Seven antifeedant sesquiterpene lactones (STLs), 4,5-dihydroniveusin A, argophyllin B, argophyllin A, 15-hydroxy-3-dehydrodesoxytifruticin, niveusin B, 1,2-anhydridoniveusin A, and an unidentified epoxide, in cultivated sunflower (Helianthus annuus L.) have been quantified by a highperformance thin-layer chromatography and UV-reflectance scanning densitometry analysis. Age-related expression of STL content in sunflower reveals a heretofore undescribed pattern in which nonpolar STLs such as 15-hydroxy-3-dehydrodesoxytifruticin predominate up to an age of three weeks, but are subsequently displaced by polar STLs, especially argophyllin A, through later foliar stages and anthesis. This leaf pattern of STL ontogeny is maintained in three widely differentH. annuus cultivars (Giant Gray Stripe, Royal Hybrid 2141, Hybrid 7111), which in turn had similar total contents of STLs. Antifeedant activity for western corn rootworm was positively correlated with STL content, particularly with argophyllin A and its isomer argophyllin B, in respective tissue extracts. Enhanced amounts of highly antifeedant argophyllins, especially in newly grown leaf and floral tissues yielding sunflower progeny, strongly suggest that these epoxy-STLs are a chemical defense against insect herbivory.
Indole-3-acetyl-amino acid conjugate hydrolases are believed to be important in the regulation of indole-3-acetic acid (IAA) metabolism in plants and therefore have potential uses for the alteration of plant IAA metabolism. To isolate bacterial strains exhibiting significant indole-3-acetyl-aspartate (IAA-Asp) hydrolase activity, a sewage sludge inoculation was cultured under conditions in which IAA-Asp served as the sole source of carbon and nitrogen. One isolate, fnterobacfer agglomerans, showed hydrolase activity inducible by I A A -L -A s~ or N-acetyl-i-Asp but not by IAA, (NH,),SO,, urea, or indoleacetamide. Among a total of 1 7 IAA conjugates tested as potential substrates, the enzyme had an exclusively high substrate specificity for IAA-i-Asp. Substrate concentration curves and Lineweaver-Burk plots of the kinetic data showed a Michaelis constant value for IAA-i-Asp of 13.5 mM. The optimal p H for this enzyme was between 8.0 and 8.5. I n extraction buffer containing 0.8 mM Mg2+ the hydrolase activity was inhibited t o 80% by 1 mM dithiothreitol and to 60% by 1 mm CuSO,; the activity was increased by 40% with 1 mM MnSO,. However, in extraction buffer with no trace elements, the hydrolase activity was inhibited to 50% by either 1 mM dithiothreitol or 1 % Triton X-100 (Sigma). These results suggest that disulfide bonding might be essential for enzyme activity. Purification of the hydrolase by hydroxyapatite and TSKphenyl (HP-Cenenchem, South San Francisco, CA) preparative high-performance liquid chromatography yielded a major 45-kD polypeptide as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
A 5.5-kb DNA fragment containing the indole-3-acetyl-aspartic acid (IAA-asp) hydrolase gene (iaaspH) was isolated from Enterobacter agglomerans strain GK12 using a hybridization probe based on the N-terminal amino acid sequence of the protein. The DNA sequence of a 2.4-kb region of this fragment was determined and revealed a 1311-nucleotide ORF large enough to encode the 45-kDa IAA-asp hydrolase. A 1.5-kb DNA fragment containing iaaspH was subcloned into the Escherichia coli expression plasmid pTTQ8 to yield plasmid pJCC2. Extracts of IPTG-induced E. coli cultures containing the pJCC2 recombinant plasmid showed IAA-asp hydrolase levels 5 to 10-fold higher than those in E. agglomerans extracts. Homology searches revealed that the IAA-asp hydrolase was similar to a variety of amidohydrolases. In addition, IAA-asp hydrolase showed 70% sequence identity to a putative thermostable carboxypeptidase of E. coli.
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