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.