Abstract
M.Sc.
On exposure to abiotic or biotic stresses, plants initiate a cascade of metabolic reactions, some
of which lead to the biosynthesis of secondary metabolites with roles in self defense.
Phytoalexins are a class of secondary metabolites synthesized de novo in response to microbial
attack by activation of certain biosynthetic pathways. Cruciferae phytoalexins are all indole
based with a carbon, nitrogen and sulfur containing constituent on the 3’ position of the
indole ring. This common similarity of all Cruciferae phytoalexins suggests that the plants all
share a common indole precursor. Camalexin is the primary phytoalexin of Arabidopsis thaliana.
De novo synthesis of camalexin upon infection, as well as its antimicrobial nature supports its
role in disease resistance. Evidence exists that suggests the inducible biosynthesis of camalexin
involves steps of the tryptophan pathway, along with an increase in transcript and protein
levels of the tryptophan pathway enzymes after microbial infection.
Bacterial LPS (lipopolysaccharide) has been described as one of the microbe/pathogenassociated
molecular patterns (M/PAMPs) capable of eliciting the activation of the plant
innate immune system. LPS is an integral component of the cell surface of Gram-negative
bacteria. It is a complex which is exposed to the external environment, and is thus involved
with external interactions of the bacteria.
The hypothesis investigated in this dissertation is that LPS, as a lipoglycan PAMP, results in
activation of signal transduction pathways involved in defense that lead to the production of
the defense metabolite, camalexin. Furthermore, that the genes CYP71B15, CYP79B2 and
TSB are up-regulated in response to LPS during camalexin biosynthesis via the tryptophan
pathway. To test this hypothesis, camalexin production was investigated through a
combination of analytical techniques including thin layer chromatography (TLC), high
performance liquid chromatography (HPLC), gas chromatography (GC), ultra pressure liquid
chromatography-mass spectrometry (UPLC-MS) and fluorescence spectroscopy. Genes in the
camalexin biosynthetic pathway were investigated by two-step reverse transcription
polymerase chain reaction (PCR), GUS reporter gene assays and quantitative real time PCR
(RT-qPCR).