Genome-wide analysis of the mulberry (Morus abla L.) GH9 gene family and the functional characterization of MaGH9B6 during the development of the abscission zone.

Plant glycoside hydrolase family 9 genes (GH9s) are widely distributed in plants and involved in a variety of cellular and physiological processes. In the current study, nine GH9 genes were identified in the mulberry and were divided into two subfamilies based on the phylogenetic analysis. Conserved motifs and gene structure analysis suggested that the evolution of the two subfamilies is relatively conserved and the glycoside hydrolase domain almost occupy the entire coding region of the GH9s gene. Only segmental duplication has played a role in the expansion of gene family. Collinearity analysis showed that mulberry GH9s had the closest relationship with poplar GH9s. MaGH9B1, MaGH9B6, MaGH9B5, and MaGH9B3 were detected to have transcript accumulation in the stalk of easy-to drop mature fruit drop, suggesting that these could play a role in mulberry fruit drop. Multiple cis-acting elements related to plant hormones and abiotic stress responses were found in the mulberry GH9 promoter regions and showed different activities under exogenous abscisic acid (ABA) and 2,4- dichlorophenoxyacetic acid (2,4-D) stresses. We found that the lignin content in the fruit stalk decreased with the formation of the abscission zone (AZ), which could indirectly reflect the formation process of the AZ. These results provide a theoretical basis for further research on the role of GH9s in mulberry abscission.

MaABI5 and MaABF1 transcription factors regulate the expression of MaJOINTLESS during fruit abscission in mulberry (Morus alba L.).

Mulberry holds significant economic value. However, during the ripening stage of its fruit, the phenomenon of abscission, resulting in heavy fruit drop, can severely impact the yield. The formation of off-zone structures is a critical factor in the fruit abscission process, and this process is regulated by multiple transcription factors. One such key gene that plays a significant role in the development of the off-zone in the model plant tomato is JOINTLESS, which promotes the expression of abscission-related genes and regulates the differentiation of abscission zone tissue cells. However, there is a lack of information about fruit abscission mechanism in mulberry. Here, we analyzed the MaJOINTLESS promoter and identified the upstream regulators MaABF1 and MaABI5. These two regulators showed binding with MaJOINTLESS promoter MaABF1 (the ABA Binding Factor/ABA-Responsive Element Binding Proteins) activated the expression of MaJOINTLESS, while MaABI5 (ABSCISIC ACID-INSENSITIVE 5) inhibited the expression of MaJOINTLESS. Finally, the differentially expressed genes (DEGs) were analyzed by transcriptome sequencing to investigate the expression and synergistic relationship of endogenous genes in mulberry during abscission. GO classification and KEGG pathway enrichment analysis showed that most of the DEGs were concentrated in MAPK signaling pathway, flavonoid biosynthesis, citric acid cycle, phytohormone signaling, amino acid biosynthesis, and glycolysis. These results provide a theoretical basis for subsequent in-depth study of physiological fruit abscission in mulberry.