Expression response of duplicated metallothionein 3 gene to copper stress in Silene vulgaris ecotypes
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Metallothioneins (MTs) were identified as important players in metal metabolism. MT3 gene presents a key metallothionein controlling copper homeostasis in plants. We have selected one cupricolous and one non-cupricolous ecotype to isolate and analyse the MT3 gene in Silene vulgaris. For expression data comparison, we have also included other metal-tolerant ecotypes. Based on a S. vulgaris BAC library screening, we have identified and sequenced a genomic clone containing MT3 gene (SvMT3). We found that SvMT3 gene has been locally duplicated in a tandem arrangement. Expression analysis and complementation studies using yeast mutants showed that both copies of the SvMT3 gene were functional. Moreover, we examined the expression of MT3 gene(s) in selected ecotypes under different copper treatments to show the tissue-specific expression response to copper stress. We demonstrated that higher copper concentrations specifically affected MT3 expression among ecotypes. Our analysis shows that MT3a has similar expression pattern in cupricolous ecotypes while MT3b has common expression features shared by all metallophyte S. vulgaris ecotypes. Our data indicate that down-regulation of MT3b root expression in higher copper concentrations is associated with copper stress. We propose that there might be a specific regulation of SvMT3s transcription depending on the type of heavy metal tolerance.
KeywordsCopper Gene duplication Metallothionein Heavy metal tolerance Silene vulgaris
Metallothionein 3 of S. vulgaris
Quantitative real-time PCR
Reactive oxygen species
Atomic absorption spectroscopy
The work was supported by the IGA AF MENDELU (grant no. DP3/2009 and TP 1/2010), Czech Science Foundation (grant nos. P501/12/2220, P501/10/0102, P501/12/G090, 13-34962P and 522/09/0083), Centre of the Region Haná for Biotechnological and Agricultural Research (grant no. ED0007/01/01), and grant CZ.1.05/2.1.00/03.0058. We thank Prof. Dennis Thiele for providing copper-sensitive yeast mutants, Dr. Jelena Samardzic for yeast expression vector and Dr. Martin Lysak for N. caerulescens plants. Particularly, we are grateful to Prof. Dr. Henk Schat for providing heavy metal-tolerant ecotypes Imsbach and Plombieres. The authors are indebted to Dr. A. Oulton for language correction.
Conflict of interest
The authors declare that they have no competing interests.
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