The Five AhMTP1 Zinc Transporters Undergo Different Evolutionary Fates towards Adaptive Evolution to Zinc Tolerance in Arabidopsis halleri

Abstract

Gene duplication is a major mechanism facilitating adaptation to changing environments. From recent genomic analyses, the acquisition of zinc hypertolerance and hyperaccumulation characters discriminating Arabidopsis halleri from its zinc sensitive/non-accumulator closest relatives Arabidopsis lyrata and Arabidopsis thaliana was proposed to rely on duplication of genes controlling zinc transport or zinc tolerance. Metal Tolerance Protein 1 (MTP1) is one of these genes. It encodes a Zn²⁺/H⁺ antiporter involved in cytoplasmic zinc detoxification and thus in zinc tolerance. MTP1 was proposed to be triplicated in A. halleri, while it is present in single copy in A. thaliana and A. lyrata. Two of the three AhMTP1 paralogues were shown to co-segregate with zinc tolerance in a BC1 progeny from a cross between A. halleri and A. lyrata. In this work, the MTP1 family was characterized at both the genomic and functional levels in A. halleri. Five MTP1 paralogues were found to be present in A. halleri, AhMTP1-A1, -A2, -B, -C, and -D. Interestingly, one of the two newly identified AhMTP1 paralogues was not fixed at least in one A. halleri population. All MTP1s were expressed, but transcript accumulation of the paralogues co-segregating with zinc tolerance in the A. halleri X A. lyrata BC1 progeny was markedly higher than that of the other paralogues. All MTP1s displayed the ability to functionally complement a Saccharomyces cerevisiæ zinc hypersensitive mutant. However, the paralogue showing the least complementation of the yeast mutant phenotype was one of the paralogues co-segregating with zinc tolerance. From our results, the hypothesis that pentaplication of MTP1 could be a major basis of the zinc tolerance character in A. halleri is strongly counter-balanced by the fact that members of the MTP1 family are likely to experience different evolutionary fates, some of which not concurring to increase zinc tolerance. Arabidopsis halleri has developed the characters of zinc hypertolerance and hyperaccumulation as compared to its close relatives Arabidopsis thaliana and Arabidopsis lyrata. Different candidate genes were proposed to account for the appearance of these characters in A. halleri. One of them is MTP1 (Metal Tolerance Protein 1), which is involved in cytoplasmic zinc detoxification. We found that A. halleri harbored five gene copies of MTP1, whereas A. thaliana and A. lyrata possess a single copy. It is thus tempting to associate the zinc hypertolerance character of A. halleri to the pentaplication of MTP1. However, we observed that one of the five MTP1 copies is not fixed in a population growing on metal contaminated soil. Also, the different MTP1 genes are markedly differentially expressed in A. halleri, two of them being poorly expressed and repressed in response to zinc constraint. AhMTP1 copies were also demonstrated to display a differential ability to complement the zinc hypersensitivity of a yeast mutant that is dysfunctional in vacuolar zinc transporters. Our findings suggest that different evolutionary fates, some of them not concurring to increase zinc tolerance, are likely to take place for the members of the MTP1 family in A. halleri.