Purification and Immunological Identification of Metallothioneins 1 and 2 from Arabidopsis thaliana

Abstract

Gene families encoding two types of metallothioneins (MTs), MT1 and MT2, have been identified in Arabidopsis thaliana, and their respective mRNAs have been shown to be regulated by copper in a tissue-specific manner (J. Zhou and P.B. Goldsbrough [1994] Plant Cell 6: 875–884; J. Zhou and P.B. Goldsbrough [1995] Mol Gen Genet 248: 318–328; A.S. Murphy and L. Taiz [1995] Plant Physiol 109:1–10). However, to date the protein products have not been identified. To purify MT proteins from Arabidopsis, we isolated low-molecular-mass, copper-binding, thiol-rich proteins using selective precipitation followed by size-exclusion, copper-affinity, and thiol-affinity chromatographies. Polyclonal antibodies raised against Arabidopsis MT-glutathione-S-transferase fusion proteins cross-reacted with the 4.5- and 8-kD bands in immunoblots of low-molecular-mass, copper-binding proteins purified from seedling, mature leaf, and mature root tissues. The identity of the proteins was subsequently confirmed by amino acid sequencing. MT1 expression was constitutive in roots and inducible by copper in mature leaves; the reverse pattern was observed for MT2. MT2 expression was also concentrated in the growing tip of the root. The accumulation of the MT1- and MT2-encoded proteins thus parallels the regulation of their respective mRNAs with regard to tissue specificity and induction by copper. In addition, a new type of MT, designated MT3, was derived from the database, detected by reverse transcription-polymerase chain reaction, and tentatively identified at the protein level by amino acid sequencing of a 7-kD cysteine-rich polypeptide.

Examining the Specific Contributions of Individual Arabidopsis Metallothioneins to Copper Distribution and Metal Tolerance

“…Here we have demonstrated that six Arabidopsis MTs, including representatives of all four types of plant MTs, can impart metal tolerance when expressed in S. cerevisiae. Although the metal-binding capacity of some plant MTs has been previously demonstrated (Tommey et al, 1991;Evans et al, 1992;Murphy et al, 1997), this is the first report, to our knowledge, to compare all four types of MTs from a single plant species. Our results suggest that all MT isoforms are able to bind Cu ions in vivo.…”

“…It has been shown that MT1 and MT2 proteins from Arabidopsis can be purified by Cu affinity chromatography and are able to bind Cu ions in vitro (Murphy et al, 1997). When expressed in the yeast Dcup1 mutant, the MT1a and MT2a genes were also able to confer Cu and Cd tolerance (Zhou and Goldsbrough, 1994).…”

Genetic engineering in the improvement of plants for phytoremediation of metal polluted soils

“…MTs bind to Cu, Cd and Zn; they probably regulate intracellular Zn concentrations and detoxify normally lethal concentrations of Cd and Cu (Murphy et al, 1997). MTs have the highest a nity to Cu, and MT expression correlates with Cu but not Cd tolerance in Arabidopsis (Murphy and Taiz, 1995).…”

Characteristic and Expression Analysis of a Metallothionein Gene, OsMT2b, Down-Regulated by Cytokinin Suggests Functions in Root Development and Seed Embryo Germination of Rice

“…For plant, MTs can efficiently bind metals (Murphy et al, 1997;Leszczyszyn et al, 2007) and some of them are transcriptionally regulated by metals; they are also thought to play important roles in metal tolerance, detoxification, and homeostasis in plants (Cobbett and Goldsbrough, 2002;Hall, 2002;Hassinen et al, 2007). On the other hand, there were also intensive studies for the responses of MTs to stress conditions, such as draft and reactive oxidant species (Akashi et al, 2004;Wong et al, 2004).…”