Modern tomatoes have narrow genetic diversity limiting their improvement potential. We present a tomato pan-genome constructed using genome sequences of 725 phylogenetically and geographically ...representative accessions, revealing 4,873 genes absent from the reference genome. Presence/absence variation analyses reveal substantial gene loss and intense negative selection of genes and promoters during tomato domestication and improvement. Lost or negatively selected genes are enriched for important traits, especially disease resistance. We identify a rare allele in the TomLoxC promoter selected against during domestication. Quantitative trait locus mapping and analysis of transgenic plants reveal a role for TomLoxC in apocarotenoid production, which contributes to desirable tomato flavor. In orange-stage fruit, accessions harboring both the rare and common TomLoxC alleles (heterozygotes) have higher TomLoxC expression than those homozygous for either and are resurgent in modern tomatoes. The tomato pan-genome adds depth and completeness to the reference genome, and is useful for future biological discovery and breeding.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Volatile phenols possess “smoky, spicy” aromas and are routinely measured in grapes, wines and other foodstuffs for quality control. Routine analyses of volatile phenols rely on gas chromatography – ...mass spectrometry (GC-MS), but slow throughput of GC-MS can cause challenges during times of surge demand, i.e. following ‘smoke taint’ events involving forest fires near vineyards. Parallel extraction of headspace volatiles onto sorbent sheets (HS-SPMESH) followed by direct analysis in real time mass spectrometry (DART-MS) is a rapid alternative to conventional GC-MS approaches. However, HS-SPMESH extraction is poorly suited for lower volatility odorants, including volatile phenols. This work reports development and validation of an HS-SPMESH-DART-MS approach for five volatile phenols (4-ethylphenol, 4-ethylguiacol, guaiacol, 4-methylguaiacol, and cresols). Prior to HS-SPMESH extraction, volatile phenols were acetylated to facilitate their extraction. A unique feature of this work was the use of d6-Ac2O as a derivatizing agent to overcome issues with isobaric interferences inherent to chromatography-free MS techniques. The use of alkaline conditions during derivatization resulted in cumulative measurement of both free and bound forms of volatile phenols. The validated HS-SPMESH-DART-MS method achieved a throughput of 24 samples in ∼60 min (including derivatization and extraction time) with low limits of detection (<1 μg L−1) and good repeatability (3–6% RSD) in grape and wine matrices. Validation experiments with smoke-tainted grape samples indicated good correlation between total (free + bound) volatile phenols measured by HS-SPMESH-DART-MS and a gold standard GC-MS method.
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•Acetylation of volatile phenols in grapes and wine to increase volatility.•Parallel extraction of derivatized volatile phenols with sorbent sheets.•Rapid quantitation of extracted volatile phenols using DART-MS.•Resolution of isobaric interferences with a deuterated derivatizing agent.•Validation of new method against gold standard GC-MS method.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Copper–sulfhydryl complexes in wine can be disrupted by addition of brine to release free hydrogen sulfide (H2S), and the resulting “brine-releasable H2S” is reported to correlate with formation of ...H2S during bottle storage. However, both the mechanism of the brine-release assay and factors affecting the stability of copper sulfhydryls under brine-release conditions are not well understood. By varying brine composition and concentration, it is shown that release of copper-complexed H2S requires the presence of a halide (Cl– and Br–) and is not due to a general “salting-out” effect. Release of copper-complexed H2S by the brine dilution assay is highly temperature-dependent. When H2S and Cu(II) are added to a model wine, brine-releasable H2S decreases markedly (∼10-fold) after a 20 min incubation period prior to performing the brine-release assay. In commercial wines, the fraction of added H2S recovered through the brine-release assay was correlated with the initial glutathione (GSH) concentration (r 2 = 0.58) but not with initial Cu. Negligible additional release of H2S from organopolysulfanes was observed following addition of a disulfide-reducing agent (tris(2-carboxyethyl)phosphine). As previous studies have reported a correlation between H2S formed under brine-release conditions and normal storage, these results suggest that the susceptibility of a wine to form latent copper–sulfhydryl precursors of H2S following copper addition is dependent on the concentration of sulfhydryls like GSH.
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IJS, KILJ, NUK, PNG, UL, UM, UPUK
Poly(dimethylsiloxane)-based thin-film sorbent sheets (SPMESH) have previously been used for parallel headspace (HS) extraction prior to direct analysis in real-time mass spectrometry (DART-MS) for ...rapid quantitation of odorants in complex matrices. However, HS-SPMESH extraction is poorly suited for less volatile odorants, e.g., volatile phenols. This report describes modifications to the previous SPMESH extraction device, which make it amenable to parallel extraction of low-volatility analytes from multiwell plates under direct immersion (DI) conditions. Optimization and validation of the DI-SPMESH-DART-MS approach were performed on four volatile phenols (4-ethylphenol, 4-ethylguaiacol, 4-methylguaiacol, and guaiacol) of relevance to the quality of grape juices. Negative-ion mode DART-MS spectra showed a series of oxygenated adducts M + nO – H− for all analytes, but isobaric interferences could be limited for three of the four analytes by selecting an appropriate MS/MS transition. Signal suppression from nonvolatiles (sugars, acids) could be overcome by a rinse step. DI-SPMESH-DART-MS analysis of 24 samples could be performed in ∼45 min (30 min extraction, 16 min DART analysis) with 0.5–3 μg/L detection limits in aqueous and model juice solutions. In real grape juices (n = 5 cultivars), good accuracy (72–137%) could be achieved for two of the four volatile phenols initially investigated, 4-ethylphenol and 4-ethylguaiacol. However, poor accuracy was observed for guaiacol in some cultivars, and 4-methylguaiacol could not be quantitated due to interferences with other volatile phenols. Despite these limitations, DI-SPMESH-DART-MS/MS may be useful for prescreening a large number of samples prior to more selective conventional analyses.
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IJS, KILJ, NUK, PNG, UL, UM, UPUK
Quantitative and qualitative measurements of trace-level analytes in plants or foodstuffs, e.g., secondary metabolites like carotenoids, are often performed at centralized core facilities or off-site ...laboratories. However, preparation, storage, and/or transport of both intact samples and sample extracts may be cumbersome and complicated, especially for air-sensitive analytes. We describe the development of inexpensive swellable microextraction (SweME) devices for extraction and storage of nonpolar analytes. SweME devices consist of a thin layer of poly(dimethylsiloxane) (PDMS) grafted onto a stainless steel support. Pretreating the SweME device with small volumes of the organic solvent causes the PDMS to swell. The swollen SweME device can then be immersed directly into complex matrices for absorptive extraction of low-molecular-weight, nonpolar analytes. Following storage, analytes can be solvent-desorbed prior to characterization. Proof-of-principle work with carotenoids from tomatoes and carrots demonstrates that SweME is appropriate for semiquantitative analyses and increases the stability of air-sensitive analytes during storage at ambient temperatures as compared to the solvent extracts. Carotenoid profiles (fractional carotenoid contributions) from tomato and carrot samples were well correlated between SweME and liquid–liquid extraction (R 2 = 0.97 and 0.94). Lycopene, the most abundant carotenoid in tomatoes, saw a less than 20% decrease in extracted mass during 1 month of ambient SweME storage. Extractions and desorptions can be run in parallel using multiwell plates. In summary, swelled sorbent extraction with SweME devices is a convenient and inexpensive approach for isolation and storage of analytes in complex matrices and may be particularly well suited for evaluating large numbers of plant samples through external laboratories.
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IJS, KILJ, NUK, PNG, UL, UM, UPUK
Quantitation of trace-level (μg/L to ng/L) volatile compounds is routinely performed in a broad range of applications, including analyses of odorants, pesticide residues, or toxins in foodstuffs and ...related matrices. Conventional analyses based on gas chromatography–mass spectrometry (GC-MS) are limited by low throughput, and ambient approaches to sample introduction have typically had poor sensitivity. We prepared polydimethylsiloxane-coated stainless steel meshes for extraction and preconcentration of volatiles (Solid Phase Mesh Enhanced Sorption from Headspace, SPMESH), which could then be analyzed by Direct Analysis in Real Time (DART)-MS. The SPMESH cards were characterized by electron microscopy, and figures of merit for the approach were determined using two representative volatiles: 2-isobutyl-3-methoxypyrazine (IBMP) and linalool. Using DART-MS/MS and isotopically labeled internal standards, we achieved detection limits of 21 ng/L and 71 μg/L for IBMP and linalool in water. Good accuracy and precision could also be achieved for IBMP spikes in grape macerate, although accuracy for linalool was compromised by the presence of interferences. Detection limits could be further improved by an order of magnitude through the use of high resolution (HR) MS. Because extraction can be performed inexpensively in parallel and because it requires short data acquisition times (<1 min), SPMESH-DART-MS may be appropriate for high throughput trace level volatile analyses.
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IJS, KILJ, NUK, PNG, UL, UM
The level of hydrogen sulfide (H2S) can increase during abiotic storage of wines, and potential latent sources of H2S are still under investigation. We demonstrate that elemental sulfur (S0) residues ...on grapes not only can produce H2S during fermentation but also can form precursors capable of generating additional H2S after bottle storage for 3 months. H2S could be released from S0-derived precursors by addition of a reducing agent (TCEP), but not by addition of strong brine to induce release of H2S from metal sulfide complexes. The size of the TCEP-releasable pool varied among yeast strains. Using the TCEP assay, multiple polar S0-derived precursors were detected following normal-phase preparative chromatography. Using reversed-phase liquid chromatography and high-resolution mass spectrometry, we detected an increase in the levels of diglutathione trisulfane (GSSSG) and glutathione disulfide (GSSG) in S0-fermented red wine and an increase in the levels of glutathione S-sulfonate (GSSO3 –) and tetrathionate (S4O6 2–) in S0-fermented white wine as compared to controls. GSSSG, but not S4O6 2–, was shown to evolve H2S in the presence of TCEP. Pathways for the formation of GSSSG, GSSG, GSSO3 –, and S4O6 2– from S0 are proposed.
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IJS, KILJ, NUK, PNG, UL, UM, UPUK
Although they possess significant viticultural advantages, interspecific hybrid grapes (Vitis spp.) are reported to produce wine with lower tannin concentrations than European wine varieties (Vitis ...vinifera). However, extensive quantitative data on this phenomenon as well as mechanistic explanations for these differences are lacking. A survey of primarily commercial wines from the Finger Lakes American Viticultural Area (New York) using a protein precipitation method determined that hybrid-based wines had >4-fold lower tannin concentrations than vinifera wines. To elucidate factors responsible for differences in wine tannin, 24 wines were produced from both red hybrid and vinifera cultivars under identical conditions. Lower wine tannin in French–American hybrid- than vinifera-based wines could be partially explained by lower grape tannin. However, experiments in which cell wall material was incubated with tannin indicated that cell wall binding may be of equal or greater importance in explaining lower wine tannin concentrations in hybrid-based wines. Subsequent characterization of cell wall material revealed that protein in flesh cell walls and, to a lesser extent, pectin in skin cell walls were correlated with cell wall binding.
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IJS, KILJ, NUK, PNG, UL, UM, UPUK
Volatile sulfur compounds (VSCs), particularly low molecular weight sulfhydryls like hydrogen sulfide (H
2
S) and methanethiol (MeSH), are often observed in wines with sulfurous off-aromas. Recent ...work has shown both H
2
S and MeSH can increase up to a few µM (> 40 µg/L) during anoxic storage, but the identity of the latent sources of these sulfhydryls is still disputed. This review critically evaluates the latent precursors and pathways likely to be responsible for the loss and formation of these sulfhydryls during wine storage based on the existing enology literature as well as studies from food chemistry, geochemistry, biochemistry, and synthetic chemistry. We propose that three precursor classes have sufficient concentration and metastability to serve as latent sulfhydryl precursors in wine: 1) transition metal-sulfhydryl complexes, particularly those formed following Cu(II) addition, which are released under anoxic conditions through an unknown mechanism; 2) asymmetric disulfides, polysulfanes, and (di)organopolysulfanes formed through transition-metal mediated oxidation (e.g., Cu(II)) of sulfhydryls or pesticide degradation, and released through sulfitolysis, metal-catalyzed thiol-disulfide exchange or related reactions; 3) S-alkylthioacetates, primarily formed during fermentation, and releasable hydrolytically. Some evidence also exists for S-amino acids serving as precursors. Based on these findings, we propose a "decision tree" approach to choosing appropriate strategies for managing wines with sulfurous off-aromas.
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BFBNIB, GIS, IJS, KISLJ, NUK, PNG, UL, UM, UPUK
Parallel extraction of headspace volatiles from multiwell plates using sorbent sheets (HS-SPMESH) followed by direct analysis in real-time high-resolution mass spectrometry (DART-HRMS) can be used as ...a rapid alternative to solid-phase micro-extraction (SPME) gas-chromatography mass-spectrometry (GC-MS) for trace level volatile analyses. However, an earlier validation study of SPMESH-DART-MS using 3-isobutyl-2-methoxypyrazine (IBMP) in grape juice showed poor correlation between SPMESH-DART-MS and a gold standard SPME-GC-MS around the compound’s odor detection threshold (<10 ng/kg) in grape juice, and lacked sufficient sensitivity to detect IBMP at this concentration in grape homogenate. In this work, we report on the development and validation of an improved SPMESH extraction approach that lowers the limit of detection (LOD < 0.5 ng/kg), and regulates crosstalk between wells (<0.5%) over a calibration range of 0.5−100 ng/kg. The optimized SPMESH-DART-MS method was validated using Cabernet Sauvignon and Merlot grape samples harvested from commercial vineyards in the central valley of California (n = 302) and achieved good correlation and agreement with SPME-GC-MS (R2 = 0.84) over the native range of IBMP (<0.5−20 ng/kg). Coupling of SPMESH to a lower resolution triple quadrupole (QqQ)-MS via a new JumpShot-HTS DART source also achieved low ng/kg detection limits, and throughput was improved through positioning stage optimizations which reduced time spent on intra-well SPMESH areas.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
