我们最近的研究表明,铁氧还蛋白(Fd)和铁氧还蛋白NADP还原酶(FNR)的亚型在薄荷腺毛中萜类生物合成的2C-甲基-D-赤藓糖醇4-磷酸(MEP)途径的还原步骤中起着至关重要的作用(GT)。基于对几个转录组数据集的分析,我们证明了叶型FNR(L-FNR)的转录本的存在,叶型Fd(FdI),根型FNR(R-FNR),和薄荷科(唇形科)的几个成员中的两个根型Fds(FdII和FdIII)。本研究报告了薄荷(Mentha×piperitaL.)的所有Fd和FNR亚型的生化特征。使用光还原方法测定Fd和FNR同工型的氧化还原电位。根据心肌黄递酶测定,薄荷R-FNR对NADPH的特异性常数(kcat/Km)明显高于L-FNR。使用铁氰化物作为电子受体获得了类似的结果。当测定NADPH-细胞色素C还原酶活性时,FdII和FdIII同种型的特异性常数(与FdI相比)对于L-FNR略高,对于R-FNR则明显更高。基于代表各种薄荷器官和细胞类型的样品的实时定量PCR测定,FdII基因在代谢活跃的GTs中高度表达(但在根中也存在较低水平),而FdIII在根和GTs中均以低水平表达。我们的数据提供了证据表明FdII的高转录水平,与FdIII相比,编码酶的生化特性没有差异,可能支持薄荷GTs中通过MEP途径形成大量单萜。这项工作为进一步研究独特的R-FNR-FdII对在唇形科非光合GTs中的作用奠定了基础。
Our recent investigations indicated that isoforms of ferredoxin (Fd) and ferredoxin NADP+ reductase (FNR) play essential roles for the reductive steps of the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway of terpenoid biosynthesis in peppermint glandular trichomes (GTs). Based on an analysis of several transcriptome data sets, we demonstrated the presence of transcripts for a leaf-type FNR (L-FNR), a leaf-type Fd (Fd I), a root-type FNR (R-FNR), and two root-type Fds (Fd II and Fd III) in several members of the mint family (Lamiaceae). The present study reports on the biochemical characterization of all Fd and FNR isoforms of peppermint (Mentha × piperita L.). The redox potentials of Fd and FNR isoforms were determined using photoreduction methods. Based on a diaphorase assay, peppermint R-FNR had a substantially higher specificity constant (kcat/Km) for NADPH than L-FNR. Similar results were obtained with ferricyanide as an electron acceptor. When assayed for NADPH-cytochrome c reductase activity, the specificity constant with the Fd II and Fd III isoforms (when compared to Fd I) was slightly higher for L-FNR and substantially higher for R-FNR. Based on real-time quantitative PCR assays with samples representing various peppermint organs and cell types, the Fd II gene was expressed very highly in metabolically active GTs (but also present at lower levels in roots), whereas Fd III was expressed at low levels in both roots and GTs. Our data provide evidence that high transcript levels of Fd II, and not differences in the biochemical properties of the encoded enzyme when compared to those of Fd III, are likely to support the formation of copious amounts of monoterpene via the MEP pathway in peppermint GTs. This work has laid the foundation for follow-up studies to further investigate the roles of a unique R-FNR-Fd II pair in non-photosynthetic GTs of the Lamiaceae.