Abstract
The amounts of taste components, including those for pungency, in chili pepper fruit change depending on environmental factors. Our previous study revealed that the amount of capsaicinoid was significantly increased in chili pepper fruits that were cultivated under a drought stress condition. Therefore, the present experiment was conducted to determine the effect of drought stress on pungency and the expression levels of capsaicinoid biosynthesis genes in chili peppers. Japanese chili pepper cultivars ‘Shishito’ and ‘Sapporo’ were selected and cultivated in a greenhouse under a drought stress condition or an excess water supply condition. The fruits were used for morphological analysis, and the quantification of the capsaicinoid content in the placental septum was done using high performance liquid chromatography. Gene expression analysis was carried out using a quantitative reverse transcription polymerase chain reaction for 18 capsaicinoid biosynthesis genes. Based on the obtained gene expression patterns, we divided the 18 genes into three groups. The genes in group 1 (ACL, pAMT, Pun1, WRKY9, CaKR1, CaMYB31, FAT, and KAS I) showed higher gene expression levels in the drought stress condition than in the excess water supply condition in both cultivars at 20 DAF. The genes in group 2 (KAS III, BCKDH, ACS, BCAT, and 4CL) showed higher gene expression levels in the drought stress condition than in the excess water supply condition in only one of the cultivars at 20 DAF. The genes in group 3 (PAL, C3H, HCT, C4H, and COMT) did not show any significant differences in gene expression between the two treatments in either cultivar at all DAF. The genes in our experiment showed similar expression patterns in pungent parthenocarpic fruit and control fruit of ‘Shishito’ as in a previous study. Moreover, we found that the number of seeds tended to be lower in fruits cultivated under a drought stress condition, while capsaicinoid content was higher. It is possible that drought stress firstly affected the number of seeds in the fruits, and the decrease in the number of seeds subsequently caused changes in capsaicinoid biosynthesis.
Introduction
Capsicum annuum is a species of chili pepper that belongs to the Solanaceae family. It is widely grown for its fruit, and is consumed as a vegetable or as a spice after drying. The pungency of chili peppers is derived from a group of heat-producing alkaloids, including capsaicin (8-methyl-N-vanillyl-6-nonenamide) and several related compounds, which are collectively termed capsaicinoids (Suzuki and Iwai, 1984). The greater the concentration of capsaicin, the greater the pungency of the fruit. Capsaicinoid synthesis occurs mainly in the placenta and interlobular septum of the fruit, and the capsaicinoid is accumulated in glandular vesicles located on the surface of the placental tissue (Iwai et al., 1979). The biosynthesis of capsaicinoids is accomplished by the condensation of vanillylamine and branched-chain fatty acid moieties, and these two precursors are derived from two separate pathways that constitute the capsaicinoid biosynthesis pathway (Arce-Rodríguez and Ochoa-Alejo, 2019), i.e., the phenylpropanoid pathway and the branched-chain fatty acid pathway. Many genes are known to be involved in the capsaicinoid biosynthesis pathway (Qin et al., 2014). Not only genotype, but also cultivation environment is known to affect pungency level. Therefore, pungency level may vary even among chili pepper fruits of the same variety with the same genotype as a result of different cultivation conditions (Harvell and Bosland, 1997; Zewdie and Bosland, 2000). Also, capsaicinoid accumulation is associated with the developmental stage of the fruit (Estrada et al., 1997; Zewdie and Bosland, 2000). Furthermore, environmental factors, such as soil salinity stress, temperature, elevation, light exposure, and soil nutrients (nitrogen, phosphorus, and potassium) are known to cause changes in the pungency levels of chili pepper fruits. In water-stressed plants of C. chinense Jacq., capsaicin concentration increases in some parts of the fruit (Ruiz-Lau et al., 2011). Our previous investigations found that taste components, including capsaicinoid content, were significantly increased in chili pepper fruits that were cultivated under a drought stress condition (Rathnayaka et al., 2020). We considered that the expression of capsaicinoid biosynthesis genes may be related to drought stress-dependent fluctuation in pungency, and were interested in elucidating the genetic mechanism by conducting a gene expression analysis. Also, there have been few investigations regarding the influence of environmental factors, especially water supply, on pungency and expression of capsaicinoid biosynthesis genes in chili pepper fruit. Therefore, the present study was conducted to determine the relationship between drought stress, the content of capsaicinoids, and the expression of capsaicinoid biosynthesis genes.
Materials and Methods
Plant materials and experimental design
The experiment was conducted from April to October 2020 in a greenhouse at the experimental farm (733 m a.s.l) of the Education and Research Center of Alpine Field Science, Faculty of Agriculture, Shinshu University in Minamiminowa, Nagano, Japan. We used the local cultivars ‘Shishito’ (Takii & Co., Ltd., Kyoto, Japan), a very low pungent chili cultivar in Japan and ‘Sapporo Oonaga Nanban’ (Tsurushin Seed, Matsumoto, Japan; hereafter ‘Sapporo’); ‘Sapporo’ is a Japanese pungent variety of the pepper. Seeds were sown on March 25, 2020, and subsequently, the seedlings were transplanted to plastic pots with a 75 mm diameter and filled with a commercial potting medium (Nae-ichiban; Sumitomo Forestry Landscaping Co., Ltd., Tokyo, Japan). During the seedling rearing period, the greenhouse was heated using oil heaters at night until early April to keep the temperature above 15°C. On May 27, 2020, seedlings of approximately 150 mm in height were transplanted to clay pots (diameter of 30 cm) filled with the same commercial potting medium. During the cultivation period, slow-acting garden fertilizer (N:P:K, 10:10:10; Shizen Oyokagaku Co., Ltd., Nagoya, Japan) was applied. The stress treatments were applied 1 week after the seedlings were transplanted. All flowers that were present were removed before the stress treatments were applied.
Two kinds of stress treatment were used: drought stress treatment and excess water supply treatment. For the drought stress treatment, 150 mL of water was applied for each irrigation, and for the excess water supply treatment, 780 mL of water was applied for each irrigation based on pot size, as described by Rathnayaka et al. (2020). In the excess water supply treatment, the excess water that overflowed from the pots was retained in a dish placed under the pot to allow absorption through the pot base. The number of times water was supplied was determined depending on the daily temperature and weather. When the temperature during the day exceeded 30°C or it was sunny, water was applied three times per day. When the temperature during the day remained below 30°C or it was rainy or cloudy, water was applied twice per day. During the stress treatment, each flower was tagged 1 day before flowering, and the fruits were harvested at 20 and 30 days after flowering (DAF). Then, the placental septum weight, capsaicinoid content, and the expression levels of capsaicinoid biosynthesis genes were analyzed as described below. Five individual plants of each cultivar were used for each treatment. Data was analyzed using Student’s t-test within each DAF to reveal significant differences between Drought and Excess water treatment.
Pre-sample preparation for capsaicinoid analysis and RNA isolation
In the present study, capsaicinoid analysis and analysis of the expression levels of genes involved in the capsaicinoid biosynthesis pathway (Fig. 1) were carried out using separated placental septum tissue as described by Kondo et al. (2021b). The placental septum of fruit at 20 and 30 DAF were vertically separated; one half was lyophilized for capsaicinoid extraction, and the other half kept at −80°C for RNA isolation.
Capsaicinoid analysis
High performance liquid chromatography (HPLC) apparatus and analysis conditions
As mentioned above, half of the placental septum was lyophilized using a freeze-dryer (FDU-200; Tokyo Rikakikai Co., Ltd., Tokyo, Japan). After determining dry weight, the samples were placed into collection tubes with stainless-steel bases, and crushed using a Micro Smash TM MS-100 (Tomy Seiko Co., Ltd., Tokyo, Japan). Then, capsaicinoids (capsaicin and dihydrocapsaicin) were extracted in 5 mL of acetone. After transferring the supernatant to an eggplant-shaped flask, 2 mL of ethyl acetic was added to the residue, and the supernatant was collected. The mixed supernatant was completely evaporated at 40°C using a rotary evaporator (N-1100; Tokyo Rikakikai). Finally, the extracted capsaicinoids were dissolved in 5 mL of methanol, and 10 μL of the extracted liquid was filtered and chromatographed using HPLC (LC solution; Shimadzu Corporation, Kyoto, Japan).
In the HPLC analysis, separation was performed using a YMC-Pack ODS-A column (5 μm; 75 × 4.6 mm I. D.) coupled to a guard column (YMC-Guardpack ODS-A). The eluent consisted of methanol and distilled water (50:50), and the flow rate and temperature were set to 1.0 mL·min−1 and 40°C, respectively. For detection with an ultraviolet detector, the wavelength was set to 280 nm. The capsaicinoid concentration (μg·g−1 DW)—i.e., the capsaicinoid content per unit dry weight of placental septum—was calculated based on the peak area of capsaicin and dihydrocapsaicin. Then, we also calculated capsaicinoid content per fruit (μg/fruit) by multiplying capsaicinoid concentration (μg·g−1 DW) and dry weight of placental septum (μg).
RNA isolation and quantitative reverse transcription polymerase chain reaction (RT-qPCR) of capsaicinoid biosynthesis genes
Total RNA was isolated from the other half of the placental septum with an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). After removing genomic DNA, total RNA was used for RT-qPCR, and cDNA was synthesized using a High-Capacity RNA-to-cDNA kit (ReverTra Ace® qPCR RT Master Mix with gDNA Remover; Toyobo Co., Ltd., Osaka, Japan). Then, qPCR was performed using the Step One Real-Time PCR System (Applied Biosystems). In the analysis, 18 capsaicinoid biosynthesis genes were analyzed as target genes, and actin was used as the reference gene. For gene amplification, we used the primers described by Tanaka et al. (2017), Koeda et al. (2019), Han et al. (2018) and Kondo et al. (2021a); the complete primer sequences are listed in Table 1. For PCR, PowerUpTM SYBR Green Master Mix (Applied Biosystems) was used for the PCR mixture, and the thermal cycle conditions described by Tanaka et al. (2017) were adopted (98°C for 2 min, followed by 40 cycles of 95°C for 10 s, 60°C for 10 s, and 68°C for 60 s). Finally, the relative expression level was calculated based on the comparative threshold cycle (Ct) method (Hohjoh, 2013).
Results
Number of seeds and placental septum weight
In the drought stress condition, harvested fruits had a significantly lower number of seeds at 20 and 30 DAF in both cultivars compared with the excess water stress condition (Fig. 2). The fruits were also smaller and had a stumpy shape. The dry weight (g) of the placental septum was significantly smaller in the drought stress condition than in the excess water condition when harvested at 20 and 30 DAF in both cultivars (Fig. 3).
Capsaicinoid content
We investigated the capsaicinoid concentration (μg·g−1 DW) in the fruit harvested at 20 and 30 DAF, and compared the concentration among the two treatments. The capsaicinoid content was significantly higher in the drought stress condition than in the excess water supply condition in fruit harvested at 20 DAF (in ‘Shishito’, drought stress: 2252 μg·g−1 DW, and excess water: 387 μg·g−1 DW; in ‘Sapporo’, drought stress: 32499 μg·g−1 DW, and excess water: 2766 μg·g−1 DW). These results indicated that capsaicinoid concentration increased under drought stress condition (Fig. 4). Additionally, we also compared capsaicinoid content per fruit (μg), because capsaicinoid concentration (capsaicinoid content per unit dry weight of placental septum) may vary greatly depending on the size of the placental septum. However, similarly to the capsaicinoid concentration results, the capsaicinoid content of drought-stressed fruit tended to be significantly higher, and tended to increase with increased DAF harvest (Fig. 5).
RT-qPCR analysis was conducted for 18 genes involved in the capsaicinoid biosynthesis pathway using three placental septum samples for each treatment and each cultivar. According to the obtained gene expression patterns, we divided the 18 genes into three groups (Fig. 6). The genes in group 1 showed higher gene expression levels in the drought stress condition than in the excess water supply conditions in both cultivars at 20 DAF. The genes in group 1 were ACL (acyl carrier protein), pAMT (putative aminotransferase), Pun1 (acyltransferase), WRKY9, CaKR1 (ketoacyl-ACP reductase), CaMYB31, FAT (ketoacyl-ACP reductase), and KAS I (ketoacyl-ACP synthase I). The pAMT, CaKR1, and CaMYB31 genes also showed higher expression levels in the drought stress condition than in the excess water supply condition when harvested 30 DAF in ‘Sapporo’. The genes in group 2 showed higher gene expression levels in the drought stress condition than in the excess water supply condition in only one of the cultivars at 20 DAF. The genes in group 2 were KAS III (ketoacyl-ACP synthase III), BCKDH (branched-chain α-ketoacid dehydrogenase), ACS (acyl-CoA synthetase), BCAT (branched-chain amino acid transferase), and 4CL (4-coumaroyl-CoA ligase). Among the group 2 genes, KAS III, BCKDH, and BCAT showed significantly higher levels of expression at 20 DAF in ‘Shishito’, and BCAT showed a significantly higher level of expression at 30 DAF in ‘Sapporo’. In addition, ACS showed significantly higher levels of expression only at 20 DAF in ‘Sapporo’, and at 30 DAF in ‘Shishito’. The 4CL gene showed significantly higher expression levels in the drought stress condition than in the excess water stress condition in ‘Sapporo’ at 20 and 30 DAF; however, this was not seen in ‘Shishito’. The genes in group 3 did not show any significant differences in gene expression between the two treatments in either cultivar at all DAF. The genes in group 3 were PAL (phenylalanine ammonia lyase), C3H (coumaroyl shikimate/quinate 3-hydroxylase), HCT (hydroxycinnamoyl transferase), C4H (cinnamate 4-hydroxylase), and COMT (caffeic acid O-methyltransferase).
Within the capsaicinoid synthesis pathway, most of the genes belonging to groups 1 and 2 are involved in the branched-chain fatty acid pathway or downstream of the synthesis pathway (Fig. 1), except for CaMYB31 and WRKY9, which encode transcriptional factors. Whereas, the genes assigned to group 3 are mainly involved in the phenylpropanoid synthesis pathway.
Discussion
The capsaicinoid concentration (capsaicinoid content per unit dry weight of placental septum) was significantly increased in the drought stress condition in both ‘Shishito’ and ‘Sapporo’ (Fig. 4). This result was in line with the authors’ previous finding that capsaicinoid content in the placental septum increased under a drought stress condition in Japanese chili pepper varieties ‘Botankosho’ and ‘Sapporo’ (Rathnayaka et al., 2020). Estrada et al. (1999) also reported that chili fruit was more pungent when grown under drought stress conditions.
We also analyzed the capsaicinoid content per fruit because the size of the placental septum can affect the capsaicinoid concentration of the whole fruit. The results revealed that the capsaicinoid content per fruit was also increased in the fruits cultivated under the drought stress condition (Fig. 5), although the dry weight of the placental septum was smaller in the drought stress condition than in the excess water condition. Considering these results, drought stress on the plants appears to increase the pungency levels of the fruit by promoting capsaicinoid synthesis through the activation of genes involved in the capsaicinoid synthesis pathway.
On the other hand, from the result of the present experiment we found the number of seeds tended to be fewer in fruits cultivated under drought stress condition, while capsaicinoid contents were higher (Figs. 2, 4, and 5). Kondo et al. (2021a) reported parthenocarpy induced the fluctuation of pungency in chili pepper fruits, and parthenocarpic (non-seeded) fruits tended to have high pungency. Our result were not contradictory, so there is possibility that drought stress firstly affected the number of seeds in the fruits, and the decrease of seeds subsequently caused changes in capsaicinoid biosynthesis. Thus, drought stress may indirectly affect pungency fluctuation and further research should investigate the mechanism.
In the present study, RT-qPCR analysis was conducted for 18 capsaicinoid biosynthesis genes, and the expression patterns were compared between the drought stress condition and the excess water condition in ‘Shishito’ and ‘Sapporo’. From the results, we categorized the 18 genes into three groups (groups 1, 2, and 3) based on expression patterns.
Firstly, group 1 genes included ACL, pAMT, Pun1, WRKY9, CaKR1, CaMYB31, FAT, and KAS I, which exhibited significantly high expression in both cultivars under the drought stress condition at 20 DAF. Then, group 2 genes included KAS III, BCKDH, ACS, BCAT, and 4CL, and expression levels were higher only in either cultivar under the drought stress condition at 20 DAF. When we focused on gene location in the capsaicinoid biosynthesis pathway, there were two downstream genes, Pun1 and pAMT; Pun1 is the most downstream gene in the capsaicinoid biosynthesis pathway and pAMT is located on the most downstream in the phenylpropanoid pathway. According to Stewart et al. (2005, 2007), Pun1 is the most critical gene in capsaicinoid biosynthesis, and responsible for the final reaction in the synthesis pathway. pAMT is also an important gene, as it is required for the production of vanillylamine (Lang et al., 2009). Considering the importance of these two genes, it was reasonable to consider that expressional changes of Pun1 and pAMT contributed to pungency levels of ‘Shishito’ and ‘Sapporo’ depending on presence of the water deficit. Additionally, group 1 and 2 genes included a lot of genes involved with the branched-chain fatty acid pathway. According to Doi et al. (2013), the expression levels of pAMT, KAS, FAT, ACL, BCAT, and Pun 1 increased with increasing pungency in ‘Shishito’ fruits. Then, Kondo et al. (2021a) also reported that parthenocarpic treatment in ‘Shishito’ fruits brought increases in pungency and expression of group1 and 2 genes, including genes involved with branched-chain fatty acid pathway. These were almost consistent with the present results, so there may be a similar gene regulation mechanism involved with fluctuation of pungency in chili peppers, regardless of the cultivars or environmental factors. Group 1 genes also included two genes encoding transcriptional factors, CaMYB31 and WRKY9. The functional loss of CaMYB31 was reported to cause a drastic reduction in or the loss of pungency (Han et al., 2019). We also found that the expression levels of CaMYB31 and WRKY9 were significantly higher in the drought stress condition than in the excess water condition at 20 DAF in both cultivars. According to Zhu et al. (2019), the MYB transcriptional factor encoded by CaMYB31 directly regulates the expression levels of capsaicinoid biosynthesis genes. Thus, the transcription of group 1 and 2 genes may be controlled by CaMYB31, and its upstream gene, WRKY9, may regulate the expression levels of genes in the capsaicinoid synthesis pathway. A dual-luciferase reporter assay and chromatin immunoprecipitation-PCR analysis revealed that CaMYB31 directly targeted a set of capsaicinoid biosynthesis genes to activate their expression (Zhu et al., 2019). It appears that expression of CaMYB31 is significantly up regulated in plants under drought stress, and that expression of CaMYB31 leads to the activation of its target genes, KAS, pAMT, Pun 1, FAT, and BCAT. Moreover, genes in group 1 and most of the genes in group 2 showed similar expression patterns between the drought stress condition and excess water stress condition, indicating that the genes involved in the branched-chain fatty acid pathway are significantly activated by a drought stress condition.
Group 3 genes included PAL, C3H, HCT, C4H, and COMT; and in this group there were no significant differences in gene expression level between the drought stress and excess water stress conditions. According to Kondo et al. (2021a), there was no significant difference in the gene expression levels of ACS, BCKDH, PAL, 4CL, C4H, HCT, C3H, and COMT between pungent parthenocarpic fruit and control fruit of the ‘Shishito’ variety. These were almost consistent with the present results; we also observed there was no significant difference in the gene expression levels of ACS, PAL, C4H, HCT, C3H, and COMT between ‘Shishito’ fruit grown under the drought stress and excess water stress conditions. We also found that group 3 genes located on the phenylpropanoid pathway, contrarily to the group 1 and 2 genes. According to Liu et al. (2015), genes involved in the phenylpropanoid pathway, which were assigned to group 3 in the present study (PAL, C3H, HCT, C4H, and COMT), are also responsible for the biosynthesis of secondary metabolites, such as phenolic acid, flavonoids, and lignin. Therefore, it is assumed that there was no significant difference in the gene expression level of these genes between the stressed and non-stressed conditions in both cultivars and at all DAF. However, it was also reported that the enzyme activities of PAL and C4H were significantly higher in the placenta of ‘Beauty Zest’ hot chili peppers (C. annuum) from water-deficient plants than in controls (Sung et al., 2005). Moreover, Phimchan et al. (2014) also mentioned that the enzyme activities of PAL and C4H were higher in drought-stressed plants than in non-drought-stressed plants. In the present study, the PAL and C4H expression levels were not significantly higher in the drought stress condition, and this contradiction should be further investigated.
In conclusion, our results indicated that the drought stress on the plants increased the capsaicinoid content of the fruit by promoting capsaicinoid synthesis in the placental septum. We found that 18 genes involved in the capsaicinoid biosynthesis pathway, especially the genes in groups 1 and 2, were responsible for the increased pungency of fruit from drought-stressed plants. However, further experiments using several other stress conditions (e.g., salinity, high temperature, and soil fertility) are needed to confirm the behavior of the genes responsible for capsaicinoid synthesis under conditions of stress. In addition, the key genes responsible for promoting capsaicinoid synthesis under conditions of stress should be further explored.
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