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Transgenic apple plants overexpressing the - gene of show increased levels of 3-hydroxyphloridzin and reduced susceptibility to apple scab and fire blight
Full text linkMain conclusion
Overexpression of chalcone-3-hydroxylase provokes increased accumulation of 3-hydroxyphloridzin inMalus. Decreased flavonoid concentrations but unchanged flavonoid class composition were observed. The increased 3-hydroxyphlorizin contents correlate well with reduced susceptibility to fire blight and scab.
The involvement of dihydrochalcones in the apple defence mechanism against pathogens is discussed but unknown biosynthetic steps in their formation hamper studies on their physiological relevance. The formation of 3-hydroxyphloretin is one of the gaps in the pathway. Polyphenol oxidases and cytochrome P450 dependent enzymes could be involved. Hydroxylation of phloretin in position 3 has high similarity to the B-ring hydroxylation of flavonoids catalysed by the well-known flavonoid 3′-hydroxylase (F3′H). Using recombinant F3′H and chalcone 3-hydroxylase (CH3H) from Cosmos sulphureus we show that F3′H and CH3H accept phloretin to some extent but higher conversion rates are obtained with CH3H. To test whether CH3H catalyzes the hydroxylation of dihydrochalcones in planta and if this could be of physiological relevance, we created transgenic apple trees harbouring CH3H from C. sulphureus. The three transgenic lines obtained showed lower polyphenol concentrations but no shift between the main polyphenol classes dihydrochalcones, flavonols, hydroxycinnamic acids and flavan 3-ols. Increase of 3-hydroxyphloridzin within the dihydrochalcones and of epicatechin/catechin within soluble flavan 3-ols were observed. Decreased activity of dihydroflavonol 4-reductase and chalcone synthase/chalcone isomerase could partially explain the lower polyphenol concentrations. In comparison to the parent line, the transgenic CH3H-lines showed a lower disease susceptibility to fire blight and apple scab that correlated with the increased 3-hydroxyphlorizin contents
Untersuchungen von bioaktiven Substanzen, die mittels konventioneller und innovativen Extraktionstechniken gewonnen wurden
Full text linkKumulative Dissertation aus acht ArtikelnNatural products are the most important precursors for drug production worldwide, and one of the biggest challenges of our generation is their sustainable production. For that purpose,various biological sources (flowers, leaves, fruits, herbs, stems, roots, algae) of secondary metabolites were investigated concerning different aspects: (i) Modern and conventional extraction methods as well as the development of new extraction strategies (ii) Investigation of agricultural waste (iii) Characterisation and analysis of secondary metabolites and their respective properties.A focus was placed on the investigation of hemp (Cannabis sativa L.). Therefore, a novel extraction process for cannabinoids based on ionic liquids and supercritical CO2(scCO2) was developed. Additionally, in the frame of the doctoral college Bioactive,cannabinoid yields obtained by pressurised liquid extraction (PLE) from hemp threshingresidues were analysed. Ultimately, the lesser exploited hemp roots were investigated fortriterpenoids and their respective antioxidative capacity and extracted by scCO2.Another major part of this thesis was dedicated to the investigation of polyphenols.By using has kap berries (Lonicera caerulea L.) as model plant, a new extraction process based on scCO2 and ethanol was developed to harvest anthocyanins, iridoids and fatty acids.Furthermore, the enzyme mix snailase was utilised to hydrolyse flavonoid glycosides from different plant extracts, and the hydrolysis efficiency was evaluated. Finally, grapevine (Vitisvinifera L.) shoot residue extracts, obtained by PLE, were investigated on polyphenols andtheir respective antioxidant power for a biorefinery concept in the framework of Bioactive.Carotenoids were the focus of two further cooperations in the frame between the two doctoral colleges Bioactive and Flower Power. To shed light on the colouration of burmarigold (Bidens ferulifolia) flowers, carotenoids were obtained by ultrasound-assisted extraction, identified and analysed as well as compared with polyphenols for a better understanding of the pigmentation. In addition, cultivated glacial algae (Tetraedronminimum) was analysed on carotenoids and their respective antioxidant capacity, among other parameters
Secondary metabolites as quality factor for horticultural products
No full textSecondary metabolites play a major role as quality factors in horticultural products by significantly contributing to plant health, optical attributes, sensory attributes and health beneficial effects for the consumer. This thesis concentrated on dihydrochalcones, a rare class of secondary metabolites, which is, however present in particularly high amounts in apple (Malus × domestica), one of the major cash-cows of global fruit production. Dihydrochalcones are assumed to be particularly relevant for both plant health and health beneficial effects for the consumer. In apple, phloridzin (phloretin 2'-O-glucoside) is prevalent representing more than 90% of the soluble phenolic compounds, but small amounts of 3-hydroxyphloretin and 3-hydroxyphloridzin are also constitutively present. An involvement of the latter in defense against plant pathogens was suggested but not unambiguously demonstrated so far. Despite of the large amounts of dihydrochalcones present in apple, their physiological function in planta still remains a puzzle. For systematic investigation, sufficiently comprehensive knowledge of the underlying pathway is yet lacking. Whereas dihydrochalcone biosynthesis has been studied during the past few years, the introduction of a hydroxyl group in position 3 of dihydrochalcones was investigated in this thesis for the first time. Two types of enzymes could catalyse the reaction, polyphenol oxidases (PPOs) and/or cytochrome P450 dependent monooxygenases. 3-Hydroxyphloridzin and the corresponding aglycone 3-hydroxyphloretin occur as intermediates in the oxidation of phloridzin and phloretin by unspecific PPOs, if intact cells are damaged and the enzymes and dihydrochalcones are released from different cell compartments. It seems however unlikely that unspecific enzymes which produce a spectrum of cell-toxic compounds should be involved in the biosynthesis of constitutive 3-hydroxyphloridzin present in Malus sp. Hydroxylation in position 3 of dihydrochalcones shows high similarity to the introduction of a second hydroxyl group in the B-ring of flavonoids and chalcones which are catalysed by the cytochrome P450 dependent monooxygenases flavonoid 3'-hydroxylase (F3'H) and chalcone 3-hydroxylase (CH3H). F3´Hs usually show a broad substrate specificity, whereas CH3Hs specifically hydroxylate chalcones. Chalkones clearly show structural relation to dihydrochalcones as both are lacking the heterocyclic C ring of flavonoids. To investigate a potential involvement of CH3H and F3'H in 3-hydroxylation of dihydrochalcones, their substrate specificity was tested in vitro and in planta. Recombinant enzymes obtained by heterologous expression in yeast were able to convert dihydrochalcones to some extent, and higher conversion rates were observed with CH3H than with F3'H. The respective natural substrates were, however, much better accepted. To test whether CH3H catalyzes the hydroxylation of dihydrochalcones also in planta and if this could be of physiological relevance, we created transgenic apple trees harbouring CH3H from Cosmos sulphureus under the control of the CaMV 35S promotor in cooperation with the Institute of Fruit Breeding at the Julius Kühn Institut Dresden-Pillnitz. The three independent transgenic lines obtained showed an increase of 3-hydroxyphloridzin within the dihydrochalcones thereby unambiguously demonstrating that CH3H can mediate 3-hydroxylation of dihydrochalcones in planta. Increased levels of 3-hydroxyphloridzin correlated with reduced susceptibility to fire blight and apple scab, which are the main bacterial and fungal diseases of apple. To test a possible involvement of the F3'H of apple in the formation of constitutive 3-hydroxydihydrochalcones, cDNA clones of the two types of F3'Hs present in apple was isolated and heterologously expressed in yeast. Despite showing high F3'H activity with various flavonoid substrates, hydroxylation of dihydrochalcones was not observed at standard conditions, indicating that F3'H is not part of the dihydrochalcone pathway. A final clarification will require silencing of the Malus F3'Hs, as previous results have shown that substrate conversion in planta can occur under suitable physiological conditions, even if substrate specificity of the involved enzyme is very low. The findings from the present work will feed into new strategies for the biotechnological production of dihydrochalcones derivatives as well as to innovative plant protection approaches
Züchtung von Weihnachtssternen mit neuer Blütenfarbe mittels Genome editing
Full text linkPoinsettia (Euphorbia pulcherrima) commonly shows red bract colouration caused by theaccumulation of cyanidin-type anthocyanins. Orange-red cultivars, which accumulatepelargonidin-type anthocyanins occur rarely, whereas blue poinsettias are not naturally presentdue to the lack of delphinidin formation. Breeding is an arduous process, and a lot of effort isnecessary to obtain plants with desired traits. Some phenotypes are not possible to achieve byclassical breeding approaches due to limitations in the genetic background of the plants.Molecular breeding techniques like genome editing can help to overcome those obstacles andobtain novel poinsettia varieties with orange or blue colour in a fast and efficient way. Thecolour of anthocyanin pigments depends on the B-ring hydroxylation pattern and ranges fromorange pelargonidin (one hydroxyl group), dark red to pink cyanidin (two hydroxyl groups)and blue to violet delphinidin (three hydroxyl groups). Two factors are crucial for theestablishment of the B-ring hydroxylation pattern. First, the presence or absence of flavonoid3’-hydroxylase (F3’H) and flavonoid 3’5’-hydroxylase (F3’5’H) activity, and second, thesubstrate specificity of dihydroflavonol 4-reductase (DFR), which provides importantintermediates in the formation of anthocyanins. In this thesis, the physiological background ofcolour formation was studied in petunia as a model plant and in red, and orange poinsettiacultivars. Based on this, a strategy for molecular breeding of orange and blue poinsettia wasestablished. The investigations on the molecular background of pigment formation in orang
Biochemische und molekularbiologische Untersuchungender Chalkon 3-hydroxylase
No full textThe recently discovered cytochrome P450 depended enzyme CH3H catalyses the hydroxylation in position 3 of the B ring of chalcones. This reaction is similar to the hydroxylation of flavonoids at position 3' but cannot be catalysed by the well-known F3-H despite the broad substrate specificity of this enzyme. The objective of this work was the construction of vectors carrying F3-H and CH3H cDNA clones for the production of Strep-tagged fusion proteins, comparison of the sequences to identify putative CH3H specific sites, Mutation of the cDNA clones and testing the substrate specificity of the resulting fusion proteins and identification of specific regions in the amino acid sequence of CH3H determining the substrate specificity. Strep-tagged fusion proteins were produced from CH3H of Cosmos sulphureus, Bidens ferulifolia and Coreopsis grandiflora and F3-H of Bidens ferulifolia and Dahlia variabilis for a better purification of the recombinant enzymes. Mutations were performed in CH3Hs and F3-H of Bidens ferulifolia, Cosmos sulphureus, Dahlia variabilis and putative CH3H of Coreopsis grandiflora. The mutations at position 104 in SRS1 and 219 in SRS2 in Cosmos CH3H had characteristic influence on substrate specificity. Changes in these positions converted CH3H into F3-H. The selected amino acids for mutation in Bidens CH3H and F3-H did not have any influence on substrate specificity and all recombinant enzymes were still CH3H-s. After mutation of the inactive, putative CH3H of Coreopsis catalytic activity was observed for the first time. The mutants 53Q and 104A in SRS 1 and 211M in SRS 2 seem to be authentic CH3Hs
Biosynthese der anthochloren Pigmente und ihre biotechnologische Verwendung
Full text linkAnthochlor pigments are a group of secondary plant metabolites that, together with carotenoids and some flavonoids, produce yellow flower colour. Both, chalcones and aurones are collectively known as anthochlor pigments and serve as UV nectar guides in some plants. Especially Asteraceae accumulate two types of anthochlor pigments: on the one hand, the hydroxy types of chalcones and aurones, and on the other hand the deoxy types of chalcones and their corresponding aurones. Both types differ only in the presence or absence of a hydroxyl group in the 6’ position of the B-ring (chalcones) or the 4 position of the A-ring (aurones). Thus, hydroxychalcones do not readily accumulate in plants due to quick izomerization. Hence, our strategies in establishing yellow colouration involve the introduction of genes encoding proteins that lead to the accumulation of deoxy type anthochlors. Three chapters make up this thesis: 1.) The biochemical basis of pigmentation of different coloured Bidens ferulifolia cultivars was unravelled. The main aspects were the pigment composition and enzyme activity assays of involved proteins. The prevalent pigment in yellow cultivars was the 6’-deoxychalcone okanin and its derivates, whereas red cultivars accumulated cyanidin. Furthermore, we concluded that missing FHT and/or DFR activity created a bottleneck in anthocyanin biosynthesis. This aspect may be highly interesting for breeders of Bidens ferulifolia. 2.) Genetic and biochemical analysis of the bicoloured white-yellow Dahlia variabilis ‘Seattle’. From three putative CHRs, none were differentially expressed. Additionally, a strong difference in CHS2 expression between yellow and white petal parts may explain the bicolouration. Nonetheless, the transcriptomes that were built during the work can form the basis for further research and consolidate Dahlia variabilis ‘Seattle’ as a model plant for anthochlor research. 3.) Within this thesis, genes involved in anthochlor biosynthesis, derived from different members of the Asteraceae species, were cloned in large multigene constructs, utilizing the GoldenBraid vector system. These multigene constructs were introduced in model and target plants via transient and stable transformation, utilizing different transformation methods. We successfully created yellow petals with transient expression of a legume CHR and a glycosyltransferase in petals of Pelargonium zonale. Though the presence of transcripts was confirmed, HPLC analysis did not show traces of the expected 6’deoxychalcone isoliquiritigenin. Stable transformation of Pelargonium zonale and Petunia hybrida struggled with problems during regeneration. Only few regenerants that lacked a stable integration of the transgenes could be obtained. Other approaches to elucidate the contribution of the single genes of the anthochlor pathway included the design of constructs of the novel CRISPR/Cas9 system and virus-induced gene silencing (VIGS) methods, with the same results as the stable integration of genes.Die Anthochloren Pigmente sind eine Gruppe sekundärer Pflanzenmetabolite und tragen gemeinsam mit Carotenoiden und einigen Flavonoiden zur gelben Blütenfärbung bei. Chalcone und Aurone gemeinsam bilden hierbei die Klasse der Anthochlore und dienen als Blütenmal für UV-sensitive Insekten. Insbesondere Asteraceen, akkumulieren zwei Typen an Anthochloren. Zum einen die Hydroxy-Typen von Chalconen und Auronen, zum anderen die Deoxy-Typen von Chalconen und Auronen. Beide Typen unterscheiden sich lediglich im Vorhandensein einer Hydroxy-Gruppe an der Position 6‘ des B-Rings (Chalcone), bzw. an der Position 4 des A-Rings bei Auronen. Hydroxychalcone akkumulieren sich nicht in Pflanzen, da sie schnell isomerisieren. Daher entschieden wir uns für eine Strategie zur Schaffung gelber Blütenfarben, Gene einzuführen, welche Proteine kodieren, die an der Synthese von Anthochloren des Deoxytyps beteiligt sind.Die Arbeit umfasst drei große Themenbereiche: 1.) Die biochemische Basis der Pigmentation verschieden farbiger Kultivare von Bidens ferulifolia wurde untersucht. Der Fokus hierbei lag auf der Ergründung der Pigmentkomposition wie auch der Untersuchungen der Aktivität beteiligter Enzyme. Die vorherrschenden Pigmente waren in den gelben Kultivaren 6‘-Deoxychalcone, in roten Blütenteilen Cyanidin. Desweiteren wurde festgestellt, dass teilweise fehlende FHT und/oder DFR Aktivität zu einem Flaschenhals der Anthocyanbiosynthese führte. Dieser Aspekt kann sich als höchst interessant für Bidens-Züchter erweisen. 2.) Genetische und biochemische Analysen der zweifarbigen, weiß-gelben Dahlia variabilis ‚Seattle‘.Eine Erklärung für die Zweifarbigkeit fand sich unter anderem in einer stark reduzierten Expression von CHS2. Nichtsdestotrotz bilden die im Rahmen der Arbeit gebildeteten Transkriptome eine gute Basis für weitere Untersuchung und sprechen für die Verwendung der Dahlia variabilis ‚Seattle‘ als Modellpflanze für die Forschung an Anthochloren. 3.) Im Rahmen dieser Arbeit wurden Gene, die an der Synthese von Anthochloren des Deoxytyps beteiligt sind, aus diversen Mitgliedern der Asteraceen-Familie isoliert und in große Multigen-Konstrukte des GoldenBraid-Systems geklont. Diese Multigen-Konstrukte wurden mittels verschiedener Transformationsmethoden sowohl stabil als auch transient in verschiedenen Ziel- und Modellpflanzen eingebracht. Die daraus resultierenden Pflanzen wurden sowohl auf RNA-, DNA- und Metabolit-Ebene auf das Vorhandensein der Gene und ihrer Produkte untersucht. Es gelang die Erschaffung von einzelnen gelben Petalen bei der transienten Expression von CHR mit einer Glykosyltransferase in Pelargonium zonale. Obwohl Transkripte durch PCR bestätigt wurden, zeigten HPLC Untersuchungen jedoch keinerlei Vorhandensein von dem zu erwarteten Pigment Isoliquiritigenin. Die stabile Transformation von Pelargonium zonale und Petunia hybrida führte zu nur wenige Stecklingen, welche jedoch keinen stabilen Einbau der Transgene aufwiesen
Site directed mutagenesis for the identification of sequence differences between chalcone 3-hydroxylase and flavonoid 3'-hydroxylase
No full textChalkon 3-hydroxylase und Flavonoid 3'-hydroxylase sind Cytochrom P450 abhängige Monooxygenasen, die im B-Ring von Chalkonen und/oder verschiedenen Flavonoidklassen eine zusätzliche Hydroxylgruppe an der Position 4 (Chalkone) bzw. 4' (Flavonoide) zu der bereits vorhandenen OH-Gruppe einführen. Während die F3'H keinen Umsatz mit Chalkonen zeigen weist die CH3H eine breitere Substratakzeptanz auf, da sie sowohl Chalkone als auch Flavonoide umsetzt. Im Rahmen dieser Arbeit wurden sieben cDNÄKlone aus Bidens ferulifolia, Cosmos sulphureus, Dahlia variabilis und Coreopsis grandiflora bearbeitet. Bei den drei erstgenannten Pflanzen waren Paare aus CH3H und F3'H vorhanden, während bei Coreopsis grandiflora nur eine Aminosäuresequenz unbekannter Substratspezifität vorlag. Fünf der sieben verfügbaren cDNA Klone lagen bereits in einem Plasmid vor, der einen C-terminalen Strep-Tag aus acht Aminosäuren enthielt. Die cDNA Klone CH3H aus D. variabilis sowie F3'H aus C. sulphureus mussten erst mit dem Strep-Tag versehen werden. Sequenzunterschiede zwischen den Proteinen wurden durch zielgerichtete Mutagenese auf ihren Einfluss auf die Substratakzeptanz bzw. -spezifität abezüglich Isoliquiritigenin, Apigenin Dihydrokämpferol, Kämpferol und Naringenin untersucht. Im Rahmen der Diplomarbeit wur- den bestimmte Positionen in den Aminosäuresequenzen als potenziell relevant für die Funktionalität der Enzyme identifiziert. Insbesondere die Modifikation an der Position 136 verursachte eine erhebliche Änderung der Aktivität bei drei der untersuchten Enzyme (C. sulphureus CH3H und F3'H sowie C. grandiflora). Wie aus der Untersuchung der Sequenzen hervorging, enthielten alle Enzyme mit Ausnahme des aus C. grandiflora isolierten an Stelle 136 Prolin während sich in letzterem ein Serin an der genannten Position befand. Durch diesen gezielten Austausch bei C. grandiflora konnte ein mit nur geringem Naringenin-Umsatz fast inaktives Enzym in ein funktionelles aktives Enzym umgewandelt werden. Der entgegengesetzte Austausch von Serin in Prolin an der Position 136 führte bei C. sulphureus CH3H und F3'H zu einem inaktiven Enzym. Andere untersuchte Positionen, die sich zum größten Teil in oder in der Nähe der verschiedenen Substraterkennungsstellen befinden, erwiesen sich als wenig einflussreich auf die Substratakzeptanz bzw. -spezifität.Chalcone 3-hydroxylase (CH3H) and flavonoid 3'-hydroxylase (F3-H) are cytochrome P450 dependent monooxygenases. Chalcones and/or other flavonoidclasses carry an extra hydroxyl group at position 4 (chalcone) or 4' (flavonoid) to the already existing hydroxyl group in ring B. While F3'H shows no conversion of chalcones, CH3H accepts a broad range of substrates, and hydroxylates both chalcones and flavonoids. In this thesis, seven cDNA clones from Bidens ferulifolia, Cosmos sulphureus, Dahlia variabilis and Coreopsis grandiflora were studied. Pairs of CH3H and F3'H from the first three plant species and a single F3-H cDNA clone from C. grandiflora were available. From the seven cDNA clones, five were already present in the pIBA51 vector conveying the C-terminal strep-tag of eight amino acids. The other two cDNA clones encoding CH3H D. variabilis and F3'H C. sulphureus were subcloned into the the pIBA51 vector. Sequence differences between the translated proteins putatively determining substrate specificity were analyzed by site-directed mutagenesis. The substrate specificity of resulting recombinant mutated proteins were tested with the five (14C)-labelled substrates isoliquiritigenin, apigenin, dihydrokaempferol, kaempferol and naringenin. It could be shown that substrate specificity is not determined by single locations but must be a result of the interaction of several amino acids in the N-terminal regions. Position 136 in the C. grandiflora amino acid sequence was identified as potentially relevant for the functional activity of this enzyme type. A highly conserved serine is present in all CH3Hs and F3-Hs, whereas the C.grandiflora F3-H showed a proline in this position. This results in an inactive enzyme as shown by site-directed mutagenesis. In this work the importance of the serine in position 136 was confirmed by introducing the proline in the CH3H and F3-H of Cosmos sulphureus. This triggered the opposite effect and resulted in functionally inactive enzymes
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