Heavy metals like Cu2+ and Zn2+ are necessary for the normal plant growth and development. They important roles as cofactors of various enzymes (Broadley et al., 2007; Ishimaru et al., 2011; Tripathi et al., 2015). Despite this, high concentrations of essential and non-essential heavy metals in the soil may result in toxicity symptoms due to diverse impacts of heavy metals, such as disrupting the protein structure and inhibiting their activity, displacing of an essential element in proteins, and stimulation of the formation of reactive oxygen species (Cho et al., 2000; Van Assche et al., 1990; Sharma et al., 2008; Bhargava et al., 2012). Such toxicity symptoms result in the plant growth deficiency as well as the low yield. However, in the plants, some mechanisms have evolved tolerance in polluted soils which maintain homeostasis of heavy metals in the cells (Tripathi et al., 2015; Clemens, 2001; Hossain et al., 2012). Essential metals homeostasis, such as copper, iron, manganese and zinc, needs well-coordinated activities of transporters mediating the import into the cell, in addition to distributing to organelles and exporting from the cell (Tripathi et al., 2015; White et al., 2011; Shahpiri et al., 2015). There are also metal chelating peptides and proteins like metallothioneins (MTs) and phytochelatins that are important in the homeostasis and transport of physiologically some essential metals (such as Zn2+and Cu2+), metal detoxification (such as Cd2+and Hg2+), protection against the possible oxidative stress, intracellular redox balances, cell proliferation and apoptosis regulation, and the need for the protection against neuronal injury as well as degeneration (Cobbett, 2000; Wang et al., 2010; Palmiter, 1998; Buchanan-Wollaston, 1994; Chaudhary et al., 2018; Mierek-Adamska et al., 2019).
MTs can be regarded as a family of low molecular weight (7–10 kDa), Cys-rich proteins that can bind to metals through Cys thiol groups. According to the Cys residues arrangement, MT proteins can be divided into three groups. Class I MTs included 20 conserved cysteine residues, mainly mammalian MTs; they can also be seen in vertebrates. Class II MTs are mostly in plants, fungi and invertebrates, with no strict cysteines arrangement. Class III MTs are mostly in plants; they are non-translationally synthesized polypeptides which are composed of some repeating units of γ-Glu-Cys, such as some phytochelatins (PCs) (Cobbett et al., 2002; Rauser, 1999). Plant MTs, in turn, may be classified into four groups: MT1, MT2, MT3, and MT4, according to the Cys residue distribution pattern (Freisinger, 2011). The members of MT1, MT2 and MT3 sub-families include two Cys-rich regions in the N-terminal and C-terminal which are separated through a Cys-free linker region having a typical length that is almost between 30 and 45 amino acids (Cobbett et al., 2002; Schicht et al., 2009). The six Cys residues which are located at the C-terminus in each subfamily can be arranged in highly conserved CXCXXXCXCXXCXC motifs (X: any amino acid besides Cys) (Freisinger, 2011). On the other hand, the number as well as the distribution pattern of Cys residues in the N-terminal region can identify each subfamily; it can be applied as the main distinguishing factor for subfamily discrimination (Freisinger, 2011). The typical type 1, type2 and type3 MTs include six, eight and four Cys residues, respectively, at their N-terminal Cys-rich region (Lane et al., 2011). The members belonging to the type 4 subfamily can be described by three Cys-rich regions that are separated through shorter peptide linkers (Peroza et al., 2007).
Plants generally have different MT isoforms that belong to different types of MTs. Different MT types are differentially represented in different plant organs (Hegelund et al., 2012; Zhou et al., 2006). Type 1 MTs can be expressed mostly in roots, whereas type 2 MTs are in the leaves, type 3 MTs are in the fruits and type 4 MTs are in the seeds (Zhou et al, 2006; Hsieh et al., 1995, 1996; Guo et al., 2003; Mierek-Adamska et al., 2018).
Despite many studies already published on the MT types expression as the response to heavy metals and oxidative stresses, the knowledge still lags behind the MT types expression in responding to plant hormones. Therefore in the present work, the aleurone layer of barley seed was used as a model system to investigate the impact of hormones on the MT types expression (Finnie et al., 2011).
The aleurone layer is composed of one layer of single and unique cells around endosperm in the cereal seeds (Finnie et al., 2011). Upon seed imbibition, the embryo produces the hormones that are received by aleurone layer cells (Bønsager et al., 2007). In response to hormones, the aleurone layer synthesizes and secretes a range of enzymes, such as hydrolases, for depolymerizing endosperm cell walls and degrading endosperm storage carbohydrates and proteins (Caspers et al., 2001; Mundy et al., 1986). The barley aleurone layer may be separated from the rest of seed tissues and kept in the culture medium, thus making it possible to address the added signaling molecules in some isolated system (Finnie et al., 2011). Such qualities have encouraged its use to serve as a model system to study plant signaling and germination.
Previously, Tauris et al. 2009 and Hegelund et al., 2012 revealed that the genes encoding HvMT2b2 and HvMT4 and HvMT3 were expressed at an extremely high level in the barley aleurone layer (Tauris et al., 2009, Hegelund et al., 2012]. In the present research study, the HvMT2b2 and HvMT4 expression, the members of type2 and type 4, respectively were studied in the response to phytohormones gibberellic acid (GA), abscisic acid (ABA), salicylic acid (SA), kinetin (Kin), indole acetic acid (IAA) and ethephon (ET). Furthermore, to functionally characterize these MT isoforms, the genes which encode HvMT2b2 and HvMT4 were cloned and heterologously represented in Escherichia coli. The ability of strains expressing the recombinant forms of HvMT2b2 and HvMT4 were analyzed for remediation of Zn2+ and Cd2+ from the medium. The recombinant HvMT2b2 and HvMT4 were purified and the apo-protein was prepared. The apo-form was exposed to the metal ions and the ability of their binding to HvMT2b2 and HvMT4 were studied by in vitro experiments.