Publikationen 2009 - 2016


Transgenic apple plants overexpressing the chalcone 3-hydroxylase gene of Cosmos sulphureus show increased levels of 3-hydroxyphloridzin and reduced susceptibility to apple scab and fire blight.

Hutabarat OS, Flachowsky H, Regos I, Miosic S, Kaufmann C, Faramarzi S, Alam MZ, Gosch C, Peil A, Richter K, Hanke MV, Treutter D, Stich K, Halbwirth H.: Planta. 2016 May; 243(5):1213-24. doi: 10.1007/s00425-016-2475-9. Epub 2016 Feb 19.

MAIN CONCLUSION: Overexpression of chalcone-3-hydroxylase provokes increased accumulation of 3-hydroxyphloridzin in Malus . 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.



DNA and Flavonoids Leach out from Active Nuclei of Taxus and Tsuga after Extreme Climate Stresses.

Feucht W, Schmid M, Treutter D.: Plants (Basel). 2015 Sep 21;4(3):710-27. doi: 10.3390/plants4030710

Severe over-stresses of climate caused dramatic changes in the intracellular distribution of the flavonoids. This was studied in needles from the current year's growth of the following species and varieties: Tsuga canadensis, Taxus baccata, T. aurea, T. repens, T. nana, and T. compacta. The mode of steady changes in flavonoids was evaluated by microscopic techniques. Most of the flavonoids stain visibly yellow by themselves. The colorless flavanol subgroup can be stained blue by the DMACA reagent. In mid-summer 2013, outstanding high temperatures and intense photo-oxidative irradiation caused in a free-standing tree of Taxus baccata dramatic heat damage in a limited number of cells of the palisade layers. In these cells, the cytoplasm was burned brown. However, the nucleus maintained its healthy "blue" colored appearance which apparently was a result of antioxidant barrier effects by these flavanols. In late May 2014, excessive rainfall greatly affected all study trees. Collectively, in all study trees, a limited number of the mesophyll nuclei from the needless grown in 2013 and 2014 became overly turgid, enlarged in size and the flavanols leached outward through the damaged nuclear membranes. This diffusive stress event was followed one to three days later by a similar efflux of DNA. Such a complete dissolution of the nuclei in young tissues was the most spectacular phenomenon of the present study. As a common feature, leaching of both flavanols and DNA was markedly enhanced with increasing size and age of the cells. There is evidence that signalling flavonoids are sensitized to provide in nuclei and cytoplasm multiple mutual protective mechanisms. However, this well-orchestrated flavonoid system is broken down by extreme climate events.


The importance of the use of rootstocks with hypersensitivity resistance to PPV for the cultivation of hypersensitive European plum cultivars

Neumüller, M.; Hadersdorfer, J. ; Mühlberger, L.; Hartmann, W.; Treutter, D.: Acta horticulturae · January 2015 DOI: 10.17660/ActaHortic.2015.1063.10

The behavior of 6- to 10-year-old trees of two hypersensitive plum cultivars (‘Jojo’, ‘Hoh 4517’) grafted onto a PPV sensitive rootstock under natural PPV inoculation conditions was observed. Some of the rootstocks got infected with PPV. In case of ‘Jojo’, no effect on the phenotype of the crown part could be observed. In case of ‘Hoh 4517’, single young shoots showed necrosis on the young shoots. These lesions facilitated secondary infections with Pseudomonas bacteria which then led to the death of whole branches. In order to avoid these damages it is highly recommended to use rootstocks with hypersensitivity resistance for all scion cultivars with hypersensitivity resistance to PPV.



Phenolic contents in fruit juices of plums with different skin colors

Goldner, K.; Vio Michaelis, S.; Neumüller, M.; Treutter, D.: Journal of Applied Botany and Food Quality 88, 322 - 326 (2015), DOI:10.5073/JABFQ.2015.088.046

Polyphenols in fruits are of increasing interest for consumers and for plant scientists because of their health beneficial potential and their role in plant physiology and disease resistance. Anthocyanins contribute significantly to the attractive pigmentation of red and blue plums. Mirabelles and several reineclaudes do usually not accumulate anthocyanins in the skin. Is this linked to a general low phenolic level? Both the health aspect and the pigmentation are interesting traits for the breeder. For this purpose, rapid analytical methods are necessary. One time consuming step is the extraction of polyphenols. However, fruit juices are easily produced and are anyhow used for estimation of quality traits such as sugars and acidity. Here we show that HPLC analysis of plum juices represent the phenolic profiles of the whole fruits. We analysed the phenolic patterns of juices from 43 plum varieties with yellow, blue and dark blue fruit skins. In most cases, a weak red pigmentation co-occurs with a low total phenol level. However, there are exceptions that may help the breeder to combine yellow fruit skin with a high level of health beneficial phenolic compounds by using the appropriate donor genotypes. The method described here offers a valuable tool for selection.






Phenolic profiles in leaves of chicory cultivars (Cichorium intybus L.) as influenced by organic and mineral fertilizers.

Sinkovic L, Demšar L, Žnidarcic D, Vidrih R, Hribar J, Treutter D.: Food Chem. 2015 Jan 1;166:507-13. doi: 10.1016/j.foodchem.2014.06.024. Epub 2014 Jun 13

Chicory (Cichorium intybus L.) is a typical Mediterranean vegetable, and it shows great morphological diversity, including different leaf colours. Five cultivars commonly produced in Slovenia ('Treviso', 'Verona', 'Anivip', 'Castelfranco', 'Monivip') were grown in pots under controlled conditions in a glasshouse, with organic and/or mineral fertilizers administered to meet nitrogen requirements. HPLC analysis was carried out to study the phenolic compositions of the leaves. A total of 33 phenolic compounds were extracted from these chicory leaves and were quantitatively evaluated in an HPLC-DAD-based metabolomics study. Among the cultivars, the highest TPC was seen for 'Treviso' (300.1 mg/100 g FW), and the lowest, for 'Castelfranco' (124.9 mg/100g FW). Across the different treatments, the highest TPC was in the control samples (254.3 mg/100 g FW), and the lowest for the organic (128.6 mg/100 g FW) and mineral fertilizer (125.5 mg/100 g FW) treatments. The predominant phenolic compounds in all of the samples were hydroxycinnamic acids, including chlorogenic and cichoric acid. Fertilizer administration provides a discriminant classification of the chicory cultivars according to their phenolic compounds.



Transcriptomic analysis of Prunus domestica undergoing hypersensitive response to plum pox virus infection.

Rodamilans B, San León D, Mühlberger L, Candresse T, Neumüller M, Oliveros JC, García JA.: PLoS One. 2014 Jun 24;9(6):e100477. doi: 10.1371/journal.pone.0100477.

Plum pox virus (PPV) infects Prunus trees around the globe, posing serious fruit production problems and causing severe economic losses. One variety of Prunus domestica, named 'Jojo', develops a hypersensitive response to viral infection. Here we compared infected and non-infected samples using next-generation RNA sequencing to characterize the genetic complexity of the viral population in infected samples and to identify genes involved in development of the resistance response. Analysis of viral reads from the infected samples allowed reconstruction of a PPV-D consensus sequence. De novo reconstruction showed a second viral isolate of the PPV-Rec strain. RNA-seq analysis of PPV-infected 'Jojo' trees identified 2,234 and 786 unigenes that were significantly up- or downregulated, respectively (false discovery rate; FDR?0.01). Expression of genes associated with defense was generally enhanced, while expression of those related to photosynthesis was repressed. Of the total of 3,020 differentially expressed unigenes, 154 were characterized as potential resistance genes, 10 of which were included in the NBS-LRR type. Given their possible role in plant defense, we selected 75 additional unigenes as candidates for further study. The combination of next-generation sequencing and a Prunus variety that develops a hypersensitive response to PPV infection provided an opportunity to study the factors involved in this plant defense mechanism. Transcriptomic analysis presented an overview of the changes that occur during PPV infection as a whole, and identified candidates suitable for further functional characterization.



Utilization of real-time electrospray ionization mass spectrometry to gain further insight into the course of nucleotide degradation by intestinal alkaline phosphatase.

Kaufmann CM, Graßmann J, Treutter D, Letzel T.: Rapid Commun Mass Spectrom. 2014 Apr 30;28(8):869-78. doi: 10.1002/rcm.6855


Related with its ability to degrade nucleotides, intestinal alkaline phosphatase (iAP) is an important participant in intestinal pH regulation and inflammatory processes. However, its activity has been investigated mainly by using artificial non-nucleotide substrates to enable the utilization of conventional colorimetric methods. To capture the degradation of the physiological nucleotide substrate of the enzyme along with arising intermediates and the final product, the enzymatic assay was adapted to mass spectrometric detection. Therewith, the drawbacks associated with colorimetric methods could be overcome.


Enzymatic activity was comparatively investigated with a conventional colorimetric malachite green method and a single quadrupole mass spectrometer with an electrospray ionization source using the physiological nucleotide substrates ATP, ADP or AMP and three different pH-values in either methodological approach. By this means the enzymatic activity was assessed on the one hand by detecting the phosphate release spectrometrically at defined time points of enzymatic reaction or on the other by continuous monitoring with mass spectrometric detection.


Adaption of the enzymatic assay to mass spectrometric detection disclosed the entire course of all reaction components--substrate, intermediates and product--resulting from the degradation of substrate, thereby pointing out a stepwise removal of phosphate groups. By calculating enzymatic substrate conversion rates a distinctively slower degradation of AMP compared to ADP or ATP was revealed together with the finding of a substrate competition between ATP and ADP at alkaline pH.


The comparison of colorimetric and mass spectrometric methods to elucidate enzyme kinetics and specificity clearly underlines the advantages of mass spectrometric detection for the investigation of complex multi-component enzymatic assays. The entire course of enzymatic substrate degradation was revealed with different nucleotide substrates, thus allowing a specific monitoring of intestinal alkaline phosphatase activity.



Identification of phenolic compounds in plum fruits (Prunus salicina L. and Prunus domestica L.) by high-performance liquid chromatography/tandem mass spectrometry and characterization of varieties by quantitative phenolic fingerprints.

Jaiswal R, Karaköse H, Rühmann S, Goldner K, Neumüller M, Treutter D, Kuhnert N.: J Agric Food Chem. 2013 Dec 11;61(49):12020-31. doi: 10.1021/jf402288j. Epub 2013 Dec 2.

Plums (Prunus domestica L. and Prunus salicina L.) are edible fruits mostly consumed in America, China, and Europe. We have used LC-MS(n) to detect and characterize the phenolic compounds in plum varieties. Forty-one phenolics were detected comprising caffeoylquinic acids, feruloylquinic acid, p-coumaroylquinic acids, methyl caffeoylquinates, methyl p-coumaroylquinate, caffeoylshikimic acids, catechin, epicatechin, rutin, esculin, quercetin, quercetin-3-O-hexosides, dimeric proanthocyanidins, trimeric proanthocyanidins, caffeoyl-glucoside, feruloyl-glucoside, p-coumaroyl-glucoside, vanillic acid-glucosides, 3,4-dihydroxybenzoyl-glucoside, quercetin-3-O-pentosides, quercetin-3-O-rhamnoside, quercetin-pentoside-rhamnosides, and 3-p-methoxycinnamoylquinic acid. This is the first time when 3-p-methoxycinnamoylquinic acid is reported in nature. Chlorogenic acids and proanthocyanidins were the major phenolics present in plums. Furthermore, HPLC with DAD and chemical reaction detection was used to generate quantitative phenolic fingerprints from the fruit flesh of 33 plum varieties. The predominant compound was 3-caffeoylquinic acid in nearly all varieties studied; generally, however, the qualitative and quantitative profiles showed high diversity even among closely related progenies.


Erwinia amylovora affects the phenylpropanoid-flavonoid pathway in mature leaves of Pyrus communis cv. Conférence.

Vrancken K, Holtappels M, Schoofs H, Deckers T, Treutter D, Valcke R.: Plant Physiol Biochem. 2013 Nov;72:134-44. doi: 10.1016/j.plaphy.2013.03.010. Epub 2013 Mar 23.

Flavonoids, which are synthesized by the phenylpropanoid-flavonoid pathway, not only contribute to fruit colour and photoprotection, they also may provide antimicrobial and structural components during interaction with micro-organisms. A possible response of this pathway was assessed in both mature and immature leaves of shoots of 2-year-old pear trees cv. Conférence, which were inoculated with the gram-negative bacterium Erwinia amylovora strain SGB 225/12, were mock-inoculated or were left untreated. The phenylpropanoid-flavonoid pathway was analysed by histological studies, by gene expression using RT-qPCR and by HPLC analyses of the metabolites at different time intervals after infection. Transcription patterns of two key genes anthocyanidin reductase (ANR) and chalcone synthase (CHS) related to the phenylpropanoid-flavonoid pathway showed differences between control, mock-inoculated and E. amylovora-inoculated mature leaves, with the strongest reaction 48 h after inoculation. The impact of E. amylovora was also visualised in histological sections, and confirmed by HPLC, as epicatechin -which is produced via ANR- augmented 72 h after inoculation in infected leaf tissue. Besides the effect of treatments, ontogenesis-related differences were found as well. The increase of certain key genes, the rise in epicatechin and the visualisation in several histological sections in this study suggest a non-negligible impact on the phenylpropanoid-flavonoid pathway in Pyrus communis due to inoculation with E. amylovora. In this study, we propose a potential role of this pathway in defence mechanisms, providing a detailed analysis of the response of this system attributable to inoculation with E. amylovora.




Induction of stilbene phytoalexins in grapevine (Vitis vinifera) and transgenic stilbene synthase-apple plants (Malus domestica) by a culture filtrate of Aureobasidium pullulans.

Rühmann S, Pfeiffer J, Brunner P, Szankowski I, Fischer TC, Forkmann G, Treutter D.: Plant Physiol Biochem. 2013 Nov;72:62-71. doi: 10.1016/j.plaphy.2013.03.011. Epub 2013 Mar 23.

Products containing the epiphytic yeast Aureobasidium pullulans are commercially available and applied by fruit growers to prevent several fungal and bacterial diseases of fruit trees. The proposed beneficial mechanisms relate to limitations of space and nutrients for the pathogens in presence of the rapidly proliferating yeast cells. These explanations ignore the potential of yeasts to elicit the plant's defense. Our experiments aim at clarifying if an autoclaved and centrifuged suspension of A. pullulans may induce defense mechanisms. As a model system, the biosynthesis and accumulation of stilbene phytoalexins in callus and shoots of grapevine Vitis vinifera grown in vitro was used. Yeast application to the plant tissue stimulated stilbene biosynthesis, sometimes at the cost of flavonoids. The expression of the gene encoding stilbene synthase was enhanced and the enzyme showed higher activity while chalcone synthase activity and expression was reduced in some cases. An accumulation of stilbenes was also found in transgenic apple trees (Malus domestica cv. Holsteiner Cox) harboring the stilbene synthase-gene under control of its own promoter. These results clearly show that the application of A. pullulans may induce defense mechanisms of the treated plants.




Loss of nuclear flavanols during drought periods in Taxus baccata.

Feucht W, Treutter D, Dithmar H, Polster J.: Plant Biol (Stuttg). 2013 May;15(3):462-70. doi: 10.1111/j.1438-8677.2012.00661.x. Epub 2012 Oct 8.

Normally, needles of Taxus baccata during the growth period prominently stain blue for nuclear flavanols with the histochemical DMACA procedure. However, under excess heat and drought conditions, nuclear flavanols of current-year needles decline to zero. Nevertheless, greenish-yellow-coloured flavonols (quercetin derivatives) were still observed in nuclei. All of these yellow nuclei were in a silenced state and without mitosis. This link between drought and loss of nuclear flavanols was found in 3?years, 2003, 2007 and 2010. In 2007, exceptional drought occurred in early spring, interrupted by short rains. This, in turn, led to flushing of new sprouts, a characteristic feature in which nuclei were overloaded with flavanols. By the end of three drought periods, all nuclei developed blue-coloured nuclear flavanols. The flavanols seem to be associated with the histone proteins of chromatin. The oxidative degradation of catechin in Tris buffer (pH 8.0) containing MgCl2 was studied in the presence of the H4-core fragment TYTEHAKRKTVTAMD, modified according to the epigenetic histone code. The results show that catechin degradation can be significantly inhibited by the non-modified peptides and the methylated peptides (methylation at both lysine residues). The acetylated and formylated peptides do not show this behaviour. These observations indicate that flavanol association at chromosomes appears to be regulated by the epigenetic histone code.


Diversity of phenolic profiles in the fruit skin of Prunus domestica plums and related species.

Treutter D, Wang D, Farag MA, Baires GD, Rühmann S, Neumüller M.: J Agric Food Chem. 2012 Dec 5;60(48):12011-9. doi: 10.1021/jf303644f. Epub 2012 Nov 16.

The fruits of the European plum Prunus domestica exhibit a great diversity in appearance including skin colors. This study attempts to elucidate the phenylpropanoid and flavonoid profiles of 28 plum varieties belonging to P. domestica and related species as well as hybrids. A total of 49 phenolic compounds extracted from the fruit skin were quantitatively evaluated in an HPLC-DAD-based metabolomic study. The total phenolic contents of the cultivars varied among 0.4-29.9 mg/g fresh weight. The predominant anthocyanins were glycosides of cyanidin and peonidin, and rutin was the principal flavonol, whereas neochlorogenic acid and n-chlorogenic acid were the main hydroxycinnamic acids. Aside from these major phenolic classes, a group of tentatively identified flavones and several acylated flavonoids were also found. Principal component analysis revealed that anthocyanins and hydroxycinnamic acids contributed most to variety separation. The heterogeneity between the different varieties was also assessed using hierarchical cluster analysis of sample phenolics profile. A simple separation of species could not be found confirming the close relationship among them.





RNA-mediated gene silencing signals are not graft transmissible from the rootstock to the scion in greenhouse-grown apple plants Malus sp.

Flachowsky H, Tränkner C, Szankowski I, Waidmann S, Hanke MV, Treutter D, Fischer TC.: Int J Mol Sci. 2012;13(8):9992-10009. doi: 10.3390/ijms13089992. Epub 2012 Aug 10.


RNA silencing describes the sequence specific degradation of RNA targets. Silencing is a non-cell autonomous event that is graft transmissible in different plant species. The present study is the first report on systemic acquired dsRNA-mediated gene silencing of transgenic and endogenous gene sequences in a woody plant like apple. Transgenic apple plants overexpressing a hairpin gene construct of the gusA reporter gene were produced. These plants were used as rootstocks and grafted with scions of the gusA overexpressing transgenic apple clone T355. After grafting, we observed a reduction of the gusA gene expression in T355 scions in vitro, but not in T355 scions grown in the greenhouse. Similar results were obtained after silencing of the endogenous Mdans gene in apple that is responsible for anthocyanin biosynthesis. Subsequently, we performed grafting experiments with Mdans silenced rootstocks and red leaf scions of TNR31-35 in order to evaluate graft transmitted silencing of the endogenous Mdans. The results obtained suggested a graft transmission of silencing signals in in vitro shoots. In contrast, no graft transmission of dsRNA-mediated gene silencing signals was detectable in greenhouse-grown plants and in plants grown in an insect protection tent.


Polyphenol metabolism provides a screening tool for beneficial effects of Onobrychis viciifolia (sainfoin).

Thill, J., Regos, I., Farag, M.A., Ahmad, A.F., Kusek, J., Castro, A., Schlangen, K., Hayot Carbonero, C., Gadjev, I.Z., Smith, L.M.J., Halbwirth, H., Treutter, D.,Stich, K.: Phytochemistry 82 (2012) 67–80

Onobrychis viciifolia (sainfoin) is a traditional fodder legume showing multiple benefits for the environment, animal health and productivity but weaker agronomic performance in comparison to other legumes. Benefits can be mainly ascribed to the presence of polyphenols. The polyphenol metabolism in O. viciifolia was studied at the level of gene expression, enzyme activity, polyphenol accumulation and antioxidant activity. A screening of 37 accessions regarding each of these characters showed a huge variability between individual samples. Principal component analysis revealed that flavonols and flavan 3-ols are the most relevant variables for discrimination of the accessions. The determination of the activities of dihydroflavonol 4-reductase and flavonol synthase provides a suitable screening tool for the estimation of the ratio of flavonols to flavan 3-ols and can be used for the selection of samples from those varieties that have a specific optimal ratio of these compounds for further breeding.

Flavanols in nuclei of tree species: facts and possible functions

Feucht, W., Treutter, D., Polster, J.: Trees – Structure and Function 26 (2012) DOI 10.1007/s00468-012-0725-4

This review presents a new conceptual model for the involvement of low molecular flavanols in chromatin remodelling and genome organization. The experiments are based on the property of flavanols to associate with nuclei as revealed by blue staining after treatment with the p-dimethylaminocinnamaldehyde reagent. From a critical standpoint, this puzzling finding is nearly incompatible with current views about nuclear organization. Therefore, it was necessary to collect a whole host of data to confirm this new aspect and to gain some insight into possible regulatory roles of histone–flavanol assemblies. A lot of research has been devoted to this topic over the last 13 years. In particular, conifer nuclei were found to contain flavanols, whereas the nuclei of most angiospermous tree species investigated until now reacted negatively. Camellia sinensis (tea bush), being a broad-leaved dicotyledonous species indeed has nuclei with prominent flavanol staining. A subnuclear patterning of flavanols can be observed which is regulated by genetic and epigenetic mechanisms. Broadly speaking, flavanols of nuclei range from evenly diffuse to mosaic-like mottling and from pale to dark blue. The diffuse type is apparently characteristic of a more silenced nuclear state, whilst mild to prominent mottling implicates a transcriptionally more activated state. Dark blue flavanol blobs within the mottled mosaic state indicate a heterochromatin pattern whilst pale blue stippling tends to euchromatin. Environmental stress conditions such as drought combined with heat induce key signals for downregulation of nuclear flavanols. In this article, various aspects of nuclear flavanol localization are summarized and discussed.

In vivo and in vitro efficacy of sainfoin (Onobrychis viciifolia) against Eimeria spp in lambs.

Saratsis, A., Regos, I.,Tzanidakis, N., Voutzourakis, N., Stefanakis, A., Treutter, D., Joachim, A., Sotiraki,S .: Vet. Parasitol. (2012), doi:10.1016/j.vetpar.2012.03.014

The effect of sainfoin (Onobrychis viciifolia) against ovine coccidia was evaluated in vivo and in vitro. In 3 in vivo trials weaned lambs were allocated into two treatment groups receiving diets with either lucerne (Medicago sativa) or sainfoin. During the trials, which lasted for 7 (trial 1) or 8 weeks (trials 2 and 3), oocysts per gram of faeces (OPGs), faecal scores and weight gain were recorded. In two of the experiments (trials 1 and 3) a reduction in the mean oocyst excretion rates was observed, starting three to four weeks after sainfoin hay feeding. This reduction ranged between 21.3% (trial 1) and 61.7% (trial 3) compared to the control values. As a result, a decrease in the total number of oocysts excreted (expressed as the mean area under the curve of the OPG) was observed from week 4 to the end of the two trials, respectively (trial 1: 42.6% reduction, p = 0.05; trial 3: 52.4% reduction, p = 0.06). The results did not show any significant diet effect on lamb growth rates and faecal scores. In the in vitro experiments the effect of 39 sainfoin extracts were tested in an oocyst sporulation inhibition assay. The Eimeria oocysts sporulation inhibition throughout the experiments did not exceed 10.7%, showing that extracts of this forages do not have a significant inhibitory effect on Eimeria oocyst sporulation. This was an initial attempt to investigate a possible anticoccidial effect of sainfoin and further studies are needed in order to better understand its mode of action against Eimeria.

Effects of Apple (Malus x domestica Borkh.) Phenolic Compounds on Proteins and Cell Wall-Degrading Enzymes of Venturia inaequalis

Golba, B., Treutter, D., Kollar, A.: Trees – Structure and Function 26 (2012) 131-139

Cell wall-degrading enzymes of Venturia inaequalis are supposed to be fungal virulence factors whereas phenolic compounds of the host plant may be involved in defence. Since phenolic structures are predestined for an interaction with proteins we studied the effects on enzymes and proteins in course of in vitro culture and with preparations from culture filtrates and mycelia, respectively. The native compounds epicatechin, catechin, phloridzin, chlorogenic acid, caffeic acid, pcoumaric acid and phloridzin tested under non-oxidizing conditions had no or weak effects on enzyme activities. A significant inhibition of pectinase was only detected with the highest concentrations of procyanidins and phloretin. Aerobe conditions resulted in a fast oxidation of most phenolics which was enhanced by fungal phenoloxidases. Generally, no inhibition of fungal growth occurred in vitro but distinct irreversible effects on proteins and enzymes were detected with oxidized phenolics in course of in vitro-cultures as well as with the corresponding preparations. Efficacy of inhibitory activity in in vitro-cultures depended on media, culture technique and time course. Direct treatment of enzyme preparations with the oxidized phenolics resulted in a distinct inhibition of cellulolytic and especially pectinolytic activity. Apart from cellulase pattern altered by phenolics, in vitro-culture zymograms revealed a non-specific reduction of enzymatic activities, whereas action on total culture filtrate proteins resulted in specific effects due to phenolic compounds and incubation time. An attempt was made to characterize the oxidation products of epicatechin. Chromatographic fractionation revealed a non-resolvable complex of inhibitory compounds which were not consistent with the typical yellow oxidation products.

Differential gene expression in leaves of a scab susceptible and a resistant apple cultivar upon Venturia inaequalis inoculation.

Holzapfel, C., Meisel, B., Thümmler, F., Leser, C., Treutter, D.: Trees – Structure and Function 26 (2012) 121-129

Apple scab, one of the most damaging diseases in apple worldwide is caused by the fungal pathogen Venturia inaequalis. Pathogen-induced gene expression was analyzed in leaves of greenhouse- and field-grown trees of the scab susceptible cultivar Golden Delicious and the resistant cultivar Rewena using the macroarray-technique. The results show that the defence of the Vf resistant cultivar Rewena is based on different mechanisms than the basal defence response of the susceptible Golden Delicious: Whereas lignification seems to play an essential role in scab defence of Rewena, a thaumatin-like as well as a flavonoid gene are assumed to be involved in the mostly insufficient defensive response of Golden Delicious. Furthermore, a method was developed for quantification of the V. inaequalis in infected leaves using real-time quantitative reverse transcription-PCR.

Substrate specificity and contribution of the glycosyltransferase UGT71A15 to phloridzin biosynthesis

Gosch, C., Flachowsky, H, Halbwirth, H., Thill, J., Mjka-Wittmann, R., Treutter, D., Richter, K., Hanke, M.-V., Stich, K.: Trees – Structure and Function 26 (2012) 259-271

The dihydrochalcone phloridzin (phloretin 2´-O-glucoside) is the most abundant phenolic compound in apple (Malus x domestica). The final step in the biosynthesis of phloridzin is the glycosylation of phloretin at position 2´. Three cDNA clones from apple encoding glycosyltransferases are available which are able to catalyze the reaction in vitro. We investigated the possible role of glycosyltransferase UGT71A15 in phloridzin biosynthesis. The recombinant enzyme showed broad substrate acceptance but highest activities were observed with flavonols. Specific activities and the kinetic data indicated that phloretin it is not the main native substrate of the UGT71A15. Overexpression of UGT71A15 in transgenic apple trees did not lead to morphological changes. However, an increase of the molar ratio phloridzin:phloretin was found in all clones, indicating a physiological relevance of UGT71A15 in planta, although a decrease of the total amount of dihydrochalcones in the majority of the samples was found. Unexpectedly, the increase of the phloridzin:phloretin ratio was not reflected by an increase of the glucosyltransferase activities. In contrast, the majority of transgenic plants showed a reduced glucosylating activity with both phloretin and quercetin as a substrate, but the observed activity changes in a given sample were not consistent between the two substrates. An increased susceptibility of M. robusta against the fire blight causing bacterium E. amylovora as a result of UGT71A15 overexpression could not be observed.


Topp, B.L., Russell, D.M. , Neumüller, M. , Dalbó, M.A., Liu, W. (2012). In: M.L. Badenes and D.H. Byrne (eds.), Fruit Breeding, Handbook of Plant Breeding, Springer Science+Business Media 8, 571-621

There are 19–40 species of plum, depending on taxonomist, that have originated in Europe, Asia and America. From this great diversity only two species, the hexaploid European plum ( Prunus domestica ) and the diploid Japanese plum ( P. salicina and hybrids), are of worldwide commercial signifi cance. The European plums were cultivated in Roman times and stone remnants indicate human use 6,000 years ago. Their origin is uncertain but may have involved P. cerasifera and possibly P. spinosa as ancestors. The rich diversity and history of European plums is refl ected in the many pomological groups including Prunes, Gages, Mirabelles, Damsons, Bullaces and St Juliens. Today, European plum breeding concentrates on selection for resistance to Sharka disease caused by the Plum Pox Virus which limits production in many countries. Resistant cultivars have been developed using both conventional and genetic transformation techniques. Japanese plums originated in China but were introduced to the west, from Japan, only 150 years ago. Luther Burbank hybridized them with other plum species with the result that most modern cultivars are multispecies amalgams. This heterogeneity, plus the high heterozygosity from outcrossing, means that large seedling populations are required in cultivar development. Effi cient cross-pollination and seedling management techniques are required for these large populations. The trend of interspecifi c hybridisation continues today with four of the top 20 Californian cultivars being interspecifi cs involving plum and apricot. Fruit quality, functional food value, productivity and adaptation through disease resistance, chilling requirement and phenology are selection criteria in both Japanese and European plum breeding. Molecular markers are used for selection of self-compatibility and nematode resistance and for diversity and taxonomic studies. Most new rootstock releases are clonally propagated and of interspecifi c origin. The priorities for plum and peach rootstock breeding are similar and rootstocks developed for peach are sometimes also used for plum. American plum species, ancient Oriental cultivars and autochthonous European cultivars represent important germplasm resources that require preservation for use in future breeding.

Two-photon excitation with pico-second fluorescence lifetime imaging to detect nuclear association of flavanols

Mueller-Harvey. I., Feucht, W., Polster, J., Trnkovád, L., Burgose, P., Parkere, A.W., Botchwaye, S.W. (2012) Analytica Chimica Acta 719, 68– 75

Two-photon excitation enabled for the first time the observation and measurement of excited state fluorescence lifetimes from three flavanols in solution, which were ?1.0 ns for catechin and epicatechin, but <45 ps for epigallocatechin gallate (EGCG). The shorter lifetime for EGCG is in line with a lower fluorescence quantum yield of 0.003 compared to catechin (0.015) and epicatechin (0.018). In vivo experiments with onion cells demonstrated that tryptophan and quercetin, which tend to be major contributors of background fluorescence in plant cells, have sufficiently low cross sections for two-photon excitation at 630 nm and therefore do not interfere with detection of externally added or endogenous flavanols in Allium cepa or Taxus baccata cells. Applying two-photon excitation to flavanols enabled 3-D fluorescence lifetime imaging microscopy and showed that added EGCG penetrated the whole nucleus of onion cells. Interestingly, EGCG and catechin showed different lifetime behaviour when bound to the nucleus: EGCG lifetime increased from <45 to 200 ps, whilst catechin lifetime decreased from 1.0 ns to 500 ps. Semi-quantitative measurements revealed that the relative ratios of EGCG concentrations in nucleoli associated vesicles: nucleus: cytoplasm were ca. 100:10:1. Solution experiments with catechin, epicatechin and histone proteins provided preliminary evidence, via the appearance of a second lifetime (_2 = 1.9–3.1 ns), that both flavanols may be interacting with histone proteins. We conclude that there is significant nuclear absorption of flavanols. This advanced imaging using two-photon excitation and biophysical techniques described here will prove valuable for probing the intracellular trafficking and functions of flavanols, such as EGCG, which is the major flavanol of green tea.

Silencing of flavanone-3-hydroxylase in apple (Malus × domestica Borkh.) leads to accumulation of flavanones, but not to reduced fire blight susceptibility.

Flachowsky, H., Halbwirth, H., Treutter, D., Richter, K., Hanke, M.-V., Szankowski, I., Gosch, C., Stich, K., Fischer, T. (2012) Plant Physiology and Biochemistry 51, 18-25

Transgenic antisense flavanone-3-hydroxylase apple plants were produced to mimic the effect of the agrochemical prohexadione-Ca on apple leaves. This enzyme inhibitor for 2-oxoglutarate dependent dioxygenases is used as a growth retardant and for control of secondary fire blight of leaves. Like using the agent, silencing of flavanone-3-hydroxylase leads to an accumulation of flavanones in leaves, but in contrast not to the formation of 3-deoxyflavonoids. In prohexadione-Ca treated leaves the 3-deoxyflavonoid luteoforol is formed from accumulating flavanones, acting as an antimicrobial compound against the fire blight pathogen Erwinia amylovora. Seemingly, the silencing of just one of the 2-oxoglutarate dependent dioxygenases (in apple also flavonol synthase and anthocyanidin synthase take part downstream in the pathway) does not provide a sufficiently high ratio of flavanones to dihydroflavonols. This seems to be needed to let the dihydroflavonol-4-reductase/flavanone-4-reductase enzyme reduce flavanones to luteoforol, and to let this be reduced by the leucoanthocyanidin-4-reductase/3-deoxyleucoanthocyanidin-4-reductase, each acting with their respective weak secondary activities. Accordingly, also the intended inducible resistance to fire blight by prohexadione-Ca is not observed with the antisense flavanone-3-hydroxylase apple plants. On the other hand, for most transgenic lines with strong flavanone-4-reductase down-regulation, upregulation of gene expression for the other flavonoid genes was found. This provides further evidence for the feedback regulation of flavonoid gene expression having been previously reported for the prohexadione-Ca inhibited apple plants.

LAMP DNA Analytik mit homogener Farbreaktion

Hadersdorfer, J., Neumüller, M., Treutter, D., Fischer, T.C. (2012) GIT Labor-Fachzeitschrift 3/2012, 2-4


Fundamental and Applied Aspects of Plum (Prunus domestica) Breeding

Neumüller, M.: Fruit, Vegetable and Cereal Science and Biotechnology 5 (2011) 139-156

The hexaploid European plum (Prunus domestica L.) is one of the most important temperate fruit crops. Its origin is unclear as wild forms are missing. The genetic base which can be used for breeding is highly diverse and provides a good base for further improvement of the fruit crop. Information on the inheritance of single traits are rarely available. Breeding focuses on resistance and fruit quality. Classical breeding is the most important method applied. Very few data is available on the genome sequence. No marker assisted selection systems are available. Genetic engineering is limited to the transformation of embryonic tissue derived from seeds. Prunus domestica is the only Prunus species where genotypes completely resistant to the Plum pox virus exist. This resistance is based on a hypersensitive response of the plant cells to the virus. Interspecific hybridization becomes more important in terms of transferring resistance traits from European plum to related species and of developing hybrids with new fruit characters. Classical breeding is far from being the limit of the improvement of plum genotypes.

Acylated flavonol glycosides from the forage legume, Onobrychis viciifolia (sainfoin).

Veitch, N.C., Regos, I., Kite, G.C., Treutter, D.: Phytochemistry 72 (2011) 423-429

Ten acylated flavonol glycosides were isolated from aqueous acetone extracts of the aerial parts of the forage legume, Onobrychis viciifolia, and their structures determined using spectroscopic methods. Among these were eight previously unreported examples which comprised either feruloylated or sinapoylated derivatives of 3-O-di- and 3-O-triglycosides of kaempferol (3,5,7,4’-tetrahydroxyflavone) or quercetin (3,5,7,3’,4’-pentahydroxyflavone). The diglycosides were acylated at the primary Glc residue of O-?-Rhap(1?6)-?-Glcp (rutinose), whereas the triglycosides were acylated at the terminal Rha residues of the branched trisaccharides, O-?-Rhap(1?2)[?-Rhap(1?6)]-?-Galp or O-?-Rhap(1?2)[?-Rhap(1?6)]- ?-Glcp. Identification of the primary 3-O-linked hexose residues as either Gal or Glc was carried out by negative ion electrospray and serial MS, and cryoprobe NMR spectroscopy. Analysis of UV and MS spectra of the acylated flavonol glycosides provided additional diagnostic features relevant to direct characterisation of these compounds in hyphenated analyses. Quantitative analysis of the acylated flavonol glycosides present in different aerial parts of sainfoin revealed that the highest concentrations were in mature leaflets.

Fast and reliable detection of Plum pox virus in woody host plants using the Blue LAMP protocol

Hadersdorfer, J., Neumüller, M., Treutter, D., Fischer, T.C.: Annals of Applied Biology 2011, 456–466

Up to now, the polymerase chain reaction is the most widely used method for the amplification of nucleic acids in vitro, especially for pathogen detection because of its high sensitivity. In the recent years, however, numerous isothermal amplification methods were developed to avoid the need for thermal cycling. The most frequently applied approach seems to be loop-mediated isothermal amplification (LAMP). The great advantage of LAMP is its enormous rate of amplification paired with a very high specificity and low artefact susceptibility. This study presents a straightforward procedure for Plum pox virus (PPV) detection. A modified one-step reverse transcription loop-mediated isothermal amplification protocol of Varga and James is applied to virus suspensions from plant extracts obtained by a simplified and standardised procedure. Gel electrophoresis is substituted by a homogenous colour test upon nucleic acid amplification. This procedure takes only 2.5 h from sampling to result and requires minimal technical equipment. With amplification and visualisation homogenously taking place in non-opened tubes the risk of cross-contamination of subsequent samples by former amplification products via facilities and equipment is strongly minimised. Hence, the Blue LAMP provides a fast and reliable detection of PPV both for single samples and for large-scale surveys.

Nuclei of Tsuga canadensis: Role of Flavanols in Chromatin Organization.

Feucht, W., Schmid, M., Treutter, D.: International Journal of Molecular Sciences 12 (2011) 6834-6855

Needle primordia of Tsuga canadensis (hemlock) arising from flank meristems of a shoot apex, form cell lineages consisting of four or eight cells. Within a recently established lineage there is striking uniformity in the pattern of nuclear flavanols. This fact points to an identical transcriptional expression of these flavanols during cell cycling. However two lineages, even if located close together within the same meristem, can be very different in the expression of both cell shape and nuclear flavanol pattern, indicating that epigenetic positional signals are operating in a collective specification of cell lineage development. There is a wide range of nuclear flavanol patterning from a mosaic-like distribution in an activated cell type to a homogenous appearance in silenced cell types. Single cells deriving from lineages are desynchronized because they underlie a signaling network at a higher tissue level which results in stronger epigenetic modifications of their nuclear flavanols. As an extreme case of epigenetic modulation, transient drought conditions caused a drastic reduction of nuclear flavanols. Upon treatment with sucrose or cytokinin, these nuclear flavanols could be fully restored. Analytical determination of the flavanols revealed 3.4 mg/g DW for newly sprouting needles and 19.6 mg/g DW for anthers during meiosis. The roughly 6-fold difference in flavanols is apparently a reflection of the highly diverging organogenetic processes. Collectively, the studies provide strong evidence for combinatorial interplay between cell fate and nuclear flavanols.

Approaches to Determine the Origin of European Plum (Prunus Domestica) Based on DNA Nucleotide Sequences

Xuan , H., Spann, D., Schlottmann , P., Neumüller , M.: Acta Hort. 918 (2011) 261-267

7 nuclear SSRs and 10 chloroplast SSRs (cpSSRs) from 4 non-coding regions were chosen for DNA and cpDNA analysis from a total of about 30 individuals of P. domestica, P. spinosa, P. cerasifera, P. salicina and interspecific crosses of P. domestica × P. cerasifera, P. domestica × P. spinosa and P. cerasifera × P. salicina and used for molecular phylogenetic approaches to help clarify the origin of European plums. Primers were labelled with Cy5 and Cy5.5 and the PCR products were detected and analysed by capillary electrophoresis using a Beckman CEQ 8000 DNA sequencer (Beckman Coulter, Inc.) by comparison with internal size standards. Cluster analysis was performed with GelCluster V1.0 (BioSci-software), to obtain dendrogram grouping output. Using the 7 SSRs and 9 cpSSRs successfully separated the 30 individuals of P. domestica, P. spinosa, P. cerasifera, P. salicina and interspecific crosses of P. omestica × P. cerasifera, P. domestica × P. spinosa, P. domestica × P. armeniaca and P. cerasifera × P. salicina. ‘Tatjana’, formerly described as a P. cerasifera genotype, is likely to be a hybrid between P. salicina and P. cerasifera. ‘69 KO’ from Lauenburg is probably not a pure P. ceracifera genotype, but has P. salicina ancestors.

The Influence of Apple- or Red-Grape Pomace Enriched Piglet Diet on Blood Parameters, Bacterial Colonisation, and Marker Gene Expression in Piglet White Blood Cells.

Sehm, J., Treutter, D., Lindermayer, H., Meyer, H.H.D., Pfaffl, M.W.: Food and Nutrition Sciences (2011) 366-376


Optimization of a high-performance liquid chromatography method for the analysis of complex polyphenol mixtures and application for sainfoin extracts (Onobrychis viciifolia).

Regos, I., Treutter, D.: J. Chromatography A, 1217 (2010) 6169-6177

A pentafluorophenylpropyl (PFP) stationary phase was tested for the simultaneous determination of several classes of phenolic compounds. The chromatographic results were compared with those obtained by using a bifunctional phase constituted of octadecyl and phenylpropyl bonded silica and three conventional C18 columns. The elution gradient was optimized with 5% formic acid and sodium acetate in combination with acetic acid as additives and methanol as solvents. For these evaluations, a complex phenolic extract of Onobrychis viciifolia (sainfoin) and test mixtures containing 54 standard substances including 2 simple phenolic compounds, 1 amino acid, 4 hydroxybenzoic acids (HBA), 6 hydroxycinnamic acids (HCA), 3 flavan-3-ols, 9 anthocyanins, 2 dihydroflavonols, 1 chalcone, 4 flavones, 1 isoflavone and 21 flavonols have been assayed. The perfluorinated column showed good resolution for the studied phenolic compounds which have the following elution order: HBA, HCA, flavan-3-ols, anthocyanins, dihydroflavonols, flavones, flavonols and isoflavones. Compared with other columns, it provides longer elution ranges for HBA, HCA and flavan-3-ols and increased retention times for all compound classes except anthocyanins which were similarly retained on a C18 column. Its selectivity is different from C18 and bifunctional phases. A high-performance liquid chromatography (HPLC) method with diode array detection (DAD) and post-column derivatization with p-dimethyl-aminocinnamic aldehyde (DMACA) has been validated for the analysis of individual phenolic compounds from a sainfoin plant extract (O. viciifolia).

Rutin and proanthocyanidin contents in buckwheat grains: combined biosynthesis in interspecific hybrids between Fagopyrum esculentum Moenchh x F. homotropicum Ohnishi and their progeny. Ölschläger, C., Zeller, F.J., Treutter, D.: Eur. J. Plant Science Biotechnol. 4 (2010) 98-109

Hybrids between sporophytic self-incompatible common buckwheat F. esculentum and self-fertilizing F. homotropicum are promising with respect to an improvement of agronomic productivity. However, the nutritional potential of these hybrids regarding their flavonoid phenolics profiles exhibit wide variability which is based on the metabolic differences between the two species. In F. homotropicum the flavan 3-ol content is dominating whereas rutin – being the focus of interest for buckwheat consumers – was represented in higher amounts in F. esculentum. Four classes of phenolics compounds, namely benzoic acids, hydroxycinnamic acids, flavonols and flavan 3-ols, were quantified in the grains of breeding lines and hybrids. Based on these data it could be shown that the metabolic channeling within the flavonoid biosynthesis is inherited. Two main regulatory steps of the phenylpropanoid/flavonoid pathway are postulated which seems to be responsible for different flavonoid phenotypes, rutin- and flavan 3-ol-phenotypes, in particular. Metabolomic studies by using HPLC/DAD and HPLC/CRD methods allow the description of the flavonoid phenotypes for selection of appropriate parents and valuable hybrids as a basis for breeding of varieties with optimized phenolic profiles.

Managing phenol contents in crop plants by phytochemical farming and breeding—visions and constraints.

Treutter, D.: Int. J. Mol. Sci. 11 (2010) 807-857

Two main fields of interest form the background of actual demand for optimized levels of phenolic compounds in crop plants. These are human health and plant resistance to pathogens and to biotic and abiotic stress factors. A survey of agricultural technologies influencing the biosynthesis and accumulation of phenolic compounds in crop plants is presented, including observations on the effects of light, temperature, mineral nutrition, water management, grafting, elevated atmospheric CO2, growth and differentiation of the plant and application of elicitors, stimulating agents and plant activators. The underlying mechanisms are discussed with respect to carbohydrate availability, trade-offs to competing demands as well as to regulatory elements. Outlines are given for genetic engineering and plant breeding. Constraints and possible physiological feedbacks are considered for successful and sustainable application of agricultural techniques with respect to management of plant phenol profiles and concentrations.

Effect of defoliation and fruit thinning on fruit quality of ‘Crimson Seedless’ grape.

Abd El-Razek, E., Treutter, D., Saleh, M.M.S., El-Shammaa, M., Fouad, A.A., Abdel-Hamid, N., Abou-Rawash, M.: Res. J. Agriculture and Biological Sciences, 6 (2010) 289-295

‘Crimson Seedless’ vines were treated with 5 defoliation managements: (1) leaf basal removal (LBR), (2) leaf basal removal + hedging (LBR+H), (3) leaf basal removal + sterile shoot removal (SSR+LBR), (4) combination of treatments 2 and 3 (LBR+SSR+H), (5) leaf basal removal + fruit thinning (LBR+FTH). Defoliation improved cluster weight, size, and compactness. Berries were bigger (size, weight) with LBR + FTH, followed by the other treatments except LBR+H+SSR. Moreover, defoliation accelerated the ripening process and the berry juice was increased, whereas, fruit firmness and adherence were decreased. Also, it increased T.S.S and decreased the acidity. Total anthocyanins in the berry were improved by all treatments.

Solar UVB response of bioactives in strawberry (Fragaria x ananassa Duch. L.): a comparison of protected and open-field cultivation.

Josuttis, M., Dietrich, H., Treutter, D., Will, F., Linnemannstöns, L., Krüger, E.: J. Agric. Food Chem. 58 (2010) 12692–12702

Strawberries (Fragaria _ ananassa Duch. cvs. Everest, Elsanta) were grown in a tunnel covered with two films, which were distinguished in their ultraviolet transparency, as well as under open-field conditions. One applied film was not transparent for UVB radiation, and the second film transmitted 70% of UVB radiation. During the present study, the nutritional value and quality parameters of the fruits were evaluated. Strawberries were UV-unresponsive in view of the content of ascorbic acid and sum parameters like total anthocyanins and antioxidant capacity measured with TEAC (trolox equivalent antioxidant capacity), ORAC (oxygen radical absorbance capacity) and total phenols. These parameters were mainly affected by sampling date and cultivar. However, HPLC analysis showed that individual phenolics were affected in the absence of UV radiation. The content of the anthocyanin cyanidin 3-glucoside and the flavonols quercetin 3-glucuronide and kaempferol 3-glucoside was decreased in the fruits grown under UV blocking film compared to open-field grown strawberries. By means of the UV transparent film the content of the mentioned flavonoids could be enhanced up to similar amounts like in open-field grown strawberries. All other phenolics were not consistently affected by UV radiation. This result was independent of cultivar.

Effect of bioregulators on growth and secondary metabolism of Actinidia arguta plants.

Treutter, D., Hadersdorfer, J., Pietzner, J., Steber, M.: Acta Horticulturae 884 (2010) 689-694

The growth retardant prohexadione-Ca altered the phenolics profiles of Actinidia arguta leaves. A simultaneous application with gibberellins or cytokinin partially compensated the growth retardation induced by prohexadione-Ca and increased the content of phenolics while total biomass production was nearly unaffected by the growth regulators.

Towards an understanding of the inheritance of hypersensitivity resistance against the Sharka virus in European Plum (Prunus domestica L.): Generation of interspecific hybrids with lower ploidy levels.

Neumüller, M., Lanzl, S., Hartmann, W., Feucht, W., Treutter, D.: Acta Horticulturae , 814 (2010) 721-726

Hypersensitivity resistance to the sharka virus is one of the most promising resistance mechanisms used in breeding for European plum cultivars. Its genetic determination is poorly understood as the hexaploid genome of P. domestica hampers the investigation of the inheritance of the resistance trait. A monogenic dominant inheritance can be excluded. Hypersensitive genotypes of P. domestica were crossed with genotypes of diploid myrobalan (P. cerasifera) and tetraploid sloe (P. spinosa). The descendants were tested for hypersensitivity against PPV. In order to investigate their parentage, their chromosome number was determined using a new staining technique for chromosomes in Prunus root tips. In both crossing combinations, the interspecific hybrid nature of the seedlings could be shown. Some of them showed strong hypersensitive response when inoculated with PPV. The hypersensitive interspecific hybrids are used for further crossings in order to investigate the inheritance of the trait of interest. Moreover, they are tested for their potential use as rootstocks for different Prunus species.

In vitro germination of Prunus domestica seeds.

Paskaš. K., Neumüller, M., Treutter, D.: Acta Horticulturae 874 (2010) 249-254

Breeding of new European plum (Prunus domestica L.) cultivars faces two major problems: low fruit set in cross-pollination and low germination rate of the seeds obtained from the harvested fruit. Up to now, breeding programs reported mainly stratification in peat, soil or sand and embryo culture according to Theiler (1971) in order to avoid the need of stratification and its associated problems, such as high losses due to microbial infection. The in vitro technique used in this study combines elements of different reported stratification methods and minimizes the infection rate during the stratification. Seeds were placed in a modified Murashige and Skoog (MS) medium in test tubes. Two seed treatments were tested: 1) sowing and stratification immediately after the harvest, 2) seeds were dried and stored for four months prior to sowing and stratification. Stratification was carried out in vitro at 4°C in both cases. Germination was checked once a week. When the radicle emerged through the surrounding tissues, seedlings were removed from the test tubes and cultivated in soil in the greenhouse. Some hard seed coats were removed manually to relieve the radical from the hardened testa and to encourage seeds germinate. The germination rate ranged from 16–100%. By comparing the two treatments it is obvious that if the seeds are sown immediately after harvest, the germination percentage is higher.

Breeding for Sharka resistance and high fruit quality in European Plum (Prunus domestica) at Weihenstephan: breeding strategy and selection tools.

Neumüller, M., Hartmann, W., Treutter, D.: Acta Horticulturae 874 (2010) 221-228

In 2005, a breeding program for European plum (Prunus domestica) was established at the Technical University of Munich in Weihenstephan, Germany. Methods for increasing the yield of seedlings out of a given number of pollinated flowers as well as timesaving selection methods for different traits of interest have been developed which may help to answer important problems that plum production is faced with. The breeding program aims at combining durable and complete resistance against Plum pox virus (PPV), the causative agent of Sharka disease, with high fruit quality. As a source of Sharka resistance, descendants of the crossing combination ‘Ortenauer’ × ‘Stanley’ are used. They originate from the breeding program of the University of Hohenheim and show a hypersensitive reaction after inoculation with PPV. In order to exploit the possible sources of resistance and to combine them, a gene bank of Prunus domestica genotypes has been built up and screened for Sharka resistance to find new sources of both hypersensitivity resistance as well as quantitative resistance against PPV. Moreover, crossings between highly PPV sensitive genotypes are performed in order to create new sources of hypersensitivity resistance which can be used in further breeding cycles. To enhance the fruit quality level of Sharka resistant cultivars, crossings with large sized, specially coloured and exceptionally tasty plum genotypes originating from the Weihenstephan Prunus gene bank or from the Hohenheim breeding program are performed. Furthermore, P. domestica genotypes are crossed with large sized P. cerasifera and P. salicina selections. Some interspecific hybrids show strong resistance against the Prune rust caused by the fungus Tranzchelia pruni-spinosae. Resistance tests which allow the selection of European plum genotypes resistant to Monilinia ssp. infections leading to the well known Brown rot of the fruits are under development.

Control of Sharka by breeding.

Hartmann, W., Neumüller, M.: Acta Horticulturae 874 (2010) 229-237

PPV is the most dangerous virus in stone fruit growing and is spread worldwide. In infected areas it is recommended that only tolerant or resistant cultivars are cultivated. From the plum breeding programme at Hohenheim 6 tolerant or resistant cultivars have been produced, which are successful in production in Germany. Some promising clones will be introduced. Tolerant or resistant cultivars can be infected by PPV and will continue to be a source of infection. The first absolute virus resistant cultivar worldwide is ‘Jojo’ introduced in 1999. ‘Jojo’ reacts hypersensitivly to PPV and has continued to grow healthily for more than 20 years in totally infected orchards. In the breeding programme quantitative and hypersensitive resistance were combined. The heredity of hypersensitivity depends upon the chosen parents. Using donors from Hohenheim the hypersensitivity index has been much higher than that achieved by using the K4 hybrid or its progenies. Forty-three promising clones with different size and ripening times have been selected for tests in different regions. By pyramiding of the genes responsible for quantitative and hypersensitive PPV resistance we hope to get cultivars with a durable absolute PPV resistance and high fruit quality.

In vitro grafting as a tool for investigating the hypersensitivity of European Plum (Prunus domestica l.) to the plum pox virus.

Lichtenegger, L., M. Neumüller, D. Treutter, D.: Acta Horticulturae 874 (2010) 243-248

The hypersensitive response of Prunus domestica to the Plum pox virus (PPV) was investigated in a model system by in vitro grafting of virus free and virus infected plums with hypersensitive genotypes to confirm the results obtained ex vitro. Shoot-tips were grafted onto rootstocks with a V-cut at the scion. Successful graft union formation ranged from 80 to 90%. The defense mechanism was observed by macroscopic observations. Furthermore, the intergrowth of root and scion was microscopically observed with special respect to the occurrence of hypersensitive reactions in single cells. PPV infected scions grafted onto hypersensitive rootstocks showed a macroscopically visible hypersensitive reaction as necrotic lesions on the leaves, leading to the death of the scion. All hypersensitive genotypes showed an accumulation of phenol vacuoles in cells close-by the graft union. The cells died a short time after the formation of tissue bridges between the two grafting partners. In contrast, no abnormalities in the establishment of graft unions between virus infected plants with PPV sensitive genotypes could be observed. The macroscopically visible symptoms and the histological observations correspond with former results obtained in ex vitro graftings.

Establishing a PPV isolate collection as a prerequisite for an effective selection process in breeding plums for durable resistance against sharka. Hadersdorfer, J., Neumüller, M., Fischer, T., Treutter, D.: Acta Hort. 874 (2010) 59-62

To aid the plum breeding program for Plum pox virus (PPV) resistance at Weihenstephan a PPV isolate collection was built up. The collection is a prerequisite for establishing a selection procedure for cultivars which are completely resistant against the Sharka disease caused by PPV. Grafted plants of the most promising seedlings of the breeding program are infected with all the isolates of the collection to test their resistance against all PPV isolates. Up until now, 48 isolates have been collected originating from Europe, North America and Africa. They have been classified with one of the known PPV strains (PPV-D, PPV-M, PPV-Rec, PPV-C, PPV-EA and PPV-W) using two complementary PCR protocols.

Metabolic engineering of flavonoid biosynthesis in apple (Malus domestica Borkh.). Szankowski, I., Li, H., Flachowsky, H., Höfer, M., Hanke, M.-.V., Fischer, T., Forkmann, G., Treutter, D., Schwab, W. and Hoffmann, T.: Acta Horticulturae 814 (2010) 511-516

Flavonoids are a large family of polyphenolic compounds with manifold functions in plants including pathogen defence. Present in a wide range of vegetables and fruits, flavonoids form an integral part of the human diet and confer multiple health benefits. Modifying flavonoid biosynthesis in fruit crops, such as apple, offers the opportunity to increase plant resistance against pathogens and the health benefit potential of the fruit. Both overexpression and RNAi-based suppression strategies were used to modify flavonoid biosynthesis in apple. Introducing the maize Lc transcription factor gene, responsible for controlling the expression of structural genes of the flavonoid biosynthetic pathway in maize, into Malus domestica Borkh. cv. ‘Holsteiner Cox’ resulted in increased mRNA levels for most of the structural genes of the flavonoid pathway. Lc transgenic plants accumulated higher levels of the anthocyanin idaein, the flavan-3-ols epicatechin, catechin, and some distinct dimeric proanthocyanidins. In a second approach, the consequences of RNAi silencing of the genes for anthocyanidin synthase (ANS) and UDP-galactose:flavonoid 3-O-galactosyltransferase (FGT) gene to induce a shift towards flavan-3-ols were examined. Preliminary results obtained from transgenic plants of the cv. ‘Holsteiner Cox’ and of the red-leaved type TNR 31-35 (a descendent from Malus sieversii var. sieversii f. niedzwetzkyana (Dieck)) revealed that suppression of ANS or FGT resulted in increased amounts of flavan-3-ols and flavonols. In the red leaved type TNR 31-35, anthocyanin accumulation decreased after ANS silencing.


Identification and Quantification of Phenolic Compounds from the Forage Legume Sainfoin, (Onobrychis viciifolia).

Regos I, Urbanella A, Treutter D; J. Agr. Food Chem., (2009) 57, 5843-5852

Phenolic compounds of sainfoin (Onobrychis viciifolia) variety Cotswold Common are assumed to contribute to its nutritive value and bioactive properties. A purified acetone/water extract was separated by Sephadex LH-20 gel chromatography. Sixty-three phenolic and other aromatic compounds were identified by means of chemical, chromatographic and spectroscopic methods. Reverse phase-HPLC with diode-array and chemical reaction detection was used to investigate the phenolic composition of different plant organs. All plant parts showed specific phenolic profiles. Moreover, there were considerable variations in the phenolic content among individual plants of the same variety. The three most abundant phenolic compounds were found to be arbutin predominant in petiols, 17.7 mg/g dry weight (DW), rutin (predominant in leaves, 19.9 mg/g DW) and catechin (predominant flavanol in petiols, 3.5 mg/g DW). The present study reveals that the phenolic profile of sainfoin is even more complex than hitherto assumed.

Effects of quercetin and catechin on hepatic glutathione-S transferase (GST), NAD(P)H quinone oxidoreductase 1 (NQO1), and antioxidant enzyme activity levels in rats.

Wiegand H, Boesch-Saadatmandi C, Regos I, Treutter D, Wolffram S, Rimbach G; Nutrition and Cancer (2009) 61, 717–722

Cell culture data indicate that quercetin and catechinmay affect the activity of phase II and antioxidant enzymes. However, little is known about the impact of dietary flavonoids in vivo. Therefore, the present study aimed to investigate the in vivo effects of the flavonoids quercetin and catechin on mRNA and activity levels of phase II enzymes glutathione-S transferase (GST) and NAD(P)H quinone oxidoreductase-1 (NQO1) in rat liver. Furthermore, the activity of the hepatic antioxidant enzymes catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD) was determined. Feeding male Wistar rats (3 × 6 animals) over 3 wk with semisynthetic diets enriched with quercetin and catechin (2 g/kg diet) did not affect liver enzyme activity of CAT, GPx, and SOD as well lipid peroxidation and glutathione levels. Dietary quercetin significantly decreased activity of hepatic GST (24%), whereas dietary catechin significantly decreased NQO1 activity (26%) compared to controls. Changes in GST and NQO1 activity were partly reflected on mRNA levels. Current data indicate that dietary flavonoids have little effects on liver oxidant/antioxidant status but do significantly affect the phase II enzymes GST and NQO1 in rat liver. This in turn may affect the ability of the organism to detoxify endogenous and exogenous xenobiotics.

Flavanols in nuclei and cytoplasm: reduction of the micronuclei-inducing effect of aflatoxin b1 in v79 cells through catechin.

Bauer J; Neubauer K ; Dithmar H; Polster J; Feucht W; Advances in food sciences (2009) 31, 82-88

Nuclei of V79 cells showed that, in response to the carcinogen aflatoxin B1 (0.10 - 2.5 ?g/ml), the average number of micronuclei was significantly increased in comparison with controls. Pre-incubation of the cells in (+)-catechin at 0.01, 0.10 and 1.00 mg/L resulted in a significant reduction of micronuclei. With epigallocatechin gallate (0.001, 0.01 and 0.1 mg/L), a major flavanol in tea leaves, the reducing beneficial effect was not so pronounced. Using UV-VIS spectroscopic titration, this galloylated tea flavanol was found to associate more strongly with serum albumin than (+)-catechin; this may be one reason why epigallocatechin gallate showed a lower protective effect against the clastogenic properties of aflatoxin B1 than (+)-catechin. The intracellular localization of flavanols was assayed histochemically with the selective DMACA reagent. Nuclei of V79 cells as well as nuclei from different types of human cells showed a particularly high affinity for added (+)-catechin and (-)-epicatechin. Polymorphonuclear lymphocytes were found to bind maximal amounts of catechins along the border lines of their segments. The inhibition of the formation of aflatoxin B1-induced micronuclei by catechins and the predominance of nuclear-associated catechins supports the assumption that the nucleus itself might be the preferred site of protective mechanisms.

Nuclei of Taxus baccata: Flavanols Linked to Chromatin Remodeling Factors.

W Feucht, H Dithmar, J Polster. (2009) J. Botany pp. 1-9; doi:10.1155/2009/842869

Microscopic studies of young needles and shoot tips from Taxus baccata showed that flavanols are localized in the nuclei. This observation is based on the histochemical staining of flavanols with the DMACA reagent. The colour that is obtained with this reagent varies frompale to deep blue, depending on the amount of flavanols. This study is focused on nondifferentiated cell lineages and on differentiating cells. The key point to note is that all nuclei of a cell lineage showed a uniform DMACA staining pattern based on the amount and structural appearence of nuclear flavanols. This points to transcriptional and epigenetic programming. However, comparing various cell lineages from different shoot tips and needles revealed a lineage-specific expression of nuclear flavanols. This result implied that both positional and developmental signals from neighbouring cells were involved in the nuclear flavanol binding of lineages. The cells of a developmentally advanced lineage loose their intimate contact and, then, they separate from each other to undergo an autonomous, individual sequence of differentiation. This in turn was accompanied by differences in the nuclear flavanol patterns of the single cells. Investigating different mitotic stages revealed a wide spectrum in flavanol staining intensities of the chromosomes. These observations should be linked to UV-VIS spectroscopical kinetic results indicating that nuclear flavanols bound to histones are involved in epigenetically regulated modification of chromatin. The kinetic studies show that catechin is relatively rapidly degraded by oxygen in the presence of Mg2+-ions. However, this degradation reaction is strongly inhibited when histone proteins were added. This behaviour is a clear indication that coregulatory interactions exist between catechin and histones.

Plum breeding.

Hartmann, W.; Neumüller M. (2009): In: Jain, S.M.; Priyadarshan, P.M. (eds.; 2009), Breeding Plantation Tree Crops: Temperate Species. Springer Science + Business Media, pp. 161-230.