High expression in leaves of the zinc hyperaccumulator Arabidopsis halleri of AhMHX, a homolog of an Arabidopsis thaliana vacuolar metal/proton exchanger

Abstract

Zn hyperaccumulator plants sequester Zn into their shoot vacuoles. To date, the only transporters implicated in Zn sequestration into the vacuoles of hyperaccumulator plants are cation diffusion facilitators (CDFs). We investigated the expression in Arabidopsis halleri of a homolog of AtMHX , an A. thaliana tonoplast transporter that exchanges protons with Mg, Zn and Fe ions. A. halleri has a single copy of a homologous gene, encoding a protein that shares 98% sequence identity with AtMHX . Western blot analysis with vacuolar-enriched membrane fractions suggests localization of AhMHX in the tonoplast. The levels of MHX proteins are much higher in leaves of A. halleri than in leaves of the non-accumulator plant A. thaliana . At the same time, the levels of MHX transcripts are similar in leaves of the two species. This suggests that the difference in MHX levels is regulated at the post-transcriptional level. In vitro translation studies indicated that the difference between AhMHX and AtMHX expression is not likely to result from the variations in the sequence of their 5 ¢ untranslated regions (5 ¢ UTRs). The high expression of AhMHX in A. halleri leaves is constitutive and not significantly affected by the metal status of the plants. In both species, MHX transcript levels are higher in leaves than in roots, but the difference is higher in A. halleri . Metal sequestration into root vacuoles was suggested to inhibit hyperaccumulation in the shoot. Our data implicate AhMHX as a candidate gene in metal accumulation or tolerance in A. halleri . Key-words :AtMHX ; hyperaccumulation; magnesium; metal transporter; vacuole; zinc; 5 ′ UTR. INTRODUCTIONA few plant species, generally referred to as metal hyperaccumulators, can accumulate and tolerate very high levels of certain metals (Baker & Brooks 1989). While Zn is an essential mineral, its excess leads to toxicity. Arabidopsis halleri and Thlaspi caerulescens ( Brassicaceae ) are among the 16 known plant species that can accumulate and tolerate high levels of Zn (Baker & Brooks 1989), and are the two main model species in which Zn hyperaccumulation has been studied to date (reviewed by Baker & Whiting 2002;Assuncao, Schat & Aarts 2003). Excessive accumulation of Zn in the shoot is a constitutive property of the A. halleri species . When different populations of A. halleri plants were grown hydroponically in the presence of high Zn levels, there were no differences in the accumulating abilities of populations from sites with different degrees of contaminations (Macnair 2002).The molecular basis of metal hyperaccumulation in A. halleri and T. caerulescens was addressed in a number of studies. Transcription of genes encoding Zn uptake transporters (that are presumably localized in the plasma membrane) is higher in both roots and shoots of T. caerulescens compared with the related non-accumulator species Thlaspi arvense Pence et al . 2000;Assuncao et al . 2001). Expression of these uptake transporters is down-regulated in the presence of 1 and 50 µ M Zn in T. arvense...

Compartmentation and complexation of metals in hyperaccumulator plants

“…Protein families involved in vacuolar sequestration may be the Nramp’s (natural resistance-associated macrophage proteins), CDF’s (cation diffusion facilitator), and CAX’s (cation exchanger; reviewed by Hall and Williams, 2003) as well as CPx-type ATPases. Until now, already several transporters for vacuolar sequestration of zinc (and possibly cadmium) and nickel have been investigated and could be partially characterized (Van der Zaal et al, 1999; Desbrosses-Fonrouge et al, 2005; Elbaz et al, 2006; Haydon and Cobbett, 2007; Morel et al, 2009). Several CDF transporters for vacuolar sequestration of Zn (and possibly Cd and Co) have been characterized, all are homologous, almost identical in sequence.…”

A Novel Major Facilitator Superfamily Protein at the Tonoplast Influences Zinc Tolerance and Accumulation in Arabidopsis

“…AtMHX was cloned from Arabidopsis by PCR based on sequence similarity to a mammalian Na 1 -Ca 21 exchanger, but was shown to transport Mg 21 and Zn 21 by proton antiport and localized to vacuoles (Shaul et al, 1999). The importance of this transporter for Zn homeostasis in A. thaliana is not clear, although expression of MHX in A. halleri is elevated compared to A. thaliana and may be important for Zn tolerance in this species (Elbaz et al, 2006).…”

Recent advances in the analysis of metal hyperaccumulation and hypertolerance in plants using proteomics

“…The ability to tolerate and accumulate metals was found to be associated with the ability to cope with reactive oxygen species (ROS), the expression of metal transporters and the suppression of genes involved in defense and disease resistance. In the abovementioned studies, the modulation of gene expression was considered at the level of transcription/mRNA turnover, which may not directly correlate with the protein level, as has been shown for the putative Zn and Mg transporter protein MHX, which is more abundant in A. halleri than in A. thaliana , even though the corresponding transcript levels are not different (Elbaz et al, 2006). Moreover, RNA analyses do not consider the potential impact of protein folding, stability and localization, protein/protein interactions and post-translational modifications, which are all crucial determinants of protein function.…”