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

Abstract

Abstract Background Nitrogen (N) is very important for plant development, growth, and secondary metabolism. Lithocarpus polystachyus Rehd, also called sweet tea, is known for its medicinal and food value. It contains several bioactive components, including phlorizin, with various pharmacological activities. Research has indicated a positive correlation between phlorizin and nitrogen (N) content in L. polystachyus. However, no reports have analyzed the effect of N treatment on phlorizin content and and reveal the molecular mechanisms of phlorizin synthesis.Results We grew L. polystachyus without and with N fertilization at six levels (0, 25, 75, 125, 175, 225, and 275 mg per plant) and analyzed the plant growth status and leaf phlorizin content. These initial experiments revealed that 75 mg N/plant resulted in the best seedling height, ground diameter, crown width, and total phlorizin content in L. polystachyus. Subsequent comparison of L. polystachyus plants grown without and with 75 mg/plant N fertilization identified 150 differentially accumulated metabolites in the leaves, including 42 flavonoids, using ultra-performance liquid chromatography-tandem mass spectrometry. Transcriptomic analysis by RNA sequencing detected 162 genes involved in flavonoid biosynthesis, among which 53 were significantly different between N-treated and untreated materials. Fertilization (75 mg N/plant) upregulated the expression levels of phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase (4CL), and phlorizin synthase (PGT1) but downregulated those of trans-cinnamate 4-monooxygenase (C4H) and chalcone isomerase (CHI), were related to the phlorizin synthesis. Integrated analysis of the transcriptome and metabolome found that the increase in phlorizin via the flavonoid biosynthetic pathway after N fertilization was consistent with the the expression levels upregulation of phlorizin biosynthetic genes. Quantitative real-time PCR (qRT-PCR) analysis validated the RNA-sequencing data. Thus, these observations indicated that the expression levels of PAL, PGT1, 5-O-(4-coumaroyl)-D-quinate 3'-monooxygenase (C3'H), C4H, and shikimate O-hydroxycinnamoyltransferase (HCT) genes were related to phlorizin metabolism in L. polystachyus under N fertilization.Conclusions Overall, understanding the molecular mechanism of phlorizin synthesis will help improve plant quality and serve as a reference for further pharmacological studies.