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Both flavonols and proanthocyanidins (PAs) are widespread secondary metabolites in plant kingdom and are found in vegetables and fruits (Pietta et al., 2000). Based on their pharmaceutical properties flavonols and PAs assume important function in plants but also in human organism (Ross et al., 2002; Thompson et al., 1976; Pietta et al., 2002; Bagchi et al., 2000; Middelton et al., 2000; Pagonis et al., 1986; Harborne et al., 2000; Lee et al., 2006). This work presents the analysis of flavonolsynthesis in Arabidopsis thaliana and PA-synthesis in Fragaria x ananassa.
Plant extracts of three genotypes, wildtype (Col-0), FLS1-single mutant (fls1-2m) and ANS/FLS1-double mutant (ldox/fls1-2 m), contained flavonols, albeit at greatly different levels. This can be explained by residual FLS activity contributed by either one or more of the FLS2-6 polypeptides or by ANS side activity (Pelletier et al., 1999; Owens et al., 2008; Stracke et al., 2009). Arabidopsis FLS4 and FLS6 were recently identified as pseudogenes or non-functional copies. Therefore, FLS1, FLS2, FLS3, FLS5 and ANS were cloned and expressed in bacterial and yeast cells. The expression of all polypeptides was confirmed by Western blot analysis using FLS antiserum. The FLS activity of the recombinant polypetides was examined in standard assay. Only FLS1 and ANS converted dihydroflavonols to flavonols (Owens et al., 2008; Preuss et al., 2009). Sequence alignment with FLS polypeptides and ANS showed that FLS1, FLS3 and ANS contained the conserved iron, 2-oxoglutarate and substrate binding sites. Homology models of FLS1 and FLS3 should confirm the results of sequence analysis. Models of the ANS-NAR (2rbt) and ANS-DHQ complexes were employed as template for homology modeling of FLS1 and FLS3 (Wilmouth et al., 2002). FLS3 differs from FLS1 by replacement of 136Leu through 136Tyr and this residue could change the orientation of nearby 149His involved in substrate binding. This could impact the FLS activity of FLS3 in vitro. The activity of FLS3 was therefore re-examined through extended bioconversion studies using the respective bacterial transformant. The conversion of dihydroflavonols to flavonols was demonstrated by TLC and HPLC co-chromatographie. The results suggest that FLS3 should be annotated as a second functional gene in A. thaliana. Furthermore, FLS3 is responsible for the formation of residual flavonols in ANS/FLS1-double mutant line.
The recombinant proteins FHT, FLS, DFR, ANS, LAR, ANR and FGT from Fragaria x ananassa were expressed in E. coli and S. cerevisiae. Cell extracts preparations were obtained from transformed bacterial (FGT) and yeast cultures after induction of protein expression. The activity of the recombinant polypetides was examined in standard assay with 4-hydroxylated and 3,4-hydroxylated flavonoids as substrates. In particular, 4-hydroxylated flavonoid were the preferred subtrates of FHT, FLS, DFR and ANS. In contrast LAR and ANR showed higher activity on 3,4-hydroxylated flavonoids. A strict substrate specificity was found only for LAR. FGT demonstrated a strong 3-O-glucosyltransferase activity on both tested anthocyanidins. Furthermore, enzyme activity was studied in fruit of six cultivated strawberry genotypes grown at two Italien locations with five growth stages. Enzyme activity of CHS/CHI, FHT, DFR, LAR, ANR and FGT throughout ripening were quantified, but ANR showed in all genotypes only from G1 to W stages activity. In order to study gene-enzyme relationship for each genotype, a Pearson correlation analysis was carried out between the corresponding CHS, CHI, FHT, DFR, LAR, ANR and FGT enzyme activity and transcript data. Based on average transcript levels and enzyme activity from all genotypes, three patterns of gene expression and enzyme activity could be identified: ‘two phases’ CHS, CHI, FHT, LAR and ANS genes and enzymes showing a first peak (transcrpit level and activity) at G1 stage and a second peak at T or R stage; ‘one phase’ DFR and FGT genes and enzyme activity up-regulated at T or R stage; ‘one phase’ F3H, FLS and ANR gene and enzymes, the transcript levels and activity diminished throughout fruit ripening. The observed correlation patterns concerning regulation of the flavonoid metabolism in strawberry fruits results possibly from post-transcriptional control and other mechanisms.