Production of grains with ultra-low heavy metal accumulation by pyramiding novel Alleles of OsNramp5 and OsLsi2 in two-line hybrid rice

Abstract

Ensuring rice yield and grain safety quality are vital for human health. In this study, we developed two-line hybrid rice (TLHR) with ultra-low grain cadmium (Cd) and arsenic (As) accumulation by pyramiding novel alleles of OsNramp5 and OsLsi2. We first generated low Cd accumulation restorer (R) lines by editing OsNramp5, OsLCD, and OsLCT1 in japonica and indica. After confirming that OsNramp5 was most efficient in reducing Cd, we edited this gene in C815S, a genic male sterile line (GMSL), and screened it for alleles with low Cd accumulation. Next, we generated R and GMSL lines with low As accumulation by editing OsLsi2 in a series of YK17 and C815S lines. When cultivated in soils that were heavily polluted with Cd and As, the edited R, GMSL, and TLHR plants showed significantly reduced heavy metal accumulation, while maintaining a relatively stable yield potential. This study provides an effective scheme for the safe production of grains in As- and/or Cd-polluted paddy fields.

Keywords: Ultra‐low heavy metal accumulation; arsenic; cadmium; gene pyramiding; grain safety; two‐line hybrid rice.

Figure 1

Evaluation of Cd accumulation ability among the three key Cd genes in japonica and indica rice. (a) Structure of the key edited genes Os07g0257200 (OsNramp5), Os01g0956700 (LCD), and Os06g0579200 (OsLCT1). Red arrows indicate CRISPR/Cas9 target sites. (b) Identification of OsNramp5, LCD, and OsLCT1 mutations by sequencing the target site in T₀ transgenic lines. (c) Panicle phenotypes of homozygous lines generated by editing OsNramp5, LCD, and OsLCT1 in YK17 (left) and JH212 (right) backgrounds. Bar = 3 cm. (d) Whole plant phenotypes of homozygous lines generated by editing OsNramp5, LCD, and OsLCT1 in the YK17 (above) and JH212 (below) backgrounds. Bar = 20 cm. (e) Statistical analysis of yield‐related traits of homozygous lines generated by editing OsNramp5, LCD, and OsLCT1 in YK17 (left) and JH212 (right) backgrounds. The light‐green background is a genotype with excellent yield traits under the same genetic background. (Values are means ± SD. *P < 0.05; **P < 0.01, n = 10, two‐tailed Student's t‐test, three independent experiments). (f) Contents of Cd in grains of each edited line after cultivation in cement pools containing different Cd concentrations in the soil. Data are mean ± SD. (n = 3 biologically independent samples). Different letters indicate significant differences at P < 0.05, according to the one‐way ANOVA and Tukey's multiple comparison tests.

Figure 2

Creation of low Cd accumulation male sterile line C815S cultivar by editing OsNramp5. (a) Identification of OsNramp5 mutation by sequencing the target site in T₀ transgenic lines. (b) Whole‐plant phenotypes of homozygous lines and panicle phenotypes. Scale bar (left) = 20 cm, scale bar (right) = 2 cm. (c) Evaluation of pollen fertility among the homozygous lines (Bar =100 μm). (d) Contents of Cd in grains of OsNramp5 edited lines cultivated in cement pools containing different Cd concentrations in the soil. Data were analysed using ANOVA, followed by Tukey's multiple comparison test (P < 0.05) with three biological replicates.

Figure 3

Production of low Cd and/or low As accumulation restorer lines and male sterile lines. (a) Structure of OsLsi2 (Os03g0107300). Red arrows indicate CRISPR/Cas9 target sites. (b) Identification of OsLsi2 mutation in the T₀ generation in YK17, YK17 ^OsNramp5‐1, C815S, and C815S ^OsNramp5‐3 backgrounds. (c) Whole plant phenotypes and their respective panicle phenotypes in YK17 and YK17 ^OsNramp5‐1 backgrounds (top) and the C815S and C815S ^OsNramp5‐3 backgrounds (bottom). (Plant phenotype bar = 20 cm; panicle phenotype bar = 2 cm). (d) Statistical analysis of yield‐related traits of homozygous lines in the backgrounds of YK17, YK17 ^OsNramp5‐1, C815S, and C815S ^OsNramp5‐3. Values are means ± SD. *P < 0.05; **P < 0.01, n = 3, two‐tailed student t‐test). (e) Content of As in grains of OsLsi2 edited lines cultivated in As‐polluted soils under the background of YK17 and YK17 ^OsNramp5‐1. Data were analysed using ANOVA, followed by Tukey's multiple comparison test (P < 0.05) with three biological replicates. (f) As content in grains of OsLsi2 edited lines cultivated in As‐polluted soils in the background of C815S and C815S ^OsNramp5‐3. Data were analysed using ANOVA, followed by Tukey's multiple comparison test (P < 0.05) with three biological replicates.

Figure 4

Two‐line hybrid combinations with low Cd and/or low As accumulation. (a) Plant phenotypes of the four hybrid combinations CLY17, CLY17 ^OsNramp5 , CLY17 ^OsLsi2 and CLY17 ^{OsNramp5 OsLsi2} and their respective panicle phenotypes. Scale bar (left) = 20 cm, scale bar (right) = 2 cm. (b) Statistical analysis of yield‐related traits in hybrid combinations. (Values are means ± SD. *P < 0.05; **P < 0.01, n = 3, two‐tailed student t‐test). (c) Contents of Cd in grains of CLY17, CLY17 ^OsNramp5 , and CLY17 ^{OsNramp5 OsLsi2} hybrids cultivated in soils polluted with different Cd concentrations. Data were analysed using ANOVA, followed by Tukey's multiple comparison test (P < 0.05) with three biological replicates. (d) Contents of As in grains of CLY17, CLY17 ^OsLsi2 , and CLY17 ^{OsNramp5 OsLsi2} hybrids cultivated in soil containing 31.6 ± 1.4 mg/kg total As. Data were analysed using ANOVA, followed by Tukey's multiple comparison test (P < 0.05) with three biological replicates.

Figure 5

Determination of metal concentrations in restorer lines and two‐line hybrid rice combinations. The mutant and WT plants were cultivated together in an experimental paddy field containing 0.483 mg/kg and 26.21 mg/kg of total Cd and As concentrations in the soil, respectively. (a) Cd, (b) As, (c) Mn, (d) Si, (e) Fe, (f) Zn concentrations in grains of restorer lines (YK17, YK17 ^OsNramp5 , YK17 ^OsLsi2 and YK17 ^{OsNramp5 OsLsi2} ) and two‐line hybrid rice combinations (CLY17, CLY17 ^OsNramp5 , CLY17 ^OsLsi2 and CLY17 ^{OsNramp5 OsLsi2} ). Data were analysed using ANOVA, followed by Tukey's multiple comparison test (P < 0.05) with three biological replicates.

Figure 6

Determination of rice grain quality traits in restorer lines and two‐line hybrid rice combinations. (a) Brown rice appearance of restorer lines (YK17, YK17 ^OsNramp5 , YK17 ^OsLsi2 , and YK17 ^{OsNramp5 OsLsi2} ) and two‐line hybrid rice combinations (CLY17, CLY17 ^OsNramp5 , CLY17 ^OsLsi2 , and CLY17 ^{OsNramp5 OsLsi2} ). (b) Gelatinisation characteristics of starch in urea solution. Starch powder was mixed with varying concentrations (1–9 mol/L) of urea solution. (c) Gelatinisation temperature of endosperm starch. Values are means ± SD. **P < 0.01, n = 3, two‐tailed student t‐test). (d) Gel consistency. (e) Alkali‐spreading values. (f) Amylose content. (g) Total starch content. (h) Soluble sugar content. (i) Total protein content. (d–i) Data were analysed by ANOVA followed by Tukey's multiple comparison test (P < 0.05, three biological replicates).