Article Figures & Data
Figures
- Figure 1.
Resemblance of Pi toxicity in miR399-overexpressing, pho2, and UBC24 loss-of-function (ubc24-1) plants. A, Pi toxic phenotype shown as chlorosis and necrosis in the leaves of 24-d-old miR399b-overexpressing (b), pho2 (c), and ubc24-1 (d) plants. Pi toxicity of pho2 was rescued by transforming a genomic copy of wild-type UBC24. Two independent rescued lines, pho2-C1 (e) and pho2-C2 (f), are shown (see also Fig. 4). a, Wild-type plant. Bar = 1 cm. B, Pi concentration in the shoots of wild-type (Wt; blue), miR399b-overexpressing (miR399b; yellow), ubc24-1 (green), and pho2 (red) plants from A. Error bars indicate the sd (n = 3). C, Pi uptake activity of wild-type (Wt; blue circles), miR399b-overexpressing (miR399b; yellow triangles), ubc24-1 (green diamonds), and pho2 (red squares) plants. Error bars represent the sd (n = 3). D, Shoot-to-root ratios of the [33P]Pi taken up by wild-type (Wt; blue bars), miR399b-overexpressing (miR399b; yellow bars), ubc24-1 (green bars), and pho2 (red bars) plants from C.
- Figure 2.
Impairment of Pi remobilization in the pho2 mutant. A, Changes in Pi concentration in the leaves of wild-type plants (dotted lines) or pho2 mutants (solid lines) grown under Pi-sufficient (1 mm KH2PO4) conditions. Individual leaves were collected at the indicated times, beginning with 9-d-old seedlings. Leaves from 10 plants were pooled and two proximal leaves were collected as one sample for Pi assay. Error bars represent the sd (n = 3). B, Autoradiographs of leaf image obtained from pulse-chase labeling experiments. The first two leaves of wild-type (Wt) plants are outlined because of faint signals. Leaves with chlorosis or necrosis phenotypes in the miR399f-overxpressing (miR399f) and pho2 plants are marked with asterisks. Bar = 1 cm.
- Figure 3.
A, Mutation of the UBC24 gene in the pho2 mutant. An early termination (indicated as the asterisk) at the W671 position caused by a single nucleotide change in the sixth exon of UBC24 was identified in the pho2 mutant. The translation initiation site and the ubiquitin-conjugating conserved domain (UBC) are indicated. Five miR399 target sequences and the T-DNA insertion in the second exon of the ubc24-1 mutant are shown. B, RNA gel-blot analyses of UBC24 (4.1 kb) and miR399 (21 nt) in wild-type (Wt), miR399b-overexpressing (miR399b), and pho2 plants grown hydroponically under Pi-sufficient (+, 250 μm KH2PO4) or Pi-deficient (−) nutrient solution. 5S rRNA and tRNA and 25S and 18S rRNA staining is shown as the loading control. C, Protein gel-blot analysis of the UBC protein in the roots of wild-type (Wt), miR399b-overexpressing (miR399b), pho2, and ubc24-1 plants grown under Pi-sufficient (+) or Pi-deficient (−) media.
- Figure 4.
Complementation of pho2 phenotypes by UBC24. A, Pi concentration in the shoots of 25-d-old wild-type (Wt), miR399b-overexpressing (miR399b), pho2, and two rescued transgenic-line (pho2-C1 and pho2-C2) plants grown under Pi-sufficient soil. Error bars indicate the sd (n = 3). B, Distribution of Pi in the leaves of 19-d-old wild-type (Wt), miR399-overexpressing (miR399b), pho2, and two rescued-line (pho2-C1 and pho2-C2) plants grown under Pi-sufficient (1 mm KH2PO4) medium. Error bars indicate the sd (n = 3).
- Figure 5.
Kinetics analysis of Pi uptake activity. A, Eadie-Hofstee plots of Pi uptake rate for wild-type (Wt), pho2, and miR399b-overexpressing (miR399b) plants with 2 to 2,000 μm Pi concentration. V indicates the Pi transport activity and [S] the external Pi concentration in the uptake solution. B, Vmax and Km values of Pi uptake activity calculated from A. Error bars indicate the sd (n = 3).
- Figure 6.
Expression of members of PHT1, PHT2, and PHT3 families and PHO1 in wild-type, miR399b-overexpressing, and pho2 plants. A, RT-PCR analyses of mRNA levels in the root and shoot samples collected from Pi-sufficient (+Pi) or Pi-deficient (−Pi) conditions. B, Quantitative PCR analyses of the relative amount of PHT1;8, PHT2;1, PHT3;2, and PHT3;3 mRNA. Error bars indicate the sd (n = 2). Expression of PHT1;8 was nondetectable (nd) in the Pi-sufficient roots of wild-type plants.
- Figure 7.
Tissue and cellular localization of UBC24 and miR399 by promoter::reporter analyses. A, GUS staining in the vascular tissues of UBC24 promoter::GUS transgenic plants grown under Pi-sufficient conditions. a, Whole seedling; b, cotyledon; c, the third true leaf; d and e, root; f, flower; g, enlarged receptacle; h, cross section of the root. The strong signal in the middle of pollen indicates the central vascular tissues. B, GUS staining (a–m) or GFP florescence (n–q and s) in miR399 promoter::reporter transgenic plants. All seedlings were grown under Pi-deficient media (−Pi) except those in a, b, and c, which were grown under Pi-sufficient media (+Pi). The growth condition and expression driven by different miR399 promoters was indicated in each image. a, d, g, and j, Whole seedling; b, c, e, and k, cotyledon; f, l, and n, first true leaf; h, i, o, p, and q, root; m, r, and s, cross section of root. r, Root section from Pi-starved wild-type plants showing autofluorescence in the epidermis and xylem. s, Arrow indicates the GFP signal in the phloem of the root. n, Red is the fluorescence of chlorophyll. q, Red fluorescence of cell walls results from staining with propidium iodide. Bar = 5 mm in A, a; and B, a, d, g, and j. Bar = 1 mm in A, b, c, and f; and B, b, c, e, f, k, l, and n. Bar = 100 μm in A, d and g; and B, h, i, o, and p. Bar = 50 μm in A, e and h; and B, m, q, r, and s.
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