Summary for the new academy-funded project with acronym 'ringling' for years 2019-2023
Wood cell walls contain a large proportion of lignin polymer, which binds the cellulose and hemicellulose fibers together forming a hard material. The polymer is formed out of monolignols, the biosynthesis of which is well known but the transport mechanisms for monolignols into the apoplastic space are still enigmatic. Neither the lignification initiation sites are known. We do know that lignification starts in the middle lamella and progresses into the primary and secondary cell walls. In this project we investigate the mechanisms which transport monolignols into the cell wall space, how the initiation of lignification happens, what the apoplastic metabolome contains, and where the hydrogen peroxide needed for lignin polymerization is in the cell wall space. The results can be utilized in Norway spruce transformation to make harder wood for mechanical wood products or more soluble lignin for the needs of paper and pulp industry.

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

  1. to clone and characterize monolignol transporters in developing xylem of Norway spruce,
  2. to investigate the chemical nature of the initiation sites for lignin polymerization, and
  3. 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’.