Genome-Wide Identification and Analysis of the WRKY Gene Family in Asparagus officinalis

Abstract

In recent years, the related research of the WRKY gene family has been gradually promoted, which is mainly reflected in the aspects of environmental stress and hormone response. However, to make the study of the WRKY gene family more complete, we also need to focus on the whole-genome analysis and identification of the family. In previous studies, the whole WRKY gene family of Arabidopsis, legumes and other plants has been thoroughly studied. However, since the publication of Asparagus officinalis genome-wide data, there has never been an analysis of the whole WRKY gene family. To understand more broadly the function of the WRKY gene family, the whole genome and salt stress transcriptome data of asparagus were used for comprehensive analysis in this study, including WRKY gene family identification, phylogenetic tree construction, analysis of conserved mods and gene domains, extraction of cis-acting elements, intron/exon analysis, species collinearity analysis, and WRKY expression analysis under salt stress. The results showed that a total of 70 genes were selected and randomly distributed on 10 chromosomes and one undefined chromosome. According to the functional classification of Arabidopsis thaliana, the WRKY family of asparagus was divided into 11 subgroups (C1–C9, U1, U2). It is worth considering that the distribution rules of gene-conserved motifs, gene domains and introns/exons in the same subfamily are similar, which suggests that genes in the same subfamily may regulate similar physiological processes. In this study, 11 cis-acting elements of WRKY family were selected, among which auxin, gibberellin, abscisic acid, salicylic acid and other hormone-regulated induction elements were involved. In addition, environmental stress (such as drought stress and low-temperature response) also accounted for a large proportion. Interestingly, we analyzed a total of two tandem duplicate genes and 13 segmental duplication genes, suggesting that this is related to the amplification of the WRKY gene family. Transcriptome data analysis showed that WRKY family genes could regulate plant growth and development by up-regulating and down-regulating gene expression under salt stress. Volcanic maps showed that 3 and 15 AoWRKY genes were significantly up-regulated or down-regulated in NI&NI+S and AMF&AMF+S, respectively. These results provide a new way to analyze the evolution and function of the WRKY gene family, and can provide a reference for the production and research of asparagus.

1. Introduction

Transcription factors (TFs), proteins that regulate downstream genes by binding to specific DNA, play an important role in plant growth and development [1].

As one of the largest transcription factor families, the functions of the WRKY family in environmental stress and hormone response have been thoroughly studied [2]. WRKY TFs are divided into three groups based on the number of WRKY domains and the difference in zinc finger structure: Group 1 contains two WRKY domains and one C₂H₂ zinc finger motif. Unlike group 1, group 2 lacks one WRKY domain. Group 3, like group 2, contains a WRKY domain and a zinc finger motif; however, this zinc finger structure is C₂HC. It is also worth noting that group 2 is subdivided into five subfamilies [3]. Both WRKY amino acid domains and zinc finger motifs contain DNA-binding domains that can specifically recognize W-box (TTGACT/C) sequences. The binding of the WRKY domain to the DNA domain after mutation was significantly reduced. Interestingly, WRKY proteins can also recognize domains such as PRE4 (TGCGCTT), WT-box (GGACTTTC), and WK-box (TTTTCCAC) [2,3,4,5].

Due to the frequent exposure to various biological and abiotic stresses, such as salt stress, drought stress, pests and diseases, plants have evolved molecular mechanisms to prevent and resist stress, among which WRKY family is an important part of the resistance to environmental stress. A WRKY transcription factor named SPF1 (Sweet potato factor 1) was discovered in Ipomoea batatas more than 20 years ago [6]. Subsequently, ABF1 (ABRE binding factor 1) and ABF2 (ABRE binding factor 2) involved in seed germination were found in Avena sativ [7]. After extensive research, the researchers identified the WRKY family in several species, including A.thaliana (74 WRKY genes), Oryza sativa (102 WRKY genes), and Solanum lycopersicum (81 WRKY genes) [8,9,10]. Under drought stress, AtWRKY53 in A. thaliana controls stomatal opening and closing by binding Qua-Quine Starch promoter, thus affecting drought tolerance [11]. The overexpression of WRKY11 in rice can enhance plant heat resistance [12]. The WRKY family mainly regulates plant stress processes. However, different studies have found that the WRKY family is also involved in regulating pollen development, and fruit ripening [13,14]. The study of FUSCA3 in A. thaliana showed that AtWRKY TFs can activate FUSCA3 by regulating W-box, thus participating in seed dormancy process [15]. AtWRKY44 (TRANSPARENT TESTA GLABRA2) is the first and so far only WRKY TF that can induce trichoid development [16,17]. Interestingly, in the vernalization pathway of plants, AtWRKY can promote the accumulation of CULLIN3A promoter to activate the expression of flowering genes. However, WRKY can also inhibit flowering through the non-vernalization pathway in the latest investigation, indicating that WRKY has a dual role in the induction of flowering [18,19]. In summary, the WRKY transcription factor family regulates the plant life process from multiple dimensions. As a perennial herb, asparagus is cultivated all over the world, especially in Western developed countries. As the largest producer and exporter of asparagus, China’s asparagus planting area accounts for more than 2/5 of the world, which makes asparagus also have a certain economic value [20]. A. officinalis is best known for its medicinal properties in addition to being a food ingredient. Flavonoids, as the main components of asparagus, have anti-inflammatory, antibacterial and antioxidant effects. The leading anti-inflammatory ingredient in flavonoids is rutin, and it has also been suggested to relieve diabetes and arthritis, as well as liver disease, which is also worth mentioning [21,22,23,24,25]. The categories of asparagus can be easily classified according to color, such as green asparagus, white asparagus, etc. [26,27]. According to the type of asparagus, the global production of asparagus in 2021 reached 8.5 million metric tons [28]. The asparagus genome was published in 2017 and includes 10 chromosomes with a total length of 1187.54 Mb [29]. Salinity is an important factor affecting the physical and chemical properties of land, and its imbalance will aggravate the soil environmental stress in arid areas, resulting in crop yield reduction [30]. Quantitative analysis results show that the global saline-alkali land area is more than 424,291.05 square kilometers [31]. Therefore, it is urgent to increase the efforts to cultivate salt-resistant crops. Previous studies have shown that asparagus has a strong salt tolerance and can survive in saline soil with a concentration below 0.3% [20]. Previous studies focused on the edible and medicinal value of asparagus and rarely involved genome-wide analysis. Since the genomic information has been published, we combined the genomic information to identify the WRKY transcription factor family in order to provide a basis for the study of asparagus and WRKY in other plants. In the following studies, we conducted a comprehensive bioinformation analysis of the asparagus WRKY family, including the identification and screening of gene families, comparison of phylogenetic relationships with A. thaliana, analysis of gene structure and conserved motif, analysis of cis-acting elements, collinearity analysis within and between species, and WRKY expression under salt stress. The purpose and significance of this study is to deepen the understanding of WRKY family and explore the regulatory network of WRKY gene in asparagus, so as to provide a certain basis for salt stress resistance and the improvement of asparagus yield.

2. Materials and Methods

2.1. Identifification of the WRKY Gene Family in A. offificinalis

The A. offificinalis genome sequence file, annotation file, protein sequence and coding sequence (CDS) used in this paper were obtained from the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/, accessed on 20 August 2022). The Hidden Markov Model (HMM) file of the WRKY domain (PF03106) was obtained from the Pfam database (release 35.0; http://pfam.xfam.org/, accessed on 20 August 2022) [32]. The candidate members of A. offificinalis with the WRKY protein domain were screened using the HMMMER search (Version 3.2.1) method in two steps. First, the HMM profile in the HMMER software is used to search for and compare target members that contain the WRKY protein domain (Table S1). And in Linux, MAFFT v7.505 was used for multiple sequence alignment of candidates. Second, to avoid missing the target protein, candidate proteins with e-value < 1 × 10⁻²⁰ in the first screening were selected to reconstruct the HMM model using HMMER (version 3.2.1) [33]. The new HMM model was used to search the protein sequence with an e-value < 0.05. We considered the intersection of the two results as the final filter result. Further, we verified the existence of the WRKY domain in candidate proteins on the online website of the SMART program (http://smart.embl-heidelberg.de/, accessed on 23 August 2022), NCBI Conserved Domain Search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 23 August 2022), and Pfam Batch Sequence Search (http://pfam.xfam.org/search#tabview=tab1, accessed on 23 August 2022), and determined the final protein sequence after screening and removing redundancy [34].

2.2. Phylogenetic Analysis and Classification of AoWRKY Genes

To explore the phylogenetic relationships and taxonomy of WRKY Genes, we established a rooted neighbor-joining (NJ) phylogenetic tree between A. offificinalis (AoWRKY) and A. thaliana (AtWRKY) in MEGA11 software (version 11.0.11). The AtWRKY protein sequences from TAIR (https://www.Arabidopsis.org, accessed on 25 August 2022) were reported by Eulgem [2]. The AoWRKY family members and AtWRKY family members are aligned under the initial parameters using ClustalW in MEGA11 software. The neighbor-joining (NJ) method was adopted and 1000 replications were used for the bootstrap method, Poisson model, and pairwise deletion [35,36]. Finally, The phylogenetic tree of AoWRKY genes was glorified with online software iTOL (http://itol.embl.de/, accessed on 11 September 2022) and Adobe Illustrator 2020 software (version 24.0.1.341).

2.3. Gene Structure, Cis-Acting Regulatory and Conserved Motif Analysis of AoWRKY Genes

The online programs MEME (https://meme-suite.org/meme/tools/meme, accessed on 12 September 2022) and PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 12 September 2022) were used to analyze the conserved motif and cis-acting regulatory of the AoWRKY family, respectively. The following parameters were set for conservative motif analysis: Maximum Number of Motifs 15, Motif E-value Threshold no limit, Minimum Motif Width 6, Maximum Motif Width 50, Minimum Sites per Motif 2, and Maximum Sites per Motif 70. At the same time, the structural information of AoWRKY genes was extracted from GFF files, and the above results were visualized in the same image with TBtools software [3].

2.4. Chromosome Localization, Duplication and Synteny Analysis

The chromosome length and location information of the WRKY genes were obtained from NCBI. TBtools (v1.0987671) software was used to predict and locate the location of AoWRKY genes. Theoretical isoelectric point (pI), molecular weight (MW), amino acid numbers (aa) instability index, and aliphatic index were analyzed in ProtParam in Expasy (https://web.expasy.org/protparam/, accessed on 3 September 2022) and DNAMAN software (version 9.0). The open reading frame (ORF) lengths were searched in the ORFfinder website (https://www.ncbi.nlm.nih.gov/orffifinder, accessed on 13 September 2022) and the subcellular localization of the AoWRKY genes was confirmed in the BUSCA program (https://busca.biocomp.unibo.it, accessed on 13 September 2022) [37,38].

2.5. Analysis of AoWRKY Gene Expression Profiles under Different Conditions

AoWRKY expression levels under different salt stress conditions were experimentally treated as follows: (1) non-inoculated A. officinalis plants without salinity stress (NI); (2) inoculated A. officinalis plants without salinity stress (arbuscular mycorrhiza fungi, AMF); (3) non-inoculated A. officinalis plants subjected to salinity stress (NI + S); and (4) inoculated A. officinalis plants subjected to salinity stress (AMF + S) [39]. Sequence read archives (SRAs) were retrieved from the National Centre for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/sra/?term=SRP188664#, accessed on 14 September 2022). FASTQ files generated the pair-end data containing forward and reverse reads from SRA files. FastQC and MultiQC are used for data inspection [40,41]. We filtered low-quality fragments and process adapters through Trimmomatic [42]. We constructed a genome index file using Hisat2 and compared reads to the reference genome [43]. The DEseq2 package in the R language is used to screen differentially expressed genes [44]. Screening of differentially expressed genes was carried out at a significant adjusted p-value (p.adj) < 0.05 and an absolute value of log2FC (log of fold change) > 1 to filter out insignificantly expressed genes. The volcano plot was created by using differential expression data from ggplot. Differentially expressed AoWRKY genes were labeled in the volcano plot. The heatmap of the differentially expressed genes was constructed by the pheatmap package.

3. Results

3.1. Identification of AoWRKY Genes in A. offificinalis

After two filters by HMMMER search (Version 3.2.1), we obtained a total of 96 target genes. These 96 candidate genes were submitted to the online site of the SMART program, NCBI Conserved Domain Search, and Pfam Batch Sequence Search for the identification of WRKY domains. A total 70 final target genes were identified through domain identification and the removal of repeats. Chromosomal mapping of the target gene was performed using TBtools, which named the gene AoWRKY1-70 based on its location on the chromosome. The basic physical and chemical properties of target genes are shown in Table 1. AoWRKY52 had the smallest amino acid number (aa) and molecular weight (114aa, 12,996.32 Da). In contrast, the protein sequence with the largest amino acid number and molecular weight (MW) was AoWRKY32 (674aa and 72,655.57 Da). In addition, the theoretical isoelectric point (pI) and MW of AoWRKY4 and AoWRKY34 could not be predicted, and the pI of the remaining AoWRKY genes ranged from 4.66 (AoWRKY29) to 10.24 (AoWRKY38). Among all the protein sequences, 29 were acidic (pI < 6.5), 33 were alkaline (pI > 7.5), and six were neutral (pI = 6.5–7.5). Instability Index values above 40 are labelled as structural instability. In the AoWRKY sequence, only AoWRKY11 belongs to the stable category, and the others are all unstable. Subcellular localization prediction of the AoWRKY family revealed that only AoWRKY44 was located in the chloroplast, 47 were located in the nucleus, and 22 in extracellular space.

3.2. Systematic Classification Analysis of AoWRKY and AtWRKY

A. thaliana has been well studied as a model plant. Therefore, the Arabidopsis WRKY family was selected as an outgroup to construct a new evolutionary tree. To analyze the classification and function of the AoWRKY gene family (Table S2), MEGA11 software was used to perform multiple sequence alignments between 75 AtWRKY genes of Arabidopsis and 70 AoWRKY genes. In addition, the results were visualized by constructing a rooted neighbor-joining phylogenetic tree (Figure 1). The evolutionary tree shows that members classified into 7 subfamilies (I, II-a, II-b, II-c, II-d, II-e, III) in Arabidopsis have been more finely divided into 11 subfamilies(C1–C9, U1, U2) in Asparagus [2]. Subgroups I and II-c are subdivided into subgroups C1 & C9, C4 & C7. However, the U1–U2 subgroups included only the members of A. offificinalis (AoWRKY17, AoWRKY62, AoWRKY63). This suggested that these three genes may have mutated or evolved. It is worth noting that genes grouped into the same subfamily may regulate the same life process. For instance, AtWRKY14, AtWRKY16, AtWRKY35 and AtWRKY69 in C5 subgroups were involved in the pression of plant thermomorphogenesis, while AtWRKY28, AtWRKY50, AtWRKY51 and AtWRKY71 in C4 subgroups were responsible for inducing the biosynthesis of different hormones(TAIR, https://www.arabidopsis.org/, accessed on 15 September 2022). Interestingly, although the number of members contained in each subfamily was different (the number of subfamily members ranged from 1 to 32), the ratio of AoWRKY to AtWRKY in each subfamily was almost 1:1, indicating that the evolution of WRKY gene families in the two plants was relatively conservative, and the genetic relationship was relatively close.

3.3. Chromosome Localization and Cis-Acting Regulatory Analysis of AoWRKY

The AoWRKY family was named AoWRKY1-70 based on chromosome length information and AoWRKY location information from the genome annotation file of A. offificinalis, while the results were visualized in TBtools (Figure 2). The figure shows 70 AoWRKY genes randomly distributed across 10 chromosomes and one undefined chromosome. We subsequently found that chromosomes 1, 2, 5, 7, and 8 contain most members of the AoWRKY family (84.3%), whereas only one gene is located on chromosomes 9, 10, and Un.

There is a variety of cis-acting elements around 2000 bp upstream of the transcription start site, which regulate gene expression mainly by binding to transcription factors. The AoWRKY family 2000 bp promoter sequence was submitted in the PlantCARE online program, and the results are shown in Supplementary Figure S1. The prediction results of cis-acting elements after screening showed that 11 cis-acting elements were distributed in 70 AoWRKY genes, including 294 the MeJA (Methyl Jasmonate) responsiveness elements (distributed in 57 AoWRKY), 60 salicylic acid responsiveness elements (distributed in 41 AoWRKY), 129 anaerobic induction elements (distributed in 53 AoWRKY), 232 abscisic acid responsiveness elements (distributed in 63 AoWRKY), 29 defense and stress responsiveness elements (distributed in 24 AoWRKY), 51 gibberellin-responsive elements (distributed in 30 AoWRKY), 41 low-temperature responsiveness elements (distributed in 26 AoWRKY), 124 light-responsive elements (distributed in 57 AoWRKY), 25 auxin-responsive elements (distributed in 20 AoWRKY), 54 MYB-binding sites involved in drought inducibility (distributed in 41 AoWRKY), and six wound-responsive elements (distributed in 6 AoWRKY).It can be concluded that the AoWRKY family mainly regulated plant hormone response and abiotic stress, so as to provided reference for the functional study of the AoWRKY family.

3.4. Gene Structure and Conserved Motif Analysis of AoWRKY Genes

In order to explore the function of the AoWRKY gene family in detail, we submitted the data of the evolutionary lineage, conserved motif, gene domains, and intron/exon structure of the AoWRKY family to the Gene Structure View of TBtools (Figure 3). We found that the evolutionary relationship that differed from Figure 1 was two genes in the C9 group in Figure 3A (AoWRKY8, AoWRKY52), which is not classified in the same subgroup, but in a separate group. This phenomenon indicates that although the two genes have high homology with AtWRKY19 in C9, they have a relatively low homology with each other.

The number of conserved motifs in group C1 was the highest (8), while the number of conserved motifs in group C9 was the lowest (2). Analysis of conserved motifs showed that AoWRKY genes in the same subfamily contained almost the same conserved motifs, which meant that they participated in similar regulatory processes. Motif1 and motif2 are found in almost all AoWRKY families, while motif15 is only found in the C5 subfamily (Figure 3B).

Seven conserved domains exist in the AoWRKY family, and WRKY domains exist in the entire AoWRKY family (Figure 3C). C5, C7, C8, U1, and U2 only have WRKY domains, but C6 also contains plant_zn_clust domains. It is worth noting that AoWRKY59 is in C1 and AoWRKY29 is in C4. The AoWRKY61 in C3 has only the WRKY superfamily domain.

Figure 3D shows that each gene of AoWRKY family contains more introns, especially the C1, C2, C3, and C8 subfamilies, which provides a powerful entry point for us to better understand the gene structure.

3.5. Tandem Gene Duplication and Segmental Gene Duplication of AoWRKY Genes

Gene duplication includes whole-genome duplication, segmental duplication and tandem duplication [3]. We imported A. offificinalis genome data and the genome annotation file into the TBtools plugin MCScanX, which enabled us to obtain collinear information and gene duplication information of the A. offificinalis genome(Figure 4). Two tandem duplication gene pairs were derived from the AoWRKY family, namely AoWRKY47 and AoWRKY48 in the C4 subfamily from Chromosome 7, and AoWRKY64 and AoWRKY65 in the C8 subfamily from chromosome 8. To more intuitively display the evolutionary information of two pairs of tandem duplication genes, we calculated their Ka/Ks values (Table 2). We think that when Ka/Ks < 1, the gene evolution process has a negative selection effect, which means that the gene is subject to purification selection. In the segmental gene duplication phase, 1547 pairs were derived, of which only 13 pairs of segmental duplication genes located in Chr1, Chr3, Chr4, Chr5, Chr7, Chr8, and Chr10 were predicted by the AoWRKY family.

Not only did genetic evolution occur within A. offificinalis species, we also analyzed the homologous gene pairs of A. offificinalis with A. thaliala (11 orthologous gene pairs), Populus trichocarpa (58 orthologous gene pairs), Sesamum indicum (38 orthologous gene pairs), and Ananas comosus (31 orthologous gene pairs) in an attempt to understand the evolutionary relationships between species (Figure 5). The homologous gene pairs between A. offificinalis and the other four species are shown in the Supplementary Tables S3–S6. Remarkably, there are four homologous gene pairs shared between the five species. In addition, while there were 14 pairs of homologous gene pairs in A. offificinalis, Sesamum indicum (S. indicum), Ananas comosus (A.comosus) and Populus trichocarpa (P.trichocarpa), there were only two pairs in A. offificinalis, S. indicum and A. comosus (Figure 6).

3.6. Analysis of AoWRKY Gene Expression under Different Salt Stress

The heat map (Figure 7A,B) and the volcano plot (Figure 7C,D) were drawn in R language (R-4.3.1) editor Rstudio based on the existing RNA-seq data (NCBI). In the experiment with salinity as the only variable, there were 46 differentially expressed genes in the NI and NI + S groups. Obviously, the expression levels of 14 AoWRKY genes (AoWRKY4, AoWRKY7, AoWRKY11, AoWRK12, AoWRKY15, AoWRKY32, AoWRKY33, AoWRKY34, AoWRK35, AoWRKY36, AoWRKY40, AoWRKY44, AoWRKY49, and AoWRKY70) were higher under salt stress (NI + S). In the NI group, the expression levels of eight AoWRKY genes (AoWRKY1, AoWRK3, AoWRKY6, AoWRKY13, AoWRKY14, AoWRKY25, AoWRKY27, and AoWRKY30) were higher. Moreover, we found that the highly expressed genes in the two groups were completely different; so, we concluded that the mechanisms and functions of AoWRKY family in response to salt stress may be quite different. Compared with the salt-stress-only group (NI + S), AoWRKY11/12 in the group (NI) was significantly down-regulated (p.adj < 0.05, log2FC > 1), while AoWRKY30 was significantly up-regulated (p.adj < 0.05, log2FC < −1), which was consistent with the expression pattern in Figure 7A,C [45]. Similar to the analysis of NI and NI+S groups, we found that 11 AoWRKY genes (AoWRKY1, AoWRKY9, AoWRKY13, AoWRKY14, AoWRKY17, AoWRKY27, AoWRKY30, AoWRKY41, AoWRK51, AoWRKY64, AoWRKY69) were highly expressed in the AMF group, while 12 genes (AoWRKY2, AoWRKY11, AoWRKY15, AoWRKY16, AoWRKY18, AoWRKY21, AoWRKY31, AoWRKY43, AoWRK46, AoWRKY49, AoWRKY50, and AoWRKY68) were highly expressed in the AMF + S group, and the high-expression genes in the two groups were also not overlapping. In addition, compared with the AMF + S group, six genes in the AMF group were significantly up-regulated and nine genes were significantly down-regulated (Figure 7D).

4. Discussion

Asparagus, a lily family, has high edible and medicinal value, and is widely cultivated throughout the globe. Asparagus has some tolerance to several major environmental stresses. However, few previous studies have studied the physiological mechanism of asparagus from the whole-genome level. Based on the published whole-genome information and transcriptome information, this paper conducted a comprehensive analysis of the asparagus WRKY family, aiming to provide references for the cultivation and yield increase in asparagus [46].

As one of the largest transcription factor families, the WRKY gene family is mainly involved in plant stress response and hormone regulation [2]. With the rapid development of whole-gene sequencing technology, the WRKY family of a variety of plants has been identified and analyzed, including soybean [5], Solanum lycopersicum [47], Carica papaya [48], cotton [49], Cucumis sativus [50], Brachypodium distachyon [51], Gossypium raimondii [52], Aegilops tauschii [53], Gossypium [54], Camellia sinensis [55], and grape [56], but at present, there is little characterization and analysis of the WRKY gene family of A. offificinalis.

To understand more broadly the function of the WRKY gene family, the whole genome and salt-stress transcriptome data of A. offificinalis were used for comprehensive analysis in this study, including WRKY gene family identification, phylogenetic tree construction, analysis of conserved motifs and gene domains, extraction of cis-acting elements, intron/exon analysis, species collinearity analysis, and WRKY expression analysis under salt stress.

The most critical step in the study was to screen and identify WRKY family members from the whole A. offificinalis genome for subsequent analysis. Unlike previous studies, there were only 70 AtWRKY genes in A. offificinalis, compared to 105 in cabbage [32] and 74 in Arabidopsis [2]. In higher plants, the number of WRKY family genes is at least 22, but at most 202, indicating that the evolution of the WRKY family belonging to the middle number of A. offificinalis has a certain conservation, but also has a high gene-loss rate (http://plntfdb.bio.uni-potsdam.de/v3.0/fam_mem.php?family_id=WRKY, accessed on 13 September 2022). It is worth mentioning that the number of WRKY gene family members is not proportional to the size of the genome [57,58]. To further understand the function of AtWRKY, we localized the AtWRKY family to 10 chromosomes and one undefined chromosome and mapped 11 subfamily (C1–C9, U1, U2) phylogenetic trees of A. offificinalis and Arabidopsis by multi-sequence alignment. Except for the U1 and U2 subgroups, the number of members of Arabidopsis and asparagus in subgroups C1–C9 is almost 1:1. This suggests that the rates of gene replication and loss in the two species are similar and conserved. Notably, we found no family members of Arabidopsis in the U1 and U2 subfamilies, suggesting that our asparagus WRKY family may have mutated and evolved. Because of the structural diversity, the distribution of introns and exons can reflect the phylogenetic relationship of gene families [59]. The number of introns in the AtWRKY gene ranges from 2 to 6, and the intron/exon structure is similar in the same subfamily. Similarly, the conserved motifs of WRKY proteins in the same subfamily are similar.

It was found that the AoWRKY gene family has a variety of hormone-related regulatory elements, of which more than 57 members have methyl jasmonate and abscisic acid response elements. Methyl jasmonate can induce the expression of defense-related genes in plants, and promote the production of defense substances to resist environmental stresses such as salt stress and low temperature stress. At the same time, it can also conduct signal transduction and promote the synthesis of other hormones [60]. Abscisic acid is also a key factor in plants’ regulation of stress and can enhance plant drought tolerance [61]. In addition, elements involved in drought response were detected in 41 AoWRKY genes, and defense and stress response elements were detected in 24 AoWRKY genes.

The most critical driving force of species evolution is gene replication, including segment duplication and tandem duplication [62]. A total of two pairs of tandem duplication and 13 pairs of segment duplication were detected in the A. offificinalis WRKY family. Not surprisingly, two of the genes in each pair were from the same subfamily. It is more strongly suggested that the WRKY gene is more conserved in the same subfamily.

Arbuscular mycorrhizal fungi have been identified as soil amendments to increase plant salt tolerance due to salt stress [63]. After grouping asparagus and measuring its transcriptome data (Table S7), we found that AoWRKY11 and AoWRKY15 were up-regulated in NI+S group and AMF+S group, indicating that they were salt-stress-related genes. The genes with an up-regulated expression in the NI group were almost contained in the AMF group. In addition, the AMF group also up-regulated the expression of other genes, suggesting that AMF promotion could promote the expression of more genes. Most of the up-regulated genes of the NI + S and AMF + S groups were different, suggesting that AMF could improve gene salt tolerance and enhance gene expression. Unfortunately, the three biological duplications of some gene expression levels were not perfect, and the reproducibility of the data was not ideal. We speculated that these genes might be caused by some mistakes in the experiment, so these genes were not discussed in this study.

5. Conclusions

In this work, 70 AoWRKY genes were identified, randomly distributed across 11 chromosomes. Further analysis showed that the gene conserved motifs, gene structure and intron/exon arrangement were similar in the same subfamily, indicating highly conserved genes.

We found that the AoWRKY family has more hormone response elements and stress defense elements, which indicates that the AoWRKY family is mainly involved in hormone regulation and stress resistance. In addition, we also found that this part responds to light-response elements and low-temperature-induced response elements, suggesting that we have functional diversity in the AoWRKY family. Finally, we found that several genes were up-regulated or down-regulated under salt stress, and the most important one was that AoWRKY11/15 was finally identified as a salt-stress-resistant gene.

Author Contributions

Conceptualization, J.C.; methodology, J.C.; software, J.C., Q.Z. and Y.Z.; formal analysis, S.H. and J.D.; investigation, J.C.; resources, J.C. and J.M.; writing—original draft preparation, J.C.; writing—review and editing, Y.G. and D.L.; visualization, J.C. and C.W.; supervision, J.C.; project administration, D.L. and Y.G.; funding acquisition, Y.G. and D.L. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article or Supplementary Files.

Acknowledgments

We thank Qianqian Zhang (Chinese Institute for Brain Research, College of Biological Sciences and Technology Beijing Forestry University) and Yingying Zhang (Beijing Forestry University) for their technical support. This research was supported by the National Natural Science Foundation of China (31370669).

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

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Figures and Tables

Figure 1. Phylogenetic tree of WRKY genes in A. officinalis and A. thaliana. The neighbor-joining (NJ) method was adopted and 1000 replications for bootstrap method, Poisson model, and pairwise deletion. The AoWRKY and AtWRKY gene families were labeled with pink and purple blocks, respectively. The evolutionary tree is divided into 11 subfamilies (C1–C9 fa, U2). The Roman numerals (I, II(a,b,c,d,e), III) in parentheses indicate the subfamily classification in Arabidopsis, as well as the different domains of the WRKY family.

Figure 2. Location distribution of WRKY gene family in chromosomes in A. officinalis. The ordinate is chromosome length; Chr1-10 and Un stand for chromosome number.

Figure 3. Phylogenetic relationship, conserved motifs, gene structure and domain architecture of AoWRKY genes. (A) An unrooted NJ tree obtained using MEGA 11 based on A. offificinalis WRKY protein sequences. (B) Conserved motifs of AoWRKY gene family analyzed in MEME. (C) Conserved domains of AoWRKY family. (D) Introns and exons of the AoWRKY family. Note: Different colors indicate different meanings, see the figure note for details.

Figure 4. Duplication and synteny of AoWRKY genes. The gray lines represent tandem duplicate pairs of whole genome. The colored lines represent segmental gene duplication. The red font indicates segmental gene duplication and the blue font indicates tandem gene duplication. The segmental duplication gene pairs are shown in Table S8.

Figure 5. Schematic diagram of syntenic analysis of AoWRKY genes. Synteny of the AoWRKY genes with the WRKY genes of A. thaliana & P. trichocarpa (A), S. indicum & A. comosus (B). The gray lines represent genome-wide collinear gene pairs, and the blue lines represent collinear gene pairs in the WRKY gene family.

Figure 6. Venn diagram analysis between A. offificinalis, S. indicum, A. comosus and P. trichocarpa. The number of overlapping areas of species indicates the number of homologous genes between these species.

Figure 7. RNA-seq analysis of AoWRKY gene family under different salt stress. (A,B) Expression heatmap of AoWRKY gene family under different salt stress. (C,D) Volcanic plot of AoWRKY gene family under different salt stress. The vertical dashed lines represent the points 1 and −1. The horizontal dashed lines represent the points 0.05. Down indicates that the gene is down-regulated and Up indicates that the gene is up-regulated.

Table 1

Detailed information of AoWRKY genes.

Gene Symbol	pI	MW (Da)	Length (aa)	Instability Index	Aliphatic Index	Subcellular Localization	ORF
AoWRKY1	6.48	46,154.82	428	55.77	53.18	Nucleus	1287
AoWRKY2	9.15	32,384.89	293	59.03	62.32	Nucleus	882
AoWRKY3	8.75	28,313.14	254	44.03	64.53	Extracellular	765
AoWRKY4	undefined	undefined	241	52.57	65.19	Nucleus	726
AoWRKY5	5.24	29,765.87	264	46.71	57.99	Nucleus	795
AoWRKY6	9.44	31,856.07	290	50.58	59.83	Nucleus	873
AoWRKY7	5.41	31,262.65	285	49.21	56.18	Nucleus	858
AoWRKY8	6.04	46,486.38	413	73.69	73.20	Extracellular	1242
AoWRKY9	9.39	25,939.09	227	53.18	57.49	Nucleus	684
AoWRKY10	9.07	46,949.56	419	62.02	72.12	Nucleus	1260
AoWRKY11	8.97	13,655.35	122	33.04	51.89	Nucleus	369
AoWRKY12	7.67	20,371.41	177	56.61	49.49	Nucleus	534
AoWRKY13	5.61	27,669.79	244	51.91	63.52	Extracellular	735
AoWRKY14	8.38	21,604.16	199	55.32	55.88	Nucleus	600
AoWRKY15	6.21	63,743.09	585	49.54	56.02	Nucleus	1758
AoWRKY16	9.04	45,956.35	420	49.61	62.50	Nucleus	1263
AoWRKY17	8.53	44,932.82	399	57.93	58.87	Nucleus	1200
AoWRKY18	8.73	55,651.20	505	61.60	49.49	Nucleus	1518
AoWRKY19	8.81	21,958.55	196	60.57	51.79	Extracellular	591
AoWRKY20	5.65	30,925.36	272	50.22	57.79	Extracellular	819
AoWRKY21	9.54	20,022.42	174	40.10	58.16	Nucleus	525
AoWRKY22	6.92	40,959.89	360	52.21	59.36	Nucleus	1083
AoWRKY23	9.84	40,178.56	356	47.34	59.94	Nucleus	1071
AoWRKY24	7.67	38,127.89	345	57.86	52.87	Nucleus	1038
AoWRKY25	9.44	31,856.07	290	50.58	59.83	Nucleus	873
AoWRKY26	8.59	56,176.11	517	51.30	56.52	Nucleus	1554
AoWRKY27	5.12	28,238.23	253	52.89	61.66	Extracellular	762
AoWRKY28	8.77	24,308.45	209	48.08	60.05	Nucleus	630
AoWRKY29	4.66	19,682.85	181	49.78	51.22	Nucleus	546
AoWRKY30	5.18	34,275.83	300	76.79	65.97	Extracellular	903
AoWRKY31	8.34	26,410.43	230	58.47	61.43	Nucleus	693
AoWRKY32	5.57	72,655.57	674	61.01	53.25	Nucleus	2025
AoWRKY33	10.02	29,933.48	267	45.29	59.63	Nucleus	804
AoWRKY34	undefined	undefined	259	51.14	65.52	Nucleus	780
AoWRKY35	5.99	46,045.61	422	52.88	53.39	Nucleus	1269
AoWRKY36	10.1	17,298.73	156	56.66	52.63	Nucleus	471
AoWRKY37	5.54	32,599.17	298	59.24	49.43	Extracellular	897
AoWRKY38	10.24	24,637.92	222	58.85	65.45	Extracellular	669
AoWRKY39	5.06	31,499.98	278	58.6	73.31	Nucleus	837
AoWRKY40	4.89	32,499.36	293	41.65	66.93	Extracellular	882
AoWRKY41	8.56	25,121.05	219	43.79	55.25	Extracellular	660
AoWRKY42	5.54	32,599.17	298	59.24	49.43	Extracellular	897
AoWRKY43	5.22	35,686.71	327	60.99	68.01	Extracellular	984
AoWRKY44	6.74	54,215.42	487	54.77	49.47	Chloroplast	1464
AoWRKY45	5.36	34,350.08	313	54.95	59.87	Nucleus	942
AoWRKY46	9.62	47,107.05	428	61.23	59.77	Extracellular	1287
AoWRKY47	7.72	34,634.99	315	65.79	63.14	Nucleus	948
AoWRKY48	9.28	31,193.24	281	64.28	61	Nucleus	846
AoWRKY49	8.32	46,637.34	423	56.38	50.45	Nucleus	1272
AoWRKY50	6.31	30,633.89	277	70.53	46.14	Nucleus	834
AoWRKY51	9.9	36,191.16	318	43.8	60.13	Nucleus	957
AoWRKY52	7.78	12,996.32	114	42.92	39.3	Nucleus	345
AoWRKY53	5.49	32,548.79	299	43.69	52.51	Nucleus	900
AoWRKY54	9.17	19,282.63	169	51.94	62.31	Nucleus	510
AoWRKY55	7.05	22,006.13	200	53.76	56.55	Nucleus	603
AoWRKY56	6.2	57,636.71	529	52.79	54.97	Nucleus	1590
AoWRKY57	5.85	59,592.16	541	50.51	58.15	Nucleus	1626
AoWRKY58	9.38	14,978.79	136	76.11	53.82	Nucleus	411
AoWRKY59	6.82	14,042.8	122	70.52	58.36	Nucleus	369
AoWRKY60	6.55	27,418.73	242	44.53	64.88	Nucleus	729
AoWRKY61	6.11	25,787.13	232	60.7	75.26	Extracellular	699
AoWRKY62	4.83	32,010.46	283	57.63	60.99	Extracellular	852
AoWRKY63	6.35	27,294.78	241	52.99	68.84	Extracellular	726
AoWRKY64	6.76	30,279.76	266	60.72	61.62	Extracellular	801
AoWRKY65	5.88	31,046.72	277	57.25	78.19	Extracellular	834
AoWRKY66	6.37	26,946.25	238	54.49	61.89	Extracellular	717
AoWRKY67	5.29	40,200.29	356	43.22	78.99	Extracellular	1071
AoWRKY68	9.17	18,202.33	154	47.22	54.35	Nucleus	465
AoWRKY69	8.97	23,501.23	204	44.12	48.24	Nucleus	615
AoWRKY70	6.26	33,056.91	297	53.52	60.77	Extracellular	894

Table 2

Tandem duplication of AoWRKY family and Ka, Ks values.

Tandem-Duplicated Gene Pairs	Ka	Ks	Ka/Ks	Chromosome	Subfamily
AoWRKY47 & AoWRKY48	0.194258151	0.418275774	0.464426015	Chr7	C4
AoWRKY64 & AoWRKY65	0.272881601	0.909314382	0.300095992	Chr8	C8

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/genes14091704/s1: Figure S1: The cis-acting elements of the AoWRKY gene family; Table S1: Sequence logos for the conserved motifs of Asparagus officinalis WRKY domain proteins; Table S2: Details of the AoWRKY gene family; Table S3: One-to-one orthologous relationship of WRKY genes of Asparagus Officinalis and Arabidopsis thaliana. Table S4: One-to-one orthologous relationship of WRKY genes of Asparagus Officinalis and Sesamum indicum; Table S5: One-to-one orthologous relationship of WRKY genes of Asparagus Officinalis and Ananas comosus; Table S6: One-to-one orthologous relationship of WRKY genes of Asparagus Officinalis and Populus trichocarpa; Table S7: SRA accession number for Asparagus officinalis transcriptome data; Table S8: The segmental duplication genes pairs of AtWRKY.

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