The mechanical strength of inflorescence stems is an important trait in cut flowers. Calcium ions (Ca²⁺) play a pivotal role in maintaining stem strength, but little is known about the underlying molecular mechanisms. In this study, we treated herbaceous peony (Paeonia lactiflora Pall.) with ethyl glycol tetraacetic acid (EGTA), an effective Ca²⁺ chelator, and used morphology indicators, spectroscopic analysis, histochemical staining, electron microscopy, and proteomic techniques to investigate the role of Ca²⁺ in inflorescence stem mechanical strength. The EGTA treatment reduced the mechanical strength of inflorescence stems, triggered the loss of Ca²⁺ from cell walls, and reduced lignin in thickened secondary walls in xylem cells as determined by spectroscopic analysis and histochemical staining. Electron microscopy showed that the EGTA treatment also resulted in significantly fewer xylem cell layers with thickened secondary walls as well as in reducing the thickness of these secondary walls. The proteomic analysis showed 1065 differentially expressed proteins (DEPs) at the full-flowering stage (S4). By overlapping the Kyoto encyclopedia of genes and genomes (KEGG) and gene ontology (GO) analysis results, we identified 43 DEPs involved in signal transduction, transport, energy metabolism, carbohydrate metabolism, and secondary metabolite biosynthesis. Using quantitative real-time polymerase chain reaction (qRT-PCR) analysis, we showed that EGTA treatment inhibited Ca²⁺ sensors and secondary wall biosynthesis-related genes. Our findings revealed that EGTA treatment reduced the inflorescence stem mechanical strength by reducing lignin deposition in xylem cells through altering the expression of genes involved in Ca²⁺ binding and secondary wall biosynthesis.

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