Chlorophyll was removed from each sample to aid later imaging of GUS staining from the histochemical treatment. Samples were suspended in acidified methanol (2 ml conc. HCl +10 ml methanol +38 ml H2O) for 15 min at 50uC with intermittent gentle shaking, before decanting and re-suspending in a neutral- isation solution (7% NaOH in 60% ethanol) for 15 min at RT. Solutions were discarded and retained samples were rehydrated using a series of decreasing concentrations of ethanol (from 40, 20 and 10% v/v). Once fully rehydrated in milli-Q H2O, samples were mounted in 50% glycerol (v/v) and viewed under a stereo microscope for traces of indigo staining to indicate GUS activity. Primers employed Sequences of primers employed to isolate HMA4 promoter sequences from A. thaliana, A. halleri and N. caerulescens were from 59 to 39: NcHMA4-2 promoter_Fwd CTCCTTCTGTAACGC- CATTTCTGTA NcHMA4-2 promoter_Rev CTCCTTCTGTAACGC- CATTTCTGTA AtHMA4 promoter_Fwd ACTTACCGATCGGGTATGC- CATG AtHMA4 promoter_Rev TTTCTCTTCTTCTTTGTTT- TGTAACGCC AhHMA4-3 promoter_Fwd GTGTTTGCTGGTGCTA- CTGTCTGA AhHMA4-3 promoter_Rev TTTCTCTTCTTCTTTGTT- TTGTGACGCC Supporting Information Figure S1 Consensus of the genomic illustration of the fosmid B3P40. Yellow bar represents the entire 27978 bp genomic insert. Green arrows illustrate both tandem repeats of NcHMA4-1 and NcHMA4-2 and their transcriptional direction. Blue script and lines highlight sites in the fosmid which were 100% specific for that primer. Image created through Vector NTI 11 (Invitrogen, Paisley, UK). (TIF) Figure S2 Consensus of the genomic illustration of the fosmid P6P46. Yellow bar represents the entire 31521 bp genomic insert. Green arrows illustrate both tandem repeats of NcHMA4-3 and NcHMA4-4 and their transcriptional direction. Blue script and lines highlight sites in the fosmid which were 100% specific for that primer. Image created through Vector NTI 11 (Invitrogen, Paisley, UK). (TIF) Figure S3 Consensus of the genomic illustration of the fosmid J12P81. Yellow bar represents the entire 31218 bp genomic insert. Green arrow illustrates a single copy of NcHMA4-2 its transcriptional direction. Brown arrows illustrate flanking genes At2g19160 and At2g19170 and their transcriptional directions. Flanking genes are labelled according to their A. thaliana orthologues. Blue script and lines highlight sites in the fosmid which were 100% specific for that primer. Image created through Vector NTI 11 (Invitrogen, Paisley, UK). (TIF) Figure S4 Consensus of the genomic illustration of the fosmid N18P80. Yellow bar represents the entire 20090 bp genomic insert. Green box illustrates a single copy of the 59 end of NcHMA4-3. Brown arrows illustrate flanking genes At2g19060, At2g19070, At2g19080 and At2g19090 and their transcriptional directions. Flanking genes are labelled according to their A. thaliana HMA4 Quadruplication in Noccaea caerulescens orthologues. Blue script and lines highlight sites in the fosmid which were 100% specific for that primer. Image created through Vector NTI 11 (Invitrogen, Paisley, UK). (TIF) Figure S5 Consensus of the genomic illustration of the fosmid H2P47. Yellow bar represents the entire 20258 bp genomic insert. Green arrow illustrates a single copy of NcHMA4-4 and its transcriptional direction. Blue script and lines highlight sites in the fosmid which were 100% specific for that primer. Image created through Vector NTI 11 (Invitrogen, Paisley, UK). (TIF) Figure S6 HMA4 coding sequence identities. A) Clado- gram and B) Dot Matrix comparisons of coding sequences of HMA4 orthologues from Ah: Arabidopsis halleri, At: Arabidopsis thaliana and Nc: Noccaea caerulescens. Tandem repeats are highlight- ed by ‘‘-’’. ‘‘Pra’’ and ‘‘Her’’ refer to publicly available sequence data from N. caerulescens ecotypes Prayon and He´rault. The cladogram was created for nucleotide sequences by the DNA Sequence Parsimony Method (DNApars), and was run at default settings in Phylip version 3.68. The Dot Matrix program was run at default settings and supplied by Vector NTI 11. Numbers represent percentage sequence identities. (TIF) Figure S7 HMA4 Protein sequence identities. A) Clado- gram and B) Dot Matrix comparison of protein sequences of HMA4 orthologues from Ah: Arabidopsis halleri, At: Arabidopsis thaliana and Nc: Noccaea caerulescens. Tandem repeats are highlight- ed by ‘‘-’’. ‘‘Pra’’ and ‘‘Her’’ refer to publicly available sequence data from N. caerulescens ecotypes Prayon and He´rault. The cladogram was created for amino acid sequences through Protpars, Protein Sequence Parsimony Method and was run at default settings and supplied by Phylip version 3.68. The Dot Matrix program was run at default settings and supplied by Vector NTI 11. (TIF) Figure S8 HMA4 promoter region sequence identities. A) Cladogram and B) Dot Matrix comparisons of sequences 2000 bp upstream from the transcriptional start site of HMA4 orthologues from Ah: Arabidopsis halleri, At: Arabidopsis thaliana and Nc: Noccaea caerulescens. Tandem repeats are differentiated by ‘‘-’’. The cladogram was created for nucleotide sequences by the DNA Sequence Parsimony Method (DNApars), and was run at default settings in Phylip version 3.68. The Dot Matrix program was run at default settings and supplied by Vector NTI 11. Numbers represent percentage sequence identities. (TIF) Data S1 (DOC) Data S2 (DOC) Data S3 (DOC) Data S4 (DOC) Data S5 (DOC) Fosmid B3P40 insert sequence. Fosmid P6P46 insert sequence. Fosmid J12P81 insert sequence. Fosmid N18P80 insert sequence. Fosmid H2P47 insert sequence. Data S6 Consensus sequence of the entire NcHMA4 single genomic locus. (DOC) PLoS ONE | www.plosone.org 7 March 2011 | Volume 6 | Issue 3 | e17814