3.1. Identification and categorization of SmTCP family members
The entire putative TCPs were retrieved from the eggplant genome by querying Arabidopsis, rice and tomato TCPs against the eggplant genome using BLASTP. Altogether 30 presumed SmTCP members were determined and estimated through SMART and Pfam online tools to verify the existence of the unique TCPs domains. The presumed TCP genes were derived from the phylogenetic categorization of the Arabidopsis, rice and tomato TCP family. The 30 TCP transcription factors were featured by a highly conserved TCP domain at the N-terminus. The primary information for these SmTCPs sequences was summarized and tabulated in Table S2. The results of this analysis suggested the SmTCP protein length varied from 201 amino acid residues (Smechr0900228.1) to 538 (Smechr0100729.1) aa. The potential TCP genes encoded proteins with the predicted molecular weight (MW) ranging from 21.27 (Smechr0900228.1) to 57.64 (Smechr0100729.1) kDa and the isoelectric points (PI) varying between 5.25 (Smechr0201631.1) to 9.84 (Smechr0101558.1). Prediction of subcellular localization indicated that most SmTCP proteins (29 of 30, 96.7%) were localized in the nucleus (Table S2).
As per the differences in the TCP domains, 30 SmTCP proteins were categorized into two classes (Table S2). Concretely, 13 SmTCP proteins were classified into Class I, which contained the TCP domain with a four-amino-acids deletion compared to other SmTCPs. The remaining 17 genes were attributed to the Class II, and were further divided into the CIN subclade (13 SmTCPs) and the CYC/TB1 subclade (4 SmTCPs).
3.2. Phylogenetic analysis and categorization of SmTCP family
To assess the phylogenetic association and possible evolutionary history among identified genes in different TCP gene families, 30 eggplant, 24 Arabidopsis, 24 rice and 37 tomato TCP proteins were aligned and constructed a phylogenetic tree by the maximum likelihood method (Fig. 1). Using phylogenetic distribution, 30 TCPs were primarily divided into11 subgroups based on their sequence features: group A to K. Group A-F belonged to the Class I. Group I-K were assigned to Class II (CIN) TCP members. The remaining group G-H was for Class II CYC/TB1-type members.
3.3 Multiple sequences alignment
To characterize the sequence features of the TCP domain regions in SmTCP proteins, a multiple sequences alignment was conducted using DNAMAN software on the basis of the amino acid sequence of each SmTCP conserved domain (Fig. 2). The alignment outcomes highlighted divergences and similarities among these SmTCP proteins. For instance, the intact basic-Helix-Loop-Helix (bHLH) structure can be observed in all TCP members of Class II, thereby indicating the conservation of this group. In comparison, TCP members of Class I performed a higher level of diversity. For instance, Smechr0101558.1 had only Helix-Loop-Helix (HLH) structure without a basic region among Class I members. Additionally, the Class II members contained 4-amino-acid insertion compared with Class I in the basic region.
3.4. Domains and gene structure analysis
To better understand the diversity and similarity of the SmTCPs, the conserved domains and exon-intron structure were analyzed according to the phylogenetic distribution of the SmTCP genes from Arabidopsis, rice, eggplant and tomato. Visualization of TCP domains indicated all SmTCPs consisted of 39-59 amino-acid residues, which was a
Gene structure analysis can help reveal the evolutionary relationships underlying the genesis of the TCP family. Diverse SmTCP members were inclined to contribute to the various structural organizations. As shown in Fig. 3, Class I genes had no introns, apart from Smechr0101558.1, Smechr0201168.1, Smechr1102385.1 and Smechr0100729.1, which all showed one intron. In comparison to the class I SmTCP genes, the Class II ones were relatively longer and contained more exons. Class II CIN-type TCP genes had the numbers of introns ranging from 0 to 2. Class II CYC/TB1-type TCP genes had one intron, with the exception of Smechr0602362.1, which had two.
3.5 Motif distribution and promoter region structures of SmTCP family members
The 30 SmTCP predicted ORFs were subjected to MEME analysis to investigate the motif composition of the deduced polypeptides. Totally, 10 conserved motifs, named as motif 1-motif 10, were identified in Fig. 3 and Table S3. Motif 1 was annotated as a conserved basic helix loop helix (bHLH) structure, which was present in almost all SmTCPs, except Smechr0101558.1, demonstrating that it may be essential for SmTCPs to perform their functions. Motif 2 and 6 appeared only in Class I, while motif 4, 5, and 9 appeared only in Class II. Consequently, SmTCPs in the same class had similar motif composition, while there are differences in the two classes, indicating that SmTCPs in the same class may have similar functions, while some of the patterns may play an important role in particular functions.
To investigate the regulatory features of SmTCPs, the isolated 2.0-kb sequence upstream of the translational of start site of the SmTCPs was selected and assessed for the presence and constitutions of cis-acting regulatory elements (CAREs) using the PlantCARE tool (Fig. 4, Table S4). The result indicated that the well-characterized CAREs can be mainly categorized into three types: light-responsive, phytohormone-related, and abiotic stress. Several kinds of phytohormone-related CAREs were well-represented among SmTCPs promoter regions, with ABA, MeJA, SA, GA and Auxin responsive elements identified widely in the promoter regions of eggplant SmTCPs, such as ABRE, TGACG-motif, TCA-element, GARE-motif, and AuxRE, etc. As for the light-responsive elements category, abundant CAREs were distributed throughout the promoter region, including I-box, AE-box, Box II, Box 4, MRE, GT1-motif, G-box and so on. Abiotic stress-related CAREs mainly had defense- and stress-responsive elements (e.g. TC-rich repeats), drought-inducibility elements (e.g. MBS), low-temperature responsive elements (e.g. LTR), and wounding-responsive element (e.g. WUN-motif).
3.6 The prediction of the putative miRNA-target interactions
The putative miRNAs targeting SmTCPs were forecasted to gain the perspective relationship between plant growth, development and stress responses and miRNA regulation (Fig. 5). The outcomes indicated that a total of 21 SmTCP genes were targeted by 28 putative miRNAs, of which 10 SmTCPs were targeted by a single miRNA, 3 (Smechr0101558.1, Smechr0202786.1 and Smechr1200724.1) were targeted by double miRNAs. Also, Smechr0303405.1, Smechr0800708.1, 3 genes (Smechr0201631.1, Smechr0702127.1 and Smechr0800707.1), and 3 genes (Smechr0602388.1, Smechr0702382.1 and Smechr1000441.1) were targeted by 3, 4, 5 and 6 miRNAs, respectively. Meanwhile, multiple miRNAs targeted only one to three SmTCP genes, whereas 3 miRNAs (miR319a, miR319b and miR319c-3p) were able to target 7 SmTCPs, which may be critical for the regulation of plant growth, development and stress responses. These putative miRNA-genes interaction networks of eggplant TCPs may provide significant insights into their functions and also contribute to the determination of candidates for prospective investigations.
3.7. Chromosomal localization and gene duplication event of SmTCP genes
The 30 genes that encode SmTCPs were distributed unevenly over 12 chromosomes except chromosome 5, ranging between 1 to 6 members on each chromosome (Fig. 6). The E2 contained the maximum number of genes (20%, 6 genes), with most belonging to Class II CIN-type SmTCP genes. The E4, E9 and E12 all shared the lowest number of SmTCP genes (3.3%, 1 gene). The rest of the chromosomes harbored 2 to 4 SmTCP genes.
It has been proposed that gene families may be generated by tandem duplication and segmental duplication during biological evolution (Lv et al. 2020). To explore whether the SmTCP gene family had a duplication-based expansion, we analyzed the duplication events of SmTCP genes (Fig. 6, Table S5). It was found that Smechr0800707.1 and Smechr0800708.1 were a pair of tandem duplicated genes, their divergence might date from about 10.88 million years ago. Thus, it can be asserted that the duplication events played a major role in the expansion of the SmTCP gene family.
3.7 Evolutionary association of TCP genes among eggplant, Arabidopsis and tomato
The genomes of eggplant, tomato, and Arabidopsis were compared to determine SmTCP genes’ collinear relationship among various plant species (Fig. 7, Table S6). According to the results of the analysis, the genomes of eggplant and Arabidopsis had 5 (13.3%) syntenic gene pairs. Arabidopsis chromosomes occupied minor genes that existed collinear relationships within SmTCPs in the eggplant genome with the exception for E1, E3, E4, E5, E6, E8 and E12 chromosomes. A total of 28 (93.3%) gene pairs of SmTCPs between eggplant and tomato were syntenic. As a consequence, according to the results of this analysis, there is a strong collinear correlation between eggplant and tomato. These outcomes suggested that eggplant and tomato share compatible phylogenetic conservation of TCP genes.
3.8 Expression profiles of eggplant TCP in different tissues
An expression analysis with qRT-PCR was performed for 30 eggplant TCP genes in various eggplant organs, including root, stem, leaf, flower, peel and septal (Fig. 8, Table S7). The outcomes of this analysis revealed that SmTCPs had distinct expression patterns in diverse organs, suggesting the divergent roles of SmTCPs in plant growth and development. Plenty of Class II SmTCP genes were expressed at high level in leaf, flower and septal. Class I SmTCP genes were mainly highly expressed in root, stem and peel. Examples for highly expressed in specific organs included one (Smechr1102385.1) in root, 4 genes (Smechr0103973.1, Smechr0202786.1, Smechr0303405.1, and Smechr1002163.1) in stem, 9 genes (Smechr1001558.1, Smechr0400435.1, Smechr0602362.1, Smechr0602431.1, Smechr0702382.1, Smechr0800707.1, Smechr0800708.1, Smechr0900228.1, and Smechr1102037.1) in leaf, 6 genes (Smechr0201023.1, Smechr0201631.1, Smechr0300562.1, Smechr0602388.1, Smechr0702127.1, and Smechr1000441.1) in flower, Smechr0201168.1 in peel, 4 genes (Smechr0202855.1, Smechr0602003.1, Smechr0802294.1, and Smechr1000174.1). in septal. Furthermore, there were some genes that had the lowest expression level in different tissues, such as Smechr0203181.1 in root, Smechr1200724.1 in stem, Smechr0103973.1 in leaf, Smechr0201168.1 in flower, Smechr1002163.1 in peel, and Smechr1102385.1 in septal. Based on the above results, it was inferred that SmTCP genes may play a momentous role in plant developmental processes.
3.9 Expression pattern of SmTCPs under diverse treatments
To discern the possible roles of eggplant TCP genes in plant stress responses, the expression level of SmTCP genes in response to 4°, PEG, and ABA was examined using quantitative real-time PCR (qRT-PCR). All SmTCP genes responded to at least one type of the treatments, and 21 genes responded to the overall treatments (Fig. 9).
25 genes (Smechr0100729.1, Smechr0101558.1, Smechr0103973.1, Smechr0201023.1, Smechr0201168.1, Smechr0201631.1, Smechr0202855.1, Smechr0203181.1, Smechr0303082.1, Smechr0303405.1, Smechr04000435.1, Smechr0602003.1, Smechr0602362.1, Smechr0602388.1, Smechr0602431.1, Smechr0702382.1, Smechr0800707.1, Smechr0800708.1, Smechr0802294.1, Smechr0900228.1, Smechr1000174.1, Smechr1002163.1, Smechr1102037.1, Smechr1102385.1, and Smechr1200724.1) were activated under 4℃ condition. The transcription levels of 22 genes were up-regulated, whereas the transcript levels of 3 genes (Smechr0103973.1, Smechr0202855.1 and Smechr0800707.1) were down-regulated. Interestingly, most of the up-regulated genes belonged to Class I, in contrast to the down-regulated genes which mainly belonged to Class II. Furthermore, 9 of the up-regulated genes were remarkably induced at 3 h under 4℃ treatment.
Out of 24 genes induced by ABA treatment, including Smechr0100729.1, Smechr0103973.1, Smechr0201023.1, Smechr0201168.1, Smechr0201631.1, Smechr0302978.1, Smechr0303082.1, Smechr0303405.1, Smechr04000435.1, Smechr0602003.1, Smechr0602362.1, Smechr0602388.1, Smechr0602431.1, Smechr0702127.1, Smechr0702382.1, Smechr0800707.1, Smechr0800708.1, Smechr0802294.1, Smechr0900228.1, Smechr1000441.1, Smechr1002163.1, Smechr1102037.1, Smechr1102385.1, and Smechr1200724.1. 15 genes up-regulated, with more than half of them being Class II members, and nine genes (Smechr0103973.1, Smechr0303082.1, Smechr0303405.1, Smechr0702127.1, Smechr0800708.1, Smechr0900228.1, Smechr1102037.1, Smechr1102385.1, and Smechr1200724.1) were down-regulated. The transcript level of Smechr04000435.1 was greatly up-regulated (>350 fold) at 1 h.
A total 24 genes (Smechr0100729.1, Smechr0103973.1, Smechr0201023.1, Smechr0201168.1, Smechr0201631.1, Smechr0203181.1, Smechr0303082.1, Smechr0303405.1, Smechr04000435.1, Smechr0602003.1, Smechr0602362.1, Smechr0602388.1, Smechr0602431.1, Smechr0702127.1, Smechr0702382.1, Smechr0800708.1, Smechr0802294.1, Smechr0900228.1, Smechr1000174.1, Smechr1000441.1, Smechr1002163.1, Smechr1102037.1, Smechr1102385.1, and Smechr1200724.1) were induced by PEG treatment, of which the transcript levels of 13 genes were up-regulated, the remaining genes (Smechr0103973.1, Smechr0201023.1, Smechr0201631.1, Smechr0303082.1, Smechr0303405.1, Smechr0602431.1, Smechr0702127.1, Smechr0802294.1, Smechr1002163.1, Smechr1102385.1, and Smechr1200724.1) were down-regulated. The transcript level of Smechr0602362.1 was significantly up-regulated (>15 fold) under PEG treatment.
Out of 28 SmTCP genes, 19 genes (Smechr0100729.1, Smechr0103973.1, Smechr0201023.1, Smechr0303082.1, Smechr0303405.1, Smechr04000435.1, Smechr0602003.1, Smechr0602362.1, Smechr0602388.1, Smechr0602431.1, Smechr0702382.1, Smechr0800708.1, Smechr0802294.1, Smechr0900228.1, Smechr1000441.1, Smechr1002163.1, Smechr1102037.1, Smechr1102385.1, and Smechr1200724.1) were mediated by all three treatments , and 6 genes (Smechr0201631.1, Smechr0203181.1, Smechr0702127.1, Smechr0800707.1, Smechr1000174.1 and Smechr1000441.1) were induced by two treatments. The remaining three genes (Smechr0101558.1, Smechr0202855.1 and Smechr0302978.1) were induced activation by one treatment. Taken together, the expression profiles indicated that SmTCP genes might be involved in abiotic stress, especially 4℃ and ABA stress conditions.
3.10 Smechr0202855.1 and Smechr0602431.1could involve in the regulation of anthocyanin synthesis
As per our previous investigations of the short-term transcriptomes, we were inclined to select three SmTCP differentially expressed genes (Smechr0201023.1, Smechr0202855.1, and Smechr0602431.1) to determine their engagement in the regulation of anthocyanin synthesis (Figure S1). The subcellular localization outcomes of the three SmTCPs indicated that they were located in the nucleus, whereas the empty vector was both located in the nucleus and cell membrane, which was in accordance with the predicted outcomes (Fig. S2). Moreover, yeast one-hybrid and Dual-luciferase assays were utilized to gauge the binding of 3 TFs to anthocyanin synthetic structural genes promoters, and activation of gene expression. The results demonstrated that Smechr0202855.1 and Smechr0602431.1could directly bind to the SmCHS promoter and activate its expression, whereas Smechr0201023.1 could not (Fig.10).