Biochemical Variances Through Metabolomic Profile Analysis of Capsicum Chinense Jacq
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FOLIA HORTICULTURAE Folia Hort. 33(1) (2021): 17–26 Published by the Polish Society DOI: 10.2478/fhort-2021-0001 for Horticultural Science since 1989 RESEARCH ARTICLE Open access http://www.foliahort.ogr.ur.krakow.pl Biochemical variances through metabolomic profile analysis of Capsicum chinense Jacq. during fruit development Yaping Tang, Guoru Zhang, Tao Yang, Shengbao Yang*, Patiguli Aisimutuola, Baike Wang, Ning Li, Juan Wang, Qinghui Yu Institute of Horticulture Crops, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, Xinjiang, China ABSTRACT Capsicum chinense Jacq. is classified under the Solanaceae family, which is an extensively consumed spice and vegetable globally. Therefore, to gain more knowledge and insight into the diversity of Capsicum chinense Jacq. metabolites, a total of 18 placental tissues from various development stages were collected and untargeted metabolomics was conducted by means of ultra-performance liquid chromatography (UPLC) and mass spectrometry (MS). Principal component analysis (PCA) analysis established the existence of distinct metabolite distribution patterns as observed at 16 days post anthesis (DPA), compared with the metabolites at 36 and 48 DPA groups, whereas there was a difference in metabolites between the orange ripening period (B) and the red ripening period (C), which intersected with each other. Furthermore, 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 were significantly enriched across the fruit ripening stages. The capsaicin content was observed to be less in the early ripening stages, but gradually increased to a high concentration during the late ripening stages. In conclusion, our study findings submit a suitable approach for interpreting the biochemical variances of non-targeted metabolomics in hot pepper developmental stages, as well as offer new findings that can be applied in the development strategies in breeding of Capsicum chinense Jacq. Keywords: Capsicum chinense Jacq., chili pepper, GC-MS, LC-MS, pepper fruit diversity, pepper fruit morphology, untargeted metabolomics INTRODUCTION Pepper fruits (family Solanaceae) are widely diverse, as ingredients and patches, respectively, and also aids representing more than 200 species that vary according in digestion in the form of condiments. Hot peppers to size, colour, shape and chemical composition. They contain broad biological compounds like carotenoids are important nutritional and economical fruits which (provitamin A), capsaicinoids, flavonoids, vitamins can be consumed fresh as vegetables, and serve as (vitamins C and E), minerals, essential oils and the spices when dried because of their pungent and unique aroma of the fruits (Aniel et al., 2009; Purkayastha flavor. Additionally, it is an important pharmaceutical et al., 2012; Howard et al., 2016). These compounds resource in topical ointments and pain-relievers, serving have exhibited anticancer (Oyagbemi et al., 2010; *Corresponding author. e-mail: [email protected] (Shengbao Yang). Open Access. © 2021 Tang et al., published by Sciendo. This work is licensed under the Creative Commons Attribution alone 3.0 License. 18 Metabolomics of Capsicum chinense Jacq. Anandakumar et al., 2013), anti-inflammatory (Spiller Academy of Agricultural Sciences in Xinjiang. In March, et al., 2008), antimicrobial (Careaga et al., 2003) and the seeds were first sown in a greenhouse under typical antioxidant (Alvarez-Parrilla et al., 2011) properties. conditions (27°C day/19°C night; 16 h light/8 h dark), Previously conducted studies on pepper fruits which facilitated their germination, and thereafter, in have majorly focused on the antioxidant activity (Tan mid-May the seedlings were transferred to an open field. et al., 2012), nutritive components (Serrano et al., According to normal agronomy practices in the region, 2010) and phenolic contents (Tan et al., 2012) across the open field was properly managed. Irrigation using various fruit developmental stages. Moreover, the drips was employed to water the seedlings. Fungicides majority of these metabolomic studies focused only were applied six to eight times during the growing on the targeted metabolite analysis that evaluated season. capsaicinoids, carotenoids, flavonoids and ascorbic acid. Thus, there exists a research gap in extensive Extraction and measurement of capsaicinoid non-targeted metabolomic studies on pepper. Indeed, and other metabolites in fruit placenta non-targeted metabolomic approaches can elucidate the To study the various metabolites in the Capsicum plant responses to various environmental situations, chinense Jacq. pepper fruit, 18 placental tissues as displayed by the changes in metabolites seen in its were randomly collected during various stages of application in various plant species such as Oryza sativa development, ranging between 16, 36 and 48 days post (Jung et al., 2013; Kim et al., 2014), blueberries (Lee anthesis (DPA). Thereafter, the placental tissue samples et al., 2014a) and pitayas (Lee et al., 2014b). In pepper were dried at 50°C overnight, and then grounded using fruits, the phytochemicals generally undergo various a mortar and pestle. During capsaicinoid extraction, changes all through fruit development, which could 0.2 g of dried pepper samples were mixed with ethanol impact on significant dietary aspects concerning the (70%, 2.0 ml) in a 10 ml glass bottle covered with a consumption of pepper (Ghasemnezhad et al., 2012). Teflon-lined lid. For 4 h, capped bottles with the extract Equally, capsaicinoid compounds are found among contents were run at 80°C in a water bath. Thereafter, at the members in the genus Capsicum. Capsaicinoid is a frequency of 35 kHz, the samples were sonicated for the group of alkaloids which gives pungency or heat to 50 min. The extract was then centrifuged at 12,000×g hot peppers. Its biosynthesis entails a combination of for 15 min, and the supernatants were filtered using a pathways, namely the phenylpropanoid and branched 0.45 μm syringe-mounted membrane filter. Metabolite chain fatty acid pathways. The precursors for the analyses were conducted using UPLC and MS. formation of vanillyl aminophenylalanine are generated Wavelength detection was optimized at 280 nm with by the phenylpropanoid pathway while the branched an injection volume of 10 μL. Tests for each ripening chain fatty acid pathway provides valine or leucine for stage were done in six repetitions. The ProteoWizard 8-methyl-6-nonenoylCoA precursors. Capsaicinoids are software (version 3.0) was used to convert the original known to accumulate in the dissepiment of the placental MS data to mzXML, and then XCMS was employed tissue of the epidermal layer. Its synthesis starts after 20– for retention time correction, peak identification, 30 days of plant pollination and lasts up to the ripening extraction, integration, as well as alignment, and stage of the fruit (Stewart et al., 2005). Thus far, about metabolite profiling and annotation was conducted 23 analogues of capsaicinoids have been described. using the MWDB database (Metware biotechnology Capsaicin and dihydrocapsaicin (trans-8-methyl-N- Co., Ltd. Wuhan, China – http://www.metware.cn/), as vanillyl-6-nonenamide, 8-methyl-N-vanillylnonanamide, described elsewhere (Zhu et al., 2018). respectively) form the highest capsaicinoid constituents of up to 98% in Capscium (Zewdie et al., 2001). Other Statistical analysis less major capsaicinoids in capsicum fruits include nonivamide, homocapsaicin, homodihydrocapsaicin and Principal component analysis (PCA) and the Capsicum nordihydrocapsaicin (Huang et al., 2013). plant metabolites statistics were generated on the Therefore, ultra-performance liquid chromatography mixOmics package using R environment for statistical (UPLC) and mass spectrometry (MS) were used to computation (version 3.2.3). Mean, standard deviation, examine the metabolite profile of Capsicum chinense analysis of variance (ANOVA) at 95% confidence interval Jacq. during the development stages of the pepper fruit. and the F-value (p ≤ 0.001) significant level constituted The identified metabolites could assist in defining the the summarized statistics done. Pearson’s correlation diversity of pepper metabolites and the molecular basis method was employed to generate correlation analysis. of pepper fruit pungency. Generally, when conducting multiple analyses on the same dependent variable, the chance of committing a Type I error increases, thus increasing the likelihood of MATERIALS AND METHODS reaching at a significant result by pure chance. To correct this under this study, multiple testing was adjusted by Sample preparation and collection use of Bonferroni’s correction for accurate inference. The plants Capsicum chinense Jacq. (chili pepper) were The qRT-PCR results were evaluated using Student’s grown in an open field, in the year 2017, at Xinjiang t-test method. Tang et al. 19 RESULTS the green ripening period. During the colour transition period of the pepper fruit, 205 metabolites were Placental metabolite quantity during fruit up-regulated and 405 down-regulated, whereas 57 development metabolites were up-regulated and 45 down-regulated 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