PoSCPL61 as a negative regulator of seed size in tree peony of Paeonia ostii revealing by genome-wide identification of SCPL genes and functional validation

Paeonia ostii ，a widely cultivated oil tree peony with notable medicinal, edible, horticultural and cultural values, encounters constrained cultivation expansion primarily due to its inherently low seed yield. Despite the critical roles of serine carboxypeptidase-like (SCPL) proteins in plant growth, development, and stress response, their characteristics remain unexplored in P. osti . In this study, we employed a genome-wide approach to identify 126 putative PoSCPL genes in P. ostii . We conducted a comprehensive analysis of their protein physicochemical properties, phylogeny, chromosomal distribution, colinearity, conserved motifs, gene structure, and cis-acting elements. Futhemor, we investigated the expression profiles of PoSCPL across eight tissues, six seed development stages, and under abiotic stresses (drought, flooding, and high temperature) unveiling a complex regulatory network orchestratiing seed development and stress responses. Notably, at least three -- PoSCPL108 , PoSCPL61 , PoSCPL8 --emerged as key players in seed development, displaying elevated expression during early stages followed by a gradual decline. We prioritized PoSCPL8 and PoSCPL61 for functional validation. Subcellular localization analysis revealed that PoSCPL8 localizes to peroxisomal and PoSCPL61 localize to the endoplasmic reticulum localization. Functional validation via overexpression in Arabidopsis thaliana demonstrated that PoSCPL61 induces premature flowering and produces wrinkled, smaller seeds with significantly reduced length, width and weight compared to the WT (wild type) and empty vector (EV) controls, alongside increased silique numbers. Additionally, in vivo functional validation using a virus-induced gene silencing (VIGS) system to suppress the expression of PoSCPL61 in P. ostii leaves and seeds showed that, after 21 days of VIGS treatment, both seed length and width had significantly increased by 19.2 % and 5.7 %, respectively, compared to the TRV2 controls. This study identified PoSCPL61 as a key negative regulator of influencing seed size and potentially other agronomically important traits, providing a genetic target for improving P. ostii seed yield. • We identified 126 SCPL genes in the Paeonia ostii genome and analyzed them systematically. • PoSCPL61 , PoSCPL8 and PoSCPL108 are highly expressed during early seed development. • Overexpression of PoSCPL61 led to production of smaller, wrinkled seeds with reduced size and weight, but more siliques. • Silencing PoSCPL61 in P. ostii led to fuller seeds, increased seed length and width.