Microfilaments, formed by the polymerization of actin, constitute one of the three principal cytoskeleton components of eukaryotic cells. They are not only responsible for maintaining cell morphology through the three-dimensional structure, but also participate in regulating a number of cellular activities, including cell expansion, division, differentiation, organelle movement, and signal transduction (Porter and Day, 2016; Schaks et al., 2019). In cells, actin exists in two forms: monomeric globular actin (G-actin) and polymerized filamentous actin (F-actin). The ratio between the two populations varies with cell type and physiological status (Staiger, 2000). The latter, designated as microfilaments, represents the principal conduit through which actin exerts its biological functions (Pollard, 2016). The dynamics of actin within cells are controlled by dozens of actin-binding proteins (ABPs). In response to changes in growth stage or environmental conditions, ABPs regulate the activities of G-actin and F-actin, including polymerization, depolymerization, cross-linking and bundling. This ensures that the microfilament cytoskeleton undergoes highly dynamic changes, facilitating rapid cellular responses to intrinsic and extrinsic stimuli (Li et al., 2015; Porter and Day, 2016; Augustine et al., 2021).

Actin depolymerizing factor (ADF) is a kind of ABP with a small molecular weight (15~22 kDa) and a highly conserved sequence (Maciver and Hussey, 2002). The first ADF was isolated from chicken brain cells in 1980, and since then, ADFs have been identified in a diverse range of eukaryotic organisms, including fungi, animals, and plants (Bamburg et al., 1980; Gunning et al., 2015; Inada, 2017; Sun et al., 2023a). The biochemical activity of ADF is contingent upon its cellular concentration. Low concentrations of ADF promote severing or depolymerisation, whereas higher concentrations encourage G-actin nucleation and accelerate the release of Pi from ADP-Pi subunits in F-actin, along with the dissociation of branches formed by the actin-related protein 2/3 complex (Blanchoin et al., 2000; Andrianantoandro and Pollard, 2006). These biochemical activities make ADFs important participants in the regulation of dynamic changes in microfilaments within the cell, and thus they are involved in a number of cellular processes (Staiger, 2000; Pollard, 2016).

ADF genes are widely distributed in eukaryotic cells, with only one to three members in unicellular eukaryotes and animals, respectively. In contrast, higher plants have evolved a larger ADF gene family (Gunning et al., 2015; Sun et al., 2023a). To date, numerous plant ADF gene families have been identified, and the number of ADF genes in different species varies considerably. For example, common bean (Phaseolus vulgaris) has 9 (Ortega-Ortega et al., 2020), pigeon pea (Cajanus cajan) has 10 (Cao et al., 2021), Arabidopsis thaliana, rice (Oryza sativa), and tomato (Solanum lycopersicum) each has 11 (Feng et al., 2006; Ruzicka et al., 2007; Huang et al., 2012; Khatun et al., 2016), maize (Zea mays) has 13 (Huang et al., 2020), soybean (Glycine max) has 18 (Sun et al., 2023b), and wheat (Triticum aestivum) has 25 (Xu et al., 2021). Among plants, the expression characteristics and biological functions of the Arabidopsis ADF genes have been the subject of the most comprehensive study. Phylogenetic analysis demonstrates that the 11 AtADF genes can be classified into four groups (I~IV), with genes in each group displaying distinctive tissue expression patterns and differentiation in biochemical activities and biological functions (Feng et al., 2006; Ruzicka et al., 2007; Nan et al., 2017).

As sessile growth organisms, plants living in nature are subject to a range of biotic and abiotic stresses throughout their entire life cycle (Verma et al., 2016). Abiotic stress refers to the adverse effects on plants caused by non-living factors, including drought, water logging, salinity, extreme temperatures, and nutrient deficiency (Saijo and Loo, 2020). It has been demonstrated that plant ADF genes play a significant regulatory role in the response of plants to abiotic stress (Inada, 2017; Sun et al., 2023a). For example, AtADF1 was found to plays an important role in the salt stress response pathway (Wang et al., 2021), while AtADF5 has been confirmed to crucial for both of drought and cold tolerance ability of Arabidopsis (Qian et al., 2019; Zhang et al., 2021). In crop plants, OsADF3 of rice, ZmADF5 of maize, and GmADF13 of soybean had been confirmed to plays a positive role in the drought response process (Huang et al., 2012; Liu et al., 2024; Wang et al., 2024), and TaADF16 of wheat is crucial for the cold stress tolerance ability (Xu et al., 2021).

Alfalfa (Medicago sativa) is the most widely cultivated forage legume, with an area of cultivation exceeding 30 million hectares worldwide (Tang et al., 2021). It is a rich source of protein, vitamins, minerals, and numerous other nutrients, rendering it highly suitable for livestock feeding and earning it the designation of “king of forage” (Chen et al., 2021; Ma et al., 2022). Abiotic stresses, such as drought and salinity, have a significant detrimental impact on the growth and development of alfalfa, often resulting in considerable economic losses (Du et al., 2022; He et al., 2022). The exploration of stress-tolerance related genes in alfalfa is of great significance for the breeding of stress-tolerant cultivars, with the objective of ensuring their yield and quality. This study presents a systematic identification of the ADF gene family in alfalfa, accompanied by an investigation of their sequence characteristics, organs-specific expression, and expression patterns under salt and drought stress. The results of this study provide a foundation for the functional elucidation of ADF genes and stress-resistant breeding in alfalfa.

The microfilament cytoskeleton is one of the most dynamic cellular components that respond constantly to environmental stimuli as they modulate organizations (Staiger, 2000). ADF is a major ABP that controls the rearrangement of microfilaments in eukaryotic cells, thereby participating in a vast number of cellular processes (Pollard, 2016). To date, the genome-wide identification of the ADF gene family has been accomplished in a multitude of higher plants, with the number of genes in different species ranging from a few to dozens (reviewed by Sun et al., 2023a). Nevertheless, a comprehensive analysis of the ADF gene family at the whole-genome level remains to be conducted in alfalfa, a crucial forage grass cultivated globally (Tang et al., 2021). In comparison to other gene families, such as MYB, bHLH, and WRKY, the ADF gene family in higher plants is relatively small in number (Wang and Li, 2017; Sun et al., 2023a; Wang et al., 2023; Gao and Dubos, 2024). In this study, a total of nine MsADF genes were identified in alfalfa, and an analysis of the protein sequence revealed that all of their proteins contained the conserved ADF-H domain and regions associated with calmodulin- and the actin-binding domains ( Supplementary Figure S2 ). This number is approximately equivalent to the ADF family size in other plants, including Arabidopsis (11 members; Ruzicka et al., 2007), rice (11 members; Huang et al., 2012), tomato (11 members; Khatun et al., 2016), common bean (9 members; Ortega-Ortega et al., 2020), and pigeon pea (10 members; Cao et al., 2021). Gene duplication represents a fundamental source of new genes in the evolutionary process, contributing to the expansion of gene families and functional differentiation (Cannon et al., 2004). The primary duplication patterns for gene family expansion are tandem and segmental duplications, with the latter occurring with greater frequency due to the presence of duplicated chromosomal blocks (Panchy et al., 2016). This study revealed that three MsADF gene pairs underwent segmental duplication, with no evidence of tandem duplication. Furthermore, the Ka/Ks ratios of the three duplicated gene pairs were found to be less than 1 ( Figure 1B ; Supplementary Table S2 ), indicating the influence of purifying selection on the evolution of the MsADF gene family. Previous studies have documented the occurrence of segmental duplication events in the ADF families of wheat, Arabidopsis, maize, tomato, and soybean, with no instances of tandem duplication observed (Khatun et al., 2016; Nan et al., 2017; Huang et al., 2020; Xu et al., 2021; Sun et al., 2023b). It can therefore be postulated that the expansion of ADF gene families in higher plants is primarily attributed to segmental duplications. A collinearity analysis revealed that alfalfa shares five and 31 orthologous gene pairs with Arabidopsis and soybean, respectively ( Figure 1C ). The existence of orthologous gene pairs provides further evidence that the ADF gene family in plants exhibits a certain degree of conservation and may originate from common ancestral genes (Zheng et al., 2005). Notably, four MsADF genes (MsADF4, 6, 7, and 9) demonstrated a collinear relationship with those in Arabidopsis and soybean ( Figure 1C ), indicating that they may have been conserved during evolution and perform analogous functions in disparate plant species.

Phylogenetic analysis is a valuable tool for predicting gene function, with genes belonging to the same clade often demonstrating similar biological functions (Kapli et al., 2020). In the present study, 62 ADF proteins in alfalfa, Arabidopsis, rice, maize, and soybean were classified into five groups ( Figure 2 ), which is consistent with the classification of previous studies (Feng et al., 2006; Ruzicka et al., 2007; Huang et al., 2020; Sun et al., 2023b). With the exception of Group V, which exclusively comprised four monocot ADF proteins, the remaining four groups included ADF members from all five plant species. Furthermore, the number of ADFs in groups I–IV significantly differed between species, indicating that their ADF families underwent independent evolution and may have undergone functional differentiation, although ADFs in flowering plants are thought to have evolved from a common ancestor (Nan et al., 2017). Among higher plants, the function of Arabidopsis ADF genes has been the most thoroughly investigated, and dozens of ADF genes from other plants having been functionally characterized (see Sun et al., 2023a for a summary). The homologous relationship between MsADF and ADF genes from other plants may provide insights into their functional exploration. As a kind of evolutionary relic, the intron/exon arrangement carries the imprint of the evolution of a gene family, so analyzing exon-intron structures may provide insight into the evolutionary history of certain gene families (Wang et al., 2015). In this study, all MsADF genes were shown have three exons and two introns, and the length of three exons among different members were remarkably conserved ( Figure 3A ). This phenomenon had also been reported in Arabidopsis, rice, wheat, and soybean (Feng et al., 2006; Xu et al., 2021; Sun et al., 2023b). Moreover, the intron length of genes in the same clade were similar to each other, which further supports the analysis of the evolutionary relationship of MsADF genes.

In eukaryotic cells, the primary function of ADF has been identified as the acceleration of microfilament turnover through the facilitation of F-actin dynamics and the enhanced rate of G-actin departure from the F-actin end (Hussey et al., 2002). ADF has two potential binding sites for F-actin, one at the N-terminus and the other at the C-terminus (Tholl et al., 2011). Biochemical experiments demonstrated that several amino acid substitutions in the N-terminus resulted in significant variation of ADFs’ biochemical activity. For example, two members in group III (AtADF5 and AtADF9) have developed bundling rather than the typical depolymerizing activity; while ADFs belonging to group I (AtADF1, 2, 3, and 4) exhibit more pronounced depolymerizing activity compared to Groups II and IV (Nan et al., 2017). A motif analysis revealed that five MsADF proteins in groups I and II contain only the four motifs that constitute the ADF-H domain, whereas the other four members display a fifth motif at the N-terminus ( Figure 3B ; Supplementary Figure S2 ). This discrepancy in motif distribution suggests that MsADF may exhibit biochemical activity divergence. In AtADF9, two lysines (K4 and K17) at the N-terminus have been demonstrated to be essential for its bundling activity (Nan et al., 2017). It is noteworthy that these two homologous amino acids are also present in MsADF6, the only member of Group III. This suggests the possibility of a similar biochemical activity differentiation in this protein. In AtADFs, the H11 site is unique to Group I and plays a pivotal role in enhancing its depolymerizing activity (Nan et al., 2017). A further analysis of the sequence alignment reveals that two of the nine MsADFs, MsADF5 (belonging to Group I) and MsADF7 (belonging to Group II), contain the H11 site ( Supplementary Figure S2 ), suggesting that they may have enhanced depolymerizing activity. The activity of ADF family proteins is also regulated by the N-terminal serine residue, a fact that has been verified in both vertebrates and maize (Agnew et al., 1995; Moriyama et al., 1996; Smertenko et al., 1998). Upon phosphorylation, ADF becomes inactivated and loses its F-actin-binding ability (Blanchoin et al., 2000). The phosphorylation of this serine residue is dependent on calcium-dependent calmodulin-like substances. Phosphorylation represents a pivotal step in ADF protein function and is closely associated with calcium signalling pathways. It enables the protein to respond to environmental and developmental cues, thereby remodelling the cytoskeleton (Allwood et al., 2001). With the exception of MsADF6, all other eight MsADF proteins contain a conserved serine residue in their N-terminus, which may be subject to phosphorylation, as has been reported in other ADFs. The N-terminal serine of MsADF6 was replaced with a threonine residue ( Supplementary Figure S2 ), which had been demonstrated to undergo inhibitory phosphorylation by CDPK family members in plants and protists (Smertenko et al., 1998; Allwood et al., 2001). This substitution has been observed in numerous other ADFs belonging to group III, including AtADF5 and GmADF3, 5, 12, and 13 (Sun et al., 2023b), implying that the regulatory mechanism of group III ADFs’ biochemical activity may different from other groups. Biochemical analysis is needed in the future to determine the relationship between the phosphostatus of ADF and biological function.

The specific expression of ADF genes in different tissues and organs has been demonstrated in numerous plant species. The ADF genes belonging to the same group have been observed to exhibit a similar tissue-expression pattern (Ruzicka et al., 2007). In tomato, the primary expression of SlADF1, SlADF3, and SlADF10 in flowers, particularly in stamens, suggests a potential involvement of these genes in flower and pollen development, as well as other regulatory factors (Khatun et al., 2016). In this study, apart from MsADF4, all other MsADF genes were expressed in flowers, with notably high expression levels of MsADF2/3 from group IV and MsADF6 from group III ( Figure 5 ). This finding highlights the diversity and specificity of the MsADF gene family in the development of plant reproductive organs. Furthermore, ADFs are intimately linked with root formation and root hair development in plants (Bi et al., 2022). In Arabidopsis, AtADF5 and AtADF9 from group III, and AtADF6 from group IV, display ubiquitous expression across various tissues. AtADF5 displays robust expression in the root tip meristem, whereas AtADF9 exhibits elevated expression in the root apical zone, trichomes, shoot apical meristem, and callus (Ruzicka et al., 2007). The present study demonstrates that MsADF6, which belongs to the same group as AtADF5 and AtADF9, is strongly expressed in roots, nodules, old leaves, stems, and flowers ( Figure 5 ). Similarly, MsADF2/3 and MsADF9, which are grouped with AtADF6, exhibit widespread expression across different tissues. It is noteworthy that MsADF9 demonstrates lower expression in stems compared to other tissues ( Figure 5 ). These observations imply a significant role for these genes in the development of alfalfa, and the similarities in expression patterns between MsADF and AtADF genes suggest potential functional analogies.

ADFs play a pivotal role in plants’ response to external and internal stimuli by reorganizing the microfilament cytoskeleton, which alters cell morphology and serves as a key player in stress signaling pathways (Wang et al., 2011). Drought and salt are the most prevalent and severe abiotic stress for plant growth. In many species, the expression of ADF genes is induced by drought and salinity, and some ADF genes have been confirmed to having the potential to enhance plant resistance to stress (Sun et al., 2023a). In Arabidopsis, the expression of AtADF1 was rapidly induced by salt stress, and transgenic Arabidopsis overexpressing AtADF1 exhibited significantly increased survival rates under salt stress compared to the wild type (Wang et al., 2021). Drought stress can induce the expression of AtADF5, which in turn regulates the reorganization of microfilament structure through its F-actin bundling activity, thereby controlling stomatal movement and enhancing the plant’s ability to adapt to drought stress (Qian et al., 2019). The heterologous expression of OsADF3 of rice, a gene exhibited significantly upregulation under various abiotic stress, in Arabidopsis has been demonstrated to enhance germination rate, primary root length, and seedling survival rate under conditions of drought stress (Huang et al., 2012). Overexpression of ZmADF5 in maize resulted in a reduction in stomatal aperture, a decrease in water loss rate, an enhancement in ROS scavenging capacity, and an improvement in drought resistance in the plants (Liu et al., 2024). The enhancement of drought resistance by GmADF13 is achieved through the modulation of osmoregulatory substance accumulation, the regulation of enzyme activities, and the regulation of stress tolerance-related gene expression (Wang et al., 2024). In this study, many MsADF genes were identified as being up-regulated under conditions of drought and salt stress. For instance, in the underground part, the expression levels of MsADF4, 5, 6, 8, and 9 increased by more than four-fold in response to drought stress ( Figure 6A ); in the aboveground part, the expression levels of MsADF7 and 8 increased by fourteen-fold and twelve-fold, respectively, under salt stress ( Figure 6B ). Concurrently, under salt and drought stress, the expression levels of MsADF2/3, 4, and 6 were up-regulated in both the aboveground and underground parts ( Figure 6 ). The identification of these genes offers significant references for subsequent functional research. However, in the underground part, the expression level of MsADF7 was down-regulated under salt stress ( Figure 6A ). This phenomenon is analogous to the repression of TaADF20 expression in wheat under cold stress (Xu et al., 2021). This phenomenon can be explained by the antagonistic relationships among ADF family genes. For instance, AtADF9 has been shown to exert an antagonistic effect on AtADF1 by modulating its degradation of actin. Conversely, when these proteins are ectopically expressed in tobacco cells, a contrasting effect is observed (Tholl et al., 2011).

The cis-acting elements within gene promoters are closely associated with gene expression patterns in response to different stresses (Cao et al., 2021). Huang et al. (2012) conducted a comprehensive analysis of the promoter structure and expression patterns of all 11 rice ADFs, revealing the presence of stress-related cis-acting elements within the 1-kb promoter regions. This finding suggests that certain OsADF genes are induced by diverse environmental stresses, including low temperature, drought, abscisic acid (ABA), and salt (Huang et al., 2012). Compared to other genes, the MsADF7 promoter sequence exhibits the least number and types of stress-responsive cis-acting elements ( Figure 4 ). This characteristic may uniquely determine the expression pattern and response mechanism of MsADF7 to environmental stress. However, under specific time points of drought or salt stress, the expression level of MsADF7 is found to be up-regulated in the aboveground part ( Figure 6B ), suggesting that MsADF7 may play a role in the cross-talk of different signaling pathways under drought and salt stress (Yang et al., 2022). These findings underscore the complexity of the MsADF regulatory mechanism. Furthermore, the expression level of MsADF1 exhibited a continuous downward trend, reaching an extremely low and almost undetectable level after 24 hours. The down-regulation of gene expression may serve as a signal for plants to perceive and respond to drought stress, thereby participating in signal transmission and response processes to help plants adapt to adverse environments. Additionally, this study identified that certain genes are involved in the expression of salt and drought response, demonstrating varied reactions. For instance, under salt stress, the expression of MsADF1, 7, and 9 was up-regulated in the aboveground part, while their expression was down-regulated or did not differ significantly in the underground part ( Figure 6 ), suggesting that their biological functions are location-specific. These findings suggest that the MsADF gene family may play a pivotal role in plants’ response to diverse abiotic factors and may serve as crucial regulatory factors for plants to cope with environmental stress. Further investigation into the functions and regulatory mechanisms of these genes is anticipated to provide invaluable theoretical and practical guidance for enhancing crop stress resistance.

The present study identified a total of nine MsADF genes in the alfalfa genome. A phylogenetic analysis indicated that they could be classified into four groups, with members of the same group exhibiting analogous gene structure characteristics and conserved motifs. A number of cis-acting elements related to hormone- and stress- responsiveness were identified within their promoter region. Furthermore, gene expression profiles demonstrated that these MsADF genes exhibited disparate expression patterns in different organs. The RT-qPCR analysis demonstrated that the majority of the MsADF genes exhibited varying degrees of induction in response to salt and drought stress. Of particular note is the dramatic induction observed in MsADF1, 2/3, 6, and 9. These genes represent promising candidates for further investigation into the molecular mechanisms underlying alfalfa stress resistance, as well as for the breeding of new alfalfa cultivars with enhanced stress tolerance. Such insights could potentially inform the genetic improvement of alfalfa resistance to abiotic stresses.