Characterization of a Unique Pair of Ferredoxin and Ferredoxin NADP⁺ Reductase Isoforms That Operates in...

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 (k_cat/K_m) 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.