Tandem Quadruplication of HMA4 in the Zinc (Zn) and Cadmium (Cd) Hyperaccumulator Noccaea caerulescens Seosamh O´ Lochlainn 1 , Helen C. Bowen 2 , Rupert G. Fray1, John P. Hammond 1 , Graham J. King3,4, Philip J. White5, Neil S. Graham1, Martin R. Broadley1* 1 Plant and Crop Sciences Division, University of Nottingham, Sutton Bonington, Leicestershire, United Kingdom, 2 Warwick HRI, University of Warwick, Wellesbourne, Warwick, United Kingdom, 3 Rothamsted Research, Harpenden, United Kingdom, 4 Southern Cross Plant Science, Southern Cross University, Lismore, Australia, 5 Scottish Crop Research Institute, Invergowrie, Dundee, United Kingdom 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 pCC1FOSTM 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 b-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. Citation: O´ Lochlainn S, Bowen HC, Fray RG, Hammond JP, King GJ, et al. (2011) Tandem Quadruplication of HMA4 in the Zinc (Zn) and Cadmium (Cd) Hyperaccumulator Noccaea caerulescens. PLoS ONE 6(3): e17814. doi:10.1371/journal.pone.0017814 Editor: Ivan Baxter, United States Department of Agriculture, Agricultural Research Service, United States of America Received November 23, 2010; Accepted February 10, 2011; Published March 10, 2011 Copyright: 2011 O´ Lochlainn et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Seosamh O´ Lochlainn was funded by a UK Biotechnology and Biological Sciences Research Council (BBSRC) Studentship (BBSSE200613215). Rothamsted Research is an institute of the BBSRC. The Scottish Crop Research Institute is funded by the Scottish Government Rural and Environment Research and Analysis Directorate. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: martin.broadley@nottingham.ac.uk Introduction Transition metals, including Cu, Mn and Zn, have essential functions in plant growth and development [1]. However, when present at high concentrations, these metals, along with non- essential metals including Cd and Pb, become phytotoxic and must be prevented from interfering with cellular processes through compartmentalisation and exclusion [1-5]. Numerous transmem- brane proteins catalyse metal efflux from plant cells. These include P1B-ATPases, of which one group transports Cu/Ag and another transports Zn/Cd/Co/Pb [2]. The most widely studied P1B- ATPase in planta is the plasma membrane protein HMA4 [6], which has been shown to transport Zn and Cd in yeast [2,7] as well as confer Zn, Cd and Co tolerance in Arabidopsis thaliana [2,8]. HMA4 is thought to be involved in Zn homeostasis and Cd detoxification, via metal translocation from the root to the shoot [2,7–11]. At a subcellular level, the expression of HMA4 has been shown to localise in the plasma membranes of Arabidopsis thaliana mesophyll protoplasts [8]. At the tissue level, it has been localised to the pericycle cell layer of the root vasculature [12]. In hma4 knockout mutants, increased pericycle Zn accumulation, decreased Zn transport to the xylem parenchyma, and reduced shoot Zn accumulation have been observed [12]. In A. thaliana shoots, HMA4 expression has been localised in the phloem tissue, at the base of developing siliques, and in developing anthers, especially tapetum cells, to supply Zn to male reproductive tissue [9]. A small number of plant species have evolved that can tolerate and accumulate high concentrations of some metals in their aerial tissues under natural conditions, including Zn and Cd [13,14]. It is thought that 10-20 species of angiosperms are Zn hyperaccumu- lators (.,0.3% Zn DW), with two of these also able to accumulate Cd to similarly high levels. In the Brassicaceae, the accumulation of high levels of Zn in shoot tissues occurs within Noccaea and its sister clade Raparia [13,15,16], but not in Thlaspiceras which contains Zn hypertolerant species (e.g. Thlaspiceras oxyceras (Boiss.) F.K. Mey; [17]), and not in the non-Zn-hypertolerant Microthlaspi and Neurotropis clades, which are more distantly related. Within Noccaea, Cd hyperaccumulation occurs in a subset of N. caerulescens populations. Arabidopsis halleri is the only known Brassicaceae Zn/Cd hyperaccumulator occurring outside of the Noccaea genus [13,14]. Thus, Zn/Cd hyperaccumulation may have arisen through two evolutionary events within the Brassicaceae. In Arabidopsis halleri, QTL involved with Zn and Cd tolerance co-localize with HMA4 [18]. High expression of HMA4 in the first PLoS ONE | www.plosone.org 1 March 2011 | Volume 6 | Issue 3 | e17814