2025 Volume 94 Issue 2 Pages 255-265
Paprika (Capsicum annuum L.) is known for its abundant functional components such as carotenoids and phenolics. Growth responses to nutrient conditions including nitrogen (N) have been studied. However, there has been only limited analysis on the variations in components in response to N. This study investigated the effects of different N levels on the functional components of paprika. Paprika was grown under different N conditions and ripe fruits were harvested. Subsequently, mineral, carotenoid, phenolics, and sugar contents were measured. Higher N supply increased fruit weight and carotenoids, but reduced phenolics and glucose. The negative correlation between carotenoids and phenolics suggests complementary antioxidant functions in fruits, while the significant association between these metabolites and boron and potassium implies that these elements may have indirectly influenced metabolic pathways. Moreover, specific components like phenolics indicate a trade-off with yield. Capsanthin alone was significantly affected by N supply, suggesting downstream effects on the carotenoid biosynthetic pathway. This study will contribute to the optimization of N fertilizer application for balanced paprika production with high quality and economic yield.
Globally, pepper production is steadily increasing, leading to the expansion of the total pepper cultivation area (FAO, 2021). Research on various aspects, including genetic resources, which is expected to further expand production and improve quality (Jarret et al., 2019), is also on the rise. Paprika (Capsicum annuum L.) is a member of the pepper family and is a crop that contain high levels of functional components such as carotenoids and phenolics; in particular, high amounts of carotenoids (Howard et al., 2000; Ponder et al., 2021). Currently, the market for functional components is growing with the rising awareness of health through food, given the antioxidant capacity of these secondary metabolites (Baenas et al., 2019). Paprika exhibits a variety of colors, primarilly red and yellow, depending on the compositional variety of the carotenoids. The carotenoid compounds in red paprika include the red pigments, capsanthin and capsorubin, and the yellow-orange pigments, zeaxanthin, lutein, cryptoxanthin, and violaxanthin (Kim et al., 2004). The carotenoid biosynthetic pathway involves the condensation of two molecules of geranylgeranyl diphosphate to form the first carotenoid compound, phytoene. Subsequently, the following compounds are biosynthesized from phytoene: lycopene, β-carotene, cryptoxanthin, zeaxanthin, antheraxanthin, and capsanthin (Nisar et al., 2015).
Yield, as well as these functional components in pepper, are affected by several environmental factors, such as light, temperature, and water stress (Kabir et al., 2021; Lekala et al., 2019; Wang et al., 2015). Although studies on these stress responses and yields in paprika have been conducted, limited paprika research has been done on the nutrient conditions affecting metabolic regulation and mineral absorption, resulting in the continuous application of many fertilizers to increase yields and stabilize quality (Han et al., 2015). Among essential elements, nitrogen (N) is the main element required by plants and it has significant effects on the amount of metabolites, minerals, and yield. For example, in tomato, a reduction in N fertilization leads to the enhancement of phenolic compounds and ascorbic acid accumulation, while reducing carotene accumulation (Dumas et al., 2003). Gravel et al. (2010) found a negative correlation between the soil NO3− concentration and carotenoid compounds contents of tomato fruit, which showed inconsistent results. Limited research has been done on the variation in secondary metabolites and minerals in response to nutrient stress in paprika, a member of the Solanaceae family (same as tomatoes). Furthermore, we need to study the appropriate fertilization conditions adapted to paprika-specific characteristics, which are different from those of tomatoes, such as fruit size and fruit attaching patterns. Moreover, the conventional fertilizer composition used in paprika cultivation may lead to excessive application of nutrients, including N.
This study aimed to investigate the N responses of minerals and metabolites, such as carotenoids, phenolic compounds, and sugars in paprika fruits. The results will contribute to fundamental knowledge to apply appropriate N fertilization conditions to balance paprika production and quality.
Paprika (C. annuum L.) var. ‘ARTEGA’ (Enza Zaden, North Holland, The Netherlands) was germinated on filter paper and moistened with demineralized water at 33°C in the dark on March 9, 2020. On March 12, 2020, the germinated seeds were transplanted into cell trays and cultivated with deionized water in a plant growth chamber (28°C/23°C, 16 h/8 h (light/dark); FLI-2000; EYELA, Tokyo, Japan). On April 5, 2020, plants were transplanted into 75 × 75 × 75 mm Rockwool blocks (Nippon Rockwool Co., Ltd., Tokyo, Japan) and irrigated with 1/10 the concentration of a commercial standard liquid fertilizer (Otsuka-A solution; OAT Agrio, Tokyo, Japan). According to manufacturer’s instructions, this standard solution consisted of 18.56 mM N (consisting of NO3− and NH4+; the ratio of total NO3− concentration to NH4+ concentration = 0.09), 1.69 mM phosphorus (P), 8.60 mM potassium (K), 4.10 mM calcium (Ca), 1.49 mM magnesium (Mg), 21.15 μM manganese (Mn), 43.09 μM boron (B), 48.34 μM iron (Fe as Fe-EDTA), 1.38 μM zinc (Zn), 0.47 μM copper (Cu), and 0.31 μM molybdenum (Mo). On April 12, 2020, at Hokkaido University Greenhouse, Rockwool blocks were planted in 75 × 200 × 910 mm Rockwool slabs (Nippon Rockwool Co., Ltd.) at a density of three plants per slab and irrigated with liquid fertilizer. For about 18 weeks from April 26 to August 31, 2020, plants were irrigated with four different N concentrations of liquid fertilizer (Control: 18.56 mM N; N1/2: 9.28 mM N; N1/5: 3.71 mM N; N1/10: 1.86 mM N), keeping the concentrations of elements other than N and the ratio of NO3− to NH4+ unchanged among treatments. The pH of these solutions was manually adjusted from 5.3 to 5.5 using 1 mM H2SO4 or 1 mM KOH every two to three days. Irrigation frequency was changed according to the weeks after seeding (WAS), with an average flow rate of 13.9 mL·min−1 for 15 min per irrigation (from 6th WAS to 9th WAS: three irrigations per day; from 10th WAS to 11th WAS: four irrigations per day; from 12th WAS to 16th WAS: five irrigations per day; from 17th WAS to 24th WAS: six irrigations per day). Cultivation was carried out with nine replicates per treatment, and the plants were trimmed to two stems of even length, with one leaf and one flower per node.
Shoot height was measured weekly from 9th WAS to 24th WAS. At 24th WAS, stems and leaves were harvested and dried. The dry weight was then measured and the samples were ground for mineral analysis. Marketable ripe paprika fruits, excluding those with blossom-end rot (BER), were harvested, and their lengths and widths were initially measured, followed by their fresh weights. The fruit petiole, calyx, placenta, and seeds were removed and the fruit pericarp was immediately frozen. Fruit pericarps were ground in liquid N and lyophilized for metabolite and mineral analyses. For analysis, 10 replicates of fruit pericarp samples were randomly selected from each treatment at the same harvest time, which was the first of the continuous fruiting periods, called group 1 fruits (from the 2nd node to 5th node). Fruits from the 6th to 10th node were defined as group 2 fruits, and fruits above the 11th node were defined as group 3 fruits.
Measurement of mineralsEach dried sample (50 mg) was incubated with 2 mL of 61% (w/v) HNO3 (EL grade; Kanto Chemical, Tokyo, Japan) at room temperature for three nights. The solution was digested at 107.5°C in a DigiPREP apparatus (SCP Science, Quebec, Canada). After approximately 2 h, about 0.5 mL of H2O2 (EL grade; Kanto Chemical) was added to the tubes and further digested. The digested solution was cooled and filled with 10 mL 2% HNO3 in ultrapure water. B, Ca, Cu, Fe, K, Mg, Mn, Mo, sodium (Na), P, sulfur (S), and Zn were analyzed using inductively coupled plasma mass spectrometry (ICP-MS) (ELAN DRC-e; Perkin Elmer, Waltham, MA, USA).
Measurement of total N concentrationThirty milligrams of samples were digested in 1.25 mL of 18 M H2SO4 at 205°C, and 0.2 mL of H2O2 was added every 30 min five times. After cooling, the digested solution was diluted with demineralized water to a final volume of 25 mL. Then, the N concentration was determined by the Kjeldahl method using a Buchi 323 Distillation Unit (BÜCHI Labortechnik AG, Flawil, Switzerland).
Measurement of carotenoidThree major carotenoids (capsanthin, cryptoxanthin, and zeaxanthin) in red paprika fruit were evaluated using high-pressure liquid chromatography (HPLC). Extraction and measurement of carotenoids were performed by modifying the method of Tian et al. (2014). Specifically, a 0.5 g sample of lyophilized fruit was extracted with 5 mL of acetone containing 0.1% butylated hydroxytoluene (BHT). After shaking and incubating on ice in the dark for 10 min, the mixture was centrifuged at 2,360 × g for 10 min at room temperature. The samples were re-extracted twice with acetone (5.0 mL) containing 0.1% BHT. The pooled extracts were dried using an evaporator, and the samples were dissolved in 5 mL of methanol: acetonitrile (1:1, v/v) and stored at −20°C until HPLC analysis. For HPLC, the samples (20 μL) were analyzed using a TSKgel-80Ts HPLC column (4.6 mm × 150 mm; TOSOH Inc., Tokyo, Japan). The eluents consisted of acetonitrile: 2-propanol: water in a ratio of 39:53:8 (A) and acetonitrile:2-propanol in a ratio of 60:40 (B). The gradient profile was 0–30 min from 0 to 100% B. The flow rate was set at 0.3 mL·min−1 and the column temperature at 40°C. Carotenoids were identified based on their absorption spectra captured at 454 nm using a photodiode array detector (UV-2075 Plus; Jasco, Tokyo, Japan). For capsanthin, the relative level in each sample was calculated based on the average of the Control. β-Cryptoxanthin was purchased from Ehime Co., Ltd. (Ehime, Japan) and zeaxanthin was obtained from Funakoshi Co., Ltd. (Tokyo, Japan), and they were used as authentic standards. All the standards and samples were handled under low-light conditions on ice. Standard solutions of β-cryptoxanthin and zeaxanthin were prepared in methanol: acetonitrile (1:1, v/v). Aliquots were diluted in methanol: acetonitrile (1:1) to provide standard concentrations (0.001–0.05 mg·mL−1).
Measurement of total phenolics and DPPH radical scavenging activityLyophilized fruit samples were extracted by following Ofei-Manu et al. (2001). Total phenolics were measured based on the Folin-Denis method with slight modifications (Folin and Denis, 1915). Briefly, a mixture of 2.6 mL water, 0.8 mL sample extract, 0.4 mL of Na2CO3, and 0.2 mL Folin-Denis solution which contained 10% sodium tungstate dehydrate, 2% of phosphomolybdic acid, and 5% of phosphate were incubated for 30 min at room temperature. The solution was centrifuged at 3,500 × g for 5 min, and the absorbance of the supernatant was measured at 700 nm using catechin as a standard.
For the antioxidant activity assay, the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity was estimated by the method of Proestos et al. (2013) with a slight modification. The supernatant extracted for measurement of total phenolics was mixed with a 0.006% DPPH solution dissolved in methanol at a ratio of 1:1 (v/v). After incubating in the dark at room temperature for 30 minutes, the absorbance was measured at 517 nm. The DPPH radical scavenging activity was expressed as ascorbic acid equivalent per 1 g (dry weight) using the calibration curve of ascorbic acid.
Measurement of sugarsExtraction of soluble sugars was carried out following the method of Okamura et al. (2016). Lyophilized fruit weighing 50 mg was mixed with 1 mL 80% ethanol at 80°C and stirred, then kept at room temperature for 10 min. Subsequently, the mixture was centrifuged at 11,000 × g for 5 min at room temperature and the supernatant was collected. This process was repeated, and the supernatant was dried under a vacuum. The dried samples were dissolved in deionized water and subjected to soluble sugar (Glucose and Fructose) assays. Soluble sugars were measured using an enzymatic method with an F kit #716260 (J.K. International, Tokyo, Japan) and a Microplate Spectrophotometer (EPOCH2; BioTek Japan Inc., Tokyo, Japan).
Statistical analysesAll statistical analyses were performed using R version 4.0.4. Measurement data were tested using the Smirnov-Grubbs test at a 5% significance level to detect outliers.
From the 9th WAS, when group 1 fruits were observed, the primary stem length was measured. Growth differences among treatments were not observed up to the 11th WAS, but significant differences were observed from the 12th WAS, when all the group 1 fruits had fruited (Fig. 1a). At the 24th WAS, the Control and N1/2 showed no significant difference, but a significant decrease was shown in the N1/5 and N1/10 treatments compared to the Control.
Indicators of paprika plant and fruit cultivation traits. (a) Relative primary stem growth as an index of the primary stem length at the 9th week after seeding (WAS). Values and error bar express means ± SE (n = 9). (b) Dry weight of leaf and stem at 24th WAS. Values and error bar express means ± SE (n = 9). (c) Fruit fresh weight, (d) Fruit length, and (e) Fruit width of group 1 fruit from the 2nd node to the 5th node. Values and error bar express means ± SE (Control: n = 32, N1/2: n = 36, N1/5: n = 21, N1/10: n = 35). The asterisk indicates a significant difference between the N treatments (Student’s t-test, *P < 0.05). Different letters for respective plant organs indicate significant differences at P < 0.05 using Tukey’s multiple comparison test following a one-way ANOVA test. Dots indicate values of each sample measured.
In terms of paprika plant biomass, there was no significant difference in dried leaf weight between the Control and N1/2 plants; however, both the N1/5 and N1/10 plants showed a significant decrease in dried leaf and stem weights (Fig. 1b).
Regarding the number of marketable fruits, the amount of group 1 fruits remained similar even under low-N conditions, whereas the total number of marketable fruits decreased under low-N conditions (Fig. S1a). Furthermore, the number of marketable fruits decreased at higher sites compared to lower sites, irrespective of the N treatment. The fruit yield in N1/2 showed no significant difference compared to the Control, but both N1/5 and N1/10 exhibited a significant decrease in yield (Fig. S1b). The fresh weight of group 1 fruits was also significantly decreased in both N1/5 and N1/10 compared with the Control (Fig. 1c). Additionally, fruit length and width tended to decrease with decreasing N fertilization concentrations (Fig. 1d, e).
Variations in minerals in paprika plants under different N treatmentsFigure 2 shows the effects of N treatment on N concentration in the fruits, leaves, and stems. The N concentration in each organ significantly decreased under low-N conditions. Moreover, significant differences were observed among treatments for almost all mineral elements in the fruit, except Na and P (Table 1). Some elements, such as B and K, increased under low-N conditions, indicating that each element had a different response to the N treatment. Most mineral elements in the leaves and stems showed significant differences among the treatments, except for K and Fe (Tables S1 and S2).
N concentrations of paprika fruit (group 1 fruits), leaf, and stem (at 24th WAS). Values and error bar express means ± SE (fruit: n = 10, leaf and stem: n = 9). Different letters indicate significant differences at P < 0.05 using Tukey’s multiple comparison test following a one-way ANOVA test. Dots indicate values of each sample measured.
Ionome profile in group 1 fruits.
Metabolites (carotenoids, phenolics, and sugars) showed different responses to N treatment. Among the major carotenoid compounds (capsanthin, zeaxanthin, and cryptoxanthin) in red fruit, both capsanthin and zeaxanthin tended to increase in N1/2 and decrease in response to lower N conditions, especially in N1/10 (Fig. 3a, b). No significant differences were observed in cryptoxanthin among the treatments (Fig. 3c).
Concentrations of metabolites in fruit. Values and error bar express means ± SE. Different letters indicate significant differences at P < 0.05 using Tukey’s multiple comparison test following a one-way ANOVA test. Dots indicate values of each sample measured. (a) Relative capsanthin level, (b) Zeaxanthin, (c) Cryptoxanthin, (d) Phenolic compounds, (e) Glucose, (f) Fructose. The vertical axis in (a) shows the relative values calculated using the average value of the Control as 1.
It was found that the total phenolics and carotenoid concentrations in the fruit were affected by N treatment, but the trends were different (Fig. 3d). In N1/2, the lowest total phenolics concentration was observed. In comparison, the Control and N1/10 had a significantly higher concentration. DPPH radical scavenging activity showed the same trend as the phenolics (Fig. S2).
Figure 3e and f show the glucose and fructose concentrations in fruits, respectively. The glucose concentration tended to increase with decreasing N concentration and was the highest in N1/5.
Interrelationship between minerals and metabolites in fruitTo investigate the interactions among metabolites, N concentration, and/or yield, correlation analyses were performed for fruit fresh weight, fruit N concentration, carotenoid compounds, total phenolic compounds concentration, and sugars (Table 2). Regarding the effect of N treatment on growth, there was a significant positive correlation (r = 0.75, P < 0.05) between N concentration and fruit fresh weight. Regarding the relationship among metabolites, a slight negative correlation was observed between phenolic compounds and capsanthin (r = −0.38, P < 0.05) or zeaxanthin (r = −0.32, P = 0.053). For the correlations between fruit fresh weight and functional components, it was found that there was a significant negative correlation between fruit fresh weight and phenolic compounds (r = −0.37, P < 0.05) and a significant negative correlation between fruit fresh weight and glucose (r = −0.40, P < 0.05). Carotenoid compounds showed no significant correlation with the fruit fresh weight.
Correlation analysis among yield, N concentration, and metabolites under N treatments in fruit. An asterisk indicates a significant correlation (Pearson correlation test, *P < 0.05).
For the individual correlations between mineral and metabolite variables, B and K exhibited significant negative correlations with capsanthin (r = −0.43, P < 0.05; r = −0.52, P < 0.05) and significant positive correlations with phenolics (r = 0.32, P < 0.05; r = 0.40, P < 0.05) (Table 3). Mo and Zn showed significant negative correlations with glucose (r = −0.41, P < 0.05; r = −0.34, P < 0.05).
Correlation analysis between inorganic element concentrations and six metabolites in fruit under N treatments. The asterisk indicates a significant correlation (Pearson correlation test, *P < 0.05).
N fertilizer plays an important role in improving the yield of Solanaceae species (Scholberg et al., 2000). In this study, a strong relationship was found between the N application level and yield, as well as plant growth (Fig. 1a and Table 2). The N1/2 plants showed no significant differences in growth (Fig. 1a), biomass (Fig. 1b, c), or fruit size (Fig. 1d, e) compared with the Control. This suggests that at least half the N concentration of commercially used liquid fertilizers is sufficient for adequate growth under the cultivation conditions in this study. This is consistent with reports that N concentrations of 8.3–9.3 mM are adequate for increasing pepper yield and quality (Bar-Tal et al., 2001). Reducing N fertilizer while maintaining yield is expected to achieve cost savings and reduced environmental impacts. There is also concern that high N conditions may cause physiological disorders, such as blossom-end rot, resulting in a decrease in marketable yield (Arakawa et al., 2021). The number of harvested fruits did not change with N treatment in group 1 fruits; however, the number of fruits and marketable yield decreased under low N treatments (Fig. S1a, b), suggesting that it is necessary to consider the relationship between N fertilizer reduction and economic yield. Furthermore, the differences in N levels among treatments were more marked in the leaves and stems than in the fruits (Fig. 2). It has been reported that N translocation from leaves to fruits is enhanced when N supply is insufficient (Gomez-Lopez and del Amor, 2013). Lower plant growth under reduced N was observed from the 12th WAS, when all group 1 fruits were fruited (Fig. 1a). During periods of increased fruit load, branch growth is reduced, and photosynthetic assimilates are preferentially supplied to the fruit (Fukumoto et al., 2004; Heuvelink and Körner, 2001). Generally, amino acids (N resources) produced in leaves are transported to the sink along with assimilates (C resources). From the 12th WAS, the fruit load increased, which may have suppressed branch extension and enhanced N translocation to the fruit. Under low N conditions, the increased N translocation to the fruit along with the increased fruit load may have resulted in insufficient N for stem growth. Therefore, N translocation from the leaves and stems is considered to be more active under low-N conditions, especially during increased fruit load.
The concentrations of each mineral differed in the fruits, leaves, and stems, whereas they were responsive to N treatments in each plant organ (Tables 1, S1, and S2). In tomatoes, several environmental factors affected the mineral profiles of the sink and source organs (Benard et al., 2015; Osorio et al., 2014), and similar trends were observed in paprika. These results suggest that N has a significant effect on the interactions among multiple minerals in all organs. B concentrations in fruit showed a distinctive N response, increasing significantly with decreasing N supply (Table 1). This indicates that N may affect B retranslocation compared to other elements such as Ca. Eggplant has been suggested to retranslocate more actively than other elements such as Ca due to a higher B concentration in fruit than in leaves (Watanabe et al., 2016), while similar trends were observed in paprika. Additionally, B induces crosslinking of pectin in the cell wall, suggesting its involvement in BER (Arakawa et al., 2021). In this study, the Control had an average of 48% BER incidence, whereas N1/10 did not have any. High N supply may suppress the translocation of B, a cell wall component, into the fruit, thus reducing the cell wall stability of the fruit and possibly increasing physiological disorders such as BER. However, BER is also affected by Ca and temperature conditions, and further investigation is needed.
B and K had significant positive relationships with phenolics and negative ones with carotenoids (Table 3). This result indicates that B and K increase phenolic metabolites and decrease carotenoid metabolites in paprika. S may have a similar relationship due to its significant positive correlation with phenolics, but this needs to be investigated in further detail. In tomatoes, an excess of B led to the accumulation of soluble phenolics, whereas a reduced K supply resulted in low levels of phenolic compounds (Constán-Aguilar et al., 2014; Silva-Beltran et al., 2015). Also, a deficiency in S led to a decrease in phenolic compounds in tomatoes (Mohammed et al., 2015). However, these results, in which B, K, and S showed a negative relationship with carotenoids, contradict previous studies indicating the supply of B, K, and S leads to an increase in carotenoid compounds in tomatoes (Trudel and Ozbun, 1971; Xu et al., 2021; Zelena et al., 2009). Paprika is suggested to have different relationships among these elements and carotenoids compared to tomatoes. Red paprika is rich in capsanthin, while tomatoes are rich in lycopene (Kim et al., 2004; Xu et al., 2021). K shows a positive correlation with carotene and a negative correlation with lycopene, which is attributed to the effect of K on the carotenoid biosynthesis pathway (Taber et al., 2008; Trudel and Ozbun, 1971). The different carotenoid composition may affect the relationship with B, K or S in paprika fruits, and further investigation is needed. Additionally, Zn and Mo were negatively correlated with glucose (Table 3). Zn fertilization has been reported to increase the sugar content in tomato fruits (Gurmani et al., 2012). Ali et al. (2023) showed that low ZnSO4 treatment significantly increased glucose concentration, while higher ZnSO4 levels decreased it, suggesting an impact on glucose metabolism through enzyme activity and gene expression. Additionally, SlZIP11, a ZRT/IRT-like protein, is involved in Zn accumulation and sugar storage in tomato (Sun et al., 2024). Mo has been shown to contribute less to pepper sugar through stepwise multivariate regression analysis (Liu et al., 2021). However, Mo is thought to contribute to maintaining primary N and carbon metabolism based on integrated transcript and metabolite profiling approaches (Ide et al., 2011). Liu et al. (2017) reported that several sugar contents, including glucose, fluctuated with Mo treatment, indicating the need for further research. Thus, these results suggested that Zn and Mo may be involved in sugar metabolism in fruits. Variations in the mineral profiles due to N may indirectly affect these metabolic pathways.
Carotenoids and phenolic compounds have attracted much attention as antioxidants and are lipophilic and hydrophilic, respectively (Nisar et al., 2015; RiceEvans et al., 1997). The interactions between these metabolites have not been fully investigated, but are considered a crucial perspective (Long et al., 2006). In this study, a significantly negative correlation was found between capsanthin and total phenolics (r = −0.38, P < 0.05) (Table 2). Regarding the correlation between these metabolites and N concentration, there was a significant positive correlation with capsanthin levels (r = 0.36, P < 0.05) and a significant negative correlation with phenolics (r = −0.53, P < 0.05). These results suggest that capsanthin and phenolic compounds may have complementary antioxidant functions in fruits. N deficiency such as in N1/5 and N1/10 also implied an enhanced antioxidant effect due to phenolics rather than carotenoids. Becker et al. (2015) showed that carotenoids and chlorophyll decreased while flavonoids increased under low nitrogen deficiency in lettuce, which is consistent with the results of this study. Meanwhile, it has been reported that vitamins and other compounds are also responsible for the antioxidant capacity in plants, increasing ascorbic acid content with decreasing N supply (Lee and Kader, 2000). Additionally, oxidative stress increased in tomatoes under N deficiency (Safavi‐Rizi et al., 2021). It is necessary to consider complex factors, such as reactive oxygen species and free radicals, targeted for antioxidant activity because the functions performed by carotenoids and phenolic compounds are not completely equivalent (Sun and Li, 2020; Vogt, 2010).
Carotenoids are divided into two groups: carotenes, upstream of the biosynthetic pathway, and xanthophylls that are downstream. Carotenes are composed of only carbon and hydrogen, whereas xanthophylls are carotenoid compounds with oxygen atoms such as hydroxyl and ketone groups (Christaki et al., 2013). In fruits, the major carotenoid compounds are capsanthin, zeaxanthin, and cryptoxanthin, which are classified as xanthophylls and are downstream in the biosynthetic pathway (Rodriguez-Uribe et al., 2012). Although capsanthin levels in fruits were significantly affected by N treatments, zeaxanthin and cryptoxanthin did not show significant differences among treatments (Fig. 3a, b, c). Regarding β-carotene, a precursor of cryptoxanthin and the major carotenoid compound in paprika, it has been reported that low N supply did not significantly alter the carotene content in several vegetables, including tomatoes (Benard et al., 2009). It is suggested that N supply may affect the downstream carotenoid biosynthetic pathway more than the upstream. However, carotenoid content has been reported to increase or decrease under high N fertilization conditions with inconsistent results (Cheng et al., 2021; Poiroux-Gonord et al., 2010). This is because the carotenoid biosynthetic pathway is affected by light, temperature, and nutrient conditions such as N (Poiroux-Gonord et al., 2010). Increased N supply has been reported to expand leaf area and enhance photosynthetic activity, as well as increase carotenoid concentrations (Hernandez et al., 2020). Photosynthetic capacity decreases in response to low light and temperature conditions, while carotenoid concentrations increase (Zhang et al., 2020b). Thus, the relationship among carotenoids, photosynthesis, temperature, and light is interactive, requiring further focus on these interactions. It is also important to evaluate the effects of N on carotenoid biosynthesis in fruits under diverse environmental conditions.
Phenolic compounds are recognized as metabolites involved in plant stress protection (Samec et al., 2021). It has been suggested that phenolic compounds are increased in tomatoes to enhance defense against abiotic stresses such as low N availability (Benard et al., 2009). Phenylalanine biosynthesis has been reported to be maintained under low N availability, although amino acid levels decreased except for phenylalanine, the main precursor of phenolic biosynthesis (Zhou et al., 2021). Additionally, this increase in phenolic compounds has been shown to have a trade-off relationship with plant growth (Stefanelli et al., 2010). In this study, phenolic compound concentrations increased with reduced N supply (Fig. 3d), whereas fruit fresh weight decreased (Fig. 1c). There were significant negative correlations between total phenolics and fruit weight (r = −0.37, P < 0.05) and N concentration (r = −0.53, P < 0.05) (Table 2). As reported in previous studies, these results indicate that a lower N supply is associated with a higher accumulation of phenolic compounds to provide biological defense against abiotic stresses, instead of reducing growth. Phenolic compounds also have a tradeoff relationship with yield; thus, N fertilization may contribute to the production of paprika with enhanced specific functional components. Focusing on the Control and N1/2, the concentration of phenolics significantly increased with increasing N supply, with no significant difference in fruit weight or N concentration (Figs. 1c, 2, and 3d). The N concentration in N1/2 was sufficient for growth, while luxury N may be supplied to the Control. In potato (Solanum tuberosum L.) tubers, excessive N was found to be involved in phenylalanine metabolism, with significant increases in phenylalanine and tyrosine (Zhang et al., 2020a). Thus, luxury N absorption may stimulate phenolic biosynthesis with phenylalanine and tyrosine as precursors in paprika fruits.
Nutrient stress such as salt stress has been indicated to lead to high sugar contents in pepper and tomato fruits, as well as reduced yield (Rubio et al., 2009). It was reported that low N conditions lead to higher sugar levels in tomato fruit (Hernandez et al., 2020). Both glucose and fructose tended to increase as N treatment decreased to N1/5 (Fig. 3e, f). Additionally, a significant negative correlation was observed between fruit fresh weight and glucose concentration (r = −0.40, P < 0.05) (Table 2). Carbon and N metabolisms are closely regulated to maintain optimal growth, and N and glucose interact at the transcript level (Price et al., 2004). One of the reasons for glucose accumulation in fruits is thought to be to increase the gap fraction (the transmittance of light through the canopy to the fruit) due to decreased leaf and stem growth under N deficiency, resulting in enhanced photosynthetic activity (Benard et al., 2009). The reduced demand for carbon skeletons for amino acid and protein synthesis under N deficiency may be involved in sugar accumulation (Wingler et al., 2006; Zhang et al., 2021). It is suggested that N deficiency may lead to increased sugar concentrations in fruit in relation to the carbon metabolites, photosynthesis, and growth.
In conclusion, N treatment caused a decrease in yield and growth, while the amount of functional components in fruits, such as secondary metabolites and minerals, responded to N supply differently. Carotenoids were maximum at moderate N supply (9.28 mM), while phenolics were significantly increased under low-N conditions. Regarding plant mineral concentrations, the mineral profile of the fruit was altered by the N treatment. The correlation analysis suggests that elements such as Zn, B, S, Mo, and K may contribute to the changes in the mineral profile under N treatment. B and K may be involved in carotenoid and phenolic metabolites. Additionally, Zn and Mo may have a negative relationship with sugars. The yield decreased with N treatment, whereas these functional components had different N responses; for example, phenolics increased with reduced N supply. This study suggests interrelationships between carotenoids and phenolic compounds and indicates the effects of N downstream of the carotenoid biosynthetic pathway. In the future, it will be necessary to identify the molecular mechanisms of metabolite interactions and biosynthetic pathways that are sensitive to N treatment through a comprehensive survey of secondary metabolites. Thus, the appropriate management of N fertilizer application will contribute to the establishment of a paprika production system with a good balance between economic yield and quality. Furthermore, this study provides important insights to develop a sustainable cultivation system with low environmental load.
The authors would like to thank all members of the Laboratory of Plant Nutrition at Hokkaido University for assisting with sampling and measurement. We would like to thank Editage (www.editage.jp) for English language editing.
