Arbuscular Mycorrhizal Fungi-Mediated Reconfiguration of Poplar Leaf C-N-P Metabolic Networks: Environment-Dependent Synergies and Nutrient Interactions

Abstract

The regulatory mechanisms by which AMF modulate the integrated carbon (C)-nitrogen (N)-phosphorus (P) metabolic network in woody plant leaves remain unclear. We investigated how varying nitrate (NO₃⁻) and phosphate (Pi) supply, with or without AMF inoculation, reshapes the leaf metabolic network in poplar seedlings. Key findings reveal that AMF acts as a central metabolic hub, optimizing C-N-P coordination in an environment-dependent manner. Under low Pi, NO₃⁻ supply enhanced P remobilization and photosynthetic efficiency, boosting growth. AMF further optimized low-Pi adaptation by promoting P storage and buffering, significantly improving photosynthesis and biomass. Under high Pi, NO₃⁻ supply shifted focus towards enhancing Rubisco-mediated carbon assimilation. AMF synergistically improved carbon assimilation efficiency and suppressed non-essential P recycling. N metabolism effects of Pi were contingent on NO₃⁻ availability, and AMF reprogrammed N assimilation pathways accordingly, balancing uptake and utilization under different N regimes. Critically, AMF orchestrated environment-specific metabolic adjustments, reinforcing P buffering and photosynthetic gain under Pi limitation, and enhancing C assimilation efficiency while minimizing P waste under Pi sufficiency. This study demonstrates that poplar leaf C-N-P networks are reconfigured through N-P synergisms modulated by AMF, positioning AMF as a pivotal integrator of nutrient acquisition and allocation. These insights provide a physiological foundation for developing efficient forestry nutrient management and mycorrhizal application strategies.

1. Introduction

N and P are essential macronutrients for plant growth and development, with their distribution in agroforestry ecosystems often being uneven, which limits primary productivity [1,2]. As key organs for photosynthesis, respiration, and nutrient assimilation, leaves play a critical role in converting underground nutrient uptake into aboveground primary productivity. Their functional status, including photosynthetic efficiency and metabolic balance, directly influences this process [3,4]. The supply of N or P can significantly affect the carbon assimilation capacity and nutrient utilization and storage efficiency of leaves by altering physiological and biochemical processes such as leaf structural development, photosynthetic electron transport, enzyme activities, and metabolic networks (e.g., the synthesis, transport, and distribution of C, N, and P assimilates), which in turn impacts overall plant growth and productivity [5,6,7]. Notably, N-P interactions in plants are not independent, but closely interrelated. For instance, in Arabidopsis, NO₃⁻ supply is essential for activating the phosphate starvation response (PSR), and in rice, NO₃⁻ modulates PSR through the regulation of OsPHR2 activity [8,9]. Studies also show that Pi availability significantly regulates the expression of NO₃⁻ response genes [10,11]. However, most existing research on N-P interactions has focused on root systems in model plants and major crops [12]. Few studies on woody plants have shown that, under N-limited conditions, adding P does not significantly affect leaf N content. In contrast, N application in P-limited areas decreases leaf P concentrations while enhancing P uptake efficiency, and P application improves N uptake efficiency [13]. Furthermore, in N-limited areas, P application mitigates the inhibition of photosynthetic carbon assimilation, while in P-limited areas, N application has minimal effects on photosynthesis or hydraulic traits [14]. Overall, research on how N-P supply regulates core physiological and biochemical processes, particularly at the leaf scale in woody plants, remains underexplored.

AMF, as ancient soil symbionts, form mutualistic relationships with most terrestrial plants, serving as crucial ecological hubs for host adaptation to nutrient stress [15,16]. By establishing extensive hyphal networks, AMF significantly enhance the ability of host roots to acquire water and nutrients from nutrient-poor rhizospheres [17,18]. Importantly, AMF symbiosis alters host plants’ N and P uptake, metabolism, and distribution strategies [19,20]. Recent studies have also shown that AMF symbiosis can induce systemic signals in plants, reshaping carbon allocation patterns, transporting photosynthetic carbon to the mycorrhizal network in exchange for mineral nutrients, and finely regulating the balance of carbon assimilation and storage through sugar signal–nutrient feedback mechanisms [21,22]. Moreover, research in herbaceous systems indicates that AMF can modulate photosynthetic rates, thereby affecting plant carbon-use efficiency [23]. In contrast, among woody plants—key contributors to terrestrial carbon storage, productivity, and ecological restoration—particularly fast-growing species with high turnover rates, the mechanistic understanding of systemic physiological responses to nitrogen–phosphorus interactions under AMF symbiosis remains inadequate. At the leaf level, the primary organ driving ecosystem primary productivity, it is still unclear how this symbiosis coordinately restructures the metabolic network integrating carbon fixation and assimilation, N acquisition and metabolism, and P utilization. Such coordinated regulation could systematically enhance whole-plant adaptation strategies under multiple nutrient limitations. Addressing these gaps requires urgent and comprehensive investigation.

Poplar (Populus spp.), an important fast-growing timber species and ecological restoration pioneer, relies heavily on nutrient use efficiency during its seedling stage, which directly impacts the survival rate and early growth advantage in afforestation [24]. In natural habitats, poplars often face significant N-P imbalances, particularly the widespread lack of available soil P [25]. In production systems, large-scale applications of N and P fertilizers are commonly used to alleviate soil nutrient deficiencies and promote tree growth. However, this practice results in low fertilizer-use efficiency and causes various environmental problems [26,27]. In natural environments, poplars often form symbiotic relationships with AMF to alleviate nutrient stress. Studies have shown that poplar seedlings are significantly more dependent on mycorrhizal symbiosis than adult trees [28,29], but research on how AMF dynamically regulate photosynthetic performance, mineral nutrient homeostasis, and metabolic enzyme networks under different N-P conditions is still lacking.

Therefore, this study used 84 K poplar seedlings to examine (1) how N-P interactions under different nutritional regimes affect leaf physiology and biochemistry and the mechanisms underlying these responses, and (2) how AMF symbiosis regulates N-P interaction mechanisms in poplar leaves. Elucidating the mechanisms of nutrient co-uptake and utilization, together with the regulatory role of AMF, is essential for developing precise fertilization strategies, improving nutrient-use efficiency, and advancing molecular breeding.

2. Materials and Methods

2.1. Plant Materials and Experimental Design

One-month-old micro-cuttings of 84 K poplar were used in this study. The AMF Rhizophagus irregularis (BGC B109) was provided by the Institute of Plant Nutrition and Resources, Beijing Academy of Agriculture and Forestry Sciences, China. The AMF inoculum consisted of spores (50 spores/g), hyphae, soil, and infected root fragments. The growth substrate, a 1:1 mixture of washed sand and perlite, was sterilized by autoclaving at 121 °C for 2 h. Pots (25 × 15 cm) were filled with 2 kg of substrate and randomly arranged.

Seedlings were divided into two groups: one group was inoculated with 30 g of AMF inoculum (+AMF), while the control group received 30 g of autoclaved inoculum and 10 mL of filtrate from non-autoclaved inoculum (−AMF). After 60 d, seedlings were further divided into four subgroups subjected to different KNO₃ (N) and KH₂PO₄ (P) concentrations: P0 and N0 (−N−P), P0 and N30 (+N−P), P1.5 and N0 (−N+P), and P1.5 and N30 (+N+P). Potassium concentration was standardized by adding KCl. The treatments lasted for 45 d under greenhouse conditions (28/20 °C, day/night, 60% relative humidity). The experiment included 120 seedlings arranged in a factorial design comprising two AMF treatments (+AMF and −AMF), two N levels, and two P levels, with 15 replicates per treatment combination.

2.2. Photosynthetic Measurements

Net photosynthetic rate (A), stomatal conductance (gs), transpiration rate (E), and intercellular CO₂ concentration (Ci) were measured using a portable photosynthesis system (LiCor-6400, LI-COR Inc., Lincoln, NE, USA) between 8:00 and 11:00 a.m. Light intensity was set to 1000 μmol photon/m²/s, CO₂ concentration to 400 μmol/mol, and airflow rate to 500 mL/min. Measurements were taken after 2 min of stabilization at each measurement point.

2.3. Chlorophyll Fluorescence Parameters

Before measurement, the plants were placed in a darkroom for 60 min to allow for dark adaptation. For each functional leaf, 2 to 3 areas of interest (AOIs) were selected for determining chlorophyll fluorescence parameters using an Imaging-PAM system (Walz, Germany). The measured parameters included initial fluorescence (Fo), maximum fluorescence (Fm), initial fluorescence under light-adapted conditions (Fo’), maximum fluorescence under light-adapted conditions (Fm’), steady-state fluorescence (Fs), maximum quantum yield of PSII (Fv/Fm), actual photochemical quantum yield of PSII (ΦPSII), and photochemical quenching coefficient (qP). Based on these measurements, the following indices were calculated: maximum photochemical efficiency (Fv/Fm), actual photochemical efficiency (ΦPSII), non-photochemical quenching (NPQ), and photochemical quenching (qP):

Fv/Fm = (Fm − Fo)/Fm

ΦPSII = (Fm’ − Fs)/Fm’

NPQ = (Fm − Fm’)/Fm’

qP = (Fm’ − Fs)/(Fm’ − Fo’)

2.4. Plant Growth and Biomass

The plant height was measured using a measuring tape (Swordfish, Taizhou, China) [30]. At the end of the experiment, the shoot and root dry weights were measured after drying in a hot-air oven at 80 °C to a constant weight [31]. The remaining leaf samples were flash-frozen in liquid nitrogen, finely ground into powder, and stored in an ultra-low temperature freezer at −80 °C until analysis.

2.5. Mycorrhizal Colonization

Fresh roots were first stained with trypan blue according to the method of Kiheri et al. [32]. Mycorrhizal colonization was then assessed under an Olympus BX43F light microscope (200× magnification; Olympus, Tokyo, Japan) using the gridline intersection method [33].

2.6. Quantification of Leaf N and P Parameters

Following the procedure of Wang et al. [34], the dry powders of seedling leaves were digested using H₂SO₄ and H₂O₂. According to the method described by Guo et al. [35], a continuous flow analyzer (AA3, Bran-Luebbe, Hamburg, Germany) was used to determine N and P concentrations in the digests at 660 nm and 700 nm, respectively. N utilization efficiencies (NUEs) and P utilization efficiencies (PUEs) were calculated using the formula NUE or PUE = tissue biomass/amount of N or P in the tissue, following the guidelines of Gan et al. [2]. Instantaneous photosynthetic phosphorus use efficiency (PPUEi) and instantaneous photosynthetic nitrogen use efficiency (PNUEi) were calculated according to the methods described by Gan et al. [2] and [36], respectively. The equations used were PPUEi = specific leaf area × A/leaf P amount and PNUEi = specific leaf area × A/leaf N amount. The N/P ratio was defined as the nitrogen-to-phosphorus ratio in leaves.

2.7. Chlorophyll and Anthocyanin Content

Chlorophyll content was measured spectrophotometrically using 80% acetone, with absorbance at 663 and 646 nm for chlorophylls, and 470 nm for carotenoids [37,38].

2.8. Organic Acids

Malic and citric acid levels were determined using an HPLC method [39,40]. Fresh root samples were extracted in 80% ethanol, evaporated to dryness, re-dissolved, and filtered before analysis. Chromatographic conditions included a Shim-pack VP-ODS C18 column and a mobile phase of 20 mM KH₂PO₄ (pH 2.8)-methanol (95:5) at a flow rate of 0.8 mL/min, with detection at 210 nm.

2.9. Soluble Sugar and Starch Content

Soluble sugars were measured using the anthrone-sulfuric acid method [41], and starch content was determined using the GOD-POD method [42].

2.10. ATP, Free Amino Acid, and Soluble Protein Content

ATP content was measured using the method of Beutler and Mathai [43] and the Solarbio BC0300 assay kit. Free amino acids were quantified using the ninhydrin colorimetric method [44]. Soluble protein content was measured using the Bradford method [45].

2.11. Enzyme Activity

The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) was assayed following the methods of Spreitzer and Salvucci [46]. The activities of acid phosphatase (APs, EC 3.1.3.2), phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), malate dehydrogenase (MDH, EC 1.1.1.37), glutamine synthetase (GS, EC 6.3.1.2), nitrate reductase (NR, EC 1.7.99.4), glutamate synthase (GOGAT, EC 1.4.7.1), and glutamate dehydrogenase (GDH, EC 1.4.1.2) were determined according to the methods of Lei et al. [47], Gajewska et al. [48], Lü et al. [49], and Luo et al. [25], respectively.

2.12. Gene Expression Analysis

The transcriptional levels of C-N-P-related genes were measured by quantitative PCR (qPCR) as described by Gan et al. [2]. Thirty-one key genes were selected for mRNA assessment. Quantitative PCR was performed using SYBR Green and primers listed in Table S1, and β-actin 2/7 was used as the reference gene. Six independent biological replicates were analyzed, and the PCR efficiencies ranged from 95% to 108%.

2.13. Statistical Analysis

Data were analyzed using SPSS 25.0 (Statistical Product and Service Solutions, SPSS Inc., Chicago, IL, USA). A two-way analysis of variance (ANOVA) was performed to assess the effects of N treatment, P treatment, and their interaction (N×P) on all measured variables. The influence of AMF inoculation was also assessed within this model framework. Differences among the experimental variants (treatment combinations) were evaluated using Duncan’s multiple range test as a post hoc procedure, at a significance level of p < 0.05. Values were means ± SE (n = 6). Quantitative PCR Cq values were normalized following the method of Long et al. [50], and fold changes in transcript abundance were calculated. The fold change represented the relative expression of target genes compared with the reference gene, and the relative expression level was determined using the 2^−ΔΔCt method. Genes with |log₂(fold change)| > 1 were considered significantly differentially expressed. Graphical visualization of the results was performed using Origin 2021 (OriginPro, OriginLab Corporation, Northampton, MA, USA).

Structural equation modeling (SEM) was used to examine the effects of N or P addition and AMF inoculation on leaf traits and plant growth. Path coefficients were applied to quantify the relationships among the latent variables, including nutrient addition, physiological and biochemical characteristics, AMF inoculation, photosynthesis, gene expression levels, and plant growth. These coefficients were estimated using the maximum likelihood approach. Model adequacy was determined based on several criteria: a chi-square value < 3, a root mean square error of approximation (RMSEA) < 0.08, a goodness-of-fit index (GFI) > 0.85, a comparative fit index (CFI) > 0.90 and close to 1, and a chi-square p-value > 0.05. Prior to SEM construction, principal component analysis (PCA) was conducted to reduce the dimensionality of diverse data types. All SEM analyses were performed using AMOS version 29.0 (Amos Development Corporation, Chicago, IL, USA).

3. Results

3.1. AMF Colonization Under Different N-P Treatments

AMF colonization of poplar seedlings was significantly influenced by NO₃⁻, Pi, and their interaction (Figure 1A). NO₃⁻ supply increased hyphal, arbuscular, vesicular, and total colonization under both Pi levels. Pi supply enhanced colonization under low NO₃⁻ in all structures, but under high NO₃⁻ it only increased hyphal and vesicular colonization.

3.2. Effects of AMF-Mediated N-P Supply on Poplar Seedling Growth

Seedling height, basal diameter, leaf dry weight, and root- shoot ratio were significantly affected by NO₃⁻, Pi, and AMF inoculation (Figure 2). NO₃⁻ supply consistently increased height, diameter, and leaf biomass across AMF treatments. Pi addition also enhanced height under both NO₃⁻ levels, but its effects on diameter were AMF-dependent: Pi increased diameter under high NO₃⁻ in −AMF plants, whereas in +AMF plants the increase occurred only under low NO₃⁻.

For leaf dry weight, Pi and NO₃⁻ both showed positive effects, though AMF strengthened the Pi-induced increase under high NO₃⁻ and weakened it under low NO₃⁻. Root-tshoot allocation showed contrasting patterns between AMF treatments. In −AMF seedlings, NO₃⁻ reduced the ratio under low Pi but increased it under high Pi. In +AMF seedlings, NO₃⁻ had no significant influence, and Pi increased the ratio only under low NO₃⁻. These results indicate that AMF inoculation modifies N–P interactive effects on both biomass accumulation and carbon allocation.

3.3. Photosynthetic Parameters Under AMF-N-P Treatments

A, gs, Ci, and E were significantly affected by NO₃⁻, Pi, and AMF inoculation (Figure 3). NO₃⁻ increased A at both Pi levels; the effect was stronger under low Pi in +AMF plants but weaker under high Pi. gs and E increased with NO₃⁻ only under high Pi, with AMF reducing these gains. Ci responses differed: NO₃⁻ lowered Ci under low Pi in +AMF plants, but had no effect under high Pi. Pi consistently enhanced A and E and lowered Ci; AMF amplified Ci reduction under low NO₃⁻ and reversed gs responses between AMF groups.

3.4. Chlorophyll Fluorescence Responses

NO₃⁻ and Pi treatments, their interaction, and AMF inoculation significantly influenced the chlorophyll fluorescence parameters of poplar seedlings (Figure 4). NO₃⁻ increased Fv/Fm, ΦPSII, qP and reduced NPQ across Pi levels. Pi elevated Fv/Fm and ΦPSII in both AMF groups, but AMF reduced these gains under low NO₃⁻. Pi increased qP in −AMF plants at both NO₃⁻ levels, whereas in +AMF plants the increase occurred only under low NO₃⁻.

3.5. Leaf Nutrient Status

Variations in NO₃⁻ and inorganic Pi supply, together with their interactions and inoculation with AMF, exerted significant effects on the leaf N-P status of poplar seedlings (Figure 5). NO₃⁻ generally decreased N/P ratio and PPUEi, with responses varying by Pi level and AMF. Under low Pi, NO₃⁻ increased P content without affecting concentration or PUE; under high Pi, it raised P concentration and content but lowered PUE, especially in +AMF plants. Pi’s effects on N traits depended on NO₃⁻: it consistently reduced PNUEi, but less so in +AMF plants; N/P ratio increases were more pronounced in −AMF under low NO₃⁻.

3.6. N-P Metabolic Enzyme Activities

NO₃⁻ and inorganic Pi supply, their interactions, and inoculation with AMF significantly influenced the activities of NR, GS, GOGAT, GDH, APs, MDH, and PEPC in the leaves of poplar seedlings (Figure 6). NO₃⁻ enhanced APs, MDH, and PEPC under low Pi, with larger MDH and PEPC increases in +AMF plants; under high Pi, only MDH and PEPC remained responsive. Pi promoted NR, GS, and GDH under low NO₃⁻, and GOGAT under high NO₃⁻, with stronger induction in +AMF plants.

3.7. Rubisco Activity

NO₃⁻ and Pi treatments, along with their interactions and AMF inoculation, significantly influenced the activity of Rubisco in the leaves of poplar seedlings (Figure 7). Pi increased Rubisco only under high NO₃⁻ in −AMF plants, but in +AMF plants Pi stimulated Rubisco under both NO₃⁻ levels, indicating AMF broadened Pi responsiveness.

3.8. Gene Expression of C-N-P Metabolism

NO₃⁻ and Pi treatments, along with their interactions and AMF inoculation, significantly affected the transcription of C-N-P metabolism-related genes in poplar seedling leaves (Figure 8A). The regulatory effects of NO₃⁻ were strongly dependent on Pi availability. Under low Pi, NO₃⁻ markedly induced PAP10, PHR1, PHO2, PLDZETA1, MIPS, RCA1, RBCS, PEPC1, MDH, SPS, and TPS1, while repressing PHO1.H1, SPX2, and BAM1. Under high Pi, these induction patterns were weakened; several genes (e.g., PHT1.4, PHT1.9, ITPK1) shifted from no response to downregulation, and PAP10, PHR1, PHO2, and SPS became suppressed. Meanwhile, NO₃⁻ enhanced RPS6 expression and relieved SPX2 inhibition.

AMF inoculation substantially modulated NO₃⁻-dependent transcription. Under low Pi, AMF maintained the induction of PAP10, PHR1, PHO2, PLDZETA1, MIPS, RCA1, RBCS, PEPC1, MDH, and SPS, with stronger PEPC1 and MDH responses, and reversed SPX2 repression. AMF also activated PHT1.4, PHT1.9, ITPK1, and RPS6, which showed weaker responses in −AMF plants. Under high Pi, NO₃⁻ still induced SPX2, RCA1, RBCS, PEPC1, MDH, and RPS6 in the +AMF group, but effects on MIPS, ITPK1, SPS, and BAM1 were diminished.

Pi-induced transcriptional changes were also regulated by environmental NO₃⁻. Under low NO₃⁻, Pi strongly upregulated NLP2, NRT2.5, NRT1.1, GS2, GDH2, AAP2, AAP3, NiR, HHO2, ATG8, PEPC1, MDH, SPS, and TPS1, while repressing ASN1 and BAM1. Under high NO₃⁻, Pi maintained induction of GS2, NiR, and MDH, but repressed NLP2, AAP2, AAP3, HHO2, SPS, and TPS1, while inducing Fd-GOGAT, NADH-GOGAT, RCA1, RBCS, and RPS6.

AMF increased the sensitivity of many genes to Pi. Under low NO₃⁻, AMF enhanced the induction of NLP2, NRT2.5, NRT1.1, GS2, GDH2, AAP2, AAP3, NiR, PEPC1, and MDH, and additionally activated Fd-GOGAT, NADH-GOGAT, RCA1, RBCS, and RPS6. AMF reversed Pi-induced activation of HHO2, SPS, and TPS1 and increased BAM1 expression. Under low NO₃⁻ and high Pi, AMF further strengthened the upregulation of GS2, Fd-GOGAT, NADH-GOGAT, RCA1, RBCS, and RPS6 and intensified repression of NLP2 and AAP2, while maintaining AAP3, SPS, and TPS1 downregulation.

4. Discussion

4.1. Pi-Environment-Dependent Regulation of Leaf P Turnover and Allocation by NO₃⁻ Supply

Leaves, as primary sites of photosynthesis and biomass formation, require optimal N and P. P deficiency reduces chlorophyll and disrupts mesophyll ultrastructure, impairing photosynthesis [51,52,53], while N supply modulates plant P status via P-metabolic enzymes [54,55]. SEM in this study indicates that, under varying Pi environments, NO₃⁻ supply affects leaf-related indicators through distinct regulatory pathways, ultimately shaping plant growth(Figure 8B,C).

Under low Pi conditions (Figure 8J), NO₃⁻ supply regulates leaf P homeostasis and allocation in poplar seedlings, markedly enhancing carbon assimilation capacity and photosynthetic efficiency. This regulation likely involves upregulation of the PSR core transcription factor PHR1 and downregulation of its inhibitor SPX, thereby facilitating more efficient P turnover and utilization [8,56,57]. Elevated PHR1 may induce PLDZETA1 to degrade phospholipids as emergency P sources [58], while increased PAP10 and APs activity accelerate organic P hydrolysis, releasing inorganic P for photosynthetic demand [59,60]. Leaf total P remained stable, but PPUEis dropped sharply, indicating preferential P allocation compared to C assimilation [61], which contrasts with strategies where N enhances P uptake to raise total P [62]. However, dependence on phospholipid degradation and organic P hydrolysis as emergency P sources may compromise membrane integrity and deplete P reserves.

Conversely, under high Pi conditions (Figure 8C,K), NO₃⁻ supply appears to attenuate P perception and uptake by downregulating PSR core transcription factors, forming an adaptive mechanism to avoid P toxicity [63]. Although leaf P increased, declined PUE and PPUEis suggest reduced P metabolic investment under P abundance [64,65]. Upregulation of MIPS may promote membrane synthesis/adaptation, thereby supporting membrane system construction [66], and facilitating a metabolic shift toward carbon assimilation and growth.

4.2. Pi-Dependent Regulation of Leaf N–C Metabolism in Poplar Seedlings Under NO₃⁻ Supply

Under low Pi, NO₃⁻ supply not only optimizes P metabolism, but also enhances the photosynthetic apparatus (Figure 8J). Adequate ATP increases Rubisco activity (Figure S1), supporting higher A [67,68]. At the molecular level, NO₃⁻ upregulates Calvin cycle genes (RBCS, RCA1) and enzymes (PRK, FBP), boosting CO₂ fixation. Concurrent PEPC1 induction may improve C flux via malate synthesis (Figure S2A) [69,70]. Accumulation of photosynthetic pigments and N metabolites (soluble protein and free amino acids) (Figures S3 and S4) underscores N’s role in enhancing photosynthesis [71,72]. Together with improved P use, these changes increase light capture and energy conversion [73,74]. Coordinated upregulation of PK and ME1 increases the supply of carbon skeletons and reducing power for N assimilation [75]. Furthermore, upregulation of AGP and TOR helps buffer metabolic demands under low Pi and coordinate resource allocation toward biosynthesis [76], ultimately increasing aboveground biomass and reducing the root-shoot ratio (Figures S5 and S6).

Under high Pi, NO₃⁻ also enhances photosynthesis. Increased Rubisco activity with RBCS and RCA1 expression forms the basis for higher A [77,78]. Elevated pigments and photochemical efficiency indicate improved light capture and energy use (Figure S4) [79,80,81]. The responsiveness of PRK and FBP is lost, suggesting high Pi removes C-fixation bottlenecks. Metabolism shifts toward rapid use: SPS downregulation coincides with higher soluble sugars and sharply lower starch (Figure S5), alongside reduced AGP and BAM1 expression. This reallocates C from storage to active pools [82,83,84], favoring fast growth [85]. N assimilation remains crucial: greater protein/amino acid accumulation (Figure S3) plus RPS6 induction indicate enhanced protein synthesis. PK and ME1, working with MDH, supply C skeletons and reducing power. This synergy increases biomass (Figure S6), while a higher root-shoot ratio reflects investment in roots to sustain rapid shoot growth.

4.3. AMF Symbiosis Reshapes the Pi-Environment–Dependent Regulation of Leaf N–C Metabolism Under NO₃⁻ Supply

AMF enhance host adaptation under low-P stress [86], and modulate N metabolism to increase stress resistance [87]. Under low-Pi, NO₃⁻ supply greatly increased AMF colonization, consistent with their low-P adaptability [88]. SEM showed AMF inoculation altered N addition effects (Figure 8F).

As in -AMF, NO₃⁻ likely activates PHR1 to promote P turnover (Figure 8J). Higher colonization improves intracellular P homeostasis via a leaf P network: MIPS drives inositol synthesis; ITPK1 upregulation generates IP_S signals and facilitates PP–IP_S degradation; and RPS6 upregulation may enhance translation of P-turnover enzymes [89,90,91]. Although leaf P did not increase significantly, less decline in PPUEis indicates improved allocation of NO₃⁻-mobilized P to photosynthetic organs [92], aligning with AMF enhancing P transport and allocation efficiency [93,94].

Under high-Pi, AMF symbiosis markedly modifies leaf responses (Figure 8G,K), suppressing NO₃⁻-induced PSR. NO₃⁻ downregulates PAP10 and inhibits PLDZETA1, minimizing unnecessary endogenous P recycling. Unchanged APs activity suggests targeted downregulation to conserve energy/maintain P homeostasis [95,96,97]. Physiologically, leaf P concentration/content rise, while PUEs, PPUEis and N/P ratios drop markedly (more than −AMF). Consistently, P storage genes (MIPS, ITPK1) are unaffected by NO₃⁻ under AMF. NO₃⁻ increases hyphal density, but leaves arbuscule abundance and colonization stable, implying primarily regulatory effects [15,98].

4.4. AMF Symbiosis Modulates Pi-Dependent Regulation of Leaf N–C Metabolism by NO₃⁻

Under low Pi, AMF markedly amplify NO₃⁻ promotion of photosynthetic C assimilation and energy metabolism, enhancing pigment synthesis and PSII performance [99], and increasing light conversion efficiency (Figure 8F,J). AMF maintain strong NO₃⁻-induced upregulation of Calvin cycle (RBCS) and glycolysis and organic acid genes (PEPC1, MDH). Alongside RPS6 upregulation and RCA1 activation, this boosts PEPC and MDH activity, increasing malate synthesis (Figure S2), supplying C skeletons/reducing power for N assimilation, leading to higher soluble protein and amino acids (Figure S3). Greater ATP supports Rubisco (Figure S1), yielding larger photosynthetic gains. Growth strategy shifts: +AMF plants, with enhanced photosynthesis, balance soluble sugar homeostasis and increase starch accumulation (Figure S5). SPS upregulation directs photosynthates into starch, while ITPK1-MIPS activation enables phytate accumulation, forming a dual starch–phytate storage system mitigating osmotic stress/buffering P; increased vacuolar ratios strengthen P stability [100,101,102,103]. This restructuring drives biomass gains without altering root/shoot ratio (Figure S6).

Under high Pi, AMF reshape NO₃⁻ regulation (Figure 8G,K). They strengthen NO₃⁻ stimulation of Rubisco via RBCS and RCA1 co-upregulation [104,105,106]. NO₃⁻ increases chlorophyll fluorescence/content, but less than in −AMF plants (Figure S4), suggesting more stable photosynthesis [107]. NO₃⁻ still upregulates PK, ME1, and TOR, sustaining glycolysis, malate synthesis and nutrient sensing, while FBP and AGP responsiveness disappears—consistent with reduced starch/greater soluble sugars (Figure S5). Unchanged SPS and BAM1 levels indicate sugar accumulation from accelerated substrate turnover (e.g., PK, ME1 induction). This supports N assimilation, with elevated protein/amino acids (Figure S3), and RPS6 upregulation [108], reinforced by MDH and PEPC1 activation [109]. Selective gene responsiveness and C flux reallocation increase biomass, while maintaining root/shoot ratio (Figure S6).

4.5. NO₃⁻-Dependent Effects of Pi Supply on Leaf N Metabolism

N deficiency impairs photosynthesis and yield [110,111], while P deficiency weakens photosynthetic capacity [112,113]. P supply strongly affects plant N uptake and utilization efficiency: adequate P enhances N accumulation and uptake [113,114], whereas excessive P lowers physiological N-use efficiency [115].

SEM showed that under different NO₃⁻ conditions, Pi supply regulates leaf parameters through distinct pathways, thereby affecting growth (Figure 8D,E). Under low NO₃⁻ (Figure 8L), Pi supply appears to enhance NLP2 transcription, thereby activating NO₃⁻ transporter genes, and is corroborated by HHO2 upregulation [11,116]. Concurrently, upregulation of N assimilation genes (GS2, GDH2, NiR) increases N metabolism efficiency [117,118,119]. ASN1 downregulation suggests preferential allocation of N to active metabolism and ATP conservation for photosynthetic carbon processes (Figure S1), though potentially reducing NUE through feedback inhibition [120,121,122]. Overall, these promote protein accumulation (Figure S3), providing a strong N basis for photosynthesis, aligning with declined PNUEi. Under high NO₃⁻ (Figure 8M), Pi supply does not enhance N uptake, but instead maximizes assimilation efficiency via upregulation of GS2, NiR, Fd-GOGAT, and NADH-GOGAT, reinforcing the glutamate–glutamine cycle [123,124,125]. This, plus RPS6 upregulation, promoted soluble protein/free amino acid accumulation (Figure S3) [126]. This strategy coordinates N-P supply to drive C assimilation and biomass accumulation (Figure S6).

4.6. NO₃⁻-Level–Dependent Effects of Pi Supply on Leaf C–N Regulation

Under low NO₃⁻, Pi substantially enhanced photosynthetic C assimilation and metabolic flux (Figure 8L). Stimulated expression/activity of organic acid synthesis genes promoted malate/citrate accumulation for N assimilation skeletons (Figure S2) [127]. Broad upregulation of key C metabolism genes strengthened C fixation. Downregulation of AGP and BAM1 redirected C from storage (Figure S5). Upregulation of PK and ME1 promoted C flux toward N assimilation. Increased chlorophyll fluorescence/chlorophyll content collectively improved A (Figure S4) [128,129]. TOR upregulation indicated activated N metabolism. Synergistic enhancement increased plant height, leaf mass, and total biomass (Figure S6) [130,131]. However, this strategy risks C-N reserve depletion and energy and ROS imbalance under prolonged low N and fluctuating P.

Under high NO₃⁻ (Figure 8M), Pi-induced regulation differed markedly. Pi still improved photosynthesis via increased Rubisco activity and gene upregulation [46]. However, PK response was attenuated, suggesting reduced need for pyruvate for amino acid synthesis. Pi no longer strongly promoted C fixation/storage or substantially activated TOR-mediated signaling [132], reflecting weakened C-N-driven growth stimulation. Although MDH was upregulated, malate and citrate declined (Figure S2), indicating rapid consumption for intense N assimilation [133]. C allocation shifted toward “reduced storage, enhanced efficiency”: increased ATP (Figure S1), greatly reduced starch, and stable soluble sugars (Figure S5). Declines in NUE and PNUEi suggest Pi addition can exacerbate nutrient redundancy. Biomass increased (plant height, stem diameter, leaf weight, and total biomass; while root–shoot ratio decreased (Figure S5).

4.7. AMF Symbiosis Reshapes the NO₃⁻-Environment–Dependent Regulation of Leaf N–C Metabolism Under Pi Supply

AMF are integral to plant N metabolism, enhancing N uptake, utilization efficiency, and stress tolerance [134,135]. AMF optimize P fertilization effects on leaf N metabolism by enhancing N assimilation [136]. SEM revealed that AMF inoculation significantly alters Pi-supply effects on leaf N metabolism (Figure 8H,I).

Under low NO₃⁻ conditions, Pi-treated +AMF plants exhibited regulatory strategies distinct from those of −AMF plants (Figure 8H,L). AMF symbiosis amplified the alleviating effect of Pi on N stress. Pi treatment synergistically upregulated GS2, Fd-GOGAT, and NADH-GOGAT, promoting a highly efficient GS-GOGAT cycle, replacing the GS-GDH pathway in -AMF seedlings [137,138]. Enhanced N assimilation flux, coupled with sustained upregulation of transporter genes and repression of ATG8 and ASN1, resulted in increased soluble protein (Figure S3), prioritizing N for growth [139,140]. This led to decreased leaf N content and elevated N/P ratio (tissue N dilution), with unchanged NUE (prioritizing growth).

Under high NO₃⁻ + Pi supply (Figure 8M), AMF reprogrammed host adaptation to N-rich environments by reinforcing N assimilation. Compared to -AMF, seedlings exhibited significant upregulation of GS2, Fd-GOGAT, and NADH-GOGAT (enhancing assimilation), while downregulating transporter genes, forming a “suppressed uptake-enhanced assimilation” strategy. Stronger repression of NLP2 mitigated redundant N sensing and investment [141]. Soluble protein/free amino acids increased (Figure S3), and leaf N content remained stable. Moderate ATP elevation/RPS6 upregulation suggested AMF share energy burden (Figure S1). Although NUE and N/P were unchanged, a smaller decline in PNUEi indicated AMF mitigated nutrient imbalance under high Pi [142].

4.8. AMF Symbiosis Modulates NO₃⁻-Dependent Regulation of Leaf N–C Metabolism by Pi

Under low NO₃⁻ conditions (Figure 8H,L), −AMF seedlings primarily relied on organic acid metabolism, whereas +AMF plants exhibited stronger induction of Calvin cycle genes, increased Rubisco activity, and upregulation of RBCS and RCA1, enhancing carboxylation efficiency and providing reducing power and C skeletons for N assimilation [78,143,144]. This mechanism explains the observed Pi-induced increases in A and E in +AMF seedlings, with a greater decline in Ci compared to uninoculated plants. AMF also amplified Pi-induced PK and ME1 expression, promoting pyruvate production for amino acid synthesis, while lower malate accumulation confirmed a redirection of C flux toward N metabolism (Figure S2). AMF-specific suppression of Pi-induced AGP regulation reduced soluble sugars while maintaining starch stability (Figure S5), shifting C flow toward the mycorrhiza in exchange for N [145], and reinforcing TOR signaling for C-N coordination [146]. These adjustments resulted in enhanced root–shoot ratio, increased stem diameter, and greater dry matter accumulation (Figure S6).

Under high NO₃⁻ conditions, AMF shifted Pi responses from growth promotion toward metabolic efficiency by reconfiguring C–N allocation (Figure 8I,M). AMF enhanced PRK, FBP, and TOR transcription, converting Pi-induced Rubisco activation into higher C fixation efficiency without increasing stomatal conductance and water loss [147]. The antagonistic regulation between AGP upregulation and BAM1 activation redirected C from starch storage to immediate metabolic use (Figure S4), supporting mycorrhizal energy demands, but reducing C storage capacity. Optimized chlorophyll fluorescence and increased chlorophyll content ensured high light-use efficiency (Figure S4) [148,149,150]. In parallel, sustained ME1 activation and suppressed PK response reduced malate and citrate (Figure S2), while upregulation of PEPE and MDH ensured continued C skeleton supply for N assimilation [151]. Although this reconfiguration alleviated PNUE reduction, the decline in root–shoot ratio indicated excessive C allocation to aboveground tissues. Increased reliance on mycorrhiza-mediated P acquisition may heighten host vulnerability in C metabolism under fluctuating soil P availability.

5. Conclusions

This study shows that synergistic N–P utilization in poplar seedling leaves depends strongly on environmental nutrient availability. Under low Pi, NO₃⁻ supply rewires P perception and recycling, enabling preferential allocation of P to photosynthetic tissues. Coupled with upregulated C assimilation, this coordination increases photochemical efficiency and C fixation, although it remains constrained by limited P buffering capacity. In high-Pi environments, NO₃⁻ suppresses P-uptake signaling to mitigate toxicity, and redirects C flux from storage toward growth. AMF symbiosis systemically remodels this interaction. Under low Pi, AMF integrate P storage with C allocation to establish a dual-pool buffering system that substantially enhances resource resilience. Under high Pi, AMF attenuate nonessential endogenous P recycling to optimize energy–nutrient balance. Regardless of Pi supply, AMF also reconfigure host N metabolism: they promote an efficient assimilation cycle under low NO₃⁻, whereas under high NO₃⁻ they maintain homeostasis by dampening both uptake and excessive assimilation. Collectively, these results indicate that plants dynamically integrate N–P signaling with AMF-mediated regulation to achieve multiscale nutrient coordination adapted to environmental heterogeneity. This insight can inform the design of symbiosis-based precision management to improve nutrient-use efficiency in forestry species.

Nonetheless, critical gaps remain: genetic validation of N–P signaling; long-term evaluation of emergency P depletion and C reserve attrition; inclusion of field water–microbe interactions; and clarification of functional differentiation among AMF taxa. Particularly urgent is targeted dissection of the root system—the primary interface for AMF–host interactions—in orchestrating aboveground metabolism. Future work will combine genetic improvement, multi-AMF inoculation, and controlled field trials, with emphasis on root architecture and signaling mechanisms to advance understanding and application. A root-centric research agenda will therefore be prioritized.