Fatty acids with a hydrocarbon chain lengths of 20 or more carbon atoms are known as VLCFAs (Scott et al., 2022). The VLCFAs and their derivatives are the main components of numerous lipids that function in specific cell/tissue types, such as triacylglycerols that accumulate in the seeds, sphingolipids, and phospholipids that are essential for membrane homeostasis, cuticular waxes deposited on plant aerial surfaces, and suberin monomers that are indispensable for root development (Haslam and Kunst, 2013). Cuticular wax forms a hydrophobic layer covering most aerial plant surfaces. It plays a vital role in preventing excessive water loss, regulating transpiration, resistance against pathogen invasion, and tolerance against various environmental stresses (Kong et al., 2020; Arya et al., 2021; Batsale et al., 2021). The biosynthesis of VLCFAs is catalyzed by a fatty acid acyl-CoA elongase (FAE) multienzyme complex comprising four enzymes, including 3-ketoacyl-CoA synthase (KCS), β-ketoacyl-CoA reductase (KCR), β-hydroxy acyl-CoA dehydratase (HCD) and trans-2,3-enoyl-CoA reductase (ECR) (Bach and Faure, 2010; Kim et al., 2013). Using the plastid-generated C16 or C18 fatty acids as substrates, the endoplasmic reticulum-localized FAE sequentially adds two carbon units to a growing acyl chain through a repetitive cycle of condensation, keto-reduction, dehydration and enoyl-reduction (Kunst and Samuels, 2009). During the elongation process of VLCFAs, KCS catalyzes the condensation of C16 or C18 fatty acids with malonyl-CoA. Moreover, KCS exhibits strict substrate specificity and tissue specificity, and it is the key rate-limiting enzyme that determines the ultimate chain length and contents of VLCFAs (Shi et al., 2017; Ozseyhan et al., 2018). In contrast, the other three enzymes do not directly control VLCFA synthesis (Kunst and Samuels, 2009).

The first identified and characterized gene in the KCS family was FAE1/AtKCS18, specifically expressed in developing seeds, which catalyzes the synthesis of C20 and C22 fatty acids for energy storage in Arabidopsis thaliana (James et al., 1995; Rossak et al., 2001). Subsequently, numerous KCS genes have been cloned and functionally characterized in plants. The KCS proteins have two essential and conserved domains: the FAE1/Type III polyketide synthase-like protein domain (FAE1_CUT1_RppA) and the 3- Oxoacyl-[acyl-carrier-protein (ACP)] synthase III C-terminal domain (ACP_syn_III_C) (Sagar et al., 2015). KCS gene families have been identified in various higher plants, and the number of these genes varies greatly between species (Guo et al., 2016). For instance, 21 KCS genes were identified in Arabidopsis thaliana (Costaglioli et al., 2005; Joubès et al., 2008), 22 in rice (Oryza sativa; Yang et al., 2023), 25 in sorghum (Sorghum bicolor; Zhang et al., 2022a), 26 in maize (Zea mays; Campbell et al., 2019), 28 in apple (Malus × domestica Borkh.; Lian et al., 2020), 30 in peanut (Arachis hypogaea; Huai et al., 2020) and cabbage (Brassica oleracea; Xue et al., 2020), 33 in turnip (Brassica rapa; Xue et al., 2020) and barley (Hordeum vulgare; Tong et al., 2021), and as many as 58 in upland cotton (Gossypium hirsutum; Xiao et al., 2016) and rapeseed (Brassica napus; Xue et al., 2020).

The KCS gene family in Arabidopsis thaliana has been the most thoroughly studied and characterized in terms of physiological and molecular functions. It is divided into four classes (KCS1-like, FAE-like, FDH-like, CER6) or eight subclasses (α, β, γ, δ, ε, ζ, η, and θ) according to their phylogeny and duplication history (Costaglioli et al., 2005; Joubès et al., 2008). Previous studies have demonstrated that they catalyze the formation of diverse VLCFAs and play important roles in Arabidopsis developmental and metabolic processes, such as cuticular wax production (AtKCSs 1, 2, 3, 4, 5, 6, 9, 16 and 20) (Todd et al., 1999; Fiebig et al., 2000; Kim et al., 2013; Hegebarth et al., 2017; Kim et al., 2021; Huang et al., 2022; Huang et al., 2023), suberin metabolism in roots (AtKCSs 2, 9 and 20) (Franke et al., 2009; Lee et al., 2009; Kim et al., 2013), oil biosynthesis in seeds (AtKCS18) (James et al., 1995), and development of epidermis (AtKCS10 and AtKCS13) (Gray et al., 2000; Pruitt et al., 2000). Moreover, several AtKCS genes have been confirmed to play crucial roles in abiotic stress responses and tolerance. For example, AtKCS2 and AtKCS20 expression levels were drastically elevated under various abiotic stresses, indicating that they may be involved in stress adaptation and tolerance (Lee et al., 2009; Lee and Suh, 2015a). AtKCS1 was reported to be an important player involved in chilling tolerance (Chen et al., 2020c), and AtKCS4 plays a key role in the regulation of lipid metabolism under heat and dark conditions (Luzarowska et al., 2023).

In other species, particularly in crop plants, numerous KCS genes have been demonstrated to be vital for developmental and stress tolerance mechanisms. LeCER6 is an essential KCS gene that regulates fruit cuticular wax production in tomato (Smirnova et al., 2013). The maize GL4, a homolog of AtKCS6, is involved in cuticular wax accumulation in seedling leaves (Lee and Suh, 2015b). HMS1 expression increased pollen viability under low moisture stress in rice (Chen et al., 2020b). Overexpression of GhKCS13 enhanced the sensitivity to cold stress of transgenic cotton plants by modulating lipid and oxylipin biosynthesis (Wang et al., 2020a). Ectopic expression of VvKCS11 from grape (Vitis vinifera) or CcKCS6 from navel orange (Citrus clementina) enhanced the tolerance of transgenic Arabidopsis plants to various abiotic stresses (Guo et al., 2020; Yang et al., 2020). MtKCS12, predominantly expressed in the Medicago truncatula seed coat, controls the production of very long-chain lipid species in the seed coat and is essential for seed physical dormancy (Chai et al., 2021).

Soybean (Glycine max) is an economically important food and oil crop with high nutritional value, with its seed oil accounting for about 30% of all vegetable oil consumption worldwide (Zhan et al., 2020). It provides abundant nutrients for human nutrition, has various industrial uses, and is considered a suitable candidate crop for biodiesel production (Woyann et al., 2019). Moreover, soybean is one of the most commonly cultivated crops in arid and semi-arid areas where its growth and yield are usually hindered by various abiotic stresses, such as water deficiency, salt, and temperature extremes (Deshmukh et al., 2014; Gavili et al., 2018). To the best of our knowledge, there are limited studies regarding the soybean KCS gene family to date. As the KCS family genes are known to be crucial players in regulatory networks controlling biological and developmental processes as well as stress tolerance of plants, a thorough investigation of the KCS gene family in soybean is of great importance. In this study, 31 soybean KCS genes were identified using bioinformatics techniques. Their chromosomal distribution, gene structure, conserved motif distribution, gene synteny, and phylogenetic relationships were systematically analyzed. To further analyze the prospective functions of soybean’s KCS genes, their tissue-specific expression profiles were examined, as well as their response to cold, heat, salt, and drought stresses. These findings establish the groundwork for understanding the functions of the GmKCS gene family and offer crucial insights into their involvement in soybean’s stress tolerance mechanisms.

Cuticular wax forms the first protective barrier for the aerial parts of all terrestrial plants and plays a vital role in plant resistance against various biotic and abiotic stresses (Kong et al., 2020; Batsale et al., 2021). VLCFAs and their derivatives, including alkanes, ketones, alcohols, aldehydes, and wax esters, are the main components of cuticular wax. Thus, their content and composition are tightly associated with plant growth, development, and stress tolerance capability (Haslam and Kunst, 2013; Scott et al., 2022). In Arabidopsis, drought, salt and abscisic acid stress treatments lead to a significant increase in the amount of leaf cuticular wax (Kosma et al., 2009). Total leaf cuticular wax of tree tobacco (Nicotiana glauca) increased approximately 2-fold under drought stress, and leaves subjected to drying stress were more resistant to water loss than that of control (Cameron et al., 2006). Various stress conditions can elicit diverse impacts on the content, composition, and structure of plant cuticular wax (Shepherd and Wynne, 2006). The systematic identification and analysis of genes involved in plant cuticular wax synthesis and their expression patterns under stress are of great significance for understanding the molecular regulatory mechanisms of plant response to stress.

3-ketoacyl-CoA synthases, encoded by the KCS gene family, have been confirmed to be the key rate-limiting enzymes that determine the tissue and substrate specificities during VLCFA production, which are present in nearly all plants (Ozseyhan et al., 2018). The genome-wide identification and functional characterization of the KCS gene family have been described in many plant species, and large differences have been observed in the number of KCS family genes (ranging from 7 to 58) in higher plants (Guo et al., 2016; Campbell et al., 2019; Lian et al., 2020; Xue et al., 2020; Zhang et al., 2022a). However, our understanding of the structural and functional properties of the KCS gene family in soybean, one of the most important food and oil crops worldwide, is greatly limited. Identification and characterization of the soybean KCS family genes at the whole genome level could provide fundamental information for their functions and facilitate the genetic improvement of soybean.

In this study, a total of 31 KCS genes were identified in the soybean genome ( Table 1 ; Figure 1 ), which is the highest number among all the diploid plants analyzed to date (Joubès et al., 2008; Guo et al., 2016; Campbell et al., 2019; Zhang et al., 2022a; Yang et al., 2023). A higher gene number occurrence has also been reported for other gene families in soybean, such as the B-box (Shan et al., 2022), the cytokinin oxidase (Du et al., 2023), and the actin-depolymerizing factor gene families (Sun et al., 2023). The KCS gene family expansion observed in soybean is possibly due to the two whole genome duplication events that occurred during soybean evolution (Schmutz et al., 2010). It appears that the KCS family genes have been significantly subjected to duplication events and expansion in soybean. Tandem and segmental duplications are considered to be the two main modes of gene family expansion during species evolution (Lynch and Conery, 2000). The former often results in a cluster of genes in the same or adjacent intergenic regions (Lynch and Conery, 2000), which has been observed among KCS genes in many species, such as Arabidopsis (Joubès et al., 2008), apple (Lian et al., 2020), sorghum (Zhang et al., 2022a) and barely (Tong et al., 2021). However, segmental duplications are the main driving force in KCS gene family expansion. In this study, a synteny analysis showed that 27 duplicated gene pairs covering 28 GmKCS genes were found to be generated by segmental duplication ( Figure 1 ), which was also supported by their similar exon-intron structures ( Figure 3 ). However, no tandem duplication event was identified as responsible for the expansion of the GmKCS family in soybean. Therefore, segmental duplications were primarily responsible for the KCS family’s expansion during soybean evolution. Moreover, these duplicated gene pairs were not only clustered into the same branches of the phylogenetic tree but also exhibited low Ka/Ks ratios ( Figure 1 ; Figure 2 ; Supplementary Table S3 ), implying that GmKCS genes underwent a strong purifying selection during evolution and their functions might be evolutionarily conserved.

Generally, genes clustered in the same evolutionary clade are thought to perform similar biological functions (Liu et al., 2018). Based on their phylogenetic relationships, 131 KCS proteins from Arabidopsis, turnip, soybean, rice and sorghum were clustered into nine groups ( Figure 2 ), consistent with previously published reports (Joubès et al., 2008; Xiao et al., 2016; Xue et al., 2020; Yang et al., 2023). Group λ and η KCS genes are unique to monocotyledonous and dicotyledonous plants, respectively, suggesting that these genes might be formed after the separation of monocots and dicots and functions between these genes may have differentiated ( Figure 2 ; Table 2 ). Moreover, we found that KCSs in group β only existed in in two cruciferous plants, Arabidopsis and turnip. To explore whether group β KCS genes have been lost only in soybean, rice and sorghum, we searched for articles on the KCS gene family in other plant species. Interestingly, the loss of the β group KCS gene was also reported in cotton (Xiao et al., 2016), apple (Lian et al., 2020), peanut (Huai et al., 2020), barely (Tong et al., 2021), yellow horn (Liu et al., 2022), sorghum (Zhang et al., 2022a), passion fruit (Rizwan et al., 2022), and rice (Yang et al., 2023). Consistent with our results, KCS genes in group β have also been found in rapeseed and cabbage (Xue et al., 2020), implying that group β KCS genes may be unique to cruciferous plants. Genes in the other six groups might have undergone similar evolutionary diversification across plant families and species. The GmKCS gene homologs of known function in Arabidopsis provided clues or references for predicting and exploring the biological function of GmKCS genes ( Figure 2 ). For example, the group α GmKCS genes were clustered with AtKCS4 and AtKCS9, which are widely involved in the biosynthesis of cuticular wax, suberin, sphingolipids, and phospholipids (Kim et al., 2013; Kim et al., 2021). GmKCS2 in the group δ is closely related to AtKCS1, which has been shown to regulate cuticular wax biosynthesis and tolerance to chilling stress in Arabidopsis (Todd et al., 1999; Chen et al., 2020c). The group ζ GmKCS genes are closely related to AtKCS2 and AtKCS20, which are involved in the biosynthesis of cuticular wax and root suberin and tolerance to osmotic stress (Lee et al., 2009; Lee and Suh, 2015a), GmKCS3 and GmKCS8, classified in group ε, are closely related to AtKCS10, which is required for normal development of the epidermis (Pruitt et al., 2000).

FAE1_CUT1_RppA and ACP_syn_III_C are the two major conserved domains in KCS proteins. The former had been reported to contain the motif that specifically binds to the substrate and thus may be related to the substrate specificity of each KCS enzyme, whereas the latter is important for initiating the fatty acid synthase chain reactions (Funa et al., 2002; Sagar et al., 2015). Our motif analysis showed that although the ACP_syn_III_C domain is conserved among GmKCS proteins, diverse sequence variants were found in the FAE1_CUT1_RppA domain ( Figure 3C ). Moreover, we also found the N-terminal was poorly conserved among GmKCS proteins. For example, there is a significant difference in sequence length before the FAE1_CUT1_RppA domain, and some of the GmKCS contain motifs 6 and 7. In contrast, other gene family members don’t contain any of the motifs ( Figure 3C ). The different distribution of motifs within KCS proteins was also reported in previous studies (Lian et al., 2020; Liu et al., 2022; Rizwan et al., 2022). Differences in GmKCS protein sequence characteristics imply that functional differentiation through evolution may have occurred, and further research on these sequence variations is needed for their functional elucidation and exploration.

Cis-acting elements play critical roles in signal transduction and regulation of gene transcription initiation (Narusaka et al., 2003). The mechanisms underlying the expression regulation of each KCS gene could be better understood by identifying the upstream cis-acting elements of the GmKCS genes. In agreement with previous reports concerning the KCS gene family (Lian et al., 2020; Rizwan et al., 2022; Zhang et al., 2022a), cis-acting elements involved in hormone- and stress-responsiveness were found to be widely distributed in GmKCS promoters, and all GmKCS promoters contain at least one type of hormone- and stress-responsive elements ( Figure 4 ). Previous studies demonstrated that these elements are involved in gene expression regulation under various stresses (Jiang et al., 2022; Dong et al., 2023; Zhang et al., 2023). Our results suggest that GmKCS expression is closely associated with abiotic stress and hormone-signaling responses.

The diverse expression patterns of KCS genes in different tissues have been described in many plants. For example, all 21 AtKCS genes were found to be expressed but had diverse expression levels and tissue distributions (Joubès et al., 2008). Furthermore, certain rice KCS genes exhibit specific expression in different organs or different stages of growth and development (Yang et al., 2023). Exploring the expression profiles of GmKCS genes may help uncover their potential roles in plant physiological processes and development. We obtained the expression patterns of each GmKCS gene in nine tissues/organs from a public database. 27 out of the 31 GmKCS genes were found to be expressed in at least one tissue/organ, and six of which (i.e., GmKCSs 2, 5, 7, 20, 25 and 27) were differentially expressed throughout the different organs of the soybean plants ( Figure 4 ; Supplementary Table S3 ). Several members that were constitutive expressed throughout the plants (GmKCSs 2, 5, 7, 20, 25, and 27) or predominantly expressed in specific tissue/organs (GmKCSs 6, 15, 17, and 26) were identified ( Figure 4 ; Supplementary Table S3 ). The differential expression of GmKCS genes in different tissues/organs, and for certain genes, their tissue specificity, may indicate that they perform different functions in different plant organs and remains to be further investigated.

Unfavorable abiotic stress environmental conditions are often detrimental to growth and development. Drought, salt, and temperature stresses are the main environmental factors that limit the productivity of agricultural plants and threaten food security (Zhang et al., 2022b). Increasing evidence suggests that plant KCS genes are involved in abiotic stress responses and adaptation (Batsale et al., 2021). For instance, Arabidopsis kcs1 mutants exhibited reduced resistance to low humidity conditions at a young age (Todd et al., 1999), while overexpression of drought-induced KCS genes, such as CsKCS6 from orange, BnKCS1 from rapeseed, or AhKCS1 from groundnut, enhanced the drought tolerance of transgenic plants (Lokesh et al., 2019; Guo et al., 2020; Wang et al., 2020b). These genes can be targeted in crop breeding to improve drought tolerance. In our study, we examined the transcriptional profiles of the 15 leaf-expressed GmKCS gene family members under four abiotic stress conditions. Our qRT-PCR results showed that the expression levels of 9, 5, 6, and 13 GmKCS genes were altered more than two-fold under cold, heat, salt, and drought stress, respectively ( Figure 6 ). Some of them exhibited a greater than 5 fold up-regulation under stress conditions, i.e., GmKCS5 under cold stress, GmKCS4 under salt stress, and GmKCSs 1, 3, 5, 12, and 27 under drought stress ( Figure 6 ). The results indicated that the GmKCS genes may widely be involved in abiotic stress adaptation and resistance of soybean. Currently, there is limited evidence regarding the potential link between the differential expression of GmKCS genes and the accumulation of cuticular wax in leaves, as well as the response to stress in soybean. Further research is required to investigate the alterations in VLCFAs content and components in soybean plants following stress treatment. Additionally, it is important to identify the specific KCS genes that are instrumental in regulating soybean stress resistance. By targeting these key GmKCS genes, soybean breeding practices can enhance the plant’s tolerance to stress.

In this study, a total of 31 KCS genes were identified in the soybean genome, and their physicochemical properties, chromosomal location, collinearity, evolutionary relationships, gene structure, the presence of motifs and cis-acting elements, and gene expression patterns in different tissues/organs and under abiotic stress conditions were analyzed. The GmKCS genes were distributed on 14 chromosomes, and segmental duplication during evolution was primarily responsible for their expansion during soybean genome evolution. GmKCS genes could be divided into seven groups based on their phylogenetic relationships. GmKCS genes exhibited diverse expression patterns in the various tissues/organs of soybean plants. Many of them were found to respond to varied abiotic stresses, implying that they may be crucial for stress adaptation and tolerance in soybean. These findings will contribute to future research regarding the functions and regulatory mechanisms underlying soybean KCS genes in response to abiotic stress and their potential exploitation towards improving soybean stress tolerance.

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

YG: Investigation, Methodology, Writing – original draft. DW: Investigation, Software, Writing – original draft. HX: Conceptualization, Formal Analysis, Writing – original draft. ZZ: Investigation, Writing – original draft. YC: Investigation, Writing – original draft. DZ: Investigation, Writing – original draft. YJ: Investigation, Writing – original draft. MS: Software, Writing – original draft. PL: Software, Writing – original draft. QS: Software, Writing – original draft. JY: Investigation, Software, Writing – original draft. PC: Writing – original draft. YS: Investigation, Methodology, Software, Writing – original draft, Writing – review & editing.