Cloning and functional analysis of the promoter of a UDP-glycosyltransferase gene from Panax quinquefolium L.

Pq3-O-UGT2 is a key UDP-glycosyltransferase gene in the ginsenoside biosynthesis pathway of Panax quinquefolium L. Functional analysis of promoter sequences plays an important role in understanding the regulation of functional gene expression. In this study, chromosome walking technology was used to isolate the 1399 bp sequence upstream of the ATG initiation codon of Pq3-O-UGT2. Bioinformatics analysis shows that the promoter sequence contains a large number of putative cis-acting elements responsive to exogenous and endogenous factors. The full-length promoter and six 5′ terminal truncations were fused with the GUS reporter gene to test their activities. The results of histochemical staining showed that a strong GUS activity were observed in flowers, siliques, leaves, stems and roots of transgenic Arabidopsis containing the full length Pq3-O-UGT2 promoter. Fluorometric assays showed that the highest enzyme activity is the full-length promoter with 4370 pmol 4-MU/min/mg protein, and the shortest promoter containing P-198::GUS with 45 pmol 4-MU/min/mg protein was sufficient to activate GUS expression. In addition, extended light, low temperatures, MeJA, ABA, NAA and GA3 were selected to investigate the Pq3-O-UGT2 promoter in response to abiotic stress and hormone treatment. The GUS activity of Pq3-O-UGT2 full-length promoter (P-1399) was 2.12 times that of the control plant after MeJA treatment, suggesting that P-1399 is a MeJA-inducible promoter. Furthermore, the assay results showed that ABA could extensively induce GUS expression in five 5′ truncated promoter transgenic plants, except P-198. These experimental results will help us to better explore the regulatory mechanisms in the promoter region of the Pq3-O-UGT2 gene and contribute to further studies of the molecular mechanisms of glycosylation in ginseng plants. The Pq3-O-UGT2 promoter was successfully cloned. Seven 5′ truncations could drive the expression of GUS gene in transgenic Arabidopsis plants and the full-length promoter is a MeJA-inducible promoter.

The most important active ingredient in the pharmacologically active substances of P. quinquefolium is ginsenosides, which are triterpenoid saponins modified by glycosylation (Lu et al. 2017;He et al. 2019). To date, more than 150 ginsenoside monomers have been identified from the Panax species, such as P. quinquefolium, P. ginseng and P. notoginseng (Li et al. 2022). Among them, 24(R)-pseudoginsenoside F11 (P-F11) is considered to be the characteristic component in P. quinquefolium (Cao et al. 2020). Various ginsenosides can be divided into three categories based on the differences on basis structural: pentacyclic triterpenoid oleanane saponins, octylene-type saponins and tetracyclic triterpenoid dammarane saponins, which content more than 75% of the total saponins. According to the type and position of the substituents type, the tetracyclic triterpenoid dammarane saponins are divided into protopanaxadiol (PPD) and protopanaxatriol (PPT) type ginsenosides. (Lu et al. 2018;Cao et al. 2020).
The biosynthesis of ginsenosides in P. quinquefolium is the result of the interaction of multiple genes (Fig. 1). The current understanding of biosynthesis of ginsenosides includes the mevalonic acid (MVA) pathway in the cytoplasm and the methylerythritol phosphate (MEP) pathway in plastids. Relevant studies have shown that the MVP Fig. 1 The ginsenosides biosynthesis pathway in P. quinquefolius L. MVA: mevalonic acid; MEP: methylerythritol 4-phosphate; IPP: isopentenyldiphosphate; β-AS: β-amyrin synthase; GTs: Glycosyltransferases pathway is the main pathway for the biosynthesis of ginsenosides (Hou et al. 2021;Zhao et al. 2014;Liao et al. 2016). The downstream biosynthesis of ginsenosides starts form cyclization of 2,3-oxidosqualene by oxidosqualene cyclases (OSCs), followed by hydroxylation by cytochrome p450s (CYP450s) and glycosylation by UDP-glycosyltransferases (UGTs) (Han et al. 2006).
As the last step in ginsenoside biosynthesis, glycosylation modification is an important stage in determining the activity of saponins (Xiao et al. 2019). Glycosylation modification affect the biological activity, water solubility and stability of the molecule, has great significance to the structure and pharmacological activity of plant secondary metabolites. Glycosyltransferases (GTs) are a class of highly differentiated, multi-member metabolic enzymes that belong to the multigene transferase family (Wu et al. 2021). In our previous research, a UDP-glycosyltransferase gene, Pq3-O-UGT2 (Genbank accession No: KR106207) have been isolated and identified from P. quinquefolius. In vitro enzymatic activity experiments biochemically confirmed that Pq3-O-UGT2 could produce ginsenoside Rg3 and Rd by transfer a glucose moiety to the C3-O-glucoside of ginsenoside Rh2 and F2 (Lu et al. 2017). In addition, the amino acid sequence of Pq3-O-UGT2 had high similarity with that of PgUGT94Q2 in P. ginseng, but showed more efficient than PgUGT94Q2 in enzymatic catalysis (Yao et al. 2022). Those recent related studies have shown that the mechanism of Pq3-O-UGT2 in ginsenoside biosynthesis is worthy of further study, but the transcriptional regulation mechanisms and activity of the promoter Pq3-O-UGT2 remain largely unknown, and there is no reported on the transcriptional regulation of Pq3-O-UGT2 yet.
In this study, the upstream sequence of Pq3-O-UGT2 gene from P. quinquefolium was cloned and sequenced by chromosome walking technology. The plant expression vector including full-length and six 5′ terminal series truncations were constructed to verify the activity of the promoter. Extended light, low temperatures, Methyl jasmonate (MeJA), abscisic acid (ABA), naphthylacetic acid (NAA) and gibberellin (GA3) were selected to investigate the Pq3-O-UGT2 promoter in response to abiotic stress and hormone treatment. This article can enrich and improve the understanding of the transcriptional regulation of Pq3-O-UGT2 gene from P. quinquefolium.

Construction of the plant expression vectors
The full-length promoter and six 5′ terminal series truncations were fused in the plant binary expression vector pBI121 to construct seven plant expression vectors (Fig. 2). All seven fragments inserted into the expression vector were obtained by PCR. Each forward primer carrying HindIII restriction site and reverse primer carrying SamI restriction site were designed ( Table 1). The PCR amplified products were double digested (HindIII and SamI) and inserted into the corresponding site of the HindIII/SamIdigested binary expression vector pBI121 at the upstream of the β-glucuronidase (GUS) reporter gene, replacing the complete sequence of CaMV35S promoter. Then recombinant plasmids weres transferred into E. coli DH5α competent cells for sequencing. Positive clones were selected and named as: P-1399::GUS, P-1197::GUS, P-998::GUS, P-801::GUS, P-605::GUS, P-402::GUS, and P-198::GUS.

Genetic transformation
To investigate the activity of Pq3OUGT2 promoter, the positive control vector pBI121 (CK+) and the seven confirmed recombinant plasmids were introduced into Agrobacterium tumefaciens strain GV3101 using a freeze-thaw method. Transgenic Arabidopsis plants were generated by the floral dip method. Transformations were cultured on Murashige and Skoog (MS) medium containing 50 µg/mL kanamycin and harvest each kanamycin-resistant transgenic line. The positive lines were identified by PCR analysis, and primers were showed in Table 1. The gDNA isolated from the putative transgenic Arabidopsis plants by cetyltrimethylammonium bromide (CTAB) method, and the wild type of Arabidopsis plants (col 0) was used as the negative controls (CK-). Three positive lines and three negative lines were Vector construction and positive clone verification 5′-GATGCCATGTTCATCT-GCCCAGT-3′ Restriction sites of HindIII in forward primers and SamI in reverse primers were underlined for each treatment and at least 12 transgenic Arabidopsis plants were selected for each treatment.

Cloning and sequence analysis of the Pq3OUGT2 promoter
Using the gDNA of P. quinquefolium as a template, the 1453 bp of the PCR products were cloned after four rounds of TAIL-PCR. The sequencing results were compared with the cDNA information of Pq3-O-UGT2, and the comparison results showed that the sequence obtained by TAIL-PCR included the 1399 bp promoter upstream of Pq3-O-UGT2 and part of the Pq3-O-UGT2 gene sequence. The sequence of Pq3-O-UGT2 promoter was analyzed by PlantCARE and newPLACE, and the results showed that Pq3-O-UGT2 promoter contained two typical promoter elementss (TATA and CAAT boxes), and a number of cis-acting regulatory elements (Table 2). Multiple light responsive motifs were found, including Box 4, G-box, GT1-motif, GA-motif, Box І, Box II, TCCC-motif, I-box, TCT-motif and chs-CMA1a. The distribution of several hormone-responsive elements carried in the promoter of Pq3OUGT2 is shown in Fig. 3, such as GARE motif for gibberellin, TGACG-motif and CGTCA-motif for MeJA-responsiveness, TGA element responsive to auxin and ABRE responsive to ABA. Many motifs linked to stress-responsive elements were also predicted, such as W-box involved in elicitation, wounding, pathogen responsiveness was located at -1245 bp and − 396 bp, TC-rich involved in defense responsiveness was located at -649 bp. In the Pq3-O-UGT2 promoter, the − 418 bp position contained an O 2 -site which was a cis-acting regulatory element involved in zein metabolism regulation . The prediction results of these cis-acting elements of light-, hormone-and stress-response expressing genes in the promoter region we cloned provide a theoretical basis for the future study of Pq3-O-UGT2 gene regulation mechanism.

Construction of plant expression vector and functional analysis of the Pq3-0UGT2 promoter
To characterize promoter regions regulating Pq3-O-UGT2 gene, the full-length promoter and a series of 5' truncations (P-1399, P-1197, P-998, P-801, P-605, P-402, and P-198) promoter sequences were ligated to the GUS reporter gene and transformed into Arabidopsis plants. Then, the T1, T2 and T3 transgenic Arabidopsis lines were screened on kanamycin medium and further confrmed by PCR using the primer sets, respectively. There were no obvious propagated to T3 homozygous seeds. T3 homozygous transgenic Arabidopsis plants were used for further experiments.

Gus assay
Histochemical GUS staining and fluorometric GUS assays were used to test the activity of the promoter. 7-day-old and 8-week-old Arabidopsis plants were used for GUS histochemical staining. The seedlings on the 7st day were stained with the whole plant, and the plants on the 8-week-old day were stained with roots, stems, leaves, flowers and siliques, respectively. Plants that need to be stained were immersed in X-Gluc solution buffer overnight at room temperature. Then, transfer the materials to 70% alcohol to decolorize until the negative material is white. Subsequently photographed to observe the expression of GUS gene.
For fluorometric GUS assays, about 100 mg of 4-weekold Arabidopsis materials were ground in liquid nitrogen, then mixed the powders with 1mL freshly GUS extraction buffer [50 mM phosphate buffer (7.0), 0.1% (v/v) Triton X-100, 10 mM Na 2 EDTA, 0.1% (w/v) SDS, 10 mM 2-Hydroxy-1-ethanethiol]. The mixture was centrifuged at 12,000 g for 10 min at 4 °C, and the supernatants were used for subsequent analysis. Bradford method was used to measure total protein concentration using bovine serum albumin (BSA) as a standard. GUS fluorometric assays was determined by measuring the amount of 4-methylumbelliferone (4-MU) using 4-methyl-umbelliferyl-glucuronide (4-MUG) (Sigma-Aldrich, St. Louis, USA) as substrate. Whole assays were held at 37 °C and added 0.2 M Na 2 CO 3 solution when terminated. The fluorescence wavelengths were set at 365 nm (excitation) and 455 nm (emission) respectively. GUS enzymatic activity was normalized as pmol of 4-MU per minute per milligram g protein. Three independent replicates experiments were calculated for each sample. Error bars indicate the mean ± standard error.

Hormones and abiotic stress treatments
The 6-week-old of T3 homozygous transgenic Arabidopsis plants were used for hormones and abiotic stress treatments analyses. the seedlings for low temperatures treatment were cultured at 4 °C in the dark and the seedlings for extended light treatment were continually illuminated for 48 h. For hormone treatments, the seedlings were sprayed evenly with 100 µM NAA, 100 µM MeJA, 100 µM ABA, and 100 µM GA3 until all branches and leaves were completely moistened. After 48 h, seedlings of all treatment groups and control materials were ground in liquid nitrogen, then stored at − 80 °C for subsequent protein extraction and GUS quantification. Three parallel biological repetitions were performed the flowers, siliques, leaves, stems and roots of transgenic arabidopsis plants (Fig. 6).
In order to quantitatively distinguish the expression of the GUS report gene in different transformed lines, GUS fluorometric assays were performed. The results are shown in Fig. 7. The highest GUS activity of transformed lines was P-1399::GUS (4370 pmol 4-MU/min/mg protein), which is 80.01% of the CaMV35S::GUS. Comparative analysis of GUS activity in the different transformed lines showed that the GUS activity of P-1399::GUS was 2.79-fold higher than that of the P-1197::GUS and P-998::GUS, P-1197::GUS was similar to the P-998::GUS, and P-801::GUS was 38.08% higher than the P-998::GUS. These results suggested that there exist some negative regulatory motifs in -1197 to -998 and − 998 to -801, which can inhibit the expression of GUS report gene. Moreover, the results of fluorometric quantification and histochemical analysis revealed that the shortest promoter, P-198, could drive the expression of the GUS morphological differences between the transgenic and wild type Arabidopsis plants under normal growth conditions (Fig. 4). After one week of culture, the transgenic lines with different promoter fragments were subjected to GUS staining. As shown in Fig. 5, different GUS staining were observed in transgenic lines with different promoter fragments. Strong GUS activity was observed in the P-1399 transformed lines and certain blue staining was found in the shortest construct (P-198) even if the GUS signal was weak. In addition, relatively low GUS activity were observed in the P-801, P-605 and P-402 transformed lines when compared to that of the full-length promoter, indicating that the length of the promoter or cis-acting elements in the deletion affected its GUS activity. To investigate the transcriptional activity of the full-length promoter, histochemical GUS assays were performed on more than five 8-week-old transformants. GUS staining results show that the full-length promoter strongly drives the expression of the GUS gene in  Once this region was removed, GUS activity increased less in the other transformed lines after MeJA treatment. When the transformants were treated with ABA, NAA and low temperatures, the promoter activity of P-1399 transformed lines were also significantly increased by 54%, 46% and 66%, respectively. Compared with other abiotic factors, extended light and GA3 slightly induced the GUS activity of P-1399 transformants. After the transformants were treated with different environmental factors, the results of Fig. 7 gene, suggesting that the 198 bp upstream of the ATG is likely to be the minimum core fragment for transcription.

Response of various promoter constructs to abiotic stresses and hormones
To further explore the Pq3-O-UGT2 promoter in response to abiotic stress and hormone treatment, homozygous T3 transgenic Arabidopsis plants were treated with extended light, low temperatures, MeJA, ABA, NAA and GA3. Results showed in Fig. 8, the GUS activity of full-length Pq3-O-UGT2 promoter (P-1399) was 2.12-fold higher than control plants after MeJA treatment, demonstrating Characterization of P-1399 transformants lines (P-1 P-2 and P-3) and wild type Arabidopsis (C-1 C-2 and C-3) at different developmental stages. P-1 and C-1: 7-day-old seedlings. P-2 and C-2: 2-week-old plants. P-3 and C-3: 8-week-old plants

Discussion
Glycosylation modification has important implications for the structure and pharmacological activity of plant secondary metabolites, and plays an indispensable role in different stages of plant growth and development (Ren et al. 2022). In our previous research, a UDP-glycosyltransferase gene, Pq3-O-UGT2 has been identified and has been shown to play an important role in the biosynthesis of ginsenosides in P. quinquefolius. Recent studies have suggested that Pq3-O-UGT2 showed more efficiency than PgUGT94Q2 (Yao et al. 2022), but the transcriptional regulation mechanism showed that ABA could broadly induced GUS expression in the five 5′ truncated promoters transgenic plants except for P-198. Among them, the promoter activity of P-1197, P-801 and P-605 transformed lines were significantly increased by 43%, 47% and 39%, respectively. However, no significant response signal was observed for the transformed lines containing the shortest promoter construct (P-198) to different environmental stimuli. Fig. 6 Histochemical analysis of GUS activity present in flowers(a), siliques(b), leaves(c), stems(d) and roots(e) for the fulllength transgenic Arabidopsis of 8 weeks old inhibit promoter activity, or that there may be a putative suppressor. These results of fluorometric quantification and histochemical analysis imply that the activity of the deletion fragment is not directly proportional to its length. This promoter sequence composition model was also found in AhLEC1A promoter of peanut, TeLCYe promoter of marigold, SaBS promoter of Santalum album (Zhang et al. 2019;Tang et al. 2021;Yan et al. 2020).
Mechanisms of transcriptional regulation in plants are complex, which are coordinated by many cis-acting elements and trans-acting factors (Chen and Qiu 2020). Promoters are the centers of transcriptional regulation, located upstream of gene coding regions and contain multiple cisacting elements in different regions of a promoter, which determine the expression activity of a target gene to a certain degree by combing with trans-acting factors (Yan et al. 2020;Zhou et al. 2020). Sequence analysis showed multiple cis-acting elements were predicted in the Pq3-O-UGT2 promoter related to different environmental stimuli, including light-, hormone-and stress-responsive elements. MeJA-responsive elements play an important role in the plant pathogen interaction gene, and have been demonstrated to improve plant resistance to environmental stresses by activating defense mechanisms (Zheng et al. 2011;Su et al. 2011;Li et al. 2021). Relevant studies have shown that MeJA treatment produced a significant induction effect on the GmTIP1;6 gene promoter of the vegetable soybean (Feng et al. 2022), and AtPEPR promoter from Arabidopsis thaliana (Safaeizadeh and Boller 2019). Similar results were found in this study, MeJA treatment for 48 h can induce the GUS activity of Pq3-O-UGT2 promoter deletion fragments, and activity of the promoter Pq3-O-UGT2 are still largely unknown, and no studies on the transcriptional regulation of Pq3-O-UGT2 have been reported. Moreover, promoters are important regions for regulating gene expression, and the study of promoters provides important evidence to shed light on the mechanism of gene expression regulation.
In this study, a 1399 bp nucleic acid fragment was isolated by four rounds of Tail-PCR using genome walking method. After comparison with both cDNA and gDNA sequences of Pq3-O-UGT2, the obtained fragment was identified as the sequence upstream of the ATG initiation codon of Pq3-O-UGT2, which is sufficient for the analysis of the promoter function of coding genes. The results of bioinformatics analysis indicated that multiple cis-acting elements were involved in the Pq3-O-UGT2 promoter sequence, and various cis-acting elements suggested the complexity of the promoter regulation. The promoter sequence and sequence analysis results provide a theoretical basis for its functional investigation. Seven fragments including the full-length promoter and six 5′ terminal series truncations were fused with the GUS reporter gene to test the activities of the promoter. Histochemical GUS staining results indicated that all the promoter constructs could stably drive GUS gene expression in transgenic Arabidopsis plants, and the shortest promoter construct (P-198::GUS) was sufficient to activate GUS expression. Fluorometric analysis showed that P-1399::GUS had the highest GUS activity with 4370 pmol 4-MU/min/mg protein, and the GUS activity of P-1197 and P-998 was 35.81% and 34.14% lower than that of P-1399, respectively. These results indicated that one or more cisacting elements located between − 1197 to -801 bp may Fig. 7 Quantitative GUS activity assays of transgenic Arabidopsis driven by the full-length promoter and six deletion promoter fragments. Each column represents the mean value with the standard error obtained from three independent experiments. Binary vector pBI121 containing CaMV35S promoter was used as a positive control. untransformed plants were used as CK.

Fig. 8
Quantitative GUS activity assays of transgenic Arabidopsis driven by the full-length promoter and six deletion promoter fragments under hormones and abiotic stresses. Each column represents the mean value with the standard error obtained from three independent experi-ments. Each asterisk above the error bar indicates significant difference (*p < 0.05, **p < 0.01). CaMV35S::GUS was used as a positive control the transcriptional regulation mechanism of the Pq3-O-UGT2 promoter in response to ABA signaling.
In this study, the genome walking method was used to isolate the 1399 sequence upstream of the ATG initiation codon of Pq3-O-UGT2 from P. quinquefolium. Histochemical staining has shown that the GUS gene can be stably expressed in transgenic Arabidopsis plants. The promoter deletion analysis revealed that P-1399::GUS has the highest GUS activity. The shortest construct containing P-198::GUS was sufficient to activate GUS expression, and the activity of the deletion fragment is not directly proportional to its length. In addition, GUS activity of different promoter fragments had different response to different environmental factors. The results obtained in this paper on the promoter of Pq3-O-UGT2 will facilitate the understanding of the complex regulatory mechanisms of glycosyltransferase gene and triterpene biosynthesis in ginseng plants.
among which the full-length promoter (P-1399) increased by 2.12 times. However, other six 5′ terminal series truncations did not observe a significant increase in GUS activity after MeJA treatment. These results demonstrate that the location of MeJA-responsive elements play an important role when the promoter P-1399 responds to MeJA treatment and the full-length promoter (P-1399) is an MeJA-inducible promoter. In addition, treatment with 0.25 mmol/L MeJA in P. quinquefolium increased the activity of squalene synthase gene promoter and affected the accumulation of saponin (Kochan et al. 2018). When treated with 0.1 mmol/L MeJA in P. ginseng, the gene expression of transcription factor MYB2 was up-regulated by 4.66 times. MYB2 up-regulated the expression of dammarediol II synthase (DS) gene by binding to the promoter of DS, ultimately increasing the accumulation of ginsenosides . Moreover, under the induction of MeJA, the expression of transcription factors such as WRKY and MYC were up-regulated, and the expression of key enzyme genes is up-regulated through interacting with transcription factors, and finally increasing the accumulation of triterpenoids Sharma et al. 2019). In this paper, the results showed that the GUS activity of Pq3-O-UGT2 promoter increased after MeJA treatment, and whether there are transcription factors involved in the signal transduction mechanism requires more experiments to verify.
ABA is used as a stress-inducing factor in plants to enhance plant resistance. The researchers found that ABA can effectively regulate plant material metabolism and play an important role in the signaling pathways associated with plant defense against salinity and drought stress (Maymon et al. 2022;Cheng et al. 2021;Gulzar et al. 2021). The TaNRX1-D promoter can be induced by ABA to drive GUS expression (Cheng et al. 2021), and similar results were also found in this study. After 48 h of ABA treatment, the GUS activity of the Pq3-O-UGT2 promoter deletion fragments were significantly enhanced. Unlike P-1399::GUS, ABA has a greater impact on the other five 5′ terminal series truncations (except for P-198::GUS) than extended light, low temperatures, MeJA, NAA, and GA3. Therefore, the cis-acting element in response to ABA contained in the Pq3-O-UGT2 promoter has a positive regulatory effect on promoter activity. Moreover, many reports have revealed that exogenous ABA treatment is beneficial to the accumulation of saponins in ginseng plants. After treatment with ABA, the expression of PqHMGR increased by 9.4 times, and the accumulation of monomeric saponin Rg2 increased by 17.38 times in the hairy root of P. quinquefolius. Therefore, further analysis of the ABA-responsiveness regulatory elements in the Pq3-O-UGT2 promoter through site-directed deletion analysis and site-specific sequence mutations may help us to understand Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.