Gene duplication and functional diversification is a central driving force in the evolution of plant biochemical diversity. However, the latter process is not well understood. Here the diversification of the plant shikimate/quinate dehydrogenase (S/QDH) gene family was investigated in order to shed light on how duplicate genes functionally diversify. The shikimate pathway is the major biosynthetic route towards the aromatic amino acids, linking vital protein biosynthesis with the production of aromatic secondary metabolites. Dehydroquinate dehydratase/shikimate dehydrogenase (SDH) encodes the central enzyme of this pathway, catalyzing the production of shikimate. Quinate is a secondary metabolite synthesized using the same precursors as shikimate by quinate dehydrogenase (QDH). Gene duplication prior to the gymnosperm / angiosperm split generated two distinct clades in seed plants separating SDH and QDH functions whereas non-seed plants have a single copy SDH. In vitro biochemical characterization of a reconstructed ancestral enzyme was performed alongside extant members separated prior to duplication (from a lycopod, a bryophyte, and a chlorophyte) and afterwards (from a gymnosperm and an angiosperm). This revealed that novel quinate biosynthetic activity was gained in seed plants, providing evidence for the diversification of gene function via neofunctionalization. However, the ability to use both NAD(H) and NADP(H) seems to have developed in both SDH and QDH clade members of angiosperms. Finally, a method is described for analysing quinate and its derivative, chlorogenic acid in transgenic Arabidopsis. / Graduate / 2021-05-11

The shikimate pathway links carbohydrate metabolism to biosynthesis of the aromatic amino acids in plants, fungi, bacteria and apicomplexan parasites. The pathway has seven enzymatic steps which convert erythrose-4-phosphate and phosphoenolpyruvate to chorismate, the precursor of tyrosine, tryptophan and phenylalanine. Due to the absence of the pathway in mammalian species, the enzymes are attractive targets for herbicides and antimicrobials. Shikimate dehydrogenase (SDH) catalyses the fourth step, the NADP-dependent reversible reduction of 3-dehydroshikimate to shikimate. Five SDH homologs – AroE, Ael1, YdiB, RifI and SdhL – have been identified through kinetic analysis and phylogenetic studies in the bacterium Pseudomonas putida. SDH homolog gene knockouts (KO) were used to characterize their functions. The AroE KO and Ael1 KO were successfully constructed via gene SOEing of the SDH homolog with a gentamycin antibiotic cassette and homologous recombination via electroporation into WT P. putida KT2440. Preliminary characterization tested KO growth, auxotroph recovery and fluorescent activity.

The shikimate pathway links carbohydrate metabolism to biosynthesis of the aromatic amino acids in plants, fungi, bacteria and apicomplexan parasites. The pathway has seven enzymatic steps which convert erythrose-4-phosphate and phosphoenolpyruvate to chorismate, the precursor of tyrosine, tryptophan and phenylalanine. Due to the absence of the pathway in mammalian species, the enzymes are attractive targets for herbicides and antimicrobials. Shikimate dehydrogenase (SDH) catalyses the fourth step, the NADP-dependent reversible reduction of 3-dehydroshikimate to shikimate. Five SDH homologs – AroE, Ael1, YdiB, RifI and SdhL – have been identified through kinetic analysis and phylogenetic studies in the bacterium Pseudomonas putida. SDH homolog gene knockouts (KO) were used to characterize their functions. The AroE KO and Ael1 KO were successfully constructed via gene SOEing of the SDH homolog with a gentamycin antibiotic cassette and homologous recombination via electroporation into WT P. putida KT2440. Preliminary characterization tested KO growth, auxotroph recovery and fluorescent activity.

This diploma thesis deals with the development of an HPLC method for the determination of selected compounds participating in enzymatic reaction leading to the formation of gallic acid. The analysed reaction mixture contains the following reagents: shikimic acid, NADP+ and shikimatedehydrogenase (SDH) extracted from parsley; the presumed product of the reaction is gallic acid. Two chromatographic methods for the determination of the above mentioned compounds were developed using C18 HPLC column and porous graphitic carbon Hypercarb column. Molecular absorption spectrometric detection in the UV range was used in all measurements. Separation on the C18 column was found particularly suitable for analysing the composition of the end products of the reaction. Because of the NADP+ and shikimic acid peak overlap it is necessary to observe absorbance at 212 and 260 nm. Shikimic acid and NADP+ can be quantified due to the fact that shikimic acid does not absorb at 260 nm while NADP+ absorb radiation at both wavelengths. Separation via Hypercarb column was found particularly suitable for analysing the process of the reaction; additional products or intermediates can be seen in chromatograms compared to the C18 method. Determination with Hypercarb column is characterized by higher sensitivity and lower limit...

Biotic stress in the form of viral infection, as well as abiotic salt stress, cause leaves injuries, stomata closure and decreased rate of photosynthesis. These factors lead to the limitation of plant growth and to reduced amount of coenzyme NADPH. However NADPH is an important coenzyme for many metabolic pathways such as synthesis of fatty acids, amino acids and secondary metabolites involved in stress responses. NADPH is also a coenzyme for key enzymes of antioxidant system and for many regulatory enzymes. NADP-dependent enzymes are alternative source of NADPH in plants under stress conditions. In this work, activities of four NADP-dependent enzymes: Glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), NADP-isocitrate dehydrogenase (NADP-ICDH, EC 1.1.1.42), NADP-malic enzyme (decarboxylating) (NADP-ME, EC 1.1.1.40) and Shikimate dehydrogenase (SDH, EC 1.1.1.25) were studied. Activities of all these enzymes but SDH increased in leaves of tobacco plants (Nicotiana tabacum L.) infected by PVYNTN , The most sensitive enzymes to viral infection were NADP-ICDH and NADP-ME, whose activity was increased in comparison with control plants 3-fold and 2,4-fold, respectively. Changes in activity of studied enzymes were also determined in plants exposed to viral infection in combination with heat-shock...