The actin cytoskeleton which is ubiquitous to all eukaryotic cells, not only provides mechanical support for the maintenance of the basic cellular structure, but is also involved in diverse biological processes that are necessary for plant growth, development, and response to various environmental changes (Porter and Day, 2016; Melak et al., 2017; Schaks et al., 2019). The organization and dynamics of the actin cytoskeleton are specific and directly regulated by numerous actin-binding proteins (ABPs) (Pollard, 2016). The actin-depolymerizing factors (ADFs) are an important class of low-molecular-mass (15 – 22 kDa) ABPs that play essential roles in many actin-remodeling processes. Their structure is characterized by the presence of a conserved motif known as the actin-depolymerizing factor homology (ADF-H) domain (Inada, 2017). The classic form of ADF can cleave or depolymerize filamentous actin (F-actin) into either shorter fragments or globular actin (G-actin), which provides new sites for the initiation of novel actin filaments and supplies more actin monomers for polymerization. Thus it regulates the reorganization and rearrangement of the actin cytoskeleton which in turn facilitates the responses to various developmental and environmental stimuli (Wioland et al., 2017).

The ADF protein was first isolated from the brains of chick embryos (Bamburg et al., 1980), and since then the ADF genes have been identified in a wide range of eukaryotes (Gunning et al., 2015). The organisms such as simple eukaryotes and those of animal lineages typically possess a maximum of three isoforms of ADF, whereas higher plants possess an enlarged ADF family (Roy-Zokan et al., 2015). The genome-wide identification of the ADF family has been accomplished in several plant species. For instance, the genomes of Arabidopsis thaliana (Feng et al., 2006; Ruzicka et al., 2007), rice (Oryza sativa; Feng et al., 2006; Huang et al., 2012), and tomato (Solanum lycopersicum; Khatun et al., 2016) each contained 11 ADFs. In addition, 9, 10, 13, 14, 25, and 27 ADFs were identified from the genomes of common bean (Phaseolus vulgaris; Ortega-Ortega et al., 2020), pigeon pea (Cajanus cajan; Cao et al., 2021), maize (Zea Mays; Huang et al., 2020), poplar (Populus trichocarpa; Roy-Zokan et al., 2015), wheat (Triticum aestivum; Xu et al., 2021), and banana (Musa acuminata; Nan et al., 2017), respectively. The expansion of gene number may allow plant ADF genes expressed in a complicated profile and differentiated into multiple biological functions.

Based on their phylogenetic relationship, the ADFs in Arabidopsis have been categorized into four groups (I – IV), with each group exhibiting a unique expression profile (Feng et al., 2006; Ruzicka et al., 2007). The members of group I (AtADFs 1, 2, 3, and 4) were expressed at a relatively higher level throughout the plant except in the pollen. Genes of group II were specifically expressed in the polar cells: AtADF7 and AtADF10 in the mature pollen grains; whereas those of AtADF8 and AtADF11 was limited to the root-trichoblasts and root hair. The genes of Group III (AtADF5 and AtADF9) were weakly expressed in the vegetative tissues, but strongly in the rapidly growing and/or differentiating cells. AtADF6, a group IV gene was constitutively expressed at moderate or lower levels in all the organs and tissues (Dong et al., 2001; Ruzicka et al., 2007). This diversity in the tissue-specific expression patterns of plant ADFs indicated their evolution into divergent physiological functions. Indeed, biochemical experiments indicated that the ADFs belonging to groups I, II, and IV possessed a conserved F-actin cleaving/depolymerizing (D-type) activity, while the members of group III demonstrated an F-actin bundle-formation (B-type) activity, this variation resulted from certain crucial amino acid substitutions, suggesting that functional divergence had occurred in the ADF family of plants (Nan et al., 2017).

ADFs in higher plants have been proven to be involved in numerous biological processes, including the growth and expansion of organs, flowering, stomatal movement, and responses to various stresses (Burgos-Rivera et al., 2008; Huang et al., 2012; Fu et al., 2014; Sengupta et al., 2019). AtADF1 is involved in regulating hypocotyl elongation and responses to salt-induced stress (Zhao et al., 2015; Wang et al., 2021). The actin dynamics mediated by AtADF2 are essential for the infection of Arabidopsis roots by the root-knot nematode (Clement et al., 2009). AtADF3 and AtADF4 play vital roles in regulating hypocotyl growth, the morphology of epidermal cells, tolerance to drought- and osmotic-induced stress, and attacks by various pathogens (Porter et al., 2012; Henty-Ridilla et al., 2014; Zhao et al., 2016; Inada, 2017; Yao et al., 2022). In response to ABA- and drought-induced stresses, the expression of AtADF5 was induced, which then participated in regulating stomatal closure (Qian et al., 2019). ADFs in crop plants have proven to be essential for the responses and tolerance to various stress. The expression of TaADF (Genebank No. U58278) was much higher in the freezing-tolerant wheat cultivars when compared with that in the sensitive ones (Ouellet et al., 2001). TaADF3 and TaADF4 demonstrated contrasting effects on the resistance of wheat plants to infection by Puccinia striiformis f. sp. tritici (Tang et al., 2016; Zhang et al., 2017). Overexpression of OsADF3 and TaADF16 improved the drought- and freezing-tolerance of transgenic Arabidopsis seedlings, respectively (Huang et al., 2012; Xu et al., 2021). GmADF2 interacted with the P3 protein of soybean mosaic virus (SMV), which may facilitate its infection process (Lu et al., 2015). Downregulation of GhADF6 enhanced the abundance of actin filaments and bundles in the root cells, and rendered cotton plants tolerant to infection by the fungal pathogen, Verticillium dahliae (Sun et al., 2021).

Soybean is an important food and cash crop cultivated worldwide, which serves as an excellent source of plant-based proteins and edible oils which are rich in various beneficial nutrients, such as isoflavones and vitamins. Adverse environmental stresses, such as drought, salt, and extreme temperatures, greatly hindered the growth of soybean plants and thus severely diminished the yield (Deshmukh et al., 2014). To the best of our knowledge, there has been only one report regarding the soybean ADF gene to date (Lu et al., 2015). As the ADF family is known to be critical for the growth, development, and stress tolerance of plants, investigation of the family in soybean is of great importance. In this study, 18 soybean ADFs were identified from the genome of the soybean cultivar “Williams 82”. Next, the structures of these genes were analyzed, their locations in the genome were determined, the duplication events and phylogenetic relationships were identified, and the conserved motifs within the sequences of the encoded proteins were recognized. Tissue-specific expression profiles of these genes and in response to heat, cold, drought, and salt-induced stresses were also analyzed to determine their putative functions. The data provided in this study are reliable to screen key candidate genes from the ADF family in soybean for further functional investigation at molecular level, and for the molecular breeding of soybean with stress tolerance.

The ADFs are among the most important ABPs with a proven involvement in various biological processes including stress tolerance in plants (Inada, 2017; Sun et al., 2021; Xu et al., 2021). Genome-wide identification and functional characterization of the ADF genes have been achieved in several plant species (Ruzicka et al., 2007; Khatun et al., 2016; Huang et al., 2020; Xu et al., 2021). However, our understanding of the ADF family in soybean is greatly limited. Hence, these genes in soybean were identified along with an analysis of their phylogeny, duplication relationship, sequences of the genes and their encoded proteins, and expression profiles. A total of 18 ADFs were identified in the soybean genome ( Table 1 , Figure 1 ), which is the highest among all the diploid plants analyzed to date (Feng et al., 2006; Ruzicka et al., 2007; Khatun et al., 2016; Huang et al., 2020). It has been noted that this phenomenon of the existence of a high number also occurs in other soybean gene families, such as the growth regulating factor (Chen et al., 2019) and B-box gene families (Shan et al., 2022), and may have mainly resulted from two whole genome duplication events that had occurred during the evolution of soybean (Schmutz et al., 2010). Tandem and segmental duplications are considered to be the major driving forces behind the multiplication of gene families during evolution (Cannon et al., 2004). In Arabidopsis, two duplicate gene pairs (AtADFs 1 and 2; and AtADFs 3 and 4) are arranged in tandem (Nan et al., 2017), while one tandem duplication event (TaADFs 17 and 18) had been identified in wheat (Xu et al., 2021). In contrast, only segmental duplication events were identified within ADFs from tomato (Khatun et al., 2016) and maize (Huang et al., 2020). In this study, all 29 pairs of duplicated GmADFs were identified to be generated by segmental duplication, suggesting that it was primarily responsible for the expansion of the ADF family in soybean during evolution ( Figure 1 ; Supplementary Table S2 ). Further analysis of the evolutionary selective pressure showed that GmADFs underwent a strong purifying selection during evolution, suggesting that their functions might be evolutionarily conserved ( Supplementary Table S3 ).

Previous studies have shown that the ADFs in flowering plants had most likely evolved from a common ancestor (Nan et al., 2017). Based on their phylogenetic relationship, 53 ADF proteins from Arabidopsis, rice, maize, and soybean were clustered into five groups ( Figure 2 ); this was in agreement with the structure of their genes and the distribution of motifs within them ( Figure 3 ) and also the published data (Ruzicka et al., 2007; Huang et al., 2012; Khatun et al., 2016; Huang et al., 2020). It was observed that the number of genes in the subfamilies varied among the different plant spp. For example, the distribution of ADFs to the groups I, II, III, IV, and V in Arabidopsis were 4, 4, 2, 1, and 0 respectively; which were 4, 6, 4, 4, and 0 in soybean; 1, 2, 1, 4, and 3 in rice; and 1, 3, 2, 3, and 4 in maize ( Figure 2 ). These results suggested that although the ADF families in different species might have had a common ancestor, the subsequent evolutionary processes were relatively independent of each other. Correspondingly, the gene sequences and their expression patterns were also reasonably varied as described next.

Simple eukaryotes and organisms of animal lineages possess only a few ADF isoforms which have been reported to be highly conserved both structurally and functionally (Gunning et al., 2015). However, the functional divergence occurred in the higher plants as the number of individual gene members expanded to a dozen or dozens (Inada, 2017). The biochemical analysis of the ADFs from Arabidopsis showed that the group III ADFs displayed a B-type but not the classic D-type activity, and that the D-type activity of group I ADFs was stronger than those of groups II and IV (Nan et al., 2017). Multiple sequence alignment revealed that group III GmADFs had the same amino acid substitution as was identified in Arabidopsis ( Supplementary Figure S2 ), suggesting that the evolution of the B-type activity of ADFs may also have occurred in soybean. It is worth noting that not only the GmADFs of group I, but also the GmADFs 1, 4, 6, and 11 of group II harbored the H11 substitution, which is critical for the enhanced D-type activity of group I AtADFs ( Supplementary Figure S2 ), implying that functional differentiation through evolution may have occurred among the group II genes. Phosphorylation of a Ser at the N-terminus is critical for the modulation of the biochemical activity of ADFs in various organisms and is linked to the Ca-signaling pathway; thereby it reorganizes the cytoskeleton in response to environmental and developmental signals (Blanchoin et al., 2000; Porter et al., 2012; Dong and Hong, 2013). Multiple sequence alignment demonstrated the occurrence of an S10T substitution in the group III GmADFs, which may influence the roles played by them in biological processes such as signal transduction. These observations may indicate the existence of a diverse functional divergence within the GmADF family, which needs further investigation.

The expression patterns of genes in various organs or tissues may reflect to a certain extent the specific functional differences of the members of that gene family. The actin genes in Arabidopsis were grouped into the vegetative and reproductive classes due to their tissue-specific pattern (McKinney and Meagher, 1998). Similarly, previous studies have mentioned that the genes of the ADF family can also be divided into two classes that differ in their expression patterns, i.e., reproductive or constitutive/vegetative (Ruzicka et al., 2007). In this study, the group II GmADFs were found to specifically express in the flowers ( Figure 4 , Supplementary Table S4 ), which was in agreement with the published data concerning Arabidopsis, rice, maize, and wheat (Ruzicka et al., 2007; Huang et al., 2012; Khatun et al., 2016; Huang et al., 2020). Thus, it can be speculated that the GmADFs 1, 4, 6, 10, 11, and 17 may be critical for reproduction in soybean. The AtADFs of Group I have been proven to be highly expressed in all the tissues except in pollen and play important roles in diverse biological processes including organ growth and responses to various stresses (Clement et al., 2009; Wang et al., 2021; Yao et al., 2022). Similarly, in this study the expression levels of GmADFs 7, 8, 14, and 15 of group I and GmADFs 9 and 18 of group II were higher than those of the other GmADFs ( Figure 4 , Supplementary T able S4 ), implying their essential roles in soybean development.

The rearrangement of the actin cytoskeleton of plant cells can be a target for the signaling pathways induced by numerous developmental and environmental stimuli (Melak et al., 2017; Schaks et al., 2019). Previous studies have shown that the ADFs of plants actively respond to various biotic and abiotic stresses and participate in developing resistance to them, via the remodeling of the actin cytoskeleton (Inada, 2017). Previous studies concerning the genome-wide characterization of ADFs in plants have suggested that their expression levels were significantly altered under abiotic stresses such as cold, drought, high salt, abscisic acid, jasmonic acid, wounding, etc (Khatun et al., 2016; Huang et al., 2020; Xu et al., 2021). Certain ADFs had been confirmed to possess the potential to improve stress resistance in crop plants. Overexpression of TaADF16 and OsADF3 enhanced the tolerance to freezing- and drought-induced stresses in transgenic Arabidopsis plants (Huang et al., 2012; Xu et al., 2021). SaADF2 from Spartina alterniflora, a model halophyte for monocotyledonous grass crops, significantly enhanced the tolerance to drought- and salinity-induced stress when expressed in rice plants to a greater extent than its rice homolog OsADF2 (Sengupta et al., 2019). Similarly, DaADF3 isolated from Deschampsia antarctica, a plant native to Antarctica, played an important role in the enhancement of cold tolerance when expressed in rice plants (Byun et al., 2021). In this study, the expression levels of GmADFs changed to varying degrees under heat, cold, drought, and salt-induced stresses. For example, the expression levels of GmADFs 2, 5, 12, 13, and 16 were upregulated by > 20-fold under drought treatment ( Figure 5 ). Considering the critical roles of ADFs in modulating stress tolerance in plants, the exploration of the GmADFs regarding their functions is needed for the elucidation of the molecular mechanisms underlying stress tolerance in soybean.

In this study, a total of 18 ADF genes were identified in the soybean genome, which was unevenly distributed on 14 chromosomes. Segmental duplication during evolution was primarily responsible for the expansion of the soybean ADF family. Based on their phylogenetic relationships, these genes could be divided into four groups with those of each group showing similar structures and motif distribution in the encoded proteins. Several amino acids that are critical for the divergence of their biochemical activities were identified in the GmADFs, and biochemical analyses were needed in the future to clarify their functional properties. GmADF genes exhibited diverse expression patterns in the various tissues or organs of soybean plants, and many of them were found to respond to varied abiotic stresses, especially those induced by drought and salt, implying that they may crucial for generating stress tolerance in soybean. These genes are important candidates for the investigation of the molecular mechanisms concerning stress resistance of soybean, and also for the breeding of new soybean varieties with enhanced stress tolerance, which may enlighten soybean genetic improvement in resistance to abiotic stresses.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

YS and SG designed this study. YS and DW performed the bioinformatics analysis and the most experimental work. DW, MS, YG, SY, and YJ grew the plants and performed the stress treatment. YS and DW analyzed the data and wrote this manuscript. All authors have read and approved the final manuscript.