Integrated metabolomics and transcriptomics reveal that plant hormone signaling, phenylpropanoid biosynthesis, and flavonoid metabolism promote callus browning in Rheum tanguticum

Rheum tanguticum is a high-value medicinal crop with notable ecological functions, yet unsustainable harvesting and habitat changes have severely depleted wild stocks. The development of in vitro propagation systems is urgently required, but recurrent callus browning substantially limits protocol efficiency and scalability. This study aimed to systematically unravel the structural, physiological, and molecular mechanisms governing callus browning. Brown and green callus were comparatively analyzed using electron microscopy, transcriptomics, metabolomics, and physiological assays. Metabolomic profiling confirmed significant accumulation of (+)-gallocatechin, hesperetin, and vitexin in the brown callus. Transcriptomic profiling revealed the upregulated expression of key genes associated with flavonoid biosynthesis, including chalcone synthase, bifunctional dihydroflavonol 4-reductase/flavanone 4-reductase, anthocyanidin reductase, and leucoanthocyanidin reductase. Metabolomics and transcriptomics integration and physiological measurements showed marked accumulation of salicylic acid, abscisic acid, and jasmonic acid, as well as activation of ethylene signaling, during the callus browning. Although the expression of genes related to ROS scavenging was upregulated, the activities of superoxide dismutase were decreased, resulting in ROS imbalance and severe oxidative stress. Excessive ROS accumulation accelerated lipid peroxidation, which compromised the integrity and increased the permeability of cellular and vacuolar membranes. Rupture of the vacuolar membrane enabled phenolic and flavonoid compounds, which are normally sequestered within the vacuole, to interact with cytoplasmic polyphenol oxidase, thereby triggering enzymatic browning. In summary, callus browning in R. tanguticum is mediated by a coordinated interaction between hormone-induced ROS accumulation, biosynthesis and accumulation of phenolics and flavonoids, and structural impairment of the vacuolar membrane. These findings support the design of effective anti-browning treatments and improved tissue culture systems for medicinal and food plants.