Regulation of the first enzyme of the shikimate pathway in plants

Jianmin Zhao, Purdue University

Abstract

Three potato (Solanum Tuberosum L) cDNAs encoding DAHP synthase, shkA, shkB and shkBB, were cloned and sequenced. There are only 18 bp differences between shkB and shkBB cDNA sequences. For convenience, I will refer to shkB and shkBB as shkB(B). The cDNA sequences were partially confirmed by protein sequence and by mass spectrometric analyses of proteolytic fragments of DAHP synthase that was purified from potato tuber. The shkA and shkB deduced amino acid sequences show about 80% identity in the mature protein and 40% identity in the transit peptide region. The alignment of plant DAHP synthases with homologues from microorganisms reveals several common domains, but only a few conserved residues. DNA probes, specific for shkA and shkB(B), detected at least two distinct genes for potato DAHP synthases, the shkA gene and the shkB(B) gene. Using these probes, I found differential expression of the two genes. The shkA gene was highly expressed in roots, moderately in stems and tubers, and poorly in leaves, while the shkB(B) gene was expressed throughout the plant without much variation. Mechanical wounding or exposure to glyphosate induced expression of shkA but not shkB(B). These results indicate that shkA and shkB(B) serve to satisfy different requirements for carbon flow into the shikimate pathway. I propose that the function of the shkB(B) gene is "housekeeping" to satisfy the need for aromatic amino acids in protein biosynthesis, and that the shkA gene might respond to the need for precursors in the biosynthesis of secondary metabolites, like lignin and flavonoids, and to general stress. To study the intracellular transport of plant DAHP synthases, shkA and shkB(B) cDNAs were placed downstream of a T7 or SP6 promoter. DAHP synthase mRNAs were synthesized in vitro and used as templates for the synthesis of radiolabelled DAHP synthase precursors. The precursors were processed to their mature forms by isolated chloroplasts. The mature DAHP synthases were found in the soluble fraction of the chloroplasts and shown to be resistant to protease treatment. DAHP synthase precursors were also processed to their mature forms by organelle-free transit peptidase. The cleavage sites of these processings were determined by radiosequencing. The results suggest that both shkA- and shkB(B)-encoded proteins are localized in the stroma of chloroplasts.

Degree

Ph.D.

Advisors

Herrmann, Purdue University.

Subject Area

Biochemistry

COinS