Supplementary figure legends
Figure S1. Analysis of whole genome sequencing (WGS) and RNA-sequencing quality. (a) WGS data filtered by NGS QC Toolkit. (b) The short reads mapped on the Nipponbare reference genome. (c) RNA-seq data filtered by NGS QC Toolkit.
Figure S2. Assessment of salinity tolerance 100 core collection lines. Seeds of 100 rice mutant lines (M10) with wild-type (WT) were germinated and grown in hydroponic solution for 7 days under a 16-h photoperiod. One-week-old seedlings were treated with hydroponic solution containing 100 mM NaCl for 1 week. Lengths of shoots and roots were measured to evaluate salinity sensitivity with 3 biological replicates. Red lines indicate average lengths of shoot and root of WT plants. Values represent means ± SD, bars with or without asterisks indicate significant difference or non-significant, respectively. *p < 0.05, **p < 0.01, and ***p < 0.001, one-way ANOVA with Sidak’s multiple comparison test.
Figure S3. Seed germination rates of wild-type (WT) andsitl1 mutant . Seeds were germinated in hydroponic solution for 7 days under a 16-h photoperiod. Seed germination rates of WT andsitl1 mutant were scored every day for 7days (n = 3 biological replicates with 50 seeds per replicate)
Figure S4. Alleviation of reduced root growth and leaf chlorophyll content by Mg2+ supply in sitl1mutant. Rice seeds of sitl1 mutant and wild-type (WT) were germinated and grown in half-strength KimuraB nutrient solution containing 0, 10, 100, and 500 µM Mg2+ for 7 days. (a) Representative seedling images of sitl1 mutant and WT plants exposed to nutrient solution containing 0 or 500 µM Mg2+. (b) Comparison of fresh weight of root (n= 30 with 3 replicates). (c) Fresh weight of shoot (n = 30 with 3 replicates). (d) Representative leaf images of sitl1 mutant and WT exposed to a nutrient solution containing 500 µM Mg2+. (e) Comparison of total chlorophyll content of leaves of sitl1mutant and WT (n = 6 with 3 replicates). Value represent means ± SD, ns = non‐significant, *p < 0.05, **p< 0.01, and ***p < 0.001, two-way ANOVA with Sidak’s multiple comparison test.
Figure S5. Assessment of salinity and drought tolerance ofsitl1 mutant. Rice seeds of sitl1 mutant and wild-type (WT) were germinated and grown in soil mix for 7 days under a 16-h photoperiod. For the salinity treatment, one-week-old seedlings were irrigated with half-strength nutrient solution containing 0 or 50 mM NaCl for 2 weeks. For the drought treatment, one-week-old seedlings were withheld for 7 days and re-watered for 7 days. (a) Representative images of sitl1 mutant and WT plants exposed to nutrient solution containing 0 (control), 50 mM NaCl (salinity) or drought stress. (b) Comparison of fresh weight of shoot under normal growth condition (n = 30 with 3 replicates). (c) Fresh weight of shoot under salinity stress condition (n = 30 with 3 replicates). (d) Fresh weight of shoot under drought stress condition (n = 30 with 3 replicates). Value represent means ± SD, ns = non‐significant, ***p < 0.001, Student’s t-test.
Figure S6. Heat map analysis and relative fold expression of the genes encoding antioxidant defense enzymes, Na+, and K+ transporters in sitl1 mutant. One-week-old seedlings of sitl1 mutant and wild-type (WT) were used to sample leaf and root tissues. (a) Heat map of genes encoding antioxidant defense enzymes, Na+ and K+transporters in roots of sitl1 and WT (n = 3 replicates). Values of log2 fold-change and q-value were obtained from the RNA-sequencing analyses. (b) Relative expression levels of selected genes encoding antioxidant defense enzymes in roots and leaves ofsitl1 and WT (n = 6 with 3 replicates). (c) Relative expression levels of selected genes encoding Na+ and K+ transporters in roots and leaves of sitl1and WT (n = 6 with 3 replicates). Value represent means ± SD, ns = non‐significant, *p < 0.05, **p < 0.01, and ***p < 0.001, two-way ANOVA with Sidak’s multiple comparison test.
Figure S7. Shared variants of sitl1 mutant and WT via whole-genome sequencing (WGS) analysis. One-week-old seedlings ofsitl1 mutant and wild-type (WT) were used to determine shared variants of sitl1 mutant and WT. The shared SNPs and Indels between sitl1 mutant and WT (Donganbyeo) were determined based on rice reference genome (Nipponbare) according to their genome locations.
Figure S8. Scatter dot plot of DEGs in the sitl1 mutant.Illumina-based RNA-seq was performed to profile mRNA expression in roots of one-week-old seedlings of the sitl1 mutant and wild-type (WT). DEGs with statistical significance were obtained (n = 3 replicates, q value < 0.05). A set of 767 and 438 genes was identified whose mRNAs showed significantly increased and decreased transcript abundance, respectively, in roots in the sitl1 mutant.
Figure S9. The over-represented gene functions of differentially expressed genes (DEGs) in the sitl1 mutant. Pageman analysis of the sitl1 mutant versus WT in roots. The different colors represent the degree of change in the gene expression level (log2 fold change) according to Fisher’s exact test with default parameter. Red represents the significant enrichment of DEGs, blue represents the significant depletion of DEGs, and white represents no significance.
Figure S10. Metabolism overview of differentially expressed genes (DEGs) in the sitl1 mutant. Blue or Red colors represent mRNA expression levels (log2 fold change) of the upregulated or downregulated genes, respectively, in the sitl1mutant.
Figure S11. A protein sequence alignment of OsMTP1 in WT and thesitl1 mutant. OsMTP1 sequences were confirmed by rice cDNA sequences in WT and the sitl1 mutant. Alignment was performed using CLC Main Workbench software ver. 8.0.1 (https://digitalinsights.qiagen.com/products-overview/analysis-and-visualization/qiagen-clc-main-workbench/). The putative metal ion transporter CorA-like cation transporter domain was marked by red rectangles. Predicted two C-terminal transmembrane (TM) domains were indicated as green arrows. Red arrow indicates T insertion (1044_1045insT) and blue arrow indicates predicted STOP codon of OsMTP1 in sitl1 mutant. The domain structure of the OSMTP1 protein was predicted using the InterproScan server (https://www.ebi.ac.uk/interpro/). The present of TM domain was predicted using InterProScan and TMHMM server v.2.0 (http://www.cbs.dtu.dk/services/TMHMM/).
Figure S12. Phylogenetic tree of O. sativa OsMTP1 and its orthologous genes . (a) Protein sequences of OsMTP1 and its orthologous genes in A. thaliana , Z. mays , and S. bicolor . Multiple alignment was performed using CLC Main Workbench software ver. 8.0.1 (https://digitalinsights.qiagen.com/products-overview/analysis-and-visualization/qiagen-clc-main-workbench/). The putative metal ion transporter CorA-like cation transporter domain was marked by red rectangles. Predicted two C-terminal transmembrane (TM) domains were indicated as green rectangles. (b) A Neighbor-Joining tree of protein sequences constructed with CLC Main Workbench software ver. 8.0.1 (https://digitalinsights.qiagen.com/products-overview/analysis-and-visualization/qiagen-clc-main-workbench/). Asterisk indicates OsMTP1. Bootstrap analysis was performed with 1,000 replicates. Bootstrap percentages are indicated at branches. The domain structure of the OSMTP1 protein was predicted using the InterproScan server (https://www.ebi.ac.uk/interpro/). The present of TM domain was predicted using InterProScan and TMHMM server v.2.0 (http://www.cbs.dtu.dk/services/TMHMM/).
Figure S13. A protein sequence alignment of O. sativa andA. thaliana MRS2 proteins with OsMTP1 (and its orthologous genes) in O. sativa, A. thaliana, Z. mays, and S. bicolor. Protein sequences of MRS2 family and OsMTP1 orthologous genes in O. sativa , A. thaliana , and Z. mays were obtained from The Arabidopsis Information Resource (TAIR,https://www.arabidopsis.org/) and phytozome 12 (https://phytozome.jgi.doe.gov/pz/portal.html). A Neighbor-Joining tree of protein sequences constructed with CLC Main Workbench software ver. 8.0.1 (https://digitalinsights.qiagen.com/products-overview/analysis-and-visualization/qiagen-clc-main-workbench/). Red box indicates GM(I)N-motif in the first transmembrane domain. Bootstrap analysis was performed with 1,000 replicates. Bootstrap percentages are indicated at branches.
Figure S14. Phylogenetic tree of O. sativa and A. thaliana MRS2 proteins with OsMTP1 (and its orthologous genes) inO. sativa, A. thaliana, Z. mays, and S. bicolor. Protein sequences of MRS2 family and OsMTP1 orthologous genes in O. sativa , A. thaliana , and Z. mays were obtained from The Arabidopsis Information Resource (TAIR,https://www.arabidopsis.org/) and phytozome 12 (https://phytozome.jgi.doe.gov/pz/portal.html). A Neighbor-Joining tree of protein sequences constructed with CLC Main Workbench software ver. 8.0.1 (https://digitalinsights.qiagen.com/products-overview/analysis-and-visualization/qiagen-clc-main-workbench/). Asterisk indicates OsMTP1. Bootstrap analysis was performed with 1,000 replicates. Bootstrap percentages are indicated at branches.