Lignin constitutes 20–32% of woody plant cell walls, being the second most abundant biopolymer after cellulose. In a growing tree, wood formation with the final phase of lignin deposition occurs in a thin cell layer inside the cambium. The spatial isolation of the developing wood tissue makes the studies related to wood development difficult in muro. In my group, both native trees and a cell culture of Picea abies (Norway spruce) are used to study lignin biosynthesis. The cell culture is a unique system where some cells differentiate into tracheids (ca. 3% of total cells), whereas most of the cells remain undifferentiated. Lignin polymer with a structural similarity to native lignin is constitutively formed in the culture medium (Simola et al. 1992, Warinowski et al. 2016; Giummarella et al. 2019).

The spruce tissue culture line has been an object for several studies focusing on biosynthesis and structure of the extracellular lignin (e.g. Kärkönen et al. 2002; Koutaniemi et al. 2007; Laitinen et al. 2017). The emphasis has been on the enzymes involved in activation of lignin precursors (peroxidases and / or laccases), as well as in the characterisation of the genes for enzymes responsible for monolignol biosynthesis and their polymerisation.

My research interests originated from the observation that H₂O₂ removal from the culture medium strongly reduces the amounts of extracellular lignin. As H₂O₂ is needed by peroxidases this data suggest that peroxidases have the main role in activation of monolignols for lignin biosynthesis in the spruce cell culture. This led to the question of the origin of H₂O₂ in the cell wall during lignin formation. The role of redox active enzymes in plasma membranes, like respiratory burst oxidase homolog, Rboh, have been studied (Kärkönen and Kuchitsu 2015). To study regulation of lignin biosynthesis, a large scale phenolic and transciptomic profiling was conducted using the extracellular lignin-forming cell culture with lignin formation and when lignin formation was inhibited by scavenging of H₂O₂. This inhibited the action of peroxidases (Laitinen et al. 2017). The results show that apoplastic redox state regulates not only phenolic metabolism, but the whole cellular metabolism. The work also identified several novel proteins (e.g. carbohydrate oxidoreductases) for further evaluation.

In addition to the tissue culture, developing xylem of spruce is used as a research material. The origin of monolignols used in developing tracheids for cell wall lignification has been investigated by RNA-Seq and single cell metabolomics. For RNA-Seq, developing ray parenchymal cells and tracheids were separately collected by laser capture microdissection. In addition, single cell metabolome analysis was conducted in living plantlets, and both monolignols and monolignol glucosides were detected in both cell types (Blokhina et al. 2019). The results show that similarly to that in dicotyledonous species, in Norway spruce ray parenchymal cells contribute in monolignol production in addition to developing tracheids. Currently, the mechanism of transport of monolignols, the precursors for lignin into the apoplast is being studied (Väisänen et al. 2020).

1. Blokhina O, Laitinen T, Hatakeyama Y, Delhomme N, Paasela T, Zhao L, Street NR, Wada H, Kärkönen A, Fagerstedt KV (2019) Ray cells contribute to monolignol biosynthesis in developing xylem of Norway spruce. Plant Physiol 181: 1552-1572
   
   
2. Giummarella N, Koutaniemi S, Balakshin M, Kärkönen A, Lawoko M (2019) Nativity of lignin carbohydrate bonds substantiated by novel biomimetic synthesis. J Exp Bot 70: 5591-5601
   
   
3. Kärkönen A, Koutaniemi S, Mustonen M, Syrjänen K, Brunow G, Kilpeläinen I, Teeri TH, Simola LK (2002) Lignification related enzymes in Picea abies suspension cultures. Physiol Plant 114: 343-353
   
   
4. Kärkönen A, Kuchitsu K (2015) Reactive oxygen species in cell wall metabolism and development in plants. Phytochemistry, 112: 22–32
   
   
5. Koutaniemi S, Warinowski T, Kärkönen A, Alatalo E, Fossdal CG, Saranpää P, Laakso T, Fagerstedt KV, Simola LK, Paulin L, Rudd S, Teeri TH (2007) Expression profiling of the lignin biosynthetic pathway in Norway spruce using EST sequencing and real-time RT-PCR. Plant Mol Biol 65: 311-328
   
   
6. Laitinen T, Morreel K, Delhomme N, Gauthier A, Schiffthaler B, Nickolov K, Brader G, Lim KJ, Teeri TH, Street NR, Boerjan W, Kärkönen A (2017) A key role for apoplastic H₂O₂ in Norway spruce phenolic metabolism. Plant Physiol 174: 1449–1475
   
   
7. Simola, LK, Lemmetyinen J, Santanen A (1992) Lignin release and photomixotrophism in suspension cultures of Picea abies. Physiol Plant 84: 374-379
   
   
8. Väisänen E#, Takahashi J, Obudulu O, Bygdell J, Karhunen P, Blokhina O, Laitinen T, Teeri TH, Wingsle G, Fagerstedt KV, Kärkönen A (2020) Hunting monolignol transporters: membrane proteomics and biochemical transport assays with membrane vesicles of Norway spruce. J Exp Bot 71: 6379–6395
   
   
9. Warinowski T, Koutaniemi S, Kärkönen A, Sundberg I, Toikka M, Simola LK, Kilpeläinen I, Teeri TH (2016) Peroxidases bound to the growing lignin polymer produce natural-like extracellular lignin in a cell culture of Norway spruce. Front Plant Sci 7: 1523. doi: 10.3389/fpls.2016.01523.

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