Molekularbiologische und biochemische Untersuchungen zu 2-Oxoglutarat abhängigen Dioxygenasen in Equisetum arvense L. und Petroselinum crispum (Mill.) Nyman & A.W. Hill

2-oxoglutarate-dependent dioxygenases (2-ODDs) involved in flavonoid biosynthesis (FNS I, FHT, FLS and ANS) were investigated in Equisetum arvense (horsetail) and Petroselinum crispum (parsley). E. arvense is a phylogenetically ancient genus with a large and well described diversity of flavonoids (Saleh et al., 1971; Veit et al., 1995). To date however, the flavonoid biosynthesis pathway in horsetail is still unknown. In the present study, activities of different 2-ODDs were detected in crude plant enzyme extracts. In addition to FHT- and FLS activities, a FNS I-type activity could be detected. This finding represents the first evidence of a FNS I-type-activity outside of the family of Apiaceae. The isolation of the corresponding enzyme via classical protein purification techniques failed due to the instability of the enzyme. The rapidly decreasing activity present in the crude extract could also not be enriched by affinity chromatography on 2-oxoglutarate bounded sepharose or metal-chelate affinity chromatography. Using a molecular biological approach, two 2-ODD sequences were successfully cloned by RT-PCR and cDNA-based approaches. They were expressed in heterologous recombinant systems. However, no enzyme activity could be detected with the tested flavonoid intermediate substrates, evolutionary analysis within the flavonoid biosynthesis pathway was not possible. As the overall identity of the cloned sequences was only 30% to other known ODDs, no hypothesis regarding the putative enzymatic function could be generated. Whereas the cloned sequences clearly code for 2-ODDs, no evidence could be obtained for their functional role in flavoinoid biosynthesis in E. arvense. Secondly, the flavonoid biosynthesis was studied in P. crispum. Upon drought stress, red pigments accumulate in stems of parley plants. These pigments were identified as anthocyanins. They were only detected in stressed plant tissues and were identified as glycosides of cyanidin and peonidin, respectively. The cDNA of the corresponding enzyme (ANS, anthocyanidin synthase) was successfully cloned via RT-PCR and RACE methods. Subsequent expression in yeast and bacteria allowed the detection of the well-described ANS side activity (conversion of dihydroflavonols to flavonols, Welford et al., 2001; Turnbull et al., 2004) by bioconversions in cultures of transformed bacteria. However the main ANS reaction, converting leucoanthocyanidins to anthocyanidins, could be detected neither in in vitro assays nor in bioconversions. To delineate ANS sequences from FLS sequences the cloned cDNA sequence was analyzed by sequence alignments and the corresponding polypeptide sequence was compared with different known polypeptide sequences of FLS and ANS other plants. Alignments of PcFLS and AtANS, combined with phylogenetic analysis as well as the analysis of intron positions in 2-ODDs confirmed the identity of the cloned cDNA as an ANS. Furthermore, semi-quantitative PCR verified that some genes (PAL, 4-CL, CHS, FHT, FNS I, FLS) expressed in unstressed plants were transcribed less or not at all in drought stressed parsley stems and leaves. The ANS transcript was detected equally in unstressed and stressed, while the transcript of the DFR gene was only detectable in drought stress. These results corroborated at expression level the de novo synthesis of anthocyanins in drought stressed parsley as detected by metabolite analysis.