Antibacterial Effect of Pomegranate Juice on Listeria innocua and E. coli in Different Media.

The antibacterial effect of pomegranate juice (PJ) at six concentrations (0, 10, 20, 30, 40, and 50%) against Listeria innocua and Escherichia coli was investigated in distilled water (DW) and bacterial culture broth. L. innocua and E. coli at approximately 105 cfu mL-1 were inoculated in PJ samples and incubated at 4, 25, and 37 °C for 0, 6, 24, and 48 h. The bacterial population and pH of culture media were measured at each removal. Results indicated that the antibacterial effect of PJ was dependent upon bacteria species, juice concentration, incubation temperature, and growth medium. Higher juice concentration and incubation temperature resulted in increased antibacterial effects. Bacterial populations were decreased more significantly in DW systems than in the culture broth, while L. innocua was more sensitive to PJ than E. coli in the DW systems. Regardless of PJ concentrations in DW systems, L. innocua, initially inoculated at approximately 105 cfu mL-1, was reduced to undetectable levels at 25 and 37 °C within 24 h. The growth of L. innocua and E. coli was significantly inhibited in bacterial culture broth containing ≥ 20% PJ (p < 0.001). This study provides insight into the potential application of PJ in food and beverage products for food protection.

Genome-wide genetic architecture for plant maturity and drought tolerance in diploid potatoes.

Cultivated potato (Solanum tuberosum) is known to be highly susceptible to drought. With climate change and its frequent episodes of drought, potato growers will face increased challenges to achieving their yield goals. Currently, a high proportion of untapped potato germplasm remains within the diploid potato relatives, and the genetic architecture of the drought tolerance and maturity traits of diploid potatoes is still unknown. As such, a panel of 384 ethyl methanesulfonate-mutagenized diploid potato clones were evaluated for drought tolerance and plant maturity under field conditions. Genome-wide association studies (GWAS) were conducted to dissect the genetic architecture of the traits. The results obtained from the genetic structure analysis of the panel showed five main groups and seven subgroups. Using the Genome Association and Prediction Integrated Tool-mixed linear model GWAS statistical model, 34 and 17 significant quantitative trait nucleotides (QTNs) were found associated with maturity and drought traits, respectively. Chromosome 5 carried most of the QTNs, some of which were also detected by using the restricted two-stage multi-locus multi-allele-GWAS haploblock-based model, and two QTNs were found to be pleiotropic for both maturity and drought traits. Using the non-parametric U-test, one and three QTNs, with 5.13%-7.4% phenotypic variations explained, showed favorable allelic effects that increase the maturity and drought trait values. The quantitaive trait loci (QTLs)/QTNs associated with maturity and drought trait were found co-located in narrow (0.5-1 kb) genomic regions with 56 candidate genes playing roles in plant development and senescence and in abiotic stress responses. A total of 127 potato clones were found to be late maturing and tolerant to drought, while nine were early to moderate-late maturing and tolerant to drought. Taken together, the data show that the studied germplasm panel and the identified candidate genes are prime genetic resources for breeders and biologists in conventional breeding and targeted gene editing as climate adaptation tools.

Comparison of Volatile Compounds Contributing to Flavor of Wild Lowbush (Vaccinium augustifolium) and Cultivated Highbush (Vaccinium corymbosum) Blueberry Fruit Using Gas Chromatography-Olfactometry.

The flavor of blueberry fruit products is an important parameter determining consumer satisfaction. Wild lowbush blueberries are primarily processed into products, but their flavor chemistry has not been characterized. The objective of this study was to characterize the aroma chemistry of lowbush blueberries and compare it with that of highbush. Aroma volatiles of lowbush blueberries from four Canadian provinces and five highbush blueberry cultivars were isolated using headspace solid-phase microextraction (SPME) and characterized using gas chromatography-olfactometry (GC-O) and 2-dimensional gas chromatography-time of flight-mass spectrometry (GC×GC-TOF-MS). Lowbush fruit volatiles were composed of 48% esters, 29% aldehydes and 4% monterpenoids compared to 48% aldehydes, 26% monoterpenoids and 3% esters in highbush fruit. Twenty-three aroma-active peaks were identified in lowbush compared to forty-two in highbush fruit using GC-O. The most aroma-active compounds in lowbush fruit were ethyl 2-methylbutanoate, methyl 2-methylbutanoate, methyl 3-methylbutanoate, ethyl 3-methylbutanoate and ethyl propanoate compared to geraniol, (Z)-3-hexen-1-ol, 1-octen-3-one, α-terpineol and linalool in highbush fruit. The aroma volatile composition was more consistent among lowbush fruit samples than the five highbush cultivars. Aroma-active GC-O peaks were described more frequently as "floral", "fruity", "sweet" and "blueberry" in lowbush than in highbush fruit. Results suggest wild lowbush blueberries would provide "fruitier" and "sweeter" flavors to food products than cultivated highbush fruit.

Effect of natural antimicrobial agent (MicroGARD) combined with edible coating (NatureSeal) treatment on fresh-cut butternut squash.

To improve fresh-cut produce quality and shelf life, 0.5% or 1.0% MicroGARD® 730 (MG) as a natural alternative to synthetic chemical preservatives, 2.5% NatureSeal® (NS) product (vitamin/mineral-based blends), 0.5% MG combined with 2.5% NS, and 1% MG combined with 2.5% NS were used to treat fresh-cut butternut squash (Cucurbita moschata). The 240 g samples were put into food grade bags and stored at 4 or 7 °C. Microbial population, including aerobic plate counts (APCs), yeast and molds, total coliforms, and quality parameters, including head space O2 /CO2 concentration in package, pH, soluble solids, color, and conductivity, were evaluated after 0, 3, 6, 9, 12, and 20 days of storage. Results showed that after 6 days of storage at 7 °C, APC of check and control samples reached to 2.6 × 108 and 1.5 × 107 CFU/g, respectively; while they were kept at 104 CFU/g (3 to 4 log reduction) in the squash samples treated with 0.5% or 1% MG combined with NS at 7 °C. Similar results were found on squash samples stored at 4 °C for 9 days. The cut squash treated with MG combined with NS had APC ≤ 107 CFU/g at 4 °C for about 20 days compared to 9 days in controls or 0.5% MG-treated samples, and 12 days in 1% MG-treated or NS-treated samples, respectively. Considering overall quality and extended shelf life, MG combined with NS was recommended to apply to cut squashes stored at 4 °C. PRACTICAL APPLICATION: This research provided useful information and practical treatment application for developing fresh-cut produce with good quality and extended shelf life up to 20 days at 4 °C.

Tracking the development of the superficial scald disorder and effects of treatments with diphenylamine and 1-MCP using an untargeted metabolomic approach in apple fruit.

Superficial scald is a physiological storage disorder that significantly reduces the marketability of apple fruit. To gain fundamental knowledge about the biochemical pathways leading to the development of the disorder and mechanisms of treatments for prevention, an untargeted metabolomics experiment employing liquid chromatography and mass spectrometry with data independent acquisition was performed. Metabolomic changes of two apple cultivars 'Cortland' and 'Red Delicious' with scald development and scald control treatments, using diphenylamine and 1-MCP, at 0-1 °C for up to 7 months was investigated. In total, 833 features/compounds were analyzed, and among them 59 were found to change significantly in controls involved in scald development, and in response to DPA and 1-MCP treatments. Our results provide new evidence that metabolites in association with phenylpropanoid metabolism, antioxidant and redox systems, and amino acid metabolism are related closely to scald development and response to potential treatments.

Metabolic Profile of Strawberry Fruit Ripened on the Plant Following Treatment With an Ethylene Elicitor or Inhibitor.

Strawberry is the most studied nonclimacteric fruit for understanding the role ethylene has in ripening regulation. However, previous studies on the effects of ethylene on strawberry ripening were conducted with detached fruit. Thus, the aim of this work was to determine the effect of ethylene and the ethylene-action inhibitor 1-methylcyclopropene (1-MCP) applied at different developmental stages on important physical-chemical attributes of ripe 'Albion' strawberry. Fruit at four developmental stages that remained attached to the plant were dipped in one of three treatment solutions (Ethephon, 1-methylcyclopropene, and water), plus one absolute control that received no dip. Following treatment, when immature fruit were fully red or 24 h after treatment for red-treated fruit, strawberry fruit were assessed for physicochemical properties (mass, length, diameter, firmness, color, titratable acidity, soluble solids, pH, total phenolics, sugar, organic acid, amino acid, and volatile composition). The days following treatment required for fruit to ripen were also recorded. Treatments did not affect the rate of ripening nor fruit color, titratable acidity, pH, soluble solids, total phenolics, sugars, or organic acids of ripe fruit. Ethephon affected fruit mass, diameter, length, firmness, anthocyanins, amino acids, and volatiles, but these effects were dependent on fruit developmental stage at which the treatment was applied. When green fruit were treated with ethephon, ripe fruit had larger diameter and mass. Ethephon treatment of white fruit resulted in ripe fruit having greater anthocyanin content. Treatment of pink fruit resulted in ripe fruit having smaller diameter, length, and mass and greater firmness. Treatment of red fruit with ethephon altered fruit volatile composition, increasing concentrations of ethyl- and acetate-esters, which were reduced by the 1-MCP treatment. Ethephon treatment increased concentrations of 11 of the 19 free amino acids measured in ripe fruit with treatment of green and white fruit having the greatest effect. A total of 41 volatile compounds had significant correlations with 14 amino acids. While ethylene did not stimulate typical ripening of strawberry fruit, it does appear to alter fruit development and metabolism. The physiological effects of ethylene on strawberry fruit appear to depend on the developmental stage of the fruit.

Comparative transcriptome expression analysis in susceptible and resistant potato (Solanum tuberosum) cultivars to common scab (Streptomyces scabies) revealed immune priming responses in the incompatible interaction.

Common scab disease in potato has become a widespread issue in major potato production areas, leading to increasing economic losses. Varietal resistance is seen as a viable and long-term scab management strategy. However, the genes and mechanisms of varietal resistance are unknown. In the current study, a comparative RNA transcriptome sequencing and differential gene signaling and priming sensitization studies were conducted in two potato cultivars that differ by their response to common scab (Streptomyces scabies), for unraveling the genes and pathways potentially involved in resistance within this pathosystem. We report on a consistent and contrasted gene expression pattern from 1,064 annotated genes differentiating a resistant (Hindenburg) and a susceptible (Green Mountain) cultivars, and identified a set of 273 co-regulated differentially expressed genes in 34 pathways that more likely reflect the genetic differences of the cultivars and metabolic mechanisms involved in the scab pathogenesis and resistance. The data suggest that comparative transcriptomic phenotyping can be used to predict scab lesion phenotype in breeding lines using mature potato tuber. The study also showed that the resistant cultivar, Hindenburg, has developed and maintained a capacity to sense and prime itself for persistent response to scab disease over time, and suggests an immune priming reaction as a mechanism for induced-resistance in scab resistant potato cultivars. The set of genes identified, described, and discussed in the study paves the foundation for detailed characterizations towards tailoring and designing procedures for targeted gene knockout through gene editing and phenotypic evaluation.

Proteomic Changes in Antioxidant System in Strawberry During Ripening.

To investigate the strawberry antioxidant defense system during fruit ripening, a targeted quantitative proteomic approach using multiple reaction monitoring (MRM) was developed to investigate targeted proteins in the antioxidant enzyme system in strawberry fruit. We investigated 46 proteins and isoforms with 73 identified peptides which may be involved in this antioxidant enzyme system. Among the proteins that changed during ripening, aldo/keto reductase (AKR), superoxide dismutase (SOD) and glutathione transferase (GT) increased significantly, while dehydroascorbate reductase, 2-Cys peroxiredoxin, catalase (CAT), 1-Cys peroxiredoxin and L-ascorbate peroxidase (APX) decreased significantly. These results suggest that fruit ripening of strawberry activates the enzymes of an SOD/glutathione metabolism system. The methodologies used in this study will be useful for systematically characterizing the role of antioxidant enzymes in fruit ripening of other plants.

Reaction of Surrogate Escherichia coli Serotype O157:H7 and Non-O157 Strains to Nutrient Starvation: Variation in Phenotype and Transcription of Stress Response Genes and Behavior on Lettuce Plants in the Field.

Preharvest contamination with bacteria borne by irrigation water may result in leafy vegetables serving as vehicles for transmission of Shiga toxin-producing Escherichia coli (STEC) to humans. The influence of starvation-associated stress on the behavior of non-toxin-producing strains of E. coli serotype O157:H7 and serotypes O26, O103, O111, and O145 was examined subsequent to their introduction to the phyllosphere of field-grown romaine lettuce as inocula simulating starved (96 h in sterile deionized water) and nutrient-depleted (24 h broth culture) cells. As with E. coli O157:H7, leaf populations of the non-O157 strains declined rapidly during the first 72 h postinoculation, displaying the biphasic decay curve typical of serotype O157:H7 isolates. Preinoculation treatment appeared not to influence decay rates greatly (P > 0.5), but strain-specific differences (persistence period and attachment proficiency) indicated that serotype O103:H2 strain PARC445 was a better survivor. Also assessed was the impact of preinoculation treatment on phenotypes key to leaf colonization and survival and the expression of starvation stress-associated genes. The 96-h starvation period enhanced biofilm formation in one strain but reduced motility and autoinducer 2 formation in all five study strains relative to those characteristics in stationary-phase cells. Transcription of rpoS, dps, uspA, and gapA was reduced significantly (P < 0.05) in starvation-stressed cells relative to that for exponential- and stationary-phase cultures. Strain-specific differences were observed; serotype O103:H2 PARC445 had greater downturns than did serotype O157:H7 and other non-O157 strains. Within this particular cohort, the behavior of the representative serotype O157:H7 strain, PARC443 (ATCC 700728), was not predictive of behavior of non-O157 members of this STEC group.

Impact of Listeria Inoculation and Aerated Steam Sanitization on Volatile Emissions of Whole Fresh Cantaloupes.

Rapid methods to detect bacterial pathogens on food and strategies to control them are needed to mitigate consumer risk. This study assessed volatile emissions from whole cantaloupe melons (Cucumis melo) as an indicator of Listeria contamination and in response to steam vapor decontamination. Cantaloupe were inoculated with Listeria innocua, a nonpathogenic surrogate for L. monocytogenes, then exposed to 85 °C steam for 240 s (4 min) followed by rapid chilling and storage for 0, 7, 10, or 14 days at 4, 7, or 10 °C. Volatile emissions from whole melons were collected on Carbopack B/Carboxen 1000 headspace collection tubes and analyzed by gas chromatography-mass spectroscopy following thermal desorption. Introduction of L. innocua to cantaloupe rind resulted in a reduction of aromatic compound emission. However, this response was not unique to Listeria contamination in that steam vapor treatment also reduced emission of these compounds. As well, steam vapor treatment diminished the number of viable Listeria and indigenous microflora while causing physiological injury to melon rind. Heat treatment had no significant effects on flesh firmness, color, titratable acidity, or soluble solids, but the production of typical aroma volatiles during postharvest ripening was inhibited. No unique volatile compounds were detected in Listeria contaminated melons. While changes in volatile emissions were associated with Listeria inoculation, they could not be differentiated from heat treatment effects. Results indicate that volatile emissions cannot be used as a diagnostic tool to identify Listeria contamination in whole cantaloupe melons. PRACTICAL APPLICATION: The detection of pathogen contamination on fresh produce is a continuing challenge. Using a nondestructive screening method, the presence of surrogate Listeria innocua on fresh whole cantaloupes was shown to alter the emissions of aromatic volatiles from whole cantaloupes. However, these altered emissions were not found to be unique to Listeria spp. and therefore cannot be used as a definitive indicator of Listeria contamination.

Characterization of laccase from apple fruit during postharvest storage and its response to diphenylamine and 1-methylcyclopropene treatments.

To gain better understanding on laccase in apples and reveal its role in browning color formation during storage, laccases in apples were investigated. The full-length complementary DNAs encoding laccase genes were obtained from different tissues of apple including flowers, calyx, leaves and fruit peel of 'Red Delicious' and 'Cortland'. The apple laccases were compared to those in other plant species and found to have up to 99% homology to Arabidopsis and litchi. qRT-PCR analysis revealed changes in transcript abundance of LAC genes (2, 7, 9, 12, 14, 15 and 16) during storage and in response to DPA and 1-MCP treatments. Enzyme activity of laccase protein in apple peel increased with storage in control fruit, while decreased significantly with DPA or 1-MCP. Changes in phenolic compounds in pericarp tissues decreased generally during storage, but no significant effect of DPA and 1-MCP treatments on the phenolic compounds was found.

Microbial Quality and Shelf Life of Blueberry Purée Developed Using Cavitation Technology.

Blueberry purée was developed using hydrodynamic cavitation technology. The product was made from entire blueberries without adding any food additives. In this study, microbial reduction following each processing stage (at the industry setting) and after product pasteurization at 86, 88, 90, 92, 94, and 96 °C was investigated. Microbial quality including total plate counts, yeast and molds, and heat-resistant molds counts was determined. Shelf life of pasteurized products stored for up to 24 weeks at room temperature were assessed for microbial quality, soluble solids (°Brix), titratable acidity (citric acid %), pH, viscosity (cP) and flow rate (cm/30 s). Our results indicated that heat-resistant molds, initially present in frozen blueberries with counts at 2.03 log CFU/200g, were totally inactivated at 94 to 96 °C with 1 to 2 min holding time. Shelf life study showed that no product spoilage was caused by bacteria, yeasts and heat-resistant molds along with non-significant changes of textural characteristics. This study provided useful information for the food industry to develop variety of fruit purée products with no wastes of fruit materials. PRACTICAL APPLICATION: This study provides useful information for the food industry to develop safe liquid food products using cavitation technology without wasting any raw materials.

Influence of culture media, pH and temperature on growth and bacteriocin production of bacteriocinogenic lactic acid bacteria.

There has been continued interest in bacteriocins research from an applied perspective as bacteriocins have potential to be used as natural preservative. Four bacteriocinogenic lactic acid bacteria (LAB) strains of Lactobacillus curvatus (Arla-10), Enterococcus faecium (JFR-1), Lactobacillus paracasei subsp. paracasei (JFR-5) and Streptococcus thermophilus (TSB-8) were previously isolated and identified in our lab. The objective of this study was to determine the optimal growth conditions for both LAB growth and bacteriocins production. In this study, various growth conditions including culture media (MRS and BHI), initial pH of culture media (4.5, 5.5, 6.2, 7.4 and 8.5), and incubation temperatures (20, 37 and 44 °C) were investigated for LAB growth measured as optical density (OD), bacteriocin activity determined as arbitrary unit and viability of LAB expressed as log CFU ml-1. Growth curves of the bacteriocinogenic LAB were generated using a Bioscreen C. Our results indicated that Arla-10, JFR-1, and JFR-5 strains grew well on both MRS and BHI media at growth temperature tested whereas TSB-8 strain, unable to grow at 20 °C. LAB growth was significantly affected by the initial pH of culture media (p < 0.001) and the optimal pH was found ranging from 6.2 to 8.5. Bacteriocin activity was significantly different in MRS versus BHI (p < 0.001), and the optimal condition for LAB to produce bacteriocins was determined in MRS broth, pH 6.2 at 37 °C. This study provides useful information on potential application of bacteriocinogenic LAB in food fermentation processes.

Human anthocyanin bioavailability: effect of intake duration and dosing.

While in vitro and animal evidence supports a role for anthocyanins in human health, future opportunities in berry health benefits will rest upon evidence from clinical intervention trials. Because little is known about the behaviour of anthocyanins during long term intake in humans, several clinical design factors were examined. Urine from volunteers (n = 17) who consumed blueberry juice daily was analysed using LC-MS/MS for predicted flavonoid-based products of anthocyanins in relation to a 5-day anthocyanin-free run-in, 28 days of blueberry juice intake, a 7-day washout and two dosing regimens. Total and parent anthocyanin content in urine varied 10-fold among the 17 participants. A high 24-0 h total anthocyanin excretion was associated with high anthocyanin retention (i.e. 0 h, before blueberry juice intake). Total anthocyanin excretion was not different before and after up to 7 days of washout indicative of a slow release of anthocyanins. Urinary excretion of anthocyanins declined during the 36-day study. The 24-0 h excretion was greater for total anthocyanins but not for parent anthocyanins when daily blueberry juice was taken all at once rather than as ⅓ doses taken thrice daily. However parent anthocyanins were retained better (higher 0 h) with 1× dosing. These findings could aid in the design of clinical research on anthocyanins and health.

Flavonoid Metabolites in Human Urine during Blueberry Anthocyanin Intake.

The human health benefits of anthocyanins (Anc) and other flavonoids are widely recognized. However, the flavonoid-based urinary metabolites arising in vivo after Anc intake are not well described. Human (n = 17) urine was collected while blueberry juice (BJ) was consumed daily for 28 days and once after a 7 day washout. MS/MS scanning of 664 urine samples for 18 parent Anc (PAnc) and 42 predicted Anc metabolites (AncM) yielded 371 products (i.e., MS/MS × retention time (RT)). Flavonoid-based AncM, which were likely underestimated, were almost 20 times more abundant than PAnc. Together, PAnc and AncM accounted for about 1% of the daily Anc dose. Aglycone forms were >94% of the total. Cluster analysis of the 371 Anc identified about 55 major Anc that contributed about 80% to the total Anc. The abundance of flavonoid-based Anc-derived products in the gastrointestinal tract could contribute to the health benefits of Anc-rich berries.

Proteome changes in banana fruit peel tissue in response to ethylene and high-temperature treatments.

Banana (Musa AAA group) is one of the most consumed fruits in the world due to its flavor and nutritional value. As a typical climacteric fruit, banana responds to ethylene treatment, which induces rapid changes of color, flavor (aroma and taste), sweetness and nutritional composition. It has also been reported that ripening bananas at temperatures above 24 °C inhibits chlorophyll breakdown and color formation but increases the rate of senescence. To gain fundamental knowledge about the effects of high temperature and ethylene on banana ripening, a quantitative proteomic study employing multiplex peptide stable isotope dimethyl labeling was conducted. In this study, green (immature) untreated banana fruit were subjected to treatment with 10 µL L(-1) of ethylene for 24 h. After ethylene treatment, treated and untreated fruit were stored at 20 or 30 °C for 24 h. Fruit peel tissues were then sampled after 0 and 1 day of storage, and peel color and chlorophyll fluorescence were evaluated. Quantitative proteomic analysis was conducted on the fruit peels after 1 day of storage. In total, 413 common proteins were identified and quantified from two biological replicates. Among these proteins, 91 changed significantly in response to ethylene and high-temperature treatments. Cluster analysis on these 91 proteins identified 7 groups of changed proteins. Ethylene treatment and storage at 20 °C induced 40 proteins that are correlated with pathogen resistance, cell wall metabolism, ethylene biosynthesis, allergens and ribosomal proteins, and it repressed 36 proteins that are associated with fatty acid and lipid metabolism, redox-oxidative responses, and protein biosynthesis and modification. Ethylene treatment and storage at 30 °C induced 32 proteins, which were mainly similar to those in group 1 but also included 8 proteins in group 3 (identified as chitinase, cinnamyl alcohol dehydrogenase 1, cysteine synthase, villin-2, leucine-transfer RNA ligase, CP47 protein and calmodulin) and repressed 43 proteins in 4 groups (groups 4-7), of which 6 were associated with photosynthesis II oxygen-evolving protein, the photosynthesis I reaction center, sugar metabolism, the redox-oxidative system and fatty acid metabolism. Differences in the response to ethylene and holding temperature at 30 °C were also revealed and have been discussed. The identities and quantities of the proteins found were linked with quality changes. This study demonstrates that ethylene and high temperature influence banana fruit ripening and senescence at the proteomic level and reveals the mechanisms by which high temperature accelerates banana fruit ripening.

Ethylene and 1-MCP regulate major volatile biosynthetic pathways in apple fruit.

The effects of ethylene and 1-methylcyclopropene (1-MCP) on apple fruit volatile biosynthesis and gene expression were investigated. Statistical analysis identified 17 genes that changed significantly in response to ethylene and 1-MCP treatments. Genes encoding branched-chain amino acid aminotransferase (BCAT), aromatic amino acid aminotransferase (ArAT) and amino acid decarboxylases (AADC) were up-regulated during ripening and further enhanced by ethylene treatment. Genes related to fatty acid synthesis and metabolism, including acyl-carrier-proteins (ACPs), malonyl-CoA:ACP transacylase (MCAT), acyl-ACP-desaturase (ACPD), lipoxygenase (LOX), hydroperoxide lyase (HPL), alcohol dehydrogenase (ADH), pyruvate decarboxylase (PDC2), ß-oxidation, acyl-CoA synthetase (ACS), enoyl-CoA hydratase (ECHD), acyl-CoA dehydrogenase (ACAD), and alcohol acyltransferases (AATs) also increased during ripening and in response to ethylene treatment. Allene oxide synthase (AOS), alcohol dehydrogenase 1 (ADH1), 3-ketoacyl-CoA thiolase and branched-chain amino acid aminotransferase 2 (BCAT2) decreased in ethylene-treated fruit. Treatment with 1-MCP and ethylene generally produced opposite effects on related genes, which provides evidence that regulation of these genes is ethylene dependent.

Targeted quantitative proteomic investigation employing multiple reaction monitoring on quantitative changes in proteins that regulate volatile biosynthesis of strawberry fruit at different ripening stages.

A targeted quantitative proteomic investigation employing the multiple reaction monitoring (MRM, SRM) technique was conducted on strawberry fruit at different development stages. We investigated 22 proteins and isoforms from 32 peptides with 111 peptide transitions, which may be involved in the volatile aroma biosynthesis pathway. The normalized protein abundance was significantly changed in coincidence with increased volatile production and advanced fruit maturities. Among them, alcohol acyltransferase (AAT), quinone oxidoreductase (QR), malonyl Co-A decarboxylase, (MLYCD), pyruvate decarboxylase (PDC), acetyl Co-A carboxylase (ACCase), and acyl Co-A synthetase (ACAs) were increased significantly. Several alcohol dehydrogenases (ADHs), and 3-oxoacyl-ACP synthase were significantly decreased. Furthermore, the expression of seven genes related to strawberry volatile production was also investigated using real-time qPCR. Among the tested genes, QR, AAT, ACCase, OMT, PDC and ADH showed increased up-regulation during fruit ripening, while 3-isopropylmalate dehydrogenase (IMD) decreased. Strong correlation between quantitative proteomic data and gene expression suggested that AAT, QR, ACCase, and PDC played critical roles in volatile biosynthesis of strawberry during fruit ripening. Poor correlation between protein abundance and gene expression of ADH was found.

Quantitative changes in proteins responsible for flavonoid and anthocyanin biosynthesis in strawberry fruit at different ripening stages: A targeted quantitative proteomic investigation employing multiple reaction monitoring.

To better understand the regulation of flavonoid and anthocyanin biosynthesis, a targeted quantitative proteomic investigation employing LC-MS with multiple reaction monitoring was conducted on two strawberry cultivars at three ripening stages. This quantitative proteomic workflow was improved through an OFFGEL electrophoresis to fractionate peptides from total protein digests. A total of 154 peptide transitions from 47 peptides covering 21 proteins and isoforms related to anthocyanin biosynthesis were investigated. The normalized protein abundance, which was measured using isotopically-labeled standards, was significantly changed concurrently with increased anthocyanin content and advanced fruit maturity. The protein abundance of phenylalanine ammonia-lyase; anthocyanidin synthase, chalcone isomerase; flavanone 3-hydroxylase; dihydroflavonol 4-reductase, UDP-glucose:flavonoid-3-O-glucosyltransferase, cytochrome c and cytochrome C oxidase subunit 2, was all significantly increased in fruit of more advanced ripeness. An interaction between cultivar and maturity was also shown with respect to chalcone isomerase. The good correlation between protein abundance and anthocyanin content suggested that a metabolic control point may exist for anthocyanin biosynthesis. This research provides insights into the process of anthocyanin formation in strawberry fruit at the level of protein concentration and reveals possible candidates in the regulation of anthocyanin formation during fruit ripening. BIOLOGICAL SIGNIFICANCE: To gain insight into the molecular mechanisms contributing to flavonoids and anthocyanin biosynthesis and regulation of strawberry fruit during ripening is challenging due to limited molecular biology tools and established hypothesis. Our targeted proteomic approach employing LC-MS/MS analysis and MRM technique to quantify proteins in relation to flavonoids and anthocyanin biosynthesis and regulation in strawberry fruit during fruit ripening is novel. The identification of peptides and proteins provided reliable design and validation of quantitative approaches using SRM on targeted proteins proposed involved in strawberry fruit. Our data revealed the identifying candidate proteins and their quantitative changes in relation to fruit ripening and flavonoids and anthocyanin biosynthesis and regulation. More importantly, this quantitative proteomic data is also compared with chemical analysis to reveal possible control levels of this important quality trait. Although, MRM approach is not new in plant biology research, the application has been very rare. This is the first systematic multi-targeted interrogation of the possible regulation of entire pathway of flavonoids and anthocyanin biosynthesis in strawberry fruit at different ripening stages using quantitative MRM technique on mass spectrometry. Our results demonstrate the power of targeted quantitative mass spectrometry data for analysis of proteins in biological regulation. These results indicate that distinct and diverse control of flavonoids and anthocyanin biosynthesis mechanisms at metabolism and proteins levels. This important and complementary knowledge will be useful for systematically characterizing the flavonoids and anthocyanin biosynthesis pathway of any fruit/plant species.

Blueberry effects on dark vision and recovery after photobleaching: placebo-controlled crossover studies.

Clinical evidence for anthocyanin benefits in night vision is controversial. This paper presents two human trials investigating blueberry anthocyanin effects on dark adaptation, functional night vision, and vision recovery after retinal photobleaching. One trial, S2 (n = 72), employed a 3 week intervention and a 3 week washout, two anthocyanin doses (271 and 7.11 mg cyanidin 3-glucoside equivalents (C3g eq)), and placebo. The other trial, L1 (n = 59), employed a 12 week intervention and an 8 week washout and tested one dose (346 mg C3g eq) and placebo. In both S2 and L1 neither dark adaptation nor night vision was improved by anthocyanin intake. However, in both trials anthocyanin consumption hastened the recovery of visual acuity after photobleaching. In S2 both anthocyanin doses were effective (P = 0.014), and in L1 recovery was improved at 8 weeks (P = 0.027) and 12 weeks (P = 0.030). Although photobleaching recovery was hastened by anthocyanins, it is not known whether this improvement would have an impact on everyday vision.