Hunting monolignol transporters: membrane proteomics and biochemical transport assays with membrane vesicles of Norway spruce

Monolignol transport during lignification is a partially solved puzzle: both the mechanism(s) and the transported form of monolignols are unknown in developing xylem of trees. We tested a hypothesis of an active, plasma membrane (PM)-localized transport of monolignol monomers, dimers, and/or glucosidic forms with membrane vesicles prepared of developing xylem and lignin-forming tissue-cultured cells of Norway spruce (Picea abies L. Karst.), as well as of control materials, non-lignifying Norway spruce phloem and tobacco (Nicotiana tabacum L.) BY-2 cells. Xylem and BY-2 vesicles transported both coniferin and p-coumaryl alcohol glucoside, but inhibitor assays suggested this transport being over the tonoplast. Also membrane vesicles prepared from the lignin-forming spruce cells showed coniferin transport, but the Km for coniferin was much higher than those of xylem and BY-2 cells. Liquid chromatography-mass spectrometry (LC-MS/MS) analysis of membrane proteins isolated from spruce developing xylem, phloem and lignin-forming cultured cells revealed multiple transporters. These were compared to a transporter gene set that was gained by a correlation analysis with a selected set of spruce monolignol biosynthesis genes. Biochemical membrane vesicle assays showed no support for the ABC-transporter-mediated monolignol transport but point to secondary active transporters (such as MFS or MATE transporters). In contrast, proteomic and co-expression analyses suggest a role for ABC transporters and MFS transporters.

Keywords: lignin biosynthesis; monolignol transport; plasma membrane; proteomics; transporter proteins.