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Article

Genome-Wide Exploration of the WD40 Gene Family in Eggplant (Solanum melongena L.) and Analysis of Its Function in Fruit Color Formation

by
Yanbo Yang
1,†,
Qihang Cai
2,3,†,
Yimei Wang
2,†,
Liping Li
4 and
Zhenghai Sun
2,3,*
1
College of Geography and Ecotourism, Southwest Forestry University, Kunming 650224, China
2
College of Landscape and Horticulture, Southwest Forestry University, Kunming 650224, China
3
Yunnan International Joint R&D Center for Intergrated Utilization of Ornamental Grass, International Technological Cooperation Base of High Effective Economic Forestry Cultivating of Yunnan Province, South and Southeast Asia Joint R&D Center of Economic Forest Full Industry Chain of Yunnan Province, College of Landscape and Horticulture, Southwest Forestry University, Kunming 650224, China
4
College of Wetland, Southwest Forestry University, Kunming 650224, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2024, 14(3), 521; https://doi.org/10.3390/agronomy14030521
Submission received: 25 January 2024 / Revised: 28 February 2024 / Accepted: 1 March 2024 / Published: 3 March 2024
(This article belongs to the Section Plant-Crop Biology and Biochemistry)
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Abstract

:
The WD40 gene family is a highly conserved protein family in plants that plays a crucial role in various life activities. Although eggplant (Solanum melongena L.) genome sequencing has been completed, there is limited research on the WD40 family in eggplant, and the regulatory mechanism of its involvement in anthocyanin synthesis remains poorly understood. The research identified the eggplant WD40 gene family, comprising 187 SmWD40 members that are unevenly distributed across 12 chromosomes of the eggplant. Phylogenetic analysis classified them into 11 subgroups, with members within the same subgroup having similar motifs and gene structures. The promoter of the SmWD40 genes contains a high number of light, stress, and hormone response elements. The expression patterns of 20 SmWD40 members of the S5 subgroup were analyzed during the formation of fruit color in long purple eggplant. Subsequently, we used virus-induced gene silencing (VIGS) to confirm the significance of the TTG1 (SmWD40-56) gene in subgroup S5 for anthocyanin synthesis in eggplant fruit. To investigate the molecular mechanism of SmWD40-56 in eggplant fruit color formation, we analyzed the expression patterns of structural genes for anthocyanin synthesis in eggplant fruit silenced for SmWD40-56. Finally, we predicted the protein interaction network of the SmWD40-56 gene to understand its potential regulatory mechanisms. The result showed that SmWD40-56 may regulate the structural genes involved in anthocyanin biosynthesis and plays an important role in eggplant fruit color formation. This study provides some basis for studying the mechanism of eggplant fruit color formation.

1. Introduction

Anthocyanin is a natural water-soluble pigment that belongs to a class of flavonoids [1,2,3]. It is widely distributed in most plant organs and plays a crucial role in the development of blue, purple, and red traits in plant tissues [1,4]. Anthocyanins are important indicators for evaluating fruit quality and contribute significantly to plant resistance against various stresses [5,6,7]. The regulation of anthocyanin synthesis is complex and involves both structural genes and transcription factors [8].
The MBW complex is a complex formed by the MYB transcription factor, helix-loop-helix (bHLH) transcription factor and a WD40-repeat protein [9,10], which is widely involved in flavonoid synthesis, morphogenesis, and resistance physiology in plants [11,12,13]. Anthocyanin regulation by MBW complexes is complex and sophisticated [14]. MBW complexes can regulate the structural genes of anthocyanin synthesis such as dihydroflavonol 4-reductase (DFR), leucoanthocyanidin dioxygenase (LDOX), anthocyanidin synthase (ANS), flavanone 3-hydroxylase (F3H), and so on, and thus play an important role in regulating anthocyanin synthesis [15]. In addition, the MBW complex can activate anthocyanin accumulation induced by jasmonic acid (JA) [16]. WD40 proteins are a type of transcription factor with unique structures [9]. They are one of the most diverse protein families in plants [17,18]. The WD40 domain is unique and folds into a β-propeller structure, making it one of the most diverse structures in the eukaryotic genome. This domain serves as a platform for protein interaction and assembly [19,20,21]. WD40 proteins play an active role in regulating anthocyanin synthesis by enhancing and activating gene expression. Transparent testa glabra 1 (TTG1) and AN11 belong to the WD40 gene family and are frequently reported as anthocyanin-regulated genes [22,23]. TTG1 and AN11 can bind to R2R3-MYB transcription factors such as MYB113/114 and polynucleotide adenylyltransferase 1/2 (PAP1/2), thereby regulating the expression levels of anthocyanin synthesizing structural genes such as ANS, F3H, and DFR, which ultimately have an impact on anthocyanin biosynthesis [24,25]. In Petunia hybrida Vilm, the PhAN11 mutants showed the reduced expression of DFR. Further studies in Arabidopsis have revealed that the homologous gene of AN11, AtTTG1, functions similarly to AN11 [23,26]. Several studies have highlighted the regulatory function of WD40 genes in the coloration of flowers and fruits. For example, the expression of the cotton TTG1 gene in the MiTTG1 mutant of Matthiola incana L. can partially restore some of the anthocyanin phenotypes [27]. Similarly, the apple MdTTG1 gene can form a complex with bHLH to initiate the transcription of downstream genes involved in anthocyanin synthesis, such as MdDFR and MdUFGT [28].
Eggplant (Solanum melongena L.) stands as a vital vegetable with global cultivation potential. The peel color of eggplant, a crucial quality trait, greatly influences the evaluation of fruit maturity and consumer preferences [29,30]. The peel color of eggplant exhibits a diverse range, including common purple, white, green, and other colors. Research indicates a direct correlation between eggplant fruit color and the contents of anthocyanin and chlorophyll. A higher anthocyanin content results in a darker peel color, and the fruit is green when anthocyanin content is low, or white when chlorophyll content is also low [31,32]. The molecular mechanism of eggplant fruit color formation has been a hot topic of research. Expression analyses of structural (CHS, DFR and ANS) and regulatory genes (Myc, MYbB and MYbC) for anthocyanin synthesis in eggplants of different fruit colors revealed that the expression levels of anthocyanin biosynthesis genes were higher in purple fruits than in white fruits, with the upregulation of Myc and MYbC genes showing a trend consistent with that of anthocyanin biosynthesis genes [33]. Expression profiling of the structural genes for anthocyanin synthesis in eggplants (SmCHS, SmCHI, SmF3H, SmDFR, and SmANS) for different tissues, light conditions, and temperatures revealed that all genes were expressed at higher levels in the pericarp tissues except SmF3H; the expressions of SmCHI and SmANS were completely dependent on light. At low temperatures, all the structural genes were upregulated to a certain extent [34]. The MBW complex also plays an important role in fruit coloration in eggplant, with the MYB transcription factor being the most intensively studied. MYB transcription factors such as SmMYB1, SmMYB2, SmMYB75, SmMYB44, SmMYB86, and SmMYB35 have been identified to contribute to anthocyanin synthesis and accumulation in eggplant [35,36,37,38]. Some MYB transcription factors, like SmMYB86, inhibited anthocyanin synthesis by binding to the promoters of SmCHS, SmF3H, and SmANS. Silencing SmMYB86 can significantly increase the accumulation level of anthocyanin accumulation [39]. In eggplant, the bHLH regulation of anthocyanin synthesis involves two main branches. One branch involves AN1, which activates anthocyanin biosynthesis through the MYB-AN1-WD40 (MBW) complex, while the other branch involves JAF13, which forms the MYB-JAF13-WD40 (MBW) complex to regulate downstream AN1, thereby activating anthocyanin biosynthesis [40]. WD40 provides a stable platform for MYB and bHLH proteins in the MBW complex. The overexpression of the WD40 protein STAN11 in potato upregulates the STDFR gene and increases anthocyanin accumulation [41]. In tomato, SlAN11 can interact with bHLH transcription factors (SLT8 and SLGL3) to form an MBW complex with SLANT or SLGL3, thereby regulating SlDFR expression and anthocyanin synthesis [42].
In recent years, with the continuous increase in people’s demand for healthy food and product quality, more and more attention has been paid to the influence of anthocyanin content on the quality of eggplant fruit, and the research on the coloring mechanism of eggplant fruit has been deepened [43,44]. Currently, research on the mechanisms of color formation in eggplant is primarily focused on the structural genes that encode synthases and transcription factors within the MBW complex [45]. While studies on anthocyanin synthetase and the MBW complex involving the MYB and bHLH gene families have reached a certain level of maturity, so far there have been fewer studies on WD40 in eggplant [35,46]. Further exploration is warranted on the intricate interactions between WD40, MYB, and bHLH in regulating anthocyanin synthesis and influencing eggplant fruit color. Therefore, the comprehensive analysis of the WD40 family in eggplant assumes great importance in unraveling the mechanisms behind anthocyanin synthesis in eggplant fruit and enhancing the quality of eggplant. In this research, 187 members of the eggplant WD40 family were identified based on genome-wide data. The physicochemical properties analysis, phylogenetic analysis, chromosome mapping, cis-acting element analysis, and collinearity analysis were performed. This research examined the expression pattern of WD40 in different growth stages of long purple eggplant fruits and validated the function of the SmTTG1 (SmWD56) gene through virus-induced gene silencing (VIGS). Additionally, expression levels of several primary structural genes in anthocyanin synthesis were measured after SmTTG1 (SmWD40-56) silencing, and the interaction protein of the SmWD40-56 gene was predicted to delve into its role in anthocyanin synthesis and fruit color formation in eggplant. In conclusion, this study provides a thorough analysis of the eggplant WD40 family. The results are significant for the investigation of eggplant fruit color formation and the cultivation of superior varieties.

2. Materials and Methods

2.1. Identification and Physicochemical Property of SmWD40 Gene Family

Genomic data for eggplant were sourced from the eggplant genome database (http://www.eggplant-hq.cn/Eggplant) (accessed on 8 August 2023). The Hidden Markov Model (HMM) file (PF00400) of the WD40 conserved domain was obtained by downloading from Pfam (http://pfam-legacy.xfam.org) (accessed on 8 August 2023) to characterize the SmWD40 members. Protein sequence analysis of candidate genes was conducted using NCBI’s CD-Search functional (https://www.ncbi.nlm.nih.gov) (accessed on 8 August 2023) analysis tool to screen for SmWD40 members with complete WD40 structures. The final set of SmWD40 members was derived by excluding structurally incomplete genes. Subsequently, SmWD40 protein sequences were submitted for an analysis of the physicochemical properties of SmWD40 members to ExpasyProteparam (https://web.expasy.org/proteparam/) (accessed on 8 August 2023).

2.2. Chromosomal Location and Phylogenetic Analysis of SmWD40

The chromosome localization of SmWD40 members was performed by TBtools v2.052 software [47]. The Arabidopsis WD40 member protein sequence was obtained from TAIR (https://www.arabidopsis.org/) (accessed on 10 August 2023). The WD40 gene family protein sequences of eggplant and Arabidopsis were aligned by MEGA11 v11.0.10 software. The phylogenetic tree was constructed using the NJ method, and the bootstrap value was set at 1000.

2.3. Conserved Motif Analysis and Cis-Acting Element Analysis of SmWD40

MEME (https://meme-suite.org/meme/) (accessed on 14 August 2023) was utilized to analyze the motif characteristics of SmWD40 members, while their conserved domain information underwent analysis through the CD-Search function of NCBI (https://www.ncbi.nlm.nih.gov) (accessed on 14 August 2023). Upstream 2000 bp nucleic acid sequences from each SmWD40 member were extracted and uploaded to PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (accessed on 14 August 2023) for cis-acting element analysis in promoter regions. The conserved motifs and cis-acting elements, combined with phylogenetic relationships, were visually presented by the TBtools v2.052 software [48].

2.4. Collinearity Analysis of SmWD40

SmWD40 collinearity analysis was performed by the MCScanX plug-in in TBtools v2.052 software, and the collinearity relationship was visualized by TBtools v2.052 software [48].

2.5. Plant Materials and Treatments

The eggplant used in this experiment was the long purple eggplant (9211: A strain from Solanum melongena cv. Kmzc-7), the seeds of which were saved by our research group. At the beginning of the experiment, the seeds were soaked in distilled water for 24 h, and then the seeds were germinated in 15 mm glass petri dishes lined with moistened filter paper. Subsequently, they were sown in peat and vermiculite mixed medium (3:1), and then transplanted to a 15 cm diameter planting pot where they grew to 2 pairs of true leaves. Next, they were cultivated in the planting base of Southwest Forestry University (102.454308° E, 25.035229° N), with a temperature of 25 °C, relative humidity maintained at 60–70%, and a light–dark cycle of 14 h to 10 h. After the flower buds appeared, they were utilized for subsequent experiments.
To investigate the potential mechanism of the S5 subgroup in eggplant fruit color formation, gene expression levels in eggplant peel (0.5–0.8 mm) were analyzed by qRT-PCR at 5, 15, and 25 d after the end of flowering. The stage I (5 d) represents the early phase of eggplant fruit development, when the fruit color was full green. The stage II (15 d) refers to the middle stage of eggplant fruit development, when the fruit color began to appear purple, and there were still large areas of green. In stage III (25 d), the eggplant fruit became mature, and the fruit was covered with purple. The surface of the fruit was washed with ddH2O and frozen in liquid nitrogen, and then stored at −80 °C.

2.6. Virus-Induced Gene Silencing

The experimental treatments were slightly adapted based on previous research on eggplant [49]. When the eggplant material grew to the early stage of flower bud development, two pairs of mature leaves below the flower bud were infected by injection. The bacterial solution had a concentration of OD600 = 0.8, with equal proportions of TRV1 and TRV2 bacterial solutions mixed in equal proportions 1 h prior injection. To control the experimental variables, two control groups were established, consisting of empty TRV2 and H2O. When the eggplant fruit reached maturity, TRV2 detection primers were adopted to assess the virus transmission within the fruit, and the amplified fragment length was determined using the 5000 bp marker (Biomed Biotech Co., Ltd., Beijing, China). The expression level of SmTTG1 was detected by qRT-PCR.

2.7. Analysis of Anthocyanin Synthesis Structural Gene Expression Level

The anthocyanin synthesis structural genes in eggplant were acquired from the eggplant genome database (http://www.eggplant-hq.cn/Eggplant) (accessed on 12 September 2023). Expression levels of structural genes were measured by qRT-PCR, including CHS (Smechr0500387), CHI (Smechr0500261), F3H (Smechr0101736), DFR (Smechr0202337), flavonol synthase (FLS, Smechr0401078), and PAL (Smechr0400589).

2.8. qRT-PCR and Viral Vector Construction

RNA was extracted using FastPure Universal Plant Total RNA Isolation Kit (Vazyme Biotech Co., Ltd., Nanjing, China). RNA integrity was detected using 1% agarose gel electrophoresis, and the OD260/280 values of RNA were detected using a nucleic acid proteometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) to ensure that the quality of RNA met the requirements of subsequent experiments. Subsequently, RNA was reverse-transcribed into cDNA using All-In-One 5X RT MasterMix (Applied Biological Materials Inc., Shanghai, China). The qRT-PCR primers were designed using the primer-Blast function of NCBI database (Table A1). To prevent the presence of gDNA contamination in RNA, negative control experiments were performed during qRT-PCR using unreversed transcribed RNA as a template, and no contamination was considered to have occurred with no significant amplification of the negative control. qRT-PCR was performed using ArtiCanCEO SYBR qPCR Mix (Tsingke Biotechnology Co., Ltd., Beijing, China). The qRT-PCR reaction process was as follows: pre-denaturation at 95 °C for 1 min, followed by cycling for 10 s at 95 °C and 20 s at 60 °C for 40 cycles. Fluorescence was collected at the 60 °C reaction step. The Actin gene (Smechr1001310.1) served as the internal reference for a calculation of the gene relative expression using 2−ΔΔCt. The primer amplification efficiencies for qRT-PCR are shown in Table A1. The VIGS experiment was performed using tobacco rattle virus vectors. The 300 bp length fragment was identified as the silencing fragment using the VIGS online prediction tool (https://vigs.solgenomics.net/) (accessed on 16 August 2023), and the target fragment was amplified by MegaFi TM Fidelity 2X PCR MasterMix (Applied Biological Materials Inc., Shanghai, China). TRV2 was linearized using KpnI endonuclease (New England Biolabs Inc., Beijing, China). The ClonExpress II One Step Cloning Kit (Vazyme Biotech Co., Ltd., Nanjing, China) was employed to homologously recombine the ligated fragment and linearized vector. Leveraging recombinant plasmids, E. coli (Top 10) was transformed, and the bacterial solution was sequenced at Tsingke Biotech Co., Ltd. (Tsingke Biotechnology Co., Ltd., Beijing, China). Using the Trelief® reg Plasmid Mini Kit Plus (Beijing Tsingke Biotech Co., Ltd.) kit, the correct bacterial liquid vector was extracted, and the agrobacterium (GV3101) was transformed by the thermal stimulation method.

2.9. Interaction Network Analysis of SmWD40-56

To investigate the potential regulatory mechanism of SmWD40-56 in eggplant, we performed protein sequencing of the eggplant whole genome interaction protein by SmWD40-56 via STRING (https://cn.string-db.org) (accessed on 3 October 2023).

3. Results

3.1. Identification of the WD40 Genes in Eggplant

WD40 members were systematically identified in the entire eggplant genome utilizing a Hidden Markov Model. After manually removing the ineligible genes, we obtained a total of 187 compliant WD40 genes. The genes were designated as SmWD40-1 through SmWD40-187 in the order of their arrangement on the chromosomes. An analysis of physicochemical properties revealed the diversity within the SmWD40 protein family. The length of SmWD40 proteins varied from 169 amino acids (SmWD40-164) to 3249 amino acids (SmWD40-83), while the molecular weight ranged from 18,088.63 Da (SmWD40-164) to 364,048.07 Da (SmWD40-83). The analysis of the theoretical isoelectric point (pI) revealed that 125 SmWD40 proteins have a pI value of less than 7, indicating a negative charge in an alkaline pH environment. One protein (SmWD40-143) had a pI of 7, indicating no charge in a neutral environment, and 61 proteins exhibited a pI value greater than 7, suggesting a positive charge in an acidic pH range. Instability index analysis categorized 81 SmWD40 proteins as stable, with an instability coefficient of below 40, while the remaining 106 SmWD40 proteins were deemed unstable, boasting an instability coefficient surpassing 40. Aliphatic Index predictions ranged from 54.81 (SmWD40-141) to 103.1 (SmWD40-168). The grand average of hydropathicity prediction revealed that with the exception of SmWD40-65, SmWD40-78, and SmWD40-168, the grand average of hydropathicity for the remaining 184 SmWD40 proteins was less than 1, indicating a hydrophilic nature (Table A2).

3.2. Chromosome Localization of SmWD40 Genes

The analysis of chromosomal localization for the 187 identified WD40 genes indicated that the SmWD40 gene family members are distributed across 12 chromosomes, primarily concentrated at the chromosome ends. The analysis of gene density further indicated that the SmWD40 genes are mainly located in regions with high gene density on the chromosomes. Chromosomes 01, 02, and 03 have a higher abundance of SmWD40 genes, with 29, 19, and 27 members, respectively. In contrast, the numbers of SmWD40 genes on chromosomes 09, 11, and 12 were relatively low, with 10, 10, and 6 genes, respectively (Figure 1).

3.3. Phylogenetic Analysis of SmWD40 Proteins

To investigate the phylogenetic relationships within the SmWD40 gene family, the phylogenetic tree of 187 WD40 members in eggplant and 162 in Arabidopsis was constructed using the neighbor-joining (NJ) method (Figure 2). The study revealed that the phylogenetic relationship can be divided into 11 distinct subgroups. Subgroups S1 and S9 had the largest numbers of members (44), while subgroup S8 had the least (20). Notably, each subgroup included both eggplant and Arabidopsis proteins; this result indicates that WD40 is highly conserved in eggplant and Arabidopsis.

3.4. Conserved Domains and Cis-Acting Elements Prediction of SmWD40s

Motifs and conserved domains in SmWD40 were integrated based on phylogenetic relationships for further analysis. The results reveal a distribution of similarities in motifs and domains among members of the same subgroup in phylogenetic development, with motif 1 being the most prevalent across the majority of members (Figure 3A). The conserved domain analysis (Figure 3B) demonstrated that all members exhibited the WD40-specific domain, confirming the accurate identification of SmWD40 members. In addition, a parallel distribution of motif 10 and the CTLH domain regions was observed in the subgroups S3 and S5, suggesting that there may be functional similarity related to the structure.
Analyzing cis-acting elements in the upstream 2000 bp promoter region of the SmWD40 genes (Figure 3C) showed that light response elements were the most abundant among SmWD40 promoters. Among the hormone response elements, MeJA response elements were the most prevalent, with SmWD40-135 having the largest number of members (20). SmWD40-50 exhibited the largest number of auxin response elements (four members), while SmWD40-168 and SmW40-162 had the largest numbers of gibberellin response elements (five members). Among the salicylic acid response elements, SmWD40-3, SmWD40-20, and SmWD40-177 had the highest counts, with three members each. SmWD40-41 had the largest number of abscisic acid response elements, with nine members.

3.5. Collinearity Analysis of SmWD40 Genes

In order to understand the amplification mechanism of SmWD40 genes, the collinearities of SmWD40 genes were analyzed (Figure 4). The results show that there were 16 pairs of fragment duplication genes in SmWD40. Collinear gene pairs were distributed across most chromosomes, except Chr10 and Chr11, with a predominant concentration in high-gene-density regions at both ends of the chromosomes. Notably, SmWD40-121 showed a collinear relationship with SmWD40-112 and SmWD40-43, and SmWD40-94 displayed collinear relationships with SmWD40-81 and SmWD40-58. Meanwhile, SmWD40-71 was collinearly associated with SmWD40-7, SmWD40-33, and SmWD40-84, and SmWD40-43 exhibited a collinear correlation with both SmWD40-121 and SmWD40-90.

3.6. Expression Pattern Analysis of S5 Subgroup Genes in Fruit Color Development

Previous studies underscored the pivotal role of WD40 in the synthesis and accumulation of anthocyanins in plants. Current studies in Arabidopsis have shown that several genes in the S5 subgroup, such as AT5G24520 (AtTTG1) and AT1G12910 (AtAN11), have regulatory effects on anthocyanins. There may be similarities in gene function in the same subgroups [11], so we hypothesize that the SmWD40 genes in the S5 subgroup may be involved in the regulation of anthocyanin synthesis in eggplant. To explore the potential influence mechanism of the SmWD40 family in eggplant fruit color formation, we analyzed the expression patterns of S5 subgroup genes in eggplant fruits at different growth times (5 d, 15 d, 25 d) after the end of flowering by qRT-PCR (Figure 5A). The results show that these SmWD40 members exhibited different expression patterns (Figure 5B). To be specific, two distinct categories emerged: Firstly, genes exhibiting high levels in stage I and subsequently decreasing expression in stages II and III, such as SmWD40-21, SmWD40-27, SmWD40-43, SmWD40-56, SmWD40-58, SmWD40-70, SmWD40-128, SmWD40-145, SmWD40-153, SmWD40-160 and SmWD40-182. It is suggested that these genes have a potential regulatory role in the initiation of anthocyanin synthesis in eggplant fruits. On the other hand, genes with lower expressions in stage I but elevated expressions in stages II and III were identified. Examples include SmWD40-30, SmWD40-92, SmWD40-105, SmWD40-106, SmWD40-132, SmWD40-152 and SmWD40-181. These genes may exert potential regulatory functions in pigment accumulation and transport in eggplant fruits.

3.7. Analysis of the Effects of Silencing SmWD40-56 on Fruit Color and the Expression Pattern of Structural Genes for Anthocyanin Synthesis after Silencing

TTG1 plays a crucial role in anthocyanin formation in various plant species. Based on its expression patterns, the eggplant TTG1 gene, known as SmWD40-56, may contribute to the early stages of anthocyanin formation in eggplant fruit. To investigate the regulatory role of the TTG1 gene in anthocyanin synthesis in eggplant fruits, we reduced the expression levels of SmWD40-56 genes using VIGS technology. Detection primers specific to tobacco rattle virus (TRV) were used to assess mature fruits of infected plants. The results show that both Empty TRV2-infected plants and TRV2-SmWD40-56 were able to detect the amplification bands of TRV. Noticeably, the amplified fragments of TRV2-SmWD40-56 were approximately 300 bp longer in comparison to those of Empty TRV2 (Figure 6A). This observation confirms that the TRV was successfully transmitted in eggplant fruits, and that the SmWD40-56 silenced fragment was successfully transmitted with viral vectors.
After the qRT-PCR assay, it was evident that VIGS significantly downregulated the expression of the SmWD40-56 gene in eggplant fruits (Figure 6B). The colors of the mature fruits in plants with silenced SmWD40-56 were significantly different from those of the control plants (Figure 6C). The analysis focused on the expression levels of structural genes responsible for anthocyanin synthesis in fruit that had been silenced for SmWD40-56. The results show that chalcone-flavanone isomerase (CHI), chalcone synthase (CHS), F3H, and DFR expression levels decreased significantly after SmWD40-56 silencing, indicating that SmWD40-56 has a potential regulatory effect on these anthocyanin synthases (Figure 6D).

3.8. Protein Interaction Network of SmWD40-56 in Eggplant

To investigate the regulation mechanism of SmWD40-56, we used STRING to predict the interaction network of the SmWD40-56 protein in eggplant (Figure 7). The results show that SmWD40-56 interacted with genes related to flavonoid synthesis (Smechr0501995.1, Smechr0202337.1, Smechr1000237.1, Smechr0500261.1, Smechr0202240.1, Smechr0302994.1 and Smechr0401078.1), the anthocyanin-containing compound metabolic process (Smechr0401078.1, Smechr1000237.1, Smechr0202337.1) and the jasmonic acid mediated signaling pathway (Smechr0802367.1, Smechr0101484.1). In addition, SmWD40-56 interacted with genes related to pathways such as cell differentiation, protein ubiquitination, and the regulation of biosynthesis processes. The KEGG pathway prediction for the interacting proteins indicates their potential involvement in flavonoid biosynthesis. Local cluster analysis highlighted a group of interacting genes including SmWD40-56 and Smechr0202337.1, Smechr0302994.1, Smechr0202240.1, Smechr0500261.1, as well as Smechr0401078.1. This group may regulate plant anthocyanin synthesis through its naringenin 3-dioxygenase activity, chalcone isomerase activity, L-ascorbic acid binding, and oxidoreductase activity.

4. Discussion

At present, with the advancement in sequencing technology, the genome-wide identification of numerous gene families has been successfully conducted in a variety of plants. However, the eggplant WD40 gene family is still unclear [50]. In this study, the WD40 gene family of eggplant was characterized at the genome-wide level and subjected to the analysis of predicted physicochemical properties of proteins, sequence characterization, chromosomal localization, cis-acting element analysis, and gene duplication events. Expression patterns for members in subgroup S5 of the long purple eggplant fruit were investigated at distinct stages of color formation using qRT-PCR. It was found that these genes have complex expression patterns during fruit development. The eggplant TTG1 gene (SmWD40-56) is highly expressed in the early stages of fruit color formation and has potential regulatory functions in the initiation of anthocyanin synthesis. Finally, we used VIGS technology to verify the function of SmWD40-56 gene. Expression levels of structural genes in anthocyanin synthesis in eggplant fruits after silencing were analyzed by qRT-PCR, and we analyzed the protein interaction network of the SmWD40-56 gene. The silencing of the SmWD40-56 gene resulted in significant changes in the color of long purple eggplant fruits and a significant decrease in the expression levels of CHS, CHI, F3H and DFR. Combined with protein interaction network analysis, it was found that the SmWD40-56 gene may affect eggplant fruit color by interacting with multiple genes and thus regulating the expression levels of structural genes for anthocyanin synthesis.
Anthocyanins hold great potential in many aspects, such as human health and improving the ornamental value of plants [51,52]. Meanwhile, they find widespread applications in horticultural traits, food production, and health care [47,53,54]. In addition, anthocyanins are extremely critical in addressing adverse environmental conditions like drought and hypothermia [55,56]. Consequently, anthocyanin biosynthesis remains a focal point of research [57,58,59]. The crucial involvement of R2R3-MYB, bHLH, and WD40 in anthocyanin synthesis is well-established [60,61,62]. As transcription factors, they collectively form the MBW complex and regulate gene expression in anthocyanin biosynthetic pathways by binding to corresponding cis-acting elements in the promoters of structural gene [63,64]. The previous study identified the WD40 gene family in different types of plants. Specifically, in tomatoes, 207 WD40 members were identified and classified into 12 subgroups, and the analysis revealed that the WD40 protein may be involved in tomato fruit development [65]. A total of 258 WD40 genes were identified in Rhododendron simsii Planch, and transcriptome analysis revealed that these genes play potential regulatory roles in anthocyanin synthesis [66]. A total of 178 WD40 genes were identified in the potato genome, among which TTG1 and its interactions with proteins play an important role in potato anthocyanin synthesis [67]. Previous studies have highlighted the significance of TTG1, a member of the WD40 family, in anthocyanin biosynthesis in different plants, such as fabe bean [68], pomegranate [69], and Arabidopsis [70]. This research analyzed cis-acting elements and found that SmWD40 contains numerous light-, hormone-, and stress-response elements. This suggests that SmWD40 plays a significant role in eggplant photomorphogenesis, hormone response, and resistance to physiological processes. These findings are consistent with previous research on anthocyanin function [57,71,72]. The examination of the SmWD40 gene at different stages of eggplant color formation highlighted the potential regulatory role of members from subgroup S5 in fruit anthocyanin formation. The significance of SmWD40-56 in the formation of anthocyanin in eggplants was subsequently confirmed by employing the VIGS of the eggplant TTG1 gene (SmWD40-56).
At present, the process of synthesizing anthocyanin in plants is well understood, and it is a branch of the flavonoid synthesis pathway. Phenylalanine serves as the precursor for anthocyanin synthesis [51,73]. Under the influence of enzymes like phenylalanine ammonia-lyase (PAL) and CHS, phenylalanine transforms into chalcone [74]. Chalcone, in turn, forms naringenin through the activity of CHI, and further converts to dihydrokaempferol under the action of F3H [75]. Eventually, dihydrokaempferol undergoes a series of reactions catalyzed by DFR, leading to the formation of leucoanthocyanidin [74]. In this study, the protein interaction network analysis of the SmWD40-56 gene showed that this gene was highly correlated with the synthesis of chalcone and naringenin. Moreover, gene expression levels of CHS, CHI, F3H and DFR were significantly reduced after the silencing of SmWD40-56. Therefore, it could be speculated that the SmWD40-56 gene may interact with the above proteins to regulate and activate anthocyanin synthesis-related genes, thereby affecting the color formation of eggplant fruits.

5. Conclusions

In this study, 187 WD40 genes were identified in the whole genome of eggplant from the HMM file, which were irregularly distributed on 12 chromosomes of eggplant. Phylogenetic analysis showed that these SmWD40 proteins could be categorized into 11 subgroups. Gene motif and conserved domain analyses indicated similarities in gene structure among the same subgroups of phylogeny, suggesting the possibility of similar gene functions. The analysis of cis-acting elements revealed that all SmWD40 genes contain light-responsive elements, indicating a strong correlation between their gene expression and light. In addition, a large number of hormone-responsive elements and resistance-responsive elements were identified, suggesting that SmWD40 is regulated by a complex signaling network involved in the physiological process of resistance in eggplant. The analysis of the expression pattern of the S5 subgroup of genes during eggplant fruit development suggests that this subgroup of genes may play a regulatory role in processes such as the initiation of eggplant fruit color formation and anthocyanin accumulation. After applying VIGS technology to decrease the expression levels of SmWD40-56 genes in long purple eggplant fruit, the intensity of purple color in the fruits decreased significantly. The qRT-PCR results indicate that silencing SmWD40-56 significantly reduced the expression levels of CHS, CHI, F3H, and DFR genes. Additionally, protein interaction network analysis revealed a strong correlation between the SmWD40-56 gene and anthocyanin synthesis. This study serves as a reference for comprehending the mechanism of eggplant fruit color formation and enhancing eggplant quality.

Author Contributions

Conceptualization, Y.Y., Q.C. and Z.S.; formal analysis, Y.Y. and Q.C.; investigation, Y.Y., Q.C. and Y.W.; methodology, Y.Y., Q.C., Y.W., Z.S. and L.L.; project administration, Y.Y. and Q.C.; resources, Z.S. and L.L.; supervision, Z.S. and L.L.; validation, Y.Y. and Q.C.; visualization, Y.Y. and Q.C.; writing—original draft, Y.Y.; writing—review and editing, Y.Y., Q.C., Z.S. and L.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by The Key Research and Development Program of Yunnan Province (202202AEO90012-04), International cooperation project of Yunnan Province (2019IB011).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

Appendix A

Table A1. The primer sequences used in this research.
Table A1. The primer sequences used in this research.
Primer NameGene IDForward Primer (5′->3′)Reverse Primer (5′->3′)FunctionAmplification Efficiency
SmWD40-21Smechr0102060.1GGATGGGGATGCAAGGACAACATGCATCAAGTTCTCGCCGSmWD40-21 qRT-PCR0.9612
SmWD40-27Smechr0102379.1TGGAATAGGTGTGGGGGACTTGTGCATAACCACCAAACTGGSmWD40-27 qRT-PCR0.9956
SmWD40-30Smechr0200037.1AGAGGAATCTGGGGTTGGGAAGCCAAACGGTAAGGGACAGSmWD40-30 qRT-PCR0.9835
SmWD40-43Smechr0202538.1ATGACTTCACCTACCGAGCCAGAAGGGATCCATCGGAAACGSmWD40-43 qRT-PCR0.9744
SmWD40-56Smechr0302292.1TGAGTGCCGAAAATAACAATAAAACGGGCTATCAATTGGGTTTCGACSmWD40-56 qRT-PCR0.9725
SmWD40-58Smechr0302644.1ACAGTCATGTTACCGCAGGTTAGGTAGCCAGAGGTTGACCASmWD40-58 qRT-PCR0.9882
SmWD40-70Smechr0303426.1GCTGGGACCTCTCGGTCAACCATCGGCATGTGTCTCTTSmWD40-70 qRT-PCR0.9673
SmWD40-92Smechr0500827.1AGCAGGTTCTAGCTGGGATGAGGGCCTGTTCAACGAGATGSmWD40-92 qRT-PCR0.9956
SmWD40-105Smechr0502667.1TGATATTTCATCCCTGCACGAAGGCCTTTTCCAGGTGAGTCSmWD40-105 qRT-PCR1.0014
SmWD40-106Smechr0600010.1CATCGGTCGACCATTCGAGATTTGGAGGGAGTGGTGCTTCSmWD40-106 qRT-PCR0.9892
SmWD40-108Smechr0600126.1TTTGCTCCTTGGCACAACAATGCAACTTTCTCGTCTGCCCSmWD40-108 qRT-PCR0.9766
SmWD40-128Smechr0701564.1TGCAAAAGATTGGTCTGGTGGTGTTTATGTAAAGAGGCGCGASmWD40-128 qRT-PCR0.9883
SmWD40-132Smechr0702707.1ATGTCGTGCTGCCATGAACAGCTCTCAAAGCCAAATACACGSmWD40-132 qRT-PCR0.9965
SmWD40-145Smechr0802330.1AGAGTACGATCAACATCTCGGAGGCGCAATAGATGGCATGGSmWD40-145 qRT-PCR0.9746
SmWD40-152Smechr0901596.1GTGCAGACCCTGAAAAAGGCCACCACCTGTCCCCATGTTGSmWD40-152 qRT-PCR1.0233
SmWD40-153Smechr0901745.1CGGGGACATACTGGCTTTGTTCACCGGCATAGAATGAGCCSmWD40-153 qRT-PCR0.9516
SmWD40-160Smechr1000926.1CCCCCTCTTATTGTCAGCGGGAGGCGTCAGAGAAACAGACASmWD40-160 qRT-PCR0.9777
SmWD40-176Smechr1100624.1ATGACCGCACAACTGCCAAATAGCCGGATGGTCCGATCTTSmWD40-176 qRT-PCR0.1023
SmWD40-181Smechr1102707.1AACAACCCTTTGGTCCCAGCGTTAGAAGCAGCCGTTGTGCSmWD40-181 qRT-PCR0.9761
SmWD40-182Smechr1200022.1CAGGTCATAACGGCCCTGTGTGGAGCCCATTCACACGAAASmWD40-182 qRT-PCR0.9552
SmCHISmechr0500261.1CGATCGCTTAGCAGAAGCCGGCGAACGATATCTTTGCACTACTSmCHI qRT-PCR0.9765
SmCHSSmechr0500387.1GCTCGTCGTCTGTGCTGAGATCCCCGTTAGGCACGAATGSmCHS qRT-PCR0.9838
SmF3HSmechr0101736.1AGCAATGGGATATACAAAGGCATCCGCTAACGCAGGTCCAACTSmF3H qRT-PCR0.9689
SmDFRSmechr0202337.1AGTCAGGGGAATGTTGAGCATCCAGCTAGTCTCGTCATAGASmDFR qRT-PCR1.0332
SmFLSSmechr0401078.1AAGGTCAGAGCACAACAGCCGTCGATGACTGGCACCTCAASmFLS qRT-PCR0.9777
SmPALSmechr0400589.1TGGAACTTTCGAGGGCGAATCGTCGGGCGATCGTACTTTTSmPAL qRT-PCR0.9923
SmActinSmechr1001310.1GGTCGGAATGGGACAGAAGGGGTGCCTCAGTCAGGAGAACReference Gene0.9936
SmWD40-56-VIGSSmechr0302292.1ctccatggggatccgcCCAATGACATTCTTGagacgcgtgagctcgATCAGCAGAAACGGAAGSmWD40-56 amplification of silencing fragments
TRV2noneTGGGAGATGATACGCTGTTCCTAAAACTTCAGACACGDetect infected plants
Table A2. Identification and physicochemical analysis of SmWD40 protein.
Table A2. Identification and physicochemical analysis of SmWD40 protein.
Gene NameGene IDNumber of Amino AcidsMolecular WeightTheoretical pIInstability IndexAliphatic IndexGrand Average of Hydropathicity
SmWD40-1Smechr0100054.153860030.644.3434.2770.46−0.589
SmWD40-2Smechr0100084.140345571.64.6940.377.59−0.516
SmWD40-3Smechr0100138.144048400.935.4536.5887.09−0.189
SmWD40-4Smechr0100203.149355068.179.0448.0572.17−0.398
SmWD40-5Smechr0100250.137740950.027.1929.5676.47−0.233
SmWD40-6Smechr0100269.175782965.535.5132.2594.66−0.151
SmWD40-7Smechr0100290.146951874.128.7241.876.46−0.486
SmWD40-8Smechr0100456.134037891.448.5135.3768.5−0.521
SmWD40-9Smechr0100664.142448291.914.746.2478.4−0.548
SmWD40-10Smechr0100732.174784865.955.1151.9773.31−0.732
SmWD40-11Smechr0100835.11412150253.476.9239.4288.53−0.101
SmWD40-12Smechr0100970.11132124636.896.642.7284.89−0.214
SmWD40-13Smechr0101114.1982106620.955.4252.7575.51−0.339
SmWD40-14Smechr0101146.175983050.866.8834.3882.71−0.321
SmWD40-15Smechr0101232.152657618.084.8435.1187.05−0.282
SmWD40-16Smechr0101322.12513276904.986.342.1286.42−0.061
SmWD40-17Smechr0101516.148453493.779.1739.4172.77−0.477
SmWD40-18Smechr0101622.144147647.58.9232.2286.73−0.199
SmWD40-19Smechr0102034.1907101447.448.0142.5793.84−0.141
SmWD40-20Smechr0102054.144249465.819.0437.1582.62−0.238
SmWD40-21Smechr0102060.11126121602.135.2349.6478.53−0.288
SmWD40-22Smechr0102098.141946256.059.0340.9984.39−0.19
SmWD40-23Smechr0102184.11382154085.286.1835.9486.11−0.15
SmWD40-24Smechr0102204.11667186041.556.5546.4373.11−0.597
SmWD40-25Smechr0102232.135339736.775.7235.5375.38−0.292
SmWD40-26Smechr0102378.120823235.256.8552.2673.94−0.32
SmWD40-27Smechr0102379.152859948.589.0650.6473.86−0.476
SmWD40-28Smechr0102778.11698190580.356.3950.367.02−0.738
SmWD40-29Smechr0103230.149655498.418.6347.2776.05−0.406
SmWD40-30Smechr0200037.146252158.938.1730.2190.65−0.177
SmWD40-31Smechr0200130.159865181.096.6541.0991.79−0.08
SmWD40-32Smechr0200257.182090126.368.3538.287.24−0.286
SmWD40-33Smechr0200514.123926251.654.6924.93101.17−0.029
SmWD40-34Smechr0200897.177386581.587.0651.2589.5−0.232
SmWD40-35Smechr0201236.136940099.457.0424.6379.7−0.189
SmWD40-36Smechr0201464.151658044.796.650.0169.38−0.766
SmWD40-37Smechr0201698.167173205.325.7544.5379.66−0.261
SmWD40-38Smechr0201774.11027113880.836.4155.7780.68−0.452
SmWD40-39Smechr0201777.133636529.36.8639.4281.25−0.208
SmWD40-40Smechr0201803.143248118.814.8647.858.5−0.526
SmWD40-41Smechr0202123.11051115331.916.8442.392.12−0.186
SmWD40-42Smechr0202247.133236903.656.0937.4878.07−0.228
SmWD40-43Smechr0202538.131434577.846.3622.9580.99−0.291
SmWD40-44Smechr0202756.147152145.578.942.3571−0.524
SmWD40-45Smechr0202868.144048281.75.5336.3287.55−0.189
SmWD40-46Smechr0202967.149954952.247.3141.382.65−0.244
SmWD40-47Smechr0203005.130232550.545.8422.7479.11−0.216
SmWD40-48Smechr0203082.151456664.048.7750.8174.36−0.388
SmWD40-49Smechr0300525.146051307.378.640.5670.93−0.491
SmWD40-50Smechr0301092.143247227.719.1541.9387.59−0.168
SmWD40-51Smechr0301403.152859266.69.5636.3778.52−0.52
SmWD40-52Smechr0301606.143447495.089.0137.0383.53−0.235
SmWD40-53Smechr0301684.142147215.627.9442.5782.16−0.144
SmWD40-54Smechr0301928.143047321.278.1332.3571.56−0.381
SmWD40-55Smechr0302236.171478581.526.4346.3975.84−0.369
SmWD40-56Smechr0302292.141747092.844.8949.6679.28−0.433
SmWD40-57Smechr0302364.187196184.76.0541.8387.3−0.181
SmWD40-58Smechr0302644.138542188.436.4932.6683.32−0.242
SmWD40-59Smechr0302647.139244101.147.1743.0289.97−0.243
SmWD40-60Smechr0302855.140444930.378.8635.5884.41−0.215
SmWD40-61Smechr0302946.1980108146.295.7947.379.45−0.408
SmWD40-62Smechr0302999.138742791.764.9732.5376.02−0.42
SmWD40-63Smechr0303123.11132124471.516.5739.0782.47−0.249
SmWD40-64Smechr0303179.11130124653.996.7639.3479.57−0.35
SmWD40-65Smechr0303180.139142538.275.2252.8689.050.017
SmWD40-66Smechr0303262.156463489.278.575683.67−0.459
SmWD40-67Smechr0303326.175985606.126.6339.5994.18−0.162
SmWD40-68Smechr0303331.160966187.926.1524.4289.26−0.139
SmWD40-69Smechr0303392.11821202371.416.3141.2497.76−0.075
SmWD40-70Smechr0303426.135438347.795.438.0274.92−0.329
SmWD40-71Smechr0303432.1903101063.156.2338.0785.2−0.162
SmWD40-72Smechr0303452.131335957.795.1342.8485.59−0.286
SmWD40-73Smechr0303453.1916103343.754.8732.3484.49−0.286
SmWD40-74Smechr0303517.130132399.35.0932.5681.96−0.158
SmWD40-75Smechr0303588.145950643.585.641.0976.86−0.449
SmWD40-76Smechr0400216.187496741.35.5847.6870.55−0.581
SmWD40-77Smechr0401256.132635928.77.2330.6182.24−0.313
SmWD40-78Smechr0401335.185494768.785.3139.3994.670.025
SmWD40-79Smechr0401384.11484166473.855.6851.0689.97−0.285
SmWD40-80Smechr0401851.145050649.48538.4474.56−0.414
SmWD40-81Smechr0401950.178986855.866.2751.9979.15−0.397
SmWD40-82Smechr0401952.166573574.836.2146.5478.32−0.376
SmWD40-83Smechr0401998.13249364048.076.0947.6396−0.116
SmWD40-84Smechr0402112.181489327.636.3139.7684.9−0.191
SmWD40-85Smechr0402166.158065695.445.6124.6768.57−0.767
SmWD40-86Smechr0402222.144849231.76.0348.389.44−0.212
SmWD40-87Smechr0402308.144749013.248.5936.177.56−0.4
SmWD40-88Smechr0402434.11218137204.736.6333.3391.33−0.261
SmWD40-89Smechr0500012.11895211341.885.3648.6289.18−0.294
SmWD40-90Smechr0500094.139742640.624.347.475.42−0.271
SmWD40-91Smechr0500782.143849316.616.014594.18−0.174
SmWD40-92Smechr0500827.158164645.735.7542.1993.87−0.199
SmWD40-93Smechr0501136.134838447.58.6936.168.65−0.412
SmWD40-94Smechr0501240.130232668.95.929.1883.54−0.135
SmWD40-95Smechr0501281.162369179.425.8438.0284.06−0.154
SmWD40-96Smechr0501342.143748304.698.3735.3182.59−0.288
SmWD40-97Smechr0501500.133436872.715.9941.9183.8−0.14
SmWD40-98Smechr0501683.145550098.938.0644.2682.68−0.168
SmWD40-99Smechr0501744.1929104910.344.9332.2586.76−0.302
SmWD40-100Smechr0501824.120524016.198.4649.6462.73−1.172
SmWD40-101Smechr0502137.148653616.159.1541.2172.61−0.531
SmWD40-102Smechr0502285.182190061.987.3338.1885.7−0.162
SmWD40-103Smechr0502290.145349769.868.6942.9276.87−0.385
SmWD40-104Smechr0502356.141045405.38.3430.574.85−0.383
SmWD40-105Smechr0502667.11663184193.336.1951.0289.99−0.146
SmWD40-106Smechr0600010.148755330.287.6948.6976.86−0.441
SmWD40-107Smechr0600111.143447259.875.424191.68−0.058
SmWD40-108Smechr0600126.182691024.236.0841.8187.19−0.123
SmWD40-109Smechr0600145.165071617.275.4547.0170.52−0.661
SmWD40-110Smechr0600493.147353158.716.3552.3878.9−0.465
SmWD40-111Smechr0601395.169577092.815.9844.3983.41−0.31
SmWD40-112Smechr0601940.167374624.116.2848.3578.66−0.39
SmWD40-113Smechr0601947.190599310.056.4254.4365.31−0.713
SmWD40-114Smechr0602039.151458190.366.5348.1382.47−0.361
SmWD40-115Smechr0602212.1917103580.794.8931.6586.65−0.284
SmWD40-116Smechr0602338.132335657.397.0330.1587.77−0.16
SmWD40-117Smechr0602339.131534997.66.6533.7186.6−0.194
SmWD40-118Smechr0602689.141647296.756.1231.0383.92−0.143
SmWD40-119Smechr0603088.177884819.86.4349.7867.98−0.539
SmWD40-120Smechr0700242.11134125041.546.4637.6975.56−0.343
SmWD40-121Smechr0700811.11300146022.85.843.6499−0.109
SmWD40-122Smechr0700862.144348787.038.8530.7778.28−0.373
SmWD40-123Smechr0700863.11219136975.436.5531.7491.23−0.244
SmWD40-124Smechr0700946.147251755.655.0848.575.4−0.434
SmWD40-125Smechr0701007.150755962.595.4543.8678.42−0.403
SmWD40-126Smechr0701091.136940180.435.3740.8883.79−0.132
SmWD40-127Smechr0701168.132334828.155.637.4286.04−0.074
SmWD40-128Smechr0701564.158065174.626.2839.9395.5−0.206
SmWD40-129Smechr0701747.139142415.35.3436.3995.73−0.109
SmWD40-130Smechr0702099.145251980.049.5943.5969.87−0.662
SmWD40-131Smechr0702444.1931102681.236.2643.3270−0.628
SmWD40-132Smechr0702707.131334969.476.524.5678.21−0.377
SmWD40-133Smechr0702744.181689440.776.4944.0187.76−0.112
SmWD40-134Smechr0800148.173481426.565.7239.1773.27−0.401
SmWD40-135Smechr0800182.142247143.089.145.0377.65−0.405
SmWD40-136Smechr0800245.141746766.298.8446.5381.77−0.252
SmWD40-137Smechr0800403.165672721.726.149.674.94−0.387
SmWD40-138Smechr0800720.144048200.455.8340.7985.11−0.081
SmWD40-139Smechr0800881.11088121328.95.8543.489.59−0.173
SmWD40-140Smechr0801440.147353597.88.7350.1475.39−0.374
SmWD40-141Smechr0801468.175683339.828.4855.3954.81−0.728
SmWD40-142Smechr0801696.11909207813.615.2748.4384.89−0.365
SmWD40-143Smechr0801889.11137124958.9744.3877.12−0.319
SmWD40-144Smechr0802284.148453175.719.2551.8575.72−0.408
SmWD40-145Smechr0802330.158967263.636.3145.8362.43−0.787
SmWD40-146Smechr0802536.172881007.966.9138.1871.57−0.49
SmWD40-147Smechr0900060.1908100784.218.7650.272.28−0.565
SmWD40-148Smechr0900415.1911102220.868.7649.8371.68−0.678
SmWD40-149Smechr0900611.192599984.428.0638.8288.01−0.001
SmWD40-150Smechr0900982.11353148539.395.9546.2490.78−0.088
SmWD40-151Smechr0901524.176183468.496.5744.5467.95−0.554
SmWD40-152Smechr0901596.151658319.575.5232.192−0.233
SmWD40-153Smechr0901745.131834965.648.8326.1282.67−0.191
SmWD40-154Smechr0902013.134938922.345.0325.9272.84−0.378
SmWD40-155Smechr0902163.159964872.516.4240.4191.65−0.08
SmWD40-156Smechr0902418.152156569.946.1829.884.09−0.254
SmWD40-157Smechr1000020.130033350.699.7536.872.17−0.586
SmWD40-158Smechr1000478.137742581.039.1241.5778.7−0.528
SmWD40-159Smechr1000759.121224088.848.7528.4993.3−0.193
SmWD40-160Smechr1000926.134137563.787.1136.7572.02−0.358
SmWD40-161Smechr1001004.131535276.695.9936.4978−0.321
SmWD40-162Smechr1001354.178384904.316.547.5276.87−0.368
SmWD40-163Smechr1001468.139143489.138.6350.5281.3−0.401
SmWD40-164Smechr1001592.116918088.639.142.4792.9−0.034
SmWD40-165Smechr1001624.1924102745.946.7153.4264.89−0.716
SmWD40-166Smechr1001754.11345148416.16.0644.4289.62−0.097
SmWD40-167Smechr1001849.153359967.069.5842.6683.32−0.38
SmWD40-168Smechr1001886.118720631.098.5749.91103.10.107
SmWD40-169Smechr1001952.134338023.915.3845.5686.91−0.145
SmWD40-170Smechr1001992.132535950.375.7737.2480.37−0.229
SmWD40-171Smechr1002755.155561974.656.0740.0480.22−0.611
SmWD40-172Smechr1100180.138041235.456.2337.8479.34−0.309
SmWD40-173Smechr1100321.135438754.144.7233.5988.95−0.342
SmWD40-174Smechr1100584.144348823.456.541.2983.43−0.227
SmWD40-175Smechr1100594.165072901.546.3344.6466.37−0.547
SmWD40-176Smechr1100624.129833074.386.8538.7684.97−0.233
SmWD40-177Smechr1101663.143047675.999.1642.5680−0.29
SmWD40-178Smechr1102321.182189984.916.4939.3188.33−0.292
SmWD40-179Smechr1102445.158063841.265.4349.882.79−0.37
SmWD40-180Smechr1102608.149353785.194.5240.3184.12−0.326
SmWD40-181Smechr1102707.142446942.138.3841.4984.03−0.262
SmWD40-182Smechr1200022.158465408.146.1239.2899.61−0.099
SmWD40-183Smechr1200183.132334904.165.6438.7183.62−0.071
SmWD40-184Smechr1201542.147653542.496.4744.7676.16−0.501
SmWD40-185Smechr1201594.155763080.158.5538.7886.62−0.32
SmWD40-186Smechr1201735.11071116791.176.2438.8773.52−0.419
SmWD40-187Smechr1201743.167276211.316.8441.7783.79−0.294

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Figure 1. Chromosomal localization of the SmWD40 genes. Each bar corresponds to the length of a chromosome, with red and blue indicating regions with high and low gene density, respectively.
Figure 1. Chromosomal localization of the SmWD40 genes. Each bar corresponds to the length of a chromosome, with red and blue indicating regions with high and low gene density, respectively.
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Figure 2. Phylogenetic trees for WD40 proteins in eggplant and Arabidopsis, with different colors representing various subgroups. AT: Arabidopsis thaliana. Sm: Solanum melongena.
Figure 2. Phylogenetic trees for WD40 proteins in eggplant and Arabidopsis, with different colors representing various subgroups. AT: Arabidopsis thaliana. Sm: Solanum melongena.
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Figure 3. SmWD40 gene conserved domain, motif, and the cis-acting elements analysis. (A) Motif compositions. (B) Conserved domains. (C) Cis-acting elements.
Figure 3. SmWD40 gene conserved domain, motif, and the cis-acting elements analysis. (A) Motif compositions. (B) Conserved domains. (C) Cis-acting elements.
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Figure 4. Collinearity analysis of SmWD40s. The red line represents collinear genes. The colors of the bars represent the gene density on the chromosome, with red and blue signifying high and low density, respectively.
Figure 4. Collinearity analysis of SmWD40s. The red line represents collinear genes. The colors of the bars represent the gene density on the chromosome, with red and blue signifying high and low density, respectively.
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Figure 5. Expression pattern analysis of S5 subgroup genes in fruit color development. (A) Eggplant fruits at different growth times after end of flowering. Stages I, II, and III represent 5, 15, and 25 d after the end of flowering, respectively. In stage I, the fruits were completely green; in stage II, the fruits began to show purple color, but were still partially green; and in stage III, the fruits were covered with purple coloring. (B) Expression patterns within the subgroup S5 at distinct stages of fruit color development. The y-axis depicts the relative gene expression, while the x-axis corresponds to various stages of fruit color development. The data, presented as mean ± standard deviation from three biological replicates, reveal remarkable differences between samples, as indicated by distinct capital letters (p < 0.01) based on one-way ANOVA and Tukey’s test. Error bars represent the standard deviation (SD).
Figure 5. Expression pattern analysis of S5 subgroup genes in fruit color development. (A) Eggplant fruits at different growth times after end of flowering. Stages I, II, and III represent 5, 15, and 25 d after the end of flowering, respectively. In stage I, the fruits were completely green; in stage II, the fruits began to show purple color, but were still partially green; and in stage III, the fruits were covered with purple coloring. (B) Expression patterns within the subgroup S5 at distinct stages of fruit color development. The y-axis depicts the relative gene expression, while the x-axis corresponds to various stages of fruit color development. The data, presented as mean ± standard deviation from three biological replicates, reveal remarkable differences between samples, as indicated by distinct capital letters (p < 0.01) based on one-way ANOVA and Tukey’s test. Error bars represent the standard deviation (SD).
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Figure 6. Analysis of the effect of silencing SmWD40-56 on fruit color and the expression pattern of structural genes for anthocyanin synthesis after silencing. (A) Gel electrophoresis of different silencing treatment groups. M: 5000 bp marker. 1–5: TRV2-SmWD40-56. 7–11: Empty TRV2. 13–17: H2O. (B) The expression level of SmWD40-56 under distinct silencing treatments. (C) Fruit phenotype of long purple eggplant under varying silencing treatments. (D) Effect of silencing SmWD40-56 on anthocyanin synthesis structural genes in long purple eggplant fruit. The results in (B,D) are based on one-way ANOVA and Tukey’s test (p < 0.01), with capital letters showing significant differences and error bars showing standard deviation.
Figure 6. Analysis of the effect of silencing SmWD40-56 on fruit color and the expression pattern of structural genes for anthocyanin synthesis after silencing. (A) Gel electrophoresis of different silencing treatment groups. M: 5000 bp marker. 1–5: TRV2-SmWD40-56. 7–11: Empty TRV2. 13–17: H2O. (B) The expression level of SmWD40-56 under distinct silencing treatments. (C) Fruit phenotype of long purple eggplant under varying silencing treatments. (D) Effect of silencing SmWD40-56 on anthocyanin synthesis structural genes in long purple eggplant fruit. The results in (B,D) are based on one-way ANOVA and Tukey’s test (p < 0.01), with capital letters showing significant differences and error bars showing standard deviation.
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Figure 7. Protein interaction network of SmWD40-56. Each node corresponds to a distinct protein. The colored and uncolored nodes signify proteins potentially forming the first and the second shell of interactions with SmWD40-56, respectively. Lines connecting nodes denote known or predicted protein–protein interactions. The red arrow identifies the SmWD40-56 protein.
Figure 7. Protein interaction network of SmWD40-56. Each node corresponds to a distinct protein. The colored and uncolored nodes signify proteins potentially forming the first and the second shell of interactions with SmWD40-56, respectively. Lines connecting nodes denote known or predicted protein–protein interactions. The red arrow identifies the SmWD40-56 protein.
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Yang, Y.; Cai, Q.; Wang, Y.; Li, L.; Sun, Z. Genome-Wide Exploration of the WD40 Gene Family in Eggplant (Solanum melongena L.) and Analysis of Its Function in Fruit Color Formation. Agronomy 2024, 14, 521. https://doi.org/10.3390/agronomy14030521

AMA Style

Yang Y, Cai Q, Wang Y, Li L, Sun Z. Genome-Wide Exploration of the WD40 Gene Family in Eggplant (Solanum melongena L.) and Analysis of Its Function in Fruit Color Formation. Agronomy. 2024; 14(3):521. https://doi.org/10.3390/agronomy14030521

Chicago/Turabian Style

Yang, Yanbo, Qihang Cai, Yimei Wang, Liping Li, and Zhenghai Sun. 2024. "Genome-Wide Exploration of the WD40 Gene Family in Eggplant (Solanum melongena L.) and Analysis of Its Function in Fruit Color Formation" Agronomy 14, no. 3: 521. https://doi.org/10.3390/agronomy14030521

APA Style

Yang, Y., Cai, Q., Wang, Y., Li, L., & Sun, Z. (2024). Genome-Wide Exploration of the WD40 Gene Family in Eggplant (Solanum melongena L.) and Analysis of Its Function in Fruit Color Formation. Agronomy, 14(3), 521. https://doi.org/10.3390/agronomy14030521

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