Cloning and expression analysis of metallothionein family genes in response to heavy metal stress in sugarcane (<italic>Saccharum officinarum</italic> L.)

Abstract

Metallothioneins (MTs) are cysteine-rich, low-molecular-weight proteins. Plant MTs play important roles in detoxification and cellular redox regulation. In this study, hydroponic experiments of sugarcane ( Saccharum officinarum L. cv. Badila) were carried out to study the effects of CdCl2, ZnSO4, and CuCl2 treatments on plantlet growth. And then three kinds of heavy metal content in both shoots and roots of sugarcane were detected, showing an enrichment and tolerance ability to Cd2+, Zn2+, and Cu2+ in Badila seedlings. Three metallothionein genes, termed as ScMT 1 (accession number: KJ504373), ScMT 2-1-5 (accession number: MH191346), and ScMT 3 (accession number: KJ5043704), were isolated from Badila. ScMT 1 contained an open reading frame (ORF) of 228 bp encoding 75 amino acids residues. The first (52 bp) and second (176 bp) extrons of ScMT 1 were separated by an intron (283 bp). ScMT 2-1-5 contained an ORF of 246 bp encoding 81 amino acids residues. The first (64 bp), second (87 bp), and third (101 bp) extrons of ScMT2-1-5 were separated by two introns (483 bp and 853 bp). ScMT 3 contained an ORF of 198 bp encoding 65 amino acids residues. The first (50 bp) and second (148 bp) extrons of ScMT 3 were separated by an intron (259 bp). The deduce protein ScMT1, ScMT2-1-5, and ScMT3 were categorized into the subfamily of plant type1, type2, and type3 MTs, respectively. The expression profiles of the three genes under different heavy-metal stresses were investigated by real-time quantitative PCR (qRT-PCR) analysis. When treated with Cd2+, the expression of ScMT 2-1-5 was continuously and significantly up-regulated in both shoots and roots, while that of ScMT 1 showed a delayed up-regulation pattern. The expression of ScMT 3 showed a delayed up-regulation pattern in shoots and a trend of first raising and then suppressing in roots. The results suggested that ScMT 2-1-5 might play a more active role in response to Cd2+ in sugarcane while ScMT 3 might not, and ScMT 1 is involved in the molecular responses of Cd2+ stress at the later stage. When treated with Cu2+, the expression of ScMT 1 was continuously and significantly up-regulated, and that of ScMT 2-1-5 and ScMT 3 general up-regulated in shoots. As in the roots, the expression of both ScMT 1 and ScMT 2-1-5 showed a delayed up-regulation pattern which was significantly up-regulated at the later stage, while that of ScMT 3 significantly up-regulated at the early stage. It revealed that ScMT 1 cooperated with ScMT 2-1-5 and ScMT 3 is involved in the positive response to Cu2+ stress in roots, and might play a more positive role than ScMT 2-1-5 and ScMT 3, and the three genes are successively involved in the molecular responses of Cu2+ stress in roots. When treated with Zn2+, the expression of ScMT 1 and ScMT 3 increased only in shoots and roots, respectively. The expression of ScMT 2-1-5 was up-regulated at the early stage and then suppressed in both shoots and roots. It suggests that ScMT 1 and ScMT 3 mainly function in shoots and roots, respectively, when exposed to Zn2+ stress. ScMT 2-1-5 was involved in the molecular responses of Cd2+ stress at the early stage. ScMT 1, ScMT 2-1-5, and ScMT 3 showed similar or complementary expression patterns in different tissues of sugarcane when exposed to heavy metals, which revealed the functional diversity of sugarcane MTs in detoxification and cellular redox regulation, and their spatiotemporal coordination in mitigating or even preventing tissue injury caused by excessive heavy metals. The results provide a basic information for further research on mechanisms in the synergistic enhancement of tolerance to heavy metals for MTs family genes in the highly polyploid sugarcane.