טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentGodo Itamar
SubjectThe Elucidation of Factors Regulating Methionine Content in
Tobacco and Soybean Seeds, and the Effect of High
Methionine Level in Seeds on Primary
Metabolites
DepartmentDepartment of Biology
Supervisors Professor Gadi Schuster
Professor Rachel Amir
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Abstract

Human beings and livestock are unable to synthesize 10 out of the 20 protein amino acids, called "essential" amino acids and must therefore obtain them from their diet. Major food supply for humans and livestock comes from plant seeds, which contain very low proportions of some of mostly aspartate family belonged amino acids lysine, methionine and threonine.  Methionine and its metabolites play a significant role in metabolic processes in plants, humans and domesticated animals, thus making methionine a limiting factor in diet.  The goal of the current study is to study the factors regulating methionine content in seeds.  In order to determine the limiting factors in methionine biosynthesis in plant seeds, A. thaliana origined cystathionine gamma synthase (CGS) was expressed under a seed-specific promoter in N. tabacum. Expression of this gene, both with and without its N-terminal regulatory region, did not lead to higher soluble methionine content in tobacco seeds. However, a significant reduction was found in the levels of the other amino acids whose metabolism is connected to methionine, suggesting that they were incorported to proteins and that CGS plays a role in methionine biosynthesis regulation in seeds. To verify if the level of methionine can further increase, plants expressing feedback inhibition insensitive bacterial aspartate kinase (AK) were used to cross with those expressing the CGS. Seeds expressing both genes have significantly higher levels of free methionine compared to their parents, suggesting that the carbo-amino skeleton level also regulate the level of methionine.  Methionine is a precursor to different metabolites in plant seeds. Therefore, in order to examine the effect of higher concentration of methionine on the accumulation of primary metabolites in tobacco seeds, we carried out metabolic profiling. We were able to isolate 103 primary metabolites using GC-MS separation, 12 of which showed a significant contribution to the diversity between genotypes according to a PCA analysis, as well as significant statistical difference between genotypes.  Methionine is the major compound limiting the nutritional value of legume seeds. Therefore, in order to reveal if the CGS level limits methionine synthesis in soybean seeds, soybean plants were transformed with the feedback inhibition insensitive form of CGS using a seed-specific promoter, resulting in high soluble methionine content in seeds.  Total methionine content determined after seed protein hydrolysis showed that the level of methionine was not significantly altered.