Dihydroflavonol 4-reductase (DFR) genes contribute greatly to anthocyanin biosynthesis in plants. Up to now, however, research on the DFR gene family and the key anthocyanin-related DFR members in blueberries (Vaccinium corymbosum) has been limited. In this study, we performed a genome-wide identification of the blueberry DFR gene family, identifying 36 VcDFR genes categorized into five subfamilies. Gene expression analysis showed that three Subfamily III members (VcDFR11/29/34) and four Subfamily V members (VcDFR4/7/30/33) are highly expressed in blueberry fruits, particularly at late ripening stages. Transient overexpression analysis in apple fruits verified the contributions of VcDFR11 and VcDFR30 to anthocyanin biosynthesis, with VcDFR11 showing better promoting effects. Blueberry fruit-based transient overexpression further confirmed the promoting effects of VcDFR11 on anthocyanin accumulation and the expression of anthocyanin-related structural genes (especially its downstream anthocyanindin synthase (ANS) and UDP-glucose: flavonoid 3-O-glycosyltransferase (UFGT) genes). The VcDFR11 promoter contains binding sites for both bHLH and MYB transcription factors (TFs). Consistently, yeast one-hybrid and dual-luciferase assays confirmed that anthocyanin-related VcMYB-1 and VcbHLHs can bind to and activate the VcDFR11 promoter. Furthermore, co-overexpressing VcMYB-1/VcbHLHs with VcDFR11 led to much higher anthocyanin accumulation than overexpressing VcDFR11 alone, indicating that these TFs positively regulate anthocyanin biosynthesis by upregulating VcDFR11. In summary, our study characterized the blueberry DFR gene family and demonstrated the role of VcDFR11 in anthocyanin biosynthesis.

Piriformospora indica, a broad-spectrum plant growth-promoting fungus, has been successfully applied in blueberry (Vaccinium corymbosum L.). In this study, through an integrated transcriptomic and biochemical analyses, we investigated the effects of P. indica colonization on blueberry root growth under long-term tap water (EC ≈ 1500 μs/cm) irrigation. Comparative transcriptomic analysis revealed that P. indica colonization greatly influenced the expression of genes involved in RNA biosynthesis, solute transport, response to external stimuli, phytohormone action, carbohydrate metabolism, cell wall organization, and secondary metabolism pathways. Consistently, the fungal colonization significantly improved the nutrient absorption ability, and increased the contents of sucrose, starch, trehalose, total phenolic, total flavonoids, and indole-3-acetic acid (IAA), while suppressing the accumulations of jasmonic acid (JA), abscisic acid (ABA), 1-aminocyclopropane-1-carboxylic acid (ACC), and strigolactone (SL) in blueberry roots. Quantitative real-time PCR verification also confirmed the fungal influences on genes associated with these pathways/parameters, such as auxin homoeostasis-associated WAT1, cell wall metabolism-related EXP, phenylpropanoid biosynthesis-related PAL and CHS, carotenoid degradation-related CCD8, transportation-related CNGC, trehalose metabolism-related TPP, and so on. Our study demonstrated that P. indica improved blueberry adaptability to mild salt stress by synergistically regulating cell wall metabolism, secondary metabolism, stress responses, hormone homeostasis, sugar and mineral element transportation, and so on.

C-terminally encoded peptides (CEPs), a class of post-translationally modified peptides, have been discovered to be potential mediators of abiotic stress responses in several plants. In this study, to explore the possible roles of CEPs in blueberry salt and osmotic stress responses, a genome-wide identification of CEP genes was first conducted. In total, we identified 12 VcCEPs that can be further categorized into three subfamilies in blueberry. Collinearity analysis identified 14 segmental duplicated gene pairs involving four Subfamily I members and five Subfamily III members among VcCEPs, indicating that the expansion of the blueberry CEP gene family was promoted by the segmental duplication events of Subfamily I and Subfamily III members. Promoter analysis revealed that all the VcCEPs’ promoters contain drought-inducible and defence and stress-related elements, suggesting that they might contribute to the stress responses of blueberry. Gene expression analysis showed that, of the 12 VcCEPs, VcCEP1, VcCEP4, VcCEP5 and VcCEP6 expressed in blueberry root and/or leaf, with varied expression levels under salt and drought treatments, while others showed no expression or very low expression. We further identified CEP mature peptide (CEPm) sequences in VcCEPs, and found that nine VcCEPs contain one mature peptide, while VcCEP7, VcCEP2 and VcCEP10 contain 3, 2, and 2 mature peptide sequences, respectively. The CEPms of Subfamily III members (VcCEP7~11) share more than 60% similarity with reported stress-related CEP mature peptides from other plant species, indicating that they might function in blueberry stress responses. Additionally, exogenous application of different synthesized CEP mature peptides alleviated blueberry leaf yellowing/browning under 150 mM NaCl/500 mM mannitol treatments, with CEP7a*2 and CEP7a*2 + 7c showing the best effects. These findings indicate that their functions are concentration-dependent to some extent. Collectively, our study characterized the CEP genes in blueberry and can provide a basis for the future applications of CEPs in enhancing blueberry stress tolerance.

Delayed transplanting of plug seedlings frequently causes root coiling and growth retardation in strawberry (Fragaria × ananassa), compromising transplant quality and commercial nursery performance. While plant growth-promoting bacteria (PGPB) are widely used to enhance crop growth and resilience, their effects on delayed-transplanted strawberry remain poorly understood. This study investigated the individual and combined effects of two Bacillus strains (KP3P9 and K13C) on the growth and root architecture of 12-week-old ‘Benihoppe’ strawberry plug seedlings, which had been delayed-transplanted for approximately 5–8 weeks. Moreover, root metabolomic profiles were analyzed to uncover metabolic reprogramming associated with Bacillus inoculation. Results showed that all Bacillus treatments promoted plant growth, enhanced fresh weight, and improved root development of delayed-transplanted strawberry. Metabolomic analysis revealed that all Bacillus treatments increased steroid levels but decreased flavonoids, ketones/aldehydes/esters, lignans, nucleotides, phenylpropanoids, and sugars in roots of delayed-transplanted strawberry plants. Furthermore, distinct metabolic signatures were found among treatments: the single KP3P9/K13C treatment elevated polyphenols and terpenoids; K13C uniquely increased alcohols, alkaloids, and coumarins, while their co-application (KK) specifically enhanced amino acid accumulation. KEGG enrichment analysis further revealed varied metabolic reprogramming: KP3P9 significantly enriched pantothenate and CoA biosynthesis, cornified envelope formation, and aminoacyl-tRNA biosynthesis; K13C enriched tyrosine metabolism and alpha-linolenic acid (ALA) metabolism, whereas KK specifically enriched flavonoid metabolism. This study reveals the promoting effects of single and combined KP3P9 and K13C applications and provides novel insights into the metabolic basis of Bacillus-mediated growth enhancement on delayed-transplanted strawberry.