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.

How metal hyperaccumulating plants can advance Zn biofortification

“…It remains to be analyzed whether these observations can be generalized. The recently reported absence of AtZIP4 promoter activation in hairy root-transformed N. caerulescens supplied with sufficient Zn indicates that strong HMA4 activity does not always trigger Zn deficiency responses in roots (Lin et al 2016). Still, the documented domino effects of transcriptional changes partly explain at least in A. halleri why the genetic architecture of Zn hyperaccumulator traits appears to be less complex than suggested by the results of comparative transcriptome studies.…”

Phylogenetic analysis of F-bZIP transcription factors indicates conservation of the zinc deficiency response across land plants

“…The Atbzip19/23 double mutant was introgressed into A. thaliana Col-0 stably transformed with a construct harbouring the AtZIP4 gene promoter fused to the reporter gene GUS ( pAtZIP4::GUS ) 53 . Homozygous lines were determined by genotyping the F3 generation using the primers depicted in Supplementary Table S6.…”

An evolutionary and functional genomics study of Noccaea caerulescens, a heavy metal hyperaccumulating plant species

“…The ZIP-like transporter encoded by ZNT1 is highly expressed under Zn/Cd stress conditions but it is single copy gene in N. caerulescens (Hammond et al, 2006;van de Mortel et al, 2006). Genetic variation in the ZNT1 promoter corresponding to variations in Zn and Cd accumulation has been reported in N. caerulescens (Lin et al, 2016).…”

“…The comparative genome sequencing of six N. caerulescens populations revealed intra-species CNV, including the well-known examples of HMA4, which plays an essential role in Cd uptake and transport from the root to the shoot (Hanikenne et al, 2008;Halimaa et al, 2014a;Lin et al, 2014Lin et al, , 2016, and…”

“…This stress response gene may therefore be under Zn/Cd conditions may also be under selection, but this is still under investigation. If detected, the essential cis-regulatory region of the promoter should be identified by promoter deletion techniques as described for the metal homeostasis gene ZNT1 (Lin et al 2016). The sequence database from this study will provide a valuable resource for future investigations involving N. caerulescens and should also be compared to related taxa to achieve a better understanding of evolutionary scenarios.…”