Biochemical variances through metabolomic profile analysis of Capsicum chinense Jacq. during fruit development

Pepper fruits (family Solanaceae) are widely diverse, representing more than 200 species that vary according to size, colour, shape and chemical composition. They are important nutritional and economical fruits which can be consumed fresh as vegetables, and serve as spices when dried because of their pungent and unique flavor. Additionally, it is an important pharmaceutical resource in topical ointments and pain-relievers, serving as ingredients and patches, respectively, and also aids in digestion in the form of condiments. Hot peppers contain broad biological compounds like carotenoids (provitamin A), capsaicinoids, flavonoids, vitamins (vitamins C and E), minerals, essential oils and the aroma of the fruits (Aniel et al., 2009; Purkayastha et al., 2012; Howard et al., 2016). These compounds have exhibited anticancer (Oyagbemi et al., 2010; Anandakumar et al., 2013), anti-inflammatory (Spiller et al., 2008), antimicrobial (Careaga et al., 2003) and antioxidant (Alvarez-Parrilla et al., 2011) properties.

Previously conducted studies on pepper fruits have majorly focused on the antioxidant activity (Tan et al., 2012), nutritive components (Serrano et al., 2010) and phenolic contents (Tan et al., 2012) across various fruit developmental stages. Moreover, the majority of these metabolomic studies focused only on the targeted metabolite analysis that evaluated capsaicinoids, carotenoids, flavonoids and ascorbic acid. Thus, there exists a research gap in extensive non-targeted metabolomic studies on pepper. Indeed, non-targeted metabolomic approaches can elucidate the plant responses to various environmental situations, as displayed by the changes in metabolites seen in its application in various plant species such as Oryza sativa (Jung et al., 2013; Kim et al., 2014), blueberries (Lee et al., 2014a) and pitayas (Lee et al., 2014b). In pepper fruits, the phytochemicals generally undergo various changes all through fruit development, which could impact on significant dietary aspects concerning the consumption of pepper (Ghasemnezhad et al., 2012).

Equally, capsaicinoid compounds are found among the members in the genus Capsicum. Capsaicinoid is the group of alkaloids which gives pungency or heat to hot peppers. Its biosynthesis entails a combination of pathways, namely the phenylpropanoid and branched chain fatty acid pathways. The precursors for the formation of vanillyl aminophenylalanine are generated by the phenylpropanoid pathway while the branched chain fatty acid pathway provides valine or leucine for 8-methyl-6-nonenoylCoA precursors. Capsaicinoids are known to accumulate in the dissepiment of the placental tissue of the epidermal layer. Its synthesis starts after 20–30 days of plant pollination and lasts up to the ripening stage of the fruit (Stewart et al., 2005). Thus far, about 23 analogues of capsaicinoids have been described. Capsaicin and dihydrocapsaicin (trans-8-methyl-N-vanillyl-6-nonenamide, 8-methyl-N-vanillylnonanamide, respectively) form the highest capsaicinoid constituents of up to 98% in Capscium (Zewdie et al., 2001). Other less major capsaicinoids in capsicum fruits include nonivamide, homocapsaicin, homodihydrocapsaicin and nordihydrocapsaicin (Huang et al., 2013).

Therefore, ultra-performance liquid chromatography (UPLC) and mass spectrometry (MS) were used to examine the metabolite profile of Capsicum chinense Jacq. during the development stages of the pepper fruit. The identified metabolites could assist in defining the diversity of pepper metabolites and the molecular basis of pepper fruit pungency.

Fruits are a significant part of the dietary needs in humans due to their fiber, vitamins, minerals and flavour (Giovannoni, 2004). During their development and ripening, fruits undergo various changes associated with their nutrient composition, colour, aroma and even texture. These developmental changes occur due to various alterations associated with the biochemical and physiological processes that involve enzymatic activity, gene expression and formation of metabolites, in response to environmental perturbations (Osorio et al., 2012). Numerous scientific studies have aimed to investigate the developmental, maturation, ripening stages and organogenesis of fruits (Giovannoni, 2007). Herein, we applied the metabolomics approach in assessing the various cascade profile changes of Capsicum chinense Jacq. pepper fruit during its development stages. From our study findings, we established that variation in the pericarp colours and metabolite content were diverse and distinct for each developmental stage (16, 36 and 48 DPA).

During the three fruit developmental phases, the pericarp colours of the pepper fruits transformed as shown in Figure 1A–C. In pepper fruit development, carotenoids are known as the visual markers in maturation. This is attributed to the change in colour of the green pepper fruit to yellow and finally red or yellow, depending on the carotenoids synthesized and accumulated by the fruits (Gómez-García et al., 2013). PCA established that the metabolites at 16 DPA were highly distinct, compared with the 36 and 48 DPA developmental phases. Equally, the metabolites in the last two developmental phases exhibited a high correlation between them (Figure 2). This phenomenon can be attributed to the diverse gene transcriptomic and metabolite variations of the fruit during its development.

During the early developmental stages (16 DPA), the levels of most organic acids, such as dicaffeoylputrescine and maltose, which were initially high, gradually decreased. Generally, organic acids are known to play a crucial role in contributing to the flavour, taste and quality of the fruits (Shin et al., 2015). However, organic acid constituents are diverse, depending on the species of the plant, its developmental stage and tissue type (López-Bucio et al., 2000). A major polyamine, putrescine, is directly produced from ornithine through the action of ornithine decarboxylase enzymes, and the ornithine levels expressed showed consistency with the metabolic levels of dicaffeoylputrescine. Our findings were in agreement with those of other researchers (Aizat et al., 2014), where their teams had studied putrescine compounds extensively, because it is linked to biotic and abiotic stressors, ethylene production, plant growth, flowering and fruit development (Malik and Singh, 2004; Choi et al., 2012). Most flavonoid compounds like apigenin 7-O-(6″-O-acetylglucoside) also showed high levels at 16 and 28 DPA.

During development, the colour of the pepper fruit generally changes from green to red, while an orange colour signifies the breaker stage or changing point (Sun et al., 2006). The amino acids, compounds that are naturally found in fruits and vegetables, play an important role in the maintenance of quality and nutritional value of the fruits (Glew et al., 2003). The precursor L-valine amino acid, found at one end of the capsaicin chain structure, through its pathway, plays an essential role in the biosynthesis of capsaicin (Keum et al., 2012).

At the later stages (48 DPA), the hydrophobic amino acids, L-leucine, L-phenylalanine L-aspartic acid and dihydrocapsaicin levels were higher significantly than at the earlier stages. The precursor of the phenylpropanoid biosynthetic pathway, L-phenylalanine, is associated with the production of secondary metabolites in plant species. In addition, PAL (CA09g02410) is a major enzyme during biosynthesis of phenolic compounds, which is derived by converting phenylalanine to transcinnamic acid in the initial stages of the phenylpropanoid pathway; it is also a precursor in both flavonoid pathways and biosynthesis of capsaicinoid (Sutoh et al., 2006).

During pepper fruit maturation, the contents of capsaicinoid compounds change with each developmental stage (Howard et al., 2000). Capsaicinoid metabolites undergo biosynthesis through condensing of vanillylamine, a derivative of the phenylpropanoid pathway, and a series of branched-chain fatty acid moieties, which originated from the branched-chain fatty acid pathway (Zhang et al., 2016). The precursors of these aforementioned pathways include Phenylalanine, and valine or leucine. In the pepper fruit, capsaicinoids are biosynthesized and accumulate to the maximum value during the ripening period in the placental tissue. In the present study, the capsaicinoid levels gradually increased from 16 DPA up to 48 DPA, while the peak accumulation was observed at 846.64 ± 157.48 mg · g⁻¹ at the colour transition stage, which is developmental phase C. These findings are in agreement with a previously conducted study by Bae et al. (2014), which established an increase of capsaicin and dihydrocapsaicin levels significantly in Cayenne pepper developmental stages. Furthermore, an analogous reflection was observed in Shimmatogarashi peppers, where there was an increase in the levels of capsaicin from 1071 ± 16.7 μg · g⁻¹ during the immature stage, to 4363 ± 23.1 μg · g⁻¹ during the fruit maturation stage (Menichini et al., 2008).

In conclusion, we established that the metabolite distribution, expressed genes and activity of antioxidant were differential in the early stage (16 DPA), but more correlated in the breaker and later (28 and 48 DPA) stages. Thus, our findings submit that a suitable approach in interpreting biochemical variances is a non-targeted metabolomics in hot pepper developmental stages.