Comprehensive Analysis of Highbush Blueberry Plants Propagated In Vitro and Conventionally.

In vitro culture allows the production of numerous plants with both desirable and undesirable traits. To investigate the impact of the propagation method on highbush blueberry plants, an analysis was performed on four groups of differentially propagated plants: in vitro with axillary (TC-Ax) or adventitious shoots (TC-Ad), conventionally (SC) and using a mixed method (TC/SC). The analysis included plant features (shoot length and branching, chlorophyll and fluorescence and DNA methylation) and fruit properties (antioxidant compounds). The data obtained indicated significant differences between plants propagated conventionally and in vitro, as well as variations among plants derived from in vitro cultures with different types of explants. SC plants generally exhibited the lowest values of morphological and physiological parameters but produced fruits richest in antioxidant compounds. TC/SC plants were dominant in length, branching and fluorescence. Conversely, TC-Ax plants produced fruits with the lowest levels of antioxidant compounds. The methylation-sensitive amplified polymorphism (MSAP) technique was employed to detect molecular differences. TC-Ad plants showed the highest methylation level, whereas SC plants had the lowest. The overall methylation level varied among differentially propagated plants. It can be speculated that the differences among the analysed plants may be attributed to variations in DNA methylation.

Methyl jasmonate affects phenolic metabolism and gene expression in blueberry (Vaccinium corymbosum).

Blueberry (Vaccinium corymbosum) is a fruit very much appreciated by consumers for its antioxidant potential and health-promoting traits. Its beneficial potential properties are mainly due to a high content of anthocyanins and their amount can change after elicitation with methyl jasmonate. The aim of this work is to evaluate the changes in expression of several genes, accumulation of phenolic compounds and alterations in antioxidant potential in two different blueberry cultivars ('Duke' and 'Blueray') in response to methyl jasmonate (0.1 mM). Results showed that 9 h after treatment, the expression of phenylalanine ammonium lyase, chalcone synthase and anthocyanidin synthase genes was stimulated more in the 'Blueray' variety. Among the phenols measured an increase was recorded also for epicatechin and anthocyanin concentrations. 'Duke' is a richer sourche of anthocyanins compared to 'Blueray', treatment with methyl jasmonate promoted in 'Blueray' an increase in pigments as well as in the antioxidant potential, especially in fully ripe berries, but treated 'Duke' berries had greater levels, which were not induced by methyl jasmonate treatment. In conclusion, methyl jasmonate was, in some cases, an effective elicitor of phenolic metabolism and gene expression in blueberry, though with different intensity between cultivars.

Effect of Elicitor Treatments on Quality Attributes in Blueberry: Implications of Cultivar and Environmental Conditions.

Elicitors of plant defence responses can trigger defence mechanisms that are able to protect plant tissues from biotic or abiotic stresses. Since one defence response involves the activation of secondary metabolites' biosynthesis, the purpose of this study was to evaluate the effect of chitosan and melatonin pre-harvest treatments on the quality and the nutritional parameters of the fruits of blueberry (Vaccinium corymbosum L.). Across the two years of experiment, three different cultivars (cv.s. 'Cosmopolitan', 'Hortblue Poppins' and 'Legacy') were treated with 1% chitosan or 100 µM melatonin every two weeks during the ripening season and ripe fruits were progressively harvested and analysed. The treatment with both elicitors had only slight effects on dry matter, soluble solids content, titratable acidity and pH, with a cultivar-dependent response. On the other hand, elicitors significantly affected the levels of phenylpropanoid and antioxidant compounds in all cvs. in both years, with a higher accumulation of total anthocyanins and phenolics and the enhancement of the antioxidant capacity, with positive effects on the nutraceutical quality of fruits. The anthocyanin profile in terms of both absolute concentrations and the relative proportion of single anthocyanins was affected by both harvest year and cv., highlighting the role of the genetic background in the plant response to environmental conditions (with particular reference to summer heat stress) and to elicitor treatments.

Benzothiadiazole enhances ascorbate recycling and polyphenols accumulation in blueberry in a cultivar-dependent manner.

Benzothiadiazole (BTH) is a functional analogue of salicylic acid able to induce systemic acquired resistance in many horticultural crops. The aim of the work was to investigate how BTH may affect i) fruit quality, ii) ascorbic acid (AsA) oxidation and recycling metabolism and iii) phenolic compounds accumulation, during development and ripening of berries from the two selected cultivars. Blueberry (Vaccinium corymbosum L.) plants (cv 'Brigitta' and 'Duke') were treated with 0.118 mM BTH every two weeks during ripening, then all fruits of each plant were harvested and divided in four developmental stages. Results indicated that BTH had no marked effects on fruit quality parameters. During the first developmental stage, BTH negatively affected dry matter in both cv, while soluble solids and AsA content were affected in 'Duke'. In fully ripe berries, BTH reduced dry matter in 'Duke' and enhanced soluble solids content in 'Brigitta', while diminishing titratable acidity. AsA content was positively affected by BTH in 'Duke', but not in 'Brigitta'. The effect of BTH on the enzymes involved in AsA recycling was recorded in berries at the third (fruit more than half pigmented) and fourth developmental stages. After treatment, in both cv ascorbate peroxidase (APX) activity increased in fully ripe berries, while monodehydroascorbate reductase (MDHAR) activity was stimulated at the third ripening stage. Conversely, the activities of dehydroascorbate reductase (DHAR) and glutathione reductase (GR) were enhanced only in 'Brigitta' and in 'Duke', respectively. BTH stimulated total polyphenols, flavonoid and anthocyanin accumulation in 'Brigitta' and in 'Duke' at the third and fourth ripening stages. In fully ripe berries, BTH enhanced the accumulation of delphinidins, cyanidins, petunidins and peonidins in 'Brigitta', while in 'Duke' it increased all classes of anthocyanidins, including malvidin. On the contrary, the relative proportion of the individual anthocyanins was only slightly affected by BTH treatment, mainly regarding delphinidin and malvidin at the third and fourth stage of ripening of 'Duke' and 'Brigitta', respectively. These results show that preharvest BTH application can positively impact on fruit bioactive compounds levels, affecting AsA recycling and content and increasing polyphenols accumulation in fruit, but partly depending on cv and ripening stage.

Variation of Anthocyanin Content and Profile Throughout Fruit Development and Ripening of Highbush Blueberry Cultivars Grown at Two Different Altitudes.

Blueberry (Vaccinium corymbosum L.) is a widely consumed fruit and a rich source of bioactive compounds, namely, the polyphenol class of anthocyanins. Little information is available about the influence of internal (genetic and developmental) and external (environmental) factors on the levels of phenolic metabolites in blueberry fruit. In light of this consideration, total polyphenolic and flavonoid content, anthocyanin accumulation and composition were evaluated in cv. "Duke" and "Brigitta" grown at two different altitudes in Valtellina, a valley of the Alps in Northern Italy. During berry ripening, there is a developmentally coordinated shift from cyanidin-type, di-substituted anthocyanins toward delphinidin-based, tri-substituted pigments. At the lower altitude location, higher temperatures, not exceeding optimum, resulted in a more quickly berry developmental pattern and in higher anthocyanin concentrations in the early phases of ripening. At later stages of ripening, berries of both cultivars at higher altitude compensate for these initial temperature effects, and no differences were recorded in ripe fruit grown in the two locations. We conclude that anthocyanin accumulation is strongly regulated by development and genotype, and the environmental factors, associated to the altitude gradient, exert in the trial conditions only a fine-tuning influence. Fruits reach the full-ripening stage simultaneously at both sites because the initial gap in pigment levels is counterbalanced at the higher altitude by a faster rate of accumulation at later phases of the ripening process.

A complex interaction between pre-harvest and post-harvest factors determines fresh-cut melon quality and aroma.

Melons are prized for their characteristic aroma, however, pre-harvest growth, stage of ripening at harvest, post-harvest processing and storage conditions lead to quality changes in fresh-cut fruit. We considered changes in metabolites and gene expression over 14 days storage to assess underlying mechanisms and identify potential quality markers. Overall, 99 volatile organic compounds (VOCs) were detected and VOC profiles discriminated between two melon seasons, cut-size, storage temperatures and storage time, although season affected their discriminatory power. Abundance of two VOCs fell rapidly and was not associated with cut size, indicating their use as markers for early changes post-processing. Non-acetate to acetate ester ratio differed between the seasons and correlated with changes in alcohol acyl-transferase (CmAAT1) gene expression. Furthermore, CmAAT1 expression clustered with two ester VOCs that may be potential new products of this enzyme. Season also strongly affected post-harvest sugar content, most likely attributable to meteorological differences during growth. Storage temperature and cut size affected expression of transcription factors ERF71, ERF106, and TINY, whose expression generally rose during storage, probably related to increased stress. Thus, although time × temperature of storage are key factors, pre-harvest conditions and fruit processing impact significantly gene expression and aroma loss post-harvest.

Evaluation of Borage Extracts As Potential Biostimulant Using a Phenomic, Agronomic, Physiological, and Biochemical Approach.

Biostimulants are substances able to improve water and nutrient use efficiency and counteract stress factors by enhancing primary and secondary metabolism. Premise of the work was to exploit raw extracts from leaves (LE) or flowers (FE) of Borago officinalis L., to enhance yield and quality of Lactuca sativa 'Longifolia,' and to set up a protocol to assess their effects. To this aim, an integrated study on agronomic, physiological and biochemical aspects, including also a phenomic approach, has been adopted. Extracts were diluted to 1 or 10 mL L-1, sprayed onto lettuce plants at the middle of the growing cycle and 1 day before harvest. Control plants were treated with water. Non-destructive analyses were conducted to assess the effect of extracts on biomass with an innovative imaging technique, and on leaf photosynthetic efficiency (chlorophyll a fluorescence and leaf gas exchanges). At harvest, the levels of ethylene, photosynthetic pigments, nitrate, and primary (sucrose and total sugars) and secondary (total phenols and flavonoids) metabolites, including the activity and levels of phenylalanine ammonia lyase (PAL) were assessed. Moreover, a preliminary study of the effects during postharvest was performed. Borage extracts enhanced the primary metabolism by increasing leaf pigments and photosynthetic activity. Plant fresh weight increased upon treatments with 10 mL L-1 doses, as correctly estimated by multi-view angles images. Chlorophyll a fluorescence data showed that FEs were able to increase the number of active reaction centers per cross section; a similar trend was observed for the performance index. Ethylene was three-fold lower in FEs treatments. Nitrate and sugar levels did not change in response to the different treatments. Total flavonoids and phenols, as well as the total protein levels, the in vitro PAL specific activity, and the levels of PAL-like polypeptides were increased by all borage extracts, with particular regard to FEs. FEs also proved efficient in preventing degradation and inducing an increase in photosynthetic pigments during storage. In conclusion, borage extracts, with particular regard to the flower ones, appear to indeed exert biostimulant effects on lettuce; future work will be required to further investigate on their efficacy in different conditions and/or species.

Identification of innovative potential quality markers in rocket and melon fresh-cut produce.

Ready-to-eat fresh cut produce are exposed to pre- and postharvest abiotic stresses during the production chain. Our work aimed to identify stress responsive genes as new molecular markers of quality that can be widely applied to leaves and fruits and easily determined at any stage of the production chain. Stress responsive genes associated with quality losses were isolated in rocket and melon fresh-cut produce and their expression levels analyzed by quantitative real time PCR (qRT-PCR) at different time points after harvest at 20 °C and 4 °C. qRT-PCR results were supported by correlation analysis with physiological and biochemical determinations evaluated at the same conditions such as chlorophyll a fluorescence indices, total, reducing sugars, sucrose, ethylene, ascorbic acid, lipid peroxidation and reactive oxygen species. In both species the putative molecular markers increased their expression soon after harvest suggesting a possible use as novel and objective quality markers of fresh-cut produces.

Ascorbic acid metabolism during bilberry (Vaccinium myrtillus L.) fruit development.

Bilberry (Vaccinium myrtillus L.) possesses a high antioxidant capacity in berries due to the presence of anthocyanins and ascorbic acid (AsA). Accumulation of AsA and the expression of the genes encoding the enzymes of the main AsA biosynthetic route and of the ascorbate-glutathione cycle, as well as the activities of the enzymes involved in AsA oxidation and recycling were investigated for the first time during the development and ripening of bilberry fruit. The results showed that the AsA level remained relatively stable during fruit maturation. The expression of the genes encoding the key enzymes in the AsA main biosynthetic route showed consistent trends with each other as well as with AsA levels, especially during the first stages of fruit ripening. The expression of genes and activities of the enzyme involved in the AsA oxidation and recycling route showed more prominent developmental stage-dependent changes during the ripening process. Different patterns of activity were found among the studied enzymes and the results were, for some enzymes, in accordance with AsA levels. In fully ripe berries, both AsA content and gene expression were significantly higher in skin than in pulp.