Abstract
BackgroundHMA4 transporters are involved in the transport and binding of divalent heavy metals (Cd, Zn, Pb [lead] and Co [cobalt]). In general, as efflux pumps, HMA4 transporters can increase the heavy metal tolerance of yeast and Escherichia coli. Additional research has shown that the C-terminus of HMA4 contains a heavy metal-binding domain and that heterologous expression of a portion of peptides from this C-terminal domain in yeast provides a high level of Cd tolerance and Cd hyperaccumulation.ResultsWe cloned BjHMA4 from Brassica juncea, and quantitative real-time PCR analysis revealed that BjHMA4 was upregulated by Zn and Cd in the roots, stems and leaves. Overexpression of BjHMA4 dramatically affects Zn/Cd distribution in rice and wheat seedlings. Interestingly, BjHMA4 contains a repeat region named BjHMA4R within the C-terminal region; this repeat region is not far from the last transmembrane domain. We further characterized the detailed function of BjHMA4R via yeast and E. coli experiments. Notably, BjHMA4R greatly and specifically improved Cd tolerance, and BjHMA4R transformants both grew on solid media that contained 500 μM CdCl2 and presented improved Cd accumulation (approximately twice that of wild-type [WT] strains). Additionally, visualization via fluorescence microscopy indicated that BjHMA4R clearly localizes in the cytosol of yeast. Overall, these findings suggest that BjHMA4R specifically improves Cd tolerance and Cd accumulation in yeast by specifically binding Cd2+ in the cytosol under low heavy metal concentrations. Moreover, similar results in E. coli experiments corroborate this postulation.ConclusionBjHMA4R can specifically bind Cd2+ in the cytosol, thereby substantially and specifically improving Cd tolerance and accumulation under low heavy metal concentrations.
Highlights
HMA4 transporters are involved in the transport and binding of divalent heavy metals (Cd, Zn, Pb [lead] and Co [cobalt])
Characterization of BjHMA4 Isolation of B. juncea HMA4 cDNA Based on homology cloning strategies and the RACE technique, the BjHMA4 gene cloned from B. juncea was submitted to GenBank and was found to encode 1271 amino acids and have six transmembrane domains
Alignment with AtHMA4 indicated that BjHMA4 contains 5 highly conserved domains of HMA4: the metal-binding domain, A-domain, P-domain, N-domain and histidine-rich region (Fig. 1a) [16]
Summary
HMA4 transporters are involved in the transport and binding of divalent heavy metals (Cd, Zn, Pb [lead] and Co [cobalt]). Commonly known as Indian mustard, is a species of mustard plant. HMA4 (heavy metal ATPase 4) is a member of the type 1B heavy metal-transporting P-type ATPase subgroup, whose members transport the divalent cations Cd, Pb (lead), Zn, and Co (cobalt) [7, 8]. To date, this divalent class of transports exists only in prokaryotes and plants. The N-terminal regions of AtHMA2 and AtHMA4 are highly similar, and they exhibit similar expression patterns (predominantly in the vascular tissues of roots, stems and leaves) and subcellular localization (the plasma membrane) [11]
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