Genome-wide analysis of the bHLH gene family in Chinese jujube (Ziziphus jujuba Mill.) and wild jujube
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The bHLH (basic helix-loop-helix) transcription factor is one of the largest families of transcription factors in plants, containing a large number of members with diverse functions. Chinese jujube (Ziziphus jujuba Mill.) is the species with the highest economic value in the family Rhamnaceae. However, the characteristics of the bHLH family in the jujube genome are still unclear. Hence, ZjbHLHs were first searched at a genome-wide level, their expression levels under various conditions were investigated systematically, and their protein-protein interaction networks were predicted.
We identified 92 ZjbHLHs in the jujube genome, and these genes were classified into 16 classes according to bHLH domains. Ten ZjbHLHs with atypical bHLH domains were found. Seventy ZjbHLHs were mapped to but not evenly distributed on 12 pseudo- chromosomes. The domain sequences among ZjbHLHs were highly conserved, and their conserved residues were also identified. The tissue-specific expression of 37 ZjbHLH genes in jujube and wild jujube showed diverse patterns, revealing that these genes likely perform multiple functions. Many ZjbHLH genes were screened and found to be involved in flower and fruit development, especially in earlier developmental stages. A few genes responsive to phytoplasma invasion were also verified. Based on protein-protein interaction prediction and homology comparison, protein-protein interaction networks composed of 92 ZjbHLHs were also established.
This study provides a comprehensive bioinformatics analysis of 92 identified ZjbHLH genes. We explored their expression patterns in various tissues, the flowering process, and fruit ripening and under phytoplasma stress. The protein-protein interaction networks of ZjbHLHs provide valuable clues toward further studies of their biological functions.
KeywordsZjbHLHs Chinese jujube Tissue-specific expression Flower and fruit development Phytoplasma Protein-protein interaction
Apparently normal leaves
Coding domain sequences
Early white mature fruit stage
Full-red fruit stage
Hidden Markov model
Half-red fruit stage
Jujube witches’ broom disease
Quantitative real-time PCR
- white WM
mature fruit stage
Young fruit stage
Transcription factors (TFs) are important regulatory factors in eukaryotes that interact with cis-elements to regulate the expression of specific genes in response to environmental stresses . According to the sequence of arginine and lysine residues in the DNA binding region, the TFs of higher plants can be divided into four categories: zinc finger , helix-turn-helix (HTH) , basic leucine zipper (bZIP) , and helix-loop-helix (HLH) .
The basic helix-loop-helix (bHLH) family is one of the largest TF families in plants . The bHLH domain is composed of approximately 50–60 conserved amino acid sequences and contains two functional regions: one is the basic amino acid region with a length of approximately 15 amino acids at the N-terminal, and the other is the HLH region at the C-terminal . The basic region of approximately 15 amino acids is responsible for binding to the E-box (CANNTG) element. Studies have shown that two helices of the same transcription factor or different transcription factors interact to form homologous or heterologous dimers, which can combine with different parts of the gene promoter to regulate the target gene . Moreover, some atypical bHLHs with a less basic region that is critical for DNA binding were further identified and characterized in Arabidopsis [9, 10, 11].
As an increasing number of genome sequences are being released, a variety of bHLH superfamily genes have been identified and analyzed in a wide range of plant species, such as Arabidopsis , pear , peach , apple , grape  and cotton . Furthermore, the functions of many bHLH proteins in plants have been studied in detail. bHLHs play various roles in plant development [16, 17, 18], signal transduction , tolerance [19, 20, 21] and secondary metabolite production . Identification of bHLHs at a genome-wide level is the first step toward further studies into their biological functions. However, the bHLH transcription factors in Chinese jujube (Ziziphus jujuba Mill.) have not been reported before and the related functions of ZjbHLHs are still unknown.
Chinese jujube is a species with high economic value in the family Rhamnaceae and is also one of the most representative national fruit trees in China. Wild jujube, the wild relative species of Chinese jujube, usually has smaller trees and fruits than the Chinese jujube. Both jujube and wild jujube trees have many agronomic advantages, such as early fruit production, long flowering season and high tolerance to various biotic and abiotic stresses. And the bHLH gene family has a variety of functions such as flower and fruit development and is necessary for the normal growth and development in many plants [8, 16, 17, 18, 19, 20, 21, 22]. Compared with other TFs, bHLHs are involved in more reaction pathways and acted as some co-regulators on gene expression together with many other proteins. Therefore, we want to figure out the functions of this gene family in jujube. The jujube genome database [23, 24] provided the possibilities and resources for searching the crucial gene families related to its biological characteristics at the genome level. For the wide and diverse biological roles of bHLHs in plant development, the gene number, classification, and gene structure of this family in the jujube genome and their expression under various conditions in jujube and wild jujube were systematically analyzed in this study, and the protein-protein interaction networks were also predicted. The results provide valuable clues for further revealing the functions of this family in jujube growth and development.
Identification of ZjbHLHs
The information of bHLH gene family in Chinese jujube
Previous genome evolution studies showed that Chinese jujube is closely related to species of the Rosaceae family , so the number of bHLHs from three Rosaceae species (apple, pear and peach), grape, cotton and Arabidopsis were compared with ZjbHLHs (Additional file 2: Table S1). There were similar gene numbers in the bHLH family of jujube and peach, and this result was consistent with a previous study on the MADS-box family . Compared with the number of bHLH genes found in other plant species, the number of bHLH genes found in jujube and peach was lower. The gene numbers may be related to evolutionary differences, genome replication, or the genome size of these plants .
Phylogenetic analysis of ZjbHLHs
Multiple sequence alignment and conserved motifs in ZjbHLHs
The chromosomal location and gene structure of ZjbHLHs
Expression patterns of ZjbHLHs in various tissues/organs
In addition, the expression of ZjbHLH8 and 19 genes in the branches and leaves of jujube was significantly weaker than that in wild jujube. This differential expression indicated that some ZjbHLHs may have different functions between jujube and wild jujube.
ZjbHLHs involved in flower and fruit development
ZjbHLHs participated in jujube-phytoplasma interactions
ZjbHLH protein-protein interaction network prediction
In this study, a total of 92 bHLH genes were identified in the jujube genome. Based on phylogenetic analysis, intron-exon gene structure, conserved protein motifs and amino acid physical and chemical property prediction, these ZjbHLHs were divided into 16 categories; most sequences have the same conservative sequence except for the VI, VII, and VIII groups. Among them, there are 10 atypical sequences, and this trait has also been demonstrated in other plants . Furthermore, our BLAST results strongly supported our classifications of the ZjbHLHs, and detailed information about these orthologs was also summarized (Additional file 7: Table S2).
Many ZjbHLH proteins are involved in jujube flower development. ZjbHLH2 and ZjbHLH4 were expressed at higher levels at earlier flower development stages. These two genes belong to the ICE1 branch, which can regulate lateral bud growth . A similar function was also confirmed by homologous protein interactions (Fig. 6B-a). ICE1 can interact with the HOS1 protein, which is an important regulator of flowering time . ZjbHLH65, 83 and 87 proteins were homologous to CIB4, FBH4 and HEC2, respectively, and were three key regulatory factors in flower development (Fig. 6B-b, c, d). ZjbHLH83 is the homologous gene of FBH4 (At2g42280). Overexpression of FBH4 in Arabidopsis drastically elevated CO expression and caused early flowering regardless of the photoperiod . Here, ZjbHLH83 showed high expression at earlier flower development stages (Fig. 6a) and was also predicted to interact with CO (Fig. 6B-c). In addition, a series of CIB genes (CIB1, 2, 3, 4, and 5) in Arabidopsis can activate FT transcription by interacting with CRY2 protein and mediate the regulation of flowering time . There are also a series of CIB homologous genes in jujube, namely, ZjbHLH65 (homologs of CIB1), ZjbHLH74 and 75 (homologs of CIB3), ZjbHLH68 (homologs of CIB4), and ZjbHLH69 (homologs of CIL1). Since CIB genes have been proven to be conserved among plants , they are likely to have a similar function in flower development.
In fruit development, the expression patterns of most ZjbHLH genes detected in the two cultivars were in line with each other, which indicated that their functions in fruit development might be conserved among jujube varieties. ZjbHLH63 expression was significantly higher at the early stage of fruit development (Fig. 7a), which is the period of fruit enlargement. ZjbHLH63 is the homolog of AtPIF3 (Fig. 9), a key factor affecting light morphogenesis . The homologous comparison and protein interaction prediction (Fig. 7B-b) also indicated that it might be involved in fruit enlargement. In addition, ZjbHLH15 homologous protein in Arabidopsis was predicted to be involved in fruit anthocyanin synthesis (Fig. 7B-a), but in this study, its expression decreased significantly at the fruit coloring stages (Fig. 7a). Therefore, we hypothesized that jujube fruit color changes might not correlate with the accumulation of anthocyanin.
In addition, the expression patterns of some bHLH genes in jujube and wild jujube were not the same, indicating that ZjbHLH genes participate in different regulation pathways between jujube and wild jujube, especially in flower development. Further studies are needed to elucidate the detailed interaction network of the growth and development of jujube and wild jujube.
This study described the bHLH gene family of Chinese jujube at the genome level. Their gene structure, chromosomal distribution, phylogenetic relationship, and tissue-specific expression patterns were presented. Ten ZjbHLHs with atypical bHLH domains were identified. Many ZjbHLH genes were confirmed to involve in flower and fruit development and responsive to phytoplasma stress. An integrated ZjbHLHs protein-protein interaction network was also predicted. These results are very meaningful to the future functional analysis of ZjbHLHs.
Chinese jujube and wild jujube trees used in this study are cultivated in the Experimental Station of Chinese Jujube, Hebei Agricultural University. No specific permits are required for the sample collection. They are not endangered or protected species.
Seven tissue types (roots, young branches, old branches, leaves, flower buds, flowers and young fruits) collected from three jujube trees and three wild jujube trees were used for organ-specific expression analysis. The flowers of jujube and wild jujube were used for qRT-PCR analysis. The four development stages sampled were the bud emergence stage (FL1), inflorescence emergence stage (FL2), yellow bud stage (FL3) and petal spread stage (FL4). The fruits of two jujube cultivars (‘Lizao’ and ‘Yazao’) were used to investigate the expression pattern of ZjbHLHs. Five developmental stages, including the young fruit stage (Y), early white mature fruit stage (EWM), white mature fruit stage (WM), half-red fruit stage (HR) and full-red fruit stage (FR), were sampled. Each treatment was collected from three biological replicates.
Three kinds of tissues representing disease symptoms of different severity of Jujube witches’ broom (JWB) disease (apparently normal leaves (ANL), phyllody leaves (PL), and witches’ broom leaves (WBL)) from diseased trees and healthy leaves (HL) from healthy trees were collected in four periods (June, July, August and September). All treatments were conducted with three biological replicates.
Identification and protein structure analysis of ZjbHLHs
The hidden Markov model (HMM) file of the bHLH domain (PF00010) was downloaded from the Pfam database (http://pfam.xfam.org/), and HMMER 3.1b2 software was used to find the ZjbHLH protein sequences in the jujube genome . To further confirm our sequences, we used the online CD-search tool (NCBI database), the SMART tool (http://smart.embl-heidelberg.de/) and the website of PlantTFDB to screen sequences. Truncated and false genes were excluded from our analysis. The number of amino acids, molecular weight, and theoretical pI of ZjbHLH genes were predicted by NCBI and ProtParam (https://web.expasy.org/compute_pi/). The conserved motifs of ZjbHLH proteins were detected by MEME (http://meme-suite.org/) .
The chromosomal location and gene structure of ZjbHLHs
To determine the chromosomal location of the ZjbHLH genes, their gene sequences were used as query sequences in BLASTN searches against the jujube genome. Each ZjbHLH gene was mapped to the jujube genome according to its genome coordinates. Tandem duplications were identified as previously described .
Multiple sequence alignment and phylogenetic tree construction
Multiple sequence alignment was analyzed by using ClustalX2 and edited by BioEdit. A phylogenetic tree of 92 ZjbHLHs was constructed based on their conserved domains. bHLH proteins of six other species (Arabidopsis thaliana, Prunus persica, Malus domestica, Pyrus bretschneideri, Vitis vinifera L. and Anemone vitifolia Buch.) were downloaded from NCBI. MEGA 7 software and the neighbor-joining statistical method were used to construct a rooted phylogenetic tree [43, 44]. The evolutionary distances were obtained using the p-distance method, and these distances were used to estimate the number of amino acid substitutions per site. The reliability of each phylogenetic tree was established by conducting 1000 bootstrap sampling iterations.
RNA isolation and expression analysis
Total RNA was extracted using an RNAprep Pure Plant Kit (TIANGEN) according to the manufacturer’s protocol. After genomic DNA was removed by RNase-free DNase I (TIANGEN), the RNA concentration and purity were checked on a NanoDrop2000 spectrophotometer. First-strand cDNA was synthesized by reverse transcribing 500 ng of total RNA with a FastQuant RT Super Mix Kit (TIANGEN). The cDNA was used as the template for gene expression analysis.
Gene expression was detected by semiquantitative PCR and qRT-PCR. The primers used in this study are listed in Additional file 8: Table S3. PCR products were amplified in triplicate using Bio-Rad iQ™5 with TransStart Top Green qPCR SuperMix AQ131 (TransGen Biotech, China) in 20 μL reactions. Each reaction contained 10 μL of 2 × TransStart® Top Green qPCR SuperMix, 0.4 μL each of 10 μM primers, 8.2 μL of ddH2O and 1 μL of cDNA. The thermal profile for RT-qPCR was as follows: preincubation for 30 s at 95 °C, followed by 40 cycles of 5 s at 95 °C, 10 s at 53–58 °C, and 10 s at 72 °C. Three biological replicates were performed for each treatment. Threshold cycle values were calculated using iCycler software, and ZjACT was used as an internal control . Relative transcript levels were calculated according to the 2–ΔΔCT method .
Protein-protein interaction network prediction
Ninety-two ZjbHLH protein sequences were used as queries, and protein-protein interactions were predicted by the STRING website (https://string-db.org/). The orthologs of Arabidopsis thaliana were selected as references. After completing the BLAST step, the network was constructed using the highest score gene (bitscore). Finally, an interaction network among ZjbHLHs was constructed in this study.
JZ designed the research; HL, WG, CX, YZ and JZ performed the experiments, analyzed the data and wrote the paper. ZL, ML and YZ participated in the data analysis. YZ and XM performed RT-PCR and RT-qPCR experiments. All authors read and approved the final the manuscript.
Supported by the National Key R&D Program Project Funding (2018YFD1000607), the National Natural Science Foundation of China (31772285), Foundation for 100 Innovative Talents of Hebei Province (SLRC2019031), Hebei Distinguished Young Scholar (C2016204145), and Hebei Special Funds for Intellectual Introduction and Talent Training (A2016002054). These funding bodies had no role in the design of the study, sample collection, analysis or interpretation of data and in writing the manuscript.
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The authors declare that they have no competing interests.
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