Integration of proteomic, metabolomic, and ubiquitinomic analyses reveals potential mechanisms underlying low-phosphorus stress adaptation in soybean roots

Integration of proteomic, metabolomic, and ubiquitinomic analyses reveals potential mechanisms underlying low-phosphorus stress adaptation in soybean roots | 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 Integration of proteomic, metabolomic, and ubiquitinomic analyses reveals potential mechanisms underlying low-phosphorus stress adaptation in soybean roots Li Tan, Yuechen Tan, Jinqin Wang, Guiyang Shi, Fuli Li, Zhu Chen, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8083659/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Feb, 2026 Read the published version in BMC Plant Biology → Version 1 posted 24 You are reading this latest preprint version Abstract • Low-phosphorus (P) stress is a critical factor limiting soybean growth and yield. Ubiquitination, a post-translational protein modification, is increasingly recognised as a regulator of plant adaptive responses to nutrient limitation, including P deficiency. However, the mechanisms by which ubiquitination mediates soybean tolerance to low P remain underexplored. The present study aimed to elucidate the molecular basis of P efficiency in soybean, focusing on the role of ubiquitination. • A P-efficient soybean genotype, Qiandou 11, was hydroponically cultivated under low or normal P levels to investigate P uptake mechanisms. Proteomic, metabolomic, and ubiquitinomic analyses were performed to identify the metabolic pathways and proteins regulating the soybean root system in response to P deficiency. • The results indicated that QD11 rapidly adapted to P deficiency by increasing the levels of small-molecule-size organic acids and enhancing specific root length. A total of 377 differentially accumulated metabolites and 1,059 differentially expressed proteins (DEPs) were identified. The sample with the largest number of DEPs was selected for ubiquitination analysis, revealing 929 differential ubiquitination sites (585 upregulated and 344 downregulated) in 585 proteins. Notably, these proteins were significantly enriched in glycolysis, phenylpropane biosynthesis, and isoflavone biosynthesis pathways. Integrated multi-omics analysis revealed that phosphoenolpyruvate carboxylase and phenylalanine ammonia-lyase are hub proteins involved in carbon allocation during the soybean root response to low-P stress, and their regulation may be mediated by ubiquitination. • These findings elucidate ubiquitin-mediated regulatory mechanisms and key physiological traits associated with low-P tolerance in soybean. This study provides valuable insights for breeding P-efficient soybean varieties. metabolomics phosphorus uptake post-translational modification proteomics soybean ubiquitination Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 One-Sentence Summary Ubiquitination mediates the regulation of PEPC , PAL , and HCT , promoting the synthesis of organic acids and anthocyanins, and serving as a key mechanism of low-phosphorus tolerance in soybean root systems. Introduction Phosphorus (P), a fundamental macronutrient, supports essential plant functions, including genetic information transfer, energy fixation and release, and cell membrane formation. Soil P deficiency represents a major limitation to plant productivity worldwide [ 1 ]. Although phosphate fertilisers are commonly applied to alleviate this deficiency, their effectiveness is often restricted by P fixation with iron, aluminium, and calcium ions in the soil, which renders it unavailable to plants [ 2 ]. Moreover, excessive P use depletes non-renewable P resources and contributes to environmental pollution [ 3 ]. Therefore, revealing the molecular mechanisms underlying efficient P acquisition and utilisation is crucial for sustainable crop production and global food security [ 4 ]. In plants, P acquisition involves a multi-layered and highly coordinated system, with the phosphate transporter 1 (PHT1) family of phosphate transporters playing a central role by facilitating inorganic phosphate (Pi) uptake. For example, OsPT1 in rice [ 5 ] and PHT1;1 in Arabidopsis [ 6 ] are high-affinity Pi transporters that are strongly induced under P deficiency, thereby enhancing Pi acquisition. Moreover, certain isoforms, such as LePT1 [ 7 ], SbPT9 [ 8 ], and TaPT31-7A [ 9 ], are specifically localised in arbuscule-containing cells colonised by arbuscular mycorrhizal fungi (AMF), where they mediate Pi transfer from fungal hyphae to host plants. The extensive extraradical mycelium produced by AMF considerably expands the P foraging capacity of the roots. In addition to symbiotic adaptations, low-P conditions also induce substantial remodelling of root system architecture. This remodelling includes the formation of shallow root systems to explore the topsoil layers, increased root hair density, and lateral root proliferation, all of which amplify the absorptive surface area and improve Pi uptake efficiency [ 10 ]. These morphological adjustments are regulated by phosphate starvation-induced (PSI) genes, such as GmETO1 [ 11 ] and GmRR1 [ 12 ] in soybean. These genes modulate root architecture through hormone signalling and metabolic reprogramming. Beyond morphological adaptations and symbiotic partnerships, plants enhance P mobility and availability through root exudation. Under P stress conditions, soybean demonstrates a pronounced ability to synthesise and secrete organic acids, such as oxalate, citrate, and succinate [ 13 , 14 ]. These acids chelate metal ions or lower rhizosphere pH, thereby solubilising otherwise fixed P compounds [ 2 , 15 ]. Some organic acids, such as benzoic acid and p-hydroxybenzoic acid [ 16 ], as well as flavonoids, including genistein [ 17 ], also serve as signalling molecules. These molecules, in turn, recruit phosphate-solubilising microbes, including Burkholderia and Pseudomonas, which secrete organic acids and phosphatases that mineralise organic P. This interaction establishes a positive feedback loop that enhances P mobilisation in the rhizosphere [ 18 ]. Thus, plants form a robust P acquisition network by employing an integrated strategy combining membrane transport, architectural modifications, chemical exudation, and biological interactions. Notably, this network can be dissected using multi-omics approaches, including proteomics. Ubiquitination is a common post-translational protein modification in which ubiquitin (Ub) molecules are covalently attached to target proteins, thereby influencing their degradation, subcellular localisation, and interactions. In plants, ubiquitination plays a crucial role in controlling P uptake by regulating the transcription, localisation, and degradation of PHT1 [ 19 , 20 ]. Under sufficient P conditions, plants phosphorylate PHT1 using casein kinase II (CK2), preventing its localisation to the cell membrane. Meanwhile, the E2 Ub ligase PHO2 interacts with and degrades excess PHT1 to maintain homeostasis. However, under P deficiency, CK2 itself is ubiquitinated and degraded, leading to a decrease in ubiquitinated PHT1 levels. This reduction allows root cells to increase P update [ 21 ]. Moreover, ubiquitination plays a role in the root architecture's response to low-P stress. For example, the E3 ligase Transport Inhibitor Response1 (TIR1) modulates auxin and strigolactone signalling by targeting inhibitory proteins involved in axillary branch formation [ 22 ]. The ubiquitination system can further control root architecture by regulating the distribution of the auxin transport protein PIN [ 23 ]. Collectively, these findings underscore the essential role of ubiquitination in plant responses to P [ 21 ]. However, current knowledge on ubiquitination in P signalling is primarily derived from Arabidopsis [ 21 ]; therefore, its role in soybean adaptation to low P remains poorly understood [ 24 , 25 ]. Soybean ( Glycine max (L.) Merr.) is a P-sensitive crop that is highly susceptible to low-P stress [ 26 ], which significantly restricts its growth and potential yield [ 27 ]. This sensitivity renders the soybean an ideal model for investigating P-efficient mechanisms. Therefore, based on earlier field and pot trials that screened soybean germplasm [ 28 ], we utilised the high-P-efficiency genotype “Qiandou 11” (QD11) in hydroponic experiments to investigate physiological dynamics, metabolic profiles, and proteomic responses under low-P stress in the present study. We aimed to elucidate the molecular basis of P efficiency in soybean, focusing on ubiquitination-mediated regulatory mechanisms, and underscore novel strategies for breeding P-efficient varieties. Materials and methods 1.1 Experimental design and plant cultivation Soybean plants of the cultivar Qiandou 11, officially released by the “Institute of Oil Crops, Guizhou Academy of Agricultural Sciences”, were used in this study. Seeds were obtained from the same institute (Guiyang, China), and the cultivar was formally identified and registered with the authorization number “Qian Shen Dou 2016002” by the Guizhou Provincial Crop Variety Approval Committee. No wild collection or CITES-regulated materials were involved. Soybeans were cultivated under hydroponic conditions and subjected to two phosphate treatments: normal P supply (+ Pi, 0.5 mM) or low P stress (–Pi, 0.02 mM). Treatments were based on the soybean hydroponic solution described by Liao et al [ 29 ], with KH₂PO₄ as the P source. Each plant was grown individually in a separate hydroponic container to ensure biological independence and uniformity. To maintain an adequate oxygen supply, the nutrient solution was aerated for 15 min every hour. The pH was stabilised at 5.9 by adjusting with KOH every 48 h. Nutrient solution was refreshed weekly, and plants were maintained under a 16 h light (26°C)/8 h dark (20°C) regime. The plants were first grown under sufficient P supply until the three-leaf stage, after which samples were collected at 0 h (CK), 12 h (H12), 1 day (D1), 3 days (D3), and 7 days (D7) following treatment initiation. For each treatment, triplicate biological replicates were obtained, with each replicate comprising pooled tissues from five uniformly grown plants. This study does not involve human subjects or clinical trials and thus requires no clinical trial registration. 1.2 Determination of plant physiological and biochemical indicators Chlorophyll fluorescence was measured on the youngest fully expanded leaf using a JUNIOR-PAM-modulated chlorophyll fluorometer (Zequan Technology Co., Ltd., Shanghai, China). The roots of two plants per treatment were rinsed with deionised water and immersed in 100 mL of 0.5 mmol L⁻¹ CaCl₂ solution (with two drops of 20 g L⁻¹ thymol) for 4 h. Filtered (0.22 µm) exudates were analysed using high-performance liquid chromatography (HPLC; 1260 Infinity; Agilent Technologies, Santa Clara, CA, USA) to determine carboxylate content, as previously described [ 30 ]. Superoxide dismutase and peroxidase activities were determined using the nitroblue tetrazolium and guaiacol methods, respectively. Malondialdehyde and free proline (Pro) contents were measured using the thiobarbituric acid colourimetric and acid ninhydrin methods, respectively. The plants were divided into shoots and roots at the cotyledonary node. Root morphology was recorded using a scanner (Perfection V850 Pro; Epson, Nagano, Japan) and analysed with Win-RHIZO software (Regent Instruments, Quebec, Canada). Tissue samples were dried, sieved, and analysed for P content using the molybdenum blue method. P accumulation was calculated by multiplying the dry weight of the aboveground or belowground parts by their respective P concentrations. 1.3 Metabolomic analysis Samples for metabolomic, proteomic, and ubiquitinomic analyses were stored at − 80°C. Approximately 200 mg of tissue was extracted with methanol containing 2-chlorophenylalanine (4 ppm), homogenised and centrifuged. The supernatant was filtered (0.22 µm) and transferred for analysis. For quality control, 20 µL aliquots of each sample were pooled. Metabolites were analysed using an HPLC system (Thermo Ultimate 3000, Thermo Fisher Scientific, Waltham, MA, USA) coupled with a Q Exactive Focus mass spectrometer (Thermo Fisher). An ACQUITY UPLC® HSS T3 column (2.1 × 150 mm, 1.8 µm; Waters Co., Milford, MA, USA) was used for HPLC analysis. Gradient elution employed formic acid–water/acetonitrile (positive mode) and ammonium formate–water/acetonitrile (negative mode) at 0.25 mL/min. The Orbitrap system (Thermo Fisher) operated in both positive (3.5 kV) and negative (–2.5 kV) ionisation modes, with standard gas flow and a capillary temperature of 325°C. Full MS scans (m/z 81–1000; resolution 70,000) and data-dependent HCD MS/MS were acquired with dynamic exclusion. Metabolites with ≥ 2-fold changes and VIP ≥ 1 were considered differentially accumulated. 1.4 Protein extraction, digestion, and quantitative proteomics Soybean root samples were ground in liquid nitrogen. The powder was homogenised in extraction buffer (four volumes, w/v; containing 10 mM dithiothreitol, 1% protease inhibitor cocktail, and 50 µM PR-619) by ultrasonication and centrifuged (12,000 × g, 10 min, 4°C). The supernatant was precipitated with 20% trichloroacetic acid at 4°C for 2 h, washed three times with ice-cold acetone (centrifugation at 4,500 × g for 5 min per wash), and dissolved in 8 M urea. Protein concentration was determined using the BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China). Proteins were reduced with 5 mM dithiothreitol (56°C, 30 min), alkylated with 11 mM iodoacetamide (25°C, 15 min, in the dark), diluted with 200 mM triethylammonium bicarbonate (final urea < 2 M), and digested overnight at 37°C with trypsin (enzyme:substrate = 1:50). For proteomic and ubiquitinomic analyses, three biological replicates per treatment and time point were processed, with duplicate technical runs for each. Quantitative proteomics, ubiquitinomics, and parallel reaction monitoring (PRM) were performed by PTM-Biolabs Cd., Ltd. (Hangzhou, China). Detailed liquid chromatography-tandem mass spectrometry (LC-MS/MS) procedures are provided in Additional file 1: Table S1 . Functional annotation was performed using eggnog-mapper (v2.0) for Gene Ontology (GO) terms and the Kyoto Encyclopaedia of Genes and Genomes (KEGG) database for pathway mapping. Differentially expressed proteins (DEPs) were defined as those with ≥ 1.5-fold change ( p < 0.05). Protein structural domain annotation was performed using the PfamScan tool in the Pfam database. Subcellular localisation was annotated using the PSORTb software (v3.0). 1.5 Ubiquitinated peptide enrichment and quantitative ubiquitinomics Peptides were dissolved in IP buffer (pH 8.0) and incubated with pre-washed Ub affinity resin (PTM Biolabs) overnight at 4°C under gentle rotation. After incubation, resins were washed four times with IP buffer and twice with deionised water. Bound peptides were eluted three times with 0.1% trifluoroacetic acid, and the combined eluates were lyophilised. Desalting was performed using C18 ZipTips (MilliporeSigma, Burlington, MA, USA) according to the manufacturer's instructions. Detailed LC-MS/MS procedures are provided in Additional file 1: Table S1 . 1.6 PRM analysis To validate proteomics results, 17 target proteins were selected. For each, one or two characteristic peptides (detection frequency > 80%, p < 0.05) were chosen for PRM analysis. LC separation was performed using an EASY-nLC 1000U HPLC system (Thermo Fisher). Mobile phase A comprised 0.1% formic acid and 2% acetonitrile; mobile phase B comprised 0.1% formic acid and 90% acetonitrile. The gradient for mobile phase B was: 0–16 min, 6–25%; 16–22 min, 25–35%; 22–26 min, 35–80%; 26–30 min, 80%. The flow rate was 500 nL/min. The separated peptides were ionised using an NSI ion source (2.0 kV) and subsequently analysed using a Q Exactive™ Plus Mass Spectrometer (Thermo Fisher). Full MS scans (m/z 360–1305, resolution 70,000) were acquired, followed by MS/MS with normalised collision energy of 27. Fragment ions were detected in the Orbitrap at 35,000 resolution. Automatic gain control thresholds were set at 3E6 for MS1 and 1E5 for MS2, with a 1.6 m/z isolation window. Fragment ion spectra were quantified using Skyline (v21.1). 1.7 Data analysis The effects of low-P stress duration on physiological parameters were assessed using repeated-measures analysis of variance (ANOVA) with time as the within-subject factor, performed with Data Processing System software (v7.05; Zhejiang University, Hangzhou, China). Tukey’s HSD post-hoc tests were used for pairwise comparisons ( p < 0.05). The Circlize package in R was used to generate GO enrichment plots (v0.4.16 [ 31 ]). To analyse the protein motif characteristics, the Motif-x algorithm-based MoMo analysis tool was applied with a threshold motif score of > 16. Principal component analysis (PCA), KEGG pathway enrichment, heatmap, Mfuzz, and motif analyses were performed using the Jingjie Bioinformatics platform ( http://www.ptmbiolab.com ). Results 2.1 Physiological response of soybean to low-P stress To assess the response of QD11 to P deficiency, we examined dynamic changes in the agronomic traits and physiological indices of plants following exposure to the two P treatments. We considered plant P accumulation a sensitive indicator of P status. On day 1 after the induction of low-P stress, marked reductions in P accumulation (Fig. 1 A, p < 0.05) were observed in QD11 plants subjected to low-P (–Pi) conditions compared to those under the normal (+ Pi) condition. Root architecture changed after 3 days of low-P stress. Specifically, root dry weight (Fig. 1 C, p < 0.05), root volume (Additional file 1: Table S2 , p < 0.05), and specific root length (SRL; Fig. 1 D, p < 0.05) significantly increased, whereas root diameter significantly decreased (Additional file 1: Table S2 , p < 0.05). Moreover, the secretion of root carboxylic acids, including oxalic, acetic, lactic, and citric acids (Additional file 1: Table S3), was significantly increased (Fig. 1 E, p < 0.05). These observations suggest that in response to low-P stress, QD11 soybeans allocate large quantities of photosynthetic assimilates to belowground tissues. They also promote P activation and interception by secreting large amounts of carboxylic acids and developing finer and longer roots. After 3 days of treatment, the levels of malondialdehyde were higher in plants under –Pi conditions than in those under + Pi conditions (Fig. 1 F, p < 0.05), indicating an elevated level of cell membrane peroxidation in the stressed plants. Compared with + Pi plants, –Pi-treated QD11 plants were characterised by the rapid activation of the antioxidant system in vivo , exhibiting elevated superoxide dismutase and peroxidase levels after 12 h and 3 days, respectively (Additional file 1: Table S4). Chlorophyll fluorescence parameters were also affected by low-P stress conditions (Additional file 1: Table S5). Specifically, the –Pi group exhibited lower values for Φ PSII , qP, and Fv/Fm than the + Pi group after 12 h of stress. After 1 day of stress exposure, significant reductions were observed in the values of ETR and Fv/Fo, while NPQ was activated to protect the Φ PSII reaction centre. 2.2 Temporal changes in soybean root metabolites under low-P stress A total of 469 metabolites were detected among 15 samples, of which 377 were differentially accumulated (Fig. 2 A). The number of differentially accumulated metabolites (DAMs) gradually increased with prolonged exposure to low-P stress, peaking at 247 on D7 (183 upregulated and 64 downregulated). PERMANOVA revealed significant differences between the treatments (R = 0.78, p = 0.001). Furthermore, PCA revealed that the metabolites of the CK and H12 samples were distributed in the left-hand quadrant of the ordination plot, whereas those of the D1 and D3 samples were distributed in the lower-right-hand quadrant. For D7 samples, the metabolites were tightly clustered in the upper-right-hand corner (Fig. 2 B). Notably, a heatmap analysis revealed a similar pattern (Additional file 2: Figure S1 ). An MFuzz analysis categorised all the detected metabolites into four clusters (Fig. 2 C), among which clusters 1 and 4 contained 142 and 143 metabolites, respectively; their relative abundances declined throughout the 7-day treatment period. These metabolites were primarily enriched in pathways related to pyrimidine, arginine, proline, and tyrosine metabolism (Fig. 2 D), suggesting that low-P stress tends to impair nucleic acid and protein metabolism. Cluster 2 contained 63 metabolites, whose relative abundances decreased rapidly after 12 h of stress, reaching their lowest levels on D1. However, these metabolites exhibited a strong resurgence thereafter, eventually exceeding the levels observed in the control plants on D7. These metabolites were mainly enriched in tyrosine metabolism and aminoacyl-tRNA biosynthesis pathways. These results suggest an active modulation of cellular functions in response to stress conditions after 1 day of exposure. Moreover, Cluster 3 contained 71 metabolites, which were enriched in the flavonoid biosynthesis pathway. The abundance of these 71 metabolites increased following treatment and peaked at D1. 2.3 Temporal proteomic analysis of soybean roots in response to low-P stress Of the 8,883 total identified proteins, 7,027 were quantified, including 1,059 DEPs (Additional file 2: Figure S2 ). PCA (Fig. 2 A) and Pearson’s correlation (Additional file 2: Figure S3) analyses highlighted good reproducibility of the samples. The number of DEPs (478), including those that were downregulated (252), was the highest on D3 (Fig. 3 B). We subjected these DEPs to GO functional annotation, which revealed changes in biological functions in the plants exposed to low-P conditions. Compared with those in the control plants, the DEPs detected in the plants after 12 h of low-P exposure were mainly enriched in terms related to nitrate transmembrane transporter activity (GO:0015112) and nitrite reductase (NO-forming) activity (GO:0050421) (Fig. 3 C; Additional file 1: Table S6). However, after 1 and 3 days of stress, the DEPs were enriched in the cellular carbohydrate biosynthetic process, including the cellular glucan metabolic process, cellulase and xylanase activity, which mediate cell wall assembly (GO:0070726), and the regulation of root morphogenesis (GO:2000067; Fig. 3 D, E; Additional file 1: Table S6). After 7 days of exposure, the DEPs were mainly enriched in cellular responses related to nutrient levels (GO:0031669) and the phenylpropanoid catabolic process (GO:0046271, Fig. 3 F; Additional file 1: Table S6). An MFuzz analysis classified 7027 proteins into six clusters (Additional file 2: Figure S4; Additional file 1: Table S7). Proteins in clusters 1 and 3 showed similar trends. The relative abundances of these proteins declined during the early stress stages, reaching their lowest levels on D3, but rapidly increased thereafter. These proteins were particularly enriched in enzymes involved in flavonoid biosynthesis, phenylalanine, and galactose metabolism pathways. Moreover, GO analysis revealed that cluster 1 and 3 proteins were enriched in terms related to root development and response to gibberellin, aligning with the observed changes in root morphology (Fig. 1 D). In contrast, proteins in clusters 2 and 4 exhibited opposite trends, with their levels peaking on D3 and H12, respectively, before subsequently declining (Additional file 2: Figure S4). These proteins were primarily enriched in nitrogen, alanine, aspartate, and glutamate metabolism, as well as alpha-amino acid biosynthetic processes and ATP-binding processes. This pattern was consistent with the metabolomic results (Fig. 2 C), further indicating that low-P stress impaired the capacity of root cells for amino acid and protein synthesis. Cluster 5 contained 74 proteins, including components of the proteasomes, Ub family (Additional file 2: Figure S4), and ribosomes. The expression of these proteins declined during the early stages of exposure to stress but increased after 12 h, peaking on D3. We performed PRM validation for 36 peptides from 17 proteins associated with P uptake, glycolysis, and phenylpropanoid and flavonoid metabolism. This analysis identified 34 peptides with quantitative results, the peptide fragmentation ion peak area distributions of which are presented in Additional file 1: Table S8. Notably, the consistency observed between the PRM results for the candidate peptides and the trend from the label-free quantitative proteomics analysis ( p < 0.05, n = 15) confirmed the reliability of the proteomic data (Additional file 2: Figure S5). 2.4 Functional analysis of ubiquitinated differential soybean proteins under low-P stress We selected the root samples collected on D3 for quantitative ubiquitinomic profiling and quantified 1,772 proteins with 5,610 peptides containing ubiquitinated lysine sites (Kub). Using a threshold of 1.5-fold change and p < 0.05, we identified 585 upregulated and 344 downregulated ubiquitination sites. Among the identified proteins, the DEPs containing a single ubiquitination site accounted for 67%, and those containing four or more sites accounted for 5%. Ubiquitination-modified differential proteins were primarily enriched in pathways associated with carbohydrate metabolism, energy supply, and phenylpropanoid and flavonoid metabolism (Additional file 2: Figure S6). Proteins enriched in the glycolysis, pyruvate, pentose phosphate, and starch metabolism pathways were characterised by increased ubiquitination at specific Kub sites (Fig. 4 A). In contrast, proteins enriched in the phenylpropanoid biosynthesis pathway exhibited downregulation of the Kub site ubiquitination (Fig. 4 B). Moreover, Ub-modified DEPs were mainly associated with the cytoplasm (38%), plasmodesmata (23%), nucleus (19%), and cytoplasmic membrane (12%) (Fig. 4 C). Notably, the results of the EuKaryotic Orthologous Group (KOG) functional annotations were consistent with those of the KEGG pathway enrichment analysis (Additional file 1: Table S9). Protein structural domain enrichment analysis indicated that proteins with upregulated ubiquitination at Kub sites were enriched in domains such as fructose-bisphosphate aldolase class I, pathogenesis-related proteins of the Bet v I family, NOP5NT domain, and glutathione S -transferase (Additional file 2: Figure S7A). In contrast, proteins with downregulated Kub site ubiquitination were enriched in domains such as cation transporter/ATPase, N terminus, lipoxygenase, ABC transporter and cytochrome, P450 (Additional file 2: Figure S7B). We selected the following six motif sequences: A-4A-1KubE + 3, E-4KubA + 2, KubA + 1A + 2, E-3KubQ + 1, K-7D-1Kub, and R-6KubG + 1 (Additional file 2: Figure S8A). These sequences comprised alanine (A), glutamate (E), L -glutamine (Q), lysine (K), L -aspartic acid (D), and glycine (G) residues. Our results indicate that E, K, and R were mainly located upstream of the ubiquitination site; A was distributed both upstream and downstream; and Q and G were located downstream. Furthermore, a motif enrichment heatmap revealed that the amino acids A, D, E, K, and R were significantly enriched in the regions surrounding the ubiquitination sites (Additional file 2: Figure S8B). Secondary structure analyses of the identified lysine-ubiquitylated proteins indicated high levels of lysine ubiquitylation in the unstructured regions of the proteins, accounting for 67% of the total ( p = 8.57e-04; Fig. 5 A). We further observed that 40% of the ubiquitylated lysine was located on the surfaces of the proteins ( p = 1.21e-05; Fig. 5 B). The changes observed in the glycolysis pathway in response to low-P stress were categorised into two phases (Fig. 6 ). The first phase (0–24 h) involved the transport of sucrose in the leaves to the roots. During this period, we observed upregulated expression of two key enzymes, invertase and 6-phosphofructokinase (PFK), which catalyse irreversible reactions and drive the reaction towards glycolysis. Consequently, we detected elevated levels of sucrose, fructose, UDP-glucose, glucose 1-phosphate, and pyruvate in the root system. However, with prolonged exposure to the low-P stress conditions, the glycolysis pathway entered phase 2 (3–7 days). This phase was mainly characterised by a reduction in the PPi content and glycolytic products. Conversely, the contents of carboxylic acids, such as malate and citrate, were significantly increased. In addition, we observed a downregulation in the expression of invertase, PFK, and phosphoenolpyruvate carboxykinase (PEPCK) in phase 2. This was accompanied by the upregulated expression of phosphoenolpyruvate carboxylase (PEPC), sucrose synthase, UTP-glucose-1-phosphate uridylyltransferase, phosphoglucomutase (PGM), and diphosphate-dependent phosphofructokinase (PFP). In the glycolytic pathway, the protein PEPC4 exhibited the greatest reduction in ubiquitylation levels (Lys-629, D3/CK ratio = 0.16, p < 0.001). These results suggest that under –Pi conditions, ubiquitination may facilitate the substitution of PEPCK, which uses Pi as a substrate with PEP, an enzyme involved in the synthesis of organic acids rather than their direct release. Among the identified proteins, phenylalanine ammonia-lyase (PAL; GLYMA_19G182300) comprised the largest number of ubiquitination sites, with 13 significantly downregulated sites (Fig. 7 ). In the flavonoid biosynthesis (map00941) and isoflavonoid biosynthesis (map00943) pathways, we detected 6, 1, and 4 downregulated ubiquitination sites in chalcone synthase (CHS), isoflavone synthase (IFS), and isoflavone 7-O-glucosyltransferase (IF7G), respectively (Fig. 7 ). The expression of all these proteins peaked in plants subjected to stress for 7 days, coinciding with substantial anthocyanin synthesis (Fig. 7 ). The ubiquitination sites of hydroxycinnamoyl-CoA shikimate hydroxycinnamoyl transferase (HCT), cinnamoyl-CoA reductase (CCR), and isoflavone O-methyltransferase (IOMT) were also upregulated; however, their expression declined during the late stage of stress exposure (Fig. 7 ). These findings suggest that changes in the ubiquitination of key enzymes, such as PAL, CHS, and HCT, may be associated with enhanced carbon allocation towards the anthocyanin synthesis pathway. Discussion In this study, we integrated physiological, metabolomic, proteomic, and ubiquitinomic analyses to identify the potential roles of ubiquitination in regulating soybean root metabolism under P-deficient conditions. Multi-omics analyses revealed that ubiquitination modifications are widely involved in the response to P stress and exhibit significant temporal correlations with the synthesis and accumulation of root secretions. We identified multiple genes that undergo ubiquitination modifications in pathways such as glycolysis, phenylpropanoid, and flavonoid metabolism. Our findings provide novel mechanistic insights into post-translational regulatory strategies for P-efficient adaptation in crops and offer valuable candidate targets for future molecular breeding efforts. The ability of plants to acquire P is positively correlated with organic anion secretion [ 32 , 33 ]. In legumes [ 34 ], such as soybeans [ 35 , 36 ], this ability is particularly pronounced (Fig. 1 E). In the present study, exposure to low-P stress for 3 days significantly increased the contents of six organic acids in root exudates, including malic acid (+ 165%, p < 0.001) and tartaric acid (+ 145%, p < 0.001; Additional file 1: Table S3). Metabolomic analyses further confirmed that the tricarboxylic acid cycle in roots responded rapidly to low-P stress (Fig. 6 ). These results are consistent with a previous study on other P-efficient soybean cultivars, including Maetsue, Kurotome, and Fukuutaka [ 14 ]. Notably, the small organic carboxylic acids secreted by plants help chelate cations, including those of Fe, Al, and Ca, thereby releasing bound soil phosphate in the soil and increasing the available P for root uptake (Wang and Lambers, 2020). PEPC is a key rate-limiting gene that controls the release of organic acids in plants [ 37 , 38 ]. In our study, ubiquitinomic analysis revealed a strong reduction in the ubiquitylation levels of PEPC4 on Lys-629 (D3/CK ratio = 0.16, p < 0.001). Correspondingly, proteomic analysis indicated that the abundance of PEPC4 (D3/CK ratio = 4.10, p < 0.001) and MDH (I1MTU1, D3/CK ratio = 2.59, p < 0.001; I1JZP0, D3/CK ratio = 1.69, p < 0.001) was significantly upregulated in D3 (Fig. 6 ). Under soil sufficient P conditions, PEPC activity is inhibited by the downstream product malate, which is associated with the mono-ubiquitination of the p107 subunit. In contrast, during P depletion, a de-ubiquitinating enzyme converts the PEPC p110 subunit to the p107 subunit [ 39 ]. Consequently, PEPC is no longer inhibited by malic acid, facilitating the catalysis of PEP with HCO 3 – to yield large amounts of Pi and oxaloacetate [ 39 ]. Notably, Pi can replenish the metabolic P pool, while oxaloacetate is metabolised into carboxylic acids that are subsequently secreted from the cell via aluminium-activated malate transporters (Fig. 6 ). Overall, these results suggest that ubiquitination may be involved in modulating PEPC activity, thereby influencing the rate of organic carboxylic acid production. Moreover, genome-wide association studies and comparative transcriptome analyses have demonstrated that PEPC plays a crucial role in regulating root development under low-P stress in soybeans [ 40 ], potentially through its involvement in organic acid secretion [ 41 ]. In summary, PEPC plays a central role in the secretion of organic acids in plants and may also be involved in regulating root architecture. Under low-P stress, the ubiquitination system is likely involved in modulating the catalytic activity of PEPC in soybean roots. In the present study, both the metabolomic and proteomic data consistently indicated that during the later stages of low-P stress exposure (D3–D7), the phenylpropanoid and flavonoid biosynthesis pathways were activated in the soybean roots (Figs. 2 and S3). This activation ultimately led to the substantial accumulation of pelargonidin in the roots (D3/CK ratio = 832.85, p < 0.001; D7/CK ratio = 951.09, p < 0.001; Fig. 7 ). These findings align with previous metabolomic studies in cotton [ 42 ], tobacco [ 43 ], and the high-P-efficiency soybean cultivar YC03-3 [ 44 ]. These results suggest that the phenylpropanoid and flavonoid biosynthesis pathways, which are associated with anthocyanin synthesis, represent key adaptive responses to low-P stress in plant roots. Anthocyanins, such as pelargonidin, are characteristic plant metabolites that respond to low-P stress, helping to protect aboveground chloroplasts from photoinhibition [ 45 ]. However, their role in belowground tissues remains unclear. We observed that low-P stress led to increased levels of malondialdehyde (Fig. 1 F) and antioxidant enzymes (Additional file 1: Table S4), indicating aggravated oxidative stress in the roots. Low P availability induced rhizosphere acidification, increasing the solubility and activity of aluminium and heavy metals in the soil [ 29 ]. This increased solubility impeded the mobility of P within the plant and continuously triggered the production of reactive oxygen species (ROS). Therefore, we propose that the primary function of anthocyanins in roots is to chelate metal ions, whose availability increases owing to rhizosphere acidification, and to scavenge ROS, thereby maintaining cellular homeostasis. Another intriguing hypothesis is that anthocyanins may be secreted into the rhizosphere. In our study, proteomics analysis revealed that the expression of the ABC transporters I1LTM3, I1LZP7, and K7MZ73 significantly increased in response to low-P stress (Additional file 2: Figure S7B). Anthocyanins can be secreted into the soil via ABC transporter proteins [ 46 ]. If the hypothesis is confirmed, root-secreted anthocyanins may support organic acids in activating soil P [ 47 ] and protect these acids from rapid degradation via their antibacterial properties [ 48 ]. We found that flavonoids, such as apigenin (D7/CK ratio = 2.02, p < 0.01), naringenin (D7/CK ratio = 1.94, p < 0.01), and liquiritigenin (D7/CK ratio = 1.87, p < 0.01), also accumulated in the roots under low-P stress conditions (Fig. 7 ). Flavonoids play various roles in mediating plant–microbe interactions, including promoting rhizobial and arbuscular mycorrhizal colonisation and mediating the assembly and function of inter-root microbiomes [ 48 ]. Thus, the ability to synthesise and secrete flavonoids, such as anthocyanins, may serve as a potential indicator for screening P-efficient plants. Anthocyanin synthesis is further regulated by ubiquitination. In our study, among all the identified proteins, PAL exhibited the highest number of ubiquitination sites (13), which also exhibited the most substantial decrease in ubiquitination levels (D3/CK ratio: approximately 0.087–0.443). Moreover, under low-P stress, the ubiquitination levels of the key rate-limiting enzymes of anthocyanin synthesis, including PAL, CHS, and DFR, decreased. In contrast, those of HCT and CCR, which are crucial enzymes in lignin synthesis, significantly increased. By further integrating the results of metabolomic and proteomic analyses, we propose that this ubiquitin-mediated regulation is likely associated with the redistribution of carbon flux within the P-efficient soybean roots. Specifically, carbon is redirected towards the phenylpropanoid-flavonoid synthesis pathway, whereas carbon flux towards the lignin synthesis pathway is suppressed (Fig. 7 ). Notably, the ubiquitination levels of glutathione and the glutathione transferases involved in anthocyanin transport were also significantly upregulated under low-P stress (Additional file 2: Figure S7B). Collectively, these results suggest that a regulatory signalling system may be shared among the synthesis and transport of anthocyanins to ensure rapid post-translational responses to low-P stress. In general, ubiquitination appears to function as a regulatory mechanism modulating carbon flow in the high-P-efficiency soybean genotype. Through this mechanism, soybeans may achieve their low-P response by adjusting the abundance or activity of certain rate-limiting enzymes. Unlike transcriptional regulation, which requires the de novo synthesis of multiple proteins, ubiquitination modifies pre-existing proteins directly. Given that low-P conditions severely limit plant primary metabolism (Additional file 1: Table S5), transcriptional regulation would demand substantial amounts of already scarce energy and photosynthetic products (Fig. 2 C). Therefore, ubiquitination, by targeting pre-existing proteins, may provide a more rapid and energy-saving mechanism for regulating metabolic responses than transcriptional regulation. Conclusion This study provides a systematic overview of molecular responses in the P-efficient soybean genotype “QD11” under low-P stress using integrated physiological, metabolomic, proteomic, and ubiquitinomic analyses. Our results indicated that the low-P tolerance phenotype of QD11 is likely associated with its efficient root system architectural remodelling and rhizosphere acidification capacity. Under low-P stress, QD11 optimised its root system architecture by increasing its root weight, root volume, and SRL to expand its P absorption capacity. Concurrently, its root system specifically secreted organic acids, such as oxalic acid and citric acid, which effectively chelate soil metal ions and activate insoluble P, providing the physiological basis for its efficient P acquisition. In addition, multi-omics analyses revealed the temporal characteristics of soybean root systems. During the early stages of stress (1–3 days), carbon metabolic flux is primarily directed towards organic acid synthesis to support rhizosphere acidification. During the late stages of stress (3–7 days), the phenylpropanoid and flavonoid metabolic pathways are significantly activated, leading to the substantial accumulation of anthocyanins and other flavonoids. We identified proteins regulated by ubiquitination, providing valuable targets that can be explored as genetic resources for breeding P-efficient soybean varieties through gene editing or molecular marker-assisted selection technologies. In future studies, these proteins should be subjected to further functional validation through molecular experiments, such as overexpression, RNA interference, and in vitro ubiquitination assays. Abbreviations P phosphorus QD11 Qiandou 11 DEP differentially expressed protein Pi inorganic phosphate AMF arbuscular mycorrhizal fungi PSI phosphate starvation-induced Ub ubiquitin PHT1 phosphate transporter 1 CK2 casein kinase II SOD superoxide dismutase Pro proline DAM differentially accumulated metabolite PRM parallel reaction monitoring GO Gene Ontology KEGG Kyoto Encyclopaedia of Genes and Genomes SRL specific root length KOG EuKaryotic Orthologous Group PFK 6-phosphofructokinase PPi pyrophosphate PAL phenylalanine ammonia-lyase ROS reactive oxygen species Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Data availability Data is provided within the manuscript or supplementary information files. Funding This work was supported by the National Natural Science Foundation of China (32260804) and the Guizhou Key Technologies for Mountainous Agriculture Research Project (GZNYGJHX-2025001). Authors' contributions LT and YT participated in manuscript writing and figure preparation. JW, GS and FL conducted the soybean soil pot cultivation experiments and contributed to data analysis. ZC, SY and JH designed the research, performed data analysis, reviewed the literature, and contributed to manuscript writing. WZ funding acquisition. RB writing – review & editing. All authors read and approved the final manuscript. Acknowledgements Not applicable. References Cong W-F, Suriyagoda LDB, Lambers H. Tightening the Phosphorus Cycle through Phosphorus-Efficient Crop Genotypes. Trends Plant Sci. 2020;25:967–75. Li H, He K, Zhang Z, Hu Y. Molecular mechanism of phosphorous signaling inducing anthocyanin accumulation in Arabidopsis. Plant Physiol Biochem. 2023;196:121–9. López-Arredondo D, Leyva-González M, González-Morales S, López-Bucio J, Herrera-Estrella L. Phosphate Nutrition: Improving Low-Phosphate Tolerance in Crops. Annu Rev Plant Biol. 2014;65. White P, George T, Gregory P, Bengough A, Hallett P, McKenzie B. Matching roots to their environment. Ann Bot. 2013;112:207–22. Sun S, Gu M, Cao Y, Huang X, Zhang X, Ai P, et al. A Constitutive Expressed Phosphate Transporter, OsPht1;1, Modulates Phosphate Uptake and Translocation in Phosphate-Replete Rice. Plant physiol. 2012;159:1571–81. Chien P-S, Chao Y-T, Chou C-H, Hsu Y-Y, Chiang S-F, Tung C-W, et al. Phosphate transporter PHT1;1 is a key determinant of phosphorus acquisition in Arabidopsis natural accessions. Plant Physiol. 2022;190:682–97. Rosewarne GM, Barker SJ, Smith SE, Smith FA, Schachtman DP. A Lycopersicon esculentum phosphate transporter (LePT1) involved in phosphorus uptake from a vesicular-arbuscular mycorrhizal fungus. New Phytol. 1999;144:507–16. Walder F, Brulé D, Koegel S, Wiemken A, Boller T, Courty P-E. Plant phosphorus acquisition in a common mycorrhizal network: regulation of phosphate transporter genes of the Pht1 family in sorghum and flax. New Phytol. 2015;205:1632–45. Zhang Y, Yang Y, Ma Y, Danfeng W, Wang X, Zhang L et al. A Mycorrhiza-Induced Phosphate Transporter TaPT31-7A Regulating Inorganic Phosphate Uptake, Arbuscular Mycorrhiza Symbiosis, and Plant Growth in Wheat. J Agric Food Chem. 2025;73. Aslam MM, Karanja JK, Dodd IC, Waseem M, Weifeng X. Rhizosheath: An adaptive root trait to improve plant tolerance to phosphorus and water deficits? Plant Cell Environ. 2022;45:2861–74. Zhang H, Yang Y, Sun C, Liu X, Lv L, Hu Z, et al. Up-regulating GmETO1 improves phosphorus uptake and use efficiency by promoting root growth in soybean. Plant Cell Environ. 2020;43:2080–94. Yang Y, Wang L, Zhang D, Zhijun C, Wang Q, Cui R et al. Soybean type-B response regulator GmRR1 mediates phosphorus uptake and yield by modifying root architecture. Plant Physiol. 2023;194. Qin X, Hao S, Hu C, Yu M, Shabala S, Tan Q et al. Revealing the Mechanistic Basis of Regulation of Phosphorus Uptake in Soybean (Glycine max) Roots by Molybdenum: An Integrated Omics Approach. J Agric Food Chem. 2023;71. Tantriani, Cheng W, Oikawa A, Tawaraya K. Phosphorus deficiency alters root length, acid phosphatase activity, organic acids, and metabolites in root exudates of soybean cultivars. Physiol Plant. 2023;175:e14107. Chen ZC, Liao H. Organic acid anions: An effective defensive weapon for plants against aluminum toxicity and phosphorus deficiency in acidic soils. J Genet Genomics. 2016;43:631–8. Zheng J, Shi G, Dini-Andreote F, Yang Y, Jiang Y. Root-derived low molecular weight organic acids modulate keystone microbial taxa impacting plant phosphorus acquisition. J Adv Res. 2025. Zhang B, Jiang H, Zheng G, Zhang Z, Wang T, Chen Q, et al. Genistein Exudation Drives Spatial Root Allocation and Heterogeneous Microbial Communities to Enhance Phosphorus Acquisition in Soybean-Maize Intercropping. Plant Cell Environ. 2025;48:7297–312. Li C, Ma X, Wang Y, Sun Q, Chen M, Zhang C, et al. Root-mediated acidification, phosphatase activity and the phosphorus-cycling microbial community enhance phosphorus mobilization in the rhizosphere of wetland plants. Water Res. 2024;255:121548. Gao H, Wang T, Zhang Y, Li L, Wang C, Guo S, et al. GTPase ROP6 negatively modulates phosphate deficiency through inhibition of PHT1;1 and PHT1;4 in Arabidopsis thaliana. J Integr Plant Biol. 2021;63:1775–86. Yang J, Xie MY, Wang L, Yang ZL, Tian ZH, Wang ZY, et al. A phosphate-starvation induced ring-type e3 ligase maintains phosphate homeostasis partially through osspx2 in rice. Plant Cell Physiol. 2018;59:2564–75. Pan W, Wu Y, Xie Q. Regulation of Ubiquitination Is Central to the Phosphate Starvation Response. Trends Plant Sci. 2019;24:755–69. Pérez-Torres C, López-Bucio J, Cruz-Ramírez A, Ibarra-Laclette E, Dharmasiri S, Estelle M, et al. Phosphate Availability Alters Lateral Root Development in Arabidopsis by Modulating Auxin Sensitivity via a Mechanism Involving the TIR1 Auxin Receptor. Plant Cell. 2009;20:3258–72. Abas L, Benjamins R, Malenica N, Paciorek T, Wiśniewska J, Moulinier-Anzola JC, et al. Intracellular trafficking and proteolysis of the Arabidopsis auxin-efflux facilitator PIN2 are involved in root gravitropism. Nat Cell Biol. 2006;8:249–56. Mo X, Liu G, Zhang Z, Lu X, Liang C, Tian J. Mechanisms Underlying Soybean Response to Phosphorus Deficiency through Integration of Omics Analysis. Int J Mol Sci. 2022;23:4592. Xiong E, Qu X, Li J, Liu H, Ma H, Zhang D et al. The soybean ubiquitin-proteasome system: Current knowledge and future perspective. Plant Genome. 2022;16. Herridge DF, Peoples MB, Boddey RM. Global inputs of biological nitrogen fixation in agricultural systems. Plant Soil. 2008;311:1–18. Cordell D, Drangert J-O, White S. The story of phosphorus: Global food security and food for thought. Glob Environ Change. 2009;19:292–305. Yang T, Yang S, Chen Z, Tan Y, Bol R, Duan H, et al. Global transcriptomic analysis reveals candidate genes associated with different phosphorus acquisition strategies among soybean varieties. Front Plant Sci. 2022;13:1080014. Liao H, Wan H, Shaff J, Wang X, Yan X, Kochian LV. Phosphorus and Aluminum Interactions in Soybean in Relation to Aluminum Tolerance. Exudation of Specific Organic Acids from Different Regions of the Intact Root System. Plant Physiol. 2006;141:674–84. Shen J, Rengel Z, Tang C, Zhang F. Role of phosphorus nutrition in development of cluster roots and release of carboxylates in soil-grown Lupinus albus. Plant Soil. 2003;248:199–206. Gu Z, Gu L, Eils R, Schlesner M, Brors B. circlize Implements and enhances circular visualization in R. Bioinformatics. 2014;30:2811–2. Ding W, Cong W-F, Lambers H. Plant phosphorus-acquisition and -use strategies affect soil carbon cycling. Trends Ecol Evol. 2021;36:899–906. Wang Y, Lambers H. Root-released organic anions in response to low phosphorus availability: recent progress, challenges and future perspectives. Plant Soil. 2020;447:135–56. Dinkelaker B, Römheld V, Marschner H. Citric acid excretion and precipitation of calcium citrate in the rhizosphere of white lupin (Lupinus albus L). Plant Cell Environ. 1989;12:285–92. Krishnapriya V, Pandey R. Root exudation index: screening organic acid exudation and phosphorus acquisition efficiency in soybean genotypes. Crop Pasture Sci. 2016;67:1096–109. Valizadeh G, Rengel Z, Rate A. Wheat genotypes differ in growth and phosphorus uptake when supplied with different sources and rates of phosphorus banded or mixed in soil in pots. Aust J Exp Agric. 2002;42:1103–11. Lu C, Lu Y, Zhao Y. Overexpression of the GsPPC4 gene from Gleditsia sinensis enhances low phosphate tolerance in tobacco by promoting organic acid secretion. Biochem Biophys Res Commun. 2025;779:152426. Toyota K, Koizumi N, Sato F. Transcriptional activation of phosphoenolpyruvate carboxylase by phosphorus deficiency in tobacco. J Exp Bot. 2003;54:961–9. Shane MW, Fedosejevs ET, Plaxton WC. Reciprocal control of anaplerotic phosphoenolpyruvate carboxylase by in vivo monoubiquitination and phosphorylation in developing proteoid roots of phosphate-deficient harsh hakea. Plant Physiol. 2013;161:1634–44. Yu Z, Zheng M, Yan W, Ding X, Zhao T, Yang S. Phosphoenolpyruvic Carboxylase Gene GmPPC6 Negatively Regulates Root System Development Under Low Phosphorus Stress in Soybean. Plant Cell Environ. 2025;48:7886–99. Ham B-K, Chen J, Yan Y, Lucas WJ. Insights into plant phosphate sensing and signaling. Curr Opin Biotechnol. 2018;49:1–9. Iqbal A, Qiang D, Xiangru W, Huiping G, Hengheng Z, Xiling Z, et al. Integrative physiological, transcriptome and metabolome analysis reveals the involvement of carbon and flavonoid biosynthesis in low phosphorus tolerance in cotton. Plant Physiol Biochem. 2023;196:302–17. Jia H, Wang J, Yang Y, Liu G, Bao Y, Cui H. Changes in flavonol content and transcript levels of genes in the flavonoid pathway in tobacco under phosphorus deficiency. Plant Growth Regul. 2015;76:225–31. Mo X, Zhang M, Liang C, Cai L, Tian J. Integration of metabolome and transcriptome analyses highlights soybean roots responding to phosphorus deficiency by modulating phosphorylated metabolite processes. Plant Physiol Biochem. 2019;139:697–706. Landi M, Tattini M, Gould KS. Multiple functional roles of anthocyanins in plant-environment interactions. Environ Exp Bot. 2015;119:4–17. Cesco S, Neumann G, Tomasi N, Pinton R, Weisskopf L. Release of plant-borne flavonoids into the rhizosphere and their role in plant nutrition. Plant Soil. 2010;329:1–25. Tomasi N, Weisskopf L, Renella G, Landi L, Pinton R, Varanini Z, et al. Flavonoids of white lupin roots participate in phosphorus mobilization from soil. Soil Biol Biochem. 2008;40:1971–4. Wang L, Chen M-X, Lam L, Dini-Andreote F, Dai L, Wei Z. Multifaceted roles of flavonoids mediating plant-microbe interactions. Microbiome. 2022;10. Additional Declarations No competing interests reported. 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14:33:03","extension":"html","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":151436,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8083659/v1/d9952daf5a196eb258c4a339.html"},{"id":97456209,"identity":"1adc5206-e848-4f69-9bd6-127482601e62","added_by":"auto","created_at":"2025-12-04 14:33:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":106345,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of phosphorus (P) availability on soybean physiology.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEffects of P availability on six soybean varieties: (A) total phosphorus (P) accumulation in plants, (B) total plant dry weight, (C) total root dry weight, (D) specific root length (SRL), (E) secretion rate of carboxylates, and (F) malondialdehyde (MDA) content. +Pi, normal soil P availability; –Pi, low soil P availability. * denotes a significant difference between P treatments (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8083659/v1/9e478c612f5cd36d48566e82.png"},{"id":97669329,"identity":"4599c9d1-5479-45ad-bdd0-7aa2fc716024","added_by":"auto","created_at":"2025-12-08 09:27:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":662440,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMetabolomic analysis of soybean roots under low-phosphorus (P) stress.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Number of differentially accumulated metabolites (DAMs) among treatments. (B) Principal component analysis. (C) MFuzz analysis based on DAM change patterns. The trend of protein expression in successive treatments is depicted on the left. The horizontal axis is the continuous sample; the vertical axis is the protein expression. DAMs with similar fold-change patterns are grouped into four clusters. Heatmaps of DAMs are plotted on the right side for each cluster. (D) Kyoto Encyclopaedia of Genes and Genomes enrichment analysis of clusters from Mfuzz.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8083659/v1/27a1c41204d65e33999b2e77.png"},{"id":97456210,"identity":"1db81059-368a-408d-a011-320f7b396a79","added_by":"auto","created_at":"2025-12-04 14:33:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":745469,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProteomic analysis of soybean roots.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Principal component analysis. (B) Number of differentially expressed proteins (DEPs) among treatments; Gene Ontology enrichment Circlize plots of DEPs after 12 h (C), 1 day (D), 3 days (E), and 7 days (F) of low-P exposure. From the outside to the inside, the first circle indicates the name of the enriched pathway and the total number of background genes; the second indicates the number of enriched genes and \u003cem\u003ep\u003c/em\u003e-value; and the third represents the number of DEPs enriched in the pathway. The fourth circle presents the enrichment factors for each pathway.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8083659/v1/fcbc1b2a6ffb3c7a914b7b1c.png"},{"id":97668044,"identity":"6de52b1a-ce58-4104-ac4f-e41104b08e5d","added_by":"auto","created_at":"2025-12-08 09:24:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":193648,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKyoto Encyclopaedia of Genes and Genomes enrichment analysis of differentially upregulated (A) and downregulated (B) ubiquitinated proteins.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSubcellular structure localisation and distribution of differentially expressed proteins modified by ubiquitination (C).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8083659/v1/e120d5e54cd7fed2d1cf953a.png"},{"id":97456213,"identity":"8bcc93a9-4749-4fa9-932d-821fc0c29b53","added_by":"auto","created_at":"2025-12-04 14:33:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":112065,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSecondary structure analysis of ubiquitinated proteins under low-phosphorus stress.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Probability of ubiquitination in the α-helix and β-sheet regions; (B) Prediction of ubiquitination site affinity. All lysine sites are presented in green, and ubiquitinated lysine sites are depicted in red.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8083659/v1/f082b4f4bb8b3d259e473b4b.png"},{"id":97669334,"identity":"cc0812ca-2300-4cec-b8aa-07a1bb0e829e","added_by":"auto","created_at":"2025-12-08 09:27:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":278470,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential metabolites, proteins, and ubiquitinated proteins in the glycolysis pathway.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMetabolomic results are presented in the heatmap alongside metabolite names. Proteomic and ubiquitinomic results are depicted in the heatmap alongside the gene ID. The heatmap to the left of the horizontal line presents changes in differential proteins and that to the right shows changes in differential ubiquitin-labelled proteins. High to low expression is indicated by a change in colour from red to blue. PGM, phosphoglucomutase; HK, hexokinase; PFK, 6-phosphofructokinase; PFP, diphosphate-dependent phosphofructokinase; ALDO, fructose-bisphosphate aldolase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PGK, phosphoglycerate kinase; PKc, pyruvate kinase; PEPC, phosphoenolpyruvate carboxylase; PEPCK, phosphoenolpyruvate carboxykinase; MDH, malate dehydrogenase; IDH1, isocitrate dehydrogenase; ACLY, ATP citrate (pro-S)-lyase; ACO, aconitate hydratase.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8083659/v1/4fb77418c5ef871577fbd434.png"},{"id":97667734,"identity":"308023b2-4e80-4e62-a643-355b47c956db","added_by":"auto","created_at":"2025-12-08 09:24:11","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":192665,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential metabolites, proteins, and ubiquitinated proteins in the phenylpropanoid, flavonoid, and isoflavonoid pathways.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMetabolomic results are presented in the heatmap alongside metabolite names. Proteomic and ubiquitinomic results are depicted in the heatmap alongside the gene ID. The heatmap to the left of the horizontal line shows changes in differential proteins, while that to the right illustrates changes in differential ubiquitin-labelled proteins. High to low expression is indicated by a change in colour from red to blue. PAL, phenylalanine ammonia-lyase; HCT, hydroxycinnamoyl-CoA shikimate hydroxycinnamoyl transferase; CCR, cinnamoyl-CoA reductase; CHS, chalcone synthase; CHI, chalcone isomerase; DFR, dihydroflavonol 4-reductase; CHR, chalcone reductase; IFS, isoflavone synthase; HIDH, 2-hydroxyisoflavanone dehydratase; IF7GT, isoflavone 7-O-glucosyltransferase; IOMT, isoflavone O-methyltransferase; IF7MAT, isoflavone 7-O-glucoside-6ʹʹ-O-malonyltransferase; I2ʹH, isoflavone 2ʹ-hydroxylase.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8083659/v1/e930a23b4540f3a52f632308.png"},{"id":103765671,"identity":"9a150d96-6839-4c12-ad6b-98a3e01eaa05","added_by":"auto","created_at":"2026-03-02 16:07:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3006680,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8083659/v1/552afe7f-64fb-4050-8fa3-10e3f8974559.pdf"},{"id":97668402,"identity":"b2bb6d80-be2d-4d65-a435-f5bcc2fa490b","added_by":"auto","created_at":"2025-12-08 09:25:27","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":141538,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1TablesS1S9.docx","url":"https://assets-eu.researchsquare.com/files/rs-8083659/v1/de89796b02fd5603beccd6e6.docx"},{"id":97456218,"identity":"9fbaa0ec-8f85-470e-a1cd-76908faf3254","added_by":"auto","created_at":"2025-12-04 14:33:02","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2178776,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile2FigureS1S8.docx","url":"https://assets-eu.researchsquare.com/files/rs-8083659/v1/54aefd64c70e86acea0deb18.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Integration of proteomic, metabolomic, and ubiquitinomic analyses reveals potential mechanisms underlying low-phosphorus stress adaptation in soybean roots","fulltext":[{"header":"One-Sentence Summary","content":"\u003cp\u003eUbiquitination mediates the regulation of \u003cem\u003ePEPC\u003c/em\u003e, \u003cem\u003ePAL\u003c/em\u003e, and \u003cem\u003eHCT\u003c/em\u003e, promoting the synthesis of organic acids and anthocyanins, and serving as a key mechanism of low-phosphorus tolerance in soybean root systems.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003ePhosphorus (P), a fundamental macronutrient, supports essential plant functions, including genetic information transfer, energy fixation and release, and cell membrane formation. Soil P deficiency represents a major limitation to plant productivity worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Although phosphate fertilisers are commonly applied to alleviate this deficiency, their effectiveness is often restricted by P fixation with iron, aluminium, and calcium ions in the soil, which renders it unavailable to plants [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Moreover, excessive P use depletes non-renewable P resources and contributes to environmental pollution [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, revealing the molecular mechanisms underlying efficient P acquisition and utilisation is crucial for sustainable crop production and global food security [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn plants, P acquisition involves a multi-layered and highly coordinated system, with the phosphate transporter 1 (PHT1) family of phosphate transporters playing a central role by facilitating inorganic phosphate (Pi) uptake. For example, OsPT1 in rice [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] and PHT1;1 in Arabidopsis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] are high-affinity Pi transporters that are strongly induced under P deficiency, thereby enhancing Pi acquisition. Moreover, certain isoforms, such as LePT1 [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], SbPT9 [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], and TaPT31-7A [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], are specifically localised in arbuscule-containing cells colonised by arbuscular mycorrhizal fungi (AMF), where they mediate Pi transfer from fungal hyphae to host plants. The extensive extraradical mycelium produced by AMF considerably expands the P foraging capacity of the roots. In addition to symbiotic adaptations, low-P conditions also induce substantial remodelling of root system architecture. This remodelling includes the formation of shallow root systems to explore the topsoil layers, increased root hair density, and lateral root proliferation, all of which amplify the absorptive surface area and improve Pi uptake efficiency [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. These morphological adjustments are regulated by phosphate starvation-induced (PSI) genes, such as \u003cem\u003eGmETO1\u003c/em\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and \u003cem\u003eGmRR1\u003c/em\u003e [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] in soybean. These genes modulate root architecture through hormone signalling and metabolic reprogramming.\u003c/p\u003e\u003cp\u003eBeyond morphological adaptations and symbiotic partnerships, plants enhance P mobility and availability through root exudation. Under P stress conditions, soybean demonstrates a pronounced ability to synthesise and secrete organic acids, such as oxalate, citrate, and succinate [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. These acids chelate metal ions or lower rhizosphere pH, thereby solubilising otherwise fixed P compounds [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Some organic acids, such as benzoic acid and p-hydroxybenzoic acid [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], as well as flavonoids, including genistein [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], also serve as signalling molecules. These molecules, in turn, recruit phosphate-solubilising microbes, including Burkholderia and Pseudomonas, which secrete organic acids and phosphatases that mineralise organic P. This interaction establishes a positive feedback loop that enhances P mobilisation in the rhizosphere [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Thus, plants form a robust P acquisition network by employing an integrated strategy combining membrane transport, architectural modifications, chemical exudation, and biological interactions. Notably, this network can be dissected using multi-omics approaches, including proteomics.\u003c/p\u003e\u003cp\u003eUbiquitination is a common post-translational protein modification in which ubiquitin (Ub) molecules are covalently attached to target proteins, thereby influencing their degradation, subcellular localisation, and interactions. In plants, ubiquitination plays a crucial role in controlling P uptake by regulating the transcription, localisation, and degradation of PHT1 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Under sufficient P conditions, plants phosphorylate PHT1 using casein kinase II (CK2), preventing its localisation to the cell membrane. Meanwhile, the E2 Ub ligase PHO2 interacts with and degrades excess PHT1 to maintain homeostasis. However, under P deficiency, CK2 itself is ubiquitinated and degraded, leading to a decrease in ubiquitinated PHT1 levels. This reduction allows root cells to increase P update [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Moreover, ubiquitination plays a role in the root architecture's response to low-P stress. For example, the E3 ligase Transport Inhibitor Response1 (TIR1) modulates auxin and strigolactone signalling by targeting inhibitory proteins involved in axillary branch formation [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The ubiquitination system can further control root architecture by regulating the distribution of the auxin transport protein PIN [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Collectively, these findings underscore the essential role of ubiquitination in plant responses to P [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, current knowledge on ubiquitination in P signalling is primarily derived from Arabidopsis [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]; therefore, its role in soybean adaptation to low P remains poorly understood [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSoybean (\u003cem\u003eGlycine max\u003c/em\u003e (L.) Merr.) is a P-sensitive crop that is highly susceptible to low-P stress [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], which significantly restricts its growth and potential yield [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. This sensitivity renders the soybean an ideal model for investigating P-efficient mechanisms. Therefore, based on earlier field and pot trials that screened soybean germplasm [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], we utilised the high-P-efficiency genotype \u0026ldquo;Qiandou 11\u0026rdquo; (QD11) in hydroponic experiments to investigate physiological dynamics, metabolic profiles, and proteomic responses under low-P stress in the present study. We aimed to elucidate the molecular basis of P efficiency in soybean, focusing on ubiquitination-mediated regulatory mechanisms, and underscore novel strategies for breeding P-efficient varieties.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e1.1 Experimental design and plant cultivation\u003c/h2\u003e\u003cp\u003eSoybean plants of the cultivar Qiandou 11, officially released by the \u0026ldquo;Institute of Oil Crops, Guizhou Academy of Agricultural Sciences\u0026rdquo;, were used in this study. Seeds were obtained from the same institute (Guiyang, China), and the cultivar was formally identified and registered with the authorization number \u0026ldquo;Qian Shen Dou 2016002\u0026rdquo; by the Guizhou Provincial Crop Variety Approval Committee. No wild collection or CITES-regulated materials were involved.\u003c/p\u003e\u003cp\u003eSoybeans were cultivated under hydroponic conditions and subjected to two phosphate treatments: normal P supply (+\u0026thinsp;Pi, 0.5 mM) or low P stress (\u0026ndash;Pi, 0.02 mM). Treatments were based on the soybean hydroponic solution described by Liao et al [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], with KH₂PO₄ as the P source. Each plant was grown individually in a separate hydroponic container to ensure biological independence and uniformity. To maintain an adequate oxygen supply, the nutrient solution was aerated for 15 min every hour. The pH was stabilised at 5.9 by adjusting with KOH every 48 h. Nutrient solution was refreshed weekly, and plants were maintained under a 16 h light (26\u0026deg;C)/8 h dark (20\u0026deg;C) regime. The plants were first grown under sufficient P supply until the three-leaf stage, after which samples were collected at 0 h (CK), 12 h (H12), 1 day (D1), 3 days (D3), and 7 days (D7) following treatment initiation. For each treatment, triplicate biological replicates were obtained, with each replicate comprising pooled tissues from five uniformly grown plants. This study does not involve human subjects or clinical trials and thus requires no clinical trial registration.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e1.2 Determination of plant physiological and biochemical indicators\u003c/h2\u003e\u003cp\u003eChlorophyll fluorescence was measured on the youngest fully expanded leaf using a JUNIOR-PAM-modulated chlorophyll fluorometer (Zequan Technology Co., Ltd., Shanghai, China). The roots of two plants per treatment were rinsed with deionised water and immersed in 100 mL of 0.5 mmol L⁻\u0026sup1; CaCl₂ solution (with two drops of 20 g L⁻\u0026sup1; thymol) for 4 h. Filtered (0.22 \u0026micro;m) exudates were analysed using high-performance liquid chromatography (HPLC; 1260 Infinity; Agilent Technologies, Santa Clara, CA, USA) to determine carboxylate content, as previously described [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSuperoxide dismutase and peroxidase activities were determined using the nitroblue tetrazolium and guaiacol methods, respectively. Malondialdehyde and free proline (Pro) contents were measured using the thiobarbituric acid colourimetric and acid ninhydrin methods, respectively.\u003c/p\u003e\u003cp\u003eThe plants were divided into shoots and roots at the cotyledonary node. Root morphology was recorded using a scanner (Perfection V850 Pro; Epson, Nagano, Japan) and analysed with Win-RHIZO software (Regent Instruments, Quebec, Canada). Tissue samples were dried, sieved, and analysed for P content using the molybdenum blue method. P accumulation was calculated by multiplying the dry weight of the aboveground or belowground parts by their respective P concentrations.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e1.3 Metabolomic analysis\u003c/h2\u003e\u003cp\u003eSamples for metabolomic, proteomic, and ubiquitinomic analyses were stored at \u0026minus;\u0026thinsp;80\u0026deg;C. Approximately 200 mg of tissue was extracted with methanol containing 2-chlorophenylalanine (4 ppm), homogenised and centrifuged. The supernatant was filtered (0.22 \u0026micro;m) and transferred for analysis. For quality control, 20 \u0026micro;L aliquots of each sample were pooled. Metabolites were analysed using an HPLC system (Thermo Ultimate 3000, Thermo Fisher Scientific, Waltham, MA, USA) coupled with a Q Exactive Focus mass spectrometer (Thermo Fisher).\u003c/p\u003e\u003cp\u003eAn ACQUITY UPLC\u0026reg; HSS T3 column (2.1 \u0026times; 150 mm, 1.8 \u0026micro;m; Waters Co., Milford, MA, USA) was used for HPLC analysis. Gradient elution employed formic acid\u0026ndash;water/acetonitrile (positive mode) and ammonium formate\u0026ndash;water/acetonitrile (negative mode) at 0.25 mL/min. The Orbitrap system (Thermo Fisher) operated in both positive (3.5 kV) and negative (\u0026ndash;2.5 kV) ionisation modes, with standard gas flow and a capillary temperature of 325\u0026deg;C. Full MS scans (m/z 81\u0026ndash;1000; resolution 70,000) and data-dependent HCD MS/MS were acquired with dynamic exclusion. Metabolites with \u0026ge;\u0026thinsp;2-fold changes and VIP\u0026thinsp;\u0026ge;\u0026thinsp;1 were considered differentially accumulated.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e1.4 Protein extraction, digestion, and quantitative proteomics\u003c/h2\u003e\u003cp\u003eSoybean root samples were ground in liquid nitrogen. The powder was homogenised in extraction buffer (four volumes, w/v; containing 10 mM dithiothreitol, 1% protease inhibitor cocktail, and 50 \u0026micro;M PR-619) by ultrasonication and centrifuged (12,000 \u0026times; g, 10 min, 4\u0026deg;C). The supernatant was precipitated with 20% trichloroacetic acid at 4\u0026deg;C for 2 h, washed three times with ice-cold acetone (centrifugation at 4,500 \u0026times; \u003cem\u003eg\u003c/em\u003e for 5 min per wash), and dissolved in 8 M urea. Protein concentration was determined using the BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China). Proteins were reduced with 5 mM dithiothreitol (56\u0026deg;C, 30 min), alkylated with 11 mM iodoacetamide (25\u0026deg;C, 15 min, in the dark), diluted with 200 mM triethylammonium bicarbonate (final urea\u0026thinsp;\u0026lt;\u0026thinsp;2 M), and digested overnight at 37\u0026deg;C with trypsin (enzyme:substrate\u0026thinsp;=\u0026thinsp;1:50). For proteomic and ubiquitinomic analyses, three biological replicates per treatment and time point were processed, with duplicate technical runs for each. Quantitative proteomics, ubiquitinomics, and parallel reaction monitoring (PRM) were performed by PTM-Biolabs Cd., Ltd. (Hangzhou, China). Detailed liquid chromatography-tandem mass spectrometry (LC-MS/MS) procedures are provided in Additional file 1: Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eFunctional annotation was performed using eggnog-mapper (v2.0) for Gene Ontology (GO) terms and the Kyoto Encyclopaedia of Genes and Genomes (KEGG) database for pathway mapping. Differentially expressed proteins (DEPs) were defined as those with \u0026ge;\u0026thinsp;1.5-fold change (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Protein structural domain annotation was performed using the PfamScan tool in the Pfam database. Subcellular localisation was annotated using the PSORTb software (v3.0).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e1.5 Ubiquitinated peptide enrichment and quantitative ubiquitinomics\u003c/h2\u003e\u003cp\u003ePeptides were dissolved in IP buffer (pH 8.0) and incubated with pre-washed Ub affinity resin (PTM Biolabs) overnight at 4\u0026deg;C under gentle rotation. After incubation, resins were washed four times with IP buffer and twice with deionised water. Bound peptides were eluted three times with 0.1% trifluoroacetic acid, and the combined eluates were lyophilised. Desalting was performed using C18 ZipTips (MilliporeSigma, Burlington, MA, USA) according to the manufacturer's instructions. Detailed LC-MS/MS procedures are provided in Additional file 1: Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e1.6 PRM analysis\u003c/h2\u003e\u003cp\u003eTo validate proteomics results, 17 target proteins were selected. For each, one or two characteristic peptides (detection frequency\u0026thinsp;\u0026gt;\u0026thinsp;80%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were chosen for PRM analysis. LC separation was performed using an EASY-nLC 1000U HPLC system (Thermo Fisher). Mobile phase A comprised 0.1% formic acid and 2% acetonitrile; mobile phase B comprised 0.1% formic acid and 90% acetonitrile. The gradient for mobile phase B was: 0\u0026ndash;16 min, 6\u0026ndash;25%; 16\u0026ndash;22 min, 25\u0026ndash;35%; 22\u0026ndash;26 min, 35\u0026ndash;80%; 26\u0026ndash;30 min, 80%. The flow rate was 500 nL/min. The separated peptides were ionised using an NSI ion source (2.0 kV) and subsequently analysed using a Q Exactive\u0026trade; Plus Mass Spectrometer (Thermo Fisher). Full MS scans (m/z 360\u0026ndash;1305, resolution 70,000) were acquired, followed by MS/MS with normalised collision energy of 27. Fragment ions were detected in the Orbitrap at 35,000 resolution. Automatic gain control thresholds were set at 3E6 for MS1 and 1E5 for MS2, with a 1.6 m/z isolation window. Fragment ion spectra were quantified using Skyline (v21.1).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e1.7 Data analysis\u003c/h2\u003e\u003cp\u003eThe effects of low-P stress duration on physiological parameters were assessed using repeated-measures analysis of variance (ANOVA) with time as the within-subject factor, performed with Data Processing System software (v7.05; Zhejiang University, Hangzhou, China). Tukey\u0026rsquo;s HSD post-hoc tests were used for pairwise comparisons (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The Circlize package in R was used to generate GO enrichment plots (v0.4.16 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]). To analyse the protein motif characteristics, the Motif-x algorithm-based MoMo analysis tool was applied with a threshold motif score of \u0026gt;\u0026thinsp;16. Principal component analysis (PCA), KEGG pathway enrichment, heatmap, Mfuzz, and motif analyses were performed using the Jingjie Bioinformatics platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ptmbiolab.com\u003c/span\u003e\u003cspan address=\"http://www.ptmbiolab.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Physiological response of soybean to low-P stress\u003c/h2\u003e\u003cp\u003eTo assess the response of QD11 to P deficiency, we examined dynamic changes in the agronomic traits and physiological indices of plants following exposure to the two P treatments. We considered plant P accumulation a sensitive indicator of P status. On day 1 after the induction of low-P stress, marked reductions in P accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were observed in QD11 plants subjected to low-P (\u0026ndash;Pi) conditions compared to those under the normal (+\u0026thinsp;Pi) condition. Root architecture changed after 3 days of low-P stress. Specifically, root dry weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), root volume (Additional file 1: Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and specific root length (SRL; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) significantly increased, whereas root diameter significantly decreased (Additional file 1: Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Moreover, the secretion of root carboxylic acids, including oxalic, acetic, lactic, and citric acids (Additional file 1: Table S3), was significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These observations suggest that in response to low-P stress, QD11 soybeans allocate large quantities of photosynthetic assimilates to belowground tissues. They also promote P activation and interception by secreting large amounts of carboxylic acids and developing finer and longer roots.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAfter 3 days of treatment, the levels of malondialdehyde were higher in plants under \u0026ndash;Pi conditions than in those under +\u0026thinsp;Pi conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating an elevated level of cell membrane peroxidation in the stressed plants. Compared with +\u0026thinsp;Pi plants, \u0026ndash;Pi-treated QD11 plants were characterised by the rapid activation of the antioxidant system \u003cem\u003ein vivo\u003c/em\u003e, exhibiting elevated superoxide dismutase and peroxidase levels after 12 h and 3 days, respectively (Additional file 1: Table S4). Chlorophyll fluorescence parameters were also affected by low-P stress conditions (Additional file 1: Table S5). Specifically, the \u0026ndash;Pi group exhibited lower values for Φ\u003csub\u003ePSII\u003c/sub\u003e, qP, and Fv/Fm than the +\u0026thinsp;Pi group after 12 h of stress. After 1 day of stress exposure, significant reductions were observed in the values of ETR and Fv/Fo, while NPQ was activated to protect the Φ\u003csub\u003ePSII\u003c/sub\u003e reaction centre.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Temporal changes in soybean root metabolites under low-P stress\u003c/h2\u003e\u003cp\u003eA total of 469 metabolites were detected among 15 samples, of which 377 were differentially accumulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The number of differentially accumulated metabolites (DAMs) gradually increased with prolonged exposure to low-P stress, peaking at 247 on D7 (183 upregulated and 64 downregulated). PERMANOVA revealed significant differences between the treatments (R\u0026thinsp;=\u0026thinsp;0.78, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001). Furthermore, PCA revealed that the metabolites of the CK and H12 samples were distributed in the left-hand quadrant of the ordination plot, whereas those of the D1 and D3 samples were distributed in the lower-right-hand quadrant. For D7 samples, the metabolites were tightly clustered in the upper-right-hand corner (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Notably, a heatmap analysis revealed a similar pattern (Additional file 2: Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAn MFuzz analysis categorised all the detected metabolites into four clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), among which clusters 1 and 4 contained 142 and 143 metabolites, respectively; their relative abundances declined throughout the 7-day treatment period. These metabolites were primarily enriched in pathways related to pyrimidine, arginine, proline, and tyrosine metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), suggesting that low-P stress tends to impair nucleic acid and protein metabolism. Cluster 2 contained 63 metabolites, whose relative abundances decreased rapidly after 12 h of stress, reaching their lowest levels on D1. However, these metabolites exhibited a strong resurgence thereafter, eventually exceeding the levels observed in the control plants on D7. These metabolites were mainly enriched in tyrosine metabolism and aminoacyl-tRNA biosynthesis pathways. These results suggest an active modulation of cellular functions in response to stress conditions after 1 day of exposure. Moreover, Cluster 3 contained 71 metabolites, which were enriched in the flavonoid biosynthesis pathway. The abundance of these 71 metabolites increased following treatment and peaked at D1.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Temporal proteomic analysis of soybean roots in response to low-P stress\u003c/h2\u003e\u003cp\u003eOf the 8,883 total identified proteins, 7,027 were quantified, including 1,059 DEPs (Additional file 2: Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). PCA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and Pearson\u0026rsquo;s correlation (Additional file 2: Figure S3) analyses highlighted good reproducibility of the samples. The number of DEPs (478), including those that were downregulated (252), was the highest on D3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). We subjected these DEPs to GO functional annotation, which revealed changes in biological functions in the plants exposed to low-P conditions. Compared with those in the control plants, the DEPs detected in the plants after 12 h of low-P exposure were mainly enriched in terms related to nitrate transmembrane transporter activity (GO:0015112) and nitrite reductase (NO-forming) activity (GO:0050421) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC; Additional file 1: Table S6). However, after 1 and 3 days of stress, the DEPs were enriched in the cellular carbohydrate biosynthetic process, including the cellular glucan metabolic process, cellulase and xylanase activity, which mediate cell wall assembly (GO:0070726), and the regulation of root morphogenesis (GO:2000067; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, E; Additional file 1: Table S6). After 7 days of exposure, the DEPs were mainly enriched in cellular responses related to nutrient levels (GO:0031669) and the phenylpropanoid catabolic process (GO:0046271, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF; Additional file 1: Table S6).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAn MFuzz analysis classified 7027 proteins into six clusters (Additional file 2: Figure S4; Additional file 1: Table S7). Proteins in clusters 1 and 3 showed similar trends. The relative abundances of these proteins declined during the early stress stages, reaching their lowest levels on D3, but rapidly increased thereafter. These proteins were particularly enriched in enzymes involved in flavonoid biosynthesis, phenylalanine, and galactose metabolism pathways. Moreover, GO analysis revealed that cluster 1 and 3 proteins were enriched in terms related to root development and response to gibberellin, aligning with the observed changes in root morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). In contrast, proteins in clusters 2 and 4 exhibited opposite trends, with their levels peaking on D3 and H12, respectively, before subsequently declining (Additional file 2: Figure S4). These proteins were primarily enriched in nitrogen, alanine, aspartate, and glutamate metabolism, as well as alpha-amino acid biosynthetic processes and ATP-binding processes. This pattern was consistent with the metabolomic results (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), further indicating that low-P stress impaired the capacity of root cells for amino acid and protein synthesis. Cluster 5 contained 74 proteins, including components of the proteasomes, Ub family (Additional file 2: Figure S4), and ribosomes. The expression of these proteins declined during the early stages of exposure to stress but increased after 12 h, peaking on D3.\u003c/p\u003e\u003cp\u003eWe performed PRM validation for 36 peptides from 17 proteins associated with P uptake, glycolysis, and phenylpropanoid and flavonoid metabolism. This analysis identified 34 peptides with quantitative results, the peptide fragmentation ion peak area distributions of which are presented in Additional file 1: Table S8. Notably, the consistency observed between the PRM results for the candidate peptides and the trend from the label-free quantitative proteomics analysis (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;15) confirmed the reliability of the proteomic data (Additional file 2: Figure S5).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Functional analysis of ubiquitinated differential soybean proteins under low-P stress\u003c/h2\u003e\u003cp\u003eWe selected the root samples collected on D3 for quantitative ubiquitinomic profiling and quantified 1,772 proteins with 5,610 peptides containing ubiquitinated lysine sites (Kub). Using a threshold of 1.5-fold change and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, we identified 585 upregulated and 344 downregulated ubiquitination sites. Among the identified proteins, the DEPs containing a single ubiquitination site accounted for 67%, and those containing four or more sites accounted for 5%. Ubiquitination-modified differential proteins were primarily enriched in pathways associated with carbohydrate metabolism, energy supply, and phenylpropanoid and flavonoid metabolism (Additional file 2: Figure S6). Proteins enriched in the glycolysis, pyruvate, pentose phosphate, and starch metabolism pathways were characterised by increased ubiquitination at specific Kub sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In contrast, proteins enriched in the phenylpropanoid biosynthesis pathway exhibited downregulation of the Kub site ubiquitination (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Moreover, Ub-modified DEPs were mainly associated with the cytoplasm (38%), plasmodesmata (23%), nucleus (19%), and cytoplasmic membrane (12%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Notably, the results of the EuKaryotic Orthologous Group (KOG) functional annotations were consistent with those of the KEGG pathway enrichment analysis (Additional file 1: Table S9).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eProtein structural domain enrichment analysis indicated that proteins with upregulated ubiquitination at Kub sites were enriched in domains such as fructose-bisphosphate aldolase class I, pathogenesis-related proteins of the Bet v I family, NOP5NT domain, and glutathione \u003cem\u003eS\u003c/em\u003e-transferase (Additional file 2: Figure S7A). In contrast, proteins with downregulated Kub site ubiquitination were enriched in domains such as cation transporter/ATPase, N terminus, lipoxygenase, ABC transporter and cytochrome, P450 (Additional file 2: Figure S7B).\u003c/p\u003e\u003cp\u003eWe selected the following six motif sequences: A-4A-1KubE\u0026thinsp;+\u0026thinsp;3, E-4KubA\u0026thinsp;+\u0026thinsp;2, KubA\u0026thinsp;+\u0026thinsp;1A\u0026thinsp;+\u0026thinsp;2, E-3KubQ\u0026thinsp;+\u0026thinsp;1, K-7D-1Kub, and R-6KubG\u0026thinsp;+\u0026thinsp;1 (Additional file 2: Figure S8A). These sequences comprised alanine (A), glutamate (E), \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eL\u003c/span\u003e-glutamine (Q), lysine (K), \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eL\u003c/span\u003e-aspartic acid (D), and glycine (G) residues. Our results indicate that E, K, and R were mainly located upstream of the ubiquitination site; A was distributed both upstream and downstream; and Q and G were located downstream. Furthermore, a motif enrichment heatmap revealed that the amino acids A, D, E, K, and R were significantly enriched in the regions surrounding the ubiquitination sites (Additional file 2: Figure S8B). Secondary structure analyses of the identified lysine-ubiquitylated proteins indicated high levels of lysine ubiquitylation in the unstructured regions of the proteins, accounting for 67% of the total (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.57e-04; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). We further observed that 40% of the ubiquitylated lysine was located on the surfaces of the proteins (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.21e-05; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe changes observed in the glycolysis pathway in response to low-P stress were categorised into two phases (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The first phase (0\u0026ndash;24 h) involved the transport of sucrose in the leaves to the roots. During this period, we observed upregulated expression of two key enzymes, invertase and 6-phosphofructokinase (PFK), which catalyse irreversible reactions and drive the reaction towards glycolysis. Consequently, we detected elevated levels of sucrose, fructose, UDP-glucose, glucose 1-phosphate, and pyruvate in the root system. However, with prolonged exposure to the low-P stress conditions, the glycolysis pathway entered phase 2 (3\u0026ndash;7 days). This phase was mainly characterised by a reduction in the PPi content and glycolytic products. Conversely, the contents of carboxylic acids, such as malate and citrate, were significantly increased. In addition, we observed a downregulation in the expression of invertase, PFK, and phosphoenolpyruvate carboxykinase (PEPCK) in phase 2. This was accompanied by the upregulated expression of phosphoenolpyruvate carboxylase (PEPC), sucrose synthase, UTP-glucose-1-phosphate uridylyltransferase, phosphoglucomutase (PGM), and diphosphate-dependent phosphofructokinase (PFP). In the glycolytic pathway, the protein PEPC4 exhibited the greatest reduction in ubiquitylation levels (Lys-629, D3/CK ratio\u0026thinsp;=\u0026thinsp;0.16, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). These results suggest that under \u0026ndash;Pi conditions, ubiquitination may facilitate the substitution of PEPCK, which uses Pi as a substrate with PEP, an enzyme involved in the synthesis of organic acids rather than their direct release.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAmong the identified proteins, phenylalanine ammonia-lyase (PAL; GLYMA_19G182300) comprised the largest number of ubiquitination sites, with 13 significantly downregulated sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). In the flavonoid biosynthesis (map00941) and isoflavonoid biosynthesis (map00943) pathways, we detected 6, 1, and 4 downregulated ubiquitination sites in chalcone synthase (CHS), isoflavone synthase (IFS), and isoflavone 7-O-glucosyltransferase (IF7G), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The expression of all these proteins peaked in plants subjected to stress for 7 days, coinciding with substantial anthocyanin synthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The ubiquitination sites of hydroxycinnamoyl-CoA shikimate hydroxycinnamoyl transferase (HCT), cinnamoyl-CoA reductase (CCR), and isoflavone O-methyltransferase (IOMT) were also upregulated; however, their expression declined during the late stage of stress exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These findings suggest that changes in the ubiquitination of key enzymes, such as PAL, CHS, and HCT, may be associated with enhanced carbon allocation towards the anthocyanin synthesis pathway.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we integrated physiological, metabolomic, proteomic, and ubiquitinomic analyses to identify the potential roles of ubiquitination in regulating soybean root metabolism under P-deficient conditions. Multi-omics analyses revealed that ubiquitination modifications are widely involved in the response to P stress and exhibit significant temporal correlations with the synthesis and accumulation of root secretions. We identified multiple genes that undergo ubiquitination modifications in pathways such as glycolysis, phenylpropanoid, and flavonoid metabolism. Our findings provide novel mechanistic insights into post-translational regulatory strategies for P-efficient adaptation in crops and offer valuable candidate targets for future molecular breeding efforts.\u003c/p\u003e\u003cp\u003eThe ability of plants to acquire P is positively correlated with organic anion secretion [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In legumes [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], such as soybeans [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], this ability is particularly pronounced (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). In the present study, exposure to low-P stress for 3 days significantly increased the contents of six organic acids in root exudates, including malic acid (+\u0026thinsp;165%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and tartaric acid (+\u0026thinsp;145%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Additional file 1: Table S3). Metabolomic analyses further confirmed that the tricarboxylic acid cycle in roots responded rapidly to low-P stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These results are consistent with a previous study on other P-efficient soybean cultivars, including Maetsue, Kurotome, and Fukuutaka [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Notably, the small organic carboxylic acids secreted by plants help chelate cations, including those of Fe, Al, and Ca, thereby releasing bound soil phosphate in the soil and increasing the available P for root uptake (Wang and Lambers, 2020).\u003c/p\u003e\u003cp\u003e\u003cem\u003ePEPC\u003c/em\u003e is a key rate-limiting gene that controls the release of organic acids in plants [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In our study, ubiquitinomic analysis revealed a strong reduction in the ubiquitylation levels of PEPC4 on Lys-629 (D3/CK ratio\u0026thinsp;=\u0026thinsp;0.16, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Correspondingly, proteomic analysis indicated that the abundance of PEPC4 (D3/CK ratio\u0026thinsp;=\u0026thinsp;4.10, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and MDH (I1MTU1, D3/CK ratio\u0026thinsp;=\u0026thinsp;2.59, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; I1JZP0, D3/CK ratio\u0026thinsp;=\u0026thinsp;1.69, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) was significantly upregulated in D3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Under soil sufficient P conditions, PEPC activity is inhibited by the downstream product malate, which is associated with the mono-ubiquitination of the p107 subunit. In contrast, during P depletion, a de-ubiquitinating enzyme converts the PEPC p110 subunit to the p107 subunit [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Consequently, PEPC is no longer inhibited by malic acid, facilitating the catalysis of PEP with HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e to yield large amounts of Pi and oxaloacetate [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Notably, Pi can replenish the metabolic P pool, while oxaloacetate is metabolised into carboxylic acids that are subsequently secreted from the cell via aluminium-activated malate transporters (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Overall, these results suggest that ubiquitination may be involved in modulating PEPC activity, thereby influencing the rate of organic carboxylic acid production. Moreover, genome-wide association studies and comparative transcriptome analyses have demonstrated that PEPC plays a crucial role in regulating root development under low-P stress in soybeans [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], potentially through its involvement in organic acid secretion [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In summary, PEPC plays a central role in the secretion of organic acids in plants and may also be involved in regulating root architecture. Under low-P stress, the ubiquitination system is likely involved in modulating the catalytic activity of PEPC in soybean roots.\u003c/p\u003e\u003cp\u003eIn the present study, both the metabolomic and proteomic data consistently indicated that during the later stages of low-P stress exposure (D3\u0026ndash;D7), the phenylpropanoid and flavonoid biosynthesis pathways were activated in the soybean roots (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and S3). This activation ultimately led to the substantial accumulation of pelargonidin in the roots (D3/CK ratio\u0026thinsp;=\u0026thinsp;832.85, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; D7/CK ratio\u0026thinsp;=\u0026thinsp;951.09, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These findings align with previous metabolomic studies in cotton [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], tobacco [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], and the high-P-efficiency soybean cultivar YC03-3 [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. These results suggest that the phenylpropanoid and flavonoid biosynthesis pathways, which are associated with anthocyanin synthesis, represent key adaptive responses to low-P stress in plant roots. Anthocyanins, such as pelargonidin, are characteristic plant metabolites that respond to low-P stress, helping to protect aboveground chloroplasts from photoinhibition [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. However, their role in belowground tissues remains unclear. We observed that low-P stress led to increased levels of malondialdehyde (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF) and antioxidant enzymes (Additional file 1: Table S4), indicating aggravated oxidative stress in the roots. Low P availability induced rhizosphere acidification, increasing the solubility and activity of aluminium and heavy metals in the soil [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This increased solubility impeded the mobility of P within the plant and continuously triggered the production of reactive oxygen species (ROS). Therefore, we propose that the primary function of anthocyanins in roots is to chelate metal ions, whose availability increases owing to rhizosphere acidification, and to scavenge ROS, thereby maintaining cellular homeostasis.\u003c/p\u003e\u003cp\u003eAnother intriguing hypothesis is that anthocyanins may be secreted into the rhizosphere. In our study, proteomics analysis revealed that the expression of the ABC transporters I1LTM3, I1LZP7, and K7MZ73 significantly increased in response to low-P stress (Additional file 2: Figure S7B). Anthocyanins can be secreted into the soil via ABC transporter proteins [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. If the hypothesis is confirmed, root-secreted anthocyanins may support organic acids in activating soil P [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] and protect these acids from rapid degradation via their antibacterial properties [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. We found that flavonoids, such as apigenin (D7/CK ratio\u0026thinsp;=\u0026thinsp;2.02, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), naringenin (D7/CK ratio\u0026thinsp;=\u0026thinsp;1.94, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and liquiritigenin (D7/CK ratio\u0026thinsp;=\u0026thinsp;1.87, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), also accumulated in the roots under low-P stress conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Flavonoids play various roles in mediating plant\u0026ndash;microbe interactions, including promoting rhizobial and arbuscular mycorrhizal colonisation and mediating the assembly and function of inter-root microbiomes [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Thus, the ability to synthesise and secrete flavonoids, such as anthocyanins, may serve as a potential indicator for screening P-efficient plants.\u003c/p\u003e\u003cp\u003eAnthocyanin synthesis is further regulated by ubiquitination. In our study, among all the identified proteins, PAL exhibited the highest number of ubiquitination sites (13), which also exhibited the most substantial decrease in ubiquitination levels (D3/CK ratio: approximately 0.087\u0026ndash;0.443). Moreover, under low-P stress, the ubiquitination levels of the key rate-limiting enzymes of anthocyanin synthesis, including PAL, CHS, and DFR, decreased. In contrast, those of HCT and CCR, which are crucial enzymes in lignin synthesis, significantly increased. By further integrating the results of metabolomic and proteomic analyses, we propose that this ubiquitin-mediated regulation is likely associated with the redistribution of carbon flux within the P-efficient soybean roots. Specifically, carbon is redirected towards the phenylpropanoid-flavonoid synthesis pathway, whereas carbon flux towards the lignin synthesis pathway is suppressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Notably, the ubiquitination levels of glutathione and the glutathione transferases involved in anthocyanin transport were also significantly upregulated under low-P stress (Additional file 2: Figure S7B). Collectively, these results suggest that a regulatory signalling system may be shared among the synthesis and transport of anthocyanins to ensure rapid post-translational responses to low-P stress.\u003c/p\u003e\u003cp\u003eIn general, ubiquitination appears to function as a regulatory mechanism modulating carbon flow in the high-P-efficiency soybean genotype. Through this mechanism, soybeans may achieve their low-P response by adjusting the abundance or activity of certain rate-limiting enzymes. Unlike transcriptional regulation, which requires the \u003cem\u003ede novo\u003c/em\u003e synthesis of multiple proteins, ubiquitination modifies pre-existing proteins directly. Given that low-P conditions severely limit plant primary metabolism (Additional file 1: Table S5), transcriptional regulation would demand substantial amounts of already scarce energy and photosynthetic products (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Therefore, ubiquitination, by targeting pre-existing proteins, may provide a more rapid and energy-saving mechanism for regulating metabolic responses than transcriptional regulation.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study provides a systematic overview of molecular responses in the P-efficient soybean genotype \u0026ldquo;QD11\u0026rdquo; under low-P stress using integrated physiological, metabolomic, proteomic, and ubiquitinomic analyses. Our results indicated that the low-P tolerance phenotype of QD11 is likely associated with its efficient root system architectural remodelling and rhizosphere acidification capacity. Under low-P stress, QD11 optimised its root system architecture by increasing its root weight, root volume, and SRL to expand its P absorption capacity. Concurrently, its root system specifically secreted organic acids, such as oxalic acid and citric acid, which effectively chelate soil metal ions and activate insoluble P, providing the physiological basis for its efficient P acquisition. In addition, multi-omics analyses revealed the temporal characteristics of soybean root systems. During the early stages of stress (1\u0026ndash;3 days), carbon metabolic flux is primarily directed towards organic acid synthesis to support rhizosphere acidification. During the late stages of stress (3\u0026ndash;7 days), the phenylpropanoid and flavonoid metabolic pathways are significantly activated, leading to the substantial accumulation of anthocyanins and other flavonoids. We identified proteins regulated by ubiquitination, providing valuable targets that can be explored as genetic resources for breeding P-efficient soybean varieties through gene editing or molecular marker-assisted selection technologies. In future studies, these proteins should be subjected to further functional validation through molecular experiments, such as overexpression, RNA interference, and \u003cem\u003ein vitro\u003c/em\u003e ubiquitination assays.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ephosphorus\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eQD11\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eQiandou 11\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDEP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003edifferentially expressed protein\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePi\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003einorganic phosphate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAMF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003earbuscular mycorrhizal fungi\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePSI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ephosphate starvation-induced\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eUb\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eubiquitin\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePHT1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ephosphate transporter 1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCK2\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ecasein kinase II\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSOD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003esuperoxide dismutase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePro\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eproline\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDAM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003edifferentially accumulated metabolite\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePRM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eparallel reaction monitoring\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGO\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGene Ontology\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eKEGG\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eKyoto Encyclopaedia of Genes and Genomes\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSRL\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003especific root length\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eKOG\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eEuKaryotic Orthologous Group\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePFK\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e6-phosphofructokinase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePPi\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003epyrophosphate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePAL\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ephenylalanine ammonia-lyase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eROS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ereactive oxygen species\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is provided within the manuscript or supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (32260804) and the Guizhou Key Technologies for Mountainous Agriculture Research Project (GZNYGJHX-2025001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLT and YT\u0026nbsp;participated in manuscript writing and figure preparation.\u0026nbsp;JW,\u0026nbsp;GS\u0026nbsp;and\u0026nbsp;FL\u0026nbsp;conducted the soybean soil pot cultivation experiments and contributed to data analysis.\u0026nbsp;ZC,\u0026nbsp;SY\u0026nbsp;and\u0026nbsp;JH\u0026nbsp;designed the research, performed data analysis, reviewed the literature, and contributed to manuscript writing.\u0026nbsp;WZ\u0026nbsp;funding acquisition.\u0026nbsp;RB\u0026nbsp;writing\u0026nbsp;\u003cstrong\u003e\u0026ndash;\u003c/strong\u003e review \u0026amp; editing.\u0026nbsp;All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCong W-F, Suriyagoda LDB, Lambers H. Tightening the Phosphorus Cycle through Phosphorus-Efficient Crop Genotypes. Trends Plant Sci. 2020;25:967\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi H, He K, Zhang Z, Hu Y. Molecular mechanism of phosphorous signaling inducing anthocyanin accumulation in Arabidopsis. Plant Physiol Biochem. 2023;196:121\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eL\u0026oacute;pez-Arredondo D, Leyva-Gonz\u0026aacute;lez M, Gonz\u0026aacute;lez-Morales S, L\u0026oacute;pez-Bucio J, Herrera-Estrella L. Phosphate Nutrition: Improving Low-Phosphate Tolerance in Crops. Annu Rev Plant Biol. 2014;65.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWhite P, George T, Gregory P, Bengough A, Hallett P, McKenzie B. Matching roots to their environment. Ann Bot. 2013;112:207\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSun S, Gu M, Cao Y, Huang X, Zhang X, Ai P, et al. A Constitutive Expressed Phosphate Transporter, OsPht1;1, Modulates Phosphate Uptake and Translocation in Phosphate-Replete Rice. Plant physiol. 2012;159:1571\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChien P-S, Chao Y-T, Chou C-H, Hsu Y-Y, Chiang S-F, Tung C-W, et al. Phosphate transporter PHT1;1 is a key determinant of phosphorus acquisition in Arabidopsis natural accessions. Plant Physiol. 2022;190:682\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRosewarne GM, Barker SJ, Smith SE, Smith FA, Schachtman DP. A Lycopersicon esculentum phosphate transporter (LePT1) involved in phosphorus uptake from a vesicular-arbuscular mycorrhizal fungus. New Phytol. 1999;144:507\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWalder F, Brul\u0026eacute; D, Koegel S, Wiemken A, Boller T, Courty P-E. Plant phosphorus acquisition in a common mycorrhizal network: regulation of phosphate transporter genes of the Pht1 family in sorghum and flax. New Phytol. 2015;205:1632\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang Y, Yang Y, Ma Y, Danfeng W, Wang X, Zhang L et al. A Mycorrhiza-Induced Phosphate Transporter TaPT31-7A Regulating Inorganic Phosphate Uptake, Arbuscular Mycorrhiza Symbiosis, and Plant Growth in Wheat. J Agric Food Chem. 2025;73.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAslam MM, Karanja JK, Dodd IC, Waseem M, Weifeng X. Rhizosheath: An adaptive root trait to improve plant tolerance to phosphorus and water deficits? Plant Cell Environ. 2022;45:2861\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang H, Yang Y, Sun C, Liu X, Lv L, Hu Z, et al. Up-regulating GmETO1 improves phosphorus uptake and use efficiency by promoting root growth in soybean. Plant Cell Environ. 2020;43:2080\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang Y, Wang L, Zhang D, Zhijun C, Wang Q, Cui R et al. Soybean type-B response regulator GmRR1 mediates phosphorus uptake and yield by modifying root architecture. Plant Physiol. 2023;194.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eQin X, Hao S, Hu C, Yu M, Shabala S, Tan Q et al. Revealing the Mechanistic Basis of Regulation of Phosphorus Uptake in Soybean (Glycine max) Roots by Molybdenum: An Integrated Omics Approach. J Agric Food Chem. 2023;71.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTantriani, Cheng W, Oikawa A, Tawaraya K. Phosphorus deficiency alters root length, acid phosphatase activity, organic acids, and metabolites in root exudates of soybean cultivars. Physiol Plant. 2023;175:e14107.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen ZC, Liao H. Organic acid anions: An effective defensive weapon for plants against aluminum toxicity and phosphorus deficiency in acidic soils. J Genet Genomics. 2016;43:631\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZheng J, Shi G, Dini-Andreote F, Yang Y, Jiang Y. Root-derived low molecular weight organic acids modulate keystone microbial taxa impacting plant phosphorus acquisition. J Adv Res. 2025.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang B, Jiang H, Zheng G, Zhang Z, Wang T, Chen Q, et al. Genistein Exudation Drives Spatial Root Allocation and Heterogeneous Microbial Communities to Enhance Phosphorus Acquisition in Soybean-Maize Intercropping. Plant Cell Environ. 2025;48:7297\u0026ndash;312.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi C, Ma X, Wang Y, Sun Q, Chen M, Zhang C, et al. Root-mediated acidification, phosphatase activity and the phosphorus-cycling microbial community enhance phosphorus mobilization in the rhizosphere of wetland plants. Water Res. 2024;255:121548.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGao H, Wang T, Zhang Y, Li L, Wang C, Guo S, et al. GTPase ROP6 negatively modulates phosphate deficiency through inhibition of PHT1;1 and PHT1;4 in Arabidopsis thaliana. J Integr Plant Biol. 2021;63:1775\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang J, Xie MY, Wang L, Yang ZL, Tian ZH, Wang ZY, et al. A phosphate-starvation induced ring-type e3 ligase maintains phosphate homeostasis partially through osspx2 in rice. Plant Cell Physiol. 2018;59:2564\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePan W, Wu Y, Xie Q. Regulation of Ubiquitination Is Central to the Phosphate Starvation Response. Trends Plant Sci. 2019;24:755\u0026ndash;69.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eP\u0026eacute;rez-Torres C, L\u0026oacute;pez-Bucio J, Cruz-Ram\u0026iacute;rez A, Ibarra-Laclette E, Dharmasiri S, Estelle M, et al. Phosphate Availability Alters Lateral Root Development in Arabidopsis by Modulating Auxin Sensitivity via a Mechanism Involving the TIR1 Auxin Receptor. Plant Cell. 2009;20:3258\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAbas L, Benjamins R, Malenica N, Paciorek T, Wiśniewska J, Moulinier-Anzola JC, et al. Intracellular trafficking and proteolysis of the Arabidopsis auxin-efflux facilitator PIN2 are involved in root gravitropism. Nat Cell Biol. 2006;8:249\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMo X, Liu G, Zhang Z, Lu X, Liang C, Tian J. Mechanisms Underlying Soybean Response to Phosphorus Deficiency through Integration of Omics Analysis. Int J Mol Sci. 2022;23:4592.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXiong E, Qu X, Li J, Liu H, Ma H, Zhang D et al. The soybean ubiquitin-proteasome system: Current knowledge and future perspective. Plant Genome. 2022;16.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHerridge DF, Peoples MB, Boddey RM. Global inputs of biological nitrogen fixation in agricultural systems. Plant Soil. 2008;311:1\u0026ndash;18.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCordell D, Drangert J-O, White S. The story of phosphorus: Global food security and food for thought. Glob Environ Change. 2009;19:292\u0026ndash;305.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang T, Yang S, Chen Z, Tan Y, Bol R, Duan H, et al. Global transcriptomic analysis reveals candidate genes associated with different phosphorus acquisition strategies among soybean varieties. Front Plant Sci. 2022;13:1080014.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiao H, Wan H, Shaff J, Wang X, Yan X, Kochian LV. Phosphorus and Aluminum Interactions in Soybean in Relation to Aluminum Tolerance. Exudation of Specific Organic Acids from Different Regions of the Intact Root System. Plant Physiol. 2006;141:674\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShen J, Rengel Z, Tang C, Zhang F. Role of phosphorus nutrition in development of cluster roots and release of carboxylates in soil-grown Lupinus albus. Plant Soil. 2003;248:199\u0026ndash;206.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGu Z, Gu L, Eils R, Schlesner M, Brors B. circlize Implements and enhances circular visualization in R. Bioinformatics. 2014;30:2811\u0026ndash;2.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDing W, Cong W-F, Lambers H. Plant phosphorus-acquisition and -use strategies affect soil carbon cycling. Trends Ecol Evol. 2021;36:899\u0026ndash;906.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang Y, Lambers H. Root-released organic anions in response to low phosphorus availability: recent progress, challenges and future perspectives. Plant Soil. 2020;447:135\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDinkelaker B, R\u0026ouml;mheld V, Marschner H. Citric acid excretion and precipitation of calcium citrate in the rhizosphere of white lupin (Lupinus albus L). Plant Cell Environ. 1989;12:285\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKrishnapriya V, Pandey R. Root exudation index: screening organic acid exudation and phosphorus acquisition efficiency in soybean genotypes. Crop Pasture Sci. 2016;67:1096\u0026ndash;109.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eValizadeh G, Rengel Z, Rate A. Wheat genotypes differ in growth and phosphorus uptake when supplied with different sources and rates of phosphorus banded or mixed in soil in pots. Aust J Exp Agric. 2002;42:1103\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLu C, Lu Y, Zhao Y. Overexpression of the GsPPC4 gene from Gleditsia sinensis enhances low phosphate tolerance in tobacco by promoting organic acid secretion. Biochem Biophys Res Commun. 2025;779:152426.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eToyota K, Koizumi N, Sato F. Transcriptional activation of phosphoenolpyruvate carboxylase by phosphorus deficiency in tobacco. J Exp Bot. 2003;54:961\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShane MW, Fedosejevs ET, Plaxton WC. Reciprocal control of anaplerotic phosphoenolpyruvate carboxylase by in vivo monoubiquitination and phosphorylation in developing proteoid roots of phosphate-deficient harsh hakea. Plant Physiol. 2013;161:1634\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYu Z, Zheng M, Yan W, Ding X, Zhao T, Yang S. Phosphoenolpyruvic Carboxylase Gene GmPPC6 Negatively Regulates Root System Development Under Low Phosphorus Stress in Soybean. Plant Cell Environ. 2025;48:7886\u0026ndash;99.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHam B-K, Chen J, Yan Y, Lucas WJ. Insights into plant phosphate sensing and signaling. Curr Opin Biotechnol. 2018;49:1\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIqbal A, Qiang D, Xiangru W, Huiping G, Hengheng Z, Xiling Z, et al. Integrative physiological, transcriptome and metabolome analysis reveals the involvement of carbon and flavonoid biosynthesis in low phosphorus tolerance in cotton. Plant Physiol Biochem. 2023;196:302\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJia H, Wang J, Yang Y, Liu G, Bao Y, Cui H. Changes in flavonol content and transcript levels of genes in the flavonoid pathway in tobacco under phosphorus deficiency. Plant Growth Regul. 2015;76:225\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMo X, Zhang M, Liang C, Cai L, Tian J. Integration of metabolome and transcriptome analyses highlights soybean roots responding to phosphorus deficiency by modulating phosphorylated metabolite processes. Plant Physiol Biochem. 2019;139:697\u0026ndash;706.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLandi M, Tattini M, Gould KS. Multiple functional roles of anthocyanins in plant-environment interactions. Environ Exp Bot. 2015;119:4\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCesco S, Neumann G, Tomasi N, Pinton R, Weisskopf L. Release of plant-borne flavonoids into the rhizosphere and their role in plant nutrition. Plant Soil. 2010;329:1\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTomasi N, Weisskopf L, Renella G, Landi L, Pinton R, Varanini Z, et al. Flavonoids of white lupin roots participate in phosphorus mobilization from soil. Soil Biol Biochem. 2008;40:1971\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang L, Chen M-X, Lam L, Dini-Andreote F, Dai L, Wei Z. Multifaceted roles of flavonoids mediating plant-microbe interactions. Microbiome. 2022;10.\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":"metabolomics, phosphorus uptake, post-translational modification, proteomics, soybean, ubiquitination","lastPublishedDoi":"10.21203/rs.3.rs-8083659/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8083659/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u0026bull; Low-phosphorus (P) stress is a critical factor limiting soybean growth and yield. Ubiquitination, a post-translational protein modification, is increasingly recognised as a regulator of plant adaptive responses to nutrient limitation, including P deficiency. However, the mechanisms by which ubiquitination mediates soybean tolerance to low P remain underexplored. The present study aimed to elucidate the molecular basis of P efficiency in soybean, focusing on the role of ubiquitination.\u003c/p\u003e\u003cp\u003e\u0026bull; A P-efficient soybean genotype, Qiandou 11, was hydroponically cultivated under low or normal P levels to investigate P uptake mechanisms. Proteomic, metabolomic, and ubiquitinomic analyses were performed to identify the metabolic pathways and proteins regulating the soybean root system in response to P deficiency.\u003c/p\u003e\u003cp\u003e\u0026bull; The results indicated that QD11 rapidly adapted to P deficiency by increasing the levels of small-molecule-size organic acids and enhancing specific root length. A total of 377 differentially accumulated metabolites and 1,059 differentially expressed proteins (DEPs) were identified. The sample with the largest number of DEPs was selected for ubiquitination analysis, revealing 929 differential ubiquitination sites (585 upregulated and 344 downregulated) in 585 proteins. Notably, these proteins were significantly enriched in glycolysis, phenylpropane biosynthesis, and isoflavone biosynthesis pathways. Integrated multi-omics analysis revealed that phosphoenolpyruvate carboxylase and phenylalanine ammonia-lyase are hub proteins involved in carbon allocation during the soybean root response to low-P stress, and their regulation may be mediated by ubiquitination.\u003c/p\u003e\u003cp\u003e\u0026bull; These findings elucidate ubiquitin-mediated regulatory mechanisms and key physiological traits associated with low-P tolerance in soybean. This study provides valuable insights for breeding P-efficient soybean varieties.\u003c/p\u003e","manuscriptTitle":"Integration of proteomic, metabolomic, and ubiquitinomic analyses reveals potential mechanisms underlying low-phosphorus stress adaptation in soybean roots","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-04 14:32:57","doi":"10.21203/rs.3.rs-8083659/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision 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