BACKGROUND: Anthocyanins are water-soluble flavonoids in plants, which give plants bright colors and are widely used as food coloring agents, nutrients, and cosmetic additives. There are several limitations for traditional techniques of collecting anthocyanins from plant tissues, including species, origin, season, and technology. The benefits of using engineering microbial production of natural products include ease of use, controllability, and high efficiency.
RESULTS: In this study, ten genes encoding enzymes involved in the anthocyanin biosynthetic pathway were successfully cloned from anthocyanin-rich plant materials blueberry fruit and purple round eggplant rind. The Yeast Fab Assembly technology was utilized to construct the transcriptional units of these genes under different promoters. The transcriptional units of PAL and C4H, 4CL and CHS were fused and inserted into Chr. XVI and IV of yeast strain JDY52 respectively using homologous recombination to gain Strain A. The fragments containing the transcriptional units of CHI and F3H, F3\'H and DFR were inserted into Chr. III and XVI to gain Strain B1. Strain B2 has the transcriptional units of ANS and 3GT in Chr. IV. Several anthocyanidins, including cyanidin, peonidin, pelargonidin, petunidin, and malvidin, were detected by LC-MS/MS following the predicted outcomes of the de novo biosynthesis of anthocyanins in S. cerevisiae using a multi-strain co-culture technique.
CONCLUSIONS: We propose a novel concept for advancing the heterologous de novo anthocyanin biosynthetic pathway, as well as fundamental information and a theoretical framework for the ensuing optimization of the microbial synthesis of anthocyanins.

Botrytis blossom blight and fruit rot, caused by Botrytis cinerea, is a significant threat to blueberries, potentially resulting in substantial economic losses if not effectively managed. Despite the recommendation of various cultural and chemical practices to control this pathogen, there are widespread reports of fungicide resistance, leading to decreased efficacy. This study aimed to characterize the resistance profile of B. cinerea isolated from blighted blossoms and fruit in 2019, 2020 and 2022 (n = 131, 40, and 37 for the respective years). Eight fungicides (fludioxonil, thiabendazole, pyraclostrobin, boscalid, fluopyram, fenhexamid, iprodione, and cyprodinil) were tested using conidial germination at specific discriminatory doses. Additionally, 86 isolates were phylogenetically characterized using the internal transcribed spacer regions (ITS) and the protein coding genes: glyceraldehyde-3-phosphate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60), and RNA polymerase II second largest subunit (RPB2). This revealed higher fungicide resistance frequencies in 2020 and 2022 compared to 2019. Over all 3 years, over 80% of the isolates were sensitive to fludioxonil, fluopyram, and fenhexamid. Pyraclostrobin and boscalid showed the lowest sensitivity frequencies (<50%). While multi-fungicide resistance was observed in all the years, none of the isolates demonstrated simultaneous resistance to all tested fungicides. Botrytis cinerea was the most prevalent species among the isolates (74) with intraspecific diversity detected by the genes. Two isolates were found to be closely related to B. fabiopsis, B. galanthina, and B. caroliniana and 10 isolates appeared to be an undescribed species. This study reports the discovery of a potentially new species sympatric with B. cinerea on blueberries in Michigan.

In China, the Liaodong Peninsula is an important growing area for blueberries because of the high organic matter content in the soil, the abundance of light, and the large temperature difference between day and night. However, the low temperature and relative humidity of the air during the winter and early spring in the Liaodong Peninsula are the main reasons for the damage to blueberry plants. Here, we documented the transcriptome and proteome dynamics in response to cold stress in three blueberry cultivars (\'Northland\', \'Bluecrop\', and \'Berkeley\'). Functional enrichment analysis indicated that many differentially expressed genes (DEGs) and differentially abundant proteins (DAPs) were mainly involved in the pathways of protein processing in the endoplasmic reticulum, the glutathione metabolism pathway, and ribosomes. We identified 12,747 transcription factors (TFs) distributed in 20 families. Based on our findings, we speculated that cold tolerance development was caused by the expression of calcium-related genes (CDPKs and CMLs), glutathione proteins, and TFs (NAC, WRKY, and ERF). Our investigation found that three cultivars experienced cold damage when exposed to temperatures between -9 °C and -15 °C in the field. Therefore, the cold resistance of blueberries during overwintering should not only resist the influence of low temperatures but also complex environmental factors such as strong winds and low relative humidity in the air. The order of cold resistance strength in the three blueberry cultivars was \'Berkeley\', \'Bluecrop\', and \'Northland\'. These results provide a comprehensive profile of the response to cold stress, which has the potential to be used as a selection marker for programs to improve cold tolerance in blueberries.

The well-established anti-inflammatory and antioxidant properties of red fruits leave room for a biological pathway of improved muscle health promoted by berries in the diet. Our objective was to systematically review the number of trials conducted on human and animal species around the relationship between a berry diet and muscle health outcomes. Two independent examiners conducted a search for studies that utilized keywords associated with muscle health outcomes and a berry-based diet in both human and animal trials, in accordance with the PRISMA statement guidelines. The literature was searched through six electronic databases until December 2023. Screening of 152 retrieved articles resulted in a final selection of 16 reports investigating the effect of exposure to a berry-based diet and skeletal muscle health outcomes. The study protocol was registered on PROSPERO (CRD42023479682). Among the selected studies, nine involved humans and seven animal models (rats and mice). Overall, most of the studies reported positive effects on performance or muscle health. Specifically, five studies investigated the possible effects of blackcurrant on active human subjects or athletes; three studies focused on blueberry and presented results on running performance (human sample) and muscle health (rat models). The rest of the studies involved raspberries (two studies, rat models), aronia (one study, rat models), elderberry (one study, rat models), and a mixed compound (one study, rat models). In conclusion, there is some early evidence that a berry-rich diet may increase performance or muscle health, but more research is needed to fully understand the underlying biological trajectories, and thus, no firm conclusions can yet be drawn.

Blueberry (Vaccinium spp.) is among the most-consumed soft fruit and has been recognized as an important source of health-promoting compounds. Highly perishable and susceptible to rapid spoilage due to fruit softening and decay during postharvest storage, modern breeding programs are looking to maximize quality and extend the market life of fresh blueberries. However, it is uncertain how genetically controlled postharvest quality traits are in blueberries. This study aimed to investigate the prediction ability and genetic basis of the main fruit quality traits affected during blueberry postharvest to create breeding strategies for developing cultivars with an extended shelf life. To achieve this goal, we carried out target genotyping in a breeding population of 588 individuals and evaluated for several fruit quality traits after one day, one week, three weeks, and seven weeks of postharvest storage at 1 °C. Using longitudinal genome-based methods, we estimated genetic parameters and predicted unobserved phenotypes. Our results showed large diversity, moderate heritability, and consistent predictive accuracies along the postharvest storage for most of the traits. Regarding fruit quality, firmness showed the largest variation during postharvest storage, with a surprising number of genotypes maintaining or increasing their firmness even after seven weeks of cold storage. Our results suggest that we can effectively improve blueberry postharvest quality through breeding and use genomic prediction to maximize the genetic gains in the long term. We also emphasize the potential of using longitudinal genomic prediction models to predict fruit quality at extended postharvest periods by integrating known phenotypic data from harvest.

Sulfur metabolism plays a major role in plant growth and development, environmental adaptation, and material synthesis, and the sulfate transporters are the beginning of sulfur metabolism. We identified 37 potential VcSULTR genes in the blueberry genome, encoding peptides with 534 to 766 amino acids. The genes were grouped into four subfamilies in an evolutionary analysis. The 37 putative VcSULTR proteins ranged in size from 60.03 to 83.87 kDa. These proteins were predicted to be hydrophobic and mostly localize to the plasma membrane. The VcSULTR genes were distributed on 30 chromosomes; VcSULTR3;5b and VcSULTR3;5c were the only tandemly repeated genes. The VcSULTR promoters contained cis-acting elements related to the fungal symbiosis and stress responses. The transcript levels of the VcSULTRs differed among blueberry organs and changed in response to ericoid mycorrhizal fungi and sulfate treatments. A subcellular localization analysis showed that VcSULTR2;1c localized to, and functioned in, the plasma membrane and chloroplast. The virus-induced gene knock-down of VcSULTR2;1c resulted in a significantly decreased endogenous sulfate content, and an up-regulation of genes encoding key enzymes in sulfur metabolism (VcATPS2 and VcSiR1). These findings enhance our understanding of mycorrhizal-fungi-mediated sulfate transport in blueberry, and lay the foundation for further research on blueberry-mycorrhizal symbiosis.

Blueberries are vulnerable to chilling injury (CI). This can lead to limited longevity when they are subjected to cold storage conditions. This study investigated the effectiveness of a preharvest spray containing 0.02% hexanal in reducing CI and improving the postharvest storage quality of \'Star\' and \'Biloxi\' blueberries. The blueberries were stored for a period of 5 weeks at 2 °C and in 90% relative humidity (RH). The findings revealed that the preharvest hexanal spraying of both cultivars delayed senescence by mitigating CI, as evidenced by the bolstering of the antioxidant defense system through increased superoxide dismutase (SOD), ascorbate peroxidase (APX), peroxidase (POD), catalase (CAT), and phenylalanine ammonia lyase (PAL) enzyme activity. The treated fruit also maintained elevated levels of total phenol content (TPC), total flavonoids (TFC), and vitamin C, demonstrating enhanced free radical scavenging capacity (FRSC), while exhibiting reduced polyphenoloxidase (PPO) activity, and reduced malondialdehyde (MDA), and H2O2 content in comparison with the control group. The preharvest hexanal treatment also suppressed fruit softening by maintaining greater firmness and higher membrane stability index (MSI) scores, inhibiting the activity of polygalacturonase (PG), pectinmethylesterase (PME), xylanase, and α-amylase, and reducing microbial counts (MC) and incidence of decay (DI) in comparison with the control. Preharvest hexanal treatment also improved the overall storage quality by reducing weight loss, total soluble solids (TSS), pH, and the TSS/acid ratio, while increasing titratable acidity (TA) in comparison with the control during cold storage. The findings suggest that hexanal, as a preharvest application, delays senescence effectively and preserves overall quality by enhancing cold tolerance through antioxidant defense mechanisms in blueberry storage under cold conditions. © 2024 The Author(s). Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

In this study, we evaluated the potential for exogenous thymol to slow this decline by measuring the effects of thymol application on cell wall, energy, and membrane lipid metabolism. The results showed that thymol application improved the preservation of the total soluble solids, titratable acidity, decay rate, and anthocyanin content, and effectively inhibited the accumulation of O2·-, H2O2, and malondialdehyde in blueberries during storage. Thymol application also effectively maintained fruit firmness, cell wall structure, and energy levels, while delaying the degradation of membrane phospholipids and unsaturated fatty acids during the storage of post-harvest blueberries. Therefore, exogenous thymol can maintain the quality of blueberry fruits by regulating energy and membrane lipid metabolism and reducing cell wall degradation. Thus, thymol-treatment could be a suitable biocontrol agent for maintaining blueberry quality and extending blueberry fruit storage life.

To improve the color stability of anthocyanins (ACNs) in blueberry fermented beverage, the intermolecular copigmentation between ACNs and 3 different phenolic compounds, including (-)-epigallocatechin gallate (EGCG), ferulic acid (FA), and gallic acid (GA) as copigments, was compared in the model and the real blueberry fermented beverage, respectively. The copigmented ACNs by EGCG presented a high absorbance (0.34 a.u.) and redness (27.09 ± 0.17) in the model blueberry fermented beverage. The copigmentation by the participation of the 3 different phenolic compounds showed all a spontaneous exothermic reaction, and the Gibbs free energy (ΔG°) of the system was lowest (-5.90 kJ/mol) using EGCG as copigment. Furthermore, the molecular docking model verified that binary complexes formed between ACNs and copigments by hydrogen bonds and π-π stacking. There was a high absorbance (1.02 a.u.), percentage polymeric color (PC%, 68.3 %), and good color saturation (C*ab, 43.28) in the real blueberry fermented beverage aged for 90 days, and more malvidin-3-O-glucoside had been preserved in the wine using EGCG as copigment. This finding may guide future industrial production of blueberry fermented beverage with improved color.

The rhizosphere microorganisms of blueberry plants have long coexisted with their hosts under distinctively acidic soil conditions, exerting a profound influence on host performance through mutualistic symbiotic interactions. Meanwhile, plants can regulate rhizosphere microorganisms by exerting host effects to meet the functional requirements of plant growth and development. However, it remains unknown how the developmental stages of blueberry plants affect the structure, function, and interactions of the rhizosphere microbial communities. Here, we examined bacterial communities and root metabolites at three developmental stages (flower and leaf bud development stage, fruit growth and development stage, and fruit maturation stage) of blueberry plants. The results revealed that the Shannon and Chao 1 indices as well as community composition varied significantly across all three developmental stages. The relative abundance of Actinobacteria significantly increased by 10 % (p < 0.05) from stage 1 to stage 2, whereas that of Proteobacteria decreased significantly. The co-occurrence network analysis revealed a relatively complex network with 1179 edges and 365 nodes in the stage 2. Niche breadth was highest at stage 2, while niche overlap tended to increase as the plant developed. Furthermore, the untargeted metabolome analysis revealed that the number of differential metabolites of vitamins, nucleic acids, steroids, and lipids increased between stage 1 to stage2 and stage 2 to stage 3, while those for differential metabolites of carbohydrates and peptides decreased. Significant changes in expression levels of levan, L-glutamic acid, indoleacrylic acid, oleoside 11-methyl ester, threo-syringoylglycerol, gingerglycolipid B, and bovinic acid were highly correlated with the bacterial community structure. Collectively, our study reveals that significant alterations in dominant bacterial taxa are strongly correlated with the dynamics of root metabolites. These findings lay the groundwork for developing prebiotic products to enhance the beneficial effects of root microorganisms and boosting blueberry productivity via a sustainable approach.