Identification and characterization of genes encoding heavy metal tolerant protein family in Euphrates poplar (Populus. Euphratica)

Extended Abstract
Background and Objectives:
Heavy metals are a major environmental issue, severely affecting plant growth. Phytoremediation, which involves using plants to extract, separate, or eliminate pollutants, offers a practical, cost-efficient, and environmentally friendly solution. Several protein families, such as the Metal Tolerant Protein (MTP) family, play a key role in the phytoremediation of heavy metals in plants like Euphrates poplar. With advances in genomics, especially the sequencing of various organisms' genomes and data availability from public databases, researchers can now identify relevant genes and their functions. This study aims to explore the genetic diversity and relationships among the members of the MTP family in Euphrates poplar.
 
Methodology:
Members of the MTP family in the Euphrates poplar genome were identified using homologous protein sequences of the MTP family from two model plants of Arabidopsis and rice using BlastP in the NCBI database. Sequence alignment was performed, followed by phylogenetic tree construction using the Neighbor-Joining method. Various software tools were used to predict the physicochemical properties and protein localization of MTP members and to assess the selection pressure, exon-intron distribution patterns, and specific motifs within gene family members. Gene ontology was performed at three levels: biological processes, molecular function, and cellular components, including identifying unique simple sequence repeat (SSR) markers. The microRNA network regulating gene expression for each member was also identified and, the communication network between genes and MicroRNA was drawn. Gene expression was evaluated in various Euphrates poplar tissues, and RNA-seq data were normalized and analyzed for expression under 200 mM salt stress. Heat map visualizations were generated using Mev4.0 software.
 
Results:
A total of 26 MTP gene family members were identified in the P. euphratica genome through bioinformatics analysis. Phylogenetic analysis classified these into three major groups (Mn-MTP, Zn/Fe-MTP, and Zn-MTP) and seven subgroups. The encoded proteins were found to be associated with the plasma membrane and the transfer of metals to the vacuole, supporting their role in detoxifying heavy metals. Negative selection pressure was observed across all members, indicating the effect of gene divergence. Gene structure analysis showed the highest integration within groups and the lowest integration between groups. Out of the 15 gene sequences analyzed, 52 SSR markers were identified for marker-assisted selection. MTP11.2, MTP3.2, MTP12, and MTP7 genes exhibited the highest expression levels across various tissues. The MTP1.1 gene showed the most significant increase in gene expression 48 hours after salt application, indicating a potential role in plant responses to salinity and heavy metal stress.
 
Conclusion:
The presence of repeated gene sequences reflects the plant's high demand for the specific function of these genes and their differential expression during various growth stages and stress conditions. Euphrates poplar possesses numerous MTP family members, which contribute to removing metals from the soil and are linked to increased growth and wood production. This research demonstrates the utility of bioinformatics data in facilitating the identification of metal-resistant populations more efficiently and cost-effectively.