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.

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.

DNA methylation has been proposed to regulate plant stress resistance. However, the dynamic changes in DNA methylation in woody plants and their correlations with pathogenic responses are not fully understood. Here, we present single-base maps of the DNA methylomes of mulberry (Morus notabilis) leaves that were subjected to a mock treatment or inoculation with Botrytis cinerea. Compared with the former, the latter showed decreased mCG and mCHG levels and increased mCHH levels. DNA methylation inhibitors reduced resistance gene methylation levels and enhanced mulberry resistance, suggesting that the hypomethylation of resistance genes affects mulberry resistance to B. cinerea. Virus-induced gene silencing of MnMET1 enhanced the expression of mulberry-resistance genes, thereby increasing the plant’s resistance to B. cinerea. We also found that MITEs play a dominant role in controlling DNA methylation levels. MITEs appear to be the main sources of 24-nt siRNAs that regulate gene expression through the RNA-directed DNA methylation pathway.

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.

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.