Phosphorus (P) is an essential macronutrient fundamentally limiting global agricultural productivity due to its low bioavailability in soil, a constraint particularly critical for soybean (Glycine max (L.) Merr.), a major source of edible oil and protein worldwide[1, 2]. In acidic soils, such as the red soils prevalent in southwestern China (pH 4.5–5.5), phosphate is readily fixed into insoluble forms, resulting in severe P deficiency (Olsen-P < 5 mg kg⁻¹) that impairs root development, energy metabolism, and ultimately reduces yield[3, 4]. To cope with this stress, soybean orchestrates a suite of adaptive responses, including the remodeling of root system architecture (e.g., increased lateral root density and root hair length) and the exudation of organic acids to enhance P acquisition[5, 6]. These responses are coordinated by sophisticated signaling networks, with the phytohormone auxin playing a pivotal role[7].

The auxin signaling pathway, mediated by the TIR1/AFB-Aux/IAA-ARF module, is central to regulating root plasticity[8]. Aux/IAA proteins, functioning as nuclear-localized transcriptional repressors, are core components of this pathway. They feature four conserved domains that facilitate protein-protein interactions and regulate the stability and activity of ARF transcription factors, thereby modulating the expression of downstream auxin-responsive genes[9, 10]. The Aux/IAA gene family has undergone significant lineage-specific expansion, with 63 members identified in soybean, suggesting potential functional diversification in this crop compared to other species[11, 12]. Although certain Aux/IAA members in model plants like Arabidopsis and rice have been implicated in phosphate starvation responses[13, 14], the functional characterization of most soybean GmAux/IAA genes, especially their roles in regulating P homeostasis, remains largely unexplored.

Previous expression profiling analysis identified GmAux/IAA16 as a promising candidate gene that is significantly upregulated in soybean roots under low-phosphorus conditions. However, its specific functional roles and underlying molecular mechanisms in mediating soybean adaptation to low-phosphorus stress remain largely unknown. To address this knowledge gap, the present study conducted a comprehensive functional characterization of GmAux/IAA16. Its nuclear localization was confirmed, and its biological functions were investigated through the construction of heterologous overexpression lines in tobacco. Multiple experimental results collectively demonstrated that GmAux/IAA16 significantly alters root architecture, enhances phosphorus uptake efficiency, and modulates key physiological responses under low-phosphorus stress, such as the activity of reactive oxygen species (ROS)-scavenging enzymes and the accumulation of osmoprotectants. Furthermore, transcriptomic analysis revealed the downstream regulatory network governed by GmAux/IAA16. These findings establish GmAux/IAA16 as a positive regulator of low-phosphorus tolerance, offering novel insights into the molecular mechanisms underlying phosphorus use efficiency in soybean and providing valuable genetic targets for the development of phosphorus-efficient soybean varieties suitable for low-phosphorus soils.

Identification and molecular characterization of GmAux/IAA16

The soybean reference genome (Glyma v2.0) harbors 63 Aux/IAA proteins[15]. GmAux/IAA16 (XP_003530078) comprises 317 amino acids and exhibits the canonical four-domain (I–IV) architecture characteristic of the Aux/IAA family (Supplementary file 1). Sequence alignment revealed that GmAux/IAA16 is most closely related to GsIAA16 (XP_028245867) from Glycine soja (92.43% identity), followed by SsIAA18 (TKY64440) from Spatholobus suberectus (82.39%), CcIAA2 (XP_020228128) from Cajanus cajan (82.97%), an ApIAA18-like (XP_027336797) protein from Abrus precatorius (78.02%), and PvIAA16 (XP_068479571) from Phaseolus vulgaris (79.00%) (Fig. 1a) (Supplementary file 1). Phylogenetic reconstruction positioned GmAux/IAA16 and PvIAA16 within a monophyletic clade, indicating strong orthology between the two proteins (Fig. 1b). Subcellular localization of the 35S:: GmAux/IAA16–GFP fusion protein showed exclusive nuclear accumulation in tobacco epidermal cells (Fig. 1c), consistent with its predicted function as a transcriptional regulator.

Construction of GmAux/IAA16 overexpression vector and identification of positive vaccines

To investigate the role of the GmAux/IAA16 gene in plant development and growth, an overexpression vector (35S:: GmAux/IAA16-GFP) was first constructed (Supplementary file 2: Fig. S1). The GmAux/IAA16 gene was heterologously expressed in wild-type tobacco, and transgenic plants were screened using PCR. RT-PCR analysis confirmed the presence of the GmAux/IAA16 transcript in all kanamycin-resistant transgenic lines, whereas no expression was detected in wild-type plants (Supplementary file 3: Fig. S2). The gene was introduced into wild-type tobacco via the 35S:: GmAux/IAA16 -GFP expression vector (Fig. 2). Based on these results, three homozygous overexpression lines (GmAux/IAA16-1, GmAux/IAA16-3, and GmAux/IAA16-5) were selected for subsequent functional analyses.

Overexpression of GmAux/IAA16 reshaped the root structure of tobacco and enhanced nitrogen and phosphorus acquisition under low-phosphorus conditions

To investigate the biological function of GmAux/IAA16 under LP stress, stable overexpression transgenic plants were generated via Agrobacterium-mediated transformation. It was observed that LP stress markedly inhibited root development in WT tobacco plants, whereas GmAux/IAA16-overexpressing (OE) lines exhibited significant morphological and physiological advantages. Under LP conditions, the OE plants showed no notable differences in average root diameter or root volume compared to WT (Fig. 3b, c); however, they demonstrated pronounced improvements in plant height, stem diameter, number of lateral roots, total root length, and total root surface area. After 15 days of LP treatment, the OE lines exhibited increases of 12.01% in total root length, 44.56% in lateral root density, 13.24% in root surface area, and 4.23% in average root diameter relative to WT. Concurrently, plant height and stem diameter were enhanced by 35.77% and 17.96%, respectively (Fig. 3d-i). Correspondingly, the aboveground, underground, and total dry weights of OE plants increased by 27.77%, 36.36%, and 29.23%, respectively, accompanied by a 20.00% increase in root-to-shoot ratio (Fig. 3j–m), suggesting that GmAux/IAA16 promotes phosphorus foraging by modulating carbon allocation toward root development. Further quantification of nutrient content revealed that under LP stress, nitrogen and phosphorus concentrations in OE leaves increased by 20.78% and 4.93%, respectively, while those in roots rose by 17.88% and 18.60%, respectively (Fig. 3n-q). The increase in roots was particularly significant, indicating that root morphology remodeling was positively correlated with nitrogen and phosphorus accumulation. Taken together, the overexpression of GmAux/IAA16 effectively enhanced the nutrient acquisition ability of tobacco in a low-phosphorus environment.

Overexpression of GmAux/IAA16 enhances low phosphorus tolerance in tobacco

Overexpression of GmAux/IAA16 significantly enhanced the adaptability of tobacco to low-phosphorus conditions by synergistically reinforcing antioxidant capacity, energy metabolism, and osmotic homeostasis. Under low-phosphorus stress, the indole-3-acetaldehyde oxidase (IAAO) activity in leaves and roots of GmAux/IAA16-overexpressing lines was significantly upregulated—increasing by 50% and 27.75%, respectively, compared to the wild type (Fig. 4a, b). As auxin can influence ATP levels by modulating intracellular energy metabolism pathways, ATP content was measured. The results indicated that under low-phosphorus stress, OE plants maintained comparatively higher ATP levels in both leaves and roots, which were 23.44% and 52.27% higher than those of the WT, respectively (Fig. 4c, d). Furthermore, given the critical role of auxin in regulating root growth and development[16], acid phosphatase (ACP) activity was assessed. This assessment revealed that under low-phosphorus conditions, ACP activity in leaves and roots of OE plants increased by 27.55% and 42.59%, respectively, relative to the WT (Fig. 4e, f). Low-phosphorus stress typically induces excess production of reactive oxygen species (ROS), and the accumulation level of ROS serves as an important indicator of plant stress tolerance. Under low-phosphorus conditions, compared to the WT, catalase (CAT) activity in leaves and roots of OE plants increased by 170% and 123%, respectively; superoxide dismutase (SOD) activity increased by 15.24% and 11.29%; and peroxidase (POD) activity increased by 54.65% and 36.75%, respectively (Fig. 4g-l). These findings indicate a substantially enhanced antioxidant defense system in the OE lines. To further evaluate the osmoregulatory capacity of GmAux/IAA16-overexpressing plants under low-phosphorus stress, malondialdehyde (MDA) and proline contents were measured. The results demonstrated that MDA content in leaves and roots of OE plants decreased by 70.14% and 71.69%, respectively, whereas proline content increased by 129% and 87.5%, respectively, compared to the WT (Fig. 4m-p).In conclusion, these results collectively demonstrate that overexpression of GmAux/IAA16 effectively mitigated oxidative damage and membrane lipid peroxidation induced by low-phosphorus stress by synergistically enhancing antioxidant defense, energy metabolism, and osmotic adjustment, thereby significantly improving tobacco’s adaptation to low-phosphorus environments.

Analyses of endogenous hormone content under low phosphorus stress with overexpression of GmAux/IAA16

To elucidate the hormone regulatory network mediated by GmAux/IAA16, we quantified endogenous hormone levels in leaves and roots of transgenic tobacco plants under low-phosphorus (LP) and normal-phosphorus (NP) conditions using UHPLC-QTRAP-MS/MS (Fig. 5a). Indole-3-acetic acid (IAA) exhibited the most pronounced accumulation in roots, with an increase of 29.6%, while a relatively smaller increase of 8.8% was observed in leaves (Fig. 5b, c). Notable elevations were also detected in jasmonic acid derivatives: methyl jasmonate (MeJA) increased by 76.4% in roots and 66.2% in leaves (Fig. 5d, e), whereas jasmonic acid (JA) increased by 48.4% in roots and 61.8% in leaves (Fig. 5f, g). The highest increase among all measured hormones was observed for the cytokinin Zeatin (ZT), which rose by 71.1% in leaves and 56.9% in roots (Fig. 5h, i). Salicylic acid (SA) levels also increased, by 24.1% in leaves and 28.4% in roots, respectively (Fig. 5j, k). In summary, these results demonstrate that GmAux/IAA16 enhances low-phosphorus stress tolerance in tobacco by amplifying an integrated phytohormone signaling network involving auxin, jasmonates, cytokinins, and salicylic acid, thereby facilitating root architecture remodeling and improving stress defense responses.

The temporal and tissue-specific expression dynamics of GmAux/IAA16 under low phosphorus stress

To elucidate the transcriptional response of GmAux/IAA16 to phosphorus starvation, we systematically quantified its expression in wild-type (WT) and 35S:: GmAux/IAA16 transgenic tobacco at nine time points within a 48 h period under NP and LP conditions using qRT-PCR. Low phosphorus stress rapidly induced GmAux/IAA16 expression within 1 h, with peak levels observed at 12–24 h (Fig. 5a-r). Transgenic lines exhibited significantly higher expression than the WT at all time points. The folds induction under LP conditions was 2.1–3.7 in roots and 1.7–2.4 in leaves. Notably, peak expression in roots was 1.4 times higher than in leaves, indicating root-preferential activation under phosphorus deficiency. After 48 hours, expression declined in both tissues, consistent with a transient stress-induction pattern. In conclusion, GmAux/IAA16 is proposed to function as an early-response, root-biased transcriptional regulator within the low-phosphorus signaling pathway, providing a theoretical foundation for molecular strategies aimed at improving phosphorus-use efficiency in crops.

Transcriptome analyses of tobacco with overexpressed GmAux/IAA16 under low phosphorus stress

High-throughput sequencing of 24 roots cDNA libraries was conducted on the Illumina NovaSeq 6000 platform, yielding a total of 77.75 Gb of clean data. Each sample produced no less than 7.3 Gb of sequence data. Quality control metrics indicated Q20 values ≥ 97.57%, Q30 values ≥ 94.15%, and GC content consistently around 44%, collectively demonstrating high data reliability (Supplementary file 4: Table S1). Principal component analysis (PCA) revealed clear clustering of samples according to genotype and treatment conditions, with biological replicates forming tight clusters, indicating high reproducibility (Fig. 6a). Using the DESeq2 package with thresholds of |log₂FoldChange| ≥ 1 and FDR < 0.05, we identified between 853 and 5,650 differentially expressed genes (DEGs) across comparisons (Fig. 6b). Notably, after 24 hours of low-phosphorus treatment, 72.8% of DEGs were upregulated in GmAux/IAA16-overexpressing lines. Venn diagram analysis further identified 32 core DEGs common to all eight comparative groups (Fig. 6c). KEGG enrichment analysis showed that these DEGs were significantly associated with biological pathways relevant to low-phosphorus adaptation, including translation, ABC transporters, and alpha-linolenic acid metabolism (Fig. 6d). These results suggest that GmAux/IAA16 may mediate plant responses to phosphorus deficiency by modulating protein synthesis, membrane transport, and lipid-based signaling. In summary, GmAux/IAA16 induces genome-wide transcriptional reprogramming that enhances tobacco’s adaptability to low-phosphorus stress. These findings provide novel insights into the role of GmAux/IAA16 in regulating root morphology and phosphorus efficiency in soybean, and offer valuable genetic resources for molecular breeding aimed at improving phosphorus use efficiency.

Phylogenetic analyses revealed that GmAux/IAA16 and PvIAA16 from soybean clustered into a monophyletic branch, supporting the evolutionary conservation of the Aux/IAA gene family in leguminous plants[17]. Protein domain analyses revealed that GmAux/IAA16 contains four typical conserved domains, and its sequence characteristics are highly similar to those of typical Aux/IAA proteins such as Arabidopsis AtIAA7[18]. Nuclear localization experiments further confirmed the ability of GmAux/IAA16 to regulate downstream genes at the chromatin level. These results suggest that GmAux/IAA16 may play a key role in regulating the morphogenesis and phosphorus efficiency of soybean root systems.

Under low-phosphorus conditions, the lateral root density and root surface area of tobacco with GmAux/IAA16 overexpression significantly increased, which was highly consistent with the results of field studies on soybean root plasticity[19]. The simultaneous increase in root nitrogen and phosphorus contents further supports the "structure-nutrient coupling" hypothesis[20]. Transcriptome analyses revealed that the phosphorus transporter gene NtPHT1;1 was expressed in the GmAux/IA16 overexpression lines, and the expression level of NtPHT1;2 was significantly upregulated. This finding is consistent with the results of promoter sequence analyses, which indicated that the promoter regions of these genes contain cis-regulatory elements recognized by PHR1 and WRKY75[21]. In this study, we observed overexpression of GmAux/IAA16. Combined with the synergistic accumulation of indole-3-acetic acid (IAA) and jasmonic acid (JA) in the roots, this suggests that GmAux/IAA16 modulates the auxin-jasmonic acid signaling pathway. This modulation activates transcription factors such as PHR1 and WRKY75. These transcription factors subsequently upregulate NtPHT1;1 and NtPHT1;2, ultimately enhancing tobacco's phosphorus uptake capacity under low-phosphorus conditions.

RNA-seq analyses identified 4,932 differentially expressed genes whose KEGG enrichment converged on translation, ABC transport, and α-linolenate metabolism, mirroring the transcriptional signature of Pi starvation in soybean[22]. The observed enhancement of antioxidant enzyme activities and the concomitant rise in ATP levels align precisely with the established ROS-alleviation mechanisms triggered by low-phosphate stress[23]. These results indicate that GmAux/IAA16 enhances plant tolerance to low-phosphate stress by regulating the expression of multiple genes.

Based on the heterologous tobacco system, future work should employ CRISPR/Cas9-mediated knockout in soybean and promoter:: GUS reporter systems to authenticate the spatiotemporal expression pattern of GmAux/IAA16 and to dissect its genetic interactions with the PHR1–SPX module. These findings provide new insights into the role of the Aux/IAA family in soybean root development and phosphorus-use efficiency and offer a potential genetic resource for molecular breeding aimed at improving phosphorus-use efficiency. Although this research has made initial progress, there are still some limitations that require further study. Firstly, this study was mainly conducted in tobacco, and it is necessary to further verify the function of GmAux/IAA16 in soybean. Secondly, the expression pattern and regulatory network of GmAux/IAA16 in soybean are not yet fully understood. Future research should include functional validation in soybean, promoter activity analyses, protein-protein interaction network studies, and field phosphorus efficiency assessment. In addition, exploring the conservation and function of GmAux/IAA16 in other crops may help to apply this genetic resource more widely.

In conclusion, our findings unequivocally demonstrate that GmAux/IAA16 functions as a positive regulator of low-phosphate tolerance. Heterologous overexpression of GmAux/IAA16 in tobacco markedly enhanced root system architecture, improved phosphorus acquisition efficiency, and bolstered cellular homeostasis through synergistic activation of antioxidant defenses and energy metabolism. The coordinated upregulation of key phytohormones, including IAA, JA, and SA, suggests that GmAux/IAA16 mediates root morphological adaptations via an integrated hormonal network. Global transcriptomic profiling further revealed that GmAux/IAA16 orchestrates a comprehensive reprogramming of genes involved in phosphate starvation response, protein synthesis, and membrane transport. Collectively, this study provides novel insights into the molecular mechanisms by which the Aux/IAA family regulates phosphorus efficiency in soybean and identifies GmAux/IAA16 as a promising candidate gene for developing phosphorus-efficient soybean cultivars adaptable to low-phosphate soils.

The full-length cDNA sequence was translated into protein sequences using the online ExPASy ProtParam tool (https://web.expasy.org/translate/)[25]; Homologous alignment analyses was conducted using the Protein BLAST tool of NCBI (https://www.ncbi.nlm.nih.gov/)[26]; Homologous sequence alignment was performed using DNAMAN software (https://www.lynnon.com/dnaman.html)[27]; The MEGA 7 software (https://www.kent.ac.uk/software/mega-7) uses 1,000 bootstrap repetitions to construct a phylogenetic tree using the Neighbor-joining method[28].

Cloning of the GmAux/IAA16 gene, construction of overexpression vector, and subcellular localization analyses

Total RNA was extracted from the root systems of soybean near-isogenic lines and subsequently reverse transcribed into cDNA. Based on the NCBI reference genome (Glycine_max_v2.1), specific primers were designed using Primer Premier 5 software (https://www.premierbiosoft.com/primerdesign/) (Supplementary file 5: Table S2). The GmAux/IAA16 gene fragment was amplified via PCR, ligated into the pMD™18-T vector, and transformed into Escherichia coli DH5α for sequence verification. Subsequently, using the GmAux/IAA16/pMD™18-T construct as a template, a second round of PCR amplification was carried out. Following purification, the PCR products were subjected to double digestion along with the pRI101-GFP vector (which contains the 35S promoter), using the restriction enzymes SalI and EcoRI (Supplementary file 2: Fig. S2). The digested fragments were then ligated overnight using T4 DNA ligase to construct the 35S:: GmAux/IAA16-GFP fusion expression vector. This vector was introduced into Agrobacterium tumefaciens strain EHA105 by electroporation, and the resulting recombinant Agrobacterium was infiltrated into tobacco leaves. Three days post-infiltration, GFP fluorescence was observed using a (ZEISS) ZEN laser scanning confocal microscope.

The acquisition of GmAux/IAA16 genetically modified tobacco

The full-length coding sequence of GmAux/IAA16 was cloned into the pRI101-GFP skeleton vector, and the GFP gene was expressed under the regulation of the 35 S promoter. The 35S:: GmAux/IAA16-GFP recombinant plasmid was inserted into Agrobacterium root cancer strain EHA105[29]. Transformed wild-type tobacco (Nicotiana tabacum L.) Screening was performed by adding 50 mg/L kanamycin to 1/2 Murashige & Skoog (PhytoTech Labs, USA) medium. The T3 homozygous lines overexpressing 35S:: GmAux/IAA16 were identified by PCR (Supplementary file 4: Table S1).

After surface disinfection of wild-type and transgenic tobacco seeds with 40% sodium hypochlorite and Tween 20, they germinated on 1/2 MS solid medium containing 50 mg/l kanamycin. Healthy seedlings were selected for hydroponic cultivation under two phosphorus conditions: NP (500 µmol/L KH₂PO₄) and LP (20 µmol/L KH₂PO₄). After 15 days of cultivation, the root phenotypes were evaluated using the WinRHIZO (2013e version) system to determine the total root length, root surface area, root volume and average root diameter[30]. The height and stem diameter of the plants were measured using a vernier caliper, and samples were collected for dry weight and fresh weight analysis. The dry samples were analyzed for nitrogen and phosphorus contents respectively by the Kjeldahl method and the molybdenum-antimony colorimetric method in accordance with the "Agricultural Industry Standard of the People's Republic of China" (NY/T 2017 − 2011)[31].

After five days of cultivation, the seedlings with uniform growth were moved to water for three days of acclimatization. Then, phosphorus stress treatment was carried out for 15 days. The leaves and roots of tobacco were collected for physiological parameter determination. The contents of MDA, ATP, proline, as well as the activities of ACP, POD, SOD, CAT and IAAO were determined using 100 mg of leaf and root tissues. All determinations were conducted using products from Beijing Solarbio Science & Technology, Beijing, China, following the manufacturer's protocol.