Genome-wide identification and expression profile analysis of metal tolerance protein gene family in Eucalyptus grandis under metal stresses

Abstract

Metal tolerance proteins (MTPs) as Me²⁺/H⁺(K⁺) antiporters participate in the transport of divalent cations, leading to heavy metal stress resistance and mineral utilization in plants. In the present study, to obtain better knowledge of the biological functions of the MTPs family, 20 potential EgMTPs genes were identified in Eucalyptus grandis and classified into seven groups belonging to three cation diffusion facilitator groups (Mn-CDFs, Zn/Fe-CDFs, and Zn-CDFs) and seven groups. EgMTP-encoded amino acids ranged from 315 to 884, and most of them contained 4-6 recognized transmembrane domains and were clearly prognosticated to localize into the cell vacuole. Almost all EgMTP genes experienced gene duplication events, in which some might be uniformly distributed in the genome. The numbers of cation efflux and the zinc transporter dimerization domain were highest in EgMTP proteins. The promoter regions of EgMTP genes have different cis-regulatory elements, indicating that the transcription rate of EgMTP genes can be a controlled response to different stimuli in multiple pathways. Our findings provide accurate perception on the role of the predicted miRNAs and the presence of SSR marker in the Eucalyptus genome and clarify their functions in metal tolerance regulation and marker-assisted selection, respectively. Gene expression profiling based on previous RNA-seq data indicates a probable function for EgMTP genes during development and responses to biotic stress. Additionally, the upregulation of EgMTP6, EgMTP5, and EgMTP11.1 to excess Cd²⁺ and Cu²⁺ exposure might be responsible for metal translocation from roots to leaves.

Keywords: Eucalyptus grandis; Expression profile; Genome-wide identification; Heavy metals; Metal tolerance protein (MTP).

Fig. 1

Phylogenetic relationship of MTP proteins family members of Eucalyptus grandis, Populus trichocarpa, Arabidopsis thaliana, and Oryza sativa. The MTP protein sequences were aligned by ClustalX 2.0.8, and the phylogenetic trees were constructed based on the analysis of bootstrap with 100 replicates by neighbor-joining method using the MEGA 6.0 program. The identified proteins were classified into three sub-families (Fe/Zn-MTPs, Mn-MTPs, and Zn-MTPs) and seven groups based on the previous reports of phylogenetic relationships. The Zn-MTP group (violet line) contains MTP1 to MTP4, MTP5, and MTP12 groups; the Mn- MTP group (blue line) contains MTP8 and MTP9 to MTP11 groups; and the Zn/Fe- MTP group (red line) contains MTP6 and MTP7 groups

Fig. 2

Chromosomal locations and duplications of EgMTP genes in the Eucalyptus grandis genome. The location of EgMTP genes on chromosomes and the duplication relationship between them were revealed using TBtools genetic mapping software. Tandem duplication genes and segmental duplication genes are marked with pink lines and green curves, respectively

Fig. 3

Phylogenetic relationship and gene structures (exon–intron organization) of EgMTP genes. A) the neighbor joining phylogenetic tree was constructed using MEGA 6.0 with 1000 times replication. B) the exon–intron structures of EgMTP proteins, where coding DNA sequences are shown with yellow boxes. Also, thick blue lines at either terminal of the genes indicate untranslated regions (UTRs) and thin lines show introns. The same colors in the names of the genes indicate alternative splicing forms

Fig. 4

Conserved motifs were predicted by the MEME v.5.3.3 tool and displayed by the unique color mentioned in the box on the top right in different colored boxes. Among these represented motifs, four motifs possess specific functions related to MTPs. Motifs 1, 3, and 6 encode cation efflux domain and motif 8 encodes ZT dimerization domain

Fig. 5

Interaction network of EgMTP-target miRNAs. Thirty-six miRNAs are targeted for the fourteen EgMTP genes. The genes are marked in pink and miRNAs in violet

Fig. 6

Gene ontology analysis of EgMTP genes. The analysis shows biological processes, molecular function, and cellular localization of the EgMTP genes

Fig. 7

A The correlation between GC and GC3 contents in EgMTP genes. B The heat map of the relative synonymous codon usage analysis (RSCU) values of EgMTPs. The color boxes indicate RSCU values, the lowest (green) and the highest (red) codons usage. Green color indicates RSCU < 1 and dark red and distinct red represent RSCU > 1 and RSCU > 1.6, respectively

Fig. 8

A gene expression heat map diagram of all MTP genes. A E. grandis TAGoo14 clonal genotype in six tissue samples, e.g. immature xylem, phloem, young leaves, mature leaves, shoot tips, roots, and flowers. B F1 hybrid of E.grandis × E. urophylla (GUSAP1clonal genotype) in immature xylem, mature xylem, phloem, young leaves, mature leaves, and shoot tips in addition to wood xylem and upright control xylem (cambium). C Unsusceptible and resistant E. nitens seedling stems were inoculated with Phytophthora cinnamomi establishing and in mock-inoculated (control), sensitive four-month E. Grandis was split into uninfected and infested with Leptocybe invasa, and stems of one-year-old susceptible and resistant E. grandis were inoculated with Chrysoporthe austroafricana. The genes with similar profiles are grouped in hierarchical clustering. The intensity of expression is shown in the color bar, the highest (red) to lowest (green). The scale bar represents the log₂ of TPM

Fig. 9

Relative expression analysis of EgMTP5, EgMTP6, and EgMTP11 genes in the roots and leave of two-month E. garndis seedling exposed to 18-day excess metal by Cd(NO₃)₂ (10, 50, 200 mM) and CuSO₄. 5H₂O (50, 100, 300 mM). Different letters (a, b, c) indicated above the bar represent statistically significant difference at p ≤ 0.05 (Duncan's multiple range test)