Tandem Quadruplication of HMA4 in the Zinc (Zn) and Cadmium (Cd) Hyperaccumulator Noccaea caerulescens

Abstract

Zinc (Zn) and cadmium (Cd) hyperaccumulation may have evolved twice in the Brassicaceae, in Arabidopsis halleri and in the Noccaea genus. Tandem gene duplication and deregulated expression of the Zn transporter, HMA4, has previously been linked to Zn/Cd hyperaccumulation in A. halleri. Here, we tested the hypothesis that tandem duplication and deregulation of HMA4 expression also occurs in Noccaea.A Noccaea caerulescens genomic library was generated, containing 36,864 fosmid pCC1FOS™ clones with insert sizes ∼20–40 kbp, and screened with a PCR-generated HMA4 genomic probe. Gene copy number within the genome was estimated through DNA fingerprinting and pooled fosmid pyrosequencing. Gene copy numbers within individual clones was determined by PCR analyses with novel locus specific primers. Entire fosmids were then sequenced individually and reads equivalent to 20-fold coverage were assembled to generate complete whole contigs.Four tandem HMA4 repeats were identified in a contiguous sequence of 101,480 bp based on sequence overlap identities. These were flanked by regions syntenous with up and downstream regions of AtHMA4 in Arabidopsis thaliana. Promoter-reporter β-glucuronidase (GUS) fusion analysis of a NcHMA4 in A. thaliana revealed deregulated expression in roots and shoots, analogous to AhHMA4 promoters, but distinct from AtHMA4 expression which localised to the root vascular tissue.This remarkable consistency in tandem duplication and deregulated expression of metal transport genes between N. caerulescens and A. halleri, which last shared a common ancestor >40 mya, provides intriguing evidence that parallel evolutionary pathways may underlie Zn/Cd hyperaccumulation in Brassicaceae.

How metal hyperaccumulating plants can advance Zn biofortification

“…The promoters of these copies drive much stronger expression than the promoter of the A. thaliana ortholog (Hanikenne et al 2008). For N. caerulescens analogous findings with four copies in tandem were reported (Lochlainn et al 2011). The importance of changes at the HMA4 locus for the evolution of Zn hyperaccumulation was demonstrated when a selective sweep, i.e.…”

Noccaea caerulescens populations adapted to grow in metalliferous and non-metalliferous soils: Ni tolerance, accumulation and expression analysis of genes involved in metal homeostasis

“…N. caerulescens HMA4 was found expressed in roots and shoots, induced both by Zn deficiency and Zn excess, as well as in response to Cd (Papoyan and Kochian, 2004;Hammond et al, 2006;Halimaa et al, 2014). The evidence of a pivotal role of HMA4 in Zn hyperaccumulation and full tolerance to Cd/Zn in A. halleri and in N. caerulescens populations suggests a parallel evolution pathway for Zn/Cd hyperaccumulation that differs from that of the Ni hyperaccumulation (Hanikenne et al, 2008;Ó Lochlainn et al, 2011). The other member of this class of transporters is HMA3, which is the most over-expressed gene in shoots of A. halleri, both in control and in high-Zn conditions (Becher et al, 2004;Ueno et al, 2011), but no evidence of its involvement in Ni tolerance and accumulation was found in the literature so far.…”

“…In addition, HMA3 and HMA4 varied in copy number among populations of N. caerulescens adapted to grow on different soil types. Interestingly, both the copy number and the level of expression of these gene seem to be associated with N. caerulescens populations showing higher levels of Cd tolerance and accumulation (Ueno et al, 2011;Ó Lochlainn et al, 2011;Craciun et al, 2012).…”

Evolutionary aspects of elemental hyperaccumulation

“…The population with the least efficient Cd translocation had the lowest expression level and the lowest copy number (Craciun et al 2012). There can be up to four tandem copies of HMA4 in N. caerulescens (Ó Lochlainn et al 2011). In addition to HMA4, heavy metal ATPase 3 (HMA3) was implicated as conferring tolerance by being a tonoplastlocated transporter sequestering Cd into the leaf vacuoles.…”