The complementary-DNA encoding the first enzyme of the shikimate pathway in Solanum tuberosum L.
Abstract
A glyphosate-tolerant tobacco cell-line, Nicotiana tabacum L. Indiana 7, was selected from the glyphosate-sensitive Wisconsin 38 line. Tolerance and growth characteristics of Indiana 7 cells were the same for cells maintained for more than one year in the presence or absence of glyphosate. Wisconsin 38 cells accumulated 46 times more shikimate than Indiana 7 cells, when cells of both lines were exposed to the herbicide. Specific activities of 3-deoxy- sc D-arabino-heptulosonate 7-phosphate (DAHP) synthase, the first enzyme of the shikimate pathway, and 5-enolpyruvylshikimate 3-phosphate synthase, the target site of glyphosate, were elevated in Indiana 7 cells. Plants regenerated from Indiana 7 cells retained tolerance to glyphosate. A cDNA encoding potato DAHP synthase, the first from any eukaryotic source, was cloned into $\lambda$gt11. The nucleotide suquence was obtained, and the identity of the cDNA was confirmed through partial amino acid sequence analysis of DAHP synthase. The cDNA contains a 1521 bp open reading frame that encodes a polypeptide with a calculated molecular weight of 56,108. The N-terminus of the deduced amino acid sequence of potato DAHP synthase resembles a transit peptide that has regions of charged amino acid residues in common with other chloroplast transit peptide sequences. Amino acid sequence identity between the mature potato DAHP synthase and the E. coli Phe-sensitive DAHP synthase is 27%. Wounding potato tubers induced steady-state levels of translatable DAHP synthase mRNA seven-fold. The mRNA increase was followed by a comparable elevation of enzyme activity and amount. The induction was localized to the wound site, but was maximal in periderm tissue, indicating tissue-specific regulation within the potato tuber. DAHP synthase was also wound-induced in potato leaves and tomato fruit. The regulation of DAHP synthase in response to stress conditions confirms its role as a key regulatory enzyme of the shikimate pathway.
Degree
Ph.D.
Advisors
Herrmann, Purdue University.
Subject Area
Plant propagation|Biochemistry
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