African Traditional Medicine: A Dictionary of Plant Use and Applications, with Supplement: Search System for Diseases

Monoterpenoid indole alkaloids (MIA) are among the largest and most complex group
of nitrogen containing secondary metabolites that are characteristic of the Apocynaceae plant
family including the most notable Catharanthus roseus. These compounds have demonstrated
activity as successful drugs for treating various cancers, neurological disorders and
cardiovascular conditions. Due to the low yields of these compounds and high pharmacological
value, their biosynthesis is a major topic of study. Previous work highlighting the leaf epidermis
and leaf surface as a highly active area in MIA biosynthesis and MIA accumulation has made the
epidermis a major focus of this thesis. This thesis provides an in-depth analysis of the valuable
technique of RNA in situ hybridization (ISH) and demonstrates the application of the technique
to analyze the location of the biosynthetic steps involved in the production of MIAs.
The work presented in this thesis demonstrates that most of the MIAs of Eurasian Vinca
minor, African Tabernaemontana e/egans and five Amsonia species, including North American
Amsonia hubrichitii and Mediterranean A. orienta/is, accumulate in leaf wax exudates, while the
rest of the leaf is almost devoid of alkaloids. Biochemical studies on Vinca minor displayed high
tryptophan decarboxylase (TOe) enzyme activity and protein expression in the leaf epidermis
compared to whole leaves. ISH studies aimed at localizing TOe and strictosidine synthase
suggest the upper and lower epidermis of V. minor and T. e/egans as probable significant
production sites for MIAs that will accumulate on the leaf surface, however the results don't
eliminate the possibility of the involvement of other cell types.
The monoterpenoid precursor to all MIAs, secologanin, is produced through the MEP
pathway occurring in two cell types, the IPAP cells (Gl0H) and epidermal cells (LAMT and SLS).
The work presented in this thesis, localizes a novel enzymatic step, UDPG-7-deoxyloganetic acid
glucosyltransferase (UGT8) to the IPAP cells of Catharanthus longifolius. These results enable
the suggestion that all steps from Gl0H up to and including UGT8 occur in the IPAP cells of the
leaf, making the IPAP cells the main site for the majority of secologanin biosynthesis. It also
makes the IPAP cells a likely cell type to begin searching for the gene of the uncharacterized
steps between Gl0H and UGT8. It also narrows the compound to be transported from the IPAP
cells to either 7-deoxyloganic acid or loganic acid, which aids in the identification of the
transportation mechanism.

Elizabeth Edmunds

Papers

Localization of monoterpenoid indole alkaloid (MIA) biosynthesis by in situ hybridization (ISH)