Skip to main content
Download PDF

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

BMC Plant Biology volume 24, Article number: 898 (2024) Cite this article

Abstract

Background

Medicago sativa, often referred to as the “king of forage”, is prized for its high content of protein, minerals, carbohydrates, and digestible nutrients. However, various abiotic stresses can hinder its growth and development, ultimately resulting in reduced yield and quality, including water deficiency, high salinity, and low temperature. The ethylene-insensitive 3 (EIN3)/ethylene-insensitive 3-like (EIL) transcription factors are key regulators in the ethylene signaling pathway in plants, playing crucial roles in development and in the response to abiotic stresses. Research on the EIN3/EIL gene family has been reported for several species, but minimal information is available for M. sativa.

Results

In this study, we identified 10 MsEIN3/EIL genes from the M. sativa genome (cv. Zhongmu No.1), which were classified into three clades based on phylogenetic analysis. The conserved structural domains of the MsEIN3/EIL genes include motifs 1, 2, 3, 4, and 9. Gene duplication analyses suggest that segmental duplication (SD) has played a significant role in the expansion of the MsEIN3/EIL gene family throughout evolution. Analysis of the cis-acting elements in the promoters of MsEIN3/EIL genes indicates their potential to respond to various hormones and environmental stresses. We conducted a further analysis of the tissue-specific expression of the MsEIN3/EIL genes and assessed the gene expression profiles of MsEIN3/EIL under various stresses using transcriptome data, including cold, drought, salt and abscisic acid treatments. The results showed that MsEIL1, MsEIL4, and MsEIL5 may act as positive regulatory factors involved in M. sativa’s response to abiotic stress, providing important genetic resources for molecular design breeding.

Conclusion

This study investigated MsEIN3/EIL genes in M. sativa and identified three candidate transcription factors involved in the regulation of abiotic stresses. These findings will offer valuable insights into uncovering the molecular mechanisms underlying various stress responses in M. sativa.

Peer Review reports

Introduction

Ethylene is a gaseous plant hormone that plays a crucial role in regulating various aspects of plant growth and development, including seed germination [1,2,3], 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 and tissue damage [13, 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. In this model, 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, where it stabilizes EIN3 and its homolog EIL1. These proteins function as primary transcription factors (TFs) in the ethylene signaling pathway and further initiate a transcriptional cascade involving ETHYLENE RESPONSE FACTORs (ERFs) [15]. EIN3/EIL proteins have been identified as transcription factors localized in the nucleus, and their structural characteristics have been extensively studied in model plants [16,17,18]. These transcription factors not only exhibit DNA-binding activity but also share structural similarities across different species [19]. All identified EIN3/EIL homologous genes possess a conserved DNA-binding domain (DBD) that specifically binds to the EIN3 binding site in the promoter regions of target genes [20]. The N-terminal amino acid sequence of EIN3/EIL proteins is highly conserved and contains several important structural features, including an acidic domain (AD), a proline-rich region (PR), and a cluster of five small basic domains (BD I-V) [21, 22]. In contrast, the C-terminal amino acid sequence shows less conservation. For instance, some EIN3/EIL genes in plants like A. thaliana possess a unique poly-asparagine or poly-glutamine domain at the C-terminal end, a feature not commonly observed in other species such as Nicotiana tabacum and Lycopersicon esculentum [20, 23].

TFs are DNA-binding proteins that interact with cis-acting elements in gene promoter regions to regulate gene transcription, playing a crucial role in plant growth, development, and responses to environmental stresses [24, 25]. In 1997, Chao et al. identified the EIN3 gene along with five related EIN3-like genes in A. thaliana [21]. Among these, AtEIN3 and AtEIL1 play significant roles in responding to salt and cold stress, while the AtEIL3 gene serves as a central transcriptional regulator of sulfur response and metabolism in A. thaliana [26]. A. thaliana that overexpresses MnEIL3 demonstrated an increased tolerance to both salt and drought stress conditions [27]. Research has shown that ethylene pretreatment or the activation of EIN3 can enhance the salt tolerance of A. thaliana. Additionally, EIN3 has been found to directly regulate the expression of peroxidases (POD), thereby increasing POD activity and helping to scavenge reactive oxygen species (ROS) [28]. Conversely, ethylene has been shown to negatively impact plant responses to freezing stress in A. thaliana. Specifically, the overexpression of EIN3 results in diminished freezing tolerance, and biochemical analyses indicate that EIN3 negatively regulates the expression of C-repeat Binding Factor (CBF) and type-A Arabidopsis Response Regulator (ARR) genes [29]. Subsequent studies have demonstrated that EIN3/EIL genes are not only essential downstream regulators in the ethylene signaling pathway, but also play a pivotal role in the crosstalk between various plant hormones [14]. Therefore, an in-depth understanding of the functions of the EIN3/EIL family is essential for elucidating the relationships between different signal transduction pathways and stress responses during plant development.

M. sativa is a crucial perennial legume forage crop, ranking as the fourth largest cash crop in the USA, renowned for its high protein and nutrient content, as well as substantial biomass production [30]. However, environmental challenges often hinder its growth, affecting yield and quality. Current research on EIN3/EIL genes mainly focuses on model plants like A. thaliana, Oryza sativa, and Triticum aestivum [16, 18, 21], leaving a gap in understanding their role in M. sativa, particularly in stress responses. To address this gap, we identified 10 MsEIN3/EIL genes in the M. sativa 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 four abiotic stresses. This study aims to deepen our understanding of the function of EIN3/EIL genes in M. sativa 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 M. sativa

To identify MsEIN3/EIL gene members in M. sativa, we utilized the full-length protein sequences of EIN3/EIL genes from four species: A. thaliana (six), Medicago truncatula (10), Glycine max (12) and O. sativa (six), as queries for BLASTp search. 11 putative MsEIN3/EIL genes were identified in the genome of M. sativa. Subsequently, 10 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 10 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 M. sativa
Full size table

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 M. sativa genome sequence, and they were located on five specific chromosomes unevenly: Chr2, Chr3, Chr5, Chr6, and Chr8 (Fig. 1A). 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. 1B, 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.

Fig. 1
figure 1

Chromosomal distribution of MsEIN3/EIL genes and phylogenetic analysis of EIN3/EIL proteins. (A) The chromosomal locations of MsEIN3/EIL genes. The long blue bars represent the chromosomes and the chromosome numbers are indicated on the top side of the bars. (B) The phylogenetic relationships of the EIN3/EIL proteins from M. sativa, M. truncatula, A. thaliana and O. sativa. The phylogenetic tree was generated by the neighbor-joining method derived from MUSCLE alignment and the four clades are shaded with different colors. The red stars represent MsEIN3/EIL genes and the green stars represent AtEIN3/EIL genes

Full size image

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 10 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 A. thaliana (Fig. 2A). 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. 2B). 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. 2C, the MsEIN3/EIL genes displayed a range of zero to nine introns, with similar clustering patterns. Among the 10 genes, most genes contained two introns, including MsEIL2, MsEIL3, MsEIL6, MsEIL7, and MsEIL8 genes. In addition, MsEIL1 gene contained nine introns, MsEIL9 and MsEIL5 genes contained three introns, while MsEIL10 and MsEIL4 genes have one and zero intron, respectively.

Fig. 2
figure 2

Phylogenetic tree, conserved motif, and gene structure of MsEIN3/EIL gene family. (A) 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. (B) The conserved domain analysis of MsEIN3/EIL proteins shows different colors representing various conserved domains. (C) The gene structure of MsEIN3/EIL genes was determined. The introns, exons, and untranslated regions (UTR) are represented by gray lines, green boxes, and yellow boxes, respectively

Full size image

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 and 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 M. sativa, 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 and Table S3). The results showed that the MsEIN3/EIL genes in M. sativa underwent significant evolutionary divergence and were homologous in dicotyledons.

Fig. 3
figure 3

Collinearity analysis of MsEIN3/EIL gene family in M. sativa, the segmentally duplicated genes are connected by red lines, referring to the two genes highlighted in blue

Full size image
Fig. 4
figure 4

Syntenic analysis of MsEIN3/EIL genes in M. sativa compared with those in five plant species (A. thaliana, M. truncatula, G. max, O. sativa and Z. mays)

Full size image

Amino acid sequence alignment and secondary structure analysis of MsEIN3/EIL proteins

To evaluate the similarity of the MsEIN3/EIL protein sequences of M. sativa, a multiple sequence alignment analysis was conducted on protein sequences of AtEIN3 protein and 10 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 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 (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 regions are common transcriptional activation regions in plants, indicating that the acidic amino acid domain, the basic amino acid domain and the proline enrichment region are the transcriptional activation regions and functional regions of the MsEIN3/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 10 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).

Fig. 5
figure 5

Sequence alignment of AtEIN3 protein and all identified MsEIN3/EIL proteins. 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

Full size image
Table 2 The secondary structure of MsEIN3/EIL proteins
Full size table

Promoter region cis -acting regulatory elements analysis

The analysis of cis-acting regulatory elements identified 14 major cis-acting elements in the MsEIN3/EIL gene promoter sequences (Fig. 6 and Table S4). 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 10 identified MsEIN3/EIL gene promoter sequences in M. sativa 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.

Fig. 6
figure 6

Cis-acting elements in promoter region of MsEIN3/EIL genes

Full size image

Expression pattern of MsEIN3/EIL genes in tissues

Tissue expression patterns are crucial for elucidating the specific roles of MsEIN3/EIL genes in different tissues. To achieve this, real-time quantitative PCR (RT-qPCR) was conducted on the MsEIN3/EIL genes. The expression patterns varied across different tissues. Specifically, the MsEIL4 gene exhibited elevated expression levels in flowers and seeds, while the MsEIL5 gene was predominantly expressed in flowers. Conversely, the MsEIL10 gene, which is classified within the same group A as MsEIL4 and MsEIL5, demonstrated increased expression in roots and stems. Furthermore, the MsEIL1 gene, which falls under group C, showed significant expression in seeds. In contrast, the MsEIL3, MsEIL6, MsEIL7, and MsEIL8 genes, categorized in group D, exhibited high expression levels in both roots and stems (Fig. 7).

Fig. 7
figure 7

Tissue-specific expression of core MsEIN3/EIL genes was analyzed through RT-qPCR, with the MsActin gene serving as the internal standard. The data represent the means ± SEM for three independent experiments

Full size image

Expression profiles analysis of MsEIN3/EIL genes under stresses

Ethylene is considered a stress hormone involved in various stress responses [31], among which soil drought, low temperature, and salinization are some of the most common abiotic stresses that limit plant growth. To explore the potential functions of the MsEIN3/EIL genes under these stresses, we analyzed their expression under the three types of stress treatments based on previously published RNA-seq data (Fig. 8).

Under cold treatment conditions, the expression levels of the MsEIL1, MsEIL2, MsEIL4, and MsEIL5 genes in seedlings significantly increased at 2 h compared to the control (0 h), followed by a slight decrease at 6 h. Subsequently, MsEIL6 began to increase slowly at 24 h, while MsEIL1 and MsEIL5 continued to decline at 24 h before starting to rise again at 48 h (Fig. 8A and Table S5). Under drought stress, the expression trends of MsEIL1, MsEIL4, and MsEIL5 in the roots were generally consistent. Specifically, their expression levels significantly decreased at 1 h compared to the control (0 h), increased significantly at 3 h, and then gradually rose for the subsequent two time points, until a sharp decline occurred at 24 h. The expression levels of MsEIL2, MsEIL6, MsEIL7, and MsEIL8 were significantly upregulated only at specific time points after mannitol treatment, while MsEIL3 showed significant downregulation (Fig. 8B and Table S6). Under salt stress, the expression levels of MsEIL1, MsEIL5 and MsEIL6 in the roots significantly decreased at 1 h compared to the control (0 h), increased significantly at 3 h, and then gradually declined. In contrast, MsEIL4 exhibited a pattern of persistent fluctuation, with significant decreases at 1 h compared to the control significant increases at 3 h, slight decreases at 6 h, modest increases at 12 h, and finally a significant decrease at 24 h. The expression level of MsEIL8 gradually increased under salt treatment, reaching its peak at 6 h. MsEIL7 showed a significant upregulation only at 6 h of salt treatment. In contrast, the expression level of MsEIL3 decreased significantly after salt treatment (Fig. 8C and Table S7).

ABA signaling is generally associated with plant responses to abiotic stress [32]. Therefore, we analyzed the expression levels of MsEIN3/EIL genes based on published RNA-seq data after ABA treatment. The study found that under ABA treatment conditions, the expression level of the MsEIL1, MsEIL2, MsEIL4 and MsEIL5 genes in the roots significantly increased at 1 h compared to the control (0 h), followed by a significant decrease. Meanwhile, the expression level of MsEIL8 significantly increased after 3 h of ABA treatment, MsEIL6 and MsEIL7 showed significant upregulation after 12 h of ABA treatment. In contrast, MsEIL9 exhibited a marked downregulation under ABA treatment conditions (Fig. 8D and Table S8). The above results suggest that members of the MsEIN3/EIL family may play distinct roles in response to abiotic stresses.

Fig. 8
figure 8

Expression profiles of MsEIN3/EIL genes in response to various abiotic stress treatments and ABA treatment. (A) Seedlings were subjected to cold treatment at 4 °C for varying durations. (B) Seedlings were treated with 400 mM mannitol for varying durations and root tips were harvested for subsequent experiments. (C) Seedlings were treated with 250 mM NaCl for varying durations and root tips were harvested for subsequent experiments. (D) Seedlings were treated with 10 µM ABA for varying durations and root tips were harvested for subsequent experiments. Heat maps were generated by TBtools based on log2-transformed (FPKM) values. Higher and lower levels of transcript accumulation are indicated by red and blue respectively, and the median level is indicated by yellow. ABA, abscisic acid; FPKM, fragments per kilobase of transcript per million fragments mapped

Full size image

Discussion

EIN3/EIL transcription factors play important roles in plant growth and development, participating in phytohormone signaling [33], sulfur metabolism [34], and regulating transcriptional responses to abiotic stresses in plants. The regulatory mechanisms of these genes have been well elucidated in A. thaliana, but the molecular mechanisms in other plants remain unclear. Nowadays, comprehensive genome-wide analyses of the EIN3/EIL gene family have mainly been reported in some model crops. In this study, we conducted a comprehensive analysis of the EIN3/EIL gene family in M. sativa, laying the foundation for a deeper understanding of the functions of these genes.

The comparison of the EIN3/EIL genes in M. sativa and other species

By comparing the homologs of MsEIN3/EIL with those from other species, including protein structure, species evolution, and gene expression profiles, valuable information can be provided to predict the potential functions of the MsEIN3/EIL genes. According to phylogenetic analyses, these MsEIN3/EIL proteins can be divided into three clades (Fig. 1B). Additionally, EIN3/EIL members with similar gene structures and motif compositions cluster together (Fig. 2). Members clustered in the same clade often exhibit similar functions [35]. Clade A includes the MsEIN3/EIL genes (MsEIL4 and MsEIL5), which are in the same branch as AtEIN3 and AtEIL1, highlighting their close homology and suggesting that the two MsEIN3/EIL genes may play a significant positive regulatory role in the ethylene signaling pathway [28]. The MsEIL10 gene from clade A is located in the same branch as AtEIL2, indicating that MsEIL10 may have a function similar to that of AtEIL2. In clade C, MsEIL1 is in the same branch as AtEIL3, suggesting that MsEIL1 may have a regulatory role in sulfur responses and metabolism akin to that of AtEIL3 [34].

Motifs and conserved structural domains both reflect the conserved protein sequence [36, 37]. The analyses showed that motifs 1, 3 and 4 are shared by all MsEIN3/EIL genes, constituting the conserved structural domains and indicating a high degree of conservation in the MsEIN3/EIL gene family (Fig. 2). Genes with a comparable number of exons often share similar functions, highlighting the significance of this feature in understanding gene function and evolution [17]. 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 Z. mays, Gossypium hirsutum, and T. aestivum [17, 18, 38]. This phenomenon is likely the product of species evolution. Furthermore, gene duplication is probably one of the main drivers of genome and genetic system evolution [39]. In this study, only one pair of MsEIN3/EIL genes with evidence of gene duplication in M. sativa was identified, specifically through segmental duplication. In addition, gene duplication among different species is important for genome and genetic system evolution [40]. 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, revealed that M. sativa shares homologous genes with dicotyledonous plants, but not with monocotyledonous plants (Fig. 4). Among dicotyledonous plants, M. sativa has more homologous gene pairs with legumes than with A. thaliana. Within legumes, M. sativa displays more homologous gene pairs with G. max than with M. truncatula. These results suggest that the genetic background of M. sativa 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. This study shows that only MsEIL1, MsEIL4, and MsEIL5 genes are strongly expressed in all samples, while the expression levels of other genes in the seeds are low or nonexistent. To elucidate these findings, we conducted a comparative analysis of the tissue expression levels of the MsEIN3/EIL genes in relation to those of the AtEIN3/EIL genes. The analysis revealed that, with the exception of the AtEIL2 gene, the expression levels of the remaining four genes were notably elevated in seeds of A. thaliana [41]. Furthermore, we observed an unexpected similarity in the expression levels of the MsEIL1 gene and the AtEIL3 gene across various tissues, both exhibiting relatively high expression in seeds. The MsEIL4 and MsEIL5 genes demonstrated expression patterns akin to that of the AtEIN3 gene, characterized by relatively high levels in seeds and flowers. The MsEIL10 gene displayed tissue expression levels comparable to those of the AtEIL2 gene, with pronounced expression in roots and stems. The MsEIL2, MsEIL3, MsEIL6, MsEIL7, MsEIL8, and MsEIL9 genes, which are novel genes that emerged during the evolutionary development of the MsEIN3/EIL gene family and share significant homology, exhibited similar tissue expression profiles, with relatively high expression levels observed in roots and stems. These results provide new insights into the functions of the MsEIN3/EIL gene family.

EIN3/EIL genes act as the hub for modulating plant stress responses

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 [42]. A cis-acting element analysis of the promoter region of MsEIN3/EIL genes revealed the presence of ABA response elements, drought, low temperature, 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. In this study, we found that MsEIL1, MsEIL4, and MsEIL5 respond to drought, cold, high salinity, and ABA, possibly because their promoter regions contain specific cis-elements related to stress.

The comprehensive analysis of the data from cold, mannitol and salt treatments indicate that MsEIL1, MsEIL4 and MsEIL5 act as positive regulatory factors involved in the response of M. sativa to abiotic stress. Based on the results of ABA treatment, we speculate that the crosstalk between ethylene signaling and ABA signaling pathways plays a crucial role in plants’ ability to withstand abiotic stress. Studies have shown that the ethylene response factor 15 (ERF15), which is downstream of EIN3, positively regulates ABA-mediated cold tolerance in Solanum lycopersicum [43]. In addition, overexpression of GhEIN3 can enhance the salt stress tolerance of A. thaliana during the seedling development stage by modulating the ABA and ROS pathways [44]. These findings suggest that members of the EIN3/EIL family may play a crucial role as key nodes in communicating with other signaling pathways during plant stress responses. The coordinated response of MsEIN3/EIL family members with other signals will be a focus of future study.

Conclusion

In this study, we identified 10 members of the MsEIN3/EIL gene family in the M. sativa 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 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. MsEIL1, MsEIL4, and MsEIL5 genes, with high similarity to AtEIN3, AtEIL1, and AtEIL3, positively regulate responses to salt stress, drought stress, and cold stress, suggesting that they may play a crucial role in responding to adverse environmental stressors. In conclusion, our study provides important insights into the genome-wide characterization and expression patterns of the MsEIN3/EIL gene family. These findings contribute to a better understanding of the role of these genes in response to abiotic stresses in M. sativa.

Materials and methods

Plant materials

M. sativa seeds (cv. Zhongmu No.1) were obtained from ‘Jiuquan Daye’ Seed Industry Co., Ltd. and stored in a low-temperature and low-humidity storage cabinet. 10 seeds were placed in a Petri dish (11 cm × 11 cm) lined with three layers of filter paper, and 10 ml of distilled water was added. The seeds were germinated in a light incubator at a constant temperature of 20℃, with an 8 h light and 16 h dark cycle for 10 days. Subsequently, each seedling was transplanted individually into pots containing nutrient soil: vermiculite (3:1) and cultivated in a plant greenhouse under a light/dark cycle of 16/8 hours, at a temperature of 22℃ and humidity of 60% for approximately two months. Then the roots, elongated stems, post-elongated stems, leaves, flowers, and seeds of M. sativa were harvested. The samples were rapidly frozen with liquid nitrogen and stored at -80℃ until further use.

Identification and chromosome location of the MsEIN3/EIL genes

The genome sequence and annotation files of the Zhongmu No. 1 M. sativa 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 G. max, O. sativa and M. 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 M. sativa genome using BLASTP (E-value ≤ 10− 10) by TBtools software. To identify the EIN3/EIL genes in the M. sativa 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 using Simple HMM Search by TBtools software. 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 sequences are regarded as members of the MsEIN3/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 M. sativa 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 WoLF PSORT tool (https://www.genscript.com/wolf-psort.html/).

Phylogenetic analysis of EIN3/EIL genes family

To explore the evolutionary relationships of EIN3/EIL genes in M. sativa, a multiple sequence alignment of full-length EIN3/EIL family protein sequences was conducted using MUSCLE by MEGA11 software. 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 M. sativa 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 MsEIN3/EIL family genes using the Multiple Collinearity Scanning Toolkit (MCScanX) by TBtools software with default parameters. Additionally, we conducted a synergy analysis of EIN3/EIL genes in Zhongmu No.1 M. sativa with those in A. thaliana, M. truncatula, G. max, O. sativa, Z. mays. The duplication of the MsEIN3/EIL genes was visualized in TBtools using circular mapping. The homologous genetic relationship between the EIN3/EIL genes in M. sativa and other species was visualized using One Step MCScanX by TBtools software.

Multiple alignment and secondary structure of MsEIN3/EIL proteins

The protein sequences of AtEIN3 and MsEIN3/EIL were compared using the MEGA11 software and visualized in DNAMAN software. The secondary structure of MsEIN3/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

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 abiotic stresses. 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) [45]; (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 [46]; (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 h of treatment [47]. 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. The cycling program included 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 non-conserved sequences of the selected genes were used to design primers in Primer software, with the objective of identifying primers with a melting temperature (TM) of approximately 60℃ and a GC content of 40–60%. The list of primers used in this study is shown in Supplementary Table S9.

Data availability

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).

Abbreviations

SD:

Segmental duplication

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

References

  1. Naing AH, Xu J, Kim CK. Editing of 1-aminocyclopropane-1-carboxylate oxidase genes negatively affects petunia seed germination. Plant Cell Rep. 2022;41:209–20.

    Article  CAS  PubMed  Google Scholar 

  2. Linkies A, Leubner-Metzger G. Beyond gibberellins and abscisic acid: how ethylene and jasmonates control seed germination. Plant Cell Rep. 2012;31(2):253–70.

    Article  CAS  PubMed  Google Scholar 

  3. Linkies A, Müller K, Morris K, Turečková V, Wenk M, Cadman CSC, Corbineau F, Strnad M, Lynn JR, Finch-Savage WE, et al. Ethylene interacts with abscisic acid to regulate endosperm rupture during germination: a comparative approach using Lepidium sativum and Arabidopsis thaliana. Plant Cell. 2009;21(12):3803–22.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Huang YC, Yeh TH, Yang CY. Ethylene signaling involves in seeds germination upon submergence and antioxidant response elicited confers submergence tolerance to rice seedlings. Rice. 2019;12(1):23.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Koyama T. The roles of ethylene and transcription factors in the regulation of onset of leaf senescence. Front Plant Sci. 2014;5:650.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Qin H, Xiao M, Li Y, Huang R. Ethylene modulates rice root plasticity under abiotic stresses. Plants (Basel). 2024;13(3):432.

    CAS  PubMed  Google Scholar 

  7. Shibuya K. Molecular aspects of flower senescence and strategies to improve flower longevity. Breed Sci. 2018;68(1):99–108.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Hermann K, Meinhard J, Dobrev P, Linkies A, Pesek B, Hess B, Machácková I, Fischer U, Leubner-Metzger G. 1-Aminocyclopropane-1-carboxylic acid and abscisic acid during the germination of sugar beet (Beta vulgaris L.): a comparative study of fruits and seeds. J Exp Bot. 2007;58(11):3047–60.

    Article  CAS  PubMed  Google Scholar 

  9. Qin H, Wang J, Chen X, Wang F, Peng P, Zhou Y, Miao Y, Zhang Y, Gao Y, Qi Y, et al. Rice OsDOF15 contributes to ethylene-inhibited primary root elongation under salt stress. New Phytol. 2019;223(2):798–813.

    Article  CAS  PubMed  Google Scholar 

  10. Shi J, Zhu Z. Seedling morphogenesis: when ethylene meets high ambient temperature. aBIOTECH. 2021;3(1):40–8.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Naing AH, Campol JR, Kang H, Xu J, Chung MY, Kim CK. Role of ethylene biosynthesis genes in the regulation of salt stress and drought stress tolerance in petunia. Front Plant Sci. 2022;13:844449.

    Article  PubMed  PubMed Central  Google Scholar 

  12. Wang Y, Jiang H, Mao Z, Liu W, Jiang S, Xu H, Su M, Zhang J, Wang N, Zhang Z, et al. Ethylene increases the cold tolerance of apple via the MdERF1B-MdCIbHLH1 regulatory module. Plant J. 2021;106(2):379–93.

    Article  CAS  PubMed  Google Scholar 

  13. Sasidharan R, Voesenek LA. Ethylene-mediated acclimations to flooding stress. Plant Physiol. 2015;169(1):3–12.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Chen H, Bullock DA, Alonso JM, Stepanova AN. To fight or to grow: the balancing role of ethylene in plant abiotic stress responses. Plants (Basel). 2022;11(1):33.

    CAS  Google Scholar 

  15. Binder BM. Ethylene signaling in plants. J Biol Chem. 2020;295(22):7710–25.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Aluko OO, Ninkuu V, Ziemah J, Jianpei Y, Taiwo E, Ninkuu SB, Sabuli N, Adetunde LA, Imoro AM, Ozavize SF, et al. Genome-wide identification and expression analysis of EIN3/EIL gene family in rice (Oryza sativa). Plant Stress. 2024;12:100437.

    Article  CAS  Google Scholar 

  17. Jyoti SD, Azim JB, Robin AHK. Genome-wide characterization and expression profiling of EIN3/EIL family genes in Zea mays. Plant Gene. 2021;25:100270.

    Article  CAS  Google Scholar 

  18. He Y, Huang W, Yang L, Li Y, Lu C, Zhu Y, Ma D, Yin J. Genome-wide analysis of ethylene‐insensitive3 (EIN3/EIL) in Triticum aestivum. Crop Sci. 2020;60(4):2019–37.

    Article  CAS  Google Scholar 

  19. Mao K, Zhang M, Kong Y, Dai S, Wang Y, Meng Q, Ma N, Lv W. Origin, expansion, and divergence of ETHYLENE-INSENSITIVE 3 (EIN3)/EIN3-LIKE transcription factors during streptophytes evolution. Front Plant Sci. 2022;13:858477.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Li J, Li Z, Tang L, Yang Y, Zouine M, Bouzayen M. A conserved phosphorylation site regulates the transcriptional function of ETHYLENE-INSENSITIVE3-like1 in tomato. J Exp Bot. 2012;63(1):427–39.

    Article  CAS  PubMed  Google Scholar 

  21. Chao Q, Rothenberg M, Solano R, Roman G, Terzaghi W, Ecker JR. Activation of the ethylene gas response pathway in Arabidopsis by the nuclear protein ETHYLENE-INSENSITIVE3 and related proteins. Cell. 1997;89(7):1133–44.

    Article  CAS  PubMed  Google Scholar 

  22. Lee J, Kim WT. Molecular and biochemical characterization of VR-EILs encoding mung bean ETHYLENE INSENSITIVE3-LIKE proteins. Plant Physiol. 2003;132(3):1475–88.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Rieu I, Mariani C, Weterings K. Expression analysis of five tobacco EIN3 family members in relation to tissue-specific ethylene responses. J Exp Bot. 2003;54(391):2239–44.

    Article  CAS  PubMed  Google Scholar 

  24. Li J, Han G, Sun C, Sui N. Research advances of MYB transcription factors in plant stress resistance and breeding. Plant Signal Behav. 2019;14(8):1613131.

    Article  PubMed  PubMed Central  Google Scholar 

  25. Wang B, Sun Z, Liu Y, Xu B, Li J, Chi M, Xing Y, Yang B, Li J, Liu J, et al. A pervasive phosphorylation cascade modulation of plant transcription factors in response to abiotic stress. Planta. 2023;258(4):73.

    Article  CAS  Google Scholar 

  26. Dolgikh VA, Pukhovaya EM, Zemlyanskaya EV. Shaping ethylene response: the role of EIN3/EIL1 transcription factors. Front Plant Sci. 2019;10:1030.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Liu C, Li J, Zhu P, Yu J, Hou J, Wang C, Long D, Yu M, Zhao A. Mulberry EIL3 confers salt and drought tolerances and modulates ethylene biosynthetic gene expression. Peer J. 2019;7:e6391.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Peng J, Li Z, Wen X, Li W, Shi H, Yang L, Zhu H, Guo H. Salt-induced stabilization of EIN3/EIL1 confers salinity tolerance by deterring ROS accumulation in Arabidopsis. PLoS Genet. 2014;10(10):e1004664.

    Article  PubMed  PubMed Central  Google Scholar 

  29. Shi Y, Tian S, Hou L, Huang X, Zhang X, Guo H, Yang S. Ethylene signaling negatively regulates freezing tolerance by repressing expression of CBF and type-A ARR genes in Arabidopsis. Plant Cell. 2012;24(6):2578–95.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Fu C, Hernandez T, Zhou C, Wang ZY. Alfalfa (Medicago sativa L). Methods Mol Biol. 2015;1223:213–21.

    Article  CAS  PubMed  Google Scholar 

  31. Müller M, Munné-Bosch S. Ethylene Response factors: a Key Regulatory hub in hormone and stress signaling. Plant Physiol. 2015;169(1):32–41.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Parwez R, Aftab T, Gill SS, Naeem M. Abscisic acid signaling and crosstalk with phytohormones in regulation of environmental stress responses. Environ Exp Bot. 2022;199:104885.

    Article  CAS  Google Scholar 

  33. Wang KL, Li H, Ecker JR. Ethylene biosynthesis and signaling networks. Plant Cell. 2002;14:131–151.

  34. Wawrzyńska A, Sirko A. To control and to be controlled: understanding the Arabidopsis SLIM1 function in sulfur deficiency through comprehensive investigation of the EIL protein family. Front Plant Sci. 2014;5:575.

    PubMed  PubMed Central  Google Scholar 

  35. Dong X, Han B, Yin X, Mao P, Luo D, Zhou Q, Liu Z. Genome-wide identification of the GRAS transcription factor family in autotetraploid cultivated alfalfa (Medicago sativa L.) and expression analysis under drought stress. Ind Crop Prod. 2023;194:116379.

    Article  CAS  Google Scholar 

  36. Bailey TL, Williams N, Misleh C, Li WW. MEME: discovering and analyzing DNA and protein sequence motifs. Nucleic Acids Res. 2006;34:369–73.

  37. Marchler-Bauer A, Bo Y, Han L, He J, Lanczycki CJ, Lu S, Chitsaz F, Derbyshire MK, Geer RC, Gonzales NR, et al. CDD/SPARCLE: functional classification of proteins via subfamily domain architectures. Nucleic Acids Res. 2017;45(D1):D200–3.

    Article  CAS  PubMed  Google Scholar 

  38. Salih H, He S, Li H, Peng Z, Du X. Investigation of the EIL/EIN3 transcription factor gene family members and their expression levels in the early stage of cotton fiber development. Plants (Basel). 2020;9(1):128.

    CAS  PubMed  Google Scholar 

  39. Sun S, Ma W, Mao P. Genomic identification and expression profiling of WRKY genes in alfalfa (Medicago sativa) elucidate their responsiveness to seed vigor. BMC Plant Biol. 2023;23(1):568.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Magadum S, Banerjee U, Murugan P, Gangapur D, Ravikesavan R. Gene duplication as a major force in evolution. J Genet. 2013;92(1):155–61.

    Article  PubMed  Google Scholar 

  41. Li Q, Shen Y, Guo L, Wang H, Zhang Y, Fan C, Zheng Y. The EIL transcription factor family in soybean: genome-wide identification, expression profiling and genetic diversity analysis. FEBS Open Bio. 2019;9(4):629–42.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Fatma M, Asgher M, Iqbal N, Rasheed F, Sehar Z, Sofo A, Khan NA. Ethylene Signaling under Stressful environments: analyzing collaborative knowledge. Plants (Basel). 2022;11(17):2211.

    CAS  PubMed  Google Scholar 

  43. Hu C, Wang M, Zhu C, Wu S, Li J, Yu J, Hu Z. A transcriptional regulation of ERF15 contributes to ABA-mediated cold tolerance in tomato. Plant Cell Environ. 2024;47(4):1334–47.

    Article  CAS  PubMed  Google Scholar 

  44. Wang XQ, Han LH, Zhou W, Tao M, Hu QQ, Zhou YN, Li XB, Li DD, Huang GQ. GhEIN3, a cotton (Gossypium hirsutum) homologue of AtEIN3, is involved in regulation of plant salinity tolerance. Plant Physiol Biochem. 2019;143:83–93.

    Article  CAS  PubMed  Google Scholar 

  45. Zhou Q, Luo D, Chai X, Wu Y, Wang Y, Nan Z, Yang Q, Liu W, Liu Z. Multiple regulatory networks are activated during cold stress in Medicago sativa L. Int J Mol Sci. 2018;19(10):3169.

    Article  PubMed  PubMed Central  Google Scholar 

  46. Dong X, Deng H, Ma W, Zhou Q, Liu Z. Genome-wide identification of the MADS-box transcription factor family in autotetraploid cultivated alfalfa (Medicago sativa L.) and expression analysis under abiotic stress. BMC Genomics. 2021;22(1):603.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Luo D, Wu Y, Liu J, Zhou Q, Liu W, Wang Y, Yang Q, Wang Z, Liu Z. Comparative transcriptomic and physiological analyses of Medicago sativa L. indicates that multiple regulatory networks are activated during continuous ABA treatment. Int J Mol Sci. 2018;20(1):47.

    Article  PubMed  PubMed Central  Google Scholar 

Download references

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).

Author information

Authors and Affiliations

  1. College of Grassland Science and Technology, China Agricultural University, Beijing, 100193, China

    Xinru Su, Juan Wang, Shoujiang Sun, Wenxin Peng, Manli Li, Peisheng Mao & Liru Dou

Authors
  1. Xinru Su
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Juan Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shoujiang Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Wenxin Peng
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Manli Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Peisheng Mao
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Liru Dou
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D.L. and S.X. designed the project; S.X. and W.J. performed the data analysis; S.X., S.S., P.W. and W.J. interpreted the data and results; W.J. was responsible for planting materials; S.X. wrote the manuscript; and D.L., S.S., L.M., M.P. and W.J. carried out thin and tall revisions. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Liru Dou.

Ethics declarations

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.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Su, X., Wang, J., Sun, S. et al. Genome-wide identification of the EIN3/EIL transcription factor family and their responses under abiotic stresses in Medicago sativa. BMC Plant Biol 24, 898 (2024). https://doi.org/10.1186/s12870-024-05588-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12870-024-05588-2

Keywords

BMC Plant Biology

ISSN: 1471-2229

Contact us