Boosting selenium accumulation in rice grains by enhancing NRT1.1B-mediated MeSeCys translocation

Dear Editor,

Selenium (Se) deficiency affects over one billion people worldwide. Enhancing Se accumulation in rice grains through biofortification represents an effective strategy to boost dietary Se intake. Rice (Oryza sativa L.), a staple food for half of the global population, provides less than 70% of the daily recommended dietary Se due to its low grain Se concentration, which predominantly consists of selenomethionine (SeMet) (Williams et al., 2009; Zeng et al., 2023). SeMet has lower bioavailability than methyl-selenocysteine (MeSeCys), a compound found in rice roots that exhibits superior anticancer and antioxidant properties (Suzuki et al., 2007; Zhang et al., 2019). However, the mechanisms by which MeSeCys is transported to grains remain unclear. NRT1.1B, a member of the nitrate transporter 1/peptide transporter (NRT1/PTR) family, regulates nitrate transport and has been previously shown to transport SeMet (Hu et al., 2015; Zhang et al., 2019). In this study, we demonstrate that NRT1.1B functions as a bidirectional vascular transporter through both the xylem and phloem. It facilitates the uptake, transport, storage, and redistribution of MeSeCys, thereby increasing Se concentration in rice grains. This finding reveals a novel mechanism to enhance Se biofortification by promoting MeSeCys transport.

Through comprehensive physiological, genetic, and heterologous expression analyses, we found that the concentration- and time-dependent uptake of MeSeCys in rice roots exhibits saturation patterns consistent with Michaelis-Menten kinetics, indicating a carrier-mediated transport mechanism (Figures 1A and 1B). MeSeCys uptake is pH-dependent, with an optimal range between pH 5.0 and 6.0, and declines significantly under acidic (<5.0) or alkaline (>6.0) conditions (Figure 1C). This optimal pH range coincides with the activity range of plasma membrane H⁺-ATPase (pH 5.5–6.5), which generates the proton motive force essential for active transport (Zhou et al., 2021). Here, carbonyl cyanide m-chlorophenylhydrazone, a respiration inhibitor that dissipates the proton motive force, significantly inhibited MeSeCys uptake (Figure 1D), indicating that MeSeCys uptake is energy dependent and requires a maintained proton gradient. Competitive inhibition assays revealed that basic amino acids, such as lysine (Lys), arginine (Arg), and histidine (His), and acidic amino acids such as aspartic acid (Asp) and glutamic acid (Glu), minimally inhibited MeSeCys uptake. In contrast, neutral amino acids, particularly cysteine (Cys), methionine (Met), tyrosine (Tyr), and leucine (Leu), significantly suppressed MeSeCys uptake (Figure 1E). Previous studies have shown that NRT1.1B is involved in SeMet transport (Hu et al., 2015; Zhang et al., 2019). Here, Met, a structural analog of SeMet, inhibited MeSeCys uptake by 76%, suggesting that NRT1.1B may also transport MeSeCys (Figure 1E). Supporting this, heterologous expression of NRT1.1B in Saccharomyces cerevisiae (pYES2-NRT1.1B) significantly enhanced MeSeCys uptake, demonstrating NRT1.1B’s transport activity for MeSeCys (Figures 1F and 1G).

Figure 1.

Physiological characteristics of rice roots in methylselenocysteine (MeSeCys) uptake and the role of NRT1.1B in mediating MeSeCys uptake and transport.

(A and B) Concentration- (A) and time-dependent (B) kinetics of MeSeCys uptake in rice roots. Data are represented as mean ± SD (standard deviation) (n = 3).

(C) Effects of pH on MeSeCys uptake. Data are represented as mean ± SD (n = 3). Different letters indicate significant differences among pH treatments (one-way ANOVA with Tukey’s test; P < 0.05).

(D) Effects of the respiratory inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP) on MeSeCys uptake. Data are represented as mean ± SD (n = 3). Asterisks indicate significant differences between the CCCP treatment and the control (Student’s t-test, ∗∗∗∗P < 0.0001).

(E) Effects of amino acids on MeSeCys uptake. Data are represented as mean ± SD (n = 3). Different letters indicate significant differences from the control (Student’s t-test, ∗∗∗∗P < 0.0001).

(F and G) Relative expression of pYES2 and pYES2-NRT1.1B in yeast (F), and the rate of MeSeCys uptake (G). Data are represented as mean ± SD (n = 3). Asterisks indicate significant differences between pYES2 and pYES2-NRT1.1B treatments (Student’s t-test, ∗∗∗P < 0.001).

(H and I) MeSeCys uptake in excised roots (H) and leaf blades (I) of NRT1.1B-overexpressing and nrt1.1b mutant lines. Data are represented as mean ± SD (n = 3). Different letters indicate significant differences among transgenic lines (one-way ANOVA with Tukey’s test, P < 0.05).

(J–M) Selenium (Se) concentration (J and K) and transport ratio (L and M) in NRT1.1B-overexpressing and nrt1.1b mutant lines after 3 and 6 days of MeSeCys treatment. Data are represented as mean ± SD (n = 3). Different letters indicate significant differences among transgenic lines (one-way ANOVA with Tukey’s test, P < 0.05).

(N) Se concentrations in the roots of NRT1.1B-overexpressing and nrt1.1b mutant lines after continuous MeSeCys supply before anthesis. Data are represented as mean ± SD (n = 3). Different letters indicate significant differences (one-way ANOVA with Tukey’s test, P < 0.05).

(O) Effects of foliar MeSeCys spraying before flowering on Se concentrations in various tissues of NRT1.1B-overexpressing and nrt1.1b mutant lines. Data are represented as mean ± SD (n = 3). Different letters indicate significant differences among transgenic lines (one-way ANOVA with Tukey’s test, P < 0.05).

(P and Q) Se concentrations in leaf blades (P) and transport efficiency of MeSeCys from treated leaf blades to other parts of the rice plant (Q) in NRT1.1B-overexpressing and nrt1.1b mutant lines with foliar spray of MeSeCys. Data are represented as mean ± SD (n = 3). Different letters indicate significant differences among transgenic lines (one-way ANOVA with Tukey’s test, P < 0.05).

Furthermore, transgenic rice lines overexpressing NRT1.1B showed significantly enhanced MeSeCys uptake in both roots and leaves, whereas the nrt1.1b mutant exhibited significant reductions (Figures 1H and 1I). In roots, NRT1.1B facilitates MeSeCys entry into epidermal and cortical cells, followed by cell-to-cell transport via plasmodesmata to xylem parenchyma. This pathway bypasses the Casparian strip-regulated apoplastic barrier, enabling efficient radial transport into root xylem vessels. Similarly, foliar uptake depends on NRT1.1B-mediated transmembrane transport in mesophyll cells. The localization of this transporter within the membranes of epidermal, cortical, and mesophyll cells facilitates the influx of MeSeCys into roots and leaves (Hu et al., 2015).

Importantly, overexpression of NRT1.1B significantly enhanced Se translocation in rice when roots were supplied with MeSeCys, resulting in increased Se concentrations in the leaves of NRT1.1B-overexpressing lines NT31 and NT72 compared to the wild type (Figures 1J and 1K). The enhanced leaf blade/root, leaf sheath/root, and shoot/root Se allocation ratios—highlight the key role of NRT1.1B in promoting root-to-shoot MeSeCys transport (Figures 1L and 1M). This enhanced Se translocation correlates with NRT1.1B expression in root stelar cells (Hu et al., 2015), where it facilitates the loading of MeSeCys into xylem vessels through H⁺-coupled symport at the xylem -_parenchyma membranes. This process promotes transpiration-driven transport to the shoots, resulting in higher shoot Se concentrations in NT31 and NT72 compared to the wild type (Figures 1L and 1M).

Additionally, NRT1.1B facilitates the storage and remobilization of MeSeCys in rice through both the xylem and phloem. Leaf sheaths and internodes are recognized as transient nutrient reservoirs (Xu et al., 2021; Hu et al., 2022). The significant increase in Se concentrations in leaf sheaths and internodes of NRT1.1B-overexpressing lines, contrasted with the decline in the nrt1.1b mutant (Figures 1N and 1O), demonstrates that NRT1.1B facilitates the loading of MeSeCys into xylem vessels from root parenchyma cells and supports long-distance upward transport through the xylem. Upon reaching aerial tissues, MeSeCys is unloaded from xylem vessels into the parenchyma cells of internodes and leaf sheaths via NRT1.1B, where it is temporarily stored. During the grain-filling stage, MeSeCys stored in leaf sheaths, internodes, and leaf blades is remobilized through phloem transport. NRT1.1B mediates the loading of MeSeCys into phloem sieve tubes from parenchyma cells, ensuring efficient transport to developing grains. This process mirrors the pattern of carbohydrate remobilization, where non-structural sugars are catabolized to support grain filling (Xu et al., 2021; Hu et al., 2022). NRT1.1B thus enables continuous transport of MeSeCys in both xylem (root-to-shoot) and phloem (shoot-to-grain) pathways, optimizing MeSeCys distribution and promoting efficient Se allocation between leaves and developing grains.

Crucially, continuous application of MeSeCys through root and foliar uptake for 3 weeks prior to flowering significantly increased Se concentrations in grains, as well as in leaf blades, leaf sheaths, nodes, and internodes of NRT1.1B-overexpressing lines compared to the wild type (Figures 1N and 1O). This enhancement confirms the role of NRT1.1B in facilitating the loading of MeSeCys into xylem vessels for shoot translocation and coordinating MeSeCys redistribution from shoots to grains via the phloem. In contrast, the nrt1.1b mutant displayed significant reductions in grain Se concentrations, underscoring the key role of the transporter in MeSeCys translocation and redistribution, as disrupted loading in the xylem and phloem hinders the transport and redistribution of MeSeCys to developing grains.

Remarkably, NRT1.1B’s bidirectional transport capacity is evidenced by foliar-applied MeSeCys through both leaf soaking and spraying. Overexpression of NRT1.1B significantly enhanced MeSeCys transport from treated leaves downward to the roots and upward to the upper leaves and grains (Figures 1O–1Q). When lower leaves were soaked in MeSeCys solution, NT31 and NT72 exhibited higher Se concentrations in upper leaves and roots compared to the wild type (Figure 1P). Additionally, foliar spraying of MeSeCys increased Se concentrations in leaf blades, sheaths, nodes, internodes, grains, and roots in NT31 and NT72 (Figure 1O). In contrast, significant reductions in these tissues were observed in the nrt1.1b mutant. These results strongly demonstrate the crucial role of NRT1.1B in phloem-mediated bidirectional transport to grains and roots, emphasizing its importance in leaf-to-grain translocation. Rice’s structurally interconnected vascular system, including roots, stems, and leaves, facilitates this bidirectional MeSeCys transport, with nodes acting as hubs directing flow (Yamaji and Ma, 2017).

Our study demonstrates that NRT1.1B facilitates the uptake, transport, storage, and redistribution of MeSeCys in rice. Specifically, NRT1.1B optimizes the bidirectional, dual vascular translocation of MeSeCys, leading to greater Se accumulation in rice grains. These findings establish NRT1.1B as a strategic target to enhance Se delivery via MeSeCys transport from roots and leaves to developing grains.

Funding

This research was funded by the National Natural Science Foundation of China (U1904114 and 32030099).

Acknowledgments

We are very grateful to the Experimental Teaching Center of Agricultural University for its strong support of this experiment. No conflict of interest declared.

Author contributions

Conceptualization: Y.L., L.Z., B.H., and C.C.; methodology: Y.L., Y.W., H.Y., X.G., X.L., R.L., S.L., M.H., Z.W., F.Y., and X.W.; formal analysis: Y.L., H.Y., and L.Z.; investigation, L.Z.; writing – original draft: Y.L.; writing – review & editing: L.Z., B.H., and C.C.; supervision: L.Z., B.H., and C.C.; funding acquisition: L.Z. All authors have read and approved the final version of the manuscript.

Published: June 11, 2025