Functional characterization of a melon alcohol acyl-transferase gene family involved in the biosynthesis of ester volatiles. Identification of the crucial role of a threonine residue for enzyme activity

Volatile esters, a major class of compounds contributing to the aroma of many fruit, are synthesized by alcohol acyl-transferases (AAT). We demonstrate here that, in Charentais melon (Cucumis melo var. cantalupensis), AAT are encoded by a gene family of at least four members with amino acid identity ranging from 84% (Cm-AAT1/Cm-AAT2) and 58% (Cm-AAT1/Cm-AAT3) to only 22% (Cm-AAT1/Cm-AAT4). All encoded proteins, except Cm-AAT2, were enzymatically active upon expression in yeast and show differential substrate preferences. Cm-AAT1 protein produces a wide range of short and long-chain acyl esters but has strong preference for the formation of E-2-hexenyl acetate and hexyl hexanoate. Cm-AAT3 also accepts a wide range of substrates but with very strong preference for producing benzyl acetate. Cm-AAT4 is almost exclusively devoted to the formation of acetates, with strong preference for cinnamoyl acetate. Site directed mutagenesis demonstrated that the failure of Cm-AAT2 to produce volatile esters is related to the presence of a 268-alanine residue instead of threonine as in all active AAT proteins. Mutating 268-A into 268-T of Cm-AAT2 restored enzyme activity, while mutating 268-T into 268-A abolished activity of Cm-AAT1. Activities of all three proteins measured with the prefered substrates sharply increase during fruit ripening. The expression of all Cm-AAT genes is up-regulated during ripening and inhibited in antisense ACC oxidase melons and in fruit treated with the ethylene antagonist 1-methylcyclopropene (1-MCP), indicating a positive regulation by ethylene. The data presented in this work suggest that the multiplicity of AAT genes accounts for the great diversity of esters formed in melon

Metabolomics analysis of postharvest ripening heterogeneity of ‘Hass' avocadoes

The complex physiology of ‘Hass’ avocado renders its postharvest ripening heterogeneous and unpre-dictable. Several approaches have previously been undertaken to broaden our understanding of the causesof this postharvest ripening heterogeneity but without much success. In this study, a fruit biopsy method-ology was undertaken to sample mesocarp tissue from a series of individual avocado fruit while followingindividual fruit postharvest ripening characteristics without significantly disturbing their metabolism.Using both targeted and untargeted metabolomics approaches, we analyzed the metabolite profiles of thebiopsies in order to get more insight into the biochemical mechanisms underlying ‘Hass’ avocado ripen-ing heterogeneity. While C7sugars (mannoheptulose and perseitol), dry matter and total Ca2+were notcorrelated with time to reach edible ripeness, untargeted metabolomics profiling of polar and semi-polarcompounds (based on GC–MS and LC–MS platforms), revealed several metabolites, mainly amino acids,that were related to ripening heterogeneity. In addition, analysis of fatty acids revealed linoleic acid tobe differentially accumulating. In general, slowest ripening avocados had lower amounts of precursors ofmetabolites involved in key metabolic pathways. Our study indicates that comprehensive metabolomicsmay provide new markers for avocado ripening stage at harvest, and may give more insight into thecomplex ripening physiology of this fruit

Impact of postharvest ripening strategies on 'Hass' avocado fatty acid profiles

Persea americana Mill. cv 'Hass' is a subtropical fruit highly appreciated as a rich source of fatty acids mostly of the monounsaturated type. Commonly commercially applied postharvest ripening strategies for the ready to eat market based on high temperature (15 and 20 °C) and external ethylene (0 or 100 ppm applied for 24 h) application did not have a detrimental effect on the fatty acid profile or composition and total amount of oil recovered at edible ripeness. The results of this study have important implications for the fresh fruit and avocado oil industry. The composition of the fatty acid profile in 'Hass' avocados was mostly influenced by growing and environmental conditions. Commercially applied postharvest ripening strategies based on temperature and ethylene did not affect negatively the fatty acid composition of the fruit

Natriuretic peptides and integrated risk assessment for cardiovascular disease: an individual-participant-data meta-analysis

Background Guidelines for primary prevention of cardiovascular diseases focus on prediction of coronary heart disease and stroke. We assessed whether or not measurement of N-terminal-pro-B-type natriuretic peptide (NT-proBNP) concentration could enable a more integrated approach than at present by predicting heart failure and enhancing coronary heart disease and stroke risk assessment. Methods In this individual-participant-data meta-analysis, we generated and harmonised individual-participant data from relevant prospective studies via both de-novo NT-proBNP concentration measurement of stored samples and collection of data from studies identified through a systematic search of the literature (PubMed, Scientific Citation Index Expanded, and Embase) for articles published up to Sept 4, 2014, using search terms related to natriuretic peptide family members and the primary outcomes, with no language restrictions. We calculated risk ratios and measures of risk discrimination and reclassification across predicted 10 year risk categories (ie, <5%, 5% to <7·5%, and ≥7·5%), adding assessment of NT-proBNP concentration to that of conventional risk factors (ie, age, sex, smoking status, systolic blood pressure, history of diabetes, and total and HDL cholesterol concentrations). Primary outcomes were the combination of coronary heart disease and stroke, and the combination of coronary heart disease, stroke, and heart failure. Findings We recorded 5500 coronary heart disease, 4002 stroke, and 2212 heart failure outcomes among 95 617 participants without a history of cardiovascular disease in 40 prospective studies. Risk ratios (for a comparison of the top third vs bottom third of NT-proBNP concentrations, adjusted for conventional risk factors) were 1·76 (95% CI 1·56–1·98) for the combination of coronary heart disease and stroke and 2·00 (1·77–2·26) for the combination of coronary heart disease, stroke, and heart failure. Addition of information about NT-proBNP concentration to a model containing conventional risk factors was associated with a C-index increase of 0·012 (0·010–0·014) and a net reclassification improvement of 0·027 (0·019–0·036) for the combination of coronary heart disease and stroke and a C-index increase of 0·019 (0·016–0·022) and a net reclassification improvement of 0·028 (0·019–0·038) for the combination of coronary heart disease, stroke, and heart failure. Interpretation In people without baseline cardiovascular disease, NT-proBNP concentration assessment strongly predicted first-onset heart failure and augmented coronary heart disease and stroke prediction, suggesting that NT-proBNP concentration assessment could be used to integrate heart failure into cardiovascular disease primary prevention