Functional expression of AtHMA4, a P_1B‐type ATPase of the Zn/Co/Cd/Pb subclass

Abstract

SummaryMechanisms are required by all organisms to maintain the concentration of essential heavy metals (e.g. Zn and Cu) within physiological limits and to minimise the detrimental effects of non-essential heavy metals (e.g. Cd). Heavy-metal P-type ATPases (HMAs) are a subgroup of the P-type ATPase superfamily that may contribute to metal homeostasis in plants. We cloned and characterised a member of this family, AtHMA4, from Arabidopsis thaliana that clusters with the Zn/Co/Cd/Pb subclass of HMAs on phylogenetic analysis. Sequencing of the AtHMA4 cDNA showed that it contained the conserved motifs found in all P-type ATPases and also motifs that are characteristic of heavy-metal ATPases. Escherichia coli mutants defective in the HMAs, CopA and ZntA, were used in functional complementation studies. AtHMA4 was able to restore growth at high [Zn] in the zntA mutant but not at high [Cu] in the copA mutant, suggesting a role in zinc transport. Heterologous expression of AtHMA4 in Saccharomyces cerevisiae made the yeast more resistant to Cd but did not affect sensitivity to other metals compared with vector-transformed controls. The organ speci®city of AtHMA4 was analysed in Arabidopsis and showed that AtHMA4 was expressed in a range of tissues with highest expression in roots. AtHMA4 was upregulated in roots exposed to elevated levels of Zn and Mn but downregulated by Cd. Possible physiological roles of this transporter in Arabidopsis are discussed.

Identification of Thlaspi caerulescens Genes That May Be Involved in Heavy Metal Hyperaccumulation and Tolerance. Characterization of a Novel Heavy Metal Transporting ATPase

“…5). This response to increasing plant Cd status in T. caerulescens is quite different than what has been reported for its homolog in Arabidopsis, where it has been shown that root expression of AtHMA4 is down-regulated by plant exposure to Cd (Mills et al, 2003).…”

“…Here we present evidence that this ATPase facilitates a high degree of heavy metal tolerance in yeast by mediating the active efflux of heavy metals out of the cell. However in T. caerulescens, based on differences in tissue-specific and metal-responsive expression of this transporter compared with expression of its homolog in Arabidopsis (Arabidopsis thaliana; Mills et al, 2003;Hussain et al, 2004), we suggest that it may not be involved in metal tolerance in Thlaspi. Instead, we propose that it may play a role in xylem loading of metals and thus could be a key player in the hyperaccumulation phenotype expressed in T. caerulescens.…”

“…As mentioned above, the Arabidopsis P-type ATPase that it is most similar to is AtHMA4, which was recently shown to possibly be involved in Cd export and tolerance (Mills et al, 2003). The deduced amino acid sequence for TcHMA4 and its alignment with AtHMA4 are shown in Figure 1.…”

“…From the yeast complementation screen, we identified a P 1B -type heavy metal ATPase that is 71% identical on a protein level to the Arabidopsis heavy metal ATPase, AtHMA4 (Mills et al, 2003). The P 1B subfamily of ATPases is believed to be involved in the transport of heavy metals such as Zn, Cu, Co, Cd, Pb, and silver that are either essential micronutrients or noessential toxic metals, although only a few members of this subfamily have been well characterized.…”

“…The P 1B subfamily of ATPases is believed to be involved in the transport of heavy metals such as Zn, Cu, Co, Cd, Pb, and silver that are either essential micronutrients or noessential toxic metals, although only a few members of this subfamily have been well characterized. Genome sequencing efforts as well as isolation and analysis of individual members of this subgroup from different organisms indicate these heavy metal ATPases are expressed in a wide range of organisms, ranging from the archaea and bacteria to eukaryotic organisms including Arabidopsis (see, for example, Rensing et al, 1997;Palmgren, 1998, 2001;Tong et al, 2002;Mills et al, 2003).…”