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OsPT4 Facilitates Selenomethionine Transport and Biosynthesis to Enhance Seed Accumulation in Rice: Molecular Mechanisms and Biotechnological Potential.
Yang Y, Sun L, Wei J, Zhang F, Yang S, Zhang J, Qin Q, Wang J, Xu G, Sun S, Sun Y, Xue Y.
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Selenium (Se) is a vital micronutrient for humans, with important functions for health and anti-cancer properties. Organic Se shows higher antioxidant activity and much lower toxicity compared to inorganic Se, making it safer for use. Selenomethionine (SeMet) is one of the primary forms of organic Se. OsPT4, the high-affinity phosphate (Pi) transporter (PHT) of rice, has been investigated for its role in the transport of the different forms of Se, and its effects on the accumulation of SeMet in this study. The OsPT4 mutant and overexpression lines were used as research materials. Phenotypic analyses revealed that OsPT4 confers improved Se tolerance in shoots upon selenite exposure. Heterologous expression assays in Xenopus laevis oocytes and yeast systems and translocation assays in different transgenic lines of rice confirmed OsPT4-mediated selenite and SeMet transport activity, establishing its responsibility for root-to-shoot Se translocation. Transcriptomic profiling, amino acid quantification and qRT-PCR analyses further indicated that OsPT4 up-regulates methionine (Met) biosynthesis, the direct precursor of SeMet. Notably, OsPT4 significantly increased SeMet accumulation and promoted the formation of Se-rich micron-sized spherical particles in seeds under Se supplementation. These findings provide mechanistic insights into OsPT4-mediated SeMet trafficking and metabolism, advancing strategies for developing Se-biofortified rice cultivars with enhanced nutritional and therapeutic value.
GrantNo.B2024003 Shanghai Agricultural Science and Technology Innovation Program, 23ZR1469200 Natural Science Foundation of Shanghai, 23QB1405900 Shanghai Rising-Star Program, SK-JC 2023-1 Flagship Project of Eco-Environmental Protection Research Institute, SAAS, Hu Nong Ke AI-A(2025) 007 AI-Empowered Agriculture Project of Shanghai Academy of Agricultural Sciences, NAES035AE03 National Agricultural Experimental Station for Agricultural Environment, Hu-Nong-Ke-Zhuo 2022 (008) Outstanding Team Program of Shanghai Academy of Agricultural Science
FIGURE 1. Influence of OsPT4 on Se accumulation and translocation under different Se treatments. (a and b) The relative expression of OsPT4 under different Se concentrations (0, 0.5, 2 μM selenite) with various processing durations (12 h, 24 h, 3 days and 7 days) in roots (a) and shoots (b). OsACTIN (Loc_Os10g36650) was used as an internal control. (c and d) The total Se concentration in roots (c) and shoots (d) of different transgenic lines (ospt4, Ox1 and Ox2) under different Se concentrations. (e) Se accumulation rate of transgenic lines and WT under different Se treatments. The kinetics of Se accumulation rate of different lines within 20 min. The data are presented as means ± SE (n = 4 in a, b and e, n = 6 in c and d). Different letters showed significant differences in total Se concentration of roots and shoots under different treatments between WT, ospt4, Ox1 and Ox2 (p < 0.05, Student's t‐test).
FIGURE 2.
OsPT4 enhances the tolerance of shoots under Se‐riched conditions. (a) The growth phenotype of WT and OsPT4 transgenic lines under various Se treatments. (b and c) Roots and shoots biomass of the WT and transgenic plants under different Se treatments. Seedlings (14‐day old) of the WT were grown hydroponically in nutrient‐rich solution and then under 0, 0.2, 0.5, 1 and 2 μM Se treatments for 14 days, respectively. (d and e) Four‐parameter Logistic regression model between Se concentration in roots and shoots and roots (d) and shoots (e) biomass. Bar in (a) =8 cm. The data are presented as means ± SE (n = 6). Different letters in (b and c) showed significant differences in shoots and roots biomass under different treatments between WT, ospt4, Ox1 and Ox2 (p < 0.05, Student's t‐test).
FIGURE 3. Effects of OsPT4 on the accumulation and proportion of organic and inorganic Se in shoots. (a and b) Total organic (a) and inorganic (b) Se concentration of the transgenic lines and WT in shoots under different selenite treatments. (c) The proportion of organic and inorganic Se concentration of the transgenic lines and WT under different Se treatments. (d–g) Selenite and different forms of organic Se (SeMet, SeCys and MeSeCys) of the WT and OsPT4 transgenic plants under different Se applications. Seedlings (21‐day old) of the WT were grown hydroponically in nutrient‐rich solution and then under 0, 0.2, 0.5, 1 and 2 μM in (a–c) and 0.5 (Se1) and 2 (Se2) μM Se treatments in (d) for 14 days, respectively. Shoots were harvested for different forms of Se concentration analysis. Data are shown as mean ± SE (n = 4). Different letters in showed significant differences between WT, ospt4, Ox1 and Ox2 (p < 0.05, Student's t‐test).
FIGURE 4. OsPT4 plays a function in SeMet uptake and translocation. (a and b) SeMet (a) and selenite (b) uptake rate in yeast transformed with pY112 empty vector and pY112‐OsPT4. (c) SeMet transport rate in the oocyte injected with OsPT4 cRNA compared with that in the oocyte injected with water. Values are the means ± SD (n = 5 for yeast and n = 7 for oocyte). (d) Concentration‐dependent kinetics of SeMet uptake by roots of different transgenic lines and WT. (e) SeMet concentration in the xylem sap under 20 μM SeMet application. (f) The SeMet concentration in roots and shoots of different transgenic lines and WT under SeMet application. Values are the means ± SD (n = 4 in rice). Different letters showed significant differences between WT, ospt4, Ox1 and Ox2 (p < 0.05, Student's t‐test).
FIGURE 5. The metabolic network analysis of Cys and Met metabolism pathway in shoot of mutant and overexpression line. (a) All the down‐/up‐regulated DEGs in this pathway were shown here. The degrees of the colours indicated the standardised expression levels down/up‐regulated by OsPT4 mutation and overexpression (Log2FC) under different Se treatments comparing with WT. Seedlings (14‐day‐old) of transgenic lines (ospt4 and Ox1) and WT were grown hydroponically in nutrient rich solution and then subjected to 7 days 0 (Se0), 0.5 (Se1) and 2 (Se2) μM Se treatments. The yellow arrows highlight the up‐regulation of genes, while the red arrows emphasise the down‐regulation of genes. The red boxes and the different numbers within them represent various reaction pathways within that metabolism pathway. (b and c) The Cys and Met concentration in shoots of transgenic lines and WT under different Se concentrations. (d) The relative expression of genes related to L‐Methionine biosynthesis (Loc_Os09g28050 and Loc_Os10g28350) and degradation (Loc_Os09g25620). Shoots were harvested for qRT‐PCR analysis after treating with different Se concentrations for 7 days. OsACTIN (Loc_Os10g36650) were used as internal controls. The relative expression of Loc_Os09g28050, Loc_Os10g28350 and Loc_Os09g25620 under Se0 conditions was standardised to 1. Values are means ± SE (n = 4). Bar heights represent the average of two technical and four biological replicates. Different letters in (b) indicate that the values differ significantly within each time point separately (p < 0.05, Student's t‐test).
FIGURE 6. Effects of OsPT4 on the accumulation of Se different forms. (a–c) Total Se concentration in the leaf blade, leaf sheath and culm (a), seed (b) and husk (c) of the WT and OsPT4 transgenic plants under different Se treatments. (d–g) Organic and different forms of organic Se concentration in the seed of the WT and OsPT4 transgenic plants under different Se applications. Data are shown as mean ± SE (n = 4). Different letters in (a–g) indicate significant differences between WT, ospt4, Ox1 and Ox2 (p < 0.05, Student's t‐test).
FIGURE 7. Effects of OsPT4 on the accumulation of Se‐rich micron‐sized particles in endosperm. (a) The SEM image of endosperm and embryo of the WT and OsPT4 transgenic plants under 5 and 10 mg/kg Se application. (b and c) The diameter (b) and density (c) of spherical particles in endosperm of different lines under 5 and 10 mg/kg Se application. (d) Observation of Se enrichment in micron‐sized spherical particles in endosperm using TOF‐SIMS. Data are shown as mean ± SE (n = 4). Βap = 10 μm in (a). Asterisks in (b and c) indicated significant differences between WT and transgenic lines (“*”: p < 0.05; “**”: p < 0.01; “***”: p < 0.005, Student's t‐test).
