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
Equally, capsaicinoid compounds are found among the members in the genus
Therefore, ultra-performance liquid chromatography (UPLC) and mass spectrometry (MS) were used to examine the metabolite profile of
The plants
To study the various metabolites in the
Principal component analysis (PCA) and the
The pericarp colours of the pepper fruits transformed as fruit maturation progressed: A = green (16 DPA), B = orange (36 DPA) and C = red (48 DPA) (Figure 1). Multivariate analysis was made on the MS spectrum data to identify the differences in primary metabolites. In total, 370 metabolites were identified from the
As per PCA analysis, our study findings revealed that the metabolites present at the 16 DPA (green ripening period) were highly distinct with a clear cluster distribution, compared with the metabolites of the 36 and 48 DPA groups (orange and red ripening periods), whereas the difference in metabolites between the orange ripening period (B) and the red ripening period (C) intersected with each other (Figure 2).
For differential multiple analysis of metabolites, a total of 145 up-regulated metabolites were identified, while 320 metabolites were down-regulated during the green ripening period. During the colour transition period of the pepper fruit, 205 metabolites were up-regulated and 405 down-regulated, whereas 57 metabolites were up-regulated and 45 down-regulated during the red ripening stage of the fruit (Table 1 and Figure 3A–C).
Results of the differential metabolites (A represents 16 DPA, B represents 36 DPA and C represents 48 DPA).
Group name | All diff | Down-regulated | Up-regulated |
---|---|---|---|
A |
465 | 320 | 145 |
A |
610 | 405 | 205 |
B |
102 | 45 | 57 |
DPA, days post anthesis.
Using Venn diagram, the identified 726 expressed genes detected at each developmental stage (A = green stage, B = yellow stage and C = red stage) were analysed (Figure 4). 14 of the total 726 genes were commonly differentially expressed among the three groups while 82, 13 and 194 genes were uniquely expressed at A, B and C, respectively.
In studying the
KEGG annotation results of different metabolites in each group (A represents 16 DPA, B represents 36 DPA and C represents 48 DPA).
Group | Diff number | Diff KEGG Annotation |
---|---|---|
A |
465 | 27 |
A |
610 | 29 |
B |
102 | 6 |
DPA, days post anthesis.
The annotation results for the differential metabolite KEGG were classified according to their respective pathway type in the KEGG database. The differential metabolites of the three groups have a varied influence on all stages of fruit ripening. Several pathways, including metabolic pathways, biosynthesis of phenylpropanoids, ABC transporters, alanine, aspartate and glutamate metabolism, fatty acid biosynthesis, pentose and glucoronate pathways, secondary metabolites biosynthesis, cutin, biosynthesis of suberine and wax, biosynthesis of antibiotics, amino and nucleotide metabolism of sugars, biosynthesis of glucosinolate, biosynthesis of plant hormones, and microbial metabolism in diverse environments were significantly enriched across the fruit ripening stages (
Through typical correlation analysis (canonical correlation analysis [CCA]), we found that metabolite numbers 6,257, 10,464, 5,100, 16,349, 304 and 809 were synthesized from fatty acids associated with the synthesis of horseradish (fatty acid biosynthesis) pathway partial gene, propyl coenzyme A synthesis (malonyl-CoA biosynthesis) pathway partial gene and benzene propane metabolism (phenylpropanoid and benzenoid metabolism). Some genes in the pathway were typically related (Figure 6).
Placental capsaicin concentration for each respective developmental stage was also measured. From the obtained results, it was found that there was less capsaicin (631.00 ± 84.15 mg · kg−1) accumulation in green ripening stage A and slightly more capsaicin (633.46 ± 86.96 mg · kg−1) accumulation in the red ripening stage C. The peak capsaicin accumulation was at 846.64 ± 157.48 mg · kg−1 at the colour transition stage C.
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
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.