Localization and functional characterization of iridoid biosynthetic genes in Catharanthus roseus /

Catharanthus roseus is the sole biological source of the medicinal compounds vinblastine and vincristine. These chemotherapeutic compounds are produced in the aerial organs of the plant, however they accumulate in small amounts constituting only about 0.0002% of the fresh weight of the leaf. Their limited biological supply and high economical value makes its biosynthesis important to study. Vinblastine and vincristine are dimeric monoterpene indole alkaloids, which consists of two monomers vindoline and catharanthine. The monoterpene indole alkaloids (MIA's) contain a monoterpene moiety which is derived from the iridoid secologanin and an indole moiety tryptamine derived from the amino acid tryptophan. The biosynthesis of the monoterpene indole alkaloids has been localized to at least three cell types namely, the epidermis, the laticifer and the internal phloem assisted parenchyma. Carborundum abrasion (CA) technique was developed to selectively harvest epidermis enriched plant material. This technique can be used to harvest metabolites, protein or RNA. Sequencing of an expressed sequence tagged (EST) library from epidermis enriched mRNA demonstrated that this cell type is active in synthesizing a variety of secondary metabolites namely, flavonoids, lipids, triterpenes and monoterpene indole alkaloids. Virtually all of the known genes involved in monterpene indole alkaloid biosynthesis were sequenced from this library.This EST library is a source for many candidate genes involved in MIA biosynthesis. A contig derived from 12 EST's had high similarity (E'^') to a salicylic acid methyltransferase. Cloning and functional characterization of this gene revealed that it was the carboxyl methyltransferase imethyltransferase (LAMT). In planta characterization of LAMT revealed that it has a 10- fold enrichment in the leaf epidermis as compared to the whole leaf specific activity. Characterization of the recombinant enzyme revealed that vLAMT has a narrow substate specificity as it only accepts loganic acid (100%) and secologanic acid (10%) as substrates. rLAMT has a high Km value for its substrate loganic acid (14.76 mM) and shows strong product inhibition for loganin (Kj 215 |iM). The strong product inhibition and low affinity for its substrate may suggest why the iridoid moiety is the limiting factor in monoterpene indole alkaloid biosynthesis. Metabolite profiling of C. roseus organs shows that secologanin accumulates within these organs and constitutues 0.07- 0.45% of the fresh weight; however loganin does not accumulate within these organs suggesting that the product inhibition of loganin with LAMT is not physiologically relevant. The limiting factor to iridoid and MIA biosynthesis seems to be related to the spatial separation of secologanin and the MIA pathway, although secologanin is synthesized in the epidermis, only 2-5% of the total secologanin is found in the epidermis while the remaining secologanin is found within the leaf body inaccessable to alkaloid biosynthesis. These studies emphasize the biochemical specialization of the epidermis for the production of secondary metabolites. The epidermal cells synthesize metabolites that are sequestered within the plant and metabolites that are secreted to the leaf surface. The secreted metabolites comprise the epidermome, a layer separating the plant from its environment.

Winâ Foy : functional object-oriented programming language

This thesis will introduce a new strongly typed programming language utilizing Self types, named Win--*Foy, along with a suitable user interface designed specifically to highlight language features. The need for such a programming language is based on deficiencies found in programming languages that support both Self types and subtyping. Subtyping is a concept that is taken for granted by most software engineers programming in object-oriented languages. Subtyping supports subsumption but it does not support the inheritance of binary methods. Binary methods contain an argument of type Self, the same type as the object itself, in a contravariant position, i.e. as a parameter. There are several arguments in favour of introducing Self types into a programming language (11. This rationale led to the development of a relation that has become known as matching [4, 5). The matching relation does not support subsumption, however, it does support the inheritance of binary methods. Two forms of matching have been proposed (lJ. Specifically, these relations are known as higher-order matching and I-bound matching. Previous research on these relations indicates that the higher-order matching relation is both reflexive and transitive whereas the f-bound matching is reflexive but not transitive (7]. The higher-order matching relation provides significant flexibility regarding inheritance of methods that utilize or return values of the same type. This flexibility, in certain situations, can restrict the programmer from defining specific classes and methods which are based on constant values [21J. For this reason, the type This is used as a second reference to the type of the object that cannot, contrary to Self, be specialized in subclasses. F-bound matching allows a programmer to define a function that will work for all types of A', a subtype of an upper bound function of type A, with the result type being dependent on A'. The use of parametric polymorphism in f-bound matching provides a connection to subtyping in object-oriented languages. This thesis will contain two main sections. Firstly, significant details concerning deficiencies of the subtype relation and the need to introduce higher-order and f-bound matching relations into programming languages will be explored. Secondly, a new programming language named Win--*Foy Functional Object-Oriented Programming Language has been created, along with a suitable user interface, in order to facilitate experimentation by programmers regarding the matching relation. The construction of the programming language and the user interface will be explained in detail.