Barley metallothionein isoforms, MT2b2 and MT4, differentially respond to photohormones in barley aleurone layer and their recombinant forms show different affinity for binding to zinc and cadmium

Abstract

Metallothioneins (MTs) are metal-binding proteins that have important roles in the homeostasis of heavy metals. In this study, the two MT genes was studied in response to phytohormones using the barley aleurone layer as a kind of model system. The aleurone layer was isolated from barley embryo-less half grains and was incubated for 24 h with different phytohormones. Based on the results the genes encoding HvMT2b2 and HvMT4 were down-regulated through gibberellic acid (GA), while they were and up-regulated through salicylic acid (SA). Despite this, these two genes were differentially expressed to other hormones. Furthermore, the proteins HvMT2b2 and HvMT4 were heterologous expressed as GST-fusion proteins in E. coli. The HvMT4 and HvMT2b2 heterologous expression in E. coli gives rise to 10- and 3-fold improvements in the accumulation capacity for Zn²⁺, respectively. Whereas the transgenic E. coli strain that expresses HvMT2b2 could accumulate Cd²⁺ three-fold higher than control. The expression of HvMT4 did not affect the accumulation of Cd²⁺. HvMT4 which is known as seed-specific isoform seems to be able to bind to Zn²⁺ with good affinity and cannot bind Cd²⁺. In comparison, HvMT2b2 was able to bind both Zn²⁺ and Cd²⁺. Therefore HvMT4 could serve a noteworthy role in zinc storage in barley seeds. The expression of HvMT4 is induced by SA 30-fold, concerning the untreated aleurone layer. Such results could provide good insights for the assessment of the effects of phytohormones in the molecular mechanism involved in essential metal storage in cereal seeds.