Exogenous γ-aminobutyric acid promotes 2-acetyl-1-pyrroline biosynthesis by enriching glutamate and optimizing its metabolic flux in pumpkin

Aroma is a key quality trait determining the flavor and market competitiveness of pumpkins. Its characteristic fragrance mainly comes from 2-acetyl-1-pyrroline (2-AP), whose biosynthesis shares the common precursor with γ-aminobutyric acid (GABA) metabolic pathway. However, it remains unclear whether and how exogenous GABA regulates 2-AP synthesis in pumpkin. Given that the 2-AP biosynthetic pathway is highly conserved across various plant tissues, understanding its regulatory mechanisms in leaves provides a fundamental framework for improving fruit aroma quality. This study investigated the effects of exogenous GABA on 2-AP biosynthesis in pumpkin seedlings and elucidated the underlying regulatory mechanisms by analyzing key precursors, metabolic enzyme activities, and related gene expression. Foliar application of 500 mg L⁻¹ GABA significantly increased the 2-AP content in pumpkin leaves by 72.4%, and promoted seedling growth, with leaf area and fresh weight increasing by 57.7% and 58.4%, respectively. GABA treatment markedly elevated glutamate (Glu) content by 7.8-fold and reduced proline (Pro) levels by 13.6% at 30 h. Furthermore, GABA application enhanced the activities and gene expression of Δ¹-pyrroline-5-carboxylate synthase (P5CS) and proline dehydrogenase (ProDH), while suppressing those of betaine aldehyde dehydrogenase (BADH), thereby coordinately directing metabolic flux toward 2-AP synthesis. These findings demonstrate that exogenous GABA effectively promotes 2-AP biosynthesis in pumpkin by enriching the Glu pool and coordinately regulating key enzymes to suppress competitive metabolic pathways. This study provides a theoretical foundation for utilizing GABA as a biostimulant to improve aroma quality in pumpkin.