Genome-Wide Characterization and Analysis of bHLH Transcription Factors Related to Crocin Biosynthesis in Gardenia jasminoides Ellis (Rubiaceae).

Crocins, enriched in Gardenia jasminoides fruits, have a pharmacological activity against central nervous system diseases, cardiovascular diseases, and cancer cell growth. The biosynthesis of crocins has been widely explored, but its regulatory mechanism remains unknown. Here, the basic helix-loop-helix (bHLH) transcription factors related to crocin biosynthesis were systematically identified on the basis of the genome of G. jasminoides. A total of 95 GjbHLH transcription factor genes were identified, and their phylogenetic analysis indicated that they could be classified into 23 subfamilies. The combination of gene-specific bHLH expression patterns, the coexpression analysis of biosynthesis genes, and the analysis of promoter sequences in crocin biosynthesis pathways suggested that nine bHLHs in G. jasminoides might negatively regulate crocin biosynthesis. This study laid a foundation for understanding the regulatory mechanism of crocin biosynthesis and the improvement and breeding of G. jasminoides varieties.


Introduction
Crocins, glucosyl esters of crocetin, belong to apocarotenoids, which are highly accumulated in mature fruits of Gardenia jasminoides [1] and stigma of Crocus sativus. Crocins have a curative effect on various types of central nervous system diseases, such as neurodegenerative diseases [2], and various types of cardiovascular system disease, such as hypertension [3]. Crocins also have pharmacological activities, including anticancer cell growth [4], anti-inflammation and antioxidation [5], antiplatelet aggregation [6], and antiobesity [7]. G. jasminoides, named Zhi Zi in traditional Chinese medicine and recorded in the Chinese pharmacopoeia, is commonly used for antioxidant activity, anti-inflammation, and detoxification [8]. G. jasminoides, belonging to the coffee family, is a famous ornamental plant, widely distributed around the world [9].
In G. jasminoides fruits, the biosynthesis of crocins begins with the cleavage of carotenoids under the activity of carotenoid cleavage dioxygenase (CCD) to produce crocetin dialdehyde. Then, aldehyde dehydrogenase (ALDH) catalyzes the transformation of crocetin dialdehyde to crocetin, which is the key precursor of crocins. Lastly, the glycosylation of different crocetins under the catalysis of UDPglucosyltransferase (UGT) produces various crocins (crocins I-V) ( Figure S1). The genome of G. jasminoides has been finished, and the complete pathway of crocin biosynthesis including GjCCD4a, GjALDH2C3, GjUGT94E13, and GjUGT74F8 has been systematically elucidated (NCBI: VZDL00000000). However, the regulation of crocins produced in G. jasminoides remains unclear. The regulation of transcription factors (TFs) plays an important role in the biosynthesis of active compounds [10][11][12][13][14].
The regulation of bHLH TFs in crocin biosynthesis has not been described in G. jasminoides. Here, we systematically selected bHLH TFs and identified the candidate bHLHs related to the regulation of crocin production based on the genome of G. jasminoides. Our results provided a basis for further studying the regulatory mechanism of crocin biosynthesis and the breeding of G. jasminoides varieties.

Plant Materials.
The variety of G. jasminoides, named ZZ1-9, was selected and cultivated in the Chongqing Institute of Medicinal Implantation. The root, stem, leaf, young fruit, green fruit, and red fruit of G. jasminoides (ZZ1-9) were treated with liquid nitrogen and stored at -80°C for subsequent experiments.

2.3.
Phylogenetic, Gene Structure, and MEME Motif Analyses. A total of 638 bHLH genes from Arabidopsis, poplar, rice, moss, and algae were classified into 32 subfamilies [30]. The protein sequences of the bHLH family from G. jasminoides and Arabidopsis were aligned using MUSCLE methods [31]. Then, the multiple sequence alignments were used to construct a Maximum Likelihood (ML) tree by MEGA 6.0 with the Jones-Taylor-Thornton model and 1000 bootstrap replicates [32]. The phylogenetic tree of the MYC subfamily and the bHLH3 subfamily was constructed using the same method, and bHLH15 subfamily members, GjbHLH15.1 and GjbHLH15.7, were chosen as the outgroup. The online Gene Structure Display Server (GSDS 2.0) (http:// gsds.cbi.pku.edu.cn) was used to investigate the gene structure based on each coding sequence (CDS) and the corresponding genomic sequence. The conserved motifs in bHLH TFs of G. jasminoides were identified using MEME (suite version 5.0.3) with the following criteria: 22 motifs, any number of repetitions of a motif, and an optimum width of 10-200 amino acids [19,33].

Gene Expression
Analysis. The RNA-Seq from six organs (root, stem, leaf, young fruit, green fruit, and red fruit) of G. jasminoides was performed. The RNA-Seq reads were mapped to the G. jasminoides genome using HISAT2 [34], and the expression levels of bHLH genes were estimated on the basis of fragments per kilobase per million (FPKM) values using Cufflinks [35].
Total RNA was extracted from the root, stem, leaves, green fruits, and red fruits using an RNAprep Pure Plant Kit (TIANGEN Biotech, China) according to the manufacturer's instructions. The expression levels of GjbHLH candidate genes identified as putative regulators of crocin biosynthesis were confirmed by quantitative real-time reverse transcription PCR (qPCR) analysis in triplicate. Total RNA was reverse transcribed using a PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, China). qPCR was performed with a TB Green™ premix Ex Taq™ (Tli RNaseH Plus) (TaKaRa, China) and conducted in triplicate using an Applied Biosystems 7500 Real-Time PCR system (Life Technologies, USA). The primers were designed using Primer Premier 6 (Table S2), with an amplicon size ranging from 150 bp to 250 bp and an optimal Tm of 55 ± 5°C. The actin gene was used as an internal reference, and red fruit was examined for comparison; Ct values were calculated to analyze the relative expression levels by using the 2 −ΔΔCt method [36]. One-way ANOVA was performed with GraphPad Prism to detect the differences in candidate gene expression. * P < 0:05 was considered to indicate statistical difference in expression.

Gene Structure and Conserved Motif
Analyses. The structural analyses of the bHLH genes revealed that the number of exons varied from 1 to 12, and 8 genes were intronless (Figure 2(a)). Subfamily 23 had the most number of exons with an average of 12. The 8 intronless genes were distributed across 5 subfamilies. Of these genes, 3 belonged to subfamily 31, 2 belonged to subfamily 2, and 3 belonged to subfamilies 1, 11, and 30 (Table S4).
The MEME was used to characterize 22 conserved motifs within bHLH proteins in order to clarify the evolution of bHLH genes (Figure 2(b) and Table S5). Compared with the known HLH and bHLH-MYC_N domains, motif 1 and motif 2 belong to the HLH domain, and motifs 7, 12, and 14 belong to the bHLH-MYC_N domain. GjbHLHs in subfamilies 2 and 3 contained the largest number of motifs (6 types), whereas the GjbHLHs in subfamilies 11 and 16 comprised one motif each (motifs 1 and 2). Additionally, the average number of motifs per bHLH varied across subfamilies and ranged from 1 (subfamilies 11 and 16) to 6 (subfamily 2). Most motifs appeared only once in one bHLH gene, but GjbHLH26.2 protein had two copies of motif 3, and GjbHLH33.1 protein and subfamily 27 have two copies of motif 15. Motif 1 or 2 was conservatively distributed in all subfamilies. A total of 14 motifs uniquely appear in a specific subfamily: motifs 7 and 16 in subfamily 2; motifs 8, 11, and 13 in subfamily 3; motif 21 in subfamily 4; motifs 5, 14, and 22 in subfamily 23; motifs 18 and 19 in subfamily 24; motif 17 in subfamily 27; motif 10 in subfamily 31; and motif 19 in subfamily 33 (Figure 3(a)).
The bHLH-MYC_N domain of subfamily 2 was composed of motifs 12 and 7, and subfamily 23 comprised motifs 12 and 14 (Figures 3(a) and 3(b)). In addition, the ML phylogenetic tree of MYCs from Catharanthus roseus, Nicotiana tabacum, T. chinensis, and A. thaliana and all members of subfamilies 2 and 23 in G. jasminoides indicated that all members of subfamily 2 are clustered with MYC2 ( Figure 3(c)). Furthermore, GjbHLH2.3 is clustered with CrMYC2, and GjbHLH2.4 and GjbHLH2.5 are clustered with AtMYC2.3.

Differential Expression of bHLH Genes in Various
Organs. Among the 95 bHLH genes, the expression of 13 bHLH genes distributed across 8 subfamilies was undetected, with FPKM values of less than 1 ( Figure 4, Table S6). The expression of the other 72 bHLH genes comprising 19 subfamilies was detected, with FPKM values higher than 1 in at least one of the six organs tested. In addition, 44 GjbHLH genes showed a significantly high expression (log 2 jfold changej > 1) in at least one of the organs (root, stem, leaf, and young fruit), and 13 genes exhibited a significantly high expression in all four organs. The expression pattern of GjbHLH genes indicated that more GjbHLH genes showed a relatively low expression in mature G. jasminoides fruits. A total of 13 genes were specifically expressed in six organs. In particular, 7 genes (GjbHLH1.3, GjbHLH2.1, GjbHLH3.1, GjbHLH3.4, GjbHLH3.10, GjbHLH10.4, and GjbHLH31.2) were specifically expressed in the root, 2 genes (GjbHLH15.2 and GjbHLH25.1) were specifically expressed in the stem, and only 1 gene was expressed specifically in the leaf (GjbHLH7.2), young fruit (GjbHLH25.9), green fruit (GjbHLH15.4), and red fruit (GjJbHLH1.8).
Crocins have been indicated to be highly accumulated in mature G. jasminoides fruits [37]. The expression of the key enzyme genes, including GjCCD4a, GjALDH2C3, GjUGT94E13, and GjUGT74F8, in the crocin biosynthesis pathway was higher in mature fruits than in other organs. However, the expression of 9 genes (GjbHLH1.7, GjbHLH1.9, GjbHLH2.2, GjbHLH2.3, GjbHLH2.4, GjbHLH2.5, GjbHLH3.10, GjbHLH7.2, and GjbHLH27.3) significantly decreased in mature fruits compared with those in the four other organs; the FPKM values were higher than 50 in the four other organs and at least 2-fold higher than those in red fruit. Among them, 4 genes (GjbHLH2.2, GjbHLH2.3, GjbHLH2.4, and GjbHLH2.5) belonged to the MYC2 family. The qRT-PCR analysis of the 9 candidate genes indicated that the expression of 7 genes in red fruits was significantly low, which was consistent with the RNA-Seq results ( Figure 5, Table S7).

Discussion
bHLH TFs play an important role in plant stress resistance [38], signal transduction [39], secondary metabolism regulation [40], and growth and development [41]. They have been identified and analyzed in various plants. According to the sequence homology and phylogenetic relationships, 167, 183, 177, 169, and 127 bHLH genes have been identified in A. thaliana [30], P. trichocarpa [30], O. sativa [30], P. ginseng [20], and S. miltiorrhiza [19], respectively. Here, we first identified 95 GjbHLH TF genes in G. jasminoides and classified them into 23 subfamilies. The gene number of bHLH   bHLH-MYC_N domain of subfamily 2   1   2   3   4   1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  0   1   2   3   4   1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60  61  62  63  64  65  66  67  genes is similar with that in the V. vinifera genome (94 genes) [21]. The variable numbers of bHLH genes in these species are probably caused by whole genome duplication or tandem repeat during plant evolution. Both V. vinifera and G. jasminoides did not experience a whole genome duplication event after the ancient gamma triplication event of eudicots [42,43]; however, the genomes of A. thaliana, P. trichocarpa, O. sativa, P. ginseng, and S. miltiorrhiza underwent at least one whole genome duplication event after a shared gamma event [44]. Thence, the bHLH genes in these species showed significant expansion, compared with V. vinifera and G. jasminoides. In addition, compared with the bHLH family of Arabidopsis, the members of subfamilies 17,18,19,20,21,22, and 28 of bHLH TFs are lost in the G. jasminoides genome, and two new subfamilies 32 and 33 were identified in G. jasminoides. Gene families in plants might primarily evolve through tandem duplication [45]. In the G. jasminoides genome, we identified one tandem gene duplication, covering 7 bHLH genes in scaffold 108. All the clustered bHLH genes belonged to subfamily 3, suggesting the expansion of subfamily 3 members in G. jasminoides. The phylogenetic analysis of bHLH3 between G. jasminoides and previous reports of bHLH3 family members from other plants suggested that the expansion of subfamily 3 was species-specific after the speciation of G. jasminoides ( Figure S2). bHLH TFs directly or indirectly regulate the biosynthesis of active compounds in medicinal plants. For example, SmbHLH10 overexpression in S. miltiorrhiza enhanced the accumulation of tanshinones [46]. In Medicago truncatula, two bHLH TFs, TSARL1 and TSARL2, positively increase the expression of the triterpene biosynthetic genes, resulting in triterpene saponin accumulation [47]. In G. jasminoides, the expression profile of 9 candidate bHLH TFs in matured fruits significantly decreased and negatively correlated with the expression of GjCCD4a, GjALDH2C3, UGT74F8, and UGT94E13. Among them, 4 genes (GjbHLH2.2, GjbHLH2.3, GjbHLH2.4, and GjbHLH2.5) had bHLH-MYC_N protein domains. Promoter sequence analysis indicated that the 6 key enzyme genes in crocin biosynthesis pathways had a large number of bHLH TF binding sites, suggesting the potential regulation of bHLHs in G. jasminoides. The promoters of 7 candidate GjbHLH genes contained at least one of the motifs of T/G-box, G-box, and MYCATRD22 that  Color key Row Z-score Figure 4: Heatmaps representing the expression profiles of G. jasminoides bHLH genes in the root, stem, leaf, young fruit, green fruit, and red fruit. The FPKM value was normalized using Z-score (row). 8 BioMed Research International could be bound by MYC2. The phylogenetic analysis of MYCs revealed that GjbHLH2.3 is clustered with CrMYC2, which can regulate the biosynthesis of medicinally valuable terpenoid indole alkaloids in C. roseus [48]. Furthermore, the qPCR analysis indicated that the expression of most candidate genes in mature fruits was significantly low, which was consistent with the RNA-Seq results. These results implied that bHLH TFs might negatively regulate the biosynthesis of crocins in G. jasminoides. Candidate bHLH TFs should be functionally verified to clarify the molecular mechanism of the regulation of crocin biosynthesis.

Conclusions
In conclusion, 95 bHLH TF genes were identified and phylogenetic analyzed in the genome of G. jasminoides. These GjbHLHs could be classified into 23 subfamilies supported by phylogeny, additional protein motifs, and intron/exon structures. Gene-specific expression patterns, crocin biosynthesis pathways, and elucidation of the complete pathway of the crocin biosynthesis in mature G. jasminoides fruits revealed that 9 bHLH TF genes identified in G. jasminoides were potentially involved in the regulation of crocin biosyn-thesis. The candidate bHLH TF genes related to crocin biosynthesis should be further functionally identified via a series of in vivo and in vitro experimental procedures.

Data Availability
The bHLH transcription factor sequences of G. jasminoides have been uploaded into NCBI database, and the GenBank accession numbers of GjbHLH sequences are from MN385845 to MN385939.

Conflicts of Interest
The authors declare that there are no conflicts of interest regarding the publication of this paper.

Acknowledgments
The authors thank Prof. Kaizhi Hu (Chongqing Institute of Medicinal Plant Cultivation) for providing the Gardenia jasminoides material (ZZ1-9) used in this work. We also thank Prof. Jingyuan Song for comments on the manuscript. This research was supported by the National Natural Science

Supplementary Materials
Supplementary 1. Figure S1: the potential crocin biosynthetic pathway in G. jasminoides. Supplementary 2. Figure S2: an ML phylogenetic tree was constructed with GjbHLH3 subfamily and bHLH3 family members reported from other plants. GjbHLH15.1 and GjbHLH15.7 branches were chosen as the outgroup. bHLH3 family members were downloaded with the following sequence numbers: AT2G22770, AT2G22750, AT2G22760, AT4G37850, OS03G51580, OS03G12760, OS03G46860, OS12G43620, OS03G46790, and OS10G01530. Table S1: the accession numbers of AtbHLH genes. Table S2: primer sequences of candidate GjbHLH genes. Table S3: identification of GjbHLH genes through BLAST and HMMER analysis of the bHLH domain against the G. jasminoides genome. Table S4: analysis of the structural features and conserved motifs of bHLH genes in G. jasminoides. Table S5: conserved motifs predicted by MEME. Table S6: the FPKM values of 95 GjbHLH genes in different organs. Asterisks ( * ) represent the coexpression of four genes (GjALDH2C3, GjUGT94E13, GjUGT74F8, and CjCCD4a) in crocin biosynthesis. Table S7: the qRT-PCR analysis for candidate GjbHLH genes. Table S8: the cis-acting elements of the promoter sequences of 6 key enzyme genes (GjBCH, GjLCYB, GjALDH2C3, GjUGT94E13, GjUGT74F8, and CjCCD4a) in the crocin biosynthesis pathway. Table  S9: the cis-acting elements of the promoter sequences of candidate GjbHLH genes.