Browsing by Subject "Flavonoide"

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    Einfluss von erhöhtem atmosphärischen CO2 auf die N2-fixierende Symbiose von Trifolium repens L. und Rhizobium leguminosarum biovar trifolii
    (2007) Stöber, Sara; von Wirén, Nicolaus
    CO2 is one of the main greenhouse gases strongly influencing the climate and the terrestrial ecosystem. Up to know little is known about the impact of elevated atmospheric CO2 on symbiotic interactions in the rhizosphere, especially on the N2-fixing symbiosis between Trifolium repens and Rhizobium leguminosarum biovar trifolii. First results of a ten-year Free-Air CO2 enrichment experiment (Swiss FACE) showed that after three years of CO2 fumigation the genetic composition of the Rhizobium population in the root nodules of T. repens had changed. The first part of this thesis set out to clarify the question whether a genetic difference in the Rhizobium population of root nodules of white clover could still be detected after ten years of CO2 fumigation or if an adaptation of the nodule bacteria to elevated CO2 concentrations had occurred. Furthermore the thesis addressed the question whether elevated atmospheric CO2 leads to quantitative and qualitative changes in the root exudation of T. repens particularly with regard to exudation of signal substances during the nodulation process. In summer 2002 white clover plants were collected from plots fumigated with CO2 and control plots of the Swiss FACE. Rhizobium strains were isolated from the clover root nodules and used for rep-PCR DNA fingerprinting. Results clearly showed that after ten years of CO2 enrichment changes in the genetic composition of the R. l. bv. trifolii could no longer be observed. Thus, CO2-induced changes in the population structure of rhizobia seemed to be transient. This can be traced back to the possibility that over the experimental period a new C/N equilibrium in the grassland ecosystem has been established. At the beginning of the FACE experiment an increase in the C/N ratio of the soil was detected, which could be balanced in the course of time through enhanced symbiotic N2 fixation and consequently a higher N input into the ecosystem. The observed stabilisation of the grassland ecosystem most likely caused a reduction of the indirect CO2 impact on the microorganisms. This might explain why a change in the genetic composition of Rhizobium strains was not longer detected after ten years in the Swiss FACE. To investigate an influence of elevated atmospheric CO2 concentration on the release of signalling compounds clover plants were cultivated hydroponically in two independent climate chamber trials under axenic and non-axenic conditions at ambient and elevated CO2 concentrations (400 and 800 ppm) and different levels of N supply. Root exudates were collected over a period of seven hours and at three and four different plant ages, respectively. Phenolic compounds were extracted by solid-phase extraction and afterwards analysed with HPLC and LC-MS. Additionally, the isolated fractions were tested for their ability to induce the nodulation genes of R. l. bv. trifolii using a nod-gene induction test. The CO2 enrichment caused an increase in shoot and root growth in both experimental setups, but did not provoke a change in the C/N ratio of the roots. Besides the known signal compound 7,4?-dihydroxyflavone new phenolic substances could be detected, which have not yet been described in literature. The fractions were identified by their polarity, light absorption and molecular weight as aglyca and flavones. All of these had the ability for nod-gene induction except one fraction (fraction 2). CO2 influenced the exudation of signalling compounds quantitatively but not qualitatively. The enhanced exudation, especially of 7,4?-dihydroxyflavone, could be attributed to the higher root mass under elevated CO2 but also to a higher release rate on a root fresh weight basis. The CO2 reaction of the clover plants, for the biomass production as well as for the root exudation, was clearly dependent on the N supply and only significant under axenic conditions. In individual cases the N impact was more pronounced than the CO2 effect: with increasing N demand axenic clover plants enhanced the exudation of the nod-gene inducing fraction C. It is concluded that this fraction, identified as a hydroxyflavone, has therefore an important signal function under N limitation. Besides the CO2 concentration and N supply, root exudation by T. repens was considerably influenced by the plant age, which caused a reduction of the signal exudation in older plants and qualitative changes of the released phenols, especially under non-axenic conditions. The present study suggests that the genetic shift of R. l. bv. trifolii detected at the beginning of the Swiss FACE experiment was most likely a consequence of the enhanced exudation of phenolic signal compounds of T. repens under elevated atmospheric CO2 concentrations.
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    Metabolic engineering of flavonoid biosynthesis in hop (Humulus lupulus L.) for enhancing the production of pharmaceutically active secondary metabolites
    (2012) Gatica Arias, Andres Mauricio; Weber, Gerd
    For a long time, hop (Humulus lupulus L.) has been used in the brewing industry as flavoring and preserving agent. Moreover, the hop plant has been used for medicinal purposes. Recently, xanthohumol and desmethylxanthohumol have received special attention due to their potential cancer chemopreventive properties. Hop breeding programs have been mainly focused on the development of new cultivars with a high content of α- and β-acids in order to satisfy the demand of the brewing industry. However, due to the medical and pharmaceutical importance of hops, new breeding efforts have been done to create new cultivars with a higher content of xanthohumol and desmethylxanthohumol. In order, to complement these efforts, metabolic engineering of flavonoid biosynthesis offers tremendous potential to modify the production of these compounds. The flavonoid biosynthetic pathway has been intensively studied in plants. Often single target genes are regulated by several transcription factors. The R2R3 MYB transcription factor family plays an important role in the regulation of the biosynthesis of phenylpropanoids and flavonoids. Ectopic expression of these transcription factors in transgenic plants stimulated the production and enhanced the quantity of flavonoids. The main objective of the research presented here was to modulate the production of pharmaceutical metabolites in hop through metabolic engineering of the flavonoid biosynthesis pathway. Towards this goal, in a first approach, genetic engineering of hop cv. Tettnanger with the heterologous transcription factor PAP1/AtMYB75 from Arabidopsis thaliana L. was successfully accomplished. It was shown that PAP1/AtMYB75 was stably incorporated and expressed in the hop genome. The transgenic events showed reddish to pink female flowers and cones. Moreover, compared to the wildtype plants, the expression of the structural genes CHS_H1, CHI, and F3´H was elevated in transgenic hop plants. In addition, the production of anthocyanins, rutin, isoquercitin, kaempferol-7-O-glucoside, kaempferol-7-O-glucoside-malonate, desmethylxanthohumol, xanthohumol, α-acids, and β-acids in transgenic hop plants was influenced by the PAP1/AtMYB75 transcription factor. In a second approach, the homologous transcription factor HlMYB3 from H. lupulus L. was genetically introduced and expressed in the hop genome. The effect of the over-expression of the transgene on the expression rate of structural flavonoid and phloroglucinol biosynthetic genes, like PAL, C4H, 4CL, CHS_H1, CHI, F3H, F3´H, FLS, F3´5´H, OMT1, HlPT1, and VPS was examined. Transgenic events with an elevated expression of genes of flavonoid and phloroglucinol biosynthesis were identified. For quite some time successful plant tissue culture and Agrobacterium tumefaciens-mediated transformation procedures are available to genetically modify hop. However, these procedures are characterized by the low regeneration and transformation rates. Moreover, A. tumefaciens-mediated transformation is a laborious and time consuming process. For that reason, in order to evaluate further homologous or heterologous transcription factors with respect to the regulation of flavonoid and phloroglucinol biosynthesis in hop was highly desirable to have a simple and fast transformation system. A. rhizogenes-mediated transformation represents an alternative to express genes in hairy roots. Therefore, hop explants were genetically transformed with A. rhizogenes strains K599 and 15834. Hairy roots were only induced by A. rhizogenes 15834. The transgenity of the obtained hairy roots was confirmed by histochemical GUS assay. The integration of rolC and mgfp5 genes in transgenic hairy roots was confirmed by PCR. Particle bombardment combined with the regeneration of plants in temporary immersion bioreactors could provide another alternative for hop genetic transformation. In this study, a protocol for the micropropagation and shoot induction from organogenic calli of hop cv. Tettnanger using the temporary immersion bioreactors was developed. Furthermore, a procedure for particle bombardment was established using the following parameters: helium pressure of 900 psi, and target distance of 6 cm. The importance of hop relies on the secondary metabolites contain in the lupulin glands of the female cones. The present thesis demonstrated that the production of these secondary metabolites in transgenic hop plants could be influenced and enhanced by the expression of homologous or heterologous transcription factors. Moreover, the new developed transformation methods open the possibility for evaluating further genes that might influence the composition of secondary metabolites in the lupulin glands of hop.