Characterization of metallothionein genes from Broussonetia papyrifera: metal binding and heavy metal tolerance mechanisms

Abstract

Background: Broussonetia papyrifera is an economically significant tree with high utilization value, yet its cultivation is often constrained by soil contamination with heavy metals (HMs). Effective scientific cultivation management, which enhances the yield and quality of B. papyrifera, necessitates an understanding of its regulatory mechanisms in response to HM stress.

Results: Twelve Metallothionein (MT) genes were identified in B. papyrifera. Their open reading frames ranged from 186 to 372 bp, encoding proteins of 61 to 123 amino acids with molecular weights between 15,473.77 and 29,546.96 Da, and theoretical isoelectric points from 5.24 to 5.32. Phylogenetic analysis classified these BpMTs into three subclasses: MT1, MT2, and MT3, with MT2 containing seven members and MT3 only one. The expression of most BpMT genes was inducible by Cd, Mn, Cu, Zn, and abscisic acid (ABA) treatments, particularly BpMT2e, BpMT2d, BpMT2c, and BpMT1c, which showed significant responses and warrant further study. Yeast cells expressing these BpMT genes exhibited enhanced tolerance to Cd, Mn, Cu, and Zn stresses compared to control cells. Yeasts harboring BpMT1c, BpMT2e, and BpMT2d demonstrated higher accumulation of Cd, Cu, Mn, and Zn, suggesting a chelation and binding capacity of BpMTs towards HMs. Site-directed mutagenesis of cysteine (Cys) residues indicated that mutations in the C domain of type 1 BpMT led to increased sensitivity to HMs and reduced HM accumulation in yeast cells; While in type 2 BpMTs, the contribution of N and C domain to HMs' chelation possibly corelated to the quantity of Cys residues.

Conclusion: The BpMT genes are crucial in responding to diverse HM stresses and are involved in ABA signaling. The Cys-rich domains of BpMTs are pivotal for HM tolerance and chelation. This study offers new insights into the structure-function relationships and metal-binding capabilities of type-1 and - 2 plant MTs, enhancing our understanding of their roles in plant adaptation to HM stresses.

Keywords: Expression analysis; Heavy metal transfer; Metallothionein; Paper mulberry; Site-directed mutagenesis; Yeast transformation.

Fig. 1

Phylogenetic relationship of BpMT proteins. Alignments of the MT proteins from B. papyrifera and other 6 species were performed with Clustal W. The phylogenetic tree was constructed using Neighbor-Joining method of MEGA7. Different colors mean different type members according to sequence similarity

Fig. 2

The conserved motifs and domains of BpMT proteins. (A) The motifs distributed in the 12 BpMT proteins, the 10 conserved motifs are represented with different color boxes. The dark line shows the length of proteins. (B) Distribution of conserved domains within BpMT proteins. The relative positions of each domain are shown in color boxes, the names are indicated on the right

Fig. 3

Multiple sequence alignment of the motifs of 12 BpMT proteins (A) and the sequence logo of motif1, motif2, motif3 and motif4 (B)

Fig. 4

Expression patterns of BpMT genes under different HM stresses for 0, 6, 24, 72 h in the roots, stems and leaves. Heatmap of BpMT genes expression data was created by Tbtools with row clustering applied, and the blue/yellow/orange colors indicated the low/medium/high expression level. The expression was related to the internal reference gene and control conditions. (A) Cd stress. (B) Cu stress. (C) Mn stress. (D) Zn stress. The data followed by ‘ns’ means the difference between the time point and 0 h was not significant in the same tissue, while the data not marked were all significantly different to 0 h (P < 0.05)

Fig. 5

Expression profiles and relativities of BpMT genes under ABA treatment for 0, 6, 24, 72 h in the roots, stems and leaves. The expression is relative to the internal reference gene and at control conditions. The heatmap of expression data was created by Tbtools same as Fig. 4. All the differences of each gene between different time points and 0 h were significant in the same tissue (P < 0.05). (A), the expression files. (B), the relativities

Fig. 6

Redundancy analysis (RDA) was employed using Canoco 5.0 for gene expression and experiment treatments, including heavy metal and ABA treatments at different time points (0, 6, 24, 72 h)

Fig. 7

Analysis of the heavy metal stress response function of BpMT genes in yeast. (A) The growth performance of the control and BpMTs transformed yeasts under HM stresses. The decreasing trend triangle above represents the dilution ratio of 0⁰, 10^− 1, 10^− 2, 10^− 3, 10^− 4. (B) The yeast cell densities (OD₆₀₀) of INVSCI (pYES2) and INVSCI (pYES2-BpMTs) under four HM treatments. Control was cultivated in a normal media without HM stress. The lowercase letters in the figure indicated the significant differences (P < 0.05) among different yeast lines

Fig. 8

HMs tolerance and accumulation study of yeast BpMTs’ mutants (N- and C-domain named as mΔN and mΔC, respectively) generated by site-directed mutagenesis in yeast. (A), Serially diluted transformed yeast INVSC1containing pYES2, pYES2-BpMT1c, pYES2-BpMT1c-mΔC, pYES2-BpMT1c-mΔN, pYES2-BpMT2d, pYES2-BpMT2d-mΔN, pYES2-BpMT2d-mΔC, pYES2-BpMT2e, pYES2-BpMT2e-mΔN, pYES2-BpMT2e-mΔC spotted on Sc-U selective media with and without metal (Control), Cd, Cu, Mn, Zn. (B), HMs accumulation in yeast cells according to (A). All data are mean ± SD (n = 3). Different letters above bars indicate significant differences among different yeast strains under the same HM stress (P < 0.05)