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Article

Genome-Wide Identification and Expression Analysis of Eggplant DIR Gene Family in Response to Biotic and Abiotic Stresses

by
Kaijing Zhang
1,
Wujun Xing
1,
Suao Sheng
1,
Dekun Yang
1,
Fengxian Zhen
1,
Haikun Jiang
2,3,
Congsheng Yan
2,3 and
Li Jia
2,3,*
1
College of Agriculture, Anhui Science and Technology University, Chuzhou 233100, China
2
Institute of Horticulture, Anhui Academy of Agricultural Sciences, Hefei 230001, China
3
Key Laboratory of Genetic Improvement and Ecophysiology of Horticultural Crop, Hefei 230001, China
*
Author to whom correspondence should be addressed.
Horticulturae 2022, 8(8), 732; https://doi.org/10.3390/horticulturae8080732
Submission received: 26 July 2022 / Revised: 10 August 2022 / Accepted: 12 August 2022 / Published: 14 August 2022
(This article belongs to the Special Issue Gene Expressions in Response to Diseases, Abiotic Stresses and Pest Damage of Horticultural Products)
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Abstract

:
Dirigent proteins (DIR) play important roles in the biosynthesis of lignins and lignans, defensive responses, secondary metabolism, and disease resistance in plants. The DIR gene family has been identified and studied in many plants. However, the identification of DIR gene family in eggplant has not been conducted yet. Therefore, in this study, based on the available genome information of eggplant, the DIR family genes in eggplant were identified with bioinformatics methods. The expression pattern analyses of eggplant DIR family genes in different organs and stresses were also conducted to understand their biological functions. The results showed that a total of 24 DIR genes were identified in the eggplant, which were divided into three subfamilies (DIR-a, DIR-b/d, and DIR-e). Synteny analysis of DIR genes among eggplant, Arabidopsis, and rice showed that 15 eggplant DIR genes were colinear with 18 Arabidopsis DIR genes, and 16 eggplant DIR genes were colinear with 15 rice DIR genes. Phylogenetic tree analysis showed that 19 pairs of orthologous genes were identified between eggplant and pepper. The cis-acting elements analysis implied that the eggplant DIR genes contained a lot of cis-elements associated with stress and hormone response. The organ-specific expression analysis of eggplant DIR family genes revealed that only the SmDIR3 gene was highly expressed in all the 19 organs of eggplant. Some SmDIR genes, including SmDIR7, SmDIR8, SmDIR11, SmDIR14, SmDIR18, SmDIR19, SmDIR20, and SmDIR23, were not or were lowly expressed in the eggplant organs, while the other eggplant DIR family genes showed an organ-specific expression pattern. Furthermore, 19 of 24 SmDIR genes were differentially expressed in response to abiotic and biotic stresses. 5 SmDIR genes, including SmDIR3, SmDIR5, SmDIR6, SmDIR12, and SmDIR22, were differentially expressed under multiple types of abiotic and biotic stresses. Especially notable, the SmDIR22 gene was differentially expressed under three types of abiotic stresses and two types of biotic stresses, which indicated that the SmDIR22 gene plays an important role in the response to abiotic and biotic stresses. These results provide valuable evidence for a better understanding of the biological role of DIR genes in eggplant.

1. Introduction

In nature, plants are easily influenced by pathogens, pests, severe temperatures, drought, salt, and heavy metals, which cause a dramatic reduction of crop yield and quality [1,2]. Plants usually respond to biotic and abiotic stresses by activating a variety of genes, including the dirigent protein (DIR) gene. The DIR gene is involved in the synthesis of lignins and lignans, which play a pivotal role against biotic and abiotic stresses in plants [3,4,5]. As an important compound, lignin confers stability and hydrophobicity to the plant vascular system and forms a barrier against microbial pathogens and pests [6,7]. Lignan has antifungal properties by inhibiting microbe-derived degradative enzymes such as cellulases, laccases, polygalacturonases, and glucosidases [8,9,10]. Besides, lignans can also defend against insect attacks by disrupting the insect endocrine system [11]. DIR genes can regulate the biosynthesis of lignins and lignans, which indicates that DIR genes actively respond to biotic and abiotic stresses.
DIR proteins are widely distributed in almost all vascular plants, including ferns, gymnosperms, and angiosperms [12,13,14]. Previous studies reported that DIR proteins are divided into five subgroups, including DIR-a, DIR-b, DIR-c, DIR-d, and DIR-e [3]. With the increasing number of DIR proteins, another two new subgroups, DIR-f and DIR-g, were generated, and the DIR-b and DIR-d subfamilies are combined together as the DIR-b/d subgroup [12]. DIR proteins were first identified in Forsythia suspense [15]. Subsequently, the identification of DIR family genes were reported in a variety of monocotyledon and dicotyledon plants, such as Arabidopsis thaliana (L.) Heynh. [16], rice (Oryza sativa L.) [17,18], spruce (Picea spp.) [3,12], pear (Pyrus bretschneideri) [5], Populus [19], Chinese cabbage (Brassica rapa L.) [4], pepper (Capsicum annuum L.) [20], strawberry (Fragaria vesca) [21], Cucurbitaceae [22], and so on. Moreover, many studiers reported that DIR family genes play important regulatory roles in the response to biotic and abiotic stresses, such as high temperature [13,18], low temperature [4,18], drought [4,13,18,23,24,25], salt [18,23,25,26], heavy metals [18], H2O2 [13,23], and diseases [3,4,25,27,28,29,30,31]. In addition, DIR genes also actively respond to hormone stresses including ethylene, gibberellin (JA), salicylic acid (SA), abscisic acid (ABA), and so on [4,5,13,25].
Eggplant (Solanum melongena L.), one of the most important solanaceous crops, is widely cultivated across the world for its fruits, ranking third for total production and economic value in Solanaceae species, after potato (Solanum tuberosum L.) and tomato (Solanum lycopersicum L.) [32,33]. As early as 2014, the first genome sequencing project of eggplant was completed [34]. With the rapid development of genome sequencing technology, the improved eggplant genomes were constantly updated [35,36,37,38]. By using the high-quality genome information of eggplant, a great deal of research on gene family identification has been performed, such as the identification of R2R3-MYB [39], WRKY [40], NAC [41], ARF [42], and other gene families. However, the genome-wide identification of DIR gene family in eggplant was still not reported, which greatly limits the functional studies of DIR genes in eggplant.
Therefore, based on the high-quality genome information of eggplant, DIR family genes in eggplant (SmDIR genes) were identified for the first time in this study. The physicochemical characteristics, chromosome locations, gene structures, conserved motifs, phylogenetic tree, cis-acting elements, and synteny of SmDIR genes were analyzed via bioinformatic methods. Moreover, the analyses of organ-specific expression and stress-responsive gene expression patterns of SmDIR genes were conducted to preliminarily explore the biological functions of DIR family genes in eggplant. The results of the current study will provide future insight for further research on the biological functions of SmDIR genes, as well as a theoretical reference for the resistance breeding of eggplant.

2. Materials and Methods

2.1. Identification and Chromosomal Mapping of DIR Family Genes in Eggplant

The Hidden Markov Model (HMM) of dirigent domain (PF03018) was used to identify the putative DIR family genes in eggplant genome V4.1 [38] using HMMER 3.0 software [43] with E-value < 1 × 10−5. The protein sequences of candidate SmDIR genes were extracted with Fasta Extract program in TBtools software [44]. The Pfam (http://pfam.xfam.org/, accessed on 26 May 2022) [45] and SMART (http://smart.embl.de/smart/batch.pl, accessed on 26 May 2022) [46] websites were used to confirm the conserved dirigent domain. Genes containing the whole dirigent domain were chosen as the final confirmed members of DIR gene family in eggplant. The chromosomal locations of SmDIR genes were analyzed and visualized with TBtools software. The physicochemical characteristics such as the number of amino acids, molecular weight, theoretical isoelectric point, instability index, aliphatic index, and grand average of hydropathicity of each SmDIR protein were calculated with the online tool ExPASy (https://web.expasy.org/protparam/, accessed on 27 May 2022). The subcellular localization of SmDIR genes were predicted with the online website CELLO (http://cello.life.nctu.edu.tw/, accessed on 27 May 2022) [47].

2.2. Gene Structure Analysis and Phylogenetic Tree Construction of DIR Family Genes in Eggplant

The exon/intron structures of SmDIR genes were drawn with GFF3 file (General Feature Format 3) of eggplant genome V4.1 using TBtools software. The conserved motifs of SmDIR proteins were analyzed using the MEME software (version = 5.4.1) [48] with the following parameters: maximum number of misfits, 10; and the optimum width of each motif, 6–100 amino acids. The 1.5 kb upstream sequences of the translation initiation codon of SmDIR genes were extracted from the eggplant genome V4.1. The sequences were then submitted to PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 27 May 2022) website [49] for cis-acting elements prediction. The phylogenetic tree of DIR family genes in eggplant, pepper [20] and Arabidopsis [16] was generated using MEGA 11 software [50] with the neighbor-joining method and 1000 bootstrap replications. The final phylogenetic tree was visualized using the online website Evolview (https://www.evolgenius.info/evolview-v3/#login, accessed on 27 May 2022) [51].

2.3. Gene Duplication and Synteny Analysis of DIR Family Genes

The tandem and segmental duplications of SmDIR genes were analyzed with MCScanX software [52]. The synteny analysis of DIR family genes among eggplant, Arabidopsis and rice was conducted with MCScanX software. The syntenic relationships of DIR family genes among eggplant, Arabidopsis, and rice were visualized with Circos software [53].

2.4. RNA-Seq Reanalysis of Eggplant Transcriptome Sequencing Data

The published eggplant transcriptome sequencing data were downloaded from the SRA database using the corresponding accession numbers (https://www.ncbi.nlm.nih.gov/sra, accessed on 4 June 2022). The downloaded SRA data were converted into Fastq data by using fasterq-dump.2.11.0 tool (https://github.com/ncbi/sra-tools/wiki/HowTo:-fasterq-dump, accessed on 10 June 2022). The read quality of Fastq data was then evaluated using the FastQC software (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/, accessed on 10 June 2022). The adapter sequences and low-quality bases were removed from the Fastq data with Trimmomatic software (version = 0.39) [54], finally obtaining the filtered clean data. The index of eggplant genome V4.1 was built using STAR software (version = 2.7.10a) [55]. The filtered clean data were mapped to eggplant genome V4.1, generating the SAM files. Next, the SAM files were directly converted into the sorted BAM files using the SAMtools software (version = 1.15) [56]. The expression levels of each gene were estimated with StringTie software (v2.2.1) [57]. Finally, the differential expression analysis was performed using the count matrix in DESeq2 software [58].

2.5. Organ-Specific Expression Analysis of DIR Family Genes in Eggplant

The transcriptome sequencing data of 19 kinds of eggplant organs, including cotyledon, radicle, root, stem, flower, pistil, leaf, bud, fruit peduncle, fruit skin, fruit flesh, fruit, and so on, were retrieved and downloaded from the SRA database (PRJNA328564) [35]. The organ-specific expression analysis of eggplant DIR family genes was conducted using the above methods. The heatmap was drawn using the Heatmap program in TBtools software.

2.6. The Expression Profiles Analysis of Eggplant DIR Family Genes under Stresses

The transcriptome sequencing data of eggplant under stress treatments including high temperature (PRJNA531285) [59], low temperature (PRJNA572318) [60], salt (PRJNA477924) [61], bacterial wilt (caused by Ralstonia solanacearum, PRJNA728497) [62], Verticillium wilt (PRJNA451240) [63], and Tuta absoluta (PRJNA695350) [64] were retrieved and downloaded from the SRA database. The expression profiles of eggplant DIR family genes were analyzed using the above methods, and then drawn into heatmaps using TBtools software.

3. Results

3.1. Genome-Wide Identification of DIR Family Genes in Eggplant

A total of 24 DIR family genes were identified in the eggplant genome (Table 1). These eggplant DIR family genes were renamed from SmDIR1 to SmDIR24 according to their chromosomal locations. The detailed information of eggplant DIR family genes was summarized in Table 1. The 24 SmDIR proteins ranged from 146 (SmDIR11) to 546 (SmDIR17) amino acid (aa) in length, and the corresponding molecular weights varied from 16.14 kDa (SmDIR11) to 59.69 kDa (SmDIR17). The isoelectric points (pI) of the 24 SmDIR proteins ranged from 4.34 (SmDIR9) to 9.85 (SmDIR19). Among the 24 eggplant DIR proteins, 14 SmDIR proteins were basic proteins (pI > 7), and 10 SmDIR proteins were acidic proteins (pI < 7). 20 SmDIR proteins were stable proteins (instability index less than 40), and the other 4 SmDIR proteins (SmDIR1, SmDIR8, SmDIR9, and SmDIR20) were unstable proteins (instability index greater than 40). The aliphatic indexes of 24 SmDIR proteins varied from 66.53 (SmDIR16) to 101.55 (SmDIR22). Most of SmDIR proteins (79%) were hydrophobic proteins (grand average of hydropathicity > 0), and only five SmDIR proteins (SmDIR1, SmDIR3, SmDIR8, SmDIR16, and SmDIR20) were hydrophilic proteins (grand average of hydropathicity < 0). Subcellular localization prediction showed that most of the SmDIR genes were located in the plasma membrane, and the other DIR family members were located in the extracellular space and mitochondrion. (Table 1).
Based on the GFF3 file of eggplant genome V4.1, the chromosomal distributions of 24 SmDIR genes were mapped across the eggplant chromosomes using TBtools software (Figure 1). In the eggplant genome, the 24 SmDIR family genes were unevenly distributed on 10 of 12 eggplant chromosomes. There were no SmDIR genes located on chromosomes 3 and 10. Chromosome 12 harbored the largest number of 4 SmDIR genes, whereas chromosomes 4, 7, and 9 contained only one SmDIR gene, respectively. The chromosomes 1, 2, 5, 6, and 11 had three SmDIR genes, respectively. Two SmDIR genes were positioned on chromosome 8. In our study, five pairs of tandem duplication genes on chromosome 1 (SmDIR1/SmDIR2), chromosome 5 (SmDIR8/SmDIR9), chromosome 8 (SmDIR15/SmDIR16), and chromosome 12 (SmDIR21/SmDIR22, SmDIR22/SmDIR23) were identified in eggplant DIR gene family.

3.2. Phylogenetic Analysis of DIR Family Genes in Eggplant, Pepper and Arabidopsis

To investigate the evolutionary relationship of DIR family genes in eggplant, a phylogenetic tree was constructed with 24 SmDIR proteins, 24 pepper DIR proteins [20] and 25 Arabidopsis DIR proteins [16] by multiple sequence alignment (Figure 2). According to the clustering result of Arabidopsis DIR family genes, the phylogenetic tree was divided into three subgroups, including DIR-a, DIR-b/d, and DIR-e. Among them, the DIR-b/d subgroup harbored the largest number of DIR genes, containing a total of 40 DIR genes. The DIR-e subgroup had the second number of DIR genes, containing 19 DIR genes. The DIR-a subgroup had the least number of DIR genes, containing 14 DIR genes. In the phylogenetic tree, 19 pairs of orthologous genes were identified between eggplant and pepper, which were SmDIR1/CaDIR17, SmDIR2/CaDIR16, SmDIR3/CaDIR5, SmDIR4/CaDIR8, SmDIR5/CaDIR13, SmDIR6/CaDIR6, SmDIR8/CaDIR10, SmDIR9/CaDIR19, SmDIR10/CaDIR23, SmDIR12/CaDIR22, SmDIR13/CaDIR12, SmDIR14/CaDIR14, SmDIR15/CaDIR2, SmDIR16/CaDIR1, SmDIR17/CaDIR7, SmDIR18/CaDIR4, SmDIR19/CaDIR9, SmDIR22/CaDIR24, and SmDIR24/CaDIR20, respectively. The homologous genes clustered together in the evolutionary relationship possessed similar gene functions. Thus, the biological functions of SmDIR genes could be inferred from the molecular functions of pepper DIR family genes based on the clustering results of DIR family genes between eggplant and pepper.

3.3. Gene Structure and Conserved Motifs Analyses of Eggplant DIR Genes

The diagrams of phylogenetic tree and gene structures of SmDIR genes were drawn with TBtools software. The results of phylogenetic tree analysis showed that 24 SmDIR genes were classified into three subgroups, namely DIR-a, DIR-b/d and DIR-e (Figure 3), which was consistent with the clustering result of eggplant, pepper and Arabidopsis DIR genes (Figure 2). Among them, there were fourteen DIR genes in the DIR-b/d subgroup, six DIR genes in the DIR-e subgroup, and four DIR genes in the DIR-a subgroup. The gene structure analysis showed that most of DIR genes in subgroup DIR-b/d contained one exon. All four DIR genes in the DIR-a subgroup contained only one exon. All DIR genes except SmDIR10, SmDIR13, and SmDIR17 in subgroup DIR-e contained one exon (Figure 3). The results showed that most of the SmDIR genes (71%) contained only one exon and no introns, suggesting that the exon number of SmDIR genes was relatively conservative.
A schematic diagram of conserved motifs of SmDIR proteins was constructed with MEME analysis (Figure 3). A total of ten motifs were revealed in the eggplant DIR proteins (Table 2). The motif compositions of SmDIR proteins in different subgroups were different, while SmDIR proteins in the same subgroup shared the similar number, type, and order of motifs. For example, most of SmDIR proteins in the subgroup DIR-b/d contained motifs 3, 1, 5, 2, and 4, and exhibited the same order. In the subgroup DIR-a, motifs 1 and 2 were found in all SmDIR proteins and showed the same order. In the subgroup DIR-e, motifs 1, 9, 2, and 8 were found in all SmDIR proteins and showed the same order. These results indicated that the different distribution of conserved motifs in different subgroups might lead to the evolution of SmDIR gene functional diversity. The similar conserved motifs of SmDIR proteins in the same subgroup indicated that they have similar functions.

3.4. The Synteny Analysis of DIR Genes among Eggplant, Arabidopsis and Rice

To better understand the molecular functions of eggplant DIR family genes, the synteny analysis of DIR family genes among eggplant, Arabidopsis and rice was conducted. The results showed 29 syntenic relationships between 15 SmDIR genes (SmDIR2, SmDIR3, SmDIR6, SmDIR9, SmDIR10, SmDIR11, SmDIR12, SmDIR13, SmDIR15, SmDIR17, SmDIR19, SmDIR20, SmDIR21, SmDIR23, SmDIR24) genes and 18 Arabidopsis DIR genes (AT4G13580, AT3G24020, AT1G58170, AT1G55210, AT2G39430, AT3G13650, AT3G55230, AT2G28670, AT5G49040, AT2G21100, AT1G65870, AT1G64160, AT3G58090, AT5G42500, AT4G23690, AT3G13662, AT5G42655, AT4G11430). There were 27 syntenic relationships between 16 SmDIR genes (SmDIR2, SmDIR3, SmDIR4, SmDIR5, SmDIR6, SmDIR8, SmDIR9, SmDIR10, SmDIR12, SmDIR13, SmDIR15, SmDIR16, SmDIR17, SmDIR21, SmDIR23, SmDIR24) and 15 rice DIR genes (Os04g42720, Os03g05030, Os03g17220, Os01g06250, Os01g65700, Os11g07830, Os12g07580, Os11g27620, Os11g07690, Os03g59440, Os07g44930, Os07g44250, Os07g44280, Os01g62030, Os07g44450). It was found that five SmDIR genes (SmDIR1, SmDIR7, SmDIR14, SmDIR18, SmDIR22) were not colinear with either A. thaliana or rice, suggesting that these five SmDIR genes were conservative in eggplant. The synteny analysis of DIR family genes in eggplant showed that there were four pairs of SmDIR genes (SmDIR1/SmDIR21, SmDIR1/SmDIR24, SmDIR8/SmDIR13, SmDIR10/SmDIR17) with syntenic relationships, which were segmental duplication gene pairs (Figure 4).

3.5. The Cis-Acting Elements Analysis of Eggplant DIR Genes

The 1.5-kb upstream sequences from the transcription start site of SmDIR genes were extracted to analyze their cis-acting elements in the promoters. The results showed that 14 kinds of cis-elements were identified (Figure 5). Among them, cis-elements related to light responsiveness were the major type, including ACE, AE-box, ATCT-motif, Box 4, Box II, G-box, GT1-motif, I-box, LAMP-element, and so on, accounting for 31% of total cis-elements. In addition, some other cis-elements were also identified, including cis-elements related to hormone response (abscisic acid, MeJA, auxin, gibberellin, salicylic acid), stress response (drought, low temperature, defense), circadian control, endosperm expression, and meristem expression. The results indicated that eggplant DIR genes play vital roles in plant growth and development.

3.6. Organ-Specific Expression Analysis of Eggplant DIR Genes

To analyze the organ-specific expression profiles of SmDIR genes in eggplant, the transcriptome sequencing data of 19 kinds of eggplant organs (accession number: PRJNA328564) were reanalyzed with eggplant genome V4.1. The results indicated that the eggplant DIR gene SmDIR3 was highly expressed in all the 19 organs of eggplant. Some SmDIR genes, including SmDIR7, SmDIR8, SmDIR11, SmDIR14, SmDIR18, SmDIR19, SmDIR20, and SmDIR23, were not or were lowly expressed in all the eggplant organs. SmDIR12 gene was expressed in all eggplant organs, with the lower expression levels in senescent leaf, fruit peduncle, fruit flesh stage 3, and fruit skin stage 3. The SmDIR22 gene was expressed in all eggplant organs, with the highest expression level in root and the lowest expression levels in fruit flesh and skin at stage 3. The SmDIR6 gene was highly expressed in fruit skin stage 3, stem and fruit peduncle, but was lowly expressed in cotyledon and fruit. Some SmDIR gene was organ-specific expressed. For example, the SmDIR1 gene was highly expressed in bud, but was lowly or not expressed in the other eggplant organs. SmDIR13 gene was highly expressed in radicle, root and bud, but was lowly or not expressed in the other eggplant organs. The eggplant DIR genes SmDIR2, SmDIR4, SmDIR9, SmDIR10, SmDIR17, and SmDIR21 were highly expressed in radicle and root, but were lowly expressed in the other organs (Figure 6). In summary, the expression patterns of SmDIR genes varied in different organs, suggesting a degree of organ specificity.

3.7. The Expression Profiles of Eggplant DIR Genes under Abiotic Stresses

To determine the expression profiles of eggplant DIR family genes under high temperature, low temperature, and salt stresses, the published transcriptome sequencing data (PRJNA531285, PRJNA572318, PRJNA477924) were re-analyzed with eggplant genome V4.1. Compared with the control, four eggplant DIR genes including SmDIR3, SmDIR6, SmDIR12, and SmDIR22 genes were significantly down-regulated under high temperature stress. However, the expression patterns of these four SmDIR genes were different. Among them, the SmDIR3 gene was significantly down-regulated only at 20DAF (days after flowering). SmDIR12 gene was significantly down-regulated only at 10DAF. SmDIR6 gene was significantly down-regulated at 15DAF and 20DAF. SmDIR22 gene was significantly down-regulated at 10DAF, 15DAF, and 20DAF. The other 20 SmDIR genes were lowly or not expressed in the peel, and not affected by high temperature treatment (Figure 7A). Under low temperature stress, only two eggplant DIR genes SmDIR5 and SmDIR22 were significantly down-regulated in both sensitive and tolerant eggplant cultivars. Among the other twenty-two SmDIR genes, seventeen SmDIR genes were not or lowly expressed in the leaf, five SmDIR genes were not significantly regulated by low temperature treatment (Figure 7B). Under salt stress, only SmDIR22 genes were significantly down-regulated in the sensitive eggplant leaf. The other 23 SmDIR genes were not significantly affected by salt stress (Figure 7C).

3.8. The Expression Profiles of Eggplant DIR Genes under Biotic Stresses

Based on the eggplant genome V4.1, the published transcriptome sequencing data under bacterial wilt (PRJNA728497), Verticillium wilt (PRJNA451240), and Tuta absoluta stresses (PRJNA695350) were re-analyzed to investigate the expression profiles of eggplant DIR genes under biotic stresses. Under bacterial wilt stress, only five SmDIR genes were not affected, which were also not or lowly expressed in the samples. The other 19 SmDIR genes exhibited significantly differential expression under bacterial wilt infection. Among them, three SmDIR genes, including SmDIR6, SmDIR12, and SmDIR21, were up-regulated in both resistant and susceptible eggplant cultivars. SmDIR6 and SmDIR12 genes were up-regulated in both root and stem, while SmDIR21 was only up-regulated in the root. Thirteen SmDIR genes were down-regulated in response to bacterial wilt infection. Whereas the expression patterns of these 13 SmDIR genes were different. Only three of these 13 SmDIR genes, including SmDIR2, SmDIR3, and SmDIR18, were down-regulated in the susceptible eggplant cultivar. SmDIR3 was down-regulated in the root and stem of the susceptible eggplant cultivar. SmDIR2 and SmDIR18 were only down-regulated in the root of susceptible eggplant cultivar. Only one SmDIR gene SmDIR8 was down-regulated in the root of resistant eggplant cultivar. Two SmDIR genes including SmDIR16 and SmDIR17 were down-regulated in both root and stem in susceptible and resistant eggplant cultivars. Six SmDIR genes, including SmDIR4, SmDIR9, SmDIR10, SmDIR13, SmDIR19, and SmDIR24, were down-regulated in the root of both susceptible and resistant eggplant cultivars. The SmDIR5 gene was down-regulated in the root and stem of the susceptible eggplant line, but down-regulated only in the root of the resistant eggplant line. The expression patterns of three SmDIR genes (SmDIR7, SmDIR15 and SmDIR22) were different in different samples, containing both up-regulation and down-regulation (Figure 8A).
Under Verticillium wilt stress, a total of nine SmDIR genes exhibited significantly differential expression compared to the control treatment. Among them, five SmDIR genes, including SmDIR2, SmDIR9, SmDIR10, SmDIR13, and SmDIR21, were down-regulated at 24 or 48 h after Verticillium wilt infection, while four SmDIR genes, including SmDIR4, SmDIR12, SmDIR16, and SmDIR18, exhibited significantly up-regulated expression after Verticillium wilt infection. SmDIR4, SmDIR16, and SmDIR12 genes were significantly up-regulated at 48 h after Verticillium wilt infection. SmDIR18 genes were significantly up-regulated at 6 h after Verticillium wilt infection (Figure 8B). Under Tuta absoluta stress, only four SmDIR genes were significantly differentially expressed compared to the control. Among them, three SmDIR genes, including SmDIR3, SmDIR6, and SmDIR22, were up-regulated expression, one SmDIR gene SmDIR12 was down-regulated expression after Tuta absoluta infection (Figure 8C).

3.9. The Regulation Patterns Analysis of Eggplant DIR Family Genes under Stresses

To analyze the expression patterns of SmDIR genes under biotic and abiotic stresses, the differentially expressed genes were labeled and drawn into a heatmap (Figure 9). The results showed that 19 of 24 SmDIR genes were differentially expressed in response to stresses. Among them, five SmDIR genes, including SmDIR3, SmDIR5, SmDIR6, SmDIR12, and SmDIR22, were differentially expressed under multiple types of abiotic and biotic stresses, indicating that these five SmDIR genes actively participate in stress responses. Especially notable, the SmDIR22 gene was differentially expressed under three types of abiotic stresses and two types of biotic stresses, which could be considered as the favorable gene for the further studies. The other 14 of 24 SmDIR genes were only differentially expressed under biotic stresses. The regulation patterns analysis of eggplant DIR family genes could provide the theoretical references for further research on the biological function of eggplant DIR genes and the resistance breeding of eggplant.

4. Discussion

Since the Arabidopsis thaliana genome was published in 2000, more and more plant genomes have been sequenced and published, thanks to the rapid development of genome sequencing technology [65]. Based on the high-quality genome information available, the identification of many important gene families has gradually been carried out for different plants. DIR proteins have been found in almost all vascular plants and often appear as a gene family [3,12]. DIR proteins are a kind of gene family involved in the biosynthesis of cell wall lignins and lignans, which play important roles in plant growth and development, abiotic stress tolerance, biotic stress response, and secondary metabolism regulation [16,66]. The DIR gene family has been successively identified in model plants, including Arabidopsis thaliana [16] and rice [17,18], and subsequently reported in horticultural plants such as Chinese cabbage [4], pepper [20], strawberry [21], and Cucurbitaceae [22]. As the most important solanaceous crop, eggplant is one of important vegetable crops widely cultivated across the world. However, the identification of the DIR gene family in eggplant has not been performed. Therefore, in this study, the DIR gene families were identified in eggplant based on the high-quality eggplant genome V4.1 [38]. In addition, the expression profiles of eggplant DIR family genes in different organs and stress responses were conducted by reanalyzing the eggplant transcriptome sequencing data, which will provide theoretical reference for further research on molecular functions of DIR genes and screen out the favorable DIR gene for eggplant resistance molecular breeding.
The number of DIR family genes varied greatly in different plants. In this study, a total of 24 members of the DIR gene family in eggplant were identified. Previous studies identified 25, 61, 35, 35, 40, 29, 24, 33, 22, 22, and 17 DIR family genes in Arabidopsis [16], rice [17,18], spruce [3,12], pear [5], Populus [19], Chinese cabbage [4], pepper [20], strawberry [21], watermelon, melon, and cucumber [22], respectively. The numbers of DIR genes in most plants that have finished the identification of their DIR gene family were more than the number of DIR genes in eggplant. The member of the DIR gene family in pepper was same with that of DIR genes in eggplant, which is probably because both pepper and eggplant belong to the crops of the Solanaceae family. The analysis of physicochemical characteristics showed that most of the DIR proteins in eggplant are stable proteins, which is similar to the physicochemical characteristics of DIR proteins in Chinese cabbage [4], pepper [20], strawberry [21], watermelon, melon, and cucumber [22]. Phylogenetic tree analysis of 24 eggplant DIR family genes were divided into three subgroups: DIR-a, DIR-b/d, and DIR-e, which was similar to the clustering results of phylogenetic analysis of DIR family genes in Arabidopsis [16], Populus [19], and pepper [20]. There were significant differences in gene structure among the DIR genes within three different subgroups, and the gene structures and conserved motifs of DIR genes in the same subgroup were similar. Most of eggplant DIR family genes (71%) only contained one exon and no introns, which were same with the exon/intron structures of DIR genes in other plants such as rice [17], pear [5], Populus [19], pepper [20], strawberry [21], watermelon, melon, and cucumber [22]. The analysis of gene duplication events of eggplant DIR family genes showed five pairs of tandemly duplicated genes and four pairs of segmentally duplicated genes, which revealed that the expansion of the DIR gene family in eggplant resulted from both tandem and segmental duplications. This phenomenon also existed in the DIR gene families of rice [17], pear [5], pepper [20] and strawberry [21], as well as some other gene families in other plants [67,68]. The synteny analysis of the DIR family genes in Arabidopsis, rice, and eggplant found that 15 eggplant DIR genes were collinear with Arabidopsis DIR genes, while 16 eggplant DIR genes were collinear with rice DIR genes, indicating that the eggplant DIR family genes have similar homology with DIR genes in both Arabidopsis and rice.
In recent years, many transcriptome sequencing projects in eggplant have been conducted with the rapid development of high-throughput sequencing technology, forming the eggplant transcriptome sequencing big data. Therefore, making good use of these transcriptome sequencing data could not only reduce the cost of research, but also enable in-depth mining of these data. Moreover, the biological functions of different gene families in eggplant could be studied using the eggplant transcriptome sequencing big data under different treatments. In this study, with the published eggplant transcriptome sequencing big data, the organ-specific expression patterns and the stress-responsive gene expression patterns of 24 eggplant DIR family genes were analyzed. The results showed that only SmDIR3 was highly expressed in all 19 of the organs of eggplant. Some SmDIR genes, including SmDIR7, SmDIR8, SmDIR11, SmDIR14, SmDIR18, SmDIR19, SmDIR20, and SmDIR23, were not or were lowly expressed in the eggplant organs. While the other eggplant DIR family genes showed an organ-specific expression pattern. Previous studies have shown that DIR genes can contribute to the lignification of plant organs, suggesting that the DIR genes are essential for healthy plant growth. In our study, most of the SmDIR genes were specifically expressed in the radicle and root, indicating that these SmDIR genes play an important role in the development of radicle and root [69]. The organ-specific expression pattern of these SmDIR genes in different organs also cooperatively regulated the plant growth and development of eggplant.
In terms of the expression profiles of SmDIR genes in response to biotic and abiotic stress, 19 of 24 SmDIR genes were differentially expressed in response to stresses, which indicated that most of SmDIR genes were involved in response to stresses. Among them, five SmDIR genes, including SmDIR3, SmDIR5, SmDIR6, SmDIR12, and SmDIR22, were differentially expressed under multiple types of abiotic and biotic stresses, indicating that these five SmDIR genes actively participate in stress responses. Especially notable, the SmDIR22 gene was differentially expressed under three types of abiotic stresses and two types of biotic stresses. In addition, it was also found that the expression patterns of eggplant DIR family genes under biotic and abiotic stresses were different. The number of differentially expressed DIR family genes in response to abiotic stresses was less than the number of differentially expressed DIR family genes in response to biotic stresses. The number of up-regulated DIR genes in response to biotic stresses was more than that in response to abiotic stresses. These results support the hypothesis that DIR genes are associated with plant-pathogen/insect infection. After the infections by pathogen and insect, more DIR family genes would be significantly up-regulated to accumulate more lignin, thus preventing infections by pathogen and insect. Many studies have demonstrated the importance of DIR genes in plant-pathogen interactions [70,71]. In addition, some eggplant DIR genes also play an important role in abiotic stresses, especially under high temperature, low temperature, and drought, which is consistent with the types of cis-elements in the promoter of DIR genes. Whereas, most of the eggplant DIR genes were down-regulated in response to stresses, which is also worthy of further investigation.

5. Conclusions

In this study, a total of 24 DIR family genes were identified in eggplant, which were divided into three subgroups of DIR-a, DIR-b/d, and DIR-e. The gene structure and protein conserved motifs in the same subgroup were highly conservative, and the distributions of exon/intron and motifs differed among different subgroups. The organ-specific expression patterns and the stress-responsive gene expression patterns of eggplant DIR family genes were studied by re-analyzing the eggplant transcriptome sequencing data with eggplant genome V4.1. The results showed that the expression patterns of eggplant DIR family genes in different organs and stress responses were different, synergistically regulating the plant growth and development of eggplant. Among the eggplant DIR family genes, the SmDIR22 gene was differentially expressed under three types of abiotic stresses and two types of biotic stresses, which indicated that the SmDIR22 gene actively participated in stress responses. The results of the current study will provide a theoretical reference for further study of the biological functions of eggplant DIR family genes, and highlight SmDIR22 as the favorable gene for molecular breeding of eggplant resistance.

Author Contributions

L.J. conceived the research and designed the experiments. K.Z. performed research, analyzed the data and wrote the manuscript. W.X., S.S. and D.Y. participated in downloading transcriptome sequencing data and helped with the bioinformatics analysis. F.Z., H.J. and C.Y. analyzed and interpreted the data. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (32002061), the Second Level Youth Development Fund from Anhui Academy of Agricultural Sciences (QNYC-202121), China Agriculture Research System of MOF and MARA (CSRS-23-G40), the College Students’ Innovative Entrepreneurial Training Plan Program (S202110879222), the Talent Foundation of Anhui Science and Technology University (NXYJ202103), and Anhui Province Vegetable industry Technology System.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data used in this study are presented in the article.

Acknowledgments

We would like to Thank Martin Kagiki Njogu of Chuka University for their help in the improvement of this manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. The chromosomal locations of DIR family genes in eggplant.
Figure 1. The chromosomal locations of DIR family genes in eggplant.
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Figure 2. Phylogenetic analysis of DIR proteins from eggplant, pepper and Arabidopsis.
Figure 2. Phylogenetic analysis of DIR proteins from eggplant, pepper and Arabidopsis.
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Figure 3. Exon-intron structures of SmDIR genes and conserved motifs of SmDIR proteins.
Figure 3. Exon-intron structures of SmDIR genes and conserved motifs of SmDIR proteins.
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Figure 4. Syntenic relationships of DIR family genes among eggplant, Arabidopsis and rice.
Figure 4. Syntenic relationships of DIR family genes among eggplant, Arabidopsis and rice.
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Figure 5. Cis-elements analysis of the promoters of eggplant DIR family genes. (A) The types and numbers of various cis-elements in the promoters of each SmDIR gene. (B) The relative proportions of different kinds of cis-elements in the promoters of SmDIR genes are displayed by pie chart.
Figure 5. Cis-elements analysis of the promoters of eggplant DIR family genes. (A) The types and numbers of various cis-elements in the promoters of each SmDIR gene. (B) The relative proportions of different kinds of cis-elements in the promoters of SmDIR genes are displayed by pie chart.
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Figure 6. The expression heatmap of eggplant DIR family gene in different organs. The data in the boxes indicated the original FPKM values.
Figure 6. The expression heatmap of eggplant DIR family gene in different organs. The data in the boxes indicated the original FPKM values.
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Figure 7. The expression heatmaps of eggplant DIR family genes under abiotic stress treatments. (A) The expression patterns of SmDIR genes under high temperature stress. s25: control treatment (25 °C); s35: high temperature treatment (35 °C); DAF: days after flowering. (B) The expression patterns of SmDIR genes under low temperature stress. S: low temperature-sensitive eggplant cultivar; T: low temperature-tolerant eggplant cultivar. 0 h: low temperature treatment for 0 h (control treatment); 6 h: low temperature treatment (4 °C) for 6 h. (C) The expression patterns of SmDIR genes under salt stress. S: salt-sensitive eggplant cultivar; T: salt-tolerant eggplant cultivar. 0 h: salt treatment for 0 h (control treatment); 12 h: salt treatment (200 mM NaCl) for 12 h; L: leaf; R: root. In each figure, the data in the boxes of left heatmap indicated the original FPKM values. Differentially expressed genes were highlighted by red (up-regulation) and green (down-regulation) color with log2 (fold-change) values in the right table.
Figure 7. The expression heatmaps of eggplant DIR family genes under abiotic stress treatments. (A) The expression patterns of SmDIR genes under high temperature stress. s25: control treatment (25 °C); s35: high temperature treatment (35 °C); DAF: days after flowering. (B) The expression patterns of SmDIR genes under low temperature stress. S: low temperature-sensitive eggplant cultivar; T: low temperature-tolerant eggplant cultivar. 0 h: low temperature treatment for 0 h (control treatment); 6 h: low temperature treatment (4 °C) for 6 h. (C) The expression patterns of SmDIR genes under salt stress. S: salt-sensitive eggplant cultivar; T: salt-tolerant eggplant cultivar. 0 h: salt treatment for 0 h (control treatment); 12 h: salt treatment (200 mM NaCl) for 12 h; L: leaf; R: root. In each figure, the data in the boxes of left heatmap indicated the original FPKM values. Differentially expressed genes were highlighted by red (up-regulation) and green (down-regulation) color with log2 (fold-change) values in the right table.
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Figure 8. The expression heatmaps of eggplant DIR family genes under biotic stress treatments. (A) The expression patterns of SmDIR genes under bacterial wilt stress. S: susceptible eggplant cultivar; R: resistant eggplant cultivar; CT: control treatment; 1 dpi: 1 day post-inoculation; edo: early disease onset stage; pdo: peak disease onset stage. (B) The expression patterns of SmDIR genes under Verticillium wilt stress. CT: control treatment; Vm-6 h, Vm-12 h, Vm-24 h and Vm-48 h represented 6, 12, 24 and 48 h after Verticillium wilt infection. (C) The expression patterns of SmDIR genes under Tuta absoluta stress. CT: control treatment; Ta: Tuta absoluta infection. In each figure, the data in the boxes of the left heatmap indicated the original FPKM values. Differentially expressed genes were highlighted by red (up-regulation) and green (down-regulation) color with log2 (fold-change) values in the right table.
Figure 8. The expression heatmaps of eggplant DIR family genes under biotic stress treatments. (A) The expression patterns of SmDIR genes under bacterial wilt stress. S: susceptible eggplant cultivar; R: resistant eggplant cultivar; CT: control treatment; 1 dpi: 1 day post-inoculation; edo: early disease onset stage; pdo: peak disease onset stage. (B) The expression patterns of SmDIR genes under Verticillium wilt stress. CT: control treatment; Vm-6 h, Vm-12 h, Vm-24 h and Vm-48 h represented 6, 12, 24 and 48 h after Verticillium wilt infection. (C) The expression patterns of SmDIR genes under Tuta absoluta stress. CT: control treatment; Ta: Tuta absoluta infection. In each figure, the data in the boxes of the left heatmap indicated the original FPKM values. Differentially expressed genes were highlighted by red (up-regulation) and green (down-regulation) color with log2 (fold-change) values in the right table.
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Figure 9. The expression patterns heatmap of eggplant DIR family genes under abiotic and biotic stresses. Gray color represents that the SmDIR genes were not differentially expressed; red color represents up-regulated expression; green color represents down-regulated expression; blue color represents that the SmDIR genes exhibited both up-regulated and down-regulated expressions.
Figure 9. The expression patterns heatmap of eggplant DIR family genes under abiotic and biotic stresses. Gray color represents that the SmDIR genes were not differentially expressed; red color represents up-regulated expression; green color represents down-regulated expression; blue color represents that the SmDIR genes exhibited both up-regulated and down-regulated expressions.
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Table
Table 1. The physiochemical characteristics of DIR family genes in eggplant.
Table 1. The physiochemical characteristics of DIR family genes in eggplant.
Gene NameGene IDNumber of Amino Acid (aa)Molecular Weight (kDa)Theoretical pIInstability IndexAliphatic IndexGrand Average of HydropathicitySubcellular Location
SmDIR1SMEL4.1_01g003540.115616.979.3645.5576.92−0.108Mitochondrion
SmDIR2SMEL4.1_01g003550.134336.236.1311.5495.540.171Plasma Membrane
SmDIR3SMEL4.1_01g031140.119121.339.5728.1479.69−0.017Plasma Membrane
SmDIR4SMEL4.1_02g003080.119021.069.6724.4294.950.065Mitochondrion
SmDIR5SMEL4.1_02g009260.118620.697.0636.8891.240.196Plasma Membrane
SmDIR6SMEL4.1_02g018810.119421.199.0828.4393.870.111Extracellular Space
SmDIR7SMEL4.1_04g004510.119021.379.4229.7591.790.022Plasma Membrane
SmDIR8SMEL4.1_05g003580.124427.035.9043.0879.88−0.167Plasma Membrane
SmDIR9SMEL4.1_05g003590.143844.864.3440.8087.990.075Extracellular Space
SmDIR10SMEL4.1_05g019870.124926.065.4636.5885.420.115Plasma Membrane
SmDIR11SMEL4.1_06g013950.114616.148.8227.0991.510.135Plasma Membrane
SmDIR12SMEL4.1_06g020190.121223.639.7329.4789.620.094Plasma Membrane
SmDIR13SMEL4.1_06g027190.137238.414.7526.3496.020.230Plasma Membrane
SmDIR14SMEL4.1_07g016780.117719.525.2227.9695.310.124Plasma Membrane
SmDIR15SMEL4.1_08g026590.119021.287.7913.3888.840.088Plasma Membrane
SmDIR16SMEL4.1_08g026600.116719.136.0226.4966.53−0.298Plasma Membrane
SmDIR17SMEL4.1_09g022870.154659.698.5238.7397.330.354Plasma Membrane
SmDIR18SMEL4.1_11g000960.116818.766.9015.6899.760.123Plasma Membrane
SmDIR19SMEL4.1_11g019830.117819.939.8535.35100.340.069Plasma Membrane
SmDIR20SMEL4.1_11g026460.136938.794.6342.9984.80−0.063Extracellular Space
SmDIR21SMEL4.1_12g003420.118420.199.5226.9890.110.123Mitochondrion
SmDIR22SMEL4.1_12g003430.119420.879.2422.53101.550.198Extracellular Space
SmDIR23SMEL4.1_12g003440.119321.216.9623.7493.370.130Extracellular Space
SmDIR24SMEL4.1_12g003620.119421.367.8719.2779.850.055Extracellular Space
Table
Table 2. The motifs information of DIR proteins in eggplant.
Table 2. The motifs information of DIR proteins in eggplant.
MotifSequenceNumber of Amino AcidPfam Annotation
motif 1FGTLTVIDDPLTIGPEPNSKJIGRAQGIYGSASQNGVSLLMALNFVFTGGKY52Dirigent
motif 2YREMAIVGGTGKFRLARGYATAKTYWFB28Dirigent
motif 3HAKEKVTKLHFYFHDILSGKNPTAIQIAQ29-
motif 4TGDAIVEYNVVVLHY15-
motif 5GSTLSILGRNPVFHE15-
motif 6MPTAQGIDLGPKAVEKWFKKL21-
motif 7MAIIQNSNSFQLKVIISYLLILALTITFATAGRILDEEVATPTVPPNNPDPTDQPPVSGATAAGAAAGAT70-
motif 8GQHTTDGVETILHITVYJTY20-
motif 9DSLSFFGVHR10-
motif 10IYSGQIPFATPLGFQPPEDGVAIPNANGAMPTFNINGVPLGTGLAGTIFAGGNN54-
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Zhang, K.; Xing, W.; Sheng, S.; Yang, D.; Zhen, F.; Jiang, H.; Yan, C.; Jia, L. Genome-Wide Identification and Expression Analysis of Eggplant DIR Gene Family in Response to Biotic and Abiotic Stresses. Horticulturae 2022, 8, 732. https://doi.org/10.3390/horticulturae8080732

AMA Style

Zhang K, Xing W, Sheng S, Yang D, Zhen F, Jiang H, Yan C, Jia L. Genome-Wide Identification and Expression Analysis of Eggplant DIR Gene Family in Response to Biotic and Abiotic Stresses. Horticulturae. 2022; 8(8):732. https://doi.org/10.3390/horticulturae8080732

Chicago/Turabian Style

Zhang, Kaijing, Wujun Xing, Suao Sheng, Dekun Yang, Fengxian Zhen, Haikun Jiang, Congsheng Yan, and Li Jia. 2022. "Genome-Wide Identification and Expression Analysis of Eggplant DIR Gene Family in Response to Biotic and Abiotic Stresses" Horticulturae 8, no. 8: 732. https://doi.org/10.3390/horticulturae8080732

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