A transgenic/cisgenic/intragenic approach to overexpress 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase to confer glyphosate resistance and to increase aromatic amino acids in Capsicum annuum (Chile)

EPSP synthase is a key enzyme in the shikimate pathway and catalyzes the penultimate step, the transfer of enolpyruvyl moiety of phosphoenolpyruvate (PEP) to the 5-hydroxyl of shikimate-3-phosphate. The shikimate pathway is the primary route that produces the precursor to synthesize aromatic amino acids and numerous secondary metabolites in plants. We hypothesized that overexpressing EPSP synthase would increase the pools of aromatic amino acids in plants. Both chile and tobacco plants were transformed with the chile EPSP synthase gene driven by the constitutive CaMV 35S promoter, and while the chile plants did not show accumulation of the transgene product, the tobacco transformants showed significant accumulation of the chile EPSP synthase protein. The tobacco transformants also showed increased pools of phenylalanine and tyrosine. An increase in these aromatic amino acids did not change the expression of other selected genes in the aromatic amino acid biosynthesis pathway.;One of the main goals of this project was to develop an intragenic/cisgenic approach to confer resistance to the most widely used herbicide, glyphosate, in chile. EPSP synthase is the target of glyphosate. We used tobacco as the transgenic system rather than chile to test the gene constructs that would allow for the development of this intragenic system, since chile transformation is very inefficient. Since overexpressing the wild type EPSP synthase gene in tobacco provided little to no resistance to glyphosate, several gene constructs were made with mutations to make certain amino acid substitutions in the catalytic site. These mutations have been reported to produce glyphosate resistant EPSP synthase in many microbes and also some plants. One of these mutated genes with substitutions: threonine 179 to isoleucine and proline 183 to serine (...