Browsing by Subject "Biosynthesis"

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Molekulare und entwicklungsbiologische Charakterisierung von Schlüsselenzymen der Naturstoffbiosynthese in Drüsenhaaren der Sonnenblume
(2008) Göpfert, Jens C.; Spring, Otmar
Glandular trichomes from anther appendages of sunflower were collected and their RNA was isolated. Sequence comparison with known plant sesquiterpene synthases was used to identify sunflower synthases in RT-PCR reactions. Three enzymes, HaGAS1, HaGAS2 and HaCS with high similarities to already characterized sesquiterpene synthases were identified. Their nucleotide sequences were completely established on the genomic level and as RNA transcripts. The nucleotide sequences as well as the deduced amino acid sequences showed typical characteristics of terpene synthases. In order to characterize the enzymes, the sesquiterpene synthase genes were cloned and expressed in E. coli. In vitro assays with the recombinant enzymes were carried out using the native substrate farnesyldiphosphate. The resulting products were extracted and analysed by GC-MS. They were identified by comparison of data base MS-data and using reference samples under identical analytical conditions. Two expressed enzymes, HaGAS1 and HaGAS2, synthesized germacrene A as a single product. Heterologous in vivo expression of both germacrene A-synthases in S. cerevisiae confirmed the in vitro result, since the analysis of the synthesized product showed a single germacrene A peak. Due to a very low in vitro activity of HaCS, the products of the third synthase could not be directly determined by MS-analysis. Therefore, the enzyme was expressed as a thioredoxin-fusion protein in vivo in transgenic yeast. This attempt resulted in a much higher rate of product yield. Two main and at least six minor products were traced in GC-analysis. They were confirmed as sesquiterpene hydrocarbons by GC-MS analysis. One of the two main products was identified as gamma-cadinene, whereas the second main peak could not be determined conclusively. Among the minor compounds alpha-copaene, alpha-muurolene und beta-caryophyllene were identified. Screening of a H. annuus EST library (established at the Berkeley Center for Synthetic Biology, University of California, Berkeley, USA) from mRNA of trichomes revealed the presence of a cytochrome P450 protein which showed high similarity to an Artemisia annua enzyme involved in artemisinic acid biosynthesis. This enzyme and another similar protein from Lactuca sativa were cloned and coexpressed with the germacrene A-synthase HaGAS2 in yeast. The resulting product was indirectly determined as germacrene A carboxylic acid using GC-MS analysis. These novel cytochrome P450 enzymes from sunflower and lettuce can be characterized as multifunctional germacrene A-monooxygenases. They catalyse a three-step oxidation leading from germacrene A to germacrene A carboxylic acid. This oxidation process represents an essential step towards the biosynthesis of sesquiterpene lactones. Semiquantitative RT-PCR analysis demonstrated that the expression of all three sesquiterpene synthases and the sunflower P450 monooxygenase occurred directly within trichome cells. The expression was highly upregulated during the secretory stage of the capitate glandular trichomes. This developmentally regulated expression was shown for the first time in trichomes. Additionally to sesquiterpene synthase activity in trichomes of anthers and leaves, it also was detected in sunflower roots. In addition, 5-deoxynevadensin was identified as a new constituent of the glandular trichomes of sunflower. This 5-deoxy-flavone is responsible for the bright blue fluorescence of sunflower trichomes detected by fluorescence microscopy. The newly identified component may act as protectant for the STL against UV-degradation.