Plant IPCS as a novel herbicide target

Herbicides play an important role in modern agriculture. However, the number of herbicides available to use is declining due to tighter environmental regulations and the rise of herbicide resistance. The discovery of novel herbicidal targets is a potential solution to this situation. This thesis investigates Inositol Phosphoryl Ceramide Synthase (IPCS) as a novel target for herbicidal applications. IPCS is an essential enzyme found in the sphingolipid biosynthetic pathway and synthesises Inositol Phosphoryl Ceramide (IPC) from phytoceramide. IPC serves as a critical biosynthetic precursor for complex sphingolipids, which are integral to numerous cellular processes. Inhibition of IPC synthesis impedes the biosynthesis of these sphingolipids and results in the accumulation of pro-apoptotic phytoceramides, making it a promising herbicidal target. CRISPR-Cas9 genome editing was employed to generate targeted knockouts of AtIPCS isoforms in order to investigate their individual functional roles. These transgenic lines were subsequently used to assess changes in gene expression, sphingolipid profiles, and responses to Pseudomonas syringae infection. Limited data was obtained; however, it was found that AtIPCS2 was the most important isoform, as disruption resulted in inhibited plant growth and pathogen defence response. Lipid profiles were also disrupted, with the most change observed in the transgenic line targeting both AtIPCS1 and AtIPCS3, with increased levels of PI and ceramide and decreased levels of GIPC. Further replication of this result is required to fully establish the significance of this observation. The isolation of the triple mutant line was interesting as it suggested that IPCS may not be essential as originally perceived. However, the lipid profile has not yet been analysed, and detecting IPC or GIPC would confirm whether the synthesis of complex sphingolipids is truly impaired. In parallel to the genetic work, N- alkylated triazinone 1.7, previously identified through a high-throughput screening, was resynthesised through a protocol obtained from Bayer Crop Science. However, this synthetic route resulted in low yields, prompt ing efforts to optimise the synthesis of 1.7 under varied conditions and alternative pathways. Following numerous attempts, the overall yield was improved from 3% to 13%.