The roles of OfEXPA2 and OfEXPA4 on petal cell expansion during flower opening in Osmanthus fragrans

• OfEXPA2 and OfEXPA4 showed increased expression levels during early flower opening in Osmanthus fragrans . • OfEXPA2 and OfEXPA4 proteins were localized in the cell wall. • Overexpression of OfEXPA2 and OfEXPA4 altered stem diameter and flower morphology by promoting cell enlargement. • OfMYB5 positively promoted the expression of OfEXPA2 and OfEXPA4. Expansin (EXP) is a unique protein in plant cell walls that promotes cell expansion and wall relaxation. Transcriptome data and qRT-PCR results revealed that OfEXPA2 and OfEXPA4 show increased expression levels during early flower opening in Osmanthus fragrans , identifying them as candidate genes involved in this process. The bioinformatics analysis revealed the presence of the DPBB (Double-Psi Beta-Barrel) domain and pollen allergen domain in both OfEXPA2 and OfEXPA4 protein sequences. However, the amino acid sequences of OfEXPA2 and OfEXPA4 exhibited low sequence identity and did not cluster on the same branch of the evolutionary tree. Functional analysis demonstrated that the overexpression of OfEXPA2 and OfEXPA4 in tobacco plants resulted in an increase in stem diameter and enlargement of parenchyma cells in the stem cortex and pith. Furthermore, significant changes were observed in the corolla morphology of tobacco plants overexpressing OfEXPA2 and OfEXPA4, including an enlargement of petal epidermal cells. Remarkably, the petal cells of tobacco plants overexpressing 35SproOfEXPA4 exhibited atrophy. Both transgenic plants also resulted in a decrease in hemicellulose content in tobacco petals. Additionally, we have discovered a nuclear-localized MYB transcription factor, which can bind to the promoters of OfEXPA2 and OfEXPA4 , promoting their expression with a more pronounced effect on OfEXPA2 . The transient transformation of sweet osmanthus flower buds and calli with OfMYB5 resulted in a significant upregulation of OfEXPA2 and OfEXPA4 . In conclusion, this study establishes a theoretical framework for the functionality of expansin genes and lays a solid foundation for investigating the mechanism underlying flower opening of sweet osmanthus. OfMYB5 can regulate OfEXPA2 and OfEXPA4 to promote petal cell expansion represents the process of facilitation.