Figure 1.
Southern hybridization of A. halleri MTP1 harbouring BAC clones.
BAC DNA digested with EcoRI (panel A) or PstI (panel B) was probed with a full length AhMTP1 probe. Names of the BAC clones are given at the top of the lanes. The α, β, γ, and δ symbols are the names given to the four distinct hybridisation profiles. Sizes of ladder are shown on the right of each panel.
Figure 2.
Mapping of the AhMTP1 paralogues on the A. halleri × A. lyrata petraea BC1 linkage map.
The mapping was performed from the analysis of 199 plants and the four parents of an A. halleri × A. lyrata petraea BC1 population. Positions of BAC clones 7G24, 12L21, 2B14, and 1F18 that harbour the AhMTP1-A1 & -A2, AhMTP1-B, AhMTP1-C, and AhMTP1-D paralogues, respectively, are indicated in red letters (1F18rev and 1F18for are two independent markers). The other markers presented on the map have been previously described [20]. Only linkage groups (LG) 1, 4, and 6 of the A. halleri genome are shown. Colours of the bars representing the three linkage groups refer to the conserved synteny between the A. halleri and the A. thaliana genomes as inferred [22]. The regions showing no conserved synteny with A. thaliana are indicated in grey colour. The map was constructed using Joinmap 3.0. The scale to the left of figure represents centi-morgan distances.
Figure 3.
Phylogenetic analysis of MTP1 gene sub-family from A. thaliana, A. lyrata, and A. halleri.
(A) Maximum likelihood tree of MTP1 protein sequences from A. thaliana, A. lyrata and A. halleri. Bootstrap values are indicated in percentage (100 replicates). (B) Alignment of predicted amino acid sequences of MTP1s from A. thaliana, A. lyrata and A. halleri. Sequences are represented in 80 amino acids long blocks. The AtMTP1 protein sequence is from accession NP_850459 and the AlMTP1 one was extracted from scaffold 4 of the A. lyrata sequencing project available on http://genome.jgi-psf.org/cgi-bin/runAlignment?db=Araly1&advanced=1. Identities with AtMTP1 are represented by a dot and differences are written in alphabets. Dashes (–) signify deletions. Six transmembrane domains (TMDI to TMDVI) predicted using TMHMM server v. 2.0 [34] are shaded. The histidine rich loop is located between TMDIV and TMDV.
Figure 4.
Physical maps comparing the putative promoter plus 5′ UTR regions among A. thaliana, A. lyrata, and A. halleri MTP1 homologues.
Regions sharing >80% identity are shown by same coloured rectangular boxes or by same shapes. Dashed lines between different gene structures enable the relative positioning of the similar regions. Dotted lines below the gene structures indicate the position of introns. Small triangles present below or above the gene structures indicate 11 bp to 17 bp insertions (see text). Putative transcription start sites and translation start sites are indicated by +1 and ATG respectively. The Retrovirus-related Pol-polyprotein from transposon TNT 1–94 located in the putative promoter region of MTP1-A is represented by a thick line below the gene structure. Scale is shown at the bottom, relative to the ATG initiation codon.
Figure 5.
Analysis of the presence of the MTP1-D paralogue in different A. halleri genotypes.
(A) PCR analysis of two plants of the A. halleri Auby accession (D and SAF2 plants) using AhMTP1-A, -B, -C, and -D gene specific primer pairs. DNA from BAC clones 1F18, 2B14, 7G24 and 12L21 are positive controls for demonstrating the specific amplification of AhMTP1-D, -C, -A, and -B, respectively. Corresponding sizes of amplicons are indicated on the right of each lane. (B) Southern analysis of the D and SAF2 plants of the Auby accession. Plant genomic DNAs and mixed BAC clone DNAs (positive control) were digested with PstI restriction enzyme. The probe was amplified from the AhMTP1-D harbouring BAC clone. Sizes deduced from a ladder are shown to right of the panel. (C) Production of AhMTP1-D specific amplicons from plants belonging to 14 different A. halleri populations. The 1 through 14 numbers above the lanes represent the following A. halleri populations, respectively: M Auby, M Sauerland, NM Regen, M Harz, NM CZ8-13, NM nord Tyrol, M Katowice-Weinowice, NM Zakopane, NM Appusenes, NM Fagaras Ro-12-6, NM Fagaras Ro-ovirensis, NM Southern Tyrol, M Lombardie and NM Tessin, where M = metallicolous and NM = non metallicolous. AhMTP1-B specific primer pairs were used as a control.
Figure 6.
Functional complementation of the zinc-hypersensitivity of the zrc1 cot1 yeast mutant by AhMTP1s and AtMTP1.
Wild-type BY4741 (WT) and mutant zrc1 cot1 yeast strains harboured the empty pFL38H (EV1) that brought histidine autotrophy and either empty pYX212 vector (EV2) or pYX212 bearing one of MTP1s. Serial dilutions were spotted on modified selective LSP medium supplemented with different concentrations of ZnSO4 as indicated above the panels. Each spot was made with 10 µl of a yeast culture diluted at the OD600nm mentioned below the drops. Pictures were taken after 2 days for control and 4 days for other treatments.
Figure 7.
AhMTP1s transcript accumulation in plants submitted to different zinc treatments.
Roots and shoots were collected from plants of the A. halleri SAF2 genotype issued from the Auby accession that were exposed to 10 (control), 100, 300, or 1000 µM ZnSO4 for 4 days. Real-time quantitative RT–PCR was performed using gene copy specific primer pairs separately for shoots and roots. Data shown are transcript levels of AhMTP1s relative to Actin. Each data point in the graph is the average of three PCR repetitions for each of six biological replicates. Errors bars correspond to confidence intervals at the 0.05 threshold.
Table 1.
Gene-specific primer pairs used to characterise the different AhMTP1 paralogues in real-time quantitative RT–PCR analyses.