3.1. Identification of MTP genes in M. truncatula
In total, 27 genes were identified via blast analysis; subsequently, genes with incomplete functional domain were excluded for the next study, and finally, 12 candidate genes were selected for further analysis. Every gene was assigned with a specific name i.e., MtMTP1.1, MtMTP1.2, MtMTP2, MtMTP4, MtMTP5, MtMTP7, MtMTP8.1, MtMTP8.2, MtMTP9, MtMTP10.1, MtMTP10.2and MtMTP11. Characteristics of all 12 genes such as gene locus, molecular weight, number of amino acids, grand average of hydropathicity, and isoelectric points (Table 1). Except chromosome 6, all other 7 chromosomes of M. truncatula were the locus of MTP genes. The molecular weight of all MTP protein molecules varied from 39491.59 to 53259.81 kDa. The total number of inter and intra protein ionic residues were variable i.e., the highest anionic residues were in MTP1.1, and lowest in MTP5. Similarly, the highest cationic residues were in MTP10.2 and lowest in MTP4 and MTP11. All MtMTP members harbor a variable number of introns, but MtMTP1.1, MtMTP1.2 and MtMTP4 were without any intron.
3.2. Phylogenetic analysis of MTP gene families
In order to unreveal the evolutionary footprints of the MTP gene family in M. truncatuala, comparison among MTP gene families in different species was performed. We retrieved 12 AtMTP genes of Arabidopsis thaliana, 9 CsMTP genes of Cucumis sativus, 21 PtMTP genes of Populus trichocarpa, 10 OsMTP genes of Oryza sativa, and 8 TaMTP genes of Triticum aestivum were aligned against 12 MtMTP genes of M. truncatulaand phylogenetic tree was constructed for comparison of evolutionary relationship. All MTPgene families were divided into seven groups i.e., Group1, 5, 6, 7, 8, 10, and 12 (Fig. 1).
The highest number of MTPs were pooled in Group10 such as MtMTP9, MtMTP10.1, MtMTP10.2, and MtMTP11 along with AtMTP9, AtMTP10, and AtMTP11; than in Group 1 such as MtMTP1.1, MtMTP1.2, and MtMTP4along with AtMTP1, AtMTP2,AtMTP3, and AtMTP4; than in Group 8 such as MtMTP8.1, and MtMTP8.2 along with AtMTP8; than in Group 7 such as MtMTP7, along with AtMTP7, than in Group 6 such as MtMTP2, along with AtMTP6, and finally in Group 5 such as MtMTP5, along with AtMTP5, and no any MtMTP was placed in Group 12. Noticeably, ionic clustering revealed that 4 MtMTPs were clustered in Zn-CDFs group, 2 MtMTPs were clustered in Fe/Zn-CDFs group, and 6 MtMTPs were clustered in Mn-CDFs group (Fig. 1).
3.3 Chromosomal locations and synteny analysis of MtMTP gene family
Synteny analyses were performed to unreveal the distribution of genes on different chromosomes. We observed that MtMTP genes are distributed among all seven chromosomes. Furthermore, for evaluation of gene family expansion and novel functions, we also investigated gene duplication and divergence with the help of circos. We observed only segmental gene pair duplication from PGDD (Plant Genome Duplication Database). Collinearity due to excision of segmental duplication was observed in many gene pairs with 70-100% identity percentage (Table S2). Segment duplication resulted in many homologies of MTP genes between M. truncatula chromosome pairs, such as what occurs with the genes, MtMTP1.1/MtMTP1.2,MtMTP4/MtMTP5,and MtMTP8.2/MtMTP10.2 (Fig.2). Except for MtMTP7, all rest of MtMTP genes in M. truncatula displayed single and multiple genetic duplications. Noticeably, we did not observe any obvious tandem duplication among all MtMTPs.
3.4 Gene structures and motif analyses
All MTP family genes were further divided into six subfamilies (A, B, C, D, E, and F) (Fig. 3a). Subfamilies A and F were the largest among all subfamilies with six members in each, followed by subfamily B with 2 members, whereas subfamilies C, D, and Eeach contain only one gene (Fig.3a). Intron and exon analysis of all MTP genes revealed that each retrieved sequence of the MtMTP gene family is a correct and true member of six subfamilies (Fig.3c). Although there was variation in size and location of intron and exon of all MtMTP genes, the similarity index was higher among all subfamilies, which proved a close evolutionary relationship among MtMTP gene family members. All MTP family genes contain a variable number of introns, but all members of subfamily F were without any intron. Amino acid sequence-based conserved motifs of MTP were analyzed using MEME (Fig.3b and Table S3). All conserved motifs comprised 50 amino acids except motif 10which was comprised of only 41 amino acids. The largest motifs were 3 and 6, which were observed in all subfamilies, followed by motif 10, 83.3% of aforementioned. Noticeably, the number, type, and order of motifs were similar in intrasubfamily than inter subfamilies.
3.5 Protein modeling, sub-cellular localization, and GO enrichment analysis
Phyre 2 web portal (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) was employed for protein modeling using all MTP aminoacid sequences (Fig.4, and Table S4). All twelve predicted models for MTP proteins were 100% based on c6xpdB, c3j1zP, c2qfiB, and d2qfia2 templates. Similarly, sub-cellular localization, molecular function, and biological process were predicted by GO enrichment analysis (Fig.5 and Table S5). In sub-cellular localization analysis, the predicted distribution scores of MTP proteins were as following;12/60% in all membranes, 3/15% in the plasma membrane and vacuole, and 1/5% in Golgi apparatus and root hair. Noticeably, the MtMTP1.2 gene was localized in 12 sub-cellular compartments out of all 14, which underlined the significant role of MtMTP1.2in metal stress resistance. Collective scores of MTP protein molecules during biological processes were as following; trans-membrane transport of Zn+ and Mn+ ions was 3/43%, while trans-membrane transport of cations was 1/14%. More precisely, MtMTP1.1, MtMTP1.2, and MtMTP4 play a key role in transmembrane transport of Zn+, while MtMTP8.1, MtMTP8.2, and MtMTP11 play a crucial role in transmembrane transport of Mn+. Molecular function analysis revealed significant roles of MtMTP8.2 and MtMTP11 in heavy metal processes.
3.6 Gene expression analysis by RNA-seq data
MtMTP family genes expression profiling was performed by analyzing previously sequenced RNA-seq data (https://mtgea.noble.org/v3/) of the following tissues of M. truncatula; leaf, bud, shoot, hypocotyl, stem, flower, seed coat, pod, root, and root tip (Table S6). A heatmap diagram was constructed to show the differential expression level of each MtMTP gene in all tissues (Fig. 6). Comparatively, MtMTP1.1 displayed the highest expression level in a pod, while MtMTP1.2 displayed the highest expression level in the root tip. Similarly, the highest expression level of MtMTP2 was observed in plant shoots, while the expression level of MtMTP5 was mild in hypocotyl, root, and root tip. The expression level of MtMTP7 was also mild in buds while higher in the shoot. MtMTP11 displayed the highest expression level in approximately all tissues, while MtMTP10.1only in hypocotyl and seed coat, MtMTP8.1 only in the root, and MtMTP9 only in flower tissue. Noticeably, MtMTP8.2 and MtMTP10.2 displayed the lowest expression level in all tissues but the highest expression in root.
3.7 qRT-PCR analysis of MtMTPs under the effect of heavy metals
All MtMTPgenes displayed differential gene expression levels under treatment of different types of heavy metals investigated in the following tissues; root, stem, and leaf (Fig. 7). In roots, MtMTP1.2 and MtMTP4 displayed the highest expression level, while MtMTP5, MtMTP7, and MtMTP9 displayed the lowest expression level under the treatment Cd2+. Similarly, MtMTP1.1 and MtMTP11 displayed the highest expression level, while MtMTP7 displayed the lowest expression level under the treatment of Co2+.MtMTP1.1, MtMTP4, MtMTP5, MtMTP8.1, MtMTP8.2 and MtMTP11 displayed highest expression level, while MtMTP10.2 displayed lowest expression level under the treatment of Fe2+. MtMTP1.1 and MtMTP4 displayed the highest expression level, while MtMTP5 displayed the lowest expression level under the treatment of Mn2+. MtMTP1.1, MtMTP1.2 and MtMTP4 displayed highest expression level, while MtMTP2, MtMTP5, MtMTP7 and MtMTP11 displayed lowest expression level under the treatment of Zn2+.
In stem, Cd2+ treatment resulted in increased expression of MtMTP1.2 and MtMTP4, but a significant halt in expression of MtMTP2 and MtMTP5. Similarly, Co2+ treatment significantly increased the expression of MtMTP11, but decreased the expression of MtMTP7. Fe2+ treatment increased the expression of MtMTP4, MtMTP5 and MtMTP11 but resulted in decrease in expression of MtMTP2 and MtMTP10.2. Mn2+ treatment resulted in increased expreesion of MtMTP4 and MtMTP10.1, but decreased expression of MtMTP5, MtMTP7 and MtMTP10.2. Finally, Zn2+ treatment resulted in enhanced expression of MtMTP1.1, MtMTP1.2 and MtMTP4, but decresed expression of MtMTP5, MtMTP7 and MtMTP11.
In leaf, Cd2+ treatment resulted in increased expression of MtMTP4, but decresed expression of MtMTP5. Similarly, Co2+ treatment resulted in increased expression of MtMTP1.1, MtMTP4 and MtMTP5, but decreased expression of MtMTP7. Fe2+ treatment displayed increased expression of MtMTP1.2, but decreased expression of MtMTP2. Mn2+ treatment resulted in increased expreesion of MtMTP1.1 and MtMTP4, while decreased expression of MtMTP1.2, MtMTP5 and MtMTP7. Finally, Zn2+ treatment resulted in increased expression of MtMTP1.1, MtMTP1.2 and MtMTP4, while decresed expression of MtMTP2, MtMTP5 and MtMTP7.