Insights in understanding the regulation of methionine biosynthesis; Engineering alfalfa (Medicago sativa) for increased methionine content

Alfalfa is a forage legume used as a principal feed source for livestock throughout the world because it provides much of the protein required in animal diets. Alfalfa, however, is deficient in the essential amino acid, methionine (Met). One strategy of improving the Met content of alfalfa has involved introducing the corn genes encoding the Met-rich and rumen stable beta- and delta-zein seed storage proteins. However, preliminary results indicate that the synthesis of the zein proteins in alfalfa leaves is limited by the availability of free Met. Our primary research goal is to increase Met levels in alfalfa by genetically modifying Met metabolic pathway. In an effort to better understand how the pathway is regulated in alfalfa, we have isolated cDNA clones of the key genes involved in Met metabolism from alfalfa and studied their expression in the leaves and nodules of alfalfa. Cystathionine gamma-synthase (CGS), S-adenosylmethionine synthase (SAMS), and Threonine synthase (TS) are all expressed at higher levels in nodules compared to leaves, suggesting a higher level of Met metabolism in this specialized organ. We also found that Met S-methyltransferase (MMT), the enzyme responsible for Met to S-methylmethionine (SMM) conversion, is expressed predominately in the leaves of alfalfa. Expression of an Arabidopsis CGS transgene in alfalfa caused downregulation of the endogenous CGS gene in the leaves, but not the nodules of transgenic plants. There was also increased expression of MMT in the leaves of CGS overexpressing alfalfa that accompanied an increase in SMM. In order to further modulate the Met metabolic pathway for increased synthesis of Met, we expressed a mutant form of the CGS transgene that lacked a feedback regulatory control mechanism to express the CGS gene at higher levels. We also expressed an antisense TS gene in alfalfa in an effort to downregulate this competing enzyme to favor Met biosynthesis over that of Thr. Metabolite data from these transgenic plants showed that carbon flux into Met biosynthesis can be increased. However, free Met remained low in transgenic plants likely due to an increased flow toward the synthesis of SMM and SAM.