A partner protein helps OsNRAMP3 win a novel function to transport Fe 2+ in rice

Abstract

Rice (Oryza sativa L.) is a staple food for approximately 50% of the world's population, and yet, global food insecurity and malnutrition, especially iron (Fe) deficiency, remain major challenges. Nitrate transporter partner protein 2.1 (OsNAR2.1) is a small protein initially identified as a partner protein for high-affinity nitrate transporters (Liu et al., 2014). Overexpression of OsNAR2.1 significantly improves rice nitrate uptake, thus enhancing nitrogen-use efficiency and yield (Chen et al., 2017). Song et al. (2020) reported that OsNAR2.1 interacts with two nitrilase proteins to regulate root growth response to nitrate and ammonium. Previous studies showed that OsNAR2.1 interacts with MADS-box transcription factor 25 (OsMADS25). Under nitrate conditions, OsMADS25 translocates from the cytoplasm to the nucleus in an OsNAR2.1-dependent manner, thereby influencing rice root development (Wu et al., 2023). Recent work shows that Nodule Inception-Like Protein 3 (OsNLP3) and Phosphate Starvation Response 2 (OsPHR2) orchestrate direct and mycorrhizal nitrate uptake pathways by regulating OsNAR2.1 in rice (Wang et al., 2025). A previous pull-down assay indicated that OsNAR2.1 might interact with Natural Resistance-Associated Macrophage Protein 3 (OsNRAMP3) (Song et al., 2020), which is highly expressed in Node I, and facilitates manganese (Mn) allocation to young leaves but does not transport Fe2+ (Yamaji et al., 2013; Yang et al., 2013). However, the role of OsNAR2.1 in Fe2+ or Mn2+ transport remained unexplored. In this study, we demonstrate that OsNAR2.1 confers Fe2+ transport function of OsNRAMP3 in rice. Expression of pOsNAR2.1:OsNAR2.1 significantly increased Fe2+ and Mn2+ concentrations and contents under NO3− conditions (Figure S1). Additionally, osnar2.1 mutants showed reduced root and shoot Fe2+ concentrations at 0.2 mM NO3− but not at 2.5 mM NH4+ (Figure S2). To explore OsNAR2.1's molecular mechanism in Fe2+ and Mn2+ transport, a previous pull-down assay (Song et al., 2020) suggested its potential interaction with OsNRAMP3, which was further confirmed by yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC), split-luciferase complementation (LCI), and co-immunoprecipitation (Co-IP) assays (Figure 1A–D). Yeast complementation assays demonstrated that OsNAR2.1 interacts with OsNRAMP3 to confer novel Fe2+ transport activity (Figure 1E, F). Co-expressing OsNAR2.1 and OsNRAMP3 in Xenopus laevis oocytes significantly enhanced Fe2+ accumulation in the presence of 1 mM Fe2+ plus 1 mM NO3−, but not with Fe2+ alone (Figure 1G). These results indicate that the interaction between OsNAR2.1 and OsNRAMP3 promotes OsNRAMP3-mediated Fe2+ transport in a NO3−-dependent manner. Functional characterization of OsNRAMP3 in rice Fe2+ transport (A) Interaction between OsNAR2.1 and OsNRAMP3 tested in yeast cells grown on selective (−Ade, −His, −Leu, −Trp) or control (−Leu, −Trp) media. (B) Transient expression of split-YFP constructs in rice protoplasts. Scale bar = 10 μm. (C) Split-luciferase complementation assay showing the OsNRAMP3–OsNAR2.1 interaction in N. benthamiana leaves. cLUC, C-terminal luciferase; nLUC, N-terminal luciferase. (D) Co-IP assay confirming the interaction between OsNRAMP3 and OsNAR2.1 in rice protoplasts. (E) Complementation assay using a Mn-uptake-deficient yeast mutant strain. Yeast mutant Δsmf1 cells carrying pYES2 (vector control), OsNAR2.1, OsNRAMP3, AtNRAMP1 (positive control), or a combination of OsNAR2.1 and OsNRAMP3 and wild-type (BY4741) cells carrying pYES2 (vector control) grown on synthetic defined (SD)-Ura plates with varying EGTA (Mn2+ chelator) concentrations and 2% galactose. (F) Complementation assay in an Fe-uptake-deficient yeast mutant strain. Yeast mutant DEY1453 cells carrying pYES2, OsNAR2.1, OsNRAMP3, OsNRAMP1 (positive control), or a combination of OsNAR2.1 and OsNRAMP3 and wild-type (BY4741) cells carrying pYES2 grown on SD-Ura plates containing varying BPDS (Fe2+ chelator) concentrations and 2% galactose. (G) Fe2+ uptake assay in Xenopus laevis oocytes under different conditions. Oocytes injected with water (negative control), OsNAR2.1, OsNRT2.1, OsNRAMP3, a combination of OsNAR2.1 and OsNRT2.1, a combination of OsNAR2.1 and OsNRAMP3, or OsIRT1 (positive control) cultured in ND96 medium supplemented with either 1 mM Fe2+ alone or 1 mM Fe2+ plus 1 mM NO3−. Error bars indicate SD (n = 4 oocytes). (H) Growth comparison of WT and osnramp3 mutants under control and Fe-deficient conditions supplied with 0.2 mM NO3−. L.B, leaf blade; BN.S, basal node and sheath; and R, root. Bar = 10 cm. (I) Growth comparison of WT and osnramp3 mutants under control and Fe-deficient conditions with 2.5 mM NH4+. Bar = 10 cm. (J) Phenotype of hybrid lines. Scale bar = 10 cm. (K) Seed-setting phenotype of hybrid lines. Bar = 10 cm. (L) Seed-setting rate of hybrid lines. (M) Grain yield per plant of hybrid lines. (N) Grain Mn concentration in hybrid lines. (O) Grain Fe concentration in hybrid lines. Error bars represent SE (n = 4 plants). Statistical significance was determined by one-way ANOVA with Tukey's HSD (E, H, I, L-O). Different letters indicate significant differences (P < 0.05). Further analysis revealed that both OsNAR2.1 and OsNRAMP3 were predominantly expressed in Node I, with the highest levels in Nipponbare and Dongjin cultivars (Figures S3A–D, S4). Their expression followed rhythmic patterns (Figure S3E), was downregulated under Fe2+ deficiency (Figures S3F, G), and was specifically induced under low-nitrate conditions (0.2 mM NO3−) (Figures S3H, I). These findings further confirm that OsNAR2.1 interacts with OsNRAMP3 to mediate Fe2+ accumulation under low-nitrate conditions. To investigate the biological function of OsNRAMP3 in Fe2+ uptake, we obtained three homozygous Tos17 and T-DNA insertion mutants. qRT-PCR analysis confirmed nearly complete knockout of OsNRAMP3 in NG3081 and 3A-01364, and an approximately 60% reduction in NG4081 (Figure S5). Under 0.2 mM NO3−, all mutants displayed reduced biomass and total nitrogen accumulation (Figure S6F, N), though only NG3081 and 3A-01364 showed significantly decreased tissue nitrogen concentrations (Figure S6J). After nitrogen starvation, 15N uptake significantly decreased under low-nitrate (L15NO3−) conditions with increased 15N concentration in roots but decreased in shoots, leading to significantly lower shoot-to-root 15N ratios in mutants (Figure S7A–C). These results indicate that OsNRAMP3 is essential for nitrate uptake translocation under low-nitrate conditions. Next, under Fe2+ or Mn2+ deficiency at 0.2 mM NO3−, all mutants displayed significantly reduced biomass (Figures S8A, S9A). Under Fe2+ deficiency, the root Fe2+ concentration of osnramp3 mutants significantly decreased, whereas the Fe2+ concentration markedly increased in leaf blades, resulting in a significant reduction of the total Fe2+ content in osnramp3 mutants (Figure S8A). Under Mn2+ deficiency, the Mn2+ concentration increased in roots, basal nodes, and sheaths, and leaf blades compared to WT-N and WT-DJ, and yet, total accumulation decreased due to lower biomass (Figure S9A). No significant differences were observed at 2.5 mM NH4+ under either control or Fe2+-deficient conditions (Figure S8B). Mn2+ deficiency caused biomass reduction but did not alter Mn2+ accumulation (Figure S9B). Therefore, OsNRAMP3 is critical for Fe2+ and Mn2+ accumulation and allocation under low-nitrate conditions. OsNRAMP3 expression was downregulated in osnar2.1 mutants and upregulated in OsNAR2.1-overexpression lines (OE1/OE2), whereas OsNAR2.1 expression was unchanged in osnramp3 mutants (Figure S10), indicating that OsNRAMP3 acts downstream of OsNAR2.1. In hybrids between OE1 and osnramp3 mutants (OE1/NG3081, OE1/NG4081), OsNRAMP3 expression remained suppressed in OE1/NG3081 but partially recovered in OE1/NG4081 (Figure S11). At the seedling stage, OE1 displayed superior dry weight in roots, leaf blades, basal nodes, and sheaths compared to other plants under control and Fe2+-deficient conditions under low-nitrate conditions. OE1/NG4081 outperformed NG4081 but not WT, whereas OE1/NG3081 resembled NG3081 (Figure 1H). OE1 showed elevated Fe2+ concentrations in the roots, basal nodes, and sheaths, while these were partially restored in OE1/NG4081 under Fe2+ deficiency (Figure 1H). No significant differences were observed under NH4+ supply (Figure 1I). Furthermore, OE1 increased the grain yield and the seed-setting rate, whereas OE1/NG3081 showed no improvement over NG3081, and OE1/NG4081 partially rescued the NG4081 phenotype (Figure 1L, M). Overexpressing OsNAR2.1 increased grain Mn2+ concentration in OE1/NG3081 and OE1/NG4081, but enhanced grain Fe2+ concentration only in OE1/NG4081 (Figure 1N, O). These results demonstrate that OsNAR2.1 regulates OsNRAMP3 to enhance Fe2+ transport requiring OsNRAMP3 under nitrate conditions. Moreover, the OsNAR2.1-overexpression phenotype was largely maintained in the OE1/NG4081 hybrid, where OsNRAMP3 expression was reduced by only ~60% (i.e., ~40% remaining) compared to the full knockout. This suggests that ~40% of OsNRAMP3 expression is sufficient for interaction with OsNAR2.1, while the remaining OsNRAMP3 may support other roles such as Mn2+ distribution. OsNRAMP3 mediates Mn2+ redistribution in rice but lacks inherent Fe2+ transport (Yamaji et al., 2013; Yang et al., 2013). While its heterologous expression rescued the yeast Mn2+-uptake mutant Δsmf1, co-expression with OsNAR2.1 did not further enhance this Mn2+ transport (Figure 1E), consistent with prior studies (Yamaji et al., 2013; Yang et al., 2013). Neither OsNRAMP3 nor OsNAR2.1 individually complemented Fe2+ transport in yeast mutant DEY1453, but their co-expression restored Fe2+ transport (Figure 1F), indicating that their interaction confers a novel Fe2+ transport activity. This was supported by hydroponic data showing that osnramp3 mutants had disturbed Fe2+ distribution under Fe2+ deficiency (Figure S8A). Furthermore, OsNRAMP3 localization shifts from the plasma membrane (WT) to the endoplasmic reticulum in osnar2.1 mutants (Figure S12). Previous studies have reported that OsNAR2.1 regulates OsMADS25 translocation from the cytoplasm to the nucleus (Wang et al., 2025). Therefore, we propose that OsNAR2.1 similarly promotes OsNRAMP3 trafficking from the ER to the plasma membrane to facilitate Fe2+ uptake. Expression of pOsNAR2.1:OsNAR2.1 enhances nitrate absorption, nitrogen use efficiency, and yield (Chen et al., 2017). Under nitrate supply, transgenic lines accumulated more Fe2+ and Mn2+ in the roots and shoots (Figure S1), whereas osnramp3 mutants demonstrated impaired accumulation of these metals at 0.2 mM NO3− (Figures S8, S9), disrupting Fe2+ transport and growth (Figure S8A). Partial (but not full) OsNRAMP3 expression in hybrid lines (OE1/NG4081) recovered Fe2+ levels and grain yield, whereas full knockout lines (OE1/NG3081) failed to achieve these recoveries (Figures 1L–O), confirming that OsNAR2.1-mediated Fe2+ transport depends on OsNRAMP3. In this study, we focus on Fe/Mn; OsNAR2.1—a known partner for multiple transporters—may have broader roles, as its expression responded to deficiencies of Ca2+, Mg2+, K+, Fe2+, and Mn2+, and its knockout/overexpression altered Mg, Ca, and K contents, pointing to possible avenues of research in the future. Given the global prevalence of iron deficiency anemia, the OsNAR2.1-OsNRAMP3 module presents a promising Fe-biofortification strategy to develop high-yield, high-Fe rice, addressing ‘hidden hunger’. This work was financially supported by the National Key Research and Development Program of China (2021YFF1000404), the National Natural Science Foundation of China (32172665 and 32573135), the Innovation ability construction plan of Zhongshan biological breeding Laboratory (bm2022008-02), the Key Re-search and Development Project of Hainan Province in 2024 (Science and Technology Com missioner Category) (ZDYF2024KJTPY006), 2024 Sanya Science and Technology ‘Fanxing’ Special Scientific Research Project (2024KJFX032), and the Guangdong Basic and Applied Basic Research Foundation (2024A1515030094). We would like to thank Dr. Meng Yang (college of agriculture, Guangxi University, Nanning) and Luqing Zheng (Nanjing Agriculture University) for providing the yeast strains and vector for the complementation assay in yeast and Ms. Xiaoli Dai from MOA Key Laboratory of Plant Nutrition and Fertilization in Lower-Middle Reaches of the Yangtze River, Nanjing Agriculture University, for technical support in N concentration assessments of osnramp3 mutants and its relative materials. We also thank Mr. Xianyang Li and Miss Jie Yao for data analysis. The authors declare no conflicts of interest. X.F., J.X., and J.C. planned and designed this study. J.X. and J.C. performed most of the experiments. Bq.L. helped with the Xenopus laevis oocyte experiment. M.Q., X.Z., M.L., Bq.L., and K.Q. helped with the field experiments. Y.M. and Bb.L. extracted RNA and performed the qRT-PCR experiments. H.F., Q.S., and G.X. provided suggestions during the experiments. X.F., J.X., and J.C. wrote the manuscript. All authors helped with the experiments and contributed to the finalization of the manuscript. All authors have read and approved the contents of this paper. Additional Supporting Information may be found online in the supporting information tab for this article: http://onlinelibrary.wiley.com/doi/10.1111/jipb.70105/suppinfo Figure S1 Total concentration and content of Fe2+ and Mn2+ of WT and pOsNAR2.1:OsNAR2.1 transgenic plants at different N supply levels. Figure S2 Effect of osnar2.1 knockout on iron transport under 2.5 mM NH4+ or 0.2 mM NO 3 − ${<mpadded xmlns="http://www.w3.org/1998/Math/MathML">\mathrm{NO}</mpadded>}_{3}^{-}$ supply. Figure S3 Expression patterns of OsNAR2.1 and OsNRAMP3. Figure S4 Expression patterns of OsNAR2.1 and OsNRAMP3 in Dongjin and Nipponbare. Figure S5 Identification of osnramp3 mutants. Figure S6 Comparison of growth of WT and osnramp3 mutants at different nitrogen supply levels. Figure S7 NH4+ and NO 3 − ${<mpadded xmlns="http://www.w3.org/1998/Math/MathML">\mathrm{NO}</mpadded>}_{3}^{-}$ influx rates of WT and osnramp3 mutants measured using 15N-enriched sources. Figure S8 Comparison of growth of WT and osnramp3 mutants under control and Fe2+-deficient conditions. Figure S9 Comparison of growth of WT and osnramp3 mutants under control and Mn2+-deficient conditions. Figure S10 Expression of OsNRAMP3 and OsNAR2.1 in WT, osnar2.1 knockdown mutants, OsNAR2.1 overexpression lines, and osnramp3 mutants. Figure S11 Identification of the hybrid lines. Figure S12 OsNRAMP3 subcellular localization in rice protoplast of WT and the osnar2.1 mutant. Table S1 Primers used for qRT-PCR. Table S2 PCR primers used for homozygous identification of osnramp3 mutants. Table S3 PCR primers used for Co-IP. Table S4 PCR primers used for the experiment on Fe2+ absorption in Xenopus laevis oocytes. 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