Cloning and characterization of the DIR1 promoter from Eucommia ulmoides Oliv and its response to hormonal and abiotic stress

The lignans of Eucommia ulmoides have been extensively studied and shown to have a dual mechanism of regulating blood pressure. Studies have shown that DIR1 is a key gene in the biosynthetic pathway of lignans in Eucommia ulmoides Oliv. In this study, a 2000 bp upstream promoter sequence was cloned, and part of the sequence (1495 bp) and its 5′-end truncated segment were constructed into the pCAMBIA1391Z expression plasmid upstream of ß-glucuronidase (GUS). Agrobacterium-mediated genetic transformation produced stable transgenic tobacco lines. The results showed that although both full-length and truncated promoters could initiate GUS expression, their levels were influenced by the degree of deletion at the 5′ end. GUS histochemical staining showed that the core promoter region was located in the region containing the transcription initiation site (TIS) within 212 bp. In addition, the DIR1 promoter responded to environmental and hormonal stressors, such as jasmonic acid (MeJA), abscisic acid (ABA), d-mannitol (drought mimic), and high concentrations of NaCl. In transgenic tobacco seedlings, MeJA, d-mannitol, and ABA could activate the DIR1 promoter, whereas high concentrations of NaCl could inhibit it. In E. ulmoides Oliv seedlings, MeJA, NaCl, and d-mannitol activated the DIR1 promoter, whereas ABA had an inhibitory effect. In summary, our findings provide a theoretical basis for the use of the DIR1 promoter in plant genetic engineering, indicating its potential. Our study also presents novel insights for lignan biosynthesis and sheds light on the mechanisms of E. ulmoides Oliv in response to stress. We successfully cloned the DIR1 gene promoter and studied its function in regulating the expression of the DIR1 gene.


Introduction
Eucommia ulmoides Oliver (E. ulmoides), a deciduous tree belonging to the Eucommiaceae family, is a traditional and valuable source of Chinese medicine (Luo et al. 2020). E. ulmoides is also a rich source of collagen and has attracted considerable attention (Nakazawa and Toda 1995;Zhao 2019). Over 200 compounds have been isolated from E. ulmoides, including lignin, cycloolefin ethers, and terpenes, as well as pharmacologically active phenylpropanoids, iridoids, and pinoresinol diglucoside (Wang et al. 2019;Zou et al. 2019;Liu et al. 2020). Pinoresinol diglycoside is a lignan from E. ulmoides that is known to have medicinal properties (Zou et al. 2019). E-coniferyl alcohol is a general precursor for the synthesis of lignans, and pinoresinol is formed by the dimerization of two molecules of E-coniferyl alcohol. The formation of pinoresinol is a key . The DIR1 coding gene plays a key role in the biosynthesis of lignans, including pinoresinol diglycoside .DIRIGENT (DIR) proteins promote the stereospecific coupling of monolignol units to form either ( +)-pinoresinol or ( −)-pinoresinol, the first intermediates of the lignan biosynthetic pathway Gasper et al. 2016). Studies have shown that DIR proteins are encoded by heterologous genes and that they are involved in the synthesis of lignans in several plant species (Lv and Zhang 2017;Nobile et al. 2017;Uchida et al. 2017). In addition, the DIR proteins of higher plants, such as pear (Cheng et al. 2018), soybean GmDIR22 (Li et al. 2017), Cucumber ) and alfalfa (Behr et al. 2015) have been isolated. The promoter region of a gene controls the intensity, location, and pattern of gene expression (Tang and Tu 2015;Zhang et al. 2004). Although constitutive promoters are usually used to drive plant transgene expression (Srivastava et al. 2014), they are associated with a few drawbacks. For instance, they sometimes cannot achieve time-controlled gene expression (Gurr and Rushton 2005;Zheng et al. 2007). Therefore, researchers are focusing on the use of core and gene upstream promoter elements to achieve timecontrolled gene expression (Franssen et al. 2017;Yu et al. 2019). In the gene function verification, the tobacco model is already a mature research model, with a complete genetic transformation system, convenient and quick transformation, and can quickly obtain resistant buds from callus. This experiment uses the tobacco model to study the function of promoter fragments of different lengths, so as to better verify the function and structure of the promoter. In this study, the DIR1 gene promoter was cloned, bioinformatics analysis was performed, and the response elements were analyzed for different promoter fragments deleted at the 5′ end.

Bioinformatics analysis and cloning of the DIR1 promoter
Based on the known DIR1 gene sequence, Primer Premier 5.0 and oligo 7.0 were used to design primers specific for the promoter, and a Sal I and EcoR I restriction site was introduced at the 5′ and 3′ ends ( Table 1). The 2000 bp promoter sequence was amplified and cloned by BGI (Beijing, China). Briefly, genomic DNA was extracted from E. ulmoides using the CTAB method, and amplified using KOD high-fidelity  ACT TCC GGA CAT CTG AAC CT 60 enzyme with the following reaction parameters: pre-denaturation at 98 °C for 30 s, followed by 35 cycles of denaturation at 98 °C for 15 s, annealing at 61 °C for 30 s, and extension at 68 °C for 1 min, and a final extension of 10 min at 68 °C. The polymerase chain reaction (PCR) products were electrophoresed using a 1.2% gel, and the target fragment was cut and purified using an E.Z.N.A. Gel Extraction Kit. The cloned 2000 bp promoter sequence was ligated to the pClone007 vector and sequenced using BLAST against the genomic DNA. The 2000 bp DIR1 promoter sequence obtained by cloning and sequencing was submitted to BDGP to predict the transcription start site. The PlantCARE (http:// bioin forma tics. psb. ugent. be/ webto ols/ plant care/ html/) (Lescot et al. 2002) online website was used to analyze and predict the cis-acting regulatory elements in the promoter region and for the initial determination of the core region of the promoter.
The respective EuDIR1p fragments were cut using Sal I and EcoRI and ligated to the double-digested pCAM-BIA1391Z expression vector (0.4 μL Sal I, 0.4 μL EcoRI, 4 μL plasma, 2 μL of 10 × buffer H, made up with ddH 2 O to a total volume of 13.2 μL at 37 °C for 6-12 h) with T4-ligase overnight. Competent E. coli DH5α cells were transformed with the EuDIR1p-pCAMBIA1391Z constructs using the heat shock method, and the respective plasmids were harvested using the alkaline cleavage method, verified by SalI and EcoRI digestion, and then sequenced. Next, the verified plasmids were used to transform Agrobacterium LBA4404 competent cells using the heat shock method. Finally, transgenic Nicotiana tabacum explants were generated by incubating the leaf discs with the genetically engineered Agrobacterium.

GUS staining verification of genetically modified tobacco
The integration of EuDIR1p sequences into the tobacco genome was verified using PCR using the primers specific for EuDIR1p-4. Wild-type tobacco DNA was used as a negative control, whereas pCAMBIA1391Z-pEuDIR1-4::GUS plasmid served as a positive control. The in situ expression of GUS was determined using histochemical staining. Briefly, the buds, roots, stems, and leaves of mature plants were cut into small pieces (2-3 cm) after 20-40 days of rooting and incubated overnight with the GUS staining solution at 37 °C. The solution was discarded and the leaves were decolorized using 75% alcohol at 15-25 °C to completely remove the chlorophyll and visualize the coloring effect of the GUS dye solution.

Stress induction
Seedlings of E. ulmoides and different transgenic tobacco were grown at 28 and 25 °C, respectively (12 h day-night culture), and 70% relative humidity, and seedlings with similar growth rates were selected for subsequent experiments. Appropriate pressure induction chemical treatments were performed on the same batch of plants using distilled water treatment as a control. Each experiment used three biological and technical replications. To induce hormonal stress in E. ulmoides, 100 μmol/L each of methyl jasmonate (MeJA) and abscisic acid (ABA) were sprayed on the leaves. To induce drought stress and osmotic stress, the seedlings were grown in 100 ± 0.37 g of uniform high-quality soil. When the soil moisture was 4.8 ± 0.2%, 300 mmol/L mannitol and 10 g/L NaCl were used for irrigation treatment. To induce stress in tobacco seedlings, the samples were treated with the same concentration of chemical reagents used for the induction of stress in E. ulmoides. EuDIR1p-1 tobacco was treated with MeJA and NaCl, EuDIR1p-2 tobacco was treated with mannitol, and the EuDIR1p-3 tobacco was treated with ABA. Tobacco seedlings were sampled at 0, 3, 6, 12, 24, and 48 h, and the RNA was extracted and reverse-transcribed for GUS expression analysis. DIR1 gene expression was analyzed in E. ulmoides seedlings at 0, 3, 6, 12, and 24 h after RNA extraction and reverse transcription (Singh et al. 2020;Zhou et al. 2020).

Real-time fluorescence quantitative analysis
To compare the relative expression level of GUS in transgenic tobacco with DIR1 in E. ulmoides to better verify the cis-acting element function of different promoter fragments, real-time fluorescence quantitative analysis was performed. Apart from GUS histochemical staining, fluorescence quantitative analysis is also used to monitor the relative expression of GUS. RNA was extracted from the E. ulmoides and tobacco seedlings using the RNA Pure kit and then reversed transcribed to cDNA. The relative expression levels of EuD-IR1p and GUS were analyzed using RT-PCR with EuActin (Ye et al. 2018) and β-actin as the respective housekeeping genes for E. ulmoides and tobacco. The reaction parameters were as follows: pre-denaturation at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 10 s, and annealing at 60 °C. The primers are listed in Table 1. Three biological and three technical replicates were tested per sample, and the gene expression levels were calculated as previously described (Hellemans et al. 2007), using SPSS 22.0 and GraphPad Prism 8.3.0.

Cloning and bioinformatics analysis of DIR1 promoter
We successfully cloned a promoter sequence of 2000 bp upstream of the DIR gene. The sequenced 2000 bp promoter sequence was submitted to the PlantCARE database for bioinformatics analysis. As shown in Fig. 1 Based on the results of bioinformatics analysis, the sequence containing the important cis-acting elements were selected and cloned into expression vectors for further research. As seen in Online Resource 1, the sequence of 1495 bp upstream of the DIR1 gene was successfully cloned using PCR. The putative positions and functions of the cisacting elements in EuDIR1p-1 and its 5′-end truncated constructs are shown in Table 2. The elements contained in EuDIR1 promoter fragments of different lengths and their positions are shown in Fig. 2.

GUS staining verification of genetically modified tobacco
Different amplified EuDIR1p fragments were cloned into expression vectors and transformed into tobacco seedlings using the Agrobacterium-mediated leaf disc method (Online Resources 2 and 3). The transgenic plants were screened using PCR (Online Resource 4). The transgenic tobacco with different truncated fragments was stained using GUS staining solution and the staining results are shown in Fig. 3. All EuDIR1p-1, -2, -3, and -4 transgenic tobacco plants exhibited positive GUS immunostaining. It can be seen that the core elements present in EuDIR1p-1 and truncated promoters promoted GUS expression. However, unlike the truncated promoter, stronger staining intensity was observed in plants expressing EuDIR1p-1.

Different DIR1 promoter fragments stimulate GUS expression under hormonal and abiotic stress
The EuDIR1p-1 transgenic tobacco plantlets harboring the CGTCA promoter element were stimulated with 100 µM MeJA. Compared to the baseline level, GUS gene expression increased significantly by 5.065-fold in a time-dependent manner at 3 h. Then, the expression of the GUS gene gradually decreased and showed an upward trend after 24 h; however, after 48 h, the expression was inhibited (Fig. 4a). At 3 h after ABA treatment, GUS in EuDIR1p-3 transgene was significantly upregulated by 3.1 times, and recovered in about 24 h (Fig. 4b). In this study, we exposed the EuD-IR1p-1 transgenic plants to NaCl (10 g/L), which markedly suppressed GUS levels in a time-dependent manner. Compared to the baseline expression, GUS was downregulated 0.104-fold and 0.258-fold after 12 and 24 h of NaCl irrigation, respectively (Fig. 4c). Mannitol initially increased the expression of GUS in EuDIR1p-3 transgenic tobacco by 2.071 times at 6 h, but downregulated GUS expression within 12-24 h (Fig. 4d).

DIR1 promoter stimulates DIR1 gene expression in E. ulmoides seedlings under hormonal and abiotic stress
Bioinformatics analysis indicated the presence of stressrelated response elements including CGTCA-motif, MYB, Myb, and MYC in EuDIR1p. Consistent with this observation, E. ulmoides seedlings treated with MeJA or ABA showed time-dependent changes in DIR1 expression. After 3 h of MeJA treatment, DIR1 levels increased 7.56 times compared to that at the baseline, slowly decreased to the baseline level, and was followed by an upward trend after 24 h (Fig. 5a). The observations were similar to those observed in genetically modified tobacco seedlings. On the other hand, 100 μM ABA downregulated DIR1 0.061 times relative to the baseline levels after 6 h, which then recovered after 24 h (Fig. 5b). These findings were the opposite of those seen in genetically modified tobacco. To determine the effect of abiotic stresses on the DIR1 promoter, E. ulmoides seedlings were treated with 40% mannitol and 10 g/L NaCl to simulate drought and high-salt conditions, respectively. Contrary to the results seen in genetically modified tobacco, it was observed that osmotic stress significantly upregulated the expression of DIR1 in E. ulmoides seedlings; the expression level was 7.98 times that at the baseline level after 6 h, which recovered after 24 h (Fig. 5c). Drought simulation, on the other hand, upregulated DIR1 within the first 12 h of mannitol exposure, with a maximum increase of 2.98-fold at 12 h, which recovered after 24 h (Fig. 5d). In transgenic tobacco, mannitol also upregulated gene expression, but the difference was that it emerged inhibition after 12 h.

Discussion
The biosynthesis of lignans mediated by the DIR protein is mainly initiated in the cambium of stems, the starting area of ray cells, and the palisade layer of leaves, and is consistent with the appearance of lignans for defense (Burlat et al. 2001). The DIR protein itself does not have a catalytic center and cannot directly catalyze the synthesis of lignans; however, it confers optical rotation to the lignans. It can capture the monomer free radicals of lignans in the presence of oxidases, such as laccase, resulting in the biosynthesis of structure-specific dimers and macromolecular lignans (Davin and Lewis 2000;Borges et al. 2013;Seneviratne et al. 2015). Flax (Linum usitatissimum L.) and isatis (Isatis indigotica Fort.) are two important model plants for studying lignan biosynthesis. Previous studies have confirmed that DIR responds to biotic and abiotic stresses. Li first discovered and analyzed the gene family of the DIR protein in Isatis. They used MeJA to induce stress, and then tested the transcription abundance of DIRs and found that some DIR genes responded to exogenous MeJA (Li et al. 2014). In flax, Corbin studied the transcriptional response of developmentrelated plant hormones, including naphthaleneacetic acid, Stress induction using salt and insect feeding has also been carried out in plants such as cotton and spruce. These related studies have shown that DIR genes can respond to adversity stress, thereby mediating plants to synthesize lignans against these elements of stress (Ralph et al. 2007;Li et al. 2015).
There have been related studies that have constructed the promoter as a deletion fragment expression vector, followed by studying the response elements in the promoter (Qin et al. 2019). In the current study, 2000 bp sequences of DIR1 gene were successfully cloned and the missing fragment was used to identify its response elements. GUS staining revealed that stronger staining was observed in transgenic tobacco with the 1495 bp promoter. This result indicates that an enhancer was probably in the region from − 1495 to − 804 bp (Fig. 5).
Genes can be either upregulated or downregulated by various biotic and abiotic stresses Lv and Zhang 2017). It is known that CGTCA is a putative response element of MeJA, and that MYB is involved in the stress response to droughts, jasmonic acid, and powdery mildew infections (Baumann et al. 2007;Kamiya et al. 2015;Ni et al. 2019). MYC regulates the response to abiotic stresses (such as drought) and hormones (such as ABA) ). In plants, Lac and DIR protein usually co-catalyze coniferyl alcohol. First, Lac catalyzes the substrate to synthesize three free radical precursors, and then under the catalysis of a specific DIR protein, regio selectivity and stereo selectivity are performed at the same time (Pickel et al. 2010). For example, in the synthesis process of pinoresinol, the position is selected so that coniferol mainly produces racemic ( ±)-pinoresinol instead of neolignan. The results from our study showed that after treatment with MeJA or d-mannitol, the expression of GUS in transgenic tobacco increased significantly, which was consistent with the DIR1 expression in E. ulmoides. The MeJA and d-mannitol response elements in the promoter likely had a positive regulatory role at that time. However, the results obtained following ABA and NaCl treatment in transgenic tobacco and E. ulmoides were different, which indicated that stress treatment had certain differences in the activation or inhibition of the DIR1 promoter in different species. A possible reason is that the expression of genes in E. ulmoides is more complicated, and the expression of other genes is inconsistent with the expression of the GUS gene in transgenic tobacco after interaction with other genes. In addition, there exists another possibility that in plants, DIR genes are part of the defense response to biological stress (Paxton 1980). Its induction may have evolved in different plant lineages, leading to differences in expression between plants (Ralph et al. 2006). Previous studies have also shown that certain transcription factors have inconsistent effects in different species. Similarly, the functions of MYB may be completely opposite in different species, probably owing to high functional diversity in the MYB family members. (Fang et al. 2020). In summary, further studies are necessary using similar treatment to verify the reasons for inconsistency in gene regulation and expression in E. ulmoides and tobacco.

Conclusions
In this study, the DIR1 gene promoter was cloned, constructed as a deletion fragment expression vector, and introduced into tobacco. It was found that in tobacco plants, the promoter could successfully initiate the expression of the GUS gene. Using bioinformatics analysis, the transcription start site and the related cis-acting elements of the promoter were predicted. Additionally, we discovered that in the E. ulmoides seedlings, the DIR1 gene promoter could be activated by MeJA, NaCl, and d-mannitol, and inhibited by ABA. However, the results of GUS gene expression in tobacco are slightly different from those observed in E. ulmoides. These findings indicated that the promoter activates or inhibits gene expression, although with certain differences among species. Overall, our study provides a theoretical basis not only for the use of DIR1 gene promoter but also sheds light on the stress response mechanism of E. ulmoides to adversity stress and in the synthetic regulation pathway of lignan pinoresinol.
Authors' contributions ZYC and ZDG conceived and planned the experiments. LZY and LB carried out the experiments. LZY wrote the manuscript, all authors edited and approved the manuscript.