Genome-wide identification of the PLD gene family and its response to multiple abiotic stresses in soybean (Glycine max)

Genome-wide identification of the PLD gene family and its response to multiple abiotic stresses in soybean (Glycine max) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genome-wide identification of the PLD gene family and its response to multiple abiotic stresses in soybean (Glycine max) Mengshi He, Mengjun Xu, Fang Wang, Huifang Zuo, Lina Zhang, Yifei Yang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7469438/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Dec, 2025 Read the published version in BMC Plant Biology → Version 1 posted 10 You are reading this latest preprint version 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. soybean GmPLD phosphorus deficiency drought stress low nitrogen stress salt stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 1. Introduction Phospholipase D (PLD) is an enzyme that catalyzes the hydrolysis of phosphodiester bonds and facilitates base exchange reactions. The hydrolysis product of PLD, phosphatidic acid (PA), functions as a second messenger and plays a crucial role in the signal transduction processes within plant cells [ 1 ]. As the predominant phospholipase in plants, PLD is widely distributed across various tissues, including seeds, fruits, roots, leaves, and stems. Its expression is notably elevated during critical phases of plant growth and development as well as metabolic processes such as seed development and maturation, along with seedling germination [ 2 , 3 ]. The C-terminus of the protein structure of plant PLD contains two highly conserved HKD (HxKxxxxD) structural domains, with H being histidine, K being lysine, D being aspartic acid, and x being a non-conserved arbitrary amino acid [ 4 ]. These two HKD domains are separated by approximately 320 amino acids [ 5 ]. In plants, site-directed mutagenesis experiments targeting amino acid residues have demonstrated that the HKD residues are essential for the enzymatic activity of PLD [ 6 – 8 ]. The two HKD domains interact to form the catalytic active site of PLD, making them indispensable for its function [ 9 ]. In addition to these two HKD structural domains, some plant PLDs contain a highly conserved C2 structural domain at the N-terminus of the protein structure. The C2 structural domain is composed of approximately 130 amino acids, and it is a Ca 2+ -dependent phospholipid-binding domain, so PLDs containing the C2 structural domain require a certain concentration of Ca 2+ in order to exert their maximal enzymatic activity [ 10 ]. These PLD genes are known as C2-PLD s. Some plant PLD genes contain PX/PH domains and are thus classified as PX/PH-PLDs . The PX domain exhibits the ability to bind phosphatidylinositol [ 11 ], while the PH domain interacts with various phosphatidylinositol phosphates [ 12 ]. A class of PLD genes in plants, termed SP-PLDs , lack both C2 and PX/PH domains but contain a signal peptide at the N-terminus [ 13 ]. Plant PLD s are classified into seven distinct types: α, β, γ, δ, ε, ζ, and φ. Among these, PLDα , PLDβ , PLDγ , PLDδ , and PLD ε are categorized as C2-PLD s; PLDζ is classified as a PX/PH-PLD ; and PLDφ is identified as an SP-PLD . PLD plays a crucial role in the growth and development of plants, regulating processes such as seed germination, seedling growth, root hair development, pollen tube germination, apical growth, senescence, and the plant's response to abscisic acid (ABA) [ 14 – 17 ]. PLD can influence the quality, yield, and vigor of seeds by modulating the content of glycerolipid fractions in plants. Additionally, PLD is involved in regulating plant growth and development under conditions of nutrient deficiency. For instance, in Arabidopsis thaliana , PA derived from PLD interacts with ABI1 (a protein phosphatase 2C), a key negative regulator of the ABA signaling pathway, to modulate ABA signal transduction, thereby affecting seed germination and seedling growth [ 18 ]; PLDα1 and its product, PA, may influence the accumulation of Ca 2+ at the tip of root hairs by mediating the generation of reactive oxygen species (ROS) in that region [ 19 ], and this process subsequently regulates the growth of Arabidopsis root hairs. Overexpression of TaPLDδ in wheat resulting in an earlier heading and flowering. This alteration leads to an expedited transition into the reproductive growth phase and also enhances the plant's antioxidant capacity [ 20 ]. The overexpression of soybean GmPLDγ in A. thaliana can alter glycerolipid metabolism, thereby promoting the synthesis of oils rich in unsaturated fatty acids and long-chain fatty acids in seeds [ 21 ]. PLD can also respond to various abiotic stresses, including salt, low-temperature, drought, low phosphorus, low-potassium, mechanical injury, heavy metal, disease pressure, and osmotic stress. Currently, the functions of numerous PLD genes in higher plants have been identified [ 22 – 25 ]. In A. thaliana , PLDα1/PLDδ and their hydrolysis product PA positively regulate potassium uptake in the root system during low potassium stress responses. Following low potassium treatment, the enzymatic activity of PLDα1/PLDδ increases, leading to the generation of PA signals [ 26 ]. Additionally, PLDζ2 enhances root hair growth by promoting sugar-lipid synthesis and lipid remodeling, thereby improving the plant's adaptability to phosphorus deficiency [ 27 ]. In rice, OsPLDζ1 plays a positive regulatory role under salt stress conditions. The absence of OsPLDζ1 leads to impaired plant growth and reduced plant height in response to salt stress. Conversely, OsPLDα3 exhibits a negative regulatory effect during the response to salt stress [ 28 ]. In soybean, overexpression of GmPLDγ enhances seed germination rate and accelerates germination under high-salinity stress conditions [ 29 ]. When winter wheat subjected to drought stress, the growth of seedling leaves was inhibited after the addition of the PLD inhibitor butylated hydroxytoluene (n-butanol, BA) compared to the control. The levels of malondialdehyde (MDA), a product of membrane lipid peroxidation, increased, while the activity of antioxidant enzyme peroxidase (POD) decreased. These findings indicate that PLD is involved in regulating POD activity under drought stress conditions [ 30 ]. In Chorispora bungeana , CbPLDα , CbPLDβ , and CbPLDδ are involved in the plant's response to low-temperature stress, enhancing the cold resistance of tobacco seedlings [ 31 , 32 ]. However, there are currently relatively few reports on PLD genes in soybean ( Glycine max ). Therefore, it is essential to investigate the GmPLD gene family in this crop. Zhao et al. identified 18 GmPLD genes based on the W82.v1 genome and analyzed their expression patterns in various tissues and under salt stress conditions [ 33 ]. We aim to investigate the role of the GmPLD gene family in different abiotic stresses and explore whether the number of genes in the GmPLD gene family has changed with the update of the genome. Utilizing transcriptome data and qRT-PCR experiment from soybeans subjected to phosphorus and nitrogen deficiency, drought stress, and salt stress, we observed significant differences in the expression levels of certain GmPLD genes under such conditions. Building upon this observation, we initiated a comprehensive study of the GmPLD gene family. In this research, we employed bioinformatics techniques to identify 25 GmPLD genes and conducted analyses on their gene structures, three-dimensional protein conformations, tissue-specific expression patterns, and responses to abiotic stressors. Through these investigations, we aim to enhance our understanding of the functional roles played by the GmPLD gene family. This knowledge will facilitate more effective utilization of GmPLD s in soybean cultivation and contribute to increased soybean yields. 2. Materials and methods 2.1. Identification and characterization of PLD genes in soybean Using the soybean-specific database SoyBase ( https://www.soybase.org/ ; accessed February 1, 2024), the GmPLD gene family members were screened by comprehensive analysis of the W82.v2 reference genome. Subsequently, the domain analysis of the selected genes was performed using the SMART online tool ( http://smart.embl-heidelberg.de/ ; accessed February 28, 2024) to identify the members of the GmPLD gene family. Corresponding NCBI accession numbers and complete protein sequences were retrieved for each identified gene.Based on the obtained login number, UniProtKB of GmPLD gene family was obtained on Pfam database ( https://www.uniprot.org/ ; accessed March 16, 2024). Based on the obtained protein sequences and UniProtKB, the protein sequences were analysed using Expasy-ProtParam tool online analysis website ( https://web.expasy.org/protparam/ ; accessed March 16, 2024)to obtain the protein primary structure prediction results of amino acid length, relative molecular mass, isoelectric point and average hydrophobicity of the GmPLD gene family [ 34 ]; and the Expasy-ProtScale ( https://web.expasy.org/protscale/ ; accessed March 18, 2024) online analysis website to obtain the hydrophobicity of GmPLD genes [ 34 ]. The protein sequences were used for subcellular localisation analysis of the GmPLD genes on the PSORT online analysis website ( https://wolfpsort.hgc.jp/ ; accessed March 21, 2024) and Cell-PLoc 2.0 online prediction website ( http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/ ; accessed March 22, 2024) [ 35 ]. 2.2. Phylogenetic trees of the PLD gene family Based on the obtained protein sequences of AtPLD , OsPLD , and GmPLD genes, we constructed a maximum likelihood (ML) phylogenetic tree of the PLD gene family using TBtools software [ 36 ]. Then the phylogenetic tree was visualized and optimized by using iTOL online tool ( https://itol.embl.de/ ; accessed April 16, 2024). 2.3. Chromosome localisation and covariance analysis of GmPLD genes The chromosomal location of the GmPLD genes and the length of each chromosome of soybean obtained from the soybean database SoyBase ( https://www.soybase.org/ ; accessed March 17, 2024), and these data were used to generate the chromosomal localization map of GmPLD genes using MapChart software. The genome annotation file and genome sequence of soybean cultivar Wm82 were obtained from NCBI ( https://www.ncbi.nlm.nih.gov/ ; accessed March 17, 2024). After data processing, TBtools software [ 36 ] was employed to calculate the length of each soybean chromosome, determine chromosome gene density, and analyze the collinearity relationships within the GmPLD gene family; these results were then visualized using TBtools. 2.4. Pan-genomic analysis of the GmPLD family The genomic data of the GmPLD genes in 29 soybean accessions, including Williams 82 (W82), ZhongHuang (ZH13), and W05, were retrieved from the SoyOmics database ( https://ngdc.cncb.ac.cn/soyomics/index ; accessed September 6, 2024) to analyse the number of distinct PLD subfamilies present in these materials [ 37 , 38 ]. 2.5. Sequence comparison and structural domain analysis of GmPLD genes Using NCBI database ( https://www.ncbi.nlm.nih.gov/ ; accessed February 23, 2024) and Expasy-ProtParam online tool ( https://web.expasy.org/protparam/ ; accessed February 23, 2024) the obtained GmPLD genes protein sequences were used to identify their domains, so as to obtain the conserved structural domains of GmPLD gene family proteins. The conserved domains of the GmPLD gene family proteins were determined by ClustalX software, and then the sequence alignment of these conserved domains of the GmPLD genes was carried out by using ClustalX software. Based on the protein sequences of the GmPLD genes, the structural domains were analysed on the SMART online analysis website ( http://smart.embl-heidelberg.de/ ; accessed February 28, 2024) characterize the domains of the GmPLD genes, employing default parameters (E-value < 0.1). Then the domain architectures of GmPLD family proteins were plotted with the help of TBtools software [ 36 ]. 2.6. Characterization of gene structures and conserved motif distributions of GmPLD genes The genome annotation files of the GmPLD family were retrieved using NCBI ( https://www.ncbi.nlm.nih.gov/ ; accessed March 17, 2024). Using TBtools software [ 36 ], gene structure analysis of the GmPLD family was then conducted to map the CDS-UTR structure of the GmPLD gene family. Based on the acquired GmPLD protein sequences, motif analysis was performed via the MEME online tool ( https://meme-suite.org/meme/ ; accessed May 3, 2024), with the maximum number of motifs set to 7. 2.7. The 3D structure prediction of GmPLD proteins Based on the protein sequences of GmPLD genes, the three-dimensional structure were predicted using the SWISS-MODEI website ( https://swissmodel.expasy.org/ ; accessed August 28, 2024). Via the automated template screening system, high-confidence prediction models with a Global Model Quality Estimation (GMQE) score > 0.75 were rigorously selected for subsequent analyses. 2.8. Promoter cis-regulatory elements of the GmPLD genes A 2000 bp sequence upstream of the initiation codon (ATG) of GmPLD genes was retrieved from the Phytozome13 database ( https://phytozome-next.jgi.doe.gov/ ; accessed April 23, 2024). This sequence was analyzed using the PlantCARE promoter analysis tool ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ; accessed April 23, 2024) to predict cis-acting elements in the GmPLD gene promoter regions, and the results were subsequently visualized using TBtools software [ 36 ]. 2.9. Tissue expression pattern of the GmPLD genes The transcriptome data of GmPLD genes in different tissues of Williams 82 at different developmental periods were extracted from SoyOmics database ( https://ngdc.cncb.ac.cn/soyomics/transcriptome/tissues ; accessed September 5, 2024), and the expression pattern of GmPLD genes in different tissues of Williams 82 at different developmental periods was analysed [ 37 ]. These included root (root), cotyledon (cotyledon-1), leadbud (leafbud-1) and hypocotyl (stem-1) at the 5-day emergence stage (VE5); and cotyledon (cotyledon-2), leadbud (leafbud-2), leaf (leaf-1) and hypocotyl (stem-2) at the cotyledon stage (VC); Leadbud (leafbud-3), compound leaf (leaf-2), flower (flower-1), and lateral bud (axillary_bud) at the three-node stage (V3); compound leaf (leaf-3) at the tenth week of the growth period; Flowers prior to anthesis (flower-2) as well as flowers on day 0 of anthesis (flower-3), flowers on day 5 (flower-4), and wilted flowers (flower-5); Pods and seeds in the second week of seed development (pod & seed-1), pods (pod-1) and pods and seeds in the third week (pod & seed-2), pods (pod-2) and seeds (seed-1), pods and seeds(pod & seed-3) in the fourth week, pods (pod-3) and seeds (seed-2) in the fifth week, seeds (seed-3) in the sixth week, seeds (seed-4) in the eighth week, and seeds (seed-5) in the tenth week. The expression pattern of GmPLD genes in different tissues of Williams 82 at different developmental periods was analysed, and a heat map of GmPLD genes expression was drawn based on Log2-normalized FPKM values using TBtools software [ 36 ]. 2.10. Single-cell RNA sequencing of the GmPLD genes Using the Soybean Multi-Omic Atlas online database ( https://soybean-atlas.com/ ; accessed September 13, 2024), single-cell RNA sequencing (scRNA-seq) data were selected to examine the expression levels of different genes in cotyledon-stage seeds, a specific tissue region [ 39 ]. Additionally, via the SoyOmics database ( https://ngdc.cncb.ac.cn/soyomics/transcriptome ; accessed March 15, 2025), we analyzed the expression profiles of GmPLD genes in various regions of nodules, roots, stems, leaves, and shoot apical meristems (SAM) of soybean cultivar ZH 13. These analyses revealed the tissue-specific and region-specific expression patterns of GmPLD genes, further clarifying their potential roles in plant growth, development, and stress responses [ 40 ]. 2.11. Abiotic stress-induced expression of the GmPLD genes Using FPKM values from RNA-seq data of published studies[ 41 ], we obtained the expression profiles of GmPLD genes in leaves and roots of W82 under LP stress, as well as their expression profiles in W82 subjected to drought treatments (8% and 10% soil water content) and corresponding control treatments over the same time periods. Using TBtools software, based on log2-standardized FPKM values, the Heatmaps of GmPLD genes expression in leaves and roots of W82 under low phosphorus stress and W82 under 8% and 10% drought conditions (including their respective concurrent control) were generated [ 36 ]. 2.12. qRT-PCR analysis Roots, stems, cotyledons, and leaves of W82 (provided by Nanjing Agricultural University) were collected on the 20th day of its growth cycle; pods and seeds were collected at the 9th week of growth. Root samples of Nannong 94–156 hydroponically cultured for 9 days under different phosphorus levels were collected, including LP treatment (5 µM Pi) and NP treatment (500 µM Pi, NP). Root samples subjected to drought treatment for 24 hours were collected, including the control group (non-drought treatment) and the 20% PEG-simulated drought treatment group. Root samples of W82 hydroponically cultured for 5 days under different nitrogen levels were collected, including LN treatment (1 mM KNO₃) and NN treatment (7.5 mM KNO₃). For salt stress treatment samples, 150 mM KCl was used as the control, and 150 mM NaCl was used as the salt stress treatment; roots and leaves were collected separately after 48 hours of treatment. All materials were set with 3 biological replicates, and each biological replicate included 3 technical replicates. Total RNA was extracted using the RNA extraction kit (TianGen, DP419), and the RNA was reverse transcribed into first-strand cDNA using the reverse transcription kit (Yisheng, NO. 11123ES) [ 42 ]. Based on the cDNA sequences of GmPLDϕ1 and GmPLDϕ2 , specific primers were designed using Primer 5.0 (Table S11). The internal reference gene is Tubulin (GenBank accession number AY907703) [ 43 ]. The total reaction volume for qRT-PCR is 20.0 µL, including 1 µL of cDNA, 10.0 µL of 2 × FAST SYBR Mix, 1 µL each of the forward and reverse primers (10 µmol·L − 1 ), and 7 µL of RNase-Free H 2 O. qRT-PCR was performed on the CFX96 Touch system with the following reaction program: 2 min of pre-denaturation at 94 ℃; 40 cycles of 20 s denaturation at 95℃, 30 s annealing at 56℃, and 40 s extension at 72 ℃. The relative expression levels of the genes were calculated by the 2 −ΔΔCt method [ 44 ], and the average value and standard deviation of each sample were calculated. Subsequently, the data were visualized using GraphPad Prism 8 software. The significant differences between the two datasets were analyzed using the independent samples t-test. 2.13. Haplotype analysis The genotype data and plant materials utilized in this study were obtained from the research by Lu et al. [ 45 ], encompassing a total of 559 soybean accessions. These accessions are categorized into 121 wild soybeans, 207 landraces, and 231 cultivated soybeans​. For data analysis, haplotype analysis of the GmPLD gene was conducted using TASSEL 5.0 software to identify marker-trait associations [ 46 , 47 ], with the significance threshold set at p ≤ 0.05. Concurrently, Haploview 4.1 software was employed to analyze the haplotypes of 11 genes. In addition, during the preliminary laboratory work, 376 out of the 559 soybean materials were subjected to different phosphorus level treatments. Traits such as RRA, RPC, RRV, RRL, RRN, and RPAE were measured, with each line replicated three times [ 41 ]. Furthermore, the distribution of these 559 materials across different ecological regions was clarified. Subsequently, the data were visualized using GraphPad Prism 8 software, and the phenotypic differences corresponding to different haplotypes were analyzed by letter labeling method. 2.14. Identification of GmPLDϕ1 and GmPLDϕ2 enzyme activities The target vector pCold was digested with restriction endonucleases BamHI and SacI to obtain the linearised vector pCold, and the CDS of GmPLDϕ1 and GmPLDϕ2 were ligated to the linearised vector pCold at full length, and then their recombinant plasmids were transferred into BL21 sensory state. The BL21 sensory state of GmPLDϕ1 -Pcold and GmPLDϕ2 -Pcold was shaken to OD600 = 0.6. The number of cells of BL21 sensory bacillus solution of GmPLDϕ1 -Pcold and GmPLDϕ2 -Pcold with OD600 = 0.6 was calculated according to the method of dilution coated plate [ 48 ]. Finally, the phospholipase D activity of GmPLDϕ1 and GmPLDϕ2 was detected according to the PhospholipaseD (PLD) Activity Assay Kit (Suzhou Grace Biotechnology Co., Ltd., Item No. G0925W). 3. Results 3.1. Identification and characterization of PLD genes in soybean A total of 39 putative GmPLD genes were identified through genome-wide screening of the soybean W82.v2 reference genome assembly. Among these, 9 genes lacked both C2 or PX/PH structural domains and HKD structural domains. 2 genes contained only C2 structural domains, while 3 genes possessed a single HKD structural domain. The remaining 25 genes all exhibited two highly conserved HKD structural domains. Chen et al. (2012) [ 33 ] identified 18 GmPLD genes in the soybean W82.v1 genome assembly, whereas our study detected 7 additional GmPLD genes, totaling 25. This increase in gene number likely reflects improved gene annotation and assembly quality in the updated genome version. Based on the analysis of the structural domains of the GmPLD gene family, along with previously classified GmPLD genes [ 33 ], evolutionary relationships, and chromosomal locations, the 25 members of the GmPLD gene family are designated as GmPLDα1 to GmPLDα6 , GmPLDβ1 to GmPLDβ4 , GmPLDδ1 to GmPLDδ6 , GmPLDε1 to GmPLDε3 , GmPLDγ1 , GmPLDζ1 to GmPLDζ3 , and GmPLDφ1 to GmPLDφ2 (Table 1 ). The 25 GmPLD genes are distributed across 14 chromosomes (Fig. S1 ). The amino acid lengths of the GmPLD gene family, based on protein sequences queried from the Expasy online analysis website, ranged from 519 to 1126. The isoelectric points varied between 5.48 and 7.62, while the relative molecular masses ranged from 58,551.85 to 128,451.94 Da, with an average of 97,455.72 Da. The average hydrophobicity values for this gene family ranged from − 0.532 to -0.202. The subcellular localization prediction of the 25 members of the GmPLD gene family showed that 10 genes were located in the nucleus, 6 genes were located in the cytoplasm, 7 genes were located in the chloroplasts, 1 gene was located in the cytoskeleton, and 1 gene was located in the vesicles. Table 1 The characteristics of members of GmPLD gene family Gene ID Gene Name Chromosome location Size (aa) MW (kDa) pI Average hydrophobicity Subcellular localization Glyma.08g211700 GmPLDα1 Gm08: 17098305...17101929 788 89300.01 5.49 -0.409 Cytoplasm Glyma.07g031100 GmPLDα2 Gm07: 2461423...2465693 809 91550.6 5.48 -0.384 Cytoplasm Glyma.13g364900 GmPLDα3 Gm13: 45119805...45125074 807 92078.64 5.9 -0.398 Cytoplasm Glyma.06g068600 GmPLDα4 Gm06: 5257871...5262176 826 94258.93 5.77 -0.404 Nucleus Glyma.06g068700 GmPLDα5 Gm06: 5264111...5268817 821 93469.59 6.16 -0.389 Chloroplasts Glyma.15g008500 GmPLDα6 Gm15: 679168...684356 711 81216.42 6.01 -0.374 Cytoplasm Glyma.18g288600 GmPLDβ1 Gm18: 56840393...56849007 1097 123221.67 6.64 -0.532 Chloroplasts Glyma.02g093500 GmPLDβ2 Gm02: 8320963...8329508 1106 124092.7 6.63 -0.509 Chloroplasts Glyma.07g080400 GmPLDβ3 Gm07: 7312327...7319873 1047 117361.38 6.63 -0.482 Chloroplasts Glyma.03g018900 GmPLDβ4 Gm03: 1886324...1892076 759 85047.12 6.72 -0.422 Cytoskeleton Glyma.01g215100 GmPLDγ1 Gm01: 54579928...54585779 853 96097.42 7.65 -0.381 Chloroplasts Glyma.11g081500 GmPLDδ1 Gm11: 6113534...6121086 866 98442.89 6.56 -0.422 Nucleus Glyma.01g162100 GmPLDδ2 Gm01:50010865...50019215 864 98119.54 6.56 -0.43 Nucleus Glyma.05g168300 GmPLDδ3 Gm05: 35880140...35891738 857 96970.6 7.24 -0.383 Nucleus Glyma.06g020500 GmPLDδ4 Gm06: 1546983...1555296 847 96410.7 7.09 -0.396 Nucleus Glyma.04g020400 GmPLDδ5 Gm04: 1603209...1610515 847 96095.22 6.94 -0.396 Chloroplasts Glyma.08g126700 GmPLDδ6 Gm08: 9760166...9769637 857 96935.73 7.22 -0.387 Nucleus Glyma.07g010900 GmPLDε1 Gm07: 830443...834361 769 88231.65 6.11 -0.422 Nucleus Glyma.15g023500 GmPLDε2 Gm15: 1855931...1859297 759 87080.18 6.75 -0.474 Cytoplasm Glyma.08g194100 GmPLDε3 Gm08: 15635256... 5639129 776 89339.89 6.23 -0.42 Nucleus Glyma.20g238000 GmPLDζ1 Gm20: 46972128...46987629 1120 128451.94 5.85 -0.413 Nucleus Glyma.15g152100 GmPLDζ2 Gm15: 12613312...12626308 1123 127634.88 6.28 -0.414 Nucleus Glyma.09g041400 GmPLDζ3 Gm09: 3462605...3475431 1126 127843.18 6.31 -0.407 Nucleus Glyma.04g060900 GmPLDφ1 Gm04: 4966912...4970792 519 58590.32 7.02 -0.202 Chloroplasts Glyma.06g061500 GmPLDφ2 Gm06: 4631416...4635351 520 58551.85 6.49 -0.273 Vesicles 3.2. Phylogenetic relationship, covariance, pan-genomic analysis of the GmPLD genes To gain a deeper understanding of the evolutionary relationships and functions of the GmPLD gene family, we constructed a phylogenetic tree (Fig. 1 A) by integrating the GmPLD genes with A. thaliana PLD genes (12) (Table S1 ) [ 11 ] and rice PLD genes (17) (Table S2) [ 49 ]. The evolutionary relationships are categorized into seven distinct clades. Among these, the PLDδ gene and rice OsPLDκ1 form one cluster, with OsPLDκ1 and OsPLDδ1 being the closest relatives. The PLDβ and PLDγ genes constitute a single clade; however, this clade can be further subdivided into two subcommunities based on the subclasses of PLD . The PLDα genes, with the exception of OsPLDα8 , constitute one clade; the OsPLDα8 and PLD ε genes form another clade; the PLDδ genes represent a distinct clade; the PLDζ genes comprise one clade; and the PLDφ genes are classified as another clade. According to the phylogenetic tree, it is evident that PLDα and PLDε are closely related, as are PLDβ and PLDγ . Therefore, we can infer that PLDα and PLDε share similar structures and functions, just as PLDβ and PLDγ do. Covariance analysis of the GmPLD genes across soybean species (Fig. 1 B) identified a total of 13 fragment duplication events involving the GmPLD genes. These include duplications between GmPLDα1 and GmPLDα4 , GmPLDα2 and GmPLDα4 , as well as GmPLDα4 and GmPLDα6 . Additionally, duplications were observed between GmPLDβ1 and GmPLDβ2 , GmPLDβ3 and GmPLDβ4 , along with pairs such as GmPLDδ1 and GmPLDδ2 ; GmPLDδ1 and GmPLDδ4 ; and finally, between both GmPLDδ1 and GmPLDδ5 ; as well as combinations of other gene pairs: GmPLDδ2 with GmPLDδ4 ; GmPLDδ2 with GmPLDδ5 ; GmPLDδ3 with GmPLDδ6 ; GmPLDδ4 with GmPLDδ5 ; as well as GmPLDφ1 in conjunction with GmPLDφ2 (Table S3). It is evident from this that the amplification of the GmPLD gene family is primarily driven by events of fragment duplication. We analyzed the GmPLD genes in 29 different type soybean accessions and found that the number of GmPLD genes varied from 17 to 25 (Table S4-5): the landrace SoyL01 has the fewest GmPLD genes, and the cultivated soybean Williams 82 has the most GmPLD genes (Fig. 1 C). 3.3. Structural Analysis of the GmPLD genes All GmPLD proteins possess two HKD domains, and the sequence must include two HxKxxxxD motifs. The HKD1 domain of GmPLDα, GmPLDβ/δ/γ, GmPLDε, and GmPLDζ/φ contains 39, 36, 38, and 28 amino acids, respectively. Except for the HKD2 domain of GmPLDφ, which contains 27 amino acids, the HKD2 domains of other GmPLD proteins all contain 28 amino acids (Fig. 2 A). Notably, the domains within the same subfamily exhibit significant sequence similarity. Based on the gene structure analysis diagram of GmPLD (Fig. 2 B), all GmPLD proteins contain two HKD domains. All members of the GmPLDα, GmPLDβ, GmPLDδ, GmPLDε, and GmPLDγ subfamilies possess C2 domains, except for GmPLDβ4 and GmPLDε3. Every GmPLDζ protein contains a PH domain in addition to the two HKD domains but lack the PX domain; in contrast, GmPLDφ proteins contain neither PH nor PX domains, being characterized solely by the two HKD domains. Notably, GmPLDβ and GmPLDζ proteins exhibit the longest amino acid sequences, while GmPLDφ proteins have the shortest. Motif analysis revealed that all GmPLDα/β/ε/δ proteins contain seven motifs, while GmPLDφ1 and GmPLDφ2 only harbor Motif 1. The three GmPLDζ proteins lack Motif 3 and Motif 6, and GmPLDβ2 is deficient in Motif 6 and Motif 7 (Fig. 2 C, Table S6). Subsequent gene structure analysis (Fig. 2 C) showed that most GmPLD genes have lengths exceeding 3 kb, except for GmPLDζ3 , which exceeds 15 kb. Regarding exon numbers: GmPLDε genes contain 4 exons, GmPLDα genes contain 3–6 exons, and GmPLDφ genes contain 7 exons; GmPLDβ , GmPLDγ1 , and other GmPLDγ genes contain 10 exons, with the exceptions of GmPLDβ4 (18 exons), GmPLDδ2 (8 exons), and GmPLDδ4 (9 exons); the three GmPLDζ genes contain 18, 20, and 21 exons, respectively (Fig. 2 C). Notably, among all subfamilies, GmPLDζ genes exhibit the highest number of exons. The 3D protein structures of GmPLD proteins were predicted using SWISS-MODEL and organized according to the evolutionary tree of their encoding genes. As shown in Fig. 3 , PLDα, PLDγ, PLDδ, and PLDε proteins exhibit similar 3D structures. In contrast, PLDβ and PLDζ proteins also share structural similarities. Notably, the 3D structures of GmPLDφ1 and GmPLDφ2 differ from those of all other subfamily members. Despite these variations among different subfamilies, all GmPLD proteins share a segment with an extremely conserved structure. This finding suggests that the overall 3D structure of GmPLD proteins is relatively conserved across different subfamilies. 3.4. Cis-acting element analysis of the GmPLD genes The cis-regulatory elements within the promoters of all GmPLD genes were analyzed utilizing the online software PlantCARE ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ). Based on the soybean genome information, we extracted the sequences 2000 bp upstream of the initiation codon (ATG) of all GmPLD genes and visualised the cis-regulatory elements using TBtools software (Fig. 4 ). The 17 most common elements were obtained in the promoter region of the GmPLD genes. These include hormone-related elements (e.g., growth hormone, abscisic acid, methyl jasmonate (MeJA), gibberellin, salicylic acid, and estrogen), adversity-related elements (e.g., drought response, wounding response, dehydration defense, and stress response), endosperm-expression elements, meristem expression elements, light-response elements, anaerobically-induced elements, inducer-mediated activation elements, and circadian regulation-related elements. Notably, light-responsive cis-elements are abundant in the promoter region of GmPLD genes (Table S7). 3.5. Expression pattern of the GmPLD genes To investigate the expression pattern of GmPLD genes in soybean across various developmental stages, transcriptome data for GmPLD genes from different tissues at distinct developmental periods in W82 were obtained from the SoyOmics database ( https://ngdc.cncb.ac.cn/soyomics/index ). The expression levels of GmPLD genes varied across different tissues and developmental stages in W82 (Fig. 5 A, Table S8). GmPLDα1 , GmPLDα2 , and GmPLDα3 exhibit high expression levels in various tissues at distinct developmental periods; GmPLDζ1 exhibits moderate expression levels in leaf-1/3, cotyledon-2, leaf bud-2, flower-3/4, and pod; GmPLDβ3 demonstrates intermediate expression levels in cotyledon-1, leafbud, flower-1, flower-2, seed-1, and axillary bud; both GmPLDδ1 and GmPLDδ2 show tissue-enriched expression levels in stem, leaf, bud, flower, pod & seed, pod and axillary bud; GmPLDγ1 has intermediate expression levels in cotyledon-2, leaf-1, leaf-2, and root. The remaining genes exhibit low expression levels across various tissues during different developmental stages2. At least one gene from each of the other subfamilies exhibit a relatively high expression level during one of the developmental stages with the exception of the GmPLDφ subfamily (Fig. 5 A). To investigate the expression differences of GmPLDφ genes across various tissues and developmental stages, we collected W82 samples from roots, stems, cotyledons, and leaves on the 20th day of the growth cycle, as well as pods and seeds at the 9th week of growth [ 50 ]. We assessed the tissue-specific expression levels, and the results indicate that both GmPLDφ1 and GmPLDφ2 display relatively high expression levels in seeds (Fig. 5 B). Based on the above results, we found that GmPLDα1 , GmPLDα2 , GmPLDα3 , GmPLDφ1 , and GmPLDφ2 exhibit high expression levels in seeds. Therefore, we used single-cell RNA sequencing data [ 39 ] to observe their expression levels in different parts of the cotyledon stage seed (Fig. 5 C). GmPLDα1 , GmPLDα2 , and GmPLDα3 are expressed in different parts of the cotyledon-stage seed, with relatively high expression levels. GmPLDφ1 is expressed in End peripheral, Emb vasculature, End micropylar, SC inner parenchyma, SC epidermis, and SC outer parenchyma, but the expression level is relatively low. GmPLDφ2 is expressed in SC hilum epidermis, End chalazal, Emb vasculature, End micropylar, Emb epidermis, SC inner parenchyma, SC outer parenchyma, and SC tracheid bar, with relatively low expression levels. To investigate the expression patterns of GmPLD genes across various tissues and regions, we performed expression analysis of GmPLD genes based on the single-cell RNA sequencing (scRNA-seq) data from SoyBase [ 40 ], the tissues including nodules, roots, stems, leaves, and shoot apical meristems (SAM) of Zhonghuang 13 (ZH13). The corresponding gene IDs of GmPLD genes in ZH13 are listed in Table S7. The results reveal that GmPLDα1 and GmPLDα3 exhibit high expression levels in nearly all regions of nodules, roots, stems, leaves, and SAM (Fig. 6 ). GmPLDδ1 shows moderate expression across these tissues, while GmPLDγ1 displays elevated expression in nodules and roots, particularly in the Cortex2-pre1 region of the roots. Additionally, GmPLDδ2 and GmPLDζ1 / 2 are highly expressed in nodules, with GmPLDζ1 / 2 showing particularly significant expression in Infected cell2. Notably, GmPLDα4 / 5 and GmPLDε1 / 2 / 3 exhibit minimal or negligible expression across all regions. The distinct expression patterns of GmPLD genes across various tissues and regions suggest their potential involvement in diverse biological functions. For instance, the high expression of GmPLDγ1 in the Cortex2-pre1 region of the roots indicates its possible role in specialized processes such as cell differentiation or responses to environmental signals. Similarly, the specific expression of GmPLDζ1 and GmPLDζ2 in Infected cell2 implies their potential participation in symbiotic processes, including membrane remodeling, signal transduction, or nutrient exchange between the host plant and symbiotic bacteria. These findings highlight the functional versatility of GmPLD genes in regulating both developmental and symbiotic mechanisms in plants. 3.6. Expression analysis of GmPLD genes under phosphorus-deficient conditions The expression profiles of GmPLD genes in the leaves and roots of W82 under low phosphorus stress were derived from FPKM values obtained through RNA-seq data from published studies [ 41 ] (Fig. 7 A, Table S9). This analysis aims to investigate the response of GmPLD genes to low phosphorus stress. Based on the thermograms presented for the expression of GmPLD genes in the leaves and root systems of W82 under normal phosphorus (NP, 500 µM phosphate) and low phosphorus (LP, 5 µM phosphate) stress, it is evident that among the 12 GmPLD genes— GmPLDβ1 , GmPLDβ2 , GmPLDδ1 , GmPLDδ2 , GmPLDδ5 , GmPLDε2 , GmPLDγ1 , GmPLDφ1 , GmPLDφ2 , GmPLDζ1 , GmPLDζ2 , and GmPLDζ3 —significant differences are observed between NP and LP treatment in roots. In contrast, no significant differences are detected for the remaining 13 GmPLD genes. We conducted quantitative real-time PCR (qRT-PCR) on 12 GmPLD genes, with the relevant primer sequences provided in Table S11. Using the soybean cultivar NanNong 94–156 as our experimental material, we subjected the plants to various phosphorus concentration treatments and extracted root RNA for qRT-PCR detection. The experimental results indicate that under LP conditions, the expression levels of GmPLDβ1 , GmPLDβ2 , GmPLDδ2 , GmPLDδ6 , GmPLDγ1 , GmPLDφ2 , GmPLDζ2 , and GmPLDζ3 are significantly lower compared to those observed under NP conditions. In contrast, the expression levels of GmPLDε2 , GmPLDφ1 , and GmPLDζ1 exhibit a notable increase (Fig. 7 B). These findings suggest that these 11 GmPLD genes may play crucial roles in plant responses to LP stress through mechanisms involving differential expression regulation. 3.7. Haplotype Analysis of 11 Phosphorus-Responsive GmPLD genes in Soybean Germplasm Using 559 core soybean accessions, we performed genotyping and haplotype analysis on the 11 GmPLD genes showing significant differential responses under LP conditions. The result reveal that GmPLDδ2/δ5/ε2/γ1/φ1/φ2/ζ2 contain 3 haplotypes, GmPLDζ1/3 have 4 haplotypes, while GmPLDβ1/4 each possess 5 haplotypes (Fig. S2A). The superior haplotypes of the 11 GmPLD genes were varied in different phosphorus efficiency (PE) related traits (Fig. 8 , S2 B). Notably, GmPLDβ1 -Hap5 and GmPLDε2 -Hap2 are identified as superior haplotypes, demonstrating significantly higher root phosphorus acquisition efficiency (RPAE) and relative root tip number (RRN) compared to other haplotypes. Similarly, GmPLDβ2 -Hap5 shows optimal performance in relative phosphorus concentration (RPC), RPAE, relative root area (RRA), and relative root length (RRL). The analysis further identifies GmPLDδ2 -Hap3 as the superior haplotype with advantages in RPAE, RRL and RRN, while GmPLDδ5 -Hap3 exhibits optimal characteristics for RPC, RRA, RRL and RRN. Additionally: GmPLDγ1 -Hap3 enhances RPC and RRA; GmPLDφ1 -Hap2 improves RPC, RPAE and RRA; GmPLDζ1 -Hap4 positively affects RPC, RPAE and RRN; GmPLDζ2 -Hap2 promotes RPC, RPAE, RRA and RRN; and both GmPLDφ2 -Hap3 and GmPLDζ3 -Hap3/Hap4 simultaneously enhance all five PE related traits. Except for GmPLDγ1 , the optimal haplotypes of the other 10 GmPLD genes exhibited a high distribution frequency in wild materials (Fig S2C). Notably, the distribution of these optimal haplotypes across six geographical regions of China (Ⅰ to Ⅵ) displayed distinct regional characteristics: GmPLDβ1 -Hap5 showed an increasing trend from the central-south (Ⅳ) to the north (Ⅰ) region. The optimal haplotypes of GmPLDβ2/δ2/ζ1/ζ3 had significant advantages in the northern region (Ⅰ). The optimal haplotypes of GmPLDδ5/γ1/ζ2 showed the highest distribution frequency in the Huang-Huai-Hai region (Ⅱ). In particular, the optimal haplotypes of GmPLDε2/φ1 exhibited broad-spectrum distribution characteristics, being present in all surveyed areas. In contrast, the distribution frequency of the optimal haplotype of GmPLDφ2 in the Middle and Lower Changjiang Valley (Ⅲ) and the Huang-Huai-Hai (Ⅱ) region was significantly lower than that in the northern (Ⅰ) and southern (Ⅵ) regions (Fig. S2C). This study not only clarified the important role of GmPLD gene family in the regulation of phosphorus efficiency, but also revealed the genetic mechanism of soybean adaptation to LP stress from an evolutionary perspective, which provided new breeding ideas and genetic resources for responding to the challenge of global soil phosphorus deficiency. 3.8. Expression analysis of GmPLD genes under Nitrogen-Deficient Stress This study conducted hydroponic experiments using soybean cultivar W82 with two nitrogen treatments: normal nitrogen supply (NN, 7.5 mM KNO₃) and low nitrogen stress (LN, 1 mM KNO₃). Root samples were collected 5 days after treatment for RNA extraction, and the expression differences of 25 GmPLD genes were detected by qRT-PCR technology. The results show that all GmPLD genes exhibit significant expression differences between NN and LN treatments (p < 0.05) (Fig. 9 ). Under LN stress conditions, these genes show varying degrees of up-regulated expression, with GmPLDα1/2/3/4/5 , GmPLDβ1/2/3/4 , GmPLDδ2/4/6 , GmPLDε1 , GmPLDγ1 , GmPLDφ1/2 , and GmPLDζ2/3 demonstrating highly significant induced expression (p < 0.001). This study systematically reveals for the first time the response characteristics of GmPLD gene family members to LN stress, providing important target genes for molecular breeding of nitrogen-efficient soybean. 3.9. Expression analysis of GmPLD genes under drought stress The expression profiles of GmPLD genes under 8% and 10% drought treatments, based on soil moisture content in W82, are obtained from previous laboratory work (Fig. 10 A, Table S10) to investigate the response of GmPLD genes to drought stress. Based on the thermogram expression analysis of GmPLD genes under various drought treatments in W82, it is evident that five GmPLD genes— GmPLDδ1 / 2 / 6 , GmPLDε3 , and GmPLDζ2 —exhibit significant differences when subjected to a drought treatment with a soil moisture content of 10% (D10%). Other GmPLD genes exhibit no significant differences in expression between the drought treatment and the control. Under the drought treatment with a soil moisture content of 8% (D8%), significant differences are observed in the expression of 16 GmPLD genes, namely GmPLDα1 / 2 / 3 / 6 , GmPLDβ1 / 2 , GmPLDδ1/3 / 4 , GmPLDε1 / 3 , GmPLDγ1 , GmPLDφ1 / 2 , and GmPLDζ2 / 3 . In contrast to these findings, the expression levels of other GmPLD genes do not show any significant variation between the drought treatment and control conditions. qRT-PCR was performed on these 16 GmPLD genes, with their corresponding primer sequences detailed in Table S11. Employing the soybean cultivar W82 as the experimental material, root RNA was extracted for qRT-PCR detection subsequent to the plants being subjected to control and simulated drought treatment using 20% PEG-6000. The result indicates that under drought stress conditions, in comparison with the non-stressed control group, 13 out of the 16 genes exhibit significant differential expression under drought stress, while GmPLDδ4 and GmPLD ε 1/3 show no statistically significant alterations (Fig. 10 B). Notably, GmPLDα1 and GmPLDα6 are significantly up-regulated following drought treatment (Fig. 9 B). Based on the aforementioned research findings, the following conclusions can be drawn: Under drought stress conditions, certain members of the GmPLD gene family can respond to this environmental adversity by altering their expression levels. 3.10. Expression analysis of GmPLD genes under Salt Stress This study investigated the expression patterns of GmPLD gene family members in roots (R) and leaves (L) of soybean cultivar W82 under control (150 mM KCl) and salt stress (150 mM NaCl) treatments for 48 hours using qPCR technology. The results demonstrate that all examined GmPLD genes show significant differential expression in either roots or leaves, except for GmPLDα4/β3/δ3 in roots and GmPLDα6/β3/β4/ε2 in leaves, which show no significant changes (Fig. 11 ). Under salt stress conditions, GmPLDα1/2/4/6 , GmPLDβ3 , GmPLDδ3/4/5 , and GmPLDε1/2 are upregulated in both roots and leaves; GmPLDβ1/4 , GmPLDδ1/2/6 , GmPLDε3 , GmPLDφ1/2 , and GmPLDζ1/2/3 are downregulated; Interestingly, GmPLDα3/5 , GmPLDβ2 , and GmPLDγ1 exhibit opposite expression trends between roots and leaves (Fig. 11 ), suggesting their potential functional divergence in different tissues during salt stress response. 3.11. Identification of GmPLDφ1 and GmPLDφ2 phospholipase D enzyme activities Based on these results, we found eight genes, GmPLDβ1/2 , GmPLDδ1 , GmPLDγ1 , GmPLDφ1/2 , and GmPLDζ2/3 , that show significant expression changes under multiple stress conditions including low phosphorus, low nitrogen, drought, and salt stress. However, as shown in Fig. 4 A, both GmPLDφ1 and GmPLDφ2 exhibit relatively low expression levels across various developmental stages and tissues in W82. Remarkably, despite their low basal expression, these two genes demonstrated significant differential expression patterns under multiple stress conditions. This non-random, stress-responsive expression profile strongly suggests that GmPLDφ1 and GmPLDφ2 likely play critical roles in soybean's complex physiological responses to various biotic stresses. However, it remains uncertain whether GmPLDφ1 and GmPLDφ2 exhibit PLD activity. Therefore, we conducted the identification of the PLD enzyme activity of GmPLDφ1 and GmPLDφ2 (Table 2 ). Eventually, it was found that the enzyme activity of GmPLDφ1 and GmPLDφ2 were 0.003637 and 0.004028nmol NPPC hydrolysed per 104 cells per minute to produce PNP, respectively. This indicates that GmPLDφ1 and GmPLDφ2 have phospholipase D enzyme activity. Table 2 Values ​​of GmPLDφ1 and GmPLDφ2 control and measurement groups at a wavelength of 450nm. Gene name Control Measurement Significance GmPLDφ1 0.121 (0.111–0.132) 0.14075 (0.130–0.148) p = 0.23 GmPLDφ2 0.11275 (0.110–0.118) 0.13475 (0.127–0.138) p < 0.001 4. Discussion This study identified 25 GmPLD genes in the soybean W82.v2 genome assembly, which are distributed across 14 chromosomes (Fig. S1 ). Compared to the 18 genes reported by Chen et al. [ 33 ] in the W82.v1 assembly, our analysis revealed 7 additional members. This discrepancy likely stems from significant improvements in the W82.v2 genome assembly quality and the incorporation of full-length transcriptome data (Iso-Seq), which enables more comprehensive gene prediction. The 25 identified genes contain two highly conserved HKD structural domains (H for histidine, K for lysine, and D for aspartic acid) (Fig. 2 B). In comparison to previously identified GmPLD genes, we discovered two additional genes that lack C2 or PX/PH structural domains at the N-terminus of their protein structures. Notably, a PLDφ gene in rice was found where the N-terminus did not feature a C2 or PX/PH domain but instead contained a signal peptide [ 49 ]; this class of phospholipase D is referred to as SP-PLDs . The construction of the phylogenetic tree (Fig. 1 A) indicates that these two PLD genes are closely related to the PLDφ gene from rice. Furthermore, experiments conducted to assess the activity of the phospholipase D enzyme revealed that both GmPLD genes exhibit this enzymatic activity (Table 2 ). Therefore, it can be inferred that these two genes represent PLDφ types and have been designated as GmPLDφ1 and GmPLDφ2 respectively. The 25 members of the GmPLD gene family can be classified into seven distinct subfamilies, referred to as: α ( GmPLDα1 ~ GmPLDα6 ), β ( GmPLDβ1 ~ GmPLDβ4 ), δ ( GmPLDδ1 ~ GmPLDδ6 ), ε ( GmPLDε1 ~ GmPLDε3 ), γ ( GmPLDγ1 ), ζ ( GmPLDζ1 ~ GmPLDζ6 ), and φ ( GmPLDφ1 ~ GmPLDφ2 ) (Table 1 ). Previous studies have demonstrated that gene duplications play a crucial role in the emergence of new gene functions and the amplification of gene families [ 51 , 52 ]. For instance, the Arabidopsis WRKY transcription factor family has increased its gene number through segmental duplication, thereby enriching its functional diversity in plant growth, development, and responses to biotic and abiotic stresses [ 53 ]. Similarly, the GmPLD gene family exhibits analogous characteristics (Fig. 1 B), supporting the notion that segmental duplication represents a common mechanism underlying gene family expansion in plants. However, not all gene family expansions rely on segmental duplication. For example, the maize zein protein family primarily forms gene clusters via tandem duplication, which ensures the efficient synthesis of seed storage proteins to meet nutritional demands during seed development [ 54 ]. In contrast, no tandem duplication events were detected in the GmPLD family in this study. These observations suggest that different gene families may adopt distinct expansion strategies tailored to their functional requirements (e.g., involvement in stress responses, metabolic regulation, or seed development), ultimately shaping diverse evolutionary patterns. According to the tissue expression profile (Fig. 5 A), the expression levels of certain GmPLD genes in the shoot apices, flowers, pods, and seeds of Williams 82 were found to be higher than those observed in other tissues. This suggests that GmPLD genes may play a role in regulating various processes such as shoot apex growth, flower development, pod formation, seed maturation, nutrient accumulation, as well as aspects related to seed dormancy and germination. In the analysis of cis-acting elements within the GmPLD gene family (Fig. 4 ), several elements associated with plant hormones were identified, including auxin, ABA, methyl jasmonate (MeJA), zein, gibberellin, salicylic acid, and estrogen. Previous studies have demonstrated that PLD and its product PA are crucial for polarized cell expansion in plants—such as during pollen tube growth [ 55 ]. The suppression of OsPLDβ1 expression has been shown to decrease seed sensitivity to exogenous ABA; conversely, PLDβ1 can enhance ABA signaling by activating SAPK pathways that inhibit seed germination [ 49 ]. Therefore, we hypothesize that GmPLD genes may be integral to controlling multiple growth and developmental processes in soybeans—including apical growth regulation, pollen tube elongation dynamics, and mechanisms governing seed dormancy and germination. The shoot apices, flowers, pods, and seeds of plants are susceptible to various abiotic stresses. Genes that are highly expressed may play a critical role in the plant’s adaptation mechanisms to these challenges. For example, flowers might need to upregulate specific genes to attract pollinators or resist pests and diseases; conversely, pods and seeds must highly express certain genes to enhance their tolerance against adverse conditions such as drought, high temperatures, or low temperatures [ 56 , 57 ]. The cis-acting elements within the GmPLD gene family (Fig. 4 ) contain regulatory sequences associated with stress responses including drought inducibility, wound response, dehydration resistance, and general stress response. Based on the single-cell RNA sequencing data analysis shown in Fig. 6 , we observed significant expression heterogeneity of the GmPLD gene family across various tissues and specific regions of ZH 13. The differential expression levels of GmPLD genes in different regions of these tissues further highlight the functional diversity of this gene family in plant growth, development, and stress responses. This variability in expression patterns underscores the potential roles of GmPLD genes in regulating a wide range of biological processes, from cellular differentiation to environmental adaptation. In studies on plant stress response mechanisms, the PLD family has been well-documented to participate in regulating various abiotic stresses. For example: AtPLDα3 in A. thaliana has been implicated in the plant’s adaptive responses to salt and drought conditions [ 58 ]. AtPLDζ2 is specifically induced under phosphorus starvation, where it hydrolyzes phosphatidylcholine (PC) and phosphatidylethanolamine to release inorganic phosphate, thereby promoting galactolipid biosynthesis to alleviate PE [ 59 ]. In Brassica napus , PLDε -overexpressing lines exhibit enhanced biomass accumulation under both nitrogen-deficient and nitrogen-sufficient conditions. Field trials have further confirmed that these transgenic lines significantly increase seed yield without compromising seed oil content [ 60 ]. Based on these findings, the current study systematically analyzed the expression patterns of the GmPLD gene family under four representative abiotic stresses (LP, LN, drought, and salt stress) to elucidate their biological functions in soybean stress responses. Through transcriptome analysis and qRT-PCR verification, this study revealed that the GmPLD gene family exhibits distinct stress-specific expression patterns in response to abiotic stresses. Under low phosphorus stress, 12 GmPLD genes showed significant expression changes. Under low nitrogen stress, all GmPLD genes were consistently up-regulated, with this overall response pattern being particularly prominent; among them, differential expression was more significant in subfamily members such as α, β, and δ. Under drought stress, 13 GmPLD genes displayed significant expression alterations, with the up-regulation of GmPLDα1 and GmPLDα6 being particularly notable. Salt stress induced a complex tissue-specific expression profile within the family: 10 genes were co-up-regulated in both roots and leaves, 11 genes were co-down-regulated in both tissues, and 4 genes exhibited completely opposite expression trends between roots and leaves. Notably, GmPLDβ1/2 , GmPLDδ1 , GmPLDγ1 , GmPLDφ1/2 , and GmPLDζ2/3 showed significant expression differences across multiple stresses (low phosphorus, low nitrogen, drought, and salt stress), suggesting that these members may play a core regulatory role in soybean's cross-adaptation to combined stresses. Additionally, previous studies by Zhao et al. reported that in leaves under salt stress, GmPLDα1/2/3 and GmPLDδ3/4 were up-regulated while GmPLDγ1 was down-regulated [ 33 ]. This is highly consistent with the results of the present study, further validating the reliability of GmPLD gene expression patterns in response to salt stress. Haplotype analysis of 11 GmPLD genes responding to LP stress showed that the optimal haplotypes of different genes had significant advantages in PE-related traits. Some of the optimal haplotypes had the characteristics of 'pleiotropism due to one cause', which provided valuable genetic resources for soybean PE breeding. Except for GmPLDγ1 , the optimal haplotypes of the remaining 10 genes were highly frequent in wild materials, suggesting that artificial selection led to the loss of some wild adaptive genes in cultivated soybeans. In the future, these haplotypes can be introduced from wild resources to improve the adaptability of cultivated varieties. The geographical distribution frequency showed that the optimal haplotypes of different genes were dominant in specific ecological regions. For example, the dominant haplotypes in the north may adapt to cold and LP soils, and the Huang-Huai-Hai region may be more suitable for calcareous LP soils. This pattern indicates that these haplotypes are naturally selected by the local environment to form genetic adaptability that matches regional ecological conditions. This study systematically analyzed the expression and regulation characteristics of the GmPLD gene family under various abiotic stresses, and provided new insights into the mechanism of plant phospholipase D-mediated stress response. The results showed that GmPLD family members showed obvious functional differentiation characteristics: different subtypes showed specific response patterns to specific stresses (LP, LN, drought, and salt stress); some genes may be involved in multiple stress responses as core regulatory elements. Tissue-specific expression characteristics (such as differential expression in roots and leaves under salt stress) suggest its functional specialization in different organs. These findings not only expand our understanding of the functional diversity of the GmPLD family, but also provide potential molecular targets for genetic improvement of soybean resistance. Future studies can verify the function of key GmPLD genes by means of gene editing and analyze their downstream signaling pathways to improve the plant phospholipid-mediated stress adaptation network. 5. Conclusions In this study, 25 GmPLD genes were identified in the genome of soybean W82.v2, all of which contain a conserved HKD domain, with significant sequence similarity and a highly conserved three-dimensional structure. Two of these genes are newly discovered φ subtypes ( GmPLDφ1/2 ), and the family is divided into seven subfamilies. The expansion of this gene family mainly relies on fragment repeats, with no tandem repeats detected. Different GmPLD members exhibit distinct tissue-specific expression patterns: some are highly expressed in shoot tips, flowers, pods, and seeds, and contain various hormone response elements, suggesting their potential involvement in regulating growth and development. Single-cell RNA sequencing revealed heterogeneous expression of these genes across different tissue regions, confirming their functional diversity. In terms of abiotic stress responses, GmPLD members show significant functional differentiation: 11 genes respond to LP stress; 13 genes are sensitive to drought; all members are up-regulated under LN conditions; under salt stress, 10 genes are up-regulated in both roots and leaves, 11 genes are down-regulated, and 4 genes display tissue-specific opposite expression trends. The 11 optimal haplotypes of GmPLD genes in response to LP had the advantage of phosphorus efficiency, which was high frequency in wild materials and showed regional adaptive distribution. Additionally, phospholipase D activity assays confirmed that both GmPLDφ1 and GmPLDφ2 exhibit significant enzyme activity associated with phospholipase D function. This study provides target genes for molecular breeding and precision design breeding aimed at enhancing abiotic stress resistance in soybeans. Abbreviations PLD Phospholipase D W82 Williams 82 ZH13 ZhongHuang13 RRA Relative Root Area RRL Relative Root Length RRN Relative Root Tip Number RPC Relative Phosphorus Concentration RPAE Relative Phosphorus Acquisition Efficiency SAM Shoot Apical Meristems NP Normal phosphorus supply LP Low phosphorus supply LN low nitrogen treatment NN normal nitrogen treatment Declarations Ethics declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Funding This work was supported by the key scientific and technological project of Henan Province (252102110268), the National Natural Science Foundation of China (32272171), STI 2030-Major Projects (2023ZD04069), and the Central Plains Talents Program and the Outstanding Youth Science Fund of Henan Province (242300421031). Author Contribution MS.H., MJ. X., and F. W. conducted the majority of the experiments and performed data analysis. HF. Z., LN. Z., YF. Y., and QQ. H. contributed to sample preparation as well as data analysis. SS. C. and D. Z. assisted in designing the experiments and revising the manuscript. HY. L. and DD. H. were responsible for designing the experiments and editing the manuscript. All authors have read and approved the final version of the manuscript. Acknowledgement We would like to express our sincere gratitude to Professor Jiao Yongqing for providing the transcriptome data of soybeans under different drought treatments, and to Nanjing Agricultural University for providing us with plant material Williams 82 (W82). Data Availability The sequence information of the entire soybean genome was obtained from the NCBI (National Center for Biotechnology Information) GenBank website, with the access number GCA_000004515.4. This website is open to all researchers. The transcriptome data under drought stress were provided by the laboratory of Prof. Jiao Yongqing at Henan Agricultural University. The dataset supporting the conclusions of this article is included in this article and its supplementary files. Plant material Williams 82 (W82) was provided by Nanjing Agricultural University. References Wang XM. Regulatory functions of phospholipase D and phosphatidic acid in plant growth, development, and stress responses. 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17:59:25","extension":"png","order_by":72,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":113724,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7469438/v1/df741984514f85be0bea1059.png"},{"id":92204479,"identity":"8273f496-5865-4d6c-a4d2-eba715879db6","added_by":"auto","created_at":"2025-09-25 18:07:26","extension":"xml","order_by":73,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":243450,"visible":true,"origin":"","legend":"","description":"","filename":"c36c2ccd824e43b1a0291e3ff45fc1bf1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7469438/v1/90f33e6575d16757511f0f6f.xml"},{"id":92204239,"identity":"b6543c67-5a93-4a4a-a4e1-0926eeb4295c","added_by":"auto","created_at":"2025-09-25 17:59:25","extension":"html","order_by":74,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":268203,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7469438/v1/c79aedc31c0741ba34d9ba3f.html"},{"id":92204439,"identity":"3a73c721-d9e2-43f2-9209-ed88359fb5f0","added_by":"auto","created_at":"2025-09-25 18:07:24","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":9438760,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of PLD gene family. (A) Phylogenetic analysis of the PLD gene family. The phy-logenetic tree was constructed for the PLD gene family using 54 PLD genes from three plant species, including \u003cem\u003eArabidopsis thalian\u003c/em\u003ea (At), \u003cem\u003eOryza sativa \u003c/em\u003e(Os), \u003cem\u003eand Glycine max \u003c/em\u003e(Gm). The PLD gene family can be divided into five subfamilies α, β, γ, δ, ε, ζ, and φ. (B) Position and covariance of \u003cem\u003eGmPLD\u003c/em\u003e genes. The duplicated \u003cem\u003eGmPLD\u003c/em\u003e gene pairs were connected by bule lines. (C) Pan-genome analysis of \u003cem\u003eGmPLD\u003c/em\u003e genes.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7469438/v1/03ea52563a3a442a9f8d2046.jpg"},{"id":92204198,"identity":"019f6888-f771-494f-ad37-91a56bab99b4","added_by":"auto","created_at":"2025-09-25 17:59:24","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":9910547,"visible":true,"origin":"","legend":"\u003cp\u003eStructure of \u003cem\u003eGmPLD\u003c/em\u003e genes. (A) Comparison of \u003cem\u003eGmPLD\u003c/em\u003e genes domain sequences. (B) The domains of \u003cem\u003eGmPLD\u003c/em\u003e genes. The domains are represented by different color boxes. (C) The motifs and gene structure of \u003cem\u003eGmPLD\u003c/em\u003egenes.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7469438/v1/c54be92213d54c237c44f017.jpg"},{"id":92204441,"identity":"e5e02788-146b-4752-8cc7-671aac45bf94","added_by":"auto","created_at":"2025-09-25 18:07:24","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":10103322,"visible":true,"origin":"","legend":"\u003cp\u003ePredicted protein structure of \u003cem\u003eGmPLD\u003c/em\u003e genes.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7469438/v1/609082865bb37de32c16d70a.jpg"},{"id":92204203,"identity":"9de1c3e3-8409-4d40-afe5-58744bbaccb9","added_by":"auto","created_at":"2025-09-25 17:59:24","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":9824146,"visible":true,"origin":"","legend":"\u003cp\u003eCis-elements in the \u003cem\u003eGmPLD\u003c/em\u003e genes promoter regions. The arrangement of the cis-regulatory elements within the 2000 bp upstream genetic regions of the 25 identified \u003cem\u003eGmPLD \u003c/em\u003egenes were represented by colored boxes, each indicating a different cis-element.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7469438/v1/609ecc029d1e459be6cade28.jpg"},{"id":92204443,"identity":"833f4e75-766b-466d-b6b9-a4b256ed23e8","added_by":"auto","created_at":"2025-09-25 18:07:24","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":10025488,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of \u003cem\u003eGmPLD\u003c/em\u003e genes expression in various soybean tissues. (A) Evaluation of \u003cem\u003eGmPLD\u003c/em\u003e genes expression in different tissues during soybean development using publicly available RNA-seq data. root, cotyledon-1, leafbud-1 and stem-1 at the 5-day emergence stage (VE5); cotyledon-2, leafbud-2, leaf-1 and stem-2 at the cotyledon stage (VC); leafbud-3, leaf-2, flower-1, and axillary_bud at the three-node stage (V3); leaf-3 at the tenth week of the growth period; flower-2 represents the flowers before anthesis, flower-3 represents the flowers on day 0 of anthesis, flower-4 represents the flowers on day 5 of anthesis, and flower-5 represents the wilted flowers; pod \u0026amp; seed-1 in the second week of seed development, pod-1 and pod \u0026amp; seed-2 in the third week, pod-2 and seed-1, pod \u0026amp; seed-3 in the fourth week, pod-3 and seed-2 in the fifth week, seed-3 in the sixth week, seed-4 in the eighth week, and seed-5 in the tenth week. The heatmap depicts Log2 normalized RPKM values to represent gene expression levels. (B) \u003cem\u003eGmPLDφ1\u003c/em\u003e and \u003cem\u003eGmPLDφ2\u003c/em\u003e tissue expression. (C) Single-cell RNA sequencing of \u003cem\u003eGmPLDα1, GmPLDα2\u003c/em\u003e, \u003cem\u003eGmPLDα3\u003c/em\u003e, \u003cem\u003eGmPLDφ1\u003c/em\u003e and \u003cem\u003eGmPLDφ2 \u003c/em\u003ein cotyledon stage seed. SC: seed coat, Emb: embryo.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7469438/v1/1c8fedd984dd6e9de7541eeb.jpg"},{"id":92204442,"identity":"f794effb-af28-4657-af57-f49d0dac9f3f","added_by":"auto","created_at":"2025-09-25 18:07:24","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":8654579,"visible":true,"origin":"","legend":"\u003cp\u003eSingle-cell RNA sequencing of the \u003cem\u003eGmPLD\u003c/em\u003e gene family in Zhonghuang 13. (A) Single-cell expression of \u003cem\u003eGmPLD\u003c/em\u003e genes in Nodule. (B) Single-cell expression of \u003cem\u003eGmPLD\u003c/em\u003egenes in Root. (C) Single-cell expression of \u003cem\u003eGmPLD\u003c/em\u003e genes in Stem. (D) Single-cell expression of \u003cem\u003eGmPLD\u003c/em\u003e genes in Leaf. (E) Single-cell expression of \u003cem\u003eGmPLD\u003c/em\u003e genes in Shoot Apical Meristem. Nodule were collected 5 days after inoculation with Rhizobium strain USDA-110 at vegetative cotyledon (VC) stage. Root, Stem, Leaf and Shoot Apical Meristem were collected at the vegetative cotyledon (VC) stage. The intensity of the color represents the expression level, with darker shades indicating higher expression. The size of the circles corresponds to the percentage of cells expressing the gene within the total detected cells in that specific region.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7469438/v1/75c76026f47033ee62266169.jpg"},{"id":92204452,"identity":"bdad5f7d-b288-455b-b715-050a489bc680","added_by":"auto","created_at":"2025-09-25 18:07:24","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":8124439,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of \u003cem\u003eGmPLD\u003c/em\u003e genes expression under different phosphate level treatment. (A) Expression analyses of \u003cem\u003eGmPLD \u003c/em\u003egenes in leaves and roots of LP tolerant cultivar W82 under different phosphate (Pi) level treatments LP, low phosphorus supply (5 μM, Pi); NP, normal phosphorus supply (500 μM, Pi). Expression analyses was based on the RNA data sequenced by Hu et al. (2024). (B) Expression analyses of \u003cem\u003eGmPLD\u003c/em\u003e genes in roots of the Nannong94156 under different phosphorus level treatment. Differences were evaluated using the two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test (***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ns = no differences).\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7469438/v1/e1101e3d5c00ab719f8ce419.jpg"},{"id":92204455,"identity":"00708f87-e935-4ae5-a017-1537fac96f3b","added_by":"auto","created_at":"2025-09-25 18:07:25","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":7754174,"visible":true,"origin":"","legend":"\u003cp\u003eHaplotype analysis of 11 \u003cem\u003eGmPLD\u003c/em\u003e genes with significant differential responses to phosphorus-deficient conditions. RPC: Relative Phosphorus Concentration; RPAE: Relative Phosphorus Acquisition Efficiency.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7469438/v1/3e21e5e1683a7e5649915406.jpg"},{"id":92204444,"identity":"bd5baae5-cd45-4aaf-840d-4c05927c8349","added_by":"auto","created_at":"2025-09-25 18:07:24","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":7598634,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of \u003cem\u003eGmPLD\u003c/em\u003e genes expression under different nitrogen levels. Differential responses of \u003cem\u003eGmPLD\u003c/em\u003e genes in roots of W82 to nitrogen treatments. LN: low nitrogen treatment (1 mM KNO₃); NN: normal nitrogen treatment (7.5 mM KNO₃). Statistical significance was evaluated using two-tailed Student's \u003cem\u003et\u003c/em\u003e-test (***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ns = not significant).\u003c/p\u003e","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7469438/v1/4a244847e28cfa49f1e1fdaa.jpg"},{"id":92204194,"identity":"4612aceb-fd21-4329-b3d5-d7664a775a99","added_by":"auto","created_at":"2025-09-25 17:59:24","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":8669689,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of \u003cem\u003eGmPLD\u003c/em\u003e genes expression under drought stress-induced treatment. (A) Expression analyses of \u003cem\u003eGmPLD \u003c/em\u003egenes in W82 under different drought stress-induced treatments. D10%: Sampling from drought treatment at 10% soil moisture content. D8%: Sampling from drought treatment at 8% soil moisture content. D10%-CK: This group serves as the control for drought treatment with a soil moisture content of 10%, without any drought intervention. D8%-CK: This group functions as the control for drought treatment with a soil moisture content of 8%, also without any drought intervention. (B) Expression analyses of \u003cem\u003eGmPLD\u003c/em\u003e genes in roots of W82 under different drought stress-induced treatment. CK: No drought stress treatment; PEG: Simulated drought stress using 20% PEG-6000. Differences were evaluated using the two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test (***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ns = no differences).\u003c/p\u003e","description":"","filename":"Figure10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7469438/v1/ef878b122dcc08fca19f67a2.jpg"},{"id":92205736,"identity":"9539a716-dbfc-4c08-bd84-3b87edb573ce","added_by":"auto","created_at":"2025-09-25 18:23:27","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":8337044,"visible":true,"origin":"","legend":"\u003cp\u003eExpression analyses of \u003cem\u003eGmPLD\u003c/em\u003e genes in Root (R) and Leaf (L) under different NaCl (150 mM) level treatments. Expression Response of the GmPLD Gene in Roots (R) and Leaves (L) of W82 Soybean under 150 mM NaCl Treatment. KCl: Control Group, 150 mM KCl Treatment; NaCl: Treatment Group, 150 mM NaCl Treatment. Statistical significance was evaluated using two-tailed Student's \u003cem\u003et\u003c/em\u003e-test (***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ns = not significant).\u003c/p\u003e","description":"","filename":"Figure11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7469438/v1/45d0f0346a56b5c1d90b4500.jpg"},{"id":97724039,"identity":"c0b8bfd1-fe45-43d4-9964-2d511d93af06","added_by":"auto","created_at":"2025-12-08 16:11:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":100283568,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7469438/v1/9ecf5a2c-7bdb-4766-b633-5b129eb69fcf.pdf"},{"id":92204181,"identity":"67b4214e-4c5c-4609-b172-2986b7ed7ca4","added_by":"auto","created_at":"2025-09-25 17:59:24","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":79059,"visible":true,"origin":"","legend":"","description":"","filename":"TableS111.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7469438/v1/2ac513c9d91a79732e151004.xlsx"},{"id":92204191,"identity":"638740df-ebf0-4373-ae14-937e8a962ae2","added_by":"auto","created_at":"2025-09-25 17:59:24","extension":"jpg","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":4441056,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7469438/v1/5633b4658ebcf1aec08c50fe.jpg"},{"id":92204265,"identity":"6cb554bb-6597-45e7-abdd-4daa9395f0fe","added_by":"auto","created_at":"2025-09-25 17:59:26","extension":"jpg","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":9984796,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS21.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7469438/v1/e79bb8d10d2d95b4b519228d.jpg"},{"id":92205119,"identity":"bfc825a2-8e27-430f-a81d-c96e65be76d4","added_by":"auto","created_at":"2025-09-25 18:15:25","extension":"jpg","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":10127891,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS22.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7469438/v1/9af0a6190e9bce33848c10e6.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-wide identification of the PLD gene family and its response to multiple abiotic stresses in soybean (Glycine max)","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePhospholipase D (PLD) is an enzyme that catalyzes the hydrolysis of phosphodiester bonds and facilitates base exchange reactions. The hydrolysis product of PLD, phosphatidic acid (PA), functions as a second messenger and plays a crucial role in the signal transduction processes within plant cells [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. As the predominant phospholipase in plants, PLD is widely distributed across various tissues, including seeds, fruits, roots, leaves, and stems. Its expression is notably elevated during critical phases of plant growth and development as well as metabolic processes such as seed development and maturation, along with seedling germination [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The C-terminus of the protein structure of plant PLD contains two highly conserved HKD (HxKxxxxD) structural domains, with H being histidine, K being lysine, D being aspartic acid, and x being a non-conserved arbitrary amino acid [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These two HKD domains are separated by approximately 320 amino acids [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In plants, site-directed mutagenesis experiments targeting amino acid residues have demonstrated that the HKD residues are essential for the enzymatic activity of PLD [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The two HKD domains interact to form the catalytic active site of PLD, making them indispensable for its function [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In addition to these two HKD structural domains, some plant \u003cem\u003ePLDs\u003c/em\u003e contain a highly conserved C2 structural domain at the N-terminus of the protein structure. The C2 structural domain is composed of approximately 130 amino acids, and it is a Ca\u003csup\u003e2+\u003c/sup\u003e-dependent phospholipid-binding domain, so \u003cem\u003ePLDs\u003c/em\u003e containing the C2 structural domain require a certain concentration of Ca\u003csup\u003e2+\u003c/sup\u003e in order to exert their maximal enzymatic activity [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. These \u003cem\u003ePLD\u003c/em\u003e genes are known as \u003cem\u003eC2-PLD\u003c/em\u003es. Some plant \u003cem\u003ePLD\u003c/em\u003e genes contain PX/PH domains and are thus classified as \u003cem\u003ePX/PH-PLDs\u003c/em\u003e. The PX domain exhibits the ability to bind phosphatidylinositol [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], while the PH domain interacts with various phosphatidylinositol phosphates [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. A class of \u003cem\u003ePLD\u003c/em\u003e genes in plants, termed \u003cem\u003eSP-PLDs\u003c/em\u003e, lack both C2 and PX/PH domains but contain a signal peptide at the N-terminus [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Plant \u003cem\u003ePLD\u003c/em\u003es are classified into seven distinct types: α, β, γ, δ, ε, ζ, and φ. Among these, \u003cem\u003ePLDα\u003c/em\u003e, \u003cem\u003ePLDβ\u003c/em\u003e, \u003cem\u003ePLDγ\u003c/em\u003e, \u003cem\u003ePLDδ\u003c/em\u003e, and \u003cem\u003ePLD\u003c/em\u003eε are categorized as \u003cem\u003eC2-PLD\u003c/em\u003es; \u003cem\u003ePLDζ\u003c/em\u003e is classified as a \u003cem\u003ePX/PH-PLD\u003c/em\u003e; and \u003cem\u003ePLDφ\u003c/em\u003e is identified as an \u003cem\u003eSP-PLD\u003c/em\u003e.\u003c/p\u003e\u003cp\u003ePLD plays a crucial role in the growth and development of plants, regulating processes such as seed germination, seedling growth, root hair development, pollen tube germination, apical growth, senescence, and the plant's response to abscisic acid (ABA) [\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. PLD can influence the quality, yield, and vigor of seeds by modulating the content of glycerolipid fractions in plants. Additionally, PLD is involved in regulating plant growth and development under conditions of nutrient deficiency. For instance, in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, PA derived from PLD interacts with ABI1 (a protein phosphatase 2C), a key negative regulator of the ABA signaling pathway, to modulate ABA signal transduction, thereby affecting seed germination and seedling growth [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]; \u003cem\u003ePLDα1\u003c/em\u003e and its product, PA, may influence the accumulation of Ca\u003csup\u003e2+\u003c/sup\u003e at the tip of root hairs by mediating the generation of reactive oxygen species (ROS) in that region [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and this process subsequently regulates the growth of \u003cem\u003eArabidopsis\u003c/em\u003e root hairs. Overexpression of \u003cem\u003eTaPLDδ\u003c/em\u003e in wheat resulting in an earlier heading and flowering. This alteration leads to an expedited transition into the reproductive growth phase and also enhances the plant's antioxidant capacity [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The overexpression of soybean \u003cem\u003eGmPLDγ\u003c/em\u003e in \u003cem\u003eA. thaliana\u003c/em\u003e can alter glycerolipid metabolism, thereby promoting the synthesis of oils rich in unsaturated fatty acids and long-chain fatty acids in seeds [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePLD can also respond to various abiotic stresses, including salt, low-temperature, drought, low phosphorus, low-potassium, mechanical injury, heavy metal, disease pressure, and osmotic stress. Currently, the functions of numerous \u003cem\u003ePLD\u003c/em\u003e genes in higher plants have been identified [\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In \u003cem\u003eA. thaliana\u003c/em\u003e, \u003cem\u003ePLDα1/PLDδ\u003c/em\u003e and their hydrolysis product PA positively regulate potassium uptake in the root system during low potassium stress responses. Following low potassium treatment, the enzymatic activity of PLDα1/PLDδ increases, leading to the generation of PA signals [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Additionally, \u003cem\u003ePLDζ2\u003c/em\u003e enhances root hair growth by promoting sugar-lipid synthesis and lipid remodeling, thereby improving the plant's adaptability to phosphorus deficiency [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In rice, \u003cem\u003eOsPLDζ1\u003c/em\u003e plays a positive regulatory role under salt stress conditions. The absence of \u003cem\u003eOsPLDζ1\u003c/em\u003e leads to impaired plant growth and reduced plant height in response to salt stress. Conversely, \u003cem\u003eOsPLDα3\u003c/em\u003e exhibits a negative regulatory effect during the response to salt stress [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In soybean, overexpression of \u003cem\u003eGmPLDγ\u003c/em\u003e enhances seed germination rate and accelerates germination under high-salinity stress conditions [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. When winter wheat subjected to drought stress, the growth of seedling leaves was inhibited after the addition of the PLD inhibitor butylated hydroxytoluene (n-butanol, BA) compared to the control. The levels of malondialdehyde (MDA), a product of membrane lipid peroxidation, increased, while the activity of antioxidant enzyme peroxidase (POD) decreased. These findings indicate that \u003cem\u003ePLD\u003c/em\u003e is involved in regulating POD activity under drought stress conditions [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In \u003cem\u003eChorispora bungeana\u003c/em\u003e, \u003cem\u003eCbPLDα\u003c/em\u003e, \u003cem\u003eCbPLDβ\u003c/em\u003e, and \u003cem\u003eCbPLDδ\u003c/em\u003e are involved in the plant's response to low-temperature stress, enhancing the cold resistance of tobacco seedlings [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHowever, there are currently relatively few reports on \u003cem\u003ePLD\u003c/em\u003e genes in soybean (\u003cem\u003eGlycine max\u003c/em\u003e). Therefore, it is essential to investigate the \u003cem\u003eGmPLD\u003c/em\u003e gene family in this crop. Zhao et al. identified 18 \u003cem\u003eGmPLD\u003c/em\u003e genes based on the W82.v1 genome and analyzed their expression patterns in various tissues and under salt stress conditions [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. We aim to investigate the role of the \u003cem\u003eGmPLD\u003c/em\u003e gene family in different abiotic stresses and explore whether the number of genes in the \u003cem\u003eGmPLD\u003c/em\u003e gene family has changed with the update of the genome. Utilizing transcriptome data and qRT-PCR experiment from soybeans subjected to phosphorus and nitrogen deficiency, drought stress, and salt stress, we observed significant differences in the expression levels of certain \u003cem\u003eGmPLD\u003c/em\u003e genes under such conditions. Building upon this observation, we initiated a comprehensive study of the \u003cem\u003eGmPLD\u003c/em\u003e gene family. In this research, we employed bioinformatics techniques to identify 25 \u003cem\u003eGmPLD\u003c/em\u003e genes and conducted analyses on their gene structures, three-dimensional protein conformations, tissue-specific expression patterns, and responses to abiotic stressors. Through these investigations, we aim to enhance our understanding of the functional roles played by the \u003cem\u003eGmPLD\u003c/em\u003e gene family. This knowledge will facilitate more effective utilization of \u003cem\u003eGmPLD\u003c/em\u003es in soybean cultivation and contribute to increased soybean yields.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Identification and characterization of \u003cem\u003ePLD\u003c/em\u003e genes in soybean\u003c/h2\u003e\u003cp\u003eUsing the soybean-specific database SoyBase (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.soybase.org/\u003c/span\u003e\u003cspan address=\"https://www.soybase.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; accessed February 1, 2024), the \u003cem\u003eGmPLD\u003c/em\u003e gene family members were screened by comprehensive analysis of the W82.v2 reference genome. Subsequently, the domain analysis of the selected genes was performed using the SMART online tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://smart.embl-heidelberg.de/\u003c/span\u003e\u003cspan address=\"http://smart.embl-heidelberg.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; accessed February 28, 2024) to identify the members of the \u003cem\u003eGmPLD\u003c/em\u003e gene family. Corresponding NCBI accession numbers and complete protein sequences were retrieved for each identified gene.Based on the obtained login number, UniProtKB of \u003cem\u003eGmPLD\u003c/em\u003e gene family was obtained on Pfam database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; accessed March 16, 2024). Based on the obtained protein sequences and UniProtKB, the protein sequences were analysed using Expasy-ProtParam tool online analysis website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/protparam/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/protparam/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; accessed March 16, 2024)to obtain the protein primary structure prediction results of amino acid length, relative molecular mass, isoelectric point and average hydrophobicity of the \u003cem\u003eGmPLD\u003c/em\u003e gene family [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]; and the Expasy-ProtScale (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/protscale/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/protscale/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; accessed March 18, 2024) online analysis website to obtain the hydrophobicity of \u003cem\u003eGmPLD\u003c/em\u003e genes [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The protein sequences were used for subcellular localisation analysis of the \u003cem\u003eGmPLD\u003c/em\u003e genes on the PSORT online analysis website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://wolfpsort.hgc.jp/\u003c/span\u003e\u003cspan address=\"https://wolfpsort.hgc.jp/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; accessed March 21, 2024) and Cell-PLoc 2.0 online prediction website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/\u003c/span\u003e\u003cspan address=\"http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; accessed March 22, 2024) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Phylogenetic trees of the \u003cem\u003ePLD\u003c/em\u003e gene family\u003c/h2\u003e\u003cp\u003eBased on the obtained protein sequences of \u003cem\u003eAtPLD\u003c/em\u003e, \u003cem\u003eOsPLD\u003c/em\u003e, and \u003cem\u003eGmPLD\u003c/em\u003e genes, we constructed a maximum likelihood (ML) phylogenetic tree of the \u003cem\u003ePLD\u003c/em\u003e gene family using TBtools software [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Then the phylogenetic tree was visualized and optimized by using iTOL online tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://itol.embl.de/\u003c/span\u003e\u003cspan address=\"https://itol.embl.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; accessed April 16, 2024).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Chromosome localisation and covariance analysis of \u003cem\u003eGmPLD\u003c/em\u003e genes\u003c/h2\u003e\u003cp\u003eThe chromosomal location of the \u003cem\u003eGmPLD\u003c/em\u003e genes and the length of each chromosome of soybean obtained from the soybean database SoyBase (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.soybase.org/\u003c/span\u003e\u003cspan address=\"https://www.soybase.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; accessed March 17, 2024), and these data were used to generate the chromosomal localization map of \u003cem\u003eGmPLD\u003c/em\u003e genes using MapChart software. The genome annotation file and genome sequence of soybean cultivar Wm82 were obtained from NCBI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; accessed March 17, 2024). After data processing, TBtools software [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] was employed to calculate the length of each soybean chromosome, determine chromosome gene density, and analyze the collinearity relationships within the \u003cem\u003eGmPLD\u003c/em\u003e gene family; these results were then visualized using TBtools.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Pan-genomic analysis of the \u003cem\u003eGmPLD\u003c/em\u003e family\u003c/h2\u003e\u003cp\u003eThe genomic data of the \u003cem\u003eGmPLD\u003c/em\u003e genes in 29 soybean accessions, including Williams 82 (W82), ZhongHuang (ZH13), and W05, were retrieved from the SoyOmics database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ngdc.cncb.ac.cn/soyomics/index\u003c/span\u003e\u003cspan address=\"https://ngdc.cncb.ac.cn/soyomics/index\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; accessed September 6, 2024) to analyse the number of distinct PLD subfamilies present in these materials [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Sequence comparison and structural domain analysis of \u003cem\u003eGmPLD\u003c/em\u003e genes\u003c/h2\u003e\u003cp\u003eUsing NCBI database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; accessed February 23, 2024) and Expasy-ProtParam online tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/protparam/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/protparam/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; accessed February 23, 2024) the obtained \u003cem\u003eGmPLD\u003c/em\u003e genes protein sequences were used to identify their domains, so as to obtain the conserved structural domains of \u003cem\u003eGmPLD\u003c/em\u003e gene family proteins. The conserved domains of the \u003cem\u003eGmPLD\u003c/em\u003e gene family proteins were determined by ClustalX software, and then the sequence alignment of these conserved domains of the \u003cem\u003eGmPLD\u003c/em\u003e genes was carried out by using ClustalX software. Based on the protein sequences of the \u003cem\u003eGmPLD\u003c/em\u003e genes, the structural domains were analysed on the SMART online analysis website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://smart.embl-heidelberg.de/\u003c/span\u003e\u003cspan address=\"http://smart.embl-heidelberg.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; accessed February 28, 2024) characterize the domains of the \u003cem\u003eGmPLD\u003c/em\u003e genes, employing default parameters (E-value\u0026thinsp;\u0026lt;\u0026thinsp;0.1). Then the domain architectures of \u003cem\u003eGmPLD\u003c/em\u003e family proteins were plotted with the help of TBtools software [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Characterization of gene structures and conserved motif distributions of \u003cem\u003eGmPLD\u003c/em\u003e genes\u003c/h2\u003e\u003cp\u003eThe genome annotation files of the \u003cem\u003eGmPLD\u003c/em\u003e family were retrieved using NCBI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; accessed March 17, 2024). Using TBtools software [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], gene structure analysis of the \u003cem\u003eGmPLD\u003c/em\u003e family was then conducted to map the CDS-UTR structure of the \u003cem\u003eGmPLD\u003c/em\u003e gene family. Based on the acquired \u003cem\u003eGmPLD\u003c/em\u003e protein sequences, motif analysis was performed via the MEME online tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://meme-suite.org/meme/\u003c/span\u003e\u003cspan address=\"https://meme-suite.org/meme/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; accessed May 3, 2024), with the maximum number of motifs set to 7.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. The 3D structure prediction of \u003cem\u003eGmPLD\u003c/em\u003e proteins\u003c/h2\u003e\u003cp\u003eBased on the protein sequences of \u003cem\u003eGmPLD\u003c/em\u003e genes, the three-dimensional structure were predicted using the SWISS-MODEI website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://swissmodel.expasy.org/\u003c/span\u003e\u003cspan address=\"https://swissmodel.expasy.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; accessed August 28, 2024). Via the automated template screening system, high-confidence prediction models with a Global Model Quality Estimation (GMQE) score\u0026thinsp;\u0026gt;\u0026thinsp;0.75 were rigorously selected for subsequent analyses.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Promoter cis-regulatory elements of the \u003cem\u003eGmPLD\u003c/em\u003e genes\u003c/h2\u003e\u003cp\u003eA 2000 bp sequence upstream of the initiation codon (ATG) of \u003cem\u003eGmPLD\u003c/em\u003e genes was retrieved from the Phytozome13 database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://phytozome-next.jgi.doe.gov/\u003c/span\u003e\u003cspan address=\"https://phytozome-next.jgi.doe.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; accessed April 23, 2024). This sequence was analyzed using the PlantCARE promoter analysis tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; accessed April 23, 2024) to predict cis-acting elements in the \u003cem\u003eGmPLD\u003c/em\u003e gene promoter regions, and the results were subsequently visualized using TBtools software [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Tissue expression pattern of the \u003cem\u003eGmPLD\u003c/em\u003e genes\u003c/h2\u003e\u003cp\u003eThe transcriptome data of \u003cem\u003eGmPLD\u003c/em\u003e genes in different tissues of Williams 82 at different developmental periods were extracted from SoyOmics database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ngdc.cncb.ac.cn/soyomics/transcriptome/tissues\u003c/span\u003e\u003cspan address=\"https://ngdc.cncb.ac.cn/soyomics/transcriptome/tissues\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; accessed September 5, 2024), and the expression pattern of \u003cem\u003eGmPLD\u003c/em\u003e genes in different tissues of Williams 82 at different developmental periods was analysed [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. These included root (root), cotyledon (cotyledon-1), leadbud (leafbud-1) and hypocotyl (stem-1) at the 5-day emergence stage (VE5); and cotyledon (cotyledon-2), leadbud (leafbud-2), leaf (leaf-1) and hypocotyl (stem-2) at the cotyledon stage (VC); Leadbud (leafbud-3), compound leaf (leaf-2), flower (flower-1), and lateral bud (axillary_bud) at the three-node stage (V3); compound leaf (leaf-3) at the tenth week of the growth period; Flowers prior to anthesis (flower-2) as well as flowers on day 0 of anthesis (flower-3), flowers on day 5 (flower-4), and wilted flowers (flower-5); Pods and seeds in the second week of seed development (pod \u0026amp; seed-1), pods (pod-1) and pods and seeds in the third week (pod \u0026amp; seed-2), pods (pod-2) and seeds (seed-1), pods and seeds(pod \u0026amp; seed-3) in the fourth week, pods (pod-3) and seeds (seed-2) in the fifth week, seeds (seed-3) in the sixth week, seeds (seed-4) in the eighth week, and seeds (seed-5) in the tenth week. The expression pattern of \u003cem\u003eGmPLD\u003c/em\u003e genes in different tissues of Williams 82 at different developmental periods was analysed, and a heat map of \u003cem\u003eGmPLD\u003c/em\u003e genes expression was drawn based on Log2-normalized FPKM values using TBtools software [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10. Single-cell RNA sequencing of the \u003cem\u003eGmPLD\u003c/em\u003e genes\u003c/h2\u003e\u003cp\u003eUsing the Soybean Multi-Omic Atlas online database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://soybean-atlas.com/\u003c/span\u003e\u003cspan address=\"https://soybean-atlas.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; accessed September 13, 2024), single-cell RNA sequencing (scRNA-seq) data were selected to examine the expression levels of different genes in cotyledon-stage seeds, a specific tissue region [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Additionally, via the SoyOmics database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ngdc.cncb.ac.cn/soyomics/transcriptome\u003c/span\u003e\u003cspan address=\"https://ngdc.cncb.ac.cn/soyomics/transcriptome\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; accessed March 15, 2025), we analyzed the expression profiles of \u003cem\u003eGmPLD\u003c/em\u003e genes in various regions of nodules, roots, stems, leaves, and shoot apical meristems (SAM) of soybean cultivar ZH 13. These analyses revealed the tissue-specific and region-specific expression patterns of \u003cem\u003eGmPLD\u003c/em\u003e genes, further clarifying their potential roles in plant growth, development, and stress responses [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11. Abiotic stress-induced expression of the \u003cem\u003eGmPLD\u003c/em\u003e genes\u003c/h2\u003e\u003cp\u003eUsing FPKM values from RNA-seq data of published studies[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], we obtained the expression profiles of \u003cem\u003eGmPLD\u003c/em\u003e genes in leaves and roots of W82 under LP stress, as well as their expression profiles in W82 subjected to drought treatments (8% and 10% soil water content) and corresponding control treatments over the same time periods. Using TBtools software, based on log2-standardized FPKM values, the Heatmaps of \u003cem\u003eGmPLD\u003c/em\u003e genes expression in leaves and roots of W82 under low phosphorus stress and W82 under 8% and 10% drought conditions (including their respective concurrent control) were generated [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.12. qRT-PCR analysis\u003c/h2\u003e\u003cp\u003eRoots, stems, cotyledons, and leaves of W82 (provided by Nanjing Agricultural University) were collected on the 20th day of its growth cycle; pods and seeds were collected at the 9th week of growth. Root samples of Nannong 94\u0026ndash;156 hydroponically cultured for 9 days under different phosphorus levels were collected, including LP treatment (5 \u0026micro;M Pi) and NP treatment (500 \u0026micro;M Pi, NP). Root samples subjected to drought treatment for 24 hours were collected, including the control group (non-drought treatment) and the 20% PEG-simulated drought treatment group. Root samples of W82 hydroponically cultured for 5 days under different nitrogen levels were collected, including LN treatment (1 mM KNO₃) and NN treatment (7.5 mM KNO₃). For salt stress treatment samples, 150 mM KCl was used as the control, and 150 mM NaCl was used as the salt stress treatment; roots and leaves were collected separately after 48 hours of treatment. All materials were set with 3 biological replicates, and each biological replicate included 3 technical replicates. Total RNA was extracted using the RNA extraction kit (TianGen, DP419), and the RNA was reverse transcribed into first-strand cDNA using the reverse transcription kit (Yisheng, NO. 11123ES) [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Based on the cDNA sequences of \u003cem\u003eGmPLDϕ1\u003c/em\u003e and \u003cem\u003eGmPLDϕ2\u003c/em\u003e, specific primers were designed using Primer 5.0 (Table S11). The internal reference gene is Tubulin (GenBank accession number AY907703) [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The total reaction volume for qRT-PCR is 20.0 \u0026micro;L, including 1 \u0026micro;L of cDNA, 10.0 \u0026micro;L of 2 \u0026times; FAST SYBR Mix, 1 \u0026micro;L each of the forward and reverse primers (10 \u0026micro;mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and 7 \u0026micro;L of RNase-Free H\u003csub\u003e2\u003c/sub\u003eO. qRT-PCR was performed on the CFX96 Touch system with the following reaction program: 2 min of pre-denaturation at 94 ℃; 40 cycles of 20 s denaturation at 95℃, 30 s annealing at 56℃, and 40 s extension at 72 ℃. The relative expression levels of the genes were calculated by the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], and the average value and standard deviation of each sample were calculated. Subsequently, the data were visualized using GraphPad Prism 8 software. The significant differences between the two datasets were analyzed using the independent samples t-test.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.13. Haplotype analysis\u003c/h2\u003e\u003cp\u003eThe genotype data and plant materials utilized in this study were obtained from the research by Lu et al. [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], encompassing a total of 559 soybean accessions. These accessions are categorized into 121 wild soybeans, 207 landraces, and 231 cultivated soybeans​. For data analysis, haplotype analysis of the \u003cem\u003eGmPLD\u003c/em\u003e gene was conducted using TASSEL 5.0 software to identify marker-trait associations [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], with the significance threshold set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05. Concurrently, Haploview 4.1 software was employed to analyze the haplotypes of 11 genes. In addition, during the preliminary laboratory work, 376 out of the 559 soybean materials were subjected to different phosphorus level treatments. Traits such as RRA, RPC, RRV, RRL, RRN, and RPAE were measured, with each line replicated three times [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Furthermore, the distribution of these 559 materials across different ecological regions was clarified. Subsequently, the data were visualized using GraphPad Prism 8 software, and the phenotypic differences corresponding to different haplotypes were analyzed by letter labeling method.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.14. Identification of \u003cem\u003eGmPLDϕ1\u003c/em\u003e and \u003cem\u003eGmPLDϕ2\u003c/em\u003e enzyme activities\u003c/h2\u003e\u003cp\u003eThe target vector pCold was digested with restriction endonucleases BamHI and SacI to obtain the linearised vector pCold, and the CDS of \u003cem\u003eGmPLDϕ1\u003c/em\u003e and \u003cem\u003eGmPLDϕ2\u003c/em\u003e were ligated to the linearised vector pCold at full length, and then their recombinant plasmids were transferred into BL21 sensory state. The BL21 sensory state of \u003cem\u003eGmPLDϕ1\u003c/em\u003e-Pcold and \u003cem\u003eGmPLDϕ2\u003c/em\u003e-Pcold was shaken to OD600\u0026thinsp;=\u0026thinsp;0.6. The number of cells of BL21 sensory bacillus solution of \u003cem\u003eGmPLDϕ1\u003c/em\u003e-Pcold and \u003cem\u003eGmPLDϕ2\u003c/em\u003e-Pcold with OD600\u0026thinsp;=\u0026thinsp;0.6 was calculated according to the method of dilution coated plate [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Finally, the phospholipase D activity of \u003cem\u003eGmPLDϕ1\u003c/em\u003e and \u003cem\u003eGmPLDϕ2\u003c/em\u003e was detected according to the PhospholipaseD (PLD) Activity Assay Kit (Suzhou Grace Biotechnology Co., Ltd., Item No. G0925W).\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Identification and characterization of \u003cem\u003ePLD\u003c/em\u003e genes in soybean\u003c/h2\u003e\u003cp\u003eA total of 39 putative \u003cem\u003eGmPLD\u003c/em\u003e genes were identified through genome-wide screening of the soybean W82.v2 reference genome assembly. Among these, 9 genes lacked both C2 or PX/PH structural domains and HKD structural domains. 2 genes contained only C2 structural domains, while 3 genes possessed a single HKD structural domain. The remaining 25 genes all exhibited two highly conserved HKD structural domains. Chen et al. (2012) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] identified 18 \u003cem\u003eGmPLD\u003c/em\u003e genes in the soybean W82.v1 genome assembly, whereas our study detected 7 additional \u003cem\u003eGmPLD\u003c/em\u003e genes, totaling 25. This increase in gene number likely reflects improved gene annotation and assembly quality in the updated genome version.\u003c/p\u003e\u003cp\u003eBased on the analysis of the structural domains of the \u003cem\u003eGmPLD\u003c/em\u003e gene family, along with previously classified \u003cem\u003eGmPLD\u003c/em\u003e genes [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], evolutionary relationships, and chromosomal locations, the 25 members of the \u003cem\u003eGmPLD\u003c/em\u003e gene family are designated as \u003cem\u003eGmPLDα1\u003c/em\u003e to \u003cem\u003eGmPLDα6\u003c/em\u003e, \u003cem\u003eGmPLDβ1\u003c/em\u003e to \u003cem\u003eGmPLDβ4\u003c/em\u003e, \u003cem\u003eGmPLDδ1\u003c/em\u003e to \u003cem\u003eGmPLDδ6\u003c/em\u003e, \u003cem\u003eGmPLDε1\u003c/em\u003e to \u003cem\u003eGmPLDε3\u003c/em\u003e, \u003cem\u003eGmPLDγ1\u003c/em\u003e, \u003cem\u003eGmPLDζ1\u003c/em\u003e to \u003cem\u003eGmPLDζ3\u003c/em\u003e, and \u003cem\u003eGmPLDφ1\u003c/em\u003e to \u003cem\u003eGmPLDφ2\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The 25 \u003cem\u003eGmPLD\u003c/em\u003e genes are distributed across 14 chromosomes (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The amino acid lengths of the \u003cem\u003eGmPLD\u003c/em\u003e gene family, based on protein sequences queried from the Expasy online analysis website, ranged from 519 to 1126. The isoelectric points varied between 5.48 and 7.62, while the relative molecular masses ranged from 58,551.85 to 128,451.94 Da, with an average of 97,455.72 Da. The average hydrophobicity values for this gene family ranged from \u0026minus;\u0026thinsp;0.532 to -0.202. The subcellular localization prediction of the 25 members of the \u003cem\u003eGmPLD\u003c/em\u003e gene family showed that 10 genes were located in the nucleus, 6 genes were located in the cytoplasm, 7 genes were located in the chloroplasts, 1 gene was located in the cytoskeleton, and 1 gene was located in the vesicles.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe characteristics of members of \u003cem\u003eGmPLD\u003c/em\u003e gene family\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGene ID\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGene Name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eChromosome location\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSize (aa)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMW (kDa)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003epI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eAverage hydrophobicity\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eSubcellular localization\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlyma.08g211700\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eGmPLDα1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGm08: 17098305...17101929\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e788\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e89300.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e5.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e-0.409\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eCytoplasm\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlyma.07g031100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eGmPLDα2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGm07: 2461423...2465693\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e809\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e91550.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e5.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e-0.384\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eCytoplasm\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlyma.13g364900\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eGmPLDα3\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGm13: 45119805...45125074\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e807\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e92078.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e5.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e-0.398\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eCytoplasm\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlyma.06g068600\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eGmPLDα4\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGm06: 5257871...5262176\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e826\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e94258.93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e5.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e-0.404\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNucleus\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlyma.06g068700\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eGmPLDα5\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGm06: 5264111...5268817\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e821\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e93469.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e6.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e-0.389\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eChloroplasts\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlyma.15g008500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eGmPLDα6\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGm15: 679168...684356\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e711\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e81216.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e6.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e-0.374\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eCytoplasm\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlyma.18g288600\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eGmPLDβ1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGm18: 56840393...56849007\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1097\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e123221.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e6.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e-0.532\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eChloroplasts\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlyma.02g093500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eGmPLDβ2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGm02: 8320963...8329508\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1106\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e124092.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e6.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e-0.509\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eChloroplasts\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlyma.07g080400\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eGmPLDβ3\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGm07: 7312327...7319873\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1047\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e117361.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e6.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e-0.482\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eChloroplasts\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlyma.03g018900\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eGmPLDβ4\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGm03: 1886324...1892076\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e759\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e85047.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e6.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e-0.422\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eCytoskeleton\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlyma.01g215100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eGmPLDγ1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGm01: 54579928...54585779\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e853\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e96097.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e7.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e-0.381\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eChloroplasts\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlyma.11g081500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eGmPLDδ1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGm11: 6113534...6121086\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e866\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e98442.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e6.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e-0.422\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNucleus\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlyma.01g162100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eGmPLDδ2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGm01:50010865...50019215\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e864\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e98119.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e6.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e-0.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNucleus\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlyma.05g168300\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eGmPLDδ3\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGm05: 35880140...35891738\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e857\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e96970.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e7.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e-0.383\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNucleus\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlyma.06g020500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eGmPLDδ4\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGm06: 1546983...1555296\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e847\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e96410.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e7.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e-0.396\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNucleus\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlyma.04g020400\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eGmPLDδ5\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGm04: 1603209...1610515\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e847\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e96095.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e6.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e-0.396\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eChloroplasts\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlyma.08g126700\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eGmPLDδ6\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGm08: 9760166...9769637\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e857\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e96935.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e7.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e-0.387\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNucleus\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlyma.07g010900\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eGmPLDε1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGm07: 830443...834361\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e769\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e88231.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e6.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e-0.422\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNucleus\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlyma.15g023500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eGmPLDε2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGm15: 1855931...1859297\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e759\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e87080.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e6.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e-0.474\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eCytoplasm\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlyma.08g194100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eGmPLDε3\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGm08: 15635256... 5639129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e776\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e89339.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e6.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e-0.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNucleus\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlyma.20g238000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eGmPLDζ1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGm20: 46972128...46987629\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1120\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e128451.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e5.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e-0.413\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNucleus\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlyma.15g152100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eGmPLDζ2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGm15: 12613312...12626308\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1123\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e127634.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e6.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e-0.414\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNucleus\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlyma.09g041400\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eGmPLDζ3\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGm09: 3462605...3475431\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1126\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e127843.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e6.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e-0.407\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNucleus\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlyma.04g060900\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eGmPLDφ1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGm04: 4966912...4970792\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e519\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e58590.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e7.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e-0.202\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eChloroplasts\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlyma.06g061500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eGmPLDφ2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGm06: 4631416...4635351\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e520\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e58551.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e6.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e-0.273\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eVesicles\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Phylogenetic relationship, covariance, pan-genomic analysis of the \u003cem\u003eGmPLD\u003c/em\u003e genes\u003c/h2\u003e\u003cp\u003eTo gain a deeper understanding of the evolutionary relationships and functions of the \u003cem\u003eGmPLD\u003c/em\u003e gene family, we constructed a phylogenetic tree (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) by integrating the \u003cem\u003eGmPLD\u003c/em\u003e genes with \u003cem\u003eA. thaliana PLD\u003c/em\u003e genes (12) (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and rice \u003cem\u003ePLD\u003c/em\u003e genes (17) (Table S2) [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The evolutionary relationships are categorized into seven distinct clades. Among these, the \u003cem\u003ePLDδ\u003c/em\u003e gene and rice \u003cem\u003eOsPLDκ1\u003c/em\u003e form one cluster, with \u003cem\u003eOsPLDκ1\u003c/em\u003e and \u003cem\u003eOsPLDδ1\u003c/em\u003e being the closest relatives. The \u003cem\u003ePLDβ\u003c/em\u003e and \u003cem\u003ePLDγ\u003c/em\u003e genes constitute a single clade; however, this clade can be further subdivided into two subcommunities based on the subclasses of \u003cem\u003ePLD\u003c/em\u003e. The \u003cem\u003ePLDα\u003c/em\u003e genes, with the exception of \u003cem\u003eOsPLDα8\u003c/em\u003e, constitute one clade; the \u003cem\u003eOsPLDα8\u003c/em\u003e and \u003cem\u003ePLD\u003c/em\u003eε genes form another clade; the \u003cem\u003ePLDδ\u003c/em\u003e genes represent a distinct clade; the \u003cem\u003ePLDζ\u003c/em\u003e genes comprise one clade; and the \u003cem\u003ePLDφ\u003c/em\u003e genes are classified as another clade. According to the phylogenetic tree, it is evident that \u003cem\u003ePLDα\u003c/em\u003e and \u003cem\u003ePLDε\u003c/em\u003e are closely related, as are \u003cem\u003ePLDβ\u003c/em\u003e and \u003cem\u003ePLDγ\u003c/em\u003e. Therefore, we can infer that \u003cem\u003ePLDα\u003c/em\u003e and \u003cem\u003ePLDε\u003c/em\u003e share similar structures and functions, just as \u003cem\u003ePLDβ\u003c/em\u003e and \u003cem\u003ePLDγ\u003c/em\u003e do.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCovariance analysis of the \u003cem\u003eGmPLD\u003c/em\u003e genes across soybean species (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) identified a total of 13 fragment duplication events involving the \u003cem\u003eGmPLD\u003c/em\u003e genes. These include duplications between \u003cem\u003eGmPLDα1\u003c/em\u003e and \u003cem\u003eGmPLDα4\u003c/em\u003e, \u003cem\u003eGmPLDα2\u003c/em\u003e and \u003cem\u003eGmPLDα4\u003c/em\u003e, as well as \u003cem\u003eGmPLDα4\u003c/em\u003e and \u003cem\u003eGmPLDα6\u003c/em\u003e. Additionally, duplications were observed between \u003cem\u003eGmPLDβ1\u003c/em\u003e and \u003cem\u003eGmPLDβ2\u003c/em\u003e, \u003cem\u003eGmPLDβ3\u003c/em\u003e and \u003cem\u003eGmPLDβ4\u003c/em\u003e, along with pairs such as \u003cem\u003eGmPLDδ1\u003c/em\u003e and \u003cem\u003eGmPLDδ2\u003c/em\u003e; \u003cem\u003eGmPLDδ1\u003c/em\u003e and \u003cem\u003eGmPLDδ4\u003c/em\u003e; and finally, between both \u003cem\u003eGmPLDδ1\u003c/em\u003e and \u003cem\u003eGmPLDδ5\u003c/em\u003e; as well as combinations of other gene pairs: \u003cem\u003eGmPLDδ2\u003c/em\u003e with \u003cem\u003eGmPLDδ4\u003c/em\u003e; \u003cem\u003eGmPLDδ2\u003c/em\u003e with \u003cem\u003eGmPLDδ5\u003c/em\u003e; \u003cem\u003eGmPLDδ3\u003c/em\u003e with \u003cem\u003eGmPLDδ6\u003c/em\u003e; \u003cem\u003eGmPLDδ4\u003c/em\u003e with \u003cem\u003eGmPLDδ5\u003c/em\u003e; as well as \u003cem\u003eGmPLDφ1\u003c/em\u003e in conjunction with \u003cem\u003eGmPLDφ2\u003c/em\u003e (Table S3). It is evident from this that the amplification of the \u003cem\u003eGmPLD\u003c/em\u003e gene family is primarily driven by events of fragment duplication. We analyzed the \u003cem\u003eGmPLD\u003c/em\u003e genes in 29 different type soybean accessions and found that the number of \u003cem\u003eGmPLD\u003c/em\u003e genes varied from 17 to 25 (Table S4-5): the landrace SoyL01 has the fewest \u003cem\u003eGmPLD\u003c/em\u003e genes, and the cultivated soybean Williams 82 has the most \u003cem\u003eGmPLD\u003c/em\u003e genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Structural Analysis of the \u003cem\u003eGmPLD\u003c/em\u003e genes\u003c/h2\u003e\u003cp\u003eAll GmPLD proteins possess two HKD domains, and the sequence must include two HxKxxxxD motifs. The HKD1 domain of GmPLDα, GmPLDβ/δ/γ, GmPLDε, and GmPLDζ/φ contains 39, 36, 38, and 28 amino acids, respectively. Except for the HKD2 domain of GmPLDφ, which contains 27 amino acids, the HKD2 domains of other GmPLD proteins all contain 28 amino acids (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Notably, the domains within the same subfamily exhibit significant sequence similarity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBased on the gene structure analysis diagram of GmPLD (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), all GmPLD proteins contain two HKD domains. All members of the GmPLDα, GmPLDβ, GmPLDδ, GmPLDε, and GmPLDγ subfamilies possess C2 domains, except for GmPLDβ4 and GmPLDε3. Every GmPLDζ protein contains a PH domain in addition to the two HKD domains but lack the PX domain; in contrast, GmPLDφ proteins contain neither PH nor PX domains, being characterized solely by the two HKD domains. Notably, GmPLDβ and GmPLDζ proteins exhibit the longest amino acid sequences, while GmPLDφ proteins have the shortest.\u003c/p\u003e\u003cp\u003eMotif analysis revealed that all GmPLDα/β/ε/δ proteins contain seven motifs, while GmPLDφ1 and GmPLDφ2 only harbor Motif 1. The three GmPLDζ proteins lack Motif 3 and Motif 6, and GmPLDβ2 is deficient in Motif 6 and Motif 7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, Table S6).\u003c/p\u003e\u003cp\u003eSubsequent gene structure analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) showed that most \u003cem\u003eGmPLD\u003c/em\u003e genes have lengths exceeding 3 kb, except for \u003cem\u003eGmPLDζ3\u003c/em\u003e, which exceeds 15 kb. Regarding exon numbers: \u003cem\u003eGmPLDε\u003c/em\u003e genes contain 4 exons, \u003cem\u003eGmPLDα\u003c/em\u003e genes contain 3\u0026ndash;6 exons, and \u003cem\u003eGmPLDφ\u003c/em\u003e genes contain 7 exons; \u003cem\u003eGmPLDβ\u003c/em\u003e, \u003cem\u003eGmPLDγ1\u003c/em\u003e, and other \u003cem\u003eGmPLDγ\u003c/em\u003e genes contain 10 exons, with the exceptions of \u003cem\u003eGmPLDβ4\u003c/em\u003e (18 exons), \u003cem\u003eGmPLDδ2\u003c/em\u003e (8 exons), and \u003cem\u003eGmPLDδ4\u003c/em\u003e (9 exons); the three \u003cem\u003eGmPLDζ\u003c/em\u003e genes contain 18, 20, and 21 exons, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Notably, among all subfamilies, \u003cem\u003eGmPLDζ\u003c/em\u003e genes exhibit the highest number of exons.\u003c/p\u003e\u003cp\u003eThe 3D protein structures of GmPLD proteins were predicted using SWISS-MODEL and organized according to the evolutionary tree of their encoding genes. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e, PLDα, PLDγ, PLDδ, and PLDε proteins exhibit similar 3D structures. In contrast, PLDβ and PLDζ proteins also share structural similarities. Notably, the 3D structures of GmPLDφ1 and GmPLDφ2 differ from those of all other subfamily members. Despite these variations among different subfamilies, all GmPLD proteins share a segment with an extremely conserved structure. This finding suggests that the overall 3D structure of GmPLD proteins is relatively conserved across different subfamilies.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Cis-acting element analysis of the \u003cem\u003eGmPLD\u003c/em\u003e genes\u003c/h2\u003e\u003cp\u003eThe cis-regulatory elements within the promoters of all \u003cem\u003eGmPLD\u003c/em\u003e genes were analyzed utilizing the online software PlantCARE ( \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ). Based on the soybean genome information, we extracted the sequences 2000 bp upstream of the initiation codon (ATG) of all \u003cem\u003eGmPLD\u003c/em\u003e genes and visualised the cis-regulatory elements using TBtools software (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The 17 most common elements were obtained in the promoter region of the \u003cem\u003eGmPLD\u003c/em\u003e genes. These include hormone-related elements (e.g., growth hormone, abscisic acid, methyl jasmonate (MeJA), gibberellin, salicylic acid, and estrogen), adversity-related elements (e.g., drought response, wounding response, dehydration defense, and stress response), endosperm-expression elements, meristem expression elements, light-response elements, anaerobically-induced elements, inducer-mediated activation elements, and circadian regulation-related elements. Notably, light-responsive cis-elements are abundant in the promoter region of \u003cem\u003eGmPLD\u003c/em\u003e genes (Table S7).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.5. Expression pattern of the \u003cem\u003eGmPLD\u003c/em\u003e genes\u003c/h2\u003e\u003cp\u003eTo investigate the expression pattern of \u003cem\u003eGmPLD\u003c/em\u003e genes in soybean across various developmental stages, transcriptome data for \u003cem\u003eGmPLD\u003c/em\u003e genes from different tissues at distinct developmental periods in W82 were obtained from the SoyOmics database ( \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ngdc.cncb.ac.cn/soyomics/index\u003c/span\u003e\u003cspan address=\"https://ngdc.cncb.ac.cn/soyomics/index\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ). The expression levels of \u003cem\u003eGmPLD\u003c/em\u003e genes varied across different tissues and developmental stages in W82 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, Table S8). \u003cem\u003eGmPLDα1\u003c/em\u003e, \u003cem\u003eGmPLDα2\u003c/em\u003e, and \u003cem\u003eGmPLDα3\u003c/em\u003e exhibit high expression levels in various tissues at distinct developmental periods; \u003cem\u003eGmPLDζ1\u003c/em\u003e exhibits moderate expression levels in leaf-1/3, cotyledon-2, leaf bud-2, flower-3/4, and pod; \u003cem\u003eGmPLDβ3\u003c/em\u003e demonstrates intermediate expression levels in cotyledon-1, leafbud, flower-1, flower-2, seed-1, and axillary bud; both \u003cem\u003eGmPLDδ1\u003c/em\u003e and \u003cem\u003eGmPLDδ2\u003c/em\u003e show tissue-enriched expression levels in stem, leaf, bud, flower, pod \u0026amp; seed, pod and axillary bud; \u003cem\u003eGmPLDγ1\u003c/em\u003e has intermediate expression levels in cotyledon-2, leaf-1, leaf-2, and root. The remaining genes exhibit low expression levels across various tissues during different developmental stages2. At least one gene from each of the other subfamilies exhibit a relatively high expression level during one of the developmental stages with the exception of the \u003cem\u003eGmPLDφ\u003c/em\u003e subfamily (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). To investigate the expression differences of \u003cem\u003eGmPLDφ\u003c/em\u003e genes across various tissues and developmental stages, we collected W82 samples from roots, stems, cotyledons, and leaves on the 20th day of the growth cycle, as well as pods and seeds at the 9th week of growth [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. We assessed the tissue-specific expression levels, and the results indicate that both \u003cem\u003eGmPLDφ1\u003c/em\u003e and \u003cem\u003eGmPLDφ2\u003c/em\u003e display relatively high expression levels in seeds (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBased on the above results, we found that \u003cem\u003eGmPLDα1\u003c/em\u003e, \u003cem\u003eGmPLDα2\u003c/em\u003e, \u003cem\u003eGmPLDα3\u003c/em\u003e, \u003cem\u003eGmPLDφ1\u003c/em\u003e, and \u003cem\u003eGmPLDφ2\u003c/em\u003e exhibit high expression levels in seeds. Therefore, we used single-cell RNA sequencing data [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] to observe their expression levels in different parts of the cotyledon stage seed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). \u003cem\u003eGmPLDα1\u003c/em\u003e, \u003cem\u003eGmPLDα2\u003c/em\u003e, and \u003cem\u003eGmPLDα3\u003c/em\u003e are expressed in different parts of the cotyledon-stage seed, with relatively high expression levels. \u003cem\u003eGmPLDφ1\u003c/em\u003e is expressed in End peripheral, Emb vasculature, End micropylar, SC inner parenchyma, SC epidermis, and SC outer parenchyma, but the expression level is relatively low. \u003cem\u003eGmPLDφ2\u003c/em\u003e is expressed in SC hilum epidermis, End chalazal, Emb vasculature, End micropylar, Emb epidermis, SC inner parenchyma, SC outer parenchyma, and SC tracheid bar, with relatively low expression levels.\u003c/p\u003e\u003cp\u003eTo investigate the expression patterns of \u003cem\u003eGmPLD\u003c/em\u003e genes across various tissues and regions, we performed expression analysis of \u003cem\u003eGmPLD\u003c/em\u003e genes based on the single-cell RNA sequencing (scRNA-seq) data from SoyBase [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], the tissues including nodules, roots, stems, leaves, and shoot apical meristems (SAM) of Zhonghuang 13 (ZH13). The corresponding gene IDs of \u003cem\u003eGmPLD\u003c/em\u003e genes in ZH13 are listed in Table S7. The results reveal that \u003cem\u003eGmPLDα1\u003c/em\u003e and \u003cem\u003eGmPLDα3\u003c/em\u003e exhibit high expression levels in nearly all regions of nodules, roots, stems, leaves, and SAM (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e). \u003cem\u003eGmPLDδ1\u003c/em\u003e shows moderate expression across these tissues, while \u003cem\u003eGmPLDγ1\u003c/em\u003e displays elevated expression in nodules and roots, particularly in the Cortex2-pre1 region of the roots. Additionally, \u003cem\u003eGmPLDδ2\u003c/em\u003e and \u003cem\u003eGmPLDζ1\u003c/em\u003e/\u003cem\u003e2\u003c/em\u003e are highly expressed in nodules, with \u003cem\u003eGmPLDζ1\u003c/em\u003e/\u003cem\u003e2\u003c/em\u003e showing particularly significant expression in Infected cell2. Notably, \u003cem\u003eGmPLDα4\u003c/em\u003e/\u003cem\u003e5\u003c/em\u003e and \u003cem\u003eGmPLDε1\u003c/em\u003e/\u003cem\u003e2\u003c/em\u003e/\u003cem\u003e3\u003c/em\u003e exhibit minimal or negligible expression across all regions. The distinct expression patterns of \u003cem\u003eGmPLD\u003c/em\u003e genes across various tissues and regions suggest their potential involvement in diverse biological functions. For instance, the high expression of \u003cem\u003eGmPLDγ1\u003c/em\u003e in the Cortex2-pre1 region of the roots indicates its possible role in specialized processes such as cell differentiation or responses to environmental signals. Similarly, the specific expression of \u003cem\u003eGmPLDζ1\u003c/em\u003e and \u003cem\u003eGmPLDζ2\u003c/em\u003e in Infected cell2 implies their potential participation in symbiotic processes, including membrane remodeling, signal transduction, or nutrient exchange between the host plant and symbiotic bacteria. These findings highlight the functional versatility of \u003cem\u003eGmPLD\u003c/em\u003e genes in regulating both developmental and symbiotic mechanisms in plants.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.6. Expression analysis of \u003cem\u003eGmPLD\u003c/em\u003e genes under phosphorus-deficient conditions\u003c/h2\u003e\u003cp\u003eThe expression profiles of \u003cem\u003eGmPLD\u003c/em\u003e genes in the leaves and roots of W82 under low phosphorus stress were derived from FPKM values obtained through RNA-seq data from published studies [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, Table S9). This analysis aims to investigate the response of \u003cem\u003eGmPLD\u003c/em\u003e genes to low phosphorus stress. Based on the thermograms presented for the expression of \u003cem\u003eGmPLD\u003c/em\u003e genes in the leaves and root systems of W82 under normal phosphorus (NP, 500 \u0026micro;M phosphate) and low phosphorus (LP, 5 \u0026micro;M phosphate) stress, it is evident that among the 12 \u003cem\u003eGmPLD\u003c/em\u003e genes\u0026mdash;\u003cem\u003eGmPLDβ1\u003c/em\u003e, \u003cem\u003eGmPLDβ2\u003c/em\u003e, \u003cem\u003eGmPLDδ1\u003c/em\u003e, \u003cem\u003eGmPLDδ2\u003c/em\u003e, \u003cem\u003eGmPLDδ5\u003c/em\u003e, \u003cem\u003eGmPLDε2\u003c/em\u003e, \u003cem\u003eGmPLDγ1\u003c/em\u003e, \u003cem\u003eGmPLDφ1\u003c/em\u003e, \u003cem\u003eGmPLDφ2\u003c/em\u003e, \u003cem\u003eGmPLDζ1\u003c/em\u003e, \u003cem\u003eGmPLDζ2\u003c/em\u003e, and \u003cem\u003eGmPLDζ3\u003c/em\u003e\u0026mdash;significant differences are observed between NP and LP treatment in roots. In contrast, no significant differences are detected for the remaining 13 \u003cem\u003eGmPLD\u003c/em\u003e genes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe conducted quantitative real-time PCR (qRT-PCR) on 12 \u003cem\u003eGmPLD\u003c/em\u003e genes, with the relevant primer sequences provided in Table S11. Using the soybean cultivar NanNong 94\u0026ndash;156 as our experimental material, we subjected the plants to various phosphorus concentration treatments and extracted root RNA for qRT-PCR detection. The experimental results indicate that under LP conditions, the expression levels of \u003cem\u003eGmPLDβ1\u003c/em\u003e, \u003cem\u003eGmPLDβ2\u003c/em\u003e, \u003cem\u003eGmPLDδ2\u003c/em\u003e, \u003cem\u003eGmPLDδ6\u003c/em\u003e, \u003cem\u003eGmPLDγ1\u003c/em\u003e, \u003cem\u003eGmPLDφ2\u003c/em\u003e, \u003cem\u003eGmPLDζ2\u003c/em\u003e, and \u003cem\u003eGmPLDζ3\u003c/em\u003e are significantly lower compared to those observed under NP conditions. In contrast, the expression levels of \u003cem\u003eGmPLDε2\u003c/em\u003e, \u003cem\u003eGmPLDφ1\u003c/em\u003e, and \u003cem\u003eGmPLDζ1\u003c/em\u003e exhibit a notable increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). These findings suggest that these 11 \u003cem\u003eGmPLD\u003c/em\u003e genes may play crucial roles in plant responses to LP stress through mechanisms involving differential expression regulation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e3.7. Haplotype Analysis of 11 Phosphorus-Responsive \u003cem\u003eGmPLD\u003c/em\u003e genes in Soybean Germplasm\u003c/h2\u003e\u003cp\u003eUsing 559 core soybean accessions, we performed genotyping and haplotype analysis on the 11 \u003cem\u003eGmPLD\u003c/em\u003e genes showing significant differential responses under LP conditions. The result reveal that \u003cem\u003eGmPLDδ2/δ5/ε2/γ1/φ1/φ2/ζ2\u003c/em\u003e contain 3 haplotypes, \u003cem\u003eGmPLDζ1/3\u003c/em\u003e have 4 haplotypes, while \u003cem\u003eGmPLDβ1/4\u003c/em\u003e each possess 5 haplotypes (Fig. S2A). The superior haplotypes of the 11 \u003cem\u003eGmPLD\u003c/em\u003e genes were varied in different phosphorus efficiency (PE) related traits (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003e, \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB). Notably, \u003cem\u003eGmPLDβ1\u003c/em\u003e-Hap5 and \u003cem\u003eGmPLDε2\u003c/em\u003e-Hap2 are identified as superior haplotypes, demonstrating significantly higher root phosphorus acquisition efficiency (RPAE) and relative root tip number (RRN) compared to other haplotypes. Similarly, \u003cem\u003eGmPLDβ2\u003c/em\u003e-Hap5 shows optimal performance in relative phosphorus concentration (RPC), RPAE, relative root area (RRA), and relative root length (RRL). The analysis further identifies \u003cem\u003eGmPLDδ2\u003c/em\u003e-Hap3 as the superior haplotype with advantages in RPAE, RRL and RRN, while \u003cem\u003eGmPLDδ5\u003c/em\u003e-Hap3 exhibits optimal characteristics for RPC, RRA, RRL and RRN. Additionally: \u003cem\u003eGmPLDγ1\u003c/em\u003e-Hap3 enhances RPC and RRA; \u003cem\u003eGmPLDφ1\u003c/em\u003e-Hap2 improves RPC, RPAE and RRA; \u003cem\u003eGmPLDζ1\u003c/em\u003e-Hap4 positively affects RPC, RPAE and RRN; \u003cem\u003eGmPLDζ2\u003c/em\u003e-Hap2 promotes RPC, RPAE, RRA and RRN; and both \u003cem\u003eGmPLDφ2\u003c/em\u003e-Hap3 and \u003cem\u003eGmPLDζ3\u003c/em\u003e-Hap3/Hap4 simultaneously enhance all five PE related traits.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eExcept for \u003cem\u003eGmPLDγ1\u003c/em\u003e, the optimal haplotypes of the other 10 \u003cem\u003eGmPLD\u003c/em\u003e genes exhibited a high distribution frequency in wild materials (Fig S2C). Notably, the distribution of these optimal haplotypes across six geographical regions of China (Ⅰ to Ⅵ) displayed distinct regional characteristics: \u003cem\u003eGmPLDβ1\u003c/em\u003e-Hap5 showed an increasing trend from the central-south (Ⅳ) to the north (Ⅰ) region. The optimal haplotypes of \u003cem\u003eGmPLDβ2/δ2/ζ1/ζ3\u003c/em\u003e had significant advantages in the northern region (Ⅰ). The optimal haplotypes of \u003cem\u003eGmPLDδ5/γ1/ζ2\u003c/em\u003e showed the highest distribution frequency in the Huang-Huai-Hai region (Ⅱ). In particular, the optimal haplotypes of \u003cem\u003eGmPLDε2/φ1\u003c/em\u003e exhibited broad-spectrum distribution characteristics, being present in all surveyed areas. In contrast, the distribution frequency of the optimal haplotype of \u003cem\u003eGmPLDφ2\u003c/em\u003e in the Middle and Lower Changjiang Valley (Ⅲ) and the Huang-Huai-Hai (Ⅱ) region was significantly lower than that in the northern (Ⅰ) and southern (Ⅵ) regions (Fig. S2C).\u003c/p\u003e\u003cp\u003eThis study not only clarified the important role of \u003cem\u003eGmPLD\u003c/em\u003e gene family in the regulation of phosphorus efficiency, but also revealed the genetic mechanism of soybean adaptation to LP stress from an evolutionary perspective, which provided new breeding ideas and genetic resources for responding to the challenge of global soil phosphorus deficiency.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e3.8. Expression analysis of \u003cem\u003eGmPLD\u003c/em\u003e genes under Nitrogen-Deficient Stress\u003c/h2\u003e\u003cp\u003eThis study conducted hydroponic experiments using soybean cultivar W82 with two nitrogen treatments: normal nitrogen supply (NN, 7.5 mM KNO₃) and low nitrogen stress (LN, 1 mM KNO₃). Root samples were collected 5 days after treatment for RNA extraction, and the expression differences of 25 \u003cem\u003eGmPLD\u003c/em\u003e genes were detected by qRT-PCR technology. The results show that all \u003cem\u003eGmPLD\u003c/em\u003e genes exhibit significant expression differences between NN and LN treatments (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Under LN stress conditions, these genes show varying degrees of up-regulated expression, with \u003cem\u003eGmPLDα1/2/3/4/5\u003c/em\u003e, \u003cem\u003eGmPLDβ1/2/3/4\u003c/em\u003e, \u003cem\u003eGmPLDδ2/4/6\u003c/em\u003e, \u003cem\u003eGmPLDε1\u003c/em\u003e, \u003cem\u003eGmPLDγ1\u003c/em\u003e, \u003cem\u003eGmPLDφ1/2\u003c/em\u003e, and \u003cem\u003eGmPLDζ2/3\u003c/em\u003e demonstrating highly significant induced expression (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). This study systematically reveals for the first time the response characteristics of \u003cem\u003eGmPLD\u003c/em\u003e gene family members to LN stress, providing important target genes for molecular breeding of nitrogen-efficient soybean.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003e3.9. Expression analysis of \u003cem\u003eGmPLD\u003c/em\u003e genes under drought stress\u003c/h2\u003e\u003cp\u003eThe expression profiles of \u003cem\u003eGmPLD\u003c/em\u003e genes under 8% and 10% drought treatments, based on soil moisture content in W82, are obtained from previous laboratory work (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e10\u003c/span\u003eA, Table S10) to investigate the response of \u003cem\u003eGmPLD\u003c/em\u003e genes to drought stress. Based on the thermogram expression analysis of \u003cem\u003eGmPLD\u003c/em\u003e genes under various drought treatments in W82, it is evident that five \u003cem\u003eGmPLD\u003c/em\u003e genes\u0026mdash;\u003cem\u003eGmPLDδ1\u003c/em\u003e/\u003cem\u003e2\u003c/em\u003e/\u003cem\u003e6\u003c/em\u003e, \u003cem\u003eGmPLDε3\u003c/em\u003e, and \u003cem\u003eGmPLDζ2\u003c/em\u003e\u0026mdash;exhibit significant differences when subjected to a drought treatment with a soil moisture content of 10% (D10%). Other \u003cem\u003eGmPLD\u003c/em\u003e genes exhibit no significant differences in expression between the drought treatment and the control. Under the drought treatment with a soil moisture content of 8% (D8%), significant differences are observed in the expression of 16 \u003cem\u003eGmPLD\u003c/em\u003e genes, namely \u003cem\u003eGmPLDα1\u003c/em\u003e/\u003cem\u003e2\u003c/em\u003e/\u003cem\u003e3\u003c/em\u003e/\u003cem\u003e6\u003c/em\u003e, \u003cem\u003eGmPLDβ1\u003c/em\u003e/\u003cem\u003e2\u003c/em\u003e, \u003cem\u003eGmPLDδ1/3\u003c/em\u003e/\u003cem\u003e4\u003c/em\u003e, \u003cem\u003eGmPLDε1\u003c/em\u003e/\u003cem\u003e3\u003c/em\u003e, \u003cem\u003eGmPLDγ1\u003c/em\u003e, \u003cem\u003eGmPLDφ1\u003c/em\u003e/\u003cem\u003e2\u003c/em\u003e, and \u003cem\u003eGmPLDζ2\u003c/em\u003e/\u003cem\u003e3\u003c/em\u003e. In contrast to these findings, the expression levels of other \u003cem\u003eGmPLD\u003c/em\u003e genes do not show any significant variation between the drought treatment and control conditions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eqRT-PCR was performed on these 16 \u003cem\u003eGmPLD\u003c/em\u003e genes, with their corresponding primer sequences detailed in Table S11. Employing the soybean cultivar W82 as the experimental material, root RNA was extracted for qRT-PCR detection subsequent to the plants being subjected to control and simulated drought treatment using 20% PEG-6000. The result indicates that under drought stress conditions, in comparison with the non-stressed control group, 13 out of the 16 genes exhibit significant differential expression under drought stress, while \u003cem\u003eGmPLDδ4\u003c/em\u003e and \u003cem\u003eGmPLD\u003c/em\u003eε\u003cem\u003e1/3\u003c/em\u003e show no statistically significant alterations (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e10\u003c/span\u003eB). Notably, \u003cem\u003eGmPLDα1\u003c/em\u003e and \u003cem\u003eGmPLDα6\u003c/em\u003e are significantly up-regulated following drought treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). Based on the aforementioned research findings, the following conclusions can be drawn: Under drought stress conditions, certain members of the \u003cem\u003eGmPLD\u003c/em\u003e gene family can respond to this environmental adversity by altering their expression levels.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\u003ch2\u003e3.10. Expression analysis of \u003cem\u003eGmPLD\u003c/em\u003e genes under Salt Stress\u003c/h2\u003e\u003cp\u003eThis study investigated the expression patterns of \u003cem\u003eGmPLD\u003c/em\u003e gene family members in roots (R) and leaves (L) of soybean cultivar W82 under control (150 mM KCl) and salt stress (150 mM NaCl) treatments for 48 hours using qPCR technology. The results demonstrate that all examined \u003cem\u003eGmPLD\u003c/em\u003e genes show significant differential expression in either roots or leaves, except for \u003cem\u003eGmPLDα4/β3/δ3\u003c/em\u003e in roots and \u003cem\u003eGmPLDα6/β3/β4/ε2\u003c/em\u003e in leaves, which show no significant changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e11\u003c/span\u003e). Under salt stress conditions, \u003cem\u003eGmPLDα1/2/4/6\u003c/em\u003e, \u003cem\u003eGmPLDβ3\u003c/em\u003e, \u003cem\u003eGmPLDδ3/4/5\u003c/em\u003e, and \u003cem\u003eGmPLDε1/2\u003c/em\u003e are upregulated in both roots and leaves; \u003cem\u003eGmPLDβ1/4\u003c/em\u003e, \u003cem\u003eGmPLDδ1/2/6\u003c/em\u003e, \u003cem\u003eGmPLDε3\u003c/em\u003e, \u003cem\u003eGmPLDφ1/2\u003c/em\u003e, and \u003cem\u003eGmPLDζ1/2/3 are\u003c/em\u003e downregulated; Interestingly, \u003cem\u003eGmPLDα3/5\u003c/em\u003e, \u003cem\u003eGmPLDβ2\u003c/em\u003e, and \u003cem\u003eGmPLDγ1\u003c/em\u003e exhibit opposite expression trends between roots and leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e11\u003c/span\u003e), suggesting their potential functional divergence in different tissues during salt stress response.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003e3.11. Identification of \u003cem\u003eGmPLDφ1\u003c/em\u003e and \u003cem\u003eGmPLDφ2\u003c/em\u003e phospholipase D enzyme activities\u003c/h2\u003e\u003cp\u003eBased on these results, we found eight genes, \u003cem\u003eGmPLDβ1/2\u003c/em\u003e, \u003cem\u003eGmPLDδ1\u003c/em\u003e, \u003cem\u003eGmPLDγ1\u003c/em\u003e, \u003cem\u003eGmPLDφ1/2\u003c/em\u003e, and \u003cem\u003eGmPLDζ2/3\u003c/em\u003e, that show significant expression changes under multiple stress conditions including low phosphorus, low nitrogen, drought, and salt stress. However, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, both \u003cem\u003eGmPLDφ1\u003c/em\u003e and \u003cem\u003eGmPLDφ2\u003c/em\u003e exhibit relatively low expression levels across various developmental stages and tissues in W82. Remarkably, despite their low basal expression, these two genes demonstrated significant differential expression patterns under multiple stress conditions. This non-random, stress-responsive expression profile strongly suggests that \u003cem\u003eGmPLDφ1\u003c/em\u003e and \u003cem\u003eGmPLDφ2\u003c/em\u003e likely play critical roles in soybean's complex physiological responses to various biotic stresses.\u003c/p\u003e\u003cp\u003eHowever, it remains uncertain whether \u003cem\u003eGmPLDφ1\u003c/em\u003e and \u003cem\u003eGmPLDφ2\u003c/em\u003e exhibit PLD activity. Therefore, we conducted the identification of the PLD enzyme activity of \u003cem\u003eGmPLDφ1\u003c/em\u003e and \u003cem\u003eGmPLDφ2\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Eventually, it was found that the enzyme activity of \u003cem\u003eGmPLDφ1\u003c/em\u003e and \u003cem\u003eGmPLDφ2\u003c/em\u003e were 0.003637 and 0.004028nmol NPPC hydrolysed per 104 cells per minute to produce PNP, respectively. This indicates that \u003cem\u003eGmPLDφ1\u003c/em\u003e and \u003cem\u003eGmPLDφ2\u003c/em\u003e have phospholipase D enzyme activity.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eValues ​​of \u003cem\u003eGmPLDφ1\u003c/em\u003e and \u003cem\u003eGmPLDφ2\u003c/em\u003e control and measurement groups at a wavelength of 450nm.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGene name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMeasurement\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSignificance\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eGmPLDφ1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.121 (0.111\u0026ndash;0.132)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.14075 (0.130\u0026ndash;0.148)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.23\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eGmPLDφ2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.11275 (0.110\u0026ndash;0.118)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.13475 (0.127\u0026ndash;0.138)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study identified 25 \u003cem\u003eGmPLD\u003c/em\u003e genes in the soybean W82.v2 genome assembly, which are distributed across 14 chromosomes (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Compared to the 18 genes reported by Chen et al. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] in the W82.v1 assembly, our analysis revealed 7 additional members. This discrepancy likely stems from significant improvements in the W82.v2 genome assembly quality and the incorporation of full-length transcriptome data (Iso-Seq), which enables more comprehensive gene prediction.\u003c/p\u003e\u003cp\u003eThe 25 identified genes contain two highly conserved HKD structural domains (H for histidine, K for lysine, and D for aspartic acid) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). In comparison to previously identified \u003cem\u003eGmPLD\u003c/em\u003e genes, we discovered two additional genes that lack C2 or PX/PH structural domains at the N-terminus of their protein structures. Notably, a \u003cem\u003ePLDφ\u003c/em\u003e gene in rice was found where the N-terminus did not feature a C2 or PX/PH domain but instead contained a signal peptide [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]; this class of phospholipase D is referred to as \u003cem\u003eSP-PLDs\u003c/em\u003e. The construction of the phylogenetic tree (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) indicates that these two \u003cem\u003ePLD\u003c/em\u003e genes are closely related to the \u003cem\u003ePLDφ\u003c/em\u003e gene from rice. Furthermore, experiments conducted to assess the activity of the phospholipase D enzyme revealed that both \u003cem\u003eGmPLD\u003c/em\u003e genes exhibit this enzymatic activity (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Therefore, it can be inferred that these two genes represent \u003cem\u003ePLDφ\u003c/em\u003e types and have been designated as \u003cem\u003eGmPLDφ1\u003c/em\u003e and \u003cem\u003eGmPLDφ2\u003c/em\u003e respectively. The 25 members of the \u003cem\u003eGmPLD\u003c/em\u003e gene family can be classified into seven distinct subfamilies, referred to as: α (\u003cem\u003eGmPLDα1\u003c/em\u003e\u0026thinsp;~\u0026thinsp;\u003cem\u003eGmPLDα6\u003c/em\u003e), β (\u003cem\u003eGmPLDβ1\u003c/em\u003e\u0026thinsp;~\u0026thinsp;\u003cem\u003eGmPLDβ4\u003c/em\u003e), δ (\u003cem\u003eGmPLDδ1\u003c/em\u003e\u0026thinsp;~\u0026thinsp;\u003cem\u003eGmPLDδ6\u003c/em\u003e), ε (\u003cem\u003eGmPLDε1\u003c/em\u003e\u0026thinsp;~\u0026thinsp;\u003cem\u003eGmPLDε3\u003c/em\u003e), γ (\u003cem\u003eGmPLDγ1\u003c/em\u003e), ζ (\u003cem\u003eGmPLDζ1\u003c/em\u003e\u0026thinsp;~\u0026thinsp;\u003cem\u003eGmPLDζ6\u003c/em\u003e), and φ (\u003cem\u003eGmPLDφ1\u003c/em\u003e\u0026thinsp;~\u0026thinsp;\u003cem\u003eGmPLDφ2\u003c/em\u003e) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePrevious studies have demonstrated that gene duplications play a crucial role in the emergence of new gene functions and the amplification of gene families [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. For instance, the \u003cem\u003eArabidopsis WRKY\u003c/em\u003e transcription factor family has increased its gene number through segmental duplication, thereby enriching its functional diversity in plant growth, development, and responses to biotic and abiotic stresses [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Similarly, the \u003cem\u003eGmPLD\u003c/em\u003e gene family exhibits analogous characteristics (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), supporting the notion that segmental duplication represents a common mechanism underlying gene family expansion in plants. However, not all gene family expansions rely on segmental duplication. For example, the maize zein protein family primarily forms gene clusters via tandem duplication, which ensures the efficient synthesis of seed storage proteins to meet nutritional demands during seed development [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In contrast, no tandem duplication events were detected in the \u003cem\u003eGmPLD\u003c/em\u003e family in this study. These observations suggest that different gene families may adopt distinct expansion strategies tailored to their functional requirements (e.g., involvement in stress responses, metabolic regulation, or seed development), ultimately shaping diverse evolutionary patterns.\u003c/p\u003e\u003cp\u003eAccording to the tissue expression profile (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), the expression levels of certain \u003cem\u003eGmPLD\u003c/em\u003e genes in the shoot apices, flowers, pods, and seeds of Williams 82 were found to be higher than those observed in other tissues. This suggests that \u003cem\u003eGmPLD\u003c/em\u003e genes may play a role in regulating various processes such as shoot apex growth, flower development, pod formation, seed maturation, nutrient accumulation, as well as aspects related to seed dormancy and germination. In the analysis of cis-acting elements within the \u003cem\u003eGmPLD\u003c/em\u003e gene family (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e), several elements associated with plant hormones were identified, including auxin, ABA, methyl jasmonate (MeJA), zein, gibberellin, salicylic acid, and estrogen. Previous studies have demonstrated that PLD and its product PA are crucial for polarized cell expansion in plants\u0026mdash;such as during pollen tube growth [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. The suppression of \u003cem\u003eOsPLDβ1\u003c/em\u003e expression has been shown to decrease seed sensitivity to exogenous ABA; conversely, \u003cem\u003ePLDβ1\u003c/em\u003e can enhance ABA signaling by activating SAPK pathways that inhibit seed germination [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Therefore, we hypothesize that \u003cem\u003eGmPLD\u003c/em\u003e genes may be integral to controlling multiple growth and developmental processes in soybeans\u0026mdash;including apical growth regulation, pollen tube elongation dynamics, and mechanisms governing seed dormancy and germination.\u003c/p\u003e\u003cp\u003eThe shoot apices, flowers, pods, and seeds of plants are susceptible to various abiotic stresses. Genes that are highly expressed may play a critical role in the plant\u0026rsquo;s adaptation mechanisms to these challenges. For example, flowers might need to upregulate specific genes to attract pollinators or resist pests and diseases; conversely, pods and seeds must highly express certain genes to enhance their tolerance against adverse conditions such as drought, high temperatures, or low temperatures [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. The cis-acting elements within the \u003cem\u003eGmPLD\u003c/em\u003e gene family (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e) contain regulatory sequences associated with stress responses including drought inducibility, wound response, dehydration resistance, and general stress response. Based on the single-cell RNA sequencing data analysis shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e, we observed significant expression heterogeneity of the \u003cem\u003eGmPLD\u003c/em\u003e gene family across various tissues and specific regions of ZH 13. The differential expression levels of \u003cem\u003eGmPLD\u003c/em\u003e genes in different regions of these tissues further highlight the functional diversity of this gene family in plant growth, development, and stress responses. This variability in expression patterns underscores the potential roles of \u003cem\u003eGmPLD\u003c/em\u003e genes in regulating a wide range of biological processes, from cellular differentiation to environmental adaptation.\u003c/p\u003e\u003cp\u003eIn studies on plant stress response mechanisms, the \u003cem\u003ePLD\u003c/em\u003e family has been well-documented to participate in regulating various abiotic stresses. For example: \u003cem\u003eAtPLDα3\u003c/em\u003e in \u003cem\u003eA. thaliana\u003c/em\u003e has been implicated in the plant\u0026rsquo;s adaptive responses to salt and drought conditions [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. \u003cem\u003eAtPLDζ2\u003c/em\u003e is specifically induced under phosphorus starvation, where it hydrolyzes phosphatidylcholine (PC) and phosphatidylethanolamine to release inorganic phosphate, thereby promoting galactolipid biosynthesis to alleviate PE [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. In \u003cem\u003eBrassica napus\u003c/em\u003e, \u003cem\u003ePLDε\u003c/em\u003e-overexpressing lines exhibit enhanced biomass accumulation under both nitrogen-deficient and nitrogen-sufficient conditions. Field trials have further confirmed that these transgenic lines significantly increase seed yield without compromising seed oil content [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Based on these findings, the current study systematically analyzed the expression patterns of the \u003cem\u003eGmPLD\u003c/em\u003e gene family under four representative abiotic stresses (LP, LN, drought, and salt stress) to elucidate their biological functions in soybean stress responses.\u003c/p\u003e\u003cp\u003eThrough transcriptome analysis and qRT-PCR verification, this study revealed that the \u003cem\u003eGmPLD\u003c/em\u003e gene family exhibits distinct stress-specific expression patterns in response to abiotic stresses. Under low phosphorus stress, 12 \u003cem\u003eGmPLD\u003c/em\u003e genes showed significant expression changes. Under low nitrogen stress, all \u003cem\u003eGmPLD\u003c/em\u003e genes were consistently up-regulated, with this overall response pattern being particularly prominent; among them, differential expression was more significant in subfamily members such as α, β, and δ. Under drought stress, 13 \u003cem\u003eGmPLD\u003c/em\u003e genes displayed significant expression alterations, with the up-regulation of \u003cem\u003eGmPLDα1\u003c/em\u003e and \u003cem\u003eGmPLDα6\u003c/em\u003e being particularly notable. Salt stress induced a complex tissue-specific expression profile within the family: 10 genes were co-up-regulated in both roots and leaves, 11 genes were co-down-regulated in both tissues, and 4 genes exhibited completely opposite expression trends between roots and leaves.\u003c/p\u003e\u003cp\u003eNotably, \u003cem\u003eGmPLDβ1/2\u003c/em\u003e, \u003cem\u003eGmPLDδ1\u003c/em\u003e, \u003cem\u003eGmPLDγ1\u003c/em\u003e, \u003cem\u003eGmPLDφ1/2\u003c/em\u003e, and \u003cem\u003eGmPLDζ2/3\u003c/em\u003e showed significant expression differences across multiple stresses (low phosphorus, low nitrogen, drought, and salt stress), suggesting that these members may play a core regulatory role in soybean's cross-adaptation to combined stresses. Additionally, previous studies by Zhao et al. reported that in leaves under salt stress, \u003cem\u003eGmPLDα1/2/3\u003c/em\u003e and \u003cem\u003eGmPLDδ3/4\u003c/em\u003e were up-regulated while \u003cem\u003eGmPLDγ1\u003c/em\u003e was down-regulated [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. This is highly consistent with the results of the present study, further validating the reliability of \u003cem\u003eGmPLD\u003c/em\u003e gene expression patterns in response to salt stress.\u003c/p\u003e\u003cp\u003eHaplotype analysis of 11 \u003cem\u003eGmPLD\u003c/em\u003e genes responding to LP stress showed that the optimal haplotypes of different genes had significant advantages in PE-related traits. Some of the optimal haplotypes had the characteristics of 'pleiotropism due to one cause', which provided valuable genetic resources for soybean PE breeding. Except for \u003cem\u003eGmPLDγ1\u003c/em\u003e, the optimal haplotypes of the remaining 10 genes were highly frequent in wild materials, suggesting that artificial selection led to the loss of some wild adaptive genes in cultivated soybeans. In the future, these haplotypes can be introduced from wild resources to improve the adaptability of cultivated varieties. The geographical distribution frequency showed that the optimal haplotypes of different genes were dominant in specific ecological regions. For example, the dominant haplotypes in the north may adapt to cold and LP soils, and the Huang-Huai-Hai region may be more suitable for calcareous LP soils. This pattern indicates that these haplotypes are naturally selected by the local environment to form genetic adaptability that matches regional ecological conditions.\u003c/p\u003e\u003cp\u003eThis study systematically analyzed the expression and regulation characteristics of the \u003cem\u003eGmPLD\u003c/em\u003e gene family under various abiotic stresses, and provided new insights into the mechanism of plant phospholipase D-mediated stress response. The results showed that \u003cem\u003eGmPLD\u003c/em\u003e family members showed obvious functional differentiation characteristics: different subtypes showed specific response patterns to specific stresses (LP, LN, drought, and salt stress); some genes may be involved in multiple stress responses as core regulatory elements. Tissue-specific expression characteristics (such as differential expression in roots and leaves under salt stress) suggest its functional specialization in different organs. These findings not only expand our understanding of the functional diversity of the \u003cem\u003eGmPLD\u003c/em\u003e family, but also provide potential molecular targets for genetic improvement of soybean resistance. Future studies can verify the function of key \u003cem\u003eGmPLD\u003c/em\u003e genes by means of gene editing and analyze their downstream signaling pathways to improve the plant phospholipid-mediated stress adaptation network.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn this study, 25 \u003cem\u003eGmPLD\u003c/em\u003e genes were identified in the genome of soybean W82.v2, all of which contain a conserved HKD domain, with significant sequence similarity and a highly conserved three-dimensional structure. Two of these genes are newly discovered φ subtypes (\u003cem\u003eGmPLDφ1/2\u003c/em\u003e), and the family is divided into seven subfamilies. The expansion of this gene family mainly relies on fragment repeats, with no tandem repeats detected. Different \u003cem\u003eGmPLD\u003c/em\u003e members exhibit distinct tissue-specific expression patterns: some are highly expressed in shoot tips, flowers, pods, and seeds, and contain various hormone response elements, suggesting their potential involvement in regulating growth and development. Single-cell RNA sequencing revealed heterogeneous expression of these genes across different tissue regions, confirming their functional diversity. In terms of abiotic stress responses, \u003cem\u003eGmPLD\u003c/em\u003e members show significant functional differentiation: 11 genes respond to LP stress; 13 genes are sensitive to drought; all members are up-regulated under LN conditions; under salt stress, 10 genes are up-regulated in both roots and leaves, 11 genes are down-regulated, and 4 genes display tissue-specific opposite expression trends. The 11 optimal haplotypes of \u003cem\u003eGmPLD\u003c/em\u003e genes in response to LP had the advantage of phosphorus efficiency, which was high frequency in wild materials and showed regional adaptive distribution. Additionally, phospholipase D activity assays confirmed that both \u003cem\u003eGmPLDφ1\u003c/em\u003e and \u003cem\u003eGmPLDφ2\u003c/em\u003e exhibit significant enzyme activity associated with phospholipase D function. This study provides target genes for molecular breeding and precision design breeding aimed at enhancing abiotic stress resistance in soybeans.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePLD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePhospholipase D\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eW82\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eWilliams 82\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eZH13\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eZhongHuang13\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRRA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRelative Root Area\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRRL\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRelative Root Length\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRRN\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRelative Root Tip Number\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRPC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRelative Phosphorus Concentration\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRPAE\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRelative Phosphorus Acquisition Efficiency\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSAM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eShoot Apical Meristems\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eNormal phosphorus supply\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLow phosphorus supply\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLN\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003elow nitrogen treatment\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNN\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003enormal nitrogen treatment\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003e\u003cb\u003eEthics declarations\u003c/b\u003e\u003c/h2\u003e\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by the key scientific and technological project of Henan Province (252102110268), the National Natural Science Foundation of China (32272171), STI 2030-Major Projects (2023ZD04069), and the Central Plains Talents Program and the Outstanding Youth Science Fund of Henan Province (242300421031).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMS.H., MJ. X., and F. W. conducted the majority of the experiments and performed data analysis. HF. Z., LN. Z., YF. Y., and QQ. H. contributed to sample preparation as well as data analysis. SS. C. and D. Z. assisted in designing the experiments and revising the manuscript. HY. L. and DD. H. were responsible for designing the experiments and editing the manuscript. All authors have read and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe would like to express our sincere gratitude to Professor Jiao Yongqing for providing the transcriptome data of soybeans under different drought treatments, and to Nanjing Agricultural University for providing us with plant material Williams 82 (W82).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe sequence information of the entire soybean genome was obtained from the NCBI (National Center for Biotechnology Information) GenBank website, with the access number GCA_000004515.4. This website is open to all researchers. The transcriptome data under drought stress were provided by the laboratory of Prof. Jiao Yongqing at Henan Agricultural University. The dataset supporting the conclusions of this article is included in this article and its supplementary files. Plant material Williams 82 (W82) was provided by Nanjing Agricultural University.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWang XM. Regulatory functions of phospholipase D and phosphatidic acid in plant growth, development, and stress responses. Plant Physiol. 2005;139(2):566\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYan X, Li Y, Li L, Zhao P. Signal Transduction of Phospholipase D in Plants. Plant Physiol Commun. 2006;42(6):1183\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCui D, Wen F. THE ROLE OF PHOSPHOLIPASE D IN PLANT SIGNAL TRANSDUCTION. J Shandong Agricultural University(Natural Science). 2000;31(2):115\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eUesugi Y, Hatanaka T. Phospholipase D mechanism using Streptomyces PLD. 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Phospholipase Dε enhances growth and seed production in response to nitrogen availability. Plant Biotechnol J. 2016;14(3):926\u0026ndash;37.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"soybean, GmPLD, phosphorus deficiency, drought stress, low nitrogen stress, salt stress","lastPublishedDoi":"10.21203/rs.3.rs-7469438/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7469438/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePhospholipase 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 (\u003cem\u003eGlycine max\u003c/em\u003e), particularly its functional relevance to nutrient deficiencies, remains limited. We identified 25 \u003cem\u003eGmPLD\u003c/em\u003e 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 (\u003cem\u003eGmPLDφ1/2\u003c/em\u003e). 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 \u003cem\u003eGmPLD\u003c/em\u003e genes revealed superior haplotypes with significant advantages in phosphorus efficiency-related traits. Furthermore, we experimentally confirmed that both \u003cem\u003eGmPLDφ1\u003c/em\u003e and \u003cem\u003eGmPLDφ2\u003c/em\u003e possess enzymatic activities related to phospholipase D function. Our study provides a systematic analysis of the \u003cem\u003eGmPLD\u003c/em\u003e 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.\u003c/p\u003e","manuscriptTitle":"Genome-wide identification of the PLD gene family and its response to multiple abiotic stresses in soybean (Glycine max)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-25 17:59:18","doi":"10.21203/rs.3.rs-7469438/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-13T13:30:31+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-11T08:50:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"41712765939569144967493706533369340138","date":"2025-09-25T08:35:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-23T13:38:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"75231503339198186469060424627137751639","date":"2025-09-16T09:44:31+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-16T01:37:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-16T00:28:58+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-12T15:51:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-10T09:32:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-09-10T09:25:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d0396e87-801d-4f3b-bb2b-0764316e0f2b","owner":[],"postedDate":"September 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-08T16:04:33+00:00","versionOfRecord":{"articleIdentity":"rs-7469438","link":"https://doi.org/10.1186/s12870-025-07713-1","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2025-12-02 15:57:26","publishedOnDateReadable":"December 2nd, 2025"},"versionCreatedAt":"2025-09-25 17:59:18","video":"","vorDoi":"10.1186/s12870-025-07713-1","vorDoiUrl":"https://doi.org/10.1186/s12870-025-07713-1","workflowStages":[]},"version":"v1","identity":"rs-7469438","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7469438","identity":"rs-7469438","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}