Genome-wide Analysis of the bHLH Transcription Factor Family and Functional Identification of bHLH Genes Related to Puerarin Biosynthesis in Pueraria lobata var. thomsonii (Benth.)

doi:10.21203/rs.3.rs-1703982/v1

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Genome-wide Analysis of the bHLH Transcription Factor Family and Functional Identification of bHLH Genes Related to Puerarin Biosynthesis in Pueraria lobata var. thomsonii (Benth.)

https://doi.org/10.21203/rs.3.rs-1703982/v1

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published 28 Feb, 2023

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Plant basic helix-loop-helix (bHLH) transcription factors are involved in diverse biological process. Several bHLH transcription factors have been functionally characterized in various crop species. However, no systematic and comprehensively identification of bHLH members has been in Pueraria lobata var. thomsonii (Benth.) (P. thomsonii), a traditional Chinese herb that is an excellent source of puerarin. Phylogenetic analysis showed that 219 bHLH genes were identified in the P. thomsonii and classified into 13 subfamilies. All the PlbHLH genes were distributed on the 11 chromosomes unevenly and the number of PlbHLH genes on each chromosome varied from 12 to 44. Both of tandem and segmental duplications were key factors driving bHLH gene family expansion in P. thomsonii. The gene structure analysis indicated that 20 conserved motifs were found in PlbHLH gene family, 185 PlbHLH members contain more than two introns, and genes within the same subfamily appeared to share similar intron-exon gene structures. The Ka/Ks analysis indicated PlbHLH gene family undergo purifying selection during evolution. The hierarchical clustering analysis indicated that 41 PlbHLHs showed root-specific expression patterns, and seven of them exhibited up-regulated expression pattern after MeJA treatment within 24 h. Using qRT-PCR validation, five puarian synthesis genes had a similar expression pattern with the seven PlbHLH genes in response to MeJA within 24 h. Together, our study provided a good foundation for further research into the regulatory mechanism of the seven candidate PlbHLH proteins for pueraria biosynthesis in P. thomsonii.

Pueraria thomsonii

Genome-wide analysis

bHLH transcriptional factors

Expression analysis

Pueraria biosynthesis pathway

The basic/helix–loop–helix (bHLH) transcription factors have been widely found in all three eukaryotic kingdoms (Duek and Fankhauser 2005; Carretero-Paulet et al. 2010). bHLH transcription factors are identified by a conserved 50–60 amino acid sequence responsible for DNA binding, according to the typical bifunctional structure, namely the basic and HLH regions. The basic region is a 15–20 residues located at the N-terminal end, which determine the recognition of the cis-acting element E-box (5’-CANNTG-3’) or G-box (CACGTG) and allows the binding of the HLH region (Li et al. 2006). The basic region contains about 15 amino acids of which six are typically basic amino acid residues. In contrast, the HLH domain, located at the C-terminal end, is mainly composed of two relatively conserved amphipathic helices with a linking loop that varies in length or sequence. bHLH transcription factors play crucial role in plant second metabolic regulation (Heim et al. 2003).

Since the bHLH domain was initially defined, a variety of bHLH subgroups have been identified based on the frequencies of 19 conserved amino acids in the bHLH consensus motif (Atchley and Terhalle 1999). The first bHLH, Lc (L-myc), is required for the flavonoid/anthocyanin biosynthetic pathway in maize (Zea mays) (Ludwig et al. 1989). Subsequently, many more functions for plant bHLH genes have been identified, including the regulation of light signaling (Roig-Villanova et al. 2007); hormone signal transduction (Lee et al. 2006); responses to abiotic stress (Jin et al. 2016). In addition to this, bHLHs participate in regulating flavonoid biosynthesis (Matus et al. 2010). The heterogeneous overexpression of liverwort (Plagiochasma appendiculatum) PabHLH1 in Arabidopsis could activate the synthesis of both flavonoids and anthocyanins, through the up-regulation of early and late structural genes in the synthesis pathway of flavonoids (Zhao et al. 2019). Two bHLH genes, bHLH003 and bHLH007, were also found to be related to anthocyanin or flavonoids synthesis in grape (Vitis vinifera) (Wang et al. 2018a).

So far, extensive study on the bHLH gene family members in many crop species has increased our understanding of their transcriptional regulatory mechanism (Guo et al. 2021; Qian et al. 2021). However, characteristics of bHLH gene family in Pueraria lobata var. thomsonii Pueraria lobata var. thomsonii (Benth.) (hereinafter referred as P. thomsonii), belong to the leguminosae family, has long been used as traditional Chinese herb, have not yet been classified. The roots of P. thomsonii have long been used for treating fever, indigestion, indigestion, toxicosis, and liver damage from alcohol abuse in traditional Chinese medicine (Wang et al. 2020), which was recorded in The Divine Husbandman’s Classic of Materia Medica (Shen Nong Ben Cao Jing) complied in the Eastern Han Dynasty (25–250 AD) (Zhao and Guo 2018). Puerarin, the 8-C-glucoside of daidzein, was synthesized through the phenylpropanoid pathway under the actions of phenylalanine ammonia-lyase (PAL), cinnamic acid 4-hydroxylase (C4H), 4-coumarate: coenzyme A ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), isoflavone synthesis (IFS), 2-hydroxyisoflavanone dehydratase (HID) and then modified by glucosyltransferase (GT) (Wang et al. 2017). Although some putative structural genes involved in pueraria biosynthesis has been identified through transcriptome sequencing and biochemical analysis (Wang et al. 2015). However, it remains largely unknown whether the bHLH gene family members are involved in the regulation of puerarin biosynthesis in P. thomsonii.

Currently, we finished the genome sequencing of P. thomsonii, which provides an excellent opportunity for further genome-wide analysis of P. thomsonii genes (Shang et al. 2022). In this study, we performed a genome-wide analysis of bHLH genes in P. thomsonii and 219 PlbHLH genes were identified. Further, a comprehensive analysis including phylogenetic relationship, chromosomal location, gene duplication, collinearity, gene structure, conserved motifs was performed. Based on different tissues expression pattern of PlbHLH genes as well as up-regulated expression in response to methyl jasmonate (MeJA) treatment, several PlbHLH genes were considered as candidate regulators involving in puerarin biosynthesis.

Plant Materials and Treatment

P. thomsonii were grown in MS medium in an artificial climate at 26±2℃, with a photoperiod of 16 h light and 8 h dark, at Guangxi Agricultural Academy Sciences (Nanning, China). For MeJA treatment, 4-week-old seedlings were cultured in nutrient solutions with 100 uM MeJA treatment. Roots, stems and leaves for RNA extraction were collected at 0 h, 12 h and 24 h after the treatment, respectively, immediately frozen in liquid nitrogen and stored at −80°C for RNA-sequencing. Three seedlings were used as biology replicate.

P. thomsoniibHLH Sequence Retrieval and Analysis

We used blastp to search for candidate bHLH genes in P. thomsonii genome with an e-value cutoff of 1e-5 using 167 Arabidopsis thaliana (A. thaliana) bHLH proteins as query sequences (Tian et al. 2019). In addition, the bHLH encoding genes in P. thomsonii with Pfam00010 were identified using Hidden Markov Model (HMM) (EI-Gebali et al. 2019). The remaining sequences were checked for the conserved bHLH domain using Conserved Domain Database (CDD) (Lu et al. 2020) and SMART (Letunic and Bork 2007). The theoretical isoelectric point (PI) and molecular weight (MW) were predicted using ExPASy (Gasteiger 2003). ProtComp v 9.0 was used to predicted protein subcellular location.

Phylogenetic Analysis

Multiple sequence alignments using MUSCLE with default parameters were applied to confirm the conserved domains of predicted PlbHLH proteins. A phylogenetic tree was created using the NJ method of MEGA X version 10.1.7 with 1000 bootstrap replicates based on the sequence alignments (Kumar et al. 2018), and was visualized by the Interactive Tree of Life (iTOL) (Letunic and Bork 2007). The phylogenetic tree was visualized in MEGA5.0.

Chromosomal Location and Gene Synteny Analysis

PlbHLH genes mapping was performed based on physical location information from the database of P. thomsonii genome using TBtools (Chen et al. 2018). The gene duplication events were conducted using the Multiple Collinearity Scan toolkit (MCScanX), with the default parameters (Wang et al. 2012). The orthologous bHLH genes between P. thomsonii and A. thaliana as well as those between P. thomsonii and Glycine max were identified using OrthoVenn2 (Xu et al. 2019). Multiple Collinearity Scan toolkit (MCScanX) was used to analyze the gene duplication events with default parameters (Wang et al. 2012). Non-synonymous (ka) and synonymous (ks) substitutions of each duplicated bHLH gene were calculated using the Tbtools software (Chen et al. 2020).

Conserved Motifs and Genes Structure Characterization

MEME version 5.1.1 program was used to detect the conserved motifs of PlbHLH proteins using the parameters: Zero or One Occurrence per Sequence (zoops), maximum number of motifs, 15; and the optimum width of each motif, 6–50 residues (Bailey et al. 2009). Gene Structure Display Server (http://gsds.cbi.pku.edu.cn) was used to analyze the exon–intron structures within the coding sequences and the genomic sequences of each predicted PlbHLH.

Transcriptome Data Analysis

Expression pattern of each PlbHLH gene was analyzed from our previously reported RNA-seq data (Shang et al. 2022). The data included three tissues, including root, stem and leave. The expression level of each predicted transcript in each RNA-seq library was calculated as the fragments per kilobase of transcript per million fragments mapped (FPKM) using RSEM (Li and Dewey 2011). To assess the three biological replicates, the ratio of root/stem and root/leaf-transformed FPKM values were performed. The expression level of PlbHLH genes in roots with more than 2-fold change stem and leave were deemed as the the genes which were expressed the highest in root. Each PlbHLH gene was analyzed and normalized and displayed in heat map (Chen et al. 2018).

Quantitative Real-time RT-PCR Validation

Changes in the expression of PlbHLH genes in response to MeJA were detected by quantitative real-time polymerase chain reaction (qRT-PCR) analysis using SYBR® Premix Ex TaqTM (TaKaRa) chemistry on a Stratagene Mx3000P (Stratagene, CA, USA) instrument. For each biological replicate, quantifications were conducted in triplicate. The primers were designed using Primer 3 online (http://bioinfo.ut.ee/primer3-0.4.0/primer3/). Primer information is presented in Supplementary Table S1. The Plactin gene was used as the internal. The relative expression levels of the target genes were assessed based on 2− Ct method (Livak and Schmittgen 2001).

Statistical Analyses

All the expression data were analyzed using Excel and SPSS Statistical Software (version 17.0; Chicago, IL, United States), by ANOVA, followed by Tukey’s signi cant difference test at P<0.05.

Identification and Classification of PtbHLH Proteins in P. thomsonii genome

A total of 219 putative PlbHLHs were defined from P. thomsonii based on the methods in “Materials and methods”. Sequence analysis indicated that the PlbHLH proteins varied from 91 amino acids to 799 amino acids, with molecular weight ranging from 10356.7 to 89787.63 kDa, the theoretical isoelectric point values ranging of 4.29-9.61. A total of 197 PlbHLH proteins localized in nucleus, while 14 PlbHLH proteins were located in chloroplast, one in endoplasmic reticulum (PlbHLH199), four in mitochondrion, two in plasma membrane (PlbHLH57/169), one in peroxisome (PlbHLH196) (Supplementary Table S2).

Phylogenetic Analysis

To explore the evolutionary relationship and the classification of PlbHLHs, a neighbor-joining phylogenetic tree was constructed with conserved sequences of 219 PlbHLHs and 167 AtbHLHs. Based on this phylogenetic analysis and the previous reported classification of the AtbHLHs, we classified the PlbHLH family with 219 members into 13 subfamilies, designated as I to XIII (Fig. 1). This result corroborated the previously proposed classification of the bHLH family members (Pires and Dolan 2010). As is shown in Fig. 1, group I was the largest subfamily with 36 PlbHLH members, followed by IV with 34 members. In contrast, Group II, the smallest, has only two members (PlbHLH76/93), followed by group VI with four PlbHLHs (PlbHLH22/23/49/50).

Chromosomal Distribution and Synteny Analyses of PlbHLH gene Family

Based on their chromosomal locations, PlbHLH proteins were assigned from PlbHLH1 to PlbHLH219 (Supplementary Table S2). All the PlbHLH genes were distributed on the 11 chromosomes (chr) unevenly. The number of PlbHLH genes on each chr varied from 12 to 44. Chr11 had only 12 PtbHLH genes, both of chr6 and chr9 had 15 PlbHLH members. In contrast, chr1 contain the largest number, 44, of PlbHLH genes followed by 23 on chr4. Most members are closely tandem distributed on chromosomes. In addition, PlbHLH219 was positioned near the telomeric regions on chr11 (Fig. 2). According to a previous study, if two or more genes are present within 200 kb, the elements are considered a tandem repeat event (Holub 2001). It is shown that 35 PlbHLHs were identified to be tandem duplicated genes which were distributed on each chr. The duplicated genes were PlbHLH4/5, 12/13, 18/19, 25/26, 29/30, 33/34/35, 36/37/38, 39/40, 47/48, 54/55, 59/60, 62/63, 67/68, 70/71, 84/85/86, 87/88, 90/91/92, 102/103, 105/106, 114/115/116, 121/122, 125/126, 136/137, 138/139, 144/145, 161/162, 163/164/165/166, 173/174, 175/176, 180/181, 182/183, 197/198, 202/203, 213/214, 216/217 (Fig. 2; Supplementary Table S2). Basic local alignment search tool for proteins (BLASTP) and MCScanX were performed to construct the collinearity of the PlbHLH gene family and identify the possible relationship and potential duplication events between them. Intrachromosomal duplications of the PlbHLH gene family were observed in the P. thomsonii genome (Supplementary Fig. S1). In detail, 30 pairs of PlbHLH genes duplicated tandemly on the 11 chromosomes (Supplementary Table S3). Together, the results suggested that both of tandem and segmental duplications are key factors driving bHLH gene family expansion in P. thomsonii (Guo et al. 2013).

To further illustrate the potential evolutionary patterns of the PlbHLH gene family, a comparative orthologous analysis was performed between P. thomsonii and the other two plant species, A. thaliana and soybean (Glycine max), which belong to the Brassicaceae and leguminosae families, respectively (Fig. 3). The orthologous gene pairs between P. thomsonii and A. thaliana and P. thomsonii and Glycine max were 153 and 584, respectively (Supplementary Tables S4 and S5). These results revealed that the identified orthologous events of PlbHLH-GmbHLH were greatly more than those of PlbHLH-AtbHLH based on the close evolutionary relationship between P. thomsonii and Glycine max. An extensive level of synteny conservation and increased number of orthologous events of PlbHLH-GmbHLH indicated that PlbHLH genes in P. thomsonii shared a similar structure and function with GmbHLH genes in Glycine max.

To further investigate the driving force behind the duplication of PlbHLH gene pairs in P. thomsonii, Ka/Ks (non-synonymous/synonymous substitution ratio) calculation of the duplicated bHLH gene pairs was performed to determine whether a selective pressure acted on the bHLH genes. Interestingly, all the Ka/Ks values of orthologous PlbHLH gene pairs were less than 1, indicating that these genes were subjected to purifying selection with limited functional divergence during evolution after duplication events (Supplementary Tables S4 and S5).

Conserved Motifs, Domain and Gene structure Characterization

To support the phylogenetic analysis, motifs of PlbHLH family members was investigated. The results showed that 20 motifs were found in the PlbHLH gene family (Fig. 4a). In details, motif 1 was presented in all the PlbHLH members. All the PlbHLH members contained motif 2 except PlbHLH192. We found that motif 11 was only presented in PlbHLH51/192/133, and PlbHLH161-166. Motif 2 and Motif 9 were repeated twice in PlbHLH136/137/190/191/215, and PlbHLH19/20/121/122, respectively. Motif 19 was only presented in PlbHLH54/55/156/185, and repeated twice in PlbHLH54/55. The members that phylogenetically clustered in the same subfamily often showed a similar motif pattern. For example, the closely clustered pair PlbHLH24/45/47/48/136/137/1901/191/167 in subfamily I had identical motifs 1-3, 5, 6 and 14. All the 12 members of group IV contained motifs 2, 13 and 15 (Supplementary Table S6). Noticeable, all the PlbHLHs contained the conserved HLH domain except PlbHLH1/2/18/29/35/36/37/39/52/53 (Fig.4b).

Exon-intron structural diversity, an important part in the evolution of gene families, provides additional evidence supporting phylogenetic groupings (Shiu and Bleecker 2003). In order to better understand the structural features of PlbHLHs, the intron structure was detected based on their evolutionary relationships. As shown in Fig. 4c, the number of introns in PlbHLH members varied from 0 (PlbHLH8/9/16/17/46/61/104/113/119/120/122/135/177/205) to 16 (PlbHLH137). Twenty PlbHLH genes contain one intron. PlbHLHs genes within the same subfamily appeared to share similar intron-exon gene structures. A total of 185 PlbHLH genes contain more than two introns. Nevertheless, the number of exons and introns varied greatly among other subgroups (Fig. 4c).

Tissue Expression Pattern of PlbHLH Genes

In order to investigate the possible function of PlbHLH genes, hierarchical cluster analysis was performed to assess the tissue-specific transcript abundance of each PlbHLH gene, represented by FPKM, using the expression patterns of 219 PlbHLH genes corresponding to root, stem and leaf of P. thomsonii (Supplementary Fig. S2). The results indicated that five PlbHLH genes (PlbHLH13/15/78/129/155) showed no expression in the three tissues, in particular, 20 PlbHLHs (PlbHLH3/16/17/18/40/46/48/59/77/86/93/125/131/138/139/162/163/174/177/195) showed high expression level in the three tissues. High expression in root and stem, but low expression in leaf tissue was prevalent among in 13 PlbHLHs (PlbHLH25/31/34/47/53/100/115/121/126/127/165/195/196). Four PlbHLHs (PlbHLH60/63/67/145) showed high expression level in stem (Supplementary Table S7). In order to identify the highest expressed PlbHLH genes which were deemed as the root-specific expressed, the transcriptional abundance of PlbHLH genes in root with more than twice than stem and leaf were selected, consequently, 41 PlbHLH genes were screened (Fig. 5; Supplementary Table S8).

Identification of PlbHLH Genes Related to Pueraria Biosynthesis

In P. thomsonii, pueraria was produced in root, and pueraria content were much higher in the root than in stem and leaf (Chen and Zhang 2001; Han et al. 2015). MeJA is a signaling molecule associated with biosynthesis of flavonoids (Shelton et al. 2012; Zhou and Memelink 2016; Du et al. 2021), for instance, the pueraria yield could be increased to 25 times by different concentration MeJA treatment (Goyal and Ramawat 2008). In view of this, MeJA treatment analysis was performed to narrow down the candidate structural genes. The RNA-seq result of Shang et al. (2022) and results of the qRT-PCR detection indicated PlCHS1 (Pmon008G00376), PlCHR3 (Pmon003G00389), PlIFS1 (Pmon003G02838), PlIFS2 (Pmon003G02830) and PlUGT155 (Pmon006G01850) were up-regulated expressed after treatment with MeJA within 24 h (Fig. 6).

The regulation of isoflavone is at the transcriptional level (Ferreyra and Rius 2012; Li 2014). The positive regulators often exhibit a similar expression pattern to structural genes in biosynthetic pathway (Zhang et al. 2008; Xiang et al. 2019; Zhu et al. 2019). Using qRT-PCR detection, we found that the expression of 19 PlbHLH genes were not influenced by MeJA treatment within 24 h (Supplementary Fig. S3). Compared with untreated root, nine PlbHLH genes were down-regulated in response to MeJA treatment at 12 h and 24 h (Supplementary Fig. S3). In contrast, seven PlbHLH genes (PlbHLH6/56/73/79/94/149/196), were up-regulated in response to MeJA within 24 h of treatment compared with the untreated root (Fig. 7). Importantly, the expression of PlCHS1, PlCHR3, PlIFS1, PlIFS2, and PlUGT155 was positively correlated with the expression of the seven PlbHLH genes. Together, the seven bHLH genes were the candidate genes which acts regulator of puerarin biosynthesis in P. thomsonii.

Puerarin, an isoflavone compound, only comes from P. thomsonii which is a traditional Chinese medicinal herb. It often serves as a natural source of puerarin in medicinal products for human health. The genome sequencing of P. thomsonii was completed recently (Shang et al. 2022), providing the opportunity to perform a genome-wide analysis of bHLH gene family in P. thomsonii. Numerous studies have shown that bHLH-type transcription factors are involved in diverse biological processes in plant growth, development, and stress responses and regulating specialized secondary metabolite biosynthesis (Guo et al. 2021; Qian et al. 2021). However, a systematic characterization of bHLH genes in P. thomsonii is lacked.

The bHLH transcription factors comprised a large family in plants. Based on sequence homology and phylogenetic relationships, 128 bHLH genes in pummelo (Citrus grandis), 133 bHLH genes in alfalfa (Medicago sativa), 167 bHLH proteins in A. thaliana, 197 bHLH proteins in pear (Pyrus bretschneideri), 230 bHLH genes in Chinese cabbage (Brassics rapa ssp. pekinensis), potato (Solanum tuberosum) with 259 bHLH proteins, 319 bHLH proteins in soybean, and 602 bHLH proteins in Brassica napus (Toledo-Ortiz and Huq 2003; Li et al. 2006; Hudson and Hudson 2015; Mao et al. 2017; Wang et al. 2018b; Ke et al. 2020; Dong et al. 2021; Ndayambaza et al. 2021). In our study, there were 219 PlbHLH genes divided into 13 subfamilies. These results indicated that bHLH gene family has undergone substantial expansion, with potential subfunctionalization and neofunctionalization, as well as changes in gene expression patterns and protein–protein interactions.

The gain and loss of intron is highly related with rate of the gene molecular evolution (Pires and Dolan 2010). Intron loss might occur through recombination between the genomic copy of the gene and a reverse transcript of a spliced mRNA copy of the gene (Roy and Gibert 2006). It was demonstrated that genes with almost no intron often underwent positive selection during evolution, for instance, SAUR family (Chen and Hao 2014). In contrast, for bHLH gene family, a large number of bHLH genes with more than two introns were widely distributed in bHLH gene family of lotus (Nelumbo nucifers), cucumber (Cucumis sativus), pummelo and pear, and these bHLH genes underwent negative selection in the corresponding crop species (Mao et al. 2019; Li et al. 2020; Zhang et al. 2020; Dong et al. 2021). In the 219 PlbHLH genes identified in this study, 185 PlbHLH genes contain more than two introns, supporting the PlbHLH gene family undergo purifying selection during evolution process, which is consistent with the result of Ka/Ks analysis. Additionally, the distribution of introns in some gene families were not exactly the same because introns were constantly gained and lost (Chen and Cao 2014). Whether the PlbHLH gene family has a similar effect needs to study further in P. thomsonii.

Genes with similar expression patterns were consistent with their diverse functions in regulation of plant growth and development, and secondary metabolism. Transcriptional activators are often co-expressed with its downstream structural genes, therefore, co-expressed analysis is an effective way to identify regulators controlling secondary metabolism. For instance, the expression pattern analysis indicated that the expression of GmMYB29 was closely associated with that of IFS2, the expression of the two genes was consistent with the isoflavone content in different tissues. The GmMYB29-overexpressing transgenic lines leaded to higher isoflavones content in soybean hair roots via activating the transcription level of IFS2 (Chu et al. 2017). CsMYB31 was a transcriptional activator and positively regulates genus-specialized metabolite production in pepper, and the CsMYB31 expression pattern was similar with that of the 15 capsaicinoids-biosynthetic genes (Zhu et al. 2019). AabHLH112 could induce artemisnin biosynthesis via enhanced the promoter activity of artemisnin biosynthesis genes, ADS and CYP71AV1, and AabHLH112 was tightly associated with artemisinin biosynthesis genes (Xiang et al. 2019). In this study, the seven bHLH genes (PlbHLH6/56/73/79/94/149/196) and five key structure genes (PlCHS1, PlCHR3, PlIFS1, PlIFS2, and PlUGT155) were exhibited high expression in root. Furthermore, the five puerarin biosynthesis PlbHLH genes were responsive to MeJA, which could increase the content of puerarin. All these results implied that PlbHLH6/56/73/79/94/149/196 played an important role in the regulation of the puerarin biosynthetic pathway, and their function need further verification in P. thomsonii.

In summary, 219 bHLH genes were identified from P. thomsonii. Chromosomal distribution, conserved domains, motifs distribution, and exon-intron characteristics of PlbHLH gene family were investigated. The Ka/Ks analysis indicated PlbHLH gene family undergo purifying selection during evolution. The expression patterns of 219 PlbHLH genes in root, stem and leaf were analyzed. RNA-seq data, and qRT-PCR results revealed that seven PlbHLH genes had a similar expression patterns with five structural genes of the puerarin biosynthesis in response to MeJA treatment. The collected data from this study provided a foundation for advanced studies to evaluate the mechanisms of regulating puerarin biosynthesis for bHLH genes in P. thomsonii.

Author Contributions

HY, and LX designed the research. WZ prepared the materials. ZW and XS prepared all the Figures. SC, LL and PS prepared the supplementary Figures and Tables. LX performed the experiments and analyzed the data. LX and DH wrote the main text. HY revised the paper. All authors approved the final manuscript.

Funding

This study was supported by Guangxi Natural Science Foundation Project (2021GXNSFBA220026), Guangxi Natural Science Foundation Project (2020GXNSFAA297187), National Natural Science Foundation of China (31960420), and Special Project for Basic Scientific Research of Guangxi Academy of Agricultural Sciences (Guinongke 2021YT057).

Declaration of competing interest

The authors confirm no conflict of interest.

Data Availability

The transcriptome sequencing data can be found in DDBJ/ENA/GenBank and SRA under the umbrella of BioProject Accession PRJNA723378.

Atchley W R, Terhalle W, Dress A (1999) Positional dependence, cliques, and predictive motifs in the bHLH protein domain. J Mol Evol 48: 501–516
Bailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L et al (2009) MEME Suite: tools for motif discovery and searching. Nucleic Acids Res 37: W202–W208
Carretero-Paulet L, Galstyan A, Roig-Villanova I, Martinez-Garcia JF, Bilbao-Castro JR, Robertson DL (2010) Genome-wide classification and evolutionary analysis of the bHLH family of transcription factors in Arabidopsis, poplar, rice, moss, and algae. Plant Physiol 153: 1398–1412
Chen C, Xia R, Chen H, He Y (2018) TBtools, a toolkit for biologists integrating various biological data handling tools with a user-friendly interface. BioRxiv: 289660
Chen CJ, Chen H, Zhang Y, Thomas HR, Frank MH, He YH et al (2020) TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol Plant 13: 1194–1202
Chen G, Zhang JX, Ye JN (2001) Determination of puerarin, daidzein and rutin in Pueraria lobata (Wild.) Ohwi by capillary electrophoresis with electrochemical detection. J Chromatogr A 923: 255–262
Chen YZ, Cao J (2014) Comparative genomic analysis of the Sm gene family in rice and maize. Gene 539: 238–249
Chen YZ, Hao X, Cao J (2014) Small auxin upregulated RNA (SAUR) gene family in maize: Identification, evolution, and its phylogenetic comparison with Arabidopsis, rice, and sorghum. J Integr Plant Biol 56: 133–150
Chu S, Wang J, Zhu Y, Liu SL, Zhou XQ, Zhang HR et al (2017) An R2R3-type MYB transcription factor, GmMYB29, regulates isoflavone biosynthesis in soybean. PLOS Genet 13: e1006770
Dong HZ, Chen QM, Dai YQ, Hu WJ, Zhang SL, Huang XS (2021) Genome-wide identification of PbrbHLH family genes, and expression analysis in response to drought and cold stresses in pear (Pyrus bretschneideri). BMC Biol 21: 86
Du TT, Fan YX, Cao HY, Song ZH, Dong BY, Liu TY et al (2021) Transcriptome analysis revealed key genes involved in flavonoid metabolism in response to jasmonic acid in pigeon pea (Cajanus cajan (L.) Millsp.). Plant Physiol Biotech 168: 40–422
Duek PD, Fankhauser C (2005) bHLH class transcription factors take centre stage in phytochrome signalling. Trends Plant Sci 10: 51–54
Egan AN (2020) Economic and ethnobotanical uses of tubers in the genus Pueraria DC. Legume 19: 24
El-Gebali S, Mistry J, Bateman A, Eddy SR, Luciani A, Potter SC et al (2019) The Pfam protein families database in 2019. Nucleic Acids Res 47(Database issue): D427–D432
Ferreyra MF, Rius SP, Casati P (2012) Flavonoids: biosynthesis, biological functions, and biotechnological applications. Front Plant Sci 3: 222
Gasteiger E (2003) ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res 31: 3784–3788
Goyal S, Ramawat K (2008) Increased isoflavonoids accumulation in cell suspension cultures of Pueraria tuberosa by elicitors. Indian J Biotechnol 7: 378–382
Guo JR, Sun BX, He HR, Zhang YF, Tian HY, Wang BS (2021) Current understanding of bHLH transcription factors in plant abiotic stress tolerance. Int J Mol Sci 22: 4921
Guo RR, Xu XZ, Carole B, Li XQ, Gao M, Zheng Y et al (2013) Genome-wide identification, evolutionary and expression analysis of the aspartic protease gene superfamily in grape. BMC Genomics 14: 1–18
Han RC, Takahsshi H, Nakamura M, Yoshimoto N, Suzuki H, Shibata D et al (2015) Transcriptomic landscape of Pueraria lobata demonstrates potential for phytochemical study. Front Plant Sci 6: 426
Heim MA, Jakoby M, Werber M, Martin C, Weisshaar B, Bailey PC (2003) The basic helix-loop-helix transcription factor family in plants: a genome-wide study of protein structure and functional diversity. Mol Biol Evol 20: 735–747
Hien TT, Kim HG, Han EH, Wang KW, Jeong HG (2010) Molecular mechanism of suppression of MDR1 by puerarin from Pueraria lobata via NF-kappaB pathway and cAMP-responsive element transcriptional activity-dependent up-regulation of AMP-activated protein kinase in breast cancer MCF-7/adr cells. Mol Nutr Food Res 54: 918–928
Holub EB (2001) The arms race is ancient history in Arabidopsis, the wildflower. Nat Rev Genet 2: 516–527
Hudson KA, Hudson ME (2015) A classification of basic helix-loop-helix transcription factors of soybean. Int J Genomics 2015: 603182
Jin C, Huang XS, Li KQ, Yin H, Li LT, Yao ZH et al (2016) Overexpression of a bHLH1 transcription factor of Pyrus ussuriensis confers enhanced cold tolerance and increases expression of stress-responsive genes. Front Plant Sci 7: 441
Ke YZ, Wu YW, Zhou HJ, Chen P, Wang MM, Liu MM et al (2020) Genome-wide survey of the bHLH super gene family in Brassica napus. BMC Plant Biol 20: 115
Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35: 1547–1549
Lee S, Lee S, Yang KY, Kim YM, Park SY, Kim SY et al (2006) Overexpression of PRE1 and its homologous genes activates Gibberellin-dependent responses in Arabidopsis thaliana. Plant Cell Physiol 47: 591–600
Letunic I, Bork P (2018) 20 years of the SMART protein domain annotation resource. Nucleic Acids Res 46: D493–D496
Li B, Dewey CN (2011) RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinf 12: 323
Li JL, Wang T, Han J, Ren ZH (2020) Genome-wide identification and characterization of cucumber bHLH family genes and the functional characterization of CsbHLH041 in NaCl and ABA tolerance in Arabidopsis and cucumber. BMC Plant Biol 20: 272
Li S (2014) Transcriptional control of flavonoid biosynthesis. Plant Signal Behav 9: e27522
Li X, Duan X, Jiang H, Sun Y, Tang Y, Yuan Z et al (2006) Genome-wide analysis of basic/helix-loop-helix transcription factor family in rice and Arabidopsis. Plant Physiol 141: 1167–1184
Liu B, Wu ZY, Li YP, Ou CW, Huang ZJ, Zhang JW et al (2015) Puerarin prevents cardiac hypertrophy induced by pressure overload through activation of autophagy. Biochem Bioph Res Co 464: 908–915
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2 – ∆∆Ct method. Methods 25 402–408
Lu S, Wang J, Chitsaz F, Derbyshire MK, Geer RC, Gonzales NR et al (2020) CDD/SPARCLE: the conserved domain database in 2020. Nucleic Acids Res 48: D265–D268
Ludwig SR, Habera LF, Dellaporta SL, Wessler SR (1989) Lc, a member of the maize R gene family responsible for tissue-specific anthocyanin production, encodes a protein similar to transcriptional activators and contains the myc-homology region. Proc Natl Acad Sci USA 86: 7092–7096
Mao TY, Liu YY, Zhu HH, Zhang J, Yang JX, Fu Q et al (2019) Genome-wide analyses of the bHLH gene family reveals structural and functional characteristics in the aquatic plant Nelumbo nucifera. Peer J 7: e7153
Matus JT, Poupin MJ, Cañón P, Bordeu, Alcalde JA, Arce-Johnson P (2010) Isolation of WDR and bHLH genes related to flavonoid synthesis in grapevine (Vitisvinifera L.). Plant Mol Biol 72: 607–620
Mao K, Dong Q, Li C, Liu C, Ma F (2017) Genome wide identification and characterization of apple bHLH transcription factors and expression analysis in response to drought and salt stress. Front Plant Sci 8: 480
Meezan E, Meezan EM, Jones K, Moore R, Barnes S, Prasain JK (2005) Contrasting effects of puerarin and daidzin on glucose homeostasis in mice. J Agric Food Chem 53: 8760–8767
Ndayambaza B, Jin XY, Min XY, Lin XS, Yin XF, Liu WX (2021) Genome-wide identification and expression analysis of the barrel medic (Medicago truncatula) and alfalfa (Medicago sativa L.) basic helix-loop-helix transcription factor family under salt and drought stresses. J Plant Growth Regul 40: 2058–2078
Pires N, Dolan L (2010) Origin and diversification of basic-helix-loop-helix proteins in plants. Mol Biol Evol 27: 862–874
Qian YC, Zhang TY, Yu Y, Gou LP, Yang JT, Xu J et al (2021) Regulatory mechanisms of bHLH transcription factors in plant adaptive responses to various abiotic stresses. Front Plant Sci 12: 677611
Roig-Villanova I, Bou-Torrent J, Galstyan A, Carretero-Paulet L, Portolés S, Rodríguez-Concepción M et al (2007) Interaction of shade avoidance and auxin responses: a role for two novel atypical bHLH proteins. EMBO J 26: 4756–4767
Roy SW, Gilbert W (2006) The evolution of spliceosomal introns: patterns, puzzles and progress. Nat Rev Genetic 7: 211–221
Shang XH, Yi XX, Xiao L, Zhang YS, Huang D, Xia ZB et al (2022) Chromosomal-level genome and multi-omics dataset of Pueraria lobata var. thomsonii provide new insights into legume family and the isoflavone and puerarin biosynthesis pathways. Hortic Res 9: uhab035
Shelton D, Stranne M, Mikkelsen L, Pakseresht N, Welham T, Hiraka H et al (2012) Transcription factors of Lotus: regulation of isoflavonoid biosynthesis requires coordinated changes in transcription factor activity. Plant Physiol 159: 531–547
Shiu S H, Bleecker AB (2003) Expansion of the receptor-like kinase/Pelle gene family and receptor-like proteins in Arabidopsis. Plant Physiol 132: 530–543
Song XM, Huang ZN, Duan WK, Ren J, Liu TK, Li Y et al (2014) Genome-wide analysis of the bHLH transcription factor family in Chinese cabbage (Brassica rapa ssp. pekinensis). Mol Genet Genom 289: 77–91
Tian F, Yang DC, Meng YQ, Jin J, Gao G (2019) PlantRegMap: charting functional regulatory maps in plants. Nucleic Acids Res 48: D1104–D1113
Toledo-Ortiz G, Huq E, Quail PH (2003) The Arabidopsis basic/helix-loop-helix transcription factor family. Plant C 15: 1749–1770
Wang PF, Su L, Gao HH, Jiang XL, Wu XY, Li Y (2018a) Genome-wide characterization of bHLH genes in grape and analysis of their potential relevance to abiotic stress tolerance and secondary metabolite biosynthesis. Front Plant Sci 9: 64
Wang RQ, Zhao P, Kong NN, Lu RZ, Pei Y, Huang CX et al (2018b) Genome-wide identification and characterization of the potato bHLH transcription factor family. Genes 9: 54
Wang SG, Zhang SM, Wang SP, Gao P (2020) A comprehensive review on Pueraria: insights on its chemistry and medicinal value. Biomed Pharmacother 131: 110734
Wang X, Li CF, Zhou C, Li J, Zhang YS (2017) Molecular characterization of the C-glucosylation for puerarin biosynthesis in Pueraria lobata. Plant J 90: 535–546
Wang X, Li S, Li J, Li CF, Zhang YS (2015) De novo transcriptome sequencing in Pueraria lobato to identify putative genes involved in isoflavones biosynthesis. Plant Cell Rep 34: 733–743
Wang YP, Tang HB, DeBarry JD, Tan X, Li JP, Wang XY et al (2012) MCScanX: A toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res 40: e49
Xiang L, Jian DQ, Zhang FY, Yang CX, Bai G, Lan XZ et al (2019) The cold-induced bHLH transcription factor AabHLH112 promotes artemisinin biosynthesis indirectly via ERF1 in Artemisia annua. J Exp Bot 70: 4835–4848
Xu L, Dong Z, Fang L, Luo Y, Wei Z, Guo H et al (2019) OrthoVenn2: a web server for whole-genome comparison and annotation of orthologous clusters across multiple species. Nucleic Acids Res 47: W52–W58
Zhang GY, Chen M, Chen XP, Xu ZS, Guan S, Li LC et al (2008) Phylogeny, gene structures, and expression patterns of the ERF gene family in soybean (Glycine max L.). J Exp Bot 59: 4095–4107
Zhang XY, Qiu JY, Hui QL, Xu YY, He ZY, Peng LZ et al (2020) Systematic analysis of the basic/helix-loop- helix (bHLH) transcription factor family in pummelo (Citrus grandis) and identification of the key members involved in the response to iron deficiency. BMC Genomics 21: 233
Zhao Y, Zhang YY, Liu H, Zhang XS, Ni R, Wang PY (2019) Functional characterization of a liverworts bHLH transcription factor involved in the regulation of bisbibenzyls and flavonoids biosynthesis. BMC Plant Biol 19: 497
Zhao Z, Guo P, Brand E (2018) A concise classification of bencao (materia medica). Chin Med 13: 18
Zhou M, Memelink J (2016) Jasmonate-responsive transcription factors regulating plant secondary metabolism. Biotechnol Adv 34: 441–449
Zhou YX, Zhang H, Peng C (2014) Puerarin: a review of pharmacological effects. Phytother Res 28: 961–75
Zhu ZS, Sun BS, Cai W, Zhou X, Mao YH, Chen CJ et al (2019) Natural variations in the MYB transcription factor MYB31 determine the evolution of extremely pungent peppers. New Phytol 223: 922–938

No competing interests reported.

FigS1.jpg
Synteny analysis of the PlbHLH genes. Black lines along the circumference of the circle represent the positions of PlbHLH genes on P. thomsonii chromosomes. The lines in red inside the circle indicate collinearity relationships among PlbHLH genes.
FigS2.jpg
Hierachical cluster of heatmap for 219 PlbHLH genes expression in root, stem, and leaf of P. thomsonii. Red and blue correspond to high and low expression values, respectively, after z-score normalized transformation.
FigS3.jpeg
The relative expression pattern of the 34 root-specific PlbHLH genes in response to MeJA at 0 h, 12 h and 24 h. Mean values and SDs were obtained from three biology and three technical replicates. Different lower-case letters represent significant different at 0.05 level.
TableS1.xls
Supplementary Table 1. The primer sequences for qRT-PCR detection.
TableS2.xls
Supplementary Table 2. Summary of characteristics of the 219 identified PlbHLH genes in P. thomsonii.
TableS3.xls
Supplementary Table 3. The 34 segmentally duplicated PlbHLH gene pairs in P. thomsonii.
TableS4.xls
Supplementary Table 4. One-to-one orthologous relationships between P. thomsonii and Arabidopsis.
TableS5.xls
Supplementary Table 5. One-to-one orthologous relationships between P. thomsonii and Glycine max.
TableS6.xls
Supplementary Table 6. The 20 conserved motif of PlbHLH gene family in P. thomsonii.
TableS7.xls
Supplementary Table 7. The expression profiles of PlbHLH genes in root, stem and leaf.
TableS8.xls
Supplementary Table 8. The expression profiles of root-specific PlbHLH genes.

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Genome-wide Analysis of the bHLH Transcription Factor Family and Functional Identification of bHLH Genes Related to Puerarin Biosynthesis in Pueraria lobata var. thomsonii (Benth.)

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