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.

Mitogen-activated protein kinase kinase kinase (MAPKKK, MAP3K) is located upstream of the mitogen-activated protein kinase (MAPK) cascade pathway and is responsible for receiving and transmitting external signals to the downstream MAPKKs. Although a large number of MAP3K genes play important roles in plant growth and development, and response to abiotic and biotic stresses, only a few members' functions and cascade signaling pathways have been clarified, and the downstream MAPKKs and MAPKs of most MAP3Ks are still unknown. As more and more signaling pathways are discovered, the function and regulatory mechanism of MAP3K genes will become clearer. In this paper, the MAP3K genes in plants were classified and the members and basic characteristics of each subfamily of MAP3K were briefly described. Moreover, the roles of plant MAP3Ks in regulating plant growth and development and stress (abiotic and biotic) responses are described in detail. In addition, the roles of MAP3Ks involved in plant hormones signal transduction pathway were briefly introduced, and the future research focus was prospected.

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.

Increasing temperature is one of the major threats to maize growth and yield globally. Under heat stress conditions, intracellular protein homeostasis is seriously disturbed, leading to accumulation of abnormally folded proteins, especially in the endoplasmic reticulum (ER). Molecular chaperones are vital players in the renaturation process and in preventing protein aggregation. However, heat stress tolerance-associated chaperones are not well documented in maize. Here, we characterized the biological roles of HEAT UP-REGULATED GENE 1 (ZmHUG1) in maize. ZmHUG1 encodes a heat-inducible holdase-type molecular chaperone localized in the ER. Knockout mutant of ZmHUG1 exhibited remarkably enhanced sensitivity to heat stress. Accordingly, the zmhug1 mutant showed severe ER stress under high temperature. MAIZE PRENYLATED RAB ACCEPTOR 1.C1 (ZmPRA1.C1) was identified as a client of ZmHUG1, and heat-induced aggregation of ZmPRA1.C1 was accelerated in the zmhug1 mutant. Furthermore, the expression of ZmHUG1 was rapidly transactivated by ER stress sensor BASIC LEUCINE ZIPPER DOMAIN 60 (bZIP60) when heat stress occurred. This study reveals a ZmHUG1-based thermo-protective mechanism in maize.

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.

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.

To reveal whether the response of mulberry to phytoplasma infection is associated with DNA methylation changes, the methylome and transcriptome patterns of mulberry leaves in response to phytoplasma infection were explored. Though the average methylation level of infected leaves showed no significant difference with that of healthy leaves, there were 1253 differentially methylated genes and 1168 differentially expressed genes found in the infected leaves, and 215 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 infected leaves. Moreover, the expression of Mu-GsSRK was increased while its methylation level was reduced in mulberry treated with pathogen and SA. Expression of Mu-GsSRK in Arabidopsis enhanced transgenic plant disease resistance and the expressions of some defense genes when plants were inoculated with pathogens. In addition, the DNA methylation dynamic patterns and the roles of the differentially expressed and methylated genes were discussed. Our results suggested that DNA methylation has important roles in mulberry responses to phytoplasma infection, and the information provided there will facilitate to elucidate the epigenetic mechanisms underlying mulberry responses to phytoplasma infection.

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.

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.

To gain insight into the response of mulberry to phytoplasma-infection, the expression profiles of mRNAs and proteins in mulberry phloem sap were examined. A total of 955 unigenes and 136 proteins were found to be differentially expressed between the healthy and infected phloem sap. These differentially expressed mRNAs and proteins are involved in signaling, hormone metabolism, stress responses, etc. Interestingly, we found that both the mRNA and protein levels of the major latex protein-like 329 (MuMLPL329) gene were increased in the infected phloem saps. Expression of the MuMLPL329 gene was induced by pathogen inoculation and was responsive to jasmonic acid. Ectopic expression of MuMLPL329 in Arabidopsis enhances transgenic plant resistance to Botrytis cinerea, Pseudomonas syringae pv tomato DC3000 (Pst. DC3000) and phytoplasma. Further analysis revealed that MuMLPL329 can enhance the expression of some defense genes and might be involved in altering flavonoid content resulting in increased resistance of plants to pathogen infection. Finally, the roles of the differentially expressed mRNAs and proteins and the potential molecular mechanisms of their changes were discussed. It was likely that the phytoplasma-responsive mRNAs and proteins in the phloem saps were involved in multiple pathways of mulberry responses to phytoplasma-infection, and their changes may be partially responsible for some symptoms in the phytoplasma infected plants.

Environmental stresses are major constraints that limit the leaf productivity and quality of mulberry. LncRNAs have emerged as important regulators in response to biotic and abiotic stresses in plants. However, the functions and mechanisms of most lncRNAs remain largely unknown. A novel lncRNA designated as MuLnc1 was found to be cleaved by mul-miR3954 and produce secondary siRNAs in a 21 nt phase in mulberry. It was demonstrated that one of the siRNAs produced, si161579, can silence the expression of the calmodulin-like protein gene CML27 of mulberry (MuCML27). When MuCML27 was heterologously expressed in Arabidopsis, the transgenic plants exhibited enhanced resistance to Botrytis cinerea and Pseudomonas syringae pv tomato DC3000. In addition, the transgenic MuCML27-overexpressing Arabidopsis plants are more tolerant to salt and drought stresses. Furthermore, the network of mul-miR3954-MuLnc1-siRNAs-mRNAs was modeled to elucidate the interaction between lncRNAs and sRNAs with mRNAs. All of these, taken together, suggest that MuLnc1 was associated with environmental stress in mulberry and may be considered as a potential genetic improvement target gene of mulberry. The information provided may shed light on the complicated gene expression regulatory mechanisms in mulberry stress responses.

A wide range of miRNAs have been identified as phloem-mobile molecules that play important roles in coordinating plant development and physiology. Phytoplasmas are associated with hundreds of plant diseases, and the pathogenesis involved in the interactions between phytoplasmas and plants is still poorly understood. To analyse the molecular mechanisms of phytoplasma pathogenicity, the miRNAs profiles in mulberry phloem saps were examined in response to phytoplasma infection. A total of 86 conserved miRNAs and 19 novel miRNAs were identified, and 30 conserved miRNAs and 13 novel miRNAs were differentially expressed upon infection with phytoplasmas. The target genes of the differentially expressed miRNAs are involved in diverse signalling pathways showing the complex interactions between mulberry and phytoplasma. Interestingly, we found that mul-miR482a-5p was up-regulated in the infected phloem saps, and grafting experiments showed that it can be transported from scions to rootstock. Based on the results, the complexity and roles of the miRNAs in phloem sap and the potential molecular mechanisms of their changes were discussed. It is likely that the phytoplasma-responsive miRNAs in the phloem sap modulate multiple pathways and work cooperatively in response to phytoplasma infection, and their expression changes may be responsible for some symptoms in the infected plants.

Biotic stresses are major constraints limiting the leaf quality and productivity of mulberry. MLX56 is a unique chitin-binding protein isolated from Shin-Ichinose (Morus alba) latex that displays toxicity against lepidopteran caterpillars. In this study, the full-length cDNA encoding MLX56 was isolated from Husang 32 (M. multicaulis) and designated HMLX56. Amino acid sequence analysis and protein modeling of three MLX56 proteins showed that they were highly conserved among Morus species. Tissue expression pattern analysis showed that the HMLX56 gene was strongly expressed in mulberry bark and leaves but only slightly expressed in fruits. In addition, analysis of GUS expression indicated that the promoter of HMLX56 showed higher transcriptional activity along the vascular strands, and its activity can be regulated by various environmental factors. Like the MLX56 protein from M. alba, the HMLX56 protein showed toxicity to Plutella xylostella. Moreover, when the HMLX56 gene was ectopically expressed in Arabidopsis, the transgenic plants showed enhanced resistance to aphids, the fungal pathogen Botrytis cinerea and the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Our data suggest that the HMLX56 protein has a lectin-like molecular structure consisting of two hevein-like chitin-binding domains which provide not only chitin-binding activities but also other mechanisms of defense. The information provided here improves our understanding of the potential functions and defense mechanisms of MLX56 proteins, enabling in-depth functional analysis of latex exudates and perhaps facilitating mulberry genetic improvement in the future.

Studying the promoter of key genes related to mulberry disease resistance and its activity plays a critical role in clarifing the gene function and expressional regulatory mechanism.In present study,the promoter of pathogenesis-reiated protein gene MuPR1-2 was successfully cloned from genomic DNA of mulberry leaf by TaiI-PCR and designated as pMuPR1-2.Sequence analysis of pMuPR1-2 was performed using software PlantCARE and the results showed that it contains several cis-acting elements necessary for transcriptional initiation,multiple transcription factor binding sites,and a variety of environmental factor responsive elements.The plant expression vector containing GUS gene drived by pMuPR1-2 promoter was constructed.The activity of pMuPR1-2 was investigated by the method of Agrobacterium-mediated transient expression in tobacco leaves,and the result indicated that pMuPR1-2 could initiate the transcription of downstream GUS gene which was used as a reporter gene after tobacco leaves were inoculated by Pst DC3000 bacterial solution and could be induced upon pathogen attack.The transgenic Arabidopsis plants stably expressing pMuPR1-2 were further constructed.GUS histochemical staining and fluorescent quantitative analysis of GUS showed that pMuPR1-2 could be induced by pathogenic fungi and bacteria,and it was expressed in special tissues after induced by some hormones.