Kankunlanach KHAMPUANG, Nanthana CHAIWONG, Atilla YAZICI, Baris DEMIRER,Ismail CAKMAK, Chanakan PROM-U-THAI,4

(1Agronomy Division, Department of Plant and Soil Sciences, Faculty of Agriculture, Chiang Mai University, Chiang Mai 50200,Thailand;2Field and Renewable Energy Crops Research Institute, Department of Agriculture, Bangkok 10900, Thailand; 3Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul 34956, Turkey; 4Lanna Rice Research Center, Chiang Mai University, Chiang Mai 50200, Thailand)

Abstract: This study aimed to investigate the responses in rice (Oryza sativa cv. Osmancik 97)production and grain zinc (Zn) accumulation to combined Zn and sulfur (S) fertilization. The experiment was designed as a factorial experiment with two Zn and three S concentrations applied to the soil in a completely randomized design with four replications. The plants were grown under greenhouse conditions at low (0.25 mg/kg) and adequate (5 mg/kg) Zn rates combined with S (CaSO4·2H2O)application (low, 2.5 mg/kg; moderate, 10 mg/kg, and adequate, 50 mg/kg). The lowest rate of S at adequate soil Zn treatment increased grain yield by 68% compared with the same S rate at low Zn supply.Plants with the adequate S rate at low Zn and adequate Zn supply produced the highest grain yield, with increases of 247% and 143% compared with low S rate at low Zn and adequate Zn supply, respectively.The concentration of grain Zn and S responded differently to the applied S rates depending on the soil Zn condition. The highest grain Zn concentration, reaching 41.5 mg/kg, was observed when adequate Zn was supplied at the low S rate. Conversely, the adequate S rate at the low soil Zn conditions yielded the highest grain S concentration. The total grain Zn uptake per plant showed particular increases in grain Zn yield when adequate S rates were applied, showing increases of 208% and 111% compared with low S rate under low and adequate soil Zn conditions, respectively. The results indicated that the synergistic application of soil Zn and S improves grain production and grain Zn yield. These results highlight the importance of total grain Zn yield in addition to grain Zn concentration, especially under the growth conditions where grain yield shows particular increases as grain Zn is diluted due to increased grain yield by increasing S fertilization.

Key words: grain; rice; sulfur; zinc deficiency

Rice is the vital staple food crop for the world’s population, especially in Asia (Singh et al, 2018), but rice grains contain very low Zn content (Prom-U-Thai et al, 2020; Utasee et al, 2022). Brown rice contains only 19.1 mg/kg of Zn on average, compared with an average of 28.0 mg/kg Zn in wheat (Ram et al, 2016).Today, Zn deficiency is highly prevalent in the world,especially among rice consumers, which causes severe consequences in human health, including impairments in physical growth, brain function, and immune response to viral and bacterial attacks (Read et al,2019; Li et al, 2022). Therefore, improving grain Zn concentration in rice grains is of great importance for human nutrition.

Rice is generally cultivated in flooded soils containing low levels of plant-available Zn due to low redox potential, and consequently, grain Zn concentrations are further reduced under such soil conditions(Phattarakul et al, 2012). Zn deficiency may occur 2-3 weeks after transplanting, and in severe cases it can lead to a rapid hart in plant growth, especially in calcareous soils with high pH and bicarbonate levels due to the formation of Zn(OH)2and ZnCO3(Johnson-Beebout et al, 2009; Nadeem and Farooq,2019). Zn deficiency in rice is also an important yield-reducing factor due to the high sensitivity of rice to low Zn levels in soils (Alloway, 2008). Zn is known to play crucial roles in various physiological processes, which primarily affects the growth and reproductive development of plants, such as functional and structural integrity of cell membranes, pollen fertility, protein biosynthesis and functions, and detoxification of highly toxic reactive oxygen species(Maret, 2019; Cakmak et al, 2023).

Improving grain Zn concentration in rice can be achieved quickly and cost-effectively by biofortification strategies such as plant breeding and fertilizer strategy(i.e., agronomic biofortification) (Cakmak, 2008;Bouis and Saltzman, 2017). Applying Zn fertilizer either through soil application or foliar spray appears to be a rapid and effective solution for improving grain Zn density in rice crops (Tuiwong et al, 2022;Utasee et al, 2022). Soil Zn application represents an effective and easy way to overcome Zn-deficiency-related impairments in growth and yield formation in rice plants(Impa and Johnson-Beebout, 2012). The application of Zn to soil can increase the grain yield and grain Zn concentration in rice (Prom-U-Thai et al, 2020).However, the efficiency of Zn fertilizer application in rice crops is affected by various factors such as rice variety, soil chemical and physical properties, and crop rotation systems (Rehman et al, 2012).

Zn also engages in crucial antagonistic and synergistic interactions with other mineral nutrients that affects root uptake, transport, and cellular utilization of Zn in plants. Notably, significant antagonistic and synergistic interactions of Zn occur with phosphorus (P) (Ova et al,2015; Yazici et al, 2021) and nitrogen (N) (Kutman et al,2011; Khampaung et al, 2021; Tuiwong et al, 2022).Besides P and N, an adequate amount of sulfur (S) is essential for better root uptake and transport as well as seed deposition of Zn by affecting the root release of phytosiderophores (Astolfi et al, 2021; Chorianopoulou and Bouranis, 2022). The root release of phytosiderophores is a well-documented adaptation to low Zn and Fe conditions in many cereal crops, particularly in rice(Suzuki et al, 2021; Murata et al, 2022; Cakmak et al,2023). The S-containing amino acid methionine is the key substrate for the biosynthesis of phytosiderophores,such as 2′-deoxymugineic acid (Mori and Nishizawa,1987; Shojima et al, 1990; Cakmak et al, 2023).Therefore, plants suffering S deficiency contain lower amounts of micronutrient, such as Fe as seen in durum wheat due to the reduced production and release of phytosiderophores (Astolfi et al, 2018, 2021).

The S-containing amino acid methionine is also required for the biosynthesis of nicotianamine by nicotianamine synthase (Mori and Nishizawa, 1987;Nozoye, 2018). Nicotianamine and 2′-deoxymugineic acid are well-documented Zn- and Fe-chelating compounds and are involved in the transportation and seed deposition of these micronutrients (Curie et al,2009; Murata et al, 2022). Therefore, the S nutritional status of plants may significantly influences the transport and distribution of Zn and Fe in crop plants by affecting the biosynthesis of nicotianamine.Accordingly, increasing the levels of nicotianamine and 2′-deoxymugineic acid in rice plants rice transgenic approaches significantly elevates endosperm concentrations of Zn in rice seeds (Masuda et al, 2009;Banakar et al, 2017). By promoting biosynthesis and root exudation of phytosiderophores, increased levels of nicotianamine will also contribute to better mobilization and root uptake of Zn from soils (Lee et al, 2011;Nozoye, 2018; Murata et al, 2022). Given these findings, it can be concluded that by improving the pool of phytosiderophores and nicotianamine in plant tissue through adequate S supply, leaf and grain Zn concentrations of plants would be increased. The wellknown positive relationship between grain concentrations of S and Zn, as seen in wheat (Morgounov et al, 2007;Gomez-Becerra et al, 2010), supports this hypothesis.

This study sought to evaluate the effect of upgrading S nutritional status of rice plants on grain yield and leaf as well as grain concentrations of S and Zn under low and adequate Zn supplies. Since grain yield is significantly affected by S and Zn rates, we also calculated the total amounts of Zn and S accumulated in grains.

RESULTS

Grain yield and straw dry weight

Fig. 1. Plant appearance under low (0.25 mg/kg, LZ) and adequate (5 mg/kg, AZ) soil zinc concentrations, together with low (2.5 mg/kg, LS),moderate (10 mg/kg, MS) and adequate (50 mg/kg, AS) sulfur fertilizer rates.

Fig. 1 illustrated the growth of plants under low and adequate soil Zn levels at varying rates of S applications.The combination of Zn and S applications affected plant growth and grain yield differently. The grain yield was affected by combined Zn and S application with no significant interaction (S × F) (P＞ 0.05) (Fig. 2-A).Adequate soil Zn application produced a higher grain yield than the low Zn application. The adequate S rate generated the highest grain yield, but there was no significant difference between the low and moderate S rates. Zn and S application significantly affected straw dry weight (P＜ 0.05) (Fig. 2-B). Straw dry weight increased with rising S application rates in both the low and adequate Zn conditions, ranging from 42.7 to 123.6 g/pot, registering a 1.9-fold increase under low Zn and a 2.3-fold increase under adequate Zn, while no significant difference was found between the low and moderate S rates.

Yield components

The yield components were also affected by varying Zn and S rates. There was a significant interaction effect of Zn × S on the number of tillers per plant (P＜0.05) (Table 1). At low soil Zn levels, increasing S rates had no effect on the number of tillers per plant,but there was a significant effect of applying S under the adequate soil Zn conditions, whereas the moderate and adequate S rates increased the number of tillers per plant by 13.4% and 29.2%, respectively, compared with low S rate. The number of panicles per plant and 1000-grain weight were significantly affected by S and Zn application. The number of panicles per plant at the adequate soil Zn supply was higher than at low soil Zn supply, while the adequate S rate resulted in the highest number (2.9 panicles), followed by the moderate S rate (2.3 panicles) and the low S rate (1.7 panicles). The adequate Zn supply yielded a greater 1000-grain weight than under low Zn. The 1000-grain weight at the adequate S rate was the highest at 35.2 g,while there was no significant difference between the low and moderate S rates. By contrast, the percentage of filled grains was significantly affected by the interaction between Zn and S applications. Under the low Zn supply,the moderate and adequate S rates increased the percentage of filled grains, while the lowest the percentage filled grain was found with the low S rate.Under the adequate Zn conditions, the highest percentage of filled grains was 94.98% at the adequate S rate with no significant difference between the low and moderate S rates. The culm length differed among the Zn application rates combined with the S fertilizer rates (P＜ 0.01). At low Zn, applying the moderate and adequate S rates increased the mean culm length to 82.47 and 91.23 cm, respectively, while it was 79.95 cm under the low S rate (Table 1). A similar response was found between the adequate and low Zn concentrations. Applying moderate and adequate S rates increased the mean culm length to 86.90 and 98.30 cm, respectively, while the lowest culm length was 76.97 cm with low S rate (Table 1).

Fig. 2. Grain yield (A) and straw dry weight (B) under low (0.25 mg/kg, LZ) and adequate (5 mg/kg, AZ) soil zinc concentrations, with low(2.5 mg/kg, LS), moderate (10 mg/kg, MS) and adequate (50 mg/kg, AS) sulfur fertilizer rates.

Table 1. Yield components of rice grown under low (0.25 mg/kg) and adequate (5 mg/kg) soil zinc, with low (2.5 mg/kg), moderate (10 mg/kg),and adequate (50 mg/kg) sulfur fertilizer rates.

Grain Zn and S concentrations

Grain Zn concentration was significantly affected by the interaction between soil Zn and S (P＜ 0.05) (Fig.3-A). Applying the low S rate (2.5 mg/kg) under adequate Zn conditions resulted in higher grain Zn concentration than under the low Zn conditions (Fig.3-A). With adequate Zn, applying the low S rate resulted in the highest grain Zn concentration (41.5 mg/kg), while grain Zn concentration decreased with the rising S application rates. However, grain Zn concentration was not clearly affected by increasing S rates under low Zn conditions (Fig. 3-A). A different response was found in the case of grain S concentrations.The adequate S rate resulted in an increase in grain S under low Zn supply, while there was no significant effect of increasing S rates on grain S under adequate Zn treatment (Fig. 3-B).

Grain Zn and S yields

Since increasing S fertilization increased grain yield by more than and almost 3-fold at low and adequate Zn treatments, respectively, it is important to evaluate grain Zn and S yield (i.e., the total amount of Zn and S in grains per plant). These indexes were calculated by multiplying grain yield by grain Zn and S concentrations (Fig. 3-C and -D). The grain Zn and S yields were affected by applying Zn and S rates, with no significant interaction between the two factors (S × F)(P＞ 0.05). Increasing S application caused increases in grain Zn yield under both low and adequate Zn treatments. As expected, increasing S application increased grain S yield at low and adequate Zn treatments.

Relationship between grain yield and concentrations of Zn and S and between grain Zn and grain S yields

The correlation between grain yield and the concentrations of Zn and S in brown rice varied with the Zn application level (Fig. 4). A negative correlation was found between grain yield and grain Zn concentration under adequate Zn application, but no significant correlation was found under low Zn conditions (Fig. 4-A). In contrast, a positive correlation was found between grain yield and S concentration in the low Zn condition, but no significant correlation was found under adequate Zn application (Fig. 4-B).There was also no significant correlation between grain Zn and grain S concentration under either Zn application (Fig. 4-C). However, there was a strong positive correlation between grain Zn yield and grain S yield under both low Zn and adequate Zn conditions(Fig. 4-D).

Fig. 3. Grain zinc (Zn) concentration (A), grain sulfur (S) concentration (B), grain Zn yield (C) and grain S yield (D) grown under low (0.25 mg/kg, LZ) and adequate (5 mg/kg, AZ) soil Zn concentrations, with low (2.5 mg/kg, LS), moderate (10 mg/kg, MS) and adequate (50 mg/kg,AS) soil S fertilizer rates.

DISCUSSION

The grain yield and grain Zn yield of rice increased with S fertilization under low and adequate soil Zn conditions. The highest increases in grain yield and grain Zn yield were found when S fertilization was performed in combined with adequate soil Zn application. Similarly, the yield components, including culm length, the number of tillers per plant, and the percentage of filled grains, were significantly increased when S and Zn were applied together at adequate rates. S and Zn may essential roles in various physiological and metabolic processes which contribute to increased plant productivity. These processes include N-metabolism, protein biosynthesis, gene expression, pollination, and tolerance to biotic and abiotic stress (Cakmak et al, 2023; Hawkesford et al,2023). Zn is effective in improving pollen viability,seed set, and kernel number (Pandey et al, 2006; Liu et al,2021). These findings indicated that rice plants need both adequate S and Zn to ensure desirable growth,productivity and grain Zn yield. Further confirmation of these results through field experiment is required.

Several studies have demonstrated the positive effects of S fertilization on grain yield and nutrient uptake of rice plants grown under field conditions(Ausma et al, 2021; Zenda et al, 2021). Applying S at the rates of 17.5 and 20.0 kg/hm2increases the grain yield by 40% and 37%, respectively, compared with the control plants without S application in a rice field experiment (Islam et al, 2021). The positive effects of S fertilization on crop productivity depend on various factors, including the variety applied, interactions with other nutrients, and soil conditions such as soil organic matter and Zn availability (Zenda et al, 2021;Narayan et al, 2022). As mentioned above, the Zn nutritional status of plants plays a significant role in the impact of S fertilization on plant growth. Singh et al(2018) reported that the combined application of 25 kg/hm2S and 10 kg/hm2Zn to rice cultivation results in significant increases in grain yield, straw dry weight and harvest index. This beneficial effect may be attributed to the synergistic interactions between Zn and S at the cellular level. Thiols (i.e., compounds containing SH functional groups) are essential Zn-binding compounds in cellular systems and are required for various physiological functions including structural stability of cell membranes and proteins,organization and regulation of proteins, and protection of membranes and proteins from oxidation (Maret,2019; Hübner and Haase, 2021). In addition, by affecting the pool of methionine, an S-containing amino acid that is essential for the biosynthesis of phytosiderophores (Shojima et al, 1990), adequate S nutrition may also contribute to better root Zn uptake and shoot transport. Phytosiderophores are known to be effective in the mobilization, root uptake, and shoot transport of Zn in plants (Suzuki et al, 2021). Further research is needed for a better understanding of the synergistic interaction between Zn and S with respect to rice productivity.

Fig. 4. Relationships between grain zinc (Zn) concentration and grain yield (A), grain sulfur (S) concentration and grain yield (B), grain S concentration and grain Zn concentration (C), and grain S yield and grain Zn yield (D).

Besides improved productivity, the highest grain Zn concentration was found with adequate Zn supply (Fig.3-A and -B). However, the lowest and highest S application rates resulted in the highest and lowest grain Zn concentrations, respectively, under adequate Zn supply, indicating a clear dilution of Zn in the grain due to increased grain yield resulting from S fertilization (Fig. 2). A similar yield effect on grain Zn concentration was also found under low Zn supply(Figs. 2 and 3). Therefore, the amount of Zn absorption by the grains (i.e., grain Zn yield) per pot was calculated by multiplying grain yield by grain concentration of Zn for better evaluation of the effect of S fertilization on grain Zn accumulation. As shown in Fig. 4,increasing S application markedly enhanced grain Zn yield, especially when adequate Zn is applied. Similarly,there were also substantial increases in grain S yield(Fig. 3-C and -D). These results indicated that in the evaluation of the nutritional effects of fertilizer treatments, changes in grain concentrations of minerals should be also taken into consideration in addition to grain yield. The well-known lower grain Zn concentrations in modern wheat compared with older or wild wheat varieties were ascribed to the higher grain yield capacity of modern wheat varieties (Fan et al, 2008; Cakmak et al, 2010).

Given the potential effects of S nutrition on root Zn uptake, shoot transport, and grain accumulation,especially through phytosiderophores, nicotianamine,and Zn-binding SH-containing compounds (Maret,2019; Astolfi et al, 2021; Murata et al, 2022), S fertilization plays a key role in grain Zn accumulation.Grain Zn concentrations are often positively correlated with grain S concentrations (Morgounov et al, 2007;Gomez-Becerra et al, 2010). To encourage farmers to consider agronomic biofortification (e.g., the application of soil and foliar Zn and S fertilizers) to improve grain Zn concentrations, particular attention should be paid to grain Zn yield rather than grain quantity (DeFries et al, 2015). Therefore, there is a need for a new metric in the cereal trade that considers the nutritional quality of cereals such as grain Zn yield.

In conclusion, the soil application of Zn and S at adequate levels help improve rice growth and grain yield, indicating that Zn and S have synergistic effects on plant productivity. However, when grain yield is markedly improved by these treatments, the concentrations of nutritional compounds in grains(such as Zn) can be reduced significantly due to dilution effects. Therefore, besides grain yield, grain nutritional components should also be considered as important grain parameters in grain trade-off, and growers should be also compensated for the grain nutritional yield.

METHODS

Plant growth

The pot experiment was designed as a 2 × 3 factorial with two levels of Zn and three levels of S soil application in a completely randomized design with four replicates in a total of 24 pots. The experiment was conducted a March-July 2019 at the Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Turkey (geographic coordinates: 40°53′25′′N, 29°22′47′′ E). During the cropping season, the average daytime temperature was 26 °C, while the night time temperature averaged 23 °C. The average humidity was 51%during the day and 31% at night. Rice seeds (Oryza sativaL. cv.Osmancik 97) were soaked in deionized water for 24 h and then sown in pots containing 3.5 kg of S-deficient soil (0.016 mol/L KH2PO4extractable S: 11 mg/kg soil) that was transported from the Eskisehir region in Turkey. The soil had a loamy sand texture, pH 8.4, 1.2% organic matter, 8% CaCO3, and 0.16 mg/kg the diethylenetriamine penta-acetic acid (DTPA)-extractable Zn concentration. The seedlings were thinned to five plants per pot shortly after emergence. Seeds were sowed in two Zn concentrations [0.25 mg/kg (low) and 5 mg/kg(adequate) in the form of ZnCl2] and three S fertilizer applications[2.5 mg/kg (low), 10 mg/kg (moderate) and 50 mg/kg (adequate)in the form of CaSO4·2H2O]. Before potting, the soil was mixed homogeneously with nutrients of 200 mg/kg N and 286 mg/kg Ca in the form of Ca(NO3)2, 150 mg/kg P and 189 mg/kg K in the form of KH2PO4, and 10 mg/kg Fe in the form of sequestrene. After 14 d of growth, 100 mg/kg N and 15 mg/kg Fe were applied in the forms of NH4NO3and FeCl2,respectively. Then, 100 mg/kg N and 100 mg/kg K were applied in the forms of NH4NO3and KH2PO4, respectively at 75 d of growth to maintain adequate mineral nutrition. The plants were continuously submerged under approximately 10 cm of water above the soil surface throughout the entire growth period until harvesting.

Sample collection

At the maturity stage, each pot was harvested to determine yield,straw dry weight, and yield components (the number of tillers per hill, the number of panicles per plant, the number of spikelets per panicle, the percentage of filled grains, 1000-grain weight, and culm length). The samples were first washed with deionized water and then dried at 60 °C for 72 h for the determination of straw dry weight. The grain yield was measured at 14% moisture content of the grain.

Chemical analysis

Grain Zn and S concentrations were analyzed by inductively coupled plasma optical emission spectrometry (ICP-OES)(Vista-Pro Axial, Mulgrave, Australia). For the ICP-OES analysis of Zn and S, approximately 0.45 g of the sample was subjected to acid digestion in a closed-vessel microwave system (MarsExpress, CEM Corp., NC, USA). Each batch of Zn and S survey included a ground peach leaf sample (SRM 1547) as certified reference material. The grain Zn and S yields were calculated by multiplying the grain yield by the grain Zn and grain S concentrations, respectively.

Statistical analysis

Analysis of variance (ANOVA) was conducted to detect differences in grain yield, yield components, and grain Zn and S concentrations using Statistic 9 (analytical software SX).Data were analyzed as factorials in a completely randomized design. The least significant difference test atP＜ 0.05 was applied to compare the means for significant differences between Zn and S fertilizer rates. Pearson correlation analysis was conducted to test the pairwise relationships of each factor.

ACKNOWLEDGEMENTS

The authors thank the members of the Plant nutrition lab at Chiang Mai University, Thailand, and Sabanci University,Turkey, for their assistance in the glasshouse experiment,sample collection, and plant tissue analysis, and thank Dr. Dale TANEYHILL for his English editing for the entire manuscript. This research project was supported by Fundamental Fund 2023, Chiang Mai University, Thailand (Grant No. FF66/063).