© 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’ ( 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 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 . However, the morphological changes in epidermal cells in pericarp tissue had not been reported. In this study, microscopic analyses of the fruit of 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 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, , 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 ‘.’ 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% . 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 , dihydrocapsaicin, and . 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 ‘’ (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 in ‘Bhut Jolokia.’ D. Magnified view of epidermal cells in ‘Habanero.’ E. Connected region of pericarp and interlocular septum in ‘Bhut Jolokia.’ F. Connected region of pericarp and interlocular septum. Bar=1 mm (A, B, E, F), 75 µm (C, D). ip, inner side of pericarp; op outer side of pericarp; is, interlocular septum. 306 R. Sugiyama Cytologia 82(3) icinoid biosynthesis (Furuya and Hashimoto 1954, Ohta To accurately estimate the percentage of elongated cells 1962, Suzuki et al. 1980). During fruit development, out of total epidermal cells on the pericarp, we should the cells in the epidermal tissue that produce capsa- analyze several transverse sections from different parts icinoids in the interlocular septum become elongated of the pericarp. (Suzuki et al. 1980). In transverse sections of pericarp tissue of ‘Bhut Jolokia’ and ‘Habanero’ (Fig. 2), we ob- Acknowledgement served elongated epidermal cells on the inner surface of the pericarp of ‘Bhut Jolokia’ (Fig. 2A, C) but not The author thanks Dr. Hiroto Hirano for useful dis- ‘Habanero’ (Fig. 2B, D). When these cells were stained cussions. with safranin, their vacuoles were smaller than those of other cells (Fig. 2C). Our previous study showed that the References elongated cells on the interlocular septum of ‘Habanero’ express the gene encoding b-Ketoacyl-ACP synthase Bosland, P. W., Coon, D. and Cooke, P. H. 2015. Novel formation of (KAS), which is essential for capsaicinoid biosynthesis ectopic (nonplacental) capsaicinoid secreting vesicles on fruit walls explains the morphological mechanism for super-hot chile (Sugiyama et al. 2006b). These elongated cells were peppers. J. Am. Soc. Hortic. Sci. 140: 253–256. capsaicinoid-producing cells. Analyses of the border Furuya, T. and Hashimoto, K. 1954. Studies on Japanese capsicum. region between the pericarp and interlocular septum II. Distribution of capsaicin-secreting organs in Capsicum . (Fig. 2E, F) showed that the cells differed between these Yakugaku Zasshi 74: 771–772. (in Japanese) two tissues in ‘Habanero’ (Fig. 2F). However, elongated Lang, Y., Kisaka, H., Sugiyama, R., Nomura, K., Morita, A., Wata- nabe, T., Tanaka, Y., Yazawa, S. and Miwa, T. 2009. Functional epidermal cells were continuously distributed on the loss of pAMT results in biosynthesis of capsinoids, capsaicinoid pericarp and the interlocular septum in ‘Bhut Jolokia’ analogs, in cv. CH-19 Sweet. Plant J. 59: (Fig. 2E). The cuticle was separate from epidermal cells 953–961. in the pericarp of ‘Bhut Jolokia’ (Fig. 2E), indicating that Luo, X. J., Peng, J. and Li, Y. J. 2011. Recent advances in the study on capsaicinoids were secreted from epidermal cells in the capsaicinoids and capsinoids. Eur. J. Permacol. 650: 1–7. pericarp. It is important to note that the appearance of Ohta, Y. 1962. Physiological and genetical studies on the pungency of Capsicum, IV. Secretory organs, receptacles and distribution of the cuticle in the micrographs does not reflect its natural capsaicin in the Capsicum fruit. Japanese J. Breed. 12: 179–183. condition because of the technical limits of the prepara- (in Japanese) tion and fixation of paraffin sections. In another study, Stewart, C. J. Jr., Mazourek, M., Stellari, G. M., O’Connell, M. and stereo-fluorescence microscopic analyses showed that Jahn, M. 2007. Genetic control of pungency in C. chinense via capsaicinoids were secreted from vesicles on the inner the Pun1 locus. J. Exp. Bot. 58: 979–991. Sugiyama, R., Oda, H. and Kurosaki, F. 2006a. Two distinct phases surface of the pericarp (Bosland et al. 2015). However, of glandular trichome development in hop (Humulus lupulus L.). morphological changes in the epidermal cells in the peri- Plant Biotechnol. 23: 493–496. carp had not been reported. This is the first report dem- Sugiyama, R., Shite, M., Fujino, H., Tatsuo, Y., Nakamura, S., onstrating that morphological changes in epidermal cells Kakusho, N., Ito, M., Yokota, H., Kase, K. and Kurosaki, F. are related to capsaicinoid production in the pericarp of 2006b. Estimation of capsaicinoid biosynthetic capacity from capsaicinoid content and surface area of placental dissepiment in ‘ ’ Bhut Jolokia. Capsicum fruits. Plant Morphol. 18: 75–82. (in Japanese) Elongated cells containing capsaicinoids were not Suzuki, T., Fujiwake, H. and Iwai, K. 1980. Intracellular localization present across the whole area of the pericarp. These of capsaicin and its analogues, capsaicinoid, in Capsicum fruit elongated cells were mainly present on the upper part of 1. Microscopic investigation of the structure of the placenta of the fruit near the calyx (data not shown). The distribu- Capsicum annuum var. annuum cv. Karayatsubusa1. Plant Cell Physiol. 21: 839–853. tion of elongated cells was consistent with the results of Tanaka, Y., Nakashima, F., Kirii, E., Goto, T., Yoshida, Y. and Yasu- our previous report focusing on the interlocular septum ba, K. 2017. Difference in capsaicinoid biosynthesis gene expres- of ‘Habanero fruits’ (Sugiyama et al. 2006b). In the inter- sion in the pericarp reveals elevation of capsaicinoid contents in locular septum, 78% of epidermal cells were elongated chili peppers (Capsicum chinense). Plant Cell Rep. 36: 267–279. in ‘Habanero,’ compared with 33% in ‘Takanotsume.’ Bo- Tyler, B. J., Peterson, R. E., Lee, T. G., Draude, F., Pelster, A. and sland et al. (2015) conducted stereo-fluorescence micro- Arlinghaus, H. F. 2016. ToF-SIMS imaging of capsaicinoids in peppers (Capsicum chinense). Biointerphases 11: scopic observations and reported that secreting vesicles 02A327. covered 43% of the pericarp surface in ‘Bhut Jolokia.’