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

Release time:

2025-07-10

This study explores the role of the DoGSTF11 gene in Dendrobium officinale, a traditional medicinal herb. While previous research has focused on polysaccharides and alkaloids, this work addresses the lesser-known anthocyanin pathway. The researchers found that DoGSTF11 is highly expressed in the purplish variety of D. officinale and is localized in the nucleus and cell membrane, though it lacks transcriptional activation ability. Overexpressing DoGSTF11 in tomato led to increased anthocyanin accumulation, suggesting it plays a role in anthocyanin transport or sequestration. Protein interaction studies revealed that DoGSTF11 interacts with DoGST31, and that DoIDD12 and DoNF-YB3 may regulate its expression. Overall, the findings highlight DoGSTF11 as a positive regulator of anthocyanin accumulation and offer new insights for flavonoid metabolic engineering in D. officinale.

Two color variants of Dendrobium officinale—green and purplish—were used for analysis. Microscopy showed that anthocyanins in the purplish stems mainly accumulated in the epidermal cells, while the green stems showed little to no pigment. Quantitative analysis confirmed that the purplish variety had the highest anthocyanin content.

Figure 1. Phenotypic observation of different Dendrobium officinale varieties

To investigate the role of GST genes in anthocyanin regulation in D. officinale, expression analysis was conducted across green and purplish phenotypes. Eleven DoGST genes showed significant differential expression, with DoGSTF11 displaying the most prominent change. Phylogenetic analysis revealed that DoGSTF11 clusters with known anthocyanin-related GSTs, such as Arabidopsis TT19, while other DoGSTs formed a separate group. Sequence alignment showed that DoGSTF11 shares high similarity with these anthocyanin-related GSTs, especially grape VvGST4 (67% identity). These results suggest DoGSTF11 may play a key role in anthocyanin synthesis and metabolism.

Figure 2. DoGSTs expression and phylogenetic analysis.

Subcellular localization of DoGSTF11 was examined by transiently expressing a DoGSTF11-GFP fusion in tobacco leaves. Confocal microscopy showed that the GFP signal from both the control (35S:GFP) and DoGSTF11-GFP was present in the cell membrane and nucleus, indicating that DoGSTF11 localizes to these two cellular compartments.

To assess the function of DoGSTF11, it was stably overexpressed in tomato seedlings. Three transgenic lines were generated, and their stems and leaves showed noticeable purple pigmentation, especially in line OE3, which had the highest DoGSTF11 expression. Anthocyanin measurements confirmed significantly increased levels in all transgenic lines compared to wild type, with OE3 displaying the highest accumulation. These findings suggest that DoGSTF11 positively regulates anthocyanin synthesis.

Figure 4. Heterologous expression of DoGSTF11 in transgenic Solanum lycopersicum.

The transcriptional self-activation activity of DoGSTF11 was analyzed. As shown in Figure 5A, the negative control (pGBKT7) was able to grow on SD/-Trp plates but failed to grow on SD/-Trp/-His, SD/-Trp/-His/-Ade, and SD/-Trp/-His/-Ade + X-α-gal plates. The experimental group showed the same growth pattern as the negative control, indicating that DoGSTF11 lacks transcriptional self-activation activity.

Figure 5. Analysis of DoGSTF11-interacting proteins.

To identify upstream regulators of DoGSTF11, a pHIS2-DoGSTF11pro bait plasmid was constructed for yeast one-hybrid screening. Sequence analysis identified 21 unique positive clones after removing empty vectors and duplicates. These clones were able to grow on selective media containing 3-AT, indicating promoter-binding activity. Further analysis suggested that DoIDD12 and DoNF-YB3 are potential upstream transcriptional regulators of DoGSTF11.

Figure 6. Screening of upstream regulatory genes of DoGSTF11.

This study identifies DoGSTF11 as a key positive regulator of anthocyanin accumulation in purple-stem Dendrobium officinale. Protein interaction assays (Y2H and BiFC) showed that DoGSTF11 interacts with DoGST31, promoting anthocyanin buildup. Upstream regulators DoIDD12 and DoNF-YB3 were also identified by Y1H assays. A proposed model suggests that DoGSTF11 enhances anthocyanin synthesis in the endoplasmic reticulum and facilitates its transport to the vacuole for accumulation. These findings deepen understanding of the molecular mechanisms driving anthocyanin biosynthesis in this medicinal plant.

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2025-07-10

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

2025-07-08

Literature Sharing | Chinese cabbage orphan gene BR3 confers bolting resistance to Arabidopsis through the gibberellin pathway

Premature bolting affects yield and quality in Chinese cabbage, highlighting the importance of identifying bolting resistance genes. This study identifies an orphan gene, BR3 (BOLTING RESISTANCE 3), in Arabidopsis thaliana as a positive regulator of bolting resistance. BR3 is expressed during the seedling and flowering stages and localizes to the plasma membrane and nucleus. Overexpression of BR3 (BR3OE) delays bolting and flowering under both long-day and short-day conditions, with increased rosette leaf number and reduced plant height. Key flowering genes are downregulated in BR3OE plants. GA₃ treatment induces early flowering in both BR3OE and wild type (WT) plants, but BR3OE still flowers later than WT. In Chinese cabbage, BR3 shows co-expression with DELLA genes BrRGA1 and BrRGL3, suggesting a regulatory role through the GA pathway. This study offers new insights into bolting resistance mechanisms and provides valuable targets for breeding bolting-resistant Chinese cabbage varieties.

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Literature Sharing | Regulation of co-translational mRNA decay by PAP and DXO1 in Arabidopsis

This study investigates the regulation of the co-translational mRNA decay (CTRD) pathway in Arabidopsis, a critical mechanism for maintaining mRNA homeostasis. The researchers found that 3ʹ-phosphoadenosine 5ʹ-phosphate (PAP), an inhibitor of exoribonucleases, affects CTRD activity. Specifically, they showed that loss of FRY1 impairs XRN4-dependent CTRD and that exogenous PAP treatment stabilizes CTRD target mRNAs. Additionally, they discovered that another PAP-sensitive exoribonuclease, DXO1, also contributes to CTRD, likely by acting on NAD⁺-capped mRNAs involved in stress responses. These findings reveal new layers of regulation and additional players in the CTRD pathway in plants.

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Literature Sharing | Two pathogen-inducible UDP-glycosyltransferases, UGT73C3 and UGT73C4, catalyze the glycosylation of pinoresinol to promote plant immunity in Arabidopsis

This study uncovers a novel immune regulatory pathway in Arabidopsis thaliana involving two UDP-glycosyltransferases, UGT73C3 and UGT73C4, which are highly induced by Pseudomonas syringae infection. Overexpression of these genes enhances disease resistance, while their double mutation compromises immunity. Metabolomic analysis and biochemical assays reveal that UGT73C3/C4 glycosylate pinoresinol into its mono- and diglucoside forms, which promote immune responses by boosting ROS production and callose deposition. Additionally, the transcription factor HB34 directly activates UGT73C3/C4 expression, linking transcriptional regulation to glycosylation-mediated immunity. This work highlights the physiological significance of UGTs in plant defense through pinoresinol glycosylation.

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Literature Sharing | PbARP1 enhances salt tolerance of ‘Duli’ pear (Pyrus betulifolia Bunge) through abscisic acid signalling pathway

This study investigates salt stress tolerance in pears and identifies 35 salt-tolerant genes using a yeast expression library. Among them, PbARP1 was found to be significantly upregulated under salt stress in 'Duli' pear (Pyrus betulaefolia). Functional analyses showed that overexpression of PbARP1 in transgenic pear calli enhanced salt tolerance, while its suppression increased sensitivity to salt stress. Silencing PbARP1 also altered the expression of key ABA signaling genes, including PbPYL4, PbPYL9, PbPYL8, PbSRK2I, and PbABI5, suggesting that PbARP1 modulates salt stress responses through the ABA signaling pathway. Furthermore, PbPYL8, an ABA receptor, was identified as a protein interacting with PbARP1, highlighting its pivotal role in ABA-mediated salt stress regulation in pear.

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Literature Sharing | Phosphorylation of the strawberry MADS-box CMB1 regulates ripening via the catabolism of abscisic acid

This study uncovers a regulatory mechanism linking transcriptional control and posttranslational modification in strawberry fruit ripening. The MADS-box transcription factor FaCMB1 acts as a negative regulator of ripening, with both its transcript and protein levels decreasing during fruit development, a process enhanced by ABA. Functional manipulation of FaCMB1 significantly affected ripening and ABA content.

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Literature Sharing | Overexpression of soybean flavonoid 3′-hydroxylase enhances plant salt tolerance by promoting ascorbic acid biosynthesis

This study reveals that the flavonoid 3′-hydroxylase gene GmF3′H plays a key role in enhancing salt tolerance in soybean by regulating redox homeostasis. Using CRISPR/Cas9 knockout and overexpression approaches, the researchers demonstrated that GmF3′H competitively interacts with CSN5B, disrupting its binding to VTC1, a key enzyme in ascorbic acid biosynthesis. This redirection of metabolic flux toward the L-galactose pathway leads to increased ascorbic acid (AsA) levels, enhancing ROS scavenging capacity and improving salt stress tolerance during seed germination and seedling growth.

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Literature Sharing | ECE-CYC1 Transcription Factor CmCYC1a May Interact withCmCYC2 in Regulating Flower Symmetry and Stamen Development in Chrysanthemum morifolium

This study investigates the role of CmCYC1a, a CYC1 subclade gene from the ECE (CYC/TB1) family, in the floral development of Chrysanthemum morifolium. The gene was found to be more highly expressed in disc florets than in ray florets, with expression increasing during the flowering process. Functional analysis through overexpression in Arabidopsis thaliana revealed that CmCYC1a can alter floral symmetry from actinomorphic to zygomorphic and reduce the number of stamens. Additionally, yeast two-hybrid assays showed that CmCYC1a interacts with CmCYC2b, CmCYC2d, and CmCYC2f. These findings suggest that CmCYC1a contributes to the regulation of flower symmetry and stamen development in Chrysanthemum morifolium, and, together with CYC2 genes, may play a coordinated role in capitulum formation, providing new insights into the function of ECE genes in Asteraceae.

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