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Article

Study on the Molecular Mechanism of Arbuscular Mycorrhizal Symbiosis Regulating Polysaccharide Synthesis in Dendrobium officinale

by
Jiadong Chen
1,
Yiqun Zhang
1,
Man Zhang
1,
Ziyi Zhang
1,2,
Yingying Liu
1,
Xiaojing Duan
1,
Zhengming Tao
1,* and
Wu Jiang
1,*
1
Zhejiang Institute of Subtropical Crops, Zhejiang Academy of Agricultural Sciences, Wenzhou 325005, China
2
State Key Laboratory of Crop Genetics & Germplasm Enhancement and Utilization, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095, China
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2025, 26(19), 9298; https://doi.org/10.3390/ijms26199298
Submission received: 15 August 2025 / Revised: 19 September 2025 / Accepted: 20 September 2025 / Published: 23 September 2025
(This article belongs to the Section Molecular Plant Sciences)
Browse Figures
Versions Notes

Abstract

Mycorrhizal symbiosis represents a ubiquitous mutualistic relationship in nature, wherein mycorrhizal fungi enhance the host plant’s ability to absorb water and nutrients from the soil. In return, the host plant supplies the fungi with essential nutrients necessary for their metabolic activities. However, research focusing on the regulatory mechanisms governing mycorrhizal symbiosis in Dendrobium officinale remains limited. This study systematically investigates the regulatory mechanisms of mycorrhizal symbiosis on transcriptional synthesis in D. officinale by establishing a mycorrhizal symbiotic system, complemented by phenotypic observation, physiological measurement, and transcriptome sequencing. The results indicate that mycorrhizal symbiosis promotes both growth and nutrient absorption in D. officinale, concurrently increasing polysaccharide content. Through transcriptome analysis, we identified 59 differentially expressed genes associated with polysaccharide metabolism, alongside key genes and transcription factors integral to the regulatory network. Notably, the glycosyltransferase gene DoUGT83A1 was found to negatively regulate the mycorrhizal symbiotic system when heterologously expressed in tomato. This study provides a fundamental theoretical basis for elucidating the molecular mechanisms underlying polysaccharide synthesis in D. officinale and offers new insights for optimizing cultivation practices to enhance medicinal quality.

1. Introduction

Dendrobium officinale Kimura & Migo, a perennial epiphytic herb belonging to the genus Dendrobium in the Orchidaceae family, is a highly valued traditional Chinese medicinal herb [1]. For its ornamental value and a range of therapeutic effect, D. officinale is ranked as one of the ‘The first of nine Chinese fairy grasses’, and is also included in the Chinese Pharmacopoeia (2025, pp. 303–304) [2,3]. According to the “Compendium of Materia Medica,” D. officinale is known for its effects such as “strengthening yin and replenishing essence, supplementing internal deficiencies, enhancing intelligence and alleviating fear, and promoting longevity.” Research has identified various active components in D. officinale, including polysaccharides, alkaloids, and amino acids [4]. Modern pharmacological studies indicate that D. officinale possesses significant therapeutic effects, including enhancing human immunity, anti-aging properties, tumor inhibition, and sleep improvement [5,6]. The “Chinese Pharmacopoeia” stipulates that the mass fraction of polysaccharides in qualified D. officinale must not be less than 25%, highlighting the synthesis and transformation of these polysaccharides as a focal point in contemporary academic research [7]. The polysaccharides in D. officinale are primarily composed of glucose, mannose, xylose, rhamnose, glucuronic acid, and galacturonic acid [8]. Substantial research has been conducted on the phytochemical constituents and therapeutic mechanisms of D. officinale. Polysaccharides, as one of the primary bioactive components in D. officinale, exhibit multifaceted pharmacological effects, including immunomodulation, antitumor activity, antioxidant properties, and anti-inflammatory actions [9,10,11,12,13].
Mycorrhizae represent a symbiotic association formed between fungi and the roots of host plants, serving as a crucial mechanism for plants to adapt to environmental stressors [14,15]. Arbuscular mycorrhizal (AM) association occurs in an endosymbiotic form, characterized by the formation of highly branched hyphal structures known as arbuscules within the root cortical cells. This phenomenon is a hallmark of AM symbiosis and plays a crucial role in the interaction between the fungal partner and the host plant [16]. Mycorrhizal symbiosis aids hosts in absorbing nutrients from soil outside the rhizosphere via extrarhizosphere hyphae, thereby enhancing their uptake of essential nutrients like phosphorus, nitrogen, and potassium [17,18]. Host plants offer their own carbohydrates as the essential carbon source for the growth of mycorrhizal fungi, thereby sustaining a healthy symbiotic relationship between the two parties [19,20]. Mycorrhizal symbiosis enhances medicinal plants’ resistance to both environmental stresses and pathogen/pest infestations, while promoting the accumulation of bioactive medicinal compounds. For example, AM might play a key role in alfalfa growth and survival under harsh salt conditions [21]. Different arbuscular mycorrhizal fungal (AMF) species (particularly the mixed inoculum) improved the growth and physiological traits of Eclipta prostrata (L.) L. under both non-saline and saline conditions by enhancing antioxidant enzyme activity, promoting proline and total phenolic accumulation, and significantly regulating the spatiotemporal dynamics of polyphenols [22]. Previous research has demonstrated that AMF communities possess considerable potential in enhancing the yield and promoting the accumulation of bioactive components in Salvia miltiorrhiza Bge. [23].
This study inoculated potted D. officinale with mycorrhizal fungi to investigate the effects of mycorrhizal symbiosis on the growth and polysaccharide accumulation of D. officinale. Transcriptome sequencing was employed to analyze the transcriptional regulatory pathways associated with mycorrhizal symbiosis. A glycosyltransferase gene, DoUGT83A1, which is induced by mycorrhizal symbiosis, was identified. Through heterologous validation in transformed tomatoes, the role of DoUGT83A1 in sugar metabolism and the mycorrhizal symbiotic pathways of D. officinale was elucidated, providing a theoretical basis for understanding the transcriptional regulatory mechanisms of mycorrhizal symbiosis in D. officinale.

2. Results

2.1. Influence of Mycorrhizal Fungi on Phenotypic Characteristics of D. officinale

Mycorrhizal symbiosis positively influences plant growth by enhancing both vegetative development and the accumulation of metabolites. This study examined the phenotypic characteristics of mycorrhizal symbiosis in D. officinale, demonstrating that such symbiosis significantly promotes the growth of stems and roots. Specifically, there was a 28.51% increase in above-ground length and a 39.29% increase in below-ground length (Figure 1A,D). Observations from root staining indicated that mycorrhizal fungi invaded the roots of D. officinale, forming arbuscular structures (Figure 1B). Quantitative fluorescence PCR analysis revealed a significant enhancement in the expression of the fungal elongation factor gene RiEF1a and the glyceraldehyde-3-phosphate dehydrogenase gene (RiGAPDH) in mycorrhizal symbiotic roots (Figure 1C). Furthermore, polysaccharides, which are essential medicinal components of D. officinale and are known to enhance human immunity, were found to increase in total content due to mycorrhizal symbiosis (Figure 1E). Our findings indicate that the expression levels of DoPMM and DoCSLA6, which are essential for gluconeogenesis in D. officinale inoculated with mycorrhizal fungi, were significantly higher than those observed in the control group (Figure 1F). Nitrogen and phosphorus are essential elements for plant growth. Relevant studies have demonstrated that mycorrhizal symbiosis aids the host in nutrient absorption from the environment. In this study, we found that the nitrogen and phosphorus content in mycorrhizal symbiotic D. officinale plants was significantly higher than that in the control group (Supplementary Figure S1).

2.2. RNA Sequencing and Assembly of the D. officinale Transcriptome

Considering the histological differences between arbuscular mycorrhizal symbiosis (AM) and non-symbiotic (NM) roots of D. officinale, we collected three biological replicates of each type for the preparation of RNA-seq libraries. High-quality RNA-Seq data were obtained for each sample, yielding between 39 and 47 million unidirectional reads. In total, 273,034,882 raw reads were generated from the six libraries. After filtering out low-quality sequences, we obtained 255,989,632 clean reads, with average Q20 and Q30 values of 97.71% and 93.385%, respectively. From these data, we identified 24,023 genes, with lengths ranging from 53 to 19,125 base pairs (Table S1).
Principal Component Analysis (PCA) identified two distinct clusters, with the first two components accounting for 80.03% of the total variance, thereby revealing distinct mRNA populations across different types (Figure 2A). Our analysis indicated that 1978 genes in the AM group and 843 genes in the NM group exhibited highly stage-specific expression patterns, whereas the majority of genes (16,289) were commonly expressed in D. officinale (Figure 2C). Additionally, we evaluated gene expression profiles among biological triplicates, which demonstrated a high degree of correlation (R2 > 0.803) (Supplementary Figure S2).
Using a threshold of fold change (FC) > 1 and false discovery rate (FDR) < 0.05, we identified 4267 differentially expressed genes (DEGs), indicating that mycorrhizal symbiosis triggered significant alterations in the gene expression profile of D. officinale (Figure 2D). These results represented substantial differences in gene expression profiles across D. officinale mycorrhizal symbiosis. HCA using all DEGs revealed two discrete clusters corresponding to different types (Supplementary Figure S3), which constitute distinct gene sets, highlighting the specialized nature of suggesting a tight linkage of DEGs with mycorrhizal symbiosis responses.
The enrichment analysis of differentially expressed genes (DEGs) in the Gene Ontology (GO) database revealed that, within the Biological Process (BP) category, gene alterations were predominantly concentrated in processes such as macromolecule glycosylation (GO:0043413), glycosylation (GO:0070085), lipid metabolic processes (GO:0006629), and carbohydrate metabolic processes (GO:0005975). In the Cellular Component (CC) category, gene changes were primarily focused on the photosystem I reaction center (GO:0009538), protein-DNA complexes (GO:0032993), and vesicles (GO:0031982). In the Molecular Function (MF) category, differentially expressed genes were mainly associated with transferase activity, specifically transferring hexosyl groups (GO:0016758), catalytic activity (GO:0003824), and transferase activity, specifically transferring glycosyl groups (GO:0016757) (Figure 3A). Several KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways that are significantly enriched have been suggested to be linked to zeatin biosynthesis (ko00908), starch and sucrose metabolism (ko00500), plant hormone signal transduction (ko04075), and plant-pathogen interactions. (ko04626) (Figure 3B). In the study of plant hormone signal transduction, a total of 57 differentially expressed genes were identified (Supplementary Figure S4). Additionally, 35 differentially expressed genes were discovered in the context of plant-pathogen interactions (Supplementary Figure S5). Collectively, these results elucidate the regulatory effects of mycorrhizal symbiosis on the transcriptome of D. officinale, providing preliminary insights into the underlying biological processes.

2.3. Key Genes and Metabolic Pathways Involved in Polysaccharide Biosynthesis in D. officinale

The biosynthesis of polysaccharides in Dendrobium officinale is closely linked to carbohydrate metabolic pathways, playing crucial roles in physiological processes such as photosynthesis. In the sugar metabolism-related pathways, we identified a total of 59 DEGs, which are primarily involved in three biological processes: starch biosynthesis and metabolism, cellulose biosynthesis and metabolism, and UDP-sugar synthesis. These processes may significantly influence the biosynthesis of polysaccharides in D. officinale (Figure 4). Our results indicate that the starch and sucrose metabolism pathway (ko00500) contains the highest number of differentially expressed genes (DEGs) (Table S2).
Compared to the non-inoculated control, the inoculation treatment significantly enhanced the activities of glucan endo-1,3-beta-D-glucosidase (BG, EC 3.2.1.39), fructose-bisphosphate aldolase (FBA, EC 4.1.2.13), sucrose phosphate synthase (SPS, EC 2.4.1.14), 1,4-alpha-glucan branching enzyme (GBE, EC 2.4.1.18), fructokinase (FRK, EC 2.7.1.4), and pyrophosphate-fructose 6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90). These enzyme activities were positively correlated with the increase in polysaccharides. Conversely, the inoculation treatment reduced the activities of sucrose phosphate phosphatase (SPP, EC 3.1.3.24), mannosidase (MAN, EC 3.2.1.78), sucrose synthase (SuSy, EC 2.4.1.13), hexokinase (HXK, EC 2.7.1.1), UDP-glucose glycosyltransferase (UGGT, EC 2.7.7.13), GDP-D-mannose 3′, 5′-epimerase (GME, EC 5.1.3.18), and phosphoglucomutase (PGM, EC 5.4.2.2), with the activities of these enzymes decreasing as polysaccharide levels increased. Additionally, the treatment altered the activities of TPP (EC 3.1.3.12), FBPase (EC 3.1.3.11, EC 3.1.3.46), CB (EC 3.2.1.21, EC 3.2.1.4), TPS (EC 2.4.1.15), PFK (EC 2.7.1.11), α-amylase (EC 3.2.1.1) (Table S3). These changes demonstrate that the inoculation treatment significantly influenced the polysaccharide biosynthesis process in D. officinale through the coordinated regulation of multiple enzyme activities.
A total of twelve differentially expressed glycosyltransferase genes were identified in response to arbuscular mycorrhizal fungal (AMF) colonization. Among these, seven genes, including LOC110107015 and LOC110115451, were downregulated following fungal inoculation, while five genes, such as LOC110112706 and LOC110105079, exhibited upregulated expression (Table S4). Quantitative real-time PCR analysis demonstrated that the relative expression level of LOC110105079 was significantly higher than that of the other four glycosyltransferase genes (Supplementary Figure S6). Consequently, LOC110105079 was selected as the target gene for further functional characterization. The differential expression patterns of these genes indicate their potential roles in the biosynthesis of polysaccharides in D. officinale and may contribute to the increased polysaccharide content observed following mycorrhizal symbiosis.

2.4. Identification of Transcription Factors Responsive to D. officinale Mycorrhizal Symbiosis

Transcriptome analysis of D. officinale before and after inoculation treatment identified a total of 2010 annotated transcription factor sequences belonging to 56 transcription factor families, (Table 1). Among these families, the bHLH family (218), MYB-related family (121), NAC family (118), WRKY family (98), ERF family (94), and B3 family (94) were identified as the most abundant transcription factors. Among the 12 glycosyltransferase genes identified previously, 9 are potentially capable of interacting with various transcription factor families. Specifically, LOC110105079, LOC110091880, LOC110096304, LOC110101924, LOC110101938, LOC110110985, and LOC110111214 may interact with the Heat Shock Factor (HSF) family, LOC110103890 may interact with the Ethylene Response Factor (ERF) family, and LOC110112706 may interact with the SQUAMOSA Promoter Binding Protein (SBP) family (Table S4).

2.5. Overexpression of DoUGT83A1 Inhibits Tomato Growth and Reduces Polysaccharide Synthesis in Mycorrhizal Symbiosis

Through NCBI alignment, Gene ID LOC110105079 was identified as DoUGT83A1. To investigate the role of DoUGT83A1 in the synthesis of polysaccharides in D. officinale and its regulatory pathway in mycorrhizal symbiosis, transgenic tomato plants were generated to constitutively overexpress DoUGT83A1 under the control of the cauliflower mosaic virus 35S promoter. A total of 10 overexpression lines were established, and as anticipated, these transgenic plants exhibited high levels of DoUGT83A1 expression (Supplementary Figure S7). Consequently, two independent transgenic lines, UGT83A1-OE4 and UGT83A1-OE7, were selected for further investigation. Compared to wild-type plants, the overexpression of DoUGT83A1 significantly inhibited tomato growth (Figure 5A,B), with the length and weight of the inoculated mycorrhizal shoots and roots being notably lower than those of the control plants (Figure 5C–F).
After five weeks of R. irregularis inoculation, the soluble sugar and sucrose contents in wild-type (WT) and UGT83A1-OE lines were assessed. Our results indicated that, irrespective of mycorrhizal fungal inoculation, the soluble sugar and sucrose levels in UGT83A1-OE plants were significantly lower than those observed in the control group (Figure 6A,B). Additionally, the arbuscular mycorrhizal (AM) colonization levels were evaluated. Although the UGT83A1-OE lines were capable of being colonized by AM fungi, the arbuscular colonization rate in these plants was significantly lower compared to WT plants (Figure 6D). However, no significant differences were observed in the morphology and fully developed arbuscular mycorrhizal structures between the UGT83A1-OE lines and WT plants (Figure 6C,E). Furthermore, the transcriptional levels of the AM-specific SlPT4 in the mycorrhizae of UGT83A1-OE lines were significantly reduced when compared to WT plants (Figure 6F).

3. Discussion

3.1. Establishment of Mycorrhizal Symbiosis System in D. officinale Reveals Molecular Insights into Plant-Fungal Interactions

Mycorrhizal symbiosis is widely observed in nature. Mycorrhizal fungi facilitate the absorption of water and nutrients from the soil by their host plants, in return, the host plants supply the fungi with essential sugars and fatty acids necessary for their vital activities [24,25,26]. D. officinale is particularly dependent on this fungal symbiosis. The application of mycorrhizal fungi is critically important for enhancing both the yield and quality of D. officinale; however, the underlying molecular mechanisms governing their interaction remain poorly understood.
It is well established that the development of AM symbiosis relies on continuous signaling and nutrient exchange between the host root system and AM fungi. Recent studies have highlighted the crucial roles of plant hormones, such as strigolactones, auxins, and gibberellins, in regulating AM symbiosis [27,28,29]. Additionally, flavonoids serve as significant regulators in AM symbiosis [30]. This study reveals that the mycorrhizal symbiosis of D. officinale induces alterations in the genes associated with the host’s hormone transcription pathways, including those involved in the synthesis of auxins (25), cytokinins (4), gibberellins (3), abscisic acid (7), ethylene (3), jasmonic acid (5), and salicylic acid (7) (Supplementary Figure S4). This suggests that D. officinale may respond to the symbiosis with mycorrhizal fungi through changes in plant hormones.

3.2. Transcriptomic Analysis Reveals AMF-Induced Modulation of Sugar Metabolism Pathways in D. officinale

Carbohydrates are products of plant photosynthesis and serve as essential components for energy supply and signaling molecules during the mycorrhizal symbiosis process. According to the 2020 edition of the ‘Chinese Pharmacopoeia,’ the polysaccharide content in D. officinale must not be less than 25%, while the mannose content should range from 13.0% to 38.0% [13]. The synthesis of polysaccharides in D. officinale is a delicate and complex process that involves a series of substance and signal interactions, along with the regulation of gene expression [31]. Previous studies have shown that polysaccharides are composed of monosaccharides, including glucose, mannose, and galactose. Notably, DoPMM and DoCSLA6 have been identified as key enzymatic genes involved in the fructose and mannose metabolic pathways [32,33]. In this study, we observed that the expression levels of DoPMM and DoCSLA6 in mycorrhizal symbiotic Dendrobium officinale were significantly higher than those in the control group.
Current research on the functional genes of D. officinale identifies several genes involved in polysaccharide metabolism and transport, including UDP-Glucose Pyrophosphorylase (UGPase), Cellulose Synthase-Like A6, Sucrose Transporter, GDP-Mannose Pyrophosphorylase, DoSWEET Protein, Alkaline/Neutral Invertase Hexose Transporter, and Soluble Acid Invertase [34].
This study conducted a transcriptome analysis of D. officinale before and after inoculation, revealing a total of 33 differentially expressed genes that were enriched in the starch and sucrose metabolic pathways. Additionally, 17 other tertiary pathways related to polysaccharide synthesis were identified, including glycolysis/gluconeogenesis, the pentose phosphate pathway, pyruvate metabolism, and fructose and mannose metabolism. Notably, the genes exhibiting significantly upregulated expression included sucrose phosphate synthase (SPS), fructokinase (FRK), endo-1,3-β-D-glucanase (BG), and β-glucosidase (CB) (Table S2).

3.3. Transcriptional Regulation of Symbiotic Responses and Stress Adaptation

Transcription factors are critical regulators of gene expression, directly influencing various physiological and biochemical responses in plants. This study found that the expression levels of transcription factors such as WRKY, NAC, bZIP, and MYB-related proteins changed following inoculation treatment, indicating that the host plant responds to the invasion of exogenous microorganisms by regulating the expression of numerous genes. Members of the GRAS transcription factor family have been identified as genes induced by arbuscular mycorrhizal (AM) fungi, with tomato SlGRAS18 interacting with SlDELLA, a central regulator of AM formation, thereby participating in the establishment of AM symbiosis [35]. TMLs are involved in regulating AM symbiosis, with MtTML1 and MtTML2 in alfalfa coordinating the regulation of AM colonization, as evidenced by a slight increase in AM colonization observed in mutants lacking both TMLs [36]. In this study, the analysis of transcription factors (TFs) revealed that the Heat Shock Transcription Factor (HSF) family is most closely associated with the expression of DoUGT83A1. Heat shock proteins (HSPs) are a class of stress-related proteins that are either newly synthesized or upregulated in organisms when exposed to adverse environmental conditions. They play a crucial role in the short-term adaptation of plants to stress. Li found that the expression of the HSP70 gene in Dendrobium officinale was significantly upregulated under 4 °C conditions [37]. It is well established that the polysaccharide content in Dendrobium officinale significantly increases under low-temperature conditions [38].

3.4. DoUGT83A1 Functions as a Negative Regulator in Modulating AM Symbiosis

Glycosyltransferases represent a crucial gene family involved in the polysaccharide synthesis pathway in plants, typically functioning downstream within this pathway. Analyzing transcriptomic data from adult and seedling Dendrobium officinale, Zhang et al. [39] revealed that glycosyltransferase genes are critically linked to polysaccharide synthesis in this species. Additionally, Zhang et al. [40] found that the glycosyltransferase gene family is associated with the modification processes following polysaccharide synthesis in Dendrobium officinale. In our study, we observed significantly lower levels of soluble sugars and sucrose in the DoUGT83A1-OE strain compared to the control (Figure 6A,B). These sugars are essential substances that plants provide to maintain the mycorrhizal symbiotic state. Furthermore, we found that the colonization rate of mycorrhizae in the DoUGT83A1-OE strain was significantly lower than that of the control (Figure 6D), although the morphology and size of the arbuscules were altered (Figure 6C,E). This alteration may stem from the host plant’s efforts to dynamically regulate fungal proliferation levels, preventing excessive carbon consumption and thereby sustaining the mutualistic relationship between the two symbionts. Recently, an increasing number of components involved in arbuscular mycorrhiza self-regulation (AOM) have been identified [41,42]. We speculate that plants may develop the ability to manipulate various self-regulatory pathways to coordinate fungal colonization levels and symbiotic nutrient exchange. Based on these findings, we propose a working model (Figure 7).

4. Materials and Methods

4.1. Experimental Materials

The plant material utilized in this experiment comprised one-year-old acclimatized seedlings of the newly developed D. officinale cultivar ‘Tiefeng No.1’, bred by our research group. Tiefeng No.1 is a purple-stemmed D. officinale variety which contains abundant anthocyanins and polysaccharides in its stems and leaves and has excellent cold tolerance and robust disease resistance. The arbuscular mycorrhizal fungus Rhizophagus irregularis, employed in the study, was propagated in our laboratory to produce an inoculant containing mycelium, sporocarps, spores, yellow sand, quartz sand, and dried plant root segments. The containers used for potting were plastic flower pots with the following specifications: height 14.0 cm, upper diameter 23.0 cm, and lower diameter 11.8 cm, which were disinfected by wiping with 75% ethanol. The substrate applied was cedar sawdust, which underwent dry-heat sterilization at 180 °C for 24 h in a high-pressure steam sterilizer prior to the experiment.

4.2. Experimental Design

The pot experiment was conducted using a completely randomized design, incorporating two treatments: inoculation with Rhizophagus irregularis (AM) and a control group inoculated with an equal amount of autoclaved inoculum (NM). Each treatment was clearly labeled and replicated in five pots, with five plants per pot. The plants were cultivated in the laboratory for 90 days under alternating conditions of 28 °C for 14 h of light and 22 °C for 10 h of darkness. Watering was performed quantitatively as needed, and a 25 μM Hoagland nutrient solution was applied monthly. Watering continued until the potting substrate was thoroughly moist, following the method described by Liu et al. [43]. After three months of symbiotic cultivation, 10–15 D. officinale seedlings from both AM and NM treatments were randomly selected. The root substrate was rinsed away, and the roots were dried using absorbent paper. Plant height (cm) and whole plant fresh weight (g) were measured, and the average values were calculated.

4.3. Extraction of Total RNA and cDNA Synthesis from D. officinale

Ninety-day-old whole seedlings of D. officinale were collected, with three biological replicates each for arbuscular mycorrhizal (AM) and non-mycorrhizal (NM) groups (denoted as AM1/AM2/AM3 and NM1/NM2/NM3, respectively), totaling six samples. Total RNA was isolated using a modified CTAB method [37].
RNA concentration was quantified using a Nanodrop spectrophotometer (Thermo Scientific, Waltham, MA, USA), and single-stranded cDNA was synthesized by reverse transcription following the manufacturer’s protocol of the All-in-One First-Strand Synthesis MasterMix kit (Vazyme, Nanjing, China). The samples were flash-frozen in liquid nitrogen for 30 min and stored at −80 °C for subsequent transcriptomic analysis. Relative expression level of each gene was normalized to the Actin gene (GenBank: KC831582.1) [32].

4.4. Transcriptome Sequencing

The transcriptome sequencing of six samples (including AM-inoculated and non-mycorrhizal (NM) treatments of D. officinale) was performed by Shanghai Majorbio Bio-pharm Technology Co., Ltd. The raw reads were aligned to the D. officinale reference genome (ASM160598v2) using the STAR aligner, and the mapped reads were visualized using the Integrative Genomics Viewer (IGV). Transcript assembly was conducted using Cufflinks (V2.2.1), followed by comparison with the reference annotation via Cuffcompare (V2.0.1) to statistically evaluate the alignment results. The mapping rate was assessed to determine whether the sequencing data met the requirements for subsequent analyses.

4.5. Screening and Functional Annotation of Differentially Expressed Genes (DEGs)

Gene expression levels were quantified using the FPKM method. Differentially expressed genes (DEGs) were identified with DESeq2 software (V1.36.0) using the threshold criteria of padj < 0.05 and |log2FC| > 1. Functional annotation and enrichment analysis of DEGs were performed through GO and KEGG databases. Additionally, sequencing data were subjected to alignment analysis against the GO and KEGG databases.

4.6. Construction of Binary Vectors and Generation of Transgenic Plants

To construct the DoUGT83A1 overexpression vector, the open reading frame of DoUGT83A1 was cloned into the binary vector pCAMBIA1305 using the ClonExpress II One Step Cloning Kit (Vazyme, Nanjing, China). The resulting construct, in which DoUGT83A1 is regulated by the constitutive Cauliflower mosaic virus 35S promoter, was introduced into the Agrobacterium tumefaciens EHA105 strain and subsequently used for tomato transformation, as previously described [44].

4.7. RNA Extraction and Real-Time RT-PCR Analysis of Transgenic Tomato Lines

RNA extraction and real-time RT-PCR were conducted as described by Chen et al. [45]. Briefly, total RNA was extracted from plant tissues with the TRIzol reagent (Vazyme, Nanjing, China), and residual DNA was eliminated by treatment with DNaseI. Reverse transcription and Real-time RT-PCR were performed using the HiScript II One Step qRT-PCR SYBR Green Kit (Vazyme, Nanjing, China) with gene-specific primers listed in Table S5. Relative expression level of each gene was normalized to the Actin gene (U60481) [46].

4.8. Mycorrhizal Quantification and Analysis of Arbuscule Populations

For mycorrhizal quantification, root samples were stained with Trypan blue, and the level of fungal colonization was quantified using the gridline intersect method [47]. The measurement of arbuscule sizes within the arbuscule populations was conducted following the procedures outlined by Liu et al. [43]. To visualize the fungal structures, roots were stained in a 0.2 mg/mL solution of WGA AlexaFluor 488.

4.9. Statistical Analysis

All the data were analyzed for variance with the IBM SPSS Statistics 25.0 software, followed by Turkey’s test (p < 0.05), to test significant differences between different plant genotypes and treatments. The statistical significance (p value) was indicated in the figure legends, * p < 0.05, ** p < 0.01 and *** p < 0.001.

5. Conclusions

This study systematically investigates the regulatory mechanism of mycorrhizal symbiosis on the transcript synthesis of D. officinale by establishing a mycorrhizal symbiotic system, combined with phenotypic observation, physiological measurement, and transcriptome sequencing. The results indicate that mycorrhizal symbiosis promotes the growth and nutrient absorption of D. officinale, while also increasing polysaccharide content. Through transcriptome analysis, we identified 59 differentially expressed genes related to polysaccharide metabolism, along with key genes and transcription factors involved in the regulatory network. A glycosyltransferase gene, DoUGT83A1, which is up-regulated by mycorrhizal symbiosis, was screened, and its function was heterologously verified in transgenic tomatoes. Overexpression of DoUGT83A1 resulted in a reduction in soluble sugars and sucrose content in tomatoes, as well as a decreased colonization rate of mycorrhizal symbiosis. This suggests that DoUGT83A1 negatively regulates the mycorrhizal symbiotic system of D. officinale. Specifically, mycorrhizal fungi and D. officinale roots significantly enhance plant growth and nitrogen/phosphorus absorption, thereby providing ample nutrients and energy for polysaccharide biosynthesis. Mycorrhizal colonization alters the expression levels of genes related to sugar metabolism, particularly upregulating key enzymes involved in the synthesis and conversion of sucrose and fructose, thus supplying abundant substrates for polysaccharide production. Notably, the glycosyltransferase gene DoUGT83A1 plays a negative regulatory role in this process. This study provides a fundamental theoretical basis for further elucidating the molecular mechanisms of polysaccharide synthesis in D. officinale and offers new insights for optimizing cultivation practices and enhancing medicinal quality.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms26199298/s1.

Author Contributions

J.C., W.J. and Z.T. planned and designed the research; J.C., Y.Z., M.Z., Z.Z., Y.L. and X.D. performed the experiments; J.C., Y.Z. and M.Z. analyzed the data, and together with J.C. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (32102485). This work was also supported by the “Three Rural Areas and Nine Directions” Science and Technology Collaboration Program of Zhejiang Province (2025SNJF011) and also supported by the Wenzhou Agricultural New Varieties Breeding Collaboration Group Project (ZX2024005-1) and Wenzhou Major Science and Technology Innovation Research Project (ZN2023005).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Acknowledgments

We would like to express our gratitude to Minquan Song from Zhejiang Tiefengtang Biotechnology Co., Ltd. for his assistance in providing research materials and seedling cultivation for this study.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Phenotypic Analysis of Mycorrhizal Symbiosis in D. officinale. (A) Phenotypes of mycorrhizal symbiotic and non-symbiotic D. officinale. (B) Microscopic observation of stained roots of mycorrhizal symbiotic D. officinale, with red arrows indicating arbuscules. (C) Expression levels of marker genes RiEF1a and RiGAPDH in inoculated and non-inoculated roots. (D) Statistics on root and shoot lengths of inoculated and non-inoculated D. officinale. (E) Results of total polysaccharide content detection. (F) Expression levels of related to gluconeogenesis (DoPMM, DoCSLA6) in inoculated and non-inoculated roots. Values are means of three biological replicates with SE, * p < 0.05. AM: inoculation with Rhizophagus irregularis, NM: inoculated with an equal amount of an autoclaved inoculum. Internal reference gene: DoActin (GenBank: KC831582.1).
Figure 1. Phenotypic Analysis of Mycorrhizal Symbiosis in D. officinale. (A) Phenotypes of mycorrhizal symbiotic and non-symbiotic D. officinale. (B) Microscopic observation of stained roots of mycorrhizal symbiotic D. officinale, with red arrows indicating arbuscules. (C) Expression levels of marker genes RiEF1a and RiGAPDH in inoculated and non-inoculated roots. (D) Statistics on root and shoot lengths of inoculated and non-inoculated D. officinale. (E) Results of total polysaccharide content detection. (F) Expression levels of related to gluconeogenesis (DoPMM, DoCSLA6) in inoculated and non-inoculated roots. Values are means of three biological replicates with SE, * p < 0.05. AM: inoculation with Rhizophagus irregularis, NM: inoculated with an equal amount of an autoclaved inoculum. Internal reference gene: DoActin (GenBank: KC831582.1).
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Figure 2. Gene expression atlas of mycorrhizal symbiosis in D. officinale. (A) Principal component analysis under two treatments is shown. (B) FPKM violin plots in different treatments. (C) Venn diagram displays the number of uniquely and overlappingly expressed genes in different treatments (AM-NM). (D) Volcano plot comparing the number of up- and down-regulated genes in different treatments. AM: inoculation with Rhizophagus irregularis, NM: inoculated with an equal amount of an inactivated microbial agent.
Figure 2. Gene expression atlas of mycorrhizal symbiosis in D. officinale. (A) Principal component analysis under two treatments is shown. (B) FPKM violin plots in different treatments. (C) Venn diagram displays the number of uniquely and overlappingly expressed genes in different treatments (AM-NM). (D) Volcano plot comparing the number of up- and down-regulated genes in different treatments. AM: inoculation with Rhizophagus irregularis, NM: inoculated with an equal amount of an inactivated microbial agent.
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Figure 3. Biological Processes GO terms enrichment and The KEGG pathway analysis for upregulated and downregulated genes. (A) The most enriched Gene Ontology (GO) terms for all differentially expressed genes (DEGs) are categorized into three primary levels: biological process (BP), cellular component (CC), and molecular function (MF). The GO enrichment results have been transformed into negative logarithm scores to facilitate interpretation. (B) Enriched KEGG pathways were identified by analyzing all differentially expressed genes (DEGs) across various developmental stages using the Bioinformatics website (https://www.bioinformatics.com.cn, accessed on 15 June 2025). In the resulting figure, the columns and rows represent the enriched pathway terms and the corresponding rich factor, respectively. The size of the circles indicates the number of DEGs enriched within each specified KEGG pathway, while the color coding reflects the q-value (Corrected p-value) from the enrichment tests.
Figure 3. Biological Processes GO terms enrichment and The KEGG pathway analysis for upregulated and downregulated genes. (A) The most enriched Gene Ontology (GO) terms for all differentially expressed genes (DEGs) are categorized into three primary levels: biological process (BP), cellular component (CC), and molecular function (MF). The GO enrichment results have been transformed into negative logarithm scores to facilitate interpretation. (B) Enriched KEGG pathways were identified by analyzing all differentially expressed genes (DEGs) across various developmental stages using the Bioinformatics website (https://www.bioinformatics.com.cn, accessed on 15 June 2025). In the resulting figure, the columns and rows represent the enriched pathway terms and the corresponding rich factor, respectively. The size of the circles indicates the number of DEGs enriched within each specified KEGG pathway, while the color coding reflects the q-value (Corrected p-value) from the enrichment tests.
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Figure 4. The expression patterns of genes in the polysaccharide biosynthesis pathway of D. officinale. The grids in different colors indicate the relative expression levels.
Figure 4. The expression patterns of genes in the polysaccharide biosynthesis pathway of D. officinale. The grids in different colors indicate the relative expression levels.
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Figure 5. Phenotypic Analysis of DoUGT83A1 Overexpression in Tomato. (A,B) Phenotypic images depicting the overexpression of DoUGT83A1 in tomato plants are presented. (A) illustrates the phenotype without the inoculation of mycorrhizal fungi, (B) shows the phenotype following inoculation. (C,D) Statistical analyses of shoot (C) and root (D) lengths in the overexpressing tomato phenotype. (E,F) Statistical analyses of shoot (E) and root (F) biomass in the overexpressing tomato phenotype. Values are means of three biological replicates with SE. * p < 0.05. AM: inoculation with Rhizophagus irregularis, NM: inoculated with an equal amount of an inactivated microbial agent.
Figure 5. Phenotypic Analysis of DoUGT83A1 Overexpression in Tomato. (A,B) Phenotypic images depicting the overexpression of DoUGT83A1 in tomato plants are presented. (A) illustrates the phenotype without the inoculation of mycorrhizal fungi, (B) shows the phenotype following inoculation. (C,D) Statistical analyses of shoot (C) and root (D) lengths in the overexpressing tomato phenotype. (E,F) Statistical analyses of shoot (E) and root (F) biomass in the overexpressing tomato phenotype. Values are means of three biological replicates with SE. * p < 0.05. AM: inoculation with Rhizophagus irregularis, NM: inoculated with an equal amount of an inactivated microbial agent.
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Figure 6. Arbuscular mycorrhizal fungal colonization analysis of the DoUGT83A1-overexpressing plants. WT and two DoUGT83A1-overexpressing lines, OE4 and OE7, were inoculated with Rhizophagus irregularis for 5 weeks, and then (A) soluble sugar content, (B) sucrose content, (C) arbuscule morphology, (D) the fungal colonization level, (E) arbuscule populations, (F) the transcripts of the arbuscular mycorrhizal marker gene SlPT4. Values are means of three biological replicates with SE. Asterisks indicate significant differences. * p < 0.05. (Scale bars: 50 μm). AM: inoculation with Rhizophagus irregularis, NM: inoculated with an equal amount of an inactivated microbial agent. Internal reference gene: SlActin (LOC105127547).
Figure 6. Arbuscular mycorrhizal fungal colonization analysis of the DoUGT83A1-overexpressing plants. WT and two DoUGT83A1-overexpressing lines, OE4 and OE7, were inoculated with Rhizophagus irregularis for 5 weeks, and then (A) soluble sugar content, (B) sucrose content, (C) arbuscule morphology, (D) the fungal colonization level, (E) arbuscule populations, (F) the transcripts of the arbuscular mycorrhizal marker gene SlPT4. Values are means of three biological replicates with SE. Asterisks indicate significant differences. * p < 0.05. (Scale bars: 50 μm). AM: inoculation with Rhizophagus irregularis, NM: inoculated with an equal amount of an inactivated microbial agent. Internal reference gene: SlActin (LOC105127547).
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Figure 7. Schematic model for DoUGT83A1-mediated regulation of AM symbiosis. Positive and negative regulatory effects are indicated by arrows and flat-ended lines, respectively.
Figure 7. Schematic model for DoUGT83A1-mediated regulation of AM symbiosis. Positive and negative regulatory effects are indicated by arrows and flat-ended lines, respectively.
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Table 1. Classification and Quantity of Transcription Factors.
Table 1. Classification and Quantity of Transcription Factors.
Serial NumberFamily NameNumber of ItemsSerial NumberFamily NameNumber of Items
1bHLH21829GATA21
2MYB_related12130GeBP20
3NAC11831Dof15
4WRKY9832NF-YC15
5B39433SBP13
6ERF9434AP212
7bZIP8835NF-YA12
8C2H28636NF-X111
9MYB8537BES110
10FAR18438CO-like10
11G2-like6839CPP9
12C3H6040ARR-B8
13HD-ZIP5441BBR-BPC8
14ARF5242SRS8
15M-type4943EIL7
16HSF4844ZF-HD7
17GRAS3945GRF6
18TCP3846STAT6
19Nin-like3647WOX6
20MIKC3548DBB5
21S1Fa-like3549HB-PHD4
22TALE3350CAMTA3
23E2F/DP2851LSD3
24Trihelix2852HRT-like2
25HB-other2553RAV2
26NF-YB2454LFY1
27LBD2355VOZ1
28YABBY2356Whirly1
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Chen, J.; Zhang, Y.; Zhang, M.; Zhang, Z.; Liu, Y.; Duan, X.; Tao, Z.; Jiang, W. Study on the Molecular Mechanism of Arbuscular Mycorrhizal Symbiosis Regulating Polysaccharide Synthesis in Dendrobium officinale. Int. J. Mol. Sci. 2025, 26, 9298. https://doi.org/10.3390/ijms26199298

AMA Style

Chen J, Zhang Y, Zhang M, Zhang Z, Liu Y, Duan X, Tao Z, Jiang W. Study on the Molecular Mechanism of Arbuscular Mycorrhizal Symbiosis Regulating Polysaccharide Synthesis in Dendrobium officinale. International Journal of Molecular Sciences. 2025; 26(19):9298. https://doi.org/10.3390/ijms26199298

Chicago/Turabian Style

Chen, Jiadong, Yiqun Zhang, Man Zhang, Ziyi Zhang, Yingying Liu, Xiaojing Duan, Zhengming Tao, and Wu Jiang. 2025. "Study on the Molecular Mechanism of Arbuscular Mycorrhizal Symbiosis Regulating Polysaccharide Synthesis in Dendrobium officinale" International Journal of Molecular Sciences 26, no. 19: 9298. https://doi.org/10.3390/ijms26199298

APA Style

Chen, J., Zhang, Y., Zhang, M., Zhang, Z., Liu, Y., Duan, X., Tao, Z., & Jiang, W. (2025). Study on the Molecular Mechanism of Arbuscular Mycorrhizal Symbiosis Regulating Polysaccharide Synthesis in Dendrobium officinale. International Journal of Molecular Sciences, 26(19), 9298. https://doi.org/10.3390/ijms26199298

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