Mulberry is an economically significant crop for the sericulture industry worldwide. Stresses such as drought exposure have a significant influence on plant survival. Because metabolome directly reflects plant physiological condition, performing a global metabolomic analysis is one technique to examine this influence. Using a liquid chromatography-mass spectrometry (LC-MS) technique based on an untargeted metabolomic approach, the effect of drought stress on mulberry Yu-711 metabolic balance was examined. For this objective, Yu-711 leaves were subjected to two weeks of drought stress treatment and control without drought stress. Numerous differentially accumulated metabolic components in response to drought stress treatment were revealed by multivariate and univariate statistical analysis. Drought stress treatment (EG) revealed a more differentiated metabolite response than the control (CK). We found that the levels of total lipids, galactolipids, and phospholipids (PC, PA, PE) were significantly altered, producing 48% of the total differentially expressed metabolites. Fatty acyls components were the most abundant lipids expressed and decreased considerably by 73.6%. On the other hand, the prenol lipids class of lipids increased in drought leaves. Other classes of metabolites, including polyphenols (flavonoids and cinnamic acid), organic acid (amino acids), carbohydrates, benzenoids, and organoheterocyclic, had a dynamic trend in response to the drought stress. However, their levels under drought stress decreased significantly compared to the control. These findings give an overview for the understanding of global plant metabolic changes in defense mechanisms by revealing the mulberry plant metabolic profile through differentially accumulated compounds.

Drought stress remains one of the most detrimental environmental cues affecting plant growth and survival. In this work, the DNA methylome changes in mulberry leaves under drought stress (EG) and control (CK) and their impact on gene regulation were investigated by MethylRAD sequencing. The results show 138,464 (37.37%) and 56,241 (28.81%) methylation at the CG and CWG sites (W = A or T), respectively, in the mulberry genome between drought stress and control. The distribution of the methylome was prevalent in the intergenic, exonic, intronic and downstream regions of the mulberry plant genome. In addition, we discovered 170 DMGs (129 in CG sites and 41 in CWG sites) and 581 DMS (413 in CG sites and 168 in CWG sites). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicates that phenylpropanoid biosynthesis, spliceosome, amino acid biosynthesis, carbon metabolism, RNA transport, plant hormone, signal transduction pathways, and quorum sensing play a crucial role in mulberry response to drought stress. Furthermore, the qRT-PCR analysis indicates that the selected 23 genes enriched in the KEGG pathways are differentially expressed, and 86.96% of the genes share downregulated methylation and 13.04% share upregulation methylation status, indicating the complex link between DNA methylation and gene regulation. This study serves as fundamentals in discovering the epigenomic status and the pathways that will significantly enhance mulberry breeding for adaptation to a wide range of environments.

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 (Morus alba L.) is an economically important plant for the silk industry and has the possibility of contributing immensely to Chinese pharmacopeia because of its health benefits. Domesticated silkworms feed only on mulberry leaves, meaning that the worms’ survival depends on the mulberry tree. Mulberry production is threatened by climate change and global warming. However, the regulatory mechanisms of mulberry responses to heat are poorly understood. We performed transcriptome analysis of high-temperature-stressed (42 °C) M. alba seedlings using RNA-Seq technologies. A total of 703 differentially expressed genes (DEGs) were discovered from 18,989 unigenes. Among these, 356 were up-regulated, and 347 were down-regulated. KEGG analysis revealed that most DEGs were enriched in valine, leucine and isoleucine degradation, and in starch and sucrose metabolism, alpha-linolenic acid metabolism, carotenoid biosynthesis and galactose metabolism, among others. In addition, TFs such as the NAC, HSF, IAA1, MYB, AP2, GATA, WRKY, HLH and TCP families were actively involved in response to high temperatures. Moreover, we used RT-qPCR to confirm the expression changes of eight genes under heat stress observed in the RNA-Seq analysis. This study provides M. alba transcriptome profiles under heat stress and provides theoretical bases to researchers for better understanding mulberry heat response mechanisms and breeding heat-tolerant mulberry plants.

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.