Expression of NcZNT1 enhances the Zn and Cd concentrations of <i>A</i>. <i>thaliana</i>.

<p>Shoot (A) and root Zn (B) and Cd (C) concentrations (conc., in μmoles per gram dry weight) are shown of three independently transformed <i>A</i>. <i>thaliana</i> lines expressing a <i>35S</i>::<i>NcZNT1</i> construct (NcZNT1-1, NcZNT1-2, NcZNT1-3) and Col wild-type (WT) plants grown hydroponically for four weeks, first one week on half Hoagland’s media containing 2 μM ZnSO₄, thereafter on media containing 2 μM ZnSO₄ (sufficient Zn), 60 μM ZnSO₄ (excess Zn) and 2 μM CdSO₄ + 2 μM ZnSO₄ (Cd). Error bars represent the standard errors of the mean (n = 4). Different letters indicate significant differences (p<0.05) among plant types within treatments (transgenic lines and wild-type).</p

Expression of NcZNT1 in <i>A</i>. <i>thaliana</i> confers tolerance to excess Zn and Cd exposure.

<p>Three independently transformed lines expressing a <i>35S</i>::<i>NcZNT1</i> construct (NcZNT1-1, NcZNT1-2, NcZNT1-3) and Col wild-type plants (Col-WT) were grown hydroponically for four weeks, first one week on half Hoagland’s media containing 2 μM ZnSO₄, thereafter on media containing 2 μM ZnSO₄ (A), 60 μM ZnSO₄ (B) and 2 μM CdSO₄ + 2 μM ZnSO₄ (C).</p

<i>pAtZIP4</i>::<i>GUS</i> expression in <i>A</i>. <i>thaliana</i> plants grown under Zn deficiency.

<p>GUS expression was analysed in transgenic plants grown hydroponically under Zn deficiency (no Zn added to half Hoagland’s nutrient solution). Expression was observed in: (A) a detached lateral root; (B) close-up of (A) showing GUS expression in endodermis and pericycle; (C) root tip and root hair zone; (D) leaf; (E) close up of (D) with expression in trichomes; (F) young inflorescence, indicating buds/flowers in increasing age (1, 2, 3); (G) close up of (F, 3) with expression in stamen filaments; (H) older inflorescence showing expression in siliques. Relevant tissues and organs are indicated: xylem (x), pericycle (p), endodermis (e), cortex (c), epidermis (ep), root hairs (rh), root cap (rc), trichomes (t), stamen filament (sf), stamen anther (sa), pedicel (pe), and silique (s).</p

Zn, Cd, Fe, and Mn concentrations of <i>A</i>. <i>thaliana</i> are affected by expression of NcZNT1.

<p>Shoot (A, C) and root (B, D) concentrations (conc.) of Zn and Cd (A, B) and Fe and Mn (C, D) (in μmoles per gram dry weight) are shown of three independently transformed <i>A</i>. <i>thaliana</i> lines expressing a <i>35S</i>::<i>NcZNT1</i> construct (NcZNT1-1, NcZNT1-2, NcZNT1-3) and Col wild-type (WT) plants grown hydroponically for four weeks, first one week on half Hoagland’s media containing 2 μM ZnSO₄, thereafter on media containing 2 μM ZnSO₄, no Cd (0 Cd – 2 Zn), 5 μM CdSO₄ + 2 μM ZnSO₄ (5 Cd – 2 Zn) and 5 μM CdSO₄, no Zn (5 Cd – 0 Zn). Error bars represent the standard errors of the mean (n = 4). Different letters (small case for Zn and Fe; capitals for Cd and Mn) indicate significant differences (p<0.05) among plant types (transgenic lines and wild-type) and treatments.</p

Primers used for PCR amplifications.

<p>Restriction sites incorporated in the primers are underlined.</p

DNA fragment characteristics of <i>ZIP4</i>-orthologous promoters isolated from <i>Noccaea caerulescens</i>, <i>Cochlearia pyrenaica</i>, <i>Arabidopsis halleri</i> and <i>Arabidopsis lyrata</i>.

<p>Sequence lengths are provided. For <i>A</i>. <i>halleri</i> two fragments were amplified, differing in size due to a 154-bp InDel. Positions of identified sequence boxes, corresponding to two Zinc Deficiency Responsive Elements (ZDRE-1 and -2; consensus sequences indicated) and a predicted TATA box, are indicated relative to the predicted transcription start.</p

Comparison of 5’ ends of <i>NcZNT1</i> cDNAs isolated from <i>N</i>. <i>caerulescens</i> accessions La Calamine (LC), Prayon (PR), and Ganges (GA) to the 5’ end of the <i>AtZIP4</i> cDNA from <i>A</i>. <i>thaliana</i>.

<p>The first translational start codon (ATG) which is used as start codon in current study is at pos. 52–54. The second ATG, incorrectly interpreted as the translational start codon by [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149750#pone.0149750.ref005" target="_blank">5</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149750#pone.0149750.ref023" target="_blank">23</a>], is found at pos.154-156. Forward and reverse primers used for the amplification of these 5’ untranslated region (UTR) plus coding region cDNA fragments are shown as black arrows. GeneBank numbers of these sequences are: KU298434, KU298435 and KU298436 resp. for LC, PR and GA. The alignment was performed using MultAlin (<a href="http://multalin.toulouse.inra.fr/multalin" target="_blank">http://multalin.toulouse.inra.fr/multalin</a>).</p

Tolerance of NcZNT1-expressing <i>A</i>. <i>thaliana</i> to excess Zn and Cd exposure corresponds to increased dry biomass.

<p>Shoot (A) and root dry weights (B) are shown of three independently transformed <i>A</i>. <i>thaliana</i> lines expressing a <i>35S</i>::<i>NcZNT1</i> construct (NcZNT1-1, NcZNT1-2, NcZNT1-3) and Col wild-type (WT) plants grown hydroponically for four weeks, first one week on half Hoagland’s media containing 2 μM ZnSO₄, thereafter on media containing 2 μM ZnSO₄ (sufficient Zn), 60 μM ZnSO₄ (excess Zn) and 2 μM CdSO₄ + 2 μM ZnSO₄ (Cd). Error bars represent the standard errors of the mean. Different letters indicate significant differences (p<0.05) among plant types within treatments (transgenic lines and wild-type).</p

Confocal microscopy of GFP localization reflecting <i>AtZIP4</i> or <i>NcZNT1</i> promoter activity in <i>A</i>. <i>thaliana</i> and <i>N</i>. <i>caerulescens</i> roots.

<p>Transgenic <i>A</i>. <i>thaliana</i> plants (panels A-C, G-N) and transgenic <i>N</i>. <i>caerulescens</i> roots (panels (D-F, O-W) expressing <i>pAtZIP4</i>::<i>eGFP</i> (panels A-F) and <i>pNcZNT1</i>::<i>eGFP</i> (panels G-W) were grown hydroponically on half Hoagland’s nutrient solution to which no Zn was added (Zn deficiency, -Zn; panels A-C and G-J) or 2 μM ZnSO₄ (normal Zn, NZn; panels K-N) (for <i>A</i>. <i>thaliana</i>), or to which 0.05 μM ZnSO₄ was added (Zn deficiency, -Zn; panels D-F and O-S) or 100 μM ZnSO₄ (normal Zn, NZn) (for <i>N</i>. <i>caerulescens</i>). Panels A, D, G, K, O and T show the GFP florescence image; panels H, L, P and U show fluorescence upon propidium iodide staining; panels B, E, I, M, Q and V show the differential interference contrast (DIC) images; and panels C, F, J, N, S and W the merged images of each set. Root cell types are indicated with letters; x (xylem), p (pericycle), e (endodermis), c (cortex) and ep (epidermis). Scale bars are indicated.</p

<i>pNcZNT1</i>::<i>GUS</i> expression in <i>A</i>. <i>thaliana</i> plants grown under Zn deficiency.

<p>GUS expression was analysed in transgenic plants grown hydroponically under Zn deficiency (no Zn added to half Hoagland’s nutrient solution). Expression was observed in: (A) a detached lateral root (B) close-up of (A) showing GUS expression in endodermis and pericycle; (C) root tip and root hair zone; (D) leaf; (E) and (F) inflorescence including siliques. Relevant tissues and organs are indicated: xylem (x), pericycle (p), endodermis (e), cortex (c), epidermis (ep), root hairs (rh), root cap (rc), trichomes (t), vascular tissue (vt), pedicels (pe), and silique(s).</p