Mutants in the Biosynthesis of Amino Acids
Mutagenesis-selection procedures used in our laboratory resulted in the obtention of plants with modified regulatory mechanisms leading to a higher production of specific free amino acids. In particular, two mutants of the aspartate pathway in Nicotiana sylvestris were isolated following selection on growth inhibitory concentrations of lysine plus threonine, and on a lysine analog, S-(2-ammoethyl)-L- cysteine (AEC), respectively named RLT 70 and RAEC-l.The resistance property in bath cases was inherited as a monogenic dominant nuclear character. In RLT 70, up to 70% of the pool of free amino acids was made up of threonine, against 6% in the wild type plant. A completely desensitized aspartate kinase (AK) isoenzyme normally feedback inhibited by lysine was found to be the molecular basis of this overproduction. In RAEC-1, up to 25% of lysine could be found in the pool of free amino acids, compared to 1.5% in the wild type plant. The enzyme implicated in this mutant was dihydrodipicolinate synthase (DHDPS) totally desensitized to normal feedback inhibition by lysine. Evolution of the expression of both mutations during plant development revealed a peak of accumulation of the concerned amino acid, just before the elongation phase. In calli, overproduction was elevated and constant throughout the subcultures. Purification to homogeneity of the DHDPS subunit was achieved through a 2D polyacrylamide gel electrophoresis with a partially purified enzyme fraction. It was found to be a tetramer of four identical subunits, each of 39 kDa, and to be localized in the chloroplast. The purified protein was then microsequenced and the first 11 amino acids of the NH2-end were determined (collaboration with Dr.J.C. Guillemot, ELF Sanofi Bioresearch). Moreover the eluted protein was injected into rabbits to induce the production of anti-DHDPS antibodies. These polyclonal antibodies were used as probe to screen a cDNA library built in lambda gt11 from total poly A+ RNA fractions of N. Sylvestris. Several positive clones were isolated and are now being characterized.
KeywordsFree Amino Acid Total Free Amino Acid Lysine Content Aspartate Kinase Growth Inhibitory Concentration
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