Mulberry fruits are rich in anthocyanins, which are important secondary metabolites that give mulberries their bright color, favorable taste and high nutritional quality, making them a popular fruit. However, few studies have focused on the molecular mechanism underlying anthocyanin accumulation in mulberries and the gene regulatory networks of anthocyanin biosynthetic pathways remain largely unknown. In this study, we performed RNA sequencing to identify differentially expressed genes (DEGs) associated with anthocyanin accumulation between two mulberry genotypes (‘Zi Jing’, ZJ and ‘Zhen Zhu Bai’, ZZB, with purple and white fruit flesh, respectively) at 5, 18, 27 and 31 days after flower. Using transcriptome analysis, we explored several key DEGs involved in the anthocyanin biosynthetic pathway, including the structural genes: CHS, CHI, F3H, DFR1, DFR2 and ANS, known as MBW complex genes: MYB (M.alba_G0017209), MYB (M.alba_G0017689), bHLH (M.alba_G0012659), bHLH (M.alba_G0009347) and bHLH3 (M.alba_G0016257) and the ethylene response factor: ERF (M.alba_G0016603). Of these, changing trends related to expression pattern and anthocyanin content showed their most positive correlation at the post-flowering stage in both genotypes. Our results indicated that ethylene enhances anthocyanin accumulation in mulberry fruits. Furthermore, qRT-PCR was performed to confirm the above-mentioned genes’ expression (except for MYB (M.alba_G0017689) and bHLH (M.alba_G0009347) was significantly up-regulated under ethylene treatment at 300 mg/L. These findings help uncover the gene regulatory networks of the anthocyanin biosynthetic pathway and will contribute to engineering purposes in future mulberry breeding programs.

Mulberry (Morus L.; Moraceae; Rosales) is an economically important tree with a long history in China and valued for its rich nutrient, flavor content, medicinal value and useful ecological functions. Morus species are widely distributed in Asia, Europe, North and South America and Africa and exhibit obvious differences in morphological characteristics and phenological stages due to being distributed in diverse climate zones. The morphological description of the phenological stages of black mulberry (Morus nigra L.) has been established in Mediterranean climates and this study extended the BBCH scale for application in different Morus species, especially for mulberry trees grown in subtropical monsoon climates. In this study, we used the BBCH (Biologische Bundesanstalt, Bundessortenamt and CHemische Industrie) scale to describe in detail the phenological growth stages for Morus species in Wuhan, China (a subtropical monsoon climate). Based on this general scale, eight principal stages, i.e., bud, leaf and shoot development stage, inflorescence emergence stage, flowering stage, fruit development stage, fruit maturation stage, senescence and beginning of dormancy stage, were described. We provide photographic images of some primary and secondary developmental stages to better define and standardize morphological characteristics and phenological descriptions of these mulberries (Morus spp.). In addition, because the color of mulberry fruit varies from white, purple, black, or pink to red at maturity, with mostly monosexual flowers, we have also presented detailed descriptions of morphological traits of flower sexuality and fruit color among these four species. In addition, two late phenological germplasms (late bud sprouting associated with late blooming and late fruit ripening) were screened out according to the BBCH scale. Overall, this study will contribute to advance the field of mulberry breeding and implementation of agronomic practices and facilitate convenient communication between mulberry cultivators and researchers in different areas.

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.

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.

Protected cultivation is the sustainable approach to horticultural crop production under adverse climates. In this study, the performance of yard-long beans under three protected cultivations, including single-span polyhouse (SSP), five-span polyhouse (FSP), and insect-proof net house (IPN), is examined and compared to open field cultivation. The above protected cultivation can extend the harvest period of pods by 6–10 days, improve their quality, and increase yield by 15.6% to 25.1%, reducing the incidence and severity of thrips and Cercospora leaf spot, rust, and powdery mildew. Among them, yard-long beans grown in SSP are longer and straighter in shape and have the lowest incidence and severity of pests and diseases and the highest levels of total polyphenols, total sugar, soluble protein, starch, and fiber. This indicates that protected cultivation has broad application in the production of yard-long beans. Through full subset regression analysis (FSRA), we report here that the yield and of yard-long bean occurrences of pests and diseases were highly impacted by climatic factors, especially UV radiation intensity and air temperature. These results have considerable implications for improving pod yield and quality and green prevention and control of pests and diseases through optimizing facility structure and fertilizer management.

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.