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.