As a result of the high variability of fruit properties in the European plum Prunus domestica, a histochemical analysis of fruits at different stages of development was performed to understand the ripening process in cv. 'Colora' (yellow-red skinned) and cv. 'Topfive' (purple skinned). Histological analysis showed that carotenoids in the fruit had two different origins. In the fruit flesh, they derived from chloroplasts that turned into chromoplasts, whereas carotenoids in the fruit skin derived probably from proplastids. Flavan-3-ols and proanthocyanidins showed differential localization during ripening. They were visible in the vacuole in different fruit tissues or organized in tannosomes in the fruit flesh. Tanninoplasts were observed only in hypodermal cells of 'Colora'. Toward maturity, anthocyanins were detected in the epidermis and later in the hypodermis of both cultivars. The study forms a basis for the analysis of the biosynthesis of secondary metabolites in European plums and their biological effects.

Apples and their juices are important sources of phenolic compounds in human diet. Using the same juice processing method, we compared phenolic composition as well as antioxidant activity of juices from various table apple cultivars, grown at the same location. Antioxidant activity of apple juices was estimated by application of two assays, ABTS cation radical decolorization and by scavenging of reactive oxygen species (ROS) generated by xanthine/xanthine oxidase (O2·-, H2O2, OH·; superoxide assisted Fenton reaction), as measured by inhibition of ethylene release from KMB (α-Keto-γ-(methylthio)butyric acid). The apple juices differed in phenolic content and antioxidant activity, where cultivar differences were more relevant than environmental factors. Furthermore, to improve antioxidant performance in the XOD-test, a juice low in phenol content was supplemented with the appropriate amounts of phenols to the level of the best juice indicating that these phenolics contribute to the antioxidant activity of the apple juice. In ac­cordance to literature, the phenolic compounds re­presenting the main anti­oxidant activity in the apple juices comprise flavan-3-ols and chlorogenic acid.

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 long-term harsh climate conditions in 2015 distorted already from June up to November in all study trees of Tsuga and Taxus the intracellular organization of the needles. Intimately involved in these repressive processes were the flavanols, a small subgroup of the flavonoids. They were not only deposited in vacuoles of conifer needles but also in the nuclei and chromosomes. Among the many flavonoids the small group of catechin derivatives and polymers named flavanols can exclusively be stained blue with DMACA (dimethylaminocinnamaldehyde). From mid-July onward, the vacuolar flavanols of the epidermal cell layers were gradually diminished as evidenced by decreasing blue staining of nuclei and vacuoles. Subsequently, in August also the large spongy mesophyll cells showed the flavanols decreasing progressively. Apparently, the antioxidant flavanols operate as oxygen radical scavengers. (ROS) were used up during the harsh environmental stress conditions. Both, Tsuga and Taxus reacted in this way. However, it is quite surprising that in all study trees the palisade cells did not contain such vacuolar flavanols. Only these cells were in June the first to show a loss of chlorophyll from chloroplasts as well as an efflux of flavanols from the nuclei. Conversely, from September onward another group of phenols, the yellow-staining flavanols were newly formed in the palisade cells and later on also in the mesophyll cells. Obviously, they were assembled finally to stabilize finally the fragile cell sites. Summing up, the present study shows by cytological studies that the climatic conditions in 2015 produced the worst disturbance of subcellular structures observed since 2000 when our studies on nuclear phenols in needles of conifers were initiated.

The phenolic profiles of leaves of hydroponically forced chicons from five chicory (Cichorium intybus L.) cultivars were determined: 'Treviso', 'Verona', 'Anivip', 'Castelfranco' and 'Monivip'. The chicory roots were forced in the dark in three fertiliser solutions enriched in nitrogen (N), phosphorus (P) and/or potassium (K) (i.e., N/P/K, NIP, N alone), each applied at 10, 20 and 30 g/L. Phenolics were extracted from the chicon leaves and quantitatively evaluated in an HPLC-DAD based study. 'Castelfranco' showed the highest total phenolics (19.79 mg/g dry weight [DWI), and 'Monivip' the lowest (1531 mg/g DW). Across the nutrient solutions, the highest total phenolics was with N/K-1% (19.87 mg/g DW), followed by N/P/K-1% (18.91 mg/g DW), and the lowest with N-1% (14.22 mg/g DW). Determining these phenolics profiles of forced chicons not only contributes to the knowledge of their nutritional value, but also provides a solid foundation for further understanding of their total phenolics formation during hydroponic forcing. (C) 2016 Elsevier Ltd. All rights reserved.

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.

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.

Callus of two grapevine cultivars was cultivated for 4 weeks on MS-medium, half strength, which was additionally supplemented with abscisic acid (ABA), (+)-catechin or a combination of both. (+)-Catechin did not elevate the pool size of the callus flavanols. Application of 3 % sucrose caused a significant increase of the callus flavanols, whereas the response to ABA was quite variable. Leaves and shoots of cv. Spatburgunder (Pinot noir) were examined histologically on the mode of flavanol deposition. The flavanols of the leaf mesophyll were spread evenly throughout the central vacuole whereas those of the outer shoot cortex were confined to special intravacuolar globules. The amorphous leaf flavanols were converted into globdar ones in the vicinity of fungal infections. Apparently, the central vacuole acquires special adjustment under the influence of infection stress. When paraquat was applied to the shoots, amorphous material was attached towards the cell walls. Additionally, the globular flavanols disappeared which could be reversed by addition of (+)-catechin. Thus, (+)-catechin diminished the oxidative damage caused by the oxygen radical producing herbicide. (+)-Catechin and epicatechin are the dominant flavanols of the leaves, whereas the dimeric proanthocyanidins B3 and B1 (PAs) predominate in the callus.

Flavanols and flavonols of mitotic and post-mitotic nuclei in needles of Taxus baccata L., Tsuga canadensis L., and slow growing dwarf genotypes of both genera are investigated histochemically. The flavanols of nuclear chromatins and in the vacuoles stain blue with the p-dimethylamino-cinnamaldehyde (DMACA) reagent. Flavonols do not react with the reagent but owing to their UV absorbance they can be seen as bright yellow pigments. The nuclei in the photomicrographs obtained by microscopy were measured for flavanols at 640 nm. The vigorously sprouting Taxus baccata L. displays the most rapid cell cycling of the needles and the nuclei reveal clear blue and white mosaic structures. The flavanol component of Taxus baccata nuclei remains relatively stable most of the growing season. The dwarf genotypes also display fairly blue stained meristematic nuclei during the intense spring flush. However, after the spring flush and towards mid-summer the nuclear flavanols slowly decrease in parallel with a gradual increase in yellow staining nuclear flavonols. A mixture of blue stained flavanols and yellow flavonols results in greenish coloration of the nuclei. The greenish tint becomes more pronounced when the parenchyma cells mature and age. At the same time, the cytoplasm of the dwarf genotypes also begins to attain a more yellow tint. This trend continues towards mid-summer and autumn, particularly in the nana genotypes. It would appear that the yellow staining flavonols are linked to restricted growth conditions. In the present study, it becomes evident that the species-typical endogenous growth potential is related to both flavanol and flavonol allocation into the nuclei. The vigorously growing species of Taxus and Tsuga have a higher capacity for recruitment of flavanols into the nuclei than the very slow growing dwarf species.

Summary Modern and sustainable production of safe and healthy high quality vegetables for human consumption is nowadays the goal of many producers. Chicory (Cichorium intybus L.) has become an important crop used as food in many countries over the past decade. In this work fi eld and glasshouse trials were conducted to compare phenolic profi les of hydroponically forced leaves and leaves produced as common agronomic practice of fi ve chicory cultivars. Total phenolic content of hydroponically forced samples ranged from 60 to 140 mg/100 g fresh weight, and those of unforced grown leaves varied between 117 and 386 mg/100 g fresh weight. Red cultivar ‘Treviso’ shows the highest phenolic content for both, unforced and forced leaves extracts. In the unforced leaves a total of 33 and in those of forced a total of 44 compounds (peaks) were used for the discrimination study.

RATIONALE: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.METHODS: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.RESULTS: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.CONCLUSIONS: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.

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.

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.

The nuclei of the tea shrub (Camellia sinensis L.) contain flavanols. Based on the dynamic, fine-tuned subnuclear changes of the flavanol pattern during the cell cycle, it can be postulated that these blue staining phenols play a role in organizing basic mechanisms of chromatin remodelling. Silenced interphase nuclei of mature parenchyma cells indicate a pronounced diffuse distribution of blue stained flavanols using the selective reagent p-dimethylaminocinnam-aldehyde (DMACA). By contrast, in activated nuclei the flavanols reflect a variable, mosaic-like blue patterning enclosed by white interchromatin spaces. Subnuclear expression of euchromatin displays relative tiny blue dots of flavanols as compared with the larger-sized blobs of heterochromatin. From metaphase to telophase, the chromosomes stain a fairly dark blue on flavanols with a more or less diffuse appearance. Those nuclei running through mitotic inter-phases from G 1 to G 2 have well-defined flavanol-free nucleoli. The flavanol pattern of meristematic chromosomes found in the tea plant is basically also valid for herbaceous plants, such as Hyacinthus romanus L., Tulipa gesneriana L. and Allium cepa L., which genuinely do not contain nuclear flavanols. This was verified by incubation of their rootlets in solutions of green tea and epigallocatechin gallate (EGCG). Also human nuclei demonstrate an easy import of added flavanols and the resulting blue stained chromatin reflects in much the same way the structural modifications as already described for the plant nuclei. Flavanols have the potential to associate to the histones of chromatin which inhibits a possible oxidation. Even small fragments of histones can aggregate to catechin as shown on the basis of kinetic measurements for the H4-core peptide HAKRKT and its acetylated product HAK(ac)RK(ac)T.

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.

The pome fruits, apple and pear, represent very important fruit crops in Europe. They are grown on 490,000 ha in 15 countries with a production of more than 11 million tons per year (FAO, 2003). This corresponds to an estimated annual value of €20 billion at consumer prices. European consumers are highly concerned about health and safety aspects of food. High quality fruits in sufficient amounts can only be produced on healthy plants. Thus, one of the most important tasks in crop production is an effective and sustainable management of plant diseases. Two of the most important diseases in European pome fruit growing are fire blight which is caused by the bacterium Erwinia amylovora, and the scab on apple and pear caused by the fungi Venturia inaequalis and V. pirina, respectively. Erwinia species cause not only severe losses in apple and pear but also attack numerous native wild species and popular ornamentals of the Rosaceae. Intensive investigations on these pome fruit diseases revealed new insights into the molecular aspects of the host–pathogen interactions, into possible mechanisms of defence by the host plants, into the genetic background of plant resistance and tolerance and into the molecular basis of virulence. Nowadays, nearly all European countries are facing similar problems regarding plant protection in pome fruit growing. Thus, co-operation at the international level is required and a network of more than 200 European scientists from 25 countries was established by the COST action 864 ‘Combining traditional and advanced strategies for plant protection in pome fruit growing’ (Acronym POME FRUIT HEALTH). The action focused on different aspects includes the (i) investigation of plant–pathogen interactions, (ii) germplasm resources and breeding, (iii) production methods and (iv) biotechnological resources. This special issue of Trees provides an overview of the fruitful yield of the interactions among European experts. A series of reviews and original articles cover actual research topics mainly in the fields of germplasm resources and breeding, host–pathogen interactions and fruit tree protection. For breeding, it is crucial to identify germplasm with heritable disease resistance. The development of efficient markers is essential for marker-assisted selection which is already used in breeding programs for scab resistance but is also attempted for fire blight resistance. Resource evaluation must include the susceptibility to fire blight (E. amylovora) and scab (V. inaequalis, V. pirina), but also to other diseases such as powdery mildew (Podosphaera leucotricha), pear rust (Gymnosporangium sabinae), brown spot of pear (Stemphylium vesicarium) and storage diseases (e.g. Penicillium expansum and Monilinia spec.). The knowledge of the genes behind a resistance and of its mode of action improves breeding success and offers prospects in gene technology. Recent advances are reviewed for the Vf resistance of apples against the scab. The possible involvement of secondary plant metabolites such as phenolic compounds in the defence of V. inaequalis is still debated. Various floral traits that may play a role in the onset and progression of E. amylovora infection are compared between susceptible and tolerant apple and pear varieties and the expression pattern of stress-related genes in E. amylovora-infected pear leaves is shown. It is not the core scope of Trees to deal with bacteria. However, it completes the examination of fire blight management. For progress in pome fruit protection against this bacterial D. Treutter (&) Unit Fruit Science, Technische Universitat Munchen, Durnast 2, 85354 Freising, Germany e-mail: dieter.treutter@wzw.tum.de

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 down-regulation of nuclear flavanols. In this article, various aspects of nuclear flavanol localization are summarized and discussed.

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, p-coumaric 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.