The SPL gene is a plant-specific transcription factor involved in the regulation of plant growth and development, which have been identified in woody plants. The process of floral bud differentiation affects the timing of flowering and fruit set and regulates plant growth, however, the mechanism of regulation of flower development by SPL genes is less studied. In this study, 56 VcSPL genes were identified in the tetraploid blueberry. The VcSPL gene family was classified into six subfamilies, and analysis of cis-elements showed that VcSPL genes were regulated by light, phytohormones (abscisic acid, MeJA), and low temperature. In the evolutionary analysis, segmental replication may play an important role in VcSPL gene amplification. Interestingly, we also studied diploid blueberry (Bilberry), in which 24 SPL genes were identified, and 36 homologous pairs were found, suggesting a high degree of convergence in the syntenic relationship between blueberry (Vaccinium corymbosum L) and bilberry (Vaccinium darrowii). Based on the expression profile, VcSPL genes were expressed at high levels in flowers, shoots, and roots, indicating a diversity of gene functions. Then we selected 20 differentially-expressed SPL genes to further investigate the role of VcSPL in floral induction and initiation. It showed that the genes VcSPL40, VcSPL35, VcSPL45, and VcSPL53 may play a crucial role in the blueberry floral transition phase (from vegetative growth to flower initiation). These results provided important information for understanding and exploring the role of VcSPLs in flower morphogenesis and plant growth.

Red walnuts have been widely studied because of their strong antioxidant activity and ornamental value. However, research on the mechanism of anthocyanin biosynthesis in walnuts remains in the initial stage. The regulatory mechanism of TT2-type R2R3-MYB transcription factors in anthocyanin biosynthesis in walnuts is also unclear. Therefore, this study used ‘D2-1’ and ‘Jinghong 1’ walnuts as plant materials. The testa of ‘Jinghong 1’ was red, and its anthocyanin content was significantly higher than that of ‘D2-1’, mainly composed of cyanidin-3-O-arabinoside, cyanidin-3-O-galactoside, and cyanidin-3-O-glucoside. Differentially expressed genes between ‘D2-1’ and ‘Jinghong 1’ testa were enriched in phenylpropanoid biosynthesis and flavonoid biosynthesis. Next, this study identified a TT2-type R2R3-MYB transcription factor JrMYB1L, which was involved in regulating the anthocyanin biosynthesis in the testa of ‘Jinghong 1’. The overexpression of JrMYB1L could promote anthocyanin accumulation in walnut leaves and activate the expression of JrCHS, JrCHI, JrF3H, JrDFR, JrANS, JrUFGT, JrLAR, and JrANR. In addition, yeast two-hybrid results proved that JrMYB1L, JrbHLH42, and JrWD40 proteins could interact with each other. The results of yeast one-hybrid and dual-luciferase assays indicated that JrMYB1L could activate the expression of JrCHS and JrUFGT by binding to their promoters. Based on the above results, this study proposed a possible regulatory mechanism. JrMYB1L activated the expression of JrCHS and JrUFGT in the form of JrMYB1L-JrbHLH42-JrWD40 complex, thereby promoting anthocyanin accumulation in the testa of ‘Jinghong 1’. In summary, this study lays a theoretical foundation for revealing the regulatory mechanism of anthocyanin biosynthesis in red walnut and contributes to the breeding of new varieties of red walnuts with more edible and ornamental value.

Walnut is one of four major nuts in the world. China has abundant walnut germplasm resources, but there are still shortcomings in the identification of germplasm resources. This study used different walnut varieties as materials and developed 14 high-quality SSR molecular markers from 60 pairs of primers based on genome re-sequencing results. This study analyzed the genetic diversity of Chinese walnut genetic resources using 14 selected SSR markers. A total of 64 alleles were detected in 47 walnut resources, with an average of 4.571 alleles per locus. The variation range of polymorphism information content was 0.096~0.711, with an average value of 0.422. Cluster analysis, population genetic structure, and principal coordinate analysis divided 47 walnut resources into ordinary walnuts, Juglans hopeiensis, and Liaoyi 1. In addition, core SSR markers (Jr45, Jr40, Jr29, Jr35, and Jr11) were selected from 14 SSR markers, which were sufficient to distinguish 47 walnut resources. At the same time, 47 unique molecular fingerprints of walnuts were constructed using these core SSR markers. This study provides strong scientific support for rapid and efficient identification, germplasm innovation, and a variety of property protection of walnut germplasm.

Plant small heat shock proteins (sHSPs) are abundant and diverse proteins involved in various stresses. In this study, a small heat shock protein gene (VcHSP17.7) encoding a 160-amino acid protein was isolated from the highbush blueberry (Vaccinium    corymbosum L.). Sequence alignment and phylogenetic analysis indicated that the VcHSP17.7 is a member of the plant cytosolic class II sHSPs; this finding was supported by the subcellular localisation pattern of a VcHSP17.7-YFP fusion protein. Quantitative real-time PCR (qRT-PCR) showed that the VcHSP17.7 gene was expressed in all analysed tissues. Heat shock experiments were conducted using mature blueberry fruit. The anthocyanin content was lower in heat shock-treated fruit than in control fruit after 14 days of storage at 2°C. The malondialdehyde (MDA) content was lower in heat shock-treated fruit than in control fruit during subsequent low-temperature storage, which indicates that heat shock treatment may have alleviated chilling injury (CI) and enhanced the chilling tolerance of the blueberry fruit. Meanwhile, the expression of VcHSP17.7 in blueberry fruit was induced by heat shock treatment and gradually increased during subsequent low-temperature storage. These results suggest that VcHSP17.7 might be involved in the chilling tolerance of blueberry fruit caused by heat shock treatment.

Autumn shoot pruning is a significant production measure in Highbush blueberry (Vaccinium corymbosum L.) that promotes floral differentiation, but the mechanism is still unclear. To explore the role of carbohydrates in floral induction and differentiation promoted by short pruning, carbohydrate content, and the transport and metabolic-related genes were compared and analyzed. It is found that sucrose might be an important trigger for pruning to promote floral induction. Compared with only pruning the shoots, cutting half of the leaves significantly reduces the flower bud rate and delays the flower differentiation process, while further adding 1.5% sucrose spray alleviates this phenomenon partly. During floral induction, the soluble sugar content in buds increases significantly, while the sucrose content in leaf midvein and buds increases earlier. The starch and soluble sugar content in the stem decreases significantly. At the early flower bud morphological differentiation stage, a large number of starch granules accumulate in the buds, sucrose in leaves increases, glucose in buds decreases, and then fructose decreases. There is an upregulation of VcFT, VcSOC1, VcFUL, VcSUC, and VcINV expression in the leaf midvein, as well as an upregulation of VcFT, VcSOC1, VcTPS1, VcINV, and VcSWEET1 expression in buds during floral induction. At the same time, interaction between VcSOC1 and VcTPS1 proteins exists. The level of VcSOC1 and VcAMY can also be upregulated by VcTPS1. Sucrose plays an important role in blueberry floral induction through flowering factors and sugar-related genes, especially VcFT, VcSOC1, VcTPS1, VcSUC, and VcINV. The results of this study laid a strong foundation for further research on the regulation mechanism of flower bud differentiation in blueberry and other woody plants.