Water-conducting and support-giving xylem cell walls in plants are made rigid with lignin polymer that binds together the carbohydrate fibers to form a water resistant cell wall. A lot is known about monolignol biosynthesis and its regulation but there are still some fascinating points in the final steps of lignification that need elucidation. Among these are the transport of monolignols into the apoplastic space from the living cells in developing xylem, and the initiation point(s) where lignification starts in the cell corners and middle lamella. It is intriguing and most probably under tight regulation, that the lignin polymer starts forming in the cell corners and middle lamellae, furthest away from the plasma membrane, where the monomers are secreted into the apoplastic space. There the peroxidases and/or laccases oxidise monolignols to phenolic radicals that polymerise to form lignin.
Our aims are
- to clone and characterize monolignol transporters in developing xylem of Norway spruce,
- to investigate the chemical nature of the initiation sites for lignin polymerization, and
- to assess the conditions in the apoplast leading and regulating monolignol polymerization, i.e. the presence of hydrogen peroxide in the apoplastic space.
We are approaching these goals with several different techniques to minimize the risk of failure in the experiments to unravel this unknown part in cell wall development. To address the goals, we will use our transcriptomic, proteomic and single-cell metabolomic data to select the candidate monolignol transporter genes, clone them with the Gateway technology, characterize in E.coli, and knockout these in Norway spruce seedlings with subsequent phenotyping of the transformants.
Furthermore, the aim is to assess the lignification initiation sites with two complementary methods in wood sections: Raman spectroscopy imaging and immunocytochemistry with specific antibodies generated against prospective nucleation sites. The third aim is to analyse apoplastic metabolome with mass spectrometry and visualize hydrogen peroxide distribution in the cell wall by electron microscopy and cerium chloride staining. The project will generate knowledge on the genetic identity of monolignol transporters, their individual properties in tracheids and ray parenchymal cells, and aid in the understanding of the factors affecting spatial deposition of lignin in the cell wall, and in Norway spruce breeding programme for ‘designer lignins’.