Abstract: Crop yield is largely determined by the size of harvestable organs and understanding the mechanisms that govern organ size is therefore crucial for improving crop productivity. CYP78As are a plant‐specific subfamily of cytochrome P450 enzymes that have been identified as organ size regulators and are expressed in various plant organs. We previously identified BnaA09.CYP78A9, whose expression is markedly upregulated in long‐silique cultivars by a CACTA‐like transposable element (CTE) insertion in the regulatory region, acts as a pleiotropic regulator of yield‐related traits in long‐silique rapeseed varieties. Here, we show that BnaA09.CYP78A9^+CTE is expressed predominantly in the siliques and seeds of rapeseed cultivar ZS11. cyp78a9‐CRISPR knockout mutants exhibited reductions in silique length, seed size, and seed number per silique. Enzyme assays revealed that BnaA09.CYP78A9 converts lauric acid (LA) to 12‐hydroxylauric acid (12‐HOLA), and endogenous metabolite quantification revealed that 12‐HOLA levels were 5.01‐fold greater in long‐silique materials versus short‐silique materials. Application of exogenous 12‐HOLA significantly increased silique/pod elongation and seed weight, enhancing yield per plant by 32.77% in Arabidopsis and yield per unit area by 7.91%–30.82% in rapeseed and 30.14% in soybean compared with controls, respectively. 12‐HOLA application also stimulated fruit expansion in horticultural crops, increasing fresh fruit weight by 20.64%–22.96% in cucumber and 11.92%–24.13% in tomato. Transcriptome analyses revealed that 12‐HOLA treatment upregulated the expression of genes involved in auxin biosynthesis, transport, and signaling. 12‐HOLA treatment not only rapidly activated numerous transcription factors but also significantly promoted carbon metabolism within rapeseed siliques. Our study provides strong evidence that 12‐HOLA produced by BnaA09.CYP78A9 activates the auxin pathway, promotes cell elongation, and increases fruit size and seed weight. These findings highlight the potential use of 12‐HOLA as a natural plant growth regulator and CYP78A9 as a target for gene editing and molecular breeding.

Key words: 12‐hydroxy lauric acid, auxin, Brassica napus, CYP78A9, organ development, silique length