Genome-Wide Analysis of the bHLH Gene Family in Loropetalum chinense var. rubrum: Identification, Classification, Evolution, and Diversity of Expression Patterns under Cultivation

The basic helix–loop–helix (bHLH) transcription factor family is the second-largest transcription factor family in plants. Members of this family are involved in the processes of growth and development, secondary metabolic biosynthesis, signal transduction, and plant resistance. Loropetalum chinense var. rubrum is a critical woody plant with higher ornamental and economic values, which has been used as ornamental architecture and traditional Chinese herbal medicine plants. However, the bHLH transcription factors in Loropetalum chinense var. rubrum (L. chinense var. rubrum) have not yet been systematically demonstrated, and their role in the biosynthesis of anthocyanin is still unclear. Here, we identified 165 potential LcbHLHs genes by using two methods, and they were unequally distributed on chromosomes 1 to 12 of the genome of L. chinense var. rubrum. Based on an evolutionary comparison with proteins from Arabidopsis and Oryza sativa, these bHLH proteins were categorized into 21 subfamilies. Most LcbHLHs in a particular subfamily had similar gene structures and conserved motifs. The Gene Ontology annotation and Cis-elements predicted that LcbHLHs had many molecular functions and were involved in processes of plant growth, including the biosynthesis of flavonoids and anthocyanins. Transcriptomic analysis revealed different expression patterns among different tissues and cultivars of L. chinense var. rubrum. Many LcbHLHs were expressed in the leaves, and only a few genes were highly expressed in the flowers. Six LcbHLHs candidate genes were identified by bioinformatics analysis and expression analysis. Further Real-time quantitative PCR analysis and protein interaction network analysis showed that LcbHLH156, which is one of the candidate proteins belonging to the IIIf subfamily, could interact with proteins related to anthocyanin synthesis. Therefore, LcbHLH156 was transiently expressed in L. chinense var. rubrum to verify its function in regulating anthocyanin synthesis. Compared with the control group, red pigment accumulation appeared at the wound after injection, and the total anthocyanin content increased at the wound of leaves. These results lay a foundation for the research of the regulation mechanism of leaf colors in L. chinense var. rubrum and also provide a basis for the function of the LcbHLH family.


Introduction
Transcription factors (TFs) often serve as a central regulator to regulate the expression of target genes.They can form intricate networks through protein-protein interactions to control or affect many biological processes at the transcription level [1].Among the various TFs, the bHLH family is a relatively large family of transcription factors widely found in diverse eukaryotes.The bHLH domain is often made up of 50-60 amino acids and has two functionally distinct regions: a length of 10-15 basic amino acids (the basic region) and a stretch of roughly 40 amino acids (the helix-loop-helix region).The helix-loop-helix region contains two amphipathic α-helices separated by an intervening loop [2,3].The basic region is located at the N-terminus of the bHLH domain and is the DNA-binding region that allows the bHLH TFs to bind to the E-box (CANNTC).In contrast, the HLH region is located at the C-terminus of the bHLH domain.It acts as a dimerization domain, which can promote the formation of homodimers or heterodimers between proteins to alter the expression of target genes involved in various signaling pathways [4,5].In the bHLH protein domain, only 19 amino acids are conserved, and it possesses a highly conservative H-E-R motif.Previous studies have typically classified members of the bHLH superfamily into subfamilies or subgroups based on the conserved motifs, evolutionary relationships, and structural domains.Ledent and Atchley classified the bHLH transcription factors of animals into six subgroups (A-F) based on their sequence homology and evolutionary relationships [3,6].However, there are differences in classification between plants and animals because of the relative independence of the bHLH transcription factors' genealogy.So far, no definite categories of the families of plant bHLH transcription factors have been proposed [7].Generally, the bHLH transcription factor families in plants have been divided into 15-26 groups [3,8,9].For example, 147 and 167 bHLH family members were identified from Arabidopsis and Oryza sativa, respectively, and then classified into 24 and 23 subfamilies [9,10].Subsequently, the various functions of the bHLH gene have also been validated.
Through previous studies, scientists have classified the function of bHLH genes in Arabidopsis and found that bHLH genes play different roles in different aspects [11].In terms of the mineral nutrition of plants, AtbHLH121, AtbHLH18, AtbHLH19, AtbHLH20, and AtbHLH25 can regulate iron homeostasis by indirectly activating the FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT) [12].In growth and development, AtLP1 and AtLP2 can regulate longitudinal cell elongation [13].Moreover, AtNFL promotes flowering under short-day conditions in Arabidopsis [14].The PobHLH5 and PobHLH8 of P. ostreatus infestation in Arabidopsis verified that bHLH genes play an important role in growth and development [15].In addition, bHLHs are also involved in plants' abiotic stress responses, signal transmission, secondary metabolism, etc. [16].For example, the overexpression of TabHLH39 improved drought tolerance, salt tolerance, and frost resistance in transgenic Arabidopsis [17], and MdCIB1 in apples plays an active role in drought resistance [18].Researchers have also found that the expression of CpbHLH1 in transgenic model plants suppressed the accumulation of anthocyanin [19], and NnTT8 in the lotus has been indicated to be involved in the positive regulation of the biosynthesis of anthocyanin [20].It has been further shown that pomegranate fruits' anthocyanins can be regulated by a combination of bHLH and MYB [21], particularly the formation of the MYB-bHLH-WD40 ternary complex [22][23][24].
Loropetalum chinense var.rubrum (L.chinense var.rubrum) is a plant with characteristic vivid flowers and foliage found in Asia, Europe, and America.Ornamental plant lovers deeply enjoy its beautiful foliage and pretty shape [25].The ornamental value of L. chinense var.rubrum mainly includes its leaf color, flower color, and tree shape.Among which, leaf color is one of the essential ornamental values.Previous studies have shown that anthocyanin is a crucial compound for the leaf color of plants [26,27].Its synthesis is usually affected by the external environment, synthetic structural genes, and regulatory genes [28].High temperature and drought usually cause the regreening of L. chinense var.rubrum leaves mainly due to anthocyanin degradation [29,30].In addition, researchers have also further explored the coloration mechanism of L. chinense var.rubrum leaves at the molecular level and have identified and cloned structural genes related to the synthesis of anthocyanin, including LcCHI and LcANS [31].The bHLH gene is one of the essential regulatory genes in the anthocyanin synthesis pathway, which can encode the corresponding transcription factors, and then activate or inhibit the spatial and temporal expression of structural genes by forming the MYB-bHLH-WD40 complex, thereby regulating the synthesis of plant anthocyanins [24,32].It has been reported that the genome sequencing and identification of the bHLH family have been completed in various plants, such as Arabidopsis, potato, bamboo, grape, etc. [11,[33][34][35].However, the characteristics of the bHLH transcription factors and their roles in L. chinense var.rubrum are still unclear.
In this study, the bHLH gene family of L. chinense var.rubrum was systematically identified using bioinformatic methods.The potential bHLH genes regulating leaf color were filtered by a series of biological analyses and expression patterns.The function of LcbHLH156 in anthocyanin synthesis was preliminarily verified by transient expression.These results provided us a further understanding of the structure, function, and evolution of the bHLH family in L. chinense var.rubrum, and they may provide a potential basis for exploring the regulatory network of leaf coloration in L. chinense var.rubrum.

Identification of the LcbHLHs and Evolutionary Analysis
In total, 165 bHLH members were acquired via the method of homologous BLAST and HMMER searches of the genomic data of L. chinense var.rubrum, which were named LcbHLH1 to LcbHLH165 according to their chromosomal localization (Figure 1, Table S1).Predictions of the physicochemical properties by using ExPasy revealed that the number of amino acids contained in the bHLH protein sequence of L. chinense var.rubrum ranged from 120 (LcbHLH96) to 1186 aa (LcbHLH164), with an average of 375 aa.The molecular weight of these proteins ranged from 13,460.46 (LcbHLH96) to 130,429.73Da (LcbHLH84), with an average of 41,508.43Da, and the theoretical isoelectric points ranged from 4.70 (LcbHLH62, LcbHLH102, and LcbHLH103) to 10.12 (LcbHLH96), with 62.71% of them with isoelectric points lower than 7, which were predicted to be acidic.These results were consistent with previous studies of the patterns of the isoelectric points in Arabidopsis and Oryza sativa.The grand average of hydropathicity (GRAVY) of the proteins was from −1.037 to −0.102, showing that all LcbHLHs are hydrophilic.The instability index (II) ranged from 36.68 to 71.31, with only two proteins shown to be stable (II < 40), and the aliphatic index was between 51.42 and 105.58 (Table S2).Most of the LcbHLHs were localized in the nucleus, and only a few were distributed in the cytoplasm, chloroplasts, plasmodesmata, and Golgi apparatus.No signal peptide was found for any of the LcbHLHs using SignalP, indicating that they are non-secretory proteins.members, Subfamily XII was the largest subfamily of LcbHLHs, while Subfamilies IVd, VI, and X were the smallest, with only one member.In addition, compared with Arabidopsis [5], Ficus carica L. [36], and Oryza sativa [7,10,37], Subfamilies III(a + b + c), III(d + e), the IVa, VII(a + b), and XII were considerably expanded (Table 1).To clarify the evolutionary relationships of L. chinense var.rubrum with Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), an evolutionary bHLH proteins tree was constructed (Figure S1).Through the clustering of the evolutionary tree, we found that the bootstrap value of the root node is small.As the evolution progresses, the bootstrap value between the same subfamilies gradually increases.According to the previous classification criteria and a clustering analysis of the evolutionary tree in Arabidopsis and rice, 24 subfamilies of bHLH TFs were identified.L. chinense var.rubrum has 21 subfamilies, and only three subfamilies (XIII, XIV, and XV) contained no members (Figure 2).With 20 members, Subfamily XII was the largest subfamily of LcbHLHs, while Subfamilies IVd, VI, and X were the smallest, with only one member.In addition, compared with Arabidopsis [5], Ficus carica L. [36], and Oryza sativa [7,10,37], Subfamilies III(a + b + c), III(d + e), the IVa, VII(a + b), and XII were considerably expanded (Table 1).To further elucidate the structural features of LcbHLH proteins, a multiple sequence alignment was performed on the structural domains of bHLH.As shown in Figure 3, each LcbHLH protein displayed four conserved regions, including one basic region, one loop region, and two helix regions.Most LcbHLH proteins possessed highly conserved basic regions and two helixes, except for LcbHLH120, LcbHLH121, LcbHLH122, LcbHLH123, LcbHLH124, and LcbHLH125.LcbHLH122 and 123 had no loop region and two helix regions.LcbHLH120, LcbHLH121, LcbHLH124, and LcbHLH159 had no loop region and lacked the second helix region.Multiple sequence alignment showed that 24 amino acid residues were highly conserved (>60% consensus ratios), and two were conserved with a 100% consensus ratio.It is noteworthy that most LcbHLH proteins have the highly conserved H-ER-RR structures and Leu-27 in Helix 1 and Leu-59 in Helix 2 of the HLH region, which are considered to play an essential role in protein dimerization.Thus, we speculated that the LcbHLH proteins may be able to form protein complexes.
The conserved motifs, domain distribution, and gene structure of L. chinense var.rubrum were analyzed to obtain more information about the bHLHs.As shown in Figure S2, 10 motifs were identified, marked as 1-10 (Table 2).The different motifs correspond to their bHLH superfamily domains.For example, we can clearly see that Motif 8 represents the bHLH-MYC_N domain, and Motif 1 and Motif 2 can form various structural domains of the bHLH.In combination with protein sequence alignment, most of the LcbHLH proteins combined Motifs 1 and 2 due to the high conservation of the basic region and the two helix regions, while LcbHLH122, LcbHLH123, LcbHLH129, LcbHLH128, LcbHLH131, LcbHLH81, LcbHLH76, LcbHLH159, LcbHLH55, LcbHLH54, LcbHLH108, LcbHLH148, LcbHLH146, and LcbHLH93 had only Motif 1.The LcbHLH proteins clustered in the same subfamily often have similar motifs.For example, Motifs 1, 2, 5, 4, 9, 6, and 10 were identified in Subfamily II, and Motifs 1, 2, and 8 were identified in Subfamily III(d + e + f).
gions.LcbHLH120, LcbHLH121, LcbHLH124, and LcbHLH159 had no loop region and lacked the second helix region.Multiple sequence alignment showed that 24 amino acid residues were highly conserved (>60% consensus ratios), and two were conserved with a 100% consensus ratio.It is noteworthy that most LcbHLH proteins have the highly conserved H-ER-RR structures and Leu-27 in Helix 1 and Leu-59 in Helix 2 of the HLH region, which are considered to play an essential role in protein dimerization.Thus, we speculated that the LcbHLH proteins may be able to form protein complexes.The conserved motifs, domain distribution, and gene structure of L. chinense var.rubrum were analyzed to obtain more information about the bHLHs.As shown in Figure  The evolutionary relationship between gene family members can be revealed through the analysis of gene structure.The gene structure of LcbHLHs in the same subfamily was always consistent.The exon number of LcbHLHs varied from 1 to 13, whereas the exon-intron organizations were evolutionarily related (Figure S2).The LcbHLHs with one exon were clustered in four subfamilies (III(d + e), VIIIb, orphans, and II) and all the members of Subfamily VIIIb and the orphans had only one exon.

Analysis of Gene Duplication and Collinear Correlations
Tandem and fragment replication are critical means of amplifying a gene family.The 165 LcbHLHs in our study were unequally distributed among the 12 L. chinense var.rubrum chromosomes, with a maximum of 22 on chr3 and a minimum of 2 on chr4.Tandem replication is thought to occur when the distance between genes is less than 100 kb, and seven pairs of LcbHLHs fell into that category (Table S3).An intraspecific collinearity study revealed that fragment replication produced 56 pairs of LcbHLHs (Table S4).The findings showed that tandem replication and fragment replication were critical processes for expanding the LcbHLH gene family.In addition, the substitution rate ratio, Ka/Ks, is a criterion applicable to the selective pressure of gene duplication [38].Ka/Ks values less than 1 often represent negative selection, those equal to 1 represent neutral selection, and those greater than 1 represent positive selection.Among the 56 gene pairs from fragment replication and the seven gene pairs from tandem replication, all except the pairs of LcbHLH29/30, LcbHLH140/141, LcbHLH142/144, and LcbHLH102/103 among the fragment replication pairs were more significant than 1; the other Ka/Ks values were less than 1.It was speculated that they might have been purified and selected during evolution, indicating that most bHLH genes evolved slowly (Table S5).
To better understand the genetic differentiation, gene replication, and evolution of the bHLH gene families of L. chinense var.rubrum, Arabidopsis, Oryza sativa, Zea mays, and Vitis vinifera, MCScanX (Ver 2.0) was used to analyze the homologous bHLH genes of these species.In total, 89, 44, 41, and 123 pairs of orthologous bHLH genes were detected in three comparisons (L.chinense var.rubrum vs. Arabidopsis, L. chinense var.rubrum vs. Oryza sativa, L. chinense var.rubrum vs. Zea mays, and L. chinense var.rubrum vs. Vitis vinifera, respectively) (Figure 4, Table S6).Therefore, the bHLH genes of L. chinense var.rubrum and Vitis vinifera were considered to be more closely related than the bHLH genes of L. chinense var.rubrum and those of Oryza sativa, Zea mays, or Arabidopsis.S6).Therefore, the bHLH genes of L. chinense var.rubrum and Vitis vinifera were considered to be more closely related than the bHLH genes of L. chinense var.rubrum and those of Oryza sativa, Zea mays, or Arabidopsis.

GO Annotation and Analyses of the Cis-Regulatory Elements
Highly divergent sequences outside the conserved structural domain of bHLH suggested that the LcbHLH protein may be involved in multiple biological processes to some extent.To clarify the particular functions of the bHLH proteins, a GO annotation of the LcbHLHs was performed.As shown in Table S7, most LcbHLH proteins were annotated as being associated with transcription factors and the activity of protein dimerization, the regulation of transcription, and developmental processes.Regarding the cellular compo-

GO Annotation and Analyses of the Cis-Regulatory Elements
Highly divergent sequences outside the conserved structural domain of bHLH suggested that the LcbHLH protein may be involved in multiple biological processes to some extent.To clarify the particular functions of the bHLH proteins, a GO annotation of the LcbHLHs was performed.As shown in Table S7, most LcbHLH proteins were annotated as being associated with transcription factors and the activity of protein dimerization, the regulation of transcription, and developmental processes.Regarding the cellular components, 139 of the 158 bHLH genes from the transcriptomic data were located in the nucleus.Only a tiny number of genes were predicted to be distributed in the organelles, such as cytosol (two), chloroplast envelope (one), nucleoplasm (one), vacuole (one), and intracellular membrane-bounded organelle (one).In addition, a few LcbHLHs were also located in less common membrane structures and complexes, including the integral component of the membrane (one), the membrane (one), and the vacuolar membrane (one).It was expected that most of the LcbHLH proteins would be engaged in protein dimerization activity (152/158) according to the molecular functions, and the LcbHLHs also participated in various biological processes.Many LcbHLH proteins were annotated as being associated with DNA binding, growth regulation, and responses to stimuli.For example, 127 LcbHLHs were associated with DNA-binding TFs; some of the LcbHLH proteins were involved in growth and development, including the regulation of change (six), pollen development (ten), development of the anther wall tapetum (eleven), and negative regulation of seed germination (six).Several LcbHLHs could react to environmental stimuli such as cold (seven), iron (five), and light (two).
The function of gene family members is often associated with cis elements.We looked for cis elements in the promoter regions of the various LcbHLH genes to see whether there were any distinctions in their gene regulation.As shown in Table S8, light response elements were the most common cis elements in the LcbHLH promoter sequences, including Box 4, the Gap-box, the G-Box, the AE-box, the I-box, the GT1 motif, and the TCCC motif.In addition, the cis elements that participated in growth and development and related to different stress responses were also discovered, such as MSA-like, which is involved in cell cycle regulation; Motif I and the RY-element, which is engaged in seed-specific regulation; the GCN4 motif, which is involved in development of the endosperm, etc.Moreover, various cis elements were related to different stress responses, including the abiotic response elements involved in the responses to abscisic acid (ABRE), auxin (AuxRRcore, TGA-element, TGA-box, and AuxRE), jasmonic acid (CGTCA motif and TGACG motif), and gibberellin (GARE motif, TATC-box, and P-box), and the biological response elements related to salt stress (DRE) and low temperature (LTR).We also identified some cis elements with MYB recognition sites, which can regulate the synthesis of plant flavonoids (MBSI) by binding MYB-related genes.

Expression Profiles of LcbHLHs and qRT-PCR Analysis of Gene Expression
The expression patterns of LcbHLHs were analyzed on the basis of the transcriptome data of different Loropetalum chinense cultivars.New varieties of Loropetalum chinense include "Xiangnong Nichang" (XNNC), "Xiangnong Fengjiao" (XNFJ), "Xiangnong Xiangyun" (XNXY), and "Xiangnong Xiaojiao" (XNXJ) (Table S9, Figure 5A).To better observe the differences in the expression of LcbHLHs, we standardized the FPKM value and removed the LcbHLHs with FPKM values less than one from all samples, as this value is usually considered an invalid expression.An expression pattern map of 105 LcbHLHs was established from those that remained.
highly expressed in Stage I of different dark-leafed varieties (XNFJ, XNXJ, and XNNC) and decreased in expression as the leaves became green during Stage II.In addition, it is also worth noting that LcbHLH149/114 was also highly expressed in XNFJ, and LcbHLH117 also showed high expression levels in dark-leafed varieties (XNNC).These expression levels of these genes showed similar trends to the phenotype of leaf color changes.Thus, we further studied them as candidate genes.Expression profiles of LcbHLHs in different tissues."XNFJ," "XNXY," "XNXJ," and "XNNC" are the names of our selected materials.The red triangle is used to point out candidate genes.groups of LcbHLHs were divided according to the cluster analysis.Genes in Groups IV and V were highly expressed in the flowers and less represented in the leaves, and they were mainly predicted to involve the formation and development of floral organs.For example, LcbHLH133/145/25/102/69/43 in Group V was upregulated in the flowers of XNXY and XNFJ, while the corresponding leaves showed lower expression levels.LcbHLH84 in Group IV was highly expressed in the flowers of all varieties, LcbHLH43 was significantly expressed in the flowers of XNFJ, and LcbHLH22/51 was highly expressed in the flowers of XNXY and less expressed in its leaves.According to the GO enrichment analysis, LcbHLH84 may be involved in photoperiod and flowering processes, while LcbHLH69 can regulate pollen development.In addition, LcbHLH51/25/102/43 can participate in gibberellin-related anabolism and mediate the brassinosteroid signaling pathway.Analysis of the cis elements showed that all of these genes had light-responsive elements and some hormone-response elements, such as those related to gibberellin, jasmonic acid, salicylic acid, auxin, and abscisic acid.Previous studies have shown that plant flowering is regulated by various plant hormones.Among them, gibberellin plays an essential role in flowering, and other hormones such as abscisic acid, auxin, salicylic acid, and jasmonic acid are also involved in regulating flowering.These results were somewhat consistent with the GO enrichment and transcriptome analyses.
Since the ornamental value of L. chinense var.rubrum mainly lies in the leaf color, three genes (LcbHLH4/156/157) from Subfamily IIIf were selected as being involved in anthocyanin and flavonoid biosynthesis according to the structural and functional predictions.We preliminary analyzed the gene expression profiles of varieties with different leaf color in Stages I and II (Figure 5B).LcbHLH4 was more highly expressed in purple leaves (XNNC) than in green leaves (XNXY) and was significantly downregulated as the leaf color became green from Stage I to Stage II.However, LcbHLH156 and LcbHLH157 were highly expressed in Stage I of different dark-leafed varieties (XNFJ, XNXJ, and XNNC) and decreased in expression as the leaves became green during Stage II.In addition, it is also worth noting that LcbHLH149/114 was also highly expressed in XNFJ, and LcbHLH117 also showed high expression levels in dark-leafed varieties (XNNC).These expression levels of these genes showed similar trends to the phenotype of leaf color changes.Thus, we further studied them as candidate genes.
To further validate the relationship between LcbHLHs and leaf color in L. chinense var.rubrum, we demonstrated the expression levels of the candidate genes of XNFJ from the transcriptomic data using the qRT-PCR technique.These genes had consistent expression trends at the two developmental stages (I and II) (Figure 6, Table S10).We could clearly see that the expression of these six candidate genes was higher in the young leaf stage (I) and significantly decreased in the leaf maturation stage (II), consistent with the phenotypic trend.In addition, we compared the gene expression levels of three different colored leaves of HYJM1 under natural conditions, with green leaves (GL) as the control group (Figure 7A).The results showed that these six genes were upregulated in mixed leaves (ML), especially LcbHLH4/156/157, which was most significantly upregulated in mixed leaves and purple leaves (ML and PL).Notably, the expression levels of LcbHLH114/147/149 were slightly upregulated, but not significantly in mixed leaves (ML), and their expression was significantly reduced in purple leaves (PL).This suggests that they are not directly involved in the positive regulation of anthocyanin synthesis but may act in other ways to affect the anthocyanin synthesis pathway.These assumptions need further analysis and verification.(Figure 7B, Table S11).
(ML), especially LcbHLH4/156/157, which was most significantly upregulated in mixed leaves and purple leaves (ML and PL).Notably, the expression levels of LcbHLH114/147/149 were slightly upregulated, but not significantly in mixed leaves (ML), and their expression was significantly reduced in purple leaves (PL).This suggests that they are not directly involved in the positive regulation of anthocyanin synthesis but may act in other ways to affect the anthocyanin synthesis pathway.These assumptions need further analysis and verification.(Figure 7B, Table S11).

The Protein-Protein Interaction Network of the Candidate Genes
To further clarify the function, we constructed a protein interaction network of these six candidate genes via the Arabidopsis protein database.As shown in Figure 8, LcbHLH4 (TT8 in Arabidopsis) can coordinate with TTG1 and TT2 to ensure that DIHYDROFLAVO-NOL-4-REDUCTASE (DFR) is correctly expressed and then regulate the flavonoid pathway.LcbHLH114 (AT1G01260/bHLH13 in Arabidopsis) plays an important role in plants' growth and defense.It can interact with JAZ proteins and mediate the JA response to regulate plant growth and defense [39].In addition, it is worth noting that LcbHLH117/149 (MYC2 in Arabidopsis) can also interact with JAZ proteins [40][41][42], and the function of LcbHLH156/157 (GL3 in Arabidopsis) may be related to the regulation of trichome development [43,44].The most interesting thing is that there is still a particular relationship among these six candidate proteins involved in different pathways, which needs further analysis and verification.

The Protein-Protein Interaction Network of the Candidate Genes
To further clarify the function, we constructed a protein interaction network of these six candidate genes via the Arabidopsis protein database.As shown in Figure 8, LcbHLH4 (TT8 in Arabidopsis) can coordinate with TTG1 and TT2 to ensure that DIHYDROFLAVONOL-4-REDUCTASE (DFR) is correctly expressed and then regulate the flavonoid pathway.LcbHLH114 (AT1G01260/bHLH13 in Arabidopsis) plays an important role in plants' growth and defense.It can interact with JAZ proteins and mediate the JA response to regulate plant growth and defense [39].In addition, it is worth noting that LcbHLH117/149 (MYC2 in Arabidopsis) can also interact with JAZ proteins [40][41][42], and the function of LcbHLH156/157 (GL3 in Arabidopsis) may be related to the regulation of trichome development [43,44].The most interesting thing is that there is still a particular relationship among these six candidate proteins involved in different pathways, which needs further analysis and verification.

Overexpression of LcbHLH156
Based on the expression profile and protein interaction analysis, LcbHLH156 from the IIIf subfamily with high expression was selected for transient expression verification.The leaves of the variety "Xiangnong Fendai" (XNFD) were overexpressed by Agrobacterium injection, and the control CK was mainly injected with water.It can be observed that compared with CK, vector control (pCAMBIA1305-GFP), and an overexpression of LcbHLH156 (pCAMBIA1305-LcbHLH156) showed red accumulation at the wound, and the accumulation of overexpression plants was more evident (Figure 9A).One week after injection, the wound site of the leaves was collected by a puncher to determine the total anthocyanin content.It was found that the anthocyanin content in the leaves of overexpressing plants showed a significant increase (Figure 9B, Table S12).At the same time, compared with CK, it was found that the overexpressed leaves showed deeper red (a*), higher yellow (b*), and similar brightness (L*) around the wound (Figure 9C, Table S13).To further verify the results, we used qRT-PCR to detect the infected plant leaves.The results showed that compared with CK and vector control, the expression of the LcbHLH156 gene in overexpression plants increased significantly and reached the highest expression level on the 4th day (T4) (Figure 9D, Table S14).The above results indicate that LcbHLH156 can promote anthocyanin synthesis.

Overexpression of LcbHLH156
Based on the expression profile and protein interaction analysis, LcbHLH156 from the IIIf subfamily with high expression was selected for transient expression verification.The leaves of the variety "Xiangnong Fendai" (XNFD) were overexpressed by Agrobacterium injection, and the control CK was mainly injected with water.It can be observed that compared with CK, vector control (pCAMBIA1305-GFP), and an overexpression of LcbHLH156 (pCAMBIA1305-LcbHLH156) showed red accumulation at the wound, and the accumulation of overexpression plants was more evident (Figure 9A).One week after injection, the wound site of the leaves was collected by a puncher to determine the total anthocyanin content.It was found that the anthocyanin content in the leaves of overexpressing plants showed a significant increase (Figure 9B, Table S12).At the same time, compared with CK, it was found that the overexpressed leaves showed deeper red (a*), higher yellow (b*), and similar brightness (L*) around the wound (Figure 9C, Table S13).To further verify the results, we used qRT-PCR to detect the infected plant leaves.The results showed that compared with CK and vector control, the expression of the LcbHLH156 gene in overexpression plants increased significantly and reached the highest expression level on the 4th day (T4) (Figure 9D, Table S14).The above results indicate that LcbHLH156 can promote anthocyanin synthesis.

Systematic and Comprehensive Genome-Wide Detection of LcbHLHs in L. chinense var. Rubrum
With the completion of the whole genome sequences of a large number of species, a large number of studies have been conducted on the bHLH family of various species, such as Arabidopsis (n = 162) [8,9], rice (n = 167) [10], bamboo (n = 448) [34], grape (n = 94) [35], and maize (n = 208) [45].(The "n" here refers to the number of bHLH genes.)Compared with the number of these species, the number of LcbHLHs is appropriate, but the quantity is much smaller than that of bamboo and maize.Changes in the number of bHLH genes between species were often thought to be possibly associated with gene duplication events or genome size [46].Our study identified 165 bHLH genes of L. chinense var.rubrum.According to our evolutionary analysis, these 165 bHLHs are divided into 21 subfamilies and the number and distribution of these subfamilies are similar to those in Arabidopsis.They all have more members in VII(a + b) and XII, III(a + b) subfamilies.It is worth noting that LcbHLHs lack XIII, XIV, and XV subfamilies, but it has been a significant increase in the number of subfamilies III(a + b + c), IVa, and XII.The obtained genes offset the loss of genes to a certain extent so that the overall number of genomes remains stable [47].Protein multisequence alignment also confirmed that LcbHLHs had a typical bHLH domain.Further structural analysis revealed that most LcbHLHs were linked to DNA-binding and homodimer formation functions.Additionally, an analysis of the conserved motifs

Systematic and Comprehensive Genome-Wide Detection of LcbHLHs in L. chinense var. rubrum
With the completion of the whole genome sequences of a large number of species, a large number of studies have been conducted on the bHLH family of various species, such as Arabidopsis (n = 162) [8,9], rice (n = 167) [10], bamboo (n = 448) [34], grape (n = 94) [35], and maize (n = 208) [45].(The "n" here refers to the number of bHLH genes.)Compared with the number of these species, the number of LcbHLHs is appropriate, but the quantity is much smaller than that of bamboo and maize.Changes in the number of bHLH genes between species were often thought to be possibly associated with gene duplication events or genome size [46].Our study identified 165 bHLH genes of L. chinense var.rubrum.According to our evolutionary analysis, these 165 bHLHs are divided into 21 subfamilies and the number and distribution of these subfamilies are similar to those in Arabidopsis.They all have more members in VII(a + b) and XII, III(a + b) subfamilies.It is worth noting that LcbHLHs lack XIII, XIV, and XV subfamilies, but it has been a significant increase in the number of subfamilies III(a + b + c), IVa, and XII.The obtained genes offset the loss of genes to a certain extent so that the overall number of genomes remains stable [47].Protein multisequence alignment also confirmed that LcbHLHs had a typical bHLH domain.Further structural analysis revealed that most LcbHLHs were linked to DNA-binding and homodimer formation functions.Additionally, an analysis of the conserved motifs discovered that most of the LcbHLH family members had the associated conserved motifs (Motif 1 and Motif 2), constituting the structural domain of bHLH [5].The collinearity analysis showed that there were 89, 44, 41, and 123 bHLH collinear pairs between L. chinense var.rubrum and Arabidopsis thaliana, L. chinense var.rubrum and Oryza sativa, L. chinense var.rubrum and Zea mays, L. chinense var.rubrum, and Vitis vinifera, respectively.The evolutionary distance between species is closely related to the collinearity between plant genomes.Generally speaking, the larger the evolutionary distance is, the fewer the collinearity gene pairs identified between species are [47,48].The evolutionary distance between L. chinense var.rubrum and Vitis vinifera is shorter, and the genetic relationship is closer, which is consistent with the fact that they belong to woody plants and dicotyledons.These results further confirmed the accuracy of our analysis of the bHLH gene family in L. chinense var.rubrum provided a basis for further applications.

Functional Prediction of LcbHLHs
As plants' second-largest gene family, bHLHs always play an irreplaceable role in plants' growth, development, and survival [5,11].In our study, GO annotation and ciselement analysis predicted that LcbHLHs are widely involved in the growth and development of plants, including the regulation of secondary metabolite synthesis, the regulation of the cell cycle, and the response to various stresses.In addition, a few LcbHLHs also have some cis elements that respond to hormones, such as gibberellin, jasmonic acid, auxin, etc.To further explore the accuracy of our functional analysis, we analyzed the transcriptomic data and validated some of them by using real-time quantitative PCR.
Many LcbHLHs were expressed differently in different tissues.Some LcbHLHs were highly expressed in the flowers of the three varieties.LcbHLH106 was annotated as PIF3, a phytochrome-interacting factor necessary for photo-induced signal transduction that can further regulate flowering time [49,50].LcbHLH13 was highly expressed in the leaves and was annotated as PIL5, which can indirectly regulate the content of DELLA protein and further participate in the gibberellin pathway to regulate leaf elongation and other processes of growth and development (Table S15) [51][52][53].Moreover, the expression patterns of LcbHLHs in different stages and varieties were analyzed.Three genes (LcbHLH4/156/LcbHLH157) selected by evolutionary analysis and GO annotation showed high expression patterns in dark leaves, and LcbHLH114/117/149 were also upregulated in dark-leafed varieties.In addition, the qRT-PCR results of six genes showed the same trend in XNFJ.They all showed higher expressions in Stage I than in Stage II, which matched the varieties' transcriptomic results and leaf color characteristics.From the qPCR results of HYJM1 leaves with different colors, we also found that the expression trends of LcbHLH4/156/157 genes in HYJM1 leave with different colors were generally consistent, and these genes were significantly upregulated in mixed and purple leaves.
To further demonstrate our hypothesis, we analyzed the interactions of these six bHLH proteins through a protein interaction network.It was found that these proteins were mainly involved in two pathways.LcbHLH117/149/114 were mainly involved in JA signal transduction.Previous studies have shown the bHLHs in Subfamily III(d + e) participate in the JA signal pathway, leading to the accumulation of anthocyanin in apples [54,55] and the regulation of plant defense in Arabidopsis [56][57][58].In Arabidopsis, JAZ proteins can interact with bHLH TFs (GL3) to mediate the accumulation of anthocyanin [59,60].In addition, previous studies have shown that members of Subfamily IIIf in the bHLH family are related to anthocyanin synthesis and trichome initiation [19,61].AtbHLH042/TT8 could coordinate with TTG1 and TT2 to make DIHYDROFLAVO-NOL 4-REDUCTASE (DFR) and BANYULS (BAN) correctly expressed and then regulated the flavonoid pathway [23,62,63].AtbHLH001/GL3 could interact with TTG1 to regulate trichome development [64][65][66].It was identified as an essential regulator of the anthocyanin pathway in Arabidopsis, together chemical parameters were calculated using ExPASy (https://web.expasy.org/compute_pi/(accessed on 19 April 2022)) [73].

Chromosomal Localization, Tandem Duplication, and Collinearity of the bHLH Genes
The calculation of the chromosome distribution was performed on the GSDS website (http://gsds.gao-lab.org/(accessed on 6 March 2023)).Gene duplication analysis of L. chinense var.rubrum was conducted with the Multiple Collinearity Scan Toolkit (MCScanX) (Ver 2.0).The tools of TBtools (Ver 2.003) and MCScanX (Ver 2.0) were used to perform the interspecies collinearity analysis of bHLHs between L. chinense var.rubrum and Arabidopsis, Oryza sativa, Zea mays, and Vitis vinifera [74,75].All of these genomic data were obtained from EnsemblPlants (https://plants.ensembl.org/(accessed on 22 November 2022)).The non-synonymous replacement rate (Ka) and synonymous replacement rate (Ks) of the replicated gene pairs were calculated using KaKs Calculator 2.0 (Ver 2.0), and the environmental selection pressure was analyzed using the Ka/Ks ratio.

Evolution and Multiple Sequence Alignment of LcbHLHs
DNAMAN software (Ver 6.0) was used for a multiple sequence alignment of the proteins, and SnapGene software(Ver 4.3.6)was used to plot the amino acid site distribution of the bHLH proteins' conserved domains.The evolutionary bHLH proteins trees of Arabidopsis thaliana, Oryza sativa, and L. chinense var.rubrum were constructed by using MEGA11 software (Ver 11.0.11)

Analysis of the Gene Structure, Conserved Motifs, and Family Structural Domains
The intron/exon gene structure maps were obtained as GFF3 files via the Gene Structure Display Server (GSDS) website (http://gsds.gao-lab.org/(accessed on 1 June 2022)).The analysis of conserved motifs was conducted via the MEME website (https://memesuite.org/meme/doc/meme.html(accessed on 18 April 2022)).The number of motifs was 10; all other parameters were set to the default.The final result graph was drawn by using TBtools software (Ver 2.003).

GO Annotation, Analysis of the Cis-Acting Components, and the Protein-Protein Interactions of LcbHLHs
The NCBI database (https://www.ncbi.nlm.nih.gov/(accessed on 16 March 2023)) was selected as a reference database for further GO analysis of LcbHLHs using the Blast2GO program (Ver 6.0) [76].A 2000 bp promoter upstream of the bHLH gene family member of L. chinense var.rubrum was extracted by using TBtools software (Ver 2.003) and uploaded to the PlantCARE website (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/(accessed on 12 June 2022)) for predicting the cis-elements.To better understand the regulation of LcbHLHs in protein-protein interaction networks, the STRING database (STRING: functional protein association networks (string-db.org)(accessed on 15 August 2022)) was used to predict the protein interaction network of the candidate bHLH gene family in L. chinense var.rubrum.The calculation of the chromosome distribution was performed on the GSDS website (http://gsds.gao-lab.org/(accessed on 6 March 2023)).Gene duplication analysis of L. chinense var.rubrum was conducted with the Multiple Collinearity Scan Toolkit (MCScanX) (Ver 2.0).The tools of TBtools (Ver 2.003) and MCScanX (Ver 2.0) were used to perform the interspecies collinearity analysis of bHLHs between L. chinense var.rubrum and Arabidopsis, Oryza sativa, Zea mays, and Vitis vinifera [74,75].All of these genomic data were obtained from EnsemblPlants (https://plants.ensembl.org/(accessed on 22 November 2022)).The non-synonymous replacement rate (Ka) and synonymous replacement rate (Ks) of the replicated gene pairs were calculated using KaKs Calculator 2.0 (Ver 2.0), and the environmental selection pressure was analyzed using the Ka/Ks ratio.

Evolution and Multiple Sequence Alignment of LcbHLHs
DNAMAN software (Ver 6.0) was used for a multiple sequence alignment of the proteins, and SnapGene software(Ver 4.3.6)was used to plot the amino acid site distribution of the bHLH proteins' conserved domains.The evolutionary bHLH proteins trees of Arabidopsis thaliana, Oryza sativa, and L. chinense var.rubrum were constructed by using MEGA11 software (Ver 11.0.11)

Analysis of the Gene Structure, Conserved Motifs, and Family Structural Domains
The intron/exon gene structure maps were obtained as GFF3 files via the Gene Structure Display Server (GSDS) website (http://gsds.gao-lab.org/(accessed on 1 June 2022)).The analysis of conserved motifs was conducted via the MEME website (https: //meme-suite.org/meme/doc/meme.html(accessed on 18 April 2022)).The number of motifs was 10; all other parameters were set to the default.The final result graph was drawn by using TBtools software (Ver 2.003).

GO Annotation, Analysis of the Cis-Acting Components, and the Protein-Protein Interactions of LcbHLHs
The NCBI database (https://www.ncbi.nlm.nih.gov/(accessed on 16 March 2023)) was selected as a reference database for further GO analysis of LcbHLHs using the Blast2GO program (Ver 6.0) [76].A 2000 bp promoter upstream of the bHLH gene family member of L. chinense var.rubrum was extracted by using TBtools software (Ver 2.003) and uploaded to the PlantCARE website (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/(accessed on 12 June 2022)) for predicting the cis-elements.To better understand the regulation of LcbHLHs in protein-protein interaction networks, the STRING database (STRING: functional protein association networks (string-db.org)(accessed on 15 August 2022)) was used to predict the protein interaction network of the candidate bHLH gene family in L. chinense var.rubrum.

Gene Expression Patterns and Quantitative Real-Time PCR
All raw transcriptomic data were presented as FPKM values (fragments per kilobase of transcript per million mapped reads).We homogenized the raw FPKM values and visualized the heatmap using the R package [77,78].
RNA from the plant material was extracted using the FastPure Universal Plant Total RNA Isolation Kit (Vazyme, Nanjing, China).The first standard cDNA was synthesized by using the Evo M-ML RT Kit for qPCR (Accurate Biology, Changsha, China) and stored in a freezer at −40 • C. The primers were designed using Beacon Designer 8. (Table S16), and qRT-PCR was performed on a Bio-Rad CFX384TM (Bio-Rad, Hercules, CA, USA) with 2X SYBR Green Pro Taq HS Premix (Accurate Biology, Changsha, China).The cDNA was diluted to 500 ng, and the system was set to 10 µL to be prepared as three technical replicates.Each reaction had a 1 µL template.The conditions for qRT-PCR were as follows: 95 • C for 5 min, followed by 40 cycles of 95 • C for 15 s, 60 • C for 1 min, and 72 • C for 5 min.Quantitative PCR expression levels were calculated using the 2 −∆∆CT method, and the expression values of three replicates were normalized using LcActin as the internal control.The error bars represent the standard errors from three biological replicates.

Transient Expression of L. chinense var. rubrum and Color Measurement
The CDS sequence of LcbHLH156 was amplified by Phanta Max Super-Fidelity DNA Polymerase kit (Vazyme, Nanjing, China), and the specific primers were designed by Primer5 software (Ver 5.0) (Table S17).The CDS sequence of LcbHLH156 was ligated to the BamHI site of the pCAMBIA1305-GFP vector by homologous recombination, and the recombinant plasmid was transformed into the GV3101 strain.The bacterial liquid was cultured in a shaker at 28 • C and 200 rpm for 24 h.When the bacterial solution was shaken to golden yellow and the OD600 value reached 1.0, the cells were centrifuged at 5000 rpm for 10 minutes.The Agrobacterium cells were collected and re-suspended using the infection solution (10 mM MgCl 2 , 10 mM MES, pH 5.8, and 200 µM acetosyringone) [79].The leaves of L. chinense var.rubrum were injected with a 1 mL sterile syringe, and 5-10 branches were injected into each plant.Photos were recorded every three days by a portable fluorescent protein excitation light source LUYOR-3415 (LUYOR, New York, NY, USA).Random sampling was performed at 1, 4, and 7 days after treatment for qRT-PCR expression detection.
To analyze the phenotypic data, we used a puncher to sample the wound site on the leaves one week after injection.Leaf samples (10 mg) were extracted with 1 mL of 0.5% HCl in methanol for 1 day at 4 • C in the dark.After centrifuging at 12,000 rpm for 20 min, the absorbance at 510 nm and 700 nm was measured in pH 1.0 and 4.5 buffers by UV-2600 Spectrophotometer.Each sample was repeated three times [80].

Statistical Analysis
All the experiments included three replicates, and the results were presented as mean ± SD of three independent experiments.Student's t-test statistically analyzed the data.An asterisk (*) indicates p < 0.05, a double asterisk (**) indicates p < 0.01, and a triple asterisk (***) indicates p < 0.001.

Conclusions
In summary, we analyzed the physicochemical properties, evolution, gene structure, Ka/Ks value, and collinearity of the bHLH family members of different L. chinense var.rubrum varieties.In addition, we also predicted the function of LcbHLHs by using cis elements, evolutionary analysis, and GO annotation.Six candidate genes were selected by analyzing the evolutionary tree and the expression patterns of 105 LcbHLHs in different tissues and varieties.Through further analyses of qPCR and predictions of the protein interactions, we obtained a preliminary understanding of the function of these six candidate genes.The LcbHLH156 candidate gene of the IIIf family was selected for transient expression to verify the function.It was found to promote anthocyanin synthesis through phenotypic analysis and expression analysis.All these results strengthen our understanding of the bHLH gene family in L. chinense var.rubrum and provide a basis for further studies on the mechanism regulating the change in leaf color in L. chinense var.rubrum.

Supplementary Materials:
The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/plants12193392/s1. Figure S1.Evolutionary tree constructed with the sequences of the LcbHLHs, OsbHLHs, and AtbHLHs.The branch lines of the evolutionary tree are colored, representing different bHLH subfamilies.The LcbHLHs, OsbHLHs, and AtbHLHs of the same subfamily are respectively represented by blue, pink, and yellow circles.S1.Gene accession numbers of all LcbHLH genes.Table S2.Physicochemical properties.Table S3.The gene tandem replications on the chromosomes of L. chinense var.rubrum.Table S4.The gene fragment replications on the chromosomes of L. chinense var.rubrum.Table S5.The Ka/Ks ratios for duplicate pairs of LcbHLHs.Table S6.The regions of synteny of bHLH genes in diverse species.Table S7.GO annotations of the bHLH proteins in L. chinense var.rubrum.Table S8.Cis-element analysis of the bHLH proteins in L. chinense var.rubrum.Table S9.FPKM values of the transcriptome data of LcbHLHs.Table S10.qPCR data of Stages I and II of XNFJ.Table S11.qPCR data of GL, ML, and PL.Table S12.Total anthocyanin content.Table S13.Color measurement.Table S14 qPCR date of transient expression plants.Table S15.NR annotations of LcbHLHs.Table S16.List of primers used for qRT-PCR.Table S17.List of Cloning and vector primers.

Figure 1 .
Figure 1.Chromosomal distribution of LcbHLHs.The left-hand scale is used to assess the chromosome size.All chromosomes contain the LcbHLH genes.The scaffold is represented by chr0.

Figure 1 .
Figure 1.Chromosomal distribution of LcbHLHs.The left-hand scale is used to assess the chromosome size.All chromosomes contain the LcbHLH genes.The scaffold is represented by chr0.

Figure 2 .
Figure 2. Evolutionary bHLH proteins tree in L. chinense var.rubrum.The Evolutionary tree of L. chinense var.rubrum was constructed using MEGA11 software (Ver 11.0.11)via the neighbor-joining (NJ) method.The parameters were set as follows: method, "p-distance"; treatment of gaps in the data, partial deletion; self-expansion value, "bootstrap = 1000".Different subfamilies are represented by branches and frames of different colors.

Figure 2 .
Figure 2. Evolutionary bHLH proteins tree in L. chinense var.rubrum.The Evolutionary tree of L. chinense var.rubrum was constructed using MEGA11 software (Ver 11.0.11)via the neighbor-joining (NJ) method.The parameters were set as follows: method, "p-distance"; treatment of gaps in the data, partial deletion; self-expansion value, "bootstrap = 1000".Different subfamilies are represented by branches and frames of different colors.

Figure 3 .
Figure 3. Multiple alignment of the bHLH domains in LcbHLH family proteins in the LcbHLH domain shows conserved amino acids.Sequence identities > 60% are shown in grey or black shading."*" was used to interval 10 bases.

Figure 3 .
Figure 3. Multiple alignment of the bHLH domains in LcbHLH family proteins in the LcbHLH domain shows conserved amino acids.Sequence identities > 60% are shown in grey or black shading."*" was used to interval 10 bases.

Figure 5 .
Figure 5. Expression patterns of LcbHLHs (A) Four varieties with different colored leaves and flowers.Stage I and II represent the first−stage and second−stage leaves, respectively.The first stage is the young leaf stage; the second is the mature leaf period.(B) Expression patterns of the LcbHLHs.Expression profiles of LcbHLHs in different tissues."XNFJ," "XNXY," "XNXJ," and "XNNC" are the names of our selected materials.The red triangle is used to point out candidate genes.

Figure 5 .
Figure 5. Expression patterns of LcbHLHs (A) Four varieties with different colored leaves and flowers.Stage I and II represent the first−stage and second−stage leaves, respectively.The first stage is the young leaf stage; the second is the mature leaf period.(B) Expression patterns of the LcbHLHs.Expression profiles of LcbHLHs in different tissues."XNFJ," "XNXY," "XNXJ," and "XNNC" are the names of our selected materials.The red triangle is used to point out candidate genes.It was found that the expression levels of LcbHLHs in flowers, young leaves (Stage I), and mature leaves (Stage II) displayed significant differences, as shown in Figure5B.Five

Figure 6 .
Figure 6.Expression of six differentially expressed LcbHLHs in the variety XNFJ during Stages I and II.

Figure 6 .
Figure 6.Expression of six differentially expressed LcbHLHs in the variety XNFJ during Stages I and II.Plants 2023, 12, x FOR PEER REVIEW 12 of 23

Plants 2023 , 23 Figure 8 .
Figure 8. Interaction network for the six candidate LcbHLH proteins.The predicted results in parentheses are based on homologous genes in Arabidopsis.The colors of the lines represent the status of research into these protein interactions.

Figure 8 .
Figure 8. Interaction network for the six candidate LcbHLH proteins.The predicted results in parentheses are based on homologous genes in Arabidopsis.The colors of the lines represent the status of research into these protein interactions.

Figure 10 .
Figure 10.Process of identifying the bHLH family proteins.

Figure 10 .
Figure 10.Process of identifying the bHLH family proteins.

4. 3 .
Chromosomal Localization, Tandem Duplication, and Collinearity of the bHLH Genes Figure S2.Evolutionary relationships, conserved motifs, families of structural domains, and gene structure of the LcbHLHs.(A) Evolutionary tree of LcbHLHs.(B) Conserved motifs of LcbHLHs.The 10 predicted motifs are depicted by rectangles of various colors.(C) The distribution of family domains and types of LcbHLHs.(D) Gene structure of LcbHLHs.The blue rectangle represents the exon, the orange rectangle represents the UTR, and the black line represents the intron.The size of these genes is indicated by the bottom coordinate.Table Author Contributions: X.X., X.Y. and Y.L. (Yanlin Li) proposed the design of the article, performed the experiments, and authored or reviewed drafts of the paper; Y.L. (Yang Liu 1) and L.L. analysed the data; Y.L. (Yang Liu 2) and Q.M. prepared the figures and tables; W.L. and D.Z. authored or reviewed drafts of the article; Y.L. (Yang Liu 1) completed the manuscript.All authors have read and agreed to the published version of the manuscript.Funding: The work was funded by The Forestry Science and Technology Innovation Foundation of Hunan Province for Distinguished Young Scholarship (XLKJ202205), the Open Project of Horticulture Discipline of Hunan Agricultural University (2021YYXK001), the Hunan Province key research and development project (2022NK2026), the Foundation of Changsha Municipal Science and Technology Bureau (KQ2202227), the key project of Hunan Provincial Education Department (22A0155), the Forestry Bureau for Industrialization Management of Hunan Province (2130221), and the Graduate Innovation Project of Hunan Province (2023XC108), the College Student Innovation and Entrepreneurship Project of China (S202310537005), the College Student Innovation and Entrepreneurship Project of Hunan Province (202044604103).Data Availability Statement: Data are contained within the article and the Supplementary Materials.

Table 1 .
Quantitative distribution of each subfamily of bHLHs in L. chinense var.rubrum, A. thaliana, Ficus carica L., and Oryza sativa.

Table 1 .
Quantitative distribution of each subfamily of bHLHs in L. chinense var.rubrum, A. thaliana, Ficus carica L., and Oryza sativa.

Table 2 .
Sequences of the 10 predicted motifs of the LcbHLH proteins.