# Weighted Gene Correlation Network Analysis (WGCNA) ofArabidopsis Somatic Embryogenesis (SE) and Identification of Key Gene Modules to Uncover SE-Associated Hub Genes

Authors: Kithmee K. de Silva; Jim M. Dunwell; Anushka M. Wickramasuriya
Journal: International Journal of Genomics
 (2022)

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## Abstract

Somatic embryogenesis (SE), which occurs naturally in many plant species, serves as a model to elucidate cellular and molecular mechanisms of embryo patterning in plants. Decoding the regulatory landscape of SE is essential for its further application. Hence, the present study was aimed at employing Weighted Gene Correlation Network Analysis (WGCNA) to construct a gene coexpression network (GCN) forArabidopsis SE and then identifying highly correlated gene modules to uncover the hub genes associated with SE that may serve as potential molecular targets. A total of 17,059 genes were filtered from a microarray dataset comprising four stages of SE, i.e., stage I (zygotic embryos), stage II (proliferating tissues at 7 days of induction), stage III (proliferating tissues at 14 days of induction), and stage IV (mature somatic embryos). This included 1,711 transcription factors and 445 EMBRYO DEFECTIVE genes. GCN analysis identified a total of 26 gene modules with the module size ranging from 35 to 3,418 genes using a dynamic cut tree algorithm. The module-trait analysis revealed that four, four, seven, and four modules were associated with stages I, II, III, and IV, respectively. Further, we identified a total of 260 hub genes based on the degree of intramodular connectivity. Validation of the hub genes using publicly available expression datasets demonstrated that at least 78 hub genes are potentially associated with embryogenesis; of these, many genes remain functionally uncharacterized thus far. In silico promoter analysis of these genes revealed the presence of cis-acting regulatory elements, “soybean embryo factor 4 (SEF4) binding site,” and “E-box” of the napA storage-protein gene of Brassica napus; this suggests that these genes may play important roles in plant embryo development. The present study successfully applied WGCNA to construct a GCN for SE in Arabidopsis and identified hub genes involved in the development of somatic embryos. These hub genes could be used as molecular targets to further elucidate the molecular mechanisms underlying SE in plants.


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## Body

## 1. Introduction

The ability to produce embryos from undifferentiated somatic cellsin vitro is a unique developmental pathway found within the plant kingdom. Since the first report of somatic embryo induction from callus cells of carrot [1, 2], this developmental pathway based on cellular totipotency has been studied extensively due to its biological and scientific significance; it has been recognized as a model system for studying early plant embryogenesis. Until now, most studies have focused on the mechanism of somatic embryo development at the morphological level [2–4] or the development of optimized protocols for the generation of somatic embryos from a range of explants [5–8].Somatic embryogenesis (SE) involves a complex signaling network [9]; transcriptional regulation of a set of genes in response to stress caused by plant growth regulators, nutrients, certain stress conditions, and other signaling elements triggers cellular reprogramming and transformation of somatic cells into embryos [10, 11]. In 2007, Zeng et al. [12] developed the first draft gene regulatory network for early SE employing a set of transcriptionally regulated SE-related genes in cotton. Although a set of genes have been identified as markers for the initiation phase of SE [13, 14], for example, SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1 (SERK1) [15, 16], LEAFY COTYLEDON (LEC) [17–21], BABY BOOM (BBM) [22], and WUSCHEL (WUS) [18, 23], the current scientific knowledge on the underlying regulatory landscape of SE is limited. The use of transcriptomics has uncovered a large number of differentially expressed genes (DEGs) during SE in many crops, including Arabidopsis [24], rice [25], bread wheat [26], cotton [27], maize [28], and coconut [29]. However, the functions of many of these genes in SE are still not understood.Gene coexpression networks (GCNs) are increasingly used to understand the interactions among a set of transcriptionally regulated genes. There are many types of coexpression networks: signed/unsigned coexpression networks and weighted/unweighted coexpression networks [30]. In the present study, we have focused on weighted network construction as it is likely to produce more robust findings than unweighted networks [31]. Weighted Correlation Network Analysis (WGCNA) is one of the most popular clustering packages for GCN analysis [31, 32] and the first tool to be employed to construct GCNs from RNA-sequencing (RNA-seq) data. This coexpression tool is easy to use and can be used to find clusters (modules) of highly correlated genes and to identify biologically relevant associations between phenotypes/sample traits and modules from expression data [30]. Recently, WGCNA has been effectively used to identify stage-specific gene expression clusters associated with key stages of Arabidopsis zygotic embryo development [33]. In addition, this approach has been successfully used to discover the regulatory landscape of SE in rice [25] and several other biological pathways in plants [34–36]. Here, we have analyzed a transcriptome dataset covering four somatic embryo developmental stages in Arabidopsis using WGCNA to understand better the system-level functionality of the transcriptionally regulated genes in dicot SE.


## 2. Materials and Methods

### 2.1. Data Collection and Gene Filtering

The transcriptome data covering somatic embryo developmental stages of wild-typeArabidopsis were retrieved from the National Centre for Biotechnology Information (NCBI) GEO database (GEO accession: GSE48915) [37]. The dataset consisted of four developmental stages (zygotic embryos, proliferating tissues at 7 days of induction, proliferating tissues at 14 days of induction, and mature somatic embryos) with two replicates for each stage. Subsequently, the genes with variance greater than the second quartile of variance were filtered to eliminate low-expressed or nonvarying genes, and the remaining genes were used in GCN analysis (https://horvath.genetics.ucla.edu/html/coexpressionnetwork/rpackages/wgcna/faq.html, accessed on 11 May 2022). In addition, DEGs between consecutive embryonic stages were identified by calculating the fold change (FC) in gene expression through a simple t-test. Arbitrary FC cut-off of log2FC≥2.0 and p value of <0.05 were used to reduce false discoveries.


### 2.2. GCN Construction

“WGCNA” package in R software [32] was employed to identify significant gene modules and hub genes in Arabidopsis somatic embryo transcriptomes. A gene coexpression similarity matrix was constructed between the expression profiles of the filtered genes using the Pearson correlation. The similarity matrix was then transformed into an adjacency matrix where each entry encodes the connection strength between each pair of genes (“nodes”). The adjacency matrix defines a measure of node dissimilarity from which the nodes (genes) are clustered into network modules. Consequently, the GCN was developed using the automatic one-step network construction and module detection method with the following parameters:The soft threshold value (power parameter) was decided by the scale-free topology fit index curve.


### 2.3. GCN Visualization

The constructed modular networks were exported to Cytoscape (version 3.7.2) for visualization; gene correlations withp value <0.05 were filtered as significant gene correlations and visualized. The modular networks were analyzed by the “network analyzer” tool in Cytoscape for a concise and informative representation of nodes and edges.


### 2.4. Validation of Network Modules

The robustness of the coexpression modules was assessed through module preservation and quality statistics, which were computed using the modulePreservation function in the WGCNA package [38]. The adjacency matrix of the network was taken as the reference, and the dataset was selected as test data with 200 permutations (nPermutations=200). The stability of the modules was tested through the statistics median rank and Zsummary.


### 2.5. Inferring Module-Stage Relationships

Module-stage relationships of the GCN were evaluated through module eigengenes (MEs). The correlation relationships between the MEs and different somatic embryo developmental stages were analyzed and visualized through a heatmap. Gene significance was calculated based on thep value of the linear regression between the gene expression profile and the associated developmental stage.


### 2.6. Functional Enrichment Analysis

Functional enrichment analysis was performed to detect enriched biological processes in gene modules. Gene Ontology (GO) terms enriched in each module were elucidated using the “singular enrichment analysis” tool provided by agriGO v2.0 [39]. “Arabidopsis genome locus (TAIR10)” was used as the reference, and all other parameters were set as the default for the analysis. Overrepresented GO terms in each network module were identified using the hypergeometric test. To further explore the DEGs mapped to each gene module, the distribution of the following genes across modules was studied: SE-related marker genes [40], plant transcription factors (TFs) (http://planttfdb.cbi.pku.edu.cn/index.php), EMBRYO DEFECTIVE (EMB) genes [41], and gene encoding epigenetic regulators [42, 43].


### 2.7. Identification and Validation of Hub Genes

Genes in each module were arranged based on gene connectivity. The top 10 genes of each module were considered as hub genes. The transcriptome dataset published by Wickramasuriya and Dunwell in 2015 was retrieved from the ArrayExpress database (E-MTAB-2403) [24] to study the expression of hub genes during SE.


### 2.8.In Silico Analysis of Hub Genes

The promoter sequences of hub genes (1000 bp upstream from the transcription start site) were retrieved from “The Arabidopsis Information Resource” (TAIR) database and analyzed using the Multiple Em for Motif Elicitation (MEME) tool in the MEME Suite 5.3.3 [44]. The following parameters were used in the analysis: number of motifs: 10; motif site distribution: zero or once per occurrence (ZOOPS); minimum width: 6; maximum width: 50; and background model: zero-order model of sequences. Further, the biological significance of the predicted MEME motifs was investigated using the Gene Ontology for MOtifs (GOMo) version 5.3.3 [45] provided in the MEME Suite. Additionally, the retrieved promoter sequences were searched against the Plant cis-acting regulatory DNA elements (PLACE) database to identify overrepresented cis-acting regulatory elements (CREs; [46]).


## 2.1. Data Collection and Gene Filtering

The transcriptome data covering somatic embryo developmental stages of wild-typeArabidopsis were retrieved from the National Centre for Biotechnology Information (NCBI) GEO database (GEO accession: GSE48915) [37]. The dataset consisted of four developmental stages (zygotic embryos, proliferating tissues at 7 days of induction, proliferating tissues at 14 days of induction, and mature somatic embryos) with two replicates for each stage. Subsequently, the genes with variance greater than the second quartile of variance were filtered to eliminate low-expressed or nonvarying genes, and the remaining genes were used in GCN analysis (https://horvath.genetics.ucla.edu/html/coexpressionnetwork/rpackages/wgcna/faq.html, accessed on 11 May 2022). In addition, DEGs between consecutive embryonic stages were identified by calculating the fold change (FC) in gene expression through a simple t-test. Arbitrary FC cut-off of log2FC≥2.0 and p value of <0.05 were used to reduce false discoveries.


## 2.2. GCN Construction

“WGCNA” package in R software [32] was employed to identify significant gene modules and hub genes in Arabidopsis somatic embryo transcriptomes. A gene coexpression similarity matrix was constructed between the expression profiles of the filtered genes using the Pearson correlation. The similarity matrix was then transformed into an adjacency matrix where each entry encodes the connection strength between each pair of genes (“nodes”). The adjacency matrix defines a measure of node dissimilarity from which the nodes (genes) are clustered into network modules. Consequently, the GCN was developed using the automatic one-step network construction and module detection method with the following parameters:The soft threshold value (power parameter) was decided by the scale-free topology fit index curve.


## 2.3. GCN Visualization

The constructed modular networks were exported to Cytoscape (version 3.7.2) for visualization; gene correlations withp value <0.05 were filtered as significant gene correlations and visualized. The modular networks were analyzed by the “network analyzer” tool in Cytoscape for a concise and informative representation of nodes and edges.


## 2.4. Validation of Network Modules

The robustness of the coexpression modules was assessed through module preservation and quality statistics, which were computed using the modulePreservation function in the WGCNA package [38]. The adjacency matrix of the network was taken as the reference, and the dataset was selected as test data with 200 permutations (nPermutations=200). The stability of the modules was tested through the statistics median rank and Zsummary.


## 2.5. Inferring Module-Stage Relationships

Module-stage relationships of the GCN were evaluated through module eigengenes (MEs). The correlation relationships between the MEs and different somatic embryo developmental stages were analyzed and visualized through a heatmap. Gene significance was calculated based on thep value of the linear regression between the gene expression profile and the associated developmental stage.


## 2.6. Functional Enrichment Analysis

Functional enrichment analysis was performed to detect enriched biological processes in gene modules. Gene Ontology (GO) terms enriched in each module were elucidated using the “singular enrichment analysis” tool provided by agriGO v2.0 [39]. “Arabidopsis genome locus (TAIR10)” was used as the reference, and all other parameters were set as the default for the analysis. Overrepresented GO terms in each network module were identified using the hypergeometric test. To further explore the DEGs mapped to each gene module, the distribution of the following genes across modules was studied: SE-related marker genes [40], plant transcription factors (TFs) (http://planttfdb.cbi.pku.edu.cn/index.php), EMBRYO DEFECTIVE (EMB) genes [41], and gene encoding epigenetic regulators [42, 43].


## 2.7. Identification and Validation of Hub Genes

Genes in each module were arranged based on gene connectivity. The top 10 genes of each module were considered as hub genes. The transcriptome dataset published by Wickramasuriya and Dunwell in 2015 was retrieved from the ArrayExpress database (E-MTAB-2403) [24] to study the expression of hub genes during SE.


## 2.8.In Silico Analysis of Hub Genes

The promoter sequences of hub genes (1000 bp upstream from the transcription start site) were retrieved from “The Arabidopsis Information Resource” (TAIR) database and analyzed using the Multiple Em for Motif Elicitation (MEME) tool in the MEME Suite 5.3.3 [44]. The following parameters were used in the analysis: number of motifs: 10; motif site distribution: zero or once per occurrence (ZOOPS); minimum width: 6; maximum width: 50; and background model: zero-order model of sequences. Further, the biological significance of the predicted MEME motifs was investigated using the Gene Ontology for MOtifs (GOMo) version 5.3.3 [45] provided in the MEME Suite. Additionally, the retrieved promoter sequences were searched against the Plant cis-acting regulatory DNA elements (PLACE) database to identify overrepresented cis-acting regulatory elements (CREs; [46]).


## 3. Results

### 3.1. Hierarchical Clustering of Somatic Embryo Transcriptomes

In the present study, transcriptome datasets generated through microarray experiments were retrieved from the NCBI covering four somatic embryo developmental stages (with two replicates for each stage), referred to herein as stages I (zygotic embryos), II (proliferating tissues at 7 days of induction), III (proliferating tissues at 14 days of induction), and IV (mature somatic embryos). The hierarchical clustering of samples (Figure1(a)) confirmed that the sample replicates of each stage have a higher degree of correlation with each other than with other developmental stages; sample outliers were not detected in the dataset. The clustering heatmap clearly distinguished four discrete clusters of related expression patterns corresponding to the stages of somatic embryo development (Figure 1(b)). Further, stage I showed a poor correlation with the other three stages. This suggests that stage I may have a distinct expression profile as compared to other somatic embryo developmental stages.Figure 1
Hierarchical clustering of somatic embryo transcriptomes based on their Euclidean distance using average linkage clustering (replicates of each stage are labeled as “a” and “b”). (a) Unrooted hierarchical clustering dendrogram (the length between nodes corresponds to the distance between samples). (b) Hierarchical clustering heatmap visualizing the correlations between the samples.
(a)(b)


### 3.2. Filtering of Genes for the GCN Construction and Downstream Analysis

As recommended by Langfelder and Horvath [32], genes were filtered by the variance for the GCN construction; filtering genes for variance greater than 0.25 quantile identified a total of 17,059 genes (see Table S1). This included 445 EMB genes [41], 10 SE marker genes [40], and 1,711 Arabidopsis TFs (65.3%).In addition, DEGs were identified by a pairwise ratio of expression between consecutive stages of development. A total of 2,244 genes were identified by threshold filtering based onlog2FC≥2.0 and p value <0.05. 64 EMB genes [41], four SE marker genes [40], and 458 TFs were present within the DEGs identified (see Table S2). A total of 12 genes including the genes STRESS INDUCED FACTOR 2 (AT1G51850), LIGHT-HARVESTING-LIKE 3 : 1 (AT4G17600), BETA GLUCOSIDASE 28 (AT2G44460), FERREDOXIN C 1 (AT4G14890), and PHOTOSYSTEM II SUBUNIT Q (AT4G05180) were differentially expressed throughout SE (Figure 2(a)). In addition, a considerable number of genes were up- and downregulated during early embryo developmental stages (Figure 2(b)).Figure 2
Overview of DEGs. (a) Distribution of DEGs between consecutive somatic embryo developmental stages. (b) Number of up- or downregulated DEGs between stages.
(a)(b)


### 3.3. Construction of GCN

The expression profiles of the filtered 17,059 genes were used to construct a scale-free gene expression network with a soft threshold of 15 (Figure3(a)). The dynamic hierarchical clustering approach integrated with the WGCNA pipeline distinguishes groups of genes with coexpression patterns and clusters them into network modules. In total, 26 distinct coexpression gene modules were detected with the module size ranging from 35 to 3,418 genes (Figures 3(b) and 3(c)); each module was assigned with a unique colour. The module comprising most genes was the turquoise (3,418 genes) followed by the blue (2,973 genes) and brown (2,437 genes) (Figure 3(b)). The expression profiles of coexpressed genes clustered in each module were summarized as MEs. Among the filtered genes, 13 genes that failed to fit within a distinct group were assigned to the grey module and removed from the downstream analysis. Module preservation analysis indicated high module preservation, confirming that the modules generated here can also be found in diverse independent datasets (Figure 3(b)). Each module was exported and visualized using Cytoscape.Figure 3
Construction of the draft GCN for SE. (a) Network topology for different soft-thresholding powers. (b) Module preservation statistics. (c) Hierarchical clustering dendrogram of MEs.
(a)(b)(c)


### 3.4. Identification of Stage-Related Modules

The relationships between the gene modules and different somatic embryo developmental stages were determined by assessing the Pearson correlation coefficient (r) between the MEs and developmental stages. Module-trait correlation analyses revealed that multiple modules are related to SE (Figure 4(a)). A total of 18 modules were significantly associated with the somatic embryo developmental stages (r>0.8 and p value ≤0.01; Figure 4), and these modules were “stage-specific,” i.e., the module was significantly associated with only one particular developmental stage of SE: tan, turquoise, dark-orange, and green to stage I; grey60, magenta, brown, and light-yellow to stage II; green-yellow, dark-gray, dark-green, orange, blue, light-green, and light-cyan to stage III; and pink, dark-turquoise, salmon, and yellow to stage IV. Gene significance, the correlation between modular gene expression and each stage, is shown in Figure 4(b).Figure 4
Stage-specific gene modules detected by WGCNA. (a) Module-trait relationship heatmap. Each row corresponds to a module, and each column corresponds to a stage. The degree of correlation is illustrated with the colour legend. The numbers in the table correspond to the p value. Modules that are significantly associated with each somatic embryo development stage (r>0.8 and p value ≤0.01) are indicated by an asterisk. (b) Gene significance values of coexpression modules related to different somatic embryo developmental stages.
(a)(b)


### 3.5. Functional Enrichment Analysis of “Stage-Specific” Gene Modules

GO enrichment analysis performed on “stage-specific” modules showed that the genes in green and turquoise modules which exhibited a significant association with stage I were mainly enriched in the biological processes being involved in postembryonic development, hormone-mediated signaling pathway, biosynthesis pathways (sterol and fatty acids), DNA methylation, and transcription regulation (Figure5(a)). Genes in brown, light-yellow, and magenta modules, which showed significant association with stage II, were mainly enriched in the biological processes involved in root and shoot development, ATP synthesis, response to the metal ions, and DNA replication (Figure 5(b)), whereas genes in blue and light-cyan modules, which showed significant association with stage III, were enriched for the biological processes involved in transition postembryonic and seed development, hormone- and sugar-mediated signaling pathways, cell differentiation, protein modification, and RNA processing (Figure 5(c)). Moreover, the yellow module, which showed a significant relationship to stage IV, was mainly enriched in biological processes involved in ion transport, postembryonic development, signal transduction, lipid localization, response to oxidative and water stress, as well as response to phytohormones (abscisic acid, gibberellin, cytokinin, and jasmonic acid) (Figure 5(d)).Figure 5
Functional enrichment analysis of the stage-specific modules. (a)–(d) represent the significantly enriched GO terms (p value <0.05) in modules specifically associated with stages I–IV, respectively.
(a)(b)(c)(d)


### 3.6. Analysis of Hub Genes

Hub genes are nodes in a network often hypothesized to be functionally significant due to their high degree of intramodular connectivity. A total of 260 genes (top 10 genes of each module with high connectivity) were identified as potential hub genes; the hub gene with the highest degree of connectivity in each module is given in Table1 (the complete list of hub genes is given in Table S3). GO enrichment analysis of the hub genes revealed that they are mainly enriched for biological processes such as metabolic processes (mRNA and cellular amino acid), oxidation-reduction, protein folding, and postembryonic development.Table 1
Top 10 hub genes ordered by the degree of connectivity.
Gene identifierDegree of connectivityGene moduleGene nameDescriptionAT1G271203327TurquoiseAT1G27120 (GALT4)Galactosyltransferase family proteinAT5G528202853BlueNOTCHLESS (NLE)WD-40 repeat family protein/notchless proteinAT5G560902348BrownCYTOCHROME C OXIDASE 15 (COX15)Encodes a homolog of COX15AT2G431002134YellowISOPROPYLMALATE ISOMERASE 2 (IPMI2)Isopropylmalate isomerase 2AT1G710101958GreenFORMS APLOID AND BINUCLEATE CELLS 1C (FAB1C)Encodes a protein that is predicted to act as a phosphatidylinositol-3P 5-kinase but lacks a FYVE domainAT2G29890806RedVILLIN 1 (VLN1)Encodes a ubiquitously expressed villin-like proteinAT2G45600569BlackAT2G45600Alpha/beta-hydrolases superfamily proteinAT1G72400321MagentaAT1G72400Hypothetical proteinAT3G53980311PinkAT3G53980Bifunctional inhibitor/lipid-transfer protein/seed storage 2S albumin superfamily proteinAT5G65350243PurpleHISTONE 3 11 (HTR11)Histone 3 11AT5G27560182Green-yellowAT5G27560DUF1995 domain protein, putative (DUF1995)AT5G54855170TanAT5G54855Pollen Ole e 1 allergen and extensin family proteinAT1G75630156SalmonVACUOLAR H+-PUMPING ATPASE 16 KDA PROTEOLIPID SUBUNIT 4 (AVA-P4)Vacuolar H+-pumping ATPase 16 kD proteolipid (ava-p) mRNAAT1G74450107CyanAT1G74450BPS1-like protein (DUF793)AT2G23940107Midnight-blueAT2G23940Transmembrane protein (DUF788)AT1G3046082Light-cyanCLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR 30 (CPSF30)Encodes AtCPSF30, the 30-KDa subunit of cleavage and polyadenylation specificity factorAT1G0604074Grey60SALT TOLERANCE (STO)B-box zinc finger family protein that encodes a salt tolerance proteinAT2G1156058Light-greenAT2G11560Mutator-like transposase/similar to MURA transposase of maizeAT3G5505050Dark-greenD-CLADE TYPE 2C PROTEIN PHOSPHATASE 4 (PP2C.D4)Protein phosphatase 2C family proteinATCG0107048Dark-redNAD(P)H-QUINONE OXIDOREDUCTASE SUBUNIT 4L (NDHE)NADH dehydrogenase ND4LAT3G2595048Royal-blueAT3G25950TRAM, LAG1, and CLN8 (TLC) lipid-sensing domain containing proteinAT1G6541039Dark-turquoiseATP-BINDING CASSETTE I13 (ABCI13)Encodes a member of NAP subfamily of transportersAT5G0231038Light-yellowPROTEOLYSIS 6 (PRT6)Encodes a component of the N-end rule pathway that targets protein degradationAT3G6113037Dark-greyGALACTURONOSYLTRANSFERASE 1 (GAUT1)Encodes a protein with putative galacturonosyltransferase activityAT3G5335034Dark-orangeROP INTERACTIVE PARTNER 3 (RIP3)Encodes a microtubule-binding proteinAT5G4349034OrangeAT5G43490Myb-like protein XAmong the hub genes, only 234 genes were functionally annotated; of these, 13 were TFs:AUXIN RESPONSE FACTOR 9 (ARF9), FLOWERING BHLH 4 (FBH4), BASIC HELIX-LOOP-HELIX 39 (BHLH39), BASIC LEUCINE-ZIPPER 44 (bZIP44), bZIP19, ZIM-LIKE 2 (ZML2), AT5G60820, AT4G01270, KANADI 3 (KAN3), HOMEODOMAIN GLABROUS 4 (HDG4), CELL DIVISION CYCLE 5 (CDC5), NAC DOMAIN CONTAINING PROTEIN 80 (NAC080), and SALT TOLERANCE (STO)). In addition, five genes encoding transposable elements (i.e., AT2G11560, AT3G33066, AT5G32430, AT3G42820, and AT4G28900) were identified.In silico analysis of the promoter sequences (1000 bp upstream from the transcription start site) of the hub genes using the MEME tool identified four significant motifs ranging in length from 15 to 29 bp (Table 2). Motifs 1, 2, and 3 were detected across 229, 245, and 121 hub genes, respectively. Further analysis of the predicted motifs using the GOMo tool provided in the MEME suite indicated that motifs 1 and 3 may be involved in the DNA endoreduplication, polarity specification of axial/abaxial axis, and hormone-mediated signaling pathways; motifs 1 and 3 seem to function in association to cytokinin and gibberellic acid, respectively.Table 2
Conserved motifs identified in the promoter regions of hub genes using the MEME tool.
MotifE-valueMotif widthSitesSignificant GO enriched terms(q value<0.05)1NAVAAAAAAARAAARARAAARAAAAHMAAConsensus sequence:[AGT]A[AG]AA[AC]AAAA[AG]A[AG][AG][AGC]A[AG]AA[AG][AG]A[AG]AA[ATC][AC]A[AGT]2.8e-07729229(i) GO:0042023: DNA endoreduplication(ii) GO:0009944: Polarity specification of adaxial/abaxial axis(iii) GO:0009735: Response to cytokinin stimulus(iv) GO:0009744: Response to sucrose stimulus(v) GO:0006468: Protein amino acid phosphorylation(vi) GO:0009965: Leaf morphogenesisConsensus logo:2DTTTTTKTTTTKTTYConsensus sequence:[AGT][TG]TTTT[TG]TTTT[TG][TG]T[TCG]6.0e-02015245Consensus logo:3NDRRAGDDDRRWARRRARAGAADRRDAGConsensus sequence:[AGC][GAT][AG][AG][AT][GA][AGT][ATG][AGT][GA][AG][AT]A[GA][AG][GAC]A[AG]A[GA]A[AG][GAT][AG][AG][GTA][AT][GT]3.6e-02528121(i) GO:0009944: Polarity specification of adaxial/abaxial axis(ii) GO:0048481: Ovule development(iii) GO:0010050: Vegetative phase change(iv) GO:0010051: Xylem and phloem pattern formation(v) GO:0042023: DNA endoreduplication(vi) GO:0035196: Production of miRNAs involved in gene silencing by miRNA(vii) GO:0009740: Gibberellic acid mediated signaling pathwayConsensus logo:4CMAYCTYCTCCDTCHBCATCConsensus sequence:[CT][CA]A[TC]CT[CT]CT[CT]C[TAG][TGC][CT][ATC][GTC]C[AT][TC]C5.3e-0072044Consensus logo:


### 3.7. Validation of Hub Genes

A comparison of hub genes and DEGs showed that 31 hub genes are differentially expressed in SE (the expression values of differentially expressed hub genes are given in TableS4). Further, expression analysis of these genes using the Arabidopsis eFP browser demonstrated that two hub genes, AT1G19540 (Figure 6(a)) and AT5G44380 (Figure 6(b)), exhibit a seed-specific pattern of expression.Figure 6
Expression patterns of two hub genes,AT1G19540 (a) and AT5G44380 (b), when viewed through the Arabidopsis eFP browser. The normalized expression value for each gene is colour-coded as indicated by the legend.
(a)(b)Moreover, analysis of the expression profiles of hub genes in theArabidopsis somatic embryo transcriptome dataset (E-MTAB-2465) published by Wickramasuriya and Dunwell (2015) revealed that 62 hub genes are differentially expressed in somatic embryonic tissues compared to leaf tissues (log2FC≥2.0 and p value <0.05; Figure 7). Of these, 15 genes were identified as DEGs in the present analysis. For instance, CYSTEINE-RICH TRANSMEMBRANE MODULE 7 (ATHCYSTM7/AT2G33520), HEPTAHELICAL TRANSMEMBRANE PROTEIN2 (AT4G30850), INDOLE-3-ACETIC ACID INDUCIBLE 30 (IAA30/AT3G62100), RPS9C, VASCULATURE COMPLEXITY AND CONNECTIVITY (AT2G32280), AT2G21820, AT2G38900, and AT5G43770 showed a marked expression in somatic embryonic tissues as compared to leaf tissues. Expression analysis using the Arabidopsis eFP browser further showed that AT2G29300, AT2G21820, AT2G38900, AT5G43770, ATHCYSTM7, and AT1G19540 exhibit a seed-specific pattern of gene expression.Figure 7
Transcript abundance extracted from the somatic embryo transcriptome dataset, E-MTAB-2465 for the hub genes. The hub genes significantly upregulated (log2FC≥2.0) and downregulated (log2FC≤−2.0) in somatic embryonic tissues compared to leaf tissues are indicated with yellow asterisks and red diamonds, respectively. Transcript abundances are shown in fragments per kilobase of transcript per million mapped reads (FPKM).As expected, few hub genes highly expressed in leaf tissues were repressed in somatic embryos indicating the importance of gene regulation in SE (Figure7); for instance, CELLULOSE SYNTHASE-LIKE B4 (AT2G32540), CHOLINE/ETHANOLAMINE KINASE 3 (AT4G09760), GLUTAMATE DECARBOXYLASE 2 (AT1G65960), ISOPROPYLMALATE ISOMERASE 2 (AT2G43100), PEROXIREDOXIN Q (PRXQ/AT3G26060), PHOTOSYNTHETIC NDH SUBCOMPLEX L 4 (PnsL4/AT4G39710), PLASTID RIBOSOMAL PROTEIN S20 (AT3G15190), STO (AT1G06040), SINAPOYLGLUCOSE 1 (SNG1/AT2G22990), THYLAKOID RHODANESE-LIKE (TROL/AT4G01050), TONOPLAST INTRINSIC PROTEIN 2 (TIP2/AT3G26520), AT3G50685, AT4G33666, AT5G16010, and AT5G54540 genes showed a marked repression in somatic embryos compared to leaf tissues.In summary, the present study identified a total of 78 hub genes as potential regulators of SE (Figure8), including genes showing marked overexpression as well as repression in SE. Of these, 41 genes have not been functionally annotated thus far. The analysis of the promoter sequences of these uncharacterized hub genes using the PLACE database identified a total of 215 different plant CREs; ARR1AT, CAATBOX1, CACTFTPPCA1, DOFCOREZM, GATABOX, GT1CONSENSUS, POLLEN1LELAT52, and WRKY71OS were observed in all 41 functionally uncharacterized potential hub genes. Moreover, several CREs related to embryogenesis were identified (Figure 9). The functions of the predicted CREs are included in Table 3.Figure 8
Venn diagram indicating the intersection of hub genes and DEGs (log2FC≥2.0 and p value <0.05) obtained from E-MTAB-2465 [24] and GSE48915 [37].Figure 9
The distribution of several important plant CREs present in the promoter regions of functionally uncharacterized hub genes. The number of hub genes that contain the relevant CRE in their promoter region is indicated by thex-axis. The size of the circle depicts the occurrence of the CREs within the promoter regions of the hub genes (as indicated within the circle).Table 3
Functional roles of several important CREs detected in the functionally uncharacterized hub gene promoter sequences retrieved from the PLACE database [46].
Cis-acting regulatory elementFunction∗ABADESI1“ACGT” motif; transacting factor: TAF-1; responsive to ABA and desiccation. Expressed in seeds late during embryogenesis. Induced by ABA and osmotic stress in vegetative tissues.CACGTGMOTIF“CACGTG motif”; essential for expression of beta-phaseolin gene during embryogenesisCANBNNAPACore of “(CA)n element” in storage protein genes; embryo- and endosperm-specific transcription of napin (storage protein) geneCARGNCATNoncanonical CArG motif (CC-Wx8-GG); A relevantcis-element for the response to AGL15 (AGAMOUS-like 15) in vivoDPBFCOREDCDC3DPBF-1 and 2 (Dc3 promoter-binding factor-1 and 2) binding core sequence; Dc3 expression is embryo-specific and induced by ABADRE1COREZMRAB17“DRE1” core found in maize (Z.M.) rab17 gene promoter; “DRE1” was protected, in in vivo footprinting, by a protein in embryos specifically, but in leaves, was protected when was treated with ABA and drought; rab17 is expressed during late embryogenesis and is induced by ABADRE2COREZMRAB17“DRE2”; core sequence in rab17 gene promoter. rab17 is expressed during late embryogenesis and is induced by ABAEBOXBNNAPA“E-box” of napA storage-protein genePYRIMIDINEBOXOSRAMY1AFound in the promoter of alpha-amylase (Amy2/32b) gene which is induced in the aleurone layers in response to GA in embryoRYREPEATVFLEB4RY repeat motif; quantitative seed expression; binding site ofArabidopsis B3-domain-containing transcription factor FUS3, mediates abscisic acid-induced transcriptionSEF1MOTIF“SEF1 (soybean embryo factor 1)” binding motif; regulates the expression of genes encoding for the beta-conglycinin seed storage proteinsSEF3MOTIFGM“SEF3 binding site”; regulates the expression of genes encoding for the beta-conglycinin seed storage proteinsSEF4MOTIFGM7S“SEF4 (soybean embryo factor 4)” binding motif; regulates the expression of genes encoding for the beta-conglycinin seed storage proteinsTATABOX2“TATA box”; TATA box found in beta-phaseolin promoter which is accurate transcription initiation in the embryo stage∗Details of PLACE entries were retrieved from the https://www.dna.affrc.go.jp/place/place_seq.shtml (accessed on 19th May 2022).


### 3.8. Distribution of Embryogenesis-Related Genes across Network Modules

Further exploration of genes mapped to each network module found that 10 key regulators of SE includingLEC1, FUSCA3 (FUS3), and ABSCISIC ACID INSENSITIVE 3 (ABI3) are present among the highly connected genes in the network (Table 4); SE-related marker genes, LEC2, SERK1, WUS, BBM, and WUSCHEL RELATED HOMEOBOX 2 (WOX2) showed low variance in the present dataset and thus were not included in the GCN analysis. We also observed that the majority of previously published EMB genes [41] are localized to the blue and turquoise modules, which showed significant association with stage I and stage III, respectively (Figure 10; see Table S5).Table 4
Distribution of SE markers across network modules ordered by the number of interactors.
Gene identifierModuleGene nameNo. of interactors1AT3G26790TurquoiseFUSCA3 (FUS3)33462AT5G13790TurquoiseAGAMOUS-LIKE 15 (AGL15)33083AT1G21970TurquoiseLEAFY COTYLEDON 1 (LEC1)32974AT5G45980TurquoiseWUSCHEL RELATED HOMEOBOX 8 (WOX8)31585AT3G24650TurquoiseABSCISIC ACID INSENSITIVE 3 (ABI3)31156AT1G78080BrownWOUND INDUCED DEDIFFERENTIATION 1 (WIND1)21117AT5G57390TurquoiseAINTEGUMENTA-LIKE 5 (AIL5)7528AT4G37750RedAINTEGUMENTA (ANT)6889AT1G63470RedAT-HOOK MOTIF NUCLEAR LOCALIZED PROTEIN 5 (AHL5)52210AT5G65510PurpleAINTEGUMENTA-LIKE 7 (AIL7)216Figure 10
Distribution ofEMB genes across gene modules. The coloured bars represent the ratio between the number of EMB genes in each module and the total number of EMB genes in the network.In addition, we observed that 1,711Arabidopsis TFs are distributed across all the gene modules except in light-green and royal-blue modules, with the highest number of TFs present in the turquoise module (the complete list of TFs included in the GCN is given in Table S6). Notably, AP2/EREBP (APETALA2/ethylene-responsive element binding proteins), bHLH (basic helix–loop–helix), bZIP, C2H2 (Cys2-His2), HB (homeobox), NAC (NAM, ATAF, and CUC), MYB (MYB-domain), C3H, and WRKY TF families were highly represented (Figure 11(a)). Of these, members of AP2/EREBP, bHLH, C2H2, HB, NAC, MYB, and WRKY TF families were involved in early SE (Figure 11(b)). Interestingly, TFs that are targets of several microRNAs (miRNAs) were also recovered from the GCN (Table S7).Figure 11
Distribution of TFs in SE. (a) Overall distribution of TFs. The percentage is calculated as the ratio of TFs belonging to each family with respect to the total number of TFs in the network. (b) Distribution of SE-related TFs across different somatic embryo developmental stages. The percentage is calculated for each stage as the ratio of TFs present in each family with respect to the total number of TFs in the network.
(a)(b)Notably, several gene encoding epigenetic regulators were localized in network modules (Figure12). This included 14 genes involved in DNA modification, 51 genes involved in histone modification, 34 genes involved in chromatin remodeling, 15 genes encoding polycomb-group proteins, and 55 genes associated with RNA silencing (see Table S8). Each of these genes directly interacted with numerous modular genes forming a complex network.Figure 12
Distribution of genes encoding epigenetic regulators across the network modules.


## 3.1. Hierarchical Clustering of Somatic Embryo Transcriptomes

In the present study, transcriptome datasets generated through microarray experiments were retrieved from the NCBI covering four somatic embryo developmental stages (with two replicates for each stage), referred to herein as stages I (zygotic embryos), II (proliferating tissues at 7 days of induction), III (proliferating tissues at 14 days of induction), and IV (mature somatic embryos). The hierarchical clustering of samples (Figure1(a)) confirmed that the sample replicates of each stage have a higher degree of correlation with each other than with other developmental stages; sample outliers were not detected in the dataset. The clustering heatmap clearly distinguished four discrete clusters of related expression patterns corresponding to the stages of somatic embryo development (Figure 1(b)). Further, stage I showed a poor correlation with the other three stages. This suggests that stage I may have a distinct expression profile as compared to other somatic embryo developmental stages.Figure 1
Hierarchical clustering of somatic embryo transcriptomes based on their Euclidean distance using average linkage clustering (replicates of each stage are labeled as “a” and “b”). (a) Unrooted hierarchical clustering dendrogram (the length between nodes corresponds to the distance between samples). (b) Hierarchical clustering heatmap visualizing the correlations between the samples.
(a)(b)


## 3.2. Filtering of Genes for the GCN Construction and Downstream Analysis

As recommended by Langfelder and Horvath [32], genes were filtered by the variance for the GCN construction; filtering genes for variance greater than 0.25 quantile identified a total of 17,059 genes (see Table S1). This included 445 EMB genes [41], 10 SE marker genes [40], and 1,711 Arabidopsis TFs (65.3%).In addition, DEGs were identified by a pairwise ratio of expression between consecutive stages of development. A total of 2,244 genes were identified by threshold filtering based onlog2FC≥2.0 and p value <0.05. 64 EMB genes [41], four SE marker genes [40], and 458 TFs were present within the DEGs identified (see Table S2). A total of 12 genes including the genes STRESS INDUCED FACTOR 2 (AT1G51850), LIGHT-HARVESTING-LIKE 3 : 1 (AT4G17600), BETA GLUCOSIDASE 28 (AT2G44460), FERREDOXIN C 1 (AT4G14890), and PHOTOSYSTEM II SUBUNIT Q (AT4G05180) were differentially expressed throughout SE (Figure 2(a)). In addition, a considerable number of genes were up- and downregulated during early embryo developmental stages (Figure 2(b)).Figure 2
Overview of DEGs. (a) Distribution of DEGs between consecutive somatic embryo developmental stages. (b) Number of up- or downregulated DEGs between stages.
(a)(b)


## 3.3. Construction of GCN

The expression profiles of the filtered 17,059 genes were used to construct a scale-free gene expression network with a soft threshold of 15 (Figure3(a)). The dynamic hierarchical clustering approach integrated with the WGCNA pipeline distinguishes groups of genes with coexpression patterns and clusters them into network modules. In total, 26 distinct coexpression gene modules were detected with the module size ranging from 35 to 3,418 genes (Figures 3(b) and 3(c)); each module was assigned with a unique colour. The module comprising most genes was the turquoise (3,418 genes) followed by the blue (2,973 genes) and brown (2,437 genes) (Figure 3(b)). The expression profiles of coexpressed genes clustered in each module were summarized as MEs. Among the filtered genes, 13 genes that failed to fit within a distinct group were assigned to the grey module and removed from the downstream analysis. Module preservation analysis indicated high module preservation, confirming that the modules generated here can also be found in diverse independent datasets (Figure 3(b)). Each module was exported and visualized using Cytoscape.Figure 3
Construction of the draft GCN for SE. (a) Network topology for different soft-thresholding powers. (b) Module preservation statistics. (c) Hierarchical clustering dendrogram of MEs.
(a)(b)(c)


## 3.4. Identification of Stage-Related Modules

The relationships between the gene modules and different somatic embryo developmental stages were determined by assessing the Pearson correlation coefficient (r) between the MEs and developmental stages. Module-trait correlation analyses revealed that multiple modules are related to SE (Figure 4(a)). A total of 18 modules were significantly associated with the somatic embryo developmental stages (r>0.8 and p value ≤0.01; Figure 4), and these modules were “stage-specific,” i.e., the module was significantly associated with only one particular developmental stage of SE: tan, turquoise, dark-orange, and green to stage I; grey60, magenta, brown, and light-yellow to stage II; green-yellow, dark-gray, dark-green, orange, blue, light-green, and light-cyan to stage III; and pink, dark-turquoise, salmon, and yellow to stage IV. Gene significance, the correlation between modular gene expression and each stage, is shown in Figure 4(b).Figure 4
Stage-specific gene modules detected by WGCNA. (a) Module-trait relationship heatmap. Each row corresponds to a module, and each column corresponds to a stage. The degree of correlation is illustrated with the colour legend. The numbers in the table correspond to the p value. Modules that are significantly associated with each somatic embryo development stage (r>0.8 and p value ≤0.01) are indicated by an asterisk. (b) Gene significance values of coexpression modules related to different somatic embryo developmental stages.
(a)(b)


## 3.5. Functional Enrichment Analysis of “Stage-Specific” Gene Modules

GO enrichment analysis performed on “stage-specific” modules showed that the genes in green and turquoise modules which exhibited a significant association with stage I were mainly enriched in the biological processes being involved in postembryonic development, hormone-mediated signaling pathway, biosynthesis pathways (sterol and fatty acids), DNA methylation, and transcription regulation (Figure5(a)). Genes in brown, light-yellow, and magenta modules, which showed significant association with stage II, were mainly enriched in the biological processes involved in root and shoot development, ATP synthesis, response to the metal ions, and DNA replication (Figure 5(b)), whereas genes in blue and light-cyan modules, which showed significant association with stage III, were enriched for the biological processes involved in transition postembryonic and seed development, hormone- and sugar-mediated signaling pathways, cell differentiation, protein modification, and RNA processing (Figure 5(c)). Moreover, the yellow module, which showed a significant relationship to stage IV, was mainly enriched in biological processes involved in ion transport, postembryonic development, signal transduction, lipid localization, response to oxidative and water stress, as well as response to phytohormones (abscisic acid, gibberellin, cytokinin, and jasmonic acid) (Figure 5(d)).Figure 5
Functional enrichment analysis of the stage-specific modules. (a)–(d) represent the significantly enriched GO terms (p value <0.05) in modules specifically associated with stages I–IV, respectively.
(a)(b)(c)(d)


## 3.6. Analysis of Hub Genes

Hub genes are nodes in a network often hypothesized to be functionally significant due to their high degree of intramodular connectivity. A total of 260 genes (top 10 genes of each module with high connectivity) were identified as potential hub genes; the hub gene with the highest degree of connectivity in each module is given in Table1 (the complete list of hub genes is given in Table S3). GO enrichment analysis of the hub genes revealed that they are mainly enriched for biological processes such as metabolic processes (mRNA and cellular amino acid), oxidation-reduction, protein folding, and postembryonic development.Table 1
Top 10 hub genes ordered by the degree of connectivity.
Gene identifierDegree of connectivityGene moduleGene nameDescriptionAT1G271203327TurquoiseAT1G27120 (GALT4)Galactosyltransferase family proteinAT5G528202853BlueNOTCHLESS (NLE)WD-40 repeat family protein/notchless proteinAT5G560902348BrownCYTOCHROME C OXIDASE 15 (COX15)Encodes a homolog of COX15AT2G431002134YellowISOPROPYLMALATE ISOMERASE 2 (IPMI2)Isopropylmalate isomerase 2AT1G710101958GreenFORMS APLOID AND BINUCLEATE CELLS 1C (FAB1C)Encodes a protein that is predicted to act as a phosphatidylinositol-3P 5-kinase but lacks a FYVE domainAT2G29890806RedVILLIN 1 (VLN1)Encodes a ubiquitously expressed villin-like proteinAT2G45600569BlackAT2G45600Alpha/beta-hydrolases superfamily proteinAT1G72400321MagentaAT1G72400Hypothetical proteinAT3G53980311PinkAT3G53980Bifunctional inhibitor/lipid-transfer protein/seed storage 2S albumin superfamily proteinAT5G65350243PurpleHISTONE 3 11 (HTR11)Histone 3 11AT5G27560182Green-yellowAT5G27560DUF1995 domain protein, putative (DUF1995)AT5G54855170TanAT5G54855Pollen Ole e 1 allergen and extensin family proteinAT1G75630156SalmonVACUOLAR H+-PUMPING ATPASE 16 KDA PROTEOLIPID SUBUNIT 4 (AVA-P4)Vacuolar H+-pumping ATPase 16 kD proteolipid (ava-p) mRNAAT1G74450107CyanAT1G74450BPS1-like protein (DUF793)AT2G23940107Midnight-blueAT2G23940Transmembrane protein (DUF788)AT1G3046082Light-cyanCLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR 30 (CPSF30)Encodes AtCPSF30, the 30-KDa subunit of cleavage and polyadenylation specificity factorAT1G0604074Grey60SALT TOLERANCE (STO)B-box zinc finger family protein that encodes a salt tolerance proteinAT2G1156058Light-greenAT2G11560Mutator-like transposase/similar to MURA transposase of maizeAT3G5505050Dark-greenD-CLADE TYPE 2C PROTEIN PHOSPHATASE 4 (PP2C.D4)Protein phosphatase 2C family proteinATCG0107048Dark-redNAD(P)H-QUINONE OXIDOREDUCTASE SUBUNIT 4L (NDHE)NADH dehydrogenase ND4LAT3G2595048Royal-blueAT3G25950TRAM, LAG1, and CLN8 (TLC) lipid-sensing domain containing proteinAT1G6541039Dark-turquoiseATP-BINDING CASSETTE I13 (ABCI13)Encodes a member of NAP subfamily of transportersAT5G0231038Light-yellowPROTEOLYSIS 6 (PRT6)Encodes a component of the N-end rule pathway that targets protein degradationAT3G6113037Dark-greyGALACTURONOSYLTRANSFERASE 1 (GAUT1)Encodes a protein with putative galacturonosyltransferase activityAT3G5335034Dark-orangeROP INTERACTIVE PARTNER 3 (RIP3)Encodes a microtubule-binding proteinAT5G4349034OrangeAT5G43490Myb-like protein XAmong the hub genes, only 234 genes were functionally annotated; of these, 13 were TFs:AUXIN RESPONSE FACTOR 9 (ARF9), FLOWERING BHLH 4 (FBH4), BASIC HELIX-LOOP-HELIX 39 (BHLH39), BASIC LEUCINE-ZIPPER 44 (bZIP44), bZIP19, ZIM-LIKE 2 (ZML2), AT5G60820, AT4G01270, KANADI 3 (KAN3), HOMEODOMAIN GLABROUS 4 (HDG4), CELL DIVISION CYCLE 5 (CDC5), NAC DOMAIN CONTAINING PROTEIN 80 (NAC080), and SALT TOLERANCE (STO)). In addition, five genes encoding transposable elements (i.e., AT2G11560, AT3G33066, AT5G32430, AT3G42820, and AT4G28900) were identified.In silico analysis of the promoter sequences (1000 bp upstream from the transcription start site) of the hub genes using the MEME tool identified four significant motifs ranging in length from 15 to 29 bp (Table 2). Motifs 1, 2, and 3 were detected across 229, 245, and 121 hub genes, respectively. Further analysis of the predicted motifs using the GOMo tool provided in the MEME suite indicated that motifs 1 and 3 may be involved in the DNA endoreduplication, polarity specification of axial/abaxial axis, and hormone-mediated signaling pathways; motifs 1 and 3 seem to function in association to cytokinin and gibberellic acid, respectively.Table 2
Conserved motifs identified in the promoter regions of hub genes using the MEME tool.
MotifE-valueMotif widthSitesSignificant GO enriched terms(q value<0.05)1NAVAAAAAAARAAARARAAARAAAAHMAAConsensus sequence:[AGT]A[AG]AA[AC]AAAA[AG]A[AG][AG][AGC]A[AG]AA[AG][AG]A[AG]AA[ATC][AC]A[AGT]2.8e-07729229(i) GO:0042023: DNA endoreduplication(ii) GO:0009944: Polarity specification of adaxial/abaxial axis(iii) GO:0009735: Response to cytokinin stimulus(iv) GO:0009744: Response to sucrose stimulus(v) GO:0006468: Protein amino acid phosphorylation(vi) GO:0009965: Leaf morphogenesisConsensus logo:2DTTTTTKTTTTKTTYConsensus sequence:[AGT][TG]TTTT[TG]TTTT[TG][TG]T[TCG]6.0e-02015245Consensus logo:3NDRRAGDDDRRWARRRARAGAADRRDAGConsensus sequence:[AGC][GAT][AG][AG][AT][GA][AGT][ATG][AGT][GA][AG][AT]A[GA][AG][GAC]A[AG]A[GA]A[AG][GAT][AG][AG][GTA][AT][GT]3.6e-02528121(i) GO:0009944: Polarity specification of adaxial/abaxial axis(ii) GO:0048481: Ovule development(iii) GO:0010050: Vegetative phase change(iv) GO:0010051: Xylem and phloem pattern formation(v) GO:0042023: DNA endoreduplication(vi) GO:0035196: Production of miRNAs involved in gene silencing by miRNA(vii) GO:0009740: Gibberellic acid mediated signaling pathwayConsensus logo:4CMAYCTYCTCCDTCHBCATCConsensus sequence:[CT][CA]A[TC]CT[CT]CT[CT]C[TAG][TGC][CT][ATC][GTC]C[AT][TC]C5.3e-0072044Consensus logo:


## 3.7. Validation of Hub Genes

A comparison of hub genes and DEGs showed that 31 hub genes are differentially expressed in SE (the expression values of differentially expressed hub genes are given in TableS4). Further, expression analysis of these genes using the Arabidopsis eFP browser demonstrated that two hub genes, AT1G19540 (Figure 6(a)) and AT5G44380 (Figure 6(b)), exhibit a seed-specific pattern of expression.Figure 6
Expression patterns of two hub genes,AT1G19540 (a) and AT5G44380 (b), when viewed through the Arabidopsis eFP browser. The normalized expression value for each gene is colour-coded as indicated by the legend.
(a)(b)Moreover, analysis of the expression profiles of hub genes in theArabidopsis somatic embryo transcriptome dataset (E-MTAB-2465) published by Wickramasuriya and Dunwell (2015) revealed that 62 hub genes are differentially expressed in somatic embryonic tissues compared to leaf tissues (log2FC≥2.0 and p value <0.05; Figure 7). Of these, 15 genes were identified as DEGs in the present analysis. For instance, CYSTEINE-RICH TRANSMEMBRANE MODULE 7 (ATHCYSTM7/AT2G33520), HEPTAHELICAL TRANSMEMBRANE PROTEIN2 (AT4G30850), INDOLE-3-ACETIC ACID INDUCIBLE 30 (IAA30/AT3G62100), RPS9C, VASCULATURE COMPLEXITY AND CONNECTIVITY (AT2G32280), AT2G21820, AT2G38900, and AT5G43770 showed a marked expression in somatic embryonic tissues as compared to leaf tissues. Expression analysis using the Arabidopsis eFP browser further showed that AT2G29300, AT2G21820, AT2G38900, AT5G43770, ATHCYSTM7, and AT1G19540 exhibit a seed-specific pattern of gene expression.Figure 7
Transcript abundance extracted from the somatic embryo transcriptome dataset, E-MTAB-2465 for the hub genes. The hub genes significantly upregulated (log2FC≥2.0) and downregulated (log2FC≤−2.0) in somatic embryonic tissues compared to leaf tissues are indicated with yellow asterisks and red diamonds, respectively. Transcript abundances are shown in fragments per kilobase of transcript per million mapped reads (FPKM).As expected, few hub genes highly expressed in leaf tissues were repressed in somatic embryos indicating the importance of gene regulation in SE (Figure7); for instance, CELLULOSE SYNTHASE-LIKE B4 (AT2G32540), CHOLINE/ETHANOLAMINE KINASE 3 (AT4G09760), GLUTAMATE DECARBOXYLASE 2 (AT1G65960), ISOPROPYLMALATE ISOMERASE 2 (AT2G43100), PEROXIREDOXIN Q (PRXQ/AT3G26060), PHOTOSYNTHETIC NDH SUBCOMPLEX L 4 (PnsL4/AT4G39710), PLASTID RIBOSOMAL PROTEIN S20 (AT3G15190), STO (AT1G06040), SINAPOYLGLUCOSE 1 (SNG1/AT2G22990), THYLAKOID RHODANESE-LIKE (TROL/AT4G01050), TONOPLAST INTRINSIC PROTEIN 2 (TIP2/AT3G26520), AT3G50685, AT4G33666, AT5G16010, and AT5G54540 genes showed a marked repression in somatic embryos compared to leaf tissues.In summary, the present study identified a total of 78 hub genes as potential regulators of SE (Figure8), including genes showing marked overexpression as well as repression in SE. Of these, 41 genes have not been functionally annotated thus far. The analysis of the promoter sequences of these uncharacterized hub genes using the PLACE database identified a total of 215 different plant CREs; ARR1AT, CAATBOX1, CACTFTPPCA1, DOFCOREZM, GATABOX, GT1CONSENSUS, POLLEN1LELAT52, and WRKY71OS were observed in all 41 functionally uncharacterized potential hub genes. Moreover, several CREs related to embryogenesis were identified (Figure 9). The functions of the predicted CREs are included in Table 3.Figure 8
Venn diagram indicating the intersection of hub genes and DEGs (log2FC≥2.0 and p value <0.05) obtained from E-MTAB-2465 [24] and GSE48915 [37].Figure 9
The distribution of several important plant CREs present in the promoter regions of functionally uncharacterized hub genes. The number of hub genes that contain the relevant CRE in their promoter region is indicated by thex-axis. The size of the circle depicts the occurrence of the CREs within the promoter regions of the hub genes (as indicated within the circle).Table 3
Functional roles of several important CREs detected in the functionally uncharacterized hub gene promoter sequences retrieved from the PLACE database [46].
Cis-acting regulatory elementFunction∗ABADESI1“ACGT” motif; transacting factor: TAF-1; responsive to ABA and desiccation. Expressed in seeds late during embryogenesis. Induced by ABA and osmotic stress in vegetative tissues.CACGTGMOTIF“CACGTG motif”; essential for expression of beta-phaseolin gene during embryogenesisCANBNNAPACore of “(CA)n element” in storage protein genes; embryo- and endosperm-specific transcription of napin (storage protein) geneCARGNCATNoncanonical CArG motif (CC-Wx8-GG); A relevantcis-element for the response to AGL15 (AGAMOUS-like 15) in vivoDPBFCOREDCDC3DPBF-1 and 2 (Dc3 promoter-binding factor-1 and 2) binding core sequence; Dc3 expression is embryo-specific and induced by ABADRE1COREZMRAB17“DRE1” core found in maize (Z.M.) rab17 gene promoter; “DRE1” was protected, in in vivo footprinting, by a protein in embryos specifically, but in leaves, was protected when was treated with ABA and drought; rab17 is expressed during late embryogenesis and is induced by ABADRE2COREZMRAB17“DRE2”; core sequence in rab17 gene promoter. rab17 is expressed during late embryogenesis and is induced by ABAEBOXBNNAPA“E-box” of napA storage-protein genePYRIMIDINEBOXOSRAMY1AFound in the promoter of alpha-amylase (Amy2/32b) gene which is induced in the aleurone layers in response to GA in embryoRYREPEATVFLEB4RY repeat motif; quantitative seed expression; binding site ofArabidopsis B3-domain-containing transcription factor FUS3, mediates abscisic acid-induced transcriptionSEF1MOTIF“SEF1 (soybean embryo factor 1)” binding motif; regulates the expression of genes encoding for the beta-conglycinin seed storage proteinsSEF3MOTIFGM“SEF3 binding site”; regulates the expression of genes encoding for the beta-conglycinin seed storage proteinsSEF4MOTIFGM7S“SEF4 (soybean embryo factor 4)” binding motif; regulates the expression of genes encoding for the beta-conglycinin seed storage proteinsTATABOX2“TATA box”; TATA box found in beta-phaseolin promoter which is accurate transcription initiation in the embryo stage∗Details of PLACE entries were retrieved from the https://www.dna.affrc.go.jp/place/place_seq.shtml (accessed on 19th May 2022).


## 3.8. Distribution of Embryogenesis-Related Genes across Network Modules

Further exploration of genes mapped to each network module found that 10 key regulators of SE includingLEC1, FUSCA3 (FUS3), and ABSCISIC ACID INSENSITIVE 3 (ABI3) are present among the highly connected genes in the network (Table 4); SE-related marker genes, LEC2, SERK1, WUS, BBM, and WUSCHEL RELATED HOMEOBOX 2 (WOX2) showed low variance in the present dataset and thus were not included in the GCN analysis. We also observed that the majority of previously published EMB genes [41] are localized to the blue and turquoise modules, which showed significant association with stage I and stage III, respectively (Figure 10; see Table S5).Table 4
Distribution of SE markers across network modules ordered by the number of interactors.
Gene identifierModuleGene nameNo. of interactors1AT3G26790TurquoiseFUSCA3 (FUS3)33462AT5G13790TurquoiseAGAMOUS-LIKE 15 (AGL15)33083AT1G21970TurquoiseLEAFY COTYLEDON 1 (LEC1)32974AT5G45980TurquoiseWUSCHEL RELATED HOMEOBOX 8 (WOX8)31585AT3G24650TurquoiseABSCISIC ACID INSENSITIVE 3 (ABI3)31156AT1G78080BrownWOUND INDUCED DEDIFFERENTIATION 1 (WIND1)21117AT5G57390TurquoiseAINTEGUMENTA-LIKE 5 (AIL5)7528AT4G37750RedAINTEGUMENTA (ANT)6889AT1G63470RedAT-HOOK MOTIF NUCLEAR LOCALIZED PROTEIN 5 (AHL5)52210AT5G65510PurpleAINTEGUMENTA-LIKE 7 (AIL7)216Figure 10
Distribution ofEMB genes across gene modules. The coloured bars represent the ratio between the number of EMB genes in each module and the total number of EMB genes in the network.In addition, we observed that 1,711Arabidopsis TFs are distributed across all the gene modules except in light-green and royal-blue modules, with the highest number of TFs present in the turquoise module (the complete list of TFs included in the GCN is given in Table S6). Notably, AP2/EREBP (APETALA2/ethylene-responsive element binding proteins), bHLH (basic helix–loop–helix), bZIP, C2H2 (Cys2-His2), HB (homeobox), NAC (NAM, ATAF, and CUC), MYB (MYB-domain), C3H, and WRKY TF families were highly represented (Figure 11(a)). Of these, members of AP2/EREBP, bHLH, C2H2, HB, NAC, MYB, and WRKY TF families were involved in early SE (Figure 11(b)). Interestingly, TFs that are targets of several microRNAs (miRNAs) were also recovered from the GCN (Table S7).Figure 11
Distribution of TFs in SE. (a) Overall distribution of TFs. The percentage is calculated as the ratio of TFs belonging to each family with respect to the total number of TFs in the network. (b) Distribution of SE-related TFs across different somatic embryo developmental stages. The percentage is calculated for each stage as the ratio of TFs present in each family with respect to the total number of TFs in the network.
(a)(b)Notably, several gene encoding epigenetic regulators were localized in network modules (Figure12). This included 14 genes involved in DNA modification, 51 genes involved in histone modification, 34 genes involved in chromatin remodeling, 15 genes encoding polycomb-group proteins, and 55 genes associated with RNA silencing (see Table S8). Each of these genes directly interacted with numerous modular genes forming a complex network.Figure 12
Distribution of genes encoding epigenetic regulators across the network modules.


## 4. Discussion

Plant embryogenesis is a meticulous developmental process that requires the regulation of multiple genes. A GCN will serve as a map of statistically significant gene interactions that helps in narrowing down the transcriptome to the potential gene interactions involved in biological processes. Recently, Clercq et al. report an integrated gene regulatory network forArabidopsis covering TFs and target genes [47]. In the present study, WGCNA was employed to explore potential clusters of highly coregulated genes and hub genes associated with SE. Although WGCNA has been previously applied to construct a GCN for Arabidopsis zygotic embryogenesis (ZE) [33], to the best of our knowledge, this is the first report on the use of WGCNA to construct a GCN for Arabidopsis SE and to explore SE-related network modules and hub genes. The findings of this study provide new insights into the molecular mechanism of SE in plants.The GCN constructed for SE comprised of 26 network modules: black (674 genes), blue (2,973 genes), brown (2,437 genes), cyan (125 genes), dark-green (52 genes), dark-grey (39 genes), dark-orange (35 genes), dark-red (54 genes), dark-turquoise (52 genes), green (2,132 genes), green-yellow (189 genes), grey60 (79 genes), light-cyan (86 genes), light-green (59 genes), light-yellow (58 genes), magenta (338 genes), midnight-blue (117 genes), orange (35 genes), pink (357 genes), purple (271 genes), red (853 genes), royal-blue (56 genes), salmon (162 genes), tan (172 genes), turquoise (3,418 genes), and yellow (2,223 genes) modules. Among them, 18 modules showed strong associations with different stages of SE; module-trait relationship analysis revealed that four, four, seven, and four modules were significantly correlated with stages I, II, III, and IV of SE, respectively. This suggests that SE involves complex genetic networks.Functional enrichment analysis using GO is one of the most widely used bioinformatic methods to classify genes into functionally related groups [48–50]. GO analysis of the coexpressed gene clusters (or network modules) showed that the initial stages of SE were mainly enriched with biological processes such as hormone-mediated signaling, biosynthesis pathways, ATP synthesis, DNA methylation, and replication. Notably, genes involved in lipid transport, postembryonic development, signal transduction, and seed dormancy were enriched in later stages of SE; this indicates the developmental shift in the maturation phase with the accumulation of embryo-specific food reserves, a process that aids in withstanding dormancy and postembryonic development [2, 10, 51]. Furthermore, genes related to stress responses (e.g., oxidative and water stress), phytohormones (e.g., cytokinin, abscisic acid, gibberellin, and jasmonic acid), and metabolic processes were enriched in all stages of somatic embryo development studied, from the initiation to maturation stage. These findings further confirmed the importance of cell-cell interactions [52], signaling [9, 13, 53], and transcriptional activation of stress responses [54, 55] during plant SE.High-degree nodes or the genes with high network connectivity in GCN modules (“hub genes”) may have important biological functions [36, 56–58]; often, they may serve as biological markers. Several studies have successfully employed WGCNA to mine hub genes controlling biological processes [34, 59–62]. The present study reports 260 potential hub genes related to SE based on the degree of connectivity. These genes may play pivotal roles in the regulation of SE. Importantly, 13 TFs encoded by hub genes were identified in the coexpression network. They were ARF9, NAC080, ZML2, bHLH39, KAN3, bZIP19, bZIP44, HDG4, FBH4, STO, CDC5, AT5G60820, and AT4G01270; functional roles of many of these genes in the regulation of SE are not reported. Previous studies have reported that ARF9 represses the expression of its target genes such as TOPLESS (TPL) and TPL-related proteins [63, 64]. Wójcikowska and Gaj observed stable expression of ARF9 during SE [65]. In addition, KAN3, a member of GARP TF family, has also exhibited an embryonic expression pattern.In addition,ROOT UV-B SENSITIVE 6 (RUS6; AT5G49820), which encodes a DUF647 (DOMAIN OF UNKNOWN FUNCTION 647) containing protein, an ankyrin repeat-containing gene designated as AT5G65860 and a gene that encodes hydroxyproline-O-glycosyltransferases (Hyp-O-GALT), GALT4 (AT1G27120)), was also identified as hub genes in the coexpression network. The members of the RUS gene family play diverse roles in plant development [66]. Interestingly, knockout mutants of RUS6 have shown a strong embryo-lethal phenotype. In Arabidopsis, ankyrin repeat-containing proteins have been classified into 16 groups [67], and of these, proteins with only ankyrin repeats have been associated with disease resistance, antioxidation, embryogenesis, and development [68–70]. For instance, T-DNA mutants of the EMB 506 gene, which encodes a protein containing five ankyrin repeats, have shown defective embryo development at the globular-to-heart stage transition [70]. Moreover, Hyp-O-GALT enzymes are responsible for hydroxyproline glycosylation of arabinogalactan proteins, which are known to function in various aspects of plant growth and development including SE [71–73]. Although the hub genes identified in the present study are implicated to function in many plant developmental processes, the functions of many of the hub genes in SE remain to be elucidated. Hence, these genes could be potential targets for functional studies in the future.Promoter analysis of the functionally uncharacterized hub genes using the PLACE database revealed the overrepresentation of two motifs in many of the promoter regions. These were EBOXBNNAPA (consensus sequence: CANNTG) and SEF4MOTIFGM7S (consensus sequence: [A/G]TTTTT[A/G]). Of these, EBOXBNNAPA (“E-box” motif) is a CRE found in the regulatory region of the napin gene,napA in Brassica napus [74]; this gene encodes a storage protein. Moreover, CANNTG provides the binding site for bHLH TFs [75]. bHLH is one of the most frequently represented gene families in DEGs in ZE [76] and SE and is known to have diverse functions in plants [24] including cell proliferation [75]. The recognition sequence of SEF4MOTIFGM7S motif is known to interact with SEF3, a protein expressed in immature soybean seeds that acts as a transcriptional activator of the β-conglycinin α subunit gene [77]. Hence, the uncharacterized hub genes that showed considerable expression in embryonic tissues are more likely to play a significant role in plant embryo development.Differential gene expression analysis of hub genes revealed that 78 genes could be considered as potential regulators of SE; of these, 15 genes were differentially expressed in transcriptome datasets derived from two independent studies related to SE [24, 37]. One of the genes identified was IAA30, which is a member of one of the families of auxin signaling proteins (Aux/IAA; [78]). iaa30 mutants have displayed significantly impaired SE efficiency, producing fewer somatic embryos per explant [76] and suggesting its role in the initiation phase of SE. Moreover, IAA30 is a target of two important SE marker genes, LEC2 and AGL15 [79, 80]. In addition, two hub genes, AT1G19540 and AT5G44380, showed a marked expression in seed development, suggesting their roles in embryogenesis.To enhance our understanding of the regulatory mechanism of SE, the distribution of embryogenesis-related genes across the gene modules was examined. Horstman et al. report LEC1–LEC2–FUS3–BBM–ABI3 network to induce SE inArabidopsis [81]. Moreover, Zheng et al. suggest a MADS-domain TF encoding gene, and AGL15 may associate with LEC2, FUS3, and ABI3 during SE [82]. However, a recent study has found that AGL15 is not essential to promote SE [83]. In the present analysis, 10 key regulators of SE including LEC1, ABI3, FUS3, AGL15, and three members of the AINTEGUMENTA-LIKE/PLETHORA (AIL/PLT) subfamily (ANT, AIL5, and AIL7) were identified in the coexpression network. Consistent with previous literature, members of the AP2/EREBP, bHLH, bZIP, MYB, HB, WRKY, NAC, C3H, and C2H2 TF families were overrepresented in the GCN [76, 84]. In addition, members of the TF families (i.e., SPB (SQUAMOSA promoter binding protein-like), GRAS (GRAS-domain), trihelix, G2-like, and CAMTA (CALMODULIN BINDING TRANSCRIPTION ACTIVATOR 3)) that are not or to a lesser extent reported to be involved in SE were identified. The members of GRAS, trihelix, and CAMTA families are known to be involved in the regulation of stress responses [47, 85, 86].Further, it is reported that miRNAs (e.g., miR156, miR159, miR162, miR164, miR166, miR167, miR169, miR168, miR171, miR319, miR393, and miR396) play an important role in SE [87–91]. Consistent with previous studies, several TFs targeted by miRNAs were recovered from the SE-related GCN. This included seven miR156/157 targeting genes of the SPB TF family, seven miR169 targeting genes of the CCAAT TF family, six miR396 targeting genes of the GRF TF family, five miR166/miR165 targeting genes of the HB TF family, five miR164 targeting genes of the NAC family, and five miR159/miR319 targeting genes of the TCP TF family. These miRNA-targeted TF encoding genes may play a significant role in the regulation of SE responses.Recent studies have uncovered critical roles of epigenetic modifications in the regulation of SE, in particular, DNA methylation/demethylation [92–94] and histone modifications [91, 95, 96]. Recently, an expression study on Arabidopsis embryos at single-cell resolution has provided evidence for distinct expression patterns for many epigenetic regulators across embryonic tissues [97]. Our coexpression network also revealed that many genes encoding epigenetic regulators such as METHYLTRANSFERASE 1 (MET1), CHROMOMETHYLASE 3 (CMT3), DEMETER (DME), DEMETER-LIKE (DML1,-2), histone acetyltransferases (HISTONE ACETYLTRANSFERASE OF THE CBP FAMILY (HAC1,-4,-5,-12), histone deacetylases (i.e., HISTONE DEACETYLASE (HDA1,-2,-3,-5,-6,-8,-9,-14,-15,-17), and histone demethylases (JUMONJI DOMAIN-CONTAINING PROTEIN 16 (JMJ14,-16,-22,-27,-29) were coexpressed with key genes involved in the regulation of SE.The present study showed that the WGCNA pipeline could be used to identify biologically relevant modules of SE. However, our analysis has some limitations. The main limitations were the small sample size used in the analysis and the lack of an independent dataset to replicate the findings. Langfelder and Horvth [32] recommend using at least 15 samples to construct robust networks. However, high-quality, clean data could also result in biologically meaningful networks even with <15 samples. Therefore, further experiments are recommended to validate the hub genes discovered in the present study. Furthermore, the GCN built in the present study was based on microarray gene expression data. Although hybridization-based gene expression profiling approaches are high-throughput and relatively inexpensive, they have a number of limitations; most importantly, they provide only an indirect measure of the level of gene expression and can only be used to study the expression levels of genes that the arrays are designed to detect and are subjected to cross-hybridization biases [98]. Given the limitations of this approach, it would be recommended to perform a GCN analysis employing an expression dataset generated through high-throughput transcriptome sequencing (RNA-seq) with an appropriate number of replicates. Unlike microarrays, RNA-seq is not dependent on prior knowledge about the genome sequence and has higher sensitivity to genes expressed either at a low or very high level and also has higher levels of reproducibility than microarrays [99]. Therefore, it could generate a more suitable dataset for GCN analysis.


## 5. Conclusion

In this study, a GCN was successfully constructed for SE employing WGCNA. Gene modules and hub genes related toArabidopsis somatic embryo development were successfully mined based on their statistical significance. The findings reported here provide a unique resource to advance the regulation of SE at the molecular level.



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*Source: 7471063-2022-07-04.xml*