Background: Mulberry (Morus L.), a vital perennial woody plant with significant economic importance, is utilized for silkworm rearing, human consumption and medicinal use. The availability of mulberry’s whole-genome sequencing data has underscored the demand for an effective, user-friendly, and high-throughput protocol to facilitate the elucidation of gene functions. Methods and Results: In this investigation, we established a transient transformation approach using Agrobacterium tumefaciens-mediated sonication followed by vacuum infiltration in mulberry tissue culture seedlings. Simultaneously, we optimized the transformation conditions, including mulberry genotypes, A. tumefaciens strain, acetosyringone concentration, bacterial density, sonication time, and days after agroinfiltration. These optimizations aimed to achieve heightened transformation efficiency, employing GFP as a reporter gene to monitor transformation events. The optimized method included the use of an infiltration medium (10 mM MgCl₂, 10 mM MES (2-(N-morpholino)ethanesulfonic acid sodium salt), 150 μM acetosyringone, and OD600 0.5 of A. tumefaciens LBA4404) supplemented with the surfactant 0.02% Silwet L-77, with 20 s sonication followed by 20 min vacuum infiltration (0.07 MPa). Among the four mulberry genotypes, ‘Taiguo’ was the most responsive genotype and produced the highest levels of GFP expression at 7 d after infiltration. Furthermore, the optimized transient transformation approach has been proven to be successfully applicable for transiently overexpressing MaANS and MaDFR in mulberry fruits of ‘Taiguo’, in vitro, which distinctly enhanced fruit coloring and significantly increased anthocyanin accumulation, respectively. Conclusions: In summary, we devised a dependable, stable and highly efficient transient transformation approach suitable for rapid gene function examination in mulberry leaves and fruits, in vitro.

For tree crops, shortening the juvenile phase is a vital strategy to advance fruit bearing and enhance the breeding efficiency. Walnut (Juglans regia L.) seedlings usually take at least eight to 10 years to flower, but early-flowering (EF) types can flower one or two years after planting. In this study, RNA sequencing (RNA-Seq) and microRNA sequencing (miRNA-Seq) were used for a transcriptome-wide analysis of gene and miRNA expression in hybrids of the Xinjiang EF walnut variety ‘Xinwen 81’ and later-flowering (LF) walnut. Based on a high-quality chromosome-scale reference genome, a total of 3009 differentially expressed genes (DEGs) were identified, of which 933 were upregulated (accounting for 31%) and 2076 were downregulated (accounting for 69%). DEGs were functionally annotated, and the flowering-related genes FLOWERING LOCUS T (FT), SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), and LEAFY (LFY) showed remarkable upregulation in EF compared with in the LF walnut. In addition, miRNAs associated with floral transition were screened as candidates for flowering time regulation in the walnut. This work provides new insights into walnut floral transition, which may ultimately contribute to genetic improvement of the walnut.

Mulberry fruits are rich in bioactive components renowned for their antioxidant properties and potential health benefits. This study thoroughly investigated the impact of cultivation modes on the phenological stages, yield, bioactive components, and antioxidant activity of two mulberry cultivars, Yueshen Dashi (YS-DS) and Xinjiang Baisang (XJ-BS). Notably, greenhouse cultivation led to earlier phenology and shorter fruit development maturation durations compared to field cultivation. Despite a decrease in fruit production and firmness, the greenhouse-grown mulberries exhibited higher individual fruit fresh weight. The content of bioactive components, encompassing anthocyanins, polyphenols, flavonoids, and vitamin C, and of antioxidant activity (measured in the FRAP and DPPH radical scavenging assays) was found to be lower in the greenhouse-grown mulberries than in those cultivated in the field. The contents of total polyphenols and flavonoids showed robust positive correlations in the FRAP and DPPH radical scavenging assays, which suggests that the antioxidant activity of mulberry fruit might be primarily attributable to the bioactive components of total polyphenols and flavonoids. Interestingly, the sugar content and hydroxyl radical scavenging activity (HRSA) displayed an inverse relationship between the two cultivars in the greenhouse versus field conditions. The multivariate analysis highlighted distinct patterns for different cultivars under varying cultivation modes. This study underscores the potential to enhance bioactive components and antioxidant activity through effective manipulation of climate conditions, thereby unlocking the full nutritional potential of mulberry fruits on a large scale in greenhouse environments.

Walnut (Juglans regia L.) is an important nut fruit crop mainly grown for its high nutritional and medicinal value. In walnut fruit, the pellicle is the main source of polyphenols (such as proanthocyanidins), which are natural bioactive compounds but also cause astringency and bitterness for walnut fruit consumption. However, the gene regulatory networks of phenolic biosynthetic pathways remain largely unknown in walnut pellicles. Here, we performed RNA sequencing (RNA-seq) to identify differentially expressed genes (DEGs) associated with pellicle development in walnut. In this study, seven developmental stages (8-, 9-, 11-, 13-, 15-, 17-, and 19-week after pollination) of ‘Xinwen179’ pellicle tissues were harvested to conduct further transcriptome-wide profiles. Via RNA-seq, we explored several key DEGs involved in the phenolic biosynthetic pathway, such as dihydroflavonol-4-reductase (DFR), leucoanthocyanidin reductase (LAR), anthocyanidin synthase (ANS) and anthocyanidin reductase (ANR), which are dynamically expressed at developmental stages of the walnut pellicle. Among them, ANR may directly contribute to proanthocyanidins accumulation during walnut development. Taken together, our preliminary investigation on DEGs associated with pellicle development will not only elucidate the gene regulatory networks of the phenolic biosynthetic pathway for pellicle development, but also contribute to the broad spectrum of RNA-seq data resources for further genetic improvement of walnut.

The growing recognition of mulberries as a potent source of bioactive and nutritional compounds, coupled with their increasing global consumption, underscores the need for efficient off-season cultivation. This study explores the influence of both in-season and off-season cultivation on the yield, bioactive components, and antioxidant activity of ‘Taiwan Changguosang’ (Morus laevigata Wall.) in a greenhouse setting. Despite the lower fruit yield during the off-season, the off-season fruit exhibits higher levels of bioactive compounds, including total anthocyanins, polyphenols, and flavonoids compared to its in-season counterpart. Additionally, the off-season fruit demonstrates enhanced DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging and FRAP (ferric-reducing antioxidant power), attributed to climatic conditions during fruit development, particularly air temperature and solar radiation. Moreover, the off-season fruit proves to be more palatable, showcasing a favorable balance between sugar and acid. Principal Component Analysis (PCA) and correlation analysis revealed a close association between antioxidant activity and the chemical contents of total polyphenols and flavonoids. This study underscores the feasibility and benefits of off-season cultivation for enhancing the nutritional profile and antioxidant potential of mulberries, providing valuable insights for optimizing cultivation practices.