The <i>Arabidopsis ZIF2</i> gene encodes multiple transcripts.

<p>(A) Exon/intron organization of the <i>ZIF2</i> gene and T-DNA insertion site in the <i>zif2-1</i> mutant. Boxes and lines between boxes denote exons and introns, respectively. The triangle depicts the site of the T-DNA insertion. F1, F1′, F2, F3, R1, R2, and R3 indicate the location of the primers used to detect <i>ZIF2</i> expression in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004375#pgen-1004375-g003" target="_blank">Figures 3A, 3C</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004375#pgen-1004375-g007" target="_blank">7A, S</a>1 and S4. Scale bar, 200 bp. (B) Structure of the alternative <i>ZIF2</i> transcripts. The coding sequence (CDS) is shown in black and the UTRs in white. Grey boxes indicate portions of the 5′UTR overlapping with the retained intron. RACE1 to RACE6 indicate the location of the gene-specific primers used in the 5′RLM-RACE experiment. Occurrence of each <i>ZIF2</i> transcript was determined according to its detection (number of corresponding 5′RLM-RACE clones) or not (-) by RLM-5′RACE in the indicated tissue. Scale bar, 200 nt.</p

The ZIF2 transporter affects root-shoot partitioning in <i>Arabidopsis</i>.

<p>Zn concentration, expressed on a dry weight (DW) basis, in the shoot (upper panel) and root (middle panel), as well as the corresponding shoot/root ratio (lower panel), of 21-d old seedlings of the wild type (Col-0), the <i>zif2-1</i> mutant and <i>ZIF2</i>-overexpressing lines (<i>ZIF2.1</i>OX2 and <i>ZIF2.2</i>OX1) grown on control medium (30 µM Zn²⁺) or under excess Zn supply (125 and 250 µM Zn²⁺). Bars represent means ± SD (<i>n</i> = 4). Different letters indicate statistically significant differences between genotypes under each condition (<i>P</i><0.05; Student's <i>t</i>-test).</p

The <i>ZIF2</i> promoter is active in most <i>Arabidopsis</i> tissues.

<p>(A–H) Differential interference contrast microscopy images of GUS-stained transgenic plants carrying the <i>ProZIF2:GFP-GUS</i> reporter construct. <i>ZIF2</i> promoter activity in floral buds (A), a mature flower (B), a young leaf (C), the primary root (D), the primary root tip (E), a lateral root primordium at stage V (F) and stage VII (G) and a mature lateral root (H). Scale bars, 1 mm (A), 200 µm (B, C), or 50 µm (D–H). (I–L) Confocal laser scanning microscopy images of transgenic root tissues carrying the <i>ProZIF2:GFP-GUS</i> reporter construct. <i>ZIF2</i> promoter activity in the primary root (I, K) and a mature lateral root (J, L). ep, epidermis; c, cortex; en, endodermis; cc, central cylinder. Cell walls were stained with propidium iodide. The GFP and propidium iodide signals are visualized by green and red coloration, respectively. Scale bars, 25 µm (I, J), or 10 µm (K, L).</p

The expression and splicing patterns of the <i>Arabidopsis ZIF2</i> gene are Zn-regulated.

<p>(A) RT-PCR profile of <i>ZIF2.1 and ZIF2.2</i> expression in different wild-type (Col-0) tissues. The location of the F1 and R1 primers used is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004375#pgen-1004375-g002" target="_blank">Figure 2A</a>. Expression of the <i>ROC1</i> gene was used as a loading control. Results are representative of three independent experiments. (B) Real-time RT-PCR analysis of total <i>ZIF2</i> expression as well as of <i>ZIF2.1</i> and <i>ZIF2.2</i> transcript levels in different wild-type (Col-0) tissues, using <i>UBQ10</i> as a reference gene. Results are from two independent experiments and values represent means ± SE (<i>n</i> = 4). (C) RT-PCR profile of <i>ZIF2.1</i> and <i>ZIF2.2</i> expression in roots of 7-d old wild-type (Col-0) seedlings challenged for 48 h with various Zn supplies. The location of the F1 and R1 primers used is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004375#pgen-1004375-g002" target="_blank">Figure 2A</a>. Expression of the <i>ZIF1</i> and <i>ZIP1</i> or <i>UBQ10</i> genes is shown as plant metal status or loading controls, respectively. Results are representative of three independent experiments. (D) Real-time RT-PCR analysis of total <i>ZIF2</i> expression as well as of <i>ZIF2.1</i> and <i>ZIF2.2</i> transcript levels in roots of 7-d old wild-type (Col-0) seedlings challenged for 48 h with various Zn supplies, using <i>UBQ10</i> as a reference gene. Results are from two independent experiments and values represent means ± SE (<i>n</i> = 4).</p

An RNA secondary structure element sustains Zn regulation of translation driven by the <i>ZIF2.2</i> 5′UTR.

<p>(A) Organization of the 5′ end of the <i>ZIF2</i> gene. The orange and blue rectangles represent the intronic and coding sequences, respectively, and the region forming a stable RNA structure is indicated as a black line. (B) Secondary structure predictions for the <i>ZIF2.1</i> and <i>ZIF2.2</i> 5′UTRs flanked by 200 bp of the coding sequence. A potentially stable secondary structure element is enclosed in an oval. Four consecutive bases in the <i>ZIF2.2</i> 5′UTR (<i>ZIF2.2_5′UTR</i>) were mutated (<i>ZIF2.2_5′UTRM</i>) by site-directed mutagenesis to destabilize the structure, which was then restored by four complementary mutations (<i>ZIF2.2_5′UTRR</i>). Mutated bases are shown in grey. The two black arrows indicate the start and end of the intron while the blue arrows point to the start codon. The colour code indicates base pairing probabilities (red – high, blue – low) and the minimum free energy (MFE) of the entire 5′UTR (+ 200 nt of coding sequence) structures is indicated. (C) Transient expression of the <i>Pro35S:ZIF2.1_5′UTR-LUC</i>, <i>Pro35S:ZIF2.2_5′UTR-LUC</i>, <i>Pro35S:ZIF2.2_5′UTRM-LUC</i> (destabilized structure element) or <i>Pro35S:ZIF2.2_5′UTRR-LUC</i> (restored structure element) constructs in isolated <i>Arabidopsis</i> protoplasts under 0 or 100 µM Zn supplies. <i>Pro35S:GUS</i> was used as a transfection control and relative LUC/GUS activity for <i>Pro35S:ZIF2.1_5′UTR-LUC</i> under 0 µM Zn was set to 1. Results are from four biological repetitions and values represent means ± SD (<i>n</i> = 4). Different letters indicate statistically significant differences (<i>P</i><0.05) between the different constructs for each Zn concentration, and <i>P</i> values for the comparison of the two Zn concentrations for each construct are shown (Student's <i>t</i>-test).</p

Effect of GFP-ZIF2 expression on the zinc content of yeast cells.

<p>Total Zn concentration (pg/cell) of wild-type, <i>Δzrt1zrt2</i> and <i>Δzrc1cot1</i> mutant yeast cells, harbouring either the cloning vector <i>pGREG576</i> or the <i>pGREG576_ZIF2</i> plasmid, grown in Zn-free liquid medium supplemented with 0.062, 2.5 or 500 µM ZnSO₄ (means ± SD, <i>n</i> = 4). Statistically significant differences from the respective empty-vector control are indicated with the <i>P</i> values (in parentheses) obtained by Student's <i>t</i>-test. <i>ND</i>, not detectable.</p

The <i>Arabidopsis</i> ZIF2 transporter localises at the tonoplast.

<p>(A–C) Confocal laser scanning microscopy images of <i>Arabidopsis</i> wild-type mesophyll protoplasts transiently expressing either YFP alone (A) or the ZIF2.1-YFP (B) or ZIF2.2-YFP (C) fusions under the control of the 35S promoter. Arrowheads point to the YFP signal on the inner side of the chloroplasts and the nucleus. The YFP and chloroplast autofluorescence signals are visualized by green and red coloration, respectively. Scale bars, 10 µm. (D–F) Confocal laser scanning microscopy images of transgenic <i>Arabidopsis</i> root tips expressing either YFP alone (D) or the ZIF2.1-YFP (E) or ZIF2.2-YFP (F) fusions under the control of the 35S promoter. The YFP signal is visualized by green coloration. Scale bars, 50 µm. (G–R) Confocal laser scanning microscopy images of tobacco leaf epidermal cells transiently co-expressing the ZIF2.2-GFP fusion (G, J, M, P) with the plasma membrane marker PIP2A-mCherry (H, K) or the tonoplast marker g-TIP-mCherry (N, Q) under the control of the 35S promoter. Merged images of whole-cell views (I, O) or nucleus close-ups (L, R) are shown. Arrowheads point to transvacuolar strands and asterisks indicate fluorescence signals approaching the nucleus only on the side facing the exterior of the cell. The GFP and mCherry signals are visualized by green and red coloration, respectively. Scale bars, 20 µm.</p

An <i>Arabidopsis ZIF2</i> loss-of-function mutant is hypersensitive to Zn toxicity.

<p>(A) Representative images of 10-d old wild-type (Col-0) and mutant (<i>zif2-1</i>) seedlings grown on control medium (30 µM Zn²⁺) or under excess Zn supply (250 and 500 µM Zn²⁺). Scale bar, 1 cm. (B) Effect of Zn toxicity on shoot biomass (upper panel), chlorophyll content (middle panel) and PR elongation (lower panel) of wild-type (Col-0) and <i>zif2-1</i> or <i>zif1-2</i> mutant seedlings. Results are representative of three independent experiments and values represent means ± SD (<i>n</i> = 8 for shoot biomass/chlorophyll content and <i>n</i> = 16 for PR elongation). Different letters indicate statistically significant differences between genotypes (<i>P</i><0.05; Student's <i>t</i>-test).</p

The <i>Arabidopsis ZIF2</i> gene encodes multiple transcripts.

<p>(A) Exon/intron organization of the <i>ZIF2</i> gene and T-DNA insertion site in the <i>zif2-1</i> mutant. Boxes and lines between boxes denote exons and introns, respectively. The triangle depicts the site of the T-DNA insertion. F1, F1′, F2, F3, R1, R2, and R3 indicate the location of the primers used to detect <i>ZIF2</i> expression in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004375#pgen-1004375-g003" target="_blank">Figures 3A, 3C</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004375#pgen-1004375-g007" target="_blank">7A, S</a>1 and S4. Scale bar, 200 bp. (B) Structure of the alternative <i>ZIF2</i> transcripts. The coding sequence (CDS) is shown in black and the UTRs in white. Grey boxes indicate portions of the 5′UTR overlapping with the retained intron. RACE1 to RACE6 indicate the location of the gene-specific primers used in the 5′RLM-RACE experiment. Occurrence of each <i>ZIF2</i> transcript was determined according to its detection (number of corresponding 5′RLM-RACE clones) or not (-) by RLM-5′RACE in the indicated tissue. Scale bar, 200 nt.</p

The <i>ZIF2.1</i> and <i>ZIF2.2</i> 5′UTRs exert differential effects on translation.

<p>(A) Representative confocal laser scanning microscopy images of root tips of transgenic <i>Arabidopsis</i> 7d-old seedlings expressing either the ZIF2.1-YFP or ZIF2.2-YFP fusions under the control of the 35S promoter. Detection settings for YFP visualization were identical for all genotypes. The YFP signal is visualized by green coloration. Scale bars, 50 µm. (B) Transgene transcript and protein levels in <i>Arabidopsis ZIF2.1-YFP</i> and <i>ZIF2.2-YFP</i> overexpression lines. Quantification of <i>ZIF2-YFP</i> transcript (upper panel) and corresponding fusion protein (lower panel) levels in roots of 7-d old <i>ZIF2-YFP</i> overexpression seedlings grown under control conditions. Relative <i>ZIF2-YFP</i> gene expression was quantified by real-time RT-PCR using <i>UBQ10</i> as a reference gene (means ± SD, <i>n</i> = 4). The fluorescence intensity of the YFP signal in root tips (means ± SD, <i>n</i> = 12) was normalized to <i>ZIF2-YFP</i> transcript levels to determine the relative abundance of the ZIF2-YFP protein. Asterisks denote statistically significant differences from the <i>ZIF2.1-YFP</i>OX1 line (***<i>P</i><0.001; Student's <i>t</i>-test). (C) Quantification of ZIF2-YFP protein levels in <i>Arabidopsis ZIF2.1-YFP</i> and <i>ZIF2.2-YFP</i> overexpression lines. Western blot analysis of ZIF2-YFP protein levels in 5-d old seedlings from the wild type (Col-0) and <i>ZIF2.1-YFP</i>OX or <i>ZIF2.2-YFP</i>OX transgenic lines. The blot image is representative of two independent experiments and Coomassie staining is shown as a loading control. Bands were quantified and relative ZIF2-YFP protein levels plotted using the Coomassie control as a reference. The relative ZIF2-YFP protein level in the <i>ZIF2.2-YFP</i>OX1 line was set to 1. Results are from two independent experiments and values represent means ± SD (<i>n</i> = 4). (D) Effect of the <i>ZIF2.1</i> 5′UTR and <i>ZIF2.2</i> 5′UTR on translation of the <i>LUC</i> reporter gene. Transient expression of the <i>Pro35S:ZIF2.1_5′UTR-LUC</i> or <i>Pro35:ZIF2.2_5′UTR-LUC</i> constructs in isolated <i>Arabidopsis</i> protoplasts under various Zn supplies, using <i>Pro35S:GUS</i> as a transfection control. Results are representative of three independent experiments and values represent means ± SD (<i>n</i> = 4). Different letters indicate statistically significant differences (<i>P</i><0.01) between Zn concentrations for each construct and <i>P</i> values for the comparison of the two constructs under each condition are shown (Student's <i>t</i>-test).</p