MADS-box is a class of transcriptional regulators that are ubiquitous in plants and plays important roles in the process of plant growth and development. Identification and analysis of blueberry MADS-box genes can lay a foundation for their function investigations. In the present study, 249 putative MADS-box genes were identified in the blueberry genome. Those MADS-box genes were distributed on 47 out of 48 chromosomes. The phylogenetic and evolutionary analyses showed that blueberry MADS-box genes were divided into 131 type I members and 118 type II members. The type I genes contained an average of 1.89 exons and the type II genes contained an average of 7.83 exons. Motif analysis identified 15 conserved motifs, of which 4 were related to the MADS domain and 3 were related to the K-box domain. A variety of cis-acting elements were found in the promoter region of the blueberry MADS-box gene, indicating that the MADS-box gene responded to various hormones and environmental alterations. A total of 243 collinear gene pairs were identified, most of which had a Ka/Ks value of less than 1. Nine genes belonging to SEP, AP3/PI, and AGL6 subfamilies were screened based on transcriptomic data. The expression patterns of those nine genes were also verified using quantitative PCR, suggesting that VcMADS6, VcMADS35, VcMADS44, VcMADS58, VcMADS125, VcMADS188, and VcMADS212 had potential functions in blueberry fruit ripening. The results of this study provide references for an in-depth understanding of the biological function of the blueberry MADS-box genes and the mechanism of blueberry fruit ripening.

The glutathione S-transferases (GSTs, EC 2.5.1.18) constitute a versatile enzyme family with pivotal roles in plant stress responses and detoxification processes. Recent discoveries attributed the additional function of facilitating anthocyanin intracellular transportation in plants to GSTs. Our study identified 178 VcGST genes from 12 distinct subfamilies in the blueberry genome. An uneven distribution was observed among these genes across blueberry’s chromosomes. Members within the same subfamily displayed homogeneity in gene structure and conserved protein motifs, whereas marked divergence was noted among subfamilies. Functional annotations revealed that VcGSTs were significantly enriched in several gene ontology and KEGG pathway categories. Promoter regions of VcGST genes predominantly contain light-responsive, MYB-binding, and stress-responsive elements. The majority of VcGST genes are subject to purifying selection, with whole-genome duplication or segmental duplication serving as key processes that drive the expansion of the VcGST gene family. Notably, during the ripening of the blueberry fruit, 100 VcGST genes were highly expressed, and the expression patterns of 24 of these genes demonstrated a strong correlation with the dynamic content of fruit anthocyanins. Further analysis identified VcGSTF8, VcGSTF20, and VcGSTF22 as prime candidates of VcGST genes involved in the anthocyanin intracellular transport. This study provides a reference for the exploration of anthocyanin intracellular transport mechanisms and paves the way for investigating the spectrum of GST functions in blueberries.

BRANCHED1 (BRC1) is a crucial member of the TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) gene family and is well known for playing a central role in shoot branching by controlling buds’ paradormancy. However, the expression characteristics and molecular regulatory mechanism of BRC1 during blueberry bud dormancy are unclear. To shed light on these topics, shoots of three blueberry cultivars with different chilling requirements (CRs) were decapitated in summer to induce paradormancy release and subjected to different levels of chilling in winter to induce endodormancy release. The results showed that the high-CR cultivar ‘Chandler’ had the strongest apical dominance among the three cultivars; additionally, the expression of VcTCP18, which is homologous to BRC1, was the highest under both the decapitation treatment and low-temperature treatment. The ‘Emerald’ cultivar, with a low CR, demonstrated the opposite trend. These findings suggest that VcTCP18 plays a negative regulatory role in bud break and that there may be a correlation between the CR and tree shape. Through yeast 1-hybrid (Y1H) assays, we finally screened 21 upstream regulatory genes, including eight transcription factors: zinc-finger homeodomain protein 1/4/5/9, MYB4, AP2-like ethylene-responsive transcription factor AINTEGUMENTA (ANT), ASIL2-like, and bHLH035. It was found that these upstream regulatory genes positively or negatively regulated the expression of VcTCP18 based on the transcriptome expression profile. In summary, this study enriched our understanding of the regulatory network of BRCl during bud dormancy and provided new insights into the function of BRC1.

Endodormancy is the stage that perennial plants must go through to prepare for the next seasonal cycle, and it is also an adaptation that allows plants to survive harsh winters. Blueberries (Vaccinium spp.) are known to have high nutritional and commercial value. To better understand the molecular mechanisms of bud dormancy release, the transcriptomes of flower buds from the southern highbush blueberry variety “O’Neal” were analyzed at seven time points of the endo- and ecodormancy release processes. Pairwise comparisons were conducted between adjacent time points; five kinds of phytohormone were identified via these processes. A total of 12,350 differentially expressed genes (DEGs) were obtained from six comparisons. Gene Ontology analysis indicated that these DEGs were significantly involved in metabolic processes and catalytic activity. KEGG pathway analysis showed that these DEGs were predominantly mapped to metabolic pathways and the biosynthesis of secondary metabolites in endodormancy release, but these DEGs were significantly enriched in RNA transport, plant hormone signal transduction, and circadian rhythm pathways in the process of ecodormancy release. The contents of abscisic acid (ABA), salicylic acid (SA), and 1-aminocyclopropane-1-carboxylate (ACC) decreased in endo- and ecodormancy release, and the jasmonic acid (JA) level first decreased in endodormancy release and then increased in ecodormancy release. Weighted correlation network analysis (WGCNA) of transcriptomic data associated with hormone contents generated 25 modules, 9 of which were significantly related to the change in hormone content. The results of this study have important reference value for elucidating the molecular mechanism of flower bud dormancy release.

Auxin response factors (ARFs) are a transcription factor family that regulates the expression of auxin phase-responsive genes. Here, we performed a genome-wide investigation of the tetraploid blueberry (Vaccinium corymbosum cv. ‘Draper’) genome sequence. Physical and chemical properties, phylogenetic evolution, gene structure, conservative motifs, chromosome location, and cis-acting elements of blueberry ARF genes were comprehensively evaluated. A total of 70 blueberry ARF genes (VcARF) were found in its genome, which could be divided into six subfamilies. VcARF genes were unevenly distributed on 40 chromosomes and were observed to encode protein sequences ranging in length from 162 to 1117 amino acids. Their exon numbers range from 2 to 22. VcARF promoter regions contain multiple functional domains associated with light signaling, aerobic metabolism, plant hormones, stress, and cell cycle regulation. More family members of VcARF genes were discovered in blueberry than in previously studied plants, likely because of the occurrence of whole-genome duplication and/or tandem duplication. VcARF expression patterns were analyzed at different stages of fruit development, and VcARF3, VcARF4, VcARF14, VcARF37, and VcARF52 were observed to play important roles. VcARF3 and VcARF4 appeared to function as repressors, while VcARF14 acted as an essential factor in fruit firmness differences between firm and soft flesh cultivars.