Expressional and Functional Verification of the Involvement of CmEXPA4 in Chrysanthemum Root Development

Expansin (EXP) plays an important role in plant root formation. The EXP genes associated with chrysanthemum roots have not yet been reported. Here we isolated a root-specific EXP gene in chrysanthemum ( Chrysanthemum morifolium ), namely CmEXPA4 . Bioinformatics analysis showed that CmEXPA4 -encoded protein has a conserved DPPB (Double-Psi Beta-Barrel) domain in the N-terminal with a series of Cys residues, an HFD (His-Phe-Asp) motif in the central region, and a pollen allergen domain in the C-terminal. The protein also has a specific α-insertion of WCNP (Trp-Cys-Asn-Pro), which suggests that it belongs to the A-subgroup of the EXP family. In the present study, we cloned the 1,129 bp promoter region upstream of CmEXPA4 , and the analysis revealed an abundance of cis-acting elements associated with hormones, light and stress-related responses, and some root-specific regulatory elements in particular. Subcellular localization results indicated that CmEXPA4 locates in the cell wall. Exogenous indole butyric acid induced the up-regulation of CmEXPA4 expression, whereas exogenous abscisic acid inhibited its expression. Tissue expression analysis showed that CmEXPA4 was preferentially expressed in the roots and was synchronized with the rapid emergence of the root. These results suggested that CmEXPA4 may act on the growth and development of chrysanthemum roots. The function of CmEXPA4 was further tested by virus-induced gene silencing, and the results showed that CmEXPA4 silencing inhibited the normal development of the chrysanthemum root system. The roots appeared thinner and shorter, and several important root parameters, including total length, average diameter, surface area, total volume, and root tip number, decreased significantly. The cortical cells of the transgenic plant roots were significantly smaller and shorter than those of the control. Collectively, our results demonstrated that CmEXPA4 gene plays a key role in the growth and development of chrysanthemum roots and affects the root system by acting on the individual cells.