Capsaicinoids Production and Accumulation in Epidermal Cells on the Internal Side of the Fruit Pericarp in ‘Bhut Jolokia’ (Capsicum Chinense)
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© 2017 The Japan Mendel Society Cytologia 82(3): 303–306 Capsaicinoids Production and Accumulation in Epidermal Cells on the Internal Side of the Fruit Pericarp in ‘Bhut Jolokia’ (Capsicum chinense) Ryuji Sugiyama Department of Food Business, Faculty of Health and Human Life, Nagoya Bunri University, 365 Maeda, Inazawa-cho, Inazawa, Aichi 492–8520, Japan Received February 22, 2017; accepted April 22, 2017 Summary Capsaicinoids are pungent components that are produced only in the genus Capsicum. These func- tional compounds are produced in chili pepper fruits and have health benefits for humans. A high capsaicinoid level is one of the major parameters determining the commercial quality and health-promoting properties of chili peppers. Several reports have indicated that capsaicinoids are produced in the interlocular septum of chili pepper fruits. Microscopic analyses show that there are morphological changes in epidermal cells in the interlocular sep- tum during the capsaicinoid-biosynthesis stages of fruit development. Epidermal cells containing capsaicinoids in the interlocular septum had an elongated shape. Recently, it was reported that capsaicinoids and transcripts of genes encoding enzymes involved in capsaicinoid biosynthesis were present in the fruit pericarp of extremely pungent chili cultivars. However, the morphological changes in epidermal cells in pericarp tissue had not been reported. In this study, microscopic analyses of the fruit of Capsicum chinense cv. ‘Bhut Jolokia,’ an extremely pungent chili pepper variety, revealed elongated epidermal cells secreting capsaicinoids on the inside surface of the pericarp. Key words Capsaicinoid, Pericarp, Pungency, Morphological change, Epidermal cell. Chili peppers are used as a spice in many cuisines to the pericarp. During the capsaicinoid accumulation around the world. Capsaicinoids are responsible for the stage, the epidermal cells become elongated and are pungency of chili peppers (Capsicum spp.). The most easily distinguished from parenchyma cells (Suzuki abundant members of the capsaicinoids family are cap- et al. 1980). Tyler et al. (2016) used ToF-SIMS imaging saicin, dihydrocapsaicin, and nordihydrocpasaicin. The to reveal the cellular localization of capsaicinoids with fundamental chemical structure of capsaicinoids com- minimal sample preparation, and observed that capsa- prises an acid amide of vanillylamine combined with a icinoids were present in the intercellular spaces in both fatty acid. Capsaicinoids exhibit a wide range of bioac- the placenta and the interlocular septum. tivities; for example, they have been shown to suppress There are extremely high concentrations of capsa- fat accumulation and to have anti-cancer effects (Luo icinoids in some chili pepper cultivars; for example, et al. 2011). Therefore, the capsaicinoid concentration ‘Bhut Jolokia’ and ‘Trinidad Moruga Scorpion.’ Recently, is one of the major indicators of the commercial qual- capsaicinoid production was analyzed in the pericarp tis- ity and health-promoting properties of chili peppers. To sues of the extremely pungent pepper varieties ‘Trinidad enhance these health-promoting properties, there is a Moruga Scorpion,’ ‘Trinidad Scorpion,’ ‘Bhut Jolokia,’ demand to increase capsaicinoid concentrations in pep- and ‘Trinidad Moruga Scorpion Yellow’ (Bosland et al. per fruits. 2015, Tanaka et al. 2017). Bosland et al. (2015) reported Chili peppers start accumulating capsaicinoids in that vesicles containing capsaicinoids were produced fruits approximately 20 days post-anthesis. Morphologi- on the inner surface of the pericarp of three extremely cal observations have shown that capsaicinoid biosyn- pungent peppers; ‘Trinidad Moruga Scorpion,’ ‘Trinidad thesis is localized in the epidermal cells of the interlocu- Scorpion,’ and ‘Bhut Jolokia.’ Up-regulated expression lar septum, and then capsaicinoids are secreted into the of multiple capsaicinoid biosynthetic genes was also subcuticular space between the cell wall and the cuticle detected in pericarp tissue of ‘Trinidad Maruga Scorpion (Furuya and Hashimoto 1954, Ohta 1962, Sugiyama Yellow’ (Tanaka et al. 2017). However, the morphologi- et al. 2006b, Stewart et al. 2007). The interlocular sep- cal changes in epidermal cells in the pericarp were not tum is derived from the tissue connecting the placenta reported. In this study, therefore, we investigated the morphological changes in epidermal cells in the pericarp * Corresponding author, e-mail: [email protected] tissue of the extremely pungent pepper variety, ‘Bhut DOI: 10.1508/cytologia.82.303 Jolokia.’ 304 R. Sugiyama Cytologia 82(3) Materials and methods of capsaicinoids. HPLC analyses were performed using a Waters Alliance 2695 HPLC system equipped with Preparation of tissue sections a 2487 dual c fluorescence detector (Waters, Milford, Fruits of ‘Bhut Jolokia’ (C. chinense) were purchased MA). Samples were separated on an Inertsil HPLC from a local market in Japan. Tissues were fixed and column (particle size 3 µm, internal diameter 4.6 mm, stained as described by Sugiyama et al. (2006a, 2006b). length 250 mm; GL Sciences, Tokyo, Japan). The mo- Tissue slices were fixed for 1 h at room temperature in bile phase consisted of 66% methanol at a flow rate of 50% ethanol, 5% acetic acid, 3.7% formaldehyde. The 0.8 mL min-1. The excitation and emission wavelengths fixed tissues were dehydrated using a graded ethanol were 280 and 320 nm, respectively. The column was series (50 to 100%), embedded in paraffin, and then maintained at 40°C. Each analysis was performed twice. sliced into 7-µm sections. The sections were attached The total capsaicinoids content represents the sum of to MAS-coated glass slides, and paraffin was removed capsaicin, dihydrocapsaicin, and nordihydrocapsaicin. using a xylene and ethanol series. Tissues were stained with solutions of safranin. Photographs were taken with Results and discussion a bright-field microscope (Olympus, Tokyo, Japan) using a CCD camera (Nikon, Tokyo, Japan). Capsaicinoids accumulation in pericarp tissue Fruits of ‘Bhut Jolokia’ were separated into the peri- High performance liquid chromatography analyses of carp, seed, and combined placenta and interlocular capsaicinoids septum, and then the capsaicinoid content in each part Capsaicinoids were analyzed by high performance was determined. The placenta tissue (white arrowhead in liquid chromatography (HPLC) as described previously Fig. 1B), to which the seeds were attached, was too small (Lang et al. 2009). Dried fruits and tissue were ground and thin to be separated from the interlocular septum. using a blender, and capsaicinoids were extracted by The capsaicinoid contents were 8.2, 5.5, and 65.6 mg g-1 ethyl acetate. Extracted samples were filtered through a Dry Weight (DW) in the pericarp, seed, and combined 0.45-µm chromatodisc filter before quantitative analysis placenta and interlocular septum, respectively (Table Fig. 1. Fruit shape and dissected fruit of ‘Bhut Jolokia’ showing interlocular septum and placenta. A. Fruit shape of ‘Bhut Jolokia,’ B. Inner tissue of fruits. White arrowheads indicate placenta tissues that connect to seeds. Black arrowhead in- dicates interlocular septum. Table 1. Capsaicinoid contents in different parts of ‘Bhut Jolokia’ fruits. Pericarp Seed Placenta and interlocular septum Capsaicinoids (mg g-1 DW) 8.2 5.5 65.6 DW per fruit (mg) 792.3 224.4 237.3 Capsaicinoid weight per fruit (mg) 6.5 1.2 15.6 Proportion of total capsaicinoids (%) 27.9 5.3 66.7 2017 Elongated Cells Producing Capsaicinoids in Chili 305 1). On a DW basis, the proportion of pericarp tissue in Morphological changes in epidermal cells in pericarp the fruits was larger than the proportions of seeds and tissue placenta and interlocular septum (Table 1). The total Capsaicinoids accumulated not only in the placenta capsaicinoid content in the pericarp was estimated based and interlocular septum, but also in the pericarp of on the DW of the pericarp per fruit. The proportion of ‘Bhut Jolokia’ fruits. We also detected capsaicinoids in total capsaicinoids in each tissue, based on DW, was the seeds, although this could represent contamination 27.9% in the pericarp, 5.3% in the seeds, and 66.7% in of the seed surface during fruit dissection (Ohta 1962). the combined placenta and interlocular septum (Table 1). Gene expression analyses have shown that several capsa- A previous study on ‘Bhut Jolokia’ reported that the Sco- icinoid biosynthesis genes are not expressed in the seeds ville heat units of the pericarp and placenta were 311207 of ‘Habanero’ (C. chinense) (Sugiyama et al. 2006b). and 1332789, respectively (Bosland et al. 2015), consis- Capsaicinoids were detected in the pericarp of extremely tent with the capsaicinoids concentrations detected in pungent chili pepper fruits (Bosland et al. 2015, Tanaka our study. In the chili pepper ‘Trinidad Maruga Scorpion et al. 2017). Tanaka et al. (2017) also detected capsa- Yellow,’ the capsaicinoids concentration in the pericarp icinoids and up-regulation of capsaicinoids biosynthetic was 23.2 mg g-1 DW, and this tissue contained 84.3% of genes in the pericarp of ‘Trinidad Moruga Scorpion Yel- the total capsaicinoids in the fruit (Tanaka et al. 2017). low.’ Several reports have suggested that there are relation- ships between changes in cell morphology and capsa- Fig. 2. Tissue sections of pericarp and interlocular septum in ‘Bhut Jolokia’ and ‘Habanero.’ A. Pericarp of ‘Bhut Jolokia.’ B. Pericarp of ‘Habanero.’ C. Magnified view of elongated cells