Genome Structure of the Heavy Metal Hyperaccumulator Noccaea caerulescens and Its Stability on Metalliferous and Nonmetalliferous Soils

Noccaeacaerulescens (formerly known as Thlaspi caerulescens), an extremophile heavy metal hyperaccumulator model plant in the Brassicaceae family, is a morphologically and phenotypically diverse species exhibiting metal tolerance and leaf accumulation of zinc, cadmium, and nickel. Here, we provide a detailed genome structure of the approximately 267-Mb N. caerulescens genome, which has descended from seven chromosomes of the ancestral proto-Calepineae Karyotype (n = 7) through an unusually high number of pericentric inversions. Genome analysis in two other related species, Noccaea jankae and Raparia bulbosa, showed that all three species, and thus probably the entire Coluteocarpeae tribe, have descended from the proto-Calepineae Karyotype. All three analyzed species share the chromosome structure of six out of seven chromosomes and an unusually high metal accumulation in leaves, which remains moderate in N. jankae and R. bulbosa and is extreme in N. caerulescens. Among these species, N. caerulescens has the most derived karyotype, with species-specific inversions on chromosome NC6, which grouped onto its bottom arm functionally related genes of zinc and iron metal homeostasis comprising the major candidate genes NICOTIANAMINE SYNTHASE2 and ZINC-INDUCED FACILITATOR-LIKE1. Concurrently, copper and organellar metal homeostasis genes, which are functionally unrelated to the extreme traits characteristic of N. caerulescens, were grouped onto the top arm of NC6. Compared with Arabidopsis thaliana, more distal chromosomal positions in N. caerulescens were enriched among more highly expressed metal homeostasis genes but not among other groups of genes. Thus, chromosome rearrangements could have facilitated the evolution of enhanced metal homeostasis gene expression, a known hallmark of metal hyperaccumulation.Noccaea caerulescens (formerly known as Thlaspi caerulescens) is a diploid (2n = 14) biennial or short-living perennial plant from the family Brassicaceae. N. caerulescens is native to Europe, with a patchy distribution from the United Kingdom and France to Slovakia and from Germany and Poland southward to northern Spain and Italy. The widespread occurrence in Scandinavia is thought to represent naturalized populations over the past few hundred years (Koch et al., 1998, and refs. therein). N. caerulescens is one of the 120 Noccaea spp., which, together with two other genera, constitute the tribe Coluteocarpeae (approximately 127 species; Al-Shehbaz, 2012). However, the generic treatment of the tribe is far from settled, and up to 12 genera are recognized in Coluteocarpeae by F.K. Meyer (Meyer, 2001, 2006a, 2006b; Koch and German, 2013).Together with the metal hyperaccumulator species Arabidopsis halleri, N. caerulescens is among the most prominent plant model systems for the study of heavy metal hyperaccumulation and associated hypertolerance (Krämer, 2010; Hanikenne and Nouet, 2011; Pollard et al., 2014). N. caerulescens is a hyperaccumulator of zinc (Zn) on metalliferous as well as nonmetalliferous soils and of cadmium (Cd) on metalliferous soils (Reeves et al., 2001; Krämer, 2010), and its populations on serpentine soils are known to hyperaccumulate nickel (Ni; Reeves and Brooks, 1983; Reeves, 1988; Visioli et al., 2012; Maestri et al., 2013). N. caerulescens is a morphologically highly diverse species comprising at least two, but up to eight (http://www.gbif.org), recognized subspecies with partly overlapping distribution ranges (the simplest treatment includes ssp. caerulescens and sylvestris, the latter including populations formerly described as Thlaspi calaminare or N. caerulescens ssp. calaminare). As the morphological variation often has a clinal character, the taxonomic and biological value of various intraspecific taxa is questionable, and detailed studies will be needed to resolve this issue (for discussion, see Koch and German, 2013). In addition to pervasive morphological variation (Koch and German, 2013) apparently reinforced by geographical constraints (Besnard et al., 2009), there is also pronounced phenotypic variation in metal tolerance and accumulation (Escarré et al., 2000; Reeves et al., 2001; Assunção et al., 2006; Xing et al., 2008; Krämer, 2010; Tuomainen et al., 2010).Although N. caerulescens is the most commonly studied metal hyperaccumulator model species, with more than 210 studies published on the subject (Pollard et al., 2014), the detailed genome structure of N. caerulescens remains unresolved. Assunção et al. (2006) published the first amplified fragment-length polymorphism-based genetic linkage map and identified quantitative trait loci for Zn accumulation in roots. Another amplified fragment-length polymorphism-based genetic map based on a cross between two accessions with differential Cd accumulation and tolerance was used to identify quantitative trait loci associated with the accumulation of Cd and Zn (Deniau et al., 2006). Both maps comprised the expected seven linkage groups with dense clusters of linked markers located on each linkage group, most likely corresponding to centromeric regions with suppressed recombination rates. Apart from the tentative identification of centromeres, small numbers of orthologous markers shared with Arabidopsis thaliana and A. halleri did not allow the establishment of chromosome collinearity between these genomes for inference of the genome structure of N. caerulescens.More recently, Mandáková and Lysak (2008) reconstructed karyotype evolution in eight Brassicaceae species of tribes in extended lineage II (Franzke et al., 2011) by comparative chromosome painting (CCP) using chromosome-specific bacterial artificial chromosome (BAC) contigs of A.thaliana. They concluded that genomes of all the analyzed species with seven or 14 chromosome pairs (n = 7/14) were derived from the eight chromosomes of the Ancestral Crucifer Karyotype (ACK; n = 8) via an ancestral n = 7 genome named the proto-Calepineae Karyotype (PCK). The genome of N. caerulescens (accession Korenec), analyzed as a representative of the tribe Coluteocarpeae (formerly Noccaeeae) by Mandáková and Lysak (2008), has descended from a PCK-like ancestor but showed a remarkably high number of secondary chromosome rearrangements. By comparison with the ancestral PCK, in N. caerulescens six of the seven ancestral chromosomes were reshuffled by inversions encompassing pericentromeric regions. However, that study did not establish the detailed structure of the N. caerulescens genome, including precise positions of chromosome break points. Consequently, evolutionary steps leading to the origin of the inversion chromosomes were reconstructed only approximately, and gene content could not be estimated.Our study here provides a detailed comparative genome structure of N. caerulescens and relates the genome structure to the evolution of heavy metal-related extreme traits. Based on a more precise definition of ancestral genomic blocks within the ACK (Cheng et al., 2013) and using PCK as the most probable ancestral genome of N. caerulescens, we carried out a detailed analysis of the entire karyotype, including inversions, by means of CCP. Furthermore, considering the unusually high incidence of inversions, we were intrigued to find out whether this variation is unique to the reference accession (Mandáková and Lysak, 2008) or fixed in populations on both nonmetalliferous and metalliferous soils enriched for different heavy metal elements. Toward this aim, we constructed detailed comparative karyotypes for 13 populations of N. caerulescens from metalliferous and nonmetalliferous soils throughout the European species distribution range, including, in particular, populations from southern France known for the extraordinarily high concentrations of Zn and Cd in their leaves (St. Félix de Pallières and Viviez; Reeves et al., 2001). Next, we asked whether the inversions are specific to N. caerulescens or shared by other Coluteocarpeae species by deducing the chromosome structure and evolution of the N. caerulescens genome in comparison with two other species from the tribe Coluteocarpeae, Noccaea jankae and Raparia bulbosa. Finally, we addressed the question of whether chromosome inversions and rearrangements might have affected the physical positions of metal homeostasis candidate genes that were proposed to act in naturally selected metal hyperaccumulation and associated hypertolerance of N. caerulescens. We tested for the clustering of functionally related metal homeostasis genes in closer proximity on the chromosome toward supergene formation as well as for the relation between changes in chromosomal position and gene expression among metal homeostasis genes.