The transcription factor LaMYC4 from lavender regulates volatile Terpenoid biosynthesis

The basic helix-loop-helix (bHLH) transcription factors (TFs), as one of the largest families of TFs, are essential regulators of plant terpenoid biosynthesis and response to stresses. Lavender has more than 75 volatile terpenoids, yet few TFs have been identified to be involved in the terpenoid biosynthesis. Based on RNA-Seq, reverse transcription-quantitative polymerase chain reaction, and transgenic technology, this study characterized the stress-responsive transcription factor LaMYC4 regulates terpenoid biosynthesis. Methyl jasmonate (MeJA) treatment increased volatile terpenoid emission, and the differentially expressed gene LaMYC4 was isolated. LaMYC4 expression level was higher in leaf than in other tissues. The expression of LaMYC4 decreased during flower development. The promoter of LaMYC4 contained hormone and stress-responsive regulatory elements and was responsive to various treatments, including UV, MeJA treatment, drought, low temperature, Pseudomonas syringae infection, and NaCl treatment. LaMYC4 overexpression increased the levels of sesquiterpenoids, including caryophyllenes, in Arabidopsis and tobacco plants. Furthermore, the expression of crucial node genes involved in terpenoid biosynthesis and glandular trichome number and size increased in transgenic tobacco. We have shown that the stress-responsive MYC TF LaMYC4 from ‘Jingxun 2’ lavender regulates volatile terpenoid synthesis. This study is the first to describe the cloning of LaMYC4, and the results help understand the role of LaMYC4 in terpenoid biosynthesis.

Lavender is a model for studying the regulation of terpenoid synthesis [34]. More than 75 volatile terpenoids were identified in Lavandula angustifolia [35,36]. One hundred terpene synthases (TPSs) have been identified in lavender, of which 11 were characterized, and some are induced by methyl jasmonate (MeJA) [13,37]. Recently, a reference genome for the 'Jingxun 2' lavender cultivar was created [37].
This study isolated the MYC TF LaMYC4, which regulates terpenoid biosynthesis. The expression of LaMYC4 was upregulated by UV, low temperature, drought, MeJA, and Pseudomonas syringae infection. Moreover, LaMYC4 overexpression increased the levels of terpenoids (especially caryophyllene) and the number and size of glandular trichomes (GTs) in transgenic plants. These results demonstrate that LaMYC4 can be a candidate gene for L. angustifolia molecular breeding.

MeJA affects volatile terpenoid biosynthesis
Lavender plants were treated with or without 8 mM of MeJA, and volatile terpenoids were analyzed by solid-phase microextraction gas chromatography/mass spectrometry (SPME-GC-MS). The results revealed that MeJA induced various volatile terpenoid emission, and production was significantly higher in leaf ( Fig. 1 and Additional file 10: Table S1). Furthermore, MeJA promoted the emission of β-myrcene, β-cis-ocimene, and caryophyllene in lavender sepal and leaf (JAS and JAL) (Additional file 1: Fig. S1).

Isolation and bioinformatics analysis of LaMYC4
Twenty-six MYCs were previously identified (unpublished) in L. angustifolia based on genome data (PRJNA642976), and the MYC gene LaMYC4 was differentially expressed by MeJA treatment (Fig. 2a). The level of LaMYC4 expression was significantly higher in leaf than in other tissues and decreased during flower development (Fig. 2b, c). The 1422-bp open reading frame of LaMYC4 encoded 473 amino acids (Additional file 2: Fig.  S2). Bioinformatics analysis indicated that the LaMYC4 protein contained a bHLH-MYC sequence between amino acids 38 and 211, corresponding to the N-terminal region of MYB and MYC TFs, and DNA-binding domains between amino acids 299 and 373 (Fig. 2e). Physicochemical characterization using ExPASy showed that LaMYC4 had a molecular mass of 52.24 kDa and an isoelectric point of 5.75. LaMYC4 protein was clustered into subfamily 2 or subgroup-III(d + e) according to the classification and nomenclature of AtbHLH proteins (Additional file 3: Fig. S3). A phylogenetic tree was constructed with LaMYC4 and 22 MYCs from different plants (Additional file 11: Table S2) and showed that LaMYC4 was most closely related to NaMYC4 and BpMYC4 (Fig. 2d).

Subcellular localization and transactivation activity of LaMYC4
The subcellular localization of the LaMYC4 protein was assessed using a transient expression assay in tobacco (Nicotiana benthamiana) leaf. The results showed that  The yeast strain AH109 and the pGBKT7 vector containing the DNA-binding domain of GAL4 were used to measure the transactivation activity of LaMYC4. Yeast cells transformed with any vector were cultivated in SD/−Trp medium. Yeast cells transformed with the fusion plasmid (pGBKT7-LaMYC4) or positive control plasmid (pGBKT7-p53) and cultivated in SD/−Trp/Xα-Gal medium appeared blue, whereas yeast cells transformed with the negative control plasmid pGBKT7 did not turn blue (Fig. 4b), indicating that LaMYC4 has transactivation activity in yeast.

LaMYC4 overexpression increases sesquiterpenoid biosynthesis in A. thaliana
Under the control of the CaMV 35S promoter, LaMYC4 was overexpressed in transgenic A. thaliana by Agrobacterium tumefaciens-mediated transformation. Terpenoid levels were measured in transgenic plants from the T3 generation. The results indicated that the expression of LaMYC4 was significantly changed in transgenic lines, while the contents of total terpenoids and monoterpenoids did not change significantly ( Fig. 5a, b, e). In contrast, sesquiterpenoid levels increased 0.5-1.0-fold in transgenic lines overexpressing LaMYC4 (#2, #7) compared with the empty vector group (Fig. 5c). In addition, caryophyllene was the most abundant sesquiterpenoid in A. thaliana, and its emission was more than 2-fold Putative cis-acting regulatory elements were identified in the promoter sequence of LaMYC4 using the PlantCARE database. (b) Treatments included UV, cold, NaCl, drought, MeJA and Pst DC3000. The relative expression of LaMYC4 was quantified by qRT-PCR. Values shown are the means ± SD at least three replicates, and standard errors are indicated as vertical lines on the top of each bar. *p < 0.05; **p < 0.01; ***p < 0.001; t-test higher in transgenic A. thaliana than the control groups (wild-type and empty vector plants) (Fig. 5d and Additional file 4: Fig. S4). The expression of caryophyllene synthase (At5g23960) in transgenic A. thaliana (#7) was also significantly increased (Fig. 5f ).

Overexpression of LaMYC4 increases volatile terpenoid biosynthesis in tobacco
Under the CaMV 35S promoter, LaMYC4 was overexpressed in tobacco by Agrobacterium tumefaciensmediated transformation. Terpenoid concentrations were quantified in transgenic plants from the T2 generation using SPME-GC-MS. The results indicated that total volatiles and sesquiterpenoid contents increased 1-2-fold and 2-3-fold in transgenic tobacco, respectively, compared with the control (Fig. 6a, c), whereas monoterpenoid contents increased significantly only in transgenic line #5 (Fig. 6b). The contents of phytohormones Zr, IAA, JAs decreased in transgenic tobacco compared with the control, while the contents GA3 and ABA increased, and all changes were significant in transgenic line #5 (Additional file 6: Fig. S6). Caryophyllene contents were higher in lines #3 and #5 than in control plants (Fig. 6 d). Caryophyllene levels were ~ 5-fold higher in transgenic lines overexpressing LaMYC4 (#3 and #5) than in empty vector plants (Additional file 5: Fig. S5). Furthermore, transgenic tobacco plants (35S:: LaMYC4) showed reduced flower color and increased plant height (Additional file 7: Fig. S7) compared with control plants.

LaMYC4 overexpression upregulates genes related to terpenoid synthesis in tobacco
To assess the effect of LaMYC4 on the expression of genes related to terpene synthesis, we investigated HMGR, FPPS, DXS, DXR, and GPPS (the sequences are shown in Additional file 13: Table S4), which are key enzymes in the MVA and MEP pathways. The expression of genes HMGR, FPPS, DXS, DXR, and GPPS increased 1.3-to 3.8-fold (Fig. 7b) in LaMYC4-overexpressing transgenic tobacco flower. In addition, DXR expression was strongly associated with the expression of LaMYC4. These results indicate that LaMYC4 was involved in the regulation of terpenoids and affects the expression of several key genes (HMGR, FPPS, DXS, DXR, and GPPS) in terpenoid synthesis pathway. In addition, we found that the expression of diterpenoid-related synthase (NtCPS2 and NtABS) in transgenic tobacco (#5) was significantly decreased, while the expression of NtCBTS was significantly increased in transgenic tobacco (#3 and #5) (Additional file 8: Fig. S8).

LaMYC4 overexpression increases the number and size of GTs
GTs are a physical defense to insect herbivores in response to mechanical stimulation. Moreover, evidence indicates that glandular secretory trichomes (GSTs) synthesize and store terpenoids. Since LaMYC4 regulates terpenoid biosynthesis in transgenic lines, we examined GT morphology by scanning electron microscopy. GTs on the stems of the fourth fully grown internode of 35S::LaMYC4 tobacco plants had longer stalks and larger levels of these genes related to terpenoid synthesis were determined. The values shown are mean ± SD at least three replicates. Standard errors are indicated as vertical lines on the top of each bar, and bars annotated with different letters were significantly different according to Fisher's LSD test (P < 0.05) after ANOVA glandular heads than control plants (Fig. 8). Moreover, the number of GTs was 0.4-fold higher in 35S::LaMYC4 tobacco plants than in control plants (Fig. 8d).

Discussion
Plants utilize various physiological and biochemical processes to survive and respond to stresses [38,39]. Plant bHLH proteins play a pivotal role in stress responses. For instance, OsbHLH148 and OsbHLH006 (RERJ1) respond to drought stress through the JA signalling pathway [40,41]. Vitis vinifera bHLH1 responds to drought and salinity via the accumulation of flavonoids and is the regulation of abscisic acid (ABA) synthesis [42]. RsICE1 interacts with CBF/DREB1 in rice plants to improve cold tolerance [43]. We identified the promoter region of LaMYC4 by genomic analysis [37]. This region contains stress-related cis-elements that allow LaMYC4-encoded TFs to adapt to the environment. As shown in Table S5 (Additional file 14), most of the proteins of subfamilies 1, 2, 4, 10, 13, 14 and 18 responded to different biotic and abiotic stresses, such as drought, cold and salt [44]. In additional, the results of UV, MeJA treatment, drought, low temperature, Pseudomonas syringae infection, and NaCl treatment indicated that LaMYC4 responded to multiple stresses.
Plant bHLH TFs play vital roles in terpenoid biosynthesis. For instance, AtMYC2 binds to the promoter of the caryophyllene biosynthetic pathway genes TPS21 and TPS11 and stimulates gene expression [27], and CrBIS2 plays an essential role in the generation of monoterpenoid indole alkaloids [45]. LaMYC4 overexpression enhanced terpenoid synthesis, especially sesquiterpenoid caryophyllene (Additional files 4, 5: Fig. S4 and 5). TFs can simultaneously participate in the expression regulation of multiple key genes in terpenoid synthesis [46]. The transcript levels of the structural genes HMGR, FPPS, DXR, DXS, GPPS from the terpenoid biosynthesis pathway were significantly increased in LaMYC4-overexpressing lines (Fig. 7). However, the increase of monoterpenoids was not as significant as that of sesquiterpenoids. Previous studies have shown that the expression of monoterpene synthase At1g61680 and sesquiterpene synthases At5g23960 and At5g44630 was increased in CpMYC2-overexpressing Arabidopsis, while the expression of monoterpene synthase At3g25810 was decreased [28]. LaMYC4 overexpression enhanced the flux of terpenoid biosynthetic pathways, and the decrease in anthocyanin accumulation in transgenic plants produced light-colored flowers (Additional file 7: Fig. S7). Anthocyanin production is metabolically expensive, and the overexpression of VvmybA1 resulted in the accumulation of anthocyanins in leaf, whereas the concentration of most volatile compounds decreased in the leaf of transgenic plants [47]. The overexpression of CpbHLH13 increased the concentration of volatile terpenoids and decreased anthocyanin accumulation [28]. These results indicated that LaMYC4 modulated volatile terpenoid biosynthesis, especially sesquiterpenoid caryophyllene, and influenced carbon flow in the terpenoid pathway.
MYC3 and MYC4 activate JA-regulated responses and act synergistically with MYC2 to control different subsets of JA-dependent transcriptional activity [48]. Different volatile compounds are involved in JA-associated stress response [49][50][51][52]. MeJA treatment confirmed the result in our study. And this study found that LaMYC4 overexpression in tobacco increased ABA and GA3 contents and decreased JA and IAA levels (Additional file 6: Fig. S6). Abe et al. [30] have shown that AtMYC2 acts as a transcriptional activator in ABA signalling in Arabidopsis. Moreover, GA is involved in cell elongation [53]. Plant height increased in LaMYC4-overexpressing tobacco (Additional file 7: Fig. S7a, b). The morphology of stem epidermal cells was examined by scanning electron microscopy. The length of these cells was 0.3-fold higher in transgenic plants than in control plants (Additional file 7: Fig. S7). These results indicate that LaMYC4 promotes the elongation of epidermal cells by upregulating GA, increasing plant height.
Trichomes serve as physical barriers to insect herbivores [29]. Evidence indicates that GSTs produce and accumulate terpenoids [54]. In tomato, SlMYC1 regulates GT formation and terpenoid biosynthesis [29]. LaMYC4overexpression in tobacco confirmed the results that MYC plays a pivotal role in plant GT formation and terpenoid biosynthesis. In addition, the increase in terpenoid levels was significantly higher in LaMYC4-overexpressing tobacco than in LaMYC4-overexpressing A. thaliana, which may be because there is a lack of GTs in A. thaliana. In conclusion, we have shown that the stressresponsive MYC TF LaMYC4 from 'Jingxun 2' lavender regulates terpenoid synthesis. LaMYC4-overexpressing plants accumulated more terpenoids, especially sesquiterpenoid caryophyllene. In addition, LaMYC4 may be involved in regulating GT formation, increasing terpenoid biosynthesis and accumulation.

Conclusions
This study provides, to our knowledge, the first to describe the cloning of LaMYC4. We successfully profiled the tissue-specific expression patterns based on RNA-Seq. Different stress treatments and analysis of the LaMYC4 promoter sequence shown that LaMYC4 responds to multiple stress to adapt to the environment. Furthermore, LaMYC4-overexpression increased the levels of terpenoids (especially caryophyllene) and the number and size of GTs in transgenic plants. These results demonstrate that LaMYC4 can be a candidate gene for L. angustifolia molecular breeding. And our study served as a basis for future studies on the regulation of terpenoid synthesis and stress responses by MYCs.

Plant materials and treatments
The L. angustifolia cultivar used in this study was 'Jingxun 2' from the Institute of Botany, Chinese Academy of Sciences. The voucher specimen of 'Jingxun 2' was kept at the Chinese national herbarium, Institute of Botany, Chinese academy of sciences (voucher specimen: 02308796). All wild-type Arabidopsis and tobacco seeds used were obtained from Key Laboratory of Plant Resources. And all plant material was used in accordance with relevant guidelines and regulations. Transcriptome data were obtained from a previous study [13,37]. For Pst DC3000, UV, MeJA, salinity (NaCl), cold, and drought treatments, 12 one-year-old potted plants of the same cultivar (for each treatment) were grown in a greenhouse. Pst DC3000 inoculation was performed for 6 h as described previously [55]. UV treatment lasted 10 minutes a day for 3 days. MeJA treatment was with 8 mM for 12 h. NaCl treatment with 300 mM was once every 3 days, twice in total, sampling on the seventh day, and watering thoroughly each time. Cold (16 °C) and or drought treatments were for 7 days. Sepal, leaf and flower were removed from potted plants for further analysis. L. angustifolia, A. thaliana (Col-0), and tobacco (Nicotiana benthamiana and N. tabacum) were grown under a 16 h photoperiod at 22 ± 2 °C. Abbreviations corresponding to samples are as follows: sepal (S), leaf (L), root (R), stem (S), opening flower (F), glandular trichomes (GTs), flower bud (FB). FB0, FB1, FB2, F3, F4, and F5 correspond to different stages of flower development, as described previously [13].

RNA extraction and qPCR analysis
Total RNA was extracted from frozen samples using the HiPure Plant RNA Mini Kit (Magen, China) according to the manufacturer's instructions. RNA quality and concentration were analyzed by gel electrophoresis and spectrophotometry. RNA was stored at − 80 °C until use. cDNA was synthesized according to the manufacturer's instructions (Vazyme, China). Gene expression was measured by RT-qPCR on an Mx3000P system (Agilent Stratagene). Primers were designed using primer-BLAST (https:// www. ncbi. nlm. nih. gov/ tools/ primer-blast) (Additional file 15: Table S6). PCR and data analyses were performed as described previously [56].

LaMYC4 cloning and sequence analysis
Primers were designed based on the LaMYC4 sequence obtained from the lavender genome (PRJNA642976) [37] (Additional file 15: Table S6), and the gene was amplified by PCR. The PCR product was cloned into the pBM16K vector and sequenced by TsingKe (Tianjin, China). Amino acid sequences homologous to LaMYC4 were retrieved from the NCBI database. Phylogenetic analysis was performed in MEGA software version 7.0 using the neighbor-joining method. Full length amino acid sequences of Arabidopsis bHLH proteins (AtbHLHs) were downloaded from the TAIR database (http:// www. arabi dopsis. org). The reliability of the neighbor-joining tree was estimated by bootstrap analysis using 1000 bootstrap replications. The properties of the deduced amino acid sequence were predicted using ExPASy (http:// web. expasy. org/ compu te_ pi/).

Subcellular localization and the transactivating activity of LaMYC4
The full-length cDNA of LaMYC4 was cloned using primers (Additional file 15: Table S6) containing KpnI restriction sites and was ligated into the expression vector pCAMBIA2300 to produce a fusion protein (35S::LaMYC4-GFP). The empty vector (pCAMBIA2300) and the recombinant vector (35S::LaMYC4-GFP) were transformed into Agrobacterium tumefaciens GV3101 by heat shock. Four-weekold N. benthamiana plants were transformed with the 35S::LaMYC4-GFP vector or 35S::GFP vector, as described previously [57]. After 3 days of transformation, leaves were removed and analyzed on a confocal laser scanning microscope equipped with a standard filter set (Leica TCS SP5).
For the transactivation assay, the full-length cDNA of LaMYC4 was cloned into the pGBKT7 vector containing EcoRI and BamHI restriction sites. The negative control (pGBKT7), positive control (pGBKT7-p53), and recombinant vector were expressed in the yeast strain AH109 following the manufacturer's instructions.

Plant transformation and identification of transgenic lines
Bacterial colonies containing the 35S::LaMYC4-GFP vector were selected and transformed into the Arabidopsis Col-0 cultivar using a floral dip method [58] or tobacco plants using the leaf disk method [59]. Plants containing the empty vector served as a control. Explants were incubated in a growth chamber at 23 °C under a 16 h light/8 h dark photoperiod. Primary transformants were selected on half-strength Murashige and Skoog medium containing 50 μg mL − 1 kanamycin, and the presence of the transgene was confirmed by PCR.

Measurement of volatile terpenoid concentrations
The volatile compounds released from lavender, tobacco, and Arabidopsis plants were collected by SPME [28,37]. Fresh sepals (10 mg), fresh leaves (100 mg) from lavender, and fresh flowers (2 g) from tobacco were placed into headspace vials and kept at 40 °C (lavender sepals and leaves) or 60 °C (tobacco flowers) for 40 min and exposure to a DVB/CAR/PDMS fiber for 20 min, followed by analyte desorption at 250 °C for 3 min. A total of 0.25 μg of 3-octanol was added to these samples as an internal standard. To measure the release of volatiles by Arabidopsis plants, the plants were placed in a 25 cm × 38 cm plastic bag (EasyOven) and incubated at 23 ± 2 °C via DVB/ CAR/PDMS fiber for 3 h, followed by analyte desorption at 250 °C for 3 min. The relative concentration of the target compounds was determined using standard curves, which were generated by three repeats: y = 10-7x + 0.0024 and R 2 = 0.92 (Additional file 9: Fig. S9).
GC-MS analysis was performed via splitless injection using an Agilent 7890B GC system and an Agilent Technologies 7000C Inert XL Mass Selective Detector equipped with an HP-5MS UI column (30 m × 0.25 mm × 0.25 μm; Agilent Technologies), as described previously [37].
Products were identified based on retention times and electron ionization mass spectra obtained from the National Institute of Standards Technology (NIST) Mass Spectral Library (NIST-14.0) and literature data [35,60,61].

Trichome morphology and number
Samples were examined on a field-emission scanning electron microscope (Hitachi S-4800), and the number and size of stem trichomes from the fourth fully grown internode of each plant were determined.

Measurement of the level of anthocyanins and endogenous hormones
Twelve plants from each line were selected for measuring plant growth and total anthocyanin concentration. Total anthocyanins in tobacco flowers (500 mg) were measured as described previously [28]. GA, ABA, IAA, ZR, and JA in tobacco leaf were measured by enzyme-linked immunosorbent assay (ELISA). Hormones were extracted and purified according to He [62] and quantified by ELISA based on Yang et al. [63].

Statistical analysis
Statistical analysis was performed by one-way analysis of variance followed by independent-samples t-test or Fisher's least-significant difference test using SPSS software version 17.0. Data were expressed as the mean ± standard deviation of at least three independent experiments. P-values smaller than 0.05 were considered statistically significant.