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.

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.

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

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.

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

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.

Nuclei of Tsuga canadensis: role of flavanols in chromatin organization.

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.

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

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 CO(2), 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.

Histochemical Analysis of Anthocyanins, Carotenoids, and Flavan-3-ols/Proanthocyanidins in Prunus domestica L. Fruits during Ripening.

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.

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.

Cell culture data indicate that quercetin and catechin may 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 x 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.

Identification of galloylated propelargonidins and procyanidins in buckwheat grain and quantification of rutin and flavanols from homostylous hybrids originating from F. esculentumxF. homotropicum.

The flavonoid rich grain of buckwheat (Fagopyrum esculentum Moench, Fam. Polygonaceae) is of high nutritional value. With the aim to improve its agronomic productivity, cultivars were crossed with the wild species F. homotropicum which, however, differs in its flavonoid content. The intention of this work was to determine the flavonoid composition in developed interspecific hybrids and to elucidate the proanthocyanidin structures. Seven compounds were purified from methanol extracts of buckwheat (Fagopyrum esculentum Moench) grains by Sephadex LH-20 column chromatography. Beside the procyanidin epicatechin-[4-8]-epicatechin-3-O-(3,4)-dimethylgallate the following propelargonidins were identified: epiafzelechin-[4-6]-epicatechin, epiafzelechin-[4-8]-epiafzelechin-[4-8]-epicatechin, epiafzelechin-[4-8]-epicatechin-3-O-(3,4-dimethyl)-gallate, epiafzelechin-[4-8]-epiafzelechin-[4-8]-epicatechin-3-O-(3,4-dimethyl)-gallate, epiafzelechin-[4-8]-epicatechin-3-O-4-methyl-gallate and epiafzelechin-[4-8]-epicatechin-p-OH-benzoate on the basis of HPLC and LC-MS/MS.

Erwinia amylovora modifies phenolic profiles of susceptible and resistant apple through its type III secretion system.

Fire blight is a disease affecting Maloideae caused by the necrogenic bacterium Erwinia amylovora, which requires the type III protein secretion system (TTSS) for pathogenicity. Profiles of methanol-extractable leaf phenolics of two apple (Malus x domestica) genotypes with contrasting susceptibility to this disease were analyzed by HPLC after infection. Some qualitative differences were recorded between the constitutive compositions of the two genotypes but in both of them dihydrochalcones accounted for more than 90% of total phenolics. Principal component analysis separated leaves inoculated with a virulent wild-type strain from those inoculated with a non-pathogenic TTSS-defective mutant or with water. The changes in levels of the various groups of phenolics in response to the virulent bacterium were similar between the two genotypes, with a significant decrease of dihydrochalcones and a significant increase of hydroxycinnamate derivatives. Differences between genotypes were, however, recorded in amplitude and kinetic of variation in these groups. Occurrence of oxidation and polymerization reactions is proposed, based on the browning process of infected tissues, but whether some by-products act in defense as toxic compounds remain to be tested. Among direct antibacterial constitutive compounds present in apple leaves, the dihydrochalcone phloretin only was found at levels close to lethal concentrations in both genotypes. However, E. amylovora exhibited the ability to stabilize this compound at sublethal levels even in the resistant apple, rejecting the hypothesis of its involvement in the resistance of this genotype.

Alterations of flavonoid biosynthesis in young grapevine (Vitis vinifera L.) leaves, flowers, and berries induced by the dioxygenase inhibitor prohexadione-Ca.

Prohexadione-Ca is a structural mimic of 2-oxoglutarate, and according to this property, it is able to inhibit dioxygenase enzymes, which require 2-oxoglutarate as a cosubstrate. Such enzymes are involved in flavonoid biosynthesis; therefore, prohexadione-Ca treatment leads to alterations in the flavonoid metabolism in grapevine tissues. Because of the fact that phenolic compounds often are responsible for enhanced plant resistance, modification of phenylpropanoid metabolism using elicitation can be considered as a new potential strategy in plant protection. The phenolic compounds were analyzed by high-performance liquid chromatography combined with chemical reaction detection. Tissue treatment induced the accumulation of unusual flavonoids, which were identified as derivatives of pentahydroxyflavanone, eriodictyol, and luteoliflavan. Concentrations of constitutive flavonoids were also affected by the bioregulator treatment. The alterations of the flavonoid profiles are discussed with respect to substrate preferences of relevant enzymes.

Microspore development of three coniferous species: affinity of nuclei for flavonoids.

The nuclear localization of blue-staining flavanols was investigated histochemically throughout microsporogenesis in yellow cypress (Callitropsis nootkatensis (D. Don) Oerst., formerly Cupressus nootkatensis), juniper (Juniperus communis L.) and yew (Taxus baccata L.). During meiotic development, both the cytoplasm and nuclei of microspores of all species contained varying amounts of flavanols; however, the flavanols were largely confined to the nuclei in microspores just released from tetrads. Quantification by HPLC analysis indicated that, in all species, catechin and epicatechin were the dominant nuclear flavanols. At the early free microspore stage, the nuclear flavanols were barely detectable in all species, but they increased fivefold on incubation in the presence of 0.1 mM benzylaminopurine (BA) or zeatin. Histochemical studies revealed that, in addition to non-fluorescing flavanols, microspores contained yellow-fluorescing flavonoids, which yielded a distinct HPLC flavonoid profile for each species. In yellow cypress, the hydrolyzed flavonoids were identified as quercetin, apigenin, kaempferol and luteolin, whereas only quercetin and myricetin were found in microspores of juniper and in anthers of yew. Application of a UV-VIS titration technique revealed that the aglycone quercetin seems to interact more strongly with histone H3 than either glycoside rutin or kaempferol.

Redirection of anthocyanin synthesis in Osteospermum hybrida by a two-enzyme manipulation strategy.

Modern biotechnology has developed powerful tools for genetic engineering and flower colours are an excellent object to study possibilities and limitations of engineering strategies. Osteospermum hybrida became a popular ornamental plant within the last 20 years. Many cultivars display rose to lilac flower colours mainly based on delphinidin-derived anthocyanins. The predominant synthesis of delphinidin derivatives is referred to a strong endogenous flavonoid 3',5'-hydroxylase (F3'5'H) activity. Furthermore, since dihydroflavonol 4-reductase (DFR) of Osteospermum does not convert dihydrokaempferol (DHK) to leucopelargonidin, synthesis of pelargonidin-based anthocyanins is naturally not realised. In order to redirect anthocyanin biosynthesis in Osteospermum towards pelargonidin derivatives, we introduced cDNAs coding for DFRs which efficiently convert DHK to LPg. But neither the expression of Gerbera hybrida DFR nor of Fragaria x ananassa DFR - the latter is characterised by an unusual high substrate preference for DHK - altered anthocyanin composition in flowers of transgenic plants. However, chemical inhibition of F3'5'H activity in ray florets of dfr transgenic plants resulted in the accumulation of pelargonidin derivatives. Accordingly, retransformation of a transgenic plant expressing Gerbera DFR with a construct for RNAi-mediated suppression of F3'5'H activity resulted in double transgenic plants accumulating predominantly pelargonidin derivatives in flowers.

Two-phase flavonoid formation in developing strawberry (Fragaria x ananassa) fruit.

Flavonoids are important secondary metabolites in strawberry as they fulfill a wide variety of physiological functions. In addition, they are beneficial for human health. Previous studies have shown for selected enzymes from the flavonoid pathway that flavonoid biosynthesis shows two peaks during fruit development. We provide optimized protocols for the determination of the activities of the key flavonoid enzymes: phenylalanine ammonia lyase, chalcone synthase/chalcone isomerase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, flavonol synthase, flavonoid 3-O-glucosyltransferase, and flavonoid 7-O-glucosyltransferase. Using these protocols we were able to demonstrate two distinct activity peaks during fruit ripening at early and late developmental stages for all enzymes with the exception of flavonol synthase. The first activity peak corresponds to the formation of flavanols, while the second peak is clearly related to anthocyanin and flavonol accumulation. The results indicate that flavonoid 3-O-glucosyltransferase activity is not essential for redirection from flavanol to anthocyanin formation in strawberry.

Piceid (resveratrol glucoside) synthesis in stilbene synthase transgenic apple fruit.

A stilbene synthase gene along with the selectable marker gene bar for herbicide resistance was transferred via Agrobacterium tumefaciens mediated transformation into apple (Malus domesticaBorkh.) cvs. 'Elstar' and 'Holsteiner Cox'. The stilbene synthase catalyzes the conversion of 1 molecule of p-coumaroyl-CoA and 3 molecules of malonyl-CoA into 3,4',5-trihydroxystilbene, commonly known as resveratrol. This phytoalexin has implications in both phytopathology and human health. Greenhouse-grown transgenic and nontransformed control plants were grafted onto dwarfing rootstock M27. Flowering and fruiting occurred within the following years, offering the opportunity to analyze transgenic apple fruit and fertility of transgenic plants as well as inheritance of the transgenes into the seedling progeny. Molecular analysis revealed that the stilbene synthase is expressed in transgenic plants and in the skin and flesh of transgenic apple fruit. After formation, resveratrol is modified by the addition of a hexose sugar. The resulting component was characterized as piceid. With the aim of characterizing the influence of the novel biosynthetic pathway on the accumulation of other phenolic compounds naturally present in apple fruit, the amounts of flavanols, flavonols, phloretin derivatives and hydroxycinnamic acids in wild type and transgenic fruit were determined by HPLC. In all investigated transformed lines that accumulated piceid, no negative correlation between levels of piceid and the above-mentioned compounds was observed, except for the flavonol contents, which slightly decreased. Inheritance of the transgenes was confirmed in the seedling progeny, which were obtained after pollination of transgenic plants with nontransgenic pollen and vice versa after pollination of nontransgenic plants with pollen obtained from transgenic plants. The fertility of stilbene synthase transgenic plants was demonstrated. To the authors' knowledge this is the first time that data are available on piceid synthesis in transgenic apple fruit and the effects of its accumulation on levels of other phenolic compounds present in the fruit.

Simulated digestion of Vitis vinifera seed powder: polyphenolic content and antioxidant properties.

There is increasing evidence that reactive oxygen species arising from several enzymatic reactions are mediators of inflammatory events. Plant preparations have the potential for scavenging such reactive oxygen species. Flavans and procyanidins are bioavailable and stable during the process of cooking. This study used conditions that mimicked digestion of Vitis vinifera seed powder in the stomach (acidic preparation) and small intestine (neutral preparation). The flavonoids of these two preparations were released during simulated digestion and were determined with HPLC analysis. Biochemical model reactions relevant for the formation of reactive oxygen species in vivo at inflammatory sites were used to determine the antioxidant properties of the two preparations. The inhibition of the indicator reaction for the formation of reactive oxygen species represents a potential mechanism of the physiological activity of the corresponding preparation. The results of this work show clearly that the polyphenols released during the simulated digestion of the two preparations have good scavenging potential against superoxide radicals, hydroxyl radicals, and singlet oxygen. They protect low-density lipoprotein against copper-induced oxidation due to the copper-chelating properties and their chain-breaking abilities in lipid peroxidation.

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

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.

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

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.

Formation of novel flavonoids in apple (Malusxdomestica) treated with the 2-oxoglutarate-dependent dioxygenase inhibitor prohexadione-Ca.

Novel flavonoids were formed in young leaves of apple (Malusxdomestica) after treatment with the dioxygenase inhibitor prohexadione-Ca, which is known to reduce the incidence and severity of fire blight caused by Erwinia amylovora and other plant diseases. The compounds were isolated and identified as luteoliflavan, luteoliflavan 5-glucoside, eriodictyol 7-glucoside and 6"-O-trans-p-coumaroyleriodictyol 3'-glucoside. These flavonoids represent a novel biosynthetic pathway in apple leading to the formation of 3-deoxyflavans. Concomitantly, the content of regularly occurring phenylpropanoids is also influenced by prohexadione-Ca with increasing amounts of hydroxycinnamic acids and decreasing flavan-3-ols and flavonols. The altered flavonoid metabolism may be related to the lowered pathogen incidence though the isolated novel flavonoids do not exhibit antibacterial activity.

Induction of antimicrobial 3-deoxyflavonoids in pome fruit trees controls fire blight.

Fire blight, a devastating bacterial disease in pome fruits, causes severe economic losses worldwide. Hitherto, an effective control could only be achieved by using antibiotics, but this implies potential risks for human health, livestock and environment. A new approach allows transient inhibition of a step in the flavonoid pathway, thereby inducing the formation of a novel antimicrobial 3-deoxyflavonoid controlling fire blight in apple and pear leaves. This compound is closely related to natural phytoalexins in sorghum. The approach does not only provide a safe method to control fire blight: Resistance against different pathogens is also induced in other crop plants.

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

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.