Knocking-OutTriggers Up-Regulation ofExpression and Enhances Zinc Accumulation in Rice

Supplemental Data

Table S1. List of primers in this study.

Fig. S1. Tissue expression pattern ofat the reproductive stage.

A, Root. B, Stem. C, Node. D, Leaf sheath. E, Spikelet.

The tissues were harvest from transgenic plants expressing thegene driven by thepromoter at the reproductive stage.

Fig. S2. Subcellular localization of OsPDR7 protein determined in.

For each experiment, ≥ 20 individual cells were examined using a Zeiss LSM880 confocal laser scanning microscope (Carl Zeiss, Germany). Scale bars, 20 μm.

Fig. S3. Schematic diagram ofgene structure and CRISPR/Cas9 mutations in three mutant lines.

Exons andintrons are indicated by black rectangles and black lines, respectively.,, andplants harbor single site mutations. Nucleotide deletion and insertion sites are indicated with red arrows. WT, Wild type. The three bases of AGG in red frame represent protospacer adjacent motif (PAM).

Fig. S4. Phenotypes of wild type (WT) andknockout line plants at the grain filling stage.

A, Phenotypes of the wild type (WT) andknockout line plants.

B-E, Plant height and several yield related traits.

Values represent Mean ± SD (≥ 3). Statistical comparisons were performed with the Tukey’s HSD mean-separation test against the WT (*,< 0.05; **,< 0.01).

Fig. S5. Concentration, uptake and distribution of Zn, Fe, Mn and Cu in rice shoots and roots at vegetative stage.

A, D, G and J, Concentrations of Zn, Fe, Mn, and Cu in shoots and roots.

B, E, H and K, Uptakes of Zn, Fe, Mn, and Cu in the wild type (WT) andknockout lines.

C, F, I and L, Comparisons of distribution ratios of Zn, Fe, Mn and Cu between WT andknockout lines.

Seedlings of the WT andknockout lines were grown in 0.5× Kimura B nutrient solution for 28 d. Shoots and roots were harvested separately and ion concentrations were determined by inductively coupled plasma-optical emission spectrometer (ICP-OES-5110, Agilent, USA). Values represent Mean ± SD (= 3). Statistical comparisons were performed with the Tukey’s HSD mean-separation test against WT (*,< 0.05; **,< 0.01).

Fig. S6. Uptake, concentration, and distribution of rubidium (Rb) in shoots and roots at the vegetative stage.

A, Rb uptake of wild type (WT) andknockout line plants (to) at the vegetative stage.

B, Rb concentration in the shoots and roots of WT andmutants.

C, Rb distribution in the roots and shoots in WT andknockout line plants. The WT andmutants were grown in 0.5× Kimura B (KB) nutrient solution for 21 d, then transferred to 0.5× KB containing 0.4 μmol/L67ZnSO4for 2 d with 0.4 μmol/L RbCl2, and the shoots and roots were then harvested for elemental analysis.

Values represent Mean ± SD (= 3). Statistical comparisons were performed with the Tukey’s HSD mean-separation test against WT.

Fig. S7. Fe, Cu and Mn concentrations in different tissues inknockout lines () and wild type (WT) under field conditions.

A and B, Fe concentration in brown rice and in eight other tissues at the reproductive stage.

C and D, Cu concentration in brown rice and in eight different rice tissues.

E and F, Mn concentration in brown rice and in eight different rice tissues.

Plants of WT andmutants were grown in a paddy field until the grain was ripe. Values represent Mean ± SD (= 3). Statistical comparisons were performed with the Tukey’s HSD mean-separation test against the wild type (*,< 0.05).

Fig. S8. Genotypic identification and agronomic traits ofinsertion mutant.

A, Tos17 insertion site in. F, R and T represent the forward primer, reverse primer, andinsertion primer, respectively.

B, Genotypic identification of. The F and R primers were used to amplify the ‘A’ fragment from the Nipponbare wild type (WT) in lanes 1, 3, 5, 7, 9 and 11; the T and R primers were used to amplify the Tos17 insertion fragment ‘a(chǎn)’ in lanes 2, 4, 6, 8, 10 and 12. ‘a(chǎn)a’ represents the homozygous mutant allele; AA represents the homozygous WT allele, and Aa represents the heterozygote.

C, Plant height, tiller number per plant, 1000-grain weight, seed-setting rate ofand WT. Values represent Mean ± SD (= 3). Statistical comparisons were performed with the Tukey’s HSD mean-separation test against WT (*,< 0.05).

Fig. S9. Concentrations of Zn, Fe, Mn and Cu inmutant plants at grain filling stage.

Zn(A), Fe(C), Mn (E) and Cu (G) concentrations in brown rice. Zn(B), Fe (D), Mn (F) and Cu (H) concentrations in seven other tissues.

Plants of the wild type (WT) andmutant were grown in a paddy field until the grains were ripe.Values represent Mean ± SD (= 3). Statistical comparisons were performed with the Tukey’s HSD mean-separation test against WT (*,< 0.05).

Fig. S10. Expression level ofin roots of overexpression lines (to).

Fig. S11. Fe, Cu and Mn concentrations in different tissues inoverexpression lines (and) and the wild type (WT) under field conditions.

Fe (A), Cu (C) and Mn (G) concentrations in brown rice.

Fe (B), Cu (D) and Mn (F) concentrations in eight other tissues.

Plants of WT andoverexpression were grown in a paddy field until the grains were ripe. Values represent Mean ± SD (= 3). Statistical comparisons were performed with the Tukey’s HSD mean-separation test against WT (*,< 0.05).

Fig. S12. Expression levels of OsZIP genes andin roots of theknockout lines (to) and wild type (WT).

Plants of theknockout lines and WT were grown in 0.5 × KB nutrient solution for 28 d, and the roots were then collected to assay the expression levels ofand. Values represent Mean ± SD (= 3). Statistical comparisons were performed with the Tukey’s HSD mean-separation test against WT.

Fig. S13. Expression levels of Fe-related genes in roots ofknockout lines (to) and wild type (WT).

Plants of theknockout lines and WT were grown in 0.5 × KB nutrient solution for 28 d, and the roots were then collected to assay the expression levels of Fe-related genes. Values represent Mean ± SD (= 3). Statistical comparisons were performed with the Tukey’s HSD mean-separation test against WT.

Fig. S14. Differentially expressed genes validated by qRT-PCR.

Plants of theknockout lines () and wild type (WT) were grown in 0.5 × KB nutrient solution for 28 d, and the roots were then collected to assay the expression levels of six related genes. Values represent Mean ± SD (= 3). Statistical comparisons were performed with the Tukey’s HSD mean-separation test against WT (*,<0.05; **,< 0.01).