Solanine, the green substance in potato, Solanum tuberosum L., tubers, is a toxic glycoalkaloid that is a potential human health hazard. To control the formation of this glycoalkaloid a greater understanding of its site of synthesis is needed. Labelling of solanidine, a direct precursor in the biosynthesis of solanine, with digitonin may indirectly locate the site of solanine synthesis in tubers. A study using ultrastructural cytochemical techniques was initiated to explore this possibility. Sprouted tips and peridermal complex (periderm and cortex) tissue were fixed three different ways: (1) glutaraldehyde only, (2) osmium tetroxide and glutaraldehyde, and (3) glutaraldehyde-osmium tetroxide-digitonin mixture. The glutaraldehyde-osmium tetroxide-digitonin mixture provided the best fixation for this study and was used throughout. An alkaloid extract of potato tissues incubated with digitonin resulted in a precipitate being formed. Application of the alkaloid extract to TLC plates before and after incubating with digitonin indicated only solanidine was removed by the digitonin. In the electron micrographs the solanidine-digitonin complex was recognized as darkly stained needles or spicules. These spicules were observed mostly in the vacuoles in the sprouted tips. However, a few spicules were also noted in the cytoplasm. Relatively few spicules were observed in the peridermal tissue.

Steroidal glycoalkaloids (SGAs) are secondary metabolites produced by approximately 350 species in the Solanaceae family. SGAs are reported to be important for pest resistance and flavor enhancement at low concentrations but are toxic to humans and other mammals at high concentrations. Studies on sterol / SGA biosynthesis have implicated squalene synthase as a key regulatory enzyme because it catalyzes an irreversible step from the mevalonic acid pathway. However, the regulatory mechanisms of squalene synthase are not yet known. A study was conducted to elucidate the distribution pattern of SGAs and to clone the squalene synthase gene in order to determine a relationship between SGAs and gene expression levels. Solanum chacoense, a wild potato species was used as a model plant from which tissues were harvested at specified developmental stages and analyzed for SGA content. The results from the SGA analysis suggest a qualitative and quantitative tissue- and age-dependent accumulation of SGAs. Regenerative tissues such as, axiliary shoots, flowers and floral buds had the highest levels of 88, 49 and 63 µmole/g DW, respectively. The roots, stems and tubers showed the lowest amounts of SGAs of 1 to 8, 5 to 15 and 7 to 15 µmole/g DW, respectively. Stolons and tubers accumulated higher amounts of α-chaconine (59 to 67%) than α-solanine (61 to 64%) at all developmental stages analyzed. On the other hand, in young expanding, fully expanded, and old senescing leaves where leptine and leptinines tend to dominate, α-solanine and α-chaconine together accounted for only 8 to 15%, 7 to 15%, and 8 to 45%, respectively. Plant organs that showed the highest biosynthetic activity for SGA production also had high levels of transcripts coding for genes of isoprenoid biosynthesis. The results from the cloning and characterization of squalene synthase suggest that the cloned cDNA fragment is a putative S. chacoense squalene synthase gene with an open reading frame / predicted protein precursor of 411 amino acids. The cloned cDNA has high similarity (68-100%) to known plant squalene synthase genes and contains six deduced peptide domains observed in other species. The 3â untranslated regions of floral buds, young leaves (early vegetative stage), and fully expanded leaves (anthesis) were different in length with, 249, 335, and 202 nucleotides, respectively. The Southern blot analysis suggests a single copy gene although the existence of a gene family cannot be ruled out. / Ph. D.

La pomme de terre produit des glycoalcaloïdes comme la plupart des Solanacées. Deux composés, en particulier, l’α-solanine et l’α-chaconine, sont produits en plus grande quantité lorsque le tubercule est exposé à la lumière et subit des dégradations. Ces molécules sont toxiques et peuvent représenter un danger pour le consommateur et des nuisances pour l’environnement. Dans le cadre de cette thèse, les teneurs en α-solanine et α-chaconine dans la chaire de pomme de terre verdie, en germination ou en décomposition retrouvées sur les marchés d’Abidjan (Côte d'Ivoire) ont été analysées. Les résultats ont montré que la chaire de ces pommes de terre contient des quantités élevées de ces deux composés, dépassant 2 à 5 fois la limite recommandée. Pour des raisons de sécurité sanitaire, ces pommes de terre doivent être proscrites de l’alimentation humaine. En revanche, la teneur élevée en glycoalcaloïdes dans ces pommes de terre représente une source de solanidine, un précurseur pour la synthèse d'hormones et de composés pharmacologiquement actifs, qui mérite d’être exploitée. Dans cet objectif, nous avons développé une méthode chimio-enzymatique simple, comprenant un traitement acide partiel suivi d’une hydrolyse enzymatique par la β-glycosidase de la blatte Periplaneta americana dont le gène a été isolé à partir d’une librairie génomique de cDNA afin de détoxifier ces composés et produire la solanidine. / Potato produce glycoalkaloids as most plants of Solanaceae family. The principal glycoalkaloids, α-chaconine and α-solanine are produced in greater quantities when potato tubers are exposed to light and are subject to deteriorations. These compounds are toxic and can represent a real danger for the consumer and the environment where they are discharged during their degradation. In this work, the estimation of glycoalkaloids in the flesh of different types of decayed potatoes usually found in different market places of Abidjan (Ivory Coast) was evaluated. The results showed that turned green and also sprouting or rotting potato flesh contain high amounts of toxic solanine and chaconine, exceeding by 2 to 5-fold the recommended limit. For safety consideration, these decayed potatoes should be systematically set aside. The accumulation of α-chaconine and solanine in potatoes can be seen as an attractive source of solanidine that is an important precursor for hormone synthesis and some pharmacologically active compounds. To this end, we proposed herein a simple chemo-enzymatic protocol comprising a partial acidic hydrolysis followed by an enzymatic treatment with the β-glycosidase from Periplaneta americana whose gene was isolated from a cDNA genomic library in order to detoxify these compounds and produce solanidine.