Figure legends
Figure 1. sitl1 mutant decreases root growth and leaf
chlorophyll content. Rice seeds of the sitl1 mutant and wild-type
(WT) control were germinated and grown in half-strength KimuraB nutrient
solution or deionized water for 3 weeks (a) Representative seedling
images of the sitl1 mutant and WT plants at 2, 4, and 7 days
after germination (DAG) and 3 weeks after germination (WAG) under
nutrient solution condition. (b) Comparison of lengths of root,
coleoptile, leaf sheath, and leaf blade (n = 30 with 3
replicates). (c) Fresh weight (n = 30 with 3 replicates). (d)
Representative leaf images of the sitl1 mutant and WT plants at 7
DAG and 3 WAG. (e) Comparison of leaf chlorophyll content (n = 6
replicates). Leaves were sampled and measured total chlorophyll,
chlorophyll A, and chlorophyll B at 7 DAG and 3 WAG. (f) Representative
seedling images of the sitl1 mutant and WT plant at 7 DAG under
deionized water condition. Seeds of the sitl1 mutant and WT were
germinated and grown in deionized water for 7 days. (g) Comparison of
lengths of root, leaf sheath, and leaf blade at 7 DAG (n = 30
with 3 replicates). (h) Fresh weight (n = 30 with 3 replicates).
(i) Leaf chlorophyll content (n = 6 replicates). Value represent
means ± SD, ns = non‐significant, *p < 0.05 and
***p < 0.001, two-way ANOVA with
Sidak’s multiple comparison test.
Figure 2. Analysis of the sitl1 mutant in root
development. Rice seeds of the sitl1 mutant and wild-type (WT)
control were germinated and grown in half-strength KimuraB nutrient
solution (NS) or deionized water (DW) for 1 week. Detached roots were
used to scan and measure for root development. (a) Representative images
of 1-week-old stil1 mutant and WT roots. (b) Comparison of
lateral root number in primary roots (n = 30 with 3 replicates).
(c) Lateral root density (n = 30 with 3 replicates). (d) Average
length of lateral root (n = 30 with 3 replicates). (e) Sum of
lateral root length (n = 30 with 3 replicates). Lateral root
number, average length of lateral root, and sum of lateral root length
were analyzed within 3-cm root samples from root base. (f) Length of
root immature zone (n = 30 with 3 replicates). Root immature zone
was defined as the zone of the root where lateral roots were not
detected from the root tip. (g) Length of root mature zone (n =
30 with 3 replicates). Root mature zone was defined as the zone of the
root where lateral root initiation and development takes places to root
base. (h) Length of primary root (PR) width (n = 30 with 3
replicates). (i) Length of lateral root width (n = 30 with 3
replicates). Value represent means ± SD, ns = non‐significant,
***p < 0.001, two-way ANOVA with
Sidak’s multiple comparison test.
Figure 3. sitl1 mutant decreases cell number and size in
root tissue. Rice seeds of the sitl1 mutant and wild-type (WT)
were germinated and grown in half-strength KimuraB solution for 1 week.
One-week-old roots were detached and stained with propidium iodide (PI)
solution for 10 min. Root images were captured via confocal
laser-scanning microscopy and the epidermal cells were used to measure
cell size and number. (a) Representative images of root meristems of thesitl1 mutant and WT. The root epidermal cells are outlined with
solid lines. Green, red, and blue lines with double arrowheads represent
the lengths of the apical meristem, the basal meristem and the
elongation/differentiation zone, respectively (Hacham et al., 2011; Lim
et al., 2018). QC indicates the quiescent center. Scale bar, 40µm.
Quantification of (b) average cell number in the apical meristem, (c)
cell number in the basal meristem and (d) total cell number in the
meristem zone (n = 9 with 3 replicates). Quantification of (e)
apical meristem length, (f) basal meristem length and (g) total meristem
length (n = 9 with 3 replicates). Quantification of (h) average
cell length and (i) cell width in the apical and basal meristems
(n = 9 with 3 replicates). (j) Representative images of cortical
cells in mature root zone of the sitl1 mutant and WT.
Quantification of average (k) cortical cell length, (l) cell width and
(m) cell area in the mature root zone (n = 9 with 3 replicates).
Value represent means ± SD, ns = non‐significant, *p <
0.05, **p < 0.05 and ***p < 0.001,
Student’s t -test (b, c, d, e, f, g, k, l, and m) and two-wayANOVA with Sidak’s multiple comparison test (h and i).
Figure 4. sitl1 mutant enhances salinity insensitivity
by reducing Na+ influx across the plasma membrane. Rice seeds of thesitl1 mutant and wild-type (WT) were grown for 1 week under
half-strength KimuraB nutrient solution (NS) or deionized water (DW)
conditions. Seedlings were then treated with NS or DW containing 0, 50,
100 mM NaCl for 1 week. (a) Representative images of the sitl1mutant and WT at 7 days after salinity treatment. Boxes with broken
lines indicate the third leaf of the sitl1 mutant and WT. Scale
bar, 5 cm. Quantification of fresh weights of root, leaf sheath, and
leaf blade tissues under (b) NS condition and (c) DW condition (n= 30 with 3 replicates). Quantification of leaf chlorophyll content
under (d) NS condition and (e) condition (n = 30 with 3
replicates). (f) Representative DAB staining images of leaf blades of
the sitl1 mutant and WT. Rice seeds of the sitl1 mutant WT
were germinated and grown in half-strength NS for 1 week. Seedlings were
then treated with NS containing 0, 50, 100 mM NaCl for 1 week and leaf
blades were stained with DAB solution to assess the accumulation of
H2O2. Scale bar, 1mm. (g) Quantification
of H2O2 content in root tissues
(n = 6 with 3 replicates). (h) Quantification of
H2O2 content in leaf blade tissues
(n = 6 with 3 replicates). (i) Representative images of
Na+ accumulations in lateral roots of the sitl1mutant and WT. One-week-old seedlings of the sitl1 mutant and WT
were treated with NS containing 50 mM NaCl for 3 h. The lateral roots
were detached and stained with CoroNa-green AM and FM4-64 to visualize
the accumulations of Na+ in the vacuole. (j)
Representative images of Na+ distribution in rice protoplasts of thesitl1 mutant and WT. Leaf protoplasts were isolated and treated
with 0 and 50 mM NaCl solution for 1 h. The CoroNa-green AM was used to
visualize Na+ distribution in protoplasts. (k)
Quantification of CoroNa green intensity (n = 3 replicates with
average intensity of 50 protoplasts per replicate). Value represent
means ± SD, ns = non‐significant, *p < 0.05 and
***p < 0.001, two-way ANOVA with
Sidak’s multiple comparison test.
Figure 5. sitl1 mutant reduces the concentration of
Mg2+ and Na+ in root and leaf
tissues. One-week-old rice seedlings of the sitl1 mutant and
wild-type (WT) were treated with 0 or 50 mM NaCl solution for 1 week.
Oven-dried root and leaf samples of the sitl1 mutant and WT were
used to determine the concentrations of inorganic ions via ICP-OES
(n =3 replicates). (a) Potassium. (b) Magnesium. (c) Sodium. (d)
Calcium. (e) Phosphorus. Xylem sap was collected from 1-week-old rice
seedlings of the sitl1 mutant and WT after 0 or 50 mM NaCl
treatments. Collected sap was used to determine the concentrations of
inorganic ions via ICP-OES (n = 6 replicates). Quantification of
(f) Magnesium, (g) Sodium, and (h) Potassium concentrations in xylem
sap. Value represent means ± SD, ns = non‐significant, *p< 0.05 and ***p < 0.001, two-wayANOVA with Sidak’s multiple comparison test.
Figure 6. Relative fold
expression of the genes encoding antioxidant defense enzymes,
Na+, and K+ transporters.
One-week-old seedlings of the sitl1 mutant and WT were treated
with half-strength KimuraB solution containing 0 or 50 mM NaCl for 1 h.
Relative expression levels of selected marker genes in root and leaf
tissues were determined by qRT-PCR. (A) Relative expression of the genes
encoding antioxidant defense enzymes (OsCAT1 , Catalase isozyme A;OsCAT2 , Catalase isozyme B; OsAPX1 , Cytosolic ascorbate
peroxidase 1; OsAPX2 , Cytosolic ascorbate peroxidase 2;OsCuZnSOD1 , Cytosolic copper/zinc-superoxide dismutase 1;OsMnSOD , Mitochondrial manganese-superoxide dismutase;OsPOD , Peroxidase; OsGR1 , Cytosolic glutathione reductase
1; OsGR2 , Mitochondrial glutathione reductase; OsDHAR1 ,
Dehydroascorbate reductase; OsMDHAR1 , Cytosolic
monodehydroascorbate reductase; OsMDHAR2 , Putative
monodehydroascorbate reductase; OsP5CS ,
Delta-1-pyrroline-5-carboxylate synthase). (B) Relative expression of
the genes encoding Na+ and K+transporters (OsHKT1;5 , Sodium transporter Hkt1.5;OsLti6a , Plasma membrane protein 3 homolog; OsLti6b ,
Plasma membrane protein 3 homolog; OsHKT2;1 , High-affinity
potassium transporter; OsNHX1 , Vacuolar
Na+/H+ antiporter; OsSOS1 ,
Salt overly sensitive 1; OsAKT1 , AKT-type K+channels; HAK7 , Potassium transporter 7; OsCNGC1 ,
Non-selective cation channels 1). OsACTII was used as an internal
control (n = 6 with 3 replicates). Value represent means ± SD, ns
= non‐significant, *p < 0.05, **p <
0.01, and ***p < 0.001, two-way ANOVAwith Sidak’s multiple comparison test.
Figure 7. Whole-genome sequencing (WGS) and RNA-sequencing
(RNA-seq) analyses of the sitl1 mutant. (a) Number of sequence
variants in the sitl1 mutant compared with wild-type (WT) plant.
The SNPs and Indels between the sitl1 mutant and WT were
determined via WGS according to their chromosome locations. (b)
Characterization of SNP and Indel variants in the sitl1 mutant.
Colors in the pie chart represent the different features of variant
annotation based on genomic loci. Numbers indicate the number of SNPs
and Indels in the sitl1 mutant. (c-d) Transcript abundance
(log2 fold-change) and –log10 qvalue
analyses of the genes containing SNP (c) and Indel (d) variants in thesitl1 mutant via RNA-seq. Green and pale green round symbols
represent the relative gene expression level and statistical
significance, respectively. Gray dot lines indicate the cut-off value of
q value (0.05, -log10 value of 1.3). Red arrows indicate
that genes containing SNPs (c) and Indels (d) in the sitl1 mutant
have significantly higher or lower mRNA abundance.
Figure 8. Mutant identification, subcellular localization, and
gene expression analyses of OsMTP1 gene. (a) Schematic of the
genomic region corresponding to OsMTP1 . The position of the T
insertion (red arrows), the initiating codon (ATG), and the stop codon
(TAA) are indicated. Genomic OsMTP1 sequences are represented by
exons (black), introns (white), and untranslated 5’ and 3’ UTRs (gray).
Green arrow indicates the stop codon in coding sequence (CDS) ofOsMTP1 . Blue arrows indicate the qPCR-amplified regions for the
gene expression study. (b-c) Relative gene expression analysis ofOsMTP1 in roots and leaves of the sitl1 mutant and WT.
One-week-old seedlings of the sitl1 mutant and WT were used to
determine mRNA abundance of OsMTP1 gene using two different primer pairs
of qRT1 (b) and qRT2 (c) via qRT-PCR analysis (n = 6 with 3
replicates). OsACTII was used as an internal control. Value
represent means ± SD, ns = non‐significant, ***p <
0.001, two-way ANOVA with Sidak’s multiple comparison
test. (d) Subcellular localization analysis of 35S::OsMTP1-sGFPfusion protein with plasma membrane marker. Left column is the rice
protoplast expressing 35S::sGFP (empty-vector) construct used as
a control. Right column is the rice protoplast co-expressing35S::OsMTP1-sGFP fusion protein with pm-rk (plasma membrane
marker). (e) Relative gene expression of OsMTP1 in different
tissues and development stages in WT. The mRNA abundance ofOsMTP1 gene was determined using the qRT2 primer set via qRT-PCR
analysis. OsACTII was used as an internal control (n = 6
with 3 replicates). Value represent means ± SD. (f-g) Relative gene
expression of OsMTP1 in WT under salinity stress conditions.
One-week-old seedlings of WT were treated with half-strength KimuraB
solution containing 0 or 50 mM NaCl for 24 h. Root (f) and leaf blade
(g) tissues were harvested at 30 m, 1 h, 6, and 24h after treatments.
The mRNA abundance of OsMTP1 gene was determined using the qRT2
primer set via qRT-PCR analysis. OsACTII was used as an internal
control (n = 6 with 3 replicates). Value represent means ± SD. ns
= non‐significant, ***p < 0.001, two-wayANOVA with Sidak’s multiple comparison test.
Figure 9. Heterologous overexpression of OsMTP1 increases
ability of Mg2+ and Na+ transport in
yeast. (a-b) The wild-type yeast strain CM52 and CM62 transformed with
empty vector (EV) and OE-mOsMTP1 were used as positive and
negative controls. (a) Representative images of yeast cell growth on
solid medium containing 0, 0.1, and 1 mM MgCl2. (b)
Complementation of yeast cell growth assay in liquid medium containing
0, 0.1, and 1 mM MgCl2. Cell density
(OD600) of each yeast line was monitored every 2 h over
66 h (n = 8 replicates). Value represent means ± SD. (c-h) Yeast
wild-type cells (FM391) harboring EV, OE-mOsMTP1 , and OE-OsMTP1
was used to monitor cell growth rates in solid and liquid medium
containing 0, 0.5, or 1 M of NaCl and 1M of NaCl with 0.1 or 1 mM
MgCl2. (c) Representative images of yeast cell growth of
WT, EV, OE-mOsMTP1 , and OE-OsMTP1 on solid medium. (d-h)
Cell growth assay in liquid medium containing 0 mM (d), 500 mM (e), and
1 M (f) NaCl, 1M NaCl with 0.1 mM MgCl2 (g) and 1 mM
MgCl2 (h). Cell density (OD600) of each
yeast line was monitored every 2 h over 48 h (n = 8 replicates).
Value represent means ± SD.