Comparative Metabolomic Fingerprinting Analysis of Tomato Fruits from Physalis Species in Mexico’s Balsas Basin

Abstract

This study investigated the chemical and sensory distinctions in tomato fruits from three Physalis species (P. ixocarpa, P. angulata, and P. philadelphica) found in Michoacán, Mexico, using metabolomic fingerprinting through GC-MS analysis. The objective was to identify organoleptic differences that could influence consumer preferences, highlighting the significance of these species’ unique traits. These species represented a valuable genetic reservoir for potential hybridization or selection aimed at enhancing commercial varieties by focusing on organoleptic properties rather than traditional selection criteria like fruit size or yield. This research emphasizes the importance of preserving Mexican biodiversity and providing insights into domestication processes that prioritize flavor and sensory qualities. By analyzing metabolite profiles and their correlation with taste preferences, this study contributes to understanding how these differences could be leveraged in breeding programs to develop new tomato varieties with preferred flavors. It was suggested that variations in taste among the species are mainly due to differences in metabolite expression. This knowledge underscores the importance of organoleptic properties in the selection and domestication of edible fruits, offering a pathway toward the conservation and enhancement of tomato varieties through the exploitation of genetic diversity for organoleptic improvement.

1. Introduction

The genus Physalis is renowned for its rich diversity and its significant roles in the agriculture and food industries. It is native to and has diversified extensively in Mexico, comprising approximately 65 species [1,2]. This genus exhibits remarkable biological diversity, including numerous edible species that have been cultivated and domesticated over time [3,4]. The phenotypic variability among these species is considerable, influenced by inherent biological characteristics and selective pressures from agricultural practices aimed at optimizing agronomic yields and environmental resilience [5].

Domestication efforts have typically focused on improving fruit yield and size, as well as modifying plant and fruit morphology to enhance adaptability and productivity. Furthermore, organoleptic qualities such as acidity, sweetness, and bitterness are critically assessed, as these traits significantly impact consumer preferences and guide the selection of particular cultivars [6]. Within this genus, Physalis philadelphica stands out as the most commercially significant species, characterized by larger fruits and a mildly acidic taste profile, making it an essential component of many traditional Mexican dishes [5,7,8].

The most well-known Physalis species is P. philadelphica, which is distributed worldwide and from which commercial varieties have been developed [7]. Unlike other less domesticated species, it is characterized by a larger fruit (up to six times bigger) and a slightly acidic taste [5,8]. Despite being the only commercially cultivated Physalis species, it is a fundamental ingredient in many dishes of traditional Mexican cuisine [9].

In Mexico, it is common to find various locally consumed Physalis varieties [10]. These showcase distinct flavors and chemical properties. In the Ciénega region of Michoacán, which belongs to the Balsas Basin, it is still possible to find species, such as P. angulata and P. ixocarpa, cultivated and marketed under the name “tomate milpero” [11,12].

Previous research suggests that the domestication of these species is still ongoing, though not necessarily with a focus on increasing fruit size or yield [13]. However, both species are valued for their organoleptic properties in contrast to commercial tomatoes.

This study aims to analyze the chemical differences in the fruits of the three main Physalis species marketed in the Cienega region of Michoacán. Employing metabolomic fingerprinting techniques through Solid Phase Microextraction, Gas Chromatography–Mass Spectrometry (SPME-GC/MS), we seek to uncover variations in the organoleptic properties of the fruits. This methodology not only enables the discovery of new traits that could be integrated into commercial varieties but also supports the conservation of a vital segment of Mexican biodiversity, thereby safeguarding a valuable genetic reservoir for future hybridization and selection efforts.

2. Materials and Methods

2.1. Sample Collection

Biological samples were procured from local markets in the Cienega region of Michoacán. Fruits were sourced directly from local producers and collectors, ensuring their provenance from municipalities adjacent to the region. Selection criteria for the fruit specimens included an absence of disease or insect damage and a uniform stage of maturity. Maturity was verified through consistent indicators of fruit quality, such as turgency and uniformity of color. These stringent criteria ensured the collection of high-quality specimens suitable for subsequent analysis.

2.2. Metabolomic Fingerprinting Analisis using SPME-GC/MS

2.2.1. Sample Preparation and Extraction

Fruits were meticulously cleaned by rinsing them three times with distilled water before they were air-dried in a shaded area to prevent chemical degradation from sunlight exposure. Once dried, the fruits were rapidly frozen using liquid nitrogen to halt all metabolic activity and were stored at −80 °C to preserve their biochemical integrity. Subsequently, the fruits were lyophilized in a vacuum chamber to remove moisture without degrading sensitive compounds. The dried samples were then finely ground using a Retch mill to produce a uniform powder, facilitating consistent sample handling and extraction. A total of 100 milligrams of the lyophilized and ground tissue from each fruit sample was precisely weighed and transferred into amber vials to protect the sensitive compounds from light degradation. For statistical robustness, five replicates were prepared for each population studied. The vials were then incubated at 40 °C for one hour to equilibrate.

2.2.2. Solid-Phase Microextraction (SPME)

A 50/30 μm DVB/CAR/PDMS fiber (Supelco, Bellefonte, PA, USA) was used for extraction. The fiber was preconditioned in the GC/MS at 230 ± 1 °C for 15 min before each run. For analysis, the fiber was exposed for 30 min in the headspace of the sample at a controlled temperature of 30 ± 1 °C. The fiber was then stored in a holder (57330-u, Supelco, PA, USA) to prevent contamination and subsequently introduced into the GC/MS injector at 230 ± 1 °C to desorb the volatile organic compounds (VOCs). All analyses were performed in triplicate to ensure reproducibility.

2.2.3. Gas Chromatography–Mass Spectrometry (GC/MS)

A Clarus 680 gas chromatograph (Perkin-Elmer Inc., Massa, MA, USA) equipped with an Elite-5 MS capillary column (30 m length, 0.32 mm ID, 0.25 μm film thickness, operational temperature range from −60 to 320/350 °C) was used. Helium was employed as the carrier gas at a constant flow rate of 1 mL/min with an initial hold time of 0.05 min. The column temperature started at 30 ± 1 °C for 2 min and was then ramped up to 140 °C at 9 °C/min and held for 5 min. The injector temperature was maintained at 230 ± 1 °C. The mass spectrometer (Clarus SQ8T, Perkin-Elmer Inc., Massa, MA, USA) operated with an electron impact ionization source at 70 eV in the scan mode, analyzing a range from 30 to 400 m/z. The temperatures of the transfer line and the ionization source were set at 230 and 250 °C, respectively, optimizing the transfer and ionization of analytes. This comprehensive setup allowed for detailed profiling of the metabolomic fingerprint of each fruit sample, providing insights into their unique chemical compositions.

2.3. Data Analysis

To discern differences in the metabolomic fingerprints of the samples, we adopted a Non-Targeted Metabolomic approach. Original (.raw) files were converted to the CDF format for examination on the XCMS Online platform (https://xcmsonline.scripps.edu) [14]. This platform facilitated feature detection, retention time adjustment, and the peak alignment of original chromatograms [15]. To avoid false positives in metabolite identification, only those with q values ≤ 0.05 were considered. Post-detection by XCMS Online, data normalization, and an ANOVA test were executed to pinpoint pivotal metabolites in relation to treatments. We manually annotated the chosen metabolites using the NIST library with a threshold of 0.8. Annotated results underwent analysis in RStudio ver. 2023.03.0 utilizing the MetaboanalystR ver. 3.3.0 library [16]. Detected metabolites were categorized by their chemical groupings, followed by a comparative analysis of their relative and absolute expression proportions. A subsequent ANOVA discerned significant metabolites in each trial.

We computed a correlation matrix to detect relationships between diverse metabolites. Results were charted using the corrplot library in R, exclusively highlighting correlations with statistically significant differences. To ascertain inter-species distinctions, we constructed a heatmap utilizing the ion matrix of the most differentiated metabolites. The heatmap’s construction used automatically normalized and scaled data. Dendrograms employed the Minkowski correlation as a distance metric, paired with the Ward clustering algorithm, and set branch significance at p ≤ 0.05.

3. Results

3.1. Metabolite Profiling across Physalis Species

Across the three studied Physalis species, a total of 50 metabolites were identified, which belong to 15 chemical groups. The aldehydes group contained the highest number of metabolites, numbering 11, followed by alcohol with 10. Notably, eight chemical groups comprise only a single metabolite (Figure 1). All species exhibited identical metabolites, with no evidence of any compound’s absence or exclusivity to a particular species.

3.2. Metabolite Distribution by Chemical Group

Significant differences (p < 0.05) in metabolite expression were observed across almost all chemical groups, excluding esters and terpenes (Figure 2). The groups expressing the highest levels, in descending order, were alcohols, ketones, aldehydes, alkenes, and lactones. All other chemical groups displayed reduced expressions in comparison. In all chemical groups, P. angulata manifested the most pronounced expression compared to the other two Physalis species.

Concerning relative compound abundance in the fruits, a similar trend to absolute expression was noticed, with alcohols, aldehydes, and ketones being the most predominant groups, together comprising 75% of the total compound abundance. The remaining chemical groups contributed a lower proportion (Figure 3).

Within the alcohol group, the species P. angulata and P. philadelphica exhibited a higher relative abundance compared to P. ixocarpa. As for aldehydes, P. ixocarpa showed a marked abundance of these compounds compared to the other two species. Specifically, for P. angulata and P. philadelphica, ketones emerged as the second most abundant group, followed by aldehydes. Conversely, in P. ixocarpa, aldehydes were second in abundance, trailed by ketones. Alkenes, lactones, and sugars displayed some differences in proportion, but their overall abundance was limited.

3.3. Alcohol and Aldehyde Groups

Alcohol represents the most abundant group in the fruits of all three species. Within this group, four specific alcohols accounted for nearly 75% of the total alcohol abundance: 1-Penten-3-ol, 1-phenyl-; 2,5-Dimethylcyclohexanol; Benzenemethanol, 4-ethyl-; and 2-Isopropyl-5-methyl-1-heptanol (Figure 4). Notable differences among species were observed in less abundant alcohols, such as 2-Cyclohexen-1-ol and 1-methyl-, with P. ixocarpa displaying the highest abundance.

Aldehydes emerged as the second most abundant group, holding significant importance due to their contribution to fruit flavors (Figure 5). A greater variability was observed among the aldehydes than the alcohols, stemming from divergent proportions across species. P. angulata and P. philadelphica shared similar aldehyde distributions. A notable difference was evident in the benzaldehyde levels between these two species. Conversely, P. ixocarpa presented a distinct aldehyde profile, with furfural as the predominant aldehyde, followed by 2,5-dihydroxybenzaldehyde. This species exhibited a significant decrease in levels of octanal, benzaldehyde 4-methyl, 2-heptenal, and 2-hexenal relative to the other species while showing an increase in 2,5-dihydroxybenzaldehyde.

3.4. Metabolite Correlations

Chemical group correlations across each of the three populations were presented as a matrix, only including statistically significant correlations (p < 0.05). In P. ixocarpa, four positive correlations between chemical groups were found. Noteworthy interactions were observed between sugar concentrations with amino acids and between alcohols and ketones (Figure 6).

P. angulata exhibited both positive and negative significant correlations. Perhaps the most crucial correlation, especially regarding flavor context, was the inverse relationship between aldehydes and sugars, which showed a correlation coefficient of −1 (Figure 7).

In P. philadelphica, eight significant correlations were identified, with the interactions between sugars with amino acids and aldehydes being particularly important (Figure 8).

3.5. Heatmap Analysis

Of the 50 detected metabolites in Physalis species’ fruits, 20 displayed significant differences in relative expression (p ≤ 0.05). The heatmap was constructed using the normalized expression of metabolites, which showed significant variance. The expression levels of metabolites are color-coded, ranging from blue (indicating low expression) to red (indicating high expression) (Figure 9). In the figure, each row signifies an individual metabolite, and each column corresponds to a specific Physalis species. The top dendrogram indicates a global classification (metabolomic fingerprinting), suggesting that P. ixocarpa and P. philadelphica are more closely related than P. angulata, which exhibits a contrasting expression pattern. The side dendrogram delineates the metabolites according to their expression levels. Here, metabolites from P. ixocarpa and P. philadelphica express higher levels relative to P. angulata. Specific metabolites with heightened expression in P. ixocarpa include 2-Cyclohexen-1-ol, 1-methyl-; Terpinen-4-ol; 2-hydroxy-2,4-dimethyl-hept-6-en-3-one; (+)-4-Carene; methyl salicylate; furfural; and Benzaldehyde. In contrast, P. philadelphica showed elevated expression in 1-Undecene, 7-methyl-. In this section, P. angulata predominantly underexpressed these metabolites.

The bottom portion of the heatmap depicts an inverse trend where P. angulata shows a pronounced overexpression of metabolites like Ethanol; 4,5-Dipropenyldihydro-furan-2-one; 2-hexenal, (E)-; Heptadecane; 3-Octyne, 7-methyl-; Hexanal octyl ether; Furan, 2-ethyl-; and 1-Octen-3-one. Conversely, P. ixocarpa and P. philadelphica largely exhibit reduced or even negative expression levels.

4. Discussion

In this study, the chemical composition of the fruits from three species of the genus Physalis found in the Balsas basin region of Mexico was analyzed. Significant differences were identified in the absolute expression and relative abundance of various metabolites. However, there were no differences in the presence or absence of metabolites across the three studied species. As such, variations in taste can be attributed to the proportions of these metabolites. These observed discrepancies might primarily arise from the organoleptic characteristics selected during domestication, given that the three species undergo varying levels of selection.

The Balsas basin has a rich history of domesticating plant species with agricultural value. P. philadelphica is the most renowned species due to its global production and consumption [17]. Conversely, the species P. angulata and P. ixocarpa are fruits primarily produced and consumed in specific regions of Mexico [3,4,8], and they exhibit morphological and organoleptic characteristics distinct from P. philadelphica, which are valued by locals [13].

The lack of selection based on size suggests that the primary focus in the selection process revolves around traits associated with fruit flavors [5,13]. Both Physalis species are reported to have more pleasant tastes than their commercial counterparts. These attributes could be harnessed in breeding programs to establish commercial varieties [18].

The absence of exclusive metabolites among the three species suggests a low differentiation level, indicating no evolution of new biochemical pathways [19]. Differences are likely due to metabolic regulation, where certain metabolites are produced preferentially due to selection (domestication) processes. This phenomenon has been documented in species with extensive selection histories, where the search for novel organoleptic features remains a primary goal [20,21]. Such is the case with chili peppers and other fruit-bearing plants [22]; defining taste is complex as it involves a vast array of compounds, and their proportion is crucial.

Significant differences were observed in the relative expression of nearly all detected chemical groups. P. angulata exhibited the highest abundance in most chemical groups. However, fruits from this species are not the preferred choice for residents of the Cienega de Michoacán region. Local inhabitants report that P. angulata fruits have a more acidic and bitter taste compared to the P. ixocarpa fruits (J. R. Torres-García, pers. observ.). This flavor variation might be due to the dominant presence of alcohols, overshadowing the tastes from other pleasant-tasting compounds that are present in smaller proportions [6].

In addition to noticeable concentration differences among this species, perhaps the most critical aspect is the balance of various metabolites within the fruit. For instance, sugars and terpenes, known for their contribution to food flavor, were found in relatively smaller concentrations compared to other chemical groups [23].

In all three species, the chemical groups of aldehydes and alcohols had the highest number of metabolites, making them the most abundant in both absolute and relative metabolite expressions. As previously mentioned, all three species had the same compounds, but their proportions varied. In the case of alcohol, the most prevalent were 1-Penten-3-ol and 1-phenyl- in P. angulata and P. philadelphica, both of which impart a bitter taste. In contrast, in P. ixocarpa, higher concentrations of alcohol like Benzenemethanol 4-ethyl-, and 2-isopropyl-5-methyl-1-heptanol were detected, which contribute pleasant aromas and fruity flavors to foods [21].

Correlations indicate that even if the same metabolites are shared, their expression level and interrelation differ between species. Various studies have suggested that domestication can influence the flavors or organoleptic characteristics of plants [20,21]. In most of these, it is argued that this is due to differences in gene regulation related to flavor compound synthesis.

In the case of P. ixocarpa, the positive correlation between sugars and amino acids is relevant both nutritionally and organoleptically [24]. An increase in the fruit’s total sugars is associated with an increase in the number of amino acids, specifically lysine, which is an essential amino acid. Thus, a better taste also implies a higher nutritional value. Furthermore, the correlation between alcohols and ketones is closely linked to flavor, as both chemical groups significantly influence fruit flavor. These correlations are crucial since they affect both taste and nutrition, which are two essential characteristics for genetic improvement [18]. For P. angulata, the negative correlation found between aldehydes and sugars could be a reason why its flavor is not as popular in the Cienega de Michoacán region. Both chemical groups contribute to organoleptic characteristics, providing a pleasant taste and aroma [6]. The negative correlation suggests that it is challenging to maximize one’s concentration without compromising the other, limiting the selection of improved taste varieties. For P. philadelphica, relevant correlations were observed in both nutritional (amino acid–sugars) and organoleptic characteristics (aldehyde–sugars, aldehyde–terpenes). It is the species of Physalis genus that has the highest production and global consumption, which might explain the observed correlations, possibly due to improvement schemes [18].

Classification based solely on metabolites with significant statistical differences (p ≤ 0.05) allows for a more objective evaluation between species. It was observed that P. ixocarpa and P. philadelphica have more similar metabolic profiles compared to P. angulata.

Specifically, certain compounds were identified to be more significant than others in terms of classification and flavor attributes. In the case of P. ixocarpa, higher concentrations of certain compounds pivotal to flavor contribution were observed, such as 2-Cyclohexen-1-ol, 1-methyl-, which possesses a sweet and woody aroma [21] and is frequently found in plant essential oils. Terpinen-4-ol exudes a herbaceous and spicy aroma and has been recognized for its antimicrobial potential, potentially influencing food preservation [24]. Benzaldehyde, with its bitter almond scent, is extensively employed in the food industry and can be found in other aromatic fruits, such as the Huangjiu [25]. Another notable metabolite, (+)-4-Carene, carries a sweet and resinous scent. Among the compounds most expressed in this fruit are furfural, which emits a sweet and almondy aroma, and methyl salicylate, with its sweet minty scent, which is commonly utilized in the food and cosmetic industries. However, two compounds were also identified that do not contribute pleasant flavors. These include 2-hydroxy-2,4-dimethyl, which has a burnt scent, and Cyclopentanecarboxaldehyde, 2-methyl-3-methylene-, which emits a strong and unpleasant odor. In the case of the latter, its expression level was found to be equal to that in P. angulata.

In P. angulata, there is a higher expression of compounds such as Ethanol; 4,5-Dipropenyldihydro-furan-2-one; 2-hexenal, (E)-; Heptadecane; 3-Octyne, 7-methyl-; Hexanal octyl ether; Furan, 2-ethyl-; and 1-Octen-3-one. These compounds typically impart mild and pleasant flavors. However, at high concentrations, they can produce unpleasant tastes. On the other hand, compounds known to deliver unpalatable flavors include 3-Octyne, 7-methyl-; Heptadecane; Furan, 2-ethyl-; 1-Octen-3-one; Butanoic acid, 4-hydroxy-; and Cyclopentanecarboxaldehyde, 2-methyl-3-methylene-. The latter displays an expression level similar to that in P. ixocarpa, suggesting that domestication processes focusing on flavor have not encompassed all metabolites, and characteristics of wild relatives can still be found. Conversely, the compound Cyclopentanecarboxaldehyde, 2-methyl-3-methylene- showed the lowest expression in the P. philadelphica population. This compound is an aldehyde that can also influence the aroma and taste of tomato fruits. Nonetheless, due to its low expression, its contribution to the flavor of fruits from this population may be less significant.

In light of the observations from this comprehensive study, it becomes evident that the process of domestication and genetic selection within the Physalis genus has led to significant variations in metabolite profiles among species, even when the same metabolites are universally present. These variations are intricately connected to perceived tastes and organoleptic characteristics and are further influenced by the relative concentrations of different metabolites. It is crucial to recognize that the quest for optimizing flavor does not solely rely on the presence or absence of specific compounds but depends heavily on their relative proportions and interrelations. As the world continues to globalize and the demand for diverse and unique flavors increases, understanding these intricate relationships in crops like Physalis becomes paramount. Embracing this knowledge can pave the way for creating optimized crop varieties that cater not just to local preferences but can find acceptance on a global palate. Furthermore, this study underscores the value of revisiting and appreciating the biochemical complexity inherent in our traditional crops, as there remains a vast untapped potential to harness their genetic diversity for the betterment of global agriculture and food systems.

5. Conclusions

In conclusion, this study on the metabolomic profiling and sensory characteristics of tomato fruits from three distinct Physalis species in the Cienega region of Michoacán, Mexico, provides clear evidence of the significant chemical diversity that exists among the species studied. Utilizing advanced metabolomic fingerprinting techniques via SPME-GC/MS, we were able to identify specific variations in the organoleptic properties of the fruits, which directly impact consumer preferences. These findings highlight the importance of these species as valuable genetic reservoirs that could be exploited to enhance commercial tomato varieties, focusing on improving organoleptic qualities over traditional selection criteria such as fruit size or yield. Moreover, this study underscores the relevance of preserving Mexican biodiversity and leveraging its potential to strengthen the sustainability and acceptance of native varieties in local and global markets. Integrating these insights into breeding programs could lead to the development of tomato varieties that not only meet market demands in terms of flavor and aroma but also contribute to the conservation of native species, enriching global agricultural diversity.