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Genome-wide identification of SrbHLH transcription factors highlights its potential role in rebaudioside A (RA) biosynthesis in Stevia rebaudiana

BMC Plant Biology volume 23, Article number: 352 (2023) Cite this article

Abstract

Stevia rebaudiana Bertoni is a valuable medicinal plant and an essential source of natural sweetener, steviol glycosides (SGs), with rebaudioside A (RA) being one of the main components of SGs. bHLH transcription factors play a crucial role in plant development and secondary metabolism. In this study, 159 SrbHLH genes were identified from the S. rebaudiana genome, and each gene was named based on its chromosome location. The SrbHLH proteins were then clustered into 18 subfamilies through phylogenetic analysis. The analysis of conserved motifs and gene structure further supported the classification of the SrbHLH family. Chromosomal location and gene duplication events of SrbHLH genes were also studied. Moreover, based on the RNA-Seq data of different tissues of S. rebaudiana, 28 SrbHLHs were co-expressed with structural genes involved in RA biosynthesis. The expression pattern of candidate SrbHLH genes were confirmed by qPCR. Finally, dual luciferase reporter assays (DLAs) and subcellular localization analysis verified SrbHLH22, SrbHLH111, SrbHLH126, SrbHLH142, and SrbHLH152 are critical regulators of RA biosynthesis. This study provides new insights into the function of SrbHLHs in regulating SGs biosynthesis and lays the foundation for future applications of SrbHLH genes in molecular breeding of S. rebaudiana.

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Introduction

Transcription factors (TFs) are crucial in regulating various biological processes, including plant development, flowering, metabolism, and abiotic stress responsiveness [1, 2]. Basic helix-loop-helix (bHLH) is one of the most prominent families of transcription factors, and these transcription factors are widely distributed in both plants and animals [3]. The conserved bHLH domain consists of an essential DNA-binding region and two amphipathic α-helices separated by a loop of variable length (HLH) [4]. Based on conserved domains and phylogenetic relationships, bHLH can be divided into 15 to 25 subfamilies [5].

In recent years, with the development of whole genome sequencing, numerous bHLH gene families have been identified and analyzed in various plant species, such as Arabidopsis [6], rice [7], Erigeron breviscapus [8], Aquilaria sinensis [9], and Tartary buckwheat [10]. Many bHLH genes have been reported to be involved in regulating terpenoid biosynthesis in plants. For instance, Catharanthus roseus CrMYCl or Artemisia annua AaMYC2-like proteins regulate the expression of structural genes by binding to the G-box of their promoter regions, thereby affecting the accumulation of indole alkaloids or artemisinins, respectively [11, 12]. Meanwhile, Medicago truncatula TSAR1 and TSAR2 regulate the biosynthesis of triterpene saponins by binding to the N-box in the MtHMGR1 promoter region [13]. Additionally, some key transcription factors can regulate the expression of multiple structural genes in the same metabolic pathway, thereby affecting the accumulation of metabolites. For example, the bHLH transcription factors Bl (Bitter leaf) and Bt (Bitter fruit) in cucumber (Cucumis sativus) can bind to the promoters of oxosqualene cyclase, fatty acyltransferase, and cytochrome P450 genes for bitter C synthesis simultaneously, regulating the accumulation of bitter taste in cucumber leaves and fruits [14].

S. rebaudiana is a medicinal herb with a sweet taste and is an essential source of steviol glycosides (SGs), which are up to three hundred times sweeter than sucrose [15] and have potential applications in the control of diseases such as diabetes, obesity, or hypertension [16]. The biosynthesis of SGs involve two processes of backbone formation and glycosylation [17], with kaurene synthase (ent-KS), kaurene oxidase (ent-KO), and kaurenoic acid 13-hydroxylase (ent-KAH) catalyzing the formation of the backbone, while UDP glycosyltransferases (UGTs) are responsible for glycosylation at the C13-hydroxyl and C19-carboxylic acid positions of the skeleton [17, 18]. Among these SGs, stevioside (ST) and rebaudioside A (RA) are the main components, with RA being more valuable due to its high sweetness and good taste [19]. To date, four UGT genes, UGT74G1, UGT85C2, UGT91D2, and UGT76G1, involved in RA biosynthesis have been identified [17]. However, the molecular regulatory mechanisms of SG synthesis are poorly understood.

This study aimed to identify and characterize the bHLH gene family in the S. rebaudiana genome; the construction of phylogenetic tree was conducted to confirm the relationships between SrbHLH and AtbHLH proteins, the identification of segmentally duplication events were proceeded to explain the expansion of SrbHLH family. We also conducted co-expression analysis between SrbHLH genes and structural genes involved in RA synthesis, evaluated the relative expression levels of selected SrbHLH genes using qPCR method, and confirmed critical regulators for RA biosynthesis by transient dual luciferase reporter assays (DLA) and subcellular localization analysis. This study provides important clues for future research on the bHLH family in S. rebaudiana and sheds light on the molecular regulatory mechanisms underlying SG biosynthesis.

Methods

Plant growth and MeJA treatment

S. rebaudiana seeds (“Zhongke No. 1” (S1) and “Zhongke No. 4” (S4)) used in this study were identified and planted at South China Botanical Garden, Chinese Academy of Sciences, Guangzhou City, Guangdong Province. All study protocols for this species comply with relevant institutional, national, and international guidelines and legislations. Both S1 and S4 varieties were used for tissue expression analysis, and S4 was used for MeJA treatment experiments.

After removing the villi from the seed surface, the seeds were soaked in ddH2O for 2 h; Then, floating seeds were removed, and the remaining plump seeds were sterilized using 1% NaClO solution for 10 min; Furthermore, the sterilized seeds were washed 5 times with sterilized ddH2O, planted on MS plates, and moved to a culture room with a temperature of 25 ℃ and a photoperiod cycle of 16 h of light and 8 h of dark.

For the MeJA treatment experiment, 1-week-old seedlings were transferred to MS plates with 100 μM MeJA. After 6 h, 12 h, and 24 h, leaves from 30 seedlings were harvested and pooled as one sample, then immediately frozen with liquid nitrogen and stored at -80˚C. All experiments were performed with three biological replicates.

Identification of bHLH genes in the S. rebaudiana genome

The S. rebaudiana genome was sequenced and assembled in our laboratory. The Hidden Markov model (HMM) file of the bHLH domain (PF00010) was downloaded from the Pfam database (http://pfam.xfam.org/). Using PF00010 as bait, the SrbHLH gene family members were identified by searching the S. rebaudiana genome with the hmmsearch program (HMMER 3.1b1). To further verify the existence of the bHLH conserved domain, the PFAM and SMART (http://smart.emblheidelberg.de/) programs were used. Finally, the amino acid lengths, molecular weights, and isoelectric points of candidate SrbHLHs were analyzed via the tools on the ExPASy website (https://www.expasy.org/), and subcellular localizations of SrbHLH proteins were predicted using the WoLF PSORT website (https://wolfpsort.hgc.jp/).

Gene structure, genome distribution, and phylogenetic analysis

The generic feature format (GFF) file of the S. rebaudiana genome was used to obtain the gene structure and chromosomal location information of each SrbHLH. The conserved motif of each SrbHLH was predicted using the MEME web tool (https://meme-suite.org/meme/), and the gene structures were displayed using TBtools [20].

To construct the phylogenetic tree, protein sequences of Arabidopsis were downloaded from the Phytozome database (https://phytozome-next.jgi.doe.gov/). The bHLH proteins from Arabidopsis and S. rebaudiana were aligned using the ClustalW program, the Neighbor-Joining (NJ) method was used in MEGA 7.0, and 1000 bootstrap replicates were performed using the SrbHLH and AtbHLH proteins.

RNA-Seq co-expression analysis and candidate selection

Using RNA-Seq data from different tissues of two S. rebaudiana varieties, we identified enzyme-coding structural genes involved in RA biosynthesis, including ent-KS, ent-KO, ent-KAH, UGT74G1, UGT85C2, UGT91D2, and UGT76G1. These structural genes were used as bait to screen bHLH transcription factors that potentially regulate RA biosynthesis. Co-expression relationships between structural genes and SrbHLHs were estimated using Pearson correlation analysis. We filtered a set of co-expressed SrbHLHs genes using a Pearson coefficient threshold of 0.95 and p < 0.05. The OmicShare tools, a free online platform for data analysis (https://www.omicshare.com/tools), were applied to construct the structural-SrbHLH gene network.

RNA extraction and qPCR analysis

Total RNA extraction was conducted using a HiPure Total RNA Mini Kit (Code No. R4151-03). Vazyme HiScript Reverse Transcriptase (Cat No. R101-01) was used for cDNA synthesis using the manufacturer’s instructions. qPCR was performed in 384-well plates using Vazyme HiScript Q RT SuperMix (Code No. R122-01) with a LightCycler 480 (Roche, Switzerland). The expression levels of each tested gene relative to the internal reference gene (Sractin) were determined using the 2−ΔΔCT method [21]. All primers used in this study are listed in Table S1. Three biological replicates and three technical replicates were conducted.

Transient dual luciferase reporter assays

The promoter sequence (1500 bp of upstream of the transcription start site) of UGT76G1 was cloned into the pGreen II 0800-LUC reporter vector, and the full length coding sequence of SrbHLHs was cloned into the pCambia1300-UBQ-GFP effector vector. The constructed effector and reporter plasmids were transformed into Agrobacterium GV3101 and injected into tobacco leaves (Nicotiana benthamiana Domin) alongside an agrobacterium-mediated transient expression system [22]. An empty pCambia1300-UBQ-GFP vector was used as a negative control. Firefly and Renilla luciferase activity was measured using a dual luciferase reporter assay system (Promega, Madison, WI, USA). Results were evaluated as the ratio of firefly luciferase (LUC) to Renilla luciferase (REN) activity across three independent biological replicates. Primers for vector construction are listed in Supplemental Table S1.

Subcellular localization assays in tobacco leaves

The subcellular localizations of SrbHLHs were verified through transient expression assays in N. benthamiana leaves. The full-length CDSs of candidate SrbHLHs were cloned into pCambia1300-UBQ-GFP in-frame with a GFP reporter. The primers are listed in Table S1. The fusion constructs were co-transfected into N. benthamiana leaves alongside a nuclear localization marker (Histone3-mCherry). After incubation for 3 days, the localization of the target proteins was observed using a confocal fluorescence microscope (Leica, SP8).

Results

Identification of the SrbHLH gene family in S. rebaudiana

After using NCBI Batch CD-Search tools to eliminate redundant proteins, we obtained 159 bHLHs from the S. rebaudiana genome. To distinguish these genes, we named them SrbHLH1 to SrbHLH159 based on their location on the chromosomes of S. rebaudiana (Table 1). Next, we examined various physicochemical properties of the SrbHLHs, including amino acid lengths, isoelectric point (PI), protein molecular weight (MW), and subcellular localization. Among the 159 SrbHLH proteins, their amino acid sequences varied from 93 aa (SrbHLH44) to 661 aa (SrbHLH35). The MWs of SrbHLH proteins ranged from 10.33 kDa to 70.78 kDa, while the PIs of the SrbHLH proteins ranged from 4.38 (SrbHLH128) to 10.15 (SrbHLH159). Additionally, based on subcellular localization predictions, most of the SrbHLH proteins were expected to be located in the nucleus, cytoplasm, or chloroplast, with only SrbHLH34 likely found in the cytoskeleton (Table 1).

Table 1 Detailed information of bHLH members in S. rebaudiana
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Phylogenetic analysis and classification of SrbHLH genes

To evaluate the classification and evolutionary relationships among SrbHLHs in plants, we utilized bHLH amino acid sequences from S. rebaudiana and Arabidopsis to construct a Neighbor-Joining (NJ) tree using MEGA 7.0 software. The phylogenetic analysis results revealed that 159 SrbHLHs were categorized into 18 subfamilies (Fig. 1), including subfamilies A, B, C, E, G, I, L, M, N, O, P, R, S, T, U, V, X, and Y, consistent with previous research [7, 8]. Subfamily C contained 40 SrbHLHs, while subfamily P and subfamily V contained 18 and 14 SrbHLHs, respectively. Furthermore, subfamily E, S, and R included 13 SrbHLHs, whereas subfamily I, subfamily G, subfamily U, and subfamily T had only one assigned SrbHLH. Notably, subfamilies H, W, and F had no SrbHLHs assigned to them.

Fig.1
figure 1

Phylogenetic tree of bHLH proteins in S. rebaudiana and Arabidopsis thaliana. The phylogenetic tree was obtained using the NJ method in MEGA7. The tree shows 21 phylogenetic subfamilies, each subfamily represented by different colors. bHLH proteins from Arabidopsis are labeled in blue

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Gene structure and motif analysis of SrbHLH genes in S. rebaudiana

To investigate the characteristic regions of SrbHLH proteins, we utilized MEME to identify conserved motifs among the 159 SrbHLH proteins (Fig. 2). Ten distinct motifs were identified and designated as motif 1 through 10. Figure 2A illustrates that SrbHLH proteins contain different numbers of conserved motifs ranging from 1 to 5. Almost all SrbHLH family members contain two highly conserved motifs (motif 1 and 2). Additionally, we observed that SrbHLH proteins belonging to the same subfamily typically share similar composition and relative position. For instance, SrbHLH proteins within subfamily P contain motif 1, motif 2, motif 4, motif 7, and motif 8, whereas the members of subfamily V share common motifs 1, 2, and 5 (Fig. 2A). The fact that members of the same subfamily have similar gene structure and motif composition and tend to gather together in the phylogenetic trees supports the accuracy and authenticity of the subfamily classification.

Fig. 2
figure 2

The gene structure and motif distribution analysis of the SrbHLH proteins in S. rebaudiana. A Phylogenetic trees of SrbHLH proteins constructed by the NJ method; B Ten conserved motifs in the SrbHLH protein; C Exon and intron distribution of SrbHLH genes, green rectangles, and gray lines show the exons and introns, respectively

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To explore their structural composition, we further analyzed the UTR, exons, and introns structure of identified SrbHLH genes (Fig. 2B). By comparing the number and position of exons and introns, we observed that the 159 SrbHLH genes possess varying numbers, ranging from 1 to 11, with only one SrbHLH gene being intronless. Some subfamilies contained genes with a specific number of exons, such as subfamily A, where 3 SrbHLH genes in this clade contained 2 exons. Generally, genes clustered into the same group in the phylogenetic tree have a similar number of exons, such as SrbHLH47, SrbHLH48, SrbHLH49, and SrbHLH125, which each contain three exons, with similar relative positions to the UTR.

Chromosome distribution, gene duplication, and synteny analysis of SrbHLH genes in the S. rebaudiana genome

TBtools was utilized to investigate the regions of interest for SrbHLH genes on the chromosome. The analysis involved anchoring 159 SrbHLH genes to their corresponding chromosomes. The results demonstrated that the distribution of SrbHLH genes on the S. rebaudiana genome was irregular, and the genes were randomly located on all 11 chromosomes (Fig. 3). Chromosome 1 had the most SrbHLHs, with 40 genes, followed by chromosome 2, which had 33 genes. Conversely, chromosome 9 had the least SrbHLHs, with only 2 genes.

Fig. 3
figure 3

Chromosome distribution of SrbHLH genes in S. rebaudiana genome. The chromosome numbers are shown on the top of each chromosome

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Genome duplication events are considered primary drivers of genome evolution and gene family expansion, with tandem and segmental duplication being the primary patterns. In this study, we observed no tandem repeat events in the SrbHLH family. Still, we did identify 165 pairs of segmental duplicates that were unevenly distributed on different chromosomes in the S. rebaudiana genome (Fig. 4). Among these pairs of collinear relationships, some SrbHLHs were paired with more than one gene. For instance, SrbHLH149 had a collinear relationship with SrbHLH11, SrbHLH97, SrbHLH115 and SrbHLH122, while SrbHLH152 has collinear relationship with SrbHLH91, SrbHLH126, SrbHLH141, and SrbHLH142, respectively (Fig. 4, Table S2). These findings suggest that gene duplication events may be the primary cause of the expansion of the SrbHLH family.

Fig. 4
figure 4

Collinearity analysis of the SrbHLHs in the S. rebaudiana genome. The duplicated SrbHLHs were mapped to different chromosomes using shinyCircos. Colorful lines link the collinear relationships among SrbHLHs, and grey boxes present the chromosome

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Correlation analysis between SrbHLHs and structural genes involved in RA biosynthesis

Gene expression patterns provide valuable insights into gene function. As the content of SGs varies in different tissues of S. rebaudiana and the leaves are the main site of SG accumulation [16], we analyzed the expression of SrbHLH genes in different tissues. A heatmap was generated using Fragments Per Kilobase per Million (FPKM) values from RNA-seq data from two S. rebaudiana varieties (with higher RA content in S1 than S4) to show their tissue-specific expression patterns (Fig. 5A). Most SrbHLH genes exhibited tissue-specific expression patterns, such as SrbHLH25, SrbHLH47, SrbHLH48, and SrbHLH49, which were highly expressed in flowers, SrbHLH111, SrbHLH154, and SrbHLH155, which were highly expressed in leaves, SrbHLH57, SrbHLH72, and SrbHLH76, which were highly expressed in roots, and SrbHLH69 and SrbHLH70, which were highly expressed in stems. Furthermore, some SrbHLH genes showed differential expression between the two varieties. For example, the expression level of SrbHLH15, SrbHLH16, SrbHLH18, and SrbHLH19 in the roots of S1 was higher than that of S4, the expression level of SrbHLH57, SrbHLH58, and SrbHLH88 in the flowers of S4 was higher than that of S1, and the expression level of SrbHLH124 in the leaves of S1 was higher than that of S4 (Fig. 5A). These findings provide a basis for identifying bHLH transcription factors that regulate RA biosynthesis.

Fig. 5
figure 5

The expression patterns of SrbHLH genes and the correlation between the expression of SrbHLHs and structural genes involved in RA biosynthesis. A The expression patterns of SrHLH genes in different tissues of S. rebaudiana; B the co-expression relationship between SrbHLHs and structural genes involved in RA biosynthesis. Red circle: positively correlated; blue circle: negatively correlated. *, **, *** indicate a significant correlation at the 0.05, 0.01, or 0.001 level

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The biosynthesis pathway of RA in S. rebaudiana has been elucidated, with specificity mainly occurring in the later cyclization and modification pathways. Seven structural genes, including ent-KS, ent-KO, ent-KAH, UGT74G1, UGT85C2, UGT91D2, and UGT76G1, are involved in the formation of RA in S. rebaudiana [17, 23]. Using RNA-Seq data from different tissues of S. rebaudiana, co-expression analysis was conducted to identify essential SrbHLH genes regulating RA biosynthesis. Notably, we found that the expression levels of 11 SrbHLH genes were significantly negatively correlated with the seven structural genes, while the expression levels of 17 SrbHLH genes were significantly positively correlated with the seven structural genes (Fig. 5B). Therefore, we hypothesized that these 28 SrbHLH genes might be involved in regulating RA biosynthesis.

SrbHLH gene expression patterns in various tissues.

To validate the RNA-Seq results, we conducted qPCR assays to assess the gene expression of SrbHLHs in different tissues of S. rebaudiana varieties S1 and S4. We focused on 16 SrbHLH genes with the highest correlation with the critical RA biosynthesis genes UGT74G1 and UGT76G1. As depicted in Fig. 6, the qPCR results were consistent with the RNA-Seq data. Notably, SrbHLH22, SrbHLH111, and SrbHLH152 exhibited higher expression in leaves than other tissues, whereas SrbHLH126, SrbHLH142, and SrbHLH148 showed higher expression levels in all tissues. Additionally, only SrbHLH134 demonstrated significantly higher expression in the roots of variety S4 than that of other tissues. Overall, these results suggest that SrbHLH22, SrbHLH111, and SrbHLH152 may have crucial roles in RA biosynthesis and accumulation.

Fig.6
figure 6

The expression patterns of candidate SrbHLH genes in different tissues of S. rebaudiana measured by qPCR

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Expression of SrbHLH genes in response to MeJA treatment

MeJA has been shown to regulate the biosynthesis of multiple secondary metabolites [24], such as flavonoid and soyasaponin biosynthesis in G. uralensis [25, 26]. Therefore, we examined the expression patterns of 16 SrbHLH genes selected in our study under MeJA treatment. As depicted in Fig. 7, all SrbHLH genes, except for SrbHLH38 and SrbHLH54, were responsive to MeJA treatment. Among these 14 MeJA responsive genes, four SrbHLH genes (SrbHLH22, SrbHLH72, SrbHLH128, and SrbHLH148) were down-regulated, while ten SrbHLH genes (SrbHLH6, SrbHLH12, SrbHLH60, SrbHLH83, SrbHLH85, SrbHLH111, SrbHLH134, SrbHLH142, SrbHLH147, and SrbHLH152) were up-regulated under MeJA treatment. These results indicate that most of the SrbHLH genes are MeJA-responsive and may play a role in JA-regulated secondary metabolic processes.

Fig. 7
figure 7

The expression patterns of candidate SrbHLH genes under MeJA treatment measured by qPCR

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Functional verification of essential SrbHLH genes regulating rebaudioside A accumulation

It is well established that UGT74G1 and UGT76G1 are two major genes involved in RA biosynthesis. UGT74G1 catalyzes the conversion of steviolmonoside to rubusoside, while stevioside is converted to RA under catalysis by UGT76G1 [17, 27]. In this study, we cloned and analyzed the transcriptional activation or repression of 14 selected MeJA reponsive SrbHLH genes on UGT76G1 using a dual luciferase assay (DLA). Results showed that 5 SrbHLHs (SrbHLH22, SrbHLH111, SrbHLH126, SrbHLH142, SrbHLH152) transcriptionally activated the expression of UGT76G1, while SrbHLH6, 12, 60, 72, 83, 85, 134, 147, and 148 were implicated in regulating the expression of other structural genes involved in RA biosynthesis (Fig. 8).

Fig. 8
figure 8

Transcriptional activation of UGT76G1 by candidate SrbHLHs. The values shown are the means ± SD of the dual LUC/REN ratio. *, **, *** indicate a significant difference at the 0.05, 0.01, or 0.001 level

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Subcellular localization of key bHLHs involved in regulating rebaudioside A accumulation

Subcellular localization is a crucial factor in understanding gene function. Our prediction analysis revealed that SrbHLH proteins exhibit various subcellular localization patterns (Table 1). To further investigate this, we experimentally determined the subcellular localization of 5 SrbHLHs that regulate RA biosynthesis, as confirmed by DLA. Full-length cDNAs of each SrbHLH was fused to Green Fluorescent Protein (GFP) under the ubiquitin promoter and transiently expressed in N. benthamiana leaves. Our findings showed that SrbHLH111 and SrbHLH142 localized in both the nucleus and cytosol, while SrbHLH22, SrbHLH126, and SrbHLH152 were mainly localized in the nucleus (Fig. 9). These nuclear localization results were consistent with the bioinformatics prediction and the expected role of SrbHLHs as transcription factors.

Fig. 9
figure 9

The subcellular localization of candidate SrbHLH proteins

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Discussion

The bHLH transcription factors (TFs) play crucial roles in plants, including response to abiotic/biotic stress and regulation of metabolic pathways. Nowadays, genome-wide characterization of bHLH genes have been reported in numerous plants. However, little is known about this gene family in S. rebaudiana, prompting us to systematically examine bHLH proteins in S. rebaudiana, especially looking into their function in regulating RA biosynthesis. Here, we identified 159 SrbHLH genes by investigating the S. rebaudiana genome, and based on classification methods from previous study [28], these 159 SrbHLH proteins were clustered into 18 groups. Generally, genes in the same branch had similar functions. Many SrbHLH members were proximal to the bHLH proteins in Arabidopsis, which were predicted to be homologous genes. For example, AtbHLH genes in subfamily N were responsive to the JA signal. These genes were reported to participate in the regulation of abiotic stress and terpenoid metabolism through JA-mediated pathways in plants [29, 30]. Thus, we predicted that SrbHLH proteins in subfamily N (including SrbHLH26, SrbHLH91, SrbHLH124, SrbHLH126, SrbHLH127, SrbHLH141, SrbHLH142, and SrbHLH152) also participate in the regulation of abiotic stress and terpenoid metabolism in S. rebaudiana. These phylogenetic findings will contribute to the prediction of functions of SrbHLH genes in S. rebaudiana.

The gene expression level is critically important for regulating plant biological progress. In general, genes with the same expression patterns are more likely to be involved in producing similar bioactive compounds [31]. Based on RNA-Seq data, this study revealed that 159 SrbHLH genes presented different expression patterns, and numerous tissue-specific SrbHLH genes were identified (Fig. 5A). These findings will contribute to the screening of candidate genes regulating RA synthesis. Interestingly, co-expression analysis using RNA-Seq data from different tissue samples demonstrated that 11 SrbHLH genes were significantly negatively correlated with the seven structural genes involved in RA biosynthesis, whereas 17 SrbHLH genes were significantly positively correlated with structural genes (ent-KAH, ent-KO, ent-KS, UGT74G1, UGT76G1, UGT85C2, and UGT91D2) (Fig. 5B), which were reported to the key critical genes for RA biosynthesis [17,18,19]. Among the 28 SrbHLH genes co-expressed with structural genes involved in RA biosynthesis, three SrbHLH genes (SrbHLH22, SrbHLH111, and SrbHLH152) detected by qPCR were highly expressed in the leaves of S. rebaudiana, which were the main tissues of RA accumulation. In addition, SrbHLH152 belongs to subfamily N, the same clade as AtbHLH genes that regulating response to abiotic stress and terpenoid metabolism pathways [29, 30]. Therefore, in the future, these co-expressed SrbHLHs with structural genes and highly expressed in leaves may be served as candidate transcription factors regulating RA synthesis.

Several studies have shown that MeJA treatment induces the accumulation of secondary metabolites in various plant species. For example, terpenoids were induced in Catharanthus roseus cells [32] and soyasaponins in G. uralensis [25]. MeJA treatment also affected the expression of critical genes in the biosynthesis of SGs in S. rebaudiana [28]. It is shown that MeJA increased the accumulation of SGs by acting on UGT85C2 and UGT76G1 gene expression [33]. This study identified 14 SrbHLH genes co-expressed with structural genes and responded to MeJA treatment. Among them, the expression of 4 SrbHLHs were down-regulated, and 10 were up-regulated (Fig. 7), suggesting their essential roles in JA-mediated biological processes, including RA biosynthesis. It is worth noting that bHLH proteins have been reported to regulate metabolic processes by interacting with other transcription factors or co-factors [34]. For example, the COI1/JAZs/MYC2 module has been identified as a core regulator of JA-mediated terpenoid accumulation in Catharanthus roseus [35]. Therefore, investigating the regulatory function of SrbHLHs and their interaction with co-factors in RA biosynthesis in future studies could be of great interest.

Transcription factors typically bind to specific cis-elements in the promoter region of target genes to regulate the synthesis of compounds catalyzed by the target genes by regulating their expression. In the case of the bHLH gene family, binding sites usually include G-box, E-box, or N-box. For example, CrMYCl or AaMYC2-like proteins have been shown to regulate the expression of structural genes by binding to the G-box of their promoter regions, affecting the biosynthesis of indole alkaloids or artemisinin [11, 12]. In the current study, we used DLA to examine the binding ability of candidate SrbHLHs to the promoter regions of structural genes involved in RA synthesis. The results showed that 5 SrbHLHs (SrbHLH22, SrbHLH111, SrbHLH126, SrbHLH142, SrbHLH152) transcriptionally activated the expression of UGT76G1 (Fig. 8). Meanwhile, the expression pattern of these 5 selected SrbHLHs presented a positive correlation with that of UGT76G1 and the other structural genes involved in RA biosynthesis (Fig. 5). Furthermore, the nuclear localization of these SrbHLH proteins were consistent with their expected role as transcription factors (Fig. 9). Therefore, these 5 SrbHLHs appear to be the most promising candidates for regulating RA synthesis. In the future, transgenic plants will be constructed to evaluate the function of these 5 SrbHLHs in the RA biosynthesis pathway.

Conclusion

In this study, we identified 159 bHLH (SrbHLH) genes in the S. rebaudiana genome. We then constructed a phylogenetic tree to confirm the relationships between SrbHLH and AtbHLH proteins and found that SrbHLHs in the same group had similar protein motifs and gene structures. In addition, 263 pairs of segmental duplicated SrbHLH genes were identified, indicating that duplication events contributed to the expansion of SrbHLH family. Based on RNA-Seq data, we found that expression patterns of SrbHLHs differed across various tissues. Moreover, to identify candidate SrbHLHs which regulate RA biosynthesis, co-expression analysis between SrbHLH genes and structural genes involved in RA synthesis was conducted. The qPCR method analyzed the relative expression levels of the selected SrbHLH genes. Finally, SrbHLH22, SrbHLH111, SrbHLH126, SrbHLH142, and SrbHLH152 were confirmed as candidate regulators of RA biosynthesis through transient dual luciferase reporter assays (DLA) and subcellular localization analysis. These results may contribute to further understanding of the functions of SrbHLHs in SGs biosynthesis regulation and provide a theoretical basis for the application of SrbHLH genes in the molecular breeding of S. rebaudiana.

Availability of data and materials

The datasets generated and/or analyzed during this study are available in NGDC (https://ngdc.cncb.ac.cn/) with accession no. PRJCA016649. Other data are available in the supplementary table.

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Acknowledgements

We thank the editors and reviewers for their careful reading and valuable comments. We apologize to researchers whose studies are not cited due to space limitations.

Funding

This study was supported by the Key Laboratory of Molecular Analysis and Genetic Improvement of South China Agricultural Plants Foundation of South China Botanical Garden, Chinese Academy of Sciences (KF200204), the National Natural Science Foundation of China (32170362), the Guangdong Natural Science Funds for Distinguished Young Scholars (2022B1515020026), the Youth Innovation Promotion Association, Chinese Academy of Sciences (Y2021094), the South China Botanical Garden, the Chinese Academy of Sciences (QNXM-02), the Natural Science Foundation of Guangdong Province (2021A515110122), Basic and Applied Research Foundation of Guangzhou City (202201010756) and Innovation Training Programs for Undergraduates, CAS (KCJH-80107–2020-041).

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Authors and Affiliations

  1. Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement & Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, 510650, China

    Yuping Li, Yuan Qiu & Ming Luo

  2. University of Chinese Academy of Sciences, Beijing, 100049, China

    Yuan Qiu & Ming Luo

  3. College of Life Sciences, Southwest Forestry University, Yunnan, 650224, China

    Xin Xu

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  1. Yuping Li
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  2. Yuan Qiu
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  3. Xin Xu
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  4. Ming Luo
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Contributions

YPL and ML designed the experiments. YPL, YQ, and XX performed and analyzed the data. YPL, YQ, XX and ML wrote and edited the manuscript. All authors read and approved the final manuscript.

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Correspondence to Ming Luo.

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Supplementary Information

Additional file 1: Table S1.

Primers used in this study.

Additional file 2: Table S2.

The collinear relationships among SrbHLHs.

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Li, Y., Qiu, Y., Xu, X. et al. Genome-wide identification of SrbHLH transcription factors highlights its potential role in rebaudioside A (RA) biosynthesis in Stevia rebaudiana. BMC Plant Biol 23, 352 (2023). https://doi.org/10.1186/s12870-023-04353-1

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Keywords

BMC Plant Biology

ISSN: 1471-2229

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