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Article

Isolation and Functional Analysis of the DhMYB2 and DhbHLH1 Promoters from Phalaenopsis-Type Dendrobium Involved in Stress Responses and Tissue-Specific Expression

by
Yachen Wang
1,†,
Chonghui Li
2,†,
Wenjuan Zhu
3,
Yamei Li
2,
Xiqiang Song
4,* and
Junmei Yin
2,5,*
1
School of Agricultural and Rural Affairs, Hainan Open University, Haikou 570208, China
2
Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences/Key Laboratory of Crop Gene Resources and Germplasm Enhancement in Southern China, Ministry of Agriculture/The Engineering Technology Research Center of Tropical Ornamental Plant Germplasm Innovation and Utilization/Key Laboratory of Tropical Crops Germplasm Resources Genetic Improvement and Innovation of Hainan Province, Haikou 571101, China
3
Key Laboratory of Systems Health Science of Zhejiang Province, School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou 310024, China
4
Germplasm Innovation of Tropical Special Forest Trees and Ornamental Plants (Ministry of Education), School of Tropical Agriculture and Forestry, Hainan University, Haikou 570100, China
5
National Key Laboratory for Tropical Crop Breeding, Sanya Research Institute, Chinese Academy of Tropical Agricultural Sciences, Sanya 572024, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2025, 11(5), 550; https://doi.org/10.3390/horticulturae11050550
Submission received: 6 April 2025 / Revised: 13 May 2025 / Accepted: 15 May 2025 / Published: 19 May 2025
(This article belongs to the Special Issue Color Formation and Regulation in Horticultural Plants)
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Abstract

:
Phalaenopsis-type Dendrobium (Den-Phals) is a commercially valuable orchid, with floral color being key to its market appeal. Despite the significance of anthocyanin biosynthesis in color development, its transcriptional regulation in Den-Phals remains unclear. This study functionally characterized the promoters of DhMYB2 and DhbHLH1, two key transcription factors involved in anthocyanin biosynthesis. A 1864 bp DhMYB2 promoter and a 1995 bp DhbHLH1 promoter were isolated using genome walking. Bioinformatics analysis identified cis-acting elements associated with abiotic stress responses, phytohormone signaling, and floral-specific regulation. 5′-Deletion analysis in tobacco leaves identified core regulatory regions for the DhMYB2 promoter (−1864 to −937 bp) and DhbHLH1 promoter (−1995 to −924 bp). GUS staining and activity assays demonstrated that the activities of the DhMYB2 and DhbHLH1 promoters were significantly increased under treatments of long light, low temperature, drought, salicylic acid (SA), and abscisic acid (ABA), while the DhbHLH1 promoter was also induced by methyl jasmonate (MeJA) and indole-3-acetic acid (IAA). Furthermore, promoter activity of DhMYB2 and DhbHLH1 was detected only in transgenic Arabidopsis flowers, suggesting that these promoters exhibit floral-specific activity. This study provides the first functional analysis of Den-Phals anthocyanin promoters, revealing their stress-responsive nature and floral specificity, which will facilitate molecular breeding of novel orchid cultivars.

1. Introduction

Phalaenopsis-type Dendrobium (Den-Phals), a member of the Orchidaceae family, has become one of the most popular cut flowers and potted plants. Den-Phals holds significant commercial value due to its valuable traits, such as vibrant floral colors, appealing shapes, and extended vase life [1]. The manipulation of floral color has been a primary focus in the breeding of ornamental plants [2]. Anthocyanins, a class of flavonoid-derived secondary metabolites, are essential for determining flower color [3]. With advancements in molecular biology, the anthocyanin biosynthesis pathway has been extensively studied in orchids [4,5,6]. The regulation of transcription factors, particularly R2R3-MYB, basic helix-loop-helix (bHLH), and WD-repeat (WDR) proteins, has also been widely studied in orchids [7,8,9]. Genetic engineering techniques have emerged as pivotal breeding methods for modifying floral color by enhancing anthocyanin biosynthesis and composition. For example, transforming PhCHS5 (Chalcone synthase 5) and PhF3′5′H (Flavonoid 3′,5′-hydroxylase) from Phalaenopsis into both Petunia and Phalaenopsis plants led to a more intense coloration and a significant increase in anthocyanin content in the Petunia plants, and deepened lip color in transgenic Phalaenopsis flowers [10]. Overexpressing CmNAC25 (NAM/ATAF/CUC domain-containing protein 25) from Chrysanthemum morifolium in transgenic chrysanthemum petals produced a redder color, providing a target gene for molecular design breeding of flower color [11]. Conversely, overexpression of Gh-SOC1 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1) from Gerbera hybrida led to a lack of anthocyanin in transgenic gerbera lines [12].
The precision control of transgene expression relies on the selection of promoters, which govern the spatiotemporal patterns of transgene expression. Promoters are specific DNA sequences located upstream of the gene coding region. Promoters contain numerous cis-acting elements and function as switches that initiate and regulate gene transcription, and play an important role in responding to biotic and abiotic stresses [13,14,15]. Based on their structure, function, and gene expression patterns, promoters are classified into three types: constitutive, inducible, and tissue-specific [16].
Constitutive promoters, exemplified by the cauliflower mosaic virus (CaMV) 35S promoter, have been widely used for genetic modification in plants, owing to their strong and ubiquitous transcriptional activity [17]. However, these promoters are not always optimal for driving exogenous gene expression. Persistent overexpression of transgenes can perturb endogenous gene networks, resulting in developmental aberrations or unintended metabolic byproducts caused by pleiotropic interactions [18,19]. In contrast, inducible promoters provide a refined alternative by enabling precise temporal control of gene expression in response to environmental conditions (such as drought, extreme temperature, high salinity, heavy metals, and alkaline conditions) or phytohormones (such as abscisic acid, auxin, salicylic acid, ethylene, and jasmonate) [17,20,21]. Similarly, tissue-specific promoters restrict transgene activity to desired organs or tissues, minimizing deleterious pleiotropic effects [22].
Numerous studies have demonstrated that inducible promoters can modulate secondary metabolism in response to stress. cis-acting elements within promoters are critical in these responses, binding to transcription factors to induce or suppress the expression of downstream genes in response to varying stress conditions [23]. In Arabidopsis, various abiotic stress conditions induce the production of unique anthocyanin profiles via stress-responsive cis-elements within target promoters [24]. Phytohormone-responsive promoters have similarly been used to fine-tune flavonoid biosynthesis across diverse species [25,26,27]. Therefore, further research into the functional mechanisms of promoters responding to stress conditions may represent a promising direction for floral color breeding. On the other hand, tissue-specific promoters have been reported to enable localized metabolic engineering. For example, endosperm-specific promoters drive anthocyanin biosynthesis in rice and maize [28,29]. The overexpression of RsMYB1, driven by the flower-specific promoter InMYB1, significantly increased anthocyanin levels only in floral tissues of transgenic Petunia [30]. Thus, discovering new floral-specific promoters is essential for the genetic manipulation of ornamental plants.
Despite advancements in promoter engineering, functionally validated floral-specific promoters remain scarce for ornamental plant breeding, particularly those capable of integrating developmental specificity with environmental responsiveness. In our previous research, DhMYB2 and DhbHLH1 were identified as key transcription factors involved in anthocyanin biosynthesis and pigmentation in Den-Phals by activating the structural genes [1,31]. Notably, RT-qPCR analysis revealed that DhMYB2 and DhbHLH1 are specifically expressed in flowers [31], suggesting that their upstream promoters might exhibit floral-specific activity. However, whether these promoters combine floral-specific activity with stress/phytohormone inducibility remains unexplored. We hypothesized that the DhMYB2 and DhbHLH1 promoters inherently coordinate these dual functionalities, enabling spatiotemporal regulation of anthocyanin biosynthesis. To address this, we isolated the promoters of the DhMYB2 and DhbHLH1 genes for the first time and identified their key cis-acting elements using bioinformatics tools. Transient assays in tobacco demonstrated their activation in response to abiotic stress and phytohormones. Additionally, we found that the DhMYB2 and DhbHLH1 promoters exhibit floral-specific activity in transgenic Arabidopsis. Our findings contribute to a deeper understanding of the function of the DhMYB2 and DhbHLH1 promoters in stress responses and tissue-specific expression, laying a theoretical foundation for developing novel floral-specific promoters to breed a wider range of floral colors in Den-Phals.

2. Materials and Methods

2.1. Plant Materials

The Phalaenopsis-type Dendrobium (Den-Phals) cultivars Den. ‘Red Bull’ were cultivated under long-day conditions in Danzhou, Hainan Province, China. Flower tissues were used for gene cloning. The tobacco (Nicotiana benthamiana) plants were grown for 4–6 weeks in a peat, perlite, and vermiculite mix at 25 °C, with a 16 h light/8 h dark cycle, for transient expression assay in Danzhou, Hainan Province, China. The Arabidopsis plants were maintained at 22 °C under a 16 h light/8 h dark cycle, followed by tissue-specific expression analysis in Danzhou, Hainan Province, China.

2.2. Isolation and Bioinformatics Analysis of DhMYB2 and DhbHLH1 Promoters

Genomic DNA was extracted from Den-Phals flower tissues using the Super Plant Genomic DNA Kit (Tiangen Biotech, Beijing, China). The 5′ flanking unknown promoter regions of DhMYB2 and DhbHLH1 were isolated using the Genome Walking Kit (Takara, Dalian, China) through thermal asymmetric interlaced PCR (TAIL-PCR) with a nested primer strategy. For DhMYB2 promoter isolation, primary PCR was performed using either degenerate adaptor primer AP1 or AP2 paired with gene-specific outer primer SP1, followed by secondary PCR with the corresponding degenerate adaptor primer and nested inner primer SP2. For DhbHLH1, three-round TAIL-PCR was conducted using four degenerate adaptor primers (AP1–AP4) sequentially paired with nested gene-specific primers SP1–SP3. All PCR products were ligated into the pMD18-T vector and transformed into E. coli DH5α (Takara, Dalian, China). Positive clones were selected and subjected to bidirectional Sanger sequencing using M13 universal primers. Sequence reads were assembled and aligned in SeqMan (DNASTAR, Madison, WI, USA), and a consensus sequence was generated. To validate the genome walking results, full-length promoter regions were re-amplified from genomic DNA using terminal-specific primers designed from the obtained sequences. The validation PCR products were similarly cloned, transformed, and sequenced (three independent clones per promoter) to confirm sequence accuracy. These verified promoter sequences were then subjected to cis-acting element prediction using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 15 April 2023) and PLACE (http://www.dna.affrc.go.jp/PLACE), accessed on 15 April 2023. The primer sequences used are listed in Supplementary Table S1a.

2.3. Vector Construction

The DhMYB2 and DhbHLH1 promoters were cloned into the pCAMBIA1381z vector to drive the GUS reporter gene, with the CaMV35S promoter used as a positive control (Coling, Wuhan, China). 5′-deletion fragments of varying lengths from the DhMYB2 and DhbHLH1 promoters, containing SalI and HindIII restriction sites, were amplified using specific primers (listed in Supplementary Table S1b). High-fidelity KOD DNA polymerase (Toyobo, Osaka, Japan) was used in 30 μL PCR reactions containing 1 μL of genomic DNA and 0.5 μM of each primer. The PCR conditions included 35 cycles of 15 s at 98 °C, 15 s at 55 °C, and 2 min at 72 °C, followed by a final extension at 72 °C for 10 min. The resulting PCR fragments were ligated into the digested vector using the 2 × GenRec Master Mix seamless cloning kit (Generalbio, Hefei, China).

2.4. Transient Expression Analysis in Tobacco Leaves

Agrobacterium GV3101 was transformed with 1 μg of plasmid DNA by freeze–heat shock methods. Leaves from 6-week-old tobacco plants in the growing stage were prepared for infiltration. The Agrobacterium cells were resuspended in infiltration buffer containing 10 mM MES pH 5.7, 10 mM MgCl2, and 150 μM acetosyringone. The suspension was diluted to an OD600 of 0.8, and incubated at room temperature for 3 h. The cell suspensions were then injected into the tobacco leaf surfaces. The inoculated plants were kept in a growth chamber under a 16 h light/8 h dark cycle at 25 °C for 48 h.

2.5. Transformation of Arabidopsis thaliana

To promote the growth of lateral inflorescences, the primary main inflorescences at anthesis were removed. The Agrobacterium containing the target plasmid was suspended in a buffer solution with 10% sucrose and 400 μL/L Silwet-77. The inflorescences were submerged in this solution for 1 min; then, the plants were covered with cling film to maintain humidity, and incubated in the dark for 1 day. Approximately one week after transformation, watering was resumed, and the infestation process was repeated several times periodically. The plants were cultivated until maturity, and the seeds were collected and placed in a dry environment for about 1 week to screen for transformants. The seeds were then screened on MS medium plates containing 20 mg/L Hygromycin B. Seedlings with normal growth of true leaves were further confirmed by PCR analysis.

2.6. Abiotic Stress and Phytohormone Treatments

In order to analyze the function of DhMYB2 and DhbHLH1 promoters in response to stress, Agrobacterium-infiltrated tobacco plants were subjected to various abiotic stress conditions. For temperature treatments, plants were placed in growth chambers at 4 °C for cold stress and 42 °C for heat stress. Light stress involved maintaining plants under a 16 h light/8 h dark cycle for long-day conditions, or an 8 h light/16 h dark cycle for short-day conditions. Drought stress was simulated by floating leaves from infiltrated plants on liquid MS medium containing 18% (w/v) PEG 6000 for 48 h.
Additionally, phytohormone treatments were applied to Agrobacterium-infiltrated intact leaves on tobacco plants to assess promoter induction. Plants were treated with 1 mM salicylic acid (SA), 0.1 mM abscisic acid (ABA), 0.1 mM methyl jasmonate (MeJA) in 10% ethanol, or 1 mg/L indole-3-acetic acid (IAA) in MS medium. Control plants received either distilled water or 10% ethanol, as appropriate. After 48 h of treatment, leaves from both treated and control plants were collected for GUS staining and activity analysis. Each experiment was repeated three times.

2.7. GUS Staining and Activity Analysis

GUS staining was performed to visualize the spatial expression pattern, and GUS activity assays quantified the expression levels. The prepared tissue materials were immersed in GUS staining solution (Coolaber, Beijing, China) and stained for 1 h at room temperature. Subsequently, the stained tissues were destained in 70% ethanol for 1 h, with the process repeated four times. After destaining, the tissues were photographed using the microscope SZX16 (Olympus, Tokyo, Japan).
For the GUS activity assay, approximately 100 mg of fresh plant tissue was ground in liquid nitrogen. The resulting powder was transferred to an EP tube and 1 mL of extraction solution was added. After centrifugation at 12,000 rpm for 10 min at 4 °C, the supernatant was collected as the protein extract. A 150 μL aliquot of the protein extract was added to 150 μL of 4-MUG substrate solution and incubated in a water bath at 37 °C. To stop the reaction, 100 μL of the reaction mixture was added to 900 μL of termination solution. The fluorescence value of 4-MU generated in the reaction solution was calibrated against the fluorescence value of the standard solution, and GUS activity was calculated as MU/min/μg of total protein (Coolaber, Beijing, China).

2.8. Statistical Analysis

The data concerning GUS activity were assessed by one-way analysis of variance (ANOVA), followed by Tukey’s HSD post hoc analysis. In the figures, the lowercase letters indicate statistical significance based on one-way ANOVA (p < 0.05). Statistical tests and significance analysis were performed using GraphPad Prism 8.

3. Results

3.1. Sequence Isolation of the DhMYB2 and DhbHLH1 Promoters

The chromosome walking technique was used for isolating approximately 2 kb upstream regions from the initiation codon of both DhMYB2 and DhbHLH1 genes. For DhMYB2, two gene-specific primers (SP1 and SP2), designed based on the known coding sequence, were paired with degenerate adaptor primers (AP1 or AP2) in a single walking step. This approach successfully amplified a 2475 bp upstream fragment containing the promoter region (Figure 1A). Subsequent validation PCR using genomic DNA as a template, with specifically designed primers, yielded a confirmed 1864 bp promoter fragment (Figure 1B). The DhbHLH1 promoter was isolated through an iterative walking strategy comprising two consecutive steps. Primary TAIL-PCR using three nested specific primers (SP1–SP3) with adaptor primers AP1–AP4 generated 1416 bp of upstream sequence (Figure 1C). A secondary walking step, using the newly obtained sequence as a reference, extended the coverage by an additional 941 bp (Figure 1D). Assembly of these sequences yielded a contiguous 2265 bp upstream region. Genomic PCR verification using specific primers amplified a 1995 bp promoter fragment (Figure 1E), representing the functional promoter region. All fragments were verified by bidirectional sequencing, with the final validated promoter sequences provided in Supplementary Table S2. These promoter sequences were then used for bioinformatics analysis and functional assay experiments.

3.2. Characterization of cis-Acting Elements in DhMYB2 and DhbHLH1 Promoters

In plants, abiotic stress and phytohormones participate in anthocyanin biosynthesis and regulate the expression of functional genes. To explore the potential functions of DhMYB2 and DhbHLH1 promoters in stress response processes, cis-acting elements within these promoter regions were identified using the PlantCARE database. The promoter analysis revealed multiple light-responsive elements in the DhMYB2 promoter, including Box 4, TCT-motif, G-box, ACE, GATA-motif, and GT1-motif. Additionally, the DhMYB2 promoter contains ABA-response elements (ABRE and AAGAA motif), the hormone-response element ERE, the anaerobic-response element ARE, the drought-response element DRE core, the osmotic-stress-response element STRE, the SA-response element TCA motif, stress-related TC-rich repeats, inducible response-related WRKY binding site W box, the mechanical-injury-response elements WUN-motif and WRE3, and the growth regulation-associated element circadian. The analysis suggests that DhMYB2 gene expression may be influenced by external factors such as light, drought, hormones, anaerobiosis, osmotic stress, and mechanical damage (Figure 2A and Supplementary Figure S1A). Moreover, pollen-specific elements were identified in the DhMYB2 promoter regions using the PLACE online tool, including fifteen GTGANTG10 motifs (GTGA) and fourteen POLLEN1LELAT52 motifs (AGAAA) [32,33] (Supplementary Table S3a).
The DhbHLH1 promoter contains many stress-related and hormone-responsive cis-acting elements, similarly to the DhMYB2 promoter. In addition, the DhbHLH1 promoter contains more than eleven methyl jasmonic acid-responsive elements (CGTCA-motif/TGACG-motif), biotin-induced AuxRR-core and TGA-element, the low-temperature stress-response element LTR, growth regulation-related elements (CAT-box and MSA-like), and the damage-inducing element WRE3 (Figure 2B and Supplementary Figure S1B). Similarly, eighteen GTGANTG10 motifs and nine POLLEN1LELAT52 motifs were found in the DhbHLH1 promoter (Supplementary Table S3b). The DhMYB2 and DhbHLH1 promoters contain a large number of abiotic stress and hormone-responsive cis-acting elements, suggesting that the expression of these transcription factors may be affected by environmental conditions, potentially altering their regulation of anthocyanin biosynthesis in Den-Phals.

3.3. 5′-Deletion Analysis of Promoter Activity

To verify the promoter activity of the isolated sequences and identify the core regulatory region, we constructed full-length and truncated fragments of the DhMYB2 and DhbHLH1 promoters driving the GUS reporter and introduced them into tobacco (Figure 3A and Supplementary Table S1b). GUS staining revealed that both full-length and deletion sequences of DhMYB2 could drive downstream GUS expression, with the staining intensity being proportional to the length of the sequences. Specifically, the full-length promoter (−1864/−1 bp) exhibited strong activity, and the promoter still showed strong activity when truncated to −1433/−1 bp. However, when truncated to −937/−1 bp, the promoter activity was weak, and there was basically no activity when truncated to −473/−1 bp (Figure 3B). These results imply that the core regulatory region of the DhMYB2 promoter lies between −1864 bp and −937 bp. Similarly, GUS staining for the DhbHLH1 promoter showed strong activity at full length (−1995/−1 bp) and truncation to −1484/−1 bp, weak activity at truncation to −924/−1 bp, and no activity at truncation to −413/−1 bp. This suggests that the core regulatory region of the DhbHLH1 promoter is likely located between −1995 bp and −924 bp (Figure 3C). Transient GUS staining of tobacco leaves suggested that these sequences possess promoter activity, and the core regulatory regions of DhMYB2 and DhbHLH1 are located at −1864 bp to −937 bp and −1955 bp to −924 bp, respectively. This provides a basis for further studies on the promoter functions of these transcription factors.

3.4. DhMYB2 and DhbHLH1 Promoter Responses to Abiotic Stress and Phytohormones

To elucidate the function of putative cis-acting elements in the response of the DhMYB2 and DhbHLH1 promoters to abiotic stress and phytohormones, tobacco leaves infiltrated with Agrobacterium containing full-length promoter::GUS constructs were subjected to light, temperature, and drought stresses, and were also treated with MeJA, SA, ABA, and IAA.
As negative and positive controls, tobacco leaves were infiltrated with a GUS-empty vector lacking a promoter and a construct driven by the CaMV35S promoter (CaMV35Spro::GUS), respectively. The GUS-empty vector exhibited negligible staining under all tested conditions, while CaMV35Spro showed strong constitutive GUS activity, confirming the reliability of the assay system. In abiotic stress experiments, promoter-driven GUS activity showed diverse expression patterns. Specifically, promoter activities were enhanced under long light, whereas activities were significantly reduced under short light. Additionally, under different temperature conditions, promoter activities significantly increased under low temperature, whereas there was no obvious change under high temperature. Under drought stress, the promoter activities of DhMYB2 and DhbHLH1 were examined in leaves in MS medium supplemented with 18% PEG 6000, and the GUS activities showed intense staining compared with the mock-treated control, indicating that the DhMYB2 and DhbHLH1 promoters participate in the drought stress response (Figure 4).
In the phytohormone assays, the promoter activities of DhMYB2 and DhbHLH1 were significantly enhanced by the SA and ABA treatments, and the promoter activity of DhbHLH1 also increased under the MeJA and IAA treatments (Figure 5). Accordingly, GUS staining and activity assays demonstrated that the DhMYB2 and DhbHLH1 promoters are highly responsive to multiple abiotic stresses and phytohormones, suggesting that these genes are involved in abiotic stress- and phytohormone-regulated pathways during floral organ development and affect anthocyanin synthesis.

3.5. Tissue-Specific Expression Analysis of the Promoters

To investigate the temporal and spatial expression patterns of the promoters in plants, we transformed the full-length sequences of the DhMYB2 and DhbHLH1 promoters into Arabidopsis plants and observed the GUS expression. We performed GUS staining on seedlings at the cotyledon, true leaf, and shoot stages, as well as on roots, stems, leaves, floral tissues at the flowering stage, and pods and seeds at the fruiting stage. The staining results showed no positive signals in seedlings, roots, stems, or leaves, indicating that the promoters are not expressed in vegetative organs for both DhMYB2 and DhbHLH1. Similarly, pods and seeds also showed no staining signals for DhMYB2 and DhbHLH1 in the plants.
Interestingly, the promoters of DhMYB2 and DhbHLH1 exhibited strong activity in the floral tissues, with expression widely distributed in the sepals, petals, pistils, and stamens; in particular, the strongest expression was observed in the sepals (Figure 6 and Figure 7). These results indicate that the promoters are specifically expressed in flowers, suggesting that DhMYB2 and DhbHLH1 are closely related to the development of floral organs. Our earlier research indicated that DhMYB2 and DhbHLH1 are involved in regulating anthocyanin biosynthesis, implying that these transcription factors participate in the regulation of anthocyanin production in flowers.
In addition, to validate the specificity of GUS expression, we included a negative control (a GUS-empty vector lacking a promoter), and the results showed no GUS activity under the same staining conditions (Supplementary Figure S2), confirming the reliability of the assay and the specificity of promoter-driven expression.

4. Discussion

Phalaenopsis-type Dendrobium (Den-Phals) is a highly popular ornamental tropical orchid with significant market value. Developing new varieties with diverse floral colors and coloration patterns is a key focus in Den-Phals breeding. However, the lack of a fully sequenced genome and limited understanding of anthocyanin biosynthesis regulation present major challenges to improving floral coloration through genetic engineering techniques. In our previous research, we discovered that DhMYB2 and DhbHLH1 are specifically expressed in the floral organs of Den-Phals and play crucial roles in transcriptional regulation of anthocyanin biosynthesis [31]. In this study, we systematically investigated the promoter functions of these two genes.
Firstly, a 1864 bp promoter fragment of the DhMYB2 gene and a 1995 bp promoter fragment of the DhbHLH1 gene were isolated from Den-Phals, with an AT content of 73% and 63%, respectively. Bioinformatics analysis revealed that both promoter sequences contain abundant core elements such as TATA box and CAAT box, which are related to transcription initiation. These features are consistent with the characteristics of eukaryotic promoter sequences, indicating that both promoters possess typical promoter characteristics and functions [34]. Subsequently, the activities of the two promoters were further verified through GUS staining analysis and activity assays in transiently expressed tobacco leaves. We found that the full-length promoters drove the highest levels of GUS expression, while a series of 5′-truncated promoters drove a gradual decrease in GUS expression. This is consistent with the results of previous research, suggesting that there were no negative regulation elements in either the DhMYB2 or DhbHLH1 promoters [35,36].
Besides the classical eukaryotic elements, further bioinformatics analysis found that there were extensive cis-acting response elements to biotic stress and hormone signals in the promoter regions (Figure 2 and Supplementary Table S3). Light plays a crucial role in anthocyanin biosynthesis in plants. It has been reported that high levels of light induce the expression of MYB112, leading to increased anthocyanin accumulation [37]. Similarly, PpbHLH64 expression was upregulated under light treatment, resulting in induced anthocyanin accumulation in red pears [38]. In our work, we found that the DhMYB2 promoter contains twelve light-responsive elements, while the DhbHLH1 promoter contains five light-responsive elements, indicating that the expression of these two genes may be influenced by light. Consistent with the predicted results, GUS staining and activity analysis revealed that longer light exposure increased the activity of the DhMYB2 and DhbHLH1 promoters, whereas shortening the light-exposure time reduced the activity of these two promoters. Plants accumulate anthocyanins to defend against external environmental stimuli such as low temperatures, drought, and salinity [39]. It has been reported that transcription factors, such as MYBs and bHLHs, are upregulated under cold stress, leading to anthocyanin production [40]. Additionally, AnaMYB87, induced by drought stress, can activate the anthocyanin biosynthesis pathway in Ammopiptanthus nanus [41]. Similarly, our research findings reveal that DhMYB2 and DhbHLH1 promoters contain cis-acting elements that are responsive to low temperatures and drought. Subsequent experiments also demonstrated that both cold and drought stress can induce the activity of these promoters, indicating that the expression of these two genes may be influenced by cold and drought stress. However, the GUS activity of the two promoters was not induced by heat stress, indicating that these two promoters are not sensitive to heat stress.
Phytohormones are crucial in regulating plant growth, development, and stress response [42]. We found several cis-acting elements responsive to abscisic acid (ABA) and salicylic acid (SA), which regulate anthocyanin biosynthesis in many plant species through regulating the expression of transcription factors. For example, MdABI5 positively regulates ABA-induced anthocyanin biosynthesis by upregulating MdbHLH3 expression in apples [25]. In fig fruit, FcMYB113 is strongly upregulated by exogenous ABA, leading to enhanced anthocyanin biosynthesis [43]. It is also reported that airborne fungus induces the biosynthesis of anthocyanins in Arabidopsis through SA signaling and promotes the expression of transcription factors such as MYB75 and bHLH. This indirectly indicates a close correlation between SA signaling and transcription factor expression. In this study, we also found that the activity of the DhMYB2 and DhbHLH1 promoters was significantly induced by ABA and SA. Interestingly, the DhbHLH1 promoter contains up to eleven methyl jasmonate (MeJA) response elements, whereas none were found in the DhMYB2 promoter. Subsequent GUS activity assays also revealed that only the DhbHLH1 promoter was activated by MeJA, indicating significant regulation of DhbHLH1 expression by MeJA. Previous research has reported that ABA, SA, MeJA, and H2O2 interact to provide defense against stress [44]. In our study, the DhMYB2 promoter was activated by ABA and SA, but did not respond to MeJA, suggesting that ABA, SA, and MeJA may not interact in the regulation of DhMYB2 gene expression. Moreover, we found that auxin (IAA) enhanced the activity of the DhbHLH1 promoter, while the DhMYB2 promoter was insensitive to IAA treatment. IAA is an important phytohormone that plays various roles in regulating anthocyanin biosynthesis. It has been reported that IAA treatment inhibits anthocyanin accumulation in Arabidopsis, grapes, and apples [45,46,47,48]. Conversely, IAA treatment enhances anthocyanin pigments in sweet cherries, peaches, and wheat grains [49,50,51]. Therefore, further research is needed to fully understand the effect of IAA on anthocyanin biosynthesis in Den-Phals.
Analyzing the tissue-specific expression of promoters can provide deeper insights into gene expression patterns. In our previous reports, both the DhMYB2 and DhbHLH1 genes showed floral-specific expression patterns in Den-Phals, suggesting that their upstream promoters may possess tissue specificity. Further bioinformatics analysis of the two promoter sequences revealed a large number of pollen-specific elements, including fifteen GTGANTG10 motifs and fourteen POLLEN1LELAT52 motifs on the DhMYB2 promoter, and eighteen GTGANTG10 motifs and nine POLLEN1LELAT52 motifs on the DhbHLH1 promoter. The number of these specific elements is greater than the number reported in the floral-specific JcTM6 promoter [52]. In addition, in transgenic Arabidopsis overexpressing the DhMYB2 or DhbHLH1 promoters, respectively, GUS staining showed that these two promoters were exclusively active in flowers (Figure 6 and Figure 7), suggesting that they are floral-specific promoters. Tissue-specific promoters, as one of the most desired approaches in genetic engineering, are preferred over constitutive promoters for stable transgene expression in crops [53]. Endosperm-specific promoters have been extensively studied and successfully utilized to express and accumulate higher yields of recombinant proteins in seeds [54]. Recently, floral-specific promoters have also garnered significant attention from researchers. It has been reported that transgenic Arabidopsis expressing AtIPT4 under the control of the floral-specific JcTM6 promoter exhibits a larger flower phenotype, without any alterations in other organs [52].
While our study provides a comprehensive characterization of the DhMYB2 and DhbHLH1 promoters, several methodological limitations warrant consideration. Both promoters exhibited strong floral-specific expression in stable Arabidopsis transformants (Figure 6 and Figure 7); however, their detectable activity in Nicotiana benthamiana leaves during transient assays should be interpreted with caution. The agroinfiltration-based system may impose inherent limitations—such as a high plasmid copy number—capable of overriding endogenous transcriptional regulation and masking native tissue specificity. A similar phenomenon has been observed in the transplastomic expression of BoCCD4-3, where leaky activity from a synthetic riboswitch led to crocin accumulation and chloroplast bleaching, even without inducer application, illustrating how elevated expression can bypass regulatory control [55]. Additionally, endogenous transcription factors in Nicotiana may aberrantly recognize and bind to orchid promoter elements, resulting in ectopic activation. To confirm specificity, we included 35S-free GUS empty vector controls, which showed negligible activity under both normal and stress conditions (Figure 4 and Figure 5), supporting the fact that the observed expression was indeed driven by the introduced promoter sequences.
Nevertheless, further validation in floral-specific systems is essential. These findings underscore the importance of testing promoter activity within floral tissue contexts, as leaf-based assays may not accurately reflect floral transcriptional environments. Due to technical constraints in Dendrobium transformation and the absence of a robust floral transient assay, we were unable to directly assess promoter activity in Den-Phal flowers. Future studies employing floral protoplasts or flower-targeted stable transformation will be critical to fully elucidate the spatial and temporal regulation of DhMYB2 and DhbHLH1.

5. Conclusions

In this study, we successfully isolated a 1864 bp DhMYB2 promoter fragment and a 1995 bp DhbHLH1 promoter fragment from Phalaenopsis-type Dendrobium. Bioinformatics analysis revealed that these promoters exhibit the typical features of eukaryotic promoters and contain numerous cis-acting elements that are responsive to abiotic stress, hormone signals, and floral-specific elements. Analysis of 5′-deletion promoter constructs in tobacco leaves identified that the core regulatory region of the DhMYB2 promoter was between −1864 bp and −937 bp, and that of the DhbHLH1 promoter was between −1995 bp and −924 bp. Functional assays using GUS staining and activity assays confirmed that the activities of both promoters were significantly upregulated by long light, low temperature, drought, salicylic acid (SA), and abscisic acid (ABA). Additionally, the DhbHLH1 promoter was also responsive to methyl jasmonate (MeJA) and indole-3-acetic acid (IAA). Furthermore, the activity of both promoters was identified as floral-specific in transgenic Arabidopsis plants. These findings provide valuable insights into the function of DhMYB2 and DhbHLH1 in relation to anthocyanin biosynthesis, and lay the foundation for further research on floral-specific promoters for genetic breeding of ornamental flowers.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae11050550/s1. Table_S1a. The details of specific primer sequences for genome walking. Table_S1b. Primer sequences for construction of the vectors for step truncated 5′ terminal promoter. Table_S2a. The sequence of DhMYB2 promoter. Table_S2b. The sequence of DhbHLH1 promoter. Table_S3a. Prediction of cis-acting elements of the DhMYB2 promoter using PLANTCARE and PLACE. Table_S3b. Prediction of cis-acting elements of the DhbHLH1 promoter using PLANTCARE and PLACE database. Figure S1. Detailed information on the cis-acting elements of DhMYB2 and DhbHLH1 promoters. Figure S2. Histochemical analysis of GUS staining in transgenic Arabidopsis plants carrying the negative control plasmid (GUS-empty vector lacking a promoter). No GUS signal was detected in any tissues of the negative control plants, confirming the assay specificity. (a) Cotyledons seedlings, (b) True leaves seedlings, (c) Adult leaves seedlings, (d) Root during the flowering period, (e) Stem during the flowering period, (f) Leaf during the flowering period, (g) Flower, (h) Pod, (i) Seeds. Bars = 2 mm.

Author Contributions

Y.W.: project administration, funding acquisition, investigation, writing—original draft, writing—review and editing. C.L.: project administration, funding acquisition, supervision, writing—review and editing. W.Z. and Y.L.: investigation, methodology. X.S. and J.Y.: conceptualization, project administration, funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

1. This research was funded by the Central Public-interest Scientific Institution Basal Research Fund (grant number 1630032022002) and the earmarked fund for CARS-23-G60, the Hainan Provincial Natural Science Foundation of China (grant number 320QN186). 2. This research was funded by the Tropical Special Ornamental Plants Innovative Research and Resource Utilization Team of Hainan Open University (HKTD2024-03), and the scientific research start-up funding of Hainan Open University.

Data Availability Statement

All data generated or analyzed during this study are included in this published article and its supplementary materials. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no competing financial interests or personal relationships that could have influenced this work.

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Figure 1. TAIL-PCR amplification and validation of DhMYB2 and DhbHLH1 promoter regions. (A) TAIL-PCR amplification of DhMYB2 promoter regions. Top panel shows schematic diagram of chromosome walking strategy. Bottom panel shows amplification products after using degenerate adaptor primers AP1 or AP2 with nested gene-specific primers SP1 and SP2. (B) Genomic PCR validation of full-length DhMYB2 promoter (1864 bp) using sequence-specific primers. (C) Primary TAIL-PCR amplification of DhbHLH1 promoter region using degenerate adaptor primers AP1–AP4 with nested gene-specific primers SP1–SP3 (1416 bp product). (D) Secondary TAIL-PCR extension of DhbHLH1 promoter region using same AP1–AP4 primers paired with nested newly gene-specific primers SP1–SP3 (941 bp extension product). (E) Genomic PCR validation of full-length DhbHLH1 promoter (1995 bp) using sequence-specific primers.
Figure 1. TAIL-PCR amplification and validation of DhMYB2 and DhbHLH1 promoter regions. (A) TAIL-PCR amplification of DhMYB2 promoter regions. Top panel shows schematic diagram of chromosome walking strategy. Bottom panel shows amplification products after using degenerate adaptor primers AP1 or AP2 with nested gene-specific primers SP1 and SP2. (B) Genomic PCR validation of full-length DhMYB2 promoter (1864 bp) using sequence-specific primers. (C) Primary TAIL-PCR amplification of DhbHLH1 promoter region using degenerate adaptor primers AP1–AP4 with nested gene-specific primers SP1–SP3 (1416 bp product). (D) Secondary TAIL-PCR extension of DhbHLH1 promoter region using same AP1–AP4 primers paired with nested newly gene-specific primers SP1–SP3 (941 bp extension product). (E) Genomic PCR validation of full-length DhbHLH1 promoter (1995 bp) using sequence-specific primers.
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Figure 2. Linear schematic of functionally characterized cis-acting elements in DhMYB2 and DhbHLH1 promoters. Schematic representation of predicted cis-acting elements in promoters of DhMYB2 (A) and DhbHLH1 (B). Numbered boxes indicate cis-acting element: 1, Box 4; 2, TCT-motif; 3, G-box; 4, ACE; 5, GATA-motif; 6, GT1-motif; 7, ABRE; 8, AAGAA motif; 9, ERE; 10, ARE; 11, DRE core; 12, STRE; 13, TCA motif; 14, TC-rich repeats; 15, W box; 16, ATC-motif; 17, AuxRR-core; 18, TGA-element. Small red circles denote MeJA-responsive elements (CGTCA-motif/TGACG-motif).
Figure 2. Linear schematic of functionally characterized cis-acting elements in DhMYB2 and DhbHLH1 promoters. Schematic representation of predicted cis-acting elements in promoters of DhMYB2 (A) and DhbHLH1 (B). Numbered boxes indicate cis-acting element: 1, Box 4; 2, TCT-motif; 3, G-box; 4, ACE; 5, GATA-motif; 6, GT1-motif; 7, ABRE; 8, AAGAA motif; 9, ERE; 10, ARE; 11, DRE core; 12, STRE; 13, TCA motif; 14, TC-rich repeats; 15, W box; 16, ATC-motif; 17, AuxRR-core; 18, TGA-element. Small red circles denote MeJA-responsive elements (CGTCA-motif/TGACG-motif).
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Figure 3. GUS stain assay for full-length and truncated fragments of DhMYB2 and DhbHLH1 promoters in tobacco leaves. (A) A schematic diagram of the promoters, showing 5′-deletions of DhMYB2 and DhbHLH1. The box legends indicate the promoter and the numbers represent positions relative to the start codon. (B,C) GUS activities of full-length and various deletion constructs of the DhMYB2 promoter, showing a gradual decrease in GUS activity with truncated sequences. (D,E) GUS activities of full-length and various deletion constructs of the DhbHLH1 promoter, showing a gradual decrease in GUS activity with truncated sequences. The CaMV35S promoter was used as a positive control. Values represent the means ± SD from three replicates. Significant differences are indicated by different star symbols above the bars: * p < 0.05, ** p < 0.01.
Figure 3. GUS stain assay for full-length and truncated fragments of DhMYB2 and DhbHLH1 promoters in tobacco leaves. (A) A schematic diagram of the promoters, showing 5′-deletions of DhMYB2 and DhbHLH1. The box legends indicate the promoter and the numbers represent positions relative to the start codon. (B,C) GUS activities of full-length and various deletion constructs of the DhMYB2 promoter, showing a gradual decrease in GUS activity with truncated sequences. (D,E) GUS activities of full-length and various deletion constructs of the DhbHLH1 promoter, showing a gradual decrease in GUS activity with truncated sequences. The CaMV35S promoter was used as a positive control. Values represent the means ± SD from three replicates. Significant differences are indicated by different star symbols above the bars: * p < 0.05, ** p < 0.01.
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Figure 4. Analysis of DhMYB2 and DhbHLH1 promoter activities in tobacco leaves under abiotic stress treatments. Tobacco leaves containing DhMYB2 and DhbHLH1 promoters were treated with drought, varying temperatures, and different light conditions. (A,C) Under light (long light: 16 h light/8 h dark; short light: 8 h light/16 h dark) and temperature (high temperature: 42 °C; low temperature: 4 °C) treatments, promoter activities were enhanced under long light, reduced under short light, increased at low temperature, and showed no significant change at high temperature. (B,D) Under drought stress, leaves cultured in MS medium supplemented with 18% PEG 6000 exhibited intensely stained GUS activity compared to the mock-treated control. Leaves infiltrated with the GUS-empty vector (negative control) showed negligible GUS activity, while those expressing CaMV35Spro::GUS (positive control) displayed constitutive GUS expression under all conditions. Values represent the means ± SD from three replicates. Significant differences are indicated by different star symbols above the bars, * p < 0.05, ** p < 0.01.
Figure 4. Analysis of DhMYB2 and DhbHLH1 promoter activities in tobacco leaves under abiotic stress treatments. Tobacco leaves containing DhMYB2 and DhbHLH1 promoters were treated with drought, varying temperatures, and different light conditions. (A,C) Under light (long light: 16 h light/8 h dark; short light: 8 h light/16 h dark) and temperature (high temperature: 42 °C; low temperature: 4 °C) treatments, promoter activities were enhanced under long light, reduced under short light, increased at low temperature, and showed no significant change at high temperature. (B,D) Under drought stress, leaves cultured in MS medium supplemented with 18% PEG 6000 exhibited intensely stained GUS activity compared to the mock-treated control. Leaves infiltrated with the GUS-empty vector (negative control) showed negligible GUS activity, while those expressing CaMV35Spro::GUS (positive control) displayed constitutive GUS expression under all conditions. Values represent the means ± SD from three replicates. Significant differences are indicated by different star symbols above the bars, * p < 0.05, ** p < 0.01.
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Figure 5. Analysis of DhMYB2 and DhbHLH1 promoter activities in tobacco leaves under phytohormone treatments. Tobacco leaves containing DhMYB2 and DhbHLH1 promoters were treated with MeJA, SA, ABA, and IAA. (A,D). Tobacco leaves sprayed with 0.1 mM MeJA dissolved in 10% ethanol, and a control sprayed with 10% ethanol, showed significantly increased DhbHLH1 promoter activity. (B,E). Tobacco leaves sprayed with 1 mM SA and 0.1 mM ABA in distilled water showed significantly increased promoter activities of DhMYB2 and DhbHLH1. (C,F). Tobacco plants infiltrated for 2 days in MS medium containing 1 mg/L IAA showed significantly increased DhbHLH1 promoter activity. Leaves infiltrated with the GUS-empty vector (negative control) showed negligible GUS activity, while those expressing CaMV35Spro::GUS (positive control) displayed constitutive GUS expression under all conditions. Values represent the means ± SD from three replicates. Significant differences are indicated by different star symbols above the bars: * p < 0.05, ** p < 0.01.
Figure 5. Analysis of DhMYB2 and DhbHLH1 promoter activities in tobacco leaves under phytohormone treatments. Tobacco leaves containing DhMYB2 and DhbHLH1 promoters were treated with MeJA, SA, ABA, and IAA. (A,D). Tobacco leaves sprayed with 0.1 mM MeJA dissolved in 10% ethanol, and a control sprayed with 10% ethanol, showed significantly increased DhbHLH1 promoter activity. (B,E). Tobacco leaves sprayed with 1 mM SA and 0.1 mM ABA in distilled water showed significantly increased promoter activities of DhMYB2 and DhbHLH1. (C,F). Tobacco plants infiltrated for 2 days in MS medium containing 1 mg/L IAA showed significantly increased DhbHLH1 promoter activity. Leaves infiltrated with the GUS-empty vector (negative control) showed negligible GUS activity, while those expressing CaMV35Spro::GUS (positive control) displayed constitutive GUS expression under all conditions. Values represent the means ± SD from three replicates. Significant differences are indicated by different star symbols above the bars: * p < 0.05, ** p < 0.01.
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Figure 6. Histochemical analysis of GUS staining in transgenic Arabidopsis plants carrying the DhMYB2 promoter fusion construct. The DhMYB2 promoter showed strong activity in the floral tissues, widely distributed in the sepals, petals, pistil, and stamen. (a) Cotyledon seedlings, (b) true leaf seedlings, (c) adult leaf seedlings, (d) a root during the flowering period, (e) a stem during the flowering period, (f) a leaf during the flowering period, (g) a flower, (h) a pod, and (i) seeds. Pe, petals; Se, sepals; St, stamens. Bars = 2 mm.
Figure 6. Histochemical analysis of GUS staining in transgenic Arabidopsis plants carrying the DhMYB2 promoter fusion construct. The DhMYB2 promoter showed strong activity in the floral tissues, widely distributed in the sepals, petals, pistil, and stamen. (a) Cotyledon seedlings, (b) true leaf seedlings, (c) adult leaf seedlings, (d) a root during the flowering period, (e) a stem during the flowering period, (f) a leaf during the flowering period, (g) a flower, (h) a pod, and (i) seeds. Pe, petals; Se, sepals; St, stamens. Bars = 2 mm.
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Figure 7. Histochemical analysis of GUS staining in transgenic Arabidopsis plants carrying the DhbHLH1 promoter fusion construct. The DhbHLH1 promoter showed strong activity in the floral tissues, widely distributed in the sepals, petals, pistil, and stamen. (a) Cotyledon seedlings, (b) true leaf seedlings, (c) adult leaf seedlings, (d) a root during the flowering period, (e) a stem during the flowering period, (f) a leaf during the flowering period, (g) a flower, (h) a pod, and (i) seeds. Pe, petals; Se, sepals; St, stamens. Bars = 2 mm.
Figure 7. Histochemical analysis of GUS staining in transgenic Arabidopsis plants carrying the DhbHLH1 promoter fusion construct. The DhbHLH1 promoter showed strong activity in the floral tissues, widely distributed in the sepals, petals, pistil, and stamen. (a) Cotyledon seedlings, (b) true leaf seedlings, (c) adult leaf seedlings, (d) a root during the flowering period, (e) a stem during the flowering period, (f) a leaf during the flowering period, (g) a flower, (h) a pod, and (i) seeds. Pe, petals; Se, sepals; St, stamens. Bars = 2 mm.
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MDPI and ACS Style

Wang, Y.; Li, C.; Zhu, W.; Li, Y.; Song, X.; Yin, J. Isolation and Functional Analysis of the DhMYB2 and DhbHLH1 Promoters from Phalaenopsis-Type Dendrobium Involved in Stress Responses and Tissue-Specific Expression. Horticulturae 2025, 11, 550. https://doi.org/10.3390/horticulturae11050550

AMA Style

Wang Y, Li C, Zhu W, Li Y, Song X, Yin J. Isolation and Functional Analysis of the DhMYB2 and DhbHLH1 Promoters from Phalaenopsis-Type Dendrobium Involved in Stress Responses and Tissue-Specific Expression. Horticulturae. 2025; 11(5):550. https://doi.org/10.3390/horticulturae11050550

Chicago/Turabian Style

Wang, Yachen, Chonghui Li, Wenjuan Zhu, Yamei Li, Xiqiang Song, and Junmei Yin. 2025. "Isolation and Functional Analysis of the DhMYB2 and DhbHLH1 Promoters from Phalaenopsis-Type Dendrobium Involved in Stress Responses and Tissue-Specific Expression" Horticulturae 11, no. 5: 550. https://doi.org/10.3390/horticulturae11050550

APA Style

Wang, Y., Li, C., Zhu, W., Li, Y., Song, X., & Yin, J. (2025). Isolation and Functional Analysis of the DhMYB2 and DhbHLH1 Promoters from Phalaenopsis-Type Dendrobium Involved in Stress Responses and Tissue-Specific Expression. Horticulturae, 11(5), 550. https://doi.org/10.3390/horticulturae11050550

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