Tracking the Lignin Biosynthetic Pathway with Cellular Resolution

Overview: Lignin is a complex aromatic polymer produced in plant cell walls and the second most abundant natural polymer on the planet. It provides mechanical support, facilitates transport of water and nutrients through the vascular system and plays an important role in plant responses to biotic and abiotic stresses. Recently, we have shown the operation of a novel pathway to lignin biosynthesis, suggesting that xylem fiber and vascular bundle cells might follow distinct lignification patterns associated with different enzymatic activities or substrate availabilities. Recent EMSL advances in single-cell omics and mass spectrometry (MS) imaging offer unique opportunities to study these dynamic processes at the single-cell level. By harnessing these technologies, we aim to dissect lignin biosynthesis with precise spatial resolution, ultimately contributing to our fundamental knowledge in plant cell wall biogenesis. Approach: We propose a combined approach integrating EMSL’s cutting-edge capabilities including Nanodroplet Processing in One Pot for Trace Samples (nanoPOTS) combined with deep ultraviolet laser ablation (DUV LA) and MALDI-MS Imaging. By feeding 13C9-labeled lignin monomer precursors to sorghum plants, we will use MALDI MS Imaging to visualize the incorporation of labeled monolignols into the lignifying cell walls in situ, thus providing precise details of lignin polymer formation at cellular resolution. Furthermore, our approach will make use of a DUV LA system to isolate specific cell types into a nanoPOTS chip and complement the MS imaging data with detailed metabolic and proteomics profiles in a cell-specific manner. Products and Results: This strategy will allow us to: (i) introduce 13C-labeled lignin monomers into living plant tissues to track lignin synthesis during development; (ii) use MS imaging for the detailed spatial mapping of lignin deposition in different cell wall types; and (iii) integrate and validate the MS imaging data with metabolic and proteomic profiles obtained by DUV LA combined with nanoPOTS. Research findings will be publicly available through web-based databases. Novelty: This project is transformative in its approach since most of current lignin research work focuses on the characterization of bulk tissues, and detailed molecular analyses at the single-cell level are rarely found in the literature. The incorporation of isotopically labeled precursors into the lignin polymer enables the study of the lignin metabolic pathways in their native context, and when combined with MS imaging and DUV LA-nanoPOTS it will provide new insights into the regulatory mechanisms that govern the formation and structure of lignin at the molecular level. Understanding these biochemical processes will ultimately find applications to improve stress tolerance and increase carbon sequestration in major bioenergy crops.