HMA4 Quadruplication in Noccaea caerulescens Figure 1. Montage of two gel blot autoradiographs of NcHMA4 tandem repeats from N. caerulescens genomic DNA and genomic library fosmid insert DNA. All DNA was digested with EcoRI, HindIII or BamHI corresponding to lanes 1, 2 or 3 respectively, resolved on two 0.9% (w/v) agarose gels, blotted, and hybridized with the radiolabeled NcHMA4 library probe (represented by darkened regions). Fosmids labelled with ‘+’ contain tandem repeats of a NcHMA4 insert. The DNA marker was a 1 kb DNA ladder (Hyperladder I, Bioline). Montage was prepared using CorelDraw Graphics Suite X3. doi:10.1371/journal.pone.0017814.g001 relatively recent evolutionary event within N. caerulescens (Figure S6). NcHMA4-4 contained a truncation in exon 9 after amino acid (aa) 684 of the deduced protein sequence (Figure 4) and could indicate a functional but less efficient in planta Zn transporter, as recently reported for an AtHMA4 which contained a comparable truncation after aa 713 [43]. At the deduced amino acid level, NcHMA4 share between 92 and 98% identity, but only between 72 and 83% identity with AtHMA4 and between 74 and 84% identity with the three AhHMA4 (Figure S7). Within the first 2000 bp upstream of the translational start codon, NcHMA4 sequences shared 59 and 98% identity, but between 44–49% and 41–51% identity with A. thaliana and A. halleri promoter sequences respectively (Figure S8). AhHMA4 regions shared greater identity, 53–88% with AtHMA4, as previously reported [19]. Significant sequence divergence from A. thaliana and A. halleri in the 59-flanking regions of NcHMA4 genes indicates cis gene regulation may differ between species. In A. halleri, high HMA4 expression was regulated in cis and amplified by a triplication in gene copy number [19]. Increased expression of AhHMA4 correlated with enhanced Zn flux from the root symplasm into the xylem parenchyma as well as up-regulation of Zn deficiency response genes in roots supporting its role in Zn hyperaccumulation. Expression profile of NcHMA4 To investigate the expression profile of NcHMA4, T2 A. thaliana plants (n = 30), transformed with HMA4 promoters from A. thaliana (AtHMA4p::GUS, negative control), A. halleri (AhHMA4-3p::GUS, positive control) and N. caerulescens (NcHMA4-2p::GUS) fused to the b-glucuronidase (GUS) reporter gene, were analysed for GUS activity under identical nutrient replete conditions in vitro, 21 days after sowing (DAS). Figure 2. Agarose gel electrophoresis of PCR products from fosmid clones containing N. caerulescens HMA4 sequences and Noccaea genomic DNA. Primers were specific for NcHMA4-1, NcHMA4-1 and 4-2, NcHMA4-3 and NcHMA4-4. Lanes were labelled according to fosmid clones or ‘Genomic’ Noccaea caerulescens DNA. The molecular ladder was a 1 kb DNA ladder (Hyperladder I, Bioline). Gel contained 1% (w/v) agarose. doi:10.1371/journal.pone.0017814.g002 PLoS ONE | www.plosone.org 3 March 2011 | Volume 6 | Issue 3 | e17814