Plant IPCS as a novel herbicide target

Abstract

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%.