MeJA affects 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 volatile terpenoid emission, and production was significantly higher in leaves (Fig. 1). Furthermore, MeJA promoted the emission of β-myrcene, β-cis-ocimene, and caryophyllene in lavender sepals and leaves (JAS and JAL) (Additional file 2: Fig. S2).
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Twenty-six MYCs were previously identified (unpublished) in L. angustifolia based on genome data (PRJNA642976), and the MYC gene LaMYC4 was differentially expressed by MeJA treatment (Fig. 2a).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 leaves 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 3: Fig. S3). 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. A phylogenetic tree was constructed with LaMYC4 and 22 MYCs from different plants (Additional file 9: Table S2) and showed that LaMYC4 was most closely related to NaMYC4 and BpMYC4 (Fig. 2d).
Analysis of the LaMYC4 promoter sequence and response to stresses
The 2000-bp promoter upstream of the 5′-untranslated region (5′ UTR) was analyzed using PlantCARE software (Additional file 10: Table S3). Four abscisic acid response elements were found at +1432, -1469, -1467, and +1687 bp, three light or abscisic acid response elements (G-box) were located at −1431, +1469, and −1686 bp, four low-temperature response elements were situated at −104, −1478, +614, and −1809 bp, one TC-rich repeating element involved in defense and stress response was located at −1617 bp, and one gibberellin response element (TATC-box) was located at −1953 bp (Fig. 3a and Additional file 10: Table S3).
Gene expression studies have shown that MYC transcription increased in response to biotic and abiotic stresses. LaMYC4 expression levels were quantified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The results showed that LaMYC4 expression was significantly upregulated in lavender leaves by UV (~4-fold), cold (~3-fold), drought (~6-fold), MeJA (~5-fold), and Pst DC3000 (~6-fold) and downregulated 3-fold by NaCl (Fig. 3b).
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) leaves. The results showed that 35S::GFP was found in the cytoplasm and nucleus of plant cells, whereas LaMYC4 fusion proteins were only present in the nucleus (Fig. 4a), suggesting that LaMYC4 localizes to the nucleus.
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/X-α-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 total terpenoid and monoterpenoid contents did not change significantly in transgenic lines (Fig. 5a, b). 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 higher in transgenic A. thaliana than the control groups (wild-type and empty vector plants) (Fig. 5d and Additional file 4: Fig. S4).
Overexpression of LaMYC4 increases terpenoid biosynthesis in tobacco
Under the CaMV 35S promoter, LaMYC4 was overexpressed in tobacco by Agrobacterium tumefaciens-mediated transformation. Terpenoid concentrations were quantified in transgenic plants from the T2 generation using SPME-GC-MS. The results indicated that total terpenoid 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). Caryophyllene concentrations 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 11: 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 flowers. In addition, DXR expression was strongly associated with the expression of LaMYC4. These results indicate that LaMYC4 regulates terpenoid synthesis and DXR expression and indirectly controls the expression of HMGR, FPPS, DXS, and GPPS. However, this result needs to be validated.
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 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).