Introduction

Auxin is an important plant endogenous hormone that regulates various plant life processes and plays a key role, including tissue differentiation, organogenesis, apical dominance, root formation, tropism, and response to biotic and abiotic stresses [1, 2]. Numerous plants, including Arabidopsis thaliana, have a large number of functional genes that are auxin-regulated and play a significant role in growth and development [3,4,5]. Among these genes, auxin response factor (ARF) family members play a vital role in auxin signaling and regulating the expression of auxin-responsive genes [6, 7]. Most ARFs contain three conserved functional domains: the DNA binding domain (DBD) at the N terminus, the variable intermediate region (MR) acting as the activating domain (AD) or inhibitory domain (RD), and the C-terminal dimerization domain (CTD) [8]. DBD belongs to the b3-like family, which enables ARF to specifically bind to TGTCTC auxin response element (AuxRE) in various auxin response gene promoters, thus transcriptional regulation the expression of these genes widely involved in plant growth and development [9]. Depending on its amino acid composition, MR has fewer sequences and relies on ARF as a transcription activator or repressor. The AD is rich in glutamine, serine, and leucine residues, while the RD is rich in serine, proline, leucine, and glycine residues [8,9,10]. The CTD is responsible for protein-protein interactions, such as ARF homodimerization or ARF heterodimerization [5]. ARF transcription factors and auxin/indole-3-acetic acid (Aux/IAA) protein dimers can inhibit ARF-regulated transcriptional activity in the presence of low auxin concentrations, but the degree of expression inhibition is only moderate [11]. When the auxin concentrations are high, the 26 S proteasome releases the interacting ARF protein from the inhibitory dimer structure [12, 13].

Furthermore, recent studies have reported relevant information involving interacting proteins and target genes of ARF, elucidating the specific mechanisms by which ARF regulates plant developmental processes. Arabidopsis has 22 AtARF functional genes and one pseudogene [8, 14]. The expression of ARF is precisely and dynamically regulated in different developmental stages and tissues of Arabidopsis. For example, AtARF2-4, AtARF3, AtARF5, and AtARF8 are crucial for plant floral organ differentiation and development [15,16,17]. AtARF7 and AtARF19 are essential for auxin-mediated plant development by regulating unique and partially overlapping target gene sets [18]. In addition to regulating plant growth and development, the ARF gene family is also involved in plant responses to numerous abiotic stresses.

For example, overexpression of AtARF3 stimulates the expression of drought-stress-responsive genes in Arabidopsis. However, under drought stress, these ARF-activated genes maintain very low expression levels in the arf3 mutant [17]. Similarly, the expression levels of most SlARF were downregulated under salt stress, while some SlARF genes, such as SlARF1, SlARF4, and SlARF19, were significantly upregulated when undergoing salt stress [6]. In lettuce, the expression levels of most LsARFs are closely correlated with temperature changes, and LsARF8a is involved in regulating the timing of bolting [19]. In earlier studies, authors also found that the accumulation of LsARF2 and LsARF5 in flower stems continuously increased with the progress of lettuce bolting and discovered that the tryptophan metabolism pathway was involved in the early bolting of lettuce under high temperatures [20, 21]. In addition, Hu et al. [22] exhibited that ARF2/26/27/43 actively participates in banana resistance (Musa acuminata L.) against cold stress damage. These previous studies have determined that the ARF gene family is essential for regulating plant growth, hormone responses, and responses to abiotic stresses, including drought, salt, and temperature changes, and is a crucial gene family for understanding plant biology.

Alfalfa (Medicago sativa L.), known as the “king of forage,” has a high biomass and crude protein content and is rich in digestible nutrients and mineral elements, which dramatically reduces the cost of feed supplements for livestock production [23, 24]. Alfalfa is widely cultivated in North America, Asia, and other continents and is one of the most economically valuable crops in the world [24, 25]. Alfalfa is the fourth most cultivated crop in the United States, after wheat, corn, and soybeans [26]. In China, alfalfa cultivation is distributed through 14 provinces in the northern region of the country, with a locally cultivated alfalfa variety (“Xinjiang Daye”) that has large leaves and an autotetraploid genome (2n = 4x = 32), which is widely cultivated due to its strong resistance [23].

The complete genome data of the autotetraploid variety Xinjiang Daye was released, resulting in a chromosomal-level genome assembly containing 32 high-quality chromosomes instead of the previously assembled 8 chromosomes [26, 27]. The updated assembly of this genome provides an important foundation to facilitate scientific research on this economically important forage crop and improve the stress resistance of alfalfa through genetic engineering. To date, the function of the ARF gene family involved in plant development and abiotic stress has been widely reported in many plants, such as papaya (Carica papaya) [9], longan (Dimocarpus longan L.) [28], and apple (Malus domestica) [29]. Nonetheless, before the report of chen et al. [26], the genome that identified MsARF of the cultivated alfalfa is not available. Genomic data and many transcriptome analyses of cultivated alfalfa have been published, providing reliable experimental resources for systematic research on the MsARF [30,31,32]. Based on these data, our study used bioinformatics methods to identify ARF in autotetraploid Xinjiang Daye alfalfa at the genome-wide level. We analyzed the phylogeny, gene structure, motif composition, cis-regulatory elements, chromosome maps, tissue-specific expression patterns, and differential expression under various abiotic stresses. The results of this study lay the foundation for further investigation of the abiotic stress response and the creation of new germplasm of alfalfa with strong resistance through genetic engineering technology.

Results

Identification and functional analysis of MsARF family genes

After removing the redundant sequences, a total of 81 MsARF protein sequences were identified in the alfalfa genome and renamed from MsARF001 to MsARF081 according to the order of their occurrence in the genome (Table S1). The physicochemical properties of the MsARF proteins, including the gene name, gene ID, amino acid number, molecular weight, isoelectric point, and protein hydrophobicity coefficient, were investigated using ProtParam on the Expasy website (Table S2). Significant differences exist in the number of amino acids, molecular weight, and isoelectric points of the 81 MsARF genes. MsARF025 contains 1127 amino acids, making it the gene with the highest number of coding amino acids in the family, with a molecular weight of 126144.77 daltons (Da). MsARF020, with the family’s lowest number of amino acids, encodes 116 amino acids with a molecular weight of 13091.05 Da. The theoretical isoelectric points of these MsARF proteins ranged from 4.32 (MsARF054) to 9.58 (MsARF068). The instability coefficient of the MsARF family ranged from 22.84 (MsARF032) to 98.98 (MsARF039), but the instability index of most genes (66 out of 81) was higher than 40, indicating that they were unstable proteins. The aliphatic amino acid index of 81 MsARF proteins ranged from 48.43 (MsARF067) to 93.76 (MsARF001), all less than 100, indicating that they were hydrophilic proteins. Further subdivided with the help of protein hydrophobicity coefficients, 22 MsARF proteins with hydrophobicity coefficients less than − 0.5 were categorized as hydrophilic, and 59 MsARF proteins were between − 0.5 and 0.5, belonging to the amphotropic protein group.

The GO and KEGG annotation analysis was performed using the Alfalfa Database to determine the functional classification of all MsARF. A total of 59 annotated MsARF were classified as biological processes, molecular functions, or cellular components (Table S3). Terms like “regulation of nuclear acid template translation,” “regulation of RNA biological process,” and “regulation of translation, DNA template” served as the main annotations for biological processes. In the cellular components, the MsARF genes were mainly enriched in terms such as “nucleus”, “internal membrane-bound organelle”, and “membrane-bound organelle”. KEGG enrichment analysis found that 20 MsARF genes were enriched in the “plant hormone signal transduction pathway”, and only MsARF039 was enriched in “RNA polymerase”, indicating that ARF genes mainly function through the plant hormone signaling pathway (Table S4).

Phylogenetic analysis of the MsARF gene family

To study the phylogenetic relationship between MsARF proteins, 25 ARF proteins from rice, 23 from Arabidopsis, and 81 from alfalfa identified in this study were selected to further study the phylogenetic relationships of the MsARF gene family in alfalfa. The ARF gene family was divided into four major clades, of which clade I contained three Arabidopsis genes, six rice genes, and 52 alfalfa genes. Clade II contained 5 Arabidopsis genes, 9 rice genes, and 13 alfalfa genes. Clade III contains 2 Arabidopsis genes, 4 rice genes, and 7 alfalfa genes. Clade IV contains 13 Arabidopsis genes, 6 rice genes, and 9 alfalfa genes (Fig. 1).

Fig. 1
figure 1

Phylogenetic analysis of ARF protein in A. thaliana, O. sativa, and M. sativa

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Subcellular localization and structural analysis of MsARF protein

Subcellular localization prediction of the MsARF was performed, with 22 MsARF in the chloroplast, 9 MsARF in the cytoplasm, and the remaining 50 MsARF in the nucleus (Table S5). The results of the peptide, signal peptide, and transmembrane structure analysis showed that all MsARF had no peptides, signal peptides, or transmembrane structures, indicating that the 50 MsARF genes located in the nucleus acted as transcription factors, which are not secreted or transported but only function in the nucleus. Analysis of the secondary structures in the MsARF protein found that the largest proportion of the MsARF protein was a random coil, with the composition percentage of random coil > α -helix > extended chain > β -turn angle (Table S5). Simultaneously, the tertiary structure of the MsARF protein was constructed (Fig. 2). Due to the different percentages of secondary structure composition, the tertiary structure also showed significant differences. The specific types of spatial structures in MsARF proteins were relatively few, and most types (46) were obtained through horizontal flipping and rotation, as shown in Fig. 2A C, as well as 2B and 2D, 2E, 2G, and 2I. Another 24 MsARF proteins shared a common spatial framework, as shown in Fig. 2F. In addition, the remaining 11 MsARF proteins all had their own unique spatial framework.

Fig. 2
figure 2

Analysis of MsARF protein tertiary structures. A MsARF001; B MsARF002, 026, 034, 035, 045, 081; C MsARF003, 038, 066, 071; D MsARF004, 008,009,012,019,021,025,031,043,047,059,065,078; E MsARF005, 010, 029, 046, 050, 052, 053, 064, 080; F MsARFS006, 011, 014,015, 022, 023, 027, 032, 033, 036, 037, 040, 056, 057, 060, 061, 063, 067, 070, 074, 075, 076, 077, 079; G MsARF007, 017, 055; H MsARF013; I MsARF016, 024, 041, 042, 044, 048, 058, 062, 069, 073; J MsARF018; K MsARF020; L MsARF028; M MsARF030; N MsARF039; O MsARF049; P MsARF051; Q MsARF054; R MsARF068; S MsARF072

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Conservative motif analysis of the MsARF gene

The MsARF domain is the core of ARF transcription factors, which can activate downstream genes by interacting with their promoters. MEME tools were used to explore the distribution and structural diversity of conserved motifs in the MsARF protein and to identify conserved motifs, most of which play important roles in protein-protein interactions and transcriptional activity. A total of 10 conserved motifs were identified and renamed as motifs 1 to 10, with some of these motifs showing similar composition and position in the same subfamily of the MsARF (Fig. 3A, Fig. S1). Motif 8 was highly conserved in the MsARF gene family and was identified in all 80 MsARF except MsARF039, indicating that motif 8 is one of the most important motifs in the MsARF gene family (Fig. 3). Furthermore, motif 7 was highly conserved in the MsARF subfamilies II, III, and IV.

Fig. 3
figure 3

Analysis of conserved moti, domains, and gene structure of MsARF proteins. A Gene conservative motif analysis; B Protein conservative domain analysis; C Gene structure analysis

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Analysis of the conserved domains of the MsARF protein sequences revealed that all MsARF sequences contained an auxin response domain (Auxin_resp). Among the 81 MsARF sequences, except for 10 MsARFs (MsARF013/018/028/032/039/049/051/054/072/079), all other sequences contain a typical DBD domain (B3-like). However, phylogenetic analysis revealed that the Aux/IAA binding domain is only distributed in the clade II, III, and IV subfamilies (n = 19) (Figs. 1 and 3B). Furthermore, MsARF079 also contains a cyanobacterial aminoacyl-tRNA synthetases appended domain (CAAD). To continue to determine the structural differences among MsARFs and reveal gene function, regulation, and evolution, a gene structure analysis of the MsARF family was performed. Gene structure analysis showed that the number of CDS in the MsARF gene ranged from 1 to 15 (Fig. 3C), and no UTR regions were identified. Among the 81 MsARF genes, MsARF046 has the most CDS (n = 15), followed by MsARF005, MsARF026, MsARF029, MsARF043, MsARF050, MsARF064, and MsARF066 that have 14 CDS, while MsARF020 and MsARF030 have only one CDS. Phylogenetic analysis revealed that the MsARF genes differ in the number of CDS in different subfamilies.

Chromosomal localization and homology analysis of MsARF gene in alfalfa

This study identified and mapped the chromosomal positions of all MsARF genes to investigate whether there was a collateral homologous gene pair relationship between the MsARFs in alfalfa (Fig. 4). The 81 MsARF genes were unevenly distributed on 25 chromosomes (Chr) and seven other chromosomes (Chr 3.4, Chr 6.1, Chr 6.2, Chr 6.3, Chr 6.4, Chr 7.4, and Chr 8.3) (Fig. 4). The MsARF was distributed the most on Chromosome 5, with Chr 5.1 and Chr 5.2, each containing nine members, and Chr 5.3 and Chr 5.4, each containing seven members. Furthermore, five MsARFs were found on Chr 1.2, Chr 2.1, and Chr 2.4, each with four members on Chr 2.3; three MsARFs each on Chr 1.4, Chr 1.1, and Chr 8.2; and two MsARFs on Chr 1.1, Chr 2.2, Chr 4.1, and Chr 8.1 each. There was only one MsARF on the remaining eight chromosomes.

Fig. 4
figure 4

Distribution and location of the MsARF gene on alfalfa chromosomes. Chr 1.1 to Chr 8.4 represent the linkage group of “Xinjiang Daye” alfalfa. Each black line indicates the location of the ARF gene. The red line represents tandem duplicates of the ARF gene

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We used TBtools software to conduct a collinearity analysis to detect gene duplication events in the ARF in alfalfa [33]. 14 pairs of segment repeats and two sets of tandem repeats in the MsARF were identified (MsARF056/MsMASS057 and MsARF074/MsMASS075) (Fig. 5). The nonsynonymous substitution rate/synonymous substitution rate (Ka/Ks) ratio is usually an important indicator of selection pressure in evolution [34]. The analysis of homologous and homologous MsARF gene pairs showed that among the 14 pairs of segment repeats and two sets of tandem repeats of MsARF genes, the Ka/Ks ratio of two pairs of segment repeats (MsARF008/MsMASS042 and MsARF008/MsMASS042) was greater than one, indicating a positive selection effect. The Ka/Ks ratios of the remaining 14 pairs of MsARF genes were all less than one, indicating they underwent strong purification selection after replication (Table S6).

Fig. 5
figure 5

Synteny analysis of the MsARF gene. The gray line represents all the synteny blocks in the alfalfa genome. The green and red lines represent gene pairs, and tandem repeat genes in the ARF gene, respectively. The yellow heat map and the broken red line map represent the gene density and expression level of the MsARF gene, respectively

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Analysis of Cis-regulatory elements in the promoter region of the MsARF gene family

Cis-regulatory elements are specific deoxyribonucleic acid (DNA) sequences located upstream of gene coding sequences and regulate the expression of stress response genes by binding to transcription factors. Therefore, we explored the distribution of seven cis-regulatory elements in the promoter regions of these altered MsARF genes during abiotic stress. We explored the distribution of cis-regulatory elements related to hormones and abiotic stress in the MsARF gene promoter region (Fig. 6). cis-regulatory elements related to hormones include abscisic acid response: ABRE (n = 118), Methyl Jasmonate (MeJA) response: CGTCA-motif (n = 91), auxin response: TGA-element (n = 38) and AuxRR-core (n = 8). cis-regulatory elements related to abiotic stress include defense and stress response: TC-rich repeats (n = 49), drought induction: MBS (n = 66), and low-temperature response: LTR (n = 27). In addition, we found that there was only a cis-regulatory element in MsARF012, MsARF021, MsARF022, MsARF025, MsARF031, MsARF036, MsARF042, MsARF045 and MsARF051.

Fig. 6
figure 6

Analysis of Cis-regulatory elements in the upstream promoter of the MsARF gene

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Expression pattern analysis of the MsARF gene family

Tissue-specific expression is related to the specific function of ARF genes in specific tissues. Therefore, 81 MsARF genes were compared and identified using ‘blastn’ on the Alfalfa Database, and expression patterns of 45 MsARFs were obtained using gene expression profile data (Table S7). TBtools software was used to generate MsARF genes (gene expression data was 0 when mapping). The MsARF was expressed in eight specific tissues, including flowers, leaves, roots, post-elongating stems, nodules, elongating stems, young leaves, mature leaves, and senescent leaves of alfalfa. Group a genes were highly expressed in flowers, leaves, roots, pre-elongated stems, nodules, and elongated stems. Group b genes were highly expressed in flowers and leaves. Group c genes were highly expressed in flowers, and group d genes were highly expressed in young, mature, and senescent leaves, which may play an important role in leaf development. The two genes in group e, MsARF025 and MsARF049, were highly expressed in senescent leaves and considered putative key regulatory genes for this specific tissue type (Fig. 7A). To identify the expression patterns of MsARF family genes in response to abiotic stress, we analyzed the transcriptional expression profiles of MsARF family genes under salt stress, mannitol treatment, cold stress, and metal ion stress (aluminum and lead) (Fig. 7B, C). In Fig. 7B, group a genes were mainly upregulated after 48 h of cold stress, participating in alfalfa’s response to cold stress. MsARF012 and MsARF015 were mainly overexpressed after 3 h of salt stress. Five MsARF genes (MsARF016, MsARF014, MsARF075, MsARF049, and MsARF013) were overexpressed after 6 h of cold stress, and MsARF011 was overexpressed after 6 h of mannitol treatment, suggesting that these genes may play a vital role in alfalfa’s response to abiotic stress.

Fig. 7
figure 7

Analysis of the expression pattern of the MsARF gene family. A Tissue-specific expression pattern analysis; B Analysis of expression patterns under salt, drought, and cold stress; C Analysis of expression patterns under aluminum (Al) and lead (Pb) stress

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Real-time quantitative PCR analysis of the MsARF family genes

To verify the function of MsARF, a total of 25 MsARF genes (MsARF005, MsARF007, MsARF010, MsARF017, MsARF018, MsARF025, MsARF026, MsARF029, MsARF035, MsARF039, MsARF041, MsARF043, MsARF044, MsARF045, MsARF047, MsARF050, MsARF051, MsARF053, MsARF055, MsARF060, MsARF064, MsARF066, MsARF071, MsARF080, MsARF081) were selected to have their expression patterns analyzed during salt, drought, high-temperature, and low-temperature stresses at different time points (Table S8), based on RNA-seq data (Fig. 7B). MsARF041 and MsARF047 showed no or very low expression and were not further analyzed. The expression pattern of MsARFs under salt stress is depicted in Fig. 8. Except for MsARF051, which is upregulated under salt stress, other genes showed a downregulated trend compared to the control (CK). In leaf tissue, MsARF051 at 24 h, MsARF081 and MsARF029 at 12 h, ARF080 at 12 and 24 h, ARF010 at 6 h, MsARF018 at 6 and 24 h, and MsARF050 at 6 and 12 h were all upregulated, other genes at other times were all downregulated. In root tissue, only MsARF044 and MsARF029 at 12 h, and MsARF025 at 24 h were upregulated. In stem tissue, the expressions of MsARF genes were diverse. Figure 9 shows the expression patterns of 23 MsARF genes under drought stress. MsARF051 was upregulated under drought stress in all three tissues compared to CK. The changes in other genes were relatively diverse and tissue-specific, as well as related to the duration of treatment. Addition to, most genes (MsARF007, MsARF010, MsARF017, MsARF018, MsARF025, MsARF029, MsARF035, MsARF039, MsARF044, MsARF045, MsARF050, MsARF051, MsARF053, MsARF055, MsARF060, MsARF064, MsARF071 and MsARF080) were mainly upregulated in the leaves at 12 h.

Fig. 8
figure 8

The expression pattern analysis of ARF genes in diverse organs undergoing salt stress treatment for 0, 6, 12, and 24 h using qRT-PCR. Data are presented as the mean ± standard deviation. Lowercase letters indicate a significant difference at the 5% level. The same below

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Fig. 9
figure 9

The expression pattern analysis of ARF genes in diverse organs undergoing drought stress treatment for 0, 6, 12, and 24 h using qRT-PCR

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Under high-temperature treatment, MsARF051 was upregulated in all three tissues, and treatment time lengths compared with CK (Fig. 10). MsARF017, MsARF039 and MsARF071 were upregulated at 12 h, indicating that their heat stress regulation was tissue-independent and mainly acted at 12 h. Meanwhile, we also found that most MsARF genes were upregulated at 12 h in leaves and stems undergoing heat stress, while the expression pattern was quite varied in roots. Under low-temperature treatment, MsARF051 was upregulated in all three tissues and treatments compared with CK (Fig. 11). MsARF026, MsARF039, MsARF071 and MsARF081 were upregulated at 12 h in leaf and stem tissues, with significantly distinct expression patterns in the roots. The changes in the remaining genes were tissue-specific and related to the treatment time length, And most genes were upregulated in specific tissues only at 12 h, suggesting that the regulation of cold stress by these genes was significantly correlated with the stress length. Compared with CK, MsARF017, MsARF035, MsARF026, and MsARF043 were found in the leaves, and MsARF017 was upregulated in the stem at 12 h and was downregulated at 6 and 24 h (Fig. S2). And compared to CK, MsARF060 in roots and stems, while MsARF044 in all three tissues were downregulated. The dynamic expression patterns of the remaining genes showed diversity.

Fig. 10
figure 10

The expression pattern analysis of ARF genes in diverse organs undergoing high-temperature stress treatment for 0, 6, 12, and 24 h using qRT-PCR

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Fig. 11
figure 11

The expression pattern analysis of ARF genes in diverse organs undergoing low-temperature stress treatment for 0, 6, 12, and 24 h using qRT-PCR

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Discussion

Extreme weather conditions like drought, salt stress, and temperature have had a significant negative impact on alfalfa, one of the most economically important leguminous forages in the world. As a result, yield and geographic distribution have become major bottlenecks. As an important component of the auxin signaling pathway, ARF directly binds to and regulates the specific expression of downstream target genes during the auxin response [35]. Previous studies have shown that the ARF family plays a crucial role in plant growth, development, hormone response, and stress response [36, 37], making it a key gene family for understanding plant biology. Understanding the dynamics of this gene family provides an important resource for improving yield through variety improvement. However, there are no studies on the molecular function of alfalfa ARF transcription factors in response to abiotic stress. Therefore, studying the main structure and expression characteristics of ARF genes in alfalfa helps us gain insight into the regulatory role of ARF genes in alfalfa resistance to abiotic stress, growth, and development. This study identified 81 ARF family members in the alfalfa genome and analyzed the possible role of MsARF in different tissues and abiotic stress responses in alfalfa for further studies on the function of ARF genes.

Members of the ARF family have revealed the evolution of tetraploid cultivated alfalfa

As a prominent effector of many aspects of the auxin response in plants, ARF transcription factors convert chemical signals into the transcriptional regulation of a group of specific genes [19]. Due to its functional importance in the auxin cascade, the ARF gene family has been deeply studied in model plants and major crops. The genome size of lettuce is 2.5 gigabases (Gb), which is considered one of the largest assembled plant genomes to date [38]. Compared to lettuce, the reference genome of autotetraploid alfalfa is larger, reaching up to 2.738 Gb [26]. Autotetraploid alfalfa can encode a larger ARF gene family based on such a large genome. This study identified 81 members of the MsARF family, which was greater than several reported crops, such as soybean (51), rice (25), and maize (36) [39,40,41]. Previous studies have shown that the genetic relationship between alfalfa and Medicago truncatula is closest [26]. A total of 40 MtARF genes were previously identified from the genome of diploid alfalfa [42], which is precisely half of the results of this study. These results may be related to the polyploidy of alfalfa (diploid genome), similar to the MADS-box genome-wide analysis of wheat [43]. Genome doubling (plant polyploidization) provides primitive genetic material for biological evolution, improves species diversity and environmental adaptability [44], and plays a vital role in the adaptive evolution of angiosperms [45]. Therefore, identifying the tetraploid cultivated alfalfa ARF family from the genome level provides essential theoretical guidance and practical application value for research that examines the biological evolution, species protection, and genetic breeding of subsequent alfalfa.

Analysis of physicochemical properties of MsARF gene encoding proteins

This study’s identification and analysis of the MsARF gene family was completed using the whole genome data of the “Xinjiang Daye” alfalfa cultivar. The hydrophilicity analysis of 81 MsARF proteins showed that the instability coefficient of the MsARF protein family ranged from 22.84 (MsARF032) to 98.98 (MsARF039), but the instability index of most genes (66) was higher than 40, indicating that they were not stable proteins. The aliphatic amino acid index of the 81 MsARF proteins ranged from 48.43 (MsARF067) to 93.76 (MsARF001), all of which were less than 100, indicating that they were hydrophilic proteins. The further subdivision of the protein family was carried out using the protein hydrophobicity coefficient. Among them, 22 MsARF proteins had a hydrophobicity coefficient less than − 0.5, indicating that they were hydrophilic proteins, while the other 59 MsARF proteins were between − 0.5 and 0.5, indicating that they were amphiphilic proteins. This result indicates that the MsARF family proteins have better hydrophilicity. As transcription factors, ARF proteins usually function in the nucleus [7]. Consistent with the subcellular localization results reported in other species, such as eggplant [7], tomato [46], and maize [41], the subcellular predictions indicate that most of the MsARF genes were localized to the nucleus. In addition, no MsARF genes were found to have any conducting peptides, signal peptides, or transmembrane structures, indicating that the 50 MsARF genes located in the nucleus did not secrete or transport but only regulated gene expression in the nucleus.

MsARF gene homology analysis

A phylogenetic tree was constructed to analyze the relationship between the ARF gene family in rice, Arabidopsis, and cultivated alfalfa (Fig. 2). The ARF gene family was divided into four large clades, where clade I contained the most MsARF (52) genes and was closely related to AtARF10/16/17. AtARF10/16 controls root crown cell formation, limiting the stem cell niche and promoting columella cell differentiation [47]. AtARF10 plays an important role in regulating the formation of the primary outer wall and pollen development in Arabidopsis [48]. Therefore, we hypothesized that these 52 MsARF genes have similar functions to AtARF10/16/17. Clade II contained 13 alfalfa genes that were closely related to AtARF5/6/7/8/19, and it has been speculated that these 13 MsARF genes may be auxin-promoting factors in carrot protoplasts [49]. Clade III contained seven alfalfa genes, which were closely related to AtARF3 and AtARF4, and it has been speculated that these seven MsARF may be auxin suppressors [49]. Clade IV contained nine alfalfa genes, which were closely related to 13 Arabidopsis genes such as AtARF1, AtARF2, and AtARF11, suggesting that these nine MsARF may act as auxin repressors involved in the Abscisic Acid (ABA) pathway regulating seed germination and primary root growth [50].

The analysis of the MsARF gene structure

Based on the phylogenetic tree, analysis of ten conserved motifs of MsARF genes revealed different clades of MsARF genes containing common or specific motifs (Fig. 3). Although the number of motif members varied among clades, the group motif patterns were strongly conserved. The comparison of novel functional domains or motif sequences across multiple homologous proteins has been a widely used method to predict protein function based on evolutionary conservation [7]. We found that motif 8 was highly conserved in the MsARF gene family. In addition, we also found that motif 7 was highly conserved in the MsARF subfamily in Clades II, III, and IV. Although most of the motifs in MsARF were conserved, other motifs may have been related to novel plant functions and should be studied further. By analyzing the conserved domains of MsARF protein sequences, we found that all MsARFs contain an auxin resp domain, and 71 MsARFs contain a typical DBD domain (B3-like), while only 19 (23.46%) MsARFs contain an auxin/IAA binding domain and are mainly distributed in clades II, III, and IV. This result was similar to the conserved domain of the ARF protein sequence in Medicago truncatula, indicating that auxin can also regulate MsARF genes independently [51].

To further determine the structural differences between MsARF genes, gene structure analysis was conducted on the MsARF family. The number of CDS in the MsARF gene ranged from 1 to 15, and no UTR region was identified. This finding was consistent with the previous reports in papaya [9], maize [41], and barley [52], indicating that the gene structure of the ARF family was relatively simple. Gene structure analysis determined the structural differences among the MsARF gene family. Analysis of the distribution of 7 cis-regulatory elements in the 2000 bp (bp) upstream promoter region of the MsARF revealed that ABRE had the most cis-regulatory elements, followed by CGTCA motifs, MBS, TC-rich repeats, TGA elements, LTR, and AuxRR cores (Fig. 5), indicating that MsARF played a positive role in hormone responses to plant stressors.

Chromosomal localization and collinearity analysis of the MsARF

We found that 81 MsARF genes were unevenly distributed on 25 chromosomes (Chr) of alfalfa and were the most widely distributed on chromosome 5, indicating that MsARF genes mainly perform biological functions on chromosome 5. Gene replication is traditionally considered a way to expand and obtain functional diversity during evolution [53]. Gene duplication analysis in the MsARF gene family showed that fragment duplication had a greater impact on ARF genes, which was consistent with the results observed in rice and Arabidopsis in previous studies [54], suggesting that replication events in the ARF family within a plant genome may be a general evolutionary mechanism. Ka/Ks ratios are usually an important indicator of selection pressure in evolution [34]. The analysis of homologous MsARF gene pairs showed that only 2 pairs of fragment replicates (MsARF008/MsMASS042 and MsARF008/MsMASS042) had a Ka/Ks ratio greater than 1, while the remaining 14 pairs of MsARF had a Ka/Ks ratio less than 1, indicating that they experienced strong purification selection after duplication.

The MsARF genes function in response to abiotic stress

Since ARF genes play a vital role in controlling phytohormone signaling, studying each family member’s function is necessary. ARF transcription factors are crucial in plant growth and development and actively participate in plant resistance to stress [6, 17, 19, 20]. This study combined transcriptome data with a novel bioinformatic approach to search for MsARF genes that have functions related to the response and regulation of drought stress in alfalfa. We compared the MsARF genes with known transcriptome data and initially selected genes that were significantly differentially expressed under drought, salt, and cold stress as ARF candidate genes responding to abiotic stress. Subsequently, alfalfa seedlings were treated with salt, drought, cold, and heat stress, and qRT-PCR experimentally verified their expression patterns. As reported by Zhou et al. [55], ARF12 might play central roles in the regulation of NaCl-responsive genes. In addition, AcARF5 can positively regulate salt stress and drought stress in kiwifruit (Actinidia chinensis) [56]. When compared to CK, MsARF051 was upregulated in all treatment groups, which helped us elucidate that MsARF051 had great potential for transformation in the cultivation of reverse-resistant alfalfa varieties. In addition, MsARF017 and MsARF039, MsARF026, MsARF071 and MsARF081 were upregulated during temperature stress, and MsARF039 could regulate heat stress and cold stress in alfalfa. Furthermore, we found that most of the ARF genes were upregulated at 12 h under all abiotic stresses involved in this study, and we speculated that these ARF genes were mainly active at 12 h and belong to short-acting genes, especially in the leaves and stems. Future studies focusing on alfalfa should examine these genes and conduct further research through genetic engineering and editing techniques in our research. The results of this study on the alfalfa ARF gene family at the whole genome level create a foundation for mining stress resistance genes for alfalfa and provide an in-depth analysis of abiotic stress theory and guidance for the creation of new germplasm using genetic engineering technology for an alfalfa variety that has increased stress resistance.

Conclusion

Based on the whole genome of cultivated alfalfa, this study identified the ARF gene family, predicted the basic physical and chemical properties of the genes, and analyzed the phylogenetic relationship, gene structures, chromosomal localization, paralogous and homologous genes, and expression patterns of ARF genes in response to abiotic stress and different tissues. A total of 81 MsARF genes were identified and phylogenetically divided into four branches, analyzing 14 discovered segmental repeat pairs and two sets of tandem repeats. Compared with the transcriptomic data of alfalfa, the expression of MsARF051 under all stress treatments can be used as the candidate gene of alfalfa. MsARF039 is mainly involved in the response of alfalfa to temperature stress and can be used to improve new alfalfa varieties cultivated in areas with extreme temperatures. Future research will examine the functional verification of these genes and analyze the genetic transformation of these two genes to lay a foundation for further investigation of the abiotic stress theory and the creation of new germplasm using genetic engineering technology for an alfalfa variety that has increased stress resistance.

Materials and methods

Materials

The material used in this experiment was cultivated “Xinjiang Daye” alfalfa, and its seeds were provided by the National Livestock Husbandry Station, Ministry of Agriculture and Rural Affairs of The People’s Republic of China.

Identification of the MsARF gene family and analysis of its physical and chemical properties

The Plant Transcription Factor Database (http://planttfdb.gao-lab.org/) was used to download the ARF protein sequences from Arabidopsis and rice model plants. The genome data and protein-nucleic acid sequence of “Xinjiang DaYe” alfalfa used in this experiment were downloaded from the alfalfa genome website (https://figshare.com/projects/whole_genome_sequencing_and_assembly_of_Medicago_sativa/66380) [26]. In addition, the hidden Markov model (HMM) profile of ARF (PF06507) was downloaded from the Pfam database (http://pfam.xfam.org/), and MsARF family proteins from alfalfa genome were identified using HMMER 3.0 with an E-value set to 1.0 as the threshold and the remaining parameters set to default. The redundant protein was removed using the Expasy [57] online database (https://web.expasy.org/decrease_redundancy), with all parameters set to default values. Further characterization of the ARF domain in the MsARF protein sequences was performed using the Pfam website and NCBI-CD Search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.Cgi) to verify the accuracy of the MsARF protein sequences after the removal of the redundancy. Sequences without the ARF domain were removed [58]. A total of 81 MsARF protein sequences were subsequently analyzed for physicochemical properties using the online network ProtParam (https://web.expasy.org/protparam/) tools, including molecular weight (MW), theoretical isoelectric point (pI), instability index, and the grand average of hydropathicity (GRAVY) index. Subcellular localization (https://www.genscript.com/wolf psort.html) was predicted for all 81 MsARF genes by the online tool WoLF PSORT. The predicted genes were named MsARF001 through MsARF081 according to their order of occurrence in the genome. In addition, GO and KEGG annotations were performed using the Alfalfa Database (http://47.92.172.28:12088/) to investigate the functional role of the MsARF genes [59, 60].

Phylogenetic analysis, gene structure and motif composition of the MsARFs

To analyze the evolutionary relationship between MsARF proteins, multiple sequence alignments of MsARF, AtARF, and OsARF protein sequences were performed using MEGA 7.0 software [61] and automatically trimmed with TrimAL [62]. The maximum likelihood phylogenetic tree was constructed from IQ-TREE version 1.6.12, and the JTT + R6 model was determined to be the best sub-phylogenetic model using ModelFinder, with the Ultrafast bootstrap set to 1000 [63, 64]. Based on the evolutionary relationship between MsARF genes and AtARF, MsARF genes were classified into a clade I-IV type [65]. The MEME 4.12.0 online tool (http://meme-suite.org/) was used to identify the conserved motifs of the 81 alfalfa MsARF proteins. The parameters were set as follows: The maximum number of motifs was 10; each sequence selected a zero or one occurrence as the site distribution; the minimum and maximum motif widths were set to 6 and 200, respectively; other parameters were set to default values; and the motif features were visualized with TBtools [33].

Gene duplication and gene structure analysis

CDS sequences and gene sequences corresponding to all MsARF genes were obtained from the alfalfa genome file for prediction analysis of intron and exon structures of MsARF genes using GSDS2.0 (gene structure display server) (http://gsds.gao-lab.org/). MapGene2Chrome V2.0 (http://mg2c.iask.in/mg2c_v2.0/) was used to map chromosome localization based on the alfalfa genome annotation file (GFF3) data to explore the distribution characteristics of ARF genes on chromosomes. Based on the alfalfa genome sequence file in fasta format and the corresponding gene structure annotation file in GFF3/GTF format, referring to the method of Chen et al. [33], TBtools software was used to complete the chromosome location of genes and the collinearity analysis of gene duplications. The specific steps: first, the “Fasta Stats” function was opened, the alfalfa genome file was input, and the chromosome length information file was obtained. Next, the “Gene Density Profile” function was used, and gene structure annotation files was input to obtain gene density information. Then, by using the “one step MCScanX-super fast” function, the alfalfa genome file and corresponding gene structure annotation file were input to obtain the collinearity file of genes within the species. Subsequently, the association file between genes was obtained through the “File Merge for MCScanX” and “File Transform for MicroSynteny Viewer” functions, and the green RGB value was added to the MsARF gene pairs, as well as the red RGB value was added to the tandem repeat sequence genes. Finally, the “Advanced Circos” function was used to input the file obtained in the previous steps to draw the circos diagram.

Protein signal peptide, guide peptide, transmembrane structure and protein secondary and tertiary structure analysis

The signal peptide and transmembrane structures of the MsARF were analyzed using SignalP 5.0 (https://services.healthtech.dtu.dk/services/SignalP-5.0/) and TMHMM 2.0 (https://services.healthtech.dtu.dk/services/TMHMM-2.0/), respectively. The conducting, or guide, peptide analysis of the MsARF was also performed using the Target P-2.0 Server (https://services.healthtech.dtu.dk/services/TargetP-2.0/). The MsARF protein secondary structure was analyzed using SOPMA (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.plpage=npsa_sopma.html) to obtain the percentage of different conformations, including α -helix, extended chain, β -turn angle, and random coil. Using SWISS-MODEL (http://swissmodel.expasy.org/interactive), this structural analysis was subsequently used to analyze the tertiary structure of MsARF proteins.

Cis-regulatory element analysis

The PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) analyzed cis-regulatory elements in the 2000-bp sequence upstream of the upstream transcription start site of the 81 MsARF genes. In the promoter region of alfalfa ARF genes, seven cis-regulatory elements known to associate with stress and hormones were identified, including ABRE, TC-rich repeats, MBS, LTR, CGTCA-motif, AuxRR-core, and TGA-element, which are involved in the abscisic acid response, defense and stress response, drought induction, low-temperature response, Methyl Jasmonate (MeJA) response, auxin response, and auxin response cis-elements.

MsARF gene in response to abiotic stress

To investigate the expression level of MsARF under abiotic stress and various tissue sites, BLASTn alignment with the Alfalfa Database produced the expression profile data of MsARF genes under abiotic stress and various tissue sites. To deeply investigate the expression patterns of MsARF genes in different tissues, expression profile data from eight tissues were analyzed, including flowers, leaves, roots, post-elongating stems, nodules, elongating stems, young leaves, mature leaves, and senescent leaf tissues of alfalfa. The expression analysis of MsARF genes under abiotic stress included the transcriptional expression profiles of alfalfa under cold, drought, salt, aluminum, and lead stress. OmicShare Tools (https://www.omicshare.com/tools/), a freely available data analysis platform, was used for cluster analysis and expression quantity heat map mapping, and the expression level was expressed by Z-core normalized calculation results for the expression data.

Growth and stress treatment of plant materials

The plant material used in this experiment was “Xinjiang DaYe” alfalfa. First, we chose seeds with healthy and identical shapes. After surface sterilization of alfalfa seeds, they were planted in pots with vermiculite: nutrient soil = 1:1 [20 centimeters (cm) diameter, 20 cm high] with 5 biological replicates per treatment group. The seedlings were cultured in a 16-hour light/8-hour dark cycle with 60% relative humidity and a temperature of 22 °C. After 15 days, the alfalfa seedlings were inoculated with different stress treatments. A 15% polyethylene glycol-6000 solution was added to the seedling pots by root irrigation (until thoroughly poured) to simulate drought. To simulate salt stress, 220 micromolar (mM) sodium chloride solution was added to the seedling pots by root irrigation (until thoroughly poured). The whole alfalfa seedlings were harvested at different stress time points (6, 12, and 24 h) for the drought treatment group and the salt stress treatment group. To simulate cold stress, alfalfa seedlings to be treated were transferred to a 16-hour light/8-hour dark cycle in an artificial climate chamber with 60% relative humidity and temperatures at 5 °C. To simulate high temperature stress, alfalfa seedlings to be treated were transferred to a 16-hour light (32 °C)/8-hour dark (28 °C) cycle in an artificial climate chamber with 60% relative humidity. Whole alfalfa seedlings were collected after 6, 12, and 24 h of incubation. All samples were flash-frozen in liquid nitrogen and stored at 80 °C for downstream analysis.

RNA extraction and qRT-PCR

To verify the response pattern of MsARF genes to abiotic stress, twelve genes were selected for qRT-PCR analysis based on the expression heatmap. According to the instructions of the manufacturer, total ribonucleic acid (RNA)from the root, stem, and leaf tissues of “Xinjiang Daye” alfalfa under different stress treatment times were extracted using an RNA Simple Total RNA kit (Tiangen, Shanghai, China) and were reverse transcribed into cDNA using a FastKing cDNA kit (Tiangen, Shanghai, China). Using the Primer-BLAST tool at NCBI (https://www.ncbi.nlm.nih.gov/tools/primer-blast/), twelve gene-specific PCR primers were designed (Table S9) for gene expression analysis. The QuantStudio 5 real-time PCR system (Thermo Scientific, Massachusetts, USA) was used to amplify samples and standards with Hyperreal Premix Plus (SYBR Green) (Tiangen, Shanghai, China). Three biological replicates were used. Chen et al. described the experimental process that we used as a model for our experimentation [61]. The relative transcription levels of the selected genes were calculated with the 2−ΔΔCT method [62] and normalized to the expression levels of the Medicago truncatula ACTIN gene (AES78237. 1). The qRT-PCR data were collated and plotted in Excel 2016, and the one-way ANOVA (p < 0.05) was analyzed by IBM SPSS 24.0 software.