Mass spectrometry-based "omics" technologies for the study of gestational diabetes and the discovery of new biomarkers.

Gestational diabetes (GDM) is one of the most common complications occurring during pregnancy. Diagnosis is performed by oral glucose tolerance test, but harmonized testing methods and thresholds are still lacking worldwide. Short-term and long-term effects include obesity, type 2 diabetes, and increased risk of cardiovascular disease. The identification and validation of sensitidve, selective, and robust biomarkers for early diagnosis during the first trimester of pregnancy are required, as well as for the prediction of possible adverse outcomes after birth. Mass spectrometry (MS)-based omics technologies are nowadays the method of choice to characterize various pathologies at a molecular level. Proteomics and metabolomics of GDM were widely investigated in the last 10 years, and various proteins and metabolites were proposed as possible biomarkers. Metallomics of GDM was also reported, but studies are limited in number. The present review focuses on the description of the different analytical methods and MS-based instrumental platforms applied to GDM-related omics studies. Preparation procedures for various biological specimens are described and results are briefly summarized. Generally, only preliminary findings are reported by current studies and further efforts are required to determine definitive GDM biomarkers.

Positively Charged Organosilanes Covalently Linked to the Silica Network as Modulating Tools for the Salinity Correction of pH Values Obtained with Colorimetric Sensor Arrays (CSAs).

Seven increasing levels of water salinity from 0.029 to 0.600 M (as NaCl) were used to investigate the dependence of pH measurement, performed using colorimetric sensor arrays (CSAs), on ionic strength. The CSAs were arrays of sensing spots prepared in the form of sol-gel-embedding Bromothymol Blue (BB) and Bromocresol Green (BCG) in a porous nitrocellulose support. The support was impregnated over the entire thickness (≈100 µm), allowing for the signal (Hue) acquisition on the opposite side to the contact with the sample solution. Three CSAs were prepared, M1, M2, and M3. M1 contained a free cationic surfactant, hexadecyltrimethylammonium p-toluenesulfonate (CTApTs), for modulating the pKa of the indicators. In M2, the surfactant dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride (DTSACl) was covalently bonded to the sol-gel. M3 was prepared like M2 but using a larger amount of ethanol as the solvent for the synthesis. The modulation of the CTApTs or the DTSACl concentration enabled the tuning of the pKa. In general, the pKa modulation ability decreased with the increase in salinity. The presence of a surfactant covalently linked to the backbone partially reduced the competitiveness of the anionic species, improving the results. Nevertheless, the salt effect was still present, and a correction algorithm was required. Between pH 5.00 and 12.00, this correction could be made automatically by using spots taken as references to produce sensors independent of salinity. As the salt effect is virtually absent above 0.160 M, M2 and M3 can be used for future applications in seawater.

Gas chromatography coupled to time-of-flight mass spectrometry using parallel electron and chemical ionization with permeation tube facilitated reagent ion control for material emission analysis.

RATIONALE: Volatile organic compounds (VOCs) emitted by an artificial leather part for car interiors are determined using GC-MS (gas chromatograph coupled to a mass spectrometer) using simultaneous electron and chemical ionization (EI&CI). A device for swift reagent ion switching in CI mode between consecutive runs is presented. METHODS: VOCs emitted from the investigated material were sampled onto Tenax® absorption tubes using micro emission chambers and subsequently injected into the GC through thermal desorption. The detector was a time-of-flight mass spectrometer (TOFMS) simultaneously operating in EI and CI modes during a single chromatographic run. A custom permeation tube setup allowed for swift selection between various reagent ions in CI mode, e.g., [N2 H]+ , [H3 O]+ , [(H2 O)2 H]+ , and [NH4 ]+ . RESULTS: Different reagent ions are swiftly selectable between single GC runs without hardware changes. Differences in precursor ion survival yields and the selectivity of the various reactants were carefully assessed. Several examples for the improved identification of unknown compounds with the available complementary and comprehensive EI&CI data set are demonstrated for a relevant material emission application. CONCLUSION: The presented technique provides additional value to the standard GC-EI/MS procedure commonly used for material emission characterization. It allows for a non-targeted analysis approach with moderate analysis time.

Comparative Analysis of Volatile Organic Compound Purification Techniques in Complex Cooking Emissions: Adsorption, Photocatalysis and Combined Systems.

Volatile organic compounds (VOCs) are molecules present in our everyday life, and they can be positive, such as in the formation of odour and food flavour, or harmful to the environment and humans, and research is focusing on limiting their emissions. Various methods have been used to achieve this purpose. Firstly, we review three main degradation methods: activated carbon, photocatalysis and a synergetic system. We provide a general overview of the operative conditions and report the possibility of VOC abatement during cooking. Within the literature, none of these systems has ever been tested in the presence of complex matrices, such as during cooking processes. The aim of this study is to compare the three methods in order to understand the behaviour of filter systems in the case of realistically complex gas mixtures. Proton transfer reaction-mass spectrometry (PTR-MS) has been used in the real-time monitoring of volatilome. Due to the fact that VOC emissions are highly dependent on the composition of the food cooked, we evaluated the degradation capacity of the three systems for different burger types (meat, greens, and fish). We demonstrate the pros and cons of photocatalysis and adsorption and how a combined approach can mitigate the drawbacks of photocatalysis.

Phenotyping methods to assess heat stress resilience in grapevine.

Global warming has become an issue in recent years in viticulture, as increasing temperatures have a negative impact on grapevine (Vitis vinifera) production and on wine quality. Phenotyping for grapevine response to heat stress is, therefore, important to understand thermotolerance mechanisms, with the aim of improving field management strategies or developing more resilient varieties. Nonetheless, the choice of the phenotypic traits to be investigated is not trivial and depends mainly on the objectives of the study, but also on the number of samples and on the availability of instrumentation. Moreover, the grapevine literature reports few studies related to thermotolerance, generally assessing physiological responses, which highlights the need for more holistic approaches. In this context, the present review offers an overview of target traits that are commonly investigated in plant thermotolerance studies, with a special focus on grapevine, and of methods that can be employed to evaluate those traits. With the final goal of providing useful tools and references for future studies on grapevine heat stress resilience, advantages and limitations of each method are highlighted, and the available or possible implementations are described. In this way, the reader is guided in the choice of the best approaches in terms of speed, complexity, range of application, sensitivity, and specificity.

Exploring volatile organic compound emission from thermally modified wood by PTR-ToF-MS.

Thermal modification of wood is a well-known industrial process performed to increase the durability and dimensional stability or to change the colour of natural wood. The treatment influences many other properties of wood including the emission of volatile organic compounds (VOCs). VOC release ultimately affects the quality of indoor air and the capability of having low VOC emission is often included as a key parameter for the attribution of quality labels. In the present work, wood from six tree species was subjected to different types of treatment and VOC profiling was carried out on both treated and untreated samples by means of PTR-ToF-MS. Different types of thermal treatment were tested, involving either overpressure or vacuum and the effect of different temperature profiles was evaluated. Hardwood and softwood showed different release profiles under all tested conditions: the headspace of softwood was richer in several VOCs, such as terpenes, phenols and C6-C9 aldehydes and carboxylic acids. Upon thermal treatment, terpene emissions decreased, whereas several other VOCs, such as formic acid, formaldehyde, furfural and acetic acid, were released in higher amounts. With its high sensitivity and throughput, PTR-ToF-MS appears to be a very powerful analytical tool, useful in supporting the selection of wood materials for different end uses.

Single-Walled Carbon Nanohorns as Boosting Surface for the Analysis of Low-Molecular-Weight Compounds by SALDI-MS.

Limits of Matrix-Assisted Laser Desorption Ionization (MALDI) mass spectrometry (MS) in the study of small molecules are due to matrix-related interfering species in the low m/z range. Single-walled carbon nanohorns (SWCNH) were here evaluated as a specific surface for the rapid analysis of amino acids and lipids by Surface-Assisted Laser Desorption Ionization (SALDI). The method was optimized for detecting twenty amino acids, mainly present as cationized species, with the [M+K]+ response generally 2-time larger than the [M+Na]+ one. The [M+Na]+/[M+K]+ signals ratio was tentatively correlated with the molecular weight, dipole moment and binding affinity, to describe the amino acids' coordination ability. The SWCNH-based surface was also tested for analyzing triglycerides in olive oil samples, showing promising results in determining the percentage composition of fatty acids without any sample treatment. Results indicated that SWCNH is a promising substrate for the SALDI-MS analysis of low molecular weight compounds with different polarities, enlarging the analytical platforms for MALDI applications.

Quantitative Analysis of Plant Cytosolic Calcium Signals in Response to Water Activated by Low-Power Non-Thermal Plasma.

Non-thermal plasma technology is increasingly being applied in the plant biology field. Despite the variety of beneficial effects of plasma-activated water (PAW) on plants, information about the mechanisms of PAW sensing by plants is still limited. In this study, in order to link PAW perception to the positive downstream responses of plants, transgenic Arabidopsis thaliana seedlings expressing the Ca2+-sensitive photoprotein aequorin in the cytosol were challenged with water activated by low-power non-thermal plasma generated by a dielectric barrier discharge (DBD) source. PAW sensing by plants resulted in the occurrence of cytosolic Ca2+ signals, whose kinetic parameters were found to strictly depend on the operational conditions of the plasma device and thus on the corresponding mixture of chemical species contained in the PAW. In particular, we highlighted the effect on the intracellular Ca2+ signals of low doses of DBD-PAW chemicals and also presented the effects of consecutive plant treatments. The results were discussed in terms of the possibility of using PAW-triggered Ca2+ signatures as benchmarks to accurately modulate the chemical composition of PAW in order to induce environmental stress resilience in plants, thus paving the way for further applications in agriculture.

Species-Specific Induction of Plant Volatiles by Two Aphid Species in Apple: Real Time Measurement of Plant Emission and Attraction of Lacewings in the Wind Tunnel.

Upon damage by herbivores, plants release herbivory-induced plant volatiles (HIPVs). To find their prey, the pest's natural enemies need to be fine-tuned to the composition of these volatiles. Whereas standard methods can be used in the identification and quantitation of HIPVs, more recently introduced techniques such as PTR-ToF-MS provide temporal patterns of the volatile release and detect additional compounds. In this study, we compared the volatile profile of apple trees infested with two aphid species, the green apple aphid Aphis pomi, and the rosy apple aphid Dysaphis plantaginea, by CLSA-GC-MS complemented by PTR-ToF-MS. Compounds commonly released in conjunction with both species include nonanal, decanal, methyl salicylate, geranyl acetone, (Z)-3-hexenyl acetate, (Z)-3-hexenyl butanoate, (Z)-3-hexenyl 2-methyl-butanoate, (E)-ß-caryophyllene, ß-bourbonene and (Z)-3-hexenyl benzoate. In addition, benzaldehyde and (E)-ß-farnesene were exclusively associated with A. pomi, whereas linalool, (E)-4,8-dimethyl-1,3,7-nonatriene were exclusively associated with D. plantaginea. PTR-ToF-MS additionally detected acetic acid (AA) and 2-phenylethanol (PET) in the blends of both trees attacked by aphid species. In the wind tunnel, the aphid predator, Chrysoperla carnea (Stephens), responded strongly to a blend of AA and PET, much stronger than to AA or PET alone. The addition of common and species-specific HIPVs did not increase the response to the binary blend of AA and PET. In our setup, two host-associated volatiles AA + PET appeared sufficient in the attraction of C. carnea. Our results also show the importance of combining complementary methods to decipher the odor profile associated with plants under pest attack and identify behaviourally active components for predators.

Thermal Desorption-Vocus Enables Online Nondestructive Quantification of 2,4,6-Trichloroanisole in Cork Stoppers below the Perception Threshold.

2,4,6-Trichloroanisole (TCA) contamination of wine determines huge economic losses for the wine industry estimated to amount to several billion dollars yearly. Over 50 years of studies have determined that this problem is often caused by TCA contamination of the cork stopper, which releases TCA into the wine. The human threshold for TCA is extremely low. A wine contaminated by 1-2 ng/L TCA can be perceived as tainted. Contaminations with <0.5 ng/L TCA are commonly considered negligible and are not perceivable. The possibility of prescreening cork stoppers for TCA contamination would be an enormous advantage. Therefore, the demand for a fast, nondestructive method capable of quantifying the TCA contamination in cork stoppers is impelling. Vastly used analytical methods have so far struggled to provide a fast and reliable solution, whereas sensory analysis by trained panelists is expensive and time-consuming. Here we propose a novel approach based on chemical ionization-time-of-flight (CI-TOF) mass spectrometry employing the "Vocus" ion source and ion-molecule reactor. The technique proved capable of nondestructively quantifying TCA contamination in a single cork stopper in 3 s, with a limit of quantification below the perception threshold. A real test on the industrial scale, quantifying TCA contamination in more than 10000 cork stoppers in a few hours is presented, representing the largest data set of TCA analysis on cork stoppers within the literature and proving the possibility to apply the technique in an industrial environment. The correlation with standard methods for releasable TCA quantification is also discussed.

High-throughput screening for in planta characterization of VOC biosynthetic genes by PTR-ToF-MS.

Functional characterization of plant volatile organic compound (VOC) biosynthetic genes and elucidation of the biological function of their products often involve the screening of large numbers of plants from either independent transformation events or mapping populations. The low time resolution of standard gas chromatographic methods, however, represents a major bottleneck for in planta genetic characterization of VOC biosynthetic genes. Here we present a fast and highly-sensitive method for the high-throughput characterization of VOC emission levels/patterns by coupling a Proton Transfer Reaction Time-of-Flight Mass Spectrometer to an autosampler for automation of sample measurement. With this system more than 700 samples per day can be screened, detecting for each sample hundreds of spectrometric peaks in the m/z 15-300 range. As a case study, we report the characterization of VOC emissions from 116 independent Arabidopsis thaliana lines transformed with a putative isoprene synthase gene, confirming its function also when fused to a C-terminal 3×FLAG tag. We demonstrate that the method is more reliable than conventional characterization of transgene expression for the identification of the most highly isoprene-emitting lines. The throughput of this VOC screening method exceeds that of existing alternatives, potentially allowing its application to reverse and forward genetic screenings of genes contributing to VOC emission, constituting a powerful tool for the functional characterization of VOC biosynthetic genes and elucidation of the biological functions of their products directly in planta.

pH Colorimetric Sensor Arrays: Role of the Color Space Adopted for the Calculation of the Prediction Error.

A pH colorimetric sensor array was prepared and characterized by combining tetrabromophenol blue (TBB) and bromothymol blue (BB) embedded in organically modified silicate (OrMoSil) spots polyvinylidene fluoride (PVDF)-supported. The signal was based on the Hue profile (H). The individual calibrations of TBB and BB showed precisions with minimum values of 0.012 pH units at pH = 2.196 for TBB and 0.018 at pH = 6.692 for BB. The overall precision of 10 spots of the mixture TBB/BB increased in the pH range of 1.000-8.000 from a minimum value of pH precision of 0.009 at pH = 2.196 to 0.012 at pH = 6.692, with the worst value of 0.279 pH units at pH = 4.101. The possibility to produce an array with much more than 10 spots allows for improving precision. The H analytical performance was compared to those of other color spaces such as RGB, Lab, and XYZ. H was the best one, with prediction error in the range of 0.016 to 0.021 pH units, at least three times lower than the second-best (x coordinate), with 0.064 pH units. These results were also confirmed by the calculation of the main experimental contributions to the pH prediction error, demonstrating the consistency of the proposed calculation approach.

Unveiling the Molecular Basis of Mascarpone Cheese Aroma: VOCs analysis by SPME-GC/MS and PTR-ToF-MS.

Mascarpone, a soft-spread cheese, is an unripened dairy product manufactured by the thermal-acidic coagulation of milk cream. Due to the mild flavor and creamy consistency, it is a base ingredient in industrial, culinary, and homemade preparations (e.g., it is a key constituent of a widely appreciated Italian dessert 'Tiramisù'). Probably due to this relevance as an ingredient rather than as directly consumed foodstuff, mascarpone has not been often the subject of detailed studies. To the best of our knowledge, no investigation has been carried out on the volatile compounds contributing to the mascarpone cheese aroma profile. In this study, we analyzed the Volatile Organic Compounds (VOCs) in the headspace of different commercial mascarpone cheeses by two different techniques: Headspace-Solid Phase Microextraction-Gas Chromatography-Mass Spectrometry (HS-SPME GC-MS) and Proton-Transfer Reaction-Mass Spectrometry coupled to a Time of Flight mass analyzer (PTR-ToF-MS). We coupled these two approaches due to the complementarity of the analytical potential-efficient separation and identification of the analytes on the one side (HS-SPME GC-MS), and effective, fast quantitative analysis without any sample preparation on the other (PTR-ToF-MS). A total of 27 VOCs belonging to different chemical classes (9 ketones, 5 alcohols, 4 organic acids, 3 hydrocarbons, 2 furans, 1 ester, 1 lactone, 1 aldehyde, and 1 oxime) have been identified by HS-SPME GC-MS, while PTR-ToF-MS allowed a rapid snapshot of volatile diversity confirming the aptitude to rapid noninvasive quality control and the potential in commercial sample differentiation. Ketones (2-heptanone and 2-pentanone, in particular) are the most abundant compounds in mascarpone headspace, followed by 2-propanone, 2-nonanone, 2-butanone, 1-pentanol, 2-ethyl-1-hexanol, furfural and 2-furanmethanol. The study also provides preliminary information on the differentiation of the aroma of different brands and product types.

In Planta Recapitulation of Isoprene Synthase Evolution from Ocimene Synthases.

Isoprene is the most abundant biogenic volatile hydrocarbon compound naturally emitted by plants and plays a major role in atmospheric chemistry. It has been proposed that isoprene synthases (IspS) may readily evolve from other terpene synthases, but this hypothesis has not been experimentally investigated. We isolated and functionally validated in Arabidopsis the first isoprene synthase gene, AdoIspS, from a monocotyledonous species (Arundo donax L., Poaceae). Phylogenetic reconstruction indicates that AdoIspS and dicots isoprene synthases most likely originated by parallel evolution from TPS-b monoterpene synthases. Site-directed mutagenesis demonstrated invivo the functional and evolutionary relevance of the residues considered diagnostic for IspS function. One of these positions was identified by saturating mutagenesis as a major determinant of substrate specificity in AdoIspS able to cause invivo a dramatic change in total volatile emission from hemi- to monoterpenes and supporting evolution of isoprene synthases from ocimene synthases. The mechanism responsible for IspS neofunctionalization by active site size modulation by a single amino acid mutation demonstrated in this study might be general, as the very same amino acidic position is implicated in the parallel evolution of different short-chain terpene synthases from both angiosperms and gymnosperms. Based on these results, we present a model reconciling in a unified conceptual framework the apparently contrasting patterns previously observed for isoprene synthase evolution in plants. These results indicate that parallel evolution may be driven by relatively simple biophysical constraints, and illustrate the intimate molecular evolutionary links between the structural and functional bases of traits with global relevance.

Interference with ethylene perception at receptor level sheds light on auxin and transcriptional circuits associated with the climacteric ripening of apple fruit (Malus x domestica Borkh.).

Apple (Malus x domestica Borkh.) is a model species for studying the metabolic changes that occur at the onset of ripening in fruit crops, and the physiological mechanisms that are governed by the hormone ethylene. In this study, to dissect the climacteric interplay in apple, a multidisciplinary approach was employed. To this end, a comprehensive analysis of gene expression together with the investigation of several physiological entities (texture, volatilome and content of polyphenolic compounds) was performed throughout fruit development and ripening. The transcriptomic profiling was conducted with two microarray platforms: a dedicated custom array (iRIPE) and a whole genome array specifically enriched with ripening-related genes for apple (WGAA). The transcriptomic and phenotypic changes following the application of 1-methylcyclopropene (1-MCP), an ethylene inhibitor leading to important modifications in overall fruit physiology, were also highlighted. The integrative comparative network analysis showed both negative and positive correlations between ripening-related transcripts and the accumulation of specific metabolites or texture components. The ripening distortion caused by the inhibition of ethylene perception, in addition to affecting the ethylene pathway, stimulated the de-repression of auxin-related genes, transcription factors and photosynthetic genes. Overall, the comprehensive repertoire of results obtained here advances the elucidation of the multi-layered climacteric mechanism of fruit ripening, thus suggesting a possible transcriptional circuit governed by hormones and transcription factors.

Genome-wide association study unravels the genetic control of the apple volatilome and its interplay with fruit texture.

Fruit quality represents a fundamental factor guiding consumers' preferences. Among apple quality traits, volatile organic compounds and texture features play a major role. Proton Transfer Reaction-Time of Flight-Mass Spectrometry (PTR-ToF-MS), coupled with an artificial chewing device, was used to profile the entire apple volatilome of 162 apple accessions, while the fruit texture was dissected with a TAXT-AED texture analyzer. The array of volatile compounds was classed into seven major groups and used in a genome-wide association analysis carried out with 9142 single nucleotide polymorphisms (SNPs). Marker-trait associations were identified on seven chromosomes co-locating with important candidate genes for aroma, such as MdAAT1 and MdIGS. The integration of volatilome and fruit texture data conducted with a multiple factor analysis unraveled contrasting behavior, underlying opposite regulation of the two fruit quality aspects. The association analysis using the first two principal components identified two QTLs located on chromosomes 10 and 2, respectively. The distinction of the apple accessions on the basis of the allelic configuration of two functional markers, MdPG1 and MdACO1, shed light on the type of interplay existing between fruit texture and the production of volatile organic compounds.

Emission of Volatile Compounds from Apple Plants Infested with Pandemis heparana Larvae, Antennal Response of Conspecific Adults, and Preliminary Field Trial.

This study investigated the volatile emission from apple (Malus x domestica Borkh., cv. Golden Delicious) foliage that was either intact, mechanically-damaged, or exposed to larval feeding by Pandemis heparana (Denis and Schiffermüller) (Lepidoptera: Tortricidae). Volatiles were collected by closed-loop-stripping-analysis and characterized by gas chromatography-mass spectrometry in three time periods: after 1 h and again 24 and 48 h later. Volatiles for all treatments also were monitored continuously over a 72-h period by the use of proton transfer reaction - time of flight-mass spectrometry (PTR-ToF-MS). In addition, the volatile samples were analyzed by gas chromatography-electroantennographic detection (GC-EAD) using male and female antennae of P. heparana. Twelve compounds were detected from intact foliage compared with 23 from mechanically-damaged, and 30 from P. heparana-infested foliage. Interestingly, six compounds were released only by P. heparana-infested foliage. The emission dynamics of many compounds measured by PTR-ToF-MS showed striking differences according to the timing of herbivory and the circadian cycle. For example, the emission of green leaf volatiles began shortly after the start of herbivory, and increased over time independently from the light-dark cycle. Conversely, the emission of terpenes and aromatic compounds showed a several-hour delay in response to herbivory, and followed a diurnal rhythm. Methanol was the only identified volatile showing a nocturnal rhythm. Consistent GC-EAD responses were found for sixteen compounds, including five aromatic ones. A field trial in Sweden demonstrated that benzyl alcohol, 2-phenylethanol, phenylacetonitrile, and indole lures placed in traps were not attractive to Pandemis spp. adults, but 2-phenylethanol and phenylacetonitrile when used in combination with acetic acid were attractive to both sexes.

PTR-MS Characterization of VOCs Associated with Commercial Aromatic Bakery Yeasts of Wine and Beer Origin.

In light of the increasing attention towards "green" solutions to improve food quality, the use of aromatic-enhancing microorganisms offers the advantage to be a natural and sustainable solution that did not negatively influence the list of ingredients. In this study, we characterize, for the first time, volatile organic compounds (VOCs) associated with aromatic bakery yeasts. Three commercial bakery starter cultures, respectively formulated with three Saccharomyces cerevisiae strains, isolated from white wine, red wine, and beer, were monitored by a proton-transfer-reaction time-of-flight mass spectrometer (PTR-ToF-MS), a direct injection analytical technique for detecting volatile organic compounds with high sensitivity (VOCs). Two ethanol-related peaks (m/z 65.059 and 75.080) described qualitative differences in fermentative performances. The release of compounds associated to the peaks at m/z 89.059, m/z 103.075, and m/z 117.093, tentatively identified as acetoin and esters, are coherent with claimed flavor properties of the investigated strains. We propose these mass peaks and their related fragments as biomarkers to optimize the aromatic performances of commercial preparations and for the rapid massive screening of yeast collections.

Target metabolite and gene transcription profiling during the development of superficial scald in apple (Malus x domestica Borkh).

BACKGROUND: Fruit quality features resulting from ripening processes need to be preserved throughout storage for economical reasons. However, during this period several physiological disorders can occur, of which superficial scald is one of the most important, due to the development of large brown areas on the fruit skin surface. RESULTS: This study examined the variation in polyphenolic content with the progress of superficial scald in apple, also with respect to 1-MCP, an ethylene competitor interacting with the hormone receptors and known to interfere with this etiology. The change in the accumulation of these metabolites was further correlated with the gene set involved in this pathway, together with two specific VOCs (Volatile Organic Compounds), α-farnesene and its oxidative form, 6-methyl-5-hepten-2-one. Metabolite profiling and qRT-PCR assay showed these volatiles are more heavily involved in the signalling system, while the browning coloration would seem to be due more to a specific accumulation of chlorogenic acid (as a consequence of the activation of MdPAL and MdC3H), and its further oxidation carried out by a polyphenol oxidase gene (MdPPO). In this physiological scenario, new evidence regarding the involvement of an anti-apoptotic regulatory mechanism for the compartmentation of this phenomenon in the skin alone was also hypothesized, as suggested by the expression profile of the MdDAD1, MdDND1 and MdLSD1 genes. CONCLUSIONS: The results presented in this work represent a step forward in understanding the physiological mechanisms of superficial scald in apple, shedding light on the regulation of the specific physiological cascade.

Moderate warming in microcosm experiment does not affect microbial communities in temperate vineyard soils.

Changes in the soil microbial community structure can lead to dramatic changes in the soil ecosystem. Temperature, which is projected to increase with climate change, is commonly assumed to affect microbial communities, but its effects on agricultural soils are not fully understood. We collected soil samples from six vineyards characterised by a difference of about 2 °C in daily soil temperature over the year and simulated in a microcosm experiment different temperature regimes over a period of 1 year: seasonal fluctuations in soil temperature based on the average daily soil temperature measured in the field; soil temperature warming (2 °C above the normal seasonal temperatures); and constant temperatures normally registered in these temperate soils in winter (3 °C) and in summer (20 °C). Changes in the soil bacterial and fungal community structures were analysed by automated ribosomal intergenic spacer analysis (ARISA). We did not find any effect of warming on soil bacterial and fungal communities, while stable temperatures affected the fungal more than the bacterial communities, although this effect was soil dependent. The soil bacterial community exhibited soil-dependent seasonal fluctuations, while the fungal community was mainly stable. Each soil harbours different microbial communities that respond differently to seasonal temperature fluctuations; therefore, any generalization regarding the effect of climate change on soil communities should be made carefully.