Genome-wide analysis of basic helix–loop–helix superfamily members related to anthocyanin biosynthesis in eggplant (Solanum melongena L.)

Identification and analysis of eggplant bHLH family gene

To identify the bHLH sequence in the eggplant genome, the amino acid data of the eggplant genome was downloaded from the Eggplant Genome Database (http://eggplant.kazusa.or.jp/). A total of 152 Arabidopsis bHLH protein sequences were obtained from TAIR (https://www.arabidopsis.org/). In addition, the Hidden Markov Model profile for the bHLH binding domain (PF00010) was downloaded from the Pfam database (http://pfam.xfam.org/). The HMMER program was used to search for bHLH proteins in all eggplant proteins with a cut off E-value of 1e⁻⁵ using PF00010 as a query. Using the Arabidopsis bHLH protein sequences as the query sequence, the Blast-p program was used to search the amino acid database in the eggplant genome. Amino acid sequences of the candidate genes for the bHLH family were then obtained. To ensure that the candidate genes obtained were SmbHLH sequences, the obtained candidate sequences were placed in the Pfam (http://pfam.xfam.org/) and SMART databases (http://smart.emblheidelberg.de/) for confirmation. The absence of the bHLH domain was excluded.

Phylogenetic tree analysis, gene structure and conserved motif characterization

The complete sequences of the amino acids were aligned using MAFFT, and an unrooted phylogenetic tree was constructed using MEGA6 (Tamura et al., 2013) with the following parameters: number of bootstrap replications was 1000, model or method was P-distance, gaps or missing data treatment was pairwise deletion, and only bootstrap values greater than 50 could be displayed on the tree. The full-length gene and CDS sequences of eggplant bHLH genes were downloaded from the Eggplant Genome Website (http://eggplant.kazusa.or.jp/) to form the desired format. The online gene structure display server (http://gsds.cbi.pku.edu.cn/) was used to analyze the bHLH gene structure of the eggplant and the numbers of introns of the genes were clearly obtained. The eggplant bHLH amino acid sequences were downloaded and arranged in the desired order. The bHLH protein sequences of eggplant were uploaded to the online search tool MEME (http://meme-suite.org/tools/meme), and the protein’s conserved motif characteristics were analyzed.

Expression analysis

Eggplant (Solanum melongena L). cv. ‘Jingqiejingang’, ‘Changza NO.8’ (labeled PP, purple peel eggplant), ‘Baiqiezi101’ (labeled WP, white peel eggplant), ‘Lvyichangqie’ (labeled GP, green peel eggplant), and ‘Africa Red Eggplant’ (labeled O-RP, orange-red peel eggplant) seeds were placed on a damp filter paper and incubated in a dark 28 °C incubator until germination. The germinated seeds were sown in the greenhouse at Shandong Agricultural University. When the eggplant seedlings were at the three-true-leaf stage, three temperature treatments (28 °C, 4 °C, and 40 °C) were applied according to Wang et al. (2016). The leaves for RNA extractions were harvested at 1, 3, 6, and 12 h after the treatments.

To investigate the expression patterns in different tissues the stems, leaves, petioles, flowers in bloom, and the peels and pulps of the fruit from different developmental stages of four eggplant varieties (PP, WP, GP, and O-RP) were collected simultaneously from 8-week-old plants under natural conditions. In order to determine the response to different light-emitting diode (LED) light expression, LED red:blue light ratio was 1:1, 3:1, 6:1, and 9:1. The treatment was conducted according to the method of Di (2017). The leaves and peels of the fruit from the eggplant cv. ‘Jingqiejingang’ were harvested.

All of the samples collected were frozen immediately in liquid nitrogen and stored at −80 °C until use. To analyze the expression patterns of the SmbHLH genes, a qRT-PCR was performed using the qRT-PCR Probe Kit (VAZYME, China) according to the manufacturer’s instructions. The β-actin gene (GenBank: jX524155.1) was used as a reference gene. The primers used for qPCR analysis were designed by Primer Premier 5 and are listed in Table S1. The PCR products were sequenced to confirm the specific amplifications.

Identification and characterization of eggplant bHLH gene family

To identify putative bHLH proteins in eggplant, 152 bHLH protein sequences of Arabidopsis and the bHLH Hidden Markov Model were used. The Pfam and SMART program tests were used to remove redundant proteins. A total of 121 genes in the eggplant genome were identified as putative members of the SmbHLH family (designated as SmbHLH1-SmbHLH121) (Table 1). The gene ontology (GO) analysis of eggplant bHLH genes was performed in Table S2. The GO analysis revealed that SmbHLHs mainly functioned in protein and DNA binding. To further predict the function of these genes, PSORT online software was used to predict subcellular localization. The results showed that the probability of the genes in the nucleus was more than 90.0% (Table 1). The genes’ ID number in the eggplant genome database was used to find the amino acid number, molecular weight, and isoelectric point of the genes. The annotation information revealed that the length of the SmbHLH amino acid ranged from 67 (SmbHLH28) to 796 (SmbHLH18), and the molecular weights ranged from 7.93 kDa (SmbHLH28) to 91.16 kDa (SmbHLH18), which indicated that the SmbHLH gene family may have undergone a long historical evolution and participated in different biological processes. The predicted isoelectric point value of SmbHLH proteins was between 4.32 (SmbHLH119) and 10.11 (SmbHLH106) (Table 1).

Gene structure and conserved motif analysis of the eggplant bHLH family

A neighbor-joining phylogenetic tree was constructed with MEGA6 (Fig. 1). The SmbHLH genomic sequence and corresponding cDNA sequence of the same SmbHLH gene were submitted to GSDS together to show the gene structure. The numbers of introns ranged from 0 to 22 (Fig. 1). In addition, Table S3 shows that 120 genes (99.2%) were below 10, and 13 genes (10.7%) were without introns, 20 genes (16.5%) contained one intron, and the remaining genes contained two or more introns.

The MEME program was used to identify the conserved motif of SmbHLH proteins (Fig. 1). Ten conserved motifs were identified and the protein-conserved motifs of SmbHLH ranged from one to nine. Twenty-three genes (19.0%) had only one conserved motif (Table S3). Each of the predicted motifs were identified only once in each SmbHLH protein sequence. In general, SmbHLH proteins on close adjacent clades of the phylogenetic tree had the same or similar conserved motifs.

N-terminal conserved domain analysis of eggplant bHLH protein

To analyze the characteristics of the N-terminal DNA-binding region of the bHLH family in eggplant, the amino acid residues-conserved map of bHLH proteins located in the N-terminal were plotted by WEBLOGO (https://weblogo.berkeley.edu/logo.cgi). The conserved motifs of the bHLH proteins in eggplant were [E] -[R] -x(1) -[R] -[R] -x(9) -[L] -x(2) -[L] -x(1) -[P] -x(7) -[K] -x(6) -[L] -x(9)-[L] (Fig. 2).

Phylogenetic tree analysis of the eggplant bHLH protein family

To predict the function of the SmbHLH family members of eggplant, an unrooted phylogenetic tree was constructed using 121 SmbHLH proteins, 152 AtbHLH proteins, and 14 proteins related to anthocyanin synthesis, including 1 potato protein, 1 tomato protein, 2 bHLH proteins from grape, 2 apple proteins, 2 tobacco proteins, 1 snapdragon protein, 3 petunia proteins, 1 rice protein, and 1 maize protein. These bHLH proteins could be classified into seventeen distinct subfamilies based on the clade support values and classification from Arabidopsis (Fig. 3). Group A was the largest subfamily with 35 proteins, whereas the smallest groups, D and M, contained only six proteins. The numbers of eggplant bHLH proteins within each subfamily varied from 1 to 18. The proteins related to anthocyanin synthesis based on the phylogenetic tree were concentrated in group P. Therefore, the genes involved in anthocyanin synthesis in eggplant were probably SmbHLH1 and SmbHLH117. Further experiments are needed to explore and verify their functions in eggplant.

Expression profiles of eggplant bHLH genes in different colored tissues

Based on the phylogenetic tree (Fig. 3) and the results of previous studies in our laboratory, the genes of SmbHLH113, SmbHLH26, SmbHLH9 and SmbHLH10 were probably involved in anthocyanin biosynthesis. Thus, in order to gain insights into their roles in anthocyanin biosynthesis in differently colored tissues in eggplants, the expression profiles of six putative SmbHLH genes, namely, SmbHLH1, SmbHLH117, SmbHLH113, SmbHLH26, SmbHLH9, and SmbHLH10 were completed by qRT-PCR. As shown in Fig. 4, the six SmbHLH genes showed different patterns of tissue-specific expression in the different peel color varieties of eggplant. The transcript abundance of SmbHLH1 was higher in the purple tissues of the purple peel eggplant (PP) such as leaves (Fig. 4B), petioles (Fig. 4C), flowers (Fig. 4D) and peels (Fig. 4E), and in the leaves of orange-red peel eggplant (O-RP) (Fig. 4B) and green peel eggplant (GP) (Fig. 4E). However, it was lower in the white and green tissues, such as in the pulps of the four varieties (Fig. 4F), the stems of white peel (WP) and GP eggplant (Fig. 4A), and in the peels of WP, O-RP, and GP (Fig. 4E). Meanwhile, the transcript abundance of SmbHLH117 was higher in green tissues, such as the stems of GP (Fig. 4A), and the leaves of O-RP and GP (Fig. 4B), but lower in the flowers and peels of the four varieties. SmbHLH113 expression was higher in the stems of WP and GP (Fig. 4A), the leaves of O-RP and PP (Fig. 4B), the petioles of O-RP (Fig. 4C), and the pulps of WP, O-RP, and PP (Fig. 4F). The transcript abundance of SmbHLH9 was higher only in the stems, petioles, and flowers of O-RP (Figs. 4A, 4C, 4D). Almost no expression of SmbHLH26 and SmbHLH10 were observed in all tissues of the four varieties.

Expression profiles of eggplant bHLH genes under different LED lights

Light intensity and light quality have different effects on the expression of genes. Under different LED lights (red and blue light ratios of 1:1, 3:1, 6:1, 9:1, respectively), we examined the expressions of the above six SmbHLH genes in the leaves (Fig. 5) and peels (Fig. 6) of eggplant. The transcript abundances of SmbHLH1, SmbHLH117, SmbHLH113, SmbHLH26, SmbHLH10, and SmbHLH9 genes in leaves (Fig. 5), and SmbHLH113, SmbHLH26, and SmbHLH10 in peels of eggplant under R:B = 6:1 treatment (Figs. 6C, 6D, 6E) were higher than those in other treatments. The expression of SmbHLH1, SmbHLH117, and SmbHLH9 in the peels of eggplant (Figs. 6A, 6B, 6F) was higher under R:B = 9:1 treatment. These results suggested that all of the above genes related to anthocyanin biosynthesis could positively regulate plants to respond to LED 6 red:1 blue light ratio.

Response of eggplant bHLH genes expression to temperature

To investigate the roles of SmbHLH genes under different temperatures, trials at 28 °C (optimum temperature), 4 °C (low temperature), and 40 °C (high temperature) were conducted. The relative expression levels of the above six genes, namely, SmbHLH1, SmbHLH117, SmbHLH113, SmbHLH26, SmbHLH9, and SmbHLH10 were performed by qRT-PCR (Figs. 7–9). At the optimum growth temperature of eggplant at 28°, there were large changes in gene expression with the increase of treatment time. The expression of SmbHLH26 and SmbHLH10 peaked at 6 h (Figs. 7D, 7E) and SmbHLH9 (Fig. 7F) at 3 h, whereas the expression levels of the other genes were relatively stable. Under treatment at 4 °C, the transcript abundance levels of SmbHLH1, SmbHLH117, and SmbHLH9 were higher after 3 h of cold treatment (Figs. 8A, 8B, 8F), while SmbHLH26 and SmbHLH10 were higher after 6 h of cold treatment (Figs. 8D, 8E). The expression levels of the six genes were greatly induced by a high temperature of 40 °C (Fig. 9). The expression levels of SmbHLH1, SmbHLH117, SmbHLH26, and SmbHLH9 peaked at 6 h (Figs. 9A, 9B, 9D, 9F), while SmbHLH113 and SmbHLH10 peaked at 12 h (Figs. 9C, 9E). These results suggest that all of the above-mentioned genes that are related to anthocyanin biosynthesis positively regulate the plants response to temperature changes, especially to stress caused by low and high temperatures.