Abstract
Purple paprika (Capsicum annuum) is a vegetable with potential economic value; however, enhancing and maintaining the purple fruit color is challenging. We investigated the effects of night blue-light supplementation on fruit growth, anthocyanin content, and gene expression in purple paprika. During two duplicated experiments conducted in spring and autumn, purple paprika plants were subjected to blue-light supplementation at night. Ten days after fruits were pollinated, night blue light with photosynthetic photon flux density (PPFD) of 80 to 100 µmol·s−1·m−2 was supplied from 1800 HR to 0500 HR (11 hours). Fruit samples were harvested 15, 20, and 40 days after pollination from control (no-light treatment) and blue light–treated fruits. The fruit size, fresh weight, anthocyanin content, and expression levels of anthocyanin biosynthesis-related genes (HY5, bHLH, WDR, MYB, PAL, CHS, F3H, DFR, ANS, and UFGT) were determined. Blue-light supplementation increased the anthocyanin-related gene expressions in the fruit peel, enhancing anthocyanin synthesis and accumulation. However, there were no significant differences between the growth of control and blue light–treated fruits. These findings highlight the potential of night blue-light supplementation to enhance anthocyanin content in purple paprika fruits without affecting overall fruit growth.
Purple paprika (Capsicum annuum L.) is becoming an increasingly popular vegetable because of its unique purple fruit color and nutritional value. It is a potential economic vegetable that could broaden the market opportunity for vegetable producers; however, enhancing and maintaining its color is challenging. Its anthocyanins are purple, but changes in anthocyanin production can affect the color intensity of the fruit (Yamada et al. 2019).
Environmental factors, such as light, affect anthocyanin accumulation during growth and development in plants (Ma et al. 2021). Light control in agricultural technology involves adjusting the light quality, daylength, and light wavelength, especially when limited sunshine duration and/or cloudy conditions are expected, and this has a profound impact on plant growth and pigment composition. Zoratti et al. (2014) reported that light intensity and spectrum impact anthocyanin accumulation, and studies on the effect of blue light on anthocyanin accumulation on several plant species (such as pear and strawberry) have reported that blue light increases the anthocyanin content and red light has a lower effect (Tao et al. 2018; Zhang et al. 2018b).
Anthocyanins are synthesized through the flavonoid pathway and their accumulation predominantly dependents on light signals in most instances (Bulgakov et al. 2016; Gangappa and Botto 2016; Leng et al. 2000). Analyzing the expression levels of anthocyanin biosynthesis-related genes is the most effective method of evaluating anthocyanin accumulation. The biosynthesis pathway of the light-dependent regulation pathway of anthocyanins is composed of complex enzymes encoding structural genes. After plants receive light signals, photoreceptors such as ELONGATED HYPOCOTYL5 (HY5) bind to the anthocyanin biosynthesis-related structural genes resulting in regulating the downstream synthesis pathway (Stracke et al. 2010). The structural genes Phenylalanine ammonia lyase (PAL), Chalcone synthase (CHS), Flavanone 3-hydroxylase (F3H), Dihydroflavonol 4-reductase (DFR), Anthocyanidin synthase (ANS), and Flavonoid 3-O-glucosyltransferase (UFGT) then continue regulation (Jaakola 2013; Ma et al. 2021; Steyn et al. 2002; Sunil and Shetty 2022), and a complex of the transcription gene MYB (Aguilar-Barragán and Ochoa-Alejo 2014; Lu et al. 2019; Zhang et al. 2015), basic helix-loop-helix (bHLH) and WD40-repeat (WDR) (Koes et al. 2005; Ramsay and Glover 2005; Xu et al. 2015) activates the transcription of the structural genes. When plants receive blue-light radiation, plant secondary metabolism such as anthocyanin synthesis is promoted (Kokalj et al. 2019; Tao et al. 2018) by the blue light upregulates all the anthocyanin biosynthetic genes as observed in bilberry fruit (Samkumar et al. 2021) and strawberry fruit (Zhang et al. 2018a) which led to the higher accumulation of anthocyanin.
However, the response of plants to blue light in anthocyanin production is varied in plant species. Previously, we conducted a pre-experiment on the effect of blue-light radiation on anthocyanin production in paprika fruit during daytime (Onozawa et al. 2022). However, the intensity of blue light was not constant, as it was supplied to plants during the daytime. Thus, more experiments were required to confirm the effect of blue-light application on the anthocyanin production of paprika fruit and ensure the improved production of purple color paprika. Therefore, to stabilize the light intensity and to confirm the effect of blue light, we tested the effect of the blue-light application on anthocyanin production in purple paprika fruit by applying blue light during the nighttime. We aimed to elucidate the mechanisms underlying the anthocyanin pathway and determine the effects of night blue light on paprika fruit development and ultimately improve and maximize the production of purple paprika cultivated under protected conditions. We quantified the production of anthocyanins by i) evaluating gene expression related to anthocyanin biosynthesis, such as HY5, bHLH, WDR, MYB, PAL, CHS, F3H, DFR, ANS, and UFGT, and ii) determining the total anthocyanin concentration in the peel and flesh of paprika fruit during fruit development.
Materials and Methods
Plant materials and growing conditions.
Purple paprika (Capsicum annuum ‘Tequila’) was hydroponically cultivated in a glass greenhouse at the School of Agriculture, Meiji University, Japan. The experiment was duplicated twice in 2023, during spring (10 Mar to 4 Jul 2023) (Expt. 1) and autumn (9 Sep 2023 to 3 Jan 2024) (Expt. 2). The conditions inside the glasshouse were similar to those of Yamada et al. (2019): the roof and side windows were opened when the temperature inside the glasshouse reached 25 °C, the ceiling fan was activated at 30 °C, and a heater was operated when the temperature was below 12 °C. The climate in the area during the whole cultivation period is shown in Supplemental Fig. 1. Paprika seeds were sown onto seed sponge made of urethane foam. After 2 weeks, seedlings were transplanted into the hydroponic culture under a deep flow technique within plastic containers (59 × 39 × 21 cm) filled with hydroponic solution at an electrical conductivity of 2.5 dS·cm−1 and pH of 6.0. The hydroponic solution was made by mixing commercial nutrient solutions: OAT House 1 (10N–8P–27K) and 2 (calcium nitrate tetrahydrate) (OAT Agrio, Osaka, Japan). The number of paprika fruits was limited to two fruits per plant for light treatment management. A total of 50 paprika seedlings were grown during each cultivation period.
Light treatment and experiment setup.
In both experiments, light treatment was initiated 10 d after pollination (DAP). A blue light-emitting diode (LED) panel (World Trading Co., Ltd., Kanagawa, Japan) with dimensions of 30 cm (width) × 30 cm (length) and a wavelength of 460 nm and photosynthetic photon flux density (PPFD) of 80 to 100 µmol·s−1·m−2 was set ∼50 cm from the paprika fruits, and blue light was supplied from 1800 HR to 0500 HR (total 11 h per day) (Fig. 1A). Three LED panels were set up for 10 plants (Fig. 1B). The control was not given blue-light supplement at night. Fruits were harvested at 15, 20, and 40 DAP. Fruit development parameters, such as fruit fresh weight, length, and diameter, were evaluated at each harvest, and the peels of the paprika fruit of control and blue light treatment [on the direct blue-light-radiated side (DR) and indirect blue-light-radiated side (IR)] were collected carefully using a razor blade to analyze the anthocyanin concentration and conduct gene expression analyzes.
(A) Blue light treatment conditions for paprika fruit. (B) Cultivation bench setup. Paprika fruits were subjected to night blue-light radiation where fruit was divided into two parts: direct irradiated side (DR), and indirect irradiated side (IR). Each container contained two paprika plants, and the number of fruits was limited to two fruit per plant. LED = light-emitting diode.
Citation: J. Amer. Soc. Hort. Sci. 149, 5; 10.21273/JASHS05421-24
Total anthocyanin concentration.
After harvest, anthocyanin pigment was extracted from the peel (only exocarp) and flesh (only mesocarp) of the control and blue light–treated paprika fruits from the DR and IR using 10% (v/v) acetic acid at 4
Analysis of anthocyanin biosynthesis-related gene expressions.
RNA was extracted from the peel and flesh of paprika fruits according to the protocol described by Matsushita et al. (2016) and Okutsu et al. (2018). Frozen ground peel or flesh (1 g) was extracted using 1 M Tris-HCl buffer (pH 8.0) containing 0.5 M ethylenediaminetetraacetic acid, 5 M NaCl, 2% cetyltrimethylammonium bromide, 0.05% spermidine trihydrochloride, and 2% polyvinylpolypyrrolidone. The extracted RNA synthesized complementary DNA using the PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara Bio, Shiga, Japan). Subsequently, quantitative polymerase chain reaction (qPCR) was performed using Applied Biosystems 7300 Real-Time PCR System (Thermo Fisher Scientific). The evaluation was conducted using the obtained DNA template and SYBR™ Green PCR master mix (Thermo Fisher Scientific).
Statistical analysis.
Student’s t test at P < 0.05 was performed to test the significant difference in produce quality between treatments. The Tukey-Kramer test determined significant differences among treatments at each evaluation. All statistical analyses were performed using Microsoft Office Excel (Microsoft Corporation, Redmond, WA, USA).
Results
Developmental changes in purple paprika fruit.
The fresh weight of purple paprika fruits increased rapidly over the cultivation period, particularly after 20 DAP, but it was unaffected by blue-light treatment in either experiment (Fig. 2A and B). Fruit size changed gradually toward the developmental stage; however, night blue-light radiation had no impact on the fruit size (Fig. 2C and D) and fruit width (Fig. 2E and F).
Fresh weight (A and B), length (C and D), and width (E and F) of paprika fruits under night blue-light radiation at 15, 20, and 40 d after pollination (DAP) in Expts. 1 and 2. Vertical bars indicate standard error (A: n = 5 to 13; B: n = 4 to 6; C and E: n = 3 to 7; D and F: n = 5 to 6). n.s. indicates no significant difference between treatments according to Student’s t test (P < 0.05).
Citation: J. Amer. Soc. Hort. Sci. 149, 5; 10.21273/JASHS05421-24
Anthocyanin concentration in purple-colored paprika fruit.
Blue light significantly increased the anthocyanin concentration in the paprika fruit peel, particularly in the DR in Expt. 1 (Fig. 3). Despite no significant difference observed in the anthocyanin concentration among the control, DR, and IR treatments at 20 DAP in Expt. 2, DR showed the highest accumulation. Furthermore, both experiments consistently show similar patterns of anthocyanin concentration (Fig. 3). Hence, it can be assumed that night blue light can effectively increase anthocyanin accumulation in paprika peel. The highest anthocyanin concentration was observed in DR from early fruit development at 15 DAP to late fruit development at 40 DAP, which the anthocyanin concentration in the peels of DR significantly differed from that of the control in Expt. 1 and Expt. 2. This suggests that blue light may sustain anthocyanin production up to 40 DAP. In contrast, blue light did not affect the anthocyanin concentration in paprika flesh, as no anthocyanin was detected in the flesh samples (data not shown).
Total anthocyanin concentration of paprika fruit peel of control, direct blue-light radiated side (DR), and indirect blue-light radiated side (IR) of fruits harvested at 15, 20, and 40 d after pollination (DAP) in Expts. 1 and 2 (n = 3 to 6). Vertical bars indicate standard error. Different letters express significant differences at each harvest according to the Tukey-Kramer test (P < 0.05), where n.s. indicates no significant difference.
Citation: J. Amer. Soc. Hort. Sci. 149, 5; 10.21273/JASHS05421-24
Expression of anthocyanin biosynthesis-related genes.
In both experiments, the target gene expression showed similar changes in the expression profiles of paprika peels (Figs. 4 and 5). Night blue-light treatment increased the expression of anthocyanin synthesis in both upstream (HY5, bHLH, WDR, MYB, and PAL) and downstream (CHS, F3H, DFR, ANS, and UFGT) genes, particularly in the peel that received the direct blue-light treatment. Most of the interesting gene expressions were highest during the early stages of fruit development before the expression decreases over time. In both experimental periods, the expressions of HY5 were almost unchanged among DR, IR, and control over fruit development; however, bHLH, WDR, MYB, and PAL expressions reached their highest levels at 15 DAP and then reduced over time. Furthermore, this expression trend was observed in downstream synthesis pathways such as CHS, DFR, and ANS, where DR showed a significantly higher expression than the control in Expt. 1 and Expt. 2. Interestingly, the expression of F3H was only increased at 20 DAT and was barely detected at 40 DAT even on the DR of the paprika peel and control fruits in both experiments. At 15 DAP, UFGT expressions were similar between control and blue light–treated fruit in both experiments. However, at 20 DAP in Expt. 1, the expressions of UFGT in DR and IR were significantly higher compared with the control (Fig. 4). In Expt. 2, although UFGT expression decreased over time, by 40 DAP, UFGT expression in the DR remained significantly higher than in others (Fig. 5).
Anthocyanin biosynthesis-related gene expression in different sides of paprika fruit peel, direct blue-light radiated side (DR) and indirect blue-light radiated side (IR), cultivated under control and night blue light in Expt. 1 (n = 3). Vertical bars indicate standard error. Different letters express significant differences at each harvest according to the Tukey-Kramer test (P < 0.05), where n.s. indicates no significant difference.
Citation: J. Amer. Soc. Hort. Sci. 149, 5; 10.21273/JASHS05421-24
Anthocyanin biosynthesis-related gene expression in different sides of paprika fruit peel, direct blue-light radiated side (DR) and indirect blue-light radiated side (IR), cultivated under control and night blue light in Expt. 2 (n = 3). Vertical bars indicate standard error. Different letters express significant differences at each harvest according to the Tukey-Kramer test (P < 0.05), where n.s. indicates no significant difference.
Citation: J. Amer. Soc. Hort. Sci. 149, 5; 10.21273/JASHS05421-24
The expression levels of transcription factors and structural genes involved in anthocyanin biosynthesis in paprika flesh were unaffected by the presence or absence of blue light. As a result, these data are not shown.
Discussion
Color-attractive fruits and vegetables, such as tomatoes, apples, and strawberries, have been widely studied for their color development under blue light (Arakawa et al. 1985; Kim et al. 2021; Naznin et al. 2019; Zhang et al. 2018a, 2018b). However, studies of paprika fruits have mainly focused on postharvest aspects (Liu et al. 2022; Naznin et al. 2019); therefore, the effect of an extended photoperiod with blue light on anthocyanin production in purple paprika fruit, especially during development, is poorly understood.
In our pre-experimental study (Onozawa et al. 2022), we found that applying blue light during the daytime enhanced the anthocyanin content and expression of related genes in purple paprika fruits. In the present study, we adjusted the irradiation period to the nighttime to investigate the effect of blue light and an extended photoperiod on fruit color development. Similar results were observed for the anthocyanin concentration of peels, which peaked from 15 DAP to 40 DAP in both experimental periods, particularly at DR of the blue-light treatment (Fig. 3). Furthermore, we found the expression of biosynthesis genes in upstream and downstream pathways increased at the early fruit development in both experimental periods (Figs. 4 and 5). HY5, which is involved in light-dependent anthocyanin synthesis pathway, was observed during this study. Although there was no significant difference in the expression of HY5 in both experiment periods, DR showed a higher HY5 expression than IR and the control throughout the evaluation. The expressions of regulatory proteins (bHLH, WDR, and MYB) were the highest in DR among others at each evaluation in both experiments, and the expressions of PAL, the key enzyme that controls the anthocyanin biosynthesis (Zhang et al. 2018), CHS, F3H, and DFR were also significantly increased in DR in both experiments. This implies that a slight increase of HY5 expression could boost these regulatory and structural-gene expressions. A large increase in ANS and UFGT resulted in a higher intensity of purple color in the fruit, as these are the initial anthocyanin synthases that convert flavonoids into anthocyanins (Dincheva et al. 2015; Li et al. 2016; Zhang et al. 2018a). This is probably the reason that anthocyanin concentrations of peels at the DR and IR were peaked at 20 DAP in both experimental periods (Fig. 3). The expression level of the transcription gene was increased by blue light (Figs. 4 and 5) despite the lower daylight period during spring and autumn cultivation. When paprika fruits are radiated with blue light, they exhibit heightened transcription activity of anthocyanin synthesis genes, resulting in an increase in anthocyanin content. Our finding is similar to the studies by Zhang et al. (2014) and Wu et al. (2016) on the plant-stress response mechanism that when the plant receives stress, the anthocyanin production is activated.
However, the late biosynthesis genes, DFR, ANS, and UFGT, decreased over time as the purple color of paprika fruit faded in both experiments (Figs. 4 and 5). This was similar to the result of the anthocyanin degradation study by Yamada et al. (2019). A change in the purple color of paprika fruits to red and/or yellow color after ripening has been reported (Yamada et al. 2019), and this was observed during this study after we successfully increased the anthocyanin biosynthesis-related gene expression of the purple paprika fruit using night blue-light radiation. In future studies, we will investigate the changes in anthocyanin-related gene expressions during fruit ripening, with the aim of maintaining or extending the period of the purple color on paprika fruit.
Although night blue-light radiation had no effect on fruit development in spring and autumn cultivations (Fig. 2), it affected the expression of anthocyanin biosynthesis-related genes (Figs. 4 and 5), which led to the increase of the anthocyanin concentration of the paprika fruit peel, especially in the DR of the fruit, which suggested that anthocyanin production was highly active in both experiments (Fig. 3). This increase may have occurred because anthocyanin accumulation in the peripheral tissues is dependent on light-induced biosynthesis (Steyn et al. 2002). Moreover, according to Feng et al. (2013), different light intensities affect the anthocyanin content of apple peel, particularly on the side of the peel directly exposed to light. In a study on the photoregulation of anthocyanin synthesis by Mancinelli (1983), the anthocyanin levels in plant organs varied with the light exposure level because of the plant-inlying signal, developmental stage, and light exposure. Hence, the shaded side or IR of fruit and control fruit peels showed lower levels of anthocyanin concentration than the fully irradiated side or DR of the fruit peel.
Although we evaluated the expression of anthocyanin biosynthesis-related genes in the fruit flesh during both experimental periods (data not shown), we observed fluctuations in gene expression levels irrespective of exposure to blue light. Notably, this variation occurred despite the absence of detectable anthocyanin in the paprika flesh. This suggests that the flesh of purple paprika fruit may lack the capacity to accumulate or synthesize pigments, as evidenced by our inability to detect anthocyanin accumulation in the flesh (data not shown).
When limited sunshine duration and/or cloudy conditions are expected, purple paprika producers face challenges to maintaining the purple fruit color. Our study findings show that extending sunshine duration with night blue light can be an effective countermeasure for increasing the anthocyanin content of purple paprika fruit. Our results suggest that to maintain their purple color, paprika fruits should be harvested from 20 DAP but not later than 40 DAP.
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