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. 2020 May 30;15(7):1773664. doi: 10.1080/15592324.2020.1773664

Function analysis and stress-mediated cis-element identification in the promoter region of VqMYB15

Ruixiang Li 1, Fanding Zhu 1, Dong Duan 1,✉
PMCID: PMC8570707  PMID: 32475217

ABSTRACT

The transcription factor MYB15 plays an important role in grape basal immunity, and its promoter can be used as a potential target in resistance breeding. However, the regulatory mechanisms of cis-elements in its promoter region under a variety of stresses remain unclear. In this study, we identified some putative cis-regulatory elements present upstream of MYB15 in Vitis quinquangularis Shanyang (pVqMYB15_SY) and subsequently characterized the function of these elements using reporter assays. Our results showed that TCA-elements 1 and 2, ABRE, MYC and 3-AF1 binding site 1 are key cis-regulatory elements in pVqMYB15_SY and play important roles in plant bio/abiotic stress resistance.

KEYWORDS: Grapevine (Vitis quinquangularis), MYB15 promoter, functional analysis, GUS gene

1. Introduction

When plants are under biotic or abiotic stress, they are able to regulate the expression of specific genes to adapt to adversity. The level of transcriptional activation in eukaryotes is coordinated by upstream cis-acting elements in the regulation of gene expression, which are key links in plant environmental responses. Plant gene promoters contain a variety of important cis-acting elements that are involved in regulating the expression of corresponding downstream genes at the transcriptional level, thereby enabling plants to resist environmental stresses.1,2 Generally, the regions of a gene or gene family that code for a kind of protein in different species are relatively conserved; however, the similarity among their promoter sequences can vary.3,4 Therefore, the in-depth study of promoters from plants, including the determination of the core activating sequence and the number, type and distribution of cis-regulatory elements, can not only help us better understand the molecular mechanisms of gene regulation but also provide various functional promoters for genetic engineering.

In promoter function analysis, loss analysis is often used as an important method of identifying promoter cis-acting elements.5 At present, many regulatory roles of cis-elements have been predicted by using 5ʹ deletion analysis on the promoter.6,7 Studies have shown that the ZmRXO1 promoter might be regulated by light because its promoter sequence contains light-response elements such as BOXI and AE-box.8 ABREs are a class of elements capable of binding to strongly conserved abscisic acid (ABA)-dependent transcription factors,9 which exist in the promoter region of many stress-resistant genes and regulate the expression of related genes under abiotic stresses.10 The TCA-element between −563 bp and −249 bp upstream of OsPIANK1 may be responsible for resistance to Magnaporthe oryzae (M. oryzae) infection and exogenous salicylic acid (SA) application.6

MYB transcription factors (TFs) are an important family of TFs that are involved in various physiological processes,11 such as plant growth and development, stress responses, hormone signal regulation, and pathogen defense in plants. For example, they are related to the infection response to rice blast fungus,12 and they are involved in defense responses to grapevine pathogen infection caused by Plasmopara viticola, which is the most destructive grapevine disease in viticulture.13 Plant MYB TFs can be divided into four subfamilies: 1 R-MYB, R2R3-MYB, 3 R-MYB and 4 R-MYB.14 Among them, R2R3-MYB is the most abundant and versatile subfamily that exhibits diverse functions in plant development, biotic and abiotic stress responses, primary and secondary metabolism, hormone synthesis, and plant defense.15–17

The R2R3-MYB transcription factors MYB14 and MYB15 play positive roles in the activation of stilbene synthase, which is involved in the biosynthesis of stilbenes.18 In grapevine, stilbenes, as the main phytoalexins, can accumulate to inhibit fungal growth and resist abiotic stresses, such as powdery/downy mildew, UV or salt stress.19–22

In our previous study, we found that a calcium influx, an RboH-dependent oxidative burst, a MAPK cascade, jasmonate acid (JA) and salicylic acid (SA) co-contributed to flg22-triggered pVqMYB15_SY activation.23 In addition, we compared the promoter structures by looking for possible cis-regulatory elements and transcription factor-binding sites (TFBSs) between V. quinquangularis SY (a Chinese wild grape) and V. vinifera cv. Cabernet Sauvignon (a common European cultivar) and identified a specific MYB15 promoter allele (1972 bp) in V. quinquangularis SY that played a crucial role in plant basal immunity.23 However, the specific functions of some key cis-acting elements in pVqMYB15_SY that were predicted to be related to different stresses were not further investigated.

Therefore, in this study, based on this special MYB15 promoter from V. quinquangularis SY,23 we performed a predictive analysis with the PlantCARE (plant cis-acting regulatory DNA elements) database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). It was found that there were some key cis-acting elements in the sequence of pVqMYB15, such as TCA-elements, ABREs, MYCs and 3-AF1 binding sites. To verify the roles of these elements, we constructed missing-element mutants and further investigated their molecular functions through corresponding missing motifs triggered by SA, Phytophthora capsici, ABA and UV-C through heterologous expression in Nicotiana benthamiana and homologous expression in grapevine. Our data suggest that TCA-elements 1 and 2, ABRE, MYC and 3-AF1 binding site 1 are key cis-regulatory elements in pVqMYB15_SY and play important roles in plant bio/abiotic stress resistance.

2. Results

2.1. Promoter sequence analysis

The 1972 bp promoter sequence of VqMYB15 was submitted to PlantCARE for element prediction analysis.24 The results showed that the promoter sequence contained a large number of stress-response elements. For example, the 3-AF1 binding site, BOXI and BOX4 are related to light responses;25,26 TCA-elements are involved in SA responses and pathogen defense;27 and ABA response element ABRE,28 MYB binding site MBS,29 damage response element WUN motif,30 ethylene response element ERE,31 and 5ʹUTR Py-rich stretch are associated with high transcription levels in plants.32

2.2. The missing motif in pVqMYB15

To investigate whether the motifs in the upstream sequence of VqMYB15 were correlated with different corresponding stresses, we performed function-loss experiments as follows: a variety of β-glucuronidase (GUS) reporter gene fusion vectors with different kinds of cis-element deletions were constructed (Figure 1). The types and positions of missing elements in pVqMYB15 are shown in Figure 2.

Figure 1.

Figure 1.

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Construction of the GUS fusion vector of VqMYB15 promoter fragments with different kinds of cis-element deletions.

Figure 2.

Figure 2.

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The locations of missing cis-acting elements in pVqMYB15.

2.3. Induction analysis of pVqMYB15 exposed to SA while missing TCA-elements at different positions

The TCA-element was reported to be related to salicylic acid responses.27 As shown in Figure 2, we constructed five mutants with different TCA-element deletions in the promoter region of VqMYB15: ‘deletion TCA-element 1ʹ, ‘deletion TCA-element 2ʹ, ‘deletion TCA-element 3ʹ, ‘deletion TCA-elements 1 and 2ʹ and ‘deletion TCA-elements 1, 2 and 3ʹ. To further explore which TCA-element plays a key regulatory role in SA-induced pVqMYB15 activation, we performed an assay of promoter activity. Here, we evaluated SA-induced promoter activities by GUS transcripts (heterologous expression in Nicotiana benthamiana) and GUS protein levels (heterologous expression in Nicotiana benthamiana and homologous expression in grapevine). The activity of wild-type (pVqMYB15) after SA treatment was higher than that of the control, especially at the transcriptional level (Figure 3a, b and c). This result was consistent with our previous study and showed that SA might play a role in the activation of pVqMYB15, although its effect is not very strong.23 As shown in Figure 3, SA-induced pVqMYB15 activation was decreased for all mutants compared to that in the wild-type, but the decline was minimal in the mutant ‘deletion TCA-element 3ʹ. This means that the correlation between TCA-element 3 and SA is not significant. Therefore, we speculate that the regulatory roles of TCA-element 1 and TCA-element 2 are more closely related to the activation of pVqMYB15 than TCA-element 3.

Figure 3.

Figure 3.

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SA-induced pVqMYB15 activities for different TCA-element mutants. (a) Quantitative RT-qPCR of GUS transcripts in the leaves of Nicotiana benthamiana after SA infiltration; (b) Relative level of GUS enzyme activity in Nicotiana benthamiana; (c) Relative level of GUS enzyme activity in V. vinifera cv. Cabernet Sauvignon. Values show promoter activities relative to the corresponding solvent controls after treatment with 1 mM SA for 1 h. Values are mean values and standard errors from three independent experimental series. *P < .05 and **P < .01 indicate statistically significant differences (n = 3).

2.4. Induction analysis of pVqMYB15 exposure to Phytophthora capsici while missing TCA-elements at different positions

Phytophthora capsici (P. capsici) is a pathogenic oomycete that affects the cultivation of pepper and other crops, causing serious economic losses.33 Therefore, it is very important to clarify its pathogenic mechanism to provide theoretical and practical instructions for the prevention of Phytophthora disease. In plants, SA is usually correlated with a live parasitic bacterial infection and plays a key role in innate immunity.34,35 Increasing the concentration of endogenous SA or adding exogenous SA can increase stress resistance in plants.36 Recently, it was found that SA plays an important role in the process of pathogen infection and can significantly improve the resistance of pepper seedlings to pepper bacterial wilt through the interaction with pathogens.37

Therefore, in this study, we performed infection experiments to investigate the promoter activities of pVqMYB15 with different missing TCA-elements in response to P. capsici in Nicotiana benthamiana at different time points. As shown in Figure 4, the induction of the wild-type promoter was significantly increased after infection from 0 to 72 hours post-inoculation (hpi), especially at 12 hpi. Comparing the five missing constructs, it can be clearly seen that the promoter activity of mutant ‘deletion TCA-element 3ʹ was almost no different from that of the wild-type (Figure 4c). However, reductions in induction were substantial in the other four mutants (Figure 4a, b, d and e). This result is similar to that for the application of exogenous SA (Figure 3). Therefore, we speculate that TCA-elements in pVqMYB15 play an important role in the plant defense response to pathogens and that TCA-elements 1 and 2 are the key cis-regulatory elements.

Figure 4.

Figure 4.

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P. capsici-induced promoter activities for wild-type (pVqMYB15) plants and five mutants from 0 to 72 hour post-inoculation (hpi) in the leaves of Nicotiana benthamiana. The legends represent relative values of GUS transcripts in the wild-type and mutants compared to controls in non-inoculated leaves. Data represent mean values from three independent experimental series, and error bars represent standard errors.

2.5. The responses of ABA-related elements in pVqMYB15

ABA is an important plant hormone that can accumulate in many adverse situations and regulate the expression of downstream stress-related genes by integrating multiple stress signals.38 To investigate the role of ABA in the induction of pVqMYB15, we analyzed ABA response elements in the pVqMYB15 sequence and found ABA response elements such as MYC and ABRE (Figure 2). The detailed motif and position information of both cis-elements are shown in Figures 1 and 2. Here, we performed mutant construction experiments for pVqMYB15 (wild-type), ‘deletion ABRE’ and ‘deletion MYC’. In addition, we measured the transcript levels of GUS following heterologous expression in Nicotiana benthamiana after the addition of exogenous ABA, and we found that induction of ‘deletion ABRE’ and ‘deletion MYC’ were significantly decreased compared to that of pVqMYB15 (wild-type), which means that both ABRE and MYC play important roles in the ABA-induced activation of pVqMYB15.

2.6. The induction of photoresponse elements in pVqMYB15 after UV-C irradiation

Light is a fundamental factor in the control of many important biological processes during plant development and environmental responses. By analysis of the upstream VqMYB15 sequence, we found that a variety of cis-elements were correlated with responses to light (Figure 2). To further investigate the functions of these elements, we constructed 11 mutants with different deletions of the predicted motifs; their detailed sequence and position information can be found in Figure 1 and Figure 2. We transformed pVqMYB15 with different deletion fragments to infiltrate tobacco/grapevine leaves and adapted the fusion vectors for 2 days. After that, the leaves were treated with a short-term (10 min) UV-C treatment, and the GUS transcripts (heterologous expression in Nicotiana benthamiana) and enzyme activities (homologous expression in grapevine) were quantified (Figure 6). As shown in Figure 6a, the GUS transcripts of ‘deletion 3-AF1 binding site 1ʹ after UV-C induction were significantly reduced compared with those of pVqMYB15 (wild-type) and other light-responsive elements. Meanwhile, Figure 6b shows that, from the grape transient expression system, the GUS protein of ‘deletion 3-AF1 binding site 1ʹ was significantly decreased compared with that of the wild-type and the other light responsive elements, which is consistent with Figure 6a. This result means that 3-AF1 binding site 1 in pVqMYB15 is remarkably sensitive to light.

Figure 6.

Figure 6.

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The induction of photoresponse elements in pVqMYB15 after UV-C irradiation. (a) Quantitative RT-qPCR of GUS transcripts in the leaves of Nicotiana benthamiana; (b) Analysis of GUS enzyme activity in V. vinifera cv. Cabernet Sauvignon. Values show promoter activities relative to the corresponding untreated controls. Leaves were placed upside down on moist filter paper in Petri dishes, and the abaxial surface of an entire leaf was exposed to UV-C light (254 nm, 15 W, FSL, China) for 10 min at a distance of 12.5 cm from the light source. Data represent the means of three biological replicates, and error bars represent standard errors. *P < .05 and **P < .01 indicate statistically significant differences (n = 3).

3. Discussion

At present, promoter function analysis is mainly carried out through a combination of bioinformatic prediction and experimental verification. The type, number, sequential order and distance between multiple cis-elements in the promoter region of a gene could affect its transcription.39 In the current study, 1972 bp upstream of VqMYB15_SY was analyzed with the promoter prediction software PlantCARE. We found that in the promoter regions, in addition to some conservative elements, e.g., TATA-box and CAAT-box, there were other cis-elements, such as light response elements G-box, BOXI and LAMP-element, wounding response element WUN-motif, ABA response elements ABRE and MYC, and SA related TCA-elements. This suggests that the activation of pVqMYB15 under different biotic and abiotic stresses may depend on these corresponding cis-acting elements.

The analysis of deleted cis-acting elements in promoters is an important method for functional research in promoters.40 Therefore, in this study, we used our previous results to further discuss cis-elements in pVqMYB15, and we expected to find some key elements in plant defense responses. Endogenous SA can be used as a signal molecule to induce the immune response in plants, and this process can be mimicked by exogenous SA application.41 In our results, we constructed five mutants in addition to wild-type to investigate SA-induced promoter activities and found that the regulatory roles of TCA-element 1 and TCA-element 2 in pVqMYB15 activation are more significant than those of TCA-element 3 (Figures 3 and 4). This suggests that the regulatory effect of cis-acting elements on genes is probably related to the location of the cis-acting elements in the promoter regions.

In addition, some researchers reported that callose deposition could form a physical barrier to impede microbial penetration or inhibit the development of pathogen haustoria by preventing them from obtaining nutrients from host cells when plants are attacked by pathogens.42,43 To our surprise, SA has been reported to have a role in inducing the expression of callose-like genes.44 In addition, many studies have shown that endogenous SA accumulation is correlated with the generation of systemic acquired resistance in plants.45,46 Therefore, to explore the key role of SA-related cis-acting elements in the interaction between plants and pathogens, we performed a bacterial inoculation experiment (infection with P. capsica) in the leaves of Nicotiana benthamiana by instantaneous transformation with five mutants in addition to wild-type pVqMYB15. The results showed that the inductions of all mutants were decreased compared with that of wild-type pVqMYB15. In addition, the extent of the reduction was less in the ‘deletion TCA-element 3ʹ mutant than in the other mutants (Figure 4). This result is similar to that of the application of exogenous SA (Figure 3), indicating that the TCA-elements in pVqMYB15 play a key regulatory role in bio/abiotic stress tolerance, especially TCA-elements 1 and 2.

Furthermore, in addition to SA, ABA is one of the most important plant hormones and is also a cross-talk signaling molecule.47 In our research, pVqMYB15 was induced with ABA after ABRE or MYC deletion, and it was found that the promoter activities were significantly decreased in both mutants compared to that in the wild-type (Figure 5). This result indicated that ABRE and MYC elements are very important for the regulation of VqMYB15_SY.

Figure 5.

Figure 5.

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The induction of ABA-related elements in pVqMYB15 in the leaves of Nicotiana benthamiana. Values show promoter activities (GUS transcript abundance) relative to the corresponding solvent controls after treatment with 100 µM ABA for 1 h. Data represent the means of three biological replicates, and error bars represent standard errors. ** Differences that are statistically significant at the P < .01 level.

In the process of plant growth and development, light is not only directly involved in photosynthesis but is also related to the expression of light-sensitive genes.48,49 There is growing evidence that light is necessary for the induction of plant defense responses against some pathogens.50,51 For example, light has been shown to be responsible for accumulating SA and suppressing bacterial growth.52 Furthermore, the role of light has been investigated, and light has been demonstrated to be indispensable for basal defense responses in plants.53 Wingender54 also reported a relationship between cis-regulatory elements of soybean protoplasts that are involved in ultraviolet light and plant defense.

There are some cis-acting elements that are associated with light in the promoter regions of genes. For example, it is known that the 3-AF 1 binding site, G-box and BOX4 are ubiquitous light regulatory elements in many gene promoters.25,55 In our previous studies, we tested the short-term UV-C irradiation, a reliable stress elicitor, could stably induce the transcription of many stress resistance genes, such as STILBENE SYNTHASE.56 In addition, UV-C causes changes in chlorophyll, which indicate the ability of plants to resist stress.57 Therefore, in this study, we used UV-C as an elicitor to investigate the roles of 11 light-related elements in pVqMYB15 and found that the ‘3-AF 1 binding site 1ʹ is the most influential cis-acting element, indicating that the ‘3-AF 1 binding site’ plays an important role in pVqMYB15 activation under light regulation.

Plants regulate their normal growth and development by sensing a variety of environmental stresses, which allows them to survive. However, this process requires specific temporal and spatial gene expression through the synergistic expression of multiple cis-acting regulatory elements in its promoter. Therefore, it is very important to identify the key cis-acting elements in the promoter region of a gene, which can help us to further elucidate the mechanisms of plant stress tolerance in the future.

4. Materials and methods

4.1. Plant materials

Chinese wild Vitis quinquangularis Shanyang (V. quinquangularis_SY) and European cultivated grapevine (Vitis vinifera cv. Cabernet Sauvignon) were grown in the Life Science Experimental Park of Northwest University, Xi’an, Shaanxi, China. Tobacco (N. benthamiana) plants were grown in a growth chamber at 23°C with 16 h light/8 h dark.

4.2. Plasmid construction

PCR primers (Table S1) were designed to amplify pVqMYB15 promoter-deficient mutants with BoxI 1 deletion, BoxI 2 deletion, BoxI 3 deletion, BoxI 4 deletion, BoxI 5 deletion, 3-AF1 binding site 1 deletion, 3-AF1 binding site 2 deletion, 3-AF1 binding site 1 and 2 deletion, Box 4 deletion, LAMP element deletion, MRE deletion, ABRE deletion, MYC deletion, TCA-element 1 deletion, TCA-element 2 deletion, TCA-element 3 deletion, TCA-element 1 and 2 deletion, and TCA-element 1, 2 and 3 deletions. Then, the PCR fragments were connected with T-Vector pMDTM19 (Simple) (TaKaRa, Dalian, China). The T-vectors were then digested with the restriction enzymes HindIII and BglII, and the fragments were inserted into the pCAMBIA1301 expression vector (Miaolingbio, Wuhan, China), which contains the GUS reporter gene.

4.3. Transient expression

GUS expression vectors were introduced into Agrobacterium tumefaciens strain GV3101 by electroporation.58 The bacterial suspensions (OD600 = 0.6) were infiltrated into the leaves of 6- to 8-week-old N. benthamiana plants using a needle-free syringe.59 For V. vinifera leaves, a vacuum pump was used to pump the bacterial suspension solution into the leaves at 0.85 MPa for 30 min. After 48 h, the leaves were subjected to various treatments before assaying their GUS activity.

4.4. Treatment of N. benthamiana and grapevine leaves for transient promoter assays

The third to fifth fully expanded leaves were randomly selected from the plant apices of V. vinifera cv. Cabernet Sauvignon. For the SA (Solarbio, Beijing, China) and ABA (Solarbio, Beijing, China) treatments in grapevine and N. benthamiana, leaves were placed on filter paper of a Petri dish and immersed in freshly prepared 1 mM SA/100 µM ABA. In the control samples, all treatments were the same as described above, except that the solvents did not contain SA or ABA. All the leaves were collected at 1 h in the SA/ABA treatment and the solvent controls were collected at the corresponding time point. For the UV-C treatment, details can be found in a previous study.60

For the inoculation experiments with P. capsici in N. benthamiana, we used the process as follows: discs of agar-containing P. capsici were cut from the V8 media61 and placed on Petri dishes containing 25 ml of V8 liquid medium. The Petri dishes were placed in the dark at 25°C for 3 days. The liquid was poured out, and the agar blocks were removed. The mycelia were rinsed with sterile tap water 3 times, 10 ml sterile tap water was added, and the mixture was placed in the dark at 25°C for 12 h. Before the observation of zoospores, they were stimulated at 4°C for 20 min and then quiescent for 15 min. After that, the concentration of spores was adjusted to 1X104/ml for inoculation. Before inoculation, the nutrient zone was permeated, and a hole was made approximately 3 cm deep with a glass rod at 3 cm from the roots of the seedlings. Then, 10 ml of spore suspension was injected into the hole. The nutrient zone keeps moisture. Tobacco leaves were collected at 0 h, 12 h, 24 h, 36 h, 48 h, 60 h and 72 h after inoculation. The controls without zoospores were established in the same way as above and were collected at the corresponding time points. The above samples were immediately frozen in liquid nitrogen and then stored at −80°C for RNA extraction.

4.5. cDNA synthesis and quantitative real-time PCR

The total RNA in the N. benthamiana leaves was isolated using the EZNA® Total RNA kit (Omega Bio-tech) following the manufacturer’s instructions. The mRNA was transcribed into cDNA using Prime Script Reverse Transcriptase (TaKaRa). The RT-qPCR amplifications were performed as previously described.62 Information about gene-specific primers is given in Supplementary Table S2. NbEF1-α63 was used as a reference control.

4.6. Quantitative determination of GUS enzymatic activity

GUS enzymatic activity was analyzed as described previously;62 a brief description is provided here: After transient transformation and different treatments of the grapevine leaves, GUS enzymatic activity was determined with an enzyme microplate reader (SYNERGY2) using 4-methylumbelliferyl-beta-D-glucopyranoside (4-MUG, Sigma-Aldrich, Shanghai, China) as the substrate. The enzyme activity was evaluated in terms of enzyme activity per mg of protein, and the result was presented as 4-MU pmol/min/mg protein. Each experiment included three biological and three technical replicates. In addition, the data presented in all figures are the relative values of the treated samples to the controls.

Supplementary Material

Supplemental Material
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Supplemental Material
Click here for additional data file. (19.6KB, docx)

Funding Statement

This work was supported by the National Natural Science Foundation of China [31970348]; National Natural Science Foundation of China [31600256]; Young Talent fund of University Association for Science and Technology in Shaanxi Province of China [20190207]; Scientific Research Program Funded by Shaanxi Provincial Education Department [18JK0769]; Natural Science Basic Research Plan in Shaanxi Province of China [2017JQ3005]; Northwest University Training Program of Innovation and Entrepreneurship for Undergraduate [2020326]; Young Academic Talent Support Program of Northwest University.

Author contributions

Dong Duan conceived and designed the work. Ruixiang Li performed most experiments and wrote the manuscript. Fanding Zhu analyzed the data.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website.

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