| Summary: | Plant β-galactosidases (BGAL) function in various cell wall biogeneses and modifications, and they belong to the glycoside hydrolase family. However, the roles of BGAL family members in <i>Medicago truncatula</i> cell wall remodeling remain unclear. In this study, a total of 25 <i>MtBGAL</i> members of the glycoside hydrolase gene family 35 were identified, and they were clustered into nine sub-families. Many cis-acting elements possibly related to MeJA and abscisic acid responses were identified in the promoter region of the <i>MtBGAL</i> genes. Transcript analyses showed that these <i>MtBGAL</i> genes exhibited distinct expression patterns in various tissues and developing stem internodes. Furthermore, a stem-specific expression module associated with cell wall metabolic pathways was identified by weighted correlation network analysis (WGCNA). In particular, <i>MtBGAL1</i> and <i>MtBGAL23</i> within the stem-specific expression module were highly expressed in mature stems. In addition, several genes involved in lignin, cellulose, hemicellulose and pectin pathways were co-expressed with <i>MtBGAL1</i> and <i>MtBGAL23</i>. It was also found that <i>MtBGAL1</i> and <i>MtBGAL23</i> were localized to the cell wall at the subcellular level, indicating their roles in the modification of cell wall metabolites in <i>Medicago</i>. As a whole, these results will be useful for further functional characterization and utilization of <i>BGAL</i> genes in cell wall modifications aiming to improve the quality of legume forage crops.
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