The role of transketolase and transaldolase in carbon partitioning to lignin in Arabidopsis thaliana

Abstract

Lignocellulosic biomass is earth’s largest renewable material and is used for paper, pulp, biofuels, timber and lignin and cellulosic products. The secondary cell wall (SCW) of xylem tissue in vascular plants is comprised of cellulose, hemicellulose and lignin. Lignin fills the spaces between cellulose, hemicellulose and pectin, holding the cells together. The composition of lignin can be characterised by the monomers contained within the polymer. These monomers, primarily hydroxyphenyl (H-subunit), guaiacyl (G-subunit) and syringyl (Ssubunit), define the properties of the lignin in accordance with their presence in the SCW and on the ratios of their abundance. These lignin properties affect the efficiency of lignin removal in the industrial processing of wood. Although extensive research has been done on lignin biosynthesis and wood formation, our understanding of the metabolic flux of carbon into lignin formation is still incomplete and insufficient. A more complete understanding of monolignol biosynthesis and wood formation would allow us to optimise lignin content and composition to improve plant growth and biomass utilisation. Production of monolignols can be traced back to plastidic primary metabolism and aromatic amino acids biosynthesis, which includes a major carbon shunt via the pentose phosphate pathway and subsequently the shikimate-chorismate pathway. Photosynthates produced in source tissues are transported into sink tissues after which they are metabolised through glycolysis or the pentose phosphate pathway (PPP). Transketolase (TK) and transaldolase (TAL) are plastid localised enzymes which act in the PPP and are responsible for the production of erythrose 4-phosphate (E4P) in the sink tissues, that together with phosphoenolpyruvate (PEP), mainly from glycolysis, form the starting products for monolignol biosynthesis. E4P, together with PEP feed into the shikimate pathway where phenylalanine (Phe) is produced. Phe is exported from the plastid and is the starting metabolite to produce monolignols in the cytoplasm. TK however also plays a role in photosynthesis, in the Calvin cycle. The Calvin cycle uses the products of the light reactions, ATP and NADPH, to fix atmospheric CO2 into carbon skeletons for sucrose and starch biosynthesis. TK and TAL can both produce E4P for SCW formation, and so it is not yet known which gene is more important in carbon flux toward lignin. As these enzymes play a key role in primary carbon metabolism and possibly wood formation, it is important to get a clearer understanding of how they interact and channel carbon towards wood formation. In this MSc I aimed to investigate the role of transketolase and transaldolase in the growth, physiology, and lignin content and composition of Arabidopsis thaliana. I wanted to know how knockouts of TK and TAL would affect these traits and whether one protein contributes more to the flux of carbon towards lignin than the other. I also wanted to know if there is cross-talk between these genes. With a combination of T-DNA insertional mutants and CRISPR-Cas9, I knocked out both A. thaliana TK genes (TK1 and TK2) and TAL. I demonstrate that knocking out of of TK1 results in a lethal genotype with evidence for a lethal genotype when knocking out TK2 and that overexpressing TK2 is detrimental to growth in short photoperiods. I also show that there is cross-talk between TK1 and TK2, but not between the TKs and TAL. I also show that TAL is not essential for photosynthesis but does play a role in how carbon is partitioned to lignin. The findings of this study provide insights into how primary carbon metabolism in the pentose phosphate pathway and Calvin cycle can affect downstream lignin formation