throughout plants when driven by either the NcHMA4-2 or the AhHMA4-3 promoters (Figure 5). Conclusion The aim of this study was to test the hypothesis that tandem duplication and deregulation of HMA4 expression, which occurs in A. halleri, occurs in N. caerulescens. A fosmid library comprising 36,864,40 kb inserts was developed, representing a potentially valuable resource for future map-based cloning and genome sequencing in N. caerulescens. Following de novo sequencing, there was compelling evidence of tandem quadruplication for HMA4 in N. caerulescens. Whilst it is hypothetically feasible that allelic artefacts can occur, even in highly inbred populations, here, the sequencing of multiple fosmids (including long-reads of intergenic regions/non-coding repeats which are overlapping between fosmids) provides very strong support for tandem repeats of NcHMA4. This observation is strikingly consistent with a tandem HMA4 triplication in the A. halleri genome [19]. Noccaea caerulescens and A. halleri last shared a common ancestor .40 mya [44], and the current study provides intriguing new evidence that parallel evolutionary pathways may underlie two occurrences of Zn/Cd hyperaccumulation in the Brassicaceae. Further detailed sequenc- ing is now required in a wider number of species. An initial functional analysis was undertaken of HMA4 using promoters from both N. caerulescens and A. halleri expressed in A. thaliana. Again, results were remarkably consistent between species, and the regulation of HMA4 from both hyperaccumulator species appears to be distinct from endogenous AtHMA4 regulation. GUS expression was driven more highly throughout the plant by NcHMA4-2p and AhHMA4-3p than by AtHMA4p, consistent with high levels of HMA4 transcripts in shoots and roots of N. caerulescens [20-22] and A. halleri [19,45]. Throughout leaf tissue, promoters from both hyperaccumulator species drove enhanced GUS activity compared to AtHMA4p, which is consistent with a possible role in increasing Zn accumulation in leaf epidermal cells in N. caerulescens [46,47] and mesophyll cells in A. halleri [48]. Moreover, expression of all three AhHMA4 promoters in the xylem parenchyma and the cambium of leaves in both A. thaliana and A. halleri were hypothesised to be consistent with putative roles in Zn exclusion from particular cell types and metal distribution within the leaf blade [19]. Figure 5. The spatial expression of b-glucuronidase (GUS) fused to HMA4 promoter regions in Arabidopsis thaliana. The activity of GUS in whole leaves A–C and roots D–F from 21 day old in vitro cultured A. thaliana T2 transformants bearing pGWB3 constructs containing the GUS marker gene under the control of promoter sequences from A, D; AtHMA4, B, E; NcHMA4-2 and C, F; AhHMA4-3. Red bars represent 2 mm. doi:10.1371/journal.pone.0017814.g005 HMA4 Quadruplication in Noccaea caerulescens In contrast to the relatively high, shared HMA4 sequence identities between all three genomes, N. caerulescens HMA4 promoters (NcHMA4p) exhibited lower identities with those from both A. thaliana and A. halleri. We conclude that novel cis regulatory elements in N. caerulescens contribute to increased NcHMA4 gene expression. Further elucidating these cis regulatory regions in hyperaccumulators could enable the manipulation of HMA4 expression that may be exploited for use within crop systems to enhance Zn leaf accumulation for biofortification or phytoreme- diation strategies. Materials and Methods Library Construction DNA from a Noccaea caerulescens (J.&C. Presl) F.K.Mey (,250 Mb genome, 2n = 2x = 14) from a population originating from Saint Laurent Le Minier, southern France [30] was used to construct a genomic fosmid library by Warwick Plant Genomic Libraries Ltd. (Warwick HRI, Warwick, UK). Preparation of Noccaea DNA and bacterial cells for filter arraying DNA (2.5 mg), extracted from leaf tissue of a single N. caerulescens plant via the phenol – chloroform procedure [49], was randomly sheared to 40 kb fragments and end repaired to blunt, 59- phosphorylated ends. Fragments were size resolved and purified from a low melting point (LMP) agarose gel (without exposure to UV irradiation), before ligating to 8 kb Cloning-Ready Copy- Control pCC1FOS vectors and phage packaging (Epicentre Biotechnologies, Madison, W.I., USA). EPI300TM-T1R plating strains were streaked on solid Luria-Bertani (LB) plates and grown for 12 h at 37uC. A starter culture (5 ml LB broth) was inoculated with a single colony and incubated on a shaker at 225 rpm. for a further 12 h at 30uC. 50 ml of LB broth +10 mM MgSO4+0.2% (w/v) maltose (20% filter sterilised stock) was inoculated with 1 ml of starter culture and shaken at 37uC for 2–3 h until an optical density at 600 nm (OD600) of 0.8–1.0 was reached. Bacteria was pelleted (5006 g for 10 mins.), gently resuspended in 25 ml of 10 mM MgSO4, before being diluted to an OD600 of 0.5 with sterile 10 mM MgSO4. A 25 ml aliquot of this solution was mixed in a 2 ml microcentrifuge tube with 25 ml of the fosmid packaging reaction (diluted in phage dilution buffer according to library titre), and incubated for 30 mins at room temperature (RT). LB (200 ml) was added to each sample and incubated for 1 h at 37uC, shaking the tube gently once every 15 minutes. CopyControl fosmid clones were selected by pelleting samples (1 min. at 10,000 rpm.) and resuspending in fresh LB medium before spreading on LB agar plates supplemented with 12.5 mg ml21 chloramphenicol and incubating at 37uC for 12 h. Colonies were then picked into 384 well plates using a Q-Pix II bench top colony picker (Genetix Ltd., New Milton, Hampshire, UK). The filters were constructed using a MicroGrid II high-throughput automated microarrayer, (BioR- obotics Ltd., Cambridge, UK). Probing the N. caerulescens genomic library Noccaea caerulescens library DNA fragments were cloned into 36,864 E-coli EPI300TM-T1R host cells and stored in 966384-well microtiter plates which were arrayed evenly onto two nitrocellu- lose filters (48 plates per filter). Each well contained duplicated DNA fragments, whose arrangement indicated the plate of origin for DNA that hybridised to the HMA4 probe. Filters contained approximately 5% ribosomal and 15% chloroplast contamination. PLoS ONE | www.plosone.org 5 March 2011 | Volume 6 | Issue 3 | e17814