LcWRKY17, a WRKY Transcription Factor from Litsea cubeba, Effectively Promotes Monoterpene Synthesis

The WRKY gene family is one of the most significant transcription factor (TF) families in higher plants and participates in many secondary metabolic processes in plants. Litsea cubeba (Lour.) Person is an important woody oil plant that is high in terpenoids. However, no studies have been conducted to investigate the WRKY TFs that regulate the synthesis of terpene in L. cubeba. This paper provides a comprehensive genomic analysis of the LcWRKYs. In the L. cubeba genome, 64 LcWRKY genes were discovered. According to a comparative phylogenetic study with Arabidopsis thaliana, these L. cubeba WRKYs were divided into three groups. Some LcWRKY genes may have arisen from gene duplication, but the majority of LcWRKY evolution has been driven by segmental duplication events. Based on transcriptome data, a consistent expression pattern of LcWRKY17 and terpene synthase LcTPS42 was found at different stages of L. cubeba fruit development. Furthermore, the function of LcWRKY17 was verified by subcellular localization and transient overexpression, and overexpression of LcWRKY17 promotes monoterpene synthesis. Meanwhile, dual-Luciferase and yeast one-hybrid (Y1H) experiments showed that the LcWRKY17 transcription factor binds to W-box motifs of LcTPS42 and enhances its transcription. In conclusion, this research provided a fundamental framework for future functional analysis of the WRKY gene families, as well as breeding improvement and the regulation of secondary metabolism in L. cubeba.


Introduction
Secondary metabolites from plants are a one-of-a-kind source of industrially significant biochemicals, flavors, and medicines [1,2]. Terpenoids are an essential secondary metabolite in plants and are commonly used as chemicals in dyes, flavors, fragrances, insecticides, and drugs by humans thanks to their significant economic worth [3,4]. To control the expression of genes in plants, transcription factors (TFs) coordinate a series of intricate regulatory networks [3,5]. Because there are many genes encoding different enzymes in the metabolic pathways of plants, manipulating TFs may be easier and more efficient than controlling the expression of the specific enzyme [6][7][8]. Numerous TFs have been demonstrated to be engaged in a number of physiological procedures in higher plants since the first TF was found in corn [9].
One of the major families of TFs in higher plants is the WRKY family [10]. Members of this family all contain two extremely preserved structural domains, a WRKYGQK sequence and a zinc finger structure [11][12][13]. The classification of WRKY proteins into three distinct groups is based on variations in the number and arrangement of structural domains, as well as the composition of their zinc-finger motif. Two WRKY domains and a zinc finger structure of the C2H2-type can be found in Group I proteins. One WRKY domain is present in both Group II and Group III proteins; however, Group II has a C2H2 zinc finger structure, whereas Group III has a C2HC zinc finger structure [12,14,15]. The WRKY protein binds

Identification and Chromosomal Location of LcWRKYs
HMMER searches initially yielded 77 candidate genes. We eventually discovered 64 WRKY genes in the L. cubeba genome after manually removing the redundant genes (Table S1). These were named LcWRKY1-LcWRKY64 according to their position on the chromosome. These genes ranged from 124 amino acids to 1029 amino acids in length; these differences in gene length led to key differences in their expression.
The distribution of LcWRKY genes on the 12 chromosomes of L. cubeba was determined by chromosomal localization analysis ( Figure S1A). We can find these 64 LcWRKY genes scattered on the 12 chromosomes, with chromosome 4 having the most (14 genes), followed by chromosome 2 (nine genes), and chromosome 11 having the fewest (one gene). Six pairs of genes were also found to be closely linked in terms of their chromosomal position.

Motif Analysis and Structural Analysis of LcWRKYs
By analyzing the gene structure of LcWRKYs, it was evident that the protein-coding regions (CDS) of the WRKY gene range from 2 to 16 and that eight gene family members have no non-coding region (UTR). To research the structural characteristics of the evolutionary relationships among members of the LcWRKYs in L. cubeba, we utilized MEME to analyze the protein sequences of LcWRKYs ( Figure 1). Motif 1 and motif 3 are conserved sequences of the WRKYGQK heptapeptide motif, and motif 2 is a zinc finger structural motif that is contained in all LcWRKYs. Motifs 1, 2, and 3 are the hallmark conserved structures of LcWRKYs transcription factors. Analysis showed that LcWRKYs contained 4 to 12 motifs; these motifs were essentially the same, thus indicating that these exert similar biological functions. Some of these proteins contain unique motifs that may be linked to unique functionality. These structural changes imply that LcWRKYs have undergone significant changes during their evolution compared with the basic structure of WRKYs. evolutionary relationships among members of the LcWRKYs in L. cubeba, we utilized MEME to analyze the protein sequences of LcWRKYs ( Figure 1). Motif 1 and motif 3 are conserved sequences of the WRKYGQK heptapeptide motif, and motif 2 is a zinc finger structural motif that is contained in all LcWRKYs. Motifs 1, 2, and 3 are the hallmark conserved structures of LcWRKYs transcription factors. Analysis showed that LcWRKYs contained 4 to 12 motifs; these motifs were essentially the same, thus indicating that these exert similar biological functions. Some of these proteins contain unique motifs that may be linked to unique functionality. These structural changes imply that LcWRKYs have undergone significant changes during their evolution compared with the basic structure of WRKYs. Figure 1. Gene structure and motif analysis of LcWRKYs. The blue boxes, green boxes, and thin black lines represent the UTR, CDS, and introns, respectively. MEME analysis revealed conserved motifs of LcWRKY proteins. Colored boxes on the right denote 30 motifs.

Gene Duplication Analysis
A tandem repeat event is described as two or more genes located within 200 kb of a chromosomal region [32,33]. Our LcWRKY genes are clustered on L. cubeba chromosomes 2, 4, 5, 9, and 10 to form six tandem repeat event regions (Lcu02G_04000/04001, Lcu04G_ 11429/11430, Lcu04G_13190/13191, Lcu05G_16661/16662, Lcu09G_24736/24740, and Lcu10G_26179/26191) ( Figure S1A). Except for the above tandem repeat events, we identified 23 segmental repeat events by BLAST and MCScanX methods, which contained 39 WRKY genes ( Figure S1B). These findings imply that some LcWRKY genes may have Figure 1. Gene structure and motif analysis of LcWRKYs. The blue boxes, green boxes, and thin black lines represent the UTR, CDS, and introns, respectively. MEME analysis revealed conserved motifs of LcWRKY proteins. Colored boxes on the right denote 30 motifs.

Gene Duplication Analysis
A tandem repeat event is described as two or more genes located within 200 kb of a chromosomal region [32,33]. Our LcWRKY genes are clustered on L. cubeba chromosomes 2, 4, 5, 9, and 10 to form six tandem repeat event regions (Lcu02G_04000/04001, Lcu04G_ 11429/11430, Lcu04G_13190/13191, Lcu05G_16661/16662, Lcu09G_24736/24740, and Lcu10G_26179/26191) ( Figure S1A). Except for the above tandem repeat events, we identified 23 segmental repeat events by BLAST and MCScanX methods, which contained 39 WRKY genes ( Figure S1B). These findings imply that some LcWRKY genes may have developed by gene duplication and that segmental duplication events are the primary determinants of LcWRKY evolution.

Phylogenetic Analysis of WRKY Phylogeny in L. cubeba
To further investigate the phylogenetic status of LcWRKYs, we constructed a phylogenetic tree by multiple alignment for the protein sequences of LcWRKYs and AtWRKYs. We can see that LcWRKY proteins are clearly divided into three major groups. Group I featured 17 proteins, while Group III featured 7 proteins; these proteins may be involved in terpene synthesis (Table S2). The largest group was Group II, which was further di-vided into five subgroups, five belonging to II-a, eight to II-b, ten to II-c, nine to II-d, and eight to II-e ( Figure 2). developed by gene duplication and that segmental duplication events are the primary determinants of LcWRKY evolution.

Phylogenetic Analysis of WRKY Phylogeny in L. cubeba
To further investigate the phylogenetic status of LcWRKYs, we constructed a phylogenetic tree by multiple alignment for the protein sequences of LcWRKYs and AtWRKYs. We can see that LcWRKY proteins are clearly divided into three major groups. Group I featured 17 proteins, while Group III featured 7 proteins; these proteins may be involved in terpene synthesis (Table S2). The largest group was Group II, which was further divided into five subgroups, five belonging to II-a, eight to II-b, ten to II-c, nine to II-d, and eight to II-e ( Figure 2).  Combined with the published transcriptomic data (PRJNA763042, https://doi.org/ 10.1016/j.indcrop.2021.114423, accessed on 15 September 2022), we also analyzed the expression profiles of 64 WRKY genes at different developmental stages of the fruit (Figure 2). We found that these genes were specifically expressed in different developmental stages of the fruit and that the expression of genes was similar for genes clustered in the same group. Group I showed higher levels of expression during the later stages of fruit development, while Group III showed higher expression levels during both early and late fruit development. Some genes in Group II-d showed higher expression levels throughout the period of fruit development.

LcWRKY17, a Group I Protein, Was Co-Expressed with Genes Responsible for Terpene Synthesis
In a previous study, Group I WRKY genes were found to play an essential role in the control of terpenoid synthesis [34]. Weighted correlation network analysis (WGCNA) analysis showed that the 7 nodes genes of the terpenoid synthesis pathway showed extremely high connections with the other 69 edge genes. LcWRKY17 was considered as the hub gene because of its association with more terpene-synthesis-related genes ( Figure S2). Predicting the promoter cis-acting elements of terpene synthases (TPS) in L. cubeba, we found that these TPS promoters all contain WRKY binding element W-boxes ranging from 1 to 3 in number ( Figure 3A). The transcriptome data were combined to further construct a clustering heat map of the expression of hub genes and the TPS42 gene, a key enzyme for terpene synthesis. We could clearly see that LcWRKY17 clustered with TPS42 in the same family and that the expression profiles were correlated ( Figure 3B). Analysis suggested that LcWRKY17 may regulate the synthesis of terpenoids in L. cubeba.  . UBC gene is the internal reference, the expression value of the first sample DAF30 is set to 1, and the data are represented as the mean. The error bars represent the standard deviation of three biological repeats. Based on Student's t-test, asterisks indicate statistically significant differences from sample 1.

Subcellular Localization of LcWRKY17
TFs are usually expressed and exert functionality in the nucleus. The full-length CDS of the cloned LcWRKY17 gene was inserted into the pNC vector following removal of the stop codon (Table S3), thus enabling these genes to fuse with GFP protein driven by the 35S promoter when expressed in N. benthamiana leaves ( Figure 4A). The GFP protein in N. benthamiana (without gene insertion) was expressed throughout the entire cell; however, GFP protein fused with LcWRKY17 proteins was only expressed in the nucleus, thus demonstrating that LcWRKY17 genes are expressed and exert functionality in the nucleus ( Figure 4B,C).

Subcellular Localization of LcWRKY17
TFs are usually expressed and exert functionality in the nucleus. The full-length CDS of the cloned LcWRKY17 gene was inserted into the pNC vector following removal of the stop codon (Table S3), thus enabling these genes to fuse with GFP protein driven by the 35S promoter when expressed in N. benthamiana leaves ( Figure 4A). The GFP protein in N. benthamiana (without gene insertion) was expressed throughout the entire cell; however, GFP protein fused with LcWRKY17 proteins was only expressed in the nucleus, thus demonstrating that LcWRKY17 genes are expressed and exert functionality in the nucleus ( Figure 4B,C).

Quantitative Real-Time PCR (qRT-PCR)
The expression pattern of a gene is closely linked to its biological function. To further investigate the related functions of LcWRKY17 and TPS42, we sampled L. cubeba fruits during different developmental periods and analyzed their expression profiles using qRT-PCR ( Figure 3C). The results showed that LcWRKY17 and TPS42 have consistent expression profiles and showed peak expression levels at DAF60 and DAF120, thus corresponding to the critical period of essential oil synthesis [35,36]. These findings show that LcWRKY17 may be crucial in the synthesis of fruit terpenoids.

Transient Overexpression of LcWRKY17 in L. cubeba
In order to examine the function of LcWRKY17, we adopted a simple and efficient transient expression approach because the stable transformation of L. cubeba is complex and time-consuming. The empty vector (pNC-Cam2304-35S) and the recombinant vector containing LcWRKY17 (pNC-Cam2304-35S-LcWRKY17) were infiltrated by manual

Quantitative Real-Time PCR (qRT-PCR)
The expression pattern of a gene is closely linked to its biological function. To further investigate the related functions of LcWRKY17 and TPS42, we sampled L. cubeba fruits during different developmental periods and analyzed their expression profiles using qRT-PCR ( Figure 3C). The results showed that LcWRKY17 and TPS42 have consistent expression profiles and showed peak expression levels at DAF60 and DAF120, thus corresponding to the critical period of essential oil synthesis [35,36]. These findings show that LcWRKY17 may be crucial in the synthesis of fruit terpenoids.

Transient Overexpression of LcWRKY17 in L. cubeba
In order to examine the function of LcWRKY17, we adopted a simple and efficient transient expression approach because the stable transformation of L. cubeba is complex and time-consuming. The empty vector (pNC-Cam2304-35S) and the recombinant vector containing LcWRKY17 (pNC-Cam2304-35S-LcWRKY17) were infiltrated by manual evacuation into the sterile seedling leaves of L. cubeba and analyzed for volatility after 72 h of growth [37]. The expression of LcWRKY17 in L. cubeba leaves increased 4.3-fold and LcTPS42 expression increased 4.1-fold compared with the control following transient expression ( Figure 5A,B). The monoterpene content in the leaves of L. cubeba increased significantly when compared with the controls ( Figure 5C). Transient overexpression of LcWRKY17 enhanced the accumulation of major monoterpenes in L. cubeba leaves, such as α-pinene, camphene, β-myrcene, α-phellandrene, linalool, citronellal, neral, and geranial ( Figure 5D). It has been shown that LcTPS42 is highly expressed in the mid to late stages of L. cubeba fruit development and catalyzes the biosynthesis of the major monoterpene components geranium and linalool [38]. In this study, LcWRKY17 transient expression was followed by a significant increase in LcTPS42 expression and an increase in the content of major monoterpene components catalyzed by LcTPS42, indicating that WRKY transcription factors may catalyze the production of major monoterpene components through activation of LcTPS42. evacuation into the sterile seedling leaves of L. cubeba and analyzed for volatility after 72 h of growth [37]. The expression of LcWRKY17 in L. cubeba leaves increased 4.3-fold and LcTPS42 expression increased 4.1-fold compared with the control following transient expression ( Figure 5A, B). The monoterpene content in the leaves of L. cubeba increased significantly when compared with the controls ( Figure 5C). Transient overexpression of LcWRKY17 enhanced the accumulation of major monoterpenes in L. cubeba leaves, such as α-pinene, camphene, β-myrcene, α-phellandrene, linalool, citronellal, neral, and geranial ( Figure 5D). It has been shown that LcTPS42 is highly expressed in the mid to late stages of L. cubeba fruit development and catalyzes the biosynthesis of the major monoterpene components geranium and linalool [38]. In this study, LcWRKY17 transient expression was followed by a significant increase in LcTPS42 expression and an increase in the content of major monoterpene components catalyzed by LcTPS42, indicating that WRKY transcription factors may catalyze the production of major monoterpene components through activation of LcTPS42.  replicates. Student's t-test was used to assess confidence levels (CK = blank control) (**, p < 0.01).

LcWRKY17 Functions by Binding to the LcTPS42 Promoter Binding Element
LcWRKY17 is a WRKY TF member, which bound to the W-box element in the promoter of terpene synthase genes to regulate plant secondary metabolic synthesis. Using dual-LUC assay, we determined the regulatory effect of LcWRKY17 transcription factor on the LcTPS42 promoter. When compared with the control null, the dual-LUC activity experiment showed that the WRKY transcription factor LcWRKY17 significantly activated the LcTPS42 promoter ( Figure 6A).

LcWRKY17 Functions by Binding to the LcTPS42 Promoter Binding Element
LcWRKY17 is a WRKY TF member, which bound to the W-box element in the promoter of terpene synthase genes to regulate plant secondary metabolic synthesis. Using dual-LUC assay, we determined the regulatory effect of LcWRKY17 transcription factor on the LcTPS42 promoter. When compared with the control null, the dual-LUC activity experiment showed that the WRKY transcription factor LcWRKY17 significantly activated the LcTPS42 promoter ( Figure 6A). We used the Y1H assay to advance our understanding of the LcWRKY17 transcription factor's regulatory mechanism on the LcTPS42 promoter. Y1H assay revealed that cotransformation of three tandem repeats of the W-box element and pGADT7-LcWRKY17 vectors successfully activated AbA gene expression and enabled yeast to grow on Aureobasidin A (AbA) antibiotic plates, while mutant W-box (m W-box) element and pGADT7-LcWRKY17 co-transformation could not grow on plates containing AbA antibiotics ( Figure 6B). The results suggested that LcWRKY17 binds to the W-box motif of the LcTPS42 promoter. Thus, LcWRKY17 regulates the synthesis of monoterpenoids by binding to the W-box elements of the LcTPS42 promoter.

Discussion
In our research, we discovered 64 WRKY genes in L. cubeba and identified a significantly higher monoterpene content in L. cubeba when we overexpressed LcWRKY17, a member of Group I. Our research shows that WRKY TFs are crucial for L. cubeba's terpene production.
As more and more whole genomes are released, identifying and analyzing TFs at the whole genome level has become an important focus of genomics research. As the most essential TF in plants, WRKY has been identified in tobacco, rice, cucumber, grapevine, and poplar [13,18,[39][40][41]. However, no similar research has been conducted in L. cubeba and the specific function of the LcWRKY gene remains unclear [28]. After the L. cubeba genome assembly and sequencing were finished, LcWRKYs can be identified and analyzed more efficiently. In this research, we identified and analyzed the LcWRKYs for the first time. The first step in studying the functionality of gene families is to classify them. A classification system for the WRKY gene family was previously developed for Arabidopsis We used the Y1H assay to advance our understanding of the LcWRKY17 transcription factor's regulatory mechanism on the LcTPS42 promoter. Y1H assay revealed that cotransformation of three tandem repeats of the W-box element and pGADT7-LcWRKY17 vectors successfully activated AbA gene expression and enabled yeast to grow on Aureobasidin A (AbA) antibiotic plates, while mutant W-box (m W-box) element and pGADT7-LcWRKY17 co-transformation could not grow on plates containing AbA antibiotics ( Figure 6B). The results suggested that LcWRKY17 binds to the W-box motif of the LcTPS42 promoter. Thus, LcWRKY17 regulates the synthesis of monoterpenoids by binding to the W-box elements of the LcTPS42 promoter.

Discussion
In our research, we discovered 64 WRKY genes in L. cubeba and identified a significantly higher monoterpene content in L. cubeba when we overexpressed LcWRKY17, a member of Group I. Our research shows that WRKY TFs are crucial for L. cubeba's terpene production.
As more and more whole genomes are released, identifying and analyzing TFs at the whole genome level has become an important focus of genomics research. As the most essential TF in plants, WRKY has been identified in tobacco, rice, cucumber, grapevine, and poplar [13,18,[39][40][41]. However, no similar research has been conducted in L. cubeba and the specific function of the LcWRKY gene remains unclear [28]. After the L. cubeba genome assembly and sequencing were finished, LcWRKYs can be identified and analyzed more efficiently. In this research, we identified and analyzed the LcWRKYs for the first time. The first step in studying the functionality of gene families is to classify them. A classification system for the WRKY gene family was previously developed for Arabidopsis that is now widely accepted. WRKY genes in plants are divided into three main groups, and members of the Group II can be further separated into five subfamilies because they are not monophyletic [15,16]. Meanwhile, the variety of zinc finger structures can be linked to the diversity of WRKYs [42]. We discovered 64 LcWRKY genes in the L. cubeba genome database. The proteins of LcWRKYs and AtWRKYs were then used to create a phylogenetic tree. Group II was the one containing the most WRKY genes, accounting for 62.5% of the total. This phenomenon was similarly demonstrated in Arabidopsis, Brassica napus, and Brassica rapa [43].
Tandem duplication and segmental duplication produce distinct evolutionary processes for duplicated genes [44]. It is generally considered that duplicated genes located within a 200 Kb region on the same chromosome arise from tandem repeats, while duplicated genes on different chromosomes arise from segmental repeats [45,46]. We identified 12 tandem duplication genes and 23 segmental duplication events in L. cubeba, demonstrating the importance of segmental duplication in the expansion of the LcWRKYs. To adapt to different development environments, these duplicated LcWRKY genes most likely created novel gene functions. Repetitive genes are crucial for plants to adapt to challenging and shifting surroundings [44,47]. Repeat genes may undergo sub-functionalization, new functionalization, and loss during continuous evolution [48]. It has been previously demonstrated that two genes on the same chromosome that are close to one another, particularly two tandem repeat genes, are more likely to be co-regulated.
Expression patterns are closely related to gene function [49]. It has been shown that WRKY genes play a crucial role in controlling secondary metabolism, plant growth and development, and plant responses to various abiotic stressors [50][51][52]. Analysis of the expression pattern of the LcWRKY17 gene during different periods of fruit development in L. cubeba showed that LcWRKY17 was strongly expressed during the fruit's middle and late phases of growth. Furthermore, the expression profiles of LcWRKY17 and LcTPS42, a key enzyme for monoterpene synthesis, were consistent, both being specifically highly expressed in the middle and late stages of fruit development. Combined with these results, we believe that LcWRKY17 is essential for the terpene synthesis pathway.
WRKY TFs are crucial in the synthesis of plant secondary metabolites. Terpenoids are a significant part of plant secondary metabolites. Strategies for the synthesis of terpenoids in plants include the mevalonate (MVA) and mevalonate-independent (MEP) pathways [53,54]. These are controlled by a number of structural genes in the biosynthesis route, and TFs are responsible for secondary regulation [55,56]. The majority of artemisinin synthesis genes have been demonstrated to express themselves more frequently when AaWRKY1 is present, suggesting that the AaWRKY1 TF regulates the production of artemisinin [57,58]. AaWRKY9 regulates artemisinin biosynthesis in Artemisia annua via the mediation of light and jasmonic acid [34]. In S. lycopersicum, SlWRKY71 can regulate the expression of terpene synthase [59]. SlWRKY35 activates the MEP pathway in tomato fruit, thereby promoting carotenoid biosynthesis [35]. In this study, the transient overexpression of LcWRKY17 in L. cubeba significantly promoted monoterpenes in L. cubeba. Y1H and dual-LUC experiments further showed that LcWRKY7 directly binds to the W-box of the LcTPS42 promoter and activates its transcription, thereby promoting monoterpene synthesis [38].

Identification of WRKY Genes in L. cubeba
The Pfam number of the WRKY gene family (PF03106) was obtained through literature and the hidden Markov model (HMM) files of all gene families were downloaded from the Pfam Protein Family Database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 22 June 2022). Combined with the genomic data of L. cubeba obtained from our group's previous research [38], all WRKY gene families in L. cubeba were screened and identified using the Simple HMM Search plug-in in TBtools (https://github.com/CJ-Chen/ TBtools, accessed on 22 June 2022). Genes with an e-value < 10 −10 , along with duplicated genes, were removed to provide a final suite of target genes containing WRKY structural domains in L. cubeba.

Chromosomal Location Analysis
The WRKY gene's location on the chromosome was determined using the L. cubeba genome's chromosomal annotation file, and its distribution on the chromosome was mapped using MapGene2Chromosome V2 (http://mg2c.iask.in/mg2c_v2.0/, accessed on 24 June 2022).

Motif Analysis and Structural Gene Analysis
Protein sequences and CDS sequences of the WRKY genes were first retrieved by searching genomic protein files and CDS files. Protein sequences of the genes in the WRKY gene family were analyzed using MEME (https://memesuite.org/meme/tools/meme, accessed on 8 July 2022). The WRKY gene family's structures were examined using the Gene Structure View plug-in of the TBtools (https://github.com/CJ-Chen/TBtools, accessed on 8 July 2022), with a threshold of 30 motifs.

Gene Duplication Analysis
Gene duplication occurrences were examined using MCScanX [60]. According to geographic information from the L. cubeba genomic database, all LcWRKY genes were assigned to the L. cubeba chromosome. The results were visualized using the Advanced Circos plugin in TBtools (https://github.com/CJ-Chen/TBtools, accessed on 24 July 2022).

Evolutionary Analysis of the WRKY Gene Family
MEGA7.0 was used to perform protein sequence alignments of the LcWRKY and AtWRKY genes, and the alignment files were produced in phylip3.0 format [61]. The maximum likelihood (ML) approach of RAxML was used to build phylogenetic trees on the internet site CIPRES (https://www.phylo.org/portal2/login!input.action, accessed on 15 July 2022).

Expression Analysis
The expression patterns of the fruit development of L. cubeba were analyzed based on published RNA-seq data (PRJNA763042) by the subject group (https://doi.org/10.1016/ j.indcrop.2021.114423, accessed on 15 September 2022) [37]. Using the FPKM (fragments per kilobase million) value of 38,988 differentially expressed transcription factors (DETs) at the 12 stages of fruit development of L. cubeba, we employed the WGCNA approach of the R package with a weighted cut-off value >0.50 [62,63]. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to obtain keywords and pathways (http://www.kegg.jp/blastkoala/, accessed on 15 September 2022). We selected genes related to terpene synthesis and visualized these with Cytoscape.

Cis-Acting Element Analysis
TBtools program was used to extract the promoter sequences of L. cubeba WRKY family members, detected and identified using the online Plant CARE website (http:// bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 22 September 2022) for cis-element detection. The outcomes were then displayed using tBtools.

LcWRKY17 Gene Cloning
LcWRKY17 was amplified using cDNA from different tissues of L. cubeba. Primer 3 (https://bioinfo.ut.ee/primer3-0.4.0/, accessed on 15 September 2022) was responsible for creating the cloning primers. High-fidelity enzymes were selected for amplification (MCLAB, Beijing, China); the amplification system and conditions were in accordance with the manufacturer's instructions. A Gel Extraction Kit (OMEGA, Beijing, China) was used to purify the PCR products and ligate them to the vector for further sequencing (Supplementary Table S3).

Subcellular Localization Analysis
Subcellular localization experiments were utilized to investigate the sites of gene expression and action. The correctly amplified and sequenced complete gene (following removal of the stop codon) was introduced into the pNC-Green-SubC plant expression vector and transferred into Agrobacterium GV3101. Agrobacterium containing the target vector was then transferred into leaves of N. benthamiana cultivated for approximately four weeks. The results were observed by Echo Revolve fluorescence microscopy (Revolve FL) after two days, with a red nuclear localization marker as a positive control.

Quantitative Real-Time PCR (qRT-PCR)
RN38 EASY Spin Plus Plant kit (Aidlab, Beijing, China) was used to extract total RNA. An ABI PRISM 7500 instrument and a TB Green ® Premix Ex TaqTM II kit (TaKaRa, Tokyo, Japan) were used to perform qRT-PCR. Using the UBC gene as an internal control [38]. Primer Premier 3.0 was used to create primers for qRT-PCR reactions; the primer sequences are provided in Table S3. The relative expression levels were calculated by the 2 −∆∆CT approach [37].

Transient Overexpression of LcWRKY17 in L. cubeba
To investigate the role of LcWRKY17 overexpression on terpenoid synthesis in L. cubeba, we performed LcWRKY17 overexpression experiments on sterile L. cubeba seedlings [37]. The empty vector (pNC-Cam2304-35S) and the recombinant vector containing LcWRKY17 (pNC-Cam2304-35S-LcWRKY17) were each transformed into Agrobacterium LBA4404 strains and infiltrated by manual evacuation into the leaves of seedlings of L. cubeba with similar growth rates. Detection after culture at 26 • C for 50-72 h.

Dual-Luciferase and Yeast One-Hybrid (Y1H) Assays
Dual-LUC assay was used to detect the regulatory effect of the LcWRKY17 transcription factor on the LcTPS42 promoter. The LcTPS42 promoter (about 2000 bp upstream of the start codon) was constructed into the pGreenII0800-LUC vector and the LcWRKY17 gene was constructed into the pGreenII62-SK vector. Agrobacterium strain GV3101 (with pSoup) was individually transformed with the recombinant vector. Tobacco leaves were infested with a mixture of Agrobacterium cells. Using a dual LUC test kit (Promega, Madison, WI, USA), the LUC and REN enzyme activities were assessed after 2-3 days of incubation in the light incubator.
The LcWRKY17 transcription factor's ability to bind to the W-box elements of the LcTPS42 promoter was examined using the Y1H assay. The LcWRKY17 transcription factor was constructed into the pGADT7 vector. Three tandem repeats of the LcTPS42 promoter binding element W-box were constructed into the pAbAi vector with BstBI enzyme digestion and gum recovery. The pGADT7 vector containing the target fragment and the pAbAi vector were simultaneously transformed into yeast for validation. The transformed yeast cells were grown on SD/-Leu/-Ura medium with and without Aureobasidin A (AbA), then the plates were kept at 30 • C for 2-3 days.

Conclusions
In our study, we performed genome-wide analysis of the L. cubeba WRKY gene family with a particular focus on their regulatory role on secondary metabolism. L. cubeba possessed 64 WRKY genes, which are distributed on 12 different chromosomes. Evolutionary analysis divided these 64 WRKY genes into three groups, and Group II was further divided into five subgroups; this classification was clearly supported by their gene structures. Furthermore, transient overexpression experiments showed that LcWRKY17 specifically regulated the synthesis of monoterpenoids in L. cubeba. Dual-LUC and Y1H analyses further revealed that LcWRKY17 promotes monoterpene synthesis by binding to the W-box elements of the LcTPS42 promoter and activating its transcription. Collectively, our research offers a theoretical foundation for continued in-depth investigation into the function of LcWRKYs and their regulatory role on plant terpenoids.