Abiotic stress, especially drought stress, poses a significant threat to terrestrial plant growth, development, and productivity. Although mulberry has great genetic diversity and extensive stress-tolerant traits in agroforestry systems, only a few reports offer preliminary insight into the biochemical responses of mulberry leaves under drought conditions. In this study, we performed a comparative metabolomic and transcriptomic analysis on the “drooping mulberry” (Morus alba var. pendula Dippel) under PEG-6000-simulated drought stress. Our research revealed that drought stress significantly enhanced flavonoid accumulation and upregulated the expression of phenylpropanoid biosynthetic genes. Furthermore, the activities of superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) content were elevated. In vitro enzyme assays and fermentation tests indicated the involvement of flavonol synthase/flavanone 3-hydroxylase (XM_010098126.2) and anthocyanidin 3-O-glucosyltransferase 5 (XM_010101521.2) in the biosynthesis of flavonol aglycones and glycosides, respectively. The recombinant MaF3GT5 protein was found to recognize kaempferol, quercetin, and UDP-glucose as substrates but not 3-/7-O-glucosylated flavonols and UDP-rhamnose. MaF3GT5 is capable of forming 3-O- and 7-O-monoglucoside, but not di-O-glucosides, from kaempferol. This implies its role as a flavonol 3, 7-O-glucosyltransferase. The findings from this study provided insights into the biosynthesis of flavonoids and could have substantial implications for the future diversified utilization of mulberry.

Gastrointestinal cancers are a major global health challenge, with high mortality rates. This study investigated the anti-cancer activities of 30 monomers extracted from Morus alba L. (mulberry) against gastrointestinal cancers. Toxicological assessments revealed that most of the compounds, particularly immunotoxicity, exhibit some level of toxicity, but it is generally not life-threatening under normal conditions. Among these components, Sanggenol L, Sanggenon C, Kuwanon H, 3′-Geranyl-3-prenyl-5,7,2′,4′-tetrahydroxyflavone, Morusinol, Mulberrin, Moracin P, Kuwanon E, and Kuwanon A demonstrate significant anti-cancer properties against various gastrointestinal cancers, including colon, pancreatic, and gastric cancers. The anti-cancer mechanism of these chemical components was explored in gastric cancer cells, revealing that they inhibit cell cycle and DNA replication-related gene expression, leading to the effective suppression of tumor cell growth. Additionally, they induced unfolded protein response (UPR) and endoplasmic reticulum (ER) stress, potentially resulting in DNA damage, autophagy, and cell death. Moracin P, an active monomer characterized as a 2-arylbenzofuran, was found to induce ER stress and promote apoptosis in gastric cancer cells, confirming its potential to inhibit tumor cell growth in vitro and in vivo. These findings highlight the therapeutic potential of Morus alba L. monomers in gastrointestinal cancers, especially focusing on Moracin P as a potent inducer of ER stress and apoptosis.

1-Deoxynojirimycin (DNJ) is an iminosugar with notable pharmacological properties, and mulberry (Morus spp.) represents the richest natural source of this compound, particularly in young leaves and roots. Despite its tissue-specific distribution, the transcriptional regulatory mechanisms underlying its biosynthesis remain poorly understood. In this study, we integrated transcriptomic profiling, molecular assays, and phytohormone analyses to uncover a multilayered transcriptional network controlling DNJ biosynthesis in mulberry. Weighted gene co-expression network analysis identified a DNJ-associated module (MEblue) that includes 4 potential transcription factors: zinc-finger homeodomain-6 (ZFHD6), HOMEODOMAIN GLABROUS11 (HDG11), TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR-14 (TCP14), and ARABIDOPSIS THALIANA HOMEBOX-40 (ATHB40). ZFHD6, TCP14, and ATHB40 directly interacted with and activated the MnGutB1 promoter, a key biosynthetic gene. Although HDG11 did not target MnGutB1 directly, it transcriptionally activated ZFHD6, TCP14, and ATHB40, and formed protein complexes with TCP14 or ATHB40, thereby indirectly promoting DNJ biosynthesis. Furthermore, exogenous application of trans-zeatin riboside or abscisic acid induced HDG11 and MnGutB1 expression, concomitant with elevated DNJ levels. Our findings identify an HDG11-centered regulatory hierarchy that integrates phytohormonal and developmental signals to regulate DNJ biosynthesis, offering insights for metabolic engineering of DNJ production in plants.

Abiotic stress, especially drought stress poses a significant threat to the terrestrial plant growth, development, and productivity. Although mulberry have great genetic diversity and extensive stress tolerant traits in agroforestry systems, only a few reports offer preliminary insight into biochemical responses of mulberry leaves under drought conditions. In this study, we performed a comparative metabolomic and transcriptomic analysis on the “drooping mulberry” (Morus alba var. Pendula Dippel) under PEG-6000-simulated drought stress. Our research revealed that drought stress significantly enhanced flavonoid accumulation and up-regulated the expression of phenylpropanoid biosynthetic genes. Furthermore, the activities of superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) content were elevated. In vitro enzyme assays and fermentation tests indicated the involvement of flavonol synthase/flavanone 3-hydroxylase (XM_010098126.2) and anthocyanidin 3-O-glucosyltransferase 5 (XM_010101521.2) in the biosynthesis of flavonol aglycones and glycosides, respectively. The recombinant MaF3GT5 protein was found to recognize kaempferol, quercetin, and UDP-glucose as substrates, but not 3-/7-O-glucosylated flavonols and UDP-rhamnose. MaF3GT5 is capable of forming 3-O- and 7-O-monoglucoside, but not di-O-glucosides, form kaempferol. This implies its role as a flavonol 3, 7-O-glucosyltransferase. The findings from this study provided insights into the biosynthesis of flavonoids and could have substantial implications for the future diversified utilization of mulberry.

Uridine diphosphate (UDP) glycosyltransferases (UGTs) are a major class of detoxifying enzymes that metabolize xenobiotics through glycosylation. Although UGTs are widely implicated in insect resistance to plant secondary metabolites (PSMs), their functions in adapting to host plant flavonoids remain unclear. Revealing the function of UGT in the adaptation of the silkworm to mulberry flavonoids. In this study, we investigated the function of Bombyx mori UGT40K1 (BmUGT40K1) using a multi-methodological approach that integrated in vitro enzyme activity detected by HPLC-MS/MS, transgenic overexpression, and CRISPR/Cas9-mediated knockout. The results showed that flavonoid treatment on silkworm induces physiological and developmental impairments similar to those caused by UGT inhibitors. In vitroenzyme assays confirmed that BmUGT40K1, which is predominantly expressed in detoxification organs such as midgut during the larval feeding stage, is a crucial enzyme capable of metabolizing these flavonoids. Functional studies further revealed that BmUGT40K1 not only directly facilitates flavonoid detoxification but also modulates the activities of other detoxification enzymes and oxidoreductases. BmUGT40K1plays a central role in flavonoid resistance and likely serves as an important mediator of antioxidant capacity in silkworm. These findings deepen our fundamental understanding of insect-plant coevolution and adaptive responses to phytochemicals.

The internal transcribed spacer (ITS) is one of the most extensively utilized in the taxonomy of the genus Morus due to its generally concerted evolution. Although non-concerted evolution of nuclear ribosomal DNA (nrDNA) has been reported in some species, genome-wide nrDNA characteristics in the genus Morus remain poorly understood. In this study, 158 single-nucleotide polymorphisms (SNPs) and 15 insertions and deletions (InDels) were identified within the nrDNA regions of 542 mulberry accessions representing sixteen Morus species. These wide occurrences of heterogeneous SNPs and InDels revealed the intra-individual polymorphism within the nrDNA region of Morus, indicating the incomplete concerted evolution of nrDNA. Notably, 66 out of 158 SNPs and 13 out of 15 InDels were localized within the ITS regions (ITS1-5.8S-ITS2), indicating a high degree of polymorphism in the ITS, which was further validated through classical cloning and Sanger sequencing methodologies. The 13/16 bp InDel located in the ITS1 region was utilized to develop a rapid and reliable cleaved amplified polymorphic sequence (CAPS) marker-based method for distinguishing M. alba and M. notabilis from other Morus species, eliminating the need for a clone-based sequencing step or comparative phenotypic analysis. Phylogenetic analysis based on nrDNA SNPs from 542 mulberry accessions revealed six distinct clades, corresponding to the six Morus species. These findings offer novel new insights into the taxonomy, conservation, and breeding improvement of Morus species.