Introduction

Orchids are one of the largest families of angiosperms, and they have been widely spread from the tropics to polar regions on all continents except Antarctica. Nevertheless, horticultural cultivars are mainly classified into tropical and oriental orchids. In south Asia, tropical orchids are popular in the flower market, especially during the traditional festivals, since orchids are a symbol of wealth and prosperity (Zhang et al. 2018). In contrast, the oriental orchids are mainly spread in the mainland of China, Japan, and Korea where the latitudes are comparably higher and the temperatures are lower (Choi et al. 2020; Wei et al. 2020). Although most of the oriental orchids do not have colorful petals, the variable flower shapes and strong flower fragrance explain their great value in the orchid market (Ramya et al. 2019). Cymbidium faberi is one of the oldest varieties of oriental orchids, with a subdued flower color and strong flower fragrance, which could attract pollinating insects in the wild (Hossain et al. 2010, 2013). To investigate the biosynthesis of flower fragrance in C. faberi, RNA-seq analysis and yeast hybridization libraries were conducted, which provide great convenience for genetic research on the molecular level (Xu et al. 2019; Xu et al. 2020b).

Based on solid-phase microextraction (SPME) and GC/MS, the main component of the flower fragrance of C. faberi is methyl jasmonate (MeJA), which is also an important plant hormone (Zhou et al. 2018). The biosynthetic pathway of MeJA was elucidated in many model plants. MeJA is derived from α-linolenic acid in the chloroplasts where 12-oxo-phytodienoic acid (OPDA) is synthesized by a lipoxygenase (LOX), allene oxide synthase (AOS) and allene oxide cyclase (AOC). Then, OPDA is transferred to the peroxisomes to form the final product of jasmonic acid (JA) (Guan et al. 2019; Yang et al. 2019). JA can be converted into many derivatives, such as JA-isoleucine (JA-Ile), 12-hydroxyjasmonic acid (12-OH-JA) and MeJA. MeJA is synthesized by jasmonic acid carboxyl methyltransferase (JMT), which is the rate-limiting enzyme in the biosynthetic pathway (Seo et al. 2001). Among these enzymes, AOC is the only one responsible for the correct enantiomeric structure of JA (Riemann et al. 2013).

The promoter is a DNA fragment upstream to the coding region of a gene responsible for the specifical recruitment of the RNA polymerase when a gene is activated. In eukaryotes, many regulators can specifically recognize the cis-acting elements in the promoter sequences and induce the plant to rapidly respond to internal signals or the external environment. In general, the promoters can be categorized into three classes: constitutive, inducible and specific promoters (Chen et al. 2014). To identify how and where the MeJA biosynthetic pathway is regulated in C. faberi, the promoter sequences of the CfAOC and CfJMT genes were cloned from the genomic DNA and used to ectopically express a reporter gene in tobacco in this study.

To finely regulate the growth, development, and stress responses of the plant, MeJA is often interconnected with other plant hormones and controlled by many pivots of transcription factors (Ku et al. 2018; Yang et al. 2019). For example, the hormonal signals gibberellin (GA) and jasmonate (JA) act either antagonistically or synergistically to regulate diverse aspects of plant growth, development, and defense (Qi et al. 2014). Both DELLAs and Jasmonate ZIM-domain (JAZ) proteins interact with the WD-repeat/bHLH/MYB complex to mediate the synergism between GA and JA signaling in regulating trichome development. The antagonism between JA and ethylene (ET) during apical hook development has been intensively studied in Arabidopsis (Zhang et al. 2014). In banana fruit, the jasmonate signaling regulator MaMYC2s was found to physically interact with MaICE1 to mediate MeJA-induced chilling tolerance (Zhao et al. 2013). In this study, two bHLH transcription factors were selected to test the promoter activity of the CfAOC and CfJMT genes and identify their exact interaction sites.

To explore the promoter activity of the CfAOC and CfJMT genes from the MeJA biosynthesis pathway of C. faberi, these two promoters were cloned and their expression patterns were assessed in tobacco. The crucial interaction cis-elements with CfbHLH transcription factors in CfAOC and CfJMT promoters were studied and discussed in this study.

Materials and methods

Plant materials and growth conditions

The wild C. faberi was transplanted from Dangyang mountain (30°55′25’″N, 111°51′24″E), Hubei province, China in 2011, and preserved at the greenhouse of Wuhan University of Bioengineering. Nicotiana tabacum and Nicotiana benthamiana were kindly provided by associate professor Wenjun Huang (Wuhan Botanical Garden, Chinese Academy of Sciences). Transgenic and wild-type tobacco plants were grown in the greenhouse under a 16 h light/8 h dark cycle at 25 °C and 60% humidity.

Cloning and bioinformatic analysis of the promoter sequence of the CfAOC gene

The genomic DNA was extracted from young leaves of Cymbidium faberi using the CTAB protocol and a modified TAIL-PCR was applied to amplify the promoter of CfAOC as described previously (Xu et al. 2019). The universal primers used in the TAIL-PCR were the same as those reported by Xu et al., and the three nested specific primers PCfAOC-SP1, SP2 and SP3 are listed in Table S1. The TAIL-PCR products of the promoter sequence of the CfAOC gene were sequenced by Sangon Biotech (Shanghai, China) and then assembled using ContigExpress software. PLACE software (http://www.dna.affrc.go.jp/PLACE/signalscan.html) was used to predict the cis-acting elements and DNA-binding regions within the CfAOC promoter sequence. The promoter of the CfJMT gene was preserved in our lab from a previous study (Xu et al. 2019).

Vector construction and tobacco transformation

The vectors for the testing of the CfAOC and CfJMT promoters were constructed based on the pCambia1301-GUS backbone using the restriction endonucleases Hind III and Bgl II, or Pst I and Bgl II, respectively. The primers used to amplify the CfAOC and CfJMT promoter sequences are listed in Table S1. The resulting recombinant plasmids pC1301-PCfAOC and pC1301-PCfJMT were transformed into Agrobacterium tumefaciens GV3101, which was used to transform Nicotiana tabacum plants as described before (Huang et al. 2016).

Based on the bioinformatic analysis of the promoter sequences, a series of truncated fragments of the CfAOC and CfJMT promoters were obtained by PCR. The corresponding primers of the PCfAOC-F series, which were combined with PCfAOC-R, and the PCfJMT-F series, which were combine with PCfJMT-R, are also listed in Table S1. The resulting fragments starting at –81, –161, –217, –406, –445, –629, –862, –1038, –1197, and –1347 nt upstream of the translation start site of the CfAOC gene, as well as the fragments starting at –265, –302, –417, –593, –742, –887, –960, –1002, –1121, –1245, and –1377 nt upstream of the translation start site of the CfJMT gene, were integrated into the pGreen II 0800-Luc vector to identify the conserved cis-elements in the promoter sequences, which would influence their interactions with the corresponding transcription factors. The effector vectors pGreenII 62-SK-CfbHLH5 and pGreenII 62-SK-CfbHLH36, as well as the reporter vectors pGreen II 0800-Luc-PCfAOC truncated series and pGreen II 0800-Luc-PCfJMT truncated series were individually transformed into A. tumefaciens GV3101 together with pSoup. Then, the Agrobacteria with effector vector of pGreenII 62-SK-CfbHLH5 and reporter vectors of pGreen II 0800-Luc-PCfAOC, pGreen II 62-SK-CfbHLH36 and pGreen II 0800-Luc-PCfJMT truncated series were transiently co-transformed into N. benthamiana plants as described previously (Xu et al. 2020a). The enzymes used to amplify the promoter fragments and construct the vectors were purchased from TaKaRa (Dalian, China) and other reagents from Sinopharm (Shanghai, China).

GUS staining

The seedlings were screened for positive transformants by PCR using the same primers employed in the recombinant vector construction. T1 and T2 generations were selected on Murashige and Skoog (MS) medium with 10 µg/mL hygromycin (neoBioFroxx GmbH, Germany). GUS staining was detected in T2 tobacco seedlings using a previously described protocol (Zhou et al. 2012). The images were recorded using a Scanner (Microtek, ScanMaker i800, Zhongjing, China).

Dual-luciferase detection of LUC activity

Following 3 days of infiltration with the effector and reporter vectors, the tobacco leaves were subjected the dual-luciferase detection as described before (Xu et al. 2020a). Each sample was analyzed in biological triplicates.

Statistical analysis

Data on the relative LUC activity as determined by dual-luciferase detection were presented as the means ± SE from n = XY parallel experiments, and were subjected to analysis of variance using SigmaPlot 12.5 software.

Results

The promoter of the CfAOC gene was successfully cloned and bioinformatically analyzed

To identify the cis-elements in the promoter of the CfAOC gene, TAIL-PCR was applied to clone the promoter sequence. In the first round of TAIL-PCR, fragments with a length above 750 bp were purified for DNA sequencing (Figure S1a). The sequencing results revealed a fragment of about 320 bp adjacent to the ORF of the CfAOC gene upstream of the ATG start codon. The second round of TAIL-PCR using the PCfAOC-SP2 and AD4 primers yielded another fragment with a length of approximately 750 bp (Figure S1b). After three rounds of TAIL-PCR, the amplification products were sequenced and aligned, yielding the promoter region of the CfAOC gene spanning 1347 bp (Figure S1c). PlantCARE analysis predicted many cis-acting elements within the cloned sequence (Fig. 1 and Table 1), mainly including ERE, ABRE, CGTCA-motif, TGACG-motif, and GARE elements responsive to plant hormones, TCA-motif, STRE, and LTR responsive to stress, some G-box, BOX4 and circadian elements responsive to light, as well as some G-box, MYB, MBS, and MYC elements responsive to related transcription factors (marked background color in Fig. 1), suggesting that the expression of the CfAOC gene in C. faberi is influenced by many biotic and abiotic factors and controlled by a plethora of transcription factors, which is consistent with the pleiotropic effects of MeJA on plant physiology.

Fig. 1
figure 1

Sequence of CfAOC promoter and partial cis-elements, including G-box (in pale blue and pearl violet), MYB (in pale green and brick red) and myc (in purple)

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Table 1 Cis-acting elements in the CfAOC promoter predicted by PlantCARE
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The promoter of the CfAOC gene could induce the expression of the gus reporter gene in all the vegetative organs of transgenic tobacco, while the CfJMT promoter could not

To test the promoter activity and expression pattern of CfAOC and CfJMT genes, the T2 generation of tobacco transformants carrying the pCambia1301-GUS plasmid with the promoters of the CfAOC and CfJMT genes, respectively, were obtained by hygromycin screening. A total of 10 tobacco seedlings, respectively (named AOC# and JMT#) were subjected to GUS staining. The results showed that the AOC#7 and AOC#10 transformants were stained blue in all the vegetative organs, especially in the roots, while there was no staining in wild type (WT) (Fig. 2). However, none of the T2 generation of tobacco transformants with the promoter of the CfJMT gene could be stained blue (Fig. 2), just like the WT.

Fig. 2
figure 2

GUS staining of transgenic tobacco seedlings transformed with pC1301-PCfAOC-GUS. bar = 1 cm. AOC#7, AOC#10 and JMT#1, transgenic tobaccos; WT, wild type

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After the transformants grew up to the reproductive period, all the organs were collected for GUS staining. As shown in Fig. 3, the root, stem and leaf of AOC#7 were stained blue (Fig. 3A, B, C), but the reproductive organs were not stained (Fig. 3D, E, F). The staining in root is obvious compared to that in other organs, which is consistent with that in the seedling. The root of JMT#1 is not stained blue (Fig. 4A), but stem, leaf and petal were stained faint blue (Fig. 4B, C, D). However, the stamen and sepal were not stained blue (Fig. 4E, F). This result suggested that the CfJMT promoter has a weak activity in tobacco, because only very limited regions even a few cells in these tissues could be stained blue.

Fig. 3
figure 3

GUS staining of transgenic tobacco plants AOC#7. A root; B stem; C leaf; D petal; E anther and filament; F sepal

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Fig. 4
figure 4

GUS staining of transgenic tobacco plants JMT#1. A: root; B stem; C leaf; D petal; E anther and filament; F sepal

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The transient co-transfection of the promoter of the CfAOC gene with CfbHLH5 transcription factor could increase its promoter activity in tobacco

To identify the exact interaction sites of the promoter of the CfAOC gene with the basic helix–loop–helix (bHLH) transcription factors, a series of truncated promoter sequences of CfAOC gene were inserted into the pGreen II 0800-Luc vector. The LUC activity in the dual-luciferase assay showed that shorter promoter truncations had weaker interactions with the transcription factor (Fig. 5). In the region from –629 nt upstream of the translation start codon, the promoter activity was significantly activated by CfbHLH5. However, when the fragment between –629 nt and –1038 nt was truncated, the LUC activity decreased to a certain extent, suggesting that some cis-elements in this region could be recognized and integrated with CfbHLH5. When compared with the activity of single transformation with the LUC vector carrying the promoter of the CfAOC gene without any transcription factor, the results showed that the CfbHLH5 transcription factor could play a positive role in the expression of the CfAOC gene in tobacco.

Fig. 5
figure 5

Dual-luciferase assay of the relative LUC activity of the promoter sequence of the CfAOC gene when co-transformed with CfbHLH5. The LUC/REN ratio of the full length CfAOC promoter vector was used as control. The data represent the means and standard deviations from three biological replicates

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The transient co-transfection of the promoter of the CfJMT gene and the CfbHLH36 transcription factor decreased its promoter activity in tobacco

Similarly, a series of truncated promoter sequences of the CfJMT gene were inserted into pGreen II 0800-Luc vector to characterize the interaction sites of the CfJMT promoter. The LUC activity also showed that the core sequence of the promoter near the translation start site played a crucial role in the promoter activity (Fig. 6), the same as in the CfAOC promoter. In the region from –960 nt upstream of the translation start codon, the promoter activity was significantly activated by CfbHLH36. However, when the fragment between –960 nt and –1121 nt was truncated, the LUC activity was significantly decreased, suggesting that some cis-elements in this region could be recognized and integrated with CfbHLH36. When compared with the activity of single transformation with a LUC vector carrying the promoter of the CfJMT gene without any transcription factor, the results showed that the CfbHLH36 transcription factor had a negative effect on CfJMT promoter activity in tobacco.

Fig. 6
figure 6

Dual-luciferase assay of the relative LUC activity of the promoter sequence of CfJMT gene when co-transfected with CfbHLH36. The LUC/REN ratio of the full length CfJMT promoter vector was used as control. The data represent the means and standard deviations from three biological replicates

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Discussion

Orchids are famous for their fragrance, color, and charm, and account for a great proportion of sales in the flower market. However, their production is mostly limited to traditional propagation and breeding methods. In recent years, along with the rapid development of molecular and transgenic technology, extensive studies have focused on flower induction and development in orchids (Wang et al. 2017, 2019). Due to the complicated components of the flower fragrance of orchids, it is difficult to study fragrance production at the molecular level. In Cymbidium faberi, the main component of the flower fragrance is methyl jasmonate (MeJA) (Omata et al. 1990; Zhou et al. 2018). The genes encoding the crucial enzymes in the MeJA biosynthesis pathway, CfAOC and CfJMT, have been cloned from C. faberi and ectopically expressed in tomato. However, they were found to participate in the stress response but rather than influencing the volatile emissions of the plant (Zhou et al. 2018; Xu et al. 2019).

The promoter of a gene plays a great role in its expression and regulation at the transcriptional level. Many studies have focused on promoter modification to improve target traits in plant biotechnology (Hernandez-Garcia and Finer 2014). A chimeric promoter derived from potato and Arabidopsis can induce guard cell-specific expression of genes of interest under water deficit conditions (Na and Metzger 2014). In rice, bacterial transcription activator-like (TAL) effectors can bind to effector-binding elements (EBEs) in SWEET gene promoters. EBE variants that could not be recognized by TAL effectors abrogated this induction, causing resistance (Eom et al. 2019), Therefore, a corresponding diagnostic kit enables the analysis of the bacterial blight in the field and identification of suitable resistant lines. Many specific promoters were cloned and could induce genes of interest expressed in specific tissues or during specific developmental periods (Chen et al. 2014; Luan et al. 2019), which can provide great convenience in the genetic engineering of novel cultivars (Kaur et al. 2020); Ren et al. 2019; Tzean et al. 2020).

To intensively explore the expression patterns of the CfAOC and CfJMT genes, we cloned their promoter sequences. Many conserved cis-elements were found in the promoters of CfAOC and CfJMT, mainly responsive to hormones, biotic and abiotic stresses, indicating the important functions of MeJA in C. faberi and its interconnection with other hormones, as reported in the mango (Winterhagen et al. 2019), grapevine (Ahmad et al. 2019), and tea (Zhang et al. 2019). In the promoter sequences of both CfAOC and CfJMT, the truncation of core elements such as the TATA and CAAT boxes proximal to the ATG was found to significantly reduce the promoter activity, as was observed in other organisms (Figs. 5 and 6) (Lubliner et al. 2015; Lin et al. 2008). The CfAOC gene was highly expressed in flowers at the blooming stage, but significantly lower in vegetative tissues. And the lowest expression of CfAOC gene was in flowers at the withered stage (Zhou et al. 2018). The expression level of CfJMT gene was the highest in flowers, especially at the blooming stage, but was almost undetectable in vegetative tissues (Xu et al. 2019). Combined with the spatiotemporal expression pattern of CfAOC and CfJMT genes in C. faberi, these results suggested that the CfAOC promoter is comparatively constitutive while the CfJMT promoter is flower-specific, since the former could express the gus gene in the seedlings of transgenic tobacco, and the latter could not. In the mature plant, CfAOC and CfJMT promoters both unlikely activated the expression of gus gene strongly except that CfAOC promoter had an activity in the root at any stages. Considering the differences between monocots and dicots, CfAOC and CfJMT promoters derived from C. faberi might have different regulatory ways in tobacco. For example, lack of corresponding regulatory proteins in tobacco could result in weak activities of CfAOC and CfJMT promoters.

In eukaryotes, gene expression is frequently mediated by multi-protein complexes (Zimmermann et al. 2004). Transcription factors are closely associated with a variety of plant physiological processes, and the basic helix–loop–helix transcription factor MYC2 is involved in the regulation of the MeJA pathway (Lorenzo et al. 2004; Song et al. 2014; Shin et al. 2014; Zhuo et al. 2020). To reveal the target sites of CfbHLH transcription factors in the promoters of the CfAOC and CfJMT genes, the transient co-transfection experiments were conducted in this study. The relative LUC activity in the dual-luciferase assay showed that the fragment between –629 nt and –1038 nt of the CfAOC promoter, as well as the fragment between –960 nt and –1121 nt of the CfJMT promoter were very important for the recognition by CfbHLH transcription factors. Coincidentally, there are two G-box motifs, –734 nt upstream of the ATG codon in the CfAOC promoter and –1088 nt upstream of the ATG codon in the CfJMT promoter, which are likely recognized by CfbHLH transcription factors and induce the expression of the CfAOC and CfJMT genes in C. faberi. This interaction will be further studied by EMSA in the future. The CfbHLH5 transcription factor could increase the activity of the CfAOC promoter but the CfbHLH36 transcription factor decreased the activity of the CfJMT promoter, suggesting that CfbHLH transcription factor may play both positive and negative roles in the regulation of MeJA biosynthesis, which was consistent with other studies in Arabidopsis (Nakata et al. 2013; Shin et al. 2014).

Conclusions

The flower fragrance is an important economical and appreciative trait of C. faberi. To reveal the regulation mechanism of flower fragrance in this oriental orchid, the promoter sequences of the CfAOC and CfJMT genes from MeJA pathway were cloned and analyzed. The expression pattern of CfAOC and CfJMT promoters was assessed in transgenic tobacco plants. Additionally, the target sites of CfbHLH transcription factors in the promoters were identified in tobacco in vivo, and will be further verified by EMSA in the future. This study provides further theoretic guidance for the genetic modification of the flower fragrance trait in non-scented orchids.