Mulberry fruits and leaves contain resveratrol and flavonoids, which determine their nutritional and functional values, but the molecular mechanism balancing these two metabolites remains unclear. This study identifies a novel transcriptional regulatory module regulating their biosynthesis in mulberry. MaMYB306 functions as a central regulator that directly binds to the MaSTS7 promoter to activate resveratrol biosynthesis while suppressing MaCHS, MaF3H, MaDFR, and MaANS to suppress flavonoid production, redirecting metabolic flux and improving antioxidant capacity and resistance to Botrytis cinerea. Furthermore, MaWRKY49 physically interacts with MaMYB306 to enhance its transcriptional activity, whereas MabZIP60 represses MaMYB306 expression to promote flavonoid accumulation. This MabZIP60-MaMYB306-MaWRKY49 module represents a sophisticated regulatory mechanism that optimizes the balance between the two major phenylpropanoid branches. Our findings provide a novel transcriptional framework for the metabolic engineering of mulberry and other crops to simultaneously enhance health-promoting resveratrol and pathogen resistance, adding value to functional foods and supporting sustainable agriculture.

Abstract Mulberry fruit ripening involves complex regulatory mechanisms influenced by epigenetic modifications, yet the role of DNA methylation in this process remains poorly understood. Through integrated transcriptome and methylome analysis of mulberry fruits at different developmental stages, 9528 differentially expressed genes (DEGs) and 1797 differentially methylated genes (DMGs) were identified. Among these, 621 genes exhibited concurrent changes in both methylation and expression levels throughout fruit ripening. Notably, a prevailing genome-wide hypomethylation trend was observed. Intriguingly, the MabZIP60 gene showed increased expression accompanied by promoter hypermethylation during ripening, and this response was further enhanced by abscisic acid (ABA). MabZIP60 interacts with two unfolded protein response (UPR) components: luminal-binding protein 5 (BiP5) and subtilisin-like protease SBT1.4. ABA triggers proteolytic cleavage, resulting in the release of MabZIP60 from the membrane-bound complex and its subsequent translocation into the nucleus. Within the nucleus, MabZIP60 directly binds to the promoters of CHS, ANS, and LAR, activating their transcription to enhance anthocyanin production, and ABA treatment further enhances the MabZIP60-mediated transcriptional activation. Our findings reveal a novel regulatory module in which ABA recruits DNA methylation as an activating switch to control a central transcription factor network, thereby integrating hormonal signaling, epigenetic modification, and metabolic regulation to orchestrate ripening in mulberry and other horticultural crops.

Mulberry (Morus alba), a tree with multipurpose applications, is a promising source of bioactive flavonoids for functional foods. Mulberry-derived flavonoids possess prominent antioxidant, anti-inflammatory, and cardioprotective activities, making them valuable functional ingredients for nutraceutical products. Elucidating the transcriptional regulation of flavonoid biosynthesis is essential for targeted molecular breeding to enhance flavonoid accumulation in mulberry, thereby supporting sustainable production of high-value food ingredients. In this study, five R2R3-MYB transcription factors whose expression correlates with flavonoid accumulation during mulberry fruit development were identified. Among these, MaMYB12 (subgroup 7, SG7) functions as a transcriptional activator. It transient overexpression in mulberry leaves significantly elevated the content of 30 flavonoid metabolites by directly activating the promoters of key flavonoid biosynthetic genes. In contrast, MaMYB308 (SG4) functions as a strong repressor, reducing the accumulation of 106 flavonoid metabolites through directly suppressing of key flavonoid biosynthetic genes. The direct binding of both MaMYB12 and MaMYB308 to target gene promoters was verified by yeast one-hybrid assays and electrophoretic mobility shift assays. Their antagonistic regulatory roles were further confirmed in stably transformed mulberry hairy roots. Additionally, extracts from MaMYB12-overexpressing hairy roots exhibited significantly enhanced antioxidant activity, directly linking this genetic manipulation to improved functional food properties. Our findings uncover a key transcriptional module governing flavonoid metabolism in mulberry and present practical genetic tools for the molecular breeding of mulberry varieties with optimized flavonoid profiles, advancing their utility as functional food ingredients.

Salinity is one of the most serious threats to sustainable agriculture. The Salt Overly Sensitive (SOS) signaling pathway plays an important role in salinity tolerance in plants, and the SOS2 gene plays a critical role in this pathway. Mulberry not only has important economic value but also is an important ecological tree species; however, the roles of the SOS2 gene associated with salt stress have not been reported in mulberry. To gain insight into the response of mulberry to salt stress, SOS2 (designated MulSOS2) was cloned from mulberry (Morus atropurpurea Roxb), and sequence analysis of the amino acids of MulSOS2 showed that it shares some conserved domains with its homologs from other plant species. Our data showed that the MulSOS2 gene was expressed at different levels in different tissues of mulberry, and its expression was induced substantially not only by NaCl but also by ABA. In addition, MulSOS2 was exogenously expressed in Arabidopsis, and the results showed that under salt stress, transgenic MulSOS2 plants accumulated more proline and less malondialdehyde than the wild-type plants and exhibited increased tolerance to salt stress. Moreover, the MulSOS2 gene was transiently overexpressed in mulberry leaves and stably overexpressed in the hairy roots, and similar results were obtained for resistance to salt stress in transgenic mulberry plants. Taken together, the results of this study are helpful to further explore the function of the MulSOS2 gene, which provides a valuable gene for the genetic breeding of salt tolerance in mulberry.

Phytoplasma disease is one of the most serious infectious diseases that affects the growth and development of mulberry. Long non-coding RNAs (lncRNAs) play an important role in plants' defense systems; however, the contribution of lncRNAs in the response to phytoplasma infection in mulberry is still largely unknown. Herein, strand-specific RNA sequencing was performed to profile the mRNAs and lncRNAs involved in the response to phytoplasma infection in mulberry, and a total of 4169 genes were found to be differentially expressed (DE) between healthy and phytoplasma-infected leaves. Moreover, 1794 lncRNAs were identified, of which 742 lncRNAs were DE between healthy and infected leaves. Target prediction showed that there were 68 and 44 DE lncRNAs which may function as cis and trans-regulators, targeting 54 and 44 DE genes, respectively. These DE target genes are associated with biological processes such as metabolism, signaling, development, transcriptional regulation, etc. In addition, it was found that the expression of the antisense lncRNA (MuLRR-RLK-AS) of the leucine-rich repeat receptor-like protein kinase gene (MuLRR-RLK) was decreased in the phytoplasma-infected leaves. Interestingly, it was found that overexpression of MuLRR-RLK-AS can inhibit the expression of MuLRR-RLK. Moreover, it was found that the expression levels of PTI-related and MAPK genes in the transgenic MuLRR-RLK Arabidopsis plants were significantly higher than those in the wild-type plants when inoculated with pathogens, and the transgenic plants were conferred with strong disease resistance. Our results demonstrate that MuLRR-RLK-AS, as a trans-regulatory factor, can inhibit the expression of the MuLRR-RLK gene and is a negative regulatory factor for mulberry resistance. The information provided is particularly useful for understanding the functions and mechanisms of lncRNAs in the response to phytoplasma infection in mulberry.

Cytochrome P450 (CYP) is a crucial oxidoreductase enzyme that plays a significant role in plant defense mechanisms. In this study, a specific cytochrome P450 gene (MnCYP710A11) was discovered in mulberry (Morus notabilis). Bioinformatic analysis and expression pattern analysis were conducted to elucidate the involvement of MnCYP710A11 in combating Botrytis cinerea infection. After the infection of B. cinerea, there was a notable increase in the expression of MnCYP710A11. MnCYP710A11 is overexpressed in Arabidopsis and mulberry and strongly reacts to B. cinerea. The overexpression of the MnCYP710A11 gene in Arabidopsis and mulberry led to a substantial enhancement in resistance against B. cinerea, elevated catalase (CAT) activity, increased proline content, and reduced malondialdehyde (MDA) levels. At the same time, H2O2 and O2− levels in MnCYP710A11 transgenic Arabidopsis were decreased, which reduced the damage of ROS accumulation to plants. Furthermore, our research indicates the potential involvement of MnCYP710A11 in B. cinerea resistance through the modulation of other resistance-related genes. These findings establish a crucial foundation for gaining deeper insights into the role of cytochrome P450 in mulberry plants.

Although microRNAs (miRNAs) regulate the defense response of a variety of plant species against a variety of pathogenic fungi, the involvement of miRNAs in mulberry's defense against Botrytis cinerea has not yet been documented. In this study, we identified responsive B. cinerea miRNA mno-miR164a in mulberry trees. After infection with B. cinerea, the expression of mno-miR164a was reduced, which was fully correlated with the upregulation of its target gene, MnNAC100, responsible for encoding a transcription factor. By using transient infiltration/VIGS mulberry that overexpressed mno-miR164a or knocked-down MnNAC100, our study revealed a substantial enhancement in mulberry's resistance to B. cinerea when mno-miR164a was overexpressed or MnNAC100 expression was suppressed. This enhancement was accompanied by increased catalase (CAT) activity and reduced malondialdehyde (MDA) content. In addition, mno-miR164a-mediated inhibition of MnNAC100 enhanced the expression of a cluster of defense-related genes in transgenic plants upon exposure to B. cinerea. Meanwhile, MnNAC100 acts as a transcriptional repressor, directly suppressing the expression of MnPDF1.2. Our study indicated that the mno-miR164a-MnNAC100 regulatory module manipulates the defense response of mulberry to B. cinerea infection. This discovery has great potential in breeding of resistant varieties and disease control.

Galactitol synthetase (GolS) as a key enzyme in the raffinose family oligosaccharides (RFOs) biosynthesis pathway, which is closely related to stress. At present, there are few studies on GolS in biological stress. The expression of MnGolS2 gene in mulberry was increased under Botrytis cinerea infection. The MnGolS2 gene was cloned and ectopically expressed in Arabidopsis. The content of MDA in leaves of transgenic plants was decreased and the content of CAT was increased after inoculation with B. cinerea. In this study, the role of MnGolS2 in biotic stress was demonstrated for the first time. In addition, it was found that MnGolS2 may increase the resistance of B. cinerea by interacting with other resistance genes. This study offers a crucial foundation for further research into the role of the GolS2 gene.

The male zooid of Antheraea pernyi (A. pernyi) accumulates several nutrients and physiological activity-related substances for reproduction. Some components in the extracts of the male zooid of A. pernyi (EMZAP) have several functions, such as protecting the liver, enhancing immunity, antiatheroscloresis, anti-aging, and antitumor effects. In this study, we investigated the ameliorating effects on high-fat diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD). The EMZAP treatment could ameliorate NAFLD and effectively decrease the serum total cholesterol, triglyceride and low-density lipoprotein levels and a significant increase in serum high-density lipoprotein levels was observed. Additionally, the EMZAP treatment reduced the levels of liver-function enzymes and pro-inflammatory cytokines (i.e., IL-6, IL-8, TNF-α, TGF-β1) and also the oxidative stress indices and regulated the expression of genes associated with fatty acid metabolism (SREBP-1c, PPARα, ACOX-1, CPT-1) in the liver to prevent the development of NAFLD. Furthermore, EMZAP enhanced the diversity and richness of the beneficial intestinal microbes, suggesting its potential as a dietary supplement and functional food to combat NAFLD induced by HFD.

Gamma-aminobutyric acid (GABA) has been reported to accumulate in plants when subjected to salt stress, and GABA-transaminase (GABA-T) is the main GABA-degrading enzyme in the GABA shunt pathway. So far, the salt tolerance mechanism of the GABA-T gene behind the GABA metabolism remains unclear. In this study, the cDNA (designated MuGABA-T) of GABA-T gene was cloned from mulberry, and our data showed that MuGABA-T protein shares some conserved characteristics with its homologs from several plant species. MuGABA-T gene was constitutively expressed at different levels in mulberry tissues, and was induced substantially by NaCl, ABA and SA. In addition, our results demonstrated that exogenous application of GABA significantly reduced the salt damage index and increased plant resistance to NaCl stress. We further performed a functional analysis of MuGABA-T gene and demonstrated that the content of GABA was reduced in the transgenic MuGABA-T Arabidopsis plants, which accumulated more ROS and exhibited more sensitivity to salt stress than wild-type plants. However, exogenous application of GABA significantly increased the activities of antioxidant enzymes and alleviated the active oxygen-related injury of the transgenic plants under NaCl stress. Moreover, the MuGABA-T gene was overexpressed in the mulberry hairy roots, and similar results were obtained for sensitivity to salt stress in the transgenic mulberry plants. Our results suggest that the MuGABA-T gene plays a pivotal role in GABA catabolism and is responsible for a decrease in salt tolerance, and it may be involved in the ROS pathway in the response to salt stress. Taken together, the information provided here is helpful for further analysis of the function of GABA-T genes, and may promote mulberry resistance breeding in the future.

Six α-amylase/subtilisin inhibitor genes (MnASIs) were identified from mulberry (Morus notabilis). In this study, bioinformatics and expression pattern analysis of six MnASIs were performed to determine their roles in resistance to B. cinerea. The expression of all six MnASIs was significantly increased under Botrytis cinerea infection. MnASI1, which responded strongly to B. cinerea, was overexpressed in Arabidopsis and mulberry. The resistance of Arabidopsis and mulberry overexpressing MnASI1 gene to B. cinerea was significantly improved, the catalase (CAT) activity was increased, and the malondialdehyde (MDA) content was decreased after inoculation with B. cinerea. At the same time, H2O2 and O2− levels were reduced in MnASI1 transgenic Arabidopsis, reducing the damage of ROS accumulation to plants. In addition, MnASI1 transgenic Arabidopsis increased the expression of the salicylic acid (SA) pathway-related gene AtPR1. This study provides an important reference for further revealing the function of α-amylase/subtilisin inhibitors.

To understand the mechanism of small non-coding RNAs (miRNA)-mediated development and ripening of mulberry fruits, three small RNA libraries from mulberry fruits at different development stages were constructed, and 159 conserved miRNAs as well as 86 novel miRNAs were successfully identified. Among the miRNAs identified, there were 90 miRNAs which showed differential expression patterns at different stages of fruit development and ripening. The target genes of these differential expressed (DE) miRNAs were involved in growth and development, transcription and regulation of transcription, metabolic processes, and etc. Interestingly, it was found that the expression level of mul-miR477 was increased with fruit ripening, and it can target the antisense lncRNA (Mul-ABCB19AS) of the ATP binding cassette (ABC) transporter B 19 gene (Mul-ABCB19). Our results showed that mul-miR477 can repress the expression of Mul-ABCB19AS and increase the expression of Mul-ABCB19, and it acted as a positive regulator participating anthocyanin accumulation through the regulatory network of mul-miR477-Mul-ABCB19AS-Mul-ABCB19.

To reveal whether the response of mulberry to phytoplasma infection is associated with genome-wide DNA methylation changes, the methylome and transcriptome patterns of mulberry in response to phytoplasma infection were explored. Though the average methylation level of the infected leaves showed no significant difference from that of healthy leaves, there were 1,253 differentially methylated genes (DMGs) and 1,168 differentially expressed genes (DEGs) in the infected leaves, and 51 genes were found simultaneously to be differently methylated and expressed. It was found that the expression of G-type lectin S-receptor-like serine/threonine protein kinase gene (Mu-GsSRK) was increased, but its methylation level was decreased in the pathogen-infected or salicylic acid (SA)-treated leaves. Overexpression of Mu-GsSRK in Arabidopsis and in the hairy roots of mulberry enhanced transgenic plant resistance to the phytoplasma. Moreover, overexpression of Mu-GsSRK enhanced the expressions of pathogenesis-related protein 1, plant defensin, and cytochrome P450 protein CYP82C2 genes in transgenic plants inoculated with pathogens, which may contribute to the enhanced disease resistance against various pathogens. Finally, the DNA methylation dynamic patterns and functions of the differentially expressed and methylated genes were discussed. The results suggested that DNA methylation has important roles in mulberry responses to phytoplasma infection.

HD-Zip基因家族是近年来发现的一类在植物的生长发育过程中起到重要调节作用的转录因子,该基因家族是植物中特有的.所有HD-Zip转录因子都包含同源异型盒和亮氨酸拉链2个结构域,根据基因结构和功能可分为4个亚家族.该家族基因具有多种生物学功能.基于此,结合HD-Zip转录因子的结构和亚家族分类,综述近年来HD-Zip转录因子参与植物逆境胁迫响应的相关研究进展.

Chitinase is a hydrolase that uses chitin as a substrate. It plays an important role in plant resistance to fungal pathogens by degrading chitin. Here, we conducted bioinformatics analysis and transcriptome data analysis of the mulberry (Morus notabilis) chitinase gene family to determine its role in the resistance to Botrytis cinerea. A total of 26 chitinase genes were identified, belonging to the GH18 and GH19 families. Among them, six chitinase genes were differentially expressed under the infection of B. cinerea. MnChi18, which significantly responded to B. cinerea, was heterologously expressed in Arabidopsis (Arabidopsis thaliana). The resistance of MnChi18 transgenic Arabidopsis to B. cinerea was significantly enhanced, and after inoculation with B. cinerea, the activity of catalase (CAT) increased and the content of malondialdehyde (MDA) decreased. This shows that overexpression of MnChi18 can protect cells from damage. In addition, our study also indicated that MnChi18 may be involved in B. cinerea resistance through other resistance-related genes. This study provides an important basis for further understanding the function of mulberry chitinase.

MicroRNA (miRNA)作为一种重要的基因表达调控因子,在植物生长发育和响应环境胁迫过程中具有重要的调节功能.本研究克隆了桑树miR482基因(MulmiR482),发现该基因可以表达加工成两种成熟体mul-miR482-5p和mul-miR482-3p,两者在桑树根中均具有较高的表达丰度,而在叶片中的丰度较低.通过拟南芥嫁接试验发现mul-miR482-5p和mul-miR482-3p在植物体内具有长距离运输特性.将MulmiR482基因转入拟南芥,发现其可以在拟南芥中表达并可加工成成熟体,同时发现MulmiR482基因在拟南芥中表达降低了转基因植株对盐胁迫和丁香假单胞菌番茄致病变种DC3000的抗性.上述结果为深入研究桑树mul-miR482的生物功能和其表达调控机制奠定了基础.

为探讨非杂交桑品种嫁接苗采用高密度草本式栽培的丰产性能和养蚕效果,利用发条能力较强的农桑14号嫁接桑进行了高密度草本式栽培建立桑园(简称农桑14号草本桑,90 000株/hm2)的试验,以桂桑优12号杂交桑草本式栽培建立桑园(简称桂桑优12号草本桑,105 000株/hm2)和育71-1嫁接桑普通栽植低干养成建立桑园(简称育71-1普栽桑,24 000株/hm2)为对照,调查了3种桑园第1～5年的产叶量指标,并进行了养蚕鉴定.结果 表明,建桑园第1～3年,试验区农桑14号草本桑的片叶产量比对照区桂桑优12号草本桑低10.42％ ～ 14.87％,但第4年和第5年,农桑14号草本桑的片叶产量分别比桂桑优12号草本桑提高8.81％和5.18％,5年单株总产叶量农桑14号草本桑比桂桑优12号草本桑高16.04％,5年合计经济系数(片叶质量与条叶总质量之比)农桑14号草本桑比桂桑优12号草本桑高8.87％.无论是农桑14号草本桑还是桂桑优12号草本桑,各年度产叶量均高于育71-1普栽桑.建园第1年(2013年)秋蚕期和建园第2年(2014年)春蚕期的养蚕试验结果表明,农桑14号草本桑条桑育成绩均优于桂桑优12号草本桑,与育71-1普栽桑片叶育的效果相当.本试验结果说明,利用耐剪伐的嫁接桑品种,采用高密度草本式栽培可以实现速生丰产和桑叶优质.

In the present study, the cDNA (designated MuPR1) of PR1 gene was obtained from mulberry. Our results showed that MuPR1 shares some conserved characteristics with its homologues from different plant families. MuPR1 was localized exclusively to the extracellular spaces, and the MuPR1 gene was expressed constitutively in all selected mulberry tissues, although its expression levels varied substantially. It was also showed that MuPR1 expression was induced by pathogen, MeJA, SA and GA3, and transgenic Arabidopsis overexpressing MuPR1 showed enhanced resistance to Botrytis cinerea and Pst. DC3000. Moreover, the peptide derived from MuPR1 protein C-terminus with a PxGNxxxxxPY motif was suggested to play a role in activating plant resistance. Furthermore, the MuPR1 gene may have roles in mediating the rates of oxygen radical formation and detoxification. Therefore, the MuPR1 gene may be a candidate gene for breeding resistant mulberry varieties in the future.

大球盖菇是一种食、药用价值较高的珍稀食用菌,其人工培育具有栽培简便、低投入、高产出的经济优势.设计不同桑枝木屑含量配方的基质进行大球盖菇栽培,并利用反向高效液相色谱法测定大球盖菇子实体中1-脱氧野尻霉素(DNJ)的含量.结果 表明,基质不同桑枝木屑含量及出菇潮次对大球盖菇子实体DNJ富集量的影响差异显著,桑枝木屑质量分数为70％的栽培基质生产出的第1潮菇子实体DNJ富集量最高,大球盖菇干品中DNJ的质量分数达到0.230 2 mg/g,高于桑枝DNJ的含量,接近桑椹中DNJ的含量.研究结果为进一步提高桑枝资源利用效率及生产较高保健价值食用菌提供了理论依据.

The quality and quantity of mulberry leaves are often affected by various environmental factors. The plant NPR1 and its homologous genes are important for plant systemic acquired resistance. Here, the full-length cDNAs encoding the NPR1 and NPR4 genes (designated MuNPR1 and MuNPR4, respectively) were isolated from Morus multicaulis. Sequence analysis of the amino acids and protein modeling of the MuNPR1 and MuNPR4 proteins showed that MuNPR1 shares some conserved characteristics with its homolog MuNPR4. MuNPR1 was shown to have different expression patterns than MuNPR4 in mulberry plants. Interestingly, MuNPR1 or MuNPR4 transgenic Arabidopsis produced an early flowering phenotype, and the expression of the pathogenesis-related 1a gene was promoted in MuNPR1 transgenic Arabidopsis. The MuNPR1 transgenic plants showed more resistance to Pseudomonas syringae pv. tomato DC3000 (Pst. DC3000) than did the wild-type Arabidopsis. Moreover, the ectopic expression of MuNPR1 might lead to enhanced scavenging ability and suppress collase accumulation. In contrast, the MuNPR4 transgenic Arabidopsis were hypersensitive to Pst. DC3000 infection. In addition, transgenic Arabidopsis with the ectopic expression of either MuNPR1 or MuNPR4 showed sensitivity to salt and drought stresses. Our data suggest that both the MuNPR1 and MuNPR4 genes play a role in the coordination between signaling pathways, and the information provided here enables the in-depth functional analysis of the MuNPR1 and MuNPR4 genes and may promote mulberry resistance breeding in the future.