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Article

Genome-Wide Identification and Expression Analyses of Glycoside Hydrolase Family 18 Genes During Nodule Symbiosis in Glycine max

by
Rujie Li
†,
Chuanjie Gou
†,
Ke Zhang
,
Milan He
,
Lanxin Li
,
Fanjiang Kong
,
Zhihui Sun
* and
Huan Liu
*
Guangdong Provincial Key Laboratory of Plant Adaptation and Molecular Design, Innovative Center of Molecular Genetics and Evolution, School of Life Sciences, Guangzhou University, Guangzhou 510006, China
*
Authors to whom correspondence should be addressed.
†
These authors are contributed equally to this manuscript.
Int. J. Mol. Sci. 2025, 26(4), 1649; https://doi.org/10.3390/ijms26041649
Submission received: 11 December 2024 / Revised: 11 February 2025 / Accepted: 13 February 2025 / Published: 14 February 2025
(This article belongs to the Special Issue Genetics and Novel Techniques for Soybean Pivotal Characters)
Browse Figures
Versions Notes

Abstract

:
Glycoside hydrolase family 18 (GH18) proteins can hydrolyze the β-1,4-glycosidic bonds of chitin, which is a common structure component of insect exoskeletons and fungal cell walls. In this study, 36 GH18 genes were identified and subjected to bioinformatic analysis based on the genomic data of Glycine max. They were distributed in 16 out of 20 tested soybean chromosomes. According to the amino acid sequences, they can be further divided into five subclades. Class III chitinases (22 members) and class V chitinases (6 members) are the major two subclades. The amino acid size of soybean GH18 proteins ranges from 173 amino acids (aa) to 820 aa and the molecular weight ranges from 19.46 kDa to 91.01 kDa. From an evolutionary perspective, soybean GH18 genes are closely related to Medicago (17 collinear loci with soybean) and Lotus (23 collinear loci with soybean). Promoter analysis revealed that GH18 genes could be induced by environmental stress, hormones, and embryo development. GmGH18-15, GmGH18-24, and GmGH18-33 were screened out due to their nodulation specific expression and further verified by RT-qPCR. These results provide an elaborate reference for the further characterization of specific GH18 genes, especially during nodule formation in soybean.

1. Introduction

Chitin is the second most abundant biomolecule in nature, only led by cellulose. Chitin or chitin oligosaccharides (chitooligosaccharides) are the polymer or oligomer of N-acetylglucosamine (GlcNAc), which is linked by a β-1,4-glycosidic bond [1]. In most cases, chitin is the component of the exoskeleton of arthropods and the cell walls of fungi. Chitinases are capable of cleaving β-1,4-glycosidic bonds and thus releasing GlcNAc residues with different degrees of polymerization [2]. Distinguished by the structure of catalytic domains, chitinases can be primarily grouped into two categories, namely glycoside hydrolase family 18 and 19 (GH18 and 19). The classic feature of GH18 members is a barrel-like structure consisting of eight α-helices and eight β-sheets, while GH19 members have a lysozyme-like structure with several α-helices, which is specifically found in plants [3]. Based on amino acid sequence similarity, chitinases can also be classified into five groups, with class III and V belonging to GH18 and class I, II, and IV belonging to GH19 [4].
The active center of GH18 proteins contains a conserved module, (DN)-X-(DN)-X-E, which is crucial for their enzymatic activities [5]. Except for the catalytic domain, class III and V chitinases are quite different from each other. Class III chitinases do not have a carbohydrate-binding module (CBM), while class V chitinases often contains two CBMs and a c-terminal extension which is responsible for the vacuolar localization [1].
There is no chitin production in plants, which leads the biological function of plant chitinases to the interaction with environment cues or microbes. The chitinase activities could be induced in soybean roots by the ethylene treatments and beneficial microbes [6,7]. AtChiA, a class III chitinase gene in Arabidopsis thaliana, was specifically up-regulated in response to salt stress and wounding [8]. Class V chitinases are also considered as pathogen-related protein family 11 (PR-11) proteins [9,10]. For example, NtChiV, the first class V chitinase, was purified from Nicotiana tabacum when the leaves were inoculated with the tobacco mosaic virus, which exhibited antifungal activity [11,12]. AtChiC, a class V chitinase from A. thaliana, was reported to be induced by abscisic acid, jasmonic acid, flagellin, NaCl, and osmosis [13].
Besides the involvement in abiotic and biotic stress, GH18 proteins could also play a role in the interaction between plants and their symbionts. Through the establishment of symbiosis with mycorrhizal fungi and rhizobia, plants can take more P and N, respectively [14]. The expression levels of the tested class I, II, and IV chitinase genes were not changed in mycorrhizal roots of Medicago; however, there were three class III chitinase genes which were strongly up-regulated [15]. Similarly, QrchitIII-1, a class III chitinase gene from Quercus robur, was up-regulated in lateral roots during the pre-mycorrhization stage with Piloderma croceum [16]. The inoculation of the grapevine with the arbuscular mycorrhizal fungus Glomus versiforme induced the expression of class III chitinase gene VCH3 (Vitis amurensis class III chitinase) [17]. Class III chitinases also might take part in the nodule symbiosis. Srchi13 was considered as an early nodulin gene due to its specific and transient expression during nodule formation in Sesbania rostrata. The recombinant Srchi13 possessed the lipochitooligosaccharide degradation capacity [18], while in Casuarina glauca, Cgchi3 was specifically indued in actinorhizal nodules [19]. Unlike class III chitinases, the biological contribution of class V chitinase to the plant–microbe interaction is mainly limited to nodule symbiosis. In Medicago, MtNFH1 (Nod factor hydrolase 1) and MtCHIT5b (class V chitinase b) were not only inducible by rhizobial inoculation or Nod factor treatment, but also their gene products could degrade the Nod factors from Sinorhizobium meliloti with high substrate affinity. Nodule formation is strongly influenced in their gene mutants, exhibiting abnormal nodule branching and reduced nodule number, respectively [20,21,22,23]. In Lotus, the deficiency of LjCHIT5 (class V chitinase) also caused the abnormal elongation of infection threads within the nodule cortex and later caused mature nodule formation [24].
Soybean (Glycine max (L.) Merr.) is the most important leguminous crop. It is the largest source of food and animal feed protein and the second largest source of vegetable oil in the world [25]. Taking full advantage of nodule symbiosis is an environmentally friendly approach for sustainable agriculture with less chemical fertilizer investment. Although there are a few publications focused on the biological relevance of soybean chitinases in pathogen invasion and drought stress conditions [26,27], the role of soybean chitinases during nodule development is severely overlooked.
In this study, our primary objective is to better understand the soybean GH18 genes, particularly their relevance in nodule symbiosis. Therefore, a comprehensive bioinformatic analysis was performed with the GH18 genes in soybean, including chromosomal positioning, conserved motifs/domains, the phylogenetic relationship with other legumes, and expression profile, especially in nodulation. These results provide valuable resources for the further gene function characterization of soybean GH18 genes.

2. Results

2.1. Chromosomal Distribution of G. max GH18 Genes

According to the Pfam code (PF00704 for glycoside hydrolase family 18 catalytic domain) and HMM search result of soybean protein sequences (download from https://data.jgi.doe.gov/refine-download/phytozome?organism=Gmax, accessed on 10 December 2024), a total of 36 putative GH18 genes were identified (Table 1). Among them, Glyma.03G256800, Glyma.15G015100, Glyma.16G173000, and Glyma.20G035400 possessed multiple versions of transcripts. These genes were widely distributed in the soybean genome, which are mapped to 16 chromosomes (Figure 1). Chromosome 04, 06, 11, and 14 do not possess any GH18 genes. There are four GH18 genes on chromosomes 15, 18, and 20, respectively, while three GH18 genes were on chromosomes 13 and 17. The other chromosomes have 1–2 GH18 genes on them. Due to the integrity of Wm82.a2.v1 genome assembly, Glyma.U033800.1 had not been mapped to any chromosome by this point, but later turned out to be on chromosome 11 in Wm82.a4.v1.

2.2. Analysis of Phylogenetic Tree, Conserved Motifs and Conserved Domains

Based on the deduced amino acid sequences from Phytozome v13, a phylogenetic tree was constructed for soybean GH18 family members. Meanwhile, motif analysis and conserved domain identification were performed. Accordingly, 36 putative GH18 genes could be classified into 5 groups, namely class V chitinase, narbonin, chitinase-like superfamily, stabilin-1 interacting chitinase-like protein (SI-CLP), and xylanase inhibitor/class III chitinase (Figure 2). Narbonin contains a barrel-like structure, so it is considered to belong to GH18 [28]. The majority of GH18 proteins (22 out of 36) belong to the class III chitinase clade which showed a high homology degree. Motifs 19, 1, 3, 10, 5, 11, 2, 14, 6, 13, and 4 are frequently shown in this clade (see motif sequences in Figure S1). Motif 8 is exclusively detected in GmGH18-18 and GmGH18-32, which is correlated with the PAN/apple domain. The combination of motifs 15, 9, and 7 is another feature for GmGH18-18 and GmGH18-32, which is correlated with the serine/threonine kinase domain. There are two kinds of kinase domain involved, both STKc_IRAK and PLN00113, whose main difference is the presence of motif 12 and 16. The combination of motifs 17, 18, and 20 is the signature of class V chitinase (6 out of 36). Noteworthy, GmGH18-20 is the only class V chitinase which contains a serine/threonine kinase domain in group 1. There are three, three, and two GH18 proteins in group 2, 3, and 4, respectively. To some extent, GH18 proteins from group 2, 3, and 4 are similar to class V chitinase. However, motifs 17, 18, and 20 are not completely present in their protein sequences.
In order to have a revolutionary perspective on the significance of GH18 proteins, a more complicated phylogenetic tree was constructed, involving soybean, Medicago, and Lotus (see Medicago and Lotus GH18 gene IDs in Table S1) (Figure 3). These species are legumes, which can establish symbiosis with rhizobia. The biological function of several GH18 proteins from Medicago and Lotus have been thoroughly characterized during nodulation [18,19,20,21,22,23,24]. Nearly all GH18 proteins could be categorized into the five clades used for soybean GH18 classification; however, there are quite a lot of special GH18 proteins from Medicago (16 out of 47 Medicago GH18) that could not fit in. Therefore, they are uniformly named as Medicago-specific GH18. Class III chitinase remain to be the biggest clade of GH18, in which there are 18 and 9 (out of 18) members from Medicago and Lotus, respectively. In the class V chitinase clade, there are 11 proteins from Medicago and 7 proteins from Lotus. Noteworthy, GmGH18-15 and GmGH18-24 are most closely related to MtGH18-20 (MtNFH1) and MtGH18-21 (MtCHIT5b), while GmGH18-25 is most closely related to LjGH18-12 (LjChit5), indicating that these soybean counterparts might also play a role in nodule symbiosis [22,23,24]. In the SI-CLP clade, each contains 1 GH18 member for Medicago and Lotus. In the chitinase-like superfamily, there are no homologous proteins for Medicago and Lotus. In the narbonin clade, there are two Medicago GH18 proteins and one Lotus protein. Taken together, the GH18 proteins show more or less the same evolutionary trend among the three modern legume species.

2.3. Physicochemical Property Analysis of GH18 Proteins in G. max

To better understand the protein properties of soybean GH18 proteins, molecular size, isoelectric point, protein stability and hydropathicity were studied in detail. As shown in Table 1, soybean GH18 proteins have a wide range of amino acid size, from 173 aa (GmGH18-8) to 820 aa (GmGH18-18.2), which is mainly due to the presence of the C-terminal serine/threonine kinase. The average amino acid length is 368. Thus, the molecular weight ranges from 19.46 kDa (GmGH18-8) to 91.01 kDa (GmGH18-18.2), and the average molecular weight is 40.38 kDa. The pI ranges from 4.07 to 9.42, among which 22 GH18 proteins have a pI lower than 7.0 and the remaining 19 GH18 proteins have a pI higher than 7.0. The ratio of acidic enzymes to basic enzymes is about 1:1. As for the instability, the majority of soybean GH18 proteins have a value below 40, which is considered stable. Those from the SI-CLP clade, especially for GmGH18-5.1 and GmGH18-5.2, are unstable. The Aliphatic index of soybean GH18 proteins range from 70.99 (GmGH18-23) to 92.02 (GmGH18-20), which indicates the wide variation in thermal stability. The grand average of the hydropathicity of soybean GH18 proteins is basically negative, ranging from −0.352 (GmGH18-17) to 0.000 (GmGH18-2).
Plants cannot produce chitin, but the chitinases could be used to degrade exogenous chitin. Thus, whether the given chitinase can be secreted out of the cell after protein synthesize is a precondition for its biological function. To a large extent, the presence of a signal peptide can determinate its secretion. Signal peptide prediction was performed for soybean GH18 proteins. Most of them have a functional secretion signal, except for GmGH18-7, GmGH18-8, and those from the SI-CLP clade and chitinase-like superfamily.

2.4. Collinearity Analysis of GH18 Genes in Soybean, Medicago, and Lotus

Soybean has undergone whole genome duplication twice during evolution, which allows plants to acquire new genes and create genetic novelty [29,30]. An analysis of correlated gene arrangements could facilitate deducing a common ancestor [31]. In soybean, 19 pairs of fragment copy genes were identified in the GH18 gene family (Figure 4 and Table S2). Chromosome 13 has the most fragment replication genes (6 pairs), followed by chromosome 15, with four pairs of fragment replication genes. Chromosomes 03, 09, 10, 16, 17, 19, and 20 each have three pairs of fragment replication genes. Chromosome 01 has two pairs of fragment replication genes. Chromosomes 02, 05, 07, and 14 each have one pair of fragment replication genes. In contrast, there were none in chromosomes 04, 06, 08, 11, 12, and 18. Pairs of genes within the same chromosomes were not detected. In short, the GH18 gene pairs identified above could be a good implication for soybean whole genome duplication.
To obtain a larger evolutionary profile of GH18 genes in legumes, collinearity analysis was conducted between two species of soybean, Medicago, or Lotus. As shown in Figure 5a and Table S3, there are 17 soybean GH18 genes that have a common ancestor with Medicago. Chromosomes 13, 17, and 20 have the most gene pairs, each with two gene pairs. Five out of twenty soybean chromosomes have no gene pairs with Medicago. Furthermore, there are in total 23 soybean GH18 genes which have a source relationship with Lotus, namely 7 homologous gene pairs in chromosome 17, 5 homologous gene pairs in chromosome 15, 4 homologous gene pairs in chromosome 13, 2 homologous gene pairs in chromosome 20, and 1 homologous gene pair in chromosomes 01, 02, 07, 10, and 16 (Figure 5b and Table S4). Only 9 out of the 20 soybean chromosomes have gene pairs with Lotus. Collinearity analysis was then performed between Medicago and Lotus, whose genome sizes are much smaller than soybean. The results showed that there are only eight gene pairs, among which three were in Medicago chromosome 02 and Lotus chromosome 03 (Figure 5c and Table S5).

2.5. Analysis of Cis-Acting Elements in G. max GH18 Gene Promoter Regions

For a better understanding of the biological function of soybean GH18 genes, the cis-acting elements in their promoter regions were identified. Both abiotic stress (e.g., anaerobic responsiveness) and biotic stress (e.g., defense/stress responsiveness) conditions are included. Positions of such cis-acting elements are labeled in the region of 2000 bp upstream of the translation initiation site (Figure 6).
Light responsive elements were identified in all GH18 family members in soybean, among which GmGH18-14 contained the most elements, with up to 20. Methyl jasmonate-responsive elements were identified in 21 G. max GH18 family members, among which GmGH18-11, GmGH18-18, and GmGH18-25 contained the most elements, with 6. Abscisic acid-responsive elements were identified in 29 G. max GH18 family members, with GmGH18-34 containing 7. Anaerobic-responsive elements were identified in 31 G. max GH18 family members, among which GmGH18-27 contained the most elements, with 5. Drought-responsive elements were identified in 19 G. max GH18 family members, among which there are 1–3 elements, respectively. Gibberellin-responsive elements were identified in 16 G. max GH18 family members, among which GmGH18-10 contained the most elements, with 4. Salicylic acid-responsive elements were identified in 16 G. max GH18 family members, among which there are 1–2 elements for each gene. Auxin-responsive elements were identified in 15 G. max GH18 family members, among which GmGH18-19 contained the most elements, with 3. Defense/stress-responsive elements were identified in 11 G. max GH18 family members, among which GmGH18-12 contained the most elements, with 3. Low-temperature-responsive elements were identified in twelve G. max GH18 family members, among which the majority of them contained only one element. Meristem expression-related elements were identified in 11 G. max GH18 family members, among which there are 1–2 elements for each gene. Zein metabolism regulation-related elements were identified in 11 G. max GH18 family members, among which all genes contained only one element. Endosperm expression-related elements were identified in eight G. max GH18 family members, among which all genes contained only one element, except for GmGH18-7, with two elements.
Furthermore, the transcription binding sites were also analyzed to predict the potential regulators of the G. max GH18 genes. As shown in Figure S2, there are a number of TFBSs in the 2000 bp promoter regions. AT-Hook, TBP, Homeodomain, Myb/SANT, WRKY, bHLH, bZIP, and TCR were mainly identified due to higher frequencies. AT-Hook, TBP, Homeodomain, Myb/SANT, bZIP, and TCR were identified in all G. max GH18 gene promoter regions, with 3513, 2377, 1885, 1680, 740, and 628, respectively. There were 1159 WRKY that were identified in 37 G. max GH18 gene promoter sequences. There were 836 bHLH that were identified in 39 G. max GH18 gene promoter sequences.

2.6. The Expression of GH18 Genes During Nodule Symbiosis

To better understand their biological functions, the expression profile of soybean GH18 genes was investigated in detail. According to the different expression patterns, they can be classified into five groups (Figure 7a). Those from group 1 generally exhibit ubiquitous expression from the whole plant. GmGH18-5 shows a high expression level in all tested tissues. In contrast, members from group 2 showed extremely low expression in all tissues. Members from group 3, 4, and 5 have low expression in pods and seeds but have relative high expression in root hairs and nodules. Those from group 5 also have higher expression in shoots, like leaves, shoot apical meristem (SAM), stem, and flower. The difference between group 3 and 4 is the expression in root. Those from group 3 show low expression in root, while those from group 4 show the opposite.
Through the root system, legumes can acquire nitrogen during the interaction with rhizobia. Genes with higher expression in roots, root hairs, and nodules tend to play a role in nodule symbiosis. In other words, group 4 genes are very likely to be nodulation-related. To test this hypothesis, soybean roots (as well as nodules) inoculated with rhizobia were harvested for gene expression measurement at different time points. As shown in Figure 7b–d, the expression levels of GmGH18-15, GmGH18-24, and GmGH18-33 can be induced by rhizobial inoculation. The expression of GmGH18-15 decreased in nodules, while GmGH18-24 and GmGH18-33 remained highly up-regulated in nodules. Such results indicated that members from group 4 show good nodulation responsiveness and might be important for the establishment of symbiosis.
Since GmGH18-15 and GmGH18-24 are class V chitinase genes, the expression levels of other class V chitinase genes were further measured. GmGH18-20, GmGH18-21, GmGH18-23, and GmGH18-25 showed no rhizobial induction at the early infection stage (Figure S3). Except for GmGH18-23, others showed lower expression in nodules. Thus, rhizobial responsiveness is a unique feature for group 4 members. Furthermore, the expression patterns of GH18 genes in Medicago and Lotus during symbiosis were analyzed. The criteria includes (i) nodulation-related tissue specific expression and (ii) rhizobial induction at the early infection stage. The nodulation-specific members were highlighted. There are nine Medicago and two Lotus GH18 genes classified into this group, respectively (Figures S4 and S5).

3. Discussion

In this study, 36 GH18 genes were identified from the whole genome of soybean. Compared with a recent study about soybean chitinases, which claims that there are 19 class III chitinases and 6 class V chitinases, 11 more GH18 members have been newly found. Regarding class III chitinases, GmGH18-8, GmGH18-28, and GmGH18-29 are the new ones. Motif analysis clearly showed that they contained similar motifs to other class III chitinases (Figure 2). GH18 proteins from SI-CLP, the chitinase-like superfamily, and narbonin are firstly characterized. They do not possess too many similar motifs with class III or V chitinases, but they also contain chitinase-related domains (Figure 2). It is not clear whether they still display chitinase activity or whether they lost it during evolution. The classification of GmGH18-31 is ambiguous due to its low sequence homology with others. It is grouped together with GmGH18-5.1 and GmGH18-5.2 in the motif analysis (Figure 2), while it is also classified into class III chitinase in the phylogenetic analysis (Figure 3).
The genome of soybean (1.1 Gb) is larger than the genomes of Medicago (520 Mb) and Lotus (470 Mb). During evolution, soybean has undergone large-scale whole genome duplication twice, while Medicago and Lotus have experienced duplication before speciation [33]. Their genomes share a substantial collinearity (Figure 5). The homologous genes can be found in each sub-clade of GH18, with the exception that Medicago has its own specific GH18, which is worth further investigation. One benefit of gene duplication is the specialized neofunctionalization. GmGH18-15 and GmGH18-24 are a pair of fragment copy genes (Figure 4 and Figure S2) and both are inducible by rhizobia (Figure 7). Nevertheless, the expression levels of GmGH18-15 are much reduced in nodules, while GmGH18-24 remains highly expressed (Figure 7).
In some cases, plant chitinases are expressed at low levels and they can be specifically induced under certain conditions [34,35]. As shown in the cis-acting element analysis, environmental stress such as drought, anaerobic condition, light, abscisic acid, and cold temperature could influence their gene expression (Figure 6). Meanwhile, chitinases can also be induced by certain hormones such as gibberellin, methyl jasmonate, salicylic acid, and auxin (Figure 6) [13]. Certain biological progresses, such as embryo and meristem development, also need the expression of chitinases [36]. Furthermore, chitinases are widely considered as responsive to the microbe-associated molecular pattern, especially the cell wall components of fungi such as chitin, chitin oligosaccharides, and pectin polysaccharides. The recognition of these molecules by the corresponding receptors located on the plasma membrane initiates the signaling, and eventually, the expression of chitinases becomes a classic marker for plant defense. The synthesized chitinases can be secreted and further inhibit the growth of fungi by the hydrolysis of their cell walls. In agriculture, active chitinases are often used as biocontrol agents in the form of either purified molecules or plant gene transformation.
Legumes can obtain more nitrogen through the establishment of a symbiotic relationship with rhizobia. Nod factors, produced by rhizobia, are signal molecules, which can trigger the nodulation signaling in host legumes. Nod factors are modified chitin oligosaccharide consisting of at least a sugar backbone and an N-linked fatty acid chain at the non-reducing end [37]. The hydrolysis of Nod factors by host hydrolases has been studied for the past thirty years. Originally, the Nod factor degradation was found in Vicia sativa, Medicago sativa, M. truncatula, and pea [38,39,40]. Later, the specific enzymes were identified and characterized in M. truncatula and L. japonicus, which turned out to be class V chitinases [20,22,23,24]. Not only were these enzymes able to cleave Nod factors both in vitro and in vivo, but also the mutants deficient in these genes showed a strong nodulation phenotype. One biological significance of such hydrolysis is the inactivation of the nodulation signaling and the avoidance of overrated nodulation, which will cost a lot of energies. To date, the Nod factor hydrolases in soybean have not been identified yet. Interestingly, HgCht2, a homolog of plant class V chitinase from the soybean cyst nematode Heterodera glycines, was reported to be able to hydrolyze the Nod factors from both S. meliloti and B. japonicum USDA110 and play a role in antagonizing the colonization of beneficial symbionts [41]. Therefore, it is quite possible that there are certain class V chitinases in soybean that are capable of degrading Nod factors. Indeed, GmGH18-15 and GmGH18-24 are the homologous genes of MtNFH1 and MtCHIT5b in soybean (Figure 3). GH18 gene collinearity analysis between soybean and Medicago showed that both GmGH18-15 and GmGH18-24 had common ancestors with MtNFH1 (Figure 5, Table S3). Their expressions are inducible by rhizobia (Figure 7). On the other hand, the scenario in soybean could be similar with Lotus. GmGH18-25 is closely related to LjChit5, LjGH18-10, and LjGH18-13 (Figure 3). LjGH18-10 and LjGH18-13 have evolved as pseudogenes [24]. Synteny analysis revealed that GmGH18-25 and LjGH18-13 have common ancestors (Figure 5, Table S4). Although GmGH18-25 can barely be up-regulated by rhizobia, it might play a role in nodule symbiosis (Figure 5).
In conclusion, our informatic analysis with soybean GH18 genes provides a full picture of their chromosomal localizations, motif/domain structures, evolutionary cues, and expression patterns. These data are crucial references for the further characterization of specific GH18 genes in soybean.

4. Materials and Methods

4.1. Sequence Acquisition

The whole genome sequences, protein sequences, and gene annotations of G. max (Wm82.a2.v1), M. truncatula (Mt4.0v1), and L. japonicus (Lj1.0v1) were obtained from the Phytozome server (https://phytozome-next.jgi.doe.gov/) [32,42,43]. In order to identify GH18 genes in these three species, the catalytic domain of glycoside hydrolase family 18 (PF00704; http://pfam.xfam.org/) was used as a query code to search for in their protein sequences using the HMM Search function of TBtools-II (v2.119) [44]. The obtained hit rankings by both sequence scores and domain scores were taken as an intersection, and those with an E-value lower than 1 × 10−8 were considered as candidate GH18 genes. Then, their genomic sequences and protein sequences were further extracted using Fasta Extract function of TBtools-II.

4.2. Analysis of Chromosomal Localization

According to the soybean GFF3 file, the chromosome mapping of GH18 genes was performed using the Gene Localization Visualize function of TBtools-II.

4.3. Construction of Phylogenetic Tree

For the phylogenetic analysis of either soybean GH18 genes or all GH18 genes in three species, their full-length protein sequences were aligned firstly using Clustalx2.1 program, which was further used to construct a phylogenetic tree. The evolutionary history was inferred using the Maximum Likelihood method based on the Whelan And Goldman model [45]. Evolutionary analyses were conducted in MEGA7 with 1000 bootstrap tests [46]. Eventually, the phylogenetic tree was beautified with ITOL server (https://itol.embl.de/, accessed on 10 December 2024).

4.4. Identification of Conserved Motifs and Domains

MEME (https://meme-suite.org/meme/tools/meme, accessed on 10 December 2024) was used to identify 20 conserved motifs with other default settings in soybean GH18 protein sequences [47]. Conserved domains of soybean GH18 proteins were examined in the Conserved Domain Database of NCBI (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 10 December 2024) [48]. Finally, TBtools-II was used for the integration of the soybean GH18 evolutionary tree, conserved motifs, and conserved domains.

4.5. Analysis of Physicochemical Properties

Amino acids size, molecular weight, isoelectric point, instability index, aliphatic index, and grand average of hydropathicity were analyzed using the ProtParam online server (https://web.expasy.org/protparam/, accessed on 10 December 2024) [49]. The signal peptide was predicted using the SignalP-6.0 server (https://services.healthtech.dtu.dk/services/SignalP-6.0/, accessed on 10 December 2024) [50].

4.6. Collinearity Analysis

First, the soybean genomic sequences were analyzed by the Fasta Stats tool of TBtools-II to calculate the length of each chromosome. Next, the gene annotation file was processed by the Gene Density Profile tool of TBtools-II to obtain the gene density. Then, the Blast Compare Two Seqs tool was used to blast the soybean genomic sequences with themselves. Gene positions were acquired by the GFF3/GTF Gene Position (Info.) Parse tool. Lastly, the Advanced Circos tool was used to ornamented the result. Meanwhile, synteny analyses between two species were conducted in a similar way [51].

4.7. Analysis of Cis-Acting Elements and Transcription Factor Binding Sites

The 2000 bp sequences upstream of the start codon of soybean GH18 genes were obtained using the Gtf/Gff3 Sequences Extract function of TBtools-II. Later, these sequences were submitted to the PlantCARE website to obtain the predicted cis-acting elements (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 10 December 2024) [52]. The selected cis-acting elements were visualized using the Simple BioSequence Viewer function of TBtools-II. Similarly, the 2000 bp promoter sequences were uploaded to the PlantPan 4.0 online server to scan for the transcription factor binding sites (http://plantpan.itps.ncku.edu.tw/plantpan4/index.html, accessed on 10 December 2024) [53].

4.8. Gene Expression Analysis and Validation by Quantitative Real Time PCR

The expression data of GH18 genes were downloaded from Phytozome for soybean, Medicago, and the Lotus Base (https://lotus.au.dk/, accessed on 10 December 2024) [54]. Due to an updated version of genome assembly used in the Lotus Base, Lotus GH18 genes were searched for using a new gene ID by sequence blast, and then the corresponding expression data were extracted. The transcript levels were analyzed in different tissues under various conditions. The value was further normalized in the form of log2 and displayed in the heatmap.
For the validation experiments, G. max Wm82 seedlings were germinated in the vermiculite for 5 days and later inoculated with B. japonicum USDA110 (OD600 = 0.1 mL, 30 mL/plant). Roots were harvested at 0, 3, 5, and 7 days post inoculation, while nodules were harvested at 2 and 3 weeks post inoculation. RNA was extracted and used for gene expression analysis. The plant RNA purification kit and cDNA reverse transcription kit were purchased from Magen and Vazyme, respectively. The primers used are listed in Table S6. The expression of the Actin gene served as a reference. All statistical analyses were performed in the GraphPad Prism Statistical analysis and graphing software (Version 8.0.2). The means and significant P values were calculated using Student’s t-test.

5. Conclusions

As a key crop in global agriculture, soybean provides abundant plant-based protein for human beings. Biological nitrogen fixation is both an environmental-friendly and efficient approach for soybean to acquire nitrogen. Here, we performed bioinformatic analysis regarding GH18 gene chromosomal localizations, motif/domain structures, and evolutionary cues and highlighted their inducibility during nodulation. These findings implicate that certain GH18 proteins play a role in nodule symbiosis. Selecting and fully utilizing the best GH18 protein(s) may improve soybean nodulation efficiency. Thus, this can reduce the input of chemical fertilizer and enlarge the nitrogen acquisition, leading to increased production with higher nitrogen content. Future research should focus on characterizing the GH18 enzymatic activities towards Nod factors, which will facilitate the understanding of the Nod factor signaling between rhizobia and legumes.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms26041649/s1.

Author Contributions

All authors contributed to the study’s conception and design. H.L. and Z.S. designed the research; R.L. conducted the bioinformatics analysis, wrote, and revised the manuscript; C.G. conducted the bioinformatics analysis; K.Z., M.H., L.L. and F.K. modified the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was financially supported by the Guangdong Basic and Applied Basic Research Foundation (2023A1515110630 and 2023A1515110560).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data generated or analyzed in this study are included in the main text and its Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Chromosome mapping of the GH18 genes in G. max. Here, 20 soybean chromosomes were displayed in scale with a ruler beside. The position of each GH18 gene was clearly marked on the chromosomes. The yellow to blue gradient represents different gene density, with yellow as high gene density region and blue as low gene density region.
Figure 1. Chromosome mapping of the GH18 genes in G. max. Here, 20 soybean chromosomes were displayed in scale with a ruler beside. The position of each GH18 gene was clearly marked on the chromosomes. The yellow to blue gradient represents different gene density, with yellow as high gene density region and blue as low gene density region.
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Figure 2. Phylogenetic analysis and protein structure identification of GH18 genes in soybean. (a) A phylogenetic tree was constructed by the Maximum Likelihood method. Bootstrap tests with 1000 replicates were performed. (b) Here, 10 conserved motifs and 10 conserved domains were identified in soybean GH18 proteins, respectively.
Figure 2. Phylogenetic analysis and protein structure identification of GH18 genes in soybean. (a) A phylogenetic tree was constructed by the Maximum Likelihood method. Bootstrap tests with 1000 replicates were performed. (b) Here, 10 conserved motifs and 10 conserved domains were identified in soybean GH18 proteins, respectively.
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Figure 3. Phylogenetic analysis of GH18 proteins in soybean, Medicago, and Lotus. Based on the HMM search result of PF00704, 36, 47, and 18 putative GH18 proteins were identified in soybean, Medicago, and Lotus, respectively. They were further divided into 6 groups using the Maximum Likelihood method implemented on MEGA7.0. There were 50 genes in the class III chitinase clade, 23 genes in the class V chitinase clade, 3 genes in the stabilin-1 interacting chitinase-like protein clade (SI-CLP), 3 genes in the chitinase-like superfamily, 6 genes in the narbonin clade, and 16 genes in the Medicago-specific GH18 clade, respectively. Due to the reported biological function in legume–rhizobium symbiosis, MtGH18-20 (MtNFH1), MtGH18-21 (MtCHIT5b), and LjGH18-12 (LjChit5) are highlighted in red.
Figure 3. Phylogenetic analysis of GH18 proteins in soybean, Medicago, and Lotus. Based on the HMM search result of PF00704, 36, 47, and 18 putative GH18 proteins were identified in soybean, Medicago, and Lotus, respectively. They were further divided into 6 groups using the Maximum Likelihood method implemented on MEGA7.0. There were 50 genes in the class III chitinase clade, 23 genes in the class V chitinase clade, 3 genes in the stabilin-1 interacting chitinase-like protein clade (SI-CLP), 3 genes in the chitinase-like superfamily, 6 genes in the narbonin clade, and 16 genes in the Medicago-specific GH18 clade, respectively. Due to the reported biological function in legume–rhizobium symbiosis, MtGH18-20 (MtNFH1), MtGH18-21 (MtCHIT5b), and LjGH18-12 (LjChit5) are highlighted in red.
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Figure 4. Collinearity analysis of GH18 genes in soybean. Here, 20 soybean chromosomes were arranged in circle. Gray lines represent the gene pairs in the whole genome. Red lines highlight the collinear relationship within the GH18 gene family.
Figure 4. Collinearity analysis of GH18 genes in soybean. Here, 20 soybean chromosomes were arranged in circle. Gray lines represent the gene pairs in the whole genome. Red lines highlight the collinear relationship within the GH18 gene family.
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Figure 5. Collinearity analysis of GH18 genes between two species of soybean, Medicago, and Lotus. Collinearity analysis was performed between two given species: soybean–Medicago (a), soybean–Lotus (b), and Medicago–Lotus (c). Gray lines from the background show all collinear gene pairs between two genomes, while red lines highlight the collinear relationship within the GH18 family.
Figure 5. Collinearity analysis of GH18 genes between two species of soybean, Medicago, and Lotus. Collinearity analysis was performed between two given species: soybean–Medicago (a), soybean–Lotus (b), and Medicago–Lotus (c). Gray lines from the background show all collinear gene pairs between two genomes, while red lines highlight the collinear relationship within the GH18 family.
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Figure 6. Cis-acting element analysis of GH18 gene promoter regions in G. max. 41 GH18 gene promoter sequences (−2000 bp upstream of ATG) are obtained from G. max Wm82 genome (a2.v1). The gene symbols are listed on the left and the nucleotide positions are labeled at the bottom. In total, 13 cis-acting elements are identified, whose potential biological functions are related to drought responsiveness, anaerobic responsiveness, light responsiveness, gibberellin responsiveness, abscisic acid responsiveness, defense/stress responsiveness, low-temperature responsiveness, endosperm expression, zein metabolism regulation, meristem expression, Methyl jasmonate (MeJA) responsiveness, salicylic acid responsiveness, and auxin responsiveness.
Figure 6. Cis-acting element analysis of GH18 gene promoter regions in G. max. 41 GH18 gene promoter sequences (−2000 bp upstream of ATG) are obtained from G. max Wm82 genome (a2.v1). The gene symbols are listed on the left and the nucleotide positions are labeled at the bottom. In total, 13 cis-acting elements are identified, whose potential biological functions are related to drought responsiveness, anaerobic responsiveness, light responsiveness, gibberellin responsiveness, abscisic acid responsiveness, defense/stress responsiveness, low-temperature responsiveness, endosperm expression, zein metabolism regulation, meristem expression, Methyl jasmonate (MeJA) responsiveness, salicylic acid responsiveness, and auxin responsiveness.
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Figure 7. Expression profile of soybean GH18 genes and rhizobial responsiveness validation by RT-qPCR. (a) The GH18 gene expression pattern was extracted from the soybean RNA sequencing data [32] and displayed in the heatmap. According to their expression levels in pods, root hairs, leaves, roots, nodules, seeds, shoot apical meristems (SAM), stems, and flowers, 36 soybean GH18 genes were grouped into 5 categories. Group 4 was highlighted, because members from this group are nodulation-specific (N.S.). (b–d) RT-qPCR validation of the expression behavior of group 4 members under symbiotic conditions. G. max Wm82 plants were inoculated with Bradyrhizobium japonicum USDA110; the transcript levels of GmGH18-15, GmGH18-24, and GmGH18-33 in the roots (within 7 days post inoculation) and nodules (14 and 21 dpi) were measured (n = 3). The Actin gene served as a reference. Data indicate means ± SE of normalized expression values (mean value of control set to one). The asterisks indicate significantly increased expression compared to control roots without rhizobial inoculation (Student’s t-test; * p < 0.05, ** p < 0.01, *** p < 0.001).
Figure 7. Expression profile of soybean GH18 genes and rhizobial responsiveness validation by RT-qPCR. (a) The GH18 gene expression pattern was extracted from the soybean RNA sequencing data [32] and displayed in the heatmap. According to their expression levels in pods, root hairs, leaves, roots, nodules, seeds, shoot apical meristems (SAM), stems, and flowers, 36 soybean GH18 genes were grouped into 5 categories. Group 4 was highlighted, because members from this group are nodulation-specific (N.S.). (b–d) RT-qPCR validation of the expression behavior of group 4 members under symbiotic conditions. G. max Wm82 plants were inoculated with Bradyrhizobium japonicum USDA110; the transcript levels of GmGH18-15, GmGH18-24, and GmGH18-33 in the roots (within 7 days post inoculation) and nodules (14 and 21 dpi) were measured (n = 3). The Actin gene served as a reference. Data indicate means ± SE of normalized expression values (mean value of control set to one). The asterisks indicate significantly increased expression compared to control roots without rhizobial inoculation (Student’s t-test; * p < 0.05, ** p < 0.01, *** p < 0.001).
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Table 1. Physicochemical property analysis of GH18 proteins in G. max.
Table 1. Physicochemical property analysis of GH18 proteins in G. max.
Gene SymbolGene IDAmino Acids SizeMolecular Weight (kDa)Isoelectric PointInstability IndexAliphatic IndexGrand Average of HydropathicitySignal Peptide
GmGH18-1Glyma.01G055200.129631.745.3935.5483.48−0.113Yes
GmGH18-2Glyma.01G142400.128331.266.0026.9890.670.000No
GmGH18-3Glyma.02G113600.129631.695.1834.5479.86−0.106Yes
GmGH18-4Glyma.03G254300.130332.598.9738.4876.90−0.231Yes
GmGH18-5.1Glyma.03G256800.141646.849.1850.7790.05−0.323No
GmGH18-5.2Glyma.03G256800.241847.029.1050.3789.86−0.325No
GmGH18-6Glyma.05G075000.129832.649.4138.7282.21−0.115Yes
GmGH18-7Glyma.07G061600.128931.306.3123.1781.38−0.068No
GmGH18-8Glyma.07G061800.117319.468.4232.3377.28−0.296No
GmGH18-9Glyma.08G299700.130032.008.0838.6186.570.024Yes
GmGH18-10Glyma.08G300300.124525.864.8735.4187.310.106Yes
GmGH18-11Glyma.09G121000.130934.045.1839.0779.19−0.130Yes
GmGH18-12Glyma.09G126200.129230.884.0733.0287.57−0.016Yes
GmGH18-13Glyma.10G227700.130432.437.5839.3085.720.049Yes
GmGH18-14Glyma.12G156600.129831.515.5130.9483.26−0.050Yes
GmGH18-15Glyma.13G155800.137941.074.7816.1887.810.141Yes
GmGH18-16Glyma.13G330800.130834.155.3134.0083.25−0.061Yes
GmGH18-17Glyma.13G330900.126430.115.1042.1179.32−0.352No
GmGH18-18.1Glyma.15G015100.162468.808.2933.3589.58−0.072Yes
GmGH18-18.2Glyma.15G015100.282091.016.3135.5990.16−0.141Yes
GmGH18-18.3Glyma.15G015100.362468.808.2933.3589.58−0.072Yes
GmGH18-19Glyma.15G043300.129834.245.6730.6980.17−0.337No
GmGH18-20Glyma.15G206400.176286.086.4039.9492.02−0.167Yes
GmGH18-21Glyma.15G206800.136540.098.9234.7773.29−0.304Yes
GmGH18-22.1Glyma.16G173000.129731.775.0134.0087.74−0.043Yes
GmGH18-22.2Glyma.16G173000.229531.625.0134.1688.34−0.039Yes
GmGH18-23Glyma.17G076100.137441.258.7933.0870.99−0.102Yes
GmGH18-24Glyma.17G103500.137741.069.1118.0888.810.158Yes
GmGH18-25Glyma.17G217000.138443.296.1432.3379.79−0.228Yes
GmGH18-26Glyma.18G120200.129531.235.8735.9785.080.095Yes
GmGH18-27Glyma.18G120700.129531.277.5032.9685.080.045Yes
GmGH18-28Glyma.18G120800.121122.894.3629.2989.190.149Yes
GmGH18-29Glyma.18G120900.126127.868.1433.5485.250.026Yes
GmGH18-30Glyma.19G076200.131634.759.4237.3477.53−0.238Yes
GmGH18-31Glyma.19G255000.117520.038.6545.4990.34−0.114No
GmGH18-32.1Glyma.20G035400.180088.947.9342.2787.72−0.170Yes
GmGH18-32.2Glyma.20G035400.279888.777.9342.3587.46−0.178Yes
GmGH18-33Glyma.20G164600.130132.399.3441.7884.29−0.017Yes
GmGH18-34Glyma.20G164700.133337.034.8732.3979.97−0.163Yes
GmGH18-35Glyma.20G164900.129932.114.2738.8983.58−0.092Yes
GmGH18-36Glyma.U033800.130233.545.4433.1778.15−0.118Yes
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MDPI and ACS Style

Li, R.; Gou, C.; Zhang, K.; He, M.; Li, L.; Kong, F.; Sun, Z.; Liu, H. Genome-Wide Identification and Expression Analyses of Glycoside Hydrolase Family 18 Genes During Nodule Symbiosis in Glycine max. Int. J. Mol. Sci. 2025, 26, 1649. https://doi.org/10.3390/ijms26041649

AMA Style

Li R, Gou C, Zhang K, He M, Li L, Kong F, Sun Z, Liu H. Genome-Wide Identification and Expression Analyses of Glycoside Hydrolase Family 18 Genes During Nodule Symbiosis in Glycine max. International Journal of Molecular Sciences. 2025; 26(4):1649. https://doi.org/10.3390/ijms26041649

Chicago/Turabian Style

Li, Rujie, Chuanjie Gou, Ke Zhang, Milan He, Lanxin Li, Fanjiang Kong, Zhihui Sun, and Huan Liu. 2025. "Genome-Wide Identification and Expression Analyses of Glycoside Hydrolase Family 18 Genes During Nodule Symbiosis in Glycine max" International Journal of Molecular Sciences 26, no. 4: 1649. https://doi.org/10.3390/ijms26041649

APA Style

Li, R., Gou, C., Zhang, K., He, M., Li, L., Kong, F., Sun, Z., & Liu, H. (2025). Genome-Wide Identification and Expression Analyses of Glycoside Hydrolase Family 18 Genes During Nodule Symbiosis in Glycine max. International Journal of Molecular Sciences, 26(4), 1649. https://doi.org/10.3390/ijms26041649

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