Exogenous IAA-Associated miRN36-PlMYB33 Module Regulates Flowering Time in Paeonia lactiflora

Paeonia lactiflora Pall. is an important cut-flower crop whose flowering time directly determines commercial value. However, flowering regulation in herbaceous peony remains incompletely understood because it involves coordinated hormonal, genetic and environmental signals. We investigated an auxin-associated miRN36-PlMYB33 module involved in flowering-time regulation. A 70-nt miRN36 precursor and the full-length PlMYB33 coding sequence (1509 bp) were cloned from herbaceous peony, and overexpression vectors were constructed. Phylogenetic analysis placed PlMYB33 closest to grape VvMYB33. Degradome sequencing and dual-luciferase assays supported miRN36-mediated cleavage of PlMYB33 transcripts. Exogenous IAA increased miRN36 expression, reduced PlMYB33 expression, delayed flower-bud development and reduced buds, whereas the auxin transport inhibitor NPA showed the opposite expression pattern. These results suggest that auxin-related signals may act upstream of the miRN36-PlMYB33 module, although direct transcriptional regulation of miRN36 by auxin signaling requires further validation. Subcellular localization indicated that PlMYB33 is nuclear. Heterologous overexpression of PlMYB33 in Arabidopsis produced stable transgenic lines that flowered earlier than wild type. RNA-seq identified 2024 upregulated and 417 downregulated genes; the upregulated genes were enriched in circadian rhythm, hormone signaling, MAPK cascades and flavonoid biosynthesis. Flowering-related genes, including FT and SOC1, showed increased expression in PlMYB33-overexpressing Arabidopsis, suggesting that PlMYB33 is associated with flowering-promoting pathways. In herbaceous peony, TRV-mediated silencing of PlMYB33 and TRV-based transient overexpression of miRN36 both delayed flowering. Together, these results support a model in which auxin-associated miRN36 represses PlMYB33 to modulate flowering time in herbaceous peony. This work provides candidate molecular targets for flowering-time regulation and breeding in ornamental crops.