NRT1.1B improves selenium concentrations in rice grains by facilitating selenomethinone translocation

Abstract

Selenium (Se) is an essential trace element for humans and other animals, yet approximately one billion people worldwide suffer from Se deficiency. Rice is a staple food for over half of the world's population that is a major dietary source of Se. In paddy soils, rice roots mainly take up selenite. Se speciation analysis indicated that most of the selenite absorbed by rice is predominantly transformed into selenomethinone (SeMet) and retained in roots. However, the mechanism by which SeMet is transported in plants remains largely unknown. In this study, SeMet uptake was found to be an energy-dependent symport process involving H⁺ transport, with neutral amino acids strongly inhibiting SeMet uptake. We further revealed that NRT1.1B, a member of rice peptide transporter (PTR) family which plays an important role in nitrate uptake and transport in rice, displays SeMet transport activity in yeast and Xenopus oocyte. The uptake rate of SeMet in the roots and its accumulation rate in the shoots of nrt1.1b mutant were significantly repressed. Conversely, the overexpression of NRT1.1B in rice significantly promoted SeMet translocation from roots to shoots, resulting in increased Se concentrations in shoots and rice grains. With vascular-specific expression of NRT1.1B, the grain Se concentration was 1.83-fold higher than that of wild type. These results strongly demonstrate that NRT1.1B holds great potential for the improvement of Se concentrations in grains by facilitating SeMet translocation, and the findings provide novel insight into breeding of Se-enriched rice varieties.

Keywords: NRT1.1B; rice (Oryza sativa L.); selenite; selenomethionine; transport.

Figure 1

Assays of Se speciation in rice seedlings supplied with selenite. (a) Chromatograms of Se speciation based on HPLC‐ICP‐MS. (b) Se concentrations of different Se species in roots, leaf sheaths and leaf blades when supplied with selenite for 3 days and then cultured for another 3 days without Se. Values are the means ± SD (n = 3).

Figure 2

Assays of physiological characteristics of SeMet uptake by rice seedlings. (a) Concentration‐ and (b) time‐dependent SeMet uptake kinetics. (c) Effects of respiration inhibitors on SeMet uptake. (d) Competition assay of SeMet uptake using different amino acids. Values are the means ± SD (n = 3). Asterisks indicate significant differences between control and treatments as evaluated by Student's t‐tests: *P < 0.05 and **P < 0.01.

Figure 3

SeMet transport activity assays in yeast and oocyte. (a) Relative expression of NRT1.1B in yeast transformed with pYES2 empty vector and pYES2‐NRT1.1B. (b) SeMet transport rate in yeast transformed with pYES2 empty vector and pYES2‐NRT1.1B. (c) SeMet transport rate in the oocyte injected with NRT1.1B cRNA compared with that in the oocyte injected with water. Values are the means ± SD (n = 5 for yeast and n = 8 for oocyte). Asterisks indicate significant differences between pYES2 yeasts and pYES2‐NRT1.1B yeasts or between water and NRT1.1B cRNA injected oocytes as evaluated by Student’s t‐tests: *P < 0.05 and **P < 0.01.

Figure 4

(a) SeMet uptake was repressed in nrt1.1b mutant. (a) Concentration‐dependent kinetics of SeMet uptake by roots and (b) accumulation in shoots between wild type (ZH11) and nrt1.1b mutant. Values are the means ± SD (n = 3).

Figure 5

Overexpression of NRT1.1B enhances SeMet transport in rice seedlings. SeMet concentrations in xylem sap collected from different NRT1.1B‐overexpressing lines (a) OE‐31/OE‐72 and (c) OEvp‐21/OEvp‐43 supplied with 50 μm SeMet for 6 h and then transferred back to nutrient solutions without Se for 1 day. SeMet concentrations in rice roots, leaf sheaths and leaf blades of overexpressing lines (b) OE‐31/OE‐72 and (d) OEvp‐21/OEvp‐43 supplied with SeMet for 3 h and then transferred back to nutrient solutions without Se for 3 days. The wild type controls for OE‐31/OE‐72 and OEvp‐21/OEvp‐43 are Nipponbare (Nip) and Zhonghua 11 (ZH11) respectively. Values are the means ± SD (n = 3). Asterisks indicate significant differences between wild‐type plants and transgenic lines as evaluated by Student's t‐tests: *P < 0.05 and **P < 0.01.

Figure 6

Overexpression of NRT1.1B increases Se concentrations in transgenic rice. Se concentration assays in the seedlings of NRT1.1B overexpressing lines (a) OE‐31/OE‐72 and (b) OEvp‐21/OEvp‐4 when supplied with selenite. Se concentration assays in grains of NRT1.1B overexpressing lines (c) OE‐31/OE‐72 and (d) OEvp‐21/OEvp‐43. Values are the means ± SD (n = 3). Asterisks indicate significant differences between wild‐type plants and transgenic lines as evaluated by Student's t‐tests: *P < 0.05 and **P < 0.01.