Genetic transformation of Pinus elliottii: Association of SPX3 with coordinated nitrogen-phosphorus metabolism and antioxidant defense

The lengthy breeding cycle of Pinus elliottii Engelm., an economically important conifer widely cultivated in southern China, and the inherently low transformation efficiency of conifers present major obstacles to genetic improvement. To address these challenges, an Agrobacterium tumefaciens -mediated transformation protocol was developed using embryogenic calli as explants, with systematic optimization of pre-culture duration, co-cultivation period, bacterial concentration and infection time. This optimized protocol achieved an efficiency of 8.40 regenerated transgenic callus number per gram fresh weight (RGN/g). Integration and stable expression of the SPX domain-containing protein 3 ( SPX3 ) gene in transgenic lines were confirmed by green fluorescent protein (GFP) fluorescence, polymerase chain reaction (PCR), quantitative real-time PCR (qRT-PCR), and western blotting. Physiological and biochemical analyses demonstrated that SPX3 overexpression coordinately enhanced nitrogen and phosphorus metabolism, increased antioxidant enzyme activities and growth-promoting hormone levels, and reduced stress hormone contents and oxidative damage markers. Transcriptomic profiling revealed that differentially expressed genes were significantly enriched in plant hormone signal transduction, phenylpropanoid biosynthesis, and flavonoid biosynthesis pathways, and metabolomic analysis further identified differential accumulation of metabolites associated with the phenylpropanoid and flavonoid pathways. Collectively, this study established an efficient and stable genetic transformation system for P. elliottii and revealed a regulatory mechanism whereby SPX3 coordinates synergistic nitrogen and phosphorus metabolism with the activation of the phenylpropanoid-flavonoid pathway, thereby strengthening antioxidant defense and remodeling hormonal balance. These findings provide a technical platform and theoretical foundation for functional gene research and genetic improvement of stress tolerance in conifers.