Putative Upstream Regulators DoNF-YB3 and DoIDD12 Correlate with DoGSTF11 Expression and Anthocyanin Accumulation in Dendrobium officinale

Abstract

Dendrobium officinale is a traditional and valuable medicinal herb, with extensive research conducted on its polysaccharides, alkaloids, and other components, yet studies on anthocyanins remain limited. In this study, we analyzed the expression levels of GST family genes in green and purplish D. officinale and found that DoGSTF11 is highly expressed in the purplish variety. DoGSTF11 is localized to the nucleus and cell membrane but lacks transcriptional activation activity. Overexpression of DoGSTF11 in tomato enhances anthocyanin accumulation, suggesting a role in anthocyanin sequestration or transport. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays further revealed that DoGSTF11 interacts with DoGST31, while DoIDD12 and DoNF-YB3 are potential transcriptional regulators based on promoter-binding assays and expression correlation. In conclusion, our study demonstrates that DoGST11 positively regulates anthocyanin accumulation in D. officinale. These findings provide valuable insights into the metabolic engineering of flavonoids in D. officinale.

1. Introduction

Anthocyanin is a significant class of secondary metabolites in plants, playing a crucial role in plant defense against both abiotic and biotic stresses, while also offering notable health benefits to humans [1]. Over 600 types of anthocyanins have been isolated and identified in nature, which are primarily derived from six anthocyanidin aglycones: pelargonidin, cyanidin, delphinidin, peonidin, malvidin, and petunidin [2]. The biosynthetic pathway of anthocyanins, a branch of the flavonoid biosynthesis pathway, has been largely elucidated [3]. It involves multiple metabolic steps catalyzed by various enzymes, with phenylalanine serving as the direct precursor [4]. The site of anthocyanin synthesis in plants is the endoplasmic reticulum, but color can only be displayed when accumulated in the vacuole, indicating that there is an efficient anthocyanin transport and accumulation mechanism in plant cells [5]. Despite the comprehensive understanding of the biosynthesis process, the mechanisms governing anthocyanin accumulation in the cytoplasm remain unclear.

The transcriptional regulatory mechanism plays a crucial role in the spatiotemporal control of anthocyanin biosynthesis. Studies have shown that the expression of anthocyanin biosynthetic structural genes is tightly regulated by a transcriptional network known as the MBW complex, which consists of MYB, bHLH, and WD40 proteins [6]. In Fragaria × ananassa, FaEGL3 and FaLWD1/FaLWD1-like interact with FaMYB5 to form the MYB–bHLH–WD40 complex, which significantly enhances transcriptional regulation and positively influences the accumulation of both anthocyanins and proanthocyanidins [7]. Additionally, phytochrome-interacting factors (PIFs) can directly bind to the MYB transcription factor PAP1, disrupting the MBW complex and thereby markedly repressing anthocyanin accumulation [8]. In contrast, BZR1 physically interacts with PAP1 and cooperatively regulates the expression of PAP1 target genes, promoting anthocyanin biosynthesis [9].

Glutathione S-transferases (GSTs) are multifunctional proteins widely distributed in plants, playing key roles in processes such as the transport of secondary metabolites, detoxification of xenobiotics, hormone regulation, and apoptosis [10]. In Secale cereale, ScGST39 is significantly upregulated under various stress conditions, suggesting its involvement in stress tolerance mechanisms [11]. Similarly, DoGST5 has been shown to enhance cadmium (Cd) tolerance in Dendrobium officinale by modulating reactive oxygen species (ROS) homeostasis, indicating its potential utility in improving plant adaptability to heavy metal stress [12]. Moreover, GSTs have been identified as key enzymes involved in anthocyanin accumulation. The maize ZmBz2 gene was the first GST family member associated with anthocyanin transport and accumulation [13]. Loss of zmbz2 results in the retention of anthocyanins in the cytoplasm, preventing their transport to the vacuole [13]. Similarly, the GST protein encoded by petunia PhAN9 binds anthocyanins and facilitates their transport to the vacuole for storage [14]. In Arabidopsis, TT19 is essential for anthocyanin accumulation, and its mutant phenotype can be rescued by overexpressing petunia PhAN9 [15,16]. Additionally, DcGSTF2 in carnations and CkmGST3 in cyclamens are highly expressed in anthocyanin-rich tissues and during active anthocyanin synthesis, compensating for the loss of TT19 function in Arabidopsis [17,18]. Furthermore, MdGSTF6 in apples and CsGSTF1 in tea have also been implicated in anthocyanin synthesis and accumulation in economically important crops [19,20].

Dendrobium officinale, a valuable traditional Chinese medicinal plant, is renowned for its roles in promoting fluid production, delaying aging, and enhancing immunity [21,22]. Researchers have used UHPLC-MS and transcriptomic analysis to characterize the flavonoid synthesis metabolic network in D. officinale [23]. The gene DoUFGT was identified as a key determinant of anthocyanin synthesis differences between purple-stem and green-stem varieties [24]. Varieties with high anthocyanin content, such as those with purple and red stems, are particularly valued for their bright coloration, nutritional benefits, and expanded market potential [24,25]. Purple-stem D. officinale shows higher expression of flavanone 3-hydroxylase (F3H) and leucoanthocyanidin dioxygenase (LDOX), which enhances anthocyanin production [26]. Given the pivotal role of glutathione S-transferase (GST) genes in anthocyanin biosynthesis, this study investigated the expression patterns of GST family members in two phenotypes of D. officinale. Notably, DoGSTF11 exhibited the most significant differential expression, suggesting its potential involvement in anthocyanin transport. Further analysis identified DoNF-YB3 and DoIDD12 as putative transcriptional regulators of DoGSTF11 expression. These findings advance our understanding of secondary metabolite accumulation in plants and provide a theoretical basis for the metabolic engineering of flavonoids in D. officinale.

2. Materials and Methods

2.1. Plant Materials and Gene Expression Analysis

Nine-month-old tissue-cultured seedlings, one-year-old domesticated seedlings, and two-year-old mature seedlings of green and purplish D. officinale were used as the experimental materials. The growth temperature was 25 °C, and the shading rate was 70%. The seedlings were cultivated in the Yandang Mountain production area of southern Zhejiang. Stems and leaves from the purplish stem and green stem plants at these three growth stages were collected to determine the anthocyanin content via an anthocyanin quantitative method using spectrophotometry [27]. A mixed sampling method was employed with three biological replicates per group, each consisting of at least 10 seedlings. Total RNA was extracted from the whole plant of D. officinale using the pBIOZOL reagent following the manufacturer’s instructions. The concentration and purity of the RNA were assessed using a NanoPhotometer spectrophotometer (Thermo Fisher Scientific (China) Co., Ltd., Shanghai, China). The RNA samples were then reverse transcribed into cDNA using a PrimeScript RT Reagent Kit with a gDNA Eraser (Baori Medical Biotechnology (Beijing) Co., Ltd., Beijing, China). The primers were designed based on the corresponding gene sequences, and RT-qPCR was conducted to examine the expression of DoGSTF11 (LOC110097588) and its upstream and downstream regulatory genes in different tissues and growth stages. Actin was used as the internal reference gene, and relative gene expression levels were calculated using the 2^−ΔΔCt method [28]. All primers designed in this paper are in Table S1.

2.2. Subcellular Localization of DoGSTF11 in Nicotiana tabacum

Based on the sequence characteristics of DoGSTF11 (LOC110097588) and the pCAM-EYFP vector, specific primers were designed for PCR amplification. Double restriction enzyme digestion was performed on the PCR product and the pCAM-GFP vector. The successfully digested DoGSTF11 gene fragment was ligated with the pCAM-GFP vector to construct the 35S: DoGSTF11: GFP vector, which was subsequently introduced into Agrobacterium for genetic transformation. As a control, the pCAM-GFP empty vector was injected into tobacco epidermal cells. Co-expression markers for the cell nucleus and cell membrane, which were maintained in the laboratory, were co-injected with the 35S: DoGSTF11: GFP construct into tobacco epidermal cells. The subcellular localization of DoGSTF11 was then observed using laser confocal microscopy.

2.3. Agrobacterium Tumefaciens-Mediated Transformation in Solanum lycopersicum

Tomato seeds were surface-sterilized, sown on germination medium, and incubated in the dark for 3–4 days, followed by 4–5 days under a 16 h light/8 h dark photoperiod to promote seedling growth. Once the cotyledons were fully expanded, the petioles and tips were removed, and the middle sections were excised and used as explants. The cotyledon segments were pre-cultured on induction medium at 23 °C for 2–3 days. Agrobacterium was resuspended to an OD₆₀₀ of 0.1 in infiltration medium. Explants were immersed in the bacterial suspension for 10–15 min, air-dried, and transferred to co-cultivation medium for 2 days in the dark at 23 °C. After co-cultivation, the explants were transferred to selective screening medium and cultured under a 16 h light/8 h dark cycle at 23 °C for 15–30 days. The regenerating tissues were then transferred to differentiation medium and cultured under the same conditions for 30–40 days. Shoots reaching 2–3 cm in height were excised and transferred to rooting medium for 10–15 days under a 16 h light/8 h dark cycle at 23 °C. Total RNA and genomic DNA were extracted, and cDNA was synthesized via reverse transcription. PCR and qRT-PCR were conducted using gene-specific primers to analyze the DoGSTF11 expression levels in the resistant lines.

2.4. Analysis of DoGSTF11 Transcriptional Autoactivation Activity

The DoGSTF11 sequence was cloned into the pGBKT7 vector to construct the pGBKT7-DoGSTF11 plasmid. Both the pGBKT7-DoGSTF11 and pGBKT7 plasmids (negative control) were transformed into the AH109 yeast strain and spread on SD/-Trp plates. The plates were incubated at 30 °C for 3–4 days. Six colonies were randomly selected for PCR verification using pGBKT7 vector-specific primers, and all were confirmed to be correctly cloned. Subsequently, three colonies were then transferred onto SD/-Trp, SD/-Trp/-His, SD/-Trp/-His/-Ade, and SD/-Trp/-His/-Ade/X-α-gal plates. The plates were incubated at 30 °C for 3–5 days, and yeast growth was observed to assess transcriptional autoactivation.

2.5. Yeast Two-Hybrid Assay

Based on the predicted interactions of the proteins, the CDS of DoGSTF11 was separately cloned into the pGBKT7 vector as bait, and the CDSs of DoGST31 (LOC110109485), DoCHS (LOC110113809), DoDFR (LOC110093920), and DoANS (LOC110103723) were separately cloned into the pGADT7 vector as prey. Four various combinations of bait and prey vectors—pGBKT7-DoGSTF11/pGADT7-DoGST31, pGBKT7-DoGSTF11/pGADT7-DoCHS, pGBKT7-DoGSTF11/pGADT7-DoDFR, and pGBKT7-DoGSTF11/pGADT7-DoANS—were co-transformed into the Y2H-Gold yeast strain. The pGADT7-T/pGBKT7-p53 pair and the pGADT7-T/pGBKT7-Lam pair were used as positive and negative controls, respectively. After growth on SD/-Leu/-Trp medium at 30 °C, the clones were transferred into the selective medium at 30 °C for 3–5 d to test interactions.

2.6. Bimolecular Fluorescence Complementation Experiment

A homologous recombination method was employed to construct a bimolecular fluorescence complementation (BiFC) vector for detecting protein–protein interactions. Primers were designed based on the Gateway system and the fusion sites (attB1 and attB2) of the pDONR221 vector to amplify the target genes DoGSTF11 and DoGST31. The correctly verified fusion vectors containing the attL1 and attL2 sites were recombined with fluorescent protein vectors (YFP-N and YFP-C), which have attR1 and attR2 linkers. The successfully constructed fluorescent vectors were transformed into Agrobacterium GV3101 for culturing. Using the tobacco transient expression method, BiFC assays were performed, and the interaction strength and localization were observed under a laser confocal microscope. We analyzed a total of 10 randomly selected microscopic fields per biological replicate across 3 independent experiments.

2.7. Screening of Upstream Genes of DoGSTF11

The core promoter sequence of DoGSTF11 was cloned into the pHis2 vector and introduced into the yeast strain Y187 to create a bait strain. Competent cells were prepared from this bait strain. Subsequently, a D. officinale cDNA library was co-transformed with the pGADT7-Rec2 vector into these competent cells. Yeast one-hybrid screening was performed to identify regulatory proteins that bound upstream to the promoter.

3. Results

3.1. The Color Phenotype Analysis in D. officinale

Two color phenotypes of green and purplish D. officinale were selected as the experimental materials (Figure 1A,B). Microscopic analysis of the stem sections revealed that anthocyanins in the purple stem variety predominantly accumulated in the epidermal cells, whereas no significant pigment accumulation was observed in the epidermis of the green stem variety (Figure 1C–F). Among these, the purplish phenotype exhibited the highest anthocyanin content (Figure 1G).

3.2. Sequence Alignment and Phylogenetic Analysis

Given the critical role of GST family genes in regulating anthocyanin biosynthesis, the expression of GST genes was analyzed across the two phenotypes in D. officinale. Significant differences were observed in the expression levels of 11 DoGST genes. Notably, DoGSTF11 showed the most substantial differential expression (Figure 2A). The 11 differentially expressed DoGST genes were analyzed to construct a phylogenetic tree in conjunction with the previously reported GST genes involved in anthocyanin accumulation. The results show that DoGSTF11 clustered with known anthocyanin-related GSTs, such as Arabidopsis TT19 (phi type), while other DoGST proteins formed a separate cluster (Figure 2B). Amino acid sequence alignment revealed that DoGSTF11 shared high sequence similarity with anthocyanin-related GSTs, with the greatest similarity (67.0%) observed with grape VvGST4 (Figure 2C). These findings suggest that DoGSTF11 may be involved in anthocyanin synthesis and metabolism.

3.3. Subcellular Localization of DoGSTF11 in Nicotiana tabacum

To analyze the subcellular localization of the DoGSTF11 protein, tobacco leaves were transiently transformed via Agrobacterium injection. The pCAM-GFP empty vector and 35S:DoGSTF11:GFP construct were individually injected into tobacco epidermal cells and observed using a laser confocal microscope. As shown in Figure 3, the positive control (35S:GFP) exhibited green fluorescence at both the cell membrane and nucleus. A similar pattern was observed for 35S:DoGSTF11:GFP, indicating that DoGSTF11 was localized to the cell membrane and nucleus.

3.4. Functional Validation in Transgenic Solanum lycopersicum

To further investigate the function of DoGSTF11, we stably transferred DoGSTF11 into tomato seedlings. As shown in Figure 4A,B, three transgenic lines were successfully obtained. Upon observation, the stems and leaves of the transgenic tomato seedlings exhibited extensive purple coloration, particularly in the OE3 line, which also showed the highest expression of DoGSTF11. Subsequent analysis of the anthocyanin content revealed that the overexpressing transgenic lines had significantly higher anthocyanin levels compared with the wild-type plants, with the OE3 line showing the highest content (Figure 4C). These results preliminarily confirm that DoGSTF11 acts as a positive regulator of synthesis.

3.5. Interaction Between DoGSTF11 and DoGSTF31

The transcriptional self-activation activity of DoGSTF11 was analyzed. As shown in Figure 5A, the negative control (pGBKT7) grew on the SD/-Trp plates but failed to grow on the SD/-Trp/-His, SD/-Trp/-His/-Ade, and SD/-Trp/-His/-Ade + X-α-gal plates. Similarly, the experimental group exhibited growth patterns identical to those of the negative control, confirming that DoGSTF11 lacks self-activation activity.

To identify the interacting target proteins, a yeast two-hybrid assay was conducted using a yeast library constructed from mixed samples of stems and leaves of purple stem D. officinale. The bait protein expression vector pGBKT7-DoGSTF11 was employed. Four candidate proteins interacting with DoGSTF11 were initially identified and subsequently verified using a yeast two-hybrid assay. The results showed that yeast colonies grew normally on SD/-Trp/-Leu medium. However, only colonies co-expressing DoGSTF11 and DoGST31 grew, and they exhibited blue coloration on SD/-Trp/-Leu/-Ade/-His/X-α-gal/AbA medium, while other combinations failed to grow (Figure 5B). This suggests a potential interaction between DoGSTF11 and DoGST31.

The interaction between DoGSTF11 and DoGST31 was further validated through bimolecular fluorescence complementation (BiFC). As shown in Figure 5C, no fluorescence was observed in the tobacco leaves expressing only single fusion proteins (DoGSTF11-NE or DoGST31-CE) or having an additional YFP protein. However, when DoGSTF11-NE and DoGST31-CE were co-expressed, YFP fluorescence was detected in the nuclei of the tobacco leaves, providing further evidence of interaction between DoGSTF11 and DoGST31.

3.6. Identification of Upstream Regulatory Factors

A pHIS2-DoGSTF11pro bait plasmid was constructed, and yeast one-hybrid assays were performed to screen for upstream regulatory proteins that bind to the DoGSTF11 promoter. After aligning the sequences with Seqman (Seqman Pro version) and BLAST (Clustalx1.81 version), 21 unique sequences were identified, excluding empty vectors and duplicates (Figure 6A). These 21 positive clones grew on SD-TL-, SD-TLH-, and SD-TLH + 75 mM 3AT-deficient plates. Further analysis revealed that DoIDD12 and DoNF-YB3 are potential upstream regulatory factors of DoGSTF11.

To investigate the spatiotemporal expression patterns of these two upstream regulatory genes, the expression levels were analyzed across various developmental stages. As shown in Figure 6B, the expression of DoGSTF11 generally increased with the age of D. officinale. The expression patterns of DoGSTF11 closely matched those of DoIDD12 and DoNF-YB3, with high expression levels observed in the stems and leaves of biennial purplish D. officinale. These results suggest that DoIDD12 and DoNF-YB3 may function as upstream regulators of DoGSTF11 and play a role in the synthesis of anthocyanins.

4. Discussion

The coloration of plants is primarily determined by the accumulation of anthocyanins, chlorophyll, and carotenoids in plant tissues [29,30]. Among these, anthocyanins, which are water-soluble pigments belonging to the flavonoid class of secondary metabolites, play a key role in the coloration of plant organs such as flowers and fruits [31]. These pigments not only contribute to the visual appeal of plants but also serve various biological functions, including enhancing plant tolerance to abiotic stresses, resisting pathogens, and attracting pollinators [31]. In D. officinale, anthocyanins are mainly localized in the epidermis, and their content is an important indicator of the plant’s authenticity in medicinal applications [32]. In this study, we quantified the anthocyanin content in two different D. officinale varieties and found that the purple-stem variety exhibited the highest anthocyanin content. Microscopic examination of the stem sections revealed that anthocyanins in the purple-stem variety accumulated primarily in the epidermal cells, whereas no significant pigment accumulation was observed in the epidermis of the green-stem variety.

Anthocyanins, synthesized through the flavonoid biosynthesis pathway, are regulated by a variety of structural genes [31,33]. Glutathione S-transferases (GSTs) represent an ancient and extensive gene family, with many members identified across various plant species [34,35,36]. Previous studies have demonstrated that GSTs play a crucial role in anthocyanin accumulation in maize, morning glory, and Arabidopsis, with loss-of-function mutants displaying a lack of anthocyanin accumulation [13,37,38]. The abundance of GSTs is also linked to fruit pigmentation in several horticultural crops, including apples, litchi, peaches, and strawberries [19,39,40,41]. However, the transcriptional regulatory mechanisms of GSTs in Dendrobium officinale remain unclear. In this study, we analyzed the expression levels of GST family genes in two varieties of D. officinale: purple stem and green stem. Significant differences were observed in the expression of 11 DoGST genes, with 7 of these genes showing expression patterns consistent with the color phenotypes. Notably, DoGST11 exhibited the highest expression.

The dual localization of DoGSTF11 in the nucleus and membrane suggests that it may be involved in multiple biological processes beyond anthocyanin transport. The localization of DoGSTF11 in the nucleus (gene regulation) and membrane (ROS scavenging) suggests that it may play a role in signal transduction, stress response, and gene regulation. Anthocyanin synthesis requires the collaboration of the cytoplasm, membrane, and nucleus, and DoGSTF11 may integrate these processes.

Anthocyanins are synthesized in the cytoplasm but predominantly accumulate in the vacuole, where they are stabilized through sequestration mechanisms involving GSTs, multidrug and toxic compound extrusion (MATE) transporters, and ATP-binding cassette (ABC) transporters [42]. ABC transporters play a pivotal role in anthocyanin accumulation in plants [43]. GSTs function as ligandins or carrier proteins by binding to anthocyanin 3-O-glucosides, facilitating their transport from the cytosol to anthocyanin-containing vesicles, also known as anthocyanin vacuolar inclusions [44]. The final step of vacuolar sequestration involves membrane transport via MATE and ABC transporters [44]. In this study, phylogenetic analysis revealed that DoGSTF11 clustered with known anthocyanin-related GST genes, suggesting its involvement in the transport of anthocyanins from the endoplasmic reticulum to the vacuole. Moreover, overexpression of DoGSTF11 in Solanum lycopersicum led to a large area of purple pigmentation in transgenic seedlings, with a significantly higher anthocyanin content compared with the WT. These results indicate that DoGSTF11 acts as a positive regulator of anthocyanin accumulation. However, the specific regulatory role of DoGSTF11 remains unclear, and its potential synergistic interaction with ABC transporters warrants further investigation in future studies.

The accumulation of anthocyanins is tightly regulated by the MYB-bHLH-WD40 complex [45]. R2R3-MYB transcription factors are crucial for determining the specificity of downstream gene expression and driving tissue-specific anthocyanin accumulation [45,46]. bHLH transcription factors are essential for stabilizing the MBW protein complex or promoting its transcription, which in turn activates the function of the R2R3-MYB partner [47,48]. WDR proteins interact physically with both MYB and bHLH factors, further regulating anthocyanin biosynthesis [49,50,51]. In this study, Y2H and BiFC assays demonstrated that DoGSTF11 can form a complex with DoGST31. Studies have shown that grape GST31 is involved in the biosynthesis of anthocyanins in grapes under high temperature stress [52]. These findings provide preliminary evidence that DoGST11 may interact with DoGST31 to promote anthocyanin accumulation. Furthermore, the Y1H assays indicated that DoIDD12 and DoNF-YB3 may act as upstream regulators of DoGSTF11. Previous studies have shown that the Arabidopsis IDD12 gene plays a key role in the transcriptional regulation of tissue formation during root development [53]. Similarly, the Arabidopsis NF-YB3 gene is implicated in various plant biological processes, including flower development, as well as responses to biotic and abiotic stresses [54,55,56]. Notably, the expression patterns of DoIDD12 and DoNF-YB3 aligned closely with those of DoGSTF11, with high expression observed in the stems and leaves of biennial D. officinale. These results suggest that DoIDD12 and DoNF-YB3 may play a critical role in anthocyanin biosynthesis. A more detailed functional analysis of these genes will be the focus of our next stage of research.

The correlation between the expression of DoGSTF11, DoIDD12, DoNF-YB3, and DoGST31 genes and the anthocyanin content was analyzed. The results indicate that the anthocyanin content in the stem was significantly positively correlated with DoGSTF11, DoIDD12, DoNF-YB3, and DoGST31 (p < 0.01). In the leaves, the anthocyanin content was significantly positively correlated with DoGSTF11, DoIDD12, and DoNF-YB3 (p < 0.05). These findings suggest that DoGSTF11, DoNF-YB3, and DoGST31 play a more critical role in anthocyanin accumulation.

5. Conclusions

In this study, we analyzed the expression levels of GST family genes in Dendrobium officinale varieties with purple and green stems. Our findings identify DoGSTF11 as a positive regulator of anthocyanin accumulation in D. officinale. Y2H and BiFC assays confirmed that DoGSTF11 specifically interacts with DoGST31, facilitating anthocyanin accumulation. Additionally, Y1H assays further identified DoIDD12 and DoNF-YB3 as potential upstream regulators of DoGSTF11. Although these results point to a potential regulatory relationship, additional functional studies are required to confirm direct transcriptional regulation. Based on these results, we propose a hypothetical model for intracellular anthocyanin accumulation in D. officinale (Figure 7). The expression of DoGSTF11 stimulates anthocyanin synthesis in the endoplasmic reticulum, a process regulated by upstream genes or dependent on protein interactions. This regulation facilitates anthocyanin transport to the vacuole, leading to subsequent intracellular accumulation. This study provides new insights into the molecular mechanisms underlying the enhanced anthocyanin accumulation in purple-stem D. officinale, contributing to our understanding of secondary metabolite biosynthesis in medicinal plants.