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Article

Genome-Wide Identification of the Medicago ruthenica FTIP Gene Family and Expression Profiling Under Salt Stresses

by
Yonglei Tian
1,2,
Lin Zhu
1,*,
Maowei Guo
1,
Zhiyong Li
2,
Zinian Wu
1,
Hongyan Li
1,
Xingyue Li
3 and
Xiaolong Wang
4
1
Institute of Grassland Research, Chinese Academy of Agricultural Sciences, Hohhot 010010, China
2
Key Laboratory of Grassland Resources and Utilization of Ministry of Agriculture, Hohhot 010010, China
3
Institute of Grassland Research, Inner Mongolia Academy of Agricultural & Animal Husbandry Sciences, Hohhot 010031, China
4
Branch of Animal Husbandry and Veterinary of Heilongjiang, Academy of Agricultural Sciences, Qiqihar 161005, China
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2026, 27(4), 1633; https://doi.org/10.3390/ijms27041633
Submission received: 12 January 2026 / Revised: 28 January 2026 / Accepted: 5 February 2026 / Published: 7 February 2026
(This article belongs to the Topic Leguminomics: From Genomic Blueprints to Sustainable Agriculture)
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Abstract

FT-interacting proteins (FTIPs) function in signal transduction and metabolite transport, which are important to plant growth, development, and reproduction. Their bioinformatic characteristics and functions in Medicago ruthenica, a forage crop used for ecological restoration, remain unknown. We identified 19 MrFTIPs from the M. ruthenica genome, and they were unevenly distributed across seven chromosomes. Most of them are alkaline, labile and hydrophilic, with a structure comprising irregular coils, α-helices and extended chains. Phylogenetic analysis revealed five evolutionary clades with MrFTIPs. In total, two pairs of segmental duplication events were found, indicating a major pattern for MrFTIP expansion. Overall, 16, 11, and 22 gene pairs were identified from M. truncatula, Arabidopsis thaliana, and Glycine max, respectively. The promoter regions of MrFTIPs were enriched with abiotic stress responses and light or hormone signaling. Tissue-specific analysis revealed that 7 MrFTIPs were highly expressed in leaves, 9 MrFTIPs were highly expressed in petals, and 6 MrFTIPs were highly expressed in stigma and anthers. MrFTIP17 continues to be upregulated among tissues under salt stress, and MrFTIP8 continues to be upregulated among tissues under salt–alkali stress. Collectively, our study systematically characterized the genomic features, evolutionary patterns and cis-regulatory characteristics of the MrFTIP gene family in M. ruthenica, and identified MrFTIP8 and MrFTIP17 as candidate genes associated with salt stress responses in this species, thus providing insights into and potential targets for the molecular and conventional breeding of M. ruthenica.

1. Introduction

The multiple C2 domains and transmembrane region protein (MCTP) family is important for multiple processes across plants and functions in the regulation of plant growth and development processes, such as intercellular communication, intercellular transport, and even long-distance transport [1,2,3]. The MCTPs are enriched on plasmodesmata, and the close contact of plasmodesmata with the endoplasmic reticulum and plasma membrane provides a convenient basis for molecular transport and rapid signal transmission [4]. Several members of the MCTP gene family have been identified in plants such as Arabidopsis thaliana (16 MCTPs), Gossypium hirsutum (33 MCTPs), Oryza sativa (82 MCTPs), Zea mays (17 MCTPs) and Solanum lycopersicum (14 MCTPs) [5,6,7,8,9]. FT-interacting proteins (FTIPs) are one of the key MCTPs members with a defined phylogenetic clade, which can direct interaction with or modulate the activity of FLOWERING LOCUS T (FT) by C2 domains [10,11].
Generally, FTIP gene family members are considered to influence plants’ sexual reproduction and regulate flowering, which is crucial for progeny reproduction and seed production [12]. FT gene-encoded proteins, such as florogens, undergo long-distance movement that must rely on FTIP1 to promote flowering, which can interact with FT proteins through the first and third C2 domains and act in the vasculature of leaves to promote flowering [13,14]. Additionally, FTIP can also regulate crop flowering time by facilitating Hd3a transport from companion cells to sieve elements [15]. FTIPs affect anther dehiscence by gene interactions with functional genes. Anther dehiscence determines successful sexual reproduction of flowering plants through timely release of pollen grains for pollination and fertilization. OsFTIP7 of Oryza sativa is highly expressed in the anthers and facilitates the nuclear translocation of a homeodomain transcription factor, which directly suppresses a predominant OsYUCCA4 and thus decreases auxin accumulation to mediate anther dehiscence [16].
Interestingly, FTIPs also seem to be a central hub that integrates environmental signals, such as light, hormone and environmental changes, to assist plants in adjusting their stress survival strategies [11]. In the O. sativa mutant Osftip1 with loss of OsFTIP1 function, an obvious drought-tolerant phenotype was observed [17]. Drought treatment induced OsFTIP1 downregulation, followed by the release and transport of OsMFT1 into the nucleus, so that it can interact with OsMYB26, destroy the binding between OsMYB26 and OsLEA3, enhance OsbZIP66 binding with OsLEA3, and activate OsLEA3 transcription, so that OsFTIP1 negatively regulates the drought response in O. sativa. In total, 27 ZmFTIPs were identified in Zea mays; they can respond to a variety of abiotic stresses and plant hormones [10]. Moreover, heavy metal abiotic stress effects are also included. In O. sativa, loss of OsFTIP7 function results in up-regulating auxin biosynthesis and inducing physiological and biochemical changes to enhance heavy metal abiotic stress [18]. In summary, FTIPs might be functional genes for optimal distribution of resources for survival and successful reproduction.
Medicago ruthenica is widely used in natural grasslands and cultivated pastures for ecological restoration, owing to its excellent stress tolerance, good forage quality, and adaptability to local environments with low temperatures, saline–alkali soil, and arid and semiarid climates [19,20,21]. The excellent stress adaptation of M. ruthenica relies on precise regulatory networks balancing growth, flowering and stress responses, and MrFTIPs, as central hubs integrating environmental signals and reproductive regulation, are likely to play key roles in this process. Currently, studies of FTIP families in plants is prevalent, focused on model plants, flowers, Poaceae and cash crops. However, little is known about the identification, evolution, expression pattern and function information of high-quality forage FTIP families.
Therefore, we hypothesize that the function and diversification of the MrFTIP family underlie M. ruthenica’s enhanced stress adaptability. This study aims to perform an extensive genome-wide analysis of FTIP family members in M. ruthenica. Analyses of gene chromosomal positions, gene and protein structures, protein domains, phylogeny, motifs, and promoter elements were carried out to infer the potential biological roles of MrFTIPs. These findings will provide theoretical foundations for future functional verification, the balance strategy between reproduction and survival, as well as potential application of MrFTIPs in M. ruthenica and improvements in legume forage crop breeding.

2. Results

2.1. Identification and Distribution of MrFTIP Genes

FTIP proteins belong to the MCTP family, and a total of 19 FTIPs are found in the whole genome of M. ruthenica (Tables S1 and S2). These MrFTIPs are named from MrFTIP1 to MrFTIP19 according to their positions on chromosomes. Seventeen MrFTIPs were found to be scattered randomly across seven chromosomes of the M. ruthenica genome (Figure 1), whereas the other two MrFTIPs (MrFTIP18 and MrFTIP19) displayed no preferential distribution on a specific chromosome. MrFTIP1, MrFTIP2, and MrFTIP3 were located on chromosome (Chr)1; MrFTIP4 was located on chromosome Chr3; MrFTIP5, MrFTIP6, and MrFTIP7 were located on chromosome Chr4; MrFTIP8 and MrFTIP9 were located on chromosome Chr5; MrFTIP10, MrFTIP11, and MrFTIP12 were located on chromosome Chr6; MrFTIP13, MrFTIP14, and MrFTIP15 were located on chromosome Chr7; and MrFTIP16 and MrFTIP17 were located on chromosome Chr8.
MrFTIPs contain two transmembrane regions (N-terminal C2 domains and C-terminal transmembrane regions), but the number of regions differs (Tables S2 and S3). MrFTIP1 contains two N-terminal C2 domains; MrFTIP15 contains one N-terminal C2 domain; MrFTIP5, MrFTIP6, MrFTIP8, MrFTIP13, MrFTIP16, MrFTIP17, and MrFTIP18 each contain four N-terminal C2 domains; and all of the remaining 10 MrFTIPs contain three N-terminal C2 domains. Further, each of the MrFTIP transmembrane domains contains one to two C-terminal phosphoribosyl transferase domains (PRT_C) near the C-terminal transmembrane region (Figures S1 and S2). MrFTIPs contain multiple C2 domains and transmembrane region proteins, indicating MrFTIPs belong to the MCTP family.

2.2. Physicochemical Property Analysis, Secondary and Tertiary Structure of MrFTIP Proteins

The MrFTIP proteins range in length from 591 to 3585 bp CDS with 196–1194 amino acids and molecular weights spanning 22.87~137.04 kDa (Table 1). Their isoelectric points were between 6.66 and 9.24, and the proportion of acidic amino acids was 15.79% (theoretical isoelectric point < 7), less than the proportion of 84.21% basic amino acids (theoretical isoelectric point > 7). The instability index of MrFTIPs ranged from 28.85 to 49.68; of these, 89.5% were unstable proteins with an instability index higher than 40, and only MrFTIP8 and MrFTIP15 were stable proteins. The aliphatic index of the MrFTIP proteins ranged from 51.42 to 77.21; all of them show an average hydrophobic coefficient from −0.061 to −0.488, indicating that they are hydrophilic proteins. Predictions for subcellular localization indicate that 17 MrFTIPs are located in the plasma membrane, with a probability of MrFTIP7 to be located in the chloroplast and MrFTIP15 to be located in the cytoplasm.
The protein secondary structure results showed that MrFTIP proteins mainly comprised alpha helices (23.10% to 39.80%), random coils (49.49% to 68.65%), and extension chains (7.54% to 16.78%), without beta turns (Table S4). These distinctions may contribute to differences in the functions of MrFTIP proteins. We subsequently obtained the three-dimensional structure of MrFTIP proteins using the FTIP sequence of Glycine max as a template and SWISS-MODEL (Figure 2). The sequence coverage of MrFTIP proteins ranged from 0.91 to 1.00, the sequence identity ranged from 70.23% to 93.8%, GMQE value ranged from 0.71 to 0.88, and the Ramachandran plot showed a favored region ranging from 88.84% to 96.89%, suggesting the reliability of the MrFTIP protein model. SWISS-MODEL results confirmed the MrFTIP protein’s extensive helices and random coil regions.

2.3. Phylogenetic Characterization of MrFTIPs

To clarify the evolutionary relationships of MrFTIPs between M. ruthenica and other species like M. truncatula, G. max, A. thaliana, and O. sativa (Table S5), we used an unrooted phylogenetic tree generated with 63 FTIP sequences (Figure 3). Our findings indicate that the 19 MrFTIP proteins in M. ruthenica can be clearly classified into five groups, with group 1 containing eight MrFTIPs (MrFTIP1, MrFTIP7, MrFTIP9, MrFTIP10, MrFTIP11, MrFTIP12, MrFTIP17, and MrFTIP19), group 3 containing four MrFTIPs (MrFTIP3, MrFTIP4, MrFTIP5, and MrFTIP18), group 4 containing two MrFTIPs (MrFTIP2 and MrFTIP14), group 5 containing one MrFTIP (MrFTIP6), and group 6 containing four MrFTIPs (MrFTIP8, MrFTIP13, MrFTIP15, and MrFTIP16); group 2 only contained members of A. thaliana and O. sativa, indicating that the MrFTIP family may be distantly related to AtFTIP2 and OsFTIP10. Group 1 exhibited a greater abundance of MrFTIP members compared with those in the other groups. In group 1, the clustering implied similar roles; MrFTIP1 was clustered with MtFTIP3.3, while MrFTIP9 and MrFTIP19 were closely clustered with MtFTIP3.4. MrFTIP17 was closely clustered with AtFTIP6, and MrFTIP12 was closely clustered with AtFTIP15, suggesting that MrFTIP17 and MrFTIP12 may have originated from the same ancestor. MrFTIP10 and MrFTIP11 were closely clustered with MtFTIP7.2. In group 3, MrFTIP3 was closely related to MtFTIP1.2; MrFTIP4 was closely related to MtFTIP1; and MrFTIP5 and MrFTIP18 were closely related to MtFTIP1.1. In group 4, MrFTIP2 and MrFTIP14 were respectively closely related to their orthologs MtFTIP3.5 and MtFTIP3. MrFTIP6 in group 5 was closely clustered with MtFTIP7.1. MrFTIP8, MrFTIP13, MrFTIP15, and MrFTIP16 in group 6 were closely related to MtFTIP7, MtFTIP3.1, and MtFTIP3.2. Most MrFTIP members were found to exhibit a higher degree of similarity with their counterparts found in M. truncatula compared with those in other plant species; only MrFTIP12 and MrFTIP17 shared a common ancestor with A. thaliana.

2.4. Gene Structures and Conserved Motif Analysis of MrFTIP Proteins

Structural analysis of MrFTIP proteins showed that 13 (MrFTIP1, MrFTIP2, MrFTIP3, MrFTIP5, MrFTIP6, MrFTIP7, MrFTIP9, MrFTIP13, MrFTIP14, MrFTIP16, MrFTIP17, MrFTIP18, and MrFTIP19) have one exon and no intron, four (MrFTIP4, MrFTIP8, MrFTIP10, and MrFTIP12) have two exons and one intron, MrFTIP11 has three exons and two introns, and MrFTIP15 has four exons and three introns (Figure 4A). These 19 MrFTIP genes can be further classified into three clades, designated as group A, B, and C. Four MrFTIP members (MrFTIP1, MrFTIP7, MrFTIP9, and MrFTIP19) belonged to the same clade subgroup A1; these four members showed a similar number of motifs, similar amino acid lengths, and shared common motif compositions, arranged in a similar pattern; the other four members (MrFTIP10, MrFTIP11, MrFTIP12, and MrFTIP17) were grouped within the clade subgroup A2; they shared the same motif compositions with subgroup A1, but had one motif more than in subgroup A1, motif15 located near the N-terminal. MrFTIP8, MrFTIP13, MrFTIP15, and MrFTIP16 were classified into group B, of which MrFTIP8, MrFTIP13, and MrFTIP16 had a similar number of motifs, similar amino acid lengths, and shared common motif compositions. As members of group C, MrFTIP2 and MrFTIP14 in subgroup C1 showed high homology; MrFTIP3, MrFTIP4, MrFTIP5, and MrFTIP18 in subgroup C2 showed high homology; and MrFTIP6 in subgroup C3 showed low homology with each other.
To enhance our understanding of diversity in the MrFTIP family, we examined the conserved motifs in MrFTIPs. The protein domain close to the C-terminus was found to be more conserved than the protein domain close to the N-terminus (Figure S3). Motif analysis resulted in a total of 15 motifs (motif1–motif15). According to the classification of the MrFTIP gene family, all members of the MrFTIP protein family had motif1 and motif2 (Figure 4B). Members in subgroup A1 possessed the same motif composition of motif1 to motif14, whereas MrFTIP1 had no motif11 and MrFTIP7 had no motif13. Members from subgroup A2 contained the same 14 motifs arranged in a similar pattern, while MrFTIP11 had no motif13. Fifteen motifs (motifs 1 to 15) were found in all proteins in MrFTIP8, MrFTIP13, and MrFTIP16 of group B, and also in MrFTIP5 and MrFTIP18 of subgroup C2 and subgroup C3. Members of subgroup C1 (MrFTIP2 and MrFTIP14) and members of subgroup C2 (MrFTIP3 and MrFTIP4) had the same motifs and motif arrangements as those in subgroup A1. Another additional feature of conserved motifs was characterized by the motif11, motif12, and motif15 distribution near the N-terminal and motif2 and motif6 distribution near the C-terminal.
To further explore the conservative domain characteristics of MrFTIP proteins, the multiple alignment of 19 MrFTIP protein sequences was performed using the MEME online program, and the sequence logos of MrFTIP were generated using WebLogo (Figure S4). The results indicate that motif1 contains 13 highly conserved residues, which was the largest number of conserved amino acid residues compared with other motifs, including aspartic acid (D), tyrosine (Y), valine (V), alanine (A), lysine (K), glycine (G), arginine (R), threonine (T), proline (P), glutamic acid (E), glutamine (Q), tryptophan (W), and phenylalanine (F). Motif15 contains eight highly conserved residues, which was the second largest number of conserved amino acid residues, including aspartic acid (D), lysine (K), glycine (G), threonine (T), proline (P), glutamine (Q), phenylalanine (F), and leucine (L). The number of conserved amino acid residues in motif9, motif10, and motif13 were the least, containing two (tyrosine, Y; proline, P), one (proline, P), and two (tryptophan, W; histidine, H), respectively. Among these amino acid residues, conserved aspartic acid (D) was widely distributed in motif1, motif2, motif3, motif4, motif5, motif6, motif7, motif12, and motif15. Moreover, the aspartic acid (D) residues were often replaced by glutamic acid (E) or vice versa; this was also observed for arginine (R) and lysine (K) residues.

2.5. Simple Sequence Repeats Analysis of MrFTIP Genes

In this study, a total of 374 SSRs were predicted in 19 MrFTIP sequences (Figure 5); the MrFTIPs’ SSR loci were rich in mononucleotide, dinucleotide, trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide repeats. The number of each repeat type varied substantially, but tetranucleotide repeats dominated (137 SSRs), followed by the dinucleotide type with 98 SSRs, mononucleotide type with 46 SSRs, and hexanucleotide type with 44 SSRs. These SSR loci were mainly distributed among coding regions, and 27 SSR loci were distributed among the non-coding regions of MrFTIP4, MrFTIP8, MrFTIP10, MrFTIP11, MrFTIP12, and MrFTIP15 (Table S6). Compared with the SSRs containing C/G, mononucleotide SSRs of A/T exhibited a higher frequency with 95.65% (44), dinucleotide SSRs of GA/AG exhibited a higher frequency of 31.63% (31), trinucleotide SSRs of AAG/ACC exhibited a higher frequency with 40.00% (10), tetranucleotide SSRs of TGTT/TGTTA and AAAG/TTTG exhibited a higher frequency with 18.25% (25), pentanucleotide SSRs of GTTCA/TTTG exhibited a higher frequency with 16.67% (4), and hexanucleotide SSRs of TTGATT/AGAACA/GAAAAA exhibited a higher frequency with 22.73% (10).

2.6. Collinearity and Selection Pressure Analysis of MrFTIPs

We further investigated the intraspecific synteny of MrFTIPs. The collinearity analysis results in M. ruthenica revealed a total of two collinear gene pairs (Figure 6A) within MrFTIPs: MrFTIP2 located in Chr1 and MrFTIP14 located in Chr7, and MrFTIP13 located in Chr7 and MrFTIP16 located in Chr8, which may have undergone segmental duplication. The interspecific collinearity was analyzed for a better understanding of the evolutionary relationship between MrFTIPs and their homologs in three other species, including M. truncatula, A. thaliana, and G. max (Figure 6B). In total, 13 MrFTIPs showed collinearity with 16 homologs of M. truncatula, 11 homologous gene pairs were found between M. ruthenica and A. thaliana, and the number of homolog pairs between 9 MrFTIPs and G. max was 22, suggesting that these FTIP homologous gene pairs may share a common ancestor. Notably, none of the pairs of collinear MrFTIP18 were shared among these four species, indicating that it may be unique to M. ruthenica or that these species may have a distant phylogenetic relationship. The Ka/Ks values of MrFTIP members (Table S8) were all <1, suggesting that MrFTIPs have experienced purifying selection.

2.7. Promoter Characterization of MrFTIPs

We isolated the 2.0 kb promoter sequences of MrFTIPs and investigated their cis-regulatory elements. The identified elements included stress-responsive elements, such as ARE, MYB, MYB-like sequence, and MYC; hormone-responsive elements such as AAGAA-motif and ABRE-motif; and light-responsive elements such as Box 4, G-box, and GT1-motif (Figure 7A). Among those, MYB and Box 4 were the most frequently occurring elements in the MrFTIP family (Figure 7B), 16 MrFTIP members contain the AAGAA-motif element, and 13 contain the ABRE or ARE element. Notably, only five of the MrFTIP members contain all the identified cis-regulatory elements.

2.8. Expression Profiles of MrFTIPs Across Various Tissues, Genotypes, and Response to Stresses

For the purpose of MrFTIP gene expression patterns, we determined all 19 gene members expressed in 5 different tissues, 2 different periods, 2 different genotypes, salt stress and saline–alkali. For different tissues (Figure 8), a total of seven MrFTIP members with high expression in the leaf, including MrFTIP1, MrFTIP10, MrFTIP11, MrFTIP12, MrFTIP13, MrFTIP15 and MrFTIP17; 9 MrFTIP members (MrFTIP4, MrFTIP5, MrFTIP6, MrFTIP7, MrFTIP9, MrFTIP10, MrFTIP14, MrFTIP17 and MrFTIP18) with high expression in the petal; and 6 MrFTIP members (MrFTIP1, MrFTIP2, MrFTIP3, MrFTIP8, MrFTIP16 and MrFTIP19) with high expression in the stigma and anthers, they were tissue-specific genes.
For development stages (Figure 9), the expression of 8 MrFTIPs was increased with leaf development, including MrFTIP1, MrFTIP2, MrFTIP9, MrFTIP10, MrFTIP11, MrFTIP13, MrFTIP15 and MrFTIP19, and there was no apparent change in 4 MrFTIPs apart from others. Most of the MrFTIPs showed decreased expression during root development, in addition to MrFTIP16 and MrFTIP19. For different genotypes (Figure 10), a total of 8 MrFTIPs were expressed higher in the salt-resistant genotype, including MrFTIP2, MrFTIP4, MrFTIP7, MrFTIP13, MrFTIP14, MrFTIP15, MrFTIP16 and MrFTIP17.
For MrFTIP’s response to salt stress (Figure 11) in leaf samples, most MrFTIP members showed fluctuation changes; only MrFTIP1, MrFTIP4 and MrFTIP19 showed a decreasing trend with increasing treatment time, and MrFTIP6, MrFTIP17 and MrFTIP18 showed an increasing trend with increasing treatment time. As for stem samples, a total of 8 MrFTIP members showed an increasing trend with increasing treatment time. Most MrFTIPs showed an increasing trend with increasing treatment time in root samples. Among them, only MrFTIP17 was upregulated in leaf, stem and root samples.
As for MrFTIP’s response to salt–alkali stress (Figure 12), a total of 6 MrFTIPs showed a decreasing trend with increasing treatment time in leaf samples, and 5 MrFTIPs showed an increasing trend with increasing treatment time; others presented fluctuating changes. A total of 13 MrFTIPs showed an increasing trend with increasing treatment time in stem samples, which was in contrast to MrFTIP19. As for roots, 7 MrFTIPs showed an increasing trend with increasing treatment time; only MrFTIP5 and MrFTIP17 showed a decreasing trend, which showed a different variation with these MrFTIPs expressed under salt stress.

3. Discussion

In plants, various signaling pathways participate in regulating plant growth and development, generation, senescence, and response to biotic and abiotic stresses; cell-to-cell transmission of signaling molecules and their transduction through long-distance transport are important for these processes; notably, MCTP proteins are important regulators of cell-to-cell signal transmission [22]. Plasmodesmata are plant cell-specific intercellular communication organelles that connect plasma membranes of neighboring cells and serve as important conduits for the exchange of molecules [23]. A large number of MCTP genes have now been identified and found to be involved in membrane transport and signal transduction [24]. MCTPs localized at the PD also act as correspondents of the endoplasmic reticulum and plasma membrane [4]. These family members are characterized by multiple N-terminal C2 domains and multiple C-terminal transmembrane domains [5]. Members of the M. ruthenica FTIP family are the same as MCTP proteins, with representative features of one to three N-terminus C2 domains and at least two C-terminal transmembrane domains, indicating that the identified MrFTIPs belong to the MCTP family (Tables S1 and S2; Figures S1 and S2). C2 domains are usually common protein signaling modules, whose principal function is mediating protein binding to specific membranes through calcium ions, thereby influencing protein localization and function [25,26]. In Arabidopsis, partial C2 domain loss impairs but does not abolish function, indicating distributed contributions [5]. Among proteins with C2 domains, an increase in the number of C2 domains leads to higher activity of the 5-phosphatase domain, which is opposite to the lack of a C2 domain [27]. In addition, the more C2 domains, proteins bind to more partners; such proteins with multiple C2 domains likely evolved to enable cooperative or crosstalk-based regulation, expanding more functional repertoire [28,29,30]. Although 19 MrFTIP proteins were predicted to have transmembrane regions, MrFTIP7 and MrFTIP15 subcellular localization predictions were localized to chloroplasts and cytoplasm, respectively (Table 1). This is most likely owing to proteins having strong hydrophobic segments, which can confuse algorithms that must distinguish signal peptides, transit peptides, and generic transmembrane helices [31,32]. The hydrophobic segments of proteins dictate their localization and membrane binding ability. Hydrophobic proteins tend to be localized to membrane-bound organelles or the cell membrane, owing to their hydrophobic amino acid sequences on the cytosolic side or spanning the hydrophobic core, which interacts with the lipid bilayer and is stably embedded in lipids [33,34,35]. Or possibly due to disordered N-termini structure is hard to distinguish from chloroplast proteins with chloroplast proteins, those chloroplast proteins are synthesized in the cytosol and carry N-terminal resemble unstructured and basic regions [31,36]. Homology modeling predicted that the existing helix and strand structure in MrFTIP proteins (Figure 2, Table S3) is dominated by large irregular coils. Random coils act as flexible regions, allowing multiple substrate interactions that endow the proteins with flexibility, thus affecting protein–ligand binding events and enzymatic activities [37,38,39]. The α-helix structure and extended chain are two common secondary structure types of proteins, which are crucial for protein rigidity and structural stability [38,39]. The α-helix achieves this through internal hydrogen bonding and side-chain interactions, while extended chains (including β-strands and extended helices) provide a stable, rigid framework through inter-strand hydrogen bonds and ordered packing. The more the α-helix, the lower the protein interaction rate [38]. The shorter chain structure facilitates changes in the protein structure and enhances the functional diversity of the protein [40].
Usually, C2 domains can be divided into Ca2+-dependent and Ca2+-independent forms; not all the C2 domains bind calcium [41]. Ca2+ binding ability depends mainly on the presence of aspartic acid or glutamic acid residues in the C2 domains [42]. In M. ruthenica, MrFTIPs contain a total of 15 conserved motifs (Figure 3); among them, 9 motifs have highly conserved aspartate residues, and 5 of these 9 motifs also contain highly conserved glutamic acid residues. Ca2+ binding induces conformational changes in the C2 domain, stabilizing the loops and enabling the domain to interact with negatively charged phospholipids in membranes, allowing the C2 domain to transition to a membrane-bound state, which is essential for activating many signaling proteins [43,44]. Furthermore, we also observed a replacement phenomenon between aspartic acid and glutamic acid residues. Both aspartic acid and glutamic acid are negatively charged but differ in side-chain length and chemical properties. Replacing one with the other can subtly change the protein structure, affect calcium binding, and alter interactions with other molecules, potentially modifying the C2 domain function [45]. Proteins may evolve to either favor or avoid these residues in certain positions to optimize protein function and stability [42,43].
Polyploidy, tandem duplication, and segmental duplication are important mechanisms for plant generation and expansion of gene families [46]. Gene duplication is the main driving force for gene family expansion and functional diversification. In total, two pairs of MrFTIP members were found to have undergone duplication, MrFTIP2/MrFTIP14 and MrFTIP13/MrFTIP16 (Figure 6). These kinds of locally collinear gene family members are distributed unevenly on each chromosome, consistent with the characteristics of segmental duplication [47]. These findings indicate that segmental duplication is the major pattern for MrFTIP family expansion. Segmental duplication, often layered on whole genome duplication and tandem duplication, contributes substantially to gene family expansion by increasing copy number, dispersing duplicates, and creating structural novelty. Its impact is highly dependent on lineage, genomic context, and gene function, but in many characterized families, it is a primary engine of expansion [48,49]. Ka/Ks values represent the non-synonymous/synonymous substitution rate ratios. Such non-synonymous substitutions in the coding region are likely to have caused changes in the structure and function of the encoded protein. Further, the selective pressure on gene families can be inferred according to the Ka/Ks ratio as follows: positive selection with Ka/Ks > 1, neutral evolution with Ka/Ks = 1, and purifying selection with Ka/Ks < 1 [50]. MrFTIP members experienced strong purifying selection (Table S7). Purifying selection leads to nearly identical protein sequences among gene family members to maintain high protein sequence conservation, and strong purifying selection indicates that harmful mutations have been consistently removed, so that the gene family performs essential non-redundant functions [51,52,53].
Promoters are sequences before the start codon that control the initiation and regulation of gene expression. They serve as binding sites for transcription machinery and integrate regulatory signals, thereby determining when, where, and how much a gene is expressed. The MrFTIP gene family contains three types of cis-elements in promoters, including stress-related elements such as ARE, MYB, MYB-like sequence, and MYC; hormone-responsive elements such as AAGAA-motif and ABRE-motif; and light-responsive elements such as Box 4, G-box, and GT1-motif (Figure 7), which suggests that members of the MrFTIP family might participate in plant development regulation and abiotic stress responses through integrating diverse signals [13,17,18]. To further explore the functional characteristics of MrFTIPs, we used qRT-PCR for tissue specificity and response to salt and saline–alkali. Tissue-specific analysis reveals that MrFTIP1 was highly expressed in leaf and floral organs, reflecting their tissue-specific functions that may be involved in the development and reproduction of light signaling in street flower organs. Furthermore, MrFTIP4, MrFTIP5, MrFTIP6, MrFTIP7, MrFTIP9, MrFTIP14 and MrFTIP18 with high expression in only petals, and MrFTIP2, MrFTIP3, MrFTIP8, MrFTIP16 and MrFTIP19 with high expression in only stigmas and anthers, also indicate their function of development and reproduction of flower organs [10,13,54].
Salt stress refers to an abiotic stress caused by the excessive accumulation of salts in the soil. When such salts dissolve, they only increase the ion concentration of the soil solution without significantly altering the soil pH value. Saline–alkali stress is a combined abiotic stress induced by the coexistence of neutral salts and alkaline salts in the soil, which causes a significant increase in soil pH. Therefore, saline–alkali stress represents a dual synergistic effect of salt stress and alkali stress, and its damage mechanism is more complex and severe than that of simple salt stress. A total of 4 gene expression types respond to salt and saline–alkali stress, including continuous decline, continuous rise, first rise and then fall, first fall and then rise (Figure 11 and Figure 12). Roots are the primary site for salinity and saline–alkali sensing and thus the focus of many transcriptome studies [55,56]. In general, sustained upregulation of root genes under salt and salt–alkali stress is strongly associated with early stress perception, ion homeostasis, and osmotic regulation [57,58,59]. Leaves are the major organ contributing to photosynthesis metabolism and energy transition; they show highly differentiated gene-expression changes under stress, coordinating photosynthesis and damage to maintain basic function [60]. Of the 19 MrFTIP family members, MrFTIP17 continues the upregulated expression trend in the leaf, stem and root under salt stress, and MrFTIP8 continues the upregulated expression trend in the leaf, stem and root under salt–alkali stress; this might be due to the numerous stress-related cis-elements in promoters of MrFTIP17 and MrFTIP8. Collectively, the diverse cis-acting elements in MrFTIPs’ promoters, their distinct tissue-specific expression profiles, and expression patterns in response to salt and salt–alkali stresses, together reveal that the MrFTIP gene family plays multifaceted roles in integrating signals to coordinate plant growth and development as well as organ-specific adaptive responses to abiotic stresses.

4. Materials and Methods

4.1. Identification, Distribution and Sequence Analysis of MrFTIPs

A total of seventeen known protein sequences of the FTIP gene family in A. thaliana were downloaded from the TAIR database (https://www.arabidopsis.org/, accessed on 1 November 2024), fourteen FTIP gene sequences of Medicago truncatula and twenty FTIP gene sequences of Glycine max were downloaded from the NCBI database, were downloaded from the NCBI database (https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_003473485.1/, https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_000004515.6/, accessed on 8 November 2024), thirteen FTIP gene sequences of O. sativa were downloaded (http://rice.uga.edu/index.shtml, accessed on 10 November 2024) according to a published article [11]. These sequences were used as queries to identify FTIP gene members from the published M. ruthenica genome (https://github.com/yinm2018/Medicago_ruthenica_genome, accessed on 2 December 2024) using BLASTP 2.14.0 (e  < 10−5) [21]. Protein domains of the identified FTIP gene members were annotated using the Pfam (v33.1) database, and the Hidden Markov Model (HMM) profiles of the C2 domain (PF00168) and C-terminus of phosphoribosyltransferases (PRT_C, a domain that often appears together with calcium-ion dependent C2 domains, PF08372) were used to confirm the core domain [22]. Finally, nineteen M. ruthenica FTIP genes were confirmed and renamed (MrFTIPs). The features and the subcellular localization information of the MrFTIPs were analyzed by the online ExPASY tool (http://web.expasy.org/protparam/, accessed on 4 January 2025) and WoLF PSORT (https://wolfpsort.hgc.jp/, accessed on 5 December 2024) [61]. The physical position and chromosomal distribution information of MrFTIPs were obtained from published genome annotation data and visualized using TBtools v2.083 [62]. Transmembrane helices of MrFTIP proteins were predicted using the TMHMM-2.0 tool (https://services.healthtech.dtu.dk/service.php?TMHMM-2.0, accessed on 6 January 2025) [63].

4.2. Structure Prediction, Gene Structure and Conserved Motif Analysis of MrFTIPs

The secondary structure and three-dimensional (3D) structure of MrFTIPs were determined by the utilization of the SOPMA program (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html, accessed on 11 January 2025) and SWISS-MODEL (https://swissmodel.expasy.org/interactive, accessed on 11 January 2025) [64], respectively. The screening criteria were sequence homology ≥ 30% and coverage ≥ 80%. Online software Gene Structure Display Server (GSDS 2.0, http://gsds.cbi.pku.edu.cn, accessed on 11 January 2025) was used for exon/intron arrangement analysis of MrFTIPs [65]. Conserved motifs of MrFTIP proteins were identified using MEME (http://meme-suite.org/tools/meme, accessed on 6 February 2025), with a parameter of 15 maximum motif numbers [66]. Multiple sequence alignments of the full-length sequences of MrFTIP proteins were performed by using MAFFT 7.0 software [67].

4.3. Simple Sequence Repeat (SSR) Analysis of MrFTIPs

The SSR loci contained in DnoNAC genes were analyzed using MISA 2.1-web online software (https://webblast.ipk-gatersleben.de/misa/, accessed on 20 February 2025) with default parameters, with detection thresholds set to mono-10, di-5, tri-4, tetra-3, penta-3, and hexa-3 repeats [68].

4.4. Phylogenetic Analysis of MrFTIPs

The FTIP family member sequences of M. ruthenica, A. thaliana, M. truncatula, G. max and O. sativa were used to construct a phylogenetic tree by MEGA11 (https://www.megasoftware.net, accessed on 5 October 2023), through the method of Neighbor-Joining (NJ) [69], with the following parameters: p-distance model, partial deletion, 1000 bootstrap replicates, cutoff with 50%, which is used to test the reliability of phylogenetic tree branches.

4.5. Gene Duplication, Collinearity and Evolutionary Pressure (Ka/Ks) Analysis

Gene duplication events were analyzed by using MCScanX, with the default parameters. The syntenic relationship of MrFTIPs in M. ruthenica was visualized using MCScanX, and collinearity between species was visualized using TBtools [62,70]. Furthermore, Ka/Ks ratio analysis on MrFTIPs was calculated using Ka/Ks Calculator in TBtools [62].

4.6. Promoter Analysis of MrFTIPs

Based on the complete genome sequencing results of M. ruthenica, the 2000 bp region upstream of the MrFTIP initiation codon (ATG) was used as a candidate promoter sequence. The obtained MrFTIP promoter sequence was submitted to PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 1 March 2025) to predict cis-acting elements in the promoter region [71]. TBtools was used to visualize gene structures, motifs, and promoter cis-acting elements [62].

4.7. Plant Material Treatment and Expression Analysis of MrFTIPs

In this experiment, seeds of two M. ruthenica cultivars (‘Zhongcao No. 80’ with salt resistance, ‘Keerqinshardi’ with salt sensitivity) were used; they were cultivated by the Institute of Grassland Research, Chinese Academy of Agricultural Sciences. ‘Zhongcao No. 80‘ is an excellent salt-tolerant M. ruthenica variety with narrow and long leaves. ‘Keerqinshardi’ is a feedable cultivar with an oval leaf shape and abundant leaves. The seeds were germinated for 7 days after abrasion. Healthy seedlings with the same growth period were selected and transplanted into 6 cm × 6 cm × 11 cm flower pots (filled with nutrient soil:vermiculite:perlite = 3:1:1), grown at 25 °C 16 h light/25 °C 8 h dark conditions in a growth chamber.
Leaf, stem and root samples of two M. ruthenica cultivars were harvested at seedling stage and subjected to different genotype analyses. Leaf samples of Zhongcao No. 80 were collected at the seedling stage and flowering stage for different development period analyses. Leaf, stem, root, petal, anther and stigma of Zhongcao No. 80 were harvested at the flowering period and subjected to different tissue analyses. As for salt stress, the healthy Zhongcao No. 80 seedlings were treated in a mixed solution (Hoagland’s solution with 250 mmol/L NaCl) for 0 h, 12 h and 24 h; leaf, stem, and root samples were used. For saline–alkali compound stress, seedlings were treated in a mixed solution (Hoagland’s solution with NaCl 250 mmol/L + NaHCO3 200 mmol/L) and applied at 0 h, 12 h and 24 h; leaf, stem, and root samples were collected [72,73,74]. Each treatment used 10 plants as a biological replicate; a total of three biological replicates were set.
The RNA extraction, cDNA strand synthesis, and qRT-PCR analysis were performed according to our previous studies [72,75]. Total RNA was extracted from tissue samples using TRIzol-chloroform according to manufacturer’s protocol (Shenggong, Shanghai, China). cDNA strand synthesis was performed according to manufacturer’s instructions (Tiangen, Beijing, China). The qRT-PCR was performed using the SYBR green qRT-PCR kit according to instructions (Applied Biosystems, Warrington, UK). EF-1α was used as a reference gene [72]; all of the primer sequences are list in Table S8. Each pair of specific primers was set up with three technical replicates. The raw Ct values were used to calculate the relative level of expression following ΔCT (∆Ct equals the Ct value of each sample minus the reference gene Ct value in all samples).

5. Conclusions

In this study, systematic identification and characterization of 19 MrFTIPs revealed their evolutionary divergence and functional diversification in M. ruthenica, which are randomly allocated across 7 chromosomes. The protein physicochemical characteristics were analyzed, and they were determined to be hydrophilic proteins. Nineteen MrFTIPs belong to three groups according to different gene structures and specific motifs. The MrFTIP family has a high degree of collinearity with G.max, with 22 homologous gene pairs within the family, and are undergoing purification selection. Through the prediction of gene promoters, MrFTIPs contain cis-elements of stress response, phytohormones and light signaling. A total of seven MrFTIP members had high expression in the leaf, nine members had high expression in the petal and six members had high expression in the stigma and anthers. Eight MrFTIPs increased with leaf development, and eight MrFTIPs were upregulated in the salt-resistant genotype. MrFTIP17 continued the upregulated expression trend in the leaf, stem and root under salt stress; MrFTIP8 continued the upregulated expression trend in the leaf, stem and root under salt–alkali stress. This study provides a comprehensive genomic and bioinformatic foundation for understanding the evolution of MrFTIPs and identifies candidate genes for further functional studies in salt-stress responses.

Supplementary Materials

The following Supporting Information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms27041633/s1.

Author Contributions

Y.T. carried out the analyses and wrote the first manuscript. L.Z. designed the experiment and carried out the analyses and funding acquisition. M.G. revised the manuscript and carried out funding acquisition. Z.L. and Z.W. contributed to the interpretation of results and manuscript revision. H.L. collected the plant materials. X.L. participated in carrying out some experiments. X.W. conceived the experiments and revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

Hohhot Basic and Applied Basic Research Project (2025-Gui-Ji-51), Lin Zhu. Natural Science Foundation of Inner Mongolia (No. 2024QN03002), Lin Zhu. 2022 Annual Scientific Research Support for the Introduction of High-Level Talents by Autonomous Region-Level Public Institutions (2023NMRC001), Maowei Guo. Special Fund for Basic Scientific Research Business of Central-level Public Welfare Scientific Research Institutes (1610332022006), Maowei Guo.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data generated or analyzed during this study are included in this published article and its Supplementary information files. Further inquiries can be directed to the corresponding author.

Acknowledgments

Cultivar seeds were provided by Li Hongyan.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Localization of MrFTIPs on chromosomes. Chr1 to Chr8 represent the seven chromosomes of M. ruthenica. As no FTIP is mapped to Chr2, it is therefore not visualized in the figure.
Figure 1. Localization of MrFTIPs on chromosomes. Chr1 to Chr8 represent the seven chromosomes of M. ruthenica. As no FTIP is mapped to Chr2, it is therefore not visualized in the figure.
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Figure 2. Three-dimensional structure of MrFTIPs. SWISS-MODEL online software (https://swissmodel.expasy.org/interactive, accessed on 11 January 2025) was used for the three-dimensional modeling of MrFTIP proteins. Blue color characterizes a sequence identity > 70%, red color characterizes a sequence identity < 50%, orange color characterizes a sequence identity between 50 and 70%, and the gray surface represents the predicted transmembrane region.
Figure 2. Three-dimensional structure of MrFTIPs. SWISS-MODEL online software (https://swissmodel.expasy.org/interactive, accessed on 11 January 2025) was used for the three-dimensional modeling of MrFTIP proteins. Blue color characterizes a sequence identity > 70%, red color characterizes a sequence identity < 50%, orange color characterizes a sequence identity between 50 and 70%, and the gray surface represents the predicted transmembrane region.
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Figure 3. Phylogenetic analysis of MrFTIPs. Phylogenetic relationships among 19 MrFTIPs marked with pink pentagram stars, 14 FTIP proteins in M. truncatula marked with yellow triangles, 19 FTIP proteins in G. max marked with green circles, 17 FTIP proteins in A. thaliana marked with red circles, and 13 FTIP proteins in O. sativa marked with blue squares, Class I, class II, class III are represented by red, yellow and green, respectively, and each group has its own color. These FTIP proteins were divided into three classes and six subgroups using MEGA 11.
Figure 3. Phylogenetic analysis of MrFTIPs. Phylogenetic relationships among 19 MrFTIPs marked with pink pentagram stars, 14 FTIP proteins in M. truncatula marked with yellow triangles, 19 FTIP proteins in G. max marked with green circles, 17 FTIP proteins in A. thaliana marked with red circles, and 13 FTIP proteins in O. sativa marked with blue squares, Class I, class II, class III are represented by red, yellow and green, respectively, and each group has its own color. These FTIP proteins were divided into three classes and six subgroups using MEGA 11.
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Figure 4. Phylogenetic and structural analysis of MrFTIPs and encoded proteins in M. ruthenica. (A) The phylogenetic tree of MrFTIP encoding proteins. Orange squares represent exons and black lines represent introns. (B) Motif composition of MrFTIPs; different motifs are represented by different colors.
Figure 4. Phylogenetic and structural analysis of MrFTIPs and encoded proteins in M. ruthenica. (A) The phylogenetic tree of MrFTIP encoding proteins. Orange squares represent exons and black lines represent introns. (B) Motif composition of MrFTIPs; different motifs are represented by different colors.
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Figure 5. The number of simple sequence repeats (SSRs) among the MrFTIP family. The X-axis represents SSR types, while the Y-axis represents the number of each SSR type.
Figure 5. The number of simple sequence repeats (SSRs) among the MrFTIP family. The X-axis represents SSR types, while the Y-axis represents the number of each SSR type.
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Figure 6. Collinearity analysis: intraspecies collinearity analysis (A) and interspecies collinearity analysis of M. ruthenica with M. truncatula (B), A. thaliana (C), and G. max (D). Red lines connecting the homologous genes and gray lines represent the collinear blocks among M. ruthenica, M. truncatula, A. thaliana and G. max.
Figure 6. Collinearity analysis: intraspecies collinearity analysis (A) and interspecies collinearity analysis of M. ruthenica with M. truncatula (B), A. thaliana (C), and G. max (D). Red lines connecting the homologous genes and gray lines represent the collinear blocks among M. ruthenica, M. truncatula, A. thaliana and G. max.
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Figure 7. (A) Cis-acting elements in the promoters of MrFTIP family members. (B) Each row represents a cis-acting element, and each column represents a member. The numbers within corresponding squares denote the counts of cis-elements; the greater the number of elements, the color of the square is more inclined to red, otherwise, the color of the square is more inclined to white.
Figure 7. (A) Cis-acting elements in the promoters of MrFTIP family members. (B) Each row represents a cis-acting element, and each column represents a member. The numbers within corresponding squares denote the counts of cis-elements; the greater the number of elements, the color of the square is more inclined to red, otherwise, the color of the square is more inclined to white.
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Figure 8. Expression of nineteen MrFTIPs in different organs of M. ruthenica. Data shown are means ± SD (n = 3). Different letters indicate statistically significant differences (one-way ANOVA followed by post-hoc Tukey test; p < 0.05).
Figure 8. Expression of nineteen MrFTIPs in different organs of M. ruthenica. Data shown are means ± SD (n = 3). Different letters indicate statistically significant differences (one-way ANOVA followed by post-hoc Tukey test; p < 0.05).
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Figure 9. Expression of nineteen MrFTIPs in M. ruthenica during two developmental stages (seeding and flowing) and two genotypes (salt-resistant genotype and salt-sensitive genotype). Data shown are means ± SD (n = 3). Different letters indicate statistically significant differences (one-way ANOVA followed by post-hoc Tukey test; p < 0.05).
Figure 9. Expression of nineteen MrFTIPs in M. ruthenica during two developmental stages (seeding and flowing) and two genotypes (salt-resistant genotype and salt-sensitive genotype). Data shown are means ± SD (n = 3). Different letters indicate statistically significant differences (one-way ANOVA followed by post-hoc Tukey test; p < 0.05).
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Figure 10. Expression of nineteen MrFTIPs in two genotypes (salt-resistant genotype ‘Zhongcao No. 80’ and salt-sensitive genotype ‘Keerqinshardi’) of M. ruthenica. Data shown are means ± SD (n = 3). Different letters indicate statistically significant differences (one-way ANOVA followed by post-hoc Tukey test; p < 0.05).
Figure 10. Expression of nineteen MrFTIPs in two genotypes (salt-resistant genotype ‘Zhongcao No. 80’ and salt-sensitive genotype ‘Keerqinshardi’) of M. ruthenica. Data shown are means ± SD (n = 3). Different letters indicate statistically significant differences (one-way ANOVA followed by post-hoc Tukey test; p < 0.05).
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Figure 11. Expression of nineteen MrFTIPs in leaf, stem, and root under salt stress. Data shown are means ± SD (n = 3). Different letters indicate statistically significant differences (one-way ANOVA followed by post-hoc Tukey test; p < 0.05).
Figure 11. Expression of nineteen MrFTIPs in leaf, stem, and root under salt stress. Data shown are means ± SD (n = 3). Different letters indicate statistically significant differences (one-way ANOVA followed by post-hoc Tukey test; p < 0.05).
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Figure 12. Expression of nineteen MrFTIPs in leaf, stem, and root under saline–alkali stress. Data shown are means ± SD (n = 3). Different letters indicate statistically significant differences (one-way ANOVA followed by post-hoc Tukey test; p < 0.05).
Figure 12. Expression of nineteen MrFTIPs in leaf, stem, and root under saline–alkali stress. Data shown are means ± SD (n = 3). Different letters indicate statistically significant differences (one-way ANOVA followed by post-hoc Tukey test; p < 0.05).
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Table 1. Physicochemical properties of MrFTIPs.
Table 1. Physicochemical properties of MrFTIPs.
Gene Family IDGene IDCDS Length (bp)Protein Length
(AA)		Relative
Molecular Weight (Da)		Theoretical Isoelectric PointCoefficient
of Instability		Aliphatic Amino Acid CoefficientAverage Hydrophobic CoefficientSubcellular LocalizationProtein Molecular Formula
MrFTIP1MruG000634232577489,283.317.2341.9385.61−0.237plasC4064H6239N1073O1145S26
MrFTIP2MruG003292232577488,994.259.2047.8990.78−0.262plasC4025H6321N1105O1107S34
MrFTIP3MruG003300239479791,382.619.2440.0383.95−0.321plasC4158H6459N1115O1147S30
MrFTIP4MruG012825246982294,545.429.1447.8884.64−0.368plasC4233H6591N1179O1206S39
MrFTIP5MruG01948935851194137,040.378.8949.6881.73−0.488plasC6147H9596N1710O1777S36
MrFTIP6MruG01980231201039119,443.989.0045.8081.03−0.457plasC5369H8369N1489O1524S40
MrFTIP7MruG021463235878589,324.809.0544.6491.25−0.251chloC4019H6322N1104O1145S28
MrFTIP8MruG02600530241007115,002.289.0635.5888.30−0.268plasC5196H8094N1436O1450S35
MrFTIP9MruG031078229276387,690.477.2645.7994.01−0.070plasC3983H6175N1055O1104S37
MrFTIP10MruG0324792991996112,736.697.5448.3482.33−0.372plasC5040H7956N1370O1502S30
MrFTIP11MruG0324812955984111,192.976.8949.9583.04−0.378plasC4960H7854N1354O1484S31
MrFTIP12MruG03255230481015114,766.326.6645.3684.26−0.317plasC5176H8073N1399O1494S30
MrFTIP13MruG03661430571018116,048.319.2843.9081.04−0.375plasC5232H8161N1419O1491S39
MrFTIP14MruG037754232877589,451.819.2045.5390.37−0.260plasC4047H6344N1108O1111S36
MrFTIP15MruG03779059119622,874.098.3328.8574.03−0.465cytoC1034H1568N284O288S9
MrFTIP16MruG04081930181005114,506.379.0644.8377.79−0.392plasC5139H8001N1407O1471S46
MrFTIP17MruG04366031021033117,962.086.9649.2479.21−0.417plasC5302H8209N1459O1508S45
MrFTIP18MruG04604835851194137,040.378.8949.6881.73−0.488plasC6147H9596N1710O1777S36
MrFTIP19MruG047479229276387,654.487.2646.6294.90−0.061plasC3981H6179N1055O1103S37
Note: Plas denotes the plasma membrane, chlo signifies the chloroplast, and cyto represents the cytoplasm.
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MDPI and ACS Style

Tian, Y.; Zhu, L.; Guo, M.; Li, Z.; Wu, Z.; Li, H.; Li, X.; Wang, X. Genome-Wide Identification of the Medicago ruthenica FTIP Gene Family and Expression Profiling Under Salt Stresses. Int. J. Mol. Sci. 2026, 27, 1633. https://doi.org/10.3390/ijms27041633

AMA Style

Tian Y, Zhu L, Guo M, Li Z, Wu Z, Li H, Li X, Wang X. Genome-Wide Identification of the Medicago ruthenica FTIP Gene Family and Expression Profiling Under Salt Stresses. International Journal of Molecular Sciences. 2026; 27(4):1633. https://doi.org/10.3390/ijms27041633

Chicago/Turabian Style

Tian, Yonglei, Lin Zhu, Maowei Guo, Zhiyong Li, Zinian Wu, Hongyan Li, Xingyue Li, and Xiaolong Wang. 2026. "Genome-Wide Identification of the Medicago ruthenica FTIP Gene Family and Expression Profiling Under Salt Stresses" International Journal of Molecular Sciences 27, no. 4: 1633. https://doi.org/10.3390/ijms27041633

APA Style

Tian, Y., Zhu, L., Guo, M., Li, Z., Wu, Z., Li, H., Li, X., & Wang, X. (2026). Genome-Wide Identification of the Medicago ruthenica FTIP Gene Family and Expression Profiling Under Salt Stresses. International Journal of Molecular Sciences, 27(4), 1633. https://doi.org/10.3390/ijms27041633

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