An evolutionary perspective on differential regulation of zinc and cadmium homeostatis genes in Arabidopsis thaliana and Noccaea caerulescens

Some plants can tolerate and accumulate unusually high levels of toxic metals, and the analysis of such plants can provide insights into the ecology of environments that are polluted with heavy metals due to human industrial activities. The study of heavy metal hyperaccumulators such as Noccaea caerulescens can show how plants cope with excess metals and increase their fitness when growing in metalliferous environments. In this thesis, I compared the molecular mechanisms of zinc (Zn) homeostasis and cadmium (Cd) response in the hyperaccumulator species N. caerulescens and its non-accumulator relative Arabidopsis thalianaby investigating the regulation of the ZNT1/ZIP4gene that promotes Zn uptake and Zn/Cd tolerance.I also studied the ecological advantages of metal hyperaccumulators in nature and determined the DNA sequences of the N. caerulescens transcriptometo find candidate genes that control metal hyperaccumulation and provide an evolutionary perspective on the emergence of this trait. The functional characterization of the N. caerulescens and A. thaliana Zn-transporter genes NcZNT1 and AtZIP4 (and their promoters) showed how their differential expression pattern contributed to their role in metal tolerance and accumulation. The NcZNT1 gene is induced by Zn deficiency and the NcZNT1 protein is localized in the plasma membrane. Transgenic N. caerulescens roots containing a transgene for the green fluorescent protein (GFP) driven by the NcZNT1promoter revealed GFP fluorescence localized to pericycle and vascular tissues. This suggests that NcZNT1 contributes to metal loading into the xylem and long-distance metal transport. The overexpression of NcZNT1 in A. thaliana increased Zn and Cd tolerance and the capacity to accumulate these metals compared to wild-type plants. These results suggest that NcZNT1 plays an important role in Zn and Cd hypertolerance and hyperaccumulation in N. caerulescens, where it is expressed in both Zn-sufficient and Zn-excess conditions. The differential activity of the NcZNT1 and AtZIP4 promoters in N. caerulescens and A. thaliana implies that different cis-regulatory elements and trans-regulatory factors are present in both species. The ecological advantage of hyperaccumulators in metal-contaminated soils was investigated by studying a natural nas1mutant, in which the Nicotianamine Synthase1 gene is disrupted by a transposon insertion. This mutant allele was found in three natural N. caerulescens populations, which were compared to wild-type plants in terms of phenotype and adaptive advantage. Although the transposon disrupted the gene, the loss of NAS1 gene activity was compensated by enhanced expression of NAS3 and NAS4, resulting in increased nicotianamine (NA) production, which enhanced the Zn and Cd accumulation in the nas1 mutants. This increased their metal sensitivity compared to wild-type plants, but also made them more toxic towards Pieris rapae caterpillars, which developed more slowly and gained less weight when fed on mutant plants exposed to excess Zn or Cd. Therefore, the possible selective advantage of the nas1 mutant in nature is high metal accumulation and the protection of plants from herbivores. Differences in nas1 allele frequency among the three natural populations suggests that nas1 alleles experience different degrees of natural selection or may be at different stages on the route to fixation. Molecular evolutionary studies involve the identification of candidate genes that play a role in adaptation. Therefore, a comprehensive set of transcript sequences was obtained from N. caerulescens accession Ganges (GA) by 454 pyrosequencing. In total, the collected 23,836 isotigs (putative transcripts) were grouped into 20,378 isogroups and 93.2% of them could be matched to Brassicaceae protein sequences, which allowedtheir functional annotation. A total of 87 isogroups was annotated as metal homeostasis related genes, including metal transporter families, metal chelator biosynthesis families, and metal tolerance gene families, which are candidate genes for the molecular analysis of heavy metal homeostasis mechanisms. A group of genes required for the synthesis of glucosinolates, which are important secondary metabolites that protect plants against herbivores, were also shown to be expressed in N. caerulescens. The METALLOTHIONEIN3 (MT3) gene was found to have been duplicated in the N. caerulescens genome, when compared to related Brassicaceae. These transcript sequences will provide an important tool to annotate the genome sequence of the N. caerulescensGanges accession, which is in progress. This genome sequence will also be the basis of genome comparisons between the different N. caerulescens accessions that have different levels of metal accumulation and metal tolerance and which may therefore differ in terms of gene expression levels or modes of actions that affect metal homeostasis.