China Tobacco Gene Research Center, Zhengzhou Tobacco Research Institute of CNTC, Zhengzhou, 450001, China.
Yunnan Key Laboratory of Tobacco Chemistry, R&D Center of China Tobacco Yunnan Industrial Co. Ltd., Kunming, 650202, China.
Planta. 2023 Dec 27;259(2):31. doi: 10.1007/s00425-023-04301-1.
Chlorogenic acid (CGA) and flavonoids are important secondary metabolites, which modulate plant growth and development, and contribute to plant resistance to various environmental stresses. ERF4 has been shown to be a repressor of anthocyanin accumulation in grape, but its full roles in regulating the biosynthesis of other phenylpropanoid compounds still needs to be further studied. In the present study, two NtERF4 genes were identified from N. tabacum genome. The expression level of NtERF4a was higher than that of NtERF4b in all the tobacco tissues examined. Over-expression of NtERF4a significantly promoted the accumulation of CGA and flavonoids in tobacco leaves, while silencing of NtERF4a significantly repressed the biosynthesis of CGA and flavonoids. RNA-seq analysis of NtERF4a-OE and WT plants revealed 8 phenylpropanoids-related differentially expressed genes (DEGs), including 4 NtPAL genes that encode key enzymes in the phenylpropanoid pathway. Activation of NtERF4a-GR fusion protein in tobacco significantly induced the transcription of NtPAL1 and NtPAL2 in the presence of protein synthesis inhibitor. Chromatin immunoprecipitation and Dual-Luc assays further indicated that NtERF4a could bind to the GCC box presented in the promoters of NtPAL1 and NtPAL2, thereby activating their transcription. Moreover, ectopic expression of NtERF4a induced the transcription of NtGSK1, NtMYC2, and NtJAZ3 genes, and enhanced the resistance of tobacco seedlings to salt and drought stresses, indicating multiple roles of NtERF4a in plants. Our findings revealed new roles of NtERF4a in modulating the accumulation of phenylpropanoid compounds in tobacco, and provided a putative target for improving phenylpropanoids synthesis and stress resistance in plants.
绿原酸（CGA）和类黄酮是重要的次生代谢物，它们调节植物的生长和发育，并有助于植物抵抗各种环境胁迫。已经表明 ERF4 是葡萄中花色苷积累的抑制剂，但它在调节其他苯丙素化合物生物合成中的全部作用仍需要进一步研究。在本研究中，从烟草基因组中鉴定出两个 NtERF4 基因。在所有检查的烟草组织中，NtERF4a 的表达水平均高于 NtERF4b。过表达 NtERF4a 显著促进了烟草叶片中 CGA 和类黄酮的积累，而 NtERF4a 的沉默则显著抑制了 CGA 和类黄酮的生物合成。NtERF4a-OE 和 WT 植物的 RNA-seq 分析显示，有 8 个苯丙素相关的差异表达基因（DEGs），包括 4 个编码苯丙素途径关键酶的 NtPAL 基因。在存在蛋白质合成抑制剂的情况下，NtERF4a-GR 融合蛋白在烟草中的激活显著诱导了 NtPAL1 和 NtPAL2 的转录。染色质免疫沉淀和双荧光素酶测定进一步表明，NtERF4a 可以结合 NtPAL1 和 NtPAL2 启动子中存在的 GCC 盒，从而激活它们的转录。此外，NtERF4a 的异位表达诱导了 NtGSK1、NtMYC2 和 NtJAZ3 基因的转录，并增强了烟草幼苗对盐和干旱胁迫的抗性，表明 NtERF4a 在植物中具有多种作用。我们的研究结果揭示了 NtERF4a 在调节烟草中苯丙素化合物积累方面的新作用，并为提高植物中苯丙素合成和抗逆性提供了一个潜在的靶标。
