Regular Article
Stable Capsaicinoid Biosynthesis during the Fruit Development Stage of Capsicum baccatum
2019 Volume 84 Issue 4 Pages 309-312
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2019 Volume 84 Issue 4 Pages 309-312
Capsaicin and its analogs, which are collectively called capsaicinoids, are pungent components that are produced only in Capsicum species. Several reports have indicated that chili peppers start accumulating capsaicinoids in fruits approximately 20 days after flowering (DAF), with capsaicinoid levels continuing to increase until 40 DAF and then decreasing. In this study, a Capsicum baccatum cultivar was analyzed to investigate capsaicinoid accumulation and the morphological changes to capsaicinoid-producing cells in maturing fruits. Capsaicinoids accumulated and capsaicinoid-producing cells were detected at 10 DAF. Additionally, the capsaicinoid contents increased during the fruit development stage. At 40 DAF, the nuclear shape was unclear, and cytoplasm appears to condense in C. baccatum.
Regarding the use of chili peppers (Capsicum spp.) and their derived products in the food and pharmaceutical industries, stabilizing pungency is one of the important objectives. Capsaicin and its analogs, which are collectively called capsaicinoids, are responsible for the pungency of chili peppers. Capsaicinoids are generally accumulated in the interlocular septa of chili pepper fruits (Furuya and Hashimoto 1954, Ohta 1962, Sugiyama et al. 2006, Stewart et al. 2007). In a previous study, we used a 3-dimensional computer model of chili pepper fruits to determine that capsaicinoid abundance is related to the number of capsaicinoid-producing cells, which affects the surface area of interlocular septa (Sugiyama et al. 2006). Capsaicinoids have also been detected in the pericarp tissues of several “super-hot” cultivars (Bosland et al. 2015, Sugiyama 2017, Tanaka et al. 2017). Capsaicinoids begin to accumulate at 20 days after flowering (DAF) and continue to accumulate until 30–50 DAF. There have been some contradictory findings regarding the changes to capsaicinoid levels during the fruit development stage. Some studies suggested that capsaicinoid contents continue to increase until the fruits mature (Saga and Tamura 1970, Sukrasno and Yeoman 1993, Stewart et al. 2005), whereas other studies indicated that capsaicinoid levels decrease after reaching a maximum level (Suzuki at al. 1980, Contreras-Padilla and Yahia 1998). During the capsaicinoid accumulation stage, the epidermal cells in interlocular septa elongate and are easily distinguished from parenchyma cells (Suzuki et al. 1980). However, the morphological changes to these cells during capsaicinoid degradation stages have not been reported.
The capsaicinoid contents and morphological characteristics of chili peppers have been investigated primarily with three domesticated species (C. annuum, C. chinense, and C. frutescens); however, C. baccatum is also a domesticated Capsicum species. An earlier investigation of a C. baccatum cultivar revealed the highest capsaicinoid levels occurred at 35 DAF (Tanaka et al. 2009). Subsequent analysis indicated the capsaicinoid contents of 36 C. baccatum cultivars (Tanaka et al. 2017) ranges between 0 and 4.3 mg g−1 dry weight. There are currently no available reports describing the morphology of capsaicinoid-producing C. baccatum cells.
In this study, the changes to epidermal cell morphology and capsaicinoid contents were investigated in developing fruits of a C. baccatum cultivar that produces small fruits. Capsaicinoids accumulated until the fruit pericarp surface became wrinkled. Additionally, capsaicinoid degradation was not observed. Although the epidermal cells shrank and became hollow, the capsaicinoid contents did not decrease during the fruit ripening stage.
Chili plants of C. baccatum were grown in plastic pots using commercial potting soils at Nagoya Bunri University in Japan from May through October 2018. Fruits were collected at 10, 20, 30, and 40 DAF.
High-performance liquid chromatography analyses of capsaicinoidsCapsaicinoids were analyzed by high-performance liquid chromatography (HPLC). Dried fruits and tissues were ground with a blender, and capsaicinoids were extracted with methanol. The resulting extracted samples were passed through a 0.45-µm Chromatodisc filter before quantitative analysis of capsaicinoids. The HPLC analyses were performed using a JASCO HPLC system equipped with an FP-2020 Plus fluorescence detector (JASCO, Tokyo, Japan). Samples were separated on a CrestPac C18S column (5 µm particle size, 4.6 mm internal diameter, and 150 mm length; JASCO). The mobile phase consisted of 66% methanol at a flow rate of 1 mL min−1. The excitation and emission wavelengths were 280 and 320 nm, respectively. The column was maintained at 40°C. The total capsaicinoid content was based on the abundance of capsaicin and dihydrocapsaicin. At least five samples were independently analyzed, and the average total capsaicinoid content was calculated.
Preparation of tissue sectionsTissues were fixed and stained as described by Sugiyama et al. (2006), with minor modifications. Tissue slices were fixed for 1 h at room temperature in a solution comprising 50% ethanol, 5% acetic acid, and 3.7% formaldehyde. The fixed tissues were dehydrated with 2-propanol, embedded in paraffin, and then sliced into 4-µm sections. The sections were attached to glass slides, after which the paraffin was removed with a xylene and ethanol series. The tissues were stained with a safranin solution and then analyzed with an Olympus BH2 bright-field microscope (Olympus, Tokyo, Japan) and photographed with an Olympus DP25 digital camera. Three to five tissues were fixed and used to prepare paraffin sections.
In this study, a C. chinense cultivar that produces fruits that are the same size as the C. baccatum fruits were used as the control (Fig. 1). The fruits of C. baccatum and C. chinense were globose with a diameter of about 6–7 mm at 40 DAF. Abscised C. baccatum fruits were observed after 40 DAF. Thus, fruits were collected at 10, 20, 30, and 40 DAF for subsequent analyses. In C. chinense and C. baccatum fruits, capsaicinoids were detected starting from 10 DAF (Fig. 2A). In C. chinense, the capsaicinoid contents continued to increase until 30 DAF, after which they decreased. This observation was consistent with the results of a previous study of C. chinense (Contreras-Padilla and Yahia 1998); however, peak capsaicinoid levels in other chili pepper cultivars reportedly occur at 50 DAF. Regarding the C. baccatum fruits, the capsaicinoid contents peaked around 20 DAF and remained relatively stable at the following analyzed time points. This was in contrast to several reports describing considerable decreases in capsaicinoid contents after reaching maximum levels (Suzuki at al. 1980, Contreras-Padilla and Yahia 1998). Capsaicinoids are more stable than the other chemicals in chili pepper fruits, such as ascorbic acid, phenolic compounds, and carotenoids. An analysis of capsaicinoid content per C. baccatum fruit revealed that capsaicinoid levels continued to increase until the fruits matured (Fig. 2B). Capsaicinoids did not degrade in C. baccatum fruits.
Capsaicinoids are biosynthesized in the epidermal cells of the interlocular septum and are secreted into the subcuticular space between the cell wall and cuticle. The transverse section of an interlocular septum during the fruit development stage is presented in Fig. 3. At 10, 20, and 30 DAF, elongated and stained cells were observed (Fig. 3A–C, I–K). Capsaicinoid-producing cells had relatively small vacuoles and were filled with the cytoplasm (Fig. 3E–G, M–O). Moreover, the cuticle detached from these cells (Fig. 3A–C). At 40 DAF, the mature fruits had little dried pericarp (Fig. 1A). In C. baccatum, the capsaicinoid-producing cells were weakly stained at 40 DAF (Fig. 3D). Some cells shrank from the outer surface of the cell wall. Additionally, the cytoplasmic structure and nuclei were unclear (Fig. 3H). In C. chinense, almost all of the capsaicinoid-producing cells shrank (Fig. 3P).
Capsaicinoid accumulation and the morphological changes to capsaicinoid-producing cells were investigated in developing C. baccatum fruits. Capsaicinoids and morphological changes to epidermal cells were detected starting from 10 DAF. During the fruit development stage, the capsaicinoid contents increased.
Capsaicinoids did not degrade during the fruit development stagePrevious studies concluded that capsaicinoid contents decrease at the end of the fruit development stage (Suzuki at al. 1980, Contreras-Padilla and Yahia 1998). Additionally, Díaz et al. (2004) reported that peroxidase efficiently catalyzes the oxidation of capsaicin. Peroxidase activity was detected in the epidermal cells of placental tissue (Díaz et al. 2004, Sung et al. 2005). Earlier studies used capsaicinoid amount per fruit dry weight as the unit for analyzing capsaicinoid levels. Moreover, the percentage of the fruit dry weight corresponding to the seed weight was high at the end of the fruit development stage (Saga and Tamura 1970, Marcelis and Baan Hofman-Eijer 1995). The seed weight may affect the apparent decrease in the capsaicinoid contents per dry fruit weight. Capsaicinoids are accumulated as a result of biosynthesis rate. Capsaicinoid levels vary as the rate of its biosynthesis and degradation. In the case of capsinoids, non-pungent analogs of capsaicinoids, capsinoid contents remarkably decrease at the end of the fruit development stage (Maeda et al. 2006). Decreased biosynthesis or increased degradation of capsinoids might influence the accumulation of capsinoids in fruit mature stages. The data presented herein confirmed that capsaicinoid levels continue to increase in C. baccatum fruits during the fruit development stage. Further studies to analyze the rate of biosynthesis and degradation of capsaicinoids are required.
Morphological changes to mature fruitsAt 10 DAF, vacuoles were small and undetectable in C. baccatum and C. chinense cells (Fig. 3E, M). Between 20 and 30 DAF in the C. baccatum fruit cross-section, the regions of weak staining may be small vacuoles (Fig. 3F, G). In contrast, at 20 DAF, the regions of weak staining were not clearly observed in C. chinense fruit cross-sections (Fig. 3N). At 40 DAF, almost all capsaicinoid-producing cells shrank in C. baccatum and C. chinense (Fig. 3D, P). Because parenchyma cell shapes were still normal, the observed cell shrinkage was not due to our histochemical methods. Thus, capsaicinoid-producing cell conditions might change from 30 to 40 DAF. Nuclear was not clear and cytoplasm appears to condense at 40 DAF in C. baccatum and C. chinense (Fig. 3H, P). To determine when this cell morphological change take place, daily histochemical analyses from 30 to 40 DAF are required.
The author thanks Professor Dr. Yoshifumi Hirabayashi of the Department of Health and Nutrition, Nagoya Bunri University, for technical assistance.
