Heavy metal tolerance and accumulation in Thlaspi caerulescens, a heavy metal hyperaccumulating plant species = Zware metalen tolerantie en accumulatie in Thlaspi caerulescens, een zware metalen hyperaccumulerende plantensoort

Minerals are essential for humans, plants and animals and have an important micronutrient role in physiological and metabolic processes of plants. Next to this essential role of minerals, they can also be very toxic when available to the plant in elevated amounts. Plants therefore need to keep very tight control over the intracellular mineral concentrations in a process called metal homeostasis. Although the metal homeostasis mechanisms are supposed to be universal within plants, there are plant species that can tolerate and even accumulate large amounts of metals without any sign of toxicity. <i>Thlaspi caerulescens</i> J. &amp; C. Presl (Brassicaceae), a close relative of the plant reference species <i>Arabidopsis thaliana</i> (Arabidopsis), is one of these natural metal hyperaccumulator species. The overall aim of this project is to unravel the molecular genetic mechanism of heavy metal tolerance and hyperaccumulation of the metal hyperaccumulating plant species <i>T. caerulescens. </i>To achieve this goal heterologous transcript profiling experiments were performed, which involved comparative microarray hybridization experiments of the hyperaccumulator <i>T. caerulescens</i> and Arabidopsis. Arabidopsis is used as the reference species for heterologous transcript profiling because of the availability of genetic resources and the complete genome sequence. The micronutrient zinc has an essential role in physiological and metabolic processes in plants as a cofactor or structural element. <i>Thlaspi caerulescens</i> can accumulate up to 3% of zinc on a dry weight basis without any sign of toxicity. The question postulated here is if this has drastic effects on the zinc homeostasis mechanism. We examined in detail the transcription profiles of roots of Arabidopsis and <i>T. caerulescens </i>plants grown under deficient, sufficient and excess supply of zinc (<b>Chapter 2</b>). A total of 608 genes were detected in Arabidopsis and 352 in <i>T. caerulescens</i> that responded transcriptionally to changes in zinc supply. Only 14% of these genes were also zinc-responsive in Arabidopsis. When comparing Arabidopsis and <i>T. caerulescens </i>at comparable zinc exposures, over 2200 genes were significantly differentially expressed. While a large fraction of these genes are of yet unknown function, many genes with a different expression between Arabidopsis and <i>T. caerulescens</i> appear to function in metal homeostasis, in abiotic stress response and in lignin biosynthesis. The high expression of lignin biosynthesis genes corresponds to the deposition of lignin in the endodermis. Contrary to Arabidopsis roots, which have one endodermal cell layer, we found there are two endodermal layers in <i>T. caerulescens</i> roots. This extra physical barrier could enhance the control of metal fluxes in the plant, in addition to the higher expression of metal transporters in the root.<b/> Cadmium is a widespread, naturally occurring non-essential element that is toxic for plants in higher concentrations. In <b>chapter 3</b> we compare between and within species transcript profiles of Arabidopsis<i> </i>and <i>T. caerulescens </i>roots exposed to cadmium, with the aim to establish which genes are most likely to be relevant for the tolerance to cadmium exposure of <i>T. caerulescens</i>. The comparative transcriptional analysis of the cadmium response of roots of the <i>T. caerulescens</i> and Arabidopsis emphasizes the role of genes involved in lignin-, glutathione- and sulfate metabolism. Furthermore two transcription factors, <i>MYB72 </i>and <i>bHLH100</i>, with an altered expression after exposure to cadmium, are studied for their involvement in metal homeostasis. Analysis of a <i>myb72</i> knock-out mutant showed enhanced sensitivity to excess zinc or iron deficiency. Rather than controlling Cd tolerance, this gene appears to be involved in iron homeostasis, affecting the response to Cd indirectly. Arabidopsis<i> </i>transformants overexpressing the transcription factor <i>bHLH100</i> showed enhanced zinc and nickel tolerance, and although the exact role of this gene still needs to be resolved, the genes appears to have a role in metal homeostasis in Arabidopsis. <i>T. caerulescens</i> accessions exhibit distinct metal accumulation, translocation and tolerance characteristics. <i>T. caerulescens</i> accession Ganges can accumulate high amounts of cadmium and is extremely tolerant to cadmium, whereas the La Calamine accession is also tolerant to cadmium but accumulates much less cadmium compared to Ganges. The transcription profiles of leaves and roots of <i>T. caerulescens</i> accessions Ganges and La Calamine plants grown with and without cadmium were examined using the Qiagen-Operon Arabidopsis Genome Array and results are described in <b>chapter 4</b>. A total of 161 genes were differentially expressed between the two <i>T. caerulescens</i> accessions in response to changes in cadmium supply and 38 genes were differentially expressed in <i>T. caerulescens</i> accession Ganges leaves in response to cadmium. The comparative transcriptional analysis emphasizes that there are just minor differences between the two accessions but the genes which are differentially expressed could play an important role in the hyperaccumulation of cadmium in Ganges. The microarray data suggest that especially genes involved in cell wall modification and stress response relate to the major difference between the two accessions in cadmium hyperaccumulation. Plants have evolved a complex network of homeostatic mechanisms that serve to control the uptake, accumulation, trafficking and detoxification of metals. One potential mechanism for heavy metal detoxification in plants is the chelation of metal ions to ligands like organic acids, amino acids, peptides and polypeptides. This mechanism is important for the distribution of metal ions by keeping metal ions mobile within the plant. In plants metals are often found to be chelated to nicotianamine. Nicotianamine is formed by trimerization of S-adenosylmethionine, which is catalyzed by the enzyme nicotianamine synthase. Arabidopsis contains four nicotianamine synthase (<i>NAS</i>) genes. Also in <i>T. caerulescens</i> four full-length cDNAs encoding nicotianamine synthase members were identified (<b>Chapter 5</b>). The four genes were named <i>TcNAS1-TcNAS4</i>, analogous to the corresponding closest homologue in Arabidopsis. Arabidopsis<i> </i>plants over-expressing <i>TcNAS1</i>, <i>TcNAS2</i>, <i>TcNAS3</i> or <i>TcNAS4</i> that were tested for their response to growth on media with different zinc, iron, nickel or cadmium supply, provided evidence that the <i>Thlaspi</i> genes all have a genuine NAS function because they complement the NAS deficiency in specific triple knock-out Arabidopsis mutants. Evidence for a functional role in metal homeostasis was sought in studying the Arabidopsis<i> </i>single, double, triple and a quadruple nicotianamine synthase T-DNA insertion mutants. The combination of null mutations in three or four <i>AtNAS</i> genes, results in a severe phenotype that includes interveinal chlorosis and altered metal concentrations in leaves, roots and seeds. Arabidopsis transformants overexpressing <i>TcNAS3</i> or <i>TcNAS4</i> showed enhanced zinc and nickel tolerance compared to wild type plants. The research described in this thesis does contribute to a better understanding of heavy metal hyperaccumulation in <i>T. caerulescens</i> and it can be concluded that it seems unlikely that altered regulation and overexpression of single genes will be sufficient to convert metal nonaccumulators into hyperaccumulators. However, the possibility that overexpression of one or two key regulatory loci have this effect remains