated by the different content of ethyl esters between ‘Granny Smith’ and Red Delicious’ apples [11]. Indeed, ethanol can serve as a substrate for ethyl esters, enhancing their synthesis [136,145,146] and competitively inhibiting the formation of esters originated from other alcohols [147]. As a consequence, an imbalance between the ratio of ethyl and the remaining esters occurs during postharvest storage of fruits of ethanol-accumulating apple varieties and those of many other fruit-bearing species, most likely affecting aroma perception. The fruits of ‘Granny Smith’ and ‘Royal Gala’ apple varieties did not seem to accumulate ethanol under low-oxygen-pressure storage; however, a negative effect on ester synthesis, in particular straight-chain esters, was observed, with the impact proportional to the decrease in O₂ pressure [148–154]. This decrease can be explained by the fact that the LOX pathway requires the presence of oxygen. This effect has also been described in other apple varieties. [137,153]. In contrast, the concentration of branched-chain esters, monitored by HS-SPME–GC–MS, did not seem to be negatively affected by low oxygen, possibly because branched-chain amino acid levels were unaltered [154]. Furthermore, low oxygen suppresses the production of the hormone ethylene, which is involved in ester synthesis, as demonstrated during apple or banana storage in the presence of its antagonist 1-MCP [155–157].

## 5.2. Terpenoid Volatiles

Several studies in *Citrus* have highlighted important changes in terpenoid volatiles monitored by HS-SPME–GC–MS. These changes were related to Cl and tolerance to cold storage. In mandarins, the accumulation of terpenoid volatiles is associated with chilling-sensitive fruits [158]. This increase is temperature dependent, and the authors suggest that it was responsible for decreased fruit palatability, as terpenes can contribute to an unpleasant aroma, providing ‘musty’, ‘resinous’ and ‘oily’ notes.

Another study on volatile emission by intact grapefruits stored at 12 and 2 °C for 7 weeks outlined important differences in the profiles of the terpenoid volatiles as a consequence of temperature [98]. Interestingly, grapefruits stored at 2 °C experienced a strong increase in monoterpene content, particularly in limonene and β-myrcene levels; this group of volatiles was strongly decreased in fruits stored at 12 °C at the beginning of the postharvest period, after which their content remained unchanged. In contrast, sesquiterpene emissions were predominant in the fruits stored at 12 °C [98]. While the accumulation of the monoterpene β-myrcene under 2 °C can negatively impact negatively consumer acceptance, by providing ‘musty’ and ‘wet soil’ aroma notes to the fruits [108], sesquiterpene ketone nootkatone levels, an important volatile in grapefruit aroma, seems to be promoted (in term of it content) under moderate–intermediate-temperature storage, but not refrigerated conditions [98,159] (Table 3). Taken together, these data suggest that the aroma quality of grapefruits could be better maintained during intermediate-temperature storage, as has been previously demonstrated in mandarins [126,149].

The trend in the accumulation of the monoterpene limonene and the sesquiterpene α-farnesene, which showed a transient increase after one week of storage under the two different temperatures, could be related to cold-induced responses. Limonene release, measured both by HS-SPME–GC–MS and
capillary gas-liquid chromatography-MS, has also been described in other *Citrus* species [125-127,160] and could be a consequence of cell wall and plasmatic membrane disruptions in the oil glands [99,125]. The degree of accumulation of α-farnesene is correlated with the susceptibility of different cold-sensitive *Citrus* species to CI development at 0 °C storage [128]. Interestingly, α-farnesene stopped being emitted by grapefruits after 3 weeks of storage at 12 °C, which coincided with the decrease in the observed CI symptoms, i.e., peel injury. At 2 °C, α-farnesene emissions were maintained during the whole postharvest period, concomitant with the CI symptom progression, confirming the relation between the detection of this volatile and CI manifestation. In this sense, the detection of α-farnesene by metabolome-driven approaches could be of high value as a potential biomarker to assess *Citrus* quality during postharvest (Table 3). Similarly, the monoterpene linalool, a key component of the aroma of papaya, was negatively affected by cold postharvest storage at 10 °C, and a concomitant downregulation of linalool synthase expression was also observed, suggesting that this volatile could be used as a marker to define papaya quality during postharvest storage [129] (Table 3). Linalool is also the predominant compound responsible for flavor in Muscat table grapes, a highly appreciated quality trait [161]. The postharvest storage of ‘Shine Muscat’ grapes at different temperatures between 0 and 10 °C showed that the decrease in linalool, profiled by HS-SPME-GC-MS, was enhanced at relatively low (0, 2 and 5 °C) temperatures in both fruit skin and flesh. Concomitant with linalool levels, grapes stored for four weeks at 10 °C presented a higher Muscat flavor than grapes stored at 0 °C for the same duration [130]. A possible effect of temperature on linalool synthesis or on the interconversion of free (aroma-producing) linalool and its glycosidically (odorless) bound form could be responsible for the observed differences in its concentration. In this sense, optimal storage at 10 °C for a short period or at relatively low temperatures followed by poststorage conditioning at 10 °C is fundamental for maintaining aroma quality for consumers [130] (Table 3).

The combination of 1-MCP with high O₂ or high CO₂ seemed to favor terpene content, as did the CO₂-enriched atmosphere in lemon [162]. CA storage under an elevated CO₂ atmosphere (8% CO₂ and 2%–3% O₂) could also promote terpene accumulation in mango. Indeed, fruit injury as a result of CA can enhance the activity of glycosidases, releasing monoterpenes, such as linalool or terpineol, from their glycoside-bound forms [163]. However, all tested CA treatments resulted in a reduction in total sesquiterpenes and also enhanced levels of ethanol, acetaldehyde and esters compared to those under atmospheric conditions. In addition, it was established that mangoes should not be stored under 3%–5% O₂ to avoid excess fermentative compound accumulation and maintain fruit aroma quality [163,164].

## 6. Conclusions

Although fruit responses to postharvest storage conditions are species and even cultivar dependent, making them especially complicated to study, metabolomic approaches alone or combined with transcriptomic/proteomic analyses are highly useful for understanding how metabolic changes affect quality traits. In particular, reconfiguration of fruit metabolism as a consequence of the abiotic/biotic stress encountered during postharvest storage conditions (cold, hypoxia, pathogens, etc.) has a direct impact on the accumulation of taste- and aroma-producing metabolites, which are decisive attributes for consumers and thus for the fruit industry. Even though many molecular mechanisms active during fruit postharvest storage and senescence remain elusive, future omic studies will shed light on them to optimize fruit storage conditions.

Furthermore, the recent advances in metabolomic-driven technology allows the identification of valuable biomarkers that can be employed by the fruit industry to tightly monitor changes in quality attributes during postharvest storage. In this sense, the use of multiplatform approaches offers the possibility to select a set of metabolite markers, which could better depict the impact of postharvest storage on aroma, taste, appearance and nutritional value [165].

**Author Contributions:** All the authors did the literature searching, writing and original draft preparation. All authors have read and agreed to the submitted version of the manuscript.
**Funding:** The authors want to acknowledge funding through grants RTI2018-099797-B-100 (Ministerio de Ciencia, Innovación y Universidades, Spain) and UMA18-FEDERJA-179 (FEDER-Junta Andalucía). In addition, we acknowledge partial funding by the University of Malaga (Plan Propio) and the European Union's H2020 Programme (GoodBerry; grant number 679303). D.P. acknowledges the support by the Spanish Ministry of Science and Innovation (BES-2013-062856).

**Conflicts of Interest:** The authors declare no conflict of interest.

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