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Title: TARGETING OF A NUCLEAR ANTHRANILATE SYNTHASE ALPHA-SUBUNIT GENE (ASAA2) TO THE TOBACCO PLASTID GENOME RESULTS IN ENHANCED TRYPTOPHAN BIOSYNTHESIS

Author
item Zhang, Xing Hai
item BROTHERTON, JEFFREY - CROP SCIENCES UOFI URBANA
item WIDHOLM, JACK - CROP SCIENCES UOFI URBANA
item PORTIS JR, ARCHIE

Submitted to: Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/25/2001
Publication Date: 9/1/2001
Citation: Zhang, X., Brotherton, J.E., Widholm, J.M., Portis Jr, A.R. 2001. Targeting of a nuclear anthranilate synthase alpha-subunit gene (asaa2) to the tobacco plastid genome results in enhanced tryptophan biosynthesis. Plant Physiology.127:131-141

Interpretive Summary: Tryptophan is an essential amino acid for the human diet. In plants, tryptophan is made in the chloroplasts and a key regulatory enzyme for the biosynthetic pathway, anthranilate synthase, is inhibited by tryptophan. Furthermore the enzyme level is very low and the enzyme must be imported into the chloroplast because it is first made outside using a gene (DNA) located in the nucleus. In this study we incorporated a gene specifying a form of the enzyme less sensitive to tryptophan into the chloroplast DNA of tobacco plants in an attempt to increase tryptophan production by circumventing regulatory controls on both enzyme activity and expression. Transgenic plants were obtained, which have 10-fold higher levels of tryptophan in their leaves and a 28 percent increase in the seeds as compared to normal plants. This information will benefit scientists attempting to improve the nutritional value of plants by modifying the various metabolic pathways occurring in the chloroplast that have genes for key enzymes located in their nuclear DNA.

Technical Abstract: Anthranilate synthase (AS) is the key control enzyme of the tryptophan (Trp) biosynthetic pathway. A cDNA (ASA2) encoding the putative mature peptide of a feedback-insensitive tobacco AS alpha-subunit was independently incorporated by transformation into two different regions of the tobacco plastid genome, resulting in two lines of transplastomic plants with normal phenotype. Stable and site-specific integration of the ASA2 transgene into the plastid genome was confirmed in the progeny. A high level of ASA2 mRNA was observed in transplastomic plants but not in wild type. In one line the ASA2 gene was co-transcribed with the plastid gene ORF184 operon resulting in at least 10-fold more abundant ORF 184 mRNA than in the wild type. Transplastomic plants exhibited both a higher level of AS alpha-subnit protein and AS enzyme activity that was less sensitive to Trp-feedback inhibition. The plants were also more tolerant to AS inhibitor, 5-methyltryptophan (5MT), during seed germination. Cross-pollination tests between transplastomic and wild type plants showed both resistance to the selection agent spectinomycin and tolerance to 5MT were transmitted maternally. The cellular free Trp concentration in leaves of transplastomic plants was over 10-fold higher than in wild type plants. Our studies demonstrate feasibility of modifying biosynthetic pathways of important metabolites through transformation of the plastid genome.