Experimental Center of Subtropical Forestry, Chinese Academy of Forestry, Xinyu, 336600, China.
Research Institute of Forestry, Chinese Academy of Forestry, Beijing, 100091, China.
BMC Plant Biol. 2024 May 6;24(1):366. doi: 10.1186/s12870-024-05090-9.
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 HCT were negatively correlated with phlorizin accumulation. Therefore, we speculate that PAL, 4CL, and PGT1 participate in the phlorizin pathway under an optimal N environment, regulating phlorizin biosynthesis. These findings provide a basis for improving plant bioactive constituents and serve as a reference for further pharmacological studies.
氮（N）是植物生长和发育所必需的。在石栎（Lithocarpus polystachyus Rehd.）中，根皮苷是主要的生物活性化合物，具有药理学活性。研究表明，该物种的植物氮（N）含量与根皮苷合成之间存在正相关。然而，尚无研究分析氮施肥对根皮苷含量的影响，并阐明石栎根皮苷合成的分子机制。
比较了未施肥（0mg/株）和施肥（25、75、125、175、225 和 275mg/株）的石栎植株，结果表明，75mg N/株施肥使幼苗高度、地径、冠宽和总根皮苷含量达到最大。随后使用超高效液相色谱-串联质谱法（UPLC-MS/MS）对叶片进行分析，检测到 150 种代谢物，包括 42 种类黄酮，这些代谢物在未施肥和 75mg N/株施肥的植株之间差异积累。通过 RNA 测序对石栎植株进行转录组分析，发现了 162 个参与类黄酮生物合成的基因，其中 53 个基因在 N 处理和未处理的植株之间存在显著差异。施肥（75mg N/株）特异性地上调了苯丙氨酸解氨酶（PAL）、4-香豆酸-CoA 连接酶（4CL）和根皮苷合酶（PGT1）的基因表达，但下调了反式肉桂酸 4-单加氧酶（C4H）、莽草酸 O-羟肉桂酰转移酶（HCT）和查尔酮异构酶（CHI）的基因表达，这些基因与根皮苷合成有关。最后，对转录组和代谢组进行综合分析表明，氮施肥后根皮苷的增加与根皮苷生物合成基因的上调一致。实时荧光定量 PCR（qRT-PCR）用于验证 RNA 测序数据。因此，我们的结果表明，氮施肥通过调节 PAL、PGT1、5-O-（4-香豆酰基）-D-奎宁酸 3'-单加氧酶（C3'H）、C4H 和 HCT 基因的表达水平，增加了石栎中的根皮苷代谢。
我们的结果表明，向石栎中添加 75mg/株 N 可显著促进包括根皮苷在内的类黄酮的积累和类黄酮合成相关基因的表达。在这些条件下，基因 PAL、4CL 和 PGT1 与根皮苷的积累呈正相关，而 C4H、CHI 和 HCT 与根皮苷的积累呈负相关。因此，我们推测在最佳氮环境下，PAL、4CL 和 PGT1 参与根皮苷途径，调节根皮苷的生物合成。这些发现为提高植物生物活性成分提供了依据，并为进一步的药理学研究提供了参考。
