Evaluation of trade-off relationship between grain number and zinc concentration in rice, Oryza sativa L.

Abstract

Rice is a staple food for almost half of the world’s population. During domestication from wild rice, the selection of plants with reduced seed shattering enabled efficient harvesting. Further selection for increased grain number improved yield potential. Breeding high-yielding cultivars remains necessary to support the growing world population. Despite success in increasing yield potential, improvements in micronutrient content in rice grains have lagged. Zinc (Zn) is an essential micronutrient for crop productivity and human health. Previously, we reported that reduced seed-setting rate was associated with increased grain Zn concentration, suggesting a trade-off between grain number and Zn concentration. Based on these findings, we investigated the grain Zn concentration in high-yielding rice conferred by Gn1a, a gene involved in the regulation of grain number per panicle. Lower grain Zn concentration was observed in high-yielding plants. However, reducing the seed-setting rate by artificially trimming spikelets at the flowering stage restored grain Zn concentrations in high-yielding plants. Other mineral element concentrations were less affected under high-yielding plants. Our results showed clear trade-off between grain number and Zn concentration, emphasizing the importance of enhancing grain Zn concentrations in breeding programs of high-yielding rice.

Introduction

Rice (Oryza sativa L.) is one of the most important crops, feeding almost half of the world’s population, and is predominantly cultivated in Asian countries (Khush 1997). Cultivated rice was domesticated from wild rice (O. rufipogon) through the selection of natural variations that facilitated ease of cultivation (Ishikawa et al. 2020). During rice domestication, grain yield improved considerably to meet the needs of human development. An early domestication trait was the reduction of seed shattering, enabling efficient harvesting during seed maturation (Ishikawa 2024). Subsequent selection focused on improving grain yield by targeting three major quantitative traits: grain weight, grain number per panicle, and panicle number per plant (Xing and Zhang 2010). Among these traits, grain number per panicle has contributed the most to the higher yields observed in modern rice varieties (Lu et al. 2022).

Multiple genes that control the number of grains have been reported, most of which influence plant hormones that affect panicle branching (Lu et al. 2022). Quantitative trait locus (QTL) analysis of the difference in the number of grains between the indica cultivar Habataki and the japonica cultivar Koshihikari identified Gn1a (Grain number 1a) as the major locus that increased grain productivity (Ashikari et al. 2005). A causal mutation at Gn1a was identified in OsCKX2, which encodes a cytokinin oxidase/dehydrogenase enzyme that degrades the phytohormone cytokinin. WFP (WEALTHY FARMER’S PANICLE)/IPA (Ideal Plant Architecture 1), known as OsSPL14, was detected as a locus for increased primary branch number in panicles (Jiao et al. 2010, Miura et al. 2010). The expression of OsSPL14 is tightly controlled by a microRNA (miRNA)-targeted sequence within the coding sequence. NOG1 (NUMBER OF GRAINS 1) was identified as the locus responsible for the difference in grain number between wild rice (O. rufipogon) and high-yielding indica rice (Huo et al. 2017). NOG1 encodes an enoyl-CoA hydratase/isomerase, and the upregulation of NOG1 expression was found to increase grain yield by enhancing the grain number per panicle. Recently, brassinosteroids (BRs) were reported to regulate grain number in rice. BRASSINOSTEROID-DEFICIENT DWARF3 (BRD3), a BR catabolic gene, increases grain number (Zhang et al. 2024). A recent genome-wide association study using a panel of 317 cultivars detected GNP3 (GRAIN NUMBER PER PANICLE 3) as a locus for the number of grains per panicle. The causal mutation at GNP3 was found at the gene encoding MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE 22 (OsMKKK22) (Ma et al. 2025). Higher expression of GNP3 enhances panicle branching by lowering ethylene levels. Beyond these major loci, other loci that contribute to an increase in the number of grains have been reported (Lu et al. 2022, Xing and Zhang 2010). The utility of these valuable alleles or the manipulation of their gene expression levels for breeding high-yielding rice cultivars is required to meet the human demands for an increasing world population (Ikeda et al. 2013, Ueda et al. 2025).

Despite efforts to increase grain yield through genetic improvements, less attention has been paid to grain micronutrient density, an issue known as “hidden hunger” (Pasion et al. 2023, Welch and Graham 2004). Zinc (Zn) is one of the most important micronutrients for plant growth (Broadley et al. 2007) and human health (Roohani et al. 2013). For plants, Zn plays crucial roles for better seed viability, seedling vigour, germination, disease resistance and abiotic stress tolerance (Cakmak 2008), and affects a range of important metabolic pathways at its low and toxic level (Kolesnikov et al. 2025). For humans, Zn deficiency causes various problems, such as poor immunity, skin rashes, hair loss, appetite loss, and stunted growth in children, and is especially prevalent in developing countries (Lowe 2025, Palmer et al. 2024). As the grain Zn concentration in rice is lower than that in other cereal crops, it is necessary to boost the grain Zn concentration to ensure nutritional security.

Zn transporters from the soil to grains have been identified, and they are mainly classified based on the location of plant tissues for Zn allocation in rice (Huang et al. 2022). Zn uptake is primarily mediated by the Zn-regulated transporter and iron-regulated transporter-like protein (ZRT-IRT-related protein, ZIP) family. OsZIP1, OsZIP5, and OsZIP9 function as influx transporters for Zn uptake (Huang et al. 2020, Ramesh et al. 2003, Tan et al. 2020, Yang et al. 2020). The sequestration of Zn in the roots is controlled by OsHMA3 (also known as a cadmium (Cd) transporter; Cai et al. 2019, Sasaki et al. 2014, Ueno et al. 2010) and OsMTP1 (Menguer et al. 2013). OsHMA3 encodes an efflux transporter that transports Zn from the cytoplasm to vacuoles (Cai et al. 2019, Sasaki et al. 2014, Ueno et al. 2010). Then, root-to-shoot translocation of Zn is mediated by OsHMA2 (Takahashi et al. 2012, Yamaji et al. 2013) and possibly OsZIP7 (Tan et al. 2019), which function in pericycle cells to transport Zn into the xylem for further delivery. To support active cell growth, nodes play an important role in the preferential distribution of Zn to developing tissues, and thus contain high concentrations of Zn (Yamaji and Ma 2014). The major Zn transporters localised in the nodes are encoded by OsZIP3, OsZIP4, and OsHMA2 (Mu et al. 2021, Sasaki et al. 2015, Yamaji et al. 2013), and play specific roles in transporting Zn between different cell types (Huang et al. 2022). These transporters facilitate Zn accumulation in the embryo and aleurone layers of the grain. Although manipulation of these Zn transporters holds potential for the efficient delivery of Zn in rice grains, their regulation is tightly controlled to maintain plant homeostasis (Huang et al. 2022).

Natural variation offers a route for breeding new cultivars with elevated Zn levels. We previously surveyed A-genome wild rice species to evaluate their grain Zn concentrations and found that Australian wild rice (O. meridionalis) had higher grain Zn concentrations than Asian rice cultivars (O. sativa Nipponbare and IR36; Ishikawa et al. 2017). To understand the genetic basis of the high grain Zn concentration in O. meridionalis, we conducted QTL analysis using backcrossed recombinant inbred lines between O. sativa Nipponbare and Australian wild rice O. meridionalis W1627 and detected a QTL on chromosome 9 (qGZn9) associated with higher Zn concentration (Ishikawa et al. 2017). Further genetic analyses revealed that qGZn9 consisted of two distinct loci, qGZn9a and qGZn9b. Using the near-isogenic line (NIL) carrying W1627 chromosomal segment covering qGZn9a, which is designated as NIL(qGZn9a), we found that an increase in the grain Zn concentration of the NIL(qGZn9a) was associated with reduced fertility, which may be a consequence of grain Zn accumulation due to the reduced number of fertilised grains. The reduced seed-setting rate of NIL(qGZn9a) was partially attributed to defects in anther dehiscence, suggesting that the increased grain Zn concentration is a consequence of indirect effects (Ogasawara et al. 2021). A reduction in fertility with increased grain Zn concentration was also reported through the identification of the causal mutation at Grain Zn Content 1 (GZnC1) (Hou et al. 2023). The causal gene underlying GZnC1 was identified as EMBRYO SAC ABORTION 1 (ESA1) and in the O. rufipogon YJCWR, the allele was abolished, leading to a higher grain Zn concentration via reduction of the seed-setting rate in the O. sativa genetic background. These findings indicate that reduced fertility can lead to increased grain Zn concentration, which may account for the trade-off in Zn allocation. Based on these findings, we speculate that increasing the number of grains may cause a reduction in grain Zn levels, posing a challenge for breeding high-yielding rice cultivars (Miyazaki et al. 2021).

In this study, we aimed to evaluate the grain Zn level in high-yielding plants to confirm the trade-off between grain number and Zn concentration. We evaluated the grain Zn concentration in japonica cultivar ‘Kinuhikari’ and the NIL(Gn1a), a near-isogenic line carrying the chromosomal segment of indica cultivar Keicho 2, which exhibited an increase in grain number by Gn1a (Ashikari et al. 2005). The evaluation of grain Zn levels in a common genetic background enabled us to investigate the trade-off relationship in Zn distribution.

Materials and Methods

Plant materials

The japonica cultivar, O. sativa cv. Kinuhikari was used as the control for comparison with high-yielding rice. High-yielding rice conferred by Gn1a was previously produced by backcrossing Keicho 2, an indica cultivar, as a donor parent and Kinuhikari, as a recurrent parent. After five backcrosses, a plant (BC₅F₁) carrying a chromosomal segment that covered Gn1a was selected. Introgression of the Gn1a allele from Keicho 2 was confirmed using PCR-based genotyping using the primer set of 5ʹ-GCCACCTTGTCCCTTCTACA-3ʹ and 5ʹ-TGCCATCCTGACCTGCTCT-3ʹ. The corresponding chromosomal segment was fixed in the subsequent generation and designated as the NIL(Gn1a). Kinuhikari and NIL(Gn1a) were grown in pots filled with a uniform soil mixture consisting of 2.5L of soil supplemented with 3.75 g of compound N–P–K fertilizer containing humic substances (Fumin Hosuka; SunAgro Co., Japan) and 3.75 g of calcium silicate fertilizer (Million; SoftSilica Co., Japan). Plants were cultivated under continuously flooded conditions with a maintained water depth of approximately 4 cm. Experiments were conducted in a greenhouse at Kobe University, Japan under natural light and temperature conditions. No additional fertilizers were applied during the cultivation period.

Trimming spikelets for artificial reduction of seed-setting rate

To artificially reduce grain number in a panicle, we randomly trimmed spikelets using scissors. At the beginning of the flowering stage, approximately every third spikelet was removed including male and female gametophytes. The seed-setting rate of the panicle was calculated using fertilised seeds against the total number of spikelets, including those trimmed with scissors. Using this approach, approximate seed-setting rates of 60% were achieved for each plant.

Determination of mineral concentrations

A single panicle was selected from each plant, and 15 grains were randomly sampled from the panicle for measuring grain Zn concentration. Four to six plants for each genotype or spikelet trimming treatment were used to calculate the average grain Zn concentrations. Samples were harvested approximately 50 days after heading, when seeds had fully matured. Rice grains were de-hulled and dried in an oven for 16 h at 70°C. Subsequently, the weight of 15 grains was measured for each plant and they were digested with concentrated nitric acid (60% (w/v)) at up to 140°C in a heating block as described previously (Sasaki et al. 2012). After dilution, micronutrient concentrations (Zn, copper (Cu), iron (Fe), manganese (Mn), calcium (Ca), potassium (K), phosphorus (P), and magnesium (Mg)) were determined using inductively coupled plasma-mass spectrometry (ICP-MS, 7700X or Agilent 8900; Agilent Technologies, USA).

Results

Reduction in grain Zn concentration in high-yielding rice

Previously, we showed that a reduction in the number of grains per panicle caused an increase in grain Zn concentration, implying a trade-off between them (Ogasawara et al. 2021). To evaluate the trade-off between grain number and Zn concentration, we examined grain Zn levels in Kinuhikari and NIL(Gn1a), carrying the indica Keicho 2 allele of Gn1a. The average grain number of NIL(Gn1a) was 136.5 ± 2.6, 12.8% higher than that of Kinuhikari (121.0 ± 9.2) (Fig. 1A, 1B). Analysis of grain Zn concentration showed that Kinuhikari had an average grain Zn concentration of 39.4 ± 0.7 mg/kg, compared to 36.5 ± 0.6 mg/kg for NIL(Gn1a), which was 7.5% lower than that of Kinuhikari, with a significant difference (p < 0.01) (Fig. 1C).

Fig. 1.

Grain number and Zn concentration in high-yielding NIL(Gn1a). (A) Photographs of the main panicle in Kinuhikari and NIL(Gn1a). Scale bar: 3 cm. (B) grain number and (C) Zn concentration in Kinuhikari and NIL(Gn1a). Data are mean ± SD of four plants grown in 2017. Significant differences were determined using Student’s t-test (*; p < 0.05, **; p < 0.01).

Increase in grain Zn concentration associated with reduction in seed-setting rate

The reduction in grain Zn concentration in NIL(Gn1a) might account for the additional genetic effects of chromosomal segments conferred by a donor parent from Keicho 2, which was introgressed in NIL(Gn1a). To exclude this possibility, we conducted a physiological experiment by trimming the spikelets at the flowering stage to artificially reduce the seed-setting rate. Spikelet bases of Kinuhikari and NIL(Gn1a) plants were trimmed at approximately every third position by scissors. As a result, a 30–35% reduction in seed-setting rate was artificially achieved (Fig. 2A). The average total number of grains for Kinuhikari and NIL(Gn1a) without spikelet trimming (control) were 120.3 ± 12.8 and 180.0 ± 17.8, respectively, compared to 79.5 ± 9.1 and 121.5 ± 12.1 with spikelet trimming, respectively (Fig. 2B). The seed-setting rates of Kinuhikari and NIL(Gn1a) without spikelet trimming were 92.3 ± 1.2% and 91.4 ± 2.4%, respectively, whereas those with spikelet trimming were 59.6 ± 2.4% and 62.9 ± 1.3%, respectively. On average, 33.4% and 33.3% of grains were artificially trimmed for Kinuhikari and the NIL(Gn1a), respectively (Fig. 2C). We then compared their grain Zn concentration at seed maturation (Supplemental Fig. 1). Plants with reduced seed-setting rate displayed an increase in grain Zn concentration compared to those without spikelet trimming (Fig. 2D, Supplemental Table 1). The average grain Zn concentration of Kinuhikari without and with spikelet trimming was 40.8 ± 1.4 mg/kg and 46.9 ± 1.9 mg/kg, respectively (p < 0.01) representing a 15.2% increase following spikelet trimming. Similarly, the average grain Zn concentration of the NIL(Gn1a) without and with spikelet trimming was 34.4 ± 1.1 mg/kg and 40.3 ± 1.8 mg/kg, respectively (p < 0.01) corresponding to a 17.3% increase. The grain number of Kinuhikari without spikelet trimming and NIL(Gn1a) with spikelet trimming were similar, at 120.3 ± 12.8 and 121.5 ± 12.1, respectively (Fig. 2B). A comparison of grain Zn concentrations between them showed no significant differences (Fig. 2D), confirming that grain Zn concentration is negatively associated with grain number.

Fig. 2.

Reduction in number of grains correlated with an increase in grain Zn level. (A) Panicle photographs of Kinuhikari and NIL(Gn1a) with spikelet cut. The white triangle indicates a spikelet that received a cutting treatment. Scale bar: 5 cm. (B–D) Number of grains (B), Seed-setting rate (C), and Grain Zn concentration (D) of Kinuhikari and NIL(Gn1a) with spikelet cut. Data are mean ± SD of six plants grown in 2025. Significant differences were determined using Tukey’s HSD test (p < 0.01).

Negative correlation between grain number and Zn concentration in rice

To further elucidate the relationship between grain number and Zn concentration, we performed correlation analysis. The grain number and Zn concentration of 24 Kinuhikari and NIL(Gn1a) plants grown in 2025 with and without spikelet trimming were plotted in a correlation diagram (Fig. 3). A strong negative correlation was observed between grain number and Zn concentration (Pearson’s r = –0.92, p < 0.001). The tendency was similar to the experiments conducted in 2018 (Pearson’s r = –0.92, p < 0.001) and 2024 (Pearson’s r = –0.87, p < 0.001) (Supplemental Fig. 2). These results strongly indicate a clear trade-off between grain number and Zn concentration in rice.

Fig. 3.

Correlation analysis of grain number and Zn concentration. Values for grain number and Zn concentration for each individual grown in 2025 are plotted (n = 24), and the regression line for each plot is indicated. Correlation analysis between grain number and Zn concentration was conducted using Pearson’s correlation coefficient. The level of significance was set at p < 0.001.

Other mineral concentrations were less affected in the NIL(Gn1a)

We observed a clear negative correlation between grain number and Zn concentration over the years (Fig. 3, Supplemental Fig. 2). To determine whether similar trade-offs exist for other micronutrients using the same samples used for the evaluation of the grain Zn concentration in Kinuhikari and the NIL(Gn1a) (Fig. 1), we investigated concentrations of Cu, Fe, Mn, Ca, K, P, and Mg. No significant differences were observed, except for Mn (Table 1). We also investigated these concentrations for those grown in 2018, 2024, and 2025. The Mn concentration was not significantly different in 2018 and 2024 (Supplemental Table 2).

Table 1.Concentrations of other micronutrients in Kinuhikari and NIL(Gn1a) and their p values

Minerals	Grain mineral concentration (mg/kg)a		pb
	Kinuhikari	NIL(Gn1a)
Cu	3.86 ± 0.31	3.35 ± 0.35	0.077
Fe	14.93 ± 2.01	17.74 ± 1.30	0.057
Mn	42.62 ± 5.28	29.85 ± 3.99	0.008**
Ca	154.18 ± 11.86	149.47 ± 4.97	0.491
K	3528.40 ± 542.53	3218.73 ± 292.96	0.354
P	2612.32 ± 846.28	2097.30 ± 154.91	0.276
Mg	1042.35 ± 241.75	913.49 ± 55.96	0.339

^a Data are mean ± SD of four plants grown in 2017.

^b Significant differences were determined using Student’s t-test (**; p < 0.01).

Discussion

The domestication of wild rice, O. rufipogon, involved selection for increased grain production to meet the demands of an increasing population (Fuller 2007). However, improvements in micronutrient content have received less attention. In this study, we focused on the Zn concentration in rice grain, which is important for human health. We used Kinuhikari and NIL(Gn1a) to compare the grain number per panicle and Zn concentration. The results showed a reduction in the grain Zn concentration in the NIL(Gn1a) compared to that in Kinuhikari (Fig. 1C). Furthermore, the reduced grain Zn concentration in the NIL(Gn1a) was restored by reducing the number of grains to a number similar to that of Kinuhikari via artificial spikelet trimming (Fig. 2D). Our genetic and physiological evaluation of grain Zn levels confirmed a clear trade-off in the regulation of grain Zn allocation (Fig. 3, Supplemental Fig. 2). Future studies should investigate whether this trade-off is conserved across other high-yielding rice grains. Moreover, it is of interest to evaluate grain Zn levels across genetic backgrounds such as indica rice, which generally exhibits higher yield potential than japonica rice. A previous study showed that an introgression of a yield-positive Gn1a allele in several indica cultivars did not increase grain number, whereas introgression of the epigenetic OsSPL14^WFP allele dramatically increased grain number (Kim et al. 2018). A comparison of grain Zn concentrations in these materials would be ideal for evaluating the trade-off relationship between grain number and Zn concentration in indica cultivars. Additionally, yield increases in rice cultivars are also achieved by increasing the grain size and panicle number per plant (Lu et al. 2022). The evaluation of grain Zn concentration in these high-yielding plants is important for future studies to contribute to the biofortification of grain Zn. Furthermore, assessing the effect of Zn contents on plant growth is also vital when breeding useful biofortified plants.

High yields can also be achieved by hybrid rice through heterosis (hybrid vigour), the phenomenon in which the first filial offspring (F₁) from genetically diverse parents displays advantages in growth behaviour and yield compared to their parents (Chen et al. 2023, Gu and Han 2024). To support a growing population, the use of hybrid rice instead of inbred high-yielding varieties is increasing, especially in Asian countries. In hybrid rice, it is vital to increase or maintain micronutrient concentrations and yield potential. A recent study on the evaluation of grain Fe and Zn contents in hybrid rice grown under irrigated and aerobic conditions showed that certain combinations of parental lines exhibited higher micronutrient content and grain yield under aerobic conditions than under irrigated transplanted conditions (Anusha et al. 2021). However, evaluating micronutrient levels in hybrid rice is complex due to its multiple combining benefits. If the parental lines with general combining abilities for both grain Zn and yield levels were identified, it would have great potential to resolve the twin issues of food and nutritional security.

In the evaluation of other micronutrient concentrations (Cu, Fe, Mn, Ca, K, P, and Mg) between Kinuhikari and the NIL(Gn1a) grown in 2017, no significant differences were observed, except for Mn (Table 1). However, the Mn concentration was not significantly different in 2018 and 2024 (Supplemental Table 2), suggesting that Mn concentration is not in a trade-off relationship. Therefore, among the micronutrients examined in our study, Zn concentration was the most sensitive to the number of grains per panicle. Currently, we do not know why Zn concentration is particularly sensitive to the number of grains per panicle. The sensitivity of the Zn balance to the number of grains may account for the function or characteristics of Zn transporters compared to those of other micronutrients. Further evaluation is necessary to elucidate the precise regulation of micronutrient level in response to grain number in rice.

To increase the grain Zn concentration in high-yielding rice, natural alleles involved in higher grain Zn concentrations can be used. To search for such valuable alleles, the utility of Oryza species could be ideal, as they have greater genetic diversity (Mahender et al. 2016, Ricachenevsky and Sperotto 2016, Swamy et al. 2016). Several QTLs that have the potential to increase grain Zn concentrations have been reported using genetic resources, including wild rice O. rufipogon (Anuradha et al. 2012, Garcia-Oliveira et al. 2009, Shekhawat et al. 2025). In our previous study, we detected QTLs controlling higher grain Zn concentrations observed in the Australian wild rice O. meridionalis (Ishikawa et al. 2017). Among the QTLs, qGZn9 was genetically divided into two distinct regions: qGZn9a and qGZn9b. Because qGZn9a affects the grain Zn concentration by reducing fertility, it is unsuitable for breeding new cultivars. In contrast, the O. meridionalis allele at qGZn9b increased grain Zn level without reducing fertility, thus having potential for high-yielding rice. It would be of interest to investigate grain Zn concentrations in high-yielding cultivars with and without the qGZn9b allele of O. meridionalis. If successful, the application of qGZn9b would be ideal for elite indica cultivars or hybrid rice, which are cultivated in areas that require biofortification. Balancing the grain yield and Zn concentration is important and challenging for future rice breeding. These challenges are also important for other crops, such as wheat and maize, to biofortify human health (Breure et al. 2023, Fradgley et al. 2022).

Author Contribution Statement

SK, NM, and RI conceived and designed the study. SK, NM, RT, and RI performed the experiments. SK, NM, RT, KN, TI, JFM, and RI analysed the data. HM provided the genetic material for Kinuhikari and the NIL(Gn1a). SK and RI prepared the manuscript. All the authors have read and approved the final version of the manuscript.

 Acknowledgments

We thank Akemi Morita, Miho Kashino, and Sanae Rikiishi for determining grain Zn concentrations and Lim Sathya and Naohiro Matsubara for helping experiments. We are grateful for the support from the Joint Usage/Research Center, Institute of Plant Science and Resources, Okayama University. This research was partially supported by Grants-in-Aid from the Japan Society for the Promotion of Science 24KK0123 (R.I.) and The Public Foundation of Elizabeth Arnold-Fuji (R.I.).

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