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FIGURE 1. Complementation of growth defects of bacterial mutant strains MM281 by OsHKT2;4 under the low-Mg2+ conditions. (A) Growth of bacterial strains on N-minimal medium containing 0.01, 0.1, 0.5, 1, 2, or 10 mM Mg2+. The strains used in this assay were the strains MM281 transformed with the empty pTrc99A vector only (MM281+pTrc99A), coding sequence of MGT10 in the pTrc99A vector (MM281+MGT10), or coding sequence of OsHKT2;4 in pTrc99A vector (MM281+OsHKT2;4). From left to right is a 10-fold dilution series of bacterial cultures. (B) Growth curves of bacterial strains in liquid cultures. Bacterial cells described in (A) were grown in N-minimal liquid medium containing increasing concentrations of Mg2+ from 0.1 to 10 mM. Aliquots of the cultures were taken and monitored every 2 h by OD600 readings for the cell density from 10 to 24 h. Data are represented as the mean ± SD, n = 3.
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FIGURE 2. The oocytes expressing OsHKT2;4 produced Mg2+ currents under the high-Mg2+ conditions. (A) The typical current traces generated from the oocytes injected with water perfused with (a) 6 mM Mg2+ (Control) and from the oocytes expressing OsHKT2;4 (OsHKT2;4) perfused with (b) 1.2 mM, (c) 6 mM or (d) 20 mM Mg2+. Dotted lines represent the zero current level. (B) The current-voltage relationships deduced from the oocytes expressing OsHKT2;4 perfused with 0.3, 1.2, 6 or 20 mM Mg2+. Summarized current data are from 8 cells/condition. (C) The current amplitudes at –150 mV recorded from the oocytes injected with water (Control) and oocytes expressing OsHKT2;4 (OsHKT2;4) perfused with different Mg2+ concentrations. (D) Reversal potentials of currents generated from the oocytes expressing OsHKT2;4 in the presence of various concentrations of Mg2+ as indicated in the figure. Data in Figure 2 are presented as representative recordings or as mean ± SE of n (n = 6) observations with three repetitions, in which n is the number of samples. Asterisks indicate statistically significant differences compared with data from oocytes expressing OsHKT2;4 perfused with 1.2 mM Mg2+ (Unpaired student’s t-test, ∗P < 0.05).
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FIGURE 3. Genetic characterization and phenotypic analysis of atmgt6 mutant lines and their OsHKT2;4 overexpressed lines in low-Mg2+ conditions. (A) Scheme of AtMGT6 gene structure and position of the T-DNA insertion of SALK_203866. The gray boxes indicate 5′ and 3′ untranslated regions, and black boxes and lines indicate exons and introns, respectively. The T-DNA insertion is shown as the triangle above the gene diagram. (B) Gene fragment of OsHKT2;4, including its promoter and CDS region that was introduced into atmgt6 lines. (C) Semi-quantitative mRNA levels of AtMGT6 and OsHKT2;4 by RT-PCR analysis in wild type (Col-0), AtMGT6 knockout mutant (atmgt6), and two atmgt6 lines overexpressing OsHKT2;4 (OE29 and OE34). AtActin2 was used as the internal standard. (D) The growth of Col-0, atmgt6, OE29, and OE34 under the Mg2+-deficient conditions. After planted in half-strength Murashige and Skoog (1/2 MS) medium for 3 days, Col-0, atmgt6, OE29, and OE34 were transferred to one-sixth-strength MS (1/6 MS) medium containing 0, 0.01, 0.1, 0.25, 0.75, and 2 mM Mg2+ in total, and were photographed after growing for 7 days. Quantitative analyses of primary root length (E) and whole-plant fresh weight (F) of Col-0, atmgt6, OE29, and OE34 under the Mg2+-deficient conditions described in (D). Six independent 10-day-old seedlings of each genotype were gathered as one biological repeat for root length and fresh weight measurement. Data are represented as the mean ± SD, n = 3, in which n is the number of biological repeat.
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FIGURE 4. Growth phenotype of atmgt6 and transgenic OsHKT2;4-overexpression atmgt6 lines in high-Mg2+ conditions. (A) The growth of Col-0, atmgt6, OE29, and OE34 under the Mg2+-abundant conditions. After planted in half-strength MS (1/2 MS) medium for 3 days, Col-0, atmgt6, OE29, and OE34 were transferred to one-sixth-strength MS (1/6 MS, referred to as “Control” in the figure) containing a basal 0.25 mM Mg2+ and 1/6 MS medium supplemented with extra 2, 4, 6, 8, and 10 mM Mg2+. Plants were photographed after growing for another 7 days. Quantitative analyses of primary root length (B) and whole-plant fresh weight (C) of Col-0, atmgt6, OE29, and OE34 under the Mg2+-abundant conditions described in (A). Six independent 10-day-old seedlings of each genotype were gathered as one biological repeat for root length and fresh weight measurement. Data are represented as the mean ± SD, n = 3, in which n is the number of biological repeat. Asterisks indicate statistically significant differences compared with atmgt6 (Student’s t-test, ∗P < 0.05).
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FIGURE 5. Mg2+ content and its partitioning in shoots and roots of atmgt6 and transgenic OsHKT2;4-overexpression atmgt6 lines. Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) analysis of Mg2+ contents in shoots (A) and roots (B) of Col-0, atmgt6 and two transgenic OsHKT2;4-overexpression atmgt6 lines, OE29 and OE34. After planted in the hydroponic medium containing 0.25 mM Mg2+ for 2 weeks, Col-0, atmgt6, OE29, and OE34 were transferred to the hydroponic medium containing 0, 0.25 (Control), or 6 mM Mg2+, and were harvested for elemental analysis of roots and shoots after growing for 2 days. Data are represented as the mean ± SD, n = 3. Asterisks indicate statistically significant differences compared with atmgt6 (Student’s t-test, ∗P < 0.05). (C) Altered Mg2+ partitioning between shoot (Sh) and root (R) in Col-0, atmgt6, OE29, and OE34. Values are deduced from (A,B).
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FIGURE 6. Effects of high Ca2+ additions on the growth of atmgt6 and transgenic OsHKT2;4-overexpression atmgt6 lines in the Mg2+-abundant medium. (A) The growth of Col-0, atmgt6 and two transgenic OsHKT2;4 overexpression atmgt6 lines (OE29 and OE34) in 1/6 MS (Control) and the Mg2+-abundant medium (1/6 MS with extra 8 mM Mg2+, referred to as “+8 mM Mg2+” in the figure) containing different extra Ca2+ concentrations. After planted in 1/2 MS medium for 3 days, Col-0, atmgt6, OE29, and OE34 were transferred to the 1/6 MS medium (containing a basal 0.25 mM Mg2+ and 0.5 mM Ca2+), or the Mg2+-abundant medium with 0, 0.1, 0.5, 1, or 3 mM extra Ca2+. Plants were photographed after growing for another 7 days. Quantitative analyses of primary root length (B) and whole-plant fresh weight (C) of Col-0, atmgt6, OE29, and OE34 under the conditions described in (A). Six independent 10-day-old seedlings of each genotype were gathered as one biological repeat for root length and fresh weight measurement. Data are represented as the mean ± SD, n = 3, in which n is the number of biological repeat. Asterisks indicate statistically significant differences compared with atmgt6 (Student’s t-test, ∗P < 0.05).
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FIGURE 7. Effects of the Ca2+ depletion on the growth of atmgt6 and transgenic OsHKT2;4-overexpression atmgt6 lines in the Mg2+-abundant medium. (A) The growth of Col-0, atmgt6 and transgenic two OsHKT2;4 overexpression atmgt6 lines (OE29 and OE34) in 1/6 MS (Control) and the 1/6 MS medium depleted of Ca2+ (–Ca2+). After planted in 1/2 MS medium for 3 days, Col-0, atmgt6, OE29, and OE34 were transferred to the 1/6 MS medium (containing a basal 0.25 mM Mg2+ and 0.5 mM Ca2+), or the 1/6 MS medium depleted of Ca2+ and supplemented with extra 2, 4, 6, or 8 mM Mg2+. Plants were photographed after growing for another 7 days. Quantitative analyses of primary root length (B) and whole-plant fresh weight (C) of Col-0, atmgt6, OE29, and OE34 under the conditions described in (A). Six independent 10-day-old seedlings of each genotype were gathered as one biological repeat for root length and fresh weight measurement. Data are represented as the mean ± SD, n = 3, in which n is the number of biological repeat. Asterisks indicate statistically significant differences compared with atmgt6 (Student’s t-test, ∗P < 0.05).
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