Abstract Background Legume-based intercropping systems function in boosting crop productivity. However, the precise physiological mechanisms by which root exudates derived from these systems on crop growth have not been characterized. Here, the rhizosphere soil from a peanut/maize intercropping system was analyzed for metabolome profiles. Sucrose (SUC) and myo-inositol (MI) were significantly declined while oxalic acid (OA) was dramatically enriched compared with peanut monoculture. After concentration screening, the optimal concentrations of OA, SUC, and MI have been determined as 1.0 g/pot, 0.1 g/pot, and 0.1 g/pot, respectively. Armed with the optimal concentrations, OA, SUC, MI, and their combinations were applied to peanut soil, respectively. Results Agronomical and physiological assesses indicated that single application of SUC and the combination application of “OA + SUC” showed better performance on peanut growth, pod yield, and soil nitrogen (N) turnover processes including total N and NO[3]^−-N contents as well as activities of N turnover enzymes. Consequently, the transcriptome and metabolome profiles of SUC were further determined. A total of 1036/24 up-regulated and 797/35 down-regulated differential expressed genes (DEGs)/differential accumulated metabolites (DAMs) were detected in SUC-treated peanut roots, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis advocated that DEGs were mostly enriched in crucial pathways like Glycolysis/Gluconeogenesis-tricarboxylic acid cycle (TCA cycle) and N uptake and assimilation. Moreover, DAMs like D-Aspartic acid, L-Glutamic acid, and L-Threonine were identified in “Sucrose vs. Control”. Conclusion Application of root exudates like sucrose and oxalic acid derived from root exudates of peanut/maize intercropping system fulfil pivotal roles in enhancing peanut growth and productivity via modulating N turnover processes. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-025-06994-w. Keywords: Legume-based intercropping, Root exudates, Peanut, Plant growth, Yield Background Legume crops serve as fundamental source of high-quality proteins for humans and livestock worldwide owing to their economical and socioeconomic benefits [[36]1, [37]2]. Legume crops also play central roles in low-input cropping systems due to their capacity of biological nitrogen fixation [[38]3, [39]4]. Thus, to meet the challenge of global food security, a major re-focus of legume crops is largely required. It is widely accepted that legume-inclusive production systems (e.g. leguminous/gramineous and leguminous/grass intercropping) have long-term benefits for both crops and soil, which are suitable for modern cropping systems to lower the risk of global climate change over coming decades [[40]5–[41]9]. Legume-based intercropping strategies have been extensively adopted by farmers worldwide especially in developing countries due to the distinctive complementarity and facilitation processes between legumes and their concomitant species [[42]10–[43]13]. On the one hand, intercropped legumes could provide more nitrogen resources for their neighboring crops through biological nitrogen fixation. One the other hand, the intercropped gramineous or grass crops could also improve the utilization of nutrient resources like phosphorus (P) and boron (B), which consequently promote the growth performance of legumes crops [[44]14, [45]15]. Therefore, it is tempting to speculate that root exudates are key mediators in regulating this intercropping process. In recent years, rhizosphere ecology has been received more attention by agronomists due to its central importance in root growth [[46]16–[47]18]. Particularly, roots of legume crops secrete an enormous range of exudation into the rhizosphere soil that could affect the rhizosphere’s biological and ecological processes, thereby impacting the plant growth and, ultimately, crop productivity [[48]19–[49]21]. Enormous steps have been taken toward elucidating the functions of root exudates in mediating the interspecific interactions in legume-based intercropping systems. Accumulated evidence indicates that the symbiotic nitrogen-fixing rhizobia of legumes could help to accelerate the nutrient assimilation as well as induce growth facilitators of their neighboring crops via regulating the soil microbial communities and modulating soil physical and chemical properties [[50]14, [51]22, [52]23]. Therefore, it is tempting to speculate that some unique components of root exudates might participate in legume-based interspecific interactions, which warrant further investigation. Amino acids, organic acids, soluble sugars, and other secondary metabolites compose a larger proportion of root exudates. The multiple biological functions of these exudates are often divided into two classes, namely, positive and negative interactions [[53]21, [54]24]. In some cases, root exudates serve as allelochemicals in mediating growth inhibition and increasing sensitivity to harsh environment of their neighboring species (known as allelopathy), by which legume crops may gain an advantage over their competitors [[55]25, [56]26]. Contrastingly; however, the continuous cropping obstacle of legume crop species could be attributed to the over accumulation of residual allelochemicals in soil [[57]1, [58]27]. Thus, the root exudates of legume crops act as a double-edged sword whereby legumes establish a delicate balance between their neighboring plants and themselves via impacting the resource competition. In recent years, utilization of root exudates have come into light of the agronomists and farmers in promoting plant growth, mitigating abiotic stress, and boosting crop productivity due to their low-cost and eco-friendly properties [[59]28, [60]29]. However, insufficient research are available to determine how specific metabolites in exudates (e.g., oxalic acid and soluble sugars) directly regulate nitrogen turnover enzyme activities and gene expression in legume-based intercropping system. Peanut (Arachis hypogaea L.), an energy-dense and nutritious food, is a typical representative of legume crops [[61]30, [62]31]. To date, most researches about the promoting effects of peanut plant growth were based on fertilizers and plant growth regulators, which limits the identification of root exudations. In our multi-year field experiment, we have shown that the crop productivity of maize and peanut in the intercropping system was significantly increased compared with monoculture of maize or peanut [[63]11, [64]32]. Importantly, current literatures also advocated that peanut/maize intercropping system enhances crop productivity compared with monoculture of peanut and maize [[65]12, [66]24, [67]33]. Thus, it is tempting to speculate that some marker root exudations might play vital roles in boosting intercropping advantages. Therefore, objectives of the present work were to (1) identify the marker root exudates involved in maize/peanut intercropping system; (2) figure out the proper concentrations of the these marker exudates; (3) investigate the potential roles of these exudates on peanut growth with emphasis on the nitrogen turnover processes in both rhizosphere soil and peanut roots. It is hypothesized that exogenous application of these marker root exudates could promote the peanut growth. The current study could broaden our horizon on the distinctive functions of root exudations derived from legume-based intercropping systems and develop innovative agricultural practices for boosting peanut production in the context of sustainable agriculture. Materials Plant materials Peanut (Arachis hypogaea L.) variety Qinghua 6 obtained from Qingdao Agricultural University (Qingdao, Shandong Province, China) and maize (Zea mays L.) variety Jinhai188 provided by Jinhai Seed Industry Co., Ltd (Yantai, Shandong Province, China) were utilized as the experimental materials. These two varieties were all major-cultivated crops in Shandong Province. Experimental design Experiment I For the identification of distinctive root exudates of peanut/maize intercropping system, a pot-grown experiment was conducted at Qingdao Agricultural University, Shandong province, China (120.39^◦ E, 36.33^◦ N, altitude 57 m) from May to September, 2021. The uniform-sized peanut and maize seeds were soaked in 2% (v/v) sodium hypochlorite for 15 min and washed twice in distilled water. After germinating in the chamber for 7 days, the seeds were sown in ceramic pots (inner diameter of 26 cm and height of 20 cm). A total of 3 treatments were composed: Peanut monoculture (two peanut seeds were sown in one pot), Maize monoculture (two maize seeds were sown in one pot), and Peanut/maize intercropping (one peanut seed and one maize seed were sown in one pot). There were 30 pots in each treatment with three replicates in each treatment. Each pot was filled with equal amount of soil. The soil chemical properties were analyzed beforehand as follows: organic matter of 21.4 g · kg^−1, total nitrogen (N) of 1.12 g · kg^−1, available phosphorus (P) of 23.18 mg · kg^−1, available potassium (K) of 96.3 mg · kg^−1, and pH of 6.87. The seedlings were grown in a greenhouse with the photosynthetic photon flux density (PPFD) of 1200 µmol m^−2 s^−1, photoperiod of 16/8 h (light, 6:00–22:00/dark, 22:00–6:00), air temperature of 25/18℃ (day/night), and relative air humidity of 75%. Each pot was well watered with equal amount of distilled water every two days. The rhizosphere soil samples were collected at 10 cm below the soil level in the middle of the two plants at podding and maturity stages of peanut, respectively (Fig. [68]1). Fig. 1. [69]Fig. 1 [70]Open in a new tab Schematic diagram illustrating the experimental design of Experiment I, Experiment II, and Experiment III. SUC, Sucrose; MI, Myo-Inositol; OA, Oxalic acid Experiment II To shed light on the optimal concentrations of various root exudates on peanut growth, a pot-grown experiment was conducted at Qingdao Agricultural University, Shandong province, China (120.39^◦ E, 36.33^◦ N, altitude 57 m) from May to July, 2022. The uniform-sized peanut seeds were soaked in 2% (v/v) sodium hypochlorite for 15 min and washed twice in distilled water. After germinating in the chamber for 7 days, the seeds were sown in ceramic pots (inner diameter of 26 cm and height of 20 cm) with one seed each. The soil properties and growth conditions were identical to those in Experiment I. At 45 days after sowing, the pots were treated with 200 mL of water (Control), 0.05 g sucrose + 200 mL water (SUC 0.05), 0.10 g sucrose + 200 mL water (SUC 0.10), 1.00 g sucrose + 200 mL water (SUC 1.00), 0.05 g oxalic acid + 200 mL water (OA 0.05), 0.10 g oxalic acid + 200 mL water (OA 0.10), 1.00 g oxalic acid + 200 mL water (OA 1.00), 0.05 g myo-inositol + 200 mL water (MI 0.05), 0.10 g myo-inositol + 200 mL water (MI 0.10), and 1.00 g myo-inositol + 200 mL water (MI 1.00), respectively. At 70 days after sowing, the agronomic and physiological characters were determined for each treatment (Fig. [71]1). In the whole study, the sampling and measuring dates as well as the concentration adopted were determined based on our preliminary investigations (unpublished data) [[72]34–[73]36]. Experiment III To determine the potential roles of combined application of root exudates under optimal concentrations, a pot-grown experiment was carried out at Qingdao Agricultural University, Shandong province, China (120.39^◦ E, 36.33^◦ N, altitude 57 m) from August to December, 2022. The uniform-sized peanut seeds were soaked in 2% (v/v) sodium hypochlorite for 15 min and washed twice in distilled water. After germinating in the chamber for 7 days, the seeds were sown in ceramic pots (inner diameter of 26 cm and height of 20 cm) with one seed each. The soil properties and growth conditions were identical to those in Experiment I. At 45 days after sowing, the pots were treated with 200 mL of water (Control), 0.10 g sucrose + 200 mL water (SUC), 1.00 g oxalic acid + 200 mL water (OA), 0.10 g myo-inositol + 200 mL water (MI), 0.10 g sucrose + 0.10 g myo-inositol + 200 mL water (SUC + MI), 0.10 g sucrose + 1.00 g oxalic acid + 200 mL water (SUC + OA), 0.10 g myo-inositol + 1.00 g oxalic acid + 200 mL water (MI + OA), and 0.10 g sucrose + 0.10 g myo-inositol + 1.00 g oxalic acid + 200 mL water (SUC + MI + OA) respectively. Then, the root samples of Control and SUC were collected for the analysis of transcriptome and metabolome at 46 and 48 days after sowing, respectively. At 70 days after sowing, the agronomic, physiological, and soil characters were determined for each treatment (Fig. [74]1). At 120 days after sowing, the peanut pods were harvested. After sun-drying for 10 days, peanut yield and yield-related components were determined. Measurements and data collection Non‑targeted metabolites extraction and analysis The rhizosphere soil samples from Experiment I and the entire root samples from Experiment III were collected based on the protocol of De Vos et al. (2007). After mixing thoroughly, the samples were frozen in liquid nitrogen for 30 min and delivered to BioTree Biotechnology Co., Ltd. (Shanghai, China) for metabonomic analysis as reported by Theodoridis et al. (2008). In brief, the metabolites were firstly extracted for the UHPLC-QE-MS analysis. Chromatographic separation of target compounds was performed with a Vanquish UHPLC system (Thermo Fisher Scientific) equipped with a UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm). The mobile phase consisted of Phase A (water with 0.01% acetic acid) and Phase B (isopropanol: acetonitrile, 1: 1, v/v). The autosampler temperature was maintained at 4℃, and the injection volume was 2 µL. The gradient elution program was as follows: 1% B from 0 to 0.5 min, linear increase to 99% B from 0.5 to 4.5 min, and re-equilibration to 1% B from 4.5 to 6 min. The UHPLC system was coupled to a Q Exactive HFX mass spectrometer (Orbitrap MS, Thermo Fisher Scientific). Then, the raw data were converted to the format of mzXML with ProteoWizard and processed with an in-house program for peak detection, extraction, alignment, and integration. The high resolution MS data were further processed with MAPS software and identified through MS2 database. Supervised orthogonal projections to latent structures-discriminate analysis (OPLS-DA) was then applied in order to visualize group separation and find significantly changed metabolites. Variance was analyzed in the data with variable importance in the projection (VIP) > 1 and p-value < 0.05 as statistically significance. Moreover, commercial databases such as Kyoto Encyclopedia of Genes and Genomes (KEGG) ([75]http://www.genome.jp/kegg/) and MetaboAnalyst ([76]http://www.metaboanalyst.ca/) have been utilized for pathway enrichment analysis. In the current study, positive and negative ionization modes have been analyzed separately whereas their combination results were shown. Agronomic characters, gas exchange parameters, SPAD value, and LAI The agronomic characters including plant height and biomass of the seedlings were determined as described in our earlier reports [[77]35, [78]37]. The gas exchange parameters of peanut leaf: net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO[2] concentration (Ci), and transpiration rate (Tr) were determined by using a portable photosynthesis system (Li-COR 6800, Lincoln, NE, USA) based on our previous report [[79]34]. The third fully expanded leaves of the main stem (termed functional leaves) were analyzed with critical parameters configured as follows: ambient air temperature of 25℃, PPFD of 1,200 µmol m^−2 s^−1, CO[2] concentration of 380 µmol mol^−1, and relative air humidity of 75% in the leaf chamber. The leaf chlorophyll content was evaluated by assaying the SPAD value which was determined nondestructively with a SPAD 502 chlorophyll meter (Soil Plant Analysis Development; Minolta, Japan). Leaf area index (LAI) was determined via a portable laser area meter (CI-203, CID Bio-Science, Camas, WA, USA) and was calculated according to formula as: LAI = Total green leaf area of the sampling area/Ground area of the sampling plant [[80]38]. Soil total nitrogen, nitrate nitrogen, and ammonium nitrogen The total soil nitrogen (TN) content was determined following the method of Zhang et al. [[81]39]. Briefly, 0.3 g of the dried soil was digested with concentrated sulfuric acid and assayed by using a continuous flow analytical system (Auto Analyzer III, BRAN + LUEBBE, Hamburg, Germany) based on the standard curve. The contents of soil nitrate nitrogen (NO[3]^−-N) and ammonium nitrogen (NH[4]^+-N) were assessed as modified by the procedures of Bao et al. [[82]40]. Briefly, 5.00 g of dried soil samples were extracted with 50 mL of 2 M KCl on a rotary shaker for 1 h. After standing for 1 h at room temperature, the supernatant was collected for the determination of soil NO[3]^−-N content by assessing the absorbance at 220 nm and 275 nm. Meanwhile, the NH[4]^+-N content was also analyzed by measuring the absorbance at 625 nm. Activities of soil nitrogen metabolism enzymes The activity of soil urease (S-UE) was analyzed by extraction with KCl solution and measured at 690 nm as originally described [[83]41]. The activity of soil hydroxylamine reductase (S-HR) was assessed by using the iodometry method as modified by Shi et al. [[84]42]. The activity of soil nitrate reductase (S-NR) was determined by incubating with KNO[3] as the substrate and measured at 520 nm following the method of Schinner et al. [[85]43]. The activity of soil nitrite reductase (S-NiR) was measured based on the product (nitrite) formed according to the protocol of McNally et al. [[86]44]. The activity of soil protease (S-PT) was analyzed by incubating with sodium caseinate solution, measured at 680 nm, and calculated with the standard curve of tyrosine [[87]45]. Total RNA extraction and RNA-seq analysis The peanut root samples were collected and the total RNA was extracted via Cetyltrimethylammonium bromide (CTAB) method. The RNA integrity was determined by using a RNA Nano 6000 Assay Kit with Bioanalyzer 2100 system (Agilent Technologies). Then, the samples were delivered to Novogene Corporation Inc. for the subsequent RNA-seq analysis. Briefly, the clustering of the index-coded samples was conducted with a cBot Cluster Generation System by TruSeq PE Cluster Kit v3-cBot-HS (Illumia) and the raw data were processed with in-house perl scripts. Then, the sequencing reads of RNA-seq data were mapped to the reference genome by HISAT2 [[88]46]. Genes were annotated by searching against the PeanutBase ([89]https://peanutbase.org/). The differential expressed genes (DEGs) were identified (Padj < 0.05 and log[2]FC > 1) and categorized using the Gene Ontology (GO) enrichment analysis and the KEGG pathway database ([90]http://www.genome.jp/kegg/) [[91]47]. The raw sequencing data were deposited to Sequence Read Archive database (Accession number: PRJNA1171604). Quantitative real-time PCR The total RNA was extracted with the TRIzol reagent. Then, the cDNA was synthesized using an M-MLV Reverse Transcription Reagents Kit (Invitrogen) based on the manufacturer’s instructions. The synthesized cDNA was then taken as the template for qRT-PCR analysis with SYBR^® Green mix using a real-time PCR system (Bio-Rad). The primers used for qRT-PCR were listed in Table S1. Statistical analysis All experiments were carried out with a randomized complete block design with three independent biological replicates except for the metabolome analysis in Experiment III (four replicates). SPSS software (Version 22.0, SPSS Inc.) was utilized for the statistical analysis. Variations among treatments were determined by one-way analysis of variance (ANOVA). Significant treatment differences were tested using Tukey’s test at P < 0.05. Pearson correlation analysis was used for correlation analysis with “corrplot” R package. Results Identification of root exudates from peanut/maize intercropping system The first objective of this study was to identify the distinctive root exudates derived from peanut/maize intercropping system compared with monoculture based on the determination of non‑targeted metabolites. In Experiment I, principal component analysis (PCA) indicated that the first and second principal components were displayed on the X (PC1, 40.6%) and Y (PC2, 20.5%) axis, respectively (Fig. [92]2A). The super classes of the identified metabolites from all treatments mainly include lipid (24.78%), acid (15.93%), alcohol (15.04%), carbohydrate (9.73%), amine (6.19%), and heterocyclic compound (5.31%) (Fig. [93]2B). Volcano plots showed that 7/3 up-regulated and 3/27 down-regulated differential accumulated metabolites (DAMs) were detected in “M/P vs. M” and “M/P vs. P”, respectively, at peanut podding stage. Meanwhile, 3/0 up-upregulated and 0/22 down-regulated DAMs were observed in “M/P vs. M” and “M/P vs. P”, respectively, at peanut maturity stage (Fig. [94]2C; Table S2). Fig. 2. [95]Fig. 2 [96]Open in a new tab A Principal component analysis (PCA) of the metabolomic data from treatments of ‘Maize/Peanut-Podding stage’, ‘Maize/Peanut-Maturity stage’, ‘Maize-Podding stage’, ‘Maize-Maturity stage’, ‘Peanut-Podding stage’, and ‘Peanut-Maturity stage’. B Super classes of the identified metabolites from all treatments. C Volcano plots showing the differentially accumulated metabolites (DAMs) among treatments. “M/P vs M” and “M/P vs P” represent “Maize/Peanut vs. Maize” and “Maize/Peanut vs. Peanut”, respectively Heatmap analysis suggest that compared with intercropping, DAMs in peanut monoculture were mostly up-regulated in classes like alcohol (e.g. Myo-Inositol 3, Myo-Inositol 1, and Campesterol), carbohydrate (e.g. Sucrose), and 13 kinds of lipid compounds while down-regulated in classes of acid (Oxalic acid) and lipid (Tripropylene glycol monomethyl ether and Propylene glycol) at peanut podding stage. At peanut maturity stage, DAMs in peanut monoculture were mostly up-regulated in classes such as carbohydrate (e.g. Fructose 2), alcohol (e.g. Myo-Inositol 3, Myo-Inositol 1, and D-Pinitol), and acid (e.g. Benzoic acid), compared with intercropping (Fig. [97]3A). Notably, the enrichment analysis of KEGG further advocated that pathways associated with “Inositol phosphate metabolism”, “Galactose metabolism”, “Fructose and mannose metabolism”, and “Starch and sucrose metabolism” exhibited higher degree of alteration in “M/P vs. P” at both podding and maturity stages (Fig. [98]3B). The outcome of the striking results above prompted us to further elucidate the “Galactose metabolism” and “Inositol phosphate metabolism” in more detail. In these pathways, we noticed that some marker metabolites such as sucrose, myo-inositol 1, myo-inositol 3, and scyllo-inositol were significantly decreased in “M/P vs. P” at both growth stages whereas myo-inositol 1 and scyllo-inositol were dramatically increased in “M/P vs. M” at both growth stages of peanut (Fig. [99]3C). Fig. 3. [100]Fig. 3 [101]Open in a new tab A Heatmap analysis of abundance of identified metabolites among treatments. “*” represent differentially accumulated metabolites (DAMs) in monocultures compare to intercropping treatments at different growth stages. B Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of differentially expressed metabolites in different comparison groups. C DAMs in galactose and inositol phosphate metabolism. The value was the Log[2]FoldChange of differentially expressed metabolites. The colors of the boxes represent up-regulated (red) and down-regulated (blue) metabolites. “M/P vs. M” and “M/P vs. P” represent “Maize/Peanut vs. Maize” and “Maize/Peanut vs. Peanut”, respectively Taken together, based on the results of root exudates in Experiment I, sucrose (SUC), oxalic acid (OA), and myo-inositol (MI) have emerged as potential candidates that could be utilized to enhance plant growth and yield in peanut production. Concentration screening of sucrose, oxalic acid, and myo-inositol in response to peanut growth In Experiment II, the optimal concentrations of SUC, OA, and MI on peanut growth have been screened by using pot-grown experiments. The different concentrations of these substances were utilized based on our preliminary experiments and the literatures reported in legume crops. To evaluate the roles of these substances on some crucial physiological and growth parameters, radar maps have been conducted. Application of 0.10 g SUC per pot dramatically increased the Pn, Gs, and below-ground biomass compared with other treatments (Fig. [102]4A). Meanwhile, application of 1.00 g OA per pot showed increased Pn, LAI, and above-ground biomass compared with other treatments (Fig. [103]4B). Moreover, application of different concentrations of MI decreased the plant height and Pn compared with control; however, application of 0.10 g MI per pot showed higher SPAD value, below and above-ground biomass, and LAI, compared with other treatments (Fig. [104]4C). Fig. 4. [105]Fig. 4 [106]Open in a new tab Effect of exogenous application of different concentrations of myo-inositol A, oxalic acid B, and sucrose C on net photosynthetic rate (Pn), stomatal conductance (Gs), and SPAD value of the functional leaves, as well as LAI, below-ground biomass, and above-ground biomass of peanut seedlings. SUC, Sucrose; MI, Myo-Inositol; OA, Oxalic acid In summary, based on the above findings, we deduced that application of 0.10 g SUC, 1.00 g OA, and 0.10 g MI per pot could be the optimal concentrations in enhancing peanut growth, which have been utilized for the subsequent investigation. Effects of sucrose, oxalic acid, and myo-inositol on peanut growth, chlorophyll content, and photosynthetic rate In Experiment III, the optimal concentrations of SUC, OA, and MI have been utilized to further investigate the roles of these root exudates and their combinations on peanut growth. Application of SUC, OA + SUC (OS), and MI + OA + SUC (MOS) significantly increased the leaf biomass by 57.63, 41.00, and 24.08%, whereas application of MI and SUC significantly increased the stem biomass by 19.31 and 35.55%, respectively, compared with control. Additionally, application of OA, SUC, MI + OA (MO), MI + SUC (MS), OS, and MOS showed significant induction in root biomass except for MI (Fig. [107]5A). MI showed a dramatic decrease in plant height by 19.32% whereas OA, MO, OS, and MOS significantly increased the plant height by 17.36, 30.07, 23.47, and 23.72%, respectively, compared with control (Fig. [108]5B). For the leaf chlorophyll content, only OS significantly increased the SPAD value by 15.80% compared with control (Fig. [109]5C). Moreover, Pn was markedly decreased by 52.52% under MI and was not affected by other treatments compared with control (Fig. [110]5D). Fig. 5. [111]Fig. 5 [112]Open in a new tab Effect of single and combined application of 0.1 g pot^-1 of myo-inositol, 1.0 g pot^-1 of oxalic acid, and 0.1 g pot^-1 of sucrose on (A) total biomass of seedlings, (B) plant height, (C) SPAD value, and (D) net photosynthetic rate (Pn) of peanut functional leaves. Means denoted by different lowercase letters indicate statistical significance among treatments at P < 0.05 according to Tukey's test. Effects of sucrose, oxalic acid, and myo-inositol on nitrogen content and activities of nitrogen turnover enzymes in peanut rhizosphere soil Compared with control, MI, OA, SUC, MO, OS, and MOS significantly increased the plant total N content with only one exception of MS (Fig. [113]6A). Similarly, MI, SUC, MO, MS, OS, and MOS substantially induced the NO[3]^−-N content with one exception of OA (Fig. [114]6B). Strikingly, no significant difference was observed among treatments in NH[4]^+-N content (Fig. [115]6C). We then concentrated on the activities of enzymes involving in soil nitrogen turnover processes. As shown in Fig. [116]6D, MI, OA, SUC, MO, and MS significantly induced the activity of S-UE by 8.02, 24.15, 33.85, 7.43, and 9.37%, respectively, compared with control. However, only MI showed a significant increase in activity of S-HR by 49.68% compared with control, with no significant difference among other treatments (Fig. [117]6E). In addition, all treatments showed significant increases in activities of S-NR (Fig. [118]6F) and S-NiR (Fig. [119]6G) to varying degrees compared with control. Notably, MI, SUC, MO, MS, and MOS dramatically reduced the activity of S-PT by 46.72, 29.35, 61.91, 64.23, and 26.24%, respectively, compared with control (Fig. [120]6H). Fig. 6. [121]Fig. 6 [122]Open in a new tab Effect of single and combined application of 0.1 g pot^−1 of myo-inositol, 1.0 g pot^−1 of oxalic acid, and 0.1 g pot^−1 of sucrose on A total N content, B NH[4]^+-N content, and C NO[3]^−-N content of peanut functional leaves, and activities of D urease, E hydroxylamine reductase, F nitrate reductase, G nitrite reductase, and H protease of peanut rhizosphere soil. Means denoted by different lowercase letters indicate statistical significance among treatments at P < 0.05 according to Tukey’s test. SUC, Sucrose; MI, Myo-Inositol; OA, Oxalic acid; MO, Myo-Inositol + Oxalic acid; MS, Myo-Inositol + Sucrose; OS, Oxalic acid + Sucrose; MOS, Myo-Inositol + Oxalic acid + Sucrose Effects of sucrose, oxalic acid, and myo-inositol on peanut yield and yield related components At maturity stage, the peanut pods were manually harvested and dried, and then peanut yield was evaluated based on the pod weight per pot. OA, SUC, MO, and OS significantly increased the peanut pod weight by 30.22, 37.82, 22.78, and 41.14%, respectively, compared with control. Strikingly, no significant difference was observed among treatments in peanut pod number per plant. Similar to the pod weight data, OA, SUC, and OS showed a dramatic increase in peanut seed weight per plant by 28.54, 44.84, and 55.64%, respectively. Moreover, only OS significantly increased the full fruit rate by 16.13% compared with control (Table [123]1). Table 1. Effects of single and combined application of 0.1 g pot^−1 of myo-inositol, 1.0 g pot^−1 of oxalic acid, and 0.1 g pot^−1 of sucrose on peanut yield and yield related components Treatment Pod weight (g/plant) Pod number (No./plant) Seed weight (g/plant) Full fruit rate (%) Control 6.32 ± 0.57e 9.53 ± 1.36a 4.17 ± 0.43d 64.55 ± 7.33b MI 6.72 ± 0.53de 10.67 ± 1.15a 4.30 ± 0.42d 68.89 ± 7.88ab OA 8.23 ± 0.72abc 12.33 ± 0.67a 5.36 ± 0.6bc 69.84 ± 3.44ab SUC 8.71 ± 0.45ab 10.78 ± 1.07a 6.04 ± 0.41ab 64.35 ± 3.52b MO 7.76 ± 0.15bcd 10.11 ± 1.84a 5.10 ± 0.34 cd 63.70 ± 5.13b MS 7.26 ± 0.97cde 10.40 ± 1.44a 4.60 ± 0.33 cd 67.22 ± 7.52ab OS 8.92 ± 0.49a 10.67 ± 1.15a 6.49 ± 0.55a 74.96 ± 1.31a MOS 7.19 ± 0.54cde 10.22 ± 2.34a 4.66 ± 0.58 cd 72.44 ± 2.36ab [124]Open in a new tab Data are presented as the means ± standard deviation (SD) of three replications. Different letters in the same column indicate statistical significance among treatments at P < 0.05 according to Tukey’s test SUC Sucrose, MI Myo-Inositol, OA Oxalic acid, MO Myo-Inositol + Oxalic acid, MS Myo-Inositol + Sucrose, OS Oxalic acid + Sucrose, MOS Myo-Inositol + Oxalic acid + Sucrose A Pearson correlation analysis was further conducted and suggest that NO[3]^−-N, S-NR, and S-NiR showed significant positive correlations with some yield and yield related components. Moreover, TN, NO[3]^−-N, S-NR, and S-PT showed significant positive correlations with some physiological parameters (Fig. S1). Transcriptome and metabolome profiles revealing the role of sucrose on peanut growth The physiological and yield data outlined above implies that sucrose is a promising root exudate derived from peanut/maize intercropping system that could be utilized in peanut production. To further elucidate the profound role of sucrose application on peanut growth, the transcriptome and metabolome profiles have been determined on plant roots. A total of 1036 up-regulated and 797 down-regulated DEGs have been detected in ‘Sucrose vs. Control’ (Fig. [125]7A; Table S3). qRT-PCR assay suggested that the selected genes of interest showed similar expression patterns with the RNA-seq data (Fig. S2). The Gene Ontology (GO) annotation of DEGs indicated that most of the DEGs were enriched in pathways associated with “iron ion binding”, “peroxidase activity”, and “antioxidant activity” e.g. (Fig. [126]7B). Moreover, 24 up-regulated and 35 down-regulated DAMs were observed in ‘Sucrose vs. Control’ (Fig. [127]7C; Table S4). GO annotation showed that most of the DAMs could be categorized into pathways such as “Organic acids and derivatives”, “Organooxygen compounds”, and “Cinnamic acids and derivatives” (Fig. [128]7D). Fig. 7. [129]Fig. 7 [130]Open in a new tab A Volcano plot showing the differentially expressed genes (DEGs) in ‘Sucrose vs. Control’. B Gene Ontology (GO) annotation of DEGs in ‘Sucrose vs. Control’. Numbers above each bar represent DEG numbers. C Volcano plot showing the differentially accumulated metabolites (DAMs) in ‘Sucrose vs. Control’. D Super classes of DAMs in ‘Sucrose vs. Control’. Numbers above each bar represent up-regulated or down-regulated DAM numbers KEGG pathway analysis further indicated that most of the DEGs were enriched in pathways like “Glycolysis/Gluconeogenesis”, “Nitrogen metabolism”, “MAPK signaling”, and “Phenylpropanoid biosynthesis” (Fig. [131]8A) while DAMs could be mainly classified as “D-Amino acid”, “Alanine, aspartate and glutamate”, and “Glycine, serine, and threonine” metabolisms (Fig. [132]8B). Thus, the above data prompted us to further uncover the Glycolysis/Gluconeogenesis-tricarboxylic acid cycle (TCA cycle) pathway in more detail. DEGs including GALM, FPK, FBA, PK, PDC, MDH, AST, and IDH et al. in ‘Sucrose vs. Control’ have been detected (Fig. [133]8C). Furthermore, transcriptome profiles regarding N uptake and N assimilation have been detected. DEGs including AMT, NRT, NR, NIR, and GS were observed in ‘Sucrose vs. Control’ (Fig. [134]8D). In addition, the metabolome profiles of amino acid were further identified where DAMs such as D-Aspartic acid (Asp), L-Glutamic acid (Glu), L-Threonine (Thr), D-Glutamic acid (Glu), and D-Serine (Ser) were shown in ‘Sucrose vs. Control’ (Fig. [135]8E). Fig. 8. [136]Fig. 8 [137]Open in a new tab A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of DEGs in ‘Sucrose vs. Control’. B KEGG pathway analysis of DAMs in ‘Sucrose vs. Control’. DEGs in C Glycolysis/Gluconeogenesis-tricarboxylic acid cycle (TCA cycle) and D N uptake and assimilation metabolisms. Heatmap represent the expression levels of genes. E The profile of amino acid metabolites in each treatment. The value is the Log[2]FoldChange (Log[2]FC) of each metabolite. The colors of the boxes represent up-regulated (pink) and down-regulated (green) metabolites. Asterisks indicate significant differences (P < 0.05) according to Tukey’s test Discussion Identification of key exudates from peanut/maize intercropping system Although the distinctive functions of legume-based intercropping systems have been intensively studied [[138]13, [139]14, [140]48], the possible roles of root exudates derived from these systems are still obscure. Here, myo-inositol (MI), oxalic acid (OA), and sucrose (SUC) have been identified from the rhizosphere soil of a peanut/maize intercropping system based on the metabolome analysis. Physiological and agronomical data advocated that OA and SUC could be taken as good candidates in promoting peanut growth and yield formation. Particularly, physiological and multi-omics evidence further indicated that application of SUC could simulate the processes of N turnover in both rhizosphere soil and peanut roots. Our work would be important for deepening our understanding of the functions of root exudates derived from legume-based intercropping systems. Single-action mechanism of MI, OA, and SUC in peanut MI, OA, and SUC were all marker metabolites in the rhizosphere soil of peanut/maize intercropping system. Notably, MI and SUC were significantly declined while OA was dramatically enriched compared with peanut monoculture (Fig. [141]3A). MI is of paramount biological importance in the biogenesis of cell wall and membrane [[142]49], transportation and storage of auxin [[143]50], and vitro rooting in tissue culture systems [[144]51]. Unexpected, we observed that exogenous application of MI failed to increase the peanut growth and pod yield (Figs. [145]5 and [146]6; Table [147]1). Indeed, to the best of our knowledge, researches concerning the promoting effects of MI are scare in the literature. We therefore deduce that MI acts as a growth factor which was implicated in processes of signal transduction, rather than promote plant growth directly. Reversely, exogenous application of OA and SUC showed positive effects on peanut growth and yield formation in the current study. In previous approaches, OA, a crucial dicarboxylic acid, has been applied to promote the solubility of soil P and soil health, and thereby improving plant productivity [[148]52, [149]53]. Moreover, intercropping systems facilitate the P uptake by changing rhizosphere properties [[150]54]. Coincidently, peanut requires sufficient P to accelerate flower-bud differentiation, which is a crucial phase for yield formation [[151]55–[152]57]. In accordance with the earlier reports, OA dramatically enhanced the peanut root biomass and plant height (Fig. [153]5A and B). We therefore speculate that OA is involved in the peanut growth facilitation, at least partially, through enhancing soil P availability, which warrant further experimental validation. Combined effect analysis of MI, OA, and SUC on peanut In line with the OA application data, SUC has been shown to play promoting effects on peanut growth in the current study (Fig. [154]5; Table [155]1). Apart from the pivotal position occupied in providing carbon resources for the biosynthesis of starch in carbon metabolism, SUC is also considered to be a multifunctional nutrient factor involved in plant growth and development, signal transduction, and abiotic stress resistance [[156]58–[157]60]. Thus, it is tempting to explore the synergistic effects of OA and SUC on peanut growth responses. Unexpected, combined application of OA and SUC failed to further simulate the peanut growth and pod yield with one exception of the SPAD data, compared with their sole applications (Fig. [158]5; Table [159]1). These confused observations may arise due to some unknown antagonistic pathways exist in metabolisms of organic acids and sugar, which requires further studies. SUC modulates soil-root interactions in peanut rhizosphere soil Soil biochemical analysis advocated that exogenous SUC simulated the TN and NO[3]^−-N, as well as the activities of NR and NiR in the rhizosphere soil of peanut (Fig. [160]6A, B, F and G). Consistent with our findings, an extensive literature indicated that SUC might stimulate the biosynthesis of nitrogenous compounds via activating N metabolic enzymes including NR and NiR [[161]61–[162]63]. Moreover, Gordon et al. (1999) reported that the initial hydrolysis of SUC in nodules of pea, a typical legume crop, play essential roles in N fixation [[163]64]. Likewise, the reinforcement of total N especially N[2] in the rhizosphere soil could provide more resources for the biological N fixation of symbiotic rhizobia in peanut. Notably, peanut pods develop underground and a large percentage of nutrients were absorbed directly from the soil at podding stage [[164]27, [165]36, [166]65]. From this point of view, SUC might also contributes to the nutrients assimilation of peanut pods, and thereby increases pod yield. The data so far demonstrate that exogenous application of SUC modulates the N turnover processes in peanut rhizosphere soil. More attention has been paid to the root metabolic pathways in response to SUC. Strikingly, DEGs regarding Glycolysis/Gluconeogenesis-TCA cycle have been detected in peanut roots (Fig. [167]8C). Multiple studies have reported the dominant role of Glycolysis/Gluconeogenesis-TCA cycle in linking energy metabolism with both N and C [[168]66–[169]68], hinting a possibility that SUC helps to activate multiple genes regarding this crucial hub metabolism. Literatures advocated that SUC could help to (1) enhance the plant photosynthesis and C translocation; (2) increase the N metabolizing enzyme activities and optimize N partitioning; (3) promote the balance of N and C [[170]69, [171]70]. Moreover, crucial genes involving in N uptake like AMT1, NRT1, and NRT2 were dramatically induced in SUC treated peanut roots. Coincidently, some marker DEGs regarding N assimilation such as NR, NIR, and GS were also enriched (Fig. [172]8D), suggesting that SUC benefits in roots N turnover processes of both N uptake and N assimilation. We further noticed that SUC induced N assimilation results in some crucial DAMs involving amino acid metabolism (Fig. [173]8E), among which Asp contributes to modulating the lateral root development as well as nutrient absorption [[174]71, [175]72]; Glu occupies a central position in the biosynthesis of other amino acids [[176]73, [177]74]; Ser displayed properties of regulating photosynthesis and abiotic stress responses [[178]75, [179]76]. The above findings firmly established a regulatory pathway in peanut roots by which SUC promotes the N uptake and assimilation, activates Glycolysis/Gluconeogenesis-TCA cycle, accelerates the biosynthesis of crucial amino acids thereafter, and finally enhances plant growth and pod yield. Conclusions In general, evidence is provided in the current study to support the hypothesis that exogenous application of sucrose and oxalic acid boost the peanut growth pod yield by modulating the N turnover processes in both rhizosphere soil and roots. The outcome of the current study provides a novel approach to promote crop production by using appropriate root exudates derived from legume-based intercropping systems. Future work pertaining to the molecular mechanisms of these root exudates on plant growth and yield formation are still required to make a more comprehensive understanding of the interspecific interactions in legume-based intercropping systems. Supplementary Information [180]12870_2025_6994_MOESM1_ESM.xlsx^ (9.9KB, xlsx) Supplementary Material 1. Table S1. Primers used for qRT-PCR in this study. [181]12870_2025_6994_MOESM2_ESM.xlsx^ (41.8KB, xlsx) Supplementary Material 2. Table S2. The identified metabolites in the rhizosphere soil of different treatments. [182]12870_2025_6994_MOESM3_ESM.xlsx^ (131.5KB, xlsx) Supplementary Material 3. Table S3. Differentially expressed genes in ‘Sucrose vs. Control’. [183]12870_2025_6994_MOESM4_ESM.xlsx^ (45.5KB, xlsx) Supplementary Material 4. Table S4. Differentially accumulated metabolites in ‘Sucrose vs. Control’. [184]12870_2025_6994_MOESM5_ESM.docx^ (159.5KB, docx) Supplementary Material 5. Fig. S1 Correlation analysis of peanut yield, yield characteristics, plant physiological properties, and soil nitrogen properties under exogenous application of root exudates. ^* and ^**refer to P < 0.05 and P < 0.01, respectively. [185]12870_2025_6994_MOESM6_ESM.docx^ (111.6KB, docx) Supplementary Material 6. Fig. S2 Relative expression of genes as interest from RNA-seq data. Acknowledgements