Mulberry is a fundamental component of the global sericulture industry, and its positive impact on our health and the environment cannot be overstated. However, the mulberry reference genomes reported previously remained unassembled or unplaced sequences. Here, we report the assembly and analysis of the telomere-to-telomere gap-free reference genome of the mulberry species, Morus notabilis, which has emerged as an important reference in mulberry gene function research and genetic improvement. The mulberry gap-free reference genome produced here provides an unprecedented opportunity for us to study the structure and function of centromeres. Our results revealed that all mulberry centromeric regions share conserved centromeric satellite repeats with different copies. Strikingly, we found that M. notabilis is a species with polycentric chromosomes and the only reported polycentric chromosome species up to now. We propose a compelling model that explains the formation mechanism of new centromeres and addresses the unsolved scientific question of the chromosome fusion-fission cycle in mulberry species. Our study sheds light on the functional genomics, chromosome evolution, and genetic improvement of mulberry species.

Flavones, one of the largest classes of flavonoids in plants, have a variety of bioactivities and participate in the resistance response of plants to biotic and abiotic stresses. However, flavone synthase (FNS), the key enzyme for flavone biosynthesis, has not yet been characterized in mulberry. In this study, we report that the leaves of certain mulberry cultivars, namely BJ7, PS2, and G14, are rich in flavones. We identified a Fe²⁺/2-oxoglutarate-dependent dioxygenase from Morus notabilis (MnFNSI) that shows the typical enzymatic activity of a FNSI-type enzyme, and directly converts eriodictyol and naringenin into their corresponding flavones. Overexpression of MnFNSI in tobacco increased the flavones contents in leaves and enhanced the tolerance of tobacco to ultraviolet-B (UV-B) stress. We found that mulberry cultivars with higher flavones contents exhibit less UV-B induced damage after a UV-B treatment. Accordingly, our findings demonstrate that MnFNSI, a FNSI-type enzyme, is involved in the biosynthesis of flavones, which provide protection against UV-B radiation. These results lay the foundation for obtaining mulberry germplasm resources that are more tolerant to UV-B stress and richer in their nutritional value.

As the prevalence of diabetes continues to increase, the number of individuals living with diabetes complications will reach an unprecedented magnitude. Continuous use of some synthetic agents to reduce blood glucose levels causes severe side effects, and thus, the demand for nontoxic, affordable drugs persists. Naturally occurring compounds, such as iminosugars derived from the mulberry (Morus spp.), have been shown to reduce blood glucose levels. In mulberry, 1-deoxynojirimycin (DNJ) is the predominant iminosugar. However, the mechanism underlying DNJ biosynthesis is not completely understood. Here, we showed that DNJ in mulberry is derived from sugar and catalyzed through 2-amino-2-deoxy-D-mannitol (ADM) dehydrogenase MnGutB1. Combining both targeted and nontargeted metabolite profiling methods, DNJ and its precursors ADM and nojirimycin (NJ) were quantified in mulberry samples from different tissues. Purified His-tagged MnGutB1 oxidized the hexose derivative ADM to form the 6-oxo compound DNJ. The mutant MnGutB1 D283N lost this remarkable capability. Furthermore, in contrast to virus-induced gene silencing of MnGutB1 in mulberry leaves that disrupted the biosynthesis of DNJ, overexpression of MnGutB1 in hairy roots and light-induced upregulation of MnGutB1 enhanced DNJ accumulation. Our results demonstrated that hexose derivative ADM, rather than lysine derivatives, is the precursor in DNJ biosynthesis, and it is catalyzed by MnGutB1 to form the 6-oxo compound. These results represent a breakthrough in producing DNJ and its analogs for medical use by metabolic engineering or synthetic biology.

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.

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.