Genome-wide identification of the EIN3/EIL transcription factor family and their responses under abiotic stresses in Alfalfa (Medicago sativa)

Genome-wide identification of the EIN3/EIL transcription factor family and their responses under abiotic stresses in Alfalfa (Medicago sativa) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genome-wide identification of the EIN3/EIL transcription factor family and their responses under abiotic stresses in Alfalfa (Medicago sativa) Xinru Su, Juan Wang, Shoujiang Sun, Wenxin Peng, Manli Li, Peisheng Mao, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4513747/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Sep, 2024 Read the published version in BMC Plant Biology → Version 1 posted 11 You are reading this latest preprint version Abstract Background Alfalfa ( Medicago sativa ) is known as the "king of forage" due to its high protein, mineral, carbohydrate, and digestive nutrient content. However, various abiotic stresses inhibit the growth and development of alfalfa, ultimately leading to a decrease in yield and quality. The ethylene-insensitive 3 (EIN3)/ethylene-insensitive 3-like (EIL) transcription factors are core regulators in plant ethylene signaling, playing important roles in plant development and response to abiotic stresses. However, a comprehensive genome-wide analysis of EIN3/EIL genes in alfalfa has not yet been conducted. Results In this study, we identified ten MsEIN3/EIL genes from the alfalfa (cv.Zhongmu No.1) genome, which were classified into four clades based on phylogenetic analysis. The motif 1, motif 2, motif 3, motif 4, and motif 9 of the MsEIN3/EIL genes constitute the conserved structural domains. Gene duplication analyses suggest that segmental duplication (SD) is a major driver of the expansion of the MsEIN3/EIL gene family during evolution. The analysis of the cis -acting elements in the promoter of MsEIN3/EIL genes showed their ability to respond to various hormones and stresses. The analysis of tissue expression revealed that group A and group C members were highly expressed in flowers and seeds, while group D members were highly expressed in roots and stems. Furthermore, RNA-Seq analysis demonstrated that the expression of MsEIN3/EIL genes were responsive to ABA treatment and different abiotic stresses (e.g., salt, cold, and drought stress). Conclusion This study investigated MsEIN3/EIL genes in alfalfa and identified three candidate MsEIN3/EIL transcription factors involved in the regulation of abiotic stresses. These findings will provide valuable insights into uncovering the molecular mechanisms underlying various stress responses in alfalfa. Alfalfa EIN3/EIL gene family Transcription factors Stress Expression profiling Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Ethylene (ET) is a gaseous plant hormone that play a crucial role in regulating various aspects of plant growth and development, including seed germination[ 1 – 3 ], seedling growth[ 4 ], leaf senescence[ 5 ], root development[ 6 ], flower senescence[ 7 ], and fruit ripening[ 8 ]. Nowadays, emerging research provides increasing evidence that ethylene is involved in plant responses to abiotic stresses such as high salinity[ 9 ], high temperature[ 10 ], drought[ 11 ], low temperature stress[ 12 ], flooding[ 13 ] and tissue damage[ 14 ]. To further understand the mechanism of action of ethylene, researchers have modelled a linear signal transduction pathway for ethylene response in the model plant Arabidopsis thaliana : ethylene molecules bind to the receptor to inactivate constitutive triple response 1 (CTR1) and fail to phosphorylate the ethylene-insensitive 2 (EIN2) protein. The C-terminal end of the EIN2 is cleaved off and translocated from endoplasmic reticulum to the nucleus, stabilizing EIN3/EIL family transcription factors and ultimately activating downstream ethylene signal responses [ 15 ]. In 1997, Chao et al. identified the EIN3 gene and five related EIN3-like genes in A.thaliana , of which AtEIN3 and AtEIL1 genes play important roles in salt and cold stress, and the AtEIL3 gene is a central transcriptional regulator of sulfur response and metabolism in A.thaliana [ 16 , 17 ]. Subsequent studies have shown that EIN3/EIL genes are not only crucial downstream regulators in the ethylene signalling pathway, but also important for crosstalk between various plant hormones[ 14 ]. Therefore, an in-depth understanding of the function of EIN3/EIL family is essential for elucidating the relationship between various signal transduction pathways and stress responses during plant development. The EIN3/EIL gene family is an important transcription factor gene family in higher plants, and plays an important role in plant growth and development[ 17 ]. EIN3/EIL proteins have been identified as transcription factors localized in the nucleus, and their structural features have been well studied in model plants[ 18 – 20 ]. EIN3/EIL transcription factors are nuclear-localized proteins with DNA-binding activity, and their protein sequences exhibit structural similarities across different species[ 21 ]. All identified EIN3/EIL homologous genes contain a conserved DNA-binding domain (DBD) that specifically binds to the EIN3 binding site in the promoter region of target genes[ 22 ]. The N-terminal amino acid sequence of EIN3/EIL proteins is highly conserved and contains several significant structural features, including an amino-terminal acidic domain (AD), a proline-rich region (PR), and a cluster of five small basic domains (BD I-V)[ 16 , 23 ]. In contrast, the C-terminal amino acid sequence is less conserved than the N-terminal sequence. Some EIN3/EIL genes in plants like A.thaliana have a unique poly-asparagine or poly-glutamine region at the C-terminal end, which is not commonly observed in other species such as tobacco and tomato[ 22 , 24 ]. Transcription factors (TFs) are DNA-binding proteins that specifically bind and interact with cis -acting elements in the promoter region to activate (or repress) gene transcription[ 25 ]. Transcription factor regulation, as the first step in the regulation of gene expression, is closely linked to molecular regulatory mechanisms in plant growth and development, as well as in response to environmental stresses[ 26 ]. Furthermore, the EIN3/EIL family genes, as central transcription factors in ethylene signaling, play an important role in ethylene regulation of abiotic stress. It’s reported that ethylene pretreatment or activation of EIN3 can enhance the salt tolerance of Arabidopsis. Further research has revealed that EIN3 can directly regulate the expression of POD (Peroxidases), enhance POD activity, and thus scavenge reactive oxygen species[ 27 ]. However, ethylene negatively regulates plant responses to freezing stress in Arabidopsis. Overexpression of EIN3 results in reduced freezing tolerance and the biochemical analysis indicates that EIN3 negatively regulates the expression of CBF (C-repeat Binding Factor) and type-A ARR (Arabidopsis Response Regulator) genes[ 28 ]. Further investigation is needed to understand the role of ethylene and EIN3/EIL genes in different abiotic stresses across various species. Alfalfa ( Medicago sativa ) is a widely cultivated perennial legume forage crop and ranks as the fourth largest cash crop following wheat, maize, and soybean, spanning 320,000 hectares[ 29 ]. Known as the "king of forage", alfalfa is highly regarded for its high protein, minerals, carbohydrates and digestive nutrient content, as well as its substantial biomass production[ 30 ]. However, the growth and development of alfalfa are constantly hindered by various environmental factors, leading to a decline in yield and quality. Improving alfalfa's resilience to these challenges is a key strategy for enhancing yield. Ethylene, as a gaseous plant hormone, is involved in regulating plant responses to abiotic stress, and the EIN3/EIL genes play a crucial role in this process. Therefore, it is essential to investigate the role of EIN3/EIL genes in alfalfa to gain insights into improving its stress tolerance. Currently, research on EIN3/EIL genes primarily focuses on model plants such as A.thaliana , Oryza sativa and Triticum aestivum [ 16 , 18 , 20 ]. However, there is a notable gap in knowledge regarding alfalfa, specifically whether EIN3/EIL genes play a role in various stress responses in this species. To address this gap, we identified ten MsEIN3/EIL genes within the alfalfa genome and conducted a comprehensive analysis of their physicochemical properties, gene structure, motif composition, chromosomal distribution, interspecies relationships, and cis -acting elements. Furthermore, we investigated the tissue-specific expression patterns of MsEIN3/EIL genes and their responses to three abiotic stresses. This study aims to deepen our understanding of the function of EIN3/EIL genes in alfalfa and provide valuable insights for future research on the involvement of MsEIN3/EIL genes in conferring resistance to abiotic stress. Results Identification of MsEIN3/EIL genes in alfalfa To identify MsEIN3/EIL gene members in alfalfa, we utilized the full-length protein sequences of EIN3/EIL genes from four species: A. thaliana (6), Medicago truncatula (10), Glycine max (12) and O. sativa (6), as queries for BLASTp search. Eleven putative MsEIN3/EIL genes were identified in the genome of alfalfa. Subsequently, ten hypothetical MsEIN3/EIL proteins were identified through a combination of Hidden Markov Model (HMM) profile and BLAST search results. Further analysis using the CDD database ( https://www.ncbi.nlm.nih.gov/cdd ) and InterPro website ( https://www.ebi.ac.uk/interpro/ ) led to the identification of ten MsEIN3/EIL genes with EIN3 domain (PF04873), which were named MsEIL1 - MsEIL10 according to their chromosomal order (Table S1 ). The physical and chemical properties of these genes were collected as shown in Table 1 . The length of the MsEIN3/EIL proteins ranged from 256 amino acids of MsEIL5 to 810 amino acids of MsEIL1, and the molecular weight varied from 28.7 kDa (MsEIL5) to 91.7 kDa (MsEIL1). Among these proteins, MsEIL3 had the lowest protein isoelectric point at 4.95, while MsEIL6 had the highest at 9.04. Subcellular localization prediction indicated that only the MsEIL5 protein was localized in the cytoplasm, whereas the remaining nine proteins were found in the nucleus. This suggests that the MsEIL5 gene might exhibit altered nucleoplasmic localization, potentially leading to distinct functions or activities within the nucleus. Table 1 Physicochemical properties of identified MsEIN3/EIL genes in alfalfa. Gene Name Gene ID Chr Amino Acids Molecular Weight (Da) Isoelectric Point Subcellular Localization Instability Index Average of Hydropathicity MsEIL1 MsG0280011087 Chr2 810 91682.22 6.77 Nucleus 61.58 -0.653 MsEIL2 MsG0380011801 Chr3 566 63911.38 5.01 Nucleus 47.3 -0.703 MsEIL3 MsG0380011928 Chr3 577 64885.43 4.95 Nucleus 47.3 -0.678 MsEIL4 MsG0380015861 Chr3 638 72458.81 5.91 Nucleus 47.83 -0.758 MsEIL5 MsG0580029523 Chr5 256 28702.52 5.28 Cytoplasm 54.81 -0.504 MsEIL6 MsG0680034184 Chr6 591 67376.84 9.04 Nucleus 45.13 -0.788 MsEIL7 MsG0680034186 Chr6 619 70770.16 8.97 Nucleus 53.09 -0.74 MsEIL8 MsG0680034200 Chr6 653 74096.42 8.96 Nucleus 42.16 -0.698 MsEIL9 MsG0680034213 Chr6 694 79272.84 8.97 Nucleus 46.75 -0.683 MsEIL10 MsG0880043875 Chr8 628 71288.97 6.41 Nucleus 45.13 -0.767 Chromosome distribution and phylogenetic analysis of MsEIN3/EIL genes To determine the chromosome distribution of the identified MsEIN3/EIL genes, a chromosome map of MsEIN3/EIL genes was constructed based on the alfalfa genome sequence, and they were located on five specific alfalfa chromosomes unevenly: Chr2, Chr3, Chr5, Chr6, and Chr8 (Fig. 1 A). Among these chromosomes, Chr2, Chr5, and Chr8 each contained one MsEIN3/EIL gene, while Chr3 contained three MsEIN3/EIL genes, representing 30.0% of the total MsEIN3/EIL genes. Chr6 contained the most MsEIN3/EIL genes, accounting for 40.0%. These findings imply the diversity and complexity of the members of MsEIN3/EIL genes. To further understand the evolutionary relationships of the MsEIN3/EIL proteins, we conducted a comparative analysis of EIN3/EIL proteins from M. truncatula , A. thaliana , and O. sativa . Then the neighbor-joining method and the JTT model in MEGA11 were used to construct a rootless phylogenetic tree. As shown in Fig. 1 B, the EIN3/EIL proteins were divided into four clades, designated as A, B, C, and D. Clade A contains AtEIN3, AtEIL1, AtEIL2, MsEIL4, MsEIL5 and MsEIL10 proteins. Clade B consists of AtEIL4 and AtEIL5 proteins. Clade C includes the AtEIL3 and MsEIL1 proteins, and Clade D contains MsEIL2, MsEIL3, MsEIL6, MsEIL7, MsEIL8 and MsEIL9 proteins. Therefore, it is hypothesized that MsEIL1 , MsEIL4 , MsEIL5 and MsEIL10 genes evolved from the AtEIN3/EIL genes, while the other genes may be new genes that arose from the MsEIN3/EIL genes in the course of species evolution. Gene structure, conserved motif and domain analysis of MsEIN3/EIL proteins Gene structure analysis is essential for understanding the relationship between evolution and the functional differentiation of gene families. To study the diversity of MsEIN3/EIL protein motifs, the online server MEME was used to analyze the conserved motifs of MsEIN3/EIL protein sequences. A total of ten unique conserved motifs were found in this analysis, named motif 1 to motif 10 (Table S2). Notably, motif 1, 3, and 4 were found to be universally present in all genes and located at the N-terminal region. This finding aligns with the reported conservation of the N-terminal region among members of EIN3/EIL genes in Arabidopsis (Fig. 2 A). Furthermore, motifs 5, 7, 8, and 10 were found to be exclusive to Clade D members, suggesting that these motifs may serve a unique function that distinguishes the role of these proteins from other MsEIN3/EIL proteins. In addition, most closely related MsEIN3/EIL exhibited a similar motif composition, suggesting that they may have functional redundancy. According to the NCBI CDD database, it was observed that the Clade D members and MsEIL4 protein belong to the EIN3 family, while the protein domains of the other members belong to the EIN3 superfamily (Fig. 2 B). To further explore the structural diversity of the identified MsEIN3/EIL genes, the exon-intron structures of these genes were analyzed. As shown in Fig. 2 C, the MsEIN3/EIL genes displayed a range of 0 to 9 introns, with similar clustering patterns. Among the ten genes, most genes contained two introns, including MsEIL2 , MsEIL3 , MsEIL6 , MsEIL7 , and MsEIL8 genes. In addition, MsEIL1 gene contained 9 introns, MsEIL9 and MsEIL5 genes contained 3 introns, while MsEIL10 and MsEIL4 genes have 1 and 0 intron, respectively. Gene duplication and synteny analysis of MsEIN3/EIL genes Furthermore, we conducted a thorough examination of potential gene duplication events and found a fragment duplication event involving two MsEIN3/EIL genes located on chromosome 3 (Fig. 3 , Table S3). The results showed that some genes of MsEIN3/EIL may be caused by gene duplication events, which are the main factors contributing to the amplification of the MsEIN3/EIL gene family. These findings reveal the genomic structure and evolutionary relationship of the MsEIN3/EIL genes in alfalfa, providing important insights into its potential functional significance in stress response and plant development. To gain a deeper understanding of the gene replication mechanism of the MsEIN3/EIL gene family, we conducted an analysis of the collinearity between the MsEIN3/EIL genes and various plant species, such as dicotyledonous plants like A.thaliana , M.truncatula , G.max , and monocotyledonous plants like O.sativa and Zea mays . The results revealed multiple homologous gene pairs between M.sativa and dicotyledonous plants ( A. thaliana , M. truncatula and G. max ), but no homologous pairs with monocotyledonous plants ( O. sativa and Z. mays ), providing insights into their evolutionary relationship (Fig. 4 ,Table S4-S6). The results showed that the MsEIN3/EIL genes in alfalfa underwent significant evolutionary divergence and were homologous in dicotyledons. Amino acid sequence alignment and secondary structure analysis of MsEIN3/EIL proteins To evaluate the similarity of the MsEIN3/EIL protein sequences of alfalfa, a multiple sequence alignment analysis was conducted on protein sequences of AtEIN3 protein and ten MsEIN3/EIL proteins. The analysis revealed a high degree of conservation in the EIN3/EIL protein sequences. The protein sequences of MsEIN3/EIL displayed characteristic structural features of the EIN3/EIL protein, including a completely conserved EIN3 domain at the N-terminus, an amino-terminal acidic domain (AD), a proline-rich region (PR), and five small basic domains (BD I-V). In addition, MsEIN3/EIL proteins have poly-asparagine regions and poly-glutamine regions near the C-terminus, which is similar to that observed in other plant species such as Arabidopsis and mung beans (Fig. 5). The N-terminal sequences of MsEIN3/EIL proteins is highly conserved, whereas the C-terminal sequences show little similarity, suggesting that the changes in MsEIN3/EIL members are mainly due to variations in the C-terminal sequences. The acidic amino acid enrichment region, proline and glutamate enrichment region are common transcriptional activation regions in plants, indicating that the acidic amino acid region, the basic amino acid region and the proline enrichment region are the transcriptional activation regions and functional regions of the EIN3/EIL gene family. Studying the secondary structure of proteins is essential for comprehending their function. Therefore, we conducted an in-depth analysis of the secondary structure of all MsEIN3/EIL proteins. Among ten MsEIN3/EIL proteins, random coils accounted for the largest proportion (37.96 ~ 59.72%), followed by α-helix (23.57 ~ 41.84%), extended strand (7.05 ~ 21.18%), and β-turn (1.88 ~ 6.85%) (Table 2 ). Figure 5 Sequence alignment of AtEIN3 protein and all identified MsEIN3/EIL proteins in Alfalfa. Sequences were aligned by ClustalX, and identical or similar residues were shaded as colors. Black rectangle covers the structural features. AD: acidic domain; BD I-V: basic domain I-V; PR: proline-rich region; ploy N/Q: poly asparagine / glutamine region. Table 2 The secondary structure of MsEIN3/EIL proteins Gene Name Gene ID α-Helix(%) Extended Strand(%) β-Turn(%) Random Coil(%) MsEIL1 MsG0280011087 36.3 13.95 4.07 45.68 MsEIL2 MsG0380011801 39.4 10.95 5.48 44.17 MsEIL3 MsG0380011928 34.84 9.88 4.16 51.13 MsEIL4 MsG0380015861 31.35 7.05 1.88 59.72 MsEIL5 MsG0580029523 37.5 9.38 5.47 47.66 MsEIL6 MsG0680034184 39.09 12.69 4.57 43.65 MsEIL7 MsG0680034186 41.84 14.54 5.65 37.96 MsEIL8 MsG0680034200 41.81 11.33 6.28 40.58 MsEIL9 MsG0680034213 41.64 13.26 5.62 39.48 MsEIL10 MsG0880043875 23.57 21.18 6.85 48.41 Promoter region cis -acting regulatory elements analysis The analysis of cis -acting regulatory elements identified fourteen major cis -acting elements in the MsEIN3/EIL gene promoter sequences (Fig. 6 , Table S7-8). Among all the identified MsEIN3/EIL genes promoter sequences, light-responsive cis -acting elements accounted for the largest proportion (57.2%) and were classified into the first category. Hormone-responsive cis -acting elements including auxin, abscisic acid, gibberellin, methyl jasmonate and salicylic acid were the second largest category (15.6%), Anaerobic induction cis -acting elements formed the third largest category (9.2%). Other categories, such as low-temperature elements, binding site related elements, plant developmental elements, defense stress elements and others, accounted for 18% of the total. All ten identified MsEIN3/EIL gene promoter sequences in alfalfa contained hormone-responsive cis -acting elements, suggesting that these genes may interact with other hormones to regulate plant growth. The promoter regions of all identified MsEIN3/EIL genes contained low-temperature response elements and anaerobic induction response elements. The promoter region of MsEIL1 gene contained defense stress response elements, while the promoter regions of seven MsEIN3/EIL genes contained MYB binding sites (MBS) associated with drought induction. The results of cis -acting elements indicate that MsEIN3/EIL genes can respond to various hormones and stresses, and these response elements may directly influence the stress response ability of MsEIN3/EIL genes under stressful conditions. Expression pattern of MsEIN3/EIL genes in tissues Tissue expression pattern analysis is important to understand the specific function of MsEIN3/EIL genes in different tissues of alfalfa. The transcript abundance profiles of the MsEIN3/EIL genes in six tissues, including leaves, flowers, post-elongated stems, elongated stems, roots, and seeds, were assessed using RNA-Seq data. The expression profiles were then visualized as a heat map using TBtools software to depict the expression patterns. The results showed that only MsEIL1 , MsEIL4 and MsEIL5 genes were expressed in the various tissues, while the remaining seven MsEIN3/EIL genes were not detected in the different tissues of alfalfa (Fig. 7 ,Table S9). To validate the RNA-Seq data, real-time quantitative PCR (RT-PCR) was performed on the MsEIN3/EIL genes. The results indicate that all MsEIN3/EIL genes were expressed in various tissues except for MsEIL2 and MsEIL9 genes. The expression patterns varied across different tissues. Specifically, the MsEIL4 gene showed high expression levels in flowers and seeds, while the MsEIL5 gene exhibited high expression in flowers. On the other hand, MsEIL10 gene, which belongs to the same group A as MsEIL4 and MsEIL5 genes, displayed elevated expression levels in roots and stems, suggesting that this gene may have undergone functional changes during the process of polyploidization. Additionally, the MsEIL1 gene, categorized under group C, was highly expressed in seeds. In contrast, the MsEIL3 MsEIL6 , MsEIL7 , and MsEIL8 genes, belonging to group D, demonstrated high expression levels in both roots and stems (Fig. 8 ). Expression profiles analysis of MsEIN3/EIL genes under stresses The analysis of cis -acting elements in MsEIN3/EIL genes showed that nearly all genes had elements responsive to cold, drought, and abscisic acid (ABA) (Table S8). To further investigate the expression levels of MsEIN3/EIL genes under abiotic stresses, we analyzed their expression patterns under cold stress, drought stress, salt stress, and ABA treatments using published transcriptomic data (Fig. 9 ). To ascertain the dynamic changes in the expression level of MsEIN3/EIL genes in response to cold and drought treatments, the transcriptome data of the alfalfa seedlings subjected to different durations of cold and drought treatments were analyzed. Interestingly, only half of the MsEIN3/EIL genes showed changes in expression levels. During the cold treatment, the expression levels of MsEIL1 , MsEIL2 , MsEIL4 and MsEIL5 genes were significantly increased after 2 h of treatment and subsequently decreased after 6 h of treatment (Fig. 8 A,Table S10). In contrast, the expression of MsEIN3/EIL genes exhibited an opposite trend during drought treatment. MsEIL1 , MsEIL4 , MsEIL5 and MsEIL6 genes were significantly down-regulated after 1 h of drought treatment, and then up-regulated after 6 h of treatment (Fig. 8 B,Table S11). This suggests that these genes may be involved in the response to low temperature and drought stress. In the transcriptome data of root tips treated with salt, we observed a significant down-regulation of the expression levels of MsEIL1 , MsEIL4 , MsEIL5 and MsEIL6 genes after 1 h of salt treatment, followed by an up-regulation after 3 h of treatment (Fig. 8 C,Table S12). In the transcriptome data of ABA-treated root tips, the expression levels of MsEIL1 , MsEIL2 , MsEIL4 and MsEIL5 genes were significantly increased after 1 h and subsequently decreased after 3 h of treatment (Fig. 8 D,Table S13). Comprehensive analysis of the data from the four treatments revealed that the expression trend of MsEIN3/EIL genes during cold treatment was similar to that during ABA treatment, while the expression trend of MsEIN3/EIL genes during drought treatment was similar to that during salt treatment. Consequently, it is hypothesized that the MsEIN3/EIL genes may respond to cold stress by regulating the ABA synthesis pathway, and the processes of MsEIN3/EIL genes responding to drought and salt stress may share similar characteristics. Discussion EIN3/EIL transcription factors play important roles in plant growth and development, participating in phytohormone signaling[ 31 ], sulfur metabolism[ 32 ], and regulating transcriptional responses to abiotic stresses in plants. The EIN3/EIL gene family is significant for activating downstream genes in the ethylene signaling pathway and initiating ethylene signaling transactivation[ 17 ]. Genome-wide characterization, evolutionary relationship analysis and molecular functional analysis of the EIN3/EIL gene family have been extensively characterized in many model crops, such as A. thaliana , O. sativa and T. aestivum . However, studies of the EIN3/EIL gene family in alfalfa have not been reported. Therefore, we conducted a comprehensive analysis including genome-wide identification, physicochemical properties, subcellular localization, phylogenetic tree construction, gene structure with conserved motifs, and cis -acting elements in alfalfa. Motif is a data-based mathematical statistical sequence model in biology that reflects the conserved nature of protein sequences[ 33 ]. The analysis revealed that motifs 1, 2, 3, 4, and 9 are shared by all MsEIN3/EIL genes, indicating their conservation within the gene family. Conserved structural domains are regions that remain unchanged across biological evolution or within a protein family, often serving crucial functions that are highly conserved[ 34 ]. We were intrigued to discover that motifs 1, 2, 3, 4, and 9 constitute the MsEIN3/EIL conserved structural domain. The distribution of exons and introns is a crucial structural feature that influences the evolutionary process of the gene. Genes with a comparable number of exons often share similar functions, highlighting the significance of this feature in understanding gene function and evolution. Upon analysis of intron-exon organization, it was found that most EIN3/EIL genes exhibit a low number of introns, which is consistent with the structural features of maize[ 19 ], cotton[ 35 ], and wheat[ 20 ]. The similar number of intron numbers in these species suggest that the EIN3/EIL genes are specific to each species. The high proportion of intronless genes indicates a potential occurrence of intron loss during the evolution of the EIN3/EIL genes. In addition, phylogenetic tree analysis demonstrated that MsEIL4 and MsEIL5 were clustered with AtEIN3 and AtEIL1 , MsEIL1 was clustered with AtEIL3 , and MsEIL10 was clustered with AtEIL2 . This not only indicates the relatedness between alfalfa and AtEIN3/EIL members, but also implies functional distinctions among them. The primary mechanism driving functional diversity and the emergence of new genes is gene duplication and subsequent divergence. Of the four genes clustered with AtEIN3 , AtEIL1 , MsEIL4 , and MsEIL5 on the phylogenetic tree, MsEIL4 and MsEIL5 are more likely to have evolved from AtEIN3 and AtEIL1 . It is possible that MsEIL1 originated from AtEIL3 , and MsEIL10 may have evolved from AtEIL2 . Conversely, the remaining genes may be novel genes that arose during the re-speciation of alfalfa EIN3/EIL genes (Fig. 1 B). Most EIN3/EIL genes with a low number of introns are likely the product of species evolution, and gene duplication is probably one of the main drivers of genome and genetic system evolution. Therefore, differences in the number of EIN3/EIL gene numbers among species are primarily a result of gene duplication events. In this study, only one pair of MsEIN3/EIL genes with evidence of gene duplication in alfalfa was identified, specifically through segmental duplication (SD)[ 36 ]. Moreover, gene duplication among different species is important for genome and genetic system evolution. A comparative analysis of homologous genes was conducted among dicotyledon and monocotyledon species, including A. thaliana , M. truncatula , G. max , O. sativa , and Z. mays . The study found that alfalfa shares homologous genes with dicotyledonous plants, but not with monocotyledonous plants. Among dicotyledonous plants, alfalfa has more homologous gene pairs with legumes than with A. thaliana . Within legumes, alfalfa displays more homologous gene pairs with G. max than with M. truncatula . These results suggest that the genetic background of alfalfa clover is closer to dicotyledons and more closely related to legumes. The tissue-specific expression of gene families provides insights into their potential functions during different developmental processes. The EIN3/EIL gene family is a common superfamily in plants, and its family member genes are expressed in almost all tissues of higher plants. However, there are differences in expression levels and expression patterns among various family members. In soybean, GmEIN3/EIL genes are expressed in roots, stems, leaves and flowers. The highest expression of GmEIL1 - GmEIL4 show the highest expression in roots, with lower expression in other tissues, while GmEIL8 - GmEIL12 exhibit lower levels expression in all tissues[ 37 ]. Similar expression patterns are observed in the alfalfa EIN3/EIL gene family. For instance, MsEIL4 and MsEIL5 genes exhibited the highest expression in flowers, and MsEIL1 , MsEIL3 , MsEIL6 , MsEIL7 , MsEIL8 and MsEIL10 genes exhibited the highest expression in roots and stems. Therefore, studying the tissue-specific expression patterns of MsEIN3/EIL genes in alfalfa, can provide a foundation for understanding their specific functions. Recent studies have demonstrated that the EIN3/EIL transcription factors play crucial roles in plant development, as well as in the regulation of phytohormones and stress responses. A cis -acting element analysis of the promoter region of MsEIN3/EIL genes revealed the presence of numerous hormone response elements, including abscisic acid, gibberellin, salicylic acid, jasmonic acid lactone, and auxin, as well as stress response elements, such as drought, low temperature, anaerobic, and defence stresses. This further confirms the important functions of the MsEIN3/EIL gene family in regulating plant growth and stress response. Furthermore, we investigated the responses of MsEIN3/EIL gene family members to cold, drought, salt stress and ABA treatments and analyzed their expression patterns. The majority of MsEIN3/EIL genes were induced, with significantly increased expression levels in alfalfa seedlings treated with exogenous ABA. This suggests that MsEIN3/EIL is involved in the response to ABA signaling pathway, similar to observations in poplar treated with ABA[ 38 ]. Under mannitol, salt and cold treatments, the MsEIN3/EIL genes were induced to varying degrees, with some differences. The expression levels of MsEIL1 , MsEIL4 and MsEIL5 genes in roots of alfalfa seedlings showed a transient down-regulation followed by a significant up-regulation after drought treatment. This was similar to the result of significant up-regulation of MnEILs gene expression in roots of mulberry after PEG treatment[ 39 ], indicating their active role in coping with drought stress. The expression levels of different MsEIN3/EIL genes were found to have consistent expression trends induced by salt stress. Specifically, the expression levels of MsEIL1 , MsEIL4 and MsEIL5 genes exhibited a down-regulation followed by up-regulation trend during salt stress treatment. Studies in Arabidopsis have shown that the germination rate of the ein3eil1 double mutant was significantly decreased under salt stress, while EIN3 overexpression plants exhibited increased germination rate and enhanced seedling resistance compared to the wild type[ 40 ]. And the EIN3 gene has been demonstrated to improve tolerance to salt stress by preventing the accumulation of reactive oxygen species (ROS)[ 41 ]. Since MsEIL4 gene is a homologue of the Arabidopsis AtEIN3 gene, it is likely that MsEIL4 gene also play an active role in salt stress. Finally, the relative expression of MsEIN3/EIL genes in roots was generally upregulated under cold stress conditions, consistent with the expression pattern of GmEIN3/EIL genes in the legume soybean[ 42 ]. Additional, it has been reported that both ein3-1 as well as ein3 eil1 double mutants exhibit enhanced frost tolerance, while EIN3 overexpression plants display increased sensitivity to cold stress[ 28 ]. The aforementioned studies collectively indicate that MsEIN3/EIL genes may be involved in the response to a range of abiotic stresses. However, the specific mechanisms of their actions still require further investigation. Conclusion In this study, we identified ten members of the MsEIN3/EIL gene family in the alfalfa genome, and found that most of the MsEIN3/EIL proteins were located in the nucleus and contained conserved EIN3/EIL structures. The distribution of MsEIN3/EIL genes on the alfalfa chromosomes was not uniform, with SD being the main driver of their evolution. Phylogenetic analysis of EIN3/EIL genes from different species revealed that the EIN3/EIL genes were classified into four clades, A, B, C, and D. Expression analysis showed that the MsEIN3/EIL gene family members exhibited diverse expression patterns in different tissues. Furthermore, MsEIN3/EIL genes responded to cold, drought, and salt stresses, as well as ABA treatments. The expression patterns of MsEIL1 , MsEIL4 , and MsEIL5 genes were particularly sensitive, exhibiting the high similarity to AtEIN3 , AtEIL1 , and AtEIL3 . In conclusion, our study provides important insights into the genome-wide characterization and expression patterns of the alfalfa EIN3/EIL gene family. These findings contribute to a better understanding of the role of these genes in response to abiotic stresses in alfalfa. Materials and Methods Plant materials Alfalfa (cv. Zhongmu No.1) seeds used in this experiment were provided by ‘Jiuquan Daye’ Seed Industry Co., Ltd. (harvested in 2022), and the plants were planted in the greenhouse of China Agricultural University. For RT- PCR, samples of root, post-elongated stem, elongated stem, leaf, and flower were collected at three weeks after seedling transplantation and growth. Each sample consisted of three biological replicates. After collection, the sample was quickly frozen and ground into a fine powder using liquid nitrogen to preserve its molecular integrity. Identification and Chromosome location of the EIN3/EIL genes in Alfalfa The genome sequence and annotation files of the Zhongmu No. 1 alfalfa variety were downloaded from the figshare website ( https://figshare.com/articles/dataset/Medicago_sativa_genome_and_annottion_ files). The EIN3/EIL protein sequences from A.thaliana , were acquired from the TAIR database ( http://www.arabidopsis.org/ ). The EIN3/EIL protein sequences of Glycine max, O.sativa and Medicago truncatula were obtained from Phytozome12 ( https://phytozome-next.jgi.doe.gov/ ). The 34 EIN3/EIL protein sequences were used as queries to search for possible EIN3/EIL proteins within the alfalfa genome using BLASTP (E-value ≤ 10 − 10 ) by TBtools software. To identify the EIN3/EIL genes in the alfalfa genome accurately, the Hidden Markov Model (HMM) file of the EIN3/EIL protein domain (PF04873) was downloaded from the Pfam database( http://pfam-legacy.xfam.org/ ), and then the sequences containing MsEIN3/EIL protein domains were retrieved from the genome of Zhongmu No.1 alfalfa using HMMER 3.0. The results were re-confirmed by NCBI-CDD ( https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi ) and InterPro ( https://www.ebi.ac.uk/interpro/search/sequence/ ) to determine that it contains the conserved domain specific to EIN3/EIL, excluding the sequence that does not contain the typical domain of EIN3/EIL proteins, and the remaining protein sequence is regarded as a member of the alfalfa EIN3/EIL gene family. To study the distribution of the MsEIN3/EIL genes on chromosomes, we obtained the location information from the gene annotation file of the alfalfa genome database. The chromosome location of the MsEIN3/EIL genes was extracted from the genome annotation file GFF3. Subsequently, the distribution of MsEIN3/EIL genes on chromosomes was visualized using TBtools software. The protein sequences of MsEIN3/EIL were analyzed using ExPASy-ProtParam ( https://web.expasy ) to predict their physical and chemical characteristics. Additionally, subcellular localization was predicted using the WoLFPSORT tool ( https://www.genscript.com/wolf-psort.html/ ). Phylogenetic analysis of EIN3/EIL genes family in Alfalfa and Arabidopsis To explore the evolutionary relationships of EIN3/EIL genes in alfalfa, a multiple sequence alignment of full-length EIN3/EIL family protein sequences was conducted using MUSCLE. The phylogenetic relationship was constructed based on 1000 bootstrap replicates in MEGA11, which utilized the neighbor-joining (NJ) method and the Jones-Taylor-Thornton (JTT) model. The phylogenetic tree was visualized and optimized using Evolview ( http://www.evolgenius.info/evolview/#/ ). Analysis of motifs, gene structures and conserved domains The exon/intron sites and length information of each EIN3/EIL gene were extracted from the gene annotation file GFF3 of the alfalfa genome database. The conserved protein motifs of the MsEIN3/EIL family genes were identified using the MEME tool ( https://meme-suite.org/meme/tools/meme ), with a maximum of ten motifs and default parameters. The NCBI conserved domain database (CDD) ( https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi ) was used to perform domain analysis and determine the type and location of all MsEIN3/EIL protein sequences. Additionally, TBtools software was used to visualize the exon-intron structure of the MsEIN3/EIL genes and the conserved motifs and structural architecture domains of the MsEIN3/EIL proteins. Gene duplication and syntenic analysis To investigate potential gene duplication events in the MsEIN3/EIL gene family, we identified homologous gene pairs and relationships among alfalfa MsEIN3/EIL family genes using the Multiple Collinearity Scanning Toolkit (MCScanX) software with default parameters. Additionally, we conducted a synergy analysis of EIN3/EIL genes in Zhongmu No.1 alfalfa with those in A.thaliana , M.truncatula , G.max , O.sativa , Z.mays . The duplication of the MsEIN3/EIL genes in alfalfa was visualized in TBtools using circular mapping. The homologous genetic relationship between the EIN3/EIL genes in alfalfa and other species was visualized using One Step MCScanX. Multiple alignment and Secondary structure of MsEIN3/EIL proteins The protein sequences of Arabidopsis AtEIN3 and alfalfa EIN3/EIL were compared using the MEGA11 software and visualized in DNAMAN software. The secondary structure of EIN3/EIL proteins was predicted using the online tool SOPMA ( https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa%20_sopma.html ). Analysis of the MsEIN3/EIL genes promoter in alfalfa To investigate the role of MsEIN3/EIL genes in plant responses to various stressors, we analyzed the cis -acting elements of these genes in detail. We extracted a 2000 bp nucleotide sequence upstream of the start codon of each MsEIN3/EIL gene from the Zhongmu No.1 genome sequence and submitted it to the PlantCARE database ( https://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ) for cis -acting element prediction. The regulatory functions of the predicted cis -acting elements were classified and visualized using TBtools software. Analysis of gene expression using RNA-seq datasets Transcriptome data were downloaded from NCBI public databases to investigate MsEIN3/EIL genes expression pattern in different tissues and abiotic stresses. RNA-Seq data from different tissues, including post-elongated stem, elongated stem, flower, leaf, root, and seed, were obtained from the CADL-Gene Expression Atlas database provided by the Noble Research Institute. The expression pattern of MsEIN3/EIL genes was analyzed under different conditions, including abiotic stress and ABA treatments, using RNA-Seq datasets. The raw RNA-seq data used in this study was obtained from the NCBI database (SRR7091780-SRR7091794; SRR7160313-SRR7160357). Abiotic stress material was generated in the following treatments: 1) Seedlings were collected at 0, 2, 6, 24, and 48 h under low temperature treatment at 4°C (three replicates per time point)[ 43 ]; 2) Seedlings were treated with 400 mM mannitol for 0, 1, 3, 6, 12, and 24 h (with three biological replicates per treatment time point), the root tip of each seedling was excised and collected[ 44 ]; 3) Seedlings were treated for 0, 1, 3, 6, 12 and 24 h (with three biological replicates per treatment time point) in 250 mM NaCl. The root tip of each seedling was excised and collected. The ABA treatments were as follows: Seedlings aged 12 days were treated for 0, 1, 3 and 12 h in a 1/2 MS nutrient solution containing 10 µM ABA (pH = 5.8), respectively. The root tips were collected after 0, 1, 3 and 12 hours of treatment[ 45 ]. The raw data were filtered and converted from SRA files to FASTQ files using the SRA to Fastq application of TBtools software. Finally, the gene expression values of MsEIN3/EIL genes under abiotic stress were calculated and normalized using TBtools software to draw heat maps. RNA extraction and RT-qPCR analysis Total RNA was extracted using the RNA extraction kit (Huayueyang Biotech Co., Ltd., Beijing, China). cDNA was synthesized for reverse transcription using the SuperMix for qPCR kit (TransGen Biotech, Beijing, China), following the manufacturer's instructions provided in the kit. Then, RT-qPCR was performed on a CFX96 Real-Time Detection System. RT-qPCR was performed using a cycling program consisting of an initial step at 95°C for 3 min, followed by 40 cycles of 95°C for 15 s and 60°C for 30 s. Data were calculated using the 2^ −ΔΔCt method for gene expression levels. The final values were calculated as the average of triplicate reactions. The Ct values of MsActin were used to normalize the Ct values for each gene. The list of primers used in this study is shown in Supplementary Table S14. Abbreviations SD Segmental duplication ET Ethylene DBD DNA-binding domain AD Amino-terminal acidic domain PR Proline-rich region BD I-V Basic domains I-V TFs Transcription factors UTR Untranslated regions RT-PCR Real-time quantitative PCR ABA Abscisic acid Declarations Authors' contributions DL and SX designed the project; SX and WJ performed the data analysis; SX, SS, PW and WJ interpreted the data and results; WJ was responsible for planting materials; SX wrote the manuscript; and DL, SS, LM, MP and WJ carried out thin and tall revisions. All authors read and approved the final manuscript. Funding This research was funded by the Key Technology Researches for Seed Propagation of Alfalfa with Saline and Alkaline Tolerance and Drought Resistance (2022ZD0401105) and Chinese Universities Scientific Fund (2024TC076). Availability of data and materials Data are contained within the article and Supplementary Materials. Raw sequencing data of the transcriptome used in the current study are available in the NCBI's Sequence Read Archive (SRA, https://www.ncbi.nlm.nih.gov/sra) under the BioProject PRJNA454564 and PRJNA450305. The genomic information of the Zhongmu No.1 alfalfa variety was retrieved from the figshare website (https://figshare.com/articles/dataset/Medicago_sativa_genome_and_annotation_files). The RNA-Seq data, downloaded from the Noble Research Institute database (https://www.alfal fatoo lbox.org), were used to evaluate the transcript abundance profiles of MsEIN3/EIL encoding genes across six tissues, namely, leaves, flowers, post-elongated stems, elongated stems, seeds and roots. Ethics approval and consent to participate Study complied with local and national regulations for using plants. Consent for publication Not applicable. Competing interests The authors declare no competing interests. References Naing AH, Xu J, Kim CK. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4513747","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":314958324,"identity":"dea2763f-bbe6-4a2f-9cee-e5fc0d2c1edb","order_by":0,"name":"Xinru Su","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xinru","middleName":"","lastName":"Su","suffix":""},{"id":314958325,"identity":"b4cf60e6-679a-4734-b8f8-341905db344b","order_by":1,"name":"Juan Wang","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"","lastName":"Wang","suffix":""},{"id":314958326,"identity":"5b0f3c86-74a9-4a69-9299-5ef268c7779e","order_by":2,"name":"Shoujiang Sun","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Shoujiang","middleName":"","lastName":"Sun","suffix":""},{"id":314958327,"identity":"4c4cc76a-f283-40f5-a044-0cefef7e4497","order_by":3,"name":"Wenxin Peng","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Wenxin","middleName":"","lastName":"Peng","suffix":""},{"id":314958328,"identity":"838d43d6-66d9-495d-8879-09867c6fc352","order_by":4,"name":"Manli Li","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Manli","middleName":"","lastName":"Li","suffix":""},{"id":314958329,"identity":"1b6bef10-a439-43f8-90f6-75af2c8f19b9","order_by":5,"name":"Peisheng Mao","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Peisheng","middleName":"","lastName":"Mao","suffix":""},{"id":314958330,"identity":"0a0072e5-2f84-4cd7-bdc3-7e1d57d276c7","order_by":6,"name":"Liru Dou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYDCCAwyMBxIYGOQgPDbitDCAtBiTqAVIJTYQrYXveI/BgYd7Dqf3t58xYPhQdpiBf3YDfi2SZ84YHEh4djh3xpkcA8YZ5w4zSNw5gF+LwY0coJYDh3M3MOQYMPO2HWYwkEggTku6Af8bA+a/pGhJMJAA2sJIjBbJM8cKgFrSDWfceFZwsOdcOo/EDQJa+I43b3z444C1PH9/8sYHP8qs5fhnENCCAg4AMQ8J6kfBKBgFo2AU4AIA5eVK0hYqbFwAAAAASUVORK5CYII=","orcid":"","institution":"China Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Liru","middleName":"","lastName":"Dou","suffix":""}],"badges":[],"createdAt":"2024-06-01 13:08:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4513747/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4513747/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-024-05588-2","type":"published","date":"2024-09-30T15:57:23+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":58602699,"identity":"0e095777-9e49-4cdb-a2d6-869b6a585137","added_by":"auto","created_at":"2024-06-18 18:30:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":31459,"visible":true,"origin":"","legend":"\u003cp\u003eChromosomal distribution of \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes and phylogenetic analysis of EIN3/EIL proteins. (\u003cstrong\u003eA\u003c/strong\u003e) The chromosomal locations of \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes. The long blue bars represent the chromosomes and the chromosome numbers are indicated on the top side of the bars. (\u003cstrong\u003eB\u003c/strong\u003e) The phylogenetic relationships of the EIN3/EIL proteins from \u003cem\u003eMedicago sativa\u003c/em\u003e, \u003cem\u003eMedicago truncatula\u003c/em\u003e, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and \u003cem\u003eOryza sativa\u003c/em\u003e. The phylogenetic tree was generated by the neighbor-joining method derived from Clustal X alignment and the four clades are shaded with different colors. The red stars represent \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes and the green stars represent \u003cem\u003eAtEIN3/EIL\u003c/em\u003egenes.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4513747/v1/6e975ecef9ecf6c393fdc3ea.png"},{"id":58602694,"identity":"18ddf22a-fb08-4247-a710-2c2057b26770","added_by":"auto","created_at":"2024-06-18 18:30:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":19237,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree, conserved motif, and gene structure of \u003cem\u003eMsEIN3/EIL\u003c/em\u003e gene family. (\u003cstrong\u003eA\u003c/strong\u003e) The conserved motif of MsEIN3/EIL proteins was analyzed using the MEME tool, and the results were visualized with TBtools. The motifs are labeled as 1–10 and represented by different colored boxes. (\u003cstrong\u003eB\u003c/strong\u003e) The conserved domain analysis of MsEIN3/EIL proteins shows different colors representing various conserved domains. (\u003cstrong\u003eC\u003c/strong\u003e)The gene structure of \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes was determined. The introns, exons, and untranslated regions (UTR) are represented by gray lines, green boxes, and yellow boxes, respectively.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4513747/v1/cf0ca7ca1d70c35e810efe01.png"},{"id":58602695,"identity":"888e2343-e03e-4adf-95f8-faa0fcb39142","added_by":"auto","created_at":"2024-06-18 18:30:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":56182,"visible":true,"origin":"","legend":"\u003cp\u003eCollinearity analysis of \u003cem\u003eMsEIN3/EIL\u003c/em\u003e gene family in alfalfa, the segmentally duplicated genes are connected by red lines, referring to the two genes highlighted in blue.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4513747/v1/1169f505238cfc770bc23a4b.png"},{"id":58603070,"identity":"33611acf-3592-42d4-827d-e7ffaf6add14","added_by":"auto","created_at":"2024-06-18 18:38:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":95305,"visible":true,"origin":"","legend":"\u003cp\u003eSyntenic analysis of \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes in \u003cem\u003eM. sativa\u003c/em\u003e compared with those in five plant species (\u003cem\u003eA. thaliana\u003c/em\u003e, \u003cem\u003eM. truncatula\u003c/em\u003e, \u003cem\u003eG. max\u003c/em\u003e, \u003cem\u003eO. sativa\u003c/em\u003e and \u003cem\u003eZ. mays\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4513747/v1/81f1a000eb7a48b930a0987f.png"},{"id":58602703,"identity":"b27cbc10-983e-4f0e-b785-d7936ab96f58","added_by":"auto","created_at":"2024-06-18 18:30:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":278017,"visible":true,"origin":"","legend":"\u003cp\u003eSequence alignment of AtEIN3 protein and all identified MsEIN3/EIL proteins in Alfalfa. Sequences were aligned by ClustalX, and identical or similar residues were shaded as colors. Black rectangle covers the structural features. AD: acidic domain; BD I-V: basic domain I-V; PR: proline-rich region; ploy N/Q: poly asparagine / glutamine region.\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4513747/v1/a98a8e46b3c8eb104e7710f0.png"},{"id":58602700,"identity":"14561d67-d628-4ce5-8824-9caa7b5274ab","added_by":"auto","created_at":"2024-06-18 18:30:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":21503,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eCis\u003c/em\u003e-acting elements in promoter region of \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes.\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4513747/v1/f65401c625e0dc3401caed02.png"},{"id":58603071,"identity":"5d04a691-3f66-4cd9-b4ee-0bf69731c914","added_by":"auto","created_at":"2024-06-18 18:38:23","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":12253,"visible":true,"origin":"","legend":"\u003cp\u003eExpression pattern of ten \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes in different tissues based on RNA-seq data.\u003c/p\u003e","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4513747/v1/2e129c7acf1fb161d28f6fee.png"},{"id":58602702,"identity":"d9225877-63e3-4304-a7ef-9c693b2116bc","added_by":"auto","created_at":"2024-06-18 18:30:23","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":50402,"visible":true,"origin":"","legend":"\u003cp\u003eTissue-specific expression of core \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes in alfalfa was analyzed through RT-qPCR, with the \u003cem\u003eMsActin\u003c/em\u003e gene serving as the internal standard. The data represent the means ± SEM for three independent experiments.\u003c/p\u003e","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4513747/v1/c23685d22ad9104e077b4249.png"},{"id":58602698,"identity":"7f563a91-052f-4c36-bd22-f11c283b83ea","added_by":"auto","created_at":"2024-06-18 18:30:23","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":43175,"visible":true,"origin":"","legend":"\u003cp\u003eExpression profiles of \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes in response to various abiotic stress treatments and ABA treatment, including cold (\u003cstrong\u003eA\u003c/strong\u003e), drought (\u003cstrong\u003eB\u003c/strong\u003e), salt (\u003cstrong\u003eC\u003c/strong\u003e) and ABA (\u003cstrong\u003eD\u003c/strong\u003e), are presented. (\u003cstrong\u003eA\u003c/strong\u003e) Seedlings were subjected to cold treatment at 4°C for varying durations. (\u003cstrong\u003eB\u003c/strong\u003e) Seedlings were treated with 400 mM mannitol for varying durations and root tips were harvested for subsequent experiments. (\u003cstrong\u003eC\u003c/strong\u003e) Seedlings were treated with 250 mM NaCl for varying durations and root tips were harvested for subsequent experiments. (\u003cstrong\u003eD\u003c/strong\u003e) Seedlings were treated with 10 μM ABA for varying durations and root tips were harvested for subsequent experiments.\u003c/p\u003e","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-4513747/v1/297033e771fa35db15c1c425.png"},{"id":66097066,"identity":"cc3a02a8-d8c2-4168-a9e8-acbf84cf420b","added_by":"auto","created_at":"2024-10-07 16:13:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1799233,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4513747/v1/1b515bbe-fb5c-4725-aad3-8b07f6817a39.pdf"},{"id":58602696,"identity":"bcddd06f-53f2-406d-9629-9f7e4158922a","added_by":"auto","created_at":"2024-06-18 18:30:23","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":989442,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4513747/v1/70e17a4ecb5343297c5e1ee0.xlsx"}],"financialInterests":"Competing interest reported. The authors declare no competing interests.","formattedTitle":"Genome-wide identification of the EIN3/EIL transcription factor family and their responses under abiotic stresses in Alfalfa (Medicago sativa)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEthylene (ET) is a gaseous plant hormone that play a crucial role in regulating various aspects of plant growth and development, including seed germination[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], seedling growth[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], leaf senescence[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], root development[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], flower senescence[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and fruit ripening[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Nowadays, emerging research provides increasing evidence that ethylene is involved in plant responses to abiotic stresses such as high salinity[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], high temperature[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], drought[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], low temperature stress[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], flooding[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and tissue damage[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. To further understand the mechanism of action of ethylene, researchers have modelled a linear signal transduction pathway for ethylene response in the model plant \u003cem\u003eArabidopsis thaliana\u003c/em\u003e: ethylene molecules bind to the receptor to inactivate constitutive triple response 1 (CTR1) and fail to phosphorylate the ethylene-insensitive 2 (EIN2) protein. The C-terminal end of the EIN2 is cleaved off and translocated from endoplasmic reticulum to the nucleus, stabilizing \u003cem\u003eEIN3/EIL\u003c/em\u003e family transcription factors and ultimately activating downstream ethylene signal responses [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In 1997, Chao et al. identified the \u003cem\u003eEIN3\u003c/em\u003e gene and five related \u003cem\u003eEIN3-like\u003c/em\u003e genes in \u003cem\u003eA.thaliana\u003c/em\u003e, of which \u003cem\u003eAtEIN3\u003c/em\u003e and \u003cem\u003eAtEIL1\u003c/em\u003e genes play important roles in salt and cold stress, and the \u003cem\u003eAtEIL3\u003c/em\u003e gene is a central transcriptional regulator of sulfur response and metabolism in \u003cem\u003eA.thaliana\u003c/em\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Subsequent studies have shown that \u003cem\u003eEIN3/EIL\u003c/em\u003e genes are not only crucial downstream regulators in the ethylene signalling pathway, but also important for crosstalk between various plant hormones[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Therefore, an in-depth understanding of the function of \u003cem\u003eEIN3/EIL\u003c/em\u003e family is essential for elucidating the relationship between various signal transduction pathways and stress responses during plant development.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eEIN3/EIL\u003c/em\u003e gene family is an important transcription factor gene family in higher plants, and plays an important role in plant growth and development[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. EIN3/EIL proteins have been identified as transcription factors localized in the nucleus, and their structural features have been well studied in model plants[\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. \u003cem\u003eEIN3/EIL\u003c/em\u003e transcription factors are nuclear-localized proteins with DNA-binding activity, and their protein sequences exhibit structural similarities across different species[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. All identified \u003cem\u003eEIN3/EIL\u003c/em\u003e homologous genes contain a conserved DNA-binding domain (DBD) that specifically binds to the EIN3 binding site in the promoter region of target genes[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The N-terminal amino acid sequence of EIN3/EIL proteins is highly conserved and contains several significant structural features, including an amino-terminal acidic domain (AD), a proline-rich region (PR), and a cluster of five small basic domains (BD I-V)[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In contrast, the C-terminal amino acid sequence is less conserved than the N-terminal sequence. Some \u003cem\u003eEIN3/EIL\u003c/em\u003e genes in plants like \u003cem\u003eA.thaliana\u003c/em\u003e have a unique poly-asparagine or poly-glutamine region at the C-terminal end, which is not commonly observed in other species such as tobacco and tomato[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTranscription factors (TFs) are DNA-binding proteins that specifically bind and interact with \u003cem\u003ecis\u003c/em\u003e-acting elements in the promoter region to activate (or repress) gene transcription[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Transcription factor regulation, as the first step in the regulation of gene expression, is closely linked to molecular regulatory mechanisms in plant growth and development, as well as in response to environmental stresses[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Furthermore, the \u003cem\u003eEIN3/EIL\u003c/em\u003e family genes, as central transcription factors in ethylene signaling, play an important role in ethylene regulation of abiotic stress. It\u0026rsquo;s reported that ethylene pretreatment or activation of EIN3 can enhance the salt tolerance of Arabidopsis. Further research has revealed that EIN3 can directly regulate the expression of \u003cem\u003ePOD\u003c/em\u003e (Peroxidases), enhance POD activity, and thus scavenge reactive oxygen species[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, ethylene negatively regulates plant responses to freezing stress in Arabidopsis. Overexpression of \u003cem\u003eEIN3\u003c/em\u003e results in reduced freezing tolerance and the biochemical analysis indicates that EIN3 negatively regulates the expression of \u003cem\u003eCBF\u003c/em\u003e (C-repeat Binding Factor) and type-A \u003cem\u003eARR\u003c/em\u003e (Arabidopsis Response Regulator) genes[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Further investigation is needed to understand the role of ethylene and \u003cem\u003eEIN3/EIL\u003c/em\u003e genes in different abiotic stresses across various species.\u003c/p\u003e \u003cp\u003eAlfalfa (\u003cem\u003eMedicago sativa\u003c/em\u003e) is a widely cultivated perennial legume forage crop and ranks as the fourth largest cash crop following wheat, maize, and soybean, spanning 320,000 hectares[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Known as the \"king of forage\", alfalfa is highly regarded for its high protein, minerals, carbohydrates and digestive nutrient content, as well as its substantial biomass production[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. However, the growth and development of alfalfa are constantly hindered by various environmental factors, leading to a decline in yield and quality. Improving alfalfa's resilience to these challenges is a key strategy for enhancing yield. Ethylene, as a gaseous plant hormone, is involved in regulating plant responses to abiotic stress, and the \u003cem\u003eEIN3/EIL\u003c/em\u003e genes play a crucial role in this process. Therefore, it is essential to investigate the role of \u003cem\u003eEIN3/EIL\u003c/em\u003e genes in alfalfa to gain insights into improving its stress tolerance.\u003c/p\u003e \u003cp\u003eCurrently, research on \u003cem\u003eEIN3/EIL\u003c/em\u003e genes primarily focuses on model plants such as \u003cem\u003eA.thaliana\u003c/em\u003e, \u003cem\u003eOryza sativa\u003c/em\u003e and \u003cem\u003eTriticum aestivum\u003c/em\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, there is a notable gap in knowledge regarding alfalfa, specifically whether \u003cem\u003eEIN3/EIL\u003c/em\u003e genes play a role in various stress responses in this species. To address this gap, we identified ten \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes within the alfalfa genome and conducted a comprehensive analysis of their physicochemical properties, gene structure, motif composition, chromosomal distribution, interspecies relationships, and \u003cem\u003ecis\u003c/em\u003e-acting elements. Furthermore, we investigated the tissue-specific expression patterns of \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes and their responses to three abiotic stresses. This study aims to deepen our understanding of the function of \u003cem\u003eEIN3/EIL\u003c/em\u003e genes in alfalfa and provide valuable insights for future research on the involvement of \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes in conferring resistance to abiotic stress.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eIdentification of\u003c/b\u003e \u003cb\u003eMsEIN3/EIL\u003c/b\u003e \u003cb\u003egenes in alfalfa\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo identify \u003cem\u003eMsEIN3/EIL\u003c/em\u003e gene members in alfalfa, we utilized the full-length protein sequences of \u003cem\u003eEIN3/EIL\u003c/em\u003e genes from four species: \u003cem\u003eA. thaliana\u003c/em\u003e (6), \u003cem\u003eMedicago truncatula\u003c/em\u003e (10), \u003cem\u003eGlycine max\u003c/em\u003e (12) and \u003cem\u003eO. sativa\u003c/em\u003e (6), as queries for BLASTp search. Eleven putative \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes were identified in the genome of alfalfa. Subsequently, ten hypothetical MsEIN3/EIL proteins were identified through a combination of Hidden Markov Model (HMM) profile and BLAST search results. Further analysis using the CDD database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/cdd\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/cdd\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and InterPro website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/interpro/\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/interpro/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) led to the identification of ten \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes with EIN3 domain (PF04873), which were named \u003cem\u003eMsEIL1\u003c/em\u003e-\u003cem\u003eMsEIL10\u003c/em\u003e according to their chromosomal order (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The physical and chemical properties of these genes were collected as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The length of the MsEIN3/EIL proteins ranged from 256 amino acids of MsEIL5 to 810 amino acids of MsEIL1, and the molecular weight varied from 28.7 kDa (MsEIL5) to 91.7 kDa (MsEIL1). Among these proteins, MsEIL3 had the lowest protein isoelectric point at 4.95, while MsEIL6 had the highest at 9.04. Subcellular localization prediction indicated that only the MsEIL5 protein was localized in the cytoplasm, whereas the remaining nine proteins were found in the nucleus. This suggests that the \u003cem\u003eMsEIL5\u003c/em\u003e gene might exhibit altered nucleoplasmic localization, potentially leading to distinct functions or activities within the nucleus.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhysicochemical properties of identified \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes in alfalfa.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGene ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChr\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAmino Acids\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMolecular Weight (Da)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIsoelectric Point\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSubcellular Localization\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eInstability Index\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eAverage of Hydropathicity\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMsEIL1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMsG0280011087\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChr2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e810\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e91682.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e61.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.653\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMsEIL2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMsG0380011801\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChr3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e566\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e63911.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e47.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.703\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMsEIL3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMsG0380011928\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChr3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e577\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e64885.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e47.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.678\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMsEIL4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMsG0380015861\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChr3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e638\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e72458.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e47.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.758\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMsEIL5\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMsG0580029523\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChr5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e28702.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCytoplasm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e54.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.504\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMsEIL6\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMsG0680034184\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChr6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e591\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e67376.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e9.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e45.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.788\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMsEIL7\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMsG0680034186\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChr6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e619\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e70770.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e8.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e53.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.74\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMsEIL8\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMsG0680034200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChr6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e653\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e74096.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e8.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e42.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.698\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMsEIL9\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMsG0680034213\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChr6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e694\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e79272.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e8.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e46.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.683\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMsEIL10\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMsG0880043875\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChr8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e628\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e71288.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNucleus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e45.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.767\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eChromosome distribution and phylogenetic analysis of\u003c/b\u003e \u003cb\u003eMsEIN3/EIL\u003c/b\u003e \u003cb\u003egenes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo determine the chromosome distribution of the identified \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes, a chromosome map of \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes was constructed based on the alfalfa genome sequence, and they were located on five specific alfalfa chromosomes unevenly: Chr2, Chr3, Chr5, Chr6, and Chr8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Among these chromosomes, Chr2, Chr5, and Chr8 each contained one \u003cem\u003eMsEIN3/EIL\u003c/em\u003e gene, while Chr3 contained three \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes, representing 30.0% of the total \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes. Chr6 contained the most \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes, accounting for 40.0%. These findings imply the diversity and complexity of the members of \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes. To further understand the evolutionary relationships of the MsEIN3/EIL proteins, we conducted a comparative analysis of EIN3/EIL proteins from \u003cem\u003eM. truncatula\u003c/em\u003e, \u003cem\u003eA. thaliana\u003c/em\u003e, and \u003cem\u003eO. sativa\u003c/em\u003e. Then the neighbor-joining method and the JTT model in MEGA11 were used to construct a rootless phylogenetic tree. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, the EIN3/EIL proteins were divided into four clades, designated as A, B, C, and D. Clade A contains AtEIN3, AtEIL1, AtEIL2, MsEIL4, MsEIL5 and MsEIL10 proteins. Clade B consists of AtEIL4 and AtEIL5 proteins. Clade C includes the AtEIL3 and MsEIL1 proteins, and Clade D contains MsEIL2, MsEIL3, MsEIL6, MsEIL7, MsEIL8 and MsEIL9 proteins. Therefore, it is hypothesized that \u003cem\u003eMsEIL1\u003c/em\u003e, \u003cem\u003eMsEIL4\u003c/em\u003e, \u003cem\u003eMsEIL5\u003c/em\u003e and \u003cem\u003eMsEIL10\u003c/em\u003e genes evolved from the \u003cem\u003eAtEIN3/EIL\u003c/em\u003e genes, while the other genes may be new genes that arose from the \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes in the course of species evolution.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGene structure, conserved motif and domain analysis of MsEIN3/EIL proteins\u003c/h2\u003e \u003cp\u003eGene structure analysis is essential for understanding the relationship between evolution and the functional differentiation of gene families. To study the diversity of MsEIN3/EIL protein motifs, the online server MEME was used to analyze the conserved motifs of MsEIN3/EIL protein sequences. A total of ten unique conserved motifs were found in this analysis, named motif 1 to motif 10 (Table S2). Notably, motif 1, 3, and 4 were found to be universally present in all genes and located at the N-terminal region. This finding aligns with the reported conservation of the N-terminal region among members of \u003cem\u003eEIN3/EIL\u003c/em\u003e genes in Arabidopsis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Furthermore, motifs 5, 7, 8, and 10 were found to be exclusive to Clade D members, suggesting that these motifs may serve a unique function that distinguishes the role of these proteins from other MsEIN3/EIL proteins. In addition, most closely related MsEIN3/EIL exhibited a similar motif composition, suggesting that they may have functional redundancy.\u003c/p\u003e \u003cp\u003eAccording to the NCBI CDD database, it was observed that the Clade D members and MsEIL4 protein belong to the EIN3 family, while the protein domains of the other members belong to the EIN3 superfamily (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). To further explore the structural diversity of the identified \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes, the exon-intron structures of these genes were analyzed. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, the \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes displayed a range of 0 to 9 introns, with similar clustering patterns. Among the ten genes, most genes contained two introns, including \u003cem\u003eMsEIL2\u003c/em\u003e, \u003cem\u003eMsEIL3\u003c/em\u003e, \u003cem\u003eMsEIL6\u003c/em\u003e, \u003cem\u003eMsEIL7\u003c/em\u003e, and \u003cem\u003eMsEIL8\u003c/em\u003e genes. In addition, \u003cem\u003eMsEIL1\u003c/em\u003e gene contained 9 introns, \u003cem\u003eMsEIL9\u003c/em\u003e and \u003cem\u003eMsEIL5\u003c/em\u003e genes contained 3 introns, while \u003cem\u003eMsEIL10\u003c/em\u003e and \u003cem\u003eMsEIL4\u003c/em\u003e genes have 1 and 0 intron, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGene duplication and synteny analysis of\u003c/b\u003e \u003cb\u003eMsEIN3/EIL\u003c/b\u003e \u003cb\u003egenes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFurthermore, we conducted a thorough examination of potential gene duplication events and found a fragment duplication event involving two \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes located on chromosome 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Table S3). The results showed that some genes of \u003cem\u003eMsEIN3/EIL\u003c/em\u003e may be caused by gene duplication events, which are the main factors contributing to the amplification of the \u003cem\u003eMsEIN3/EIL\u003c/em\u003e gene family. These findings reveal the genomic structure and evolutionary relationship of the \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes in alfalfa, providing important insights into its potential functional significance in stress response and plant development.\u003c/p\u003e \u003cp\u003eTo gain a deeper understanding of the gene replication mechanism of the \u003cem\u003eMsEIN3/EIL\u003c/em\u003e gene family, we conducted an analysis of the collinearity between the \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes and various plant species, such as dicotyledonous plants like \u003cem\u003eA.thaliana\u003c/em\u003e, \u003cem\u003eM.truncatula\u003c/em\u003e, \u003cem\u003eG.max\u003c/em\u003e, and monocotyledonous plants like \u003cem\u003eO.sativa\u003c/em\u003e and \u003cem\u003eZea mays\u003c/em\u003e. The results revealed multiple homologous gene pairs between \u003cem\u003eM.sativa\u003c/em\u003e and dicotyledonous plants (\u003cem\u003eA. thaliana\u003c/em\u003e, \u003cem\u003eM. truncatula\u003c/em\u003e and \u003cem\u003eG. max\u003c/em\u003e), but no homologous pairs with monocotyledonous plants (\u003cem\u003eO. sativa\u003c/em\u003e and \u003cem\u003eZ. mays\u003c/em\u003e), providing insights into their evolutionary relationship (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e,Table S4-S6). The results showed that the \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes in alfalfa underwent significant evolutionary divergence and were homologous in dicotyledons.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAmino acid sequence alignment and secondary structure analysis of MsEIN3/EIL proteins\u003c/h2\u003e \u003cp\u003eTo evaluate the similarity of the MsEIN3/EIL protein sequences of alfalfa, a multiple sequence alignment analysis was conducted on protein sequences of AtEIN3 protein and ten MsEIN3/EIL proteins. The analysis revealed a high degree of conservation in the EIN3/EIL protein sequences. The protein sequences of MsEIN3/EIL displayed characteristic structural features of the EIN3/EIL protein, including a completely conserved EIN3 domain at the N-terminus, an amino-terminal acidic domain (AD), a proline-rich region (PR), and five small basic domains (BD I-V). In addition, MsEIN3/EIL proteins have poly-asparagine regions and poly-glutamine regions near the C-terminus, which is similar to that observed in other plant species such as Arabidopsis and mung beans (Fig.\u0026nbsp;5). The N-terminal sequences of MsEIN3/EIL proteins is highly conserved, whereas the C-terminal sequences show little similarity, suggesting that the changes in MsEIN3/EIL members are mainly due to variations in the C-terminal sequences. The acidic amino acid enrichment region, proline and glutamate enrichment region are common transcriptional activation regions in plants, indicating that the acidic amino acid region, the basic amino acid region and the proline enrichment region are the transcriptional activation regions and functional regions of the \u003cem\u003eEIN3/EIL\u003c/em\u003e gene family.\u003c/p\u003e \u003cp\u003e Studying the secondary structure of proteins is essential for comprehending their function. Therefore, we conducted an in-depth analysis of the secondary structure of all MsEIN3/EIL proteins. Among ten MsEIN3/EIL proteins, random coils accounted for the largest proportion (37.96\u0026thinsp;~\u0026thinsp;59.72%), followed by α-helix (23.57\u0026thinsp;~\u0026thinsp;41.84%), extended strand (7.05\u0026thinsp;~\u0026thinsp;21.18%), and β-turn (1.88\u0026thinsp;~\u0026thinsp;6.85%) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;5\u003c/b\u003e Sequence alignment of AtEIN3 protein and all identified MsEIN3/EIL proteins in Alfalfa. Sequences were aligned by ClustalX, and identical or similar residues were shaded as colors. Black rectangle covers the structural features. AD: acidic domain; BD I-V: basic domain I-V; PR: proline-rich region; ploy N/Q: poly asparagine / glutamine region.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe secondary structure of MsEIN3/EIL proteins\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGene ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eα-Helix(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eExtended Strand(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eβ-Turn(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRandom Coil(%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMsEIL1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMsG0280011087\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e36.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e45.68\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMsEIL2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMsG0380011801\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e39.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e44.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMsEIL3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMsG0380011928\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e34.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e51.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMsEIL4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMsG0380015861\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e31.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e59.72\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMsEIL5\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMsG0580029523\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e37.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e47.66\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMsEIL6\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMsG0680034184\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e39.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e43.65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMsEIL7\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMsG0680034186\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e41.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e14.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e37.96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMsEIL8\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMsG0680034200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e41.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e40.58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMsEIL9\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMsG0680034213\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e41.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e39.48\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMsEIL10\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMsG0880043875\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e23.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e21.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e48.41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003cb\u003ePromoter region\u003c/b\u003e \u003cb\u003ecis\u003c/b\u003e\u003cb\u003e-acting regulatory elements analysis\u003c/b\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe analysis of \u003cem\u003ecis\u003c/em\u003e-acting regulatory elements identified fourteen major \u003cem\u003ecis\u003c/em\u003e-acting elements in the \u003cem\u003eMsEIN3/EIL\u003c/em\u003e gene promoter sequences (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e, Table S7-8). Among all the identified \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes promoter sequences, light-responsive \u003cem\u003ecis\u003c/em\u003e-acting elements accounted for the largest proportion (57.2%) and were classified into the first category. Hormone-responsive \u003cem\u003ecis\u003c/em\u003e-acting elements including auxin, abscisic acid, gibberellin, methyl jasmonate and salicylic acid were the second largest category (15.6%), Anaerobic induction \u003cem\u003ecis\u003c/em\u003e-acting elements formed the third largest category (9.2%). Other categories, such as low-temperature elements, binding site related elements, plant developmental elements, defense stress elements and others, accounted for 18% of the total. All ten identified \u003cem\u003eMsEIN3/EIL\u003c/em\u003e gene promoter sequences in alfalfa contained hormone-responsive \u003cem\u003ecis\u003c/em\u003e-acting elements, suggesting that these genes may interact with other hormones to regulate plant growth. The promoter regions of all identified \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes contained low-temperature response elements and anaerobic induction response elements. The promoter region of \u003cem\u003eMsEIL1\u003c/em\u003e gene contained defense stress response elements, while the promoter regions of seven \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes contained MYB binding sites (MBS) associated with drought induction. The results of \u003cem\u003ecis\u003c/em\u003e-acting elements indicate that \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes can respond to various hormones and stresses, and these response elements may directly influence the stress response ability of \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes under stressful conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression pattern of\u003c/b\u003e \u003cb\u003eMsEIN3/EIL\u003c/b\u003e \u003cb\u003egenes in tissues\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTissue expression pattern analysis is important to understand the specific function of \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes in different tissues of alfalfa. The transcript abundance profiles of the \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes in six tissues, including leaves, flowers, post-elongated stems, elongated stems, roots, and seeds, were assessed using RNA-Seq data. The expression profiles were then visualized as a heat map using TBtools software to depict the expression patterns. The results showed that only \u003cem\u003eMsEIL1\u003c/em\u003e, \u003cem\u003eMsEIL4\u003c/em\u003e and \u003cem\u003eMsEIL5\u003c/em\u003e genes were expressed in the various tissues, while the remaining seven \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes were not detected in the different tissues of alfalfa (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003e,Table S9).\u003c/p\u003e \u003cp\u003eTo validate the RNA-Seq data, real-time quantitative PCR (RT-PCR) was performed on the \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes. The results indicate that all \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes were expressed in various tissues except for \u003cem\u003eMsEIL2\u003c/em\u003e and \u003cem\u003eMsEIL9\u003c/em\u003e genes. The expression patterns varied across different tissues. Specifically, the \u003cem\u003eMsEIL4\u003c/em\u003e gene showed high expression levels in flowers and seeds, while the \u003cem\u003eMsEIL5\u003c/em\u003e gene exhibited high expression in flowers. On the other hand, \u003cem\u003eMsEIL10\u003c/em\u003e gene, which belongs to the same group A as \u003cem\u003eMsEIL4\u003c/em\u003e and \u003cem\u003eMsEIL5\u003c/em\u003e genes, displayed elevated expression levels in roots and stems, suggesting that this gene may have undergone functional changes during the process of polyploidization. Additionally, the \u003cem\u003eMsEIL1\u003c/em\u003e gene, categorized under group C, was highly expressed in seeds. In contrast, the \u003cem\u003eMsEIL3 MsEIL6\u003c/em\u003e, \u003cem\u003eMsEIL7\u003c/em\u003e, and \u003cem\u003eMsEIL8\u003c/em\u003e genes, belonging to group D, demonstrated high expression levels in both roots and stems (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression profiles analysis of\u003c/b\u003e \u003cb\u003eMsEIN3/EIL\u003c/b\u003e \u003cb\u003egenes under stresses\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe analysis of \u003cem\u003ecis\u003c/em\u003e-acting elements in \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes showed that nearly all genes had elements responsive to cold, drought, and abscisic acid (ABA) (Table S8). To further investigate the expression levels of \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes under abiotic stresses, we analyzed their expression patterns under cold stress, drought stress, salt stress, and ABA treatments using published transcriptomic data (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003e). To ascertain the dynamic changes in the expression level of \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes in response to cold and drought treatments, the transcriptome data of the alfalfa seedlings subjected to different durations of cold and drought treatments were analyzed. Interestingly, only half of the \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes showed changes in expression levels. During the cold treatment, the expression levels of \u003cem\u003eMsEIL1\u003c/em\u003e, \u003cem\u003eMsEIL2\u003c/em\u003e, \u003cem\u003eMsEIL4\u003c/em\u003e and \u003cem\u003eMsEIL5\u003c/em\u003e genes were significantly increased after 2 h of treatment and subsequently decreased after 6 h of treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eA,Table S10). In contrast, the expression of \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes exhibited an opposite trend during drought treatment. \u003cem\u003eMsEIL1\u003c/em\u003e, \u003cem\u003eMsEIL4\u003c/em\u003e, \u003cem\u003eMsEIL5\u003c/em\u003e and \u003cem\u003eMsEIL6\u003c/em\u003e genes were significantly down-regulated after 1 h of drought treatment, and then up-regulated after 6 h of treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eB,Table S11). This suggests that these genes may be involved in the response to low temperature and drought stress.\u003c/p\u003e \u003cp\u003eIn the transcriptome data of root tips treated with salt, we observed a significant down-regulation of the expression levels of \u003cem\u003eMsEIL1\u003c/em\u003e, \u003cem\u003eMsEIL4\u003c/em\u003e, \u003cem\u003eMsEIL5\u003c/em\u003e and \u003cem\u003eMsEIL6\u003c/em\u003e genes after 1 h of salt treatment, followed by an up-regulation after 3 h of treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eC,Table S12). In the transcriptome data of ABA-treated root tips, the expression levels of \u003cem\u003eMsEIL1\u003c/em\u003e, \u003cem\u003eMsEIL2\u003c/em\u003e, \u003cem\u003eMsEIL4\u003c/em\u003e and \u003cem\u003eMsEIL5\u003c/em\u003e genes were significantly increased after 1 h and subsequently decreased after 3 h of treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eD,Table S13). Comprehensive analysis of the data from the four treatments revealed that the expression trend of \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes during cold treatment was similar to that during ABA treatment, while the expression trend of \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes during drought treatment was similar to that during salt treatment. Consequently, it is hypothesized that the \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes may respond to cold stress by regulating the ABA synthesis pathway, and the processes of \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes responding to drought and salt stress may share similar characteristics.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003eEIN3/EIL\u003c/em\u003e transcription factors play important roles in plant growth and development, participating in phytohormone signaling[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], sulfur metabolism[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], and regulating transcriptional responses to abiotic stresses in plants. The \u003cem\u003eEIN3/EIL\u003c/em\u003e gene family is significant for activating downstream genes in the ethylene signaling pathway and initiating ethylene signaling transactivation[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Genome-wide characterization, evolutionary relationship analysis and molecular functional analysis of the \u003cem\u003eEIN3/EIL\u003c/em\u003e gene family have been extensively characterized in many model crops, such as \u003cem\u003eA. thaliana\u003c/em\u003e, \u003cem\u003eO. sativa\u003c/em\u003e and \u003cem\u003eT. aestivum\u003c/em\u003e. However, studies of the \u003cem\u003eEIN3/EIL\u003c/em\u003e gene family in alfalfa have not been reported. Therefore, we conducted a comprehensive analysis including genome-wide identification, physicochemical properties, subcellular localization, phylogenetic tree construction, gene structure with conserved motifs, and \u003cem\u003ecis\u003c/em\u003e-acting elements in alfalfa.\u003c/p\u003e \u003cp\u003eMotif is a data-based mathematical statistical sequence model in biology that reflects the conserved nature of protein sequences[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The analysis revealed that motifs 1, 2, 3, 4, and 9 are shared by all \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes, indicating their conservation within the gene family. Conserved structural domains are regions that remain unchanged across biological evolution or within a protein family, often serving crucial functions that are highly conserved[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. We were intrigued to discover that motifs 1, 2, 3, 4, and 9 constitute the MsEIN3/EIL conserved structural domain. The distribution of exons and introns is a crucial structural feature that influences the evolutionary process of the gene. Genes with a comparable number of exons often share similar functions, highlighting the significance of this feature in understanding gene function and evolution. Upon analysis of intron-exon organization, it was found that most \u003cem\u003eEIN3/EIL\u003c/em\u003e genes exhibit a low number of introns, which is consistent with the structural features of maize[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], cotton[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], and wheat[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The similar number of intron numbers in these species suggest that the \u003cem\u003eEIN3/EIL\u003c/em\u003e genes are specific to each species. The high proportion of intronless genes indicates a potential occurrence of intron loss during the evolution of the \u003cem\u003eEIN3/EIL\u003c/em\u003e genes. In addition, phylogenetic tree analysis demonstrated that \u003cem\u003eMsEIL4\u003c/em\u003e and \u003cem\u003eMsEIL5\u003c/em\u003e were clustered with \u003cem\u003eAtEIN3\u003c/em\u003e and \u003cem\u003eAtEIL1\u003c/em\u003e, \u003cem\u003eMsEIL1\u003c/em\u003e was clustered with \u003cem\u003eAtEIL3\u003c/em\u003e, and \u003cem\u003eMsEIL10\u003c/em\u003e was clustered with \u003cem\u003eAtEIL2\u003c/em\u003e. This not only indicates the relatedness between alfalfa and AtEIN3/EIL members, but also implies functional distinctions among them. The primary mechanism driving functional diversity and the emergence of new genes is gene duplication and subsequent divergence. Of the four genes clustered with \u003cem\u003eAtEIN3\u003c/em\u003e, \u003cem\u003eAtEIL1\u003c/em\u003e, \u003cem\u003eMsEIL4\u003c/em\u003e, and \u003cem\u003eMsEIL5\u003c/em\u003e on the phylogenetic tree, \u003cem\u003eMsEIL4\u003c/em\u003e and \u003cem\u003eMsEIL5\u003c/em\u003e are more likely to have evolved from \u003cem\u003eAtEIN3\u003c/em\u003e and \u003cem\u003eAtEIL1\u003c/em\u003e. It is possible that \u003cem\u003eMsEIL1\u003c/em\u003e originated from \u003cem\u003eAtEIL3\u003c/em\u003e, and \u003cem\u003eMsEIL10\u003c/em\u003e may have evolved from \u003cem\u003eAtEIL2\u003c/em\u003e. Conversely, the remaining genes may be novel genes that arose during the re-speciation of alfalfa \u003cem\u003eEIN3/EIL\u003c/em\u003e genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eMost \u003cem\u003eEIN3/EIL\u003c/em\u003e genes with a low number of introns are likely the product of species evolution, and gene duplication is probably one of the main drivers of genome and genetic system evolution. Therefore, differences in the number of \u003cem\u003eEIN3/EIL\u003c/em\u003e gene numbers among species are primarily a result of gene duplication events. In this study, only one pair of \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes with evidence of gene duplication in alfalfa was identified, specifically through segmental duplication (SD)[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Moreover, gene duplication among different species is important for genome and genetic system evolution. A comparative analysis of homologous genes was conducted among dicotyledon and monocotyledon species, including \u003cem\u003eA. thaliana\u003c/em\u003e, \u003cem\u003eM. truncatula\u003c/em\u003e, \u003cem\u003eG. max\u003c/em\u003e, \u003cem\u003eO. sativa\u003c/em\u003e, and \u003cem\u003eZ. mays\u003c/em\u003e. The study found that alfalfa shares homologous genes with dicotyledonous plants, but not with monocotyledonous plants. Among dicotyledonous plants, alfalfa has more homologous gene pairs with legumes than with \u003cem\u003eA. thaliana\u003c/em\u003e. Within legumes, alfalfa displays more homologous gene pairs with \u003cem\u003eG. max\u003c/em\u003e than with \u003cem\u003eM. truncatula\u003c/em\u003e. These results suggest that the genetic background of alfalfa clover is closer to dicotyledons and more closely related to legumes.\u003c/p\u003e \u003cp\u003eThe tissue-specific expression of gene families provides insights into their potential functions during different developmental processes. The \u003cem\u003eEIN3/EIL\u003c/em\u003e gene family is a common superfamily in plants, and its family member genes are expressed in almost all tissues of higher plants. However, there are differences in expression levels and expression patterns among various family members. In soybean, \u003cem\u003eGmEIN3/EIL\u003c/em\u003e genes are expressed in roots, stems, leaves and flowers. The highest expression of \u003cem\u003eGmEIL1\u003c/em\u003e-\u003cem\u003eGmEIL4\u003c/em\u003e show the highest expression in roots, with lower expression in other tissues, while \u003cem\u003eGmEIL8\u003c/em\u003e-\u003cem\u003eGmEIL12\u003c/em\u003e exhibit lower levels expression in all tissues[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Similar expression patterns are observed in the alfalfa \u003cem\u003eEIN3/EIL\u003c/em\u003e gene family. For instance, \u003cem\u003eMsEIL4\u003c/em\u003e and \u003cem\u003eMsEIL5\u003c/em\u003e genes exhibited the highest expression in flowers, and \u003cem\u003eMsEIL1\u003c/em\u003e, \u003cem\u003eMsEIL3\u003c/em\u003e, \u003cem\u003eMsEIL6\u003c/em\u003e, \u003cem\u003eMsEIL7\u003c/em\u003e, \u003cem\u003eMsEIL8\u003c/em\u003e and \u003cem\u003eMsEIL10\u003c/em\u003e genes exhibited the highest expression in roots and stems. Therefore, studying the tissue-specific expression patterns of \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes in alfalfa, can provide a foundation for understanding their specific functions.\u003c/p\u003e \u003cp\u003eRecent studies have demonstrated that the \u003cem\u003eEIN3/EIL\u003c/em\u003e transcription factors play crucial roles in plant development, as well as in the regulation of phytohormones and stress responses. A \u003cem\u003ecis\u003c/em\u003e-acting element analysis of the promoter region of \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes revealed the presence of numerous hormone response elements, including abscisic acid, gibberellin, salicylic acid, jasmonic acid lactone, and auxin, as well as stress response elements, such as drought, low temperature, anaerobic, and defence stresses. This further confirms the important functions of the \u003cem\u003eMsEIN3/EIL\u003c/em\u003e gene family in regulating plant growth and stress response. Furthermore, we investigated the responses of \u003cem\u003eMsEIN3/EIL\u003c/em\u003e gene family members to cold, drought, salt stress and ABA treatments and analyzed their expression patterns. The majority of \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes were induced, with significantly increased expression levels in alfalfa seedlings treated with exogenous ABA. This suggests that \u003cem\u003eMsEIN3/EIL\u003c/em\u003e is involved in the response to ABA signaling pathway, similar to observations in poplar treated with ABA[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Under mannitol, salt and cold treatments, the \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes were induced to varying degrees, with some differences. The expression levels of \u003cem\u003eMsEIL1\u003c/em\u003e, \u003cem\u003eMsEIL4\u003c/em\u003e and \u003cem\u003eMsEIL5\u003c/em\u003e genes in roots of alfalfa seedlings showed a transient down-regulation followed by a significant up-regulation after drought treatment. This was similar to the result of significant up-regulation of \u003cem\u003eMnEILs\u003c/em\u003e gene expression in roots of mulberry after PEG treatment[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], indicating their active role in coping with drought stress. The expression levels of different \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes were found to have consistent expression trends induced by salt stress. Specifically, the expression levels of \u003cem\u003eMsEIL1\u003c/em\u003e, \u003cem\u003eMsEIL4\u003c/em\u003e and \u003cem\u003eMsEIL5\u003c/em\u003e genes exhibited a down-regulation followed by up-regulation trend during salt stress treatment. Studies in Arabidopsis have shown that the germination rate of the \u003cem\u003eein3eil1\u003c/em\u003e double mutant was significantly decreased under salt stress, while \u003cem\u003eEIN3\u003c/em\u003e overexpression plants exhibited increased germination rate and enhanced seedling resistance compared to the wild type[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. And the \u003cem\u003eEIN3\u003c/em\u003e gene has been demonstrated to improve tolerance to salt stress by preventing the accumulation of reactive oxygen species (ROS)[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Since \u003cem\u003eMsEIL4\u003c/em\u003e gene is a homologue of the Arabidopsis \u003cem\u003eAtEIN3\u003c/em\u003e gene, it is likely that \u003cem\u003eMsEIL4\u003c/em\u003e gene also play an active role in salt stress. Finally, the relative expression of \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes in roots was generally upregulated under cold stress conditions, consistent with the expression pattern of \u003cem\u003eGmEIN3/EIL\u003c/em\u003e genes in the legume soybean[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Additional, it has been reported that both \u003cem\u003eein3-1\u003c/em\u003e as well as \u003cem\u003eein3 eil1\u003c/em\u003e double mutants exhibit enhanced frost tolerance, while \u003cem\u003eEIN3\u003c/em\u003e overexpression plants display increased sensitivity to cold stress[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The aforementioned studies collectively indicate that \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes may be involved in the response to a range of abiotic stresses. However, the specific mechanisms of their actions still require further investigation.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we identified ten members of the \u003cem\u003eMsEIN3/EIL\u003c/em\u003e gene family in the alfalfa genome, and found that most of the \u003cem\u003eMsEIN3/EIL\u003c/em\u003e proteins were located in the nucleus and contained conserved EIN3/EIL structures. The distribution of \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes on the alfalfa chromosomes was not uniform, with SD being the main driver of their evolution. Phylogenetic analysis of \u003cem\u003eEIN3/EIL\u003c/em\u003e genes from different species revealed that the \u003cem\u003eEIN3/EIL\u003c/em\u003e genes were classified into four clades, A, B, C, and D. Expression analysis showed that the \u003cem\u003eMsEIN3/EIL\u003c/em\u003e gene family members exhibited diverse expression patterns in different tissues. Furthermore, \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes responded to cold, drought, and salt stresses, as well as ABA treatments. The expression patterns of \u003cem\u003eMsEIL1\u003c/em\u003e, \u003cem\u003eMsEIL4\u003c/em\u003e, and \u003cem\u003eMsEIL5\u003c/em\u003e genes were particularly sensitive, exhibiting the high similarity to \u003cem\u003eAtEIN3\u003c/em\u003e, \u003cem\u003eAtEIL1\u003c/em\u003e, and \u003cem\u003eAtEIL3\u003c/em\u003e. In conclusion, our study provides important insights into the genome-wide characterization and expression patterns of the alfalfa \u003cem\u003eEIN3/EIL\u003c/em\u003e gene family. These findings contribute to a better understanding of the role of these genes in response to abiotic stresses in alfalfa.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials\u003c/h2\u003e \u003cp\u003eAlfalfa (cv. Zhongmu No.1) seeds used in this experiment were provided by \u0026lsquo;Jiuquan Daye\u0026rsquo; Seed Industry Co., Ltd. (harvested in 2022), and the plants were planted in the greenhouse of China Agricultural University. For RT- PCR, samples of root, post-elongated stem, elongated stem, leaf, and flower were collected at three weeks after seedling transplantation and growth. Each sample consisted of three biological replicates. After collection, the sample was quickly frozen and ground into a fine powder using liquid nitrogen to preserve its molecular integrity.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIdentification and Chromosome location of the\u003c/b\u003e \u003cb\u003eEIN3/EIL\u003c/b\u003e \u003cb\u003egenes in Alfalfa\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe genome sequence and annotation files of the Zhongmu No. 1 alfalfa variety were downloaded from the figshare website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://figshare.com/articles/dataset/Medicago_sativa_genome_and_annottion_\u003c/span\u003e\u003cspan address=\"https://figshare.com/articles/dataset/Medicago_sativa_genome_and_annottion_\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e files). The EIN3/EIL protein sequences from \u003cem\u003eA.thaliana\u003c/em\u003e, were acquired from the TAIR database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.arabidopsis.org/\u003c/span\u003e\u003cspan address=\"http://www.arabidopsis.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The EIN3/EIL protein sequences of \u003cem\u003eGlycine max, O.sativa\u003c/em\u003e and \u003cem\u003eMedicago truncatula\u003c/em\u003e were obtained from Phytozome12 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://phytozome-next.jgi.doe.gov/\u003c/span\u003e\u003cspan address=\"https://phytozome-next.jgi.doe.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The 34 EIN3/EIL protein sequences were used as queries to search for possible EIN3/EIL proteins within the alfalfa genome using BLASTP (E-value\u0026thinsp;\u0026le;\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;10\u003c/sup\u003e) by TBtools software. To identify the \u003cem\u003eEIN3/EIL\u003c/em\u003e genes in the alfalfa genome accurately, the Hidden Markov Model (HMM) file of the EIN3/EIL protein domain (PF04873) was downloaded from the Pfam database(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://pfam-legacy.xfam.org/\u003c/span\u003e\u003cspan address=\"http://pfam-legacy.xfam.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and then the sequences containing MsEIN3/EIL protein domains were retrieved from the genome of Zhongmu No.1 alfalfa using HMMER 3.0. The results were re-confirmed by NCBI-CDD (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and InterPro (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/interpro/search/sequence/\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/interpro/search/sequence/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to determine that it contains the conserved domain specific to EIN3/EIL, excluding the sequence that does not contain the typical domain of EIN3/EIL proteins, and the remaining protein sequence is regarded as a member of the alfalfa \u003cem\u003eEIN3/EIL\u003c/em\u003e gene family.\u003c/p\u003e \u003cp\u003eTo study the distribution of the \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes on chromosomes, we obtained the location information from the gene annotation file of the alfalfa genome database. The chromosome location of the \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes was extracted from the genome annotation file GFF3. Subsequently, the distribution of \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes on chromosomes was visualized using TBtools software. The protein sequences of MsEIN3/EIL were analyzed using ExPASy-ProtParam (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy\u003c/span\u003e\u003cspan address=\"https://web.expasy\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to predict their physical and chemical characteristics. Additionally, subcellular localization was predicted using the WoLFPSORT tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genscript.com/wolf-psort.html/\u003c/span\u003e\u003cspan address=\"https://www.genscript.com/wolf-psort.html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhylogenetic analysis of\u003c/b\u003e \u003cb\u003eEIN3/EIL\u003c/b\u003e \u003cb\u003egenes family in Alfalfa and Arabidopsis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo explore the evolutionary relationships of \u003cem\u003eEIN3/EIL\u003c/em\u003e genes in alfalfa, a multiple sequence alignment of full-length EIN3/EIL family protein sequences was conducted using MUSCLE. The phylogenetic relationship was constructed based on 1000 bootstrap replicates in MEGA11, which utilized the neighbor-joining (NJ) method and the Jones-Taylor-Thornton (JTT) model. The phylogenetic tree was visualized and optimized using Evolview (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.evolgenius.info/evolview/#/\u003c/span\u003e\u003cspan address=\"http://www.evolgenius.info/evolview/#/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of motifs, gene structures and conserved domains\u003c/h2\u003e \u003cp\u003eThe exon/intron sites and length information of each \u003cem\u003eEIN3/EIL\u003c/em\u003e gene were extracted from the gene annotation file GFF3 of the alfalfa genome database. The conserved protein motifs of the \u003cem\u003eMsEIN3/EIL\u003c/em\u003e family genes were identified using the MEME tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://meme-suite.org/meme/tools/meme\u003c/span\u003e\u003cspan address=\"https://meme-suite.org/meme/tools/meme\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), with a maximum of ten motifs and default parameters. The NCBI conserved domain database (CDD) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to perform domain analysis and determine the type and location of all MsEIN3/EIL protein sequences. Additionally, TBtools software was used to visualize the exon-intron structure of the \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes and the conserved motifs and structural architecture domains of the MsEIN3/EIL proteins.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eGene duplication and syntenic analysis\u003c/h2\u003e \u003cp\u003eTo investigate potential gene duplication events in the \u003cem\u003eMsEIN3/EIL\u003c/em\u003e gene family, we identified homologous gene pairs and relationships among alfalfa \u003cem\u003eMsEIN3/EIL\u003c/em\u003e family genes using the Multiple Collinearity Scanning Toolkit (MCScanX) software with default parameters. Additionally, we conducted a synergy analysis of \u003cem\u003eEIN3/EIL\u003c/em\u003e genes in Zhongmu No.1 alfalfa with those in \u003cem\u003eA.thaliana\u003c/em\u003e, \u003cem\u003eM.truncatula\u003c/em\u003e, \u003cem\u003eG.max\u003c/em\u003e, \u003cem\u003eO.sativa\u003c/em\u003e, \u003cem\u003eZ.mays\u003c/em\u003e. The duplication of the \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes in alfalfa was visualized in TBtools using circular mapping. The homologous genetic relationship between the \u003cem\u003eEIN3/EIL\u003c/em\u003e genes in alfalfa and other species was visualized using One Step MCScanX.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMultiple alignment and Secondary structure of MsEIN3/EIL proteins\u003c/h2\u003e \u003cp\u003eThe protein sequences of Arabidopsis AtEIN3 and alfalfa EIN3/EIL were compared using the MEGA11 software and visualized in DNAMAN software. The secondary structure of EIN3/EIL proteins was predicted using the online tool SOPMA (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa%20_sopma.html\u003c/span\u003e\u003cspan address=\"https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa%20_sopma.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of the\u003c/b\u003e \u003cb\u003eMsEIN3/EIL\u003c/b\u003e \u003cb\u003egenes promoter in alfalfa\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the role of \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes in plant responses to various stressors, we analyzed the \u003cem\u003ecis\u003c/em\u003e-acting elements of these genes in detail. We extracted a 2000 bp nucleotide sequence upstream of the start codon of each \u003cem\u003eMsEIN3/EIL\u003c/em\u003e gene from the Zhongmu No.1 genome sequence and submitted it to the PlantCARE database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"https://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for \u003cem\u003ecis\u003c/em\u003e-acting element prediction. The regulatory functions of the predicted \u003cem\u003ecis\u003c/em\u003e-acting elements were classified and visualized using TBtools software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of gene expression using RNA-seq datasets\u003c/h2\u003e \u003cp\u003eTranscriptome data were downloaded from NCBI public databases to investigate \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes expression pattern in different tissues and abiotic stresses. RNA-Seq data from different tissues, including post-elongated stem, elongated stem, flower, leaf, root, and seed, were obtained from the CADL-Gene Expression Atlas database provided by the Noble Research Institute. The expression pattern of \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes was analyzed under different conditions, including abiotic stress and ABA treatments, using RNA-Seq datasets. The raw RNA-seq data used in this study was obtained from the NCBI database (SRR7091780-SRR7091794; SRR7160313-SRR7160357). Abiotic stress material was generated in the following treatments: 1) Seedlings were collected at 0, 2, 6, 24, and 48 h under low temperature treatment at 4\u0026deg;C (three replicates per time point)[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]; 2) Seedlings were treated with 400 mM mannitol for 0, 1, 3, 6, 12, and 24 h (with three biological replicates per treatment time point), the root tip of each seedling was excised and collected[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]; 3) Seedlings were treated for 0, 1, 3, 6, 12 and 24 h (with three biological replicates per treatment time point) in 250 mM NaCl. The root tip of each seedling was excised and collected. The ABA treatments were as follows: Seedlings aged 12 days were treated for 0, 1, 3 and 12 h in a 1/2 MS nutrient solution containing 10 \u0026micro;M ABA (pH\u0026thinsp;=\u0026thinsp;5.8), respectively. The root tips were collected after 0, 1, 3 and 12 hours of treatment[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The raw data were filtered and converted from SRA files to FASTQ files using the SRA to Fastq application of TBtools software. Finally, the gene expression values of \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes under abiotic stress were calculated and normalized using TBtools software to draw heat maps.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and RT-qPCR analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted using the RNA extraction kit (Huayueyang Biotech Co., Ltd., Beijing, China). cDNA was synthesized for reverse transcription using the SuperMix for qPCR kit (TransGen Biotech, Beijing, China), following the manufacturer's instructions provided in the kit. Then, RT-qPCR was performed on a CFX96 Real-Time Detection System. RT-qPCR was performed using a cycling program consisting of an initial step at 95\u0026deg;C for 3 min, followed by 40 cycles of 95\u0026deg;C for 15 s and 60\u0026deg;C for 30 s. Data were calculated using the 2^\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method for gene expression levels. The final values were calculated as the average of triplicate reactions. The Ct values of \u003cem\u003eMsActin\u003c/em\u003e were used to normalize the Ct values for each gene. The list of primers used in this study is shown in Supplementary Table S14.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eSD \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Segmental duplication\u003c/p\u003e\n\u003cp\u003eET \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Ethylene\u003c/p\u003e\n\u003cp\u003eDBD \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;DNA-binding domain\u003c/p\u003e\n\u003cp\u003eAD \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Amino-terminal acidic domain\u003c/p\u003e\n\u003cp\u003ePR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Proline-rich region\u003c/p\u003e\n\u003cp\u003eBD I-V \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Basic domains I-V\u003c/p\u003e\n\u003cp\u003eTFs \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Transcription factors\u003c/p\u003e\n\u003cp\u003eUTR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Untranslated regions\u003c/p\u003e\n\u003cp\u003eRT-PCR \u0026nbsp; \u0026nbsp; \u0026nbsp; Real-time quantitative PCR\u003c/p\u003e\n\u003cp\u003eABA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Abscisic acid\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDL and SX designed the project; SX and WJ performed the data analysis; SX, SS, PW and WJ interpreted the data and results; WJ was responsible for planting materials; SX wrote the manuscript; and DL, SS, LM, MP and WJ carried out thin and tall revisions. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Key Technology Researches for Seed Propagation of Alfalfa with Saline and Alkaline Tolerance and Drought Resistance (2022ZD0401105) and\u0026nbsp;Chinese Universities Scientific Fund (2024TC076).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are contained within the article and Supplementary Materials. Raw sequencing data of the transcriptome used in the current study are available in the NCBI\u0026apos;s Sequence Read Archive (SRA, https://www.ncbi.nlm.nih.gov/sra) under the BioProject PRJNA454564 and PRJNA450305. The genomic information of the Zhongmu No.1 alfalfa variety was retrieved from the figshare website (https://figshare.com/articles/dataset/Medicago_sativa_genome_and_annotation_files). The RNA-Seq data, downloaded from the Noble Research Institute database (https://www.alfal fatoo lbox.org), were used to evaluate the transcript abundance profiles of \u003cem\u003eMsEIN3/EIL\u003c/em\u003e encoding genes across six tissues, namely, leaves, flowers, post-elongated stems, elongated stems, seeds and roots.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudy complied with local and national regulations for using plants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNaing AH, Xu J, Kim CK. 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Int J Mol Sci. 2018;20(1):47.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Alfalfa, EIN3/EIL gene family, Transcription factors, Stress, Expression profiling","lastPublishedDoi":"10.21203/rs.3.rs-4513747/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4513747/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eAlfalfa (\u003cem\u003eMedicago sativa\u003c/em\u003e) is known as the \"king of forage\" due to its high protein, mineral, carbohydrate, and digestive nutrient content. However, various abiotic stresses inhibit the growth and development of alfalfa, ultimately leading to a decrease in yield and quality. The ethylene-insensitive 3 (EIN3)/ethylene-insensitive 3-like (EIL) transcription factors are core regulators in plant ethylene signaling, playing important roles in plant development and response to abiotic stresses. However, a comprehensive genome-wide analysis of \u003cem\u003eEIN3/EIL\u003c/em\u003e genes in alfalfa has not yet been conducted.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn this study, we identified ten \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes from the alfalfa (cv.Zhongmu No.1) genome, which were classified into four clades based on phylogenetic analysis. The motif 1, motif 2, motif 3, motif 4, and motif 9 of the \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes constitute the conserved structural domains. Gene duplication analyses suggest that segmental duplication (SD) is a major driver of the expansion of the \u003cem\u003eMsEIN3/EIL\u003c/em\u003e gene family during evolution. The analysis of the \u003cem\u003ecis\u003c/em\u003e-acting elements in the promoter of \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes showed their ability to respond to various hormones and stresses. The analysis of tissue expression revealed that group A and group C members were highly expressed in flowers and seeds, while group D members were highly expressed in roots and stems. Furthermore, RNA-Seq analysis demonstrated that the expression of \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes were responsive to ABA treatment and different abiotic stresses (e.g., salt, cold, and drought stress).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study investigated \u003cem\u003eMsEIN3/EIL\u003c/em\u003e genes in alfalfa and identified three candidate \u003cem\u003eMsEIN3/EIL\u003c/em\u003e transcription factors involved in the regulation of abiotic stresses. These findings will provide valuable insights into uncovering the molecular mechanisms underlying various stress responses in alfalfa.\u003c/p\u003e","manuscriptTitle":"Genome-wide identification of the EIN3/EIL transcription factor family and their responses under abiotic stresses in Alfalfa (Medicago sativa)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-18 18:30:18","doi":"10.21203/rs.3.rs-4513747/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-07T04:23:05+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-05T15:05:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"128570647800539870215513668255703064477","date":"2024-07-20T11:24:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"313327421580686674079026752602968553877","date":"2024-06-28T18:32:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-23T09:33:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"278738873257832242812856152478134220413","date":"2024-06-07T07:26:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"190951694209010815001275453835904333200","date":"2024-06-05T09:46:35+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-04T14:49:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-04T12:09:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-04T12:09:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2024-06-01T13:04:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5874f9f2-e35e-406c-b32a-595f2df4fecf","owner":[],"postedDate":"June 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-07T16:06:52+00:00","versionOfRecord":{"articleIdentity":"rs-4513747","link":"https://doi.org/10.1186/s12870-024-05588-2","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2024-09-30 15:57:23","publishedOnDateReadable":"September 30th, 2024"},"versionCreatedAt":"2024-06-18 18:30:18","video":"","vorDoi":"10.1186/s12870-024-05588-2","vorDoiUrl":"https://doi.org/10.1186/s12870-024-05588-2","workflowStages":[]},"version":"v1","identity":"rs-4513747","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4513747","identity":"rs-4513747","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}