Expression of the ZNT1 Zinc Transporter from the Metal Hyperaccumulator Noccaea caerulescens Confers Enhanced Zinc and Cadmium Tolerance and Accumulation to Arabidopsis thaliana

Abstract

Prompt regulation of transition metal transporters is crucial for plant zinc homeostasis. NcZNT1 is one of such transporters, found in the metal hyperaccumulator Brassicaceae species Noccaea caerulescens. It is orthologous to AtZIP4 from Arabidopsis thaliana, an important actor in Zn homeostasis. We examined if the NcZNT1 function contributes to the metal hyperaccumulation of N. caerulescens. NcZNT1 was found to be a plasma-membrane located metal transporter. Constitutive overexpression of NcZNT1 in A. thaliana conferred enhanced tolerance to exposure to excess Zn and Cd supply, as well as increased accumulation of Zn and Cd and induction of the Fe deficiency response, when compared to non-transformed wild-type plants. Promoters of both genes were induced by Zn deficiency in roots and shoots of A. thaliana. In A. thaliana, the AtZIP4 and NcZNT1 promoters were mainly active in cortex, endodermis and pericycle cells under Zn deficient conditions. In N. caerulescens, the promoters were active in the same tissues, though the activity of the NcZNT1 promoter was higher and not limited to Zn deficient conditions. Common cis elements were identified in both promoters by 5' deletion analysis. These correspond to the previously determined Zinc Deficiency Responsive Elements found in A. thaliana to interact with two redundantly acting transcription factors, bZIP19 and bZIP23, controlling the Zn deficiency response. In conclusion, these results suggest that NcZNT1 is an important factor in contributing to Zn and Cd hyperaccumulation in N. caerulescens. Differences in cis- and trans-regulators are likely to account for the differences in expression between A. thaliana and N. caerulescens. The high, constitutive NcZNT1 expression in the stele of N. caerulescens roots implicates its involvement in long distance root-to-shoot metal transport by maintaining a Zn/Cd influx into cells responsible for xylem loading.

Fig 1. Comparison of 5’ ends of NcZNT1 cDNAs isolated from N. caerulescens accessions La Calamine (LC), Prayon (PR), and Ganges (GA) to the 5’ end of the AtZIP4 cDNA from A. thaliana.

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 [5, 23], 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 (http://multalin.toulouse.inra.fr/multalin).

Fig 2. pAtZIP4::GUS expression in A. thaliana plants grown under Zn deficiency.

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).

Fig 3. pNcZNT1::GUS expression in A. thaliana plants grown under Zn deficiency.

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).

Fig 4. pAtZIP4::GUS and pNcZNT1::GUS expression in N. caerulescens roots.

GUS expression was analysed in transgenic N. caerulescens roots grown hydroponically under Zn deficiency (0.05μM ZnSO₄) (A, C, E, G) and Zn sufficiency (10 μM ZnSO₄) (B, D, F, H). Expression was observed upon three-hour GUS staining of roots expressing pAtZIP4::GUS (A-D) or pNcZNT1::GUS (E-H). Images of mature roots (A, B, E, F) and root tips (C, D, G, H) are displayed.

Fig 5. Confocal microscopy of GFP localization reflecting AtZIP4 or NcZNT1 promoter activity in A. thaliana and N. caerulescens roots.

Transgenic A. thaliana plants (panels A-C, G-N) and transgenic N. caerulescens roots (panels (D-F, O-W) expressing pAtZIP4::eGFP (panels A-F) and pNcZNT1::eGFP (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 A. thaliana), 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 N. caerulescens). 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.

Fig 6. The effect of 5’ deletions of the AtZIP4 and NcZNT1 promoters on GUS expression.

The GUS activity (pmole/min/μg protein) was tested in roots of transgenic (A) pAtZIP4::GUS and (B) pNcZNT1::GUS A. thaliana plants exposed to 0 μM Zn.

Fig 7. Expression of NcZNT1 in A. thaliana confers tolerance to excess Zn and Cd exposure.

Three independently transformed lines expressing a 35S::NcZNT1 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).

Fig 8. Tolerance of NcZNT1-expressing A. thaliana to excess Zn and Cd exposure corresponds to increased dry biomass.

Shoot (A) and root dry weights (B) are shown of three independently transformed A. thaliana lines expressing a 35S::NcZNT1 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).

Fig 9. Expression of NcZNT1 enhances the Zn and Cd concentrations of A. thaliana.

Shoot (A) and root Zn (B) and Cd (C) concentrations (conc., in μmoles per gram dry weight) are shown of three independently transformed A. thaliana lines expressing a 35S::NcZNT1 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).

Fig 10. Zn, Cd, Fe, and Mn concentrations of A. thaliana are affected by expression of NcZNT1.

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 A. thaliana lines expressing a 35S::NcZNT1 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.

Fig 11. Expression of NcZNT1 affects the transcription of Zn and Fe homeostasis genes of A. thaliana.

Relative transcription analysis of genes involved in Zn and Fe homeostasis in shoots (Shoot) and roots (Root) of a transformed A. thaliana line expressing a 35S::NcZNT1 construct (ZNT1) and Col wild-type plants (WT) in response to two weeks exposure to 2 μM ZnSO₄ (SuffZn), 60 μM ZnSO₄ (ExcessZn) and 2 μM CdSO₄ + 2 μM ZnSO₄ (ExcessCd). The relative transcription of genes in WT plants exposed to 2 μM ZnSO₄ was set to 1 (first column) and fold-changes of transcription are indicated as a heat map, with the corresponding legend included at the bottom. Transcription of AtUBP6 (At1g51710) was used as reference to normalize the quantitative reverse transcriptase PCRs.