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Article

Genome-Wide Identification of the Kinesin Gene Family in Soybean and Its Response to Salt Stress

by
Ting Jin
1,*,
Kai Zhang
2,
Xiujie Zhang
2,
Chunhua Wu
2 and
Weihua Long
1
1
College of Rural Revitalization, Jiangsu Open University, Nanjing 210036, China
2
College of Agronomy, Nanjing Agricultural University, Nanjing 211800, China
*
Author to whom correspondence should be addressed.
Agronomy 2025, 15(2), 275; https://doi.org/10.3390/agronomy15020275
Submission received: 26 December 2024 / Revised: 17 January 2025 / Accepted: 21 January 2025 / Published: 22 January 2025
(This article belongs to the Section Crop Breeding and Genetics)
Browse Figures
Versions Notes

Abstract

:
The kinesin (KIN) gene family is a subgroup of motor proteins. It plays a critical role in plant development and responses to environmental stresses. However, their function in soybean salt tolerance has yet to be clearly defined. This study employed bioinformatics approaches and identified 139 kinesin family members in the soybean genome. These 139 genes were classified into 10 subgroups, unevenly distributed across the chromosomes. The promoter regions of GmKIN genes harbored several stress-responsive elements, and segmental duplication was the primary driver of the expansion of the GmKIN gene family. Based on publicly available RNA-seq data, we studied the response patterns of 139 GmKIN genes to salt stress and found that 20 KIN genes in soybeans were upregulated after salt stress, with GmKIN114, GmKIN102, GmKIN109, and GmKIN99 showing more than a threefold increase in their expression under salt stress. Using quantitative fluorescence PCR, transgenic yeast, and a transgenic hairy root system, we preliminarily validated the salt tolerance functions of the four KIN genes in soybeans. This study probed into the GmKIN gene family in soybean, offering valuable insights into the functional roles of these genes in stress adaptation.

1. Introduction

Among numerous challenges currently confronting global agriculture, increasingly severe soil salinization has significantly constrained crop growth and productivity [1,2]. Due to inappropriate irrigation practices, excessive fertilizer use, and climate change, saline–alkaline soils are continuously expanding, posing a substantial threat to the sustainable development of agricultural systems [3,4,5]. These soils typically contain high concentrations of salts, such as NaCl and Na2SO4. Such salt accumulation increases soil osmotic pressure and hinders plants from absorbing water and nutrients, resulting in osmotic stress and ionic toxicity [1,2,6].
Soybean is a vital economic oil crop. As a significant source of oil and protein for human consumption, soybean is an indispensable raw material in food processing industries [7,8]. It holds a central position in global agricultural and food systems [7,8]. The stability and growth of soybean production are pivotal to meeting the rising demands of the global population, as soybeans are not only widely used in animal feed but also have a direct impact on human dietary patterns and nutritional intake [8,9]. However, soybeans are highly sensitive to salinity stress. Saline–alkaline soils impede the growth and development of soybean plants. The imbalance of osmotic pressure in plant cells caused by salinity stress disrupts essential physiological processes, leading to stunted growth, chlorosis, and significant reductions in yield [10,11]. Consequently, enhancing the salinity and alkalinity tolerance of soybeans has become an urgent focus in both soybean breeding and agricultural production.
Plants have evolved complex and coordinated defense mechanisms to survive in saline–alkaline environments, such as regulating ion uptake and transport, synthesizing osmotic regulators, and activating antioxidant defense systems [12]. These mechanisms collectively work to mitigate the cellular damage caused by saline–alkaline stress, maintain vital physiological functions, and facilitate survival and reproduction in harsh conditions. Several gene families, including WRKY [13], MYB [14], NAC [15], and bZIP [16], have been identified as key players in the response to salinity stress.
Kinesins are a class of motor proteins characterized by an 80 nm long rod-like region. Two heavy chains (KHCs) and two light chains (KLCs) constitute a heterotetramer. Two spherical motor domains are located at the head region. The opposite end features a fan-shaped tail composed of the C-terminal region and light chains, which are responsible for interacting with the transported “cargo”. The central region comprises the rod-shaped domain of the heavy chains [17,18,19]. The spherical motor domains at the head contain both ATP-binding sites and microtubule-binding sites, which are highly conserved. In contrast, the sequences of the tail region are more variable, enabling the transport of a wide array of “cargo” [17,18,19]. Kinesins are involved in multiple cellular processes, including intracellular transport, the dynamic assembly of the cytoskeleton, cell signaling, and stress responses [20,21]. Within the cell, kinesin proteins bind to microtubules and utilize the energy derived from ATP hydrolysis to transport various materials along microtubule tracks. This ensures the proper distribution of cellular components and facilitates normal metabolic activity [22,23]. Kinesins are classified into 14 subfamilies (kinesin-1 to kinesin-14) based on their conserved sequence motifs. These motor proteins play indispensable roles in plant growth and development, such as cell division, elongation, and the formation of tissues and organs [22,23,24]. Kinesin-1 [20] and kinesin-2 [21] primarily form dimers for autoinhibition, whereas kinesin-3 achieves autoinhibition through the interlocking of its neck and motion domains, thus minimizing ATP consumption and microtubule utilization [24]. The kinesin-5 subfamily is critical in the formation and maintenance of the bipolar mitotic spindle during cell division [25]. Kinesin-12 is closely linked to the cytokinetics apparatus, specifically the membranous structure, which is composed of microtubules and actin filaments. It directs fusion-competent vesicles toward the central region and modulates the function of the metaphase spindle in Arabidopsis mitosis, playing a pivotal role in mitotic progression [26]. BR HYPERSENSITIVE 1, a member of the kinesin-13 subfamily, is essential in signal transduction during rice development [27]. In the kinesin superfamily, most motor proteins have their motor domains localized at the N-terminus and are directed toward the microtubule end. However, kinesin-14 subfamily members possess motor domains near the C-terminus, typically showing a preference for the microtubule end. Kinesin-14 is involved in spindle formation, organelle transport, nucleus transfer, chloroplast distribution, chromosome disjunction, and plastid differentiation [22,23]. In barley, four HvKIN genes—HvKIN6 (kinesin-11 subfamily), HvKIN11 (kinesin-7 subfamily), HvKIN30 (kinesin-14 subfamily), and HvKIN40 (kinesin-7 subfamily)—have been implicated their potential roles in early plant development and flowering [28].
In recent years, the potential association between kinesin genes and the stress tolerance of plants has garnered increasing attention [29,30,31]. The kinase-related protein GhKLCR1 is specifically expressed in cotton roots. In transgenic Arabidopsis plants carrying the PGhKLCR1::GUS fusion construct, glucuronidase (GUS) activity increased with concentrations of mannitol. Compared to Columbia-0, the seed germination of 35S::GhKLCR1 plants was significantly inhibited under 300 mmol/L mannitol treatment, indicating that GhKLCR1 is sensitive to drought [29]. The AsKIN gene is implicated in the response of garlic to low temperature and osmotic stress, with its overexpression enhancing Arabidopsis’ tolerance to oxidative stress during cryopreservation. This effect is linked to changes in the expression of genes involved in cold and osmotic stress responses, ultimately promoting plant growth after stress exposure [30]. The OsTUB1-Kinesin13A complex in rice protects the plant from salinity stress by maintaining the membrane localization of the Na+ transporter OsHKT1;5 (a critical regulator of ion homeostasis) [31]. The overexpression of AhMYB30 in Arabidopsis enhances transgenic plants’ resistance to freezing and salinity stress. The stress response gene, KIN1, showed elevated expression in the transgenic plants compared to wild-type controls [32]. The growth and development of soybean directly affect its quality and yield, including seed development, organogenesis, cell division, and responses to abiotic stress—all of which are influenced by motor proteins [33,34]. For example, Glyma.13G114200 (NACK2), which encodes kinesin-like proteins, is involved in the formation of the cell plate during the late stage of anther meiosis. By utilizing CRISPR/Cas9 for targeted knockout, the edited lines are unable to undergo cytokinesis after meiosis, resulting in enlarged pollen grains that cannot germinate in vivo, leading to a complete loss of male fertility [33,34]. Planting the mutants can eliminate the need for manual emasculation, saving significant human and material resources while also enhancing the purity of hybrid seeds, creating hybrid vigor, and increasing crop yields [33,34].
In order to assess the role of the kinesin gene family in soybean development, this study conducted a genome-wide identification and analysis, identifying 139 kinesin gene family members from the soybean genome. Phylogenetic relationships, gene structure features, chromosomal distribution, conserved motifs, cis-acting elements, and duplication patterns were investigated. Additionally, the expression profiles of soybean kinesin genes under salinity stress were explored, revealing a significant upregulation of GmKIN99, GmKIN102, GmKIN109, and GmKIN114. By comparing the tissue-specific expression and relative expression levels of GmKIN99, GmKIN102, GmKIN109, and GmKIN114 in soybean seedlings and in response to salinity stress, this study analyzed the functional roles of these genes in soybean. The overexpression of GmKIN99, GmKIN102, GmKIN109, and GmKIN114 improved the salinity tolerance of the INVSc1 yeast strain. Furthermore, the overexpression of GmKIN114 significantly enhanced the salinity tolerance of soybean hairy roots. The findings of this study lay a solid foundation for future research into the role of kinesin genes in enhancing plant tolerance to abiotic stresses.

2. Materials and Methods

2.1. Identification of GmKIN Family Members

The genomic sequences, protein sequences, and annotation data of GmKIN family members were obtained from the Phytozome database (https://phytozome-next.jgi.doe.gov/) (accessed on 3 February 2024) using the soybean genome (W82.a2.v1). The Hidden Markov Model (HMM) for the kinesin family (PF00225) was downloaded from the PFAM database (http://pfam.xfam.org/family/) (accessed on 3 February 2024). Using the Simple HMM Search function in TBtools [35], all GmKIN family members were retrieved, filtering out genes with an E-value less than e−5 and lacking the conserved kinesin domain (CDD, https://www.ncbi.nlm.nih.gov/cdd) (accessed on 5 February 2024). The molecular weight (MW) and isoelectric point (pI) of all amino acid sequences were computed using the ExPASy server (https://web.expasy.org/protparam) (accessed on 10 February 2024).

2.2. Motif, Gene Structure, and Phylogenetic Analysis

The GmKIN proteins were submitted to the MEME website (version 5.5.7, http://meme-suite.org/tools/meme) (accessed on 20 February 2024) to predict conserved motifs, with the setting of ten motifs and other settings left at their defaults. Gene exon and UTR positions for GmKINs were extracted from the W82.a2.v1 genome annotation file. The Gene Structure View function in TBtools was used to visualize the motifs and gene structures. All AtKIN (Arabidopsis thaliana, TAIR10), OsKIN (Oryza sativa, MSU v7.0), and GmKIN (Glycine max, W82.a2.v1) proteins were used for phylogenetic analysis [28]. ClustalW (2.0) was adopted for multiple sequence alignment. A phylogenetic tree was constructed using the neighbor-joining method in MEGA 5.2 software [36], with 1000 bootstrap replicates [37].

2.3. Promoter Cis-Element Analysis

The 2000 bp upstream sequences of GmKIN genes were extracted. Cis-elements were analyzed using the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (accessed on 5 March 2024). The identified cis-elements were categorized and quantified, generating a heatmap of their distribution.

2.4. Chromosomal Localization

The physical positions of GmKIN genes and the chromosome lengths were extracted. The Gene Location Visualize function in TBtools was then used to map the positions of GmKIN genes on the chromosomes.

2.5. Gene Synteny Analysis

Genome sequences and gene annotation files from Arabidopsis thaliana (TAIR10), Oryza sativa (MSU v7.0), and Glycine max (W82.a2.v1) were employed in synteny analysis using MCScanX mcscanx/git-97e74f40 and TBtools v2.152.

2.6. Transcriptomic Gene Expression Analysis

Transcriptomic data from GSE93322, obtained from NCBI, were used to extract the FPKM values for GmKIN genes. Then, the log2(FPKM-T/FPKM-CK) values were calculated to generate heatmaps [38]. GSE93322: Two-week-old soybean seedlings were treated with 150 mM NaCl for 6 h. After treatment, root and leaf tissues were collected for mixing, RNA was extracted, and transcriptome analysis was conducted.

2.7. RNA Extraction and Gene Expression Analysis

Total RNA from soybean tissues was extracted using the RNAprep Fast Plant Kit (Conway Century Biotechnology, Taizhou, China). cDNA synthesis was performed using the Evo M-MLV Reverse Transcription Kit (Accurate Biotechnology, Changsha, China). Quantitative real-time PCR (qRT-PCR) primers (Figure S1) were designed using the Primer-BLAST tool available on the NCBI website (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) (accessed on 25 March 2024), with GmUKN1 selected as the internal control (Table S1). The reaction mix was prepared using the SYBR Green Pro TaqHS qPCR Kit (Accurate Biotechnology). qRT-PCR was performed on the Roche LightCycler 480 Real-Time Detection System, following the manufacturer’s protocol. Three biological replicates were conducted for each sample. The relative expression levels of the target genes were calculated using the 2−ΔΔCt method [39].

2.8. Yeast Salinity Tolerance Assay Using INVSc1 Strain

The yeast expression vector pYES2-gene and the empty vector pYES2 were transformed into the INVSc1 yeast strain [40] via the PEG/LiAc method [41]. The pYES2-transformed yeast strain served as the control. After transformation, the yeast strains were plated on an SD-Ura-deficient medium to facilitate growth. Individual colonies were selected and verified by PCR using the rTaq enzyme to screen for positive clones. Correctly transformed yeast strains were cultured in YPD medium (extract peptone dextrose) at 30 °C to an OD600 of 0.6. Cultures were then serially diluted tenfold, and 10 μL of each dilution was spotted onto YPD agar plates containing varying concentrations of NaCl (0, 0.5, 0.8, 1 mol/L) [42]. Plates were incubated at 30 °C in an inverted position.

2.9. Genetic Transformation and Salinity Tolerance Evaluation of Soybean Hairy Roots

The 35S:GmKIN114 construct and the empty vector pBinGFP4 were transformed into Agrobacterium rhizogenes strain K599 [43] for the generation of transgenic soybean hairy roots. Transgenic soybean hairy roots were cultured on Murashige and Skoog (MS) medium supplemented with 500 mg/L carbenicillin and 50 mg/L cefotaxime (Biological Industries, Shanghai, China). After 15 days, GFP-positive hairy roots were identified under a stereomicroscope (Mshot, Guangzhou, China) based on green fluorescence signals, eliminating non-transgenic root systems. Positive transgenic hairy roots with a similar length and size were selected and transferred to a white solid medium (pH = 5.8, MDBio, Qingdao, China) with 0 or 100 mM NaCl [44]. After two weeks, hairy roots were harvested, and their fresh weight, root length, number of hairy roots were measured.

2.10. Data Analysis

Data analysis was performed using SAS 9.2. The analysis of differences between the two samples was conducted using Student’s t-test.

3. Results

3.1. Identification of GmKIN Family Members and Phylogenetic Tree Analysis

Based on the motor domain (PF00225) and conserved domain classification of the GmKIN family, a total of 139 GmKINs were identified and named GmKIN1 to GmKIN139. The lengths of the encoded amino acids ranged from 118 amino acids (GmKIN120) to 2792 amino acids (GmKIN46), with molecular weights (MWs) ranging from 12,969.76 Da (GmKIN120) to 318,826.5 Da (GmKIN46). The predicted isoelectric points ranged from 4.67 (GmKIN120) to 9.83 (GmKIN30) (Table S2).
A phylogenetic tree was constructed using 139 GmKINs, 61 AtKINs, and 45 OsKINs. The phylogenetic analysis revealed that kinesin proteins from the three species could be divided into 10 subfamilies: K11, K10, K3, K8, K4, K5, K7, K12, K1, and K14 (Figure 1). Among these, the K14 subfamily was the largest, consisting of 87 kinesin proteins. It was followed by the K7 subfamily, containing 50 kinesins. The K11 subfamily was the smallest, with only four kinesins. Within the largest K14 subfamily, 48 kinesin proteins were identified from soybean, while only 2 soybean kinesins were present in the smallest K7 subfamily (Figure 1). The presence of GmKIN, AtKIN, and OsKIN proteins within the same subfamily indicates their homology and similar molecular functions, providing valuable insights into the potential biological functions of the GmKIN family.

3.2. Analysis of Conserved Motifs and Gene Structure of GmKIN

The conserved motifs and gene structures were analyzed to explore the structural characteristics of GmKIN genes. Using the MEME (Multiple Em for Motif Elicitation) tool, a total of 10 conserved motifs were identified across the GmKIN family. Most of the GmKIN members contained motifs 7, 10, 1, 5, 4, 6, 3, 2, and 9, arranged in this specific order. However, some members, such as GmKIN1 and GmKIN63, contained only motifs 7 and 5, while GmKIN62 only included motifs 6 and 3 (Figure 2).
Gene structure analysis revealed that the majority of GmKIN genes contained introns and untranslated regions (UTRs). However, certain GmKIN genes, including GmKIN14, GmKIN15, GmKIN18, GmKIN23, GmKIN29, GmKIN52, GmKIN59, GmKIN61, GmKIN74, GmKIN87, GmKIN115, GmKIN119, GmKIN120, GmKIN130, GmKIN136, and GmKIN138, presented no introns (Figure 2).

3.3. Chromosomal Localization of GmKINs

A chromosomal localization analysis of the soybean kinesin gene family showed that the 139 gene members were distributed across all 20 soybean chromosomes (Figure 3). Chromosomes 16 and 20 had the fewest genes, with only three genes each. Chromosome 9 contained the highest number, with 12 genes (8.51%), followed by chromosome 17, which encompassed 11 genes (7.80%). Chromosome 16 had only three genes, which was the fewest (2.13%).

3.4. Cis-Acting Element Analysis of Promoters

The presence of various cis-acting elements in gene promoters suggests that these genes may perform diverse functions. To explore the cis-acting elements in the promoters of the GmKIN genes, genomic sequences from 2 kb upstream of ATG of each gene were extracted. These sequences were then searched and analyzed for cis-acting elements in the PlantCARE database. The identified cis-acting elements were categorized into three groups: 1. plant growth and development, such as MRE, the GCN4 motif, the Skn-1 motif, Box-4, CAT-box, CTA-box, Circadian, CCGTCC-box and O2-site; 2. phytohormone response, including ABRE, the TCA-element, the TGACG motif, the TGA-element, the CGTCA motif, P-box, the GARE motif and ERE; and 3. biotic/abiotic stress response, such as the WUN motif, TC-rich repeats, ARE, the GC motif, DRE, MBS, LTR, HSE, WRE3, and STRE.
Most of the GmKIN genes contained a large number of Box-4, the TGACG motif, the CGTCA motif, ABRE, STRE, and ARE. These findings suggest that GmKIN genes may be involved in soybean growth and development, hormonal responses (such as to ABA and jasmonic acid), and abiotic stress responses (Figure 4).

3.5. Gene Duplication and Synteny Analysis

Gene duplication events are a major driver of gene family expansion during evolution. This study analyzed the types of GmKIN gene duplication in the soybean genome. The results showed that only two pairs of genes, GmKIN35/36 and GmKIN120/121, underwent tandem duplication, and 122 segmental duplication events were identified (Figure 5). Most of the GmKIN genes exhibited multiple-pair segmental duplication events. GmKIN4 had six-pair segmental duplication events, which was the highest (Figure 5). These findings indicate that segmental duplication is the primary force driving the large-scale expansion of the GmKIN family.
To further explore the evolutionary relationships, inter-species synteny analysis was conducted by comparing the GmKIN genes with those from two other representative plants—Arabidopsis and Rice. The results revealed that 75 and 25 GmKIN gene members showed homology with Arabidopsis and rice, respectively. Additionally, soybean GmKIN genes exhibited more collinearity with Arabidopsis, suggesting that kinesin genes in Arabidopsis, rice, and soybean may have undergone differentiation (Figure 6).

3.6. Expression Profiles of GmKIN Members Under NaCl Stress and in Different Plant Tissues

Given the presence of several abiotic stress-responsive elements in the promoter regions of GmKIN genes, this study hypothesized that these genes might mediate plant responses to abiotic stress. To investigate the response of GmKIN family members to salt stress, RNA-seq data (GSE93322) for the GmKINs were downloaded, and a heatmap was constructed to visualize the expression levels of the 139 GmKIN genes under NaCl stress. The statistical results from data processing (Figure 7a) revealed that 20 GmKIN genes were upregulated after NaCl treatment, with GmKIN114, GmKIN102, GmKIN109, and GmKIN99 showing more than a threefold increase in expression levels under salinity stress. Transcriptome data for GmKIN114, GmKIN102, GmKIN109, and GmKIN99 from the Phytozome database (Figure 7b) revealed that the expression levels of these genes were higher in the root and stem tissues compared to the leaf tissues.
In order to explore the expression profiles of these genes under salinity stress, qRT-PCR was performed on soybean plants treated with 150 mM NaCl. The relative expression levels of GmKIN114, GmKIN102, GmKIN109, and GmKIN99 were significantly upregulated in the roots after 150 mM NaCl treatment (Figure 7c). These results suggest that the GmKIN114, GmKIN102, GmKIN109, and GmKIN99 gene is responsive to NaCl stress; therefore, these four KIN genes may be involved in soybean tolerance to salt stress.

3.7. Effect of Overexpressing GmKINs on Yeast Salinity Tolerance

The growth characteristics of INVSc1 yeast strains containing pYES2:GmKIN99, pYES2:GmKIN102, pYES2:GmKIN109, pYES2:GmKIN114 and the empty vector pYES2 on YPD medium containing various concentrations of NaCl (0, 0.5, 0.8, and 1 M) were examined. After 3 days of incubation, the INVSc1 yeast strains transformed with pYES2:GmKINs grew faster than those transformed with the empty pYES2 vector. At 0.5 M NaCl, the INVSc1 yeast strains transformed with pYES2:GmKINs showed growth at dilutions of 10−4 and 10−5, while the empty vector control strains did not grow (Figure 8). At 0.8 and 1 M NaCl, the INVSc1 yeast strains transformed with pYES2:GmKIN114 exhibited growth at dilutions of 10−4 and 10−5, while strains transformed with pYES2:GmKIN99, pYES2:GmKIN102, and pYES2:GmKIN109 showed growth at dilution 10−4, whereas the empty pYES2 vector strain did not grow (Figure 8). These results demonstrated that GmKIN99, GmKIN102, GmKIN109, and GmKIN114 enhanced the tolerance of INVSc1 yeast to NaCl, with GmKIN114 having the most significant effect.

3.8. Effect of Overexpressing GmKIN114 on Salt Tolerance in Soybean Hairy Roots

Agrobacterium rhizogenes-mediated genetic transformation was employed to introduce the A. rhizogenes K599 strain containing the pBinGFP4:GmKIN114 plasmid into the cotyledons of Tianlong No. 1 soybean (Figure 9a), investigating the role of GmKIN114 in soybean salinity tolerance. After 14 days of treatment with 100 mM NaCl, the hairy roots of plants transformed with pBinGFP4:GmKIN114 exhibited better growth than those of the control plants (empty vector) (Figure 9b). The fresh weight, root length and number of the hairy roots transformed with pBinGFP4:GmKIN114 were significantly higher under 100 mM NaCl treatment (p < 0.01) compared to the control (Figure 9c). However, no significant difference was observed between the two groups under 0 mM NaCl treatment (p > 0.05). These results suggested that overexpressing GmKIN114 enhanced the salinity tolerance of soybean.

4. Discussion

The kinesin superfamily is a macromolecule motor superfamily [20,21]. A common characteristic of kinesin family members is the presence of the highly conserved kinesin motor domain [17,18,19]. In this study, an evolutionary tree was constructed using 139 soybean kinesin proteins (GmKINs), 61 Arabidopsis kinesin proteins (AtKINs), and 45 rice kinesin proteins (OsKINs) (Figure 1). Based on phylogenetic analysis, the kinesin family was divided into 10 subgroups. Compared to the kinesin gene family in Arabidopsis, that in soybean did not show independently evolved subfamilies. The two had a high degree of homology (Figure 1).
The distribution of ten kinesin subfamilies in soybean mirrored the trend observed in Arabidopsis and rice. The KIN14 and KIN7 subfamilies were the largest and second largest subtypes (Figure 1). KIN14 was a highly conserved subfamily, playing an essential role in mitotic chromosome segregation and organelle transport [22,23]. The KIN11 subfamily contained the fewest kinesin proteins, with only two in soybean and one each in Arabidopsis and rice. Members of the KIN11 subfamily were involved in core functions such as signal transduction, differentiation, and catalysis [28]. Moreover, some subfamilies, such as KIN2, KIN3, KIN6, and KIN9, were absent in land plants [45]. As a result, they were also missing in soybean.
In terms of gene structure, the kinesin gene family exhibited a variety of differences within the same subfamily and between different subfamilies. For example, there were noticeable differences in the number of introns among the subfamilies (Figure 2). Some kinesin genes, such as GmKIN14, GmKIN15, GmKIN18, GmKIN23, GmKIN29, GmKIN52, GmKIN59, GmKIN61, GmKIN74, GmKIN87, GmKIN115, GmKIN119, GmKIN120, GmKIN130, GmKIN136, and GmKIN138, had no introns.
The motif analysis revealed that the majority of soybean kinesin proteins contained typical motifs. Motif 1 harbored the highly conserved ‘EIYNE’ sequence. Motif 2 had the ‘TLKFASRV’ sequence with high conservation. Motifs 3, 6, and 7 each featured a highly conserved sequence that served as a microtubule-binding site: ‘VDLAGSER’, ‘HI/VPYR’, and ‘SSRSH’, respectively. Motif 4 contained the highly conserved sequence ‘RVRPLN’. Motif 5 encompassed an ATP binding site with the highly conserved sequence ‘FAYGQTGS’. Motif 8 displayed the highly conserved ‘FDKYF’ sequence. Motif 10 was marked by a conserved sequence composed of hydrophobic residues, repeated in the pattern ‘L/VxxxLxL’. The distribution and arrangement of these motifs exhibited significant conservation, albeit with some variations. The kinesin motor domain comprised an ATP-binding site (‘FAYGQTGS’) and a microtubule-binding domain. The latter typically contained microtubule-binding motifs, such as SSRSH, xDLAGSE, and HxPYR (Figure S2). Among all soybean kinesin proteins, ATP-binding motifs had the highly conserved peptide sequence ‘FAYGQTGS’ [46]. ‘SSRSH’ was the most frequent motif, positioning it as the most conserved microtubule-binding site [47].
Gene duplication is a primary contributor to the rapid expansion and evolution of gene families [48]. The distribution of genes across chromosomes and their synteny analysis demonstrated that the major force behind the large-scale expansion of the GmKIN family was segmental duplication, consistent with that observed in Arabidopsis and rice kinesin genes (Figure 6).
The promoters of the GmKIN gene family members were enriched with cis-acting elements, which can be categorized into three main types: growth and development-related, hormone-responsive, and biotic/abiotic stress-responsive elements. Regarding hormone-responsive cis-elements, several cis-elements were involved in response to various plant hormones: the TCA-element (salicylic acid response), the TGA-element (auxin response), the TGACG and CGTCA motifs (methyl jasmonate response), the GARE motif and P-box (gibberellin response), and the ABRE (abscisic acid response). Stress-responsive cis-elements included TC-rich repeats, GC motif, WUN motif, ARE, DRE, MBS, LTR, HSE, WRE3, and STRE (Figure 4). The TGACG motif, CGTCA motif, ABRE, and TC-rich repeats suggested a strong association with plants’ response to salinity stress [49,50]. The promoter of the cotton GhACX3 gene contained elements such as ABRE, MBS, LTR, TGACG, and CGTCA motifs, which were crucial for drought and salinity tolerance [51]. In Arabidopsis, ABRE-binding factors interacted with RHD6 and inhibited its activity to respond to salinity stress [52]. Most GmKIN genes had substantial Box-4, TGACG motif, CGTCA motif, ABRE, STRE, and ARE values (Figure 4). This suggested that the upstream promoters of GmKIN genes were likely regulated by plant hormones and stress-related cis-acting elements, thus playing a significant role in modulating the growth and development of plants.
BR HYPERSENSITIVE 1, a member of the kinase protein 13 subfamily, negatively regulated brassinosteroid signaling and influenced the structural properties of rice, thereby affecting yield [27]. The kinesin-like protein GDD1/BC12 modified the expression of the KO2 gene in the gibberellin biosynthesis pathway and adjusted microtubule rearrangement and cell elongation in rice [53]. In barley, the expression of kinesins such as HvKIN7 and HvKIN35 was significantly upregulated in response to ABA and GA3 treatments [28]. In summary, kinesins play a crucial role in phytohormone-mediated responses.
Recent studies have demonstrated that motor proteins are pivotal for plant stress responses. For example, the plant-specific kinesin-like protein 1 (KP1) interacted specifically with voltage-dependent anion channel 3 (VDAC3) on the outer mitochondrial membrane to co-regulate aerobic respiration during Arabidopsis seed germination under low-temperature stress [54]. The drought-responsive gene GhKLCR1, which encoded a kinesin light-chain-related protein rich in tetratricopeptide repeats, was upregulated under abiotic stresses such as polyethylene glycol treatment. GhKLCR1 exhibited drought sensitivity. Seed germination in 35S::GhKLCR1 transgenic plants was significantly impaired under 300 mmol L−1 mannitol treatment compared to wild-type Columbia-0 [29]. Additionally, OsTUB1 interacted with kinesin13A to form the OsTUB1-kinesin13A complex, stabilizing the localization of the Na+ transporter OsHKT1;5 in membranes. This interaction maintained Na+ homeostasis and enhanced salinity tolerance in rice [31]. Furthermore, KIN1, a marker gene associated with the ICE-CBF-COR pathway, played a pivotal role in regulating Arabidopsis responses to abiotic stresses [55]. Arabidopsis plants with overexpressed AhMYB30 presented KIN1 upregulation following exposure to freezing and salinity stress [32].
Soybean (Glycine max (L.) Merr.) is one of the most vital food and oilseed crops globally [7,56]. Its abundant protein and essential amino acids make it a key contributor to plant-derived proteins and lipids [8,56]. Salinization alters the physicochemical properties of the soil, severely impairing plant growth and development. In recent years, the progressive intensification of soil salinization has led to a constant reduction in cultivable areas, thereby destabilizing crop yields [2,6,10].
Transcriptome sequencing data (GSE93322) revealed substantial variation in the expression levels of kinesin family members in soybean seedlings (root and leaf mixed samples) under salinity stress (Figure 7a). By applying a threshold of log2(FPKM-T/FPKM-CK) ≥ 2 [38], nine soybean kinesin genes were identified to be upregulated. Among them, four kinesin genes (GmKIN114, GmKIN102, GmKIN109, and GmKIN99) exhibited log2(FPKM-T/FPKM-CK) ≥ 3. Transcriptomic data for these four genes, retrieved from the Phytozome database (https://phytozome-next.jgi.doe.gov/) (accessed on 25 March 2024), showed that their relative expression was highest in roots, followed by stems, with the lowest expression observed in leaves (Figure 7b). This suggested that GmKIN genes were predominantly expressed in the subterranean tissues of soybean, particularly in hairy roots, potentially playing a crucial role in root growth and development. The RT-qPCR analysis of the expression patterns of these four GmKIN genes under salt stress further confirmed their upregulation, with significantly higher expression levels compared to the control (Figure 7c). These findings indicated that kinesin genes were integral to soybean’s response to salinity stress.
In recent years, yeast [57] and hairy root systems [58,59] have increasingly been used as models to investigate candidate genes associated with critical plant traits and stress responses. For instance, the overexpression of the TrSAMDC1 gene in Trifolium repens has been shown to enhance the tolerance of INVSc1 yeast to drought, salt, and oxidative stresses [40]. The soybean hairy root system was utilized to explore the role of GmCAMTA12 in drought responses, where the overexpression of GmCAMTA12 under 6% PEG6000 stress resulted in increased root length, specific surface area, root volume, branching number, and projected area compared to control plants [60]. Under salt treatment, the overexpression of GmP5CS in soybean hairy roots led to superior growth, longer roots, and higher survival rates compared to the control [61].
Many genes can be expressed heterologously in yeast, and the heterologous expression system can be used to study their functions [62]. We compared the yeast genome (GCA_000146045.2) and found that GmKIN114, GmKIN102, GmKIN109, and GmKIN99 in soybeans are homologous to seven genes in yeast: kinesin-like protein KIP1 (NP_009490.1), tubulin-dependent ATPase KIP3 (NP_011299.1), Kar3p (NP_015467.1), Kip2p (NP_015170.1), kinesin motor protein CIN8 (NP_010853.2), kinesin motor protein CIN8 (NP_010853.2), and Smy1p (NP_012844.1). This suggests that proteins with functions similar to those of GmKIN114, GmKIN102, GmKIN109, and GmKIN99 in soybeans may exist in yeast. In order to verify the potential role of GmKIN114, GmKIN102, GmKIN109, and GmKIN99 in enhancing salinity tolerance, this study overexpressed these genes in yeast. Yeast strains overexpressing these GmKIN genes demonstrated better growth under salinity stress compared to control strains carrying the empty vector (Figure 8). These results suggested that the overexpression of GmKIN114, GmKIN102, GmKIN109, and GmKIN99 significantly enhanced the salinity tolerance of INVSc1 yeast strains. Notably, under 0.8 M and 1 M NaCl stress, only the GmKIN114-overexpressing yeast strain exhibited growth at a dilution of 10−5, indicating that GmKIN114 conferred the strongest salinity tolerance. Subsequently, this study investigated the impact of GmKIN114 on salinity tolerance in soybean hairy roots.
As responses to salinity stress, root systems in plants have evolved diverse mechanisms at the morphological, physiological, and molecular levels, including structural adaptations, osmoregulation, enhanced antioxidant defense, the maintenance of membrane integrity, and the expression of salt-responsive genes and proteins [12,63]. Therefore, soybean hairy roots were used to further investigate the role of GmKIN114 in conferring salinity tolerance. This study observed that the overexpression of GmKIN114 in soybean hairy roots resulted in an increase in root fresh weight, root length and the number of the hairy roots (Figure 9). Collectively, these findings suggested that the overexpression of GmKIN114 enhanced salinity tolerance in soybean hairy roots.
GmKIN114 is located on Chr17 (Figure 3) and belongs to the K12 subfamily (Figure 1). In plants, members of Kinesin-12 are involved in various developmental processes, including the development of male gametophytes, embryos, seedlings, and seeds, and they play a role in key events of cell division [64]. Currently, there are a lot of studies on the K12 subfamily member KIF15 (also known as Hklp2). KIF15 is involved in the formation of bipolar spindles during mitosis. It interacts with HDAC6, NAT10, and SIRT2 to maintain acetylation levels of tubulin, thereby preserving microtubule stability [65]. Additionally, it plays a crucial role in maintaining the capability of oocyte maturation and preventing abnormal embryo development. Mutations in KIF15 may lead to fertility issues associated with oocyte aging [66,67]. Additionally, KIF15 is important in regulating mood and neuronal function in mice, making it a potential target for future research and the treatment of depression [68]. Therefore, it is questionable whether GmKIN114, which is a member of the plant K12 subfamily, plays a role in plant growth, development, and resistance to adverse conditions. GmKIN114 contains eight conserved motifs (Figure 2). In the promoter region of GmKIN114, 22 cis-regulatory elements were identified (Figure 4), including the light response element Box-4, the MYB transcription factor binding site MRE, the growth and development element CCGTCC-box, the salicylic acid (SA) response element TCA-element, the auxin response element TGA-element, the abscisic acid (ABA) response element ABRE, the jasmonic acid (JA) response elements CGTCA motif and TGACG motif, the stress response element STRE, the oxidative stress response element ARE, and the drought response element WRE3. These motifs are largely associated with salt tolerance and drought resistance in crops [49,50,51]. Through PPI validation (Table S3), we found that ten proteins interact with GmKIN114. These interacting proteins include four kinesins, four FERM central domain proteins, one ubiquitin C-terminal hydrolase, and one ubiquitin carboxyl-terminal hydrolase. Kinesin proteins interact with each other to perform their functions. For example, the K5 subfamily member Eg5 collaborates with the K12 subfamily member Kif15 to complete spindle assembly [69]. The GO analysis of the genes encoding the interacting proteins revealed that most of the predicted interacting proteins are involved in microtubule movement, defense response regulation, redox reactions, and plant growth and development. These results suggest that GmKIN114 may interact with other redox proteins and stress-related proteins to enhance salt tolerance in soybeans.

5. Conclusions

Motor proteins are essential in plant growth, development, and response to abiotic stress. However, the specific roles of the GmKIN gene family in soybean have not been fully elucidated. This study conducted a genome-wide analysis and characterization of the GmKIN genes, revealing their physicochemical properties, chromosomal locations, phylogenetic relationships, gene structures, cis-acting elements, and expression profiles. Moreover, the expression profiling of GmKIN genes under salinity stress was performed to investigate their potential involvement in stress response. The findings indicated that nine kinesin genes were upregulated in soybean seedlings under salinity stress. Four genes (GmKIN114, GmKIN102, GmKIN109, and GmKIN99) exhibited high expression levels in soybean hairy roots and were significantly induced under salinity stress. The overexpression of these four genes enhanced the salinity tolerance of yeast strains. The overexpression of GmKIN114 was related to the promoted salinity tolerance of soybean hairy roots. Based on the findings above, we preliminarily identified that the GmKIN114 gene in soybeans plays a role in salt tolerance. To gain a deeper understanding of the function and mechanism of this gene and to provide foundational material for the molecular breeding of salt-tolerant soybeans, we plan to carry out stable transformation experiments in soybeans in the future. This will yield important materials for subsequent research, and we hope to achieve further significant discoveries in upcoming experiments.
Currently, there is a scarcity of a detailed genome-wide analysis and functional characterization of the GmKIN gene family in soybean under salt stress. This study provides valuable insights for further research into the evolutionary origins of GmKINs and contributes to the functional characterization of GmKINs as candidate genes for the molecular breeding of salt-tolerant soybean varieties.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy15020275/s1, Figure S1: The amplicon specificities of five pairs of primers for qRT-PCR in this study; Figure S2: Analysis of consensus sequence conservation in the kinesin gene family; Table S1: Primers used for qRT-PCR and vector construction; Table S2: Information of the 139 GmKIN genes identified in soybean; Table S3: The annotation of genes encoding GmKIN114 and its predicted interacting proteins.

Author Contributions

Conceptualization, T.J. and K.Z.; data curation, T.J. and K.Z.; formal analysis, T.J.; funding acquisition, T.J. and W.L.; investigation, T.J., K.Z., X.Z. and C.W.; methodology, T.J.; project administration, T.J.; resources, T.J. and W.L.; supervision, T.J.; validation, T.J., K.Z., X.Z., C.W. and W.L.; visualization, T.J. and K.Z.; writing—original draft, T.J.; writing—review and editing, T.J., X.Z., K.Z. and W.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Natural Science Foundation of Jiangsu Province (BK20230980), the National Natural Science Foundation of China (32401806).

Data Availability Statement

The data presented in this study are available in the Supplementary Material.

Acknowledgments

We would like to thank Daolong Dou at Nanjing Agricultural University for kindly providing us the vectors of pBinGFP4, and Peter Gresshoff’s laboratory (the University of Queensland) developed A. rhizogenes strain K599, which has been shared freely around the world.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
AtArabidopsis thaliana
GmGlycine max
GUSGlucuronidase
KHCKinesin heavy chain
KINKinesin
KLCKinesin light chain
KP1Kinesin-like protein 1
MEMEMultiple em for motif elicitation
MSMurashige and skoog
MWMolecular weight
OsOryza sativa
pIIsoelectric point
qRT-PCRQuantitative real-time PCR
UTRsUntranslated regions
VDAC3Voltage-dependent anion channel 3

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Figure 1. Phylogenetic relationship of kinesin proteins in soybean, Arabidopsis, and rice. The phylogenetic tree was generated using MEGA5.2 software and we applied the maximum likelihood (ML) method.
Figure 1. Phylogenetic relationship of kinesin proteins in soybean, Arabidopsis, and rice. The phylogenetic tree was generated using MEGA5.2 software and we applied the maximum likelihood (ML) method.
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Figure 2. Gene structure and conserved motif analysis of KIN genes in soybeans. Ten different motifs are represented in various colors. Green boxes indicate exons, and lines indicate introns.
Figure 2. Gene structure and conserved motif analysis of KIN genes in soybeans. Ten different motifs are represented in various colors. Green boxes indicate exons, and lines indicate introns.
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Figure 3. Chromosomal distribution of kinesin genes in soybean. The scale on the left indicates the length of the chromosomes. The bar graph represents the 20 chromosomes, Chr01–Chr20. The corresponding gene symbols are shown on the right side of the chromosomes.
Figure 3. Chromosomal distribution of kinesin genes in soybean. The scale on the left indicates the length of the chromosomes. The bar graph represents the 20 chromosomes, Chr01–Chr20. The corresponding gene symbols are shown on the right side of the chromosomes.
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Figure 4. Analysis of the cis-elements in the promoter regions of soybean KIN genes.
Figure 4. Analysis of the cis-elements in the promoter regions of soybean KIN genes.
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Figure 5. Circos diagram of the 139 genes in soybean KIN gene families. Each chromosome is labeled with its corresponding number. Lines in different colors represent gene pairs among the 139 genes within the soybean KIN gene family.
Figure 5. Circos diagram of the 139 genes in soybean KIN gene families. Each chromosome is labeled with its corresponding number. Lines in different colors represent gene pairs among the 139 genes within the soybean KIN gene family.
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Figure 6. Synteny analyses between the KIN genes of soybean and two other representative plants—Arabidopsis and rice. The gray lines in the background indicate collinear blocks within soybean and other genomes, while the red lines represent syntenic KIN gene pairs.
Figure 6. Synteny analyses between the KIN genes of soybean and two other representative plants—Arabidopsis and rice. The gray lines in the background indicate collinear blocks within soybean and other genomes, while the red lines represent syntenic KIN gene pairs.
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Figure 7. Expression profiles of GmKIN genes. (a) The heatmap of soybean KIN gene expression profiles under salt stress. The heatmap is constructed using the TBtools [35]. The expression profiles of KIN genes under salt stress are obtained by retrieving the dataset GSE93322. The color key indicates fold changes (stress vs. control) scaled by row, with red to blue representing upregulation to downregulation. (b) The expression of four GmKINs in different tissues of the soybean variety Williams 82, with data sourced from the Phytozome database (https://phytozome-next.jgi.doe.gov/, accessed on 20 September 2024). Relative expression in other tissues is calculated using the expression of KIN genes in seeds as a reference. (c) The relative expression of four GmKINs under salt stress. Moreover, 14 day-old Tianlong 1 soybean seedlings are treated with NaCl (150 mM) for 24 h, and the relative expression of the four GmKINs is measured using qRT-PCR, with untreated samples at each time point serving as controls. GmUNK1 is used as a reference gene. Data are presented as means ± SD from three biological replicates, with each replicate containing three technical replicates (n = 3 × 3 = 9). A two-tailed Student t-test is performed; ** indicates significant differences between the 0 and 150 mM NaCl treatments (p < 0.01).
Figure 7. Expression profiles of GmKIN genes. (a) The heatmap of soybean KIN gene expression profiles under salt stress. The heatmap is constructed using the TBtools [35]. The expression profiles of KIN genes under salt stress are obtained by retrieving the dataset GSE93322. The color key indicates fold changes (stress vs. control) scaled by row, with red to blue representing upregulation to downregulation. (b) The expression of four GmKINs in different tissues of the soybean variety Williams 82, with data sourced from the Phytozome database (https://phytozome-next.jgi.doe.gov/, accessed on 20 September 2024). Relative expression in other tissues is calculated using the expression of KIN genes in seeds as a reference. (c) The relative expression of four GmKINs under salt stress. Moreover, 14 day-old Tianlong 1 soybean seedlings are treated with NaCl (150 mM) for 24 h, and the relative expression of the four GmKINs is measured using qRT-PCR, with untreated samples at each time point serving as controls. GmUNK1 is used as a reference gene. Data are presented as means ± SD from three biological replicates, with each replicate containing three technical replicates (n = 3 × 3 = 9). A two-tailed Student t-test is performed; ** indicates significant differences between the 0 and 150 mM NaCl treatments (p < 0.01).
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Figure 8. Effects of overexpression of four kinesin genes on the salt tolerance of lNVSel yeast. pYES2 represents the yeast with empty vector and pYES2-gene represents the yeast overexpressing soybean KIN genes. Photographs are taken after incubation at 30 °C for 72 h. The dilution rates of YPD medium containing the yeast transformants are 10−1, 10−2, 10−3, 10−4, and 10−5.
Figure 8. Effects of overexpression of four kinesin genes on the salt tolerance of lNVSel yeast. pYES2 represents the yeast with empty vector and pYES2-gene represents the yeast overexpressing soybean KIN genes. Photographs are taken after incubation at 30 °C for 72 h. The dilution rates of YPD medium containing the yeast transformants are 10−1, 10−2, 10−3, 10−4, and 10−5.
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Figure 9. Salt tolerance analyses of transgenic soybean hairy roots. (a) GFP signal in soybean hairy roots. Bar = 2 cm. (b–e) Phenotypes and changes in fresh weight, root length, and root number of transgenic soybean hairy roots after treatment with 0 or 100 mM NaCl for 14 d. Bar = 2 cm. Data are presented as means ± SD from three biological replicates, and each replicate contains three independent transgenic hair roots (n = 9). A two-tailed Student t-test is performed; ** indicates significant differences at the 0.01 level between the empty vector and 35S: GmKIN114 under the same conditions.
Figure 9. Salt tolerance analyses of transgenic soybean hairy roots. (a) GFP signal in soybean hairy roots. Bar = 2 cm. (b–e) Phenotypes and changes in fresh weight, root length, and root number of transgenic soybean hairy roots after treatment with 0 or 100 mM NaCl for 14 d. Bar = 2 cm. Data are presented as means ± SD from three biological replicates, and each replicate contains three independent transgenic hair roots (n = 9). A two-tailed Student t-test is performed; ** indicates significant differences at the 0.01 level between the empty vector and 35S: GmKIN114 under the same conditions.
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Jin, T.; Zhang, K.; Zhang, X.; Wu, C.; Long, W. Genome-Wide Identification of the Kinesin Gene Family in Soybean and Its Response to Salt Stress. Agronomy 2025, 15, 275. https://doi.org/10.3390/agronomy15020275

AMA Style

Jin T, Zhang K, Zhang X, Wu C, Long W. Genome-Wide Identification of the Kinesin Gene Family in Soybean and Its Response to Salt Stress. Agronomy. 2025; 15(2):275. https://doi.org/10.3390/agronomy15020275

Chicago/Turabian Style

Jin, Ting, Kai Zhang, Xiujie Zhang, Chunhua Wu, and Weihua Long. 2025. "Genome-Wide Identification of the Kinesin Gene Family in Soybean and Its Response to Salt Stress" Agronomy 15, no. 2: 275. https://doi.org/10.3390/agronomy15020275

APA Style

Jin, T., Zhang, K., Zhang, X., Wu, C., & Long, W. (2025). Genome-Wide Identification of the Kinesin Gene Family in Soybean and Its Response to Salt Stress. Agronomy, 15(2), 275. https://doi.org/10.3390/agronomy15020275

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