African Hydroclimate During the Early Eocene From the DeepMIP Simulations.

The early Eocene (∼56-48 Myr ago) is characterized by high CO2 estimates (1,200-2,500 ppmv) and elevated global temperatures (∼10°C-16°C higher than modern). However, the response of the hydrological cycle during the early Eocene is poorly constrained, especially in regions with sparse data coverage (e.g., Africa). Here, we present a study of African hydroclimate during the early Eocene, as simulated by an ensemble of state-of-the-art climate models in the Deep-time Model Intercomparison Project (DeepMIP). A comparison between the DeepMIP pre-industrial simulations and modern observations suggests that model biases are model- and geographically dependent, however, these biases are reduced in the model ensemble mean. A comparison between the Eocene simulations and the pre-industrial suggests that there is no obvious wetting or drying trend as the CO2 increases. The results suggest that changes to the land sea mask (relative to modern) in the models may be responsible for the simulated increases in precipitation to the north of Eocene Africa. There is an increase in precipitation over equatorial and West Africa and associated drying over northern Africa as CO2 rises. There are also important dynamical changes, with evidence that anticyclonic low-level circulation is replaced by increased south-westerly flow at high CO2 levels. Lastly, a model-data comparison using newly compiled quantitative climate estimates from paleobotanical proxy data suggests a marginally better fit with the reconstructions at lower levels of CO2.

Identification of New Natural Sources of Flavour and Aroma Metabolites from Solid-State Fermentation of Agro-Industrial By-Products.

Increasing consumer demand for natural flavours and fragrances has driven up prices and increased pressure on natural resources. A shift in consumer preference towards more sustainable and economical sources of these natural additives and away from synthetic production has encouraged research into alternative supplies of these valuable compounds. Solid-state fermentation processes support the natural production of secondary metabolites, which represents most flavour and aroma compounds, while agro-industrial by-products are a low-value waste stream with a high potential for adding value. Accordingly, four filamentous fungi species with a history of use in the production of fermented foods and food additives were tested to ferment nine different agro-industrial by-products. Hundreds of volatile compounds were produced and identified using headspace (HS) solid-phase microextraction (SPME) coupled to gas chromatography-mass spectrometry (GC-MS). Four compounds of interest, phenylacetaldehyde, methyl benzoate, 1-octen-3-ol, and phenylethyl alcohol, were extracted and quantified. Preliminary yields were encouraging compared to traditional sources. This, combined with the low-cost substrates and the high-value natural flavours and aromas produced, presents a compelling case for further optimisation of the process.

Metabolomics Approach to Assess the Relative Contributions of the Volatile and Non-volatile Composition to Expert Quality Ratings of Pinot Noir Wine Quality.

Wine flavor and quality are determined by the assessment of multiple sensory stimuli, including aroma, taste, and mouthfeel. It is therefore important to consider the contribution of as many metabolites as possible when attempting to relate wine composition to quality. In this study, partial least squares regression of the volatile (untargeted headspace solid-phase microextraction coupled with gas chromatography time-of-flight mass spectrometry), non-volatile (untargeted reverse-phase ultra-high-performance liquid chromatography mass spectrometry), and combined metabolite profiles were used to predict Pinot Noir wine quality ratings as assessed by experts. Non-volatile metabolite profiles predicted wine quality ratings better than volatile metabolite profiles, suggesting that the non-volatile composition of Pinot Noir wines contributes to quality perception to a greater extent than the volatile composition. This was underscored by descriptive sensory analysis, which found that taste and mouthfeel attributes were better correlated with wine quality ratings than aroma attributes. Important predictors of Pinot Noir wine quality were also characterized. Some new relationships between wine metabolites and quality ratings were found: dipeptides and unsaturated fatty acids were positively related to Pinot Noir wine quality, while N-(3-methylbutyl)acetamide and xanthine were negatively associated.

Dietary palaeoecology of an Early Cretaceous armoured dinosaur (Ornithischia; Nodosauridae) based on floral analysis of stomach contents.

The exceptionally well-preserved holotype of the armoured dinosaur Borealopelta markmitchelli (Ornithischia; Nodosauridae) from the Early Cretaceous (Clearwater Formation) of northern Alberta preserves a distinct mass within the abdominal cavity. Fourteen independent criteria (including: co-allochthony, anatomical position, gastroliths) support the interpretation of this mass as ingested stomach contents-a cololite. Palynomorphs in the cololite are a subset of the more diverse external sample. Analysis of the cololite documents well-preserved plant material dominated by leaf tissue (88%), including intact sporangia, leaf cross-sections and cuticle, but also including stems, wood and charcoal. The leaf fraction is dominated (85%) by leptosporangiate ferns (subclass Polypodiidae), with low cycad-cycadophyte (3%) and trace conifer foliage. These data represent the most well-supported and detailed direct evidence of diet in an herbivorous dinosaur. Details of the dietary palaeoecology of this nodosaur are revealed, including: selective feeding on ferns; preferential ingestion of leptosporangiate ferns to the exclusion of Osmundaceae and eusporangiate ferns such as Marattiaceae; and incidental consumption of cycad-cycadophyte and conifer leaves. The presence of significant (6%) charcoal may represent the dietary use of recently burned conifer forest undergoing fern succession, early evidence of a fire succession ecology, as is associated with many modern large herbivores.

Anticancer Characteristics of Fomitopsis pinicola Extract in a Xenograft Mouse Model-a Preliminary Study.

Introduction: Medicinal mushrooms have been used for the treatment of diseases and general promotion of health for many centuries. Recent pharmacological research into medicinal mushrooms has identified various therapeutic properties, with applications in modern medicine.Aim: To evaluate the anti-cancer activities of Fomitopsis pinicola (F. pinicola) alcoholic extract in an in vivo setting.Methods: The anti-tumour effect of the F. pinicola extract was tested in a xenograft immune-compromised Rag-1 mouse model. This was followed by RT-PCR and metabolomics analyses.Results: There were no observable differences in tumor growth between treated and non-treated groups. The bioactive components were not detected in the mouse plasma or the tumor site.Conclusions: The extract was poorly absorbed; this is likely due to the timing of treatment, dosage levels and modifications made to the extract where the alcohol-based solvent was replaced with water. This, in combination with fractionation studies which identified most anti-cancer compounds to be hydrophobic, largely explained the lack of anti-cancer activities in vivo.

Reference samples guide variable selection for correlation of wine sensory and volatile profiling data.

The relationship between wine flavour and wine volatile composition is well recognised, however with thousands of compounds in wine the exact nature of individual contributions may be hard to determine due to synergistic and masking effects. Untargeted chemical analyses coupled with descriptive sensory and partial least squares regression modelling can help unravel interactions to identify groups of compounds that contribute to sensory properties. Variable selection is often applied prior to modelling to eliminate irrelevant variables. In this study, sensory references used to train the sensory panel were chemically analysed and employed to reduce the number of variables used to construct the models. This novel variable selection approach was compared against the inclusion of all variables and the most commonly applied variable selection method - analysis of variance. Models constructed from variables present in sensory references performed similarly to other models and identified interesting groups of compounds to investigate further.

The Apoplastic Secretome of Trichoderma virens During Interaction With Maize Roots Shows an Inhibition of Plant Defence and Scavenging Oxidative Stress Secreted Proteins.

In Nature, almost every plant is colonized by fungi. Trichoderma virens is a biocontrol fungus which has the capacity to behave as an opportunistic plant endophyte. Even though many plants are colonized by this symbiont, the exact mechanisms by which Trichoderma masks its entrance into its plant host remain unknown, but likely involve the secretion of different families of proteins into the apoplast that may play crucial roles in the suppression of plant immune responses. In this study, we investigated T. virens colonization of maize roots under hydroponic conditions, evidencing inter- and intracellular colonization by the fungus and modifications in root morphology and coloration. Moreover, we show that upon host penetration, T. virens secretes into the apoplast an arsenal of proteins to facilitate inter- and intracellular colonization of maize root tissues. Using a gel-free shotgun proteomics approach, 95 and 43 secretory proteins were identified from maize and T. virens, respectively. A reduction in the maize secretome (36%) was induced by T. virens, including two major groups, glycosyl hydrolases and peroxidases. Furthermore, T. virens secreted proteins were mainly involved in cell wall hydrolysis, scavenging of reactive oxygen species and secondary metabolism, as well as putative effector-like proteins. Levels of peroxidase activity were reduced in the inoculated roots, suggesting a strategy used by T. virens to manipulate host immune responses. The results provide an insight into the crosstalk in the apoplast which is essential to maintain the T. virens-plant interaction.

The relation between global palm distribution and climate.

Fossil palms provide qualitative evidence of (sub-) tropical conditions and frost-free winters in the geological past, including modern cold climate regions (e.g., boreal, or polar climates). The freeze intolerance of palms varies across different organs and life stages, with seedlings in particular less tolerant of sub-zero temperatures than adult plants, limiting successful establishment of populations while permitting adult palms to survive in cultivation outside their natural ranges. Quantitatively, palms indicate minimum cold month mean temperature (CMMT) at 2-8 °C in palaeoclimate reconstructions. These data have accentuated model-proxy mismatches for high latitudes during Paleogene hyperthermals when palms expanded poleward in both hemispheres. We constructed a manually filtered dataset of >20,000 georeferenced Arecaceae records, by eliminating cultivars. Statistically derived mean annual temperature, mean annual temperature range, and CMMT thresholds for the Arecaceae and lower rank subfamilies and tribes reveal large differences in temperature sensitivity depending on lower taxonomic classification. Cold tolerant tribes such as the Trachycarpeae produce thresholds as low as CMMT ≥ 2.2 °C. However, within the palm family, CMMT < 5 °C is anomalous. Moreover, palm expansion into temperate biomes is likely a post-Palaeogene event. We recognize a CMMT ≥ 5.2 °C threshold for the palm family, unless a lower taxonomic rank can be assigned.

The fate of linoleic acid on Saccharomyces cerevisiae metabolism under aerobic and anaerobic conditions.

INTRODUCTION: Saccharomyces cerevisiae has been widely used for fermenting food and beverages for over thousands years. Its metabolism together with the substrate composition play an important role in determining the characteristics of the final fermented products. We previously showed that the polyunsaturated fatty acid, linoleic acid, which is present in the grape juice at trace levels, significantly affected the development of aroma compounds of the wines. However, the effect of linoleic acid on the overall cell metabolism of S. cerevisiae is still not clear. Therefore, we aimed to unlock the metabolic response of S. cerevisiae to linoleic acid using metabolomics and isotope labelling experiments. METHODS: We cultured the cells on a minimal mineral medium supplementing them with linoleic acid isomers and 13C-linoleic acid. Both intracellular and extracellular metabolite profiles were determined using gas chromatography coupled to mass spectrometry (GC-MS) to investigate which S. cerevisiae pathways were affected by linoleic acid supplementation. RESULTS: The utilisation of linoleic acid by S. cerevisiae had a significant impact on the primary carbon metabolism increasing the glucose consumption and the ethanol production under anaerobic condition. The energetic state of the cell was, therefore, affected and the glycolytic pathway, the TCA cycle and the amino acid production were up-regulated. We also observed that linoleic acid was transported into the cell and converted into other fatty acids affecting their profile even under anaerobic condition. CONCLUSION: Our data clearly shows that linoleic acid supplementation in growth medium increased glucose consumption and ethanol production by S. cerevisiae under anaerobic condition. We also suggest that S. cerevisiae might be able to perform an alternative anaerobic pathway to ß-oxidation, which has not been reported yet.

The effect of linoleic acid on the Sauvignon blanc fermentation by different wine yeast strains.

The level of linoleic acid in the Sauvignon blanc (SB) grape juice affects the development of different aroma compounds during fermentation by Saccharomyces cerevisiae EC1118, including key varietal thiols such as 3-mercaptohexanol (3MH) and 3-mercaptohexyl acetate (3MHA). However, it is still unknown if linoleic acid would affect in a similar way other commonly used S. cerevisiae wine strains. Here we investigated the effect of grape juice linoleic acid on the development of aroma compounds and other metabolites of SB wines using different wine yeast strains: EC1118, AWRI796 and VIN13. Linoleic acid clearly affected the levels of acetylated aroma compounds, several amino acids, and antioxidant molecules, independent of yeast strain, but the production of 3MH was affected by linoleic acid in a strain-specific manner. Moreover, the supplementation of deuterium-labelled 3MH also affected the production of varietal thiols in a strain-specific way. Linoleic acid reduced the acetylation process probably by inhibiting an acetyltransferase, an effect that was independent of the yeast strain. However, regulation of the 3MH biosynthesis is strain-specific, which suggests a mindful consideration not only towards the wine yeast but also to the linoleic acid concentration in the grape juice in order to obtain the desired wine aroma characteristics.

Plasmin and regulators of plasmin activity control the migratory capacity and adhesion of human T cells and dendritic cells by regulating cleavage of the chemokine CCL21.

The homeostatic chemokine CCL21 has a pivotal role in lymphocyte homing and compartment localisation within the lymph node, and also affects adhesion between immune cells. The effects of CCL21 are modulated by its mode of presentation, with different cellular responses seen for surface-bound and soluble forms. Here we show that plasmin cleaves surface-bound CCL21 to release the C-terminal peptide responsible for CCL21 binding to glycosaminoglycans on the extracellular matrix and cell surfaces, thereby generating the soluble form. Loss of this anchoring peptide enabled the chemotactic activity of CCL21 and reduced cell tethering. Tissue plasminogen activator did not cleave CCL21 directly but enhanced CCL21 processing through generation of plasmin from plasminogen. The tissue plasminogen activator inhibitor neuroserpin prevented processing of CCL21 and blocked the effects of soluble CCL21 on cell migration. Similarly, the plasmin-specific inhibitor α2-antiplasmin inhibited CCL21-mediated migration of human T cells and dendritic cells and tethering of T cells to APCs. We conclude that the plasmin system proteins plasmin, tissue plasminogen activator and neuroserpin regulate CCL21 function in the immune system by controlling the balance of matrix- and cell-bound CCL21.

The Genomes of Three Uneven Siblings: Footprints of the Lifestyles of Three Trichoderma Species.

The genus Trichoderma contains fungi with high relevance for humans, with applications in enzyme production for plant cell wall degradation and use in biocontrol. Here, we provide a broad, comprehensive overview of the genomic content of these species for "hot topic" research aspects, including CAZymes, transport, transcription factors, and development, along with a detailed analysis and annotation of less-studied topics, such as signal transduction, genome integrity, chromatin, photobiology, or lipid, sulfur, and nitrogen metabolism in T. reesei, T. atroviride, and T. virens, and we open up new perspectives to those topics discussed previously. In total, we covered more than 2,000 of the predicted 9,000 to 11,000 genes of each Trichoderma species discussed, which is >20% of the respective gene content. Additionally, we considered available transcriptome data for the annotated genes. Highlights of our analyses include overall carbohydrate cleavage preferences due to the different genomic contents and regulation of the respective genes. We found light regulation of many sulfur metabolic genes. Additionally, a new Golgi 1,2-mannosidase likely involved in N-linked glycosylation was detected, as were indications for the ability of Trichoderma spp. to generate hybrid galactose-containing N-linked glycans. The genomic inventory of effector proteins revealed numerous compounds unique to Trichoderma, and these warrant further investigation. We found interesting expansions in the Trichoderma genus in several signaling pathways, such as G-protein-coupled receptors, RAS GTPases, and casein kinases. A particularly interesting feature absolutely unique to T. atroviride is the duplication of the alternative sulfur amino acid synthesis pathway.

Leukocyte integrin α4ß7 associates with heat shock protein 70.

The leukocyte integrin cell adhesion molecules α4ß7 and αEß7 mediate the homing and retention of lymphocytes to the gut, and sites of inflammation. Here we have identified heat shock protein 70 (HSP70) as a major protein that associates with the cytoplasmic domain of the integrin ß7 subunit. HSPs are molecular chaperones that protect cells from stress but more recently have been reported to also regulate cell adhesion and invasion via modulation of ß1, ß2, and ß3 integrins and integrin-associated signalling molecules. Several HSP70 isoforms including HSP70-3, HSP70-1L, HSP70-8, and HSP70-9 were specifically precipitated from T cells by a bead-conjugated ß7 subunit cytoplasmic domain peptide and subsequently identified by high-resolution liquid chromatography-tandem mass spectrometry. In confirmation, the ß7 subunit was co-immunoprecipitated from a T cell lysate by an anti-HSP70 antibody. Further, recombinant human HSP70-1a was precipitated by ß7 cytoplasmic domain-coupled beads. The HSP70 inhibitor KNK437 decreased the expression of HSP70 without affecting the expression of the ß7 integrin. It significantly inhibited α4ß7-mediated adhesion of T cells to mucosal addressin cell adhesion molecule 1 (MAdCAM-1), suggesting HSP70 is critical for maintaining ß7 integrin signalling function. The functional implications of the association of ß7 integrins with the different isoforms of HSP70 warrants further investigation.

Comprehensive lipidome profiling of Sauvignon blanc grape juice.

This study presents a comprehensive lipidome analysis of Sauvignon blanc grape juice by combining GC-MS based fatty acid profiling with shotgun lipidomics strategy. We observed that despite grape juice being a water based matrix it contains a diverse range of lipid species, including common saturated and unsaturated free and intact fatty acids as well as odd-numbered and hydroxy fatty acids. Based on GC-MS quantitative data, we found that the total lipid content of grape juice could be as high as 2.80 g/L. The majority of lipids were present in the form of complex lipids with relatively small amount of free fatty acids (<15%). Therefore we concluded that the lipidome should be considered an important component of grape juice with the potential to impact on fermentation processes as well as on the sensorial properties of fermented products. This work serves as a hypothesis generating tool, the results of which justify follow-up studies to explore the influence of the grape juice lipidome and lipid metabolism in yeast on the aroma profile of wine.

The intrinsically disordered structural platform of the plant defence hub protein RPM1-interacting protein 4 provides insights into its mode of action in the host-pathogen interface and evolution of the nitrate-induced domain protein family.

Arabidopsis thaliana (At) RPM1-interacting protein 4 (RIN4), targeted by many defence-suppressing bacterial type III effectors and monitored by several resistance proteins, regulates plant immune responses to pathogen-associated molecular patterns and type III effectors. Little is known about the overall protein structure of AtRIN4, especially in its unbound form, and the relevance of structure to its diverse biological functions. AtRIN4 contains two nitrate-induced (NOI) domains and is a member of the NOI family. Using experimental and bioinformatic approaches, we demonstrate that the unbound AtRIN4 is intrinsically disordered under physiological conditions. The intrinsically disordered polypeptide chain of AtRIN4 is interspersed with molecular recognition features (MoRFs) and anchor-identified long-binding regions, potentially allowing it to undergo disorder-to-order transitions upon binding to partner(s). A poly-l-proline II structure, often responsible for protein recognition, is also identified in AtRIN4. By performing bioinformatics analyses on RIN4 homologues from different plant species and the NOI proteins from Arabidopsis, we infer the conservation of intrinsic disorder, MoRFs and long-binding regions of AtRIN4 in other plant species and the NOI family. Intrinsic disorder and MoRFs could provide RIN4 proteins with the binding promiscuity and plasticity required to act as hubs in a pivotal position within plant defence signalling cascades.

Use of liquid chromatography-mass spectrometry (LC-MS) to detect substances of nanomolar concentration in the coronary effluent of isolated perfused hearts.

When the heart is stretched there is an abrupt increase in force that coincides with the stretch that is then followed by a second phase that develops slowly over a period of minutes, known as the "slow force response", or "SFR". The SFR is accompanied by a slow increase in the Ca(2+) transients, but the underlying cellular mechanisms are still debated. We therefore devised a simple bioassay system to investigate whether stretch released autocrine/paracrine factors that could explain the stretch-dependent Ca(2+) influx during the SFR. Our analysis was based around a metabolomics approach using tandem mass spectrometry whereby secreted compounds were analysed by liquid chromatography mass spectrometry (LC-MS/MS) at high resolution and accurate mass. Our bioassay consisted of two isolated cardiac preparations in series. A sustained stretch was applied to an "upstream" Langendorff-perfused heart, whilst monitoring the performance of an un-stretched, "downstream" trabecula perfused with coronary effluent collected from the "upstream" heart. Switching from "un-stretched" to "stretched" coronary effluent increased the amplitude of the Ca(2+) transients (340/380) fura-2 ratio) in the "downstream" trabeculae from 0.87 ± 0.04 to 0.94 ± 0.05 (p < 0.05, n = 4), and active stress (mN mm(-2)) from 15.4 ± 1.5 to 19.2 ± 1.9 (p < 0.01, n = 13), confirming the stretch-dependent release of one or more positively inotropic factor(s). To identify the factor, coronary effluent samples were concentrated by Solid Phase Extraction (SPE) treatment, lyophilised, then analysed using a Thermo Finnigan LTQ FT-ICR hybrid mass spectrometer. Differential profile data was obtained initially followed by targeting specific compounds at elevated levels in the stretched coronary effluent samples. Quantitative and statistical analyses of the differential LC-MS/MS results were then carried out using SIEVE 2.1, statistical software capable of performing differential analysis of peptides and small molecules. A number of substances were identified that were different between stretched and unstretched effluents (p < 0.01).

Fossil palm beetles refine upland winter temperatures in the Early Eocene Climatic Optimum.

Eocene climate and associated biotic patterns provide an analog system to understand their modern interactions. The relationship between mean annual temperatures and winter temperatures-temperature seasonality-may be an important factor in this dynamic. Fossils of frost-intolerant palms imply low Eocene temperature seasonality into high latitudes, constraining average winter temperatures there to >8 °C. However, their presence in a paleocommunity may be obscured by taphonomic and identification factors for macrofossils and pollen. We circumvented these problems by establishing the presence of obligate palm-feeding beetles (Chrysomelidae: Pachymerina) at three localities (a fourth, tentatively) in microthermal to lower mesothermal Early Eocene upland communities in Washington and British Columbia. This provides support for warmer winter Eocene climates extending northward into cooler Canadian uplands.

Structural model for covalent adhesion of the Streptococcus pyogenes pilus through a thioester bond.

The human pathogen Streptococcus pyogenes produces pili that are essential for adhesion to host surface receptors. Cpa, the adhesin at the pilus tip, was recently shown to have a thioester-containing domain. The thioester bond is believed to be important in adhesion, implying a mechanism of covalent attachment analogous to that used by human complement factors. Here, we have characterized a second active thioester-containing domain on Cpa, the N-terminal domain of Cpa (CpaN). Expression of CpaN in Escherichia coli gave covalently linked dimers. These were shown by x-ray crystallography and mass spectrometry to comprise two CpaN molecules cross-linked by the polyamine spermidine following reaction with the thioester bonds. This cross-linked CpaN dimer provides a model for the covalent attachment of Cpa to target receptors and thus the streptococcal pilus to host cells. Similar thioester domains were identified in cell wall proteins of other Gram-positive pathogens, suggesting that thioester domains are more widely used and provide a mechanism of adhesion by covalent bonding to target molecules on host cells that mimics that used by the human complement system to eliminate pathogens.

Ligand promiscuity within the internal cavity of Epiphyas postvittana Takeout 1 protein.

Takeout proteins are found across a diverse range of insect species and are thought to be involved in important aspects of insect physiology and behavior. These proteins act as ligand carriers, but the nature of their endogenous ligands remains unknown. The crystal structure of Epiphyas postvittana Takeout 1 (EpTo1), the only structure for any Takeout protein to date, revealed an α/ß-wrap fold with a purely hydrophobic internal cavity of tubular shape. When recombinantly expressed in Escherichia coli, we previously showed that a surrogate ubiquinone-8 ligand binds within the internal cavity of EpTo1 with excellent shape complementarity. We have now expressed EpTo1 in an insect cell expression system devoid of ubiquinone-8, and solved its crystal structure at 2.2Å resolution. Using combined information from crystallography and mass spectrometry, we identify a mixture of fatty acid moieties, mostly myristic and palmitic acid, bound inside the EpTo1 cavity, mimicking the structure of the longer ubiquinone-8 compound. No significant alteration of the internal cavity was observed regardless of the bound ligands, ubiquinone-8 or fatty acids, suggesting that the internal cavity of EpTo1 forms a rigid scaffold that imposes strict structural constraints for selectivity and specificity of ligand(s) in vivo.

The role of enoyl reductase genes in phloridzin biosynthesis in apple.

Phloridzin is the predominant polyphenol in apple (Malus × domestica Borkh.) where it accumulates to high concentrations in many tissues including the leaves, bark, roots and fruit. Despite its relative abundance in apple the biosynthesis of phloridzin and other related dihydrochalcones remains only partially understood. The key unidentified enzyme in phloridzin biosynthesis is a putative carbon double bond reductase which is thought to act on p-coumaroyl-CoA to produce the dihydro-p-coumaroyl-CoA precursor. A functional screen of six apple enoyl reductase-like (ENRL) genes was carried out using transient infiltration into tobacco and gene silencing by RNA interference (RNAi) in order to determine carbon double bond reductase activity and contribution to foliar phloridzin concentrations. The ENRL-3 gene caused a significant increase in phloridzin concentration when infiltrated into tobacco leaves whilst a second protein ENRL-5, with over 98% amino acid sequence similarity to ENRL-3, showed p-coumaroyl-CoA reductase activity in enzyme assays. Finally, an RNAi study showed that reducing the transcript levels of ENRL-3 in transgenic 'Royal Gala' led to a 66% decrease in the concentration of dihydrochalcones in the leaves in the one available silenced line. Overall these results suggest that ENRL-3, and its close homolog ENRL-5, may contribute to the biosynthesis of phloridzin in apple.