Identification and Expression Analysis of Zinc Finger A20/AN1 Stress-Associated Genes SmSAP Responding to Abiotic Stress in Eggplant

Abstract

Stress-associated proteins (SAP), a class of zinc-finger proteins, have been identified as novel stress regulatory proteins in stress responses. However, SAP genes in eggplant (SmSAP) have been little reported. It has important significance in identifying SAP members, understanding the molecular mechanisms underlying stress responses, and tolerance. We performed a comprehensive study of the A20/AN1 domains, motifs, gene structures, phylogenetic relationships, chromosomal locations, gene replications, collinearity, cis-acting elements, and expression pattern responses to various abiotic stresses. Twenty-one SAP genes were identified in eggplant (SmSAP) and were localized on 10 chromosomes. A phylogenetic analysis revealed that most of the SmSAP proteins showed a high homology with the tomato SAP members, and 21 members were divided into four groups based on the homology of the SAP members in eggplant, tomato, rice, and Arabidopsis. Further analysis revealed that SmSAP proteins contain the characteristic A20/AN1 domains, the A20 domain composed of motif 2 (ILCINNCGFFGSPATMNLCSKCYKDMJLK). Four pairs of tandem duplications were found in eggplant, and 10 SmSAP genes had collinearity with SAP genes from Arabidopsis, potato, or tomato, but only four SmSAP genes were collinear with SAP genes in the three species mentioned above. Moreover, the promoters of SmSAP genes were predicted to contain many cis-acting elements that respond to abiotic stress and hormones. A qRT-PCR analysis of the four selected SmSAP genes exhibited diverse expression levels in response to various environmental stresses. These results provided a comprehensive analysis of the SmSAP genes and lay a solid foundation for improving the understanding of the functional diversification of SAP genes under various environmental stresses in eggplant.

1. Introduction

A variety of environmental factors, such as salinity, drought, and extreme temperatures, are important causes of great damage to crop yields and the ecological environment [1]. In order to respond to various environmental stresses and reduce or eliminate all kinds of harm to plants, plants have evolved complex molecular mechanisms in the modulation of stress-responsive gene expression patterns in the signal transduction processes under these stresses.

Stress-associated proteins (SAPs), a newly identified class of zinc-finger proteins (ZFPs) including the N-terminal A20 (ZnF-A20) domain and/or a C-terminal AN1(ZnF-AN1) domain, have also been identified as key factors in the regulation of different types of abiotic and biotic stresses [2,3]. Accumulated studies have shown that SAP gene family members are widely present in different plant species, such as 14 members in Arabidopsis [4], 18 in rice [4], 13 in tomato [5], 37 in cotton [6], 27 in soybean [1], and 17 in barley [7]. Moreover, increasing evidence has shown that the SAP family members in various species of plants play important roles responding to environmental stresses. The OsSAP1 gene, first discovered in rice, can be induced by cold, drought, salt, heavy metal, wounding, and abscisic acid (ABA) [8]. Furthermore, other OsSAP genes in rice have exhibited induced expression under one or the other stress conditions, which indicates that the OsSAP gene family in rice is an important part of the stress response [4]. Similarly, all SlSAP genes in tomato could be induced by one or other type of environmental stress [5]. In addition, many SAP genes have proven to be induced by multiple stresses and phytohormones in other crops, like cotton [6], soybean [1], Brassica napus [9], cucumber [10], and barley [7].

From the above results, SAP genes in various plants are associated with variable tolerances to multiple abiotic stresses [1,7,9]. Accumulated studies have shown that SAPs containing the N-terminal A20 domain and C-terminal AN1 domain have recently been identified as novel stress regulatory proteins in plants [2]. Moreover, the overexpression or heterologous expression of SAP genes in transgenic plants can markedly enhance their tolerance to abiotic stresses such as cold, high temperature, drought, salt, and heavy metals [11,12,13]. Overexpression of the OsSAP1 gene in tobacco and rice, respectively, confer abiotic stress tolerance by affecting the expression of several endogenous genes in transgenic plants [14,15]. Similarly, the gene OsSAP8 can also significantly enhance the stress resistance of transgenic tobacco and rice [16]. In contrast, the overexpression of OsiSAP7 in Arabidopsis negatively regulates ABA and water-deficient stress signaling by acting as an E3 ubiquitin ligase [12]. AtSAP5 is confirmed to have the same activity as the E3 ubiquitin ligase, but overexpressing the AtSAP5 gene enhances the tolerance to environmental challenges, including salt stress, osmotic stress, and water deficit, which indicate that AtSAP5 acts as a positive regulator of stress responses in Arabidopsis [17]. Moreover, the overexpression of wheat gene TaSAP5 confers drought tolerance in transgenic Arabidopsis and wheat by acting as an E3 ubiquitin ligase to promote the degradation of DRIP proteins and, thus, induce the expression of drought-responsive genes [18]. Increasing studies have confirmed that overexpression of the SAP gene family in transgenic plants can not only confer a strong resistance of drought, salt, and extreme temperature but also have a strong resistance toward heavy metals and disease [19,20].

As one of most widely cultivated vegetable crops in the world, eggplant is inevitably affected by environmental stresses in the process of growth and development. The SAP gene can quickly respond to a variety of abiotic stresses and improve plant tolerance to abiotic stresses, which is a good candidate gene to improve plant stress resistance [9]. Moreover, numerous studies have made extensive efforts to investigate the biological function and regulatory mechanism of the SAP gene family, involving plant growth and development regulation, biotic and abiotic stress defense responses, and hormone regulation in various plants [2,6]. However, little has been reported on SAP genes in eggplant. Consequently, further studies involved in the regulatory network, constituent factors, and upstream or downstream regulatory genes under adverse environmental conditions are urgently needed in eggplant. Therefore, exploring the function and molecular mechanism of the SmSAP genes have important theoretical and practical significance for cultivating eggplant varieties with strong stress resistance. Moreover, the recently released eggplant genome sequence data facilitated the whole-genome analysis of the SAP gene family.

In this study, we identified 21 SmSAP gene members through a genome-wide survey in the eggplant genome downloaded from the Sol Genomics Network. Then, we comprehensively studied their A20/AN1 domain, conserved motifs, gene structures, phylogenetic relationships, chromosomal locations, gene replication events, collinearity between eggplant and other species, and cis-acting elements. These results showed that the SmSAP proteins contained the characteristic A20/AN1 domains and showed high homology with SAP members from tomato and Arabidopsis, indicating that SmSAP proteins may function similarly to SAP proteins from other species responding to abiotic stress. Therefore, to identify the expression patterns of SmSAP genes in relation with abiotic stress responses, the four selected genes (SmSAP3, SmSAP4, SmSAP16, and SmSAP21) were detected by quantitative real-time PCR (qRT-PCR). Our results suggested that SmSAP genes are involved in the regulation of abiotic stress. In summary, our results not only provide a comprehensive understanding of SmSAP genes in eggplant but also lay the foundation for the functional studies of SmSAP genes and extending the applications of novel SmSAP candidate genes for crop improvement, especially in aspects of stress resistance.

2. Materials and Methods

2.1. Identification of SAP Family Members in the Eggplant

To identify SmSAP gene members in eggplant, we downloaded the genome assembly and annotation profile of S. melongena HQ-1315 from the Sol Genomics Network (SGN, http://solgenomics.net/, 8 September 2021, date last accessed) [21]. Simultaneously, based on the corresponding published research results [4,5], we respectively obtained the SAP protein sequences of Arabidopsis, rice, and tomato from the TAIR database (https://www.arabidopsis.org/index.jsp, 8 September 2021, date last accessed) and NCBI (https://www.ncbi.nlm.nih.gov/, 8 September 2021, date last accessed). Taking these sequences as the reference sequences, we searched the proteins in the eggplant genome. The E-value cutoff was set to 10^-5. Then, the candidate SmSAP proteins could be obtained by removing redundant and repetitive sequences. For further verification of the reliability, we searched the corresponding polypeptide sequences through BLASTP in the NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome, 10 September 2021, date last accessed) and then used the Pfam (http://pfam.xfam.org/, 10 September 2021, date last accessed), NCBI Conserved Domain Database (Batch CD search, https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi, 10 September 2021, date last accessed), and SMART database (http://smart.embl-heidelberg.de/, 10 September 2021, date last accessed), searching for the A20/AN1 domain. Finally, SmSAP proteins in eggplant with an N-terminal A20 (ZnF-A20) domain and/or a C-terminal AN1 (ZnF-AN1) domain were identified.

Moreover, we made use of ExPASy (https://www.expasy.org, 11 September 2021, date last accessed) to analyze the molecular weight (MW), theoretical isoelectric point (pI), aliphatic index, and instability index and also used ProtComp 9.0 (http://linux1.softberry.com/, 11 September 2021, date last accessed) to predict the subcellular localization of the SmSAP proteins.

2.2. Phylogenetic Analysis of SAP Family Members in the Eggplant and Other Species

To further interpret the taxonomy and function of the SAP members in eggplant, tomato, rice, and Arabidopsis, we constructed an unrooted phylogenetic tree using One Step Build, a ML tree in TBtools [22]. During the period, the plug-in aligned multiple sequences using the Muscle algorithm, trimmed the comparison results using trimAl, and automatically selected amino acid substitution models using the IQ-tree [23], then built the phylogenetic tree with the maximum-likelihood (ML) method. Finally, we selected Figtree software v1.4.4 to view the phylogenetic tree.

2.3. Analysis of Motifs, Domains and Gene Structure of SAP Family Members in Eggplant

To further identify the gene function of SmSAP, we analyzed the conserved motifs, conserved domains, and gene structure. In order to determine the conserved motifs, we searched all the SmSAP protein sequences using online software MEME (http://meme-suite.org/tools/meme, 12 September 2021, date last accessed) with the default parameters and set the number of motifs to 10. Furthermore, we searched the domains of the SmSAP proteins using the Pfam (http://pfam.xfam.org/, 12 September 2021, date last accessed), NCBI Conserved Domain Database (Batch CD search, https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi, 12 September 2021, date last accessed), and SMART database (http://smart.embl-heidelberg.de/, 12 September 2021, date last accessed). Moreover, we downloaded the annotation profile of the SmSAP genes and then exhibited the motifs, domains, and gene structure of each SmSAP member with TBtools [22].

2.4. Analysis of Location on Chromosomes, Gene Duplication, and Collinearity of SAP Family Genes in Eggplant

We obtained the annotation profile from the Sol Genomics Network (https://solgenomics.net/, 8 September 2021, date last accessed) [21]. Then, we placed the SmSAP genes on chromosomes using TBtools software, according to the annotation profile and gene distribution of the eggplant genome. Simultaneously, we analyzed the gene duplication events of SmSAP with TBtools [22]. Furthermore, based on the method reported in a previous study [24], we calculated the values of nonsynonymous (Ka) and synonymous (Ks) substitution rates of duplicated genes and, further, computed the approximate dates of the SmSAP gene duplication events. We later used MCScanX in TBtools to predict the collinearity of the SmSAP genes in eggplant and other species like Arabidopsis, tomato, potato, and rice.

2.5. Identification of Putative Cis-Elements in the Promoters of SmSAP Genes

We extracted the 2.0-kb upstream sequence of the start codon (ATG) for each candidate SmSAP gene from the eggplant genome sequence for identifying the cis-regulatory elements at the promoter regions. Then, we predicted these elements by using the PlantCARE server (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, 14 September 2021, date last accessed) and visualized these cis-elements with Simple BioSequence Viewer in TBtools [22].

2.6. Plant Material, Environmental Conditions and Abiotic Stress Treatments

The plant material we studied was the eggplant variety “Sanyueqie”. The seedlings were planted in the mixtures of organic matrix and vermiculite (3:1) at 25 °C with a light cycle of 16 h (day) and 8 h (night) in the incubator for 60 days. Then, we treated all the seedlings according to the methods in previously published papers [9] with slight modifications: (1) For cold stress, the plants were kept at 4 °C for 0, 1, 6, 12, 24, and 36 h. (2) For drought stress, the plants were irrigated with 20% PEG and then incubated for 0, 1, 6, 12, 24, and 36 h at 25 °C under a photoperiod of 16-h light/8-h dark. (3) For salt stress, the plants were dipped in 200-mM NaCl and cultured at 25 °C for 0, 1, 6, 12, 24, and 36 h with a 16-h light/8-h dark cycle. At different times, we collected the second and third completely expanded leaves counted from the top, and immediately placed them in liquid nitrogen, and then, these samples were stored at −80 °C for RNA extraction. In all abiotic stress treatments, three biological replications were performed independently.

2.7. RNA Extraction, and Expression Analysis

We used the RNAprep Pure Plant Kit (TIANGEN Biotech, Beijing, China) to extract the total RNA according to the manufacturer’s specifications and then performed quantification with a two-step reaction: reverse transcription (RT) and qPCR. Reverse transcription was performed in the 2720 Thermal Cycler (Applied Biosystems^TM, Waltham, CA, USA). The qPCR expression analysis was performed on a CFX96 Real-Time PCR (Bio-Rad, Hercules, CA, USA). At the end of the PCR reaction, a melting curve was performed to analyze the expected PCR product. Gene APRT from eggplant was used as the internal control [25]. The relevant primers are listed in Table S1. All reactions were performed three times. We calculated the relative expression values of the SmSAP genes with the 2^−ΔΔCt method [26].

2.8. Statistical Analysis

We used statistical software SPSS 25.0 (SPSS, Chicago, IL, USA) to analyze the data independently repeated three times under all stress treatments, with three seedlings at each time point. The level of significance was set at p < 0.05. The column charts were drawn with Excel 2019 software. Sample variability was given as the standard deviation (SD).

3. Results

3.1. Identification of SAP Gene Family Members in Eggplant

To identify SAP genes in the eggplant genome, we used the SAPs from rice, Arabidopsis, and tomato to search the predicted proteins in eggplant. In total, 21 SmSAP genes were identified in eggplant and were then numbered from SmSAP1 to SmSAP21, according to their chromosomal locations. These SmSAP genes encoded the proteins, with 66–368 amino acids (aa) in length, with MWs from 7.56 (SmSAP8) to 41.99 (SmSAP20) kDa (Table S2). The predicted isoelectric points and GRAVY values of the SmSAP proteins varied widely from 7.48 (SmSAP3) to 9.81 (SmSAP8) and −0.788 (SmSAP9) to −0.14 (SmSAP6), respectively, suggesting that all of them were alkaline and hydrophilic proteins. The predicted subcellular localizations of these proteins showed that 15 SmSAP proteins were located in the cytoplasm, 5 proteins in the extracellular, and 1 protein in the endoplasmic reticulum (Table S2).

3.2. Phylogenetic Analysis of SAP Genes

To investigate the classification and the evolutionary characteristics of the SAP proteins, we constructed an unrooted phylogenetic tree based on the SAP protein sequences of Arabidopsis, eggplant, tomato, and rice (Figure 1). All available 66 sequences, including 13 SlSAP, 14 AtSAP, 18 OsSAP, and 21 SmSAP, were mainly clustered into four groups. Group I contained 11 SmSAP members (SmSAP4, SmSAP5, SmSAP6, SmSAP7, SmSAP8, SmSAP9, SmSAP10, SmSAP11, SmSAP14, SmSAP17, and SmSAP20) clustered to AtSAP11/12/13/14 and SlSAP11/12/13. Group II consisted of four SmSAP members, SlSAP2/3/4/5, and OsSAP4/8. Group III was mainly composed of SmSAP1/13, SlSAP1/10, AtSAP5, and OsSAP1/11. Group IV included relatively many SAP members, and four SmSAP members (SmSAP12, SmSAP15, SmSAP16, and SmSAP19) were classified into this group, out of which SmSAP12 and SlSAP6, SmSAP15 and SlSAP8, SmSAP16 and SlSAP9, and SmSAP19 and SlSAP7 were clustered together, respectively. From the above results, it was predicted that the SAP members between eggplant and other species clustered together may have similar biological functions in response to various stresses.

3.3. Analysis of Motifs, Domains, Exons and Introns of SAP Family Members in Eggplant

In order to better comprehend the SmSAP gene function, we analyzed the motifs, domains, and intron–exon structures of the SmSAP members. We used the MEME tool to analyze the conserved motifs in the SmSAP proteins and obtained the motif distributions of these SmSAP proteins (Figure 2). As shown in Figure 2 and Table S3, a great majority of the SmSAP proteins contained motif 1 and motif 3, except for SmSAP4, SmSAP14, SmSAP17, and SmSAP20. However, some SmSAP proteins included unique motifs. For instance, motif 7 only appeared in SmSAP4 and SmSAP20, and motif 8 only appeared in SmSAP17 and SmSAP20. Furthermore, SmSAP14 had no additional motifs, except for motif 2 (Figure 2 and Table S3). Then, we analyzed the conserved domains of 21 putative SmSAP proteins by Pfam, the NCBI Conserved Domain Database (Batch CD search), and the SMART database. The results showed that 11 SmSAP proteins contained both an A20 and an AN1 domain, while the other nine SmSAP proteins included AN1 domains but no A20 domains, except for SmSAP14, with only a single A20 domain. In SmSAP proteins containing only AN1 domains, seven SmSAP proteins possessed only a single AN1 domain, and the remaining SmSAP4 and SmSAP20 proteins owned two AN1 domains. In addition, two C2H2 domains were also observed in SmSAP20, which had a similar structure in the other species, like SlSAP12 [5] and MdSAP15 [27]. From the above results, we discovered that the A20 domain was made up of motif 2 (ILCINNCGFFGSPATMNLCSKCYKDMJLK), while the AN1 domain was composed of motif 1 and motif 3 or motif 5 or motif 7 independently. Further, we analyzed the exon and intron compositions of the individual SmSAP genes to investigate the structural diversity of SmSAP genes. As shown in Figure 2, the vast majority of the SmSAP genes (17 out of 21) had no introns; the other SmSAP genes (SmSAP4, SmSAP14, SmSAP17, and SmSAP20) possessed one single intron.

3.4. Location on Chromosomes, Gene Duplication, and Collinearity Analysis of SAP Family Genes in Eggplant

On the basis of the annotated information of the eggplant genomic locations, 21 eggplant SAP genes were widely distributed on 10 chromosomes, except chromosomes E06 and E08 (Figure 3). Seven SmSAP genes, including SmSAP5–SmSAP11, in eggplant were gathered on chromosome E03, and three SmSAP genes (SmSAP1–SmSAP3) were clustered on chromosome E01. Similarly, three SmSAP genes (SmSAP16–SmSAP18) were also found on chromosome E10. In addition, only one gene was found on each of the other seven chromosomes.

Gene duplication events, containing tandem duplications, fragment duplications, and whole-genome duplications, are the major driving force in plant evolution. In order to better illustrate the duplication mechanism of SmSAP genes, we analyzed all the SmSAP gene sequences with the software TBtools. Among the SAP family members in eggplant, we identified four pairs of tandem duplications, including SmSAP1/SmSAP2, SmSAP6/SmSAP7, SmSAP8/SmSAP9, and SmSAP9/SmSAP10, but found no fragment duplication in Figure 3 and Table S4. Then, we calculated the Ka and Ks values for each duplicated SmSAP gene pair using TBtools. We discovered that the Ks values of the three gene pairs were between 0.107 and 0.705, except that the Ks value of one pair of tandem duplication (SmSAP1/SmSAP2) was not obtained. Moreover, the Ks values of the three pairs were less than 1. In addition, we discovered that the Ka/Ks values of the three pairs were less than 1, indicating that these genes evolved in the selection for strong purifying. Furthermore, we obtained that the duplication events of the SmSAP genes happened from about 20.593 Mya (Ks = 0.107) to 135.516 Mya (Ks = 0.705), with an average of 79.116 Mya (Ks = 0.411). Detailed information about the duplicated gene pairs, duplication type, Ka, Ks, Ka/Ks, and approximate duplication date (Mya) of each duplicated SmSAP gene pair is shown in Table S4.

In order to detect the homology of the SAP family genes, we analyzed the collinearity between eggplant and other species with the Dual Systeny Plot for MCscanX in TBtools. We found that there existed 4 pairs of collinearity between 4 SmSAP genes and 3 AtSAP genes, 10 pairs of collinearity between 8 SmSAP genes and 8 SlSAP genes, and 12 pairs of collinearity between 10 SmSAP genes and 10 StSAP genes (Figure 4). Furthermore, 10 SmSAP genes had collinearity with SAP genes from tomato, potato, or Arabidopsis. Four SmSAP genes (SmSAP4, SmSAP15, SmSAP16 and SmSAP20) were collinear with the SAP genes in the three species mentioned above. However, there is no collinearity between the eggplant SAP genes and rice SAP genes in Figure 4. Detailed information is listed in Table S5.

3.5. Cis-Elements Analysis in the Promoters of SmSAP Genes

To detect the cis-elements involved in the mediation of gene expression, we performed an analysis of the 2.0-kb upstream sequence of the start codon (ATG) for each candidate SmSAP gene. As shown in Figure S1 and Table S6, we predicted various cis-elements in relation with plant stress and hormone responses in the promoter regions of the SmSAP genes. It was discovered that the promoter region of the SmSAP9 gene contained the most cis-elements, followed by the SmSAP11 and SmSAP20 genes. Further analysis showed that the promoter regions of all the SmSAP genes contained the cis-elements involved in the anaerobic induction; then, 20 SmSAP promoters were predicted to exist in the cis-elements related to light response and 14 SmSAP promoters included the cis-elements in relation with the MeJA response and gibberellin response, respectively. However, relatively few SmSAP genes were found to include cis-elements in relation with auxin, low temperature, and circadian. From the above results, we learned that the SmSAP genes may play diverse roles in stress and hormone responses.

3.6. Expression Analysis of Four Selected SmSAP Genes under Abiotic Stress Treatments

In order to better explore the involvement of the SmSAP genes in response to abiotic stress, we selected four SmSAP genes based on our previous transcriptome data in the NCBI SRA database (Sequence Read Archive accessions SRR15036047, SRR15036048, SRR15036049, and SRR15036050). Under cold treatment (Figure 5 and Figure S2 and Table S7), genes SmSAP3, SmSAP4, and SmSAP16 were significantly upregulated; among them, the SmSAP3 gene was significantly induced only at 6 and12 h, but the SmSAP4 gene showed a significantly induced expression level at 12, 24, and 36 h and the SmSAP16 gene at 6, 12, and 24 h. On the contrary, gene SmSAP21 was significantly inhibited at different time points, except for 24 h. Under drought treatment, the expression levels of SmSAP3, SmSAP4, SmSAP16, and SmSAP21 were induced on the whole. Moreover, the expression pattern of genes SmSAP3 and SmSAP21 exhibited similar change tendencies (first increased and then declined), and gene SmSAP3 was induced significantly at 12 and 24 h, while gene SmSAP21 showed a significantly induced expression within 24 h. The other two genes, SmSAP4 and SmSAP16, exhibited similar changes (first increased, declined, and then increased), and the SmSAP4 gene was the most significant at 36 h, while the SmSAP16 gene displayed the most significantly at 6 and 36 h. As for salt treatment, gene SmSAP3 acquired a significantly induced expression level at 24 h, while SmSAP16 obtained significantly induced expression levels at different time points, except for 1 h, and the SmSAP4 gene emerged as a significantly induced expression only at 1 h. Instead, the SmSAP4 gene was significantly inhibited at 6 h, 12 h, and 24 h, and the SmSAP21 gene showed a significantly inhibited expression level only at 1 h and 6 h. In addition, these three genes did not reach a significant difference at other time points. From the above results, we learned that the selected SmSAP genes could be induced or inhibited by cold, drought, and salt stress.

4. Discussion

Stress-associated proteins (SAPs) with A20/AN1 zinc-finger domains have been identified as important regulators involved in immune responses in humans and stress responses in plants [2,28,29]. OsiSAP1, in rice, was first identified as a regulator in response to multiple stresses [8]; then, many SAP family members were successively identified and characterized as stress-response regulators in Gramineae rice, Arabidopsis, tomato, and other species [4,5,7,9,14]. Eggplant, as an important member of Solanaceae, is little reported on in studies about stress-associated proteins. The recently published eggplant sequencing data has provided a good opportunity for a genome-wide analysis of the entire SAP gene family in eggplant [21]. In our study, we identified 21 SAP genes in eggplant (Table S2), making this family much larger than previously reported in rice, Arabidopsis, and tomato [4,5].

Stress-associated proteins are found in lots of species. Based on previous studies, the phylogenetic analysis of the SAP proteins in Solanaceae plants showed that there was very little variation within the family [5]. In a phylogenetic tree of Arabidopsis, rice, tomato, and eggplant SAP proteins, our results showed that the majority of the SAP proteins from tomato show a high homology with the eggplant SAP family members, and 21 SmSAP proteins were simply divided into four groups (Figure 1).

The SAP proteins are characterized by the existence of the zinc-finger A20 or AN1 domains and are highly conserved in all plant species [2]. Our analysis revealed that most of the eggplant SAP proteins in the same group included one A20 and one AN1 domain, which is consistent with the results of the previous reports that the A20-AN1-type is the dominant arrangement among SAP gene family members in animals and plants [30]. However, we also observed that a few clades had some exceptions in the domain-wise grouping. For example, group I had most of the proteins containing one AN1 zinc-finger domain; two proteins in this clade had one A20 and one AN1 domain; and each of the other three proteins had only a single A20 domain, two AN1 domains, or two AN1 domains, along with two C2H2 domains, respectively (Figure 2). Similar results were also discovered in rice, soybean and Amborella trichopoda but not in Arabidopsis and tomato. These cases may be due to the homology of the proteins from the same clades beyond the domain sequences [5]. These results may also indicate that the characteristic AN1 domain has an ancient origin compared with the A20 domain and that the A20-type and AN1-type members have recently evolved from a loss of A20–AN1-type members, which is consist with the research of Brassica napus [9]. We further detected the conserved motif distributions of the eggplant SAP proteins based on their evolutionary relationships. In the present study, we identified 10 conserved motifs and discovered that their distributions exhibited strong evolutionary conservation. Moreover, we found that the A20 domain was made up of motif 2, while the AN1 domain was composed of motif 1 and motif 3 or motif 5 or motif 7 independently (Figure 2 and Table S3). Moreover, exon–structural diversity often provides valuable information for understanding the evolutionary mechanisms of gene families [31,32]. SAP members in different plant species usually have the characteristics of intron-free structures [1]. For example, the majority of the SAP genes in rice and apple include no introns; a few of the other genes may contain only a single intron [4,27]. Similar results were found in our study; 17 SmSAP genes had no introns, and the other four SmSAP genes clustered into group I had only a single intron (Figure 2). These may be attributed to the fact that intron-free gene families can reduce posttranscriptional processing and rapidly adjust transcript expression [33].

Additionally, we found that 21 SmSAP genes were localized on 10 chromosomes, except chromosomes E06 and E08 (Figure 3). This is similar to the findings in rice and cotton. Gene duplication occurs in the process of organism evolution and is considered to be critical for both the diversification of gene functions and the rearrangement and expansion of genomes [34,35]. In our study, four pairs of tandem duplications were found in eggplant, but no pairs of segmental duplications were identified. For example, two A20–AN1-type SmSAP genes (SmSAP6 and SmSAP7) were found to have originated from tandem duplication with highly similar sequences (Figure 2 and Figure 3). Our findings suggest that tandem duplications may lead to the expansion of the eggplant SAP gene family and might explain why more SAP genes are identified in eggplant than in any other Solanaceae species, which was similar to the results of the SAP genes in Brassica napus [9].

Accumulated studies have suggested that stress-associated proteins (SAPs) play important roles in the mediation of different types of abiotic and biotic stresses [1,7]. This information in regard to the potential roles of SAP genes responding to various abiotic stresses provided us with the motivation to investigate the expression patterns of SmSAP genes under diverse environmental treatments. Therefore, we performed an analysis of the expression patterns of the SmSAP genes responding to abiotic stress by qRT-PCR (Figure 5). Our results indicated that all selected SmSAP genes were responsive to one or various environmental stresses, but the SmSAP genes were a little different in response to cold, drought, and salt compared to tomato. For example, the SmSAP3 gene showed significantly upregulated changes in response to cold stress within 12 h, while the ortholog of gene SmSAP3 from the tomato SlSAP3 gene exhibited downregulated expression changes within 8 h [5]. As for drought stress, the SmSAP3 gene showed no significant changes within 6 h, which was basically consistent with the results of the SlSAP3 gene. In addition, the gene SmSAP3 showed no significant changes within 12 h, but the SlSAP3 gene had a significantly upregulated expression within 8 h [5]. Our results also imply that diverse expression patterns of the four selected SmSAP genes might be related to their specific structures or the particular stress, which was consistent with studies in other species [1,27]. Taken together, our study lays a foundation for exploring the molecular roles of SAP proteins and helps to further investigate the functions of these genes in abiotic stress responses.

5. Conclusions

In conclusion, we identified 21 SmSAP proteins containing the characteristic A20/AN1 domains in eggplant. The phylogenetic analysis showed that most of the SmSAP proteins were highly homologous to the tomato SAP members, and the 21 SmSAP proteins were divided into four groups. Further analysis demonstrated that the A20 domain was made up of motif 2, while the AN1 domain was composed of motif 1 and motif 3 or motif 5 or motif 7 independently. Simultaneously, our analysis found that 21 SmSAP genes were localized on 10 chromosomes, and four pairs of tandem duplications were identified in the eggplant genome; among them, the Ka/Ks values of three pairs were less than 1, indicating that these genes had evolved during the selection of strong purification. The collinearity analysis revealed that 10 SmSAP genes were collinear with the SAP genes from Arabidopsis, potato, and tomato, and four SmSAP genes (SmSAP4, SmSAP15, SmSAP16, and SmSAP20) were collinear with the SAP genes in the three species mentioned above. Furthermore, the promoter regions of the SmSAP genes were predicted to possess lots of cis-acting elements related to abiotic stress and hormone responses. A further qRT-PCR analysis confirmed that the four selected SmSAP genes showed diverse expression patterns, indicating their involvement in the regulation of cold, drought, and salt stresses.