Sunitha Sangnak
Jeeraporn Veeradittakit1
Chanakan Prom-u-thai- 1Department of Plant and Soil Sciences, Faculty of Agriculture, Chiang Mai University, Chiang Mai, Thailand
- 2Lanna Rice Research Center, Chiang Mai University, Chiang Mai, Thailand
Increasing grain protein in rice through fertilizer management during cultivation is a promising approach to address protein deficiency, particularly in populations where rice is the staple food and access to high-protein foods is limited. This study aimed to compare the impacts of fertilizer management (control, N, S, and Zn) on grain yield, protein content, and antioxidant capacity among four rice varieties (KDML105, PTT1, KDK, and K4) under field condition for four replicates. The rice varieties responded differently to fertilizer management in terms of grain yield, protein content, and antioxidant capacity. Applying all fertilizers increased grain yield in all varieties compared with the control, although the magnitude of increase varied among varieties. The highest grain protein content in unpolished rice was observed with N fertilizer application across all varieties, while S and Zn had a smaller effect. A similar trend was found in polished rice at both 20 and 60 s of polishing, suggesting increased protein content in both the outer and inner grain layers. Fertilizer treatments increased grain nutrient concentrations but tended to have a negative impact on grain yield. Antioxidant capacity in both unpolished and polished grains was also influenced by fertilizer applications and showed a positive correlation with grain protein content, indicating a potential dual benefit for human health through improved protein and antioxidant intake. These results provide essential insights for optimizing fertilizer management to enhance both yield and high nutritional values in rice production systems.
Introduction
Rice (Oryza sativa) is a staple food crop grown and consumed by a large portion of the world’s population and plays a vital role in global food security, as well as economic and social stability, particularly in Thailand, where rice is both a primary food and a major export. In addition to being a major carbohydrate source, rice can also serve as an important plant-based protein source (Juliano and Tuaño, 2019). Rice protein is the most popular among cereal proteins due to its amino acid profile and its relatively high lysine content compared to other cereal grains. Lysine is an essential amino acid that is present in limited amounts in other plant-based foods (Krishnan et al., 2021). Therefore, increasing rice grain protein content is a significant option for adding nutritional value to rice and addressing protein deficiency among populations that rely on rice as a staple and have limited access to other dietary protein sources.
Protein has essential nutritional value for human health, but a large portion of the world’s population suffers from protein deficiency, which adversely affects their health (Black et al., 2008). This problem is especially prevalent in developing countries, where access to animal protein sources is limited. Therefore, alternative protein sources are necessary to meet consumer demands. Plant-based proteins are important alternatives that are more accessible to low-income populations and require fewer production resources, resulting in lower greenhouse gas emissions compared to animal protein production (Nasrabadi et al., 2021). Consequently, plant proteins including rice grains are gaining increasing attention from health-conscious and environmentally concerned consumers.
High-protein rice varieties, such as the tetraploid lines GD2-4x and GD4-4x, have been recently developed to combat malnutrition (Zhan et al., 2024). These lines demonstrate a substantial increase in total protein content by 40.27% and 35.15% higher, respectively, than control varieties and contain elevated levels of essential amino acids, especially glutelin, the primary rice protein and associated with increasing aleurone layer thickness and altered amyloplast morphology. In addition to breeding strategies, agronomic management through fertilizer application is an effective approach to enhance grain protein in rice. High N fertilization (200 kg/ha) can increase protein content by 37.2%–65.8%, particularly in the outer grain layers, compared to low N conditions (Shi et al., 2022; Ling et al., 2025). Additionally, S fertilization has been shown to enhance rice grain protein content, primarily because S plays a key role in synthesizing S-containing amino acids, which are essential components of protein (Okuda et al., 2009; Chaiboontha et al., 2025). Zinc fertilization, especially when combined with N can effectively enhance protein content in rice grains (Chandel et al., 2010; Soltani et al., 2023). Due to the strong relationship between these fertilizers and the protein synthesis process, previous studies have indicated that N, S, and Zn fertilization can enhance both grain yield and protein content in rice cultivation, although some side effects on milling and eating quality have not been reported. Therefore, it is particularly interesting to compare the effects of each fertilizer application on yield and grain protein accumulation and its impact during milling process to produce polished rice among different rice varieties, which are expected to respond differently to various nutrient managements. This experiment aims to evaluate the effects of N, S, and Zn fertilizer management on grain yield, protein content and its relation to antioxidant capacity in unpolished and polished rice among modern improved varieties (PTT1 and K4) and locally improved varieties (KDML105 and KDK). The results from this experiment can be used for specific nutrient management strategies in different rice varieties to enhance yield and grain protein content, thereby increasing rice value for farmers and maximizing nutritional benefits for consumers.
Materials and methods
Experiment design
The experiment was conducted under field conditions at Agronomy Research Station, Faculty of Agriculture, Chiang Mai University, Thailand. The experimental design was a split-plot with four replications. The main plots consisted of four soil fertilizer management treatments: (1) no fertilizer application (control), (2) additional application of N fertilizer (urea) at a rate of 30 kg N/ha (a total of 135 kg N/ha), N fertilizer is commonly applied by farmer practice at 60–100 kg N/ha, (3) application of Zn fertilizer (ZnO) at a rate of 40 kg Zn/ha (Phattarakul et al., 2012), and (4) application of S fertilizer (NaSO4) at 30 kg S/ha (Shivay et al., 2014; Chaiboontha et al., 2025). All fertilizers (except for the control) were applied in two equal splits: at the basal and flowering stage. The subplots consisted of four rice varieties: two traditional improved varieties with photoperiod sensitivity (Khao Dawk Mali 105; KDML105, a popular jasmine rice and Kam Doi Saket; KDK, a purple pericarp color rice) and two modern improved varieties with photoperiod insensitivity (Pathum Thani 1; PTT1) and CMU K4; K4). The initial grain protein concentration among the four rice varieties was 7.8%, 9.7%, 8.5%, and 11.5%, respectively. Soil samples were collected from the plots before the experiment was conducted by randomly sampled at 0–30 cm depth and analyzed soil properties. The properties of soil were in the following: 5.47 pH (1:2 (w/v)), 1.13% organic matter, 0.33 ds/m electrical conductivity, 4.81 cmol (+)/kg cation exchange capacity, 0.06% total N, 40.90 mg/kg available P, 30.60 mg/kg exchangeable K, 16.13 mg/kg extractable S, 3.78 mg/kg available Zn.
Crop cultivation and sample collection
Rice was cultivated during the rainy season from June to November 2023. Seedlings were prepared on 29th June by sowing rice seeds in the prepared seedbeds until almost 30 days old. Seedlings were transplanted into the plot experiment on 27th July, at 1.5 x 2.7 m for each subplot using single seedlings per hill at a 25 × 25 cm spacing. Fertilizers were applied in three splits: as a basal application, and at the tillering and flowering stages, following the standard practices of farmers in this area. For each application, 105 kg N/ha, 18.75 kg P2O5/ha, and 18.75 kg K2O/ha were applied equally. Plants were grown under flooded conditions, maintaining a water level about 10 cm above the soil surface, and drained at 14 days before harvesting.
At maturity, yield samples were randomly collected from a 1x2 m in the center of each plot to determine yield and yield components on 24th November. Seed samples of all treatments were air-dried until the moisture content was reached at approximately 14% for grain yield recorded. The leave samples were randomly collected and rinsed thoroughly with deionized water for nutrient concentration analysis. The number of tiller and panicle per plant, number of spikelets per panicle, number of filled grain, and 1000 grain weight were randomly evaluated in each replication. The percent filled grain was calculated from the number of filled grains and unfilled grains in each panicle. Rice straw was sampled and dried in a hot oven (100 °C for 76 h) for constant dry weight to determine straw dry weight.
Samples preparation and chemical analysis
About 100 g of paddy rice grains were dehulled using laboratory husking machine (Model P-1, Ngek Seng Huat Company, Thailand) to obtain the unpolished rice samples. Then, about 50 g sample of unpolished rice was polished using a laboratory polishing machine (Model K-1, Ngek Seng Huat Company, Thailand) for two different polishing durations at 20 and 60 s to yield polished rice. The grain and leave samples were freeze-dried and then ground and kept in -20°C until analysis.
Protein analysis, the samples were analyzed using the combustion method with an FP828 Series Combustion Analyzer. The samples were weighed in foil cups and then combusted at high temperatures under an oxygen flow. The N content obtained was multiplied by a factor of 5.95 (Amagliani et al., 2017) to calculate protein concentration. The percentage of yield loss was determined by the deduction of protein content in unpolished rice by polished rice grains.
For S analysis, the dried and ground samples were sieved through a 40-mesh using a Macro-Wiley Mill. A 0.5 g subsample was used to analyze S content by digestion with a di-acid mixture [perchloric acid (HClO4) and nitric acid (HNO3) in a 3:10 ratio] (Shivay et al., 2014). Zinc analysis, a 0.5 g of dried and ground samples was placed in a muffle furnace and ashed at 535 °C for 8 h. The ash was digested in a 1:1 hydrochloric acid (HCl:deionized water) solution. The digested sample was then filtered through filter paper, and the filtrate was diluted (Allan, 1961) and determined for Zn concentration using an atomic absorption spectrophotometer (AAS).
For total phenolic compounds analysis, 0.5 g samples were placed in a 50 mL plastic tube and extracted with 10 mL of 50% methanol. The mixture was shaken at 2500–3000 rpm for 60 minutes, then centrifuged at 4000 rpm for 10 minutes. The supernatant was filtered through No. 1 filter paper into a 50 mL test tube. The extraction was repeated twice with the same sample. A 1 mL aliquot of the sample extract and standard solution was added to a 10 mL test tube, followed by 0.5 mL of folin–ciocalteu reagent, 1.5 mL of sodium carbonate (Na2CO3), and 7 mL of deionized water. The mixture was shaken and left to stand for 25 m. Absorbance was measured at 760 nm using a UV-Vis spectrophotometer. For DPPH activity analysis, a 0.1 g sample was placed in a 25 mL plastic tube and extracted with 10 mL of methanol. The mixture was shaken at 2500–3000 rpm for 30 minutes, then centrifuged at 70 rpm for 5 minutes. The supernatant was filtered through a 0.45-micron nylon membrane. A 0.3 mL aliquot of the extract and standard solution was transferred to a 10 mL test tube, followed by the addition of 0.5 mL of 0.1 mM 2,2-diphenyl-1-picrylhydrazyl (DPPH) solution. The mixture was shaken and left in the dark for 20 minutes. The absorbance of the sample and blank was then measured at 517 nm using a UV-Vis spectrophotometer (Yue and Xu, 2008).
Data analysis
Data were tested for normality, and data that were not normally distributed were transformed using the rank-based inverse normal transformation before being subjected to analysis of variance (ANOVA) using Statistix for Windows, version 10. Means were compared using the Least Significant Difference (LSD) test at a 95% confidence level. Relationships between pairs of variables were analyzed by calculating the coefficient of correlation.
Results
Grain yield and yield component
A significant interaction was observed between N, S, and Zn fertilizer management and rice varieties on grain yield (p < 0.05) (Figure 1). Applying all fertilizers in all varieties increased grain yield from 18.5% to 41.7% compared with the control. For the KDML 105, the yield without fertilizer was 4.8 tons/ha, application of Zn fertilizer resulted in the highest yield of 7.2 tons/ha, 33.3% increase followed by N and S fertilizers, which did not differ significantly and had an average yield of 6.5 tons/ha, 9.7% increase from the control. For the PTT 1, S fertilizer gave the highest yield at 7.8 tons/ha, while Zn and N fertilizers yielded similarly, with an average of 6.7 tons/ha, both higher than control treatment (5.0 tons/ha) for 35.9% and 25.4%, respectively. For KDK, applying N, S, and Zn fertilizers produced no significant differences in yield, with an average of 5.1 tons/ha, 23.5% increase higher than the control treatment (3.9 tons/ha). For K4, yields were also not significantly different between N, S, and Zn fertilizer treatments (average 3.6 tons/ha), 25.0% increase higher than the control (2.7 tons/ha). The straw dry weight was also increased when applied all fertilizers in all varieties, but each variety responded differently for each fertilizer (p < 0.05) (Figure 1). The interaction effect between rice variety and fertilizer management was also observed in yield component e.g., number of tillers per plant, number of panicles per plant, percentage of filled grain, and 1000 grain weight (data not shown). Regardless of variety effect, there was the positive correlation between grain yield and number of panicles per plant under S (r= 0.54, p < 0.05) and Zn (r= 0.54, p < 0.05) fertilizer management as well as the relationship between yield and 1000 grain weight under N fertilizer management (r= 0.56, p < 0.05) (data not shown).
Figure 1. Grain yield and straw dry weight of KDML 105, PTT 1, KDK, and K4 rice varieties grown under N, S, and Zn fertilizer management compared to no fertilizer application (control). Lowercase letters indicate significant differences among rice varieties and fertilizer management treatments at p < 0.05, VxTrt indicates variety x treatment interaction. **, *** indicate significant difference at p < 0.01 and 0.001, respectively.
Grain protein concentration and its loss during polishing process
The appearance of unpolished and polished rice grains at 20 and 60 s was shown in Figure 2a). A slight removal of the bran fraction from the outer grain layers was observed after 20 s of polishing, with a greater removal occurring after 60 s of polishing. The significant interaction effects between N, S and Zn treatments and rice varieties on grain protein content were observed in the unpolished and polished rice at both 20 and 60 s (p < 0.05) (Figure 2b). Applying N fertilizer resulted in the highest increase in grain protein content across all varieties in unpolished and polished rice compared with the control, while the other fertilizer applications had a smaller effect. However, the degree of increase to each fertilizer treatment (N, S, Zn) among varieties varied from 7.4% to 16.7% in the unpolished rice, from 11.2% to 15.1% in the polished rice for 20 s and from 7.9% to 13.9% in the polished rice for 60 s compared with the control. The differing protein content across treatments for each variety, for example, the application of N significantly increased protein content in K4 in both unpolished (9.2% increase) and polished rice at 20 s (6.0% increase) and 60 s (5.8% increase) compared with the control, while other varieties such as KDM105 showed less pronounced response. Similarly, the S treatment significantly enhanced protein content in some varieties (e.g., KDK), but this effect was not observed in others.
Figure 2. Appearance of rice grain (a) and grain protein content in unpolished rice, polished rice at 20 and 60 s of KDML 105, PTT 1, KDK, and K4 rice varieties grown under N, S, and Zn fertilizer management compared to no fertilizer application (control) (b). Lowercase letters indicate significant differences among rice varieties and fertilizer management treatments at p < 0.05, VxTrt indicates variety x treatment interaction. *, **, *** indicate significant difference at p < 0.05, 0.01 and 0.001, respectively.
On the other hand, management of N, S, and Zn fertilizers affected the loss of grain protein content differently among rice varieties (p < 0.05) (Figure 3). A longer polishing period of 60 seconds (17.1 – 33.2%) resulted in a higher percentage of protein loss than a 20 s (5.9 – 16.2%) polishing periods for all varieties and fertilizer managements, with the magnitude of loss varying depending on the variety and fertilizer applied. For example, after 20 s of polishing, the highest protein loss in K4 (12.9% loss) was observed when S fertilizer was applied. However, this loss decreased to 7.9% when Zn fertilizer was applied. This pattern differed from that observed in PTT1, where the highest loss occurred with Zn fertilizer (13.3 – 25.5%), and the lowest losses were seen under the no additional fertilizer and S fertilizer applications (6.1 – 20.8%). The loss of protein in polished rice was consistent between polishing at 20 and 60 seconds, as indicated by a significant positive correlation between protein loss at these two polishing durations (p < 0.05, r = 0.80) (Figure 3).
Figure 3. Loss of grain protein (%) under polishing process for 20 and 60 s and the relationship between protein loss at 20 and 60 s polishing (n=16) of KDML 105, PTT 1, KDK, and K4 rice varieties grown under N, S, and Zn fertilizer management compared to no fertilizer application (control). Lowercase letters indicate significant differences among rice varieties and fertilizer management treatments at p < 0.05, VxTrt indicates variety x treatment interaction. **, *** indicate significant difference or correlation between each pair of parameters at p < 0.01 and 0.001, respectively.
Leave and grain nutrient concentration
Fertilization with N, S, and Zn notably increased leaves, unpolished and polished rice grain nutrient concentration differently among rice varieties (p < 0.05) (Figure 4). The concentration of N in both leaves and unpolished rice grain significantly increased in response to all treatments compared to the control, especially the effect was most pronounced with the N treatment, increase varying from 16.6 – 17.6% and 9.0 - 16.7%, respectively (Figures 4a, b). The highest increase was observed on K4, while KDML105 consistently showed the lowest N concentration across all treatments. The concentration of S in leaves and unpolished rice grain was similarly affected by variety and fertilizer treatment (p < 0.05). The S fertilizer treatment significantly enhanced tissues S concentration, particularly in KDML105, increased 15.4% from the control, but KDK and K4 were not responded to S fertilization, whereas the negative response was found in PTT1 (Figures 4a, b). Both leaf and unpolished rice Zn concentrations increased significantly under the Zn fertilization treatment in most varieties, while less or no effect were observed for the other fertilizer treatments (p < 0.05) (Figures 4a, b). In leave tissues, the highest Zn concentration was found by 39.2 mg/kg in PTT1 (20.9% increase) and 37.3 mg/kg in KDML105 (25.4% increase), followed by 32.3 mg/kg in K4 (33.1% increase) compared with the control, while no effect was found in KDK when Zn fertilizer was applied. In unpolished rice, the highest Zn concentration was found by 47.2 mg/kg in K4 (8.7% increase), followed by 39.6 mg/kg in PTT1 and KDK (8.3% increase), but no difference was found in KDML105. For the polished rice grains at both 20 s and 60 s, the lower grain S and Zn concentrations were observed compared to the unpolished rice grains for all treatments and varieties with varying degrees of response among the varieties (p < 0.05) (Figures 4c, d). For the polished rice grains at 20 s, the highest grain S concentration was found at 0.25% when applied S fertilizer in KDK compared with the control, while it was not observed for the other fertilizer applications. Applying S and Zn fertilizers in K4 resulted in a slightly higher grain S concentration compared to the control and N application, whereas no treatment effect was observed in KDML105 and PTT1. Grain Zn concentration was the highest at in K4 when applied with N and Zn fertilizer compared with the control and S fertilizer application. The effect was similar with the other three varieties with different magnitude of responses. For 60 s polished grains, grain S and Zn concentration was slightly affected by treatment fertilizers in all varieties, excepted in KDML105 for grain S concentration and K4 for grain Zn concentration.
Figure 4. Leave (a), unpolished rice (b), polished rice for 20 s (c), and polished rice for 60 s (d) nutrient concentration of KDML 105, PTT 1, KDK, and K4 rice varieties grown under N, S, and Zn fertilizer management compared to no fertilizer application (control). Lowercase letters indicate significant differences among rice varieties and fertilizer management treatments at p < 0.05, VxTrt indicates variety x treatment interaction. **, *** indicate significant difference at p < 0.01 and 0.001, respectively.
There was a significant correlation between grain yield and nutrient concentrations in both the leaves and unpolished grain for four rice varieties grown under four fertilizer managements (Figure 5). Positive correlations were observed in both leaves and unpolished grain for all four rice varieties under S fertilizer (p < 0.05, r = 0.55 and p < 0.05, r = 0.76, respectively) and Zn managements (p < 0.05, r = 0.52 and p < 0.05, r = 0.70, respectively). In contrast, a negative correlation was found between grain yield and N concentration (p < 0.05, r = -0.62 and p < 0.05, r = -0.74, respectively).
Figure 5. The relationship between grain yield and concentration of N, S and Zn in tissue of leave and unpolished rice grain of KDML 105, PTT 1, KDK, and K4 rice varieties grown under N, S, and Zn fertilizer management compared to no fertilizer application (control) (n=16). *, **, *** indicate significant correlation between each pair of parameters at p < 0.05, 0.01 and 0.001, respectively.
Grain total phenol and antioxidant capacity
The significant interaction effects were observed between variety and fertilizer management on both phenol content and DPPH radical scavenging activity across unpolished and polished rice grain at 20 and 60 s (p < 0.05) (Figure 6). The application of S and Zn fertilizers produced higher phenol contents than control or N fertilizer treatments, especially for KDK and K4 varieties in all grain forms. Among varieties, K4 consistently exhibited the highest phenol levels under all fertilizer treatments, 8.4% increase from the control, and KDK found the highest concentration, 15.3% increase when N and S were applied. KDML105 and PTT1 had the lowest phenol concentration in the unpolished rice and after prolonged polishing. Prolonged polishing for both 20 s and 60 s drastically reduced phenol concentration in all varieties and fertilizer treatments, but variety and fertilizer differences were still apparent.
Figure 6. Total phenol and DPPH activity in unpolished and polished rice grains at 20 s and 60 s polishing time of KDML 105, PTT 1, KDK, and K4 rice varieties grown under N, S, and Zn fertilizer management compared to no fertilizer application (control). Lowercase letters indicate significant differences among rice varieties and fertilizer management treatments at p < 0.05, VxTrt indicates variety x treatment interaction. **, *** indicate significant difference at p < 0.01 and 0.001, respectively.
DPPH radical scavenging activity, indicative of antioxidant capacity, mirrors the patterns observed in phenol content, being highest in less-polished grains and particularly enhanced by S and Zn fertilizers. K4 and KDK under S and Zn fertilizer treatments displayed the highest DPPH activities across all degrees of polishing. KDML105 and PTT1 consistently showed significantly lower DPPH activity regardless of fertilizer or polishing duration. Increased polishing time (60 s) substantially diminishes antioxidant activity in all varieties and treatments.
The correlation was found between grain protein and total phenol in the unpolished rice (p < 0.05, r = 0.82), polished rice at 20 s (p < 0.05, r = 0.95) and 60 s (p < 0.05, r = 0.91). Additionally, there was also correlation between grain protein and DPPH activity in the unpolished rice (p < 0.05, r = 0.79), polished rice at 20 s (p < 0.05, r = 0.96) and 60 s (p < 0.05, r = 0.93) among four rice varieties grown under different fertilizer managements (Figure 7).
Figure 7. The relationship between grain protein concentration and phenol and antioxidant capacity determined by DPPH activity in unpolished and polished rice grains at 20 and 60 s of KDML 105, PTT 1, KDK, and K4 rice varieties grown under N, S, and Zn fertilizer management compared to no fertilizer application (control) (n=16). *** indicates significant correlation between each pair of parameters at p < 0.001.
Discussion
The present study has compared the effects of fertilizer management (N, S, and Zn) on grain yield, protein content, and antioxidant capacity among different rice varieties, and demonstrated that fertilizer management influenced grain yield and quality differently across the varieties. Application of all fertilizer treatments resulted in increased grain yield across all rice varieties compared with the control. For example, in KDML105 and PTT1, the highest grain yields were observed with Zn and S fertilizer applications, respectively, while other fertilizers had less effect. In contrast, in KDK and K4, the effects of the different fertilizers on grain yield were similar. These results further confirm that different rice varieties respond variably to fertilizer managements. The first group of rice varieties (KDML105 and PTT1) consists of non-pigmented varieties, while the second group (KDK and K4) comprises pigmented varieties that are genetically related within their group. This genetic relatedness may explain why similar responses to fertilizer management on productivity were observed among rice varieties within the same group. Nitrogen plays a crucial role in enhancing grain yield in rice through several mechanisms. Applying controlled-release N fertilizer enhanced grain filling in rice by increasing the sugar–spikelet ratio, resulting in higher grain yield (Liu et al., 2025). Additionally, optimizing N fertilizer improved the emergence rate of secondary tillers, while increasing the number of primary tillers and overall biomass, and promoted panicle development (Khampuang et al., 2021; Zhou et al., 2022). This study confirmed that N fertilization particularly enhanced grain filling and contributed to higher yields, as indicated by the significant correlation between grain yield and 1000-grain weight under N fertilizer management (r = 0.56, p < 0.05). On the other hand, sulfur (S) is essential for plant growth and development (Sharma et al., 2024). It improves rice yield by enhancing the availability and uptake of essential nutrients, as well as by increasing N use efficiency (Bates, 2025; Slameto Soeparjono et al., 2025; Zhao et al., 2025). Similarly, Zn played a crucial role in improving rice yield by enhancing yield components such as panicle number, spikelets per panicle, seed-setting rate, and 1000-grain weight (Zhang et al., 2025; Nandy et al., 2023). Thus, applying all fertilizers improved soil nutrient availability for plant uptake during development until harvest with the interaction effect between rice variety and fertilizer management. However, soil and plant samples during plant growth and development were not analyzed in this study, which should be addressed in future research to further clarify the mechanisms by how the fertilizers enhance productivity.
On the other hand, among the candidate fertilizers tested in this study for increasing grain protein in rice, N emerged as the most effective. Application of N fertilizer led to increased grain protein content across all rice varieties with the greatest effect observed in the K4 variety, the highest initial grain protein content compared with the other varieties. This makes N fertilizer a strong candidate for nutrition-focused breeding or agronomic programs, especially among the varieties with initially high grain protein content. It is well documented that N plays an essential role in key enzymes involved in protein synthesis, such as glutamine synthetase, glutamate synthase, and glutamic pyruvic transaminase which are crucial for the assimilation and remobilization of N used for protein synthesis in rice grain (Fei et al., 2023; Wang et al., 2021). A previous study reported that applying an optimum N rate of 180 kg N/ha improved yield, agronomic traits, and grain protein content in buckwheat, but these benefits decreased with higher rates of N application depending on specific crop, variety and growing condition (An et al., 2024; Wan et al., 2023). This confirmed that the appropriate rate of N application enhances grain protein content depending on cropping condition. On the other hand, the increased grain protein content can negatively impact the eating quality of rice by inhibiting starch gelatinization, leading to reduced peak viscosity and poorer taste quality (Shi et al., 2023, 2022). Therefore, N is a promising fertilizer for enhancing grain protein accumulation in rice, particularly in varieties with a high potential for protein accumulation, but it is important to consider the potential impact on eating quality when producing high-protein rice. On the other hand, S and Zn fertilizers had a smaller effect on grain protein content overall, but they did promote grain protein accumulation in certain rice varieties, such as K4. Sulfur is a critical component of certain amino acids, such as cysteine and methionine, which are integral to protein synthesis directly linked to the overall protein content (Okuda et al., 2009). Its application has been shown to increase the methionine content in rice grains, which is a S-containing amino acid essential for protein synthesis (Tyagi et al., 2017). A recent study confirmed that applying S fertilizer together with N has improved grain protein in wheat depending on soil management and texture (Castellari et al., 2023; Roa et al., 2024). Additionally, applying S fertilizer together with Zn was also reported to improved yield and grain protein content in rice (Khampuang et al., 2023). Therefore, S fertilization is a promising management strategy to improve grain protein content in rice, especially when combined with other fertilizers with appropriate soil condition. On the other hand, Zn acts as a cofactor for numerous enzymes involved in protein synthesis and metabolism (Tyagi et al., 2017), but the effect of Zn application on grain protein content has been rarely reported. Previous studies have mentioned that Zn fertilizer application increases grain protein content, particularly in Zn-deficient soils, by improving the plant’s ability to synthesize proteins and enzymes involved in N metabolism. However, the effect depends on plant species, soil fertility, and the Zn application rate, as excessive Zn can lead to a decrease in grain protein content (Liu et al., 2015; Ram et al., 2024). However, future research is required to explore intensive fertilizer management strategies to further enhance grain protein content in rice for use in health food products. For example, synergistic interactions between fertilizer types and application methods such as combining N, Zn and S, as well as using both soil and foliar applications at appropriate growth stages may be beneficial. In addition, this study confirmed that the increase in grain protein resulting from fertilizer management, especially N fertilizer was not limited to the outer grain layers, but also extended to the inner layers, as indicated by the grain protein content measured after 20 and 60 seconds of polishing. A greater loss of grain protein was observed with longer polishing periods across all rice varieties and fertilizer management treatments. Crucially, our results highlight the differential impact of S and Zn fertilizer applications. In particular, S application in K4 sharply increased protein loss after polishing, while Zn supplementation minimized these effects, suggesting that Zn may enhance protein retention in grain tissues less susceptible to removal. In contrast, PTT1 responded to Zn fertilization with greater protein loss, emphasizing possible genotype-dependent mechanisms influencing nutrient localization within the grain layers. Mechanistically, these effects may arise from the roles of S and Zn in protein synthesis and stabilization, where their allocation to the outer aleurone layers renders grain protein more vulnerable to loss during polishing (Okuda et al., 2009; Tyagi et al., 2017). The significant positive correlation of protein loss between 20 and 60 s underscores the consistency of nutrient depletion patterns across different varieties and management regimes. However, the degree of nutrient loss during the polishing process can also be influenced by grain characteristics, such as grain shape, pericarp thickness, and grain hardness, as well as by the type of machine used to polish the rice, including its speed and pressure. These factors should be carefully considered when assessing nutrient loss based on the degree of polishing.
This study reveals that fertilizer management not only influences grain protein content and yield but also affects the antioxidant capacity of rice grains, with notable differences among the four rice varieties. The application of N, S, and Zn led to increases in antioxidant capacity in both unpolished and polished rice grains, though the response magnitude varied among varieties. Notably, varieties that exhibited higher grain protein content under N fertilization also tended to show enhanced antioxidant capacity, as reflected by the significant positive correlation observed between grain protein content and total phenol and DPPH activity. Previous studies have reported that the accumulation of grain protein in the outer bran layers is associated with antioxidant properties in rice grains, such as the presence of bioactive peptides and the activation of antioxidant pathways (Xiong et al., 2023; Pang et al., 2018; Goufo and Trindade, 2014). Additionally, it was mentioned that the relationship between grain protein and antioxidant capacity is complex and can vary by grain type, but proteins themselves contribute to antioxidant activity through various mechanisms, including scavenging free radicals and chelating metals (Abeynayake et al., 2022). However, amino acid composition and the presence of other compounds such as phenolic compounds can also play a significant role, while a negative correlation between protein and phenolic content also has been observed in some cases (Jimenez-Pulido et al., 2022). This association suggests that protein-rich grains may likely contain higher levels of antioxidant peptides or are accompanied by other bioactive constituents concentrated within the protein fractions. These findings imply that strategic fertilizer management can be used to simultaneously improve both the nutritional and functional (health-promoting) qualities of rice grains. Additionally, the positive relationship between protein and antioxidant capacity may offers promising potential for breeding and agronomic practices aiming to maximize the health benefits of rice consumption, particularly in populations reliant on rice as a staple food.
Conclusion
This study demonstrates that fertilizer management with N, S, and Zn significantly influences grain yield, protein content, and antioxidant capacity across different rice varieties, with notable varietal-specific responses. Nitrogen was especially effective in boosting grain protein content, although its impact on eating quality merits consideration, while S and Zn showed genotype-dependent effects on protein accumulation and retention during polishing. Additionally, the positive correlation between grain protein and antioxidant capacity highlights the potential for integrated management strategies to improve both the nutritional and functional qualities of rice. These findings underscore the importance of tailoring fertilizer practices to specific rice varieties to maximize both yield and health-promoting traits, providing valuable guidance for future breeding and agronomic programs.
Data availability statement
The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.
Author contributions
SS: Formal analysis, Investigation, Methodology, Writing – original draft. JV: Formal analysis, Methodology, Resources, Validation, Writing – original draft. CP-U-T: Conceptualization, Funding acquisition, Project administration, Supervision, Validation, Visualization, Writing – review & editing.
Funding
The author(s) declared that financial support was received for work and/or its publication. This research was funded by Chiang Mai University.
Acknowledgments
The authors would like to thank all assistance for field experiment from staffs at Agronomy Division, Faculty of Agriculture, Chiang Mai University, Thailand.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Keywords: high nutrition rice, nutrient uptake, plant base protein, protein deficiency, rice cultivation
Citation: Sangnak S, Veeradittakit J and Prom-u-thai C (2026) Comparative impacts of nitrogen, sulfur, and zinc fertilization on grain yield, protein content, antioxidant capacity and its loss during polishing process among rice varieties. Front. Agron. 7:1725441. doi: 10.3389/fagro.2025.1725441
Received: 15 October 2025; Accepted: 17 December 2025; Revised: 28 November 2025;
Published: 12 January 2026.
Edited by:
Satish Kumar Singh, Banaras Hindu University, IndiaReviewed by:
Cai Qiuliang, Baise University, ChinaShahram Mahmoud Soltani, Rice Research Institute of Iran, Iran
Copyright © 2026 Sangnak, Veeradittakit and Prom-u-thai. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Chanakan Prom-u-thai, Y2hhbmFrYW4ucEBjbXUuYWMudGg=