Molecular Cloning and Functional Characterization of CpMYC2 and CpBHLH13 Transcription Factors from Wintersweet (Chimonanthus praecox L.)

Wintersweet (Chimonanthus praecox L.) is an ornamental and economically significant shrub known for its unique flowering characteristics, especially the emission of abundant floral volatile organic compounds. Thus, an understanding of the molecular mechanism of the production of these compounds is necessary to create new breeds with high volatile production. In this study, two bHLH transcription factors (CpMYC2 and CpbHLH13) of Wintersweet H29 were functionally characterized to illustrate their possible role in the production of volatile compounds. The qRT-PCR results showed that the expression of CpMYC2 and CpbHLH13 increased from the flower budding to full bloom stage, indicating that these two genes may play an essential role in blooming and aroma production in wintersweet. Gas chromatography-mass spectroscopy (GC-MS) analysis revealed that the overexpression of CpMYC2 in arabidopsis (Arabidopsis thaliana) AtMYC2-2 mutant (Salk_083483) and tobacco (Nicotiana tabaccum) genotype Petit Havana SR1 significantly increased floral volatile monoterpene, especially linalool, while the overexpression of CpbHLH13 in Arabidopsis thaliana ecotype Columbia-0 (Col-0) and tobacco genotype SR1 increased floral sesquiterpene β-caryophyllene production in both types of transgenic plants respectively. High expression of terpene synthase (TPS) genes in transgenic A. thaliana along with high expression of CpMYC2 and CpbHLH13 in transgenic plants was also observed. The application of a combination of methyl jasmonic acid (MeJA) and gibberellic acid (GA3) showed an increment in linalool production in CpMYC2-overexpressing arabidopsis plants, and the high transcript level of TPS genes also suggested the involvement of CpMYC2 in the jasmonic acid (JA) signaling pathway. These results indicate that both the CpMYC2 and CpbHLH13 transcription factors of wintersweet are possibly involved in the positive regulation and biosynthesis of monoterpene (linalool) and sesquiterpene (β-caryophyllene) in transgenic plants. This study also indicates the potential application of wintersweet as a valuable genomic material for the genetic modification of floral scent in other flowering plants that produce less volatile compounds.


Introduction
Plants produce a flower aroma that plays a significant role in their economic value and aesthetic properties [1]. Each plant has its own unique floral aroma, consisting of volatile organic compounds, which are mainly derivatives of fatty acids, terpenoids, and phenylpropanoids or benzoids. Thus, Plants 2020, 9, 785 3 of 21 wintersweet. Here, we studied two bHLH transcription factors (CpMYC2 and CpbHLH13), selected from a transcriptomic library developed from the fully open flower developmental stage of the Wintersweet H29 (Huazhong 29) genotype. The main objectives of this study were molecular cloning and investigating the function of CpMYC2 and CpbHLH13. The effect of these bHLH genes on the production of plant floral volatiles was also evaluated.

Characterization of CpMYC2 and bHLH13
Using the Illumina RNA-Seq technique, a transcriptomic library (NCBI Sequence Read Archive (SRA) accession number PRJNA492170) was constructed from the full open flower stage of the Wintersweet H29 cultivar (Figure 1), and out of 16 identified members of the bHLH family, the desired sequence of two transcription factors CpMYC2 (2858 bp) and bHLH13 (2425 bp) was selected for further study [45]. Based on the NCBI ORF finder, the open reading frame of 1884 bp and 1779 bp nucleotide, encoding 628 and 593 amino acids, were observed in CpMYC2 and CpbHLH13, respectively. The theoretical isoelectric point (pI) and the molecular weight (Mw) of the CpMYC2 and CpbHLH13 proteins were Pi/Mw: 5.65/68707. 29 and Pi/Mw: 6.43/65502.53, respectively. Multiple sequence alignment of wintersweet CpMYC2 and CpbHLH13 proteins with their homologous protein sequences in other species, especially A. thaliana, Nicotiana tabacum, V. vinifera, Cinnamomum micranthum, Solanum lycopersicum, and Zea mays revealed the presence of a basic helix-loop-helix DNA-binding domain (446-493 amino acids for CpMYC2 and 429-476 amino acids for CpbHLH13) with an MYC-N terminal, which is a common feature of MYC family proteins (Figures 2 and 3). The three-dimensional protein structure of CpMYC2 and CpbHLH13 is shown in Supplementary Figures S1 and S2. Phylogenetic analysis demonstrated that CpMYC2 was closely related to the transcription factor Arabidopsis MYC2 (AT1G32640), whereas CpbHLH13 was most homologous to AtbHLH13 (AT1G01260), also known as JASMONATE-ASSOCIATED MYC2-like2 (JAM2) (Supplementary Figures  S3 and S4). Further phylogenetic analysis with different species revealed that CpMYC2 had a close relationship with the predicted protein of Cinnamomum micranthum (CmMYC2) with 65.13% identity and A. thaliana (AtMYC2) with 54%, while CpbHLH13 was mostly homologous to C. micranthum (CmbHLH13) with 69.77% identity followed by Nelumbo nucifera (NnMYC2) with 62.46% identity and A. thaliana AtbHLH13 with 42% identity, although they were not in the same clade. Phylogenetic analyses revealed that CpbHLH13 had a close relationship with the A. thaliana AtbHLH13/JAM2 (At1g01260) and AtbHLH17/JAM1 (At2g46510) transcription factors (See Supplementary Figures S5 and S6). A summary of the homology of wintersweet CpMYC2 and CpbHLH13 with identity% and functions of the genes from various plants is described in Tables 1 and 2. Plants 2020, 9, x FOR PEER REVIEW 3 of 25 plants is still limited [44]. Thus, the identification and characterization of functional genes through transcriptome and genome sequencing is vital to uncover the molecular mechanism behind flower fragrance production in wintersweet. Here, we studied two bHLH transcription factors (CpMYC2 and CpbHLH13), selected from a transcriptomic library developed from the fully open flower developmental stage of the Wintersweet H29 (Huazhong 29) genotype. The main objectives of this study were molecular cloning and investigating the function of CpMYC2 and CpbHLH13. The effect of these bHLH genes on the production of plant floral volatiles was also evaluated.

Characterization of CpMYC2 and bHLH13
Using the Illumina RNA-Seq technique, a transcriptomic library (NCBI Sequence Read Archive (SRA) accession number PRJNA492170) was constructed from the full open flower stage of the Wintersweet H29 cultivar (Figure 1), and out of 16 identified members of the bHLH family, the desired sequence of two transcription factors CpMYC2 (2858 bp) and bHLH13 (2425 bp) was selected for further study [45]. Based on the NCBI ORF finder, the open reading frame of 1884 bp and 1779 bp nucleotide, encoding 628 and 593 amino acids, were observed in CpMYC2 and CpbHLH13, respectively. The theoretical isoelectric point (pI) and the molecular weight (Mw) of the CpMYC2 and CpbHLH13 proteins were Pi/Mw: 5.65/68707. 29 and Pi/Mw: 6.43/65502.53, respectively. Multiple sequence alignment of wintersweet CpMYC2 and CpbHLH13 proteins with their homologous protein sequences in other species, especially A. thaliana, Nicotiana tabacum, V. vinifera, Cinnamomum micranthum, Solanum lycopersicum, and Zea mays revealed the presence of a basic helix-loop-helix DNA-binding domain (446-493 amino acids for CpMYC2 and 429-476 amino acids for CpbHLH13) with an MYC-N terminal, which is a common feature of MYC family proteins (Figures 2 and 3). The three-dimensional protein structure of CpMYC2 and CpbHLH13 is shown in Supplementary Figures S1 and S2. Phylogenetic analysis demonstrated that CpMYC2 was closely related to the transcription factor Arabidopsis MYC2 (AT1G32640), whereas CpbHLH13 was most homologous to AtbHLH13 (AT1G01260), also known as JASMONATE-ASSOCIATED MYC2-like2 (JAM2) (Supplementary Figures S3 and S4). Further phylogenetic analysis with different species revealed that CpMYC2 had a close relationship with the predicted protein of Cinnamomum micranthum (CmMYC2) with 65.13% identity and A. thaliana (AtMYC2) with 54%, while CpbHLH13 was mostly homologous to C. micranthum (CmbHLH13) with 69.77% identity followed by Nelumbo nucifera (NnMYC2) with 62.46% identity and A. thaliana AtbHLH13 with 42% identity, although they were not in the same clade. Phylogenetic analyses revealed that CpbHLH13 had a close relationship with the A. thaliana AtbHLH13/JAM2 (At1g01260) and AtbHLH17/JAM1 (At2g46510) transcription factors (See Supplementary Figures S5 and S6). A summary of the homology of wintersweet CpMYC2 and CpbHLH13 with identity% and functions of the genes from various plants is described in Tables 1 and  2.

CpMYC2 and CpbHLH13 Expression in Wintersweet
To determine whether CpMYC2 and CpbHLH13 expression was correlated with VOC biosynthesis during wintersweet inflorescence, the expression profile of  (Figure 4a,b). We also measured the expression level of both the genes in different tissues of wintersweet and found that the expression level of these genes was highest in flowers, followed by leaves and stems, and lowest in fruits (Figure 4c,d). This indicates their importance and possible involvement in producing VOCs during blooming, also indicating that these two genes may play an important role in blooming and aroma production.  CpbHLH13 expression in wintersweet. The different letters on the bars show significance between the treatments by using least significant difference (LSD) at p < 0.05.

Expression Analysis of the Terpene Synthase Genes in Wild-Type and Transgenic Plants
Real-time PCR (qRT-PCR) was used to measure the expression of terpene synthase genes in wild-type and transgenic plants. The results indicate that the expression of the linalool-producing gene At1g61680 was about one and a half times higher in the plants transformed by CpMYC2 than the other plants, while wild-type Col-0 plants, the mutant Salk_083483, and mutant plants transformed by the empty vector pCAMBIA2300S (2300S-Sk83) also showed lower expression of the At3g25810 gene as compared to 35S::CpMYC2 plants (Figure 5a). Similarly, the expression of the βcaryophyllene-producing genes At5g23960 and At5g44630 was almost threefold higher in the plants transformed by the CpbHLH13 gene than the wild-type arabidopsis plants (Col-0) and the plants transformed by the vector pCAMBIA2300S ( Figure 5b). The expression of the CpMYC2 and CpbHLH13 genes in transgenic tobacco and arabidopsis plants was also measured (Supplementary

Expression Analysis of the Terpene Synthase Genes in Wild-Type and Transgenic Plants
Real-time PCR (qRT-PCR) was used to measure the expression of terpene synthase genes in wild-type and transgenic plants. The results indicate that the expression of the linalool-producing gene At1g61680 was about one and a half times higher in the plants transformed by CpMYC2 than the other plants, while wild-type Col-0 plants, the mutant Salk_083483, and mutant plants transformed by the empty vector pCAMBIA2300S (2300S-Sk83) also showed lower expression of the At3g25810 gene as compared to 35S::CpMYC2 plants (Figure 5a). Similarly, the expression of the β-caryophyllene-producing genes At5g23960 and At5g44630 was almost threefold higher in the plants transformed by the CpbHLH13 gene than the wild-type arabidopsis plants (Col-0) and the plants transformed by the vector pCAMBIA2300S ( Figure 5b). The expression of the CpMYC2 and CpbHLH13 genes in transgenic tobacco and arabidopsis plants was also measured (Supplementary Figure S7) and the plants with almost identical expression were used for further experimentation.

Overexpression of CpMYC2 and CpbHLH13 Induces VOC Emission
The positively selected plants obtained from T3 generation were used to detect the terpenes from the inflorescence of transgenic arabidopsis and tobacco plants using GC-MS. We analyzed the emission of terpenes in arabidopsis and tobacco transgenic plants overexpressed with the CpMYC2 and CpbHLH13 gene. The results indicate that linalool was the most abundant monoterpene ( Figure  6a,b) and its emission was more than 1.5 and 2 times higher in transgenic arabidopsis and tobacco plants transformed by the CpMYC2 gene (35S::CpMYC2), respectively, compared to the wild type (Col-0 and SR1) and transgenic plants transformed by the vector pCAMBIA2300S (Figure 7a,b). Similarly, it was found that β-caryophyllene was the most abundant sesquiterpene produced in arabidopsis and tobacco plants transformed by the CpbHLH13 gene (Figure 6c

Overexpression of CpMYC2 and CpbHLH13 Induces VOC Emission
The positively selected plants obtained from T3 generation were used to detect the terpenes from the inflorescence of transgenic arabidopsis and tobacco plants using GC-MS. We analyzed the emission of terpenes in arabidopsis and tobacco transgenic plants overexpressed with the CpMYC2 and CpbHLH13 gene. The results indicate that linalool was the most abundant monoterpene (Figure 6a,b) and its emission was more than 1.5 and 2 times higher in transgenic arabidopsis and tobacco plants transformed by the CpMYC2 gene (35S::CpMYC2), respectively, compared to the wild type (Col-0 and SR1) and transgenic plants transformed by the vector pCAMBIA2300S (Figure 7a,b). Similarly, it was found that β-caryophyllene was the most abundant sesquiterpene produced in arabidopsis and tobacco plants transformed by the CpbHLH13 gene (Figure 6c

Application of MeJA and GA3 on Overexpressed CpMYC2 Plants
Following MeJA and gibberellic acid (GA3) treatment, terpenoids were detected in the inflorescence of transgenic arabidopsis plants using GC-MS. The results again indicate that the emission of linalool was more than 1.5 times higher in the plants transformed by the CpMYC2 gene (35S::CpMYC2), while the mutant plants Salk_083483, mutant plants transformed by the vector pCAMBIA2300S (2300S-Sk83), and the Col-0 wild-type plants showed a lower amount of linalool emission compared to the wild-type (35S::CpMYC2) plants (Figures 9 and 10). Further analysis using real-time PCR (qRT-PCR) indicated that the expression of the linalool-producing gene At1g61680 was about twofold higher in the plants transformed by CpMYC2 (35S::CpMYC2) than the other plants,

Application of MeJA and GA3 on Overexpressed CpMYC2 Plants
Following MeJA and gibberellic acid (GA3) treatment, terpenoids were detected in the inflorescence of transgenic arabidopsis plants using GC-MS. The results again indicate that the emission of linalool was more than 1.5 times higher in the plants transformed by the CpMYC2 gene (35S::CpMYC2), while the mutant plants Salk_083483, mutant plants transformed by the vector pCAMBIA2300S (2300S-Sk83), and the Col-0 wild-type plants showed a lower amount of linalool emission compared to the wild-type (35S::CpMYC2) plants (Figures 9 and 10). Further analysis using real-time PCR (qRT-PCR) indicated that the expression of the linalool-producing gene At1g61680 was about twofold higher in the plants transformed by CpMYC2 (35S::CpMYC2) than the other plants,  while the wild-type Col-0, mutant plants Salk_083483, and mutant plants transformed by the vector pCAMBIA2300S (2300S-Sk83) showed a lower expression of the At3g25810 gene as compared to 35S::CpMYC2 plants ( Figure 11).

Overexpression of CpbHLH13 Reduces Flower Pigmentation in Tobacco Plants
A few of the flowering transformed lines of CpbHLH13 also showed phenotypic changes in petal pigmentation. These transgenic plants showed a reduction in the pigmentation of flowers ( Figure  12a). Anthocyanin contents from the inflorescence of these tobacco plants were measured by a spectrophotometer which showed a clear reduction in the anthocyanin concentration in 35S::CpbHLH13 plants compared with those of the wild type and those transformed by the empty vector pCAMBIA2300S (2300S-SR1) (Figures12b,c). By measuring the anthocyanin contents in CpbHLH13 gene-transferred transgenic tobacco (35S::CpbHLH13) plants, we confirmed the reduction of anthocyanin contents in their inflorescence.

Discussion
Plants can store and release a wide range of volatile compounds through their specialized organs and tissues, which play a significant role in atmospheric composition [46]. These volatile compounds are also important for the formation of the characteristic floral scent in different plants which improves the aesthetic properties as well as the economic value of the plant [15]. However, the regulatory mechanisms behind the formation of the floral aroma in ornamental plants are poorly characterized. To date, no bHLH transcription factor has been identified and verified by its function in the ornamental and economically significant shrub "wintersweet". A few attempts have been made to illustrate the transcriptomic analysis of these genes, but the functional characterization remains underestimated. This might be due to the lack of genetic resources as well as the difficulties in the transformation of the genes in wintersweet [9,47]. The present study was designed to decipher the molecular mechanism behind flower fragrance production in wintersweet. The motives behind the study were to identify and find out the role and function of the bHLH transcription factors CpMYC2 and CpbHLH13 in wintersweet.
Phylogenetic analysis demonstrates that CpMYC2 is closely related to Arabidopsis MYC2 (AT1G32640), whereas CpbHLH13 is most homologous to AtbHLH13 (AT1G01260), also known as JASMONATE-ASSOCIATED MYC2-like2 (JAM2). MYC2 was previously reported to promote and activate the sesquiterpene synthase genes TPS21 and TPS11 through interacting with the DELLA proteins (MYC2 interacts with DELLA proteins and regulates the expression of the sesquiterpene synthase gene). It has also been reported that MYC2 is directly involved in the induction of JA and GA in sesquiterpene biosynthesis [48] and induces monoterpene synthase in arabidopsis, which suggests that MYC2 endorses the biosynthesis of a wider range of terpenes [49], whereas JAM2 (AtbHLH13) is involved in the negative regulation of jasmonate signaling in plants [50]. They have also been reported as novel factors regulating various metabolic pathways in arabidopsis in JA signaling [51]. It has been reported that MYC2 is differentially regulated and induced during the flower opening of wintersweet, especially at the fully-open flower stage [37]. In the present study, high expression of both the genes was found at the fully-open flower stage compared to the other flower developmental stages along with the highest expression in flowers among the other developing tissues of the plant (leaves, stems, and fruits). This indicates the possible role of these genes in volatile production during flowering. MYC2 has been identified to enhance the production of volatile compounds, especially monoterpenes and sesquiterpenes, in plants [20,52]. The expression of the linalool-and β-caryophyllene-producing genes increases in plants transformed by CpMYC2 and CpbHLH13, respectively. This illustrates the role of the transcription factors CpMYC2 and CpBHLH13 in the production of monoterpenes and sesquiterpenes in wintersweet plants.
Our findings also indicate that the overexpression of CpMYC2 and CpbHLH13 in model plants shows an increase in the production of monoterpene, i.e., linalool as well as the sesquiterpene (E)-β-caryophyllene, respectively. It is possible that the significant increment in compounds could be provoked because it is a MYC2 transcription factor and part of the bHLH family, which is involved in the regulation of many regulatory functions of the plant and also involved in plant growth and the mechanism of flower development in plants [22,30,53,54]. The results also show that CpMYC2 possibly increases the production of linalool by inducing the expression of the linalool-and limonene-producing genes At1g61680 and At3g25810 in arabidopsis. Linalool itself is one of the main produced floral volatiles, and the sequestration of linalool derivatives and the derivatives of the isoteric monoterpene alcohol geraniol were experimentally proved to be present in the floral organs of several plant species.
The present study also indicates the possible involvement of wintersweet CpbHLH genes in the signaling pathways of JA and GA, leading to the production of volatile floral terpenes. A similar phenomenon was reported in other plant transcription factors such as AtPAP1 in Rosa hybrida, WRKY in cotton (Gossypium hirsutum), ORCA3 in Catharanthus roseus, EF1 and ERF2 in Atemisia annua, and AtMYC2 in arabidopsis [20,[55][56][57][58]. These transcription factors regulate the TPS genes by interacting with the DELLA proteins. The DELLA proteins act as a repressor of the TPS gene and negatively regulate the JA and GA signaling pathways by interacting with MYC2. An increase in the concentration of JA and GA decreases the level of JAZ and DELLA proteins, thus releasing the MYC2 and inducing the TPS genes, which results in the production of more volatile compounds [20,59,60]. A combined application of JA and GA3 results in an increase in the expression of the TPS gene and volatile compound production in the transformed plants. Our findings are consistent with the previously reported experimental results where the application of MeJA induces monoterpene production and emits (E)-β-ocimine, β-myrcene, and a small increase in linalool in transgenic tobacco plants [60]. The plants transferred with the CpbHLH13 gene were found to increase sesquiterpene β-caryophyllene production in the arabidopsis and tobacco plants, and the expression of the genes At5g23960 and At5g44630, which are involved in the production of almost all the sesquiterpenes in the arabidopsis, was also higher than in non-transformed plants. It was previously reported that the arabidopsis gene At5g23960 is associated with the synthesis of humulene and β-caryophyllene, which together produce 43% of the terpene volatiles from the flower [49].
In addition to increase sesquiterpene (β-caryophyllene) production in the arabidopsis and tobacco plants, we also found a few transgenic tobacco plants (35S::CpbHLH13) showing reduced flower color compared with the wild type and those transformed by the empty vector pCAMBIA2300S (2300S-SR1). The results were also verified by measuring anthocyanin contents from the flower samples using a spectrophotometer. This significant decrease in anthocyanin production could be explained by the previously described function of the bHLH13 (JAM2) and bHLH17 (JAM1) transcription factors that act as the transcriptional repressors which negatively regulate JA responses resulting in anthocyanin reduction in transgenic plants [50,51,[61][62][63]. Furthermore, it has also been reported that bHLH17 is a bHLH subgroup III transcription factor which acts as a transcriptional repressor after binding to the promoters of the target genes in the anthocyanin-regulating pathway [64], which antagonizes the activation function of MYC2 and TT8/MYB75 to negatively regulate JA responses including flowering and anthocyanin accumulation [61].
It has also been elucidated by a functional study that JAZ interacts with transcriptional activators such as MYC2 and MYB75, along with the transcriptional repressors bHLH13 and bHLH17, to reduce its transcriptional functions [61]. The balance between activators and repressors leads to an adequate output of JA responsive genes, which results in a suitable level of JA responses such as plant defense and anthocyanin accumulation.
In this study, we characterized the functional aspects of two wintersweet bHLH transcription factors, CpMYC2 and CpbHLH13, for the production of volatile organic compounds. The results show that CpMYC2 could increase the production of monoterpenes such as linalool, and CpbHLH13 could increase the production of sesquiterpenes such as β-caryophyllene in transgenic plants by enhancing the activity of terpene-producing genes. The application of the MeJA with GA3 could promote the production of linalool and linalool-producing genes in arabidopsis. Taken together, the findings from this study reveal that bHLH transcription factors (CpMYC2 and CpbHLH13) play an essential role in the production of aroma in wintersweet. This study also indicates the future potential application of wintersweet as a valuable genomic material for the genetic modification of floral scent in other flowering plants that produce small amounts of volatile organic compounds.

Selection and Analysis of Candidate Genes
The sequences of two bHLH candidate genes predicted as MYC2 and bHLH13 were obtained from a transcriptomic library (NCBI Sequence Read Archive (SRA) accession number PRJNA492170) constructed from the fully open flower of the Wintersweet (C. praecox L.) H29 (Huazhong 29) genotype situated in the garden of Huazhong Agricultural University, Wuhan, China [45]. Moreover, these genes were verified as full-length coding sequences (CDS) from the genomic data of the H29 cultivar (unpublished data) with no introns inside the sequences using BioEdit 7.0 [65] and UltraEdit software [66]. The nucleotide sequences of both candidate genes are shown in supplementary data (Sequences S1 and S2). Multiple sequence alignment of selected bHLH transcription factors of Wintersweet and other plants was performed using the CLUSTALW program with default parameters [67]. The phylogenetic tree was constructed using the neighbor-joining method with 1000 bootstrap replicates using MEGA X software [68]. These candidate genes were confirmed as transcription factors and named CpMYC2 and CpbHLH13 as per their homology with the typical MYC2 and bHLH13 transcription factor proteins of other plants. The homology of the gene sequence with arabidopsis was also confirmed by the arabidopsis information resource (TAIR) database (https://www.arabidopsis.org/Blast/index.jsp). The conserved domain of the CpMYC2 and CpbHLH13 gene was observed (https://www.ncbi.nlm.nih.gov/guide/domains-structures) using Pfam (http://pfam.xfam.org/search/sequence), and protein structure was predicted using Swissmodel (https://swissmodel.expasy.org/interactive). The Compute pI/Mw calculation tool on the ExPASy server (https://web.expasy.org/compute_pi) was used to predict the theoretical isoelectric point (pI) and the molecular weight (Mw).

Plant Materials
Flowers of the Wintersweet H29 plant were collected from the garden of Huazhong Agriculture University in Wuhan, China. Plants of the Arabidopsis thaliana ecotype Columbia (Col-0), the T-DNA inserted homozygous myc2-2 (Salk_083483) mutant [69] obtained from the SALK institute and ABRC (Arabidopsis Biological Resource Center, Columbus, OH, USA), and the Nottingham Arabidopsis Stock Centre and tobacco (Nicotiana tabacum) cv Petit Havana SR1 [70] were used as the planting and gene-transforming material. The homozygosity of the myc2-2 (Salk_083483) mutant arabidopsis plants was detected by the specific primers listed in Table 3. Plants were grown in half-strength Murashige and Skoog (MS) media under a 16-h photoperiod in the plant growth chamber. Total RNA was extracted from 0.20g frozen flowers using TRIzol reagent (CoWin Biotech Co., Ltd., Beijing, China) as per the manufacturer's instructions. DNA contamination was removed by treating the total RNA with the DNase I enzyme. cDNA was synthesized using a one-step gDNA removal and cDNA synthesis supermix (Transgene, Wuhan, China) according to the manufacturer's instructions. The synthetic first-strand cDNAs were diluted 10-fold for gene expression analysis. . Three biological replicates were tested, and reactions carried out in triplicate. Relative transcript levels were calculated by the 2 −∆∆Ct method [71] using the previously reported most stable genes RPL8 and Tubulin (CpTublin) for wintersweet [72,73], NADH dehydrogenase (NtNADH) and chaperonin CPN60-2 (NtCPN60-2) for tobacco [74], and clathrin adaptor complex subunit (CACS) (AtCACS) and β-tubulin2 (AtTubulin) [20,75] for arabidopsis as the endogenous control genes for data normalization according to the recommendations [76] and described method [77]. The primers 35S F and 35S R were used for the expression of empty gene transformation. The transcript level of monoterpene-producing At1g61680 and At3g25810 along with the sesquiterpene-producing At5g23960 and At5g44630 genes in arabidopsis was also measured. Primer Premier 5 software was used to design the primers used in the experiments. The primers used for qRT-PCR analysis are listed in Table 4. Table 4. Primers for qRT-PCR analysis.

Isolation, Cloning, and Sequencing of Candidate Genes
The full-length open reading frame of CpMYC2 and CpbHLH13 was amplified by PCR from the cDNA of wintersweet flowers by using a specific pair of primers containing Kpn1 and Xba1 restriction sites ( Table 5). The PCR conditions were 5 min, 95 • C; 30 s, 95 • C; 30 s, 60 • C, 30 cycles; 30 s, 72 • C; 5 min, 72 • C. The PCR product was purified using a Tiangen Midi Purification Kit (Tiangen, Wuhan, China) and cloned into a pEASY-T1 cloning vector (Transgene Wuhan, China), according to the manufacturer's instructions, and three positive clones were selected and sequenced.

Expression Vector Construction and Transformation
The expression vector was constructed using full-length CpMYC2 and CpbHLH13 gene sequences on a modified pCAMBIA 2300S vector by double digestion using Kpn1 and Xba1 digestion enzymes under a CaMV 35S promoter (Supplementary Figure S8a,b); 35S::CpbHLH13 was introduced into Agrobacterium tumefaciens strain GV3101 by electroporation. The positively cloned agrobacteria were selected and transformed into tobacco by using the leaf disk method [78]. The empty vector pCAMBIA 2300S was also introduced into tobacco plants as a control. The shoots of emerged explants were transferred into rooting media as described previously [78]. These explants were incubated in a growth chamber at 25 • C under a 16 h/8 h photoperiod. Homozygous arabidopsis Salk_083483 mutant plants were transformed by CpMYC2, while CpbHLH13 was transferred into the arabidopsis Col-0 cultivar via Agrobacterium tumefaciens-mediated transformation [79]. The pots containing the wild-type and mutant arabidopsis plants were carefully inverted and immersed in the infiltration medium containing agrobacterium while stirring for 1 min. The seedlings were placed back on the shelf and covered by plastic bags. Three days after infiltration, the plastic bags were removed, and the seedlings were supported with sticks and allowed to grow till the first generation of seeds (T1). The seeds from the T1 generation of arabidopsis plants and the T0 generation from tobacco were grown in 1 /2 MS media plates with 50 µM-mL-1 kanamycin for screening the positive seedlings. The surviving plants were transferred to soil pots, and their positivity was also confirmed by PCR with gene-specific primers using plant cDNA as a template (Table 5). These plants were self-crossed, and at least three plants from tobacco and 3 groups of 6 plants from arabidopsis of the T3 generation with the highest gene expression were used for further experiments.

Volatile Organic Compounds Analysis
The released floral volatiles from tobacco plants were collected by solid-phase microextraction (SPME) [43]. In triplicate, 2 g fresh flowers were placed into a 20 mL capped SPME vial and incubated at 25 ± 2 • C for 30 min. SPME fiber (50/30 µm DVB/CAR/PDMS on a 2 cm Stable Flex fiber, Supelco Inc., Bellefonte, PA, USA) was then exposed to the headspace of the capped vial for 30 min. The fiber was injected manually and desorbed in the injection port of the gas chromatograph (GC) with helium as the carrier gas. The fiber was desorbed for 5 min at 250 • C in splitless mode. Before each set of samples was assayed, the fiber was conditioned for 1 h at 250 • C in the injection port of the GC-MS and a fiber blank recorded. To measure the release of volatiles by arabidopsis plants, the closed-loop stripping method [80] was used in which the intact rosette plants with their root balls were wrapped with aluminum foil and placed in 3-L bell jars under controlled growth conditions as described previously [49]. VOCs were collected in daytime for 8 h using 1.5 mg activated charcoal and eluted with 40 µL of CH 2 Cl 2 . The eluted samples of 1µL were injected in splitless mode into a TRACE GC Ultra GC coupled to a DSQ II mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an HP-5 MS fused-silica column (30 m × 0.25 mm × 0.25 µm) (Agilent Technologies, http://www.agilent.com). The GC-MS was performed according to the standard methods [43]. The relative quantification of the target compounds for emission was determined using standard curves which were generated by three repeats: y = 7969151.04x-4406492.42 and R 2 = 0.97 for linalool and y = 50591253.45x-45934339.38 and R 2 = 0.99 for β-caryophyllene (Supplementary Figure S9a,b).

Application of MeJA and GA3 to Arabidopsis Plants
We applied MeJA (50 µM) together with GA3 (50 µM) on the arabidopsis wild type (Col-0), mutant (Salk_083483), mutant plants transformed by the empty vector pCAMBIA2300S (2300S-SK83), and mutant plants transformed by the CpMYC2 gene (35S::CpMYC2). The pots, in which 5-week-old plants were growing (T3 generation), were soaked with MeJA (50 mM) and GA3 (50 mM) solutions, respectively, for an indicated time, accompanied with the spraying of aerial parts at intervals of 1 h. dH 2 O 2 was used as a mock treatment. Inflorescences were harvested for RNA extraction. The hormone treatments for gene analysis were performed in the daytime for 4 h (10:00 to 14:00), and those for volatile analysis were 6 h (9:00 to 15:00).

Anthocyanin Contents Measurements
Total anthocyanin contents were measured by the method described [81]. Flower material (500 mg) from each treatment was crushed in a mortar with liquid nitrogen to obtain a fine powder. The anthocyanins were extracted by transferring the fine powder to an extraction solution (5 mL) containing a methanol/HCl mixture (99:1 v/v) (Sigma, St. Louis, MO, USA). The mixture was incubated at 4 • C for 24 h and then centrifuged at 13,000 rpm for 20 min at 4 • C. The supernatant was transferred to a fresh tube and the total anthocyanin was determined by measuring the OD at A530 (λ max for anthocyanin) and A657 (peak of absorption for chlorophyll) by using a spectrophotometer (Shimadzu, Kyoto, Japan). The quantification of anthocyanins was performed using the formula Q anthocyanins = (A530 − 0.25 × A657) × M−1, where Q anthocyanins is the amount of anthocyanins, and M is the weight (g) of the plant material used for extraction, as described earlier [82].

Statistical Analysis
All the statistical analysis was conducted using the Statistix 8.1 (FL, USA) software. One-way analysis of variation (ANOVA) followed by Tukey's HSD multiple comparisons were used to compare the relative expression of transcripts. Three technical and biological repeats were selected separately for every experiment. p-value < 0.05 was considered as significant.
Supplementary Materials: The following are available online at http://www.mdpi.com/2223-7747/9/6/785/s1, Figure S1: Three-dimensional structure prediction of the protein encoded by the CpMYC2 gene. Figure S2: Three-dimensional structure prediction of the protein encoded by the CpbHLH13 gene. Figure S3: Phylogenetic analysis of wintersweet CpMYC2 with the 158 arabidopsis bHLH transcription factor gene family. Figure S4: Phylogenetic analysis of wintersweet CpbHLH13 with the arabidopsis bHLH transcription factor gene family. Figure  S5: Phylogenetic analysis of Chimonanthus praecox CpMYC2 with different plant species. Figure S6: Phylogenetic analysis of Chimonanthus praecox CpbHLH13 with different plant species. Figure S7: Expression of the CpMYC2 and CpbHLH13 genes in plants. Figure S8: Partial diagram of the pCAMBIA 2300S transformation vector with CpMYC2 and CpbHLH13 genes. Figure S9: Standard curve of linalool and caryophyllene. Sequence S1: Nucleotide sequence of the CpMYC2 candidate gene. Sequence S2: Nucleotide sequence of the CpbHLH13 candidate gene.

Conflicts of Interest:
The authors declare no conflict of interest.