The materials L. cubeba used in this study from HangZhou City, Zhejiang Province, China (30°27′94′′N, 119°58′ 43′′E). Collecting different tissues including root, stem, leaf, and flower of 5-year-old L. cubeba. The fruits of different developmental stages were collected at 10 a.m. on 30, 60, 90, 120, and 150 days after flowering, and immediately frozen in liquid nitrogen, then stored at -80°C for RNA extraction.

The CDS sequences, protein sequences needed for analysis were obtained from L. cubeba genome database (Chen et al., 2020b). The bHLH gene sequence of L. cubeba was extracted using TBtools v1.0686 (https://github.com/CJ-Chen/TBtools), with the thresholds of the screening process set to 1e-5 and 45% filtration. Furthermore, putative LcbHLH proteins were discovered by reviewing HMMER and BLAST results and manually deleting duplicated sequences. Predicted LcbHLH genes were then double-checked using batches from the NCBICDD, SMART, and PFAM databases. Finally, 173 LcbHLH TFs were identified, and a phylogenetic tree was reconstructed using PhyML 3.0 with the default parameters (Gascuel, 2010).

LcbHLH protein sequences were uploaded to the ExPASy online program (Guo et al., 2014) to calculate their molecular weights (MW), isoelectric points (pI) and GRAVY values. To identify conserved motifs, the MEME (Bailey et al., 2015) 10 suite was applied using default settings. The gff3 file for the L. cubeba genome, which provides details of gene structure and was visualized using TBtools, was used to determine the exons and introns of each bHLH gene.

BLAST programs were used to map bHLH gene sequences to L. cubeba chromosome survey sequences to determine the positions of LcbHLH genes on the 12 chromosomes. Precise gene-location results were displayed using MG2C V2.1software (http://mg2c.iask.in/mg2c_v2.1/). An interspecies collinearity analysis of bHLH genes was performed using MCscanX software.

Promoter sequences of L. cubeba terpene synthase (TPS) family members were extracted using TBtools software and used to detect and visualize cis-acting elements. Detection and identification of cis-elements was carried out using PlantTFDB (http://planttfdb.gao-lab.org/) software.

RNA of L. cubeba was extracted and reverse transcribed by the method provided by Zhao et al. (2020). qRT-PCR was carried out with the assistance of an ABI PRISM 7500 instrument and the TB Green^® Premix Ex TaqTM II kit. The actin gene from L. cubeba ubiquitin conjugating enzyme (UBC) was utilized as a reference gene (Chen et al., 2020). The total volume of the qRT-PCR system was 25 μL, which comprised the following components: 12.5 μL Green Premix Ex TaqII (Til RNaseH Plus) (2x) Mix, 1.0 μL upstream primer (10 μM), 1.0 μL downstream primer (10 μM), 2.0 μL cDNA, and 8.5 μL ddH2O. Reactions were prepared on ice. Each sample was prepared using three technical replicates in addition to a control that lacked cDNA. To calculate relative expression levels, the relative expression was calculated by 2^-ΔΔCT method. Findings are reported as the mean plus standard deviation across all three replicates. Primer Premier 3.0 was used to create primers for qRT-PCR reactions of the chosen bHLH genes, which are detailed in Table S2 .

To make a prediction regarding the subcellular localization of LcbHLH78 in L. cubeba, Euk-mPLoc 2.0 online software was utilized (Chou and Shen, 2010). Transient expression of LcbHLH78 fusion protein in tobacco epidermal cells provided conclusive evidence for this assertion. LcbHLH78 was cloned into the transient expression vector pNC-Green-SubC to generate a 35S:GFP-LcbHLH78 recombinant vector (Yan et al., 2021). Then, 35S:GFP-LcbHLH78 and the empty vector were transferred into Agrobacterium strain GV3101 by chemical conversion method (Wydro et al., 2006). The OD600 of the Agrobacterium suspension was adjusted to 0.8 using an infection solution containing 10 mM MES, 10 mM MgCl₂, and 200 mM acetosyringone at pH 5.7 and incubated for 4 h at 28°C before infiltration into Nicotiana Benthamiana leaves that were 4-week-old. Fluorescence signals were examined using a confocal laser scanning microscope between 40 and 52 h after infiltration (ZEISS LSM 880, Germany). OsRde nuclear localization protein with red fluorescence was used as a positive control.

LcbHLH78 transient overexpression analysis was performed using sterile seedlings of L. cubeba. For the preparation of sterile seedlings, cut about 6 mm with buds and stems and insert them into MS (Murashige and Skoog) basal medium containing, after 30 days of light culture, transfer to the new MS basal medium containing and continue to be cultured under light for 30-45 days. Sterile L. cubeba shoots were harvested and propagated in basal medium containing 6-BA, IBA, sugar, and agar (pH 5.8). After being individually transformed to Agrobacterium strain LBA4404, the empty vector (pNC-Cam2304-35S) and a recombinant vector containing LcbHLH78 (pNC-Cam2304-35S-LcbHLH78) were infiltrated into leaves of sterile seedlings displaying similar growth and cultured at 26°C for 50 - 72 h. Then, collect the leaves with consistent growth state for qRT-PCR study and save the remaining leaf samples at –80°C for volatile analysis. Volatiles were examined using GC-MS method (Zhao et al., 2020). Table S2 provides specific information on all primers.

To identify bHLH TFs involved in terpenoid biosynthesis of LCEO, we first used an implicit Markov model to search for the bHLH domain using hmmsearch. We identified homology with A. thaliana bHLH proteins, and further screened candidate genes using NCBI-CDD conserved domain search. 173 bHLH proteins were filtered from the L. cubeba genome, named LcbHLH1 - LcbHLH173 according to their location on chromosomes or scaffolds. The molecular weight, hydrophilicity, and isoelectric point of each protein was calculated using the ExPASy tool, and the bHLH proteins range in size from 90 aa (LcbHLH44) to 1, 098 aa (LcbHLH75), theoretical isoelectric point ranged from 4.49 (LcbHLH125) to 11.51 (LcbHLH126), and hydropathicity values of all LcbHLH proteins varied between -0.083 and -1.062. More detailed information was shown in Table S1 .

To elucidate the structure and functions of L. cubeba bHLH TFs at the genomic level, we first reconstructed phylogenetic trees of LcbHLH and AtbHLH based on HMMER domain search and homology comparison with bHLH members in A. thaliana. The 173 LcbHLHs were classified into 26 groups according to the groups defined in A. thaliana; all subfamilies comprised members from both L. cubeba and A. thaliana, but the number of proteins differed between the two species ( Figure 1A ). The largest LcbHLH subgroups were XII and Ib (2), both with 35 members, while subgroup IVb was the smallest, with only five members: three in A. thaliana and two in L. cubeba. Twelve subgroups contained the same number of family members in L. cubeba and A. thaliana. Subgroup Ib (1) showed the greatest numerical discrepancy, with half as many members in A. thaliana as in L. cubeba. It is worth noting that seven bHLH family members clustered into the III (d+e) subfamily. Of these, LcbHLH55, LcbHLH59, LcbHLH78, and LcbHLH123 belonged to the IIIe family. Proteins belonging to the subfamily IIIe are involved in the regulation of plant metabolism and induced by the defense-related hormone JA (Goossens et al., 2017).

To further study domains in LcbHLH, we analyzed the gene structure and conserved domains of 173 L. cubeba bHLH proteins. Two types of highly conserved protein motifs, denoted motif 1 and motif 2, were present in most sequences. Although there was considerable variation in the length of LcbHLHs amongst subfamilies, the lengths and positions of conserved motifs were similar, suggesting a phylogenetic relationship between them ( Figure 1B ). However, significant differences were found between the various subfamilies, and some motifs were only found in certain subfamilies. For example, motif 5 was only found in subfamilies III (d + e) and IIIf. This suggests that motif 5 may specifically function in these subfamilies.

We determined the exon-intron structure of LcbHLH genes based on their evolutionary classification. LcbHLH genes had between 0 and 12 introns, with 14 LcbHLH genes being intron-free, 15 LcbHLH genes having one intron, and the remaining genes having two or more introns. Furthermore, most LcbHLH genes belonging to the same subfamily had similar exon/intron distribution patterns. For instance, subfamily III (d + e) had 0 - 2 introns, while subfamily IIIf had 8 - 9 introns. Frequent occurrence of intron gains and losses during evolution can make gene structures more complex (Roy and Gilbert, 2005). However, exceptions were also found among these genes. For example, the members of subfamily Ib (2) had a differing number of introns and exhibited great diversity in exon length.

While 14 LcbHLH genes were localized on unassembled genomic scaffolds, 159 genes were localized unevenly on the 12 L. cubeba chromosomes. The most abundant chromosomal region was chromosome 2, harboring 36 bHLH genes, followed by Chr5 (21 genes), Chr4 (17 genes), Chr1 (15 genes), Chr3 (14 genes), Chr8 (13 genes), and Chr7 (12 genes); Chr12 had the fewest gene family members of any of the chromosomes, at 2 genes. We also discovered that the length of individual L. cubeba chromosomes varies. Chr1 has the longest arm, while Chr12 has the shortest length of any of the chromosomes. This demonstrated that there was no correlation between the distribution of LcbHLH genes and the length of chromosomes. Five pairs of LcbHLH genes mapping to chromosomes 2, 5, and 10 were characterized as tandem duplications among the 159 LcbHLH genes ( Figure 2 ). IDs and genomic positions of the LcbHLH genes are listed in Table S1 .

Genome duplication, tandem duplication, segmental duplication, and transposon duplication all contribute to the evolution of plants (Qiao et al., 2019). We created a syntenic map of L. cubeba to better understand the evolutionary process underlying the LcbHLH gene family. Many genes with collinearity were found on chromosomes 2, 3, and 5 ( Figure 3 ). These findings suggest that gene duplication, particularly segmental duplication, may be linked to LcbHLH gene family amplification and that these duplication events may be the primary driver of LcbHLH evolution.

We used PlantTDFB software to find and analyze probable cis-elements in the promoter regions of TPS genes, 2,000 bp upstream of the start codon, in order to further speculate the relationship between the TPS gene and bHLH transcription factors. The results show that the TPS promoter contains CACGTG/CATGTG sequences ( Figure 4 ). In addition, previous research has shown that bHLH proteins regulate terpenoid biosynthesis by binding to G-box motifs found in the promoters of terpenoid biosynthesis genes (Liu et al., 2015; Wang et al., 2022). Considering the distribution of cis-elements in the promoter of these genes, we speculate that the bHLH TFs in L. cubeba also regulates terpenoid biosynthesis by binding to the G-box sequence on the TPS promoter.

Monoterpenes are mainly produced in the L. cubeba pericarp. As a result, the bHLH TFs, which are abundant in the pericarp, are most likely to be candidate genes for regulating terpene synthesis. Additionally, LcTPS42 has been identified as the key enzyme for geraniol, linalool and other monoterpenoids synthesis in the previous study, therefore, LcTPS42 was also included as the reference for the co-expression analysis (Chen et al., 2020b). Transcriptome sequences (PRJNA763042) of L. cubeba pericarp at different stages of development were used to identify LcbHLH genes. The expression trends of several members of subgroup IIIe were consistent with that of LcTPS42 in the pericarp of L. cubeba ( Figure 5A ). LcbHLH78 was expressed throughout all developmental periods, with the highest expression at 120 days after full bloom highly consistent with the expression trend of LcTPS42, suggesting that LcbHLH78 may be involved in the regulation of LcTPS42 ( Figure 5B ).

TFs typically carry out transcriptional regulatory tasks in the nucleus (Zhu et al., 2020). Therefore, we investigated the subcellular distribution of LcbHLH78 protein. GFP fluorescence of the empty vector was distributed throughout the cells of N. benthamiana leaves, while the nuclear marker and GFP-fused LcbHLH78 protein were localized in the nucleus ( Figure 6 ).

Because the stable transformation of L. cubeba is challenging, we used an efficient and simple transient expression assay to investigate the function of LcbHLH78. We transiently overexpressed LcbHLH78 in L. cubeba following the transient transformation method of Wang M Y et al. (2022). After transient expression of LcbHLH78, we detected a 10-fold increase in LcbHLH78 expression relative to that in seedlings transformed with an empty vector ( Figure 7A ). Transient overexpression of LcbHLH78 enhanced the accumulation of α-phellandrene, linalool, citronellal, geraniol, neral, geranial, and camphene in L. cubeba leaves ( Figures 7B, C ). Previous studies revealed that LcTPS42 is highly expressed in the pericarp and catalyzes the biosynthesis of geraniol and linalool (main components of monoterpenoid) in L. cubeba (Zhao et al., 2020). In this study, the contents of linalool, geraniol, and α-phellandrene were significantly increased after transient expression of LcbHLH78, consistent with LcTPS42 catalyzing formation of monoterpene components. It is worth noting that geraniol is the direct precursor of citral, a key component of the essential oil in L. cubeba fruit, with linalool and α-pinene as the main monoterpene components. In addition, camphene contents were also significantly increased after transient expression of LcbHLH78. Actually, G-box elements were also found on the promoters of genes involved in MVA and MEP pathways. The overexpressing of LcbHLH78 not only activated the expression of LcTPS42, but also the pathway ( Figure S1 ). These findings suggest that LcbHLH78 as a candidate gene enhances terpenoid biosynthesis.

bHLH TFs, an important set of eukaryotic protein family members, play a critical function in the growth, development, and secondary metabolism of organisms (Zhao et al., 2018). Up to now, bHLH TFs have been identified in a variety of plants, such as Dracaena cambodiana, Pyrus bretschneideri, and Persian walnut (Zhu et al., 2020; Dong et al., 2021; Ullah et al., 2021). In our study, 173 LcbHLH genes were identified, and these were separated into 26 different subfamilies based on the phylogenetic relationships they shared with other bHLH genes found in Arabidopsis (Li et al., 2006). Among them, subgroup IIIe, which is responsible for the regulation of secondary metabolism, contains four members (LcbHLH55, LcbHLH59, LcbHLH78, LcbHLH123). In terms of the number of genes, there are more bHLH gene family members in L. cubeba than in some other species: 173 LcbHLHs compared with 162 ArbHLH genes (Bailey et al., 2003) and 152 SlybHLH genes (Wang et al., 2015). This may be because two whole-genome replication (WGD) events occurred in the L. cubeba genome (Chen et al., 2020b). In addition, other plants have a larger number of bHLH gene family members than L. cubeba; for example, wheat has 225 bHLH genes (Guo and Wang, 2017), tobacco has 190 bHLH genes (Rushton et al., 2008), and sorghum has 174 bHLH genes (Fan et al., 2021).

Tandem duplication and segmental duplications provide different evolutionary dynamics to duplicated genes (Leister, 2004; Panchy et al., 2016). Arabidopsis and rice genomes contain about 10% tandem repeat genes, which have made important contributions to the expansion of some large gene families (Blanc et al., 2000). The evolutionary history of LcbHLH genes shows that tandem duplications and segmental duplications have contributed to the expansion of LcbHLH genes. We speculate that 10 LcbHLH genes are associated with tandem repeat events, and large-fragment replication events involve different chromosomes (McGowan et al., 2020). These duplicated LcbHLH genes probably formed new gene functions to adapt to various growth conditions. Repetitive genes play an important role in plant adaptation to complex and changeable environments (Cannon et al., 2004; Wang et al., 2022). Short-term and long-term evolutionary retention mechanisms of repetitive genes include subfunctionalization, new functionalization, and loss (Chen et al., 2019). After gene replication, sequence differentiation of two homologous gene copies in the promoter region may lead to expression differentiation between them. Previous studies have shown that two genes adjacent to each other on the chromosome are more likely to be co-regulated, especially two tandem repeat genes (Wang et al., 2022). In this study, sequence alignment of gene family members in the IIIe branch of L. cubeba revealed high levels of similarity. Some amino acids had undergone mutation, however, which may be the cause of functional differentiation.

bHLH TF is one of the largest families of transcription factors in plants, which participates in the regulation of plant growth and development, signal transduction, and responding to abiotic stresses such as drought, low temperature, salt and heavy metals in plants (Goossens et al., 2017). Meanwhile, bHLH plays an important role in secondary metabolism, especially terpenoid biosynthesis. For example, transcription factor BpbHLH9 in birch can increase the content of triterpenoids and the expression of key genes (Yin et al., 2017); two tissue-specific bHLH transcription factors, BI and BT, are involved in gene expression regulation of triterpenoid synthesis, which has important contributions to the cultivation and selection of cucumber (Shang et al., 2014).

Terpenoids are one of the most significant pharmacologically active components of L. cubeba, and the amount of these compounds directly influences the economic value of this plant. However, biosynthesis of the secondary metabolite terpene is tissue-specific and spatiotemporal. As a result, tissue-specific expression of TF genes may have a significant influence on the production of terpenes. We found that many LcbHLH genes are constitutively expressed at various stages of fruits. The expression trend of several subgroup IIIe members was consistent with that of LcTPS42, which was highly expressed in the later phases of fruit peel growth ( Figure 6 ). Interestingly, essential oil is rapidly produced during the later phases of fruit peel growth in L. cubeba. We speculate that the differential expression of LcbHLH family members in different tissues leads to their different secondary metabolite contents (Wang et al., 2021). Tissue-specific expression has also been extensively studied in a variety of plants. During the development of Ficus carica L. fruit, members of distinct bHLH subfamilies are expressed differently in the female flower tissue and peel (Song et al., 2021). Four genes involved in anthocyanin biosynthesis in walnut show similar expression patterns in the leaf and peel of red and green walnut at different developmental stages (Zhao et al., 2021). bHLH genes are mostly expressed in the leaves and stems of Artemisia argyi, with reduced expression in roots (Yi et al., 2022). All these studies indicate that bHLH genes belonging to the same subfamily have similar and tissue-specific expression patterns.

bHLH TFs can regulate the production of terpenoids by directly binding to the promoter of key genes involved in the biosynthesis pathway. Such as AtMYC2 activates TPS21 and TPS11 synthase genes to increase the release of sesquiterpenes by binding to the promoter region of these genes in A. thaliana (Hong et al., 2012). SlJIG, a bHLH TF, was found to be directly downstream of MYC2, regulating the expression of TPS genes or participating in the classical JA defense pathway, and is predicted to participate in JA-induced terpenoid biosynthesis (Cao et al., 2022). In Freesia hybrida, genes encoding three TFs, FhMYB21L1, FhMYB21L2, and FhMYC2, were isolated and functionally verified as regulators of linalool biosynthesis (Yang et al., 2020). Taken together, these studies suggest that.

According to previous research, the terpenoids in L. cubeba were mainly accumulated in the pericarp. The expression level of the fruit varies with different developmental stages, and the highest expression level is generally found between 120d and 150d after flowering (Wang et al., 2022). We found the expression patterns of the bHLH gene and LcTPS42 in different tissues and developmental stages in the heat map, LcbHLH46, LcbHLH55, LcbHLH78, LcbHLH145 are co-expressed with LcTPS42, however, we choose LcbHLH78 as terpene synthesis candidate gene, mainly because LcbHLH55 has been studied before (Wang et al., 2022), while LcbHLH46 and LcbHLH145 do not belong to IIIe branch, which may not play a positive role in JA regulation of plant secondary metabolism. Furthermore, Terpenoid biosynthesis can also be promoted by exogenous MeJA (Ji et al., 2019; Wang et al., 2022). In our study, GC-MS was used to determine the main terpenoid in L. cubeba, impressively, overexpression of LcbHLH78 increased the α-pinene, linalool, geraniol, neral, and geranial. Thereinto, geraniol was the direct precursor of citral, a main component of the LECO, with linalool and α-pinene as the main monoterpene component (Chen et al., 2020). Different from LcbHLH55, GCMS volatiles that overexpressed LcbHLH78 showed significantly increased camphene content, which may be due to the different functions of different transcription factors in terpene biosynthesis. This study provides a theoretical basis for the regulation of LCEO.