Multi-omics revealed molecular mechanism of biphenyl phytoalexin formation in response to yeast extract-induced oxidative stress in Sorbus aucuparia suspension cells.

KEY MESSAGE: Yeast extract-induced oxidative stress in Sorbus aucuparia suspension cells leads to the biosynthesis of various hormones, which activates specific signaling pathways that augments biphenyl phytoalexin production. Pathogen incursions pose a significant threat to crop yield and can have a pronounced effect on agricultural productivity and food security. Biphenyl phytoalexins are a specialized group of secondary metabolites that are mainly biosynthesized by Pyrinae plants as a defense mechanism against various pathogens. Despite previous research demonstrating that biphenyl phytoalexin production increased dramatically in Sorbus aucuparia suspension cells (SASCs) treated with yeast extract (YE), the underlying mechanisms remain poorly understood. To address this gap, we conducted an in-depth, multi-omics analysis of transcriptome, proteome, and metabolite (including biphenyl phytoalexins and phytohormones) dynamics in SASCs exposed to YE. Our results indicated that exposure to YE-induced oxidative stress in SASCs, leading to the biosynthesis of a range of hormones, including jasmonic acid (JA), jasmonic acid isoleucine (JA-ILE), gibberellin A4 (GA4), indole-3-carboxylic acid (ICA), and indole-3-acetic acid (IAA). These hormones activated specific signaling pathways that promoted phenylpropanoid biosynthesis and augmented biphenyl phytoalexin production. Moreover, reactive oxygen species (ROS) generated during this process also acted as signaling molecules, amplifying the phenylpropanoid biosynthesis cascade through activation of the mitogen-activated protein kinase (MAPK) pathway. Key genes involved in these signaling pathways included SaBIS1, SaBIS2, SaBIS3, SaPAL, SaB4H, SaOMT, SaUGT1, SaLOX2, SaPR1, SaCHIB1, SaCHIB2 and SaCHIB3. Collectively, this study provided intensive insights into biphenyl phytoalexin accumulation in YE-treated SASCs, which would inform the development of more efficient disease-resistance strategies in economically significant cultivars.

[Triterpenoids in Sorbus pohuashanensis suspension cell treated with yeast extract].

This study induced biological stress in Sorbus pohuashanensis suspension cell(SPSC) with yeast extract(YE) as a bio-tic elicitor and isolated and identified secondary metabolites of triterpenoids produced under stress conditions. Twenty-six triterpenoids, including fifteen ursane-type triterpenoids(1-15), two 18,19-seco-ursane-type triterpenoids(16-17), four lupine-type triterpenoids(18-21), two cycloartane-type triterpenoids(22-23), and three squalene-type triterpenoids(24-26), were isolated and purified from the methanol extract of SPSC by chromatography on silica gel, MCI, Sephadex LH-20, and MPLC. Their structures were elucidated by spectroscopic analyses. All triterpenoids were isolated from SPSC for the first time and 22-O-acetyltripterygic acid A(1) was identified as a new compound. Selected compounds were evaluated for antifungal, antitumor, and anti-inflammatory activities, and compound 1 showed an inhibitory effect on NO production in LPS-induced RAW264.7 cells.

[The Antioxidant and Geroprotective Properties of an Extract of Mountain Ash (Sorbus aucuparia L.) Fruits].

Plant polyphenols are characterized by a wide range of biological activities, including antioxidant properties, and have a high geroprotective potential. The purpose of this work was to investigate the effect of the extract of rowan berries (Sorbus aucuparia L.) on the lifespan and stress resistance of Drosophila melanogaster with the identification of possible mechanisms of its biological activity. It has been established that an ethanol extract of S. aucuparia berries, the main components of which are rutin and cyanidin-3-rutinoside, has a pronounced antioxidant activity in vitro. At the same time, treatment with rowan berry extract increased the resistance of D. melanogaster males to starvation, but reduced resistance to hyperthermia. In females, the extract reduced resistance to oxidative stress but increased resistance to hyperthermia. The effects of rowan berry extract on longevity depended both on its concentration and on the sex of fruit flies. In response to treatment with rowan berry extract, D. melanogaster males and females showed slight differences in the background level of expression of cellular stress response genes, including heat shock genes (hsp27, hsp68, and hsp83), oxidative stress resistance genes (hif1, nrf2, and sod1), circadian rhythm genes (clk and per), and the longevity gene sirt1, which may explain the differences in the observed effects.

Exogenous Ethylene Alleviates the Inhibition of Sorbus pohuashanensis Embryo Germination in a Saline-Alkali Environment (NaHCO3).

Saline-alkali stress is a major environmental stress affecting the growth and development of plants such as Sorbus pohuashanensis. Although ethylene plays a crucial role in plant response to saline-alkaline stress, its mechanism remains elusive. The mechanism of action of ethylene (ETH) may be related to the accumulation of hormones, reactive oxygen species (ROS), and reactive nitrogen species (RNS). Ethephon is the exogenous ethylene donor. Therefore, for the present study we initially used different concentrations of ethephon (ETH) to treat S. pohuashanensis embryos and identified the best treatment concentration and method to promote the release of dormancy and the germination of S. pohuashanensis embryos. We then analyzed the physiological indexes, including endogenous hormones, ROS, antioxidant components, and reactive nitrogen, in embryos and seedlings to elucidate the mechanism via which ETH manages stress. The analysis showed that 45 mg/L was the best concentration of ETH to relieve the embryo dormancy. ETH at this concentration improved the germination of S. pohuashanensis by 183.21% under saline-alkaline stress; it also improved the germination index and germination potential of the embryos. Further analysis revealed that ETH treatment increased the levels of 1-aminocyclopropane-1-carboxylic acid (ACC), gibberellin (GA), soluble protein, nitric oxide (NO), and glutathione (GSH); increased the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), nitrate reductase (NR), and nitric oxide synthase (NOS); and decreased the levels of abscisic acid (ABA), hydrogen peroxide (H2O2), superoxide anion, and malondialdehyde (MDA) of S. pohuashanensis under saline-alkali stress. These results indicate that ETH mitigates the inhibitory effects of saline-alkali stress and provides a theoretical basis by which to establish precise control techniques for the release of seed dormancy of tree species.

Metabolic and Transcriptional Stress Memory in Sorbus pohuashanensis Suspension Cells Induced by Yeast Extract.

Plant stress memory can provide the benefits of enhanced protection against additional stress exposure. Here, we aimed to explore the responses of recurrent and non-recurrent yeast extract (YE) stresses in Sorbus pohuashanensis suspension cells (SPSCs) at metabolomics and transcriptional levels. Biochemical analyses showed that the cell wall integrity and antioxidation capacity of SPSCs in the pretreated group were evidently improved. Metabolic analysis showed that there were 39 significantly altered metabolites in the pretreated group compared to the non-pretreated group. Based on the transcriptome analysis, 219 differentially expressed genes were obtained, which were highly enriched in plant-pathogen interaction, circadian rhythm-plant, oxidative phosphorylation, and phenylpropanoid biosynthesis. Furthermore, the correlation analysis of the transcriptome and metabolome data revealed that phenylpropanoid biosynthesis involved in the production of biphenyl phytoalexins may play a critical role in the memory response of SPSC to YE, and the key memory genes were also identified, including PAL1, BIS1, and BIS3. Collectively, the above results demonstrated that the memory responses of SPSC to YE were significant in almost all levels, which would be helpful for better understanding the adaptation mechanisms of medicinal plants in response to biotic stress, and laid a biotechnological foundation to accumulate favorable antimicrobial drug candidates from plant suspension cells.

Effect of Iron Source and Medium pH on Growth and Development of Sorbus commixta In Vitro.

Sorbus commixta is a valuable hardwood plant with a high economical value for its medicinal and ornamental qualities. The aim of this work was to investigate the effects of the iron (Fe) source and medium pH on the growth and development of S. commixta in vitro. The Fe sources used, including non-chelated iron sulfate (FeSO4), iron ethylenediaminetetraacetic acid (Fe-EDTA), and iron diethylenetriaminepentaacetic acid (Fe-DTPA), were supplemented to the Multipurpose medium with a final Fe concentration of 2.78 mg·L-1. The medium without any supplementary Fe was used as the control. The pH of the agar-solidified medium was adjusted to either 4.70, 5.70, or 6.70. The experiment was conducted in a culture room for six weeks with 25 °C day and night temperatures, and a 16-h photoperiod with a light intensity of 50 mmol·m-2·s-1 photosynthetic photon flux density (PPFD). Both the Fe source and pH affected the growth and development of the micropropagated plants in vitro. The leaves were greener in the pH 4.70 and 5.70 treatments. The tissue Fe content decreased with the increase of the medium pH. The leaf chlorophyll content was similar between plants treated with FeSO4 and those with Fe-EDTA. The numbers of the shoots and roots of plantlets treated with FeSO4 were 2.5 and 2 times greater than those of the control, respectively. The fresh and dry weights of the shoot and the root were the greatest for plants treated with Fe-EDTA combined with pH 5.70. The calcium, magnesium, and manganese contents in the plantlets increased in the pH 5.70 treatments regardless of the Fe source. Supplementary Fe decreased the activity of ferric chelate reductase. Overall, although the plantlets absorbed more Fe at pH 4.70, Fe-EDTA combined with pH 5.70 was found to be the best for the growth and development of S. commixta in vitro.

Sorbus aucuparia and Sorbus aria as a Source of Antioxidant Phenolics, Tocopherols, and Pigments.

Due to its nutritive and medicinal properties, berries of some Sorbus species are used for the preparation of jams and jelly as well as in traditional medicine. On the other hand, their chemical composition is not much studied especially of those grown in Balkan Peninsula. We have analyzed individual phenolics, tocopherols, carotenoids and chlorophylls using HPLC in berries from Sorbus aucuparia and Sorbus aria collected in different localities in Serbia and Montenegro together with the amounts of total phenolics and proanthocyanidins as well as their radical scavenging activity against DPPH radical. Berries of S. aucuparia were richer source of polyphenolics in comparision with S. aria and, regardless the species and locality, caffeoylquinic acids such as neochlorogenic and chlorogenic acid were the most abundant compounds. Among analyzed tocopherols the most abundant in all samples was α-tocopherol (0.48 - 19.85 µg/g dw) as it was ß-carotene among carotenoids (mean concentration of 0.98 µg/g dw in S. aucuparia and 0.40 µg/g dw in S. aria, respectively). Correlation between total phenolics and DPPH radical scavenging activity was noticed. Our study represents comprehensive report on chemical composition of S. aucuparia and S. aria which could contribute to a better understanding of their quality.

Study of the Accumulation of Toxic and Essential Ultra-Trace Elements in Fruits of Sorbus domestica L.

In the present work, the accumulation of selected toxic and essential ultra-trace elements in fruits of service tree (Sorbus domestica L.) were determined depending on harvest time. Samples were collected from the same sampling area in two different years and within one year in September and October (maturity state). Harvesting the fruits in the same area excludes the influence of metals taken up via roots, thus the impact of airborne contamination by heavy metal translocation can be studied. All samples were dried and digested using an acidic microwave assisted digestion system prior to quantification by inductively coupled plasma-sector field mass spectrometry (ICP-SFMS). The elements chosen were Arsenic and Cadmium as well as Lithium, Molybdenum, and Selenium. The Arsenic content rose with maturity in mesocarp. Cadmium found in the mesocarp was unaffected by ripeness. For Selenium and Molybdenum, no statistically significant effect of ripeness could be found on their content in mesocarp. Lithium could not be detected in the majority of fruit samples. Differences between the metal concentrations based on the year of harvest were found for Arsenic, Molybdenum, and Selenium, depending on precipitation. The drier the season, the more Arsenic was accumulated. For Molybdenum and Selenium, the opposite effect was observed.

Biphenyl Phytoalexin in Sorbus pohuashanensis Suspension Cell Induced by Yeast Extract.

Biphenyls are unique phytoalexins de novo synthesized in plants in response to pathogen attack. These compounds are found in Maloideae, a subfamily of the Rosaceae. The anti-microbial activities of biphenyls have been reported in a number of studies and they appear to represent an important defense strategy against pathogens common in the Maloideae, such as species in Malus, Pyrus, Sorbus, and Chaenomeles. Here, cell suspension cultures of Sorbus pohuashanensis were established to study biphenyl phytoalexins formation after yeast extract (YE) treatment. An ultra-performance liquid chromatography (UPLC) method coupled with quadrupole time of flight mass spectrometry (Q-TOF-MS) LC-MS/MS was applied to determine the time course of these biphenyl biomarkers accumulation in YE-treated S. pohuashanensis suspension cells. The results of quantitative analyses show the content of Noraucuparin, 2'-Hydroxyaucuparin, and their glycosides initially increased, then decreased over time. The Noraucuparin content reached its highest (225.76 µg·g(-1)) at 18 h after treatment, 6 hours earlier than that of Noraucuparin 5-O-ß-d-glucopyranoside. The content of 2'-Hydroxyaucuparin reached its highest (422.75 µg·g(-1)) at 30 h after treatment, also earlier than that of its glycoside. The understanding of phytoalexin metabolism in this study may provide a basis for improving Maloideae resistance to pathogens.

Stem CO2 efflux in six co-occurring tree species: underlying factors and ecological implications.

Stem respiration plays a role in species coexistence and forest dynamics. Here we examined the intra- and inter-specific variability of stem CO2 efflux (E) in dominant and suppressed trees of six deciduous species in a mixed forest stand: Fagus sylvatica L., Quercus petraea [Matt.] Liebl, Quercus pyrenaica Willd., Prunus avium L., Sorbus aucuparia L. and Crataegus monogyna Jacq. We conducted measurements in late autumn. Within species, dominants had higher E per unit stem surface area (Es ) mainly because sapwood depth was higher than in suppressed trees. Across species, however, differences in Es corresponded with differences in the proportion of living parenchyma in sapwood and concentration of non-structural carbohydrates (NSC). Across species, Es was strongly and NSC marginally positively related with an index of drought tolerance, suggesting that slow growth of drought-tolerant trees is related to higher NSC concentration and Es . We conclude that, during the leafless period, E is indicative of maintenance respiration and is related with some ecological characteristics of the species, such as drought resistance; that sapwood depth is the main factor explaining variability in Es within species; and that the proportion of NSC in the sapwood is the main factor behind variability in Es among species.

[Effects of different types of elicitors on secondary metabolism of Sorbus aucuparia cell cultures].

OBJECTIVE: To investigate the effects of different types of elicitors on the secondary metabolism of Sorbus aucuparia cell). METHODS: Added three different types of elicitors [ yeast extract ( YE), heavy metal ions and allelochemicals] to the SACC, and deleted the changes in the secondary metabolism of SACC using GC-MS. RESULTS: Treatment of SACC with yeast extract in- tion of biphenyls, and some long-chain fatty acids and steroids disappeared; The addition of heavy metal ions induced the g-chain fatty acids, and some steroids and triterpenes disappeared; And the addition of allelochemicals led to the disappearance long-chain fatty acids, steroids and triterpenes. CONCLUSION: These three types of elicitors have different effects on the secondary metabolism of SACC, but different elicitors of the same type act almost the same, which implies different kinds of elicitors would activate different pathways of secondary metabolism in SACC.

[Mechanism exploration on synthesis of secondary metabolites in Sorbus aucuparia cell cultures treated with yeast extract].

Suspension cultures cell of Sorbus aucuparia (SASC) was used as materials, the changes of physiological and biochemical indexes of SASC after treatment with yeast extract (YE) were detected, and the synthetic mechanism of secondary metabolites in SASC treated with YE was preliminarily explored. The results were as follows: under the assay conditions, SASC was induced to synthesize five biphenyl compounds, and these compounds content changed differently with induction time prolonging; YE treatment inhibited cell growth, the culture medium pH was gradually reduced after treatment; water-soluble protein content showed a trend of slow decline, which was significantly increased in YE treatment group (YE group) compared with the control group (CK group), the maximum relative content was 147.76% in contrast with CK group; both YE group and CK group were extracellular Ca2+ flow influx, but the YE group flow was significantly slow than CK group. The results indicate that YE induced the cells in a stress state, which was not conducive to the growth of cells and forced the cells to synthesize biphenyl compounds against external stress; water-soluble protein may serve as intracellular enzymes involved in the synthesis of compounds regulation; Ca2+ may as signal molecule mediate cell signal transduction respond to YE stress.

Chlorophyll fluorescence induction, chlorophyll content, and chromaticity characteristics of leaves as indicators of photosynthetic apparatus senescence in arboreous plants.

Parameters of chlorophyll fluorescence induction (CFI) are widely used for assessment of the physiological state of higher plant leaves in biochemical, physiological, and ecological studies and in agricultural applications. In this work we have analyzed data on variability of some CFI parameters - ΦPSII(max) = Fv/Fm (relative value of variable fluorescence), qNPQ (non-photochemical quenching coefficient), RFd ("vitality index") - in autumnal leaves of ten arboreous plant species of the temperate climatic zone. The correlation between the chlorophyll content in the leaves and fluorescence parameters characterizing photosynthetic activity is shown for two representative species, the small-leaved linden Tilia cordata and the rowan tree Sorbus aucuparia. During the period of mass yellowing of the leaves, the ΦPSII(max) value can be used as an adequate characteristic of their photochemical activity, while in summer the qNPQ or RFd values are more informative. We have established a correlation between the ΦPSII(max) value, which characterizes the maximal photochemical activity of the photosystem II, and "chromaticity coordinates" of a leaf characterizing its color features. The chromaticity coordinates determined from the optical reflection spectra of the leaves serve as a quantitative measure of their hues, and this creates certain prerequisites for a visual expert assessment of the physiological state of the leaves.

Seasonal patterns of leaf photosynthetic and secondary xylem vascular traits in current-year stems of three Sorbus species with contrasting growth habits.

Seasonal effects of environmental variables on photosynthetic activity and secondary xylem formation provide data to demonstrate how environmental factors together with leaf ageing during the season control tree growth. In this study, we assessed physiological responses in photosynthetic behaviour to seasonal climate changes, and also identified seasonal differences in vascular traits within differentiating secondary xylem tissue from three diploid species of the taxonomically complex genus Sorbus. From sampling day 150, a clear physiological segregation of S. chamaemespilus from S. torminalis and S. aria was evident. The shrubby species S. chamaemespilus could be distinguished by a higher photosynthetic capacity between days 150 and 206. This was reflected in its associations with net CO2 assimilation rate (PN), maximum photochemical efficiency of PSII (F(v)/F(m)), variable-to-initial fluorescence ratio (F(v)/F(0)), potential electron acceptor capacity ('area') in multivariate space, and also its associations with log-transformed vessel area and log-transformed relative conductivity between days 239 and 268. The maximum segregation and differentiation among the examined Sorbus species was on sampling day 206. The largest differences between S. torminalis and S. aria were found on day 115, when the latter species clearly showed closer associations with high values of vessel density and transpiration (E). Sampling day clusters were arranged along an arch-like gradient that reflected the positioning of the entire growing season in multivariate space. This arch-like pattern was most apparent in the case of S. chamaemespilus, but was also observed in S. torminalis and S. aria.

Biosynthesis of the biphenyl phytoalexin aucuparin in Sorbus aucuparia cell cultures treated with Venturia inaequalis.

Aucuparin is the most widely distributed biphenyl phytoalexin in the rosaceous subtribe Pyrinae, which includes the economically important fruit trees apple and pear. The biphenyl scaffold is formed by biphenyl synthase, which catalyzes biosynthesis of 3,5-dihydroxybiphenyl. Conversion of this precursor to aucuparin (3,5-dimethoxy-4-hydroxybiphenyl) was studied in cell cultures of Sorbus aucuparia after treatment with an elicitor preparation from the scab-causing fungus Venturia inaequalis. The sequence of the biosynthetic steps detected was O-methylation - 4-hydroxylation - O-methylation. The two alkylation reactions were catalyzed by distinct methyltransferases, which differed in pH and temperature optima as well as stability. Biphenyl 4-hydroxylase was a microsomal cytochrome P450 monooxygenase, whose activity was appreciably decreased by the addition of established P450 inhibitors. When fed to V. inaequalis-treated S. aucuparia cell cultures, radioactively labeled 3,5-dihydroxybiphenyl was not only incorporated into aucuparin but also into the dibenzofuran eriobofuran, the accumulation of which paralleled that of aucuparin. However, biphenyl 2'-hydroxylase activity proposed to be involved in dibenzofuran formation was detected in neither microsomes nor cell-free extracts in the presence of NADPH and 2-oxoglutarate, respectively. Nevertheless, a basis for studying biphenyl biosynthesis at the gene level is provided.

Viscum album versus host (Sorbus aucuparia) as bioindicators of urban areas with various levels of pollution.

In this investigation we focus on the concentration of elements in Viscum album and its host (Sorbus aucuparia) as bioindicators of urban pollution. These broadly widespread species, very common in polluted areas may provide important information to monitor environmental quality throughout the year, especially for V. album. Cd, Co, Cr, Cu, Fe, Li, Mg, Mn, Ni, Pb and Zn concentrations were measured in the leaves and soil of the tree S. aucuparia as well as in V. album, a semi-parasite living on this tree species. The tree and the semi-parasite were studied in the urban environment of Olawa (SW Poland). This area was selected because of the influence of a zinc smelter on the level of metal pollution of soil and plants and to compare the ability of S. aucuparia and V. album to accumulate metals. V. album appeared to be a better bioaccumulator of Cd, Ni, Pb and Zn and a weaker accumulator of Co than S. aucuparia in less polluted sites of Olawa. S. aucuparia was a better bioaccumulator of Cd, Ni, Pb and Zn and a weaker accumulator of Co than V. album in more polluted sites. Cluster analysis of Cd, Co, Cr, Cu, Fe, Li, Mg, Mn, Ni, Pb and Zn concentrations in plants distinguished sites with lower and higher pollution levels which suggests a possibility of using these species for bioindication. However, the ratio of metals in V. album to S. aucuparia was different depending on the pollution level.

Endogenous hydrogen peroxide is a key factor in the yeast extract-induced activation of biphenyl biosynthesis in cell cultures of Sorbus aucuparia.

Biphenyls are unique phytoalexins produced by plants belonging to Pyrinae, a subtribe of the economically important Rosaceae family. The formation of aucuparin, a well-known biphenyl, is induced by yeast extract (YE) in cell cultures of Sorbus aucuparia. However, the molecular mechanism underlying YE-induced activation of biphenyl biosynthesis remains unknown. Here we demonstrate that the addition of YE to the cell cultures results in a burst of reactive oxygen species (ROS; H(2)O(2) and O(2) (-)), followed by transcriptional activation of the biphenyl synthase 1 gene (BIS1) encoding the key enzyme of the biphenyl biosynthetic pathway and aucuparin accumulation. Pretreatment of the cell cultures with ROS scavenger dihydrolipoic acid and NADPH oxidase-specific inhibitor diphenylene iodonium abolished all of the above YE-induced biological events. However, when the cell cultures was pretreated with superoxide dismutase specific inhibitor N,N-diethyldithiocarbamic acid, although O(2) (-) continued to be generated, the H(2)O(2) accumulation, BIS1 expression and aucuparin production were blocked. Interestingly, exogenous supply of H(2)O(2) in the range of 0.05-10 mM failed to induce aucuparin accumulation. These results indicate that endogenous generation of H(2)O(2) rather than that of O(2) (-) is a key factor in YE-induced accumulation of biphenyl phytoalexins in cell cultures of S. aucuparia.

Differential effect of elicitors on biphenyl and dibenzofuran formation in Sorbus aucuparia cell cultures.

The Rosaceous subtribe Pyrinae (formerly subfamily Maloideae) is well-known for its economically important fruit trees, such as apple and pear, and also includes Sorbus aucuparia. Elicitor-treated S. aucuparia cell cultures are used to study the biosynthesis of the Pyrinae-specific phytoalexins, biphenyls and dibenzofurans. Three biphenyls (aucuparin, noraucuparin, 2'-hydroxyaucuparin) and a dibenzofuran (eriobofuran) were isolated and structure elucidated using GC-MS and NMR. A second dibenzofuran of low abundance was tentatively assigned as noreriobofuran. Treatment of S. aucuparia cell cultures with yeast extract induced the formation of aucuparin as the major phytoalexin. In contrast, addition of preparations from the fire blight bacterium, Erwinia amylovora, and the scab-causing fungus, Venturia inaequalis, resulted in accumulation of eriobofuran as the major inducible constituent. Methyl jasmonate was a poor elicitor. The observations are suggestive of a biogenic relationship between biphenyls and dibenzofurans. Elicitor-treated S. aucuparia cell cultures provide an interesting in vitro system for studying biphenyl and dibenzofuran metabolism in the economically valuable Pyrinae.

A novel 4-hydroxycoumarin biosynthetic pathway.

Coumarin forms in melilotoside (trans-ortho-coumaric acid glucoside)-containing plant species upon cell damage. In moldy melilotoside-containing plant material, trans-ortho-coumaric acid is converted by fungi to 4-hydroxycoumarin, two molecules of which spontaneously combine with formaldehyde to give dicoumarol. Dicoumarol causes internal bleeding in livestock and is the forerunner of the warfarin group of medicinal anticoagulants. Here, we report 4-hydroxycoumarin formation by biphenyl synthase (BIS). Two new BIS cDNAs were isolated from elicitor-treated Sorbus aucuparia cell cultures. The encoded isoenzymes preferred ortho-hydroxybenzoyl (salicoyl)-CoA as a starter substrate and catalyzed a single decarboxylative condensation with malonyl-CoA to give 4-hydroxycoumarin. When elicitor-treated S. aucuparia cell cultures were fed with the N-acetylcysteamine thioester of salicylic acid, 4-hydroxycoumarin accumulated in the culture medium. Incubation of the BIS isoenzymes with benzoyl-CoA and malonyl-CoA resulted in the formation of 3,5-dihydroxybiphenyl which is the precursor of the phytoalexins of the Maloideae.