Protoplast-based engineering has become an important tool for basic plant molecular biology research and developing genome-edited crops. Uncaria rhynchophylla is a traditional Chinese medicinal plant with a variety of pharmaceutically important indole alkaloids. In this study, an optimized protocol for U. rhynchophylla protoplast isolation, purification, and transient gene expression was developed. The best protoplast separation protocol was found to be 0.8 M D-mannitol, 1.25% Cellulase R-10, and 0.6% Macerozyme R-10 enzymolysis for 5 h at 26 °C in the dark with constant oscillation at 40 rpm/min. The protoplast yield was as high as 1.5 × 10⁷ protoplasts/g fresh weight, and the survival rate of protoplasts was greater than 90%. Furthermore, polyethylene glycol (PEG)-mediated transient transformation of U. rhynchophylla protoplasts was investigated by optimizing different crucial factors affecting transfection efficiency, including plasmid DNA amount, PEG concentration, and transfection duration. The U. rhynchophylla protoplast transfection rate was highest (71%) when protoplasts were transfected overnight at 24 °C with the 40 µg of plasmid DNA for 40 min in a solution containing 40% PEG. This highly efficient protoplast-based transient expression system was used for subcellular localization of transcription factor UrWRKY37. Finally, a dual-luciferase assay was used to detect a transcription factor promoter interaction by co-expressing UrWRKY37 with a UrTDC-promoter reporter plasmid. Taken together, our optimized protocols provide a foundation for future molecular studies of gene function and expression in U. rhynchophylla.

Iron is an essential nutrient for all organisms. Feruloyl-COA 6-hydroxylase 1 (F6′H1) plays a pivotal role in iron uptake in plant roots by catalyzing the biosynthesis of iron-mobilizing scopoletin, a secondary metabolite also possessing antimicrobial activity. However, it remains unclear whether F6′H1-mediated iron uptake affects plant resistance to foliar pathogens and how such a process might be regulated. Here, we show that enhanced expression of 2-oxoglutarate-dependent dioxygenases 2 (CmOGD2), a homolog of F6′H1 in Citron C-05 (Citrus medica L.), confers resistance to citrus canker caused by Xanthomonas citri subsp. citri (Xcc). CmOGD2-mediated pathogen resistance is achieved by promoting iron uptake and the accumulation of reactive oxygen species (ROS), which likely results in ferroptosis. Furthermore, CmOGD2 interacts with the enolase CmENO2 to destabilize CmZAT10.1, a transcriptional activator of CmOGD2, thereby forming a negative feedback loop that limits CmOGD2 expression. Notably, the Xcc effector pthA4 interferes with the CmOGD2–CmENO2 interaction, likely via a decoy mechanism, leading to CmZAT10.1 accumulation. These findings reveal complex regulatory mechanisms underlying the critical role of CmOGD2 in mediating Xcc resistance through iron- and ROS-dependent ferroptosis.

This study was designed to evaluate the effects of different doses of the fibrous roots of Polygonatum cyrtonema Hua on the growth performance, slaughter parameters, meat quality, immune function, cytokines, antioxidant capacity, and intestinal morphology of white-feathered broilers. Also, the mechanism to improve immune functions of broilers was explored through network pharmacology and molecular docking technology. A total of 360 AA-white-feathered broilers were randomly divided into six groups (not separated by sex), with six repetitions per group (n = 10). The groups were as follows: basal diet (CON group), basal diet supplemented with 300 mg/kg aureomycin (ANT group), basal diet supplemented with 2%, 3%, and 4% fibrous root raw powder (LD, MD, and HD group), or basal diet supplemented with 3% fibrous root processed powder (PR group), in a 42-day experiment. The dietary inclusion of P. cyrtonema fibrous roots increased slaughter performance (p < 0.05), reduced the fat rate (p < 0.05), improved intestinal morphology (p < 0.05), and improved the immune organ index to varying degrees. It also significantly improved pH reduction, drip loss, and pressure loss of breast muscle and leg muscle (p < 0.05). Furthermore, it significantly improved immune and antioxidant functions including decreased MDA content of serum (p < 0.01), increased GSH-Px content (p < 0.01), IgG, IgA, and C4 contents (p < 0.05), and increased expression of IL-2 and IFN-γ (p < 0.01). Additionally, the mechanism by which fibrous roots improve immune function in broilers was explored using network pharmacology and molecular docking technology. Network pharmacology and molecular docking revealed that flavonoids such as baicalein, 4′,5-Dihydroxyflavone, 5,7-dihydroxy-6,8-dimethyl-3-(4′-hydroxybenzyl)-chroman-4-one, and 5,7-dihydroxy-3-(2′-hydroxy-4′-methoxybenzyl)-6,8-dimethyl-chroman-4-one were key components that enhanced immune function through the MAPK1 and other key targets involved in regulating the MAPK signaling pathway. From the findings, it can be concluded that incorporating P. cyrtonema Hua fibrous root as a natural feed supplement and growth promoter in broiler diets had a positive impact on bird health and performance.