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.
Highlights
Zinc (Zn) is an essential component of several important enzymes in plants, such as RNA polymerase, alcohol dehydrogenase, Cu/Zn superoxide dismutase and carbonic anhydrase [1, 2]
When comparing the coding sequences (CDS) of A. thaliana ZIP4 (AtZIP4), NcZNT1-LC and NcZNT1-PR (deposited in GenBank: AF275751.1, from N. caerulescens accession La Calamine, LC; AF133267.1, N. caerulescens accession Prayon, PR), we noticed that the NcZNT1-PR CDS appeared to be missing the first 5’ ~90 bp [23], when compared to the NcZNT1-LC and AtZIP4 CDS, which removes 30 amino acids from the N-terminus of the predicted translated open reading frame (S1 Fig)
We found NcZNT1 to be transcribed in both shoots and roots of N. caerulescens under Zn deficiency and sufficiency, with reduced transcription under Zn excess (S4 Fig)
Summary
Zinc (Zn) is an essential component of several important enzymes in plants, such as RNA polymerase, alcohol dehydrogenase, Cu/Zn superoxide dismutase and carbonic anhydrase [1, 2]. Zn is essential for plants, elevated concentrations of Zn are generally toxic, causing leaf chlorosis and growth reduction. Cadmium (Cd) is a non-essential element with no known biological function in plants. It can be taken up by transporters of minerals which are chemically similar to Cd, such as Fe or Zn [5]. Exposure leads to reduced water and nutrient uptake, lowered photosynthesis and leaf chlorosis and a general reduction in plant growth [6, 7]. It induces the production of ROS [8]. In response to fluctuations in metal supply concentrations, metal homeostasis mechanisms evolved in plants, which ensure proper regulation of their cellular and organellar metal concentrations to maintain a stable and constant condition [9]
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