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Flavonoids impart color and mouthfeel to grapes and wine and are very sensitive to environmental conditions. Growth chamber experiments were performed to investigate the effect of temperature regimes and the differences between day/night temperatures on anthocyanins and flavonols in Merlot grapes. Among the regimes tested, the ones with diurnal 20°C determined the highest levels of anthocyanins and flavonols. Higher diurnal temperatures decreased those levels but increased the proportion of methoxylated and acylated species. When regimes with the same day temperature but different night temperatures were compared, differences between day/night temperatures did not affect anthocyanins, unless a difference of 25°C between day and night temperatures was imposed. When regimes with the same night temperature but different day temperatures were compared, the regime with higher day temperature had a lower anthocyanin level. No relationships were observed between the effects of temperature regimes on anthocyanin level and the expression of key anthocyanin genes. However, the effects on anthocyanin acylation level were consistent with the effects on the acyltransferase expression, and the effects on flavonol level were consistent with the effects on the expression of key flavonol genes. This study indicates that, in Merlot grapes, anthocyanins and flavonols are mostly sensitive to day temperatures.

Bacterial blight (causal agent Pseudomonas syringae complex, Psc) is an endemic and economically important disease of northern highbush blueberry production in Canada and the Pacific Northwest of the USA. To date, there is no comprehensive survey of the disease in the region and detailed characterization of associated pathogens from Pacific western Canada. Therefore, we did comprehensive disease survey and characterization of associated pseudomonads population using pathogen morphology, biochemical tests, and molecular characterization. We isolated 380 strains of pseudomonads from symptomatic plants from 32 research and commercial fields in 10 diverse geographic locations in British Columbia. We used P . syringae specific (Psy) primers and identified 197 Psy-PCR positive isolates out of 380. We further sequenced Psy-PCR positive isolates of pseudomonads using four housekeeping genes and identified four phylogenomic species: P. syringae (40%), Pseudomonas avellanae (29%), Pseudomonas viridiflava (20%), and phylogenomic species A (7%). P . avellanae and P. viridiflava are new phylogenomic species of Psc causing bacterial blight in highbush blueberry. We found some patterns among geographical locations and highbush blueberry varieties in the frequency distribution of isolates of these phylogenomic species. Genetic fingerprinting with rep-PCR assays identified a very high genetic diversity of pseudomonads populations among geographical locations, varieties, and phylogenomic species. Biochemical characterization (LOPAT- levan, oxidase, pectolytic activity, arginine dihydrolase, and tobacco hypersensitivity) revealed that the vast majority of isolates were Pseudomonas Group Ia. Findings of this study provide insight into the population biology of pseudomonads infecting highbush blueberry, provide information for disease diagnosis, and exploit disease management options, including identifying sources of disease resistance. Key points • High prevalence of bacterial blight caused by P. syringae complex (Psc) in highbush blueberry in Pacific western Canada • We report two new phylogenomic species of Psc, P. viridiflava and P. avellanae, that cause bacterial blight and canker disease in highbush blueberry • The genetic diversity of the population of Psc was very high

Water deficits consistently promote higher concentrations of anthocyanins in red winegrapes and their wines. However, controversy remains as to whether there is any direct effect on berry metabolism other than inhibition of growth. Early (ED) and late (LD) season water deficits, applied before or after the onset of ripening (veraison), were imposed on field grown Vitis vinifera “Cabernet Sauvignon”, and the responses of gene expression in the flavonoid pathway and their corresponding metabolites were determined. ED accelerated sugar accumulation and the onset of anthocyanin synthesis. Both ED and LD increased anthocyanin accumulation after veraison. Expression profiling revealed that the increased anthocyanin accumulation resulted from earlier and greater expression of the genes controlling flux through the anthocyanin biosynthetic pathway, including F3H, DFR, UFGT and GST. Increases in total anthocyanins resulted predominantly from an increase of 3'4'5'-hydroxylated forms through the differential regulation of F3'H and F3'5'H. There were limited effects on proanthocyanidin, other flavonols, and on expression of genes committed to their synthesis. These results demonstrate that manipulation of abiotic stress through applied water deficits not only modulates compositional changes during berry ripening, but also alters the timing of particular aspects of the ripening process.

Grapes are one of the major fruit crops and they are cultivated in many dry environments. This study comprehensively characterizes the metabolic response of grape berries exposed to water deficit at different developmental stages. Increases of proline, branched-chain amino acids, phenylpropanoids, anthocyanins, and free volatile organic compounds have been previously observed in grape berries exposed to water deficit. Integrating RNA-sequencing analysis of the transcriptome with large-scale analysis of central and specialized metabolites, we reveal that these increases occur via a coordinated regulation of key structural pathway genes. Water deficit-induced up-regulation of flavonoid genes is also coordinated with the down-regulation of many stilbene synthases and a consistent decrease in stilbenoid concentration. Water deficit activated both ABA-dependent and ABA-independent signal transduction pathways by modulating the expression of several transcription factors. Gene-gene and gene-metabolite network analyses showed that water deficit-responsive transcription factors such as bZIPs, AP2/ERFs, MYBs, and NACs are implicated in the regulation of stress-responsive metabolites. Enrichment of known and novel -regulatory elements in the promoters of several ripening-specific/water deficit-induced modules further affirms the involvement of a transcription factor cross-talk in the berry response to water deficit. Together, our integrated approaches show that water deficit-regulated gene modules are strongly linked to key fruit-quality metabolites and multiple signal transduction pathways may be critical to achieve a balance between the regulation of the stress-response and the berry ripening program. This study constitutes an invaluable resource for future discoveries and comparative studies, in grapes and other fruits, centered on reproductive tissue metabolism under abiotic stress.

Background Red raspberry fruit color is a key driver of consumer preference and a major target of breeding programs worldwide. Screening for fruit color typically involves the determination of anthocyanin content and/or the assessment of color through a colorimeter. However, both procedures are time-consuming when the analyses involve hundreds or thousands of samples. The objectives of this study were to develop a high-throughput method for red raspberry puree color measurement and to test the correlations between color parameters and total anthocyanin content. Color coordinates were collected with a colorimeter on 126 puree samples contained in Petri dishes and with the Tomato Analyzer Color Test (TACT) module to assess the same samples prepared in Petri dishes and in 96-well plates. An additional 425 samples were analyzed using only 96-well plates. Total anthocyanins were extracted from all 551 samples. Results Regression models for L*, a*, b* measured with the colorimeter and TACT using Petri dishes were all significant ( p  < 0.001), but very consistent only for L* ( R 2  = 0.94). Significant ( p  < 0.001) and very consistent regressions ( R 2  = 0.94 for L* and b*, R 2  = 0.93 for a*) were obtained for color parameters measured with TACT using Petri dishes and TACT using plates. Of the color parameters measured with the colorimeter, only L*, a*/b*, and hue significantly correlated with total anthocyanins ( p  < 0.05), but, except for L* ( R = − 0.79), the correlations were weak ( R = − 0.23 for a*/b* and R  = 0.22 for hue). Conversely, all correlations with total anthocyanins and color parameters measured with TACT were significant ( p  < 0.001) and moderately strong (e.g., R = − 0.69 for L* and R  = 0.55 for a*/b*). These values were indicative of darker colors as total anthocyanin content increased. Conclusions While the colorimeter and TACT-based methods were not fully interchangeable, TACT better captured color differences among raspberry genotypes than the colorimeter. The correlations between color parameters measured with TACT and total anthocyanins were not strong enough to develop prediction models, yet the use of TACT with 96-well plates instead of Petri dishes would enable the high-throughput measurement of red raspberry puree color.

Societal Impact Statement In blueberry, fruit color is one of the most important quality traits affecting consumers' choices. Both pigments and waxes impact fruit color; however, their roles have not been clearly elucidated. Here, the contributions of waxes and anthocyanins to fruit color are characterized. A higher content of β‐diketones—the second largest wax group in blueberries—determines a lighter fruit color; hence revealing that, at harvest, waxes contribute more than anthocyanins in determining variations in fruit color among blueberry varieties. Summary Fruit color is one of the major quality traits determining the marketability of fruits by affecting consumers' choices. In blueberries, although fruit surface color is mainly determined by pigments (e.g., anthocyanins), cuticular waxes also play a role in modulating the surface color, and a lighter color given by a dense wax bloom is normally preferred by consumers. This study investigated the content and composition of cuticular waxes and their roles in modulating fruit surface color in 12 (seven northern highbush, three southern highbush, and two hybrids) blueberry genotypes at harvest (H1, representing the first commercial pick, and H2, representing the second commercial pick). The ultrastructural morphology of cuticular waxes was analyzed in four selected genotypes by scanning electron microscopy. The level and profile of anthocyanins and their contributions to the color were also assessed. Total cuticular wax content ranged from 27.7 to 95.8 μg cm−2 among genotypes at H1 and decreased by an average of 23.9% from H1 to H2. Triterpenoids (62.5% of the total cuticular waxes on average) and β‐diketones (22.9% on average) were the first and second largest wax groups in all genotypes, respectively. β‐Diketones were previously proven to affect leaf surface color in wheat; in this study, their content strongly correlated with the lightness of the blueberry surface. Scanning electron microscopy revealed distinct wax morphologies among genotypes. No significant relationships were found between total or individual anthocyanin concentrations and fruit surface color. Our results suggest that, at harvest, variation in the fruit surface color among blueberry genotypes is more closely related to the content and composition of cuticular waxes than the level and profile of anthocyanins, with β‐diketones being particularly important. This study provides new insights for blueberry breeding programs aiming to improve the surface color in order to meet the market demand. In blueberry, fruit color is one of the most important quality traits affecting consumers' choices. Both pigments and waxes impact fruit color; however, their roles have not been clearly elucidated. Here, the contributions of waxes and anthocyanins to fruit color are characterized. A higher content of β‐diketones—the second largest wax group in blueberries—determines a lighter fruit color; hence revealing that, at harvest, waxes contribute more than anthocyanins in determining variations in fruit color among blueberry varieties.

Background Grape leaves provide the biochemical substrates for berry development. Thus, understanding the regulation of grapevine leaf metabolism can aid in discerning processes fundamental to fruit development and berry quality. Here, the temporal alterations in leaf metabolism in Merlot grapevine grown under sufficient irrigation and water deficit were monitored from veraison until harvest. Results The vines mediated water stress gradually and involving multiple strategies: osmotic adjustment, transcript-metabolite alteration and leaf shedding. Initially stomatal conductance and leaf water potential showed a steep decrease together with the induction of stress related metabolism, e.g. up-regulation of proline and GABA metabolism and stress related sugars, and the down-regulation of developmental processes. Later, progressive soil drying was associated with an incremental contribution of Ca 2+ and sucrose to the osmotic adjustment concomitant with the initiation of leaf shedding. Last, towards harvest under progressive stress conditions following leaf shedding, incremental changes in leaf water potential were measured, while the magnitude of perturbation in leaf metabolism lessened. Conclusions The data present evidence that over time grapevine acclimation to water stress diversifies in temporal responses encompassing the alteration of central metabolism and gene expression, osmotic adjustments and reduction in leaf area. Together these processes mitigate leaf water stress and aid in maintaining the berry-ripening program.

This work focused on the development of starch-based (potato, corn, sweet potato, green bean and tapioca) edible packaging film incorporated with blueberry pomace powder (BPP). The optical, mechanical, thermal, and physicochemical properties were subsequently tested. The film color was not affected by the addition of BPP. BPP incorporated into corn and green bean starch films showed increased light barrier properties, indicating a beneficial effect to prevent UV radiation-induced food deterioration. Film thickness and transparency were not primarily affected by changing the starch type or the BPP concentration, although the corn starch films were the most transparent. Furthermore, all films maintained structural integrity and had a high tensile strength. The water vapor transmission rate of all the films was found to be greater than conventional polyethylene films. The average solubility of all the films made from different starch types was between 24 and 37%, which indicates the usability of these films for packaging, specifically for low to intermediate moisture foods. There were no statistical differences in Differential Scanning Calorimetry parameters with changes in the starch type and pomace levels. Migration assays showed a greater release of the active compounds from BPP into acetic acid medium (aqueous food simulant) than ethanol medium (fatty food simulant). The incorporation of BPP into starch-chitosan films resulted in the improvement of film performance, thereby suggesting the potential for applying BPP into starch-based films for active packaging.

The onset of ripening involves changes in sugar metabolism, softening, and color development. Most understanding of this process arises from work in climacteric fruits where the control of ripening is predominately by ethylene. However, many fruits such as grape are nonclimacteric, where the onset of ripening results from the integration of multiple hormone signals including sugars and abscisic acid (ABA). In this study, we identified ten orthologous gene families in Vitis vinifera containing components of sugar and ABA-signaling pathways elucidated in model systems, including PP2C protein phosphatases, and WRKY and homeobox transcription factors. Gene expression was characterized in control- and deficit-irrigated, field-grown Cabernet Sauvignon. Sixty-seven orthologous genes were identified, and 38 of these were expressed in berries. Of the genes expressed in berries, 68% were differentially expressed across development and/or in response to water deficit. Orthologs of several families were induced at the onset of ripening, and induced earlier and to higher levels in response to water deficit; patterns of expression that correlate with sugar and ABA accumulation during ripening. Similar to field-grown berries, ripening phenomena were induced in immature berries when cultured with sucrose and ABA, as evidenced by changes in color, softening, and gene expression. Finally, exogenous sucrose and ABA regulated key orthologs in culture, similar to their regulation in the field. This study identifies novel candidates in the control of nonclimacteric fruit ripening and demonstrates that grape orthologs of key sugar and ABA-signaling components are regulated by sugar and ABA in fleshy fruit.

Background Flavonoid 3',5'-hydroxylases (F3'5'Hs) and flavonoid 3'-hydroxylases (F3'Hs) competitively control the synthesis of delphinidin and cyanidin, the precursors of blue and red anthocyanins. In most plants, F3'5'H genes are present in low-copy number, but in grapevine they are highly redundant. Results The first increase in F3'5'H copy number occurred in the progenitor of the eudicot clade at the time of the γ triplication. Further proliferation of F3'5'H s has occurred in one of the paleologous loci after the separation of Vitaceae from other eurosids, giving rise to 15 paralogues within 650 kb. Twelve reside in 9 tandem blocks of ~35-55 kb that share 91-99% identity. The second paleologous F3'5'H has been maintained as an orphan gene in grapevines, and lacks orthologues in other plants. Duplicate F3'5'H s have spatially and temporally partitioned expression profiles in grapevine. The orphan F3'5'H copy is highly expressed in vegetative organs. More recent duplicate F3'5'H s are predominately expressed in berry skins. They differ only slightly in the coding region, but are distinguished in the structure of the promoter. Differences in cis -regulatory sequences of promoter regions are paralleled by temporal specialisation of gene transcription during fruit ripening. Variation in anthocyanin profiles consistently reflects changes in the F3'5'H mRNA pool across different cultivars. More F3'5'H copies are expressed at high levels in grapevine varieties with 93-94% of 3'5'-OH anthocyanins. In grapevines depleted in 3'5'-OH anthocyanins (15-45%), fewer F3'5'H copies are transcribed, and at lower levels. Conversely, only two copies of the gene encoding the competing F3'H enzyme are present in the grape genome; one copy is expressed in both vegetative and reproductive organs at comparable levels among cultivars, while the other is transcriptionally silent. Conclusions These results suggest that expansion and subfunctionalisation of F3'5'H s have increased the complexity and diversification of the fruit colour phenotype among red grape varieties.