Abstract
Phospholipase D (PLD) is a crucial enzyme hydrolyzing phospholipids to produce lipid messengers, which play pivotal roles in plant growth and adaptation to environmental stresses. However, a comprehensive characterization of the PLD gene family in soybean (Glycine max), particularly its functional relevance to nutrient deficiencies, remains limited. We identified 25 GmPLD genes in the soybean genome, all containing the conserved HKD catalytic domain. Phylogenetic analysis classified them into seven subfamilies, including two novelly identified φ subtypes (GmPLDφ1/2). Expression profiling revealed tissue-specific patterns, with certain genes highly expressed in reproductive organs, and single-cell RNA-seq further unveiled their spatial expression heterogeneity. Under abiotic stresses, distinct expression dynamics were observed: 11 genes responded to low phosphorus; 13 to drought; all members were significantly up-regulated under low nitrogen; and salt stress induced a complex tissue-specific response. Notably, haplotype analysis of 11 low phosphorus-responsive GmPLD genes revealed superior haplotypes with significant advantages in phosphorus efficiency-related traits. Furthermore, we experimentally confirmed that both GmPLDφ1 and GmPLDφ2 possess enzymatic activities related to phospholipase D function. Our study provides a systematic analysis of the GmPLD family, demonstrating its functional diversification in soybean development and adaptation to multiple abiotic stresses. The findings offer fundamental resources for future functional studies and molecular breeding aimed at enhancing soybean stress resilience.