Identification and Functional Characterization of Alfalfa (Medicago sativa L.) Expansins in Regulating Arabidopsis Shoot and Root Development

Expansins are known as cell wall loosening proteins and are involved in cell expansion and varieties of plant developmental progresses. However, little is known about their biological functions in alfalfa (Medicago sativa L.). In the present study, 30 MsEXP genes were identified in the alfalfa (cultivar “zhongmu-1”) genome. Phylogenetic analysis revealed that these MsEXP proteins were divided into four subfamilies, including twenty-one MsEXPAs, six MsEXPBs, one MsEXL1 and two MsEXLBs. MsEXP genes were unevenly distributed on eight chromosomes. The gene structures of the MsEXP genes and the motif composition of the MsEXP proteins were inconsistent with the phylogenetic relationship of MsEXPs. Cis-acting elements analysis indicated that MsEXP genes may respond to diverse hormonal signals involved in the developmental progress of plants. Furthermore, expression analysis suggested that MsEXP genes exhibited distinct expression patterns among different tissues of alfalfa. Overexpression of MsEXPA3 or MsEXPA4 promoted the growth of leaves, stems and roots of Arabidopsis, thereby increasing the biomass of plants. Subsequent cell morphological analysis uncovered that overexpression of MsEXPA3 or MsEXPA4 promoted the expansion of cells. Taken together, these findings illustrate the functions of MsEXP proteins in regulating the development of plants. Our results may provide a strong basis for further elucidating the roles of these EXP genes in alfalfa development and valuable genetic resources for future crop improvement.