Effects of nitrogen fertilization rate on tocopherols, tocotrienols and γ-oryzanol contents and enzymatic antioxidant activities in rice grains

Abstract

Tocopherols (Toc), tocotrienols (T3) and γ-oryzanol (GO), major bioactive compounds of rice, are known to possess potent antioxidant activity. In this study, the objective was to determine the effects of nitrogen fertilization rate on contents of Toc, T3 and GO, and activities of enzymatic antioxidants in rice grains. Experiments were conducted on five different levels of nitrogen fertilization. Among the different treatments, grains of 2 N (two-fold of the recommended amount of nitrogen fertilizer) treatment showed the highest total Toc, total T3, α-T3, β-Toc, γ-Toc and γ-T3 levels, whereas 0 N (no treatment) group had the highest GO content. Increasing nitrogen fertilization significantly reduced the rice grain catalase and ascorbate peroxidase, but not the superoxide dismutase activities. Under 0 N and 0.5 N (low N fertilization) treatments, malondialdehyde and H₂O₂ contents in rice grains were significantly higher than that of other treatments. These results suggest that a two-fold increase in nitrogen fertilization favor the accumulation of Toc and T3 but not GO in rice grains.

Introduction

Rice (Oryza sativa L.) is an important agricultural commodity, and is also the main staple diet in Asia. Its grains are known to contain a complex mixture of biologically active phytochemicals such as tocopherols (Toc), tocotrienols (T3), γ-oryzanol (GO) and others (Bergman and Xu 2003; Huang and Ng 2011a). In nature, GO is rarely found in common crops and vegetables (Miller and Engel 2006), whereas T3 is mainly present in monocot species such as O. sativa (Yang et al. 2011). Both Toc and T3 belong to the family of vitamin E or tocochromanols, with each of them comprising four analogs (α, β, γ and δ). They are good antioxidant; however, recent studies have reported that T3 possessed similar or better antioxidant activity than α-Toc (Wong and Radhakrishnan 2012). GO has also been reported to possess good antioxidant activity (Aladedunye et al. 2013). These findings have led to an increased interest in investigating their functions in plants and human health among the scientific communities.

In plants, vitamin E biosynthesis is initiated by the condensation of two biosynthetic precursors, the polar homogentisate that consist of a polar chromanol ring and a lipophilic polyprenyl pyrophosphate side chain, which varies according to the type of tocochromanols. The polyprenyl precursor of tocopherols is phytyl pyrophosphate and geranylgeranyl pyrophosphate for tocotrienols (Pellaud and Mène-Saffrané 2017). According to the degree of methylation of the chromanol ring, each type of tocochromanol exhibits up to four different forms, which are α-(three-methyl groups), β- and γ-(two-methyl groups), and δ-(one-methyl group) tocochromanols (Mène-Saffrané 2018).

Studies have shown that the contents of Toc, T3 and GO varied with rice varieties, cultivation environment, harvesting time and rates of fertilization (Bergman and Xu 2003; Huang and Ng 2011a). Nitrogen deficiency was reported to reduce the rate of photosynthesis, but increased the malondialdehyde (MDA) accumulation and enzymatic antioxidant activities in the leaves of rice plants (Huang et al. 2004). Nitrogen fertilization has varying effects on the yield of bioactive substances in different plant species; for example, increasing nitrogen application can enhance the caffeine and theobromine contents in the leaves of Ilex rotunda, but had no effect on its phenolic and flavonoid concentrations (Palumbo et al. 2007). Increasing nitrogen fertilization can also increase the betaine biosynthesis but decrease the content of polysaccharides in Lycium barbarum (Chung et al. 2010), and had no effect on the sterol and Toc contents in walnut plants (Verardo et al. 2013). Excessive nitrogen application was shown to reduce the bioavailability of phenolic compounds in wheat grains (Stumpf et al. 2015), whereas decreasing nitrogen fertilization resulted in increased flavonoid, vitamin C and anthocyanin contents in the leaves of Labisia pumila (Ibrahim et al. 2012).

Application of an appropriate amount of nitrogen nutrient can promote plant growth and increase crop yields, but the lack of or too much nitrogen will affect the normal growth, and increase oxidative stress in plants. At present, information on fertilizer effects on antioxidants such as Toc, T3 and GO contents in crops remain limited. Seeds are known to contain higher levels of Toc and lipids (mainly polyunsaturated fatty acids; PUFAs) than other plant tissues; the occurrence of high levels of Toc and PUFAs in seeds suggests that Toc may protect storage lipids from oxidation (Sattler et al. 2004). Based on the hypothesis that deficient and excessive nitrogen fertilization causes oxidative stress, and that may affect the rate of antioxidant biosynthesis and activities of enzymatic antioxidants in rice plants, this study examined the effects of nitrogen fertilization rates on Toc, T3 and GO contents, and enzymatic antioxidant activities in rice grains.

Materials and methods

Chemicals

Standards of tocopherol analogs (α-, β-, γ- and δ-Toc) and γ-oryzanol were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA), whereas tocotrienol analogs (α-, β-, γ- and δ-T3) were obtained from Davos Life Science Pte. Ltd. (Helios, Singapore). All other chemicals used were of analytical grade.

Experimental design

The rice species selected in this study was Tainung 71 (TN71) of Japonica variety, which is one of the most popularly cultivated rice varieties in Taiwan. All experiments were conducted in the greenhouse of National Taiwan University, Taipei, from 24 May 2012 to 26 September 2012. The type of soil used in the experiment was sandy loam, of which the chemical properties was pH 5.3, electrical conductivity 0.8 dS m⁻¹, organic matter 38.9 g kg⁻¹, total N 1.7 g kg⁻¹, and Mehlich III extractable P and K were 52.2 and 165 mg kg⁻¹, respectively. The experiment was conducted in Wagner pots (1/5000a) with 3.5 kg of soil in each pot.

Two rice seedlings were randomly assigned to each pot and subjected to either with or without nitrogen fertilizer treatment. All treatments were arranged in a randomized complete block design. A total of five treatments were performed with four replications per treatment. There were 0 N (no treatment), 0.5 N (0.115 g pot⁻¹ N; half the recommended amount of nitrogen fertilizer), 1 N (0.23 g pot⁻¹ N; recommended amount of nitrogen fertilizer), and high N treatments, consisted of 2 N (0.46 g pot⁻¹ N) and 3 N (0.69 g pot⁻¹ N) that were two- and three-fold of the recommended amount of nitrogen fertilizer, respectively. The recommended nitrogen fertilizer rate for rice cultivation is 100 kg ha⁻¹ N (COA 2005). Urea was used as nitrogen nutrient, whereas phosphorus was given as ordinary superphosphate (P₂O₅). As the experimental soil contained a high level of potassium, no extra potassium was applied in this study. Pots were irrigated daily to maintain soil moisture at about field capacity.

Soil samples were taken before planting and after harvesting to indicate the effect of nitrogen fertilization on chemical properties of soil. They were air-dried and sieved through 20 mesh (0.85 mm). The fine soil samples were subjected to the analysis of pH, electric conductivity of saturation extract, total nitrogen (Page 1982), Mehlich III extractable P and K (Mehlich 1985) and organic matter (Soon and Abboud 1991).

Plant sample preparation

Rice grains collected at harvesting were washed with deionized water. After measuring the fresh weights, parts of the fresh samples were stored at − 80 °C and the others were oven dried at 40 °C to constant weight. Dried samples were weighed and ground to powder form, which were then collected in plastic bags and stored in a desiccator until analysis.

Plant sample analysis

For fresh plant materials, 150 mg of rice grains were taken and frozen in liquid nitrogen, followed by grinding with 0.1 M phosphate buffer (pH 6.8, containing 0.5 mM ascorbic acid and 1 mM phenylmethanesulfonyl fluoride), and then centrifuged at 12,000×g for 20 min at 4 °C. The supernatant was collected for measuring the enzymatic antioxidant activities [i.e. superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX)], malondialdehyde (MDA; an indicator of lipid peroxidation) and hydrogen peroxide (H₂O₂) contents according to the methods described previously (Uzilday et al. 2014).

Analysis of tocopherols, tocotrienols and γ-oryzanol

All dried plant samples were analyzed for Toc, T3 and GO contents using RP-HPLC (Huang and Ng 2011b). Briefly, the HPLC system consisted of a Hitachi L-2130 pump, and a Hitachi L-2485 fluorescence detector (Hitachi, Japan) set at an excitation wavelength of 290 nm and an emission wavelength of 330 nm. Chromatographic separation was performed by a normal phase Inertsil SIL 100A (5 µm, 4.6 × 250 mm) column coupled with an Inertsil SIL 100A (5 µm, 4 mm × 10 mm) guard column (GL Sciences Inc, Tokyo, Japan), and a mobile phase composed of hexane/isopropanol/ethylacetae/acetic acid (97.6:0.8:0.8:0.8, v/v/v/v) operating at room temperature. The flow rate of isocratic elution was 0.7 mL min⁻¹. A complete separation of all compounds was achieved within 30 min. All compounds were confirmed by chromatographic comparisons with their respective authentic standards. Toc, T3 and GO in rice samples were identified by retention time and quantified by the calibration curves of external standards.

Statistical analysis

All data were expressed as mean ± standard deviation (SD). Values were evaluated by one-way analysis of variance (ANOVA), followed by Duncan’s multiple range tests. Differences between treatments were considered significant when P value was < 0.05.

Results and discussion

Results showed that with the exception of pH, the contents of total nitrogen, Mehlich III P and K, and organic matter in soils were lower at harvesting than before planting (Table 1), and there was no difference in these measurements between nitrogen treated groups and the control. This observation suggests that in addition to have been absorbed by the rice plants, some of the applied nitrogen fertilizers may have lost via denitrification, and hence resulting in a lower total nitrogen concentration after planting (Fillery and Vlek 1982; Huang et al. 2016).

Table 1.

Effects of different nitrogen fertilization rates on the soil chemical properties and rice grain weight

Treatments	pH (w/v = 1:1)	EC (dS m−1)	TN (g kg−1)	Mehlich III P (mg kg−1)	Mehlich III K (mg kg−1)	OM (g kg−1)	Grains (DW; g plant−1)
Before planting
	5.3 ± 0.1ab	0.8 ± 0.1a	1.7 ± 0.1a	52.2 ± 0.2a	165 ± 4a	38.9 ± 0.2a
After harvesting
0 N	5.2 ± 0.1b	0.3 ± 0.1b	1.4 ± 0.1b	25.9 ± 6.8b	36.3 ± 1.4b	36.8 ± 0.9b	19.2 ± 3.6b
0.5 N	5.4 ± 0.1ab	0.3 ± 0.1b	1.5 ± 0.1b	29.9 ± 4.1b	37.6 ± 5.1b	36.0 ± 2.1b	25.3 ± 2.3ab
1 N	5.4 ± 0.2ab	0.3 ± 0.1b	1.4 ± 0.1b	31.6 ± 4.3b	37.8 ± 5.9b	37.0 ± 1.8ab	21.9 ± 5.6ab
2 N	5.5 ± 0.1a	0.3 ± 0.1b	1.5 ± 0.1b	29.8 ± 1.3b	35.5 ± 4.2b	36.1 ± 2.3b	31.1 ± 8.9a
3 N	5.5 ± 0.1a	0.3 ± 0.1b	1.4 ± 0.1b	30.1 ± 2.4b	36.8 ± 5.0b	36.2 ± 2.1b	30.9 ± 2.1a

Values are mean ± standard deviation (n = 4). Averages followed by the same letter in the same column are not significantly different as analyzed by Duncan’s multiple range tests at P < 0.05. EC = Electric conductivity; TN = Total nitrogen; OM = Organic matter; DW: Dried weight

Among the different treatments, it was noted that increasing rates of nitrogen fertilization were able to increase the dried weight of rice grains, and reached the highest at the 2 N treatment. Results also showed that the 2 N treatment exhibited significantly higher total vitamin E, total Toc and total T3 contents than that of the control group (Table 2). However, increasing rates of nitrogen fertilizer application caused a significant reduction in GO concentration. A strong negative correlation was noted between soil nitrogen fertility and GO content (r = − 0.8009), whereas the levels of total vitamin E (r = 0.5441), total Toc (r = 0.6719) and total T3 (r = 0.4556) had a moderate correlation with nitrogen fertilization.

Table 2.

Effects of different nitrogen fertilization rates on the contents of total vitamin E, total tocopherols (Toc), total tocotrienols (T3), γ-oryzanol and vitamin E analogs (α-, β-, γ-, and δ-Toc and T3) in rice grains

Treatments	Contents (mg kg−1, DW)
	Total vitamin E	Total Toc	Total T3	γ-Oryzanol
0 N	43.7 ± 2.6b	19.8 ± 1.4ab	23.9 ± 2.1b	161 ± 13a
0.5 N	43.1 ± 8.3b	20.0 ± 4.6ab	23.1 ± 3.9b	112 ± 15b
1 N	39.1 ± 0.9b	18.6 ± 1.5b	20.4 ± 1.8b	115 ± 12b
2 N	55.1 ± 4.2a	24.5 ± 2.5a	30.6 ± 1.6a	93 ± 4b
3 N	47.2 ± 0.9ab	22.2 ± 1.0ab	25.0 ± 1.7b	96 ± 10b
Correlation coefficient (r)	0.5441	0.6719	0.4556	− 0.8009

	α-Toc	β-Toc	γ-Toc	δ-Toc
0 N	15.22 ± 1.25a	0.89 ± 0.05bc	3.71 ± 0.31b	0.05 ± 0.01a
0.5 N	14.73 ± 3.88a	0.86 ± 0.24c	4.32 ± 1.30b	0.07 ± 0.01a
1 N	13.16 ± 1.52a	0.70 ± 0.17c	3.89 ± 1.50b	0.05 ± 0.01a
2 N	14.50 ± 1.82a	1.52 ± 0.34a	8.44 ± 1.03a	0.07 ± 0.01a
3 N	14.78 ± 3.00a	0.89 ± 0.05bc	6.08 ± 1.66b	0.05 ± 0.01a

	α-T3	β-T3	γ-T3	δ-T3
0 N	10.26 ± 0.42ab	0.14 ± 0.04a	12.58 ± 2.05b	0.94 ± 0.17a
0.5 N	10.56 ± 0.44ab	0.20 ± 0.25a	12.60 ± 2.05b	0.89 ± 0.18ab
1 N	9.14 ± 0.88b	ND	10.68 ± 2.21b	0.63 ± 0.06ab
2 N	10.58 ± 1.37ab	ND	18.37 ± 0.63a	0.60 ± 0.07b
3 N	11.15 ± 0.57a	ND	13.19 ± 2.52b	0.69 ± 0.23ab

Values are mean ± standard deviation (n = 4). Averages followed by the same letter in the same column are not significantly different as analyzed by Duncan’s multiple range tests at P < 0.05. ND: Not detected; DW: Dried weight

The levels of β-Toc, γ-Toc and γ-T3 in the 2 N treatment were significantly higher than other treatments, with the highest increase in γ-Toc (8.44 mg kg⁻¹) and γ-T3 (18.37 mg kg⁻¹) levels; however its δ-T3 content was the lowest among the different treatments.

Toc and T3 are essential components of biological membranes where they have both antioxidant and non-antioxidant functions, their principal role is to stabilize the membrane and to remove ROS and lipid peroxides. Water stress has been reported to result in substantial increase in α-Toc in the leaves of Rosmarinus officinalis (Munné-Bosch and Alegre 2000a), Melissa officinalis (Munné-Bosch and Alegre 2000b) and Fagus sylvatica (García-Plazaola and Becerril 2000). Falk and Munné-Bosch (2010) reported that T3 increased the tolerance of transgenic plants to high light stress at low temperatures, and was suggested to play a role as antioxidant in photosynthetic membranes (Matringe et al. 2008). In this study, T3 and GO were not detected in leaves and straws of rice, they were found to be only present in rice grains. This finding suggests that the presence of T3 and GO in rice grains is probably to fulfil specific functions that differ from the function of Toc in leaves.

In nitrogen fertilization studies, Fernández-Escobar and Sánchez-Zamora (2002) reported that in olive planting, increasing nitrogen fertilization rates can increase the Toc content in olive oil but its polyphenol content was reduced. Nitrogen deficiency was reported to cause the reduction of enzymes for Toc and T3 biosynthesis in rice plants at maturity, and hence resulted in lower Toc and T3 contents (Falk and Munné-Bosch 2010). Interestingly, when rice plants were treated with two-fold (2 N) higher nitrogen fertilizer than the recommended level, the rice grains were shown to have the highest Toc and T3 contents, as well as a significant increase in γ-Toc and γ-T3 levels; this suggests that a moderate increase in the soil nitrogen fertility can improve the contents of total Toc, total T3, γ-Toc and γ-T3 in rice grains.

An enhanced GO concentration was noted in rice grains of the 0 N treatment, which was significantly higher than the nitrogen fertilizer treated groups. These results suggest that nitrogen deficiency favor the GO biosynthesis as to counter the resulting increased oxidative stress incurred in rice grains.

Results showed that the SOD activity in rice grains was not affected by the different rates of nitrogen fertilization (Fig. 1). However, the increased use of nitrogen fertilizer significantly reduced the CAT and APX activities, and their activities in the 0 N treatment were higher than other treatments.

Fig. 1.

Effects of different nitrogen fertilization rates on the enzymatic antioxidant activities in rice grains. a SOD, b APX and c CAT. Error bars represent standard deviation (n = 4) and bars with the same letter are not significantly different as analyzed by Duncan’s multiple range tests at P < 0.05

Studies have demonstrated that when oxidative stress occurs, the increase O^·−₂ will be converted by SOD to H₂O₂, which will then degrade by CAT and APX into water; hence the concentration of H₂O₂ would first be affected in the case of oxidative stress (Blokhina et al. 2003). APX is the most important peroxidase in detoxifying H₂O₂; although CAT can also reduce H₂O₂ to water, it has a lower affinity for H₂O₂ than APX (Graham and Patterson 1982). The high CAT and APX activities in the 0 N treatment might be due to the high level of H₂O₂ stress in rice grains. A decline in the CAT and APX activities under excess nitrogen observed in this study suggests a possible increase in the antioxidant (i.e. Toc and T3) levels in removal of free radicals, and hence resulting in decreased levels of H₂O₂ and lipid peroxides.

Results showed that the MDA level in rice grains of 0 N and 0.5 N treatments was significantly higher than other treatments (Fig. 2), indicating that the grain MDA was higher under nitrogen deficiency or low N conditions. Similarly, the H₂O₂ level in rice grains of the 0 N treatment was higher than other treatments, with its level significantly higher than the 1 N treatment.

Fig. 2.

Effects of different nitrogen fertilization rates on the contents of MDA and H₂O₂ in rice grains. a MDA and b H₂O₂. Error bars represent standard deviation (n = 4) and bars with the same letter are not significantly different as analyzed by Duncan’s multiple range tests at P < 0.05

Increased ROS is the key factor in increasing the lipid peroxidation and MDA production. Under our experimental conditions, nitrogen deficiency and low nitrogen treatments have resulted in higher MDA and H₂O₂ levels in rice grains, suggesting that the lack of nitrogen fertilizer may affect the rate of antioxidant biosynthesis or have increased the expense of antioxidants in rice grains, hence resulting in the occurrence of oxidative stress phenomenon, and lower Toc and T3 contents. Under nitrogen deficiency (0 N treatment) conditions, the GO content in rice grains was significantly higher than the nitrogen-treated groups, this could explain the important role of GO in reducing the oxidative stress when Toc and T3 were insufficient to remove the ROS.

Conclusion

This study demonstrates that the levels of Toc and T3 peak at the 2 N treatment and then significantly declined at the 3 N treatment in rice grains, whereas the GO content was shown to decrease with increasing nitrogen fertilization. Nitrogen deficiency has the highest GO, MDA and H₂O₂ contents, and CAT and APX activities in rice grains. These results suggest that a two-fold increase in the soil nitrogen fertility could promote the accumulation of Toc and T3 in rice grains, while the nitrogen deficiency environment was more favorable for the GO biosynthesis.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.