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.

Mulberry (Morus alba) is a significant plant with numerous economic benefits; however, its growth and development are affected by nutrient levels. A high level of magnesium (Mg) or magnesium nutrient starvation are two of the significant Mg factors affecting plant growth and development. Nevertheless, M. alba’s metabolic response to different Mg concentrations is unclear. In this study, different Mg concentrations, optimal (3 mmol/L), high (6 mmol/L and 9 mmol/L), or low (1 and 2 mmol/L) and deficient (0 mmol/L), were applied to M. alba for three weeks to evaluate their effects via physiological and metabolomics (untargeted; liquid chromatography–mass spectrometry (LC-MS)) studies. Several measured physiological traits revealed that Mg deficiency and excess Mg altered net photosynthesis, chlorophyll content, leaf Mg content and fresh weight, leading to remarkable reductions in the photosynthetic efficiency and biomass of mulberry plants. Our study reveals that an adequate supply of the nutrient Mg promoted the mulberry’s physiological response parameters (net photosynthesis, chlorophyll content, leaf and root Mg content and biomass). The metabolomics data show that different Mg concentrations affect several differential metabolite expressions (DEMs), particularly fatty acyls, flavonoids, amino acids, organic acid, organooxygen compounds, prenol lipids, coumarins, steroids and steroid derivatives, cinnamic acids and derivatives. An excessive supply of Mg produced more DEMs, but negatively affected biomass production compared to low and optimum supplies of Mg. The significant DEMs correlated positively with mulberry’s net photosynthesis, chlorophyll content, leaf Mg content and fresh weight. The mulberry plant’s response to the application of Mg used metabolites, mainly amino acids, organic acids, fatty acyls, flavonoids and prenol lipids, in the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways. These classes of compounds were mainly involved in lipid metabolism, amino acid metabolism, energy metabolism, the biosynthesis of other secondary metabolites, the biosynthesis of other amino acids, the metabolism of cofactors and vitamin pathways, indicating that mulberry plants respond to Mg concentrations by producing a divergent metabolism. The supply of Mg nutrition was an important factor influencing the induction of DEMs, and these metabolites were critical in several metabolic pathways related to magnesium nutrition. This study provides a fundamental understanding of DEMs in M. alba’s response to Mg nutrition and the metabolic mechanisms involved, which may be critical to the mulberry genetic breeding program.

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.

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