Integrated transcriptome and metabolome analysis reveals the regulation of phlorizin synthesis in Lithocarpus polystachyus under nitrogen fertilization

Nitrogen (N) is essential for plant growth and development. In Lithocarpus polystachyus Rehd., a species known for its medicinal and food value, phlorizin is the major bioactive compound with pharmacological activity. Research has revealed a positive correlation between plant nitrogen (N) content and phlorizin synthesis in this species. However, no study has analyzed the effect of N fertilization on phlorizin content and elucidated the molecular mechanisms underlying phlorizin synthesis in L. polystachyus. A comparison of the L. polystachyus plants grown without (0 mg/plant) and with N fertilization (25, 75, 125, 175, 225, and 275 mg/plant) revealed that 75 mg N/plant fertilization resulted in the greatest seedling height, ground diameter, crown width, and total phlorizin content. Subsequent analysis of the leaves using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) detected 150 metabolites, including 42 flavonoids, that were differentially accumulated between the plants grown without and with 75 mg/plant N fertilization. Transcriptomic analysis of the L. polystachyus plants via RNA sequencing revealed 162 genes involved in flavonoid biosynthesis, among which 53 significantly differed between the N-treated and untreated plants. Fertilization (75 mg N/plant) specifically upregulated the expression of the genes phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase (4CL), and phlorizin synthase (PGT1) but downregulated the expression of trans-cinnamate 4-monooxygenase (C4H), shikimate O-hydroxycinnamoyltransferase (HCT), and chalcone isomerase (CHI), which are related to phlorizin synthesis. Finally, an integrated analysis of the transcriptome and metabolome revealed that the increase in phlorizin after N fertilization was consistent with the upregulation of phlorizin biosynthetic genes. Quantitative real-time PCR (qRT‒PCR) was used to validate the RNA sequencing data. Thus, our results indicated that N fertilization increased phlorizin metabolism in L. polystachyus by regulating the expression levels of the PAL, PGT1, 5-O-(4-coumaroyl)-D-quinate 3'-monooxygenase (C3'H), C4H, and HCT genes. Our results demonstrated that the addition of 75 mg/plant N to L. polystachyus significantly promoted the accumulation of flavonoids, including phlorizin, and the expression of flavonoid synthesis-related genes. Under these conditions, the genes PAL, 4CL, and PGT1 were positively correlated with phlorizin accumulation, while C4H, CHI, and