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Article

Genome-Wide Identification of MsICE Gene Family in Medicago sativa and Expression Analysis of the Response to Abiotic Stress

by
Baiji Wang
,
Qianning Liu
,
Wen Xu
,
Yuying Yuan
,
Muzhapaer Tuluhong
,
Jinqiu Yu
* and
Guowen Cui
*
College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, China
*
Authors to whom correspondence should be addressed.
Agronomy 2024, 14(9), 2064; https://doi.org/10.3390/agronomy14092064
Submission received: 20 June 2024 / Revised: 30 July 2024 / Accepted: 4 September 2024 / Published: 9 September 2024
(This article belongs to the Collection Abiotic Stress Tolerance in Plants: Towards a Sustainable Agriculture)
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Abstract

:
To predict the role of the MsICE gene family in the response to abiotic stress, in this study, bioinformatics analysis and real-time fluorescence quantitative PCR were performed. Alfalfa (Medicago sativa) is one of the most economically valuable crops globally. Inducer of CBF expression (ICE), which is part of the basic helix–loop–helix (bHLH) transcription factor (TF) family, acts as a key regulator of cold tolerance. Despite this, there is little information available about ICE genes in alfalfa. Therefore, we studied the function of ICE TFs in alfalfa. We identified 11 MsICE genes from the alfalfa genome and classified them into two groups. Analysis of the protein motif and gene structure revealed relatively high conservation among subgroups of the tightly clustered MsICE genes. Through synteny analysis, we detected duplication events in the MsICE gene family, suggesting that the ICE gene family was formed through fragment duplications. All the MsICE proteins were located in the nucleus according to subcellular localization predictions. The promoter cis-regulatory elements of MsICE genes are largely involved in light (Box 4), hormone (ABRE), and stress (MYB) responses. The MsICE01/MsICE07/MsICE09/MsICE10/MsICE11 genes contained MYB- and MYC-binding motifs, indicating an association with abiotic stress. The specific expression patterns of MsICE genes in leaves were revealed by examining their expression patterns in different tissues. These findings suggest that these genes may sense external environmental changes through leaves. Abiotic stress can cause striking upregulation of MsICE07 (PCA score: −4.03) and MsICE10 (PCA score: −4.05) expression. In this study, candidate genes associated with cold stress were identified, and subsequent molecular biological analyses allowed elucidation of the biological functions of these genes in alfalfa. This research provides a theoretical foundation for enhancing alfalfa yield and quality under cold conditions.

1. Introduction

Alfalfa (Medicago sativa) is an important perennial leguminous forage grass that is renowned for its high biomass, rich nutritive value, good palatability, and wide adaptability [1,2]. Alfalfa cultivation in China is primarily concentrated in northern regions, where the stability of the production system is influenced by cold winters and frequent spring inversions. These climatic conditions result in challenges related to overwintering and low rates of alfalfa rejuvenation. In countries with limited farmland for alfalfa agriculture, the capacity to withstand unfavourable environmental stimuli represents a significant challenge to productivity [3].
The earliest research on inducer of CBF expression (ICE) transcription factors (TFs) can be traced back to the model plant Arabidopsis thaliana. Within 15 min of low temperature exposure, CBF transcripts begin to accumulate in plants; a TF that recognizes the CBF promoter and induces CBF expression under low-temperature stress already exists in cells at normal growth temperatures, and Gilmour named this TF “ICE” [4]. ICE, which belongs to the basic helix–loop–helix (bHLH) TF family, regulates the response to cold stress by binding to downstream CBF genes [5,6]. On the basis of previous sequence comparisons and structural analyses of the ICE gene family in different species, we found that the N-terminus has an S-rich motif, whereas the conserved bHLH and ACT structural domains are located at the C-terminus, where the bHLH structural domains contain the ICE-specificity sequence KMDRASILGDAI(D/E)YLKELL [7]. The advancement of bioinformatics has facilitated the identification of members of the ICE gene family in an increasing number of species, including tomato [8], eggplant [7], Chinese wild-growing Vitis amurensis [9], rice [10], walnut [11], Eucalyptus camaldulensis [12], and indica rice [13], which has enabled the analysis of these genes and their subsequent functional identification. The ICE-CBF-COR cascade is critical for plant survival under cold stress [14]. It has been shown that overexpression of the TaICE41 or TaICE87 genes enhances freezing tolerance in A. thaliana upon cold domestication [5]. Guo et al. utilized RNAseq and qPCR to analyze gene expression in wheat tissues subjected to a range of stress conditions, focusing on 53 genes involved in the ICE-CBF-COR signalling pathway. These findings demonstrated distinct patterns of gene expression in different tissues, highlighting the tissue-specific responses of the ICE, CBF, and COR genes to various stress stimuli. Notably, TaCBF1b, TaCBF4a, and TaCOR3b were specifically upregulated in response to cold stress [5]. Recent research has shown that StICE1 can interact with the promoter region of the StLTI6A gene, potentially playing a role in preserving cell membrane integrity and enhancing the expression of StLTI6A in cold environments. These results suggest that StICE1 may have a direct effect on the regulation of the StLTI6A, CBF, and COR genes under cold stress [15]. In addition, we found that ICE genes play regulatory roles in processes such as seed germination [16] and anther development [17]. For example, our findings indicate that ICE1 plays a role in regulating plant male fertility, which is achieved by influencing anther dehydration. Loss-of-function mutation in the ICE1 gene in A. thaliana has been shown to cause anther indehiscence and reduce pollen viability as well as germination rate [16]. ICE1 negatively regulates the ABA response during seed germination by maintaining appropriate ABA signalling levels during seed germination and thus regulates the seed germination process [17]. Consequently, investigating the biological functions of ICE TFs is highly important.
The results of the present study confirmed that ICE genes play crucial roles in regulating plant growth and development and the response to abiotic stresses. To address the difficulties associated with alfalfa overwintering, this study focused on the biological functions of ICE under cold stress [18]. The comprehensive genomic data of the autotetraploid variety “Zhongmu No. 1” have been previously published. Additionally, the chromosome-level haploid genome sequence for “Zhongmu No. 1” alfalfa, which is a heterozygous autotetraploid, has also been reported [19]. A whole-genome analysis of the MsICE gene family has become possible due to rapid advances in high-throughput sequencing technology and bioinformatics [20]. A small number of systematic studies on MsICE TFs in alfalfa have been conducted, and the MsICE gene family has not been identified in alfalfa. Using the “Zhongmu No. 1” genome of alfalfa, we systematically investigated the MsICE gene family at the whole-genome level. Furthermore, in addition to performing a comprehensive bioinformatics analysis of MsICEs, we analyzed tissue-specific expression patterns and differential expression in response to various abiotic stresses. Our study provides a systematic analysis of the MsICE gene family in alfalfa, pinpoints candidate genes associated with cold stress, and lays a foundation for breeding cold-resistant plants of this important forage crop.

2. Experimental Materials and Methods

2.1. Plant Materials and Treatments

The alfalfa material used in this experiment was “Zhongmu No. 1”, the full seeds of which were planted in a mixture of nutrient soil and vermiculite (3:1). The soil was regularly watered with 1/10-strength Hoagland nutrient solution, and at 30 d after sowing, random alfalfa plants were used to determine the tissue expression specificity and expression patterns under different stress treatments. Stems, roots, and young and mature leaves of alfalfa were collected for RNA extraction and analyzed by real-time fluorescence quantitative PCR (qRT–PCR). Alfalfa plants were treated with 15% polyethylene glycol (PEG) 6000, 150 mM NaCl, and 150 mM NaHCO3 for drought, salt, and alkali treatment and examined at a total of five time points (0, 3, 6, 12, and 24 h), where 0 h was the control. Cold stress treatment was performed on alfalfa seedlings in an artificial climate incubator at 4 °C, followed by examination at five time points, with 0 h as the control period. For each treatment, three independent replicates were conducted, and RNA was extracted from frozen tissues immediately after collection in liquid nitrogen at −80 °C.

2.2. RNA Extraction and Gene Expression Analysis

Total RNA was extracted from the samples via an Ultrapure RNA Kit (CoWin Biotech, Beijing, China) according to the protocol provided by the manufacturer. The qRT–PCR primers specific for the MsICE genes were designed via the Primer-BLAST tool on the NCBI website.
First-strand cDNA synthesis was performed via oligo (dT) 10 reverse primers according to the instructions of the HiScript II Q Select Reverse Transcriptase Kit (Vazyme Biotech, Nanjing, China). Quantitative RT–PCR was performed according to the protocol for the ChamQ™ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). The alfalfa GAPDH gene was utilized as an internal reference standard. The reaction procedure was as follows: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 60 °C for 34 s, and 95 °C for 15 s [21].

2.3. Identification of the Alfalfa ICE Gene Family

The hidden Markov model PF00010 for bHLH, a conserved structural domain of the MsICE TFs, was obtained from the InterPro database [22].
The alfalfa genome, CDSs, and protein sequence files were downloaded from (https://figshare.com/articles/dataset/Medicago_sativa_genome_and_annotation_files/12623960; accessed on 9 July 2020). The MsICE protein-encoding gene family from the alfalfa genome was identified via HMMER 3.0, with the E value set to less than 1 × 10−5. After removing duplicate entries and integrating the alfalfa ICE sequence information obtained from the above steps, the candidate genes were submitted to Pfam [23] for structural domain validation, and the candidate genes that did not contain the ACT and bHLH structural domains were excluded. Each ICE member was named according to the distribution order of MsICEs on the alfalfa chromosomes. Protein physicochemical properties such as length (L), molecular weight (MW), and isoelectric point (pI) were predicted via the ExPASy ProtParam (www.expasy.org; accessed on 15 August 2023) online analysis tool [24]. Furthermore, the subcellular localization of alfalfa ICE proteins was predicted using the WoLF PSORT tool [25].

2.4. Phylogenetic Analysis of Alfalfa ICE Genes

To elucidate the evolutionary relationships among MsICE proteins, multiple sequence alignments were conducted on the protein sequences of MsICE, AtICE, and MtICE using MEGA 7.0 [26]. An evolutionary tree was constructed via the neighbour-joining (NJ) method, with 1000 bootstrap values and other parameters set to default values. The phylogenetic tree image was enhanced via the Evolview online programme [27].

2.5. Gene Structure and Conserved Motif and Domain Analyses

The conserved domains of ICE gene family members were identified via the CDD-Search tool on the NCBI website [28]. The evolutionary relationships among candidate ICE family members were analyzed using MEGA 7.0 [26]. The online software MEME 5.0 was used to predict the conserved motifs of MsICE, in which the parameters were adjusted as follows: the motif value was 10, and default values were used for other parameters [29]. Visualization of the evolutionary tree, domains, and motifs of the MsICE gene family members was completed via TBtools software II [30].

2.6. Analysis of Alfalfa ICE Gene Promoters

Using the Plant CARE database [31], cis-acting elements were identified from the promoter regions of alfalfa ICE genes (2000 bp upstream). Visualization and analysis were performed via TBtools software [30].

2.7. Identification of Intra- and Interspecies Covariates of MsICE Family Members

The ICE genes within the alfalfa genome were analyzed and visualized via the Advanced Circos feature of TBtools software [31].
The collinearity between MsICE family members and A. thaliana was plotted separately using the Dual Systeny Plot feature of the McscanX tool in TBtools [30].

2.8. Statistical Analyses and Principal Component Analysis

Each experiment consisted of three independent biological and technical replicates, and the data were analyzed for significance via the 2−ΔΔct method to calculate the relative expression of genes at different treatment times [32].
The statistical analyses were performed via SPSS 22 software. Differences were assessed using ANOVA with a significance level of p < 0.05, as determined via the Duncan multiple range test. Heatmaps were generated via TBtools [30].
Pearson correlation was used to assess the relationships between various variables, and the Shapiro–Wilk test was conducted to confirm the normality of the variables prior to conducting principal component analysis (PCA). Bartlett’s sphericity and Kaiser–Meyer–Olkin (KMO) tests were also performed. PCA was performed via XLSTAT software version 2019 [14].

3. Results

3.1. Identification of the MsICE Gene Family in Alfalfa

By using the conserved bHLH structural domain (accession number PF00010) in an HMM profile, a total of 11 MsICE genes were identified, and these genes were named MsICE1-MsICE11 on the basis of their chromosomal locations. We analyzed the MsICE genes as well as the physicochemical properties of their encoded proteins. The proteins ranged from 254 to 525 aa in length, 28,750.39 to 59,537.9 kDa in molecular weight, and 4.97 to 9.12 in isoelectric point, with 11 MsICE genes unevenly distributed on chromosomes 1–7 (Figure 1). Chromosome 5 contained up to four MsICE genes, chromosome 3 contained three MsICE genes, and the remaining chromosomes contained only one MsICE gene each. By subcellular localization analysis, all the MsICE genes were predicted to localize to the cytoplasm. The specific results are tabulated below (Table 1).

3.2. Multiple Sequence Alignment and Phylogenetic Tree Analysis of the MsICE Gene Family in Alfalfa

Through multiple sequence alignment, we found that the alfalfa ICE contains two typical BHLH and ACT domains. Furthermore, we found that only five members contained a relatively complete ACT domain, while in the remaining members, only a partial ACT domain was present (Figure 2).
To study the evolutionary relationships and classification of MsICE proteins, a total of 28 ICE proteins (11 ICE proteins from alfalfa, 12 ICE proteins from Medicago truncatula, and 5 ICE proteins from A. thaliana.) were analyzed to further study the phylogenetic relationships of the MsICE gene family in alfalfa, and a phylogenetic tree was constructed using MEGA software 7.0 (Figure 3). The phylogenetic tree was distributed in two clear groups; Group (1) contained six MsICEs, and Group (2) contained five MsICEs. Our observations revealed that MsICE08 in Group (1) has one gene ortholog in A. thaliana. Except MsICE08, the members of the MsICE gene family are all closely related to the members of the M. truncatula ICE gene family, exhibiting close evolutionary relationships, which suggests that they may have similar functions.

3.3. Gene Structure and Motif Composition of the MsICE Gene Family in Alfalfa

To further investigate the structural features of MsICEs, the conserved motifs of the encoded proteins were predicted via MEME. According to the results, there were 4–17 conserved motifs and motif 1, motif 2, and motif 3 were shared by all MsICEs, whereas motif 5 was shared by only two MsICEs. Motif 1, motif 2, and motif 5 represented the bHLH_SF superfamily domain, the ACT_UUR-ACR-like domain, and the bHLH-MYC_N structural domain, respectively (located at the N-terminus) (Figure 4C). These results indicated that the bHLH_SF superfamily domain and the ACT_UUR-ACR-like domain are conserved within the MsICE gene family, indicating that motif 1 and motif 2 are important motifs in the MsICE gene family. Interestingly, only MsICE09 and MsICE10 contained the bHLH-MYC_N structural domain, suggesting that MsICE09 and MsICE10 may have other gene functions. The observed similarity in gene structures among the MsICE subfamily members aligns with their phylogenetic relationships, as depicted in Figure 4A. The gene structure analysis (Figure 4D) revealed that members of the MsICE gene family contained 1–6 introns and 2–7 exons, and the MsICE01 gene was the most complex, containing 6 introns and 7 exons. Genes within the same cluster had similar structures, and these findings were combined with results from conserved domain analysis to suggest that they might have similar functions.

3.4. Analysis of the Cis-Acting Elements in the Promoters of MsICE Genes

To gain a more comprehensive understanding of inducible factors and gene functions, we used TBtools software to obtain the sequences of the 2000 bp regions upstream of the start site of MsICE genes and analyzed the cis-acting elements, which were systematically classified into three categories: hormone-related, light-related, and stress-related elements. As shown in the results (Figure 5), all MsICE genes have light-responsive elements (e.g., GT1-mtoif) (class 17), and all MsICE genes except MsICE07 contain hormone-responsive elements (class 8), suggesting that these genes are involved in both light and hormone regulation. Among the stress response elements, all genes except MsICE08 contained anaerobic-induced regulatory elements (AREs), and we also predicted that MYB and MYC were associated with environmental adaptation response elements. More importantly, we found that four genes contained low-temperature-responsive regulatory (LTR) elements. Thus, it is hypothesized that the MsICE gene family is involved in the response to phytohormones and cold stress.

3.5. Analysis of the Collinearity of MsICE Genes

We performed collinearity analysis via TBtools software to identify gene duplication events in the MsICE gene family, and the results are shown in Figure 6A. We found that MsICE09 and MsICE10 were related to each other and classified into the same subclass, which suggested that the abovementioned genes were formed through fragment duplications. To further investigate the evolutionary mechanisms of the MsICE gene family, we analyzed the collinearity of MsICE genes from alfalfa and A. thaliana (Figure 6B). A covariance map of MsICE TFs in alfalfa and A. thaliana. was constructed, and the results revealed four homologous pairs between alfalfa and A. thaliana., indicating a high degree of affinity between the MsICE and AtICE genes.

3.6. Tissue-Specific Expression of MsICEs

Gene expression analysis in plant tissues and developmental phases provides crucial information about biological function [33]. To clarify the expression patterns of MsICEs in alfalfa, the expression levels of MsICEs in roots, stems, young leaves, mature leaves, and flowers were analyzed via qRT–PCR (Figure 7). The analysis showed that all MsICE genes were expressed at higher levels in vegetative organs than in reproductive organs. Nearly all the genes presented tissue-specific transcript accumulation patterns, with only one gene (MsICE11) showing analogous expression profiles in different tissues. The expression of MsICE02/MsICE06/MsICE09/MsICE10 was higher in roots than in other tissues. In addition, we found that MsICE03/MsICE05/MsICE07 presented relatively high expression in young leaves, while MsICE04/MsICE08 presented relatively high expression in mature leaves. Based on these results, MsICE genes may play different roles in alfalfa growth and development.

3.7. Expression Analysis of Alfalfa MsICE Genes under Abiotic Stress

Since the promoters of MsICE genes contain many abiotic stress-related regions, qRTP–CR was used to examine the expression patterns of MsICE genes in response to various abiotic stressors (Figure 8). After drought treatment (Figure 8A), the expression of MsICE genes, particularly MsICE07, MsICE09, and MsICE10, was significantly upregulated. In addition, we found that MsICE01/MsICE02/MsICE04/MsICE10/MsICE11 were upregulated after 1 h of drought treatment, with the levels first increasing, then decreasing and then increasing again. Under cold stress (Figure 8B), MsICE01/MsICE07/MsICE10 exhibited upregulation within 1 h of treatment, followed by a gradual decrease, in comparison with the control (0 h). These results demonstrate that ICE genes respond positively to cold stress in alfalfa. In response to salt treatment (Figure 8C), all genes were upregulated, except MsICE08, which indicated that these genes actively respond to salt stress. Under alkaline stress (Figure 8D), the expression of MsICE05/MsICE07/MsICE09/MsICE10 was significantly greater than that of other MsICE genes. In conclusion, MsICE10 presented increased expression levels in response to abiotic stress, suggesting that MsICE10 plays a pivotal role in the resistance to abiotic stresses.

3.8. Correlations between Different Stress Treatment Durations and the Level of Gene Expression

Principal component analysis (PCA) was used to evaluate the relationships between gene expression under different drought stress treatment durations (0 h, 1 h, 3 h, 12 h, and 24 h). The total value for the first two PCA components was 62.90% (PCA1 = 41.00% and PCA2 = 21.90%) after drought treatment (Figure 9A). A robust positive relationship was detected between 1 h and 24 h, as well as between 6 h and 12 h. Furthermore, the highest expression of MsICE10 under drought stress was considered for all stress treatment time points combined (Figure 9B). Under different cold stress durations, the total value for the first two PCA components was 62.90% (PCA1 = 32.80% and PCA2 = 26.50%) after drought treatment. We detected a positive relationship at 3 h, 6 h, and 12 h (Figure 9C). The highest expression level of MsICE10 under cold stress was considered for all stress treatment time points combined (Figure 9D). Under different salt stress durations, the total value for the first two PCA components was 68.10% (PCA1 = 51.40% and PCA2 = 16.70%) after salt treatment. We did not find a positive relationship at any stress treatment time point (Figure 9E). The highest expression of MsICE07 under salt stress was considered for all stress treatment time points combined (Figure 9F). Under different alkaline stress durations, the total value for the first two components of the PCA was 68.10% (PCA1 = 49.10% and PCA2 = 77.60%) after salt treatment. We did not find a positive relationship at any stress treatment time point (Figure 9G). The highest expression of MsICE07 under salt stress was considered for all stress treatment time points combined (Figure 9H).

4. Discussion

Since ICE genes were discovered in A. thaliana, a growing number of studies have confirmed that ICE genes play important roles in plant growth and development, as well as in the response to abiotic stress [15,16,17]. In this study, a total of 11 MsICE genes were identified based on the “Zhongmu No. 1” alfalfa genome, and we systematically investigated the MsICE gene family. Abiotic stress has been shown to significantly increase the expression of MsICE07 and MsICE10. These findings suggest that MsICEs may play a role in the adaptation of forage plants to environmental stimuli.
More MsICEs were detected in alfalfa than in diploid cotton [34]; this may be attributed to the tetraploid nature of the former. According to the results of the subcellular localization predictions, all the MsICE proteins were located in the nucleus. This finding is consistent with the subcellular localization results for the ICE gene family in walnut [11], which indicates that ICEs may function as nuclear TFs. In this study, we found that MsICEs identified in alfalfa shared conserved bHLH and ACT domains. However, only five MsICEs contained a complete ACT domain, while the remaining contained partial ACT domains. This could be the result of sequence breaks during evolution or sequencing errors in the alfalfa genome [35]. Thus, domain gain and domain loss are different factors driving the expansion of the ICE gene family [36,37].
Phylogenetic tree analysis revealed that the 11 identified MsICE proteins could be classified into two subgroups, which is inconsistent with previous evolutionary classifications in cotton [34]. MsICE05 was found to exhibit close homology with AtICE1 and AtICE2. AtICE1 is known to play key roles in regulating plant male fertility [15] and negatively modulating the ABA response during seed germination [17]. On the basis of this information, it is hypothesized that the MsICE05 gene may share similar functional characteristics to AtICE01. An analysis of the gene structure of the 11 MsICE genes revealed that they contained between one and six introns. However, a majority of the genes contained only one intron. The number of introns reflects the characteristics of the gene, with genes exhibiting a low number of introns being regulated more quickly [38]. Fewer introns also indicate shorter transcription duration, which may facilitate rapid response to stress [39]. Also, we found that plant species differed in the location of exons, suggesting ICE genes play a crucial role in plant evolution [40]. Furthermore, we identified a group of genes that evolved through segmental gene duplication. Thus, the occurrence of duplication events implies that fragment duplication is the main factor driving the expansion of ICE genes in alfalfa.
In the gene promoter region, cis-acting elements play a significant role in regulating gene expression. Different types of cis-acting elements in the gene promoter indicate that the gene may play a variety of stress-related functions [41,42]. In this study, we found that many cis-acting elements in upstream ICE promoter regions are associated with responsiveness to light, hormones, and stress, suggesting that MsICE genes may be involved in the regulation of photoresponses and hormone and stress responses. Phytohormones participate in numerous processes throughout the life cycle of a plant [43]. In this study, we found that MsICE01/MsICE02/MsICE03/MsICE05/MsICE06/MsICE11 contain abscisic acid (ABA)-responsive elements in their promoters. Numerous excellent reviews have indicated that the ABA signalling pathway regulates the response of plants to stress [44]. Similarly, several light-responsive cis-elements that are broadly distributed in MsICE promoter regions have been identified [45]. Furthermore, the MsICE05, MsICE08, MsICE09, and MsICE11 genes contained LTR elements, suggesting that they may participate in the response to cold stress. Studies have shown that some ICE genes, such as JreICE1b, are involved in cold stress responses when they contain specific cis-elements in their promoters [11].
It is well known that cold is a major environmental factor that limits alfalfa yield and growth in cold regions [46]; thus, investigating the genes involved in cold tolerance in alfalfa cultivation and production is critical. Since ICE genes play significant roles in plant growth and stress response, investigating the function of each ICE family member in alfalfa is necessary [47]. In this study, the MsICE03/MsICE05/MsICE07 expression patterns were similar to those in cotton [34], with high expression in young leaves, followed by mature leaves, roots, and stems. In contrast, MsICE04/MsICE08 was highly expressed in mature leaves, followed by young leaves, roots, and stems. As previous findings have shown, the leaf is believed to be the first organ that senses and responds to changes outside [48]. These results indicate that ICE genes may perceive and respond to cold stress in leaves. In addition, a majority of the genes in Group (1) were highly expressed in the roots. It can be postulated the majority of the members in Group (1) may facilitate the growth and development of the root system, thereby increasing the ability of the plant to absorb nutrients through a robust root system, which in turn stimulates the rapid growth of the plant [49]. Although ICE genes have multiple functions, we focused on their response to abiotic stress. As plants have evolved to adapt to biotic and abiotic stresses, they have expressed genes that modulate physiological response processes, which improve their ability to cope with stress [50,51]. The ICE gene has previously been identified as a key regulator of the cold acclimation process and is responsible for regulating plant responses to cold stress [6,52].
The results of this study demonstrated that the expression of MsICE01, MsICE07, and MsICE10 increased significantly within one hour of cold stress and then gradually decreased. It is hypothesized that ICE genes exhibit peak activity at 1 h, and they are classified as short-acting genes [47]. In addition, MsICE07 contains many MYB elements, and this gene is markedly upregulated under drought stress. Our findings align with those of prior studies indicating that MYB proteins serve as transcriptional activators that promote gene expression in response to abiotic stress in plants [53]. Under salt stress, the expression patterns of MsICE09 and MsICE10 were observed to be analogous, consistent with their close phylogenetic relationship. Previous studies have mainly focused on the role of ICE genes in cold, drought, and salt stress. Conversely, there is a paucity of studies examining the function of ICE genes under alkaline stress in alfalfa [54]. This study demonstrated that MsICE07 exhibited a robust response to alkali stress, suggesting a crucial role for ICE genes in mediating the response to alkaline stress. Additionally, the expression levels of MsICE07 and MsICE10 were markedly elevated under various abiotic stresses. To address the challenges associated with overwintering in alfalfa, future research should investigate the physiological response mechanisms of transgenic lines, thereby offering molecular insights into the biological functions of MsICEs in alfalfa under cold stress conditions.

5. Conclusions

A total of 11 MsICE genes were identified and subsequently classified into two primary groups in alfalfa. The gene structures and conserved motifs were similar within the groups, strongly confirming that the phylogenetic classification was reliable. Among the putative MsICE promoters, we identified cis-elements associated with the abiotic stress response. Cold stress could strikingly upregulate MsICE07 and MsICE10 expression. In future research, we will explore the functions of MsICE07 and MsICE10 and analyze the genetic transformation of MsICE07 and MsICE10, providing a basis for further investigations of the cold stress response.

Author Contributions

B.W.: writing—original draft, data curation, conceptualization. Q.L.: software, validation. W.X.: investigation, data curation, software. Y.Y.: supervision, resources, validation. M.T.: writing—review and editing, validation, data curation. J.Y.: writing—review and editing, resources. G.C.: funding acquisition, writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by research and the Natural Science Foundation of Breeding Technology Innovation and New Provenance Creation of Cold-resistant and High-yield Alfalfa [2022ZD040120401].

Data Availability Statement

The original contributions presented in the study are included in the article; further inquiries can be directed to the corresponding author/s.

Acknowledgments

I would like to thank my mother and father and my best friend, Bingbing Du, who is always my strongest supporter!

Conflicts of Interest

Researchers declare that they have no financial or commercial relationships that may pose conflicts of interest in the research.

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Figure 1. Illustration of the chromosomal location of the MsICEs, with vertical bars corresponding to the chromosomes of alfalfa and lengths indicating the relative size of the chromosomes. Left-hand side shows the scale (Mb) of chromosome length.
Figure 1. Illustration of the chromosomal location of the MsICEs, with vertical bars corresponding to the chromosomes of alfalfa and lengths indicating the relative size of the chromosomes. Left-hand side shows the scale (Mb) of chromosome length.
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Figure 2. MsICEs multi-sequence alignment of conserved domains. (A) The black box represents bHLH-ZIP domain. (B) The black box represents ACT-like domain.
Figure 2. MsICEs multi-sequence alignment of conserved domains. (A) The black box represents bHLH-ZIP domain. (B) The black box represents ACT-like domain.
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Figure 3. Phylogenetic analysis of ICE proteins in alfalfa, M. truncatula, and A. thaliana. Clades in purple and orange branches refer to Group (1) and Group (2), respectively. The stars, triangles, and circles represent ICE domains from alfalfa, M. truncatula, and A. thaliana, respectively.
Figure 3. Phylogenetic analysis of ICE proteins in alfalfa, M. truncatula, and A. thaliana. Clades in purple and orange branches refer to Group (1) and Group (2), respectively. The stars, triangles, and circles represent ICE domains from alfalfa, M. truncatula, and A. thaliana, respectively.
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Figure 4. Phylogenetic relationships and structural characteristics of ICE genes from alfalfa, as well as the architectural features of their conserved protein motifs. (A) A phylogenetic tree was constructed via the neighbour-joining (NJ) method in MEGA 7. (B) The motifs of MsICE proteins are illustrated with different coloured boxes numbered 1–10. (C) Functional domain distributions of MsICEs. The coloured rectangles represent the conserved protein domains. (D) Alfalfa ICE genes are shown in the accompanying diagram, with light-green boxes indicating exons and black lines indicating introns.
Figure 4. Phylogenetic relationships and structural characteristics of ICE genes from alfalfa, as well as the architectural features of their conserved protein motifs. (A) A phylogenetic tree was constructed via the neighbour-joining (NJ) method in MEGA 7. (B) The motifs of MsICE proteins are illustrated with different coloured boxes numbered 1–10. (C) Functional domain distributions of MsICEs. The coloured rectangles represent the conserved protein domains. (D) Alfalfa ICE genes are shown in the accompanying diagram, with light-green boxes indicating exons and black lines indicating introns.
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Figure 5. Heatmaps are presented to depict predicted cis-acting elements within the promoters of ICE family members of alfalfa, where red indicates high expression levels and blue indicates low expression levels.
Figure 5. Heatmaps are presented to depict predicted cis-acting elements within the promoters of ICE family members of alfalfa, where red indicates high expression levels and blue indicates low expression levels.
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Figure 6. Collinearity analysis of MsICE genes. (A) Analyses of ICE gene sequences in alfalfa. Grey lines indicate synteny blocks and red lines indicate ICE genes that have been duplicated. (B) Genome-wide collinearity analysis for ICE genes among alfalfa and A. thaliana. Red lines indicate orthologous gene pairs.
Figure 6. Collinearity analysis of MsICE genes. (A) Analyses of ICE gene sequences in alfalfa. Grey lines indicate synteny blocks and red lines indicate ICE genes that have been duplicated. (B) Genome-wide collinearity analysis for ICE genes among alfalfa and A. thaliana. Red lines indicate orthologous gene pairs.
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Figure 7. Expression profiles of 11 MsICE genes in different alfalfa tissues. The expression levels were determined via qRT–PCR, with red indicating high expression levels and blue indicating low expression levels.
Figure 7. Expression profiles of 11 MsICE genes in different alfalfa tissues. The expression levels were determined via qRT–PCR, with red indicating high expression levels and blue indicating low expression levels.
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Figure 8. Relative expression levels of MsICE genes in response to different stress treatments: (A) Drought; (B) Cold; (C) Nacl; (D) Alkaline. RNA extracts from treated leaves were analyzed after 1 h, 3 h, 6 h, 12 h, and 24 h of treatment, and qRT–PCR was performed with specific primers for MsICE genes. MsGAPDH was used as an internal reference. Each time point had its own control, and the 0 h expression served as the reference. As a result of Student’s t-test, lowercase letters indicate significant differences between MsICEs at p < 0.05.
Figure 8. Relative expression levels of MsICE genes in response to different stress treatments: (A) Drought; (B) Cold; (C) Nacl; (D) Alkaline. RNA extracts from treated leaves were analyzed after 1 h, 3 h, 6 h, 12 h, and 24 h of treatment, and qRT–PCR was performed with specific primers for MsICE genes. MsGAPDH was used as an internal reference. Each time point had its own control, and the 0 h expression served as the reference. As a result of Student’s t-test, lowercase letters indicate significant differences between MsICEs at p < 0.05.
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Figure 9. Piplot of the PCA (A,C,E,G) describing the relationships among different stress treatment times. Piplot of the PCA (B,D,F,H) describing the relationship between gene expression treatment time and the level of gene expression.
Figure 9. Piplot of the PCA (A,C,E,G) describing the relationships among different stress treatment times. Piplot of the PCA (B,D,F,H) describing the relationship between gene expression treatment time and the level of gene expression.
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Table 1. Alfalfa ICE gene characterization. Amino acid (aa): the length of a protein is usually expressed in amino acids (aa). Kilodalton (kDa): the molecular weight of a protein is usually expressed in kilodalton (kDa).
Table 1. Alfalfa ICE gene characterization. Amino acid (aa): the length of a protein is usually expressed in amino acids (aa). Kilodalton (kDa): the molecular weight of a protein is usually expressed in kilodalton (kDa).
Sequence IDChromosome LocationGene LocationProtein Length (aa)MW (kDa)pIPredicted Localization
MsICE01MsG0180006048.01.T01Chr199365979:9936869952559,537.904.97Nucleus
MsICE02MsG0280006829.01.T01Chr26830114:683233025628,845.747.51Nucleus
MsICE03MsG0380012767.01.T01Chr324141581:2414452237842,872.417.16Nucleus
MsICE04MsG0380015497.01.T01Chr371003358:7100537629032,874.59.12Nucleus
MsICE05MsG0380016999.01.T01Chr390463402:9046631151356,524.265.84Nucleus
MsICE06MsG0480021960.01.T01Chr466477953:6647946326029,203.245.85Nucleus
MsICE07MsG0580025575.01.T01Chr520742892:2074440733137,421.684.99Nucleus
MsICE08MsG0580027082.01.T01Chr552871922:5287505339845,171.655.24Nucleus
MsICE09MsG0580027951.01.T01Chr571650578:7165157125428,750.397.59Nucleus
MsICE10MsG0580028245.01.T01Chr577182213:7718316825528,807.387.59Nucleus
MsICE11MsG0780040061.01.T01Chr772566185:7256790931635,079.718.74Nucleus
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Wang, B.; Liu, Q.; Xu, W.; Yuan, Y.; Tuluhong, M.; Yu, J.; Cui, G. Genome-Wide Identification of MsICE Gene Family in Medicago sativa and Expression Analysis of the Response to Abiotic Stress. Agronomy 2024, 14, 2064. https://doi.org/10.3390/agronomy14092064

AMA Style

Wang B, Liu Q, Xu W, Yuan Y, Tuluhong M, Yu J, Cui G. Genome-Wide Identification of MsICE Gene Family in Medicago sativa and Expression Analysis of the Response to Abiotic Stress. Agronomy. 2024; 14(9):2064. https://doi.org/10.3390/agronomy14092064

Chicago/Turabian Style

Wang, Baiji, Qianning Liu, Wen Xu, Yuying Yuan, Muzhapaer Tuluhong, Jinqiu Yu, and Guowen Cui. 2024. "Genome-Wide Identification of MsICE Gene Family in Medicago sativa and Expression Analysis of the Response to Abiotic Stress" Agronomy 14, no. 9: 2064. https://doi.org/10.3390/agronomy14092064

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