pho2, a phosphate overaccumulator, is caused by a nonsense mutation in a microRNA399 target gene

Abstract

We recently demonstrated that microRNA399 (miR399) controls inorganic phosphate (Pi) homeostasis by regulating the expression of UBC24 encoding a ubiquitin-conjugating E2 enzyme in Arabidopsis (Arabidopsis thaliana). Transgenic plants overexpressing miR399 accumulated excessive Pi in the shoots and displayed Pi toxic symptoms. In this study, we revealed that a previously identified Pi overaccumulator, pho2, is caused by a single nucleotide mutation resulting in early termination within the UBC24 gene. The level of full-length UBC24 mRNA was reduced and no UBC24 protein was detected in the pho2 mutant, whereas up-regulation of miR399 by Pi deficiency was not affected. Several characteristics of Pi toxicity in the pho2 mutant were similar to those in the miR399-overexpressing and UBC24 T-DNA knockout plants: both Pi uptake and translocation of Pi from roots to shoots increased and Pi remobilization within leaves was impaired. These phenotypes of the pho2 mutation could be rescued by introduction of a wild-type copy of UBC24. Kinetic analyses revealed that greater Pi uptake in the pho2 and miR399-overexpressing plants is due to increased Vmax. The transcript level of most PHT1 Pi transporter genes was not significantly altered, except PHT1;8 whose expression was enhanced in Pi-sufficient roots of pho2 and miR399-overexpressing compared with wild-type plants. In addition, changes in the expression of several organelle-specific Pi transporters were noticed, which may be associated with the redistribution of intracellular Pi under excess Pi. Furthermore, miR399 and UBC24 were colocalized in the vascular cylinder. This observation not only provides important insight into the interaction between miR399 and UBC24 mRNA, but also supports their systemic function in Pi translocation and remobilization.

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, V_max and K_m 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.