Objective This study systematically identified members of the SPX gene family of the fast-growing tree Eucalyptus grandis, and analysed the functional divergence and regulatory mechanism of this family in response to nitrogen and phosphorus stress. The aim was to elucidate the molecular basis of nutrient-stress adaptation in high-growth-rate plants and to provide theoretical basis for the study of the evolution and function of the nutrient metabolism of woody plants.

Method Bioinformatics analysis was conducted to characterize gene structure, phylogenetic relationships, and promoter cis-acting elements of EgSPX gene family members. Transcriptome sequencing and real-time PCR were combined to examine expression patterns of subfamily members under nitrogen deficiency and low phosphorus stress conditions.

Result A total of 21 EgSPX genes were identified, encoding proteins of 231 - 880 amino acids with predicted isoelectric points ranging from 4.89 to 9.35. The proteins were predominantly predicted to localize to the plasma membrane. Based on C-terminal domain composition, EgSPX genes were classified into four subfamilies: SPX, SPX-EXS, SPX-MFS and SPX-RING. The SPX-RING subfamily was conserved in Eucalyptus grandis, Arabidopsis thaliana, and rice, but absent in Populus trichocarpa. Collinearity analysis showed that EgSPX gene was highly homologous to Populus trichocarpa. Promoter analysis revealed enrichment of cis-elements associated with abiotic stress (e.g., low phosphorus) and tissue differentiation, especially with hormone signalling pathways such as gibberellin and jasmonic acid, suggesting that SPX may mediate the cross-regulation of phosphorus and hormone signals. Expression analysis showed that SPX gene expression was organ-differentiated, suggesting its functional specialisation in specific tissues (e.g. roots, flowers). Under nitrogen deficiency stress, EgSPX subfamily were generally upregulated, potentially acting to suppress key phosphate-starvation-response genes to prevent excessive phosphate uptake by roots and thereby maintain phosphate homeostasis. Most genes in the EgSPX-EXS and EgSPX-MFS families were up-regulated under low-phosphorus/nitrogen deficiency stress, among which EgSPX-MFS may be involved in phosphorus transmembrane translocation; and EgSPX-RING was down-regulated under both low phosphorus/nitrogen deficiency treatments, presumably through its negative regulation of nitrogen and phosphorus metabolic homeostasis.

Conclusion The SPX gene family in Eucalyptus grandis responds to nitrogen and phosphorus stress through subfamily functional differentiation, with expression patterns synergistically regulated by hormonal and stress-related signals. The results of this study provide new insights into the evolutionary adaptation and molecular mechanisms of SPX genes in woody plants and offer a foundation for SPX gene-based strategies in forest-tree improvement.