Stuck at work? Quantitative proteomics of environmental wine yeast strains reveals the natural mechanism of overcoming stuck fermentation.

During fermentation oenological yeast cells are subjected to a number of different stress conditions and must respond rapidly to the continuously changing environment of this harsh ecological niche. In this study we gained more insights into the cell adaptation mechanisms by linking proteome monitoring with knowledge on physiological behaviour of different strains during fermentation under model winemaking conditions. We used 2D-DIGE technology to monitor the proteome evolution of two newly discovered environmental yeast strains Saccharomyces bayanus and triple hybrid Saccharomyces cerevisiae × Saccharomyces kudriavzevii × S. bayanus and compared them to data obtained for the commercially available S. cerevisiae strain. All strains examined showed (i) different fermentative behaviour, (ii) stress resistance as well as (iii) susceptibility to stuck fermentation which was reflected in significant differences in protein expression levels. During our research we identified differentially expressed proteins in 155 gel spots which correspond to 70 different protein functions. Differences of expression between strains were observed mainly among proteins involved in stress response, proteins degradation pathways, cell redox homeostasis and amino acids biosynthesis. Interestingly, the newly discovered triple hybrid S. cerevisiae × S. kudriavzevii × S. bayanus strain which has the ability to naturally restart stuck fermentation showed a very strong induction of expression of two proteolytic enzymes: Pep4 and Prc1 that appear as numerous isoforms on the gel image and which may be the key to its unique properties. This study is an important step towards the better understanding of wine fermentations at a molecular level.

In Vitro Bioaccessibility and Bioavailability of Iron from Potatoes with Varying Vitamin C, Carotenoid, and Phenolic Concentrations.

The bioaccessibility and bioavailability of iron from 12 Andean potato clones were estimated using an in vitro gastrointestinal digestion procedure and the Caco-2 cell line as a model of human intestine, with ferritin formation as a marker of iron absorption. We first showed that 63.7% (for the genotype CIP_311422.016) to 79.0% (for the genotype CIP_311575.003) of the iron is released from the potato tuber matrix during in vitro gastrointestinal digestion and is therefore available at the intestinal level. On average, 32 and 24.5% of the hydrophilic bioactive components, vitamin C and chlorogenic acid, respectively, were also bioaccessible from boiled tubers. Intestinal absorption of intrinsic iron from potato tubers could not be detected using our in vitro Caco-2 cell model. When an extrinsic source of iron (20 µM FeCl3 and 1 mM ascorbic acid) was added to the digestion mixture, iron absorption varied from 1.8 to 8% for the genotypes CIP_311422.016 and CIP_311624.021, respectively, as compared to the reference control. Principal component analysis revealed negative relationships between bioavailable iron values and phenolic concentrations, whereas vitamin C concentrations were positively associated with the ferritin values. Further controlled intervention trials would be needed to conclusively assess the bioavailability of intrinsic iron from potato tubers.

Apple russeting as seen through the RNA-seq lens: strong alterations in the exocarp cell wall.

Russeting, a commercially important defect in the exocarp of apple (Malus × domestica), is mainly characterized by the accumulation of suberin on the inner part of the cell wall of the outer epidermal cell layers. However, knowledge on the underlying genetic components triggering this trait remains sketchy. Bulk transcriptomic profiling was performed on the exocarps of three russeted and three waxy apple varieties. This experimental design was chosen to lower the impact of genotype on the obtained results. Validation by qPCR was carried out on representative genes and additional varieties. Gene ontology enrichment revealed a repression of lignin and cuticle biosynthesis genes in russeted exocarps, concomitantly with an enhanced expression of suberin deposition, stress responsive, primary sensing, NAC and MYB-family transcription factors, and specific triterpene biosynthetic genes. Notably, a strong correlation (R(2) = 0.976) between the expression of a MYB93-like transcription factor and key suberin biosynthetic genes was found. Our results suggest that russeting is induced by a decreased expression of cuticle biosynthetic genes, leading to a stress response which not only affects suberin deposition, but also the entire structure of the cell wall. The large number of candidate genes identified in this study provides a solid foundation for further functional studies.

Description of the mechanisms underlying geosmin production in Penicillium expansum using proteomics.

A 2D-DIGE proteomics experiment was performed to describe the mechanism underlying the production of geosmin, an earthy-smelling sesquiterpene which spoils wine, produced by Penicillium expansum. The strains were identified by sequencing of the ITS and beta-tubulin regions. This study was based on a selection of four strains showing different levels of geosmin production, assessed by GC-MS/MS. The proteomics study revealed the differential abundance of 107 spots between the different strains; these were picked and submitted to MALDI-TOF-TOF MS analysis for identification. They belonged to the functional categories of protein metabolism, redox homeostasis, metabolic processes (glycolysis, ATP production), cell cycle and cell signalling pathways. From these data, an implication of oxidative stress in geosmin production may be hypothesized. Moreover, the differential abundance of some glycolytic enzymes may explain the different patterns of geosmin biosynthesis. This study provides data for the characterisation of the mechanism and the regulation of the production of this off-flavour, which are so far not described in filamentous fungi. BIOLOGICAL SIGNIFICANCE: Green mould on grapes, caused by P. expansum may be at the origin of off-flavours in wine. These are characterized by earthy-mouldy smells and are due to the presence of the compound geosmin. This work aims at describing how geosmin is produced by P. expansum. This knowledge is of use for the research community on grapes for understanding why these off-flavours occasionally occur in vintages.

The impact of in vitro digestion on bioaccessibility of polyphenols from potatoes and sweet potatoes and their influence on iron absorption by human intestinal cells.

The composition of potatoes as determined by chemical extraction has been described extensively. It is thus quite well known that, among other compounds, potato is rich in polyphenols, vitamins and in some minerals. This paper underlines the important role of simulated gastro-intestinal in vitro digestion in the bioaccessibility of polyphenols (chlorogenic acid and derivatives, and rutin) from potatoes and sweet potatoes and their impact on iron uptake. Concentrations of polyphenols in the flesh of two potato cultivars (Nicola, white potato, and Vitelotte, purple potato) and sweet potato were measured by Ultra Performance Liquid Chromatography after boiling and after in vitro digestion. Chemical extraction underestimates polyphenol amounts that can be released during digestion and that are actually bioaccessible. Iron uptake, as evaluated by a ferritin assay, by intestinal human cells was decreased after incubation with the intestinal phase of in vitro digestion, presumably due to the presence of polyphenols.

Unusual immuno-modulatory triterpene-caffeates in the skins of russeted varieties of apples and pears.

Three triterpene-caffeates have been isolated from skins of a russeted apple cultivar "Merton Russet" and identified by LC-MS and NMR as betulinic acid-3-cis-caffeate, betulinic acid-3-trans-caffeate, and oleanolic acid-3-trans-caffeate. Betulinic acid-3-trans-caffeate and oleanolic acid-3-trans-caffeate were also found in russeted pear skins. These compounds have not been previously reported in apples or pears, or in any other foods. Their presence was related to suberized tissue as they were only found in russet portions of the partially russeted apple cultivar "Cox's Orange Pippin" and were not detected in the waxy apple cultivar "Royal Gala". High concentrations of betulinic acid-3-trans-caffeate were found in the bark of both "Merton Russet" and "Royal Gala" trees. The three triterpene-caffeates showed anti-inflammatory activity in vitro, inhibiting NF-κB activation with IC50's of 6-9 µM. Betulinic acid-3-trans-caffeate, the predominant compound in the apples, was immuno-modulatory at around 10 µM in the in vitro and ex vivo bioassays, boosting production of the pro-inflammatory cytokine TNFα in cells stimulated with bacterial lipopolysaccharides.

Detection and quantification of natural contaminants of wine by gas chromatography-differential ion mobility spectrometry (GC-DMS).

Rapid and direct, in situ headspace screening for odoriferous volatile organic compounds (VOCs) present in fresh grapes and in wines is a very promising method for quality control because the economic value of a wine is closely related to its aroma. Long used for the detection of VOCs in complex mixtures, miniature differential ion mobility spectrometry (DMS) seems therefore adequate for in situ trace detection of many kinds of VOCs of concern appearing in the headspace of selected foodstuffs. This work aims at a rapid detection, identification, and quantification of some natural and volatile contaminants of wine such as geosmin, 2-methylisoborneol (2-MIB), 1-octen-3-ol, 1-octen-3-one, and pyrazines (2-isopropyl-3-methoxypyrazine, IPMP, and 3-isobutyl-2-methoxypyrazine, IBMP). In the present study, these compounds were spiked at a known concentration in wine and analyzed with a hyphenated trap-GC-DMS device. The detection of all target compounds at concentrations below the human olfactory threshold was demonstrated.

Anti-inflammatory procyanidins and triterpenes in 109 apple varieties.

We evaluated the potential of apple to reduce inflammation. Phenolic compounds and triterpenes were analyzed in 109 apple cultivars. Total phenolics ranged from 29 to 7882 µg g(-1) of fresh weight (FW) in the flesh and from 733 to 4868 µg g(-1) FW in the skin, with flavanols including epicatechin and procyanidins as major components. Ursolic (44.7 to 3522 µg g(-1) FW) and oleanolic (47.2 to 838 µg g(-1) FW) acids dominated the skin triterpene profile. Five chemically contrasting cultivars were fractionated and their immune-modulating activity measured using two cell-based assays targeting key points in the inflammation process. Cultivars exhibiting high contents of procyanidins were the most potent at inhibiting NF-κB while triterpene-rich fractions reduced the promoter activity of the gene of TNFα. This study provides new insights into how apple genetic diversity could be used to alleviate inflammation.

Iron uptake and homeostasis related genes in potato cultivated in vitro under iron deficiency and overload.

Potato is one of the most important staple food in the world because it is a good source of vitamin C, vitamin B6 but also an interesting source of minerals including mainly potassium, but also magnesium, phosphorus, manganese, zinc and iron to a lesser extent. The lack of iron constitutes the main form of micronutrient deficiency in the world, namely iron deficiency anemia, which strongly affects pregnant women and children from developing countries. Iron biofortification of major staple food such as potato is thus a crucial issue for populations from these countries. To better understand mechanisms leading to iron accumulation in potato, we followed in an in vitro culture experiment, by qPCR, in the cultivar Désirée, the influence of media iron content on the expression of genes related to iron uptake, transport and homeostasis. As expected, plantlets grown in a low iron medium (1 mg L(-1) FeNaEDTA) displayed a decreased iron content, a strong induction of iron deficiency-related genes and a decreased expression of ferritins. Inversely, plantlets grown in a high iron medium (120 mg L(-1) FeNaEDTA) strongly accumulated iron in roots; however, no significant change in the expression of our set of genes was observed compared to control (40 mg L(-1) FeNaEDTA).

Polyphenol and glycoalkaloid contents in potato cultivars grown in Luxembourg.

The polyphenol (phenolic acids, flavanols and flavonols) and glycoalkaloid (α-chaconine and α-solanine) contents of potato tubers grown in Luxembourg were analyzed by UPLC-DAD and HPLC-MS/MS separately in peel (approx. 2mm), outer (approx. 1cm) and inner flesh. Polyphenol contents decreased from the peel via the outer to the inner flesh and differed among the cultivars. The cultivars Vitelotte and Luminella had the highest polyphenol contents (5202 and 572 µg/g dry weight (DW) in the outer flesh), whereas Charlotte and Bintje had the lowest contents (19.5 and 48.0 µg/g DW). Chlorogenic acid and its isomers (neo- and cryptochlorogenic acid) were the major polyphenols. Glycoalkaloid contents were highest in the peel and lowest in the inner flesh, values in the flesh were below guideline limits in all cultivars. In conclusion, potatoes contribute to the daily intake of polyphenols and their consumption, thereby, may have positive effects on health.

Ascorbic acid, phenolic acid, flavonoid, and carotenoid profiles of selected extracts from Ribes nigrum.

Small fruits such as berries have low energy contents, but high contents of vitamins, micronutrients, and dietary fibers and constitute a good source of natural antioxidant compounds that are important constituents of the human diet. This study identified a large number of compounds in an extract of black currant showing high antioxidant activity and compared their profile in various parts of the plants (leaves, buds, and berries). If it was known that berries contained very high levels of natural phenolic compounds, this study showed that leaves and buds could also be considered good sources of natural antioxidants. Indeed, they contained high amounts of phenolic acids, flavonoids, and carotenoids. An acetone mixture can extract several classes of phenolic compounds with a good yield of flavonols, flavan-3-ols, and anthocyanins.

Carbohydrate metabolism and cell protection mechanisms differentiate drought tolerance and sensitivity in advanced potato clones (Solanum tuberosum L.).

In potatoes and many other crops, drought is one of the most important environmental constraints leading to yield loss. Development of drought-tolerant cultivars is therefore required for maintaining yields under climate change conditions and for the extension of agriculture to sub-optimal cropping areas. Drought tolerance mechanisms have been well described for many crop plants including Native Andean potato. However, knowledge on tolerance traits suitable for commercial potato varieties is scarce. In order to describe drought tolerance mechanisms that sustain potato yield under water stress, we have designed a growth-chamber experiment with two Solanum tuberosum L. cultivars, the more drought tolerant accession 397077.16, and the sensitive variety Canchan. After 21 days of drought exposure, gene expression was studied in leaves using cDNA microarrays. The results showed that the tolerant clone presented more differentially expressed genes than the sensitive one, suggesting greater stress response and adaptation. Moreover, it exhibited a large pool of upregulated genes belonging to cell rescue and detoxication such as LEAs, dehydrins, HSPs, and metallothioneins. Transcription factors related to abiotic stresses and genes belonging to raffinose family oligosaccharide synthesis, involved in desiccation tolerance, were upregulated to a greater extent in the tolerant clone. This latter result was corroborated by biochemical analyses performed at 32 and 49 days after drought that showed an increase in galactinol and raffinose especially in clone 397077.16. The results depict key components for the drought tolerance of this advanced potato clone.

Identification of drought-responsive compounds in potato through a combined transcriptomic and targeted metabolite approach.

Two potato clones (Solanum tuberosum L.) of the Andean cultivar group, called Sullu and SS2613, with different drought-tolerance phenotypes were exposed to a continuously increasing drought stress in a field trial. At the physiological level, while relative leaf water contents were similar in both clones, osmotic potential was lower in Sullu and declined more strongly during drought compared with SS2613. In the drought-stressed plants, tuber yield was reduced by about 70% compared with control plants in both clones. Potato cDNA microarrays and target metabolite analysis were performed on leaves sampled at several time-points after the onset of drought. At the transcriptomic level, photosynthesis-related genes were already strongly repressed in Sullu after 28 d of withholding irrigation and even more strongly after a longer stress duration, whereas, in SS2613, repression occurred only after 49 d of soil drying; similarly, a strong perturbation of carbohydrate-related genes was observed in Sullu. At the metabolite level, differential accumulation of osmotically active solutes was observed between the two cultivars; indeed, in Sullu, contents of galactose, inositol, galactinol, proline, and proline analogues were higher upon drought stress compared with SS2613. These results point to different drought responses in the cultivars at the leaf level, with, however, similar tuber yield reductions. The previously shown tolerant clone Sullu lost part of its tolerance under the experimental conditions used here; it was, however, able to maintain an absolute yield three times higher than SS2613.

Monitoring gene expression of potato under salinity using cDNA microarrays.

The molecular response to salt exposure was studied in the leaves of a Solanum tuberosum clone using cDNA microarray. Differentially expressed genes were classified according to their known or predicted function and their expression ratio as compared to the control. The major changes upon a 150 mM NaCl exposure in potato leaves occurred in the photosystem apparatus and Calvin cycle: many transcripts coding for proteins belonging to photosystems I and II and chlorophyll synthesis were repressed. On the other hand, we observed the induction of various kinds of transcription factors implicated in osmotic stress response via ABA-dependent or ABA-independent pathways but also in plant defense pathways. This revealed a crosstalk between abiotic and biotic stress responses during salt exposure, which activated several adaptation mechanisms including heat shock proteins, late embryogenesis abundant, dehydrins and PR proteins. Gene expression changes related to carbohydrate and amino acid metabolism were also observed, pointing at putative modifications at the metabolic level.

Gene expression changes related to the production of phenolic compounds in potato tubers grown under drought stress.

Polyphenols represent a large family of plant secondary metabolites implicated in the prevention of various diseases such as cancers and cardiovascular diseases. The potato is a significant source of polyphenols in the human diet. In this study, we examined the expression of thirteen genes involved in the biosynthesis of polyphenols in potato tubers using real-time RT-PCR. A selection of five field grown native Andean cultivars, presenting contrasting polyphenol profiles, was used. Moreover, we investigated the expression of the genes after a drought exposure. We concluded that the diverse polyphenolic profiles are correlated to variations in gene expression profiles. The drought-induced variations of the gene expression was highly cultivar-specific. In the three anthocyanin-containing cultivars, gene expression was coordinated and reflected at the metabolite level supporting a hypothesis that regulation of gene expression plays an essential role in the potato polyphenol production. We proposed that the altered sucrose flux induced by the drought stress is partly responsible for the changes in gene expression. This study provides information on key polyphenol biosynthetic and regulatory genes, which could be useful in the development of potato varieties with enhanced health and nutritional benefits.

Modification of the health-promoting value of potato tubers field grown under drought stress: emphasis on dietary antioxidant and glycoalkaloid contents in five native andean cultivars (Solanum tuberosum L.).

The effects of drought stress on dietary antioxidant and glycoalkaloid contents in potato tubers were investigated using a selection of five native Andean cultivars. Both freshly harvested and 4 month-stored tubers were analyzed. Responses to drought stress were highly cultivar-specific. The antioxidant contents of the yellow tuber-bearing cultivars (Sipancachi and SS-2613) were weakly affected by the drought treatment, whereas the pigmented cultivars demonstrated highly cultivar-dependent variations. A drastic reduction of anthocyanins and other polyphenols was revealed in the red- (Sullu) and purple-fleshed (Guincho Negra) cultivars, whereas an increase was shown in the purple-skinned and yellow-fleshed cultivar (Huata Colorada). The hydrophilic antioxidant capacity (evaluated by Folin-Ciocalteu and H-oxygen radical absorbance capacity assays) was highly correlated with the polyphenol content and followed, therefore, the same behavior upon drought. Carotenoid contents, including beta-carotene, as well as vitamin E, tended to increase or remain stable following drought exposure, except for the cultivar Sullu, in which the level of these lipophilic antioxidants was decreased. Vitamin C contents were not affected by drought with the exception of Guincho Negra, in which the level was increased. These variations of health-promoting compounds were associated with increased or stable levels of the toxic glycoalkaloids, alpha-solanine and alpha-chaconine. Storage at 10 degrees C for 4 months tended to decrease the concentrations of all dietary antioxidants, except those of vitamin E. This storage also reduced the drought-induced variations observed in freshly harvested tubers. These results were discussed in terms of their implications for human diet and health as well as in plant stress defense mechanisms.

Antioxidant profiling of native Andean potato tubers (Solanum tuberosum L.) reveals cultivars with high levels of beta-carotene, alpha-tocopherol, chlorogenic acid, and petanin.

The antioxidant profile of 23 native Andean potato cultivars has been investigated from a human nutrition perspective. The main carotenoid and tocopherol compounds were studied using high-performance liquid chromatography coupled with a diode array detector (HPLC-DAD) and a fluorescence detector, respectively, whereas polyphenols (including anthocyanins in colored tubers) were identified by means of both HPLC-mass spectrometry and HPLC-DAD. Antioxidant profiling revealed significant genotypic variations as well as cultivars of particular interest from a nutritional point of view. Concentrations of the health-promoting carotenoids, lutein and zeaxanthin, ranged from 1.12 to 17.69 microg g(-1) of dry weight (DW) and from 0 to 17.7 microg g(-1) of DW, with cultivars 704353 and 702472 showing the highest levels in lutein and zeaxanthin, respectively. Whereas beta-carotene is rarely reported in potato tubers, remarkable levels of this dietary provitamin A carotenoid were detected in 16 native varieties, ranging from 0.42 to 2.19 microg g(-1) of DW. The amounts of alpha-tocopherol found in Andean potato tubers, extending from 2.73 to 20.80 microg g(-1) of DW, were clearly above the quantities generally reported for commercial varieties. Chlorogenic acid and its isomers dominated the polyphenolic profile of each cultivar. Dark purple-fleshed tubers from the cultivar 704429 contained exceptionally high levels of total anthocyanins (16.33 mg g(-1) of DW). The main anthocyanin was identified as petanin (petunidin-3-p-coumaroyl-rutinoside-5-glucoside). The results suggest that Andean potato cultivars should be exploited in screening and breeding programs for the development of potato varieties with enhanced health and nutritional benefits.

Andean potato cultivars (Solanum tuberosum L.) as a source of antioxidant and mineral micronutrients.

Potato tubers were evaluated as a source of antioxidants and minerals for the human diet. A genetically diverse sample of Solanum tuberosum L. cultivars native to the Andes of South America was obtained from a collection of nearly 1000 genotypes using microsatellite markers. This size-manageable collection of 74 landraces, representing at best the genetic diversity among potato germplasm, was analyzed for iron, zinc, calcium, total phenolic, total carotenoid, and total vitamin C contents. The hydrophilic antioxidant capacity of each genotype was also measured using the oxygen radical absorbance capacity (ORAC) assay. The iron content ranged from 29.87 to 157.96 microg g-1 of dry weight (DW), the zinc content from 12.6 to 28.83 microg g-1 of DW, and the calcium content from 271.09 to 1092.93 microg g-1 of DW. Total phenolic content varied between 1.12 and 12.37 mg of gallic acid equiv g-1 of DW, total carotenoid content between 2.83 and 36.21 microg g-1 of DW, and total vitamin C content between 217.70 and 689.47 microg g-1 of DW. The range of hydrophilic ORAC values was 28.25-250.67 micromol of Trolox equiv g-1 of DW. The hydrophilic antioxidant capacity and the total phenolic content were highly and positively correlated (r = 0.91). A strong relationship between iron and calcium contents was also found (r = 0.67). Principal component analysis on the studied nutritional contents of the core collection revealed that most potato genotypes were balanced in terms of antioxidant and mineral contents, but some of them could be distinguished by their high level in distinct micronutrients. Correlations between the micronutrient contents observed in the sample and the genetic distances assessed by microsatellites were weakly significant. However, this study demonstrated the wide variability of health-promoting micronutrient levels within the native potato germplasm as well as the significant contribution that distinct potato tubers may impart to the intake in dietary antioxidants, zinc, and iron.

Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress.

Plant stress studies are more and more based on gene expression. The analysis of gene expression requires sensitive, precise, and reproducible measurements for specific mRNA sequences. Real-time RT-PCR is at present the most sensitive method for the detection of low abundance mRNA. To avoid bias, real-time RT-PCR is referred to one or several internal control genes, which should not fluctuate during treatments. Here, the non-regulation of seven housekeeping genes (beta-tubulin, cyclophilin, actin, elongation factor 1-alpha (ef1alpha), 18S rRNA, adenine phosphoribosyl transferase (aprt), and cytoplasmic ribosomal protein L2) during biotic (late blight) and abiotic stresses (cold and salt stress) was tested on potato plants using geNorm software. Results from the three experimental conditions indicated that ef1alpha was the most stable among the seven tested. The expression of the other housekeeping genes tested varied upon stress. In parallel, a study of the variability of expression of hsp20.2, shown to be implicated in late blight stress, was realized. The relative quantification of the hsp20.2 gene varied according to the internal control and the number of internal controls used, thus highlighting the importance of the choice of internal controls in such experiments.

Differential gene expression in two potato lines differing in their resistance to Phytophthora infestans.

Horizontal resistance to late blight in the potato is a primary objective of many breeding programs. Knowledge of the physiological and biochemical mechanisms underlying it, however, is scarce. The purpose of the present study was the identification of these physiological and biochemical factors in plant material obtained by crossing a late blight resistant Solanum phureja clone with a susceptible dihaploid of S. tuberosum subsp. tuberosum. The mRNA RT-PCR differential display method was used to compare the gene expression patterns of a resistant hybrid with that of a susceptible one. By sequence homology, we identified several genes with diverse functions, including genes known to be involved in resistance or stress responses and genes known to be involved in primary or secondary metabolism.