P-Type ATPase Heavy Metal Transporters with Roles in Essential Zinc Homeostasis in Arabidopsis

Abstract

Arabidopsis thaliana has eight genes encoding members of the type 1 B heavy metal-transporting subfamily of the P-type ATPases. Three of these transporters, HMA2, HMA3, and HMA4, are closely related to each other and are most similar in sequence to the divalent heavy metal cation transporters of prokaryotes. To determine the function of these transporters in metal homeostasis, we have identified and characterized mutants affected in each. Whereas the individual mutants exhibited no apparent phenotype, hma2 hma4 double mutants had a nutritional deficiency phenotype that could be compensated for by increasing the level of Zn, but not Cu or Co, in the growth medium. Levels of Zn, but not other essential elements, in the shoot tissues of a hma2 hma4 double mutant and, to a lesser extent, of a hma4 single mutant were decreased compared with the wild type. Together, these observations indicate a primary role for HMA2 and HMA4 in essential Zn homeostasis. HMA2promoter-and HMA4promoter-reporter gene constructs provide evidence that HMA2 and HMA4 expression is predominantly in the vascular tissues of roots, stems, and leaves. In addition, expression of the genes in developing anthers was confirmed by RT-PCR and was consistent with a male-sterile phenotype in the double mutant. HMA2 appears to be localized to the plasma membrane, as indicated by protein gel blot analysis of membrane fractions using isoform-specific antibodies and by the visualization of an HMA2-green fluorescent protein fusion by confocal microscopy. These observations are consistent with a role for HMA2 and HMA4 in Zn translocation. hma2 and hma4 mutations both conferred increased sensitivity to Cd in a phytochelatin-deficient mutant background, suggesting that they may also influence Cd detoxification.

Transition metal transport

“…Both transporters have been proposed to localize to the plasma membrane [4,58]. It is possible that the A. halleri and T. caerulescens homologues of AtHMA4 perform an analogous function.…”

“…In A. thaliana, the Zn(II)/Cd(II) or divalent cation transporting P IB -type ATPases AtHMA2 and AtHMA4 play a central role in root-to-shoot zinc translocation, possibly by xylem loading or by unloading metal ions from xylem parenchyma cells [4,58,59]. Both genes are mainly expressed in the root and shoot vascular tissues, and the corresponding proteins localize to the plasma membrane [4,58]. An hma4 single mutant accumulates less zinc in the shoot than wild-type plants, and an hma2hma4 double mutant displays a strong zinc deficiency phenotype in the shoot [4,58].…”

“…Both genes are mainly expressed in the root and shoot vascular tissues, and the corresponding proteins localize to the plasma membrane [4,58]. An hma4 single mutant accumulates less zinc in the shoot than wild-type plants, and an hma2hma4 double mutant displays a strong zinc deficiency phenotype in the shoot [4,58].…”

“…Not surprisingly, Arabidopsis mutants with defects in the transport of essential transition metals have severe phenotypes [4][5][6]. The dynamic range of optimal internal concentrations, within which plants are unaffected by deficiency or toxicity, is particularly narrow for transition metals [3].…”

Common Functions or Only Phylogenetically Related? The Large Family of PLAC8 Motif-Containing/PCR Genes

“…Once taken up by the root, heavy metals are transported to the vascular tissue, loaded to the xylem and translocated to the shoot, where they have to be taken up in the different shoot organs. Also in these steps too, transporters that cannot completely discriminate between essential and purely toxic heavy metals are implicated in the different transport processes (Clemens et al, 2002;Hussain et al, 2004).…”