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Article

Systemic Effects of Nitrate on Nitrogen Fixation and Sucrose Catabolism in Soybean (Glycine max (L.) Merr.) Nodules

by
Xuelai Wang
,
Tong Guo
,
Yuchen Zhang
,
Xiaochen Lyu
,
Shuangshuang Yan
,
Chao Yan
,
Zhenping Gong
and
Chunmei Ma
*
College of Agriculture, Northeast Agricultural University, Harbin 150030, China
*
Author to whom correspondence should be addressed.
Agronomy 2025, 15(5), 1032; https://doi.org/10.3390/agronomy15051032
Submission received: 31 March 2025 / Revised: 21 April 2025 / Accepted: 24 April 2025 / Published: 25 April 2025
(This article belongs to the Section Agricultural Biosystem and Biological Engineering)
Browse Figures
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Abstract

:
Soybean (Glycine max) nitrogen fixation is inhibited by nitrate, which has been linked to a reduction in carbon allocation and metabolism within nodules. However, the underlying mechanisms remain unclear. In this study, we tested the hypothesis that the nitrate-induced suppression of nitrogen fixation is mediated through altered sucrose allocation and catabolism in nodules. Using unilaterally nodulated dual-root soybean plants in sand-based systems, we applied 200 mg·L−1 nitrate exclusively to the non-nodulated roots for 14 days. Nitrate supply enhanced the proportion of dry weight in leaves but reduced it in nodules at 3, 7, and 14 days. Similarly, nodule dry weight, nodule number, acetylene reduction activity (ARA), and specific nodule activity (SNA) all declined significantly during the same intervals. Notably, sucrose content in the nodules decreased significantly by 20.4% after 3 days but recovered at 7 and 14 days. In contrast, sucrose synthase (SuSy) cleavage activity and malate content in nodules decreased significantly following nitrate treatment, with reductions of 27.8% and 30.7% observed at 7 days, and further decreased to 38.5% and 39.2% at 14 days, respectively. These results suggest that transient sucrose scarcity may drive the initial decline in nitrogen fixation capacity, while restricted sucrose catabolism and decreased malate levels may be a consequence rather than a cause.

1. Introduction

Soybean (Glycine max) is a nutritious leguminous crop that is rich in protein and oil and is used as a source of food and feed worldwide [1]. It has a strong adaptability to soil types and climatic conditions and can grow in a variety of soils, including sandy, loamy, and clay soils. One of the most distinctive features of soybean plants is their ability to form nodules through a symbiotic relationship with rhizobia, such as Bradyrhizobium [2]. The establishment of this symbiotic relationship is a complex, multi-stage process. It begins with the recognition of rhizobial signals by the plant, followed by rhizobia infection, the development of invasion threads, the formation of rhizobia primordia, and ultimately, the growth and development of rhizobia within the nodules [3]. During nodule development, the rhizobia differentiate into nitrogen-fixing bacteroids [4]. The nitrogenase complex within these bacteroids catalyzes the reduction in atmospheric nitrogen to ammonia, which is then metabolized into amino acids. These amino acids provide essential nitrogen nutrients to the host plant, supporting its growth and development [5,6]. Nodule nitrogen fixation can meet 50–60% of a soybean’s nitrogen requirements [7]. Meanwhile, as an energy-intensive process, nitrogen fixation in nodules demands a significant amount of photoassimilates to provide both energy and carbon skeletons for nitrogen fixation reactions and ammonia assimilation [5,8].
Nitrate, a common form of nitrogen found in soils, inhibits root nodule formation, growth, and nitrogen-fixing capacity in legumes both locally and systemically [9]. The mechanisms of these inhibitions are more complex and are not yet fully understood. Previous studies have suggested that nitrate-induced suppression of nodule nitrogen fixation might be linked to carbohydrate deficiency in the nodules [10]. When nitrate is applied, it alters the distribution of photoassimilates within the plant, leading to a reduction in their transport to nodules, which limits carbon availability and consequently hinders nodule growth and nitrogen-fixing capacity [11,12]. In soybean plants, sucrose, as the primary form of carbohydrate transport, plays a crucial role in this process [13]. Xu et al. [14] found that a 4.3 mM nitrate treatment significantly reduced nitrogenase activity and sucrose content in the nodules of hydroponically grown soybeans. However, Streeter et al. [15] applied 100 mg/L nitrate to sand-cultured soybeans and observed a significant decrease in nodule nitrogenase activity, with nodule sucrose content initially decreasing but later increasing over time. In addition, Gordon et al. [16] found that a 10 mM nitrate treatment significantly reduced nodule nitrogenase activity in soybeans grown in vermiculite, with no significant change in nodule sucrose content. Nevertheless, under hydroponic conditions, a 10 mM nitrate treatment not only significantly reduced nodule nitrogenase activity but also increased nodule sucrose content [17]. It is evident that the complex interplay between nitrate, nitrogen-fixing enzyme activity, and carbon allocation in soybeans requires further investigation.
Sucrose hydrolysis and malate synthesis are essential processes for nitrogen fixation in root nodules. Malate serves as the primary carbon source for bacteroids [6,18]. In root nodules, sucrose synthase (SuSy) catalyzes the conversion of sucrose into UDP-glucose and fructose [19]. Previous studies have demonstrated that sucrose metabolism, catalyzed by nodule SuSy, is essential for nodule formation, growth, and nitrogen-fixing capacity [20,21,22]. Gordon et al. [16] found that drought and salt stress can diminish the SuSy activity of nodules, thereby limiting sucrose metabolism and impacting the activity of nitrogenase within the nodules. However, while nitrate treatment significantly inhibits both the expression levels and enzyme activity of SuSy, this does not appear to be the primary factor contributing to the initial decline in nodule nitrogenase activity [17]. Lambert et al. [23] discovered that when Medicago truncatula plants were treated with 10 mM NH4NO3, there was a decrease in nitrogenase activity in root nodules, accompanied by a significant reduction in nodule sucrose and malate levels. Similarly, Sulieman et al. [24] reported that in Medicago truncatula, treatment with 5 mM nitrate resulted in a significant decrease in nodule nitrogenase activity and malate levels, while having no significant effect on nodule sucrose levels. Lyu et al. [25] found that the supply of 100 mg/L NH4NO3 restricted the metabolic conversion of sucrose to malate in soybean nodules, thereby reducing the nitrogen-fixing capacity of the nodules. Heckmann et al. [26] demonstrated that the application of exogenous malate can effectively alleviate the reduction in nodule nitrogen fixation capacity caused by nitrate supply. Furthermore, it remains unclear whether the restricted carbon supply and metabolism in root nodules is a cause or a consequence of the nitrate-induced inhibition of nitrogen fixation [17,27].
Here, based on previous studies, we hypothesize that nitrate application will inhibit nodule nitrogen fixation in soybean plants by limiting carbon supply and catabolism in nodules. Prior studies investigating the relationship between nitrate and nitrogen fixation in soybean nodules have predominantly utilized single-root soybean plants with nodules as experimental materials. However, this approach fails to eliminate the confounding effects of direct nitrate exposure on the nodules. Although the split-root method can circumvent this issue, it often results in damage to the root system. To address these limitations and elucidate the systemic regulatory effects of nitrate on nodule nitrogen fixation, this study introduces an innovative experimental design. We employed unilaterally nodulated dual-root soybean plants generated through grafting as the experimental material. By applying nitrate exclusively to the non-nodulated roots, we avoided the direct nitrate exposure to the nodules and minimized root damage inherent in traditional split-root systems. We assessed dry matter accumulation and distribution, nodule development, and nitrogen fixation capacity, as well as sucrose distribution and metabolism within nodules under different nitrate application timings to explore the systemic regulatory mechanisms. We anticipate that the findings of this study will provide novel theoretical insights into how nitrate modulates the dynamics of nitrogen fixation in soybean nodules.

2. Materials and Methods

2.1. Experimental Site and Climate Conditions

The experiment was conducted from June to July 2023 at the Northeast Agricultural University Experimental Station (126°43′ E, 45°44′ N), under ambient environmental conditions. The region is characterized by an annual precipitation of 500–550 mm, primarily concentrated from June to September. The mean annual temperature is 5.2 °C, and the annual cumulative temperature (≥10 °C) exceeds 2700 °C. During the experimental period, the average air temperature was 22.5 °C, the average relative humidity was 74.7%, total rainfall amounted to 319 mm, and the duration of sunshine was 317.6 h.

2.2. Plant Materials and Growth Conditions

The unilaterally nodulated dual-root soybean plants were prepared through grafting between a nodulated soybean variety (Glycine max (L.) Merr. cv. Heinong 40, obtained from the Heilongjiang Academy of Agricultural Sciences, Harbin, China) and a non-nodulated soybean variety (Glycine max (L.) Merr. cv. WDD01795, L8-4858, obtained from the Chinese Academy of Agricultural Sciences, Beijing, China). These grafted dual-root soybean plants were used as the experimental materials. Dual-root soybean plants were cultivated in sand-based growth systems, with two individual plants per experimental pot. The grafting of unilaterally nodulated dual-root soybean plants and the preparation of the nitrogen-free nutrient solution followed the method outlined by Lyu et al. [28]. Detailed procedures for grafting, rhizobial inoculation, and nutrient solution preparation are provided in the Supplementary Materials (see Figures S1 and S2, Table S1). Dual-root soybean plants were irrigated differentially throughout their development. Distilled water was applied once daily until the VC stage (unfolded cotyledon stage). From the VC stage to the V4 stage (four fully expanded trifoliate leaves stage), a nitrogen-free nutrient solution was administered once daily. From the V4 stage until the end of the experiment, the nutrient solution was applied twice daily. Each root compartment received 250 mL per irrigation. Growth stages were classified according to Fehr et al. [29].

2.3. Experimental Treatments and Sampling Methods

From the VC stage to the V4 stage of unilaterally nodulated dual-root soybean, both root systems were initially supplied with a nutrient solution containing 12.5 mg·L−1 of nitrogen for preliminary cultivation. From the V4 stage onward, a nitrogen-free nutrient solution was applied to both roots for 10 days to induce nitrogen starvation. Following this pretreatment, the experiment began with two distinct treatments. In the FN (nitrogen-free) treatment, both sides of the dual-root system received nitrogen-free nutrient solution. In the HN (high nitrogen-supplied) treatment, the non-nodulated roots were supplied with a nitrogen-containing solution (200 mg·L−1), while the nodulated roots continued to receive a nitrogen-free solution. Sampling was conducted at 1, 3, 7, and 14 days after treatment, with eight biological replicates for each treatment (Figure 1). During the sampling process, roots were washed with distilled water to remove adhering sand and gently blotted dry with paper towels. For each treatment, four pots were used to efficiently collect fresh nodules. The nodules were promptly frozen in liquid nitrogen and stored at −80 °C to preserve enzyme activity and prevent degradation. Direct tissue homogenization of the frozen samples was conducted for subsequent analysis of sucrose content, malate levels, and sucrose synthase activity. The remaining four pots were used to assess plant dry matter. Plants were cut at the grafting site, and tissues were separated into leaves, petioles, stems, non-nodulated roots, and the nodulated root–nodule complex. After nitrogenase activity was measured, nodulated roots were further separated from the nodules. Nodules were counted, and all plant tissues were dried to a constant weight in a forced-air oven at 60 °C.

2.4. Analysis Methods

Nodule nitrogenase activity was determined using the acetylene reduction method [30]. Sucrose content was measured using the resorcinol colorimetric method [31]. Sucrose synthase (SuSy) cleavage activity was assessed following the protocol of Zrenner et al. [32]. Malate content was quantified based on the method of Scherer [33], with minor modifications. Briefly, 0.1 g of fresh soybean nodule samples were homogenized with 1 mL of distilled water and then centrifuged at 8000× g for 10 min. The supernatant was analyzed using an HPLC system (RIGOL L3000, Chromai Technologies, Inc., Suzhou, Jiangsu, China) equipped with a Kromasil C18-BP column (250 mm × 4.6 mm, 5 μm, Sepax Technologies, Inc., Suzhou, Jiangsu, China) and a UV-Vis detector (Chromai Technologies, Inc., Suzhou, Jiangsu, China). The mobile phase consisted of methanol and a 0.01 mol L−1 NaH2PO4 buffer. The flow rate was set at 1.0 mL/min, the detection wavelength was 214 nm, the column temperature was maintained at 30 °C, and the injection volume was 10 μL.

2.5. Data Calculation

An increase in the dry weight of various organs, nodule number, individual nodule dry weight, acetylene reduction assay (ARA), specific nitrogenase activity (SNA), sucrose content, SuSy cleavage activity, and malate content was calculated as follows:
Percentage Increase = (Treatment Value HN − Treatment Value FN)/Treatment Value FN × 100%
Treatment Value HN refers to the measurement value obtained under the HN treatment. Treatment Value FN refers to the measurement value obtained under the FN treatment.
The proportion of the dry weight of various organs was calculated as follows:
Proportion of dry weight = (Dry Weight of various organs/Dry Weight of total) × 100%
The percentage increase in the dry weight proportion of various organs was calculated as follows:
Percentage Increase = (Proportion of dry weight HN − Proportion of dry weight FN)/Proportion of dry weight FN × 100%

2.6. Data Analysis

Data were statistically analyzed using SPSS version 21.0 (IBM, Armonk, NY, USA) and visualized using Origin 2021 (OriginLab, Northampton, MA, USA). Differences between treatments were analyzed using Student’s t-test, with normality testing conducted prior to analysis.

3. Results

3.1. Effects of Nitrate on Dry Matter Accumulation in Various Organs of Dual-Root Soybean

Figure 2 shows the dynamic changes in dry weight of various organs in unilaterally nodulated dual-root soybean plants following the supply of nitrate to the non-nodulated roots over periods of 1, 3, 7, and 14 days. A progressive increase in dry weight was observed in all plant parts, including leaves, petioles, stems, non-nodulated roots, nodulated roots, nodulated root–nodule complexes, shoots, belowground, and whole plants, under both nitrogen-free (FN) and high nitrogen (HN) treatments as the duration of treatment was extended. Compared with FN treatment, the HN treatment significantly increased leaf dry weight by 20.8%, 32.9%, and 34.2% at 3, 7, and 14 days after treatment (DAT), respectively (Figure 2A). Petiole dry weight significantly increased by 25.6% and 25.3% at 7 and 14 DAT (Figure 2B), and stem dry weight increased by 28.9% and 32.0% at the same time points (Figure 2C). Similarly, shoot dry weight under HN treatment increased significantly by 14.7%, 30.4%, and 31.8% at 3, 7, and 14 DAT, respectively (Figure 2G). These results indicate that the supply of nitrate to non-nodulated roots promotes shoot dry matter accumulation, with the most pronounced effect observed in leaves, followed by petioles and stems.
In the root system, the dry weight of non-nodulated roots increased significantly under HN treatment by 24.7%, 40.3%, and 41.0% at 3, 7, and 14 DAT, respectively (Figure 2D). In contrast, the dry weight of nodulated roots showed no significant differences across all time points (Figure 2E). However, the combined dry weight of nodulated roots and their associated nodules (i.e., the nodulated root–nodule complex) decreased significantly by 10.3%, 17.3%, and 18.4% at 3, 7, and 14 DAT under HN treatment (Figure 2F). Interestingly, belowground dry weight remained unchanged across treatments and time points (Figure 2H), suggesting that nitrate application to non-nodulated roots did not alter total belowground biomass but had contrasting effects on the two root types—promoting dry matter accumulation in non-nodulated roots while inhibiting it in the nodulated root–nodule complex. As a result of these organ-specific changes, the whole plant dry weight increased by 9.7%, 21.0%, and 22.7% under HN treatment at 3, 7, and 14 DAT, respectively (Figure 2I). Collectively, these findings suggest that nitrate supplementation to non-nodulated roots enhances whole-plant dry matter accumulation primarily through increased shoot dry matter, rather than changes in belowground dry matter.

3.2. Effects of Nitrate on Dry Matter Allocation in Various Organs of Dual-Root Soybean

Figure 3 shows the dynamic changes in dry weight proportions of various organs in unilaterally nodulated dual-root soybean plants following the supply of nitrate to the non-nodulated roots over periods of 1, 3, 7, and 14 days. Compared with the FN treatment, the HN treatment significantly increased the leaf dry weight proportion by 10.3%, 9.8%, and 9.3% at 3, 7, and 14 DAT, respectively (Figure 3A). In contrast, the dry weight proportions of the petioles and stems remained unchanged throughout the sampling period (Figure 3B,C). The shoot dry weight proportion under HN treatment increased by 4.5%, 7.7%, and 7.3% at 3, 7, and 14 DAT, respectively (Figure 3G). These results suggest that nitrate application to the non-nodulated roots promotes preferential allocation of dry matter to leaves, while maintaining stable partitioning in petioles and stems, thereby contributing to improved shoot dry matter accumulation. No significant differences were observed in the dry weight proportion of non-nodulated roots between FN and HN treatments during the experimental period (Figure 3D). However, the dry weight proportion of nodulated roots under HN treatment decreased significantly by 14.7%, 21.5%, and 20.9% at 3, 7, and 14 DAT, respectively (Figure 3E). Similarly, the combined dry weight proportion of nodulated roots and their associated nodules declined significantly by 18.3%, 31.6%, and 33.5% at the same time points (Figure 3F). Additionally, the belowground dry weight proportion under HN treatment was reduced by 10.4%, 21.9%, and 22.8% at 3, 7, and 14 DAT, respectively (Figure 3H). Collectively, these findings indicate that nitrate supply to non-nodulated roots redirects dry matter partitioning away from belowground organs, primarily by diminishing allocation to nodulated roots and their symbiotic nodules.

3.3. Effects of Nitrate on Nodulation and Nitrogenase Activity in Dual-Root Soybean

Figure 4 shows the dynamic changes in nodulation and nitrogenase activity of unilaterally nodulated dual-root soybean following the supply of nitrate to the non-nodulated roots over periods of 1, 3, 7, and 14 days. Compared with the FN treatment, the HN treatment resulted in significant reductions in nodule dry weight by 15.5%, 33.9%, and 45.7% at 3, 7, and 14 DAT, respectively (Figure 4A). Additionally, the proportion of nodule dry weight decreased by 23.0%, 45.3%, and 55.5% at the same time points (Figure 4B). Nodule number also declined significantly under the HN treatment, with reductions of 17.6%, 31.7%, and 37.9% at 3, 7, and 14 DAT, respectively (Figure 4C). In contrast, individual nodule dry weight did not show a significant difference before 7 DAT but decreased by 15.9% at 14 DAT (Figure 4D). These results suggest that the reduction in total nodule dry weight and its proportion due to nitrate supply to non-nodulated roots is primarily attributed to a decrease in nodule number, with a lesser impact on individual nodule growth. Comparison to the FN treatment, acetylene reduction assay (ARA) in the HN treatment was significantly lower by 34.5%, 63.2%, and 66.0% after 3, 7, and 14 DAT, respectively (Figure 4E). Furthermore, specific nitrogenase activity (SNA) was reduced by 22.7%, 36.3%, and 26.0%, respectively (Figure 4F). Collectively, these findings indicate that nitrate supply to the non-nodulated roots diminishes the nitrogen-fixing capacity of unilaterally nodulated dual-root soybean plants.

3.4. Effects of Nitrate Supply on Sucrose Content, Sucrose Synthase Cleavage Activity, and Malate Content in Nodules of Dual-Root Soybean

Figure 5 shows the dynamic changes in sucrose content, Sucrose Synthase (SuSy) cleavage activity, and malate content in the nodules of unilaterally nodulated dual-root soybean plants following nitrate supply to the non-nodulated roots over periods of 1, 3, 7, and 14 days. Compared with the FN treatment, the HN treatment resulted in a significant reduction in nodule sucrose content by 20.4% at 3 DAT, with no significant changes observed at 7 and 14 DAT (Figure 5A). These results suggest that nitrate application transiently reduces sucrose content in nodules, but this effect diminishes with prolonged nitrate exposure. For SuSy cleavage activity, no significant differences were observed between treatments at 1 and 3 DAT. However, activity decreased by 27.8% and 38.5% at 7 and 14 DAT, respectively, under HN treatment (Figure 5B). This indicates that the nitrate supply to the non-nodulated roots of unilaterally nodulated dual-root soybeans inhibits SuSy cleavage activity in the nodules, thereby limiting sucrose catabolism. Regarding malate content, the HN treatment had no significant effect at 1 and 3 DAT compared to the FN treatment; however, it caused significant reductions of 30.7% and 39.2% at 7 and 14 DAT, respectively (Figure 5C). These findings imply that nitrate application to non-nodulated roots reduces malate content in nodules, potentially restricting the energy supply available to bacteroids and thereby impairing nitrogen fixation.

4. Discussion

4.1. Effects of Nitrate on Soybean Dry Matter Accumulation and Allocation

Patterns of dry matter accumulation and allocation reflect the dynamic partitioning of photoassimilates within plants. Nitrogen is a crucial factor influencing dry matter accumulation in soybeans [34,35]. In this study, the application of nitrate to the non-nodulated roots of unilaterally nodulated dual-root soybeans significantly increased the dry weight of leaves, petioles, stems, and shoots (Figure 2A–C,G), corroborating previous research that indicates nitrate application enhances the growth of the aerial parts of soybeans [36,37]. In contrast to single-root soybeans, where nitrate affects belowground dry weight [38], the total belowground dry weight of unilaterally nodulated dual-root soybeans remained unaffected by nitrate (Figure 2H). However, the two root types exhibited different responses: non-nodulated roots experienced an increase in dry weight (Figure 2D), while the nodulated root–nodule complex showed a decrease (Figure 2F). This suggests that under localized high nitrogen conditions, root growth is more vigorous in nitrogen-rich areas [39,40]. Furthermore, the lack of significant change in nodulated root dry weight (Figure 2E) indicates that the nitrate supplied to non-nodulated roots primarily suppresses nodule growth (Figure 4A), thereby impacting the nodulated root–nodule complex.
Nitrogen supply influences not only the quantity but also the direction of carbon resource allocation within a plant [11,41]. The application of nitrate significantly increased the dry weight proportions of leaves and shoots (Figure 3A,G), while the proportions of petioles and stems remained stable (Figure 3B,C), consistent with the findings of Fujikake et al. [36]. This indicates that supplying nitrate from non-nodulated lateral roots promotes the allocation of photosynthetic products in unilaterally nodulated dual-root soybeans to the aboveground parts, primarily the leaves. In terms of belowground allocation, supplying nitrate from non-nodulated roots reduced the dry weight proportion (Figure 3H), which generally aligns with the findings of Fujikake et al. [36]. However, unlike in single-root soybeans, the dry weight proportion of non-nodulated roots did not decrease in this study (Figure 3D). Instead, the dry weight proportions of the nodulated root, nodulated root–nodule complex and the nodules significantly decreased (Figure 3E,F and Figure 4B). This suggests that supplying nitrate from non-nodulated roots results in a decline in the partitioning of photoassimilates in unilaterally nodulated dual-root soybeans toward the root system, primarily affecting the nodulated roots and, in particular, the nodules. Clearly, applying nitrate to non-nodulated roots primarily alters the allocation pattern of photoassimilates between leaves and nodules in unilaterally nodulated dual-root soybeans, thereby adjusting how soybean plants utilize their photosynthetic carbon resources.

4.2. Effects of Nitrate on Soybean Nodulation, Nitrogen Fixation, and Sucrose Transport and Metabolism in Nodules

Nitrate has been widely reported to significantly inhibit nodule number, nodule dry weight, and nitrogenase activity in soybean nodules [42,43]. In the present study, nitrate application to the non-nodulated roots of unilaterally nodulated dual-root soybean plants for 3, 7, and 14 days resulted in significant reductions in both nodule number and total nodule dry weight (Figure 4A,C). A significant decrease in individual nodule dry weight was observed only after 14 days of treatment (Figure 4D), suggesting that nitrate primarily restricts overall nodule formation and biomass accumulation, with a delayed effect on individual nodule size. Furthermore, both acetylene reduction assay (ARA) and specific nitrogenase activity (SNA) were significantly reduced at 3, 7, and 14 days after nitrate treatment (Figure 4E,F). This demonstrates that the application of nitrate to non-nodulated roots not only systemically suppresses the overall nitrogen-fixing capacity of dual-root soybean plants but also directly impairs the intrinsic activity of nitrogenase enzymes within the nodules.
Nodules rely heavily on photoassimilates to provide carbon skeletons and energy for their growth, nitrogen fixation, ammonia assimilation, and solute transport [44,45]. Previous studies have shown that both local and systemic nitrate application reduces the translocation of newly synthesized photoassimilates to root nodules in soybean, with carbohydrate deprivation likely contributing to inhibited nodule development and reduced nitrogenase activity [10,12]. Sucrose is the primary form of carbohydrate transported over long distances in soybean plants [8]. Our findings reveal a biphasic response: short-term nitrate exposure (3 days) reduced nodule sucrose content (Figure 5A), which correlated with a decline in nitrogenase activity (Figure 4E,F). In contrast, prolonged treatment (7–14 days) restored sucrose content but failed to recover nitrogen fixation capacity. This response pattern is partially consistent with the observations of Ke et al. [46], who reported parallel reductions in sucrose content and nitrogenase activity under 2 mM nitrate. However, it starkly contrasts with the findings of Gordon et al. [17], where 10 mM nitrate suppressed nitrogenase activity while elevating nodule sucrose content. The observed discrepancies between our findings and prior studies can likely be attributed to several factors: genetic variability in nitrate sensitivity across soybean cultivars, concentration-dependent effects of nitrate, and systemic signaling mechanisms uniquely operationalized by our unilateral nodulation dual-root system. These findings suggest that sucrose availability alone cannot account for the prolonged suppression of nitrogenase activity. Instead, we propose a two-phase response model: during the short-term nitrate exposure phase (1–3 days), nitrate inhibits phloem sucrose flow to the nodules, inducing carbon starvation; in the prolonged treatment phase (7–14 days), nitrate induces systemic signaling that directly inhibits nitrogenase activity and decreases sucrose consumption.
In legume nodules, sucrose synthase (SuSy) is the key enzyme responsible for catalyzing the breakdown of sucrose [21,47]. Studies have shown that a reduction in SuSy activity significantly inhibits nodule growth and nitrogen fixation capacity [20,48]. Gordon et al. [16] reported that drought and salt stress reduce nitrogenase activity along with SuSy activity in soybean nodules, suggesting that impaired sucrose metabolism may contribute to reduced nitrogenase activity. However, unlike drought and other stressors, the supply of nitrate leads to a reduction in SuSy activity that occurs later than the initial decline in nitrogenase activity. It remains uncertain whether this delay is a cause or a consequence of the reduced nitrogen-fixing capacity of the nodules [17]. Similar findings were observed in the present study. When nitrate was applied to the non-nodulated roots of unilaterally nodulated dual-root soybeans for 7 and 14 days, SuSy activity in the nodules significantly decreased (Figure 5B). The observed changes in nodule sucrose content after 7 and 14 days of nitrate supply (Figure 5A) may be attributed to this inhibition of SuSy activity. In contrast, nodule nitrogenase activity exhibited a significant decline after just 3 days of nitrate application (Figure 4E,F). These results indicate that the supply of nitrate to non-nodulated roots systemically inhibits nitrogenase activity, which cannot be fully attributed to decreases in SuSy activity. Instead, it may be a consequence of the reduced nitrogen-fixing capacity of the nodules.
Sucrose, once broken down by SuSy, is converted into malate, which provides carbon to bacteroids for nitrogen fixation [18,49]. Lyu et al. [25] demonstrated that nitrate supply inhibits the conversion of sucrose to malate in soybean nodules, thereby limiting the carbon available to bacteroids and subsequently reducing nitrogenase activity. In this study, supplying nitrate to non-nodulated roots for 7 and 14 days significantly decreased malate content in the nodules of nodulated roots in unilaterally nodulated dual-root soybeans (Figure 5C). This is consistent with previous findings that indicate nitrate further restricts the carbon supply to bacteroids [25,26]. However, nitrogenase activity in the nodules declined significantly after just 3 days of nitrate application (Figure 4E,F), suggesting that the early reduction in nitrogenase activity due to nitrate supply is not primarily a result of limited carbon availability for bacteroids. Given the dynamics of nodule nitrogenase activity, sucrose content, SuSy activity, and malate levels under nitrate treatment, we propose that impaired sucrose metabolism in nodules, which results in reduced malate content, is likely a consequence of decreased nitrogenase activity rather than its cause. The early decline in nitrogenase activity may instead be attributed to reduced allocation of photoassimilates to the nodules or feedback mechanisms associated with nitrogen accumulation [50,51].
While our study provides new insights into the effects of nitrate on nodule nitrogen fixation in soybean plants, several limitations must be acknowledged. First, the experimental design utilized a specific soybean cultivar and a specialized dual-root system, which may restrict the generalizability of our findings to other varieties or growth conditions. Future studies could explore the responses of different cultivars to nitrate application and investigate how factors such as plant age and growth stage influence these responses. Furthermore, the analytical methods employed have certain limitations. Although we measured key physiological parameters, including nitrogenase activity and sucrose content, other underlying molecular and biochemical mechanisms remain unexplored. Advanced omics approaches, such as transcriptomics and metabolomics, could be integrated into future studies to provide a more comprehensive understanding of the complex regulatory networks involved in nitrate-induced changes in nodule nitrogen fixation.

5. Conclusions

In this study, we found that supplying 200 mg·L−1 of nitrate to the non-nodulated roots of unilaterally nodulated dual-root soybean plants significantly increases dry matter accumulation in the leaves, petioles, stems, shoots, non-nodulated roots, and the entire plant. Conversely, it reduces dry matter accumulation in the nodulated root–nodule complex and the nodules. Dry matter partitioning results indicated that nitrate supply enhances dry matter allocation toward the leaves while diverting it away from the nodules. Additionally, the number of nodules, acetylene reduction activity (ARA), and specific nodule activity (SNA) decreased after 3, 7, and 14 days of nitrate supply. Nodule sucrose content significantly declines after 3 days of nitrate supply but recovers after 7 and 14 days. In contrast, sucrose synthase cleavage activity and malate content decline after 7 and 14 days. These results suggest that nitrate supply alters dry matter accumulation and allocation in dual-root soybean plants, favoring the distribution of photoassimilates to leaves over nodules. The initial decrease in nitrogen-fixing capacity may be attributed to reduced sucrose levels, while the decline in sucrose synthase cleavage activity and malate content may be consequences of the decrease in nitrogen-fixing capacity. These findings highlight the dynamic interplay between nitrogen and photoassimilates allocation in legumes and provide essential insights into the systemic regulation of nodule function in response to nitrate supply. Future research should focus on short-term (e.g., within 3 days) changes in nitrogen fixation capacity and the role of specific sucrose transporters (e.g., SWEET and SUT families) in nitrate-induced photoassimilate allocation to nodules.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy15051032/s1, Figure S1: Schematic diagram of dual-root soybean sand cultivation; Figure S2: Preparation of the dual-root soybean system; Table S1: Composition of the nitrogen-free nutrient solution.

Author Contributions

Conceptualization, X.W. and X.L.; methodology, X.W. and S.Y.; formal analysis, X.W.; investigation, X.W. and Y.Z.; resources, C.M.; data curation, X.W.; writing—original draft preparation, X.W.; writing—review and editing, X.W. and C.M.; visualization, X.W. and T.G.; supervision, C.M. and Z.G.; project administration, C.Y.; funding acquisition, C.M. and X.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by National Natural Science Foundation of China, grant number 32401967.

Data Availability Statement

All data are included in the main text.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Mus, F.; Crook, M.B.; Garcia, K.; Garcia Costas, A.; Geddes, B.A.; Kouri, E.D.; Paramasivan, P.; Ryu, M.H.; Oldroyd, G.E.D.; Poole, P.S.; et al. Symbiotic nitrogen fixation and the challenges to its extension to nonlegumes. Appl. Environ. Microbiol. 2016, 82, 3698–3710. [Google Scholar] [CrossRef]
  2. Nakei, M.D.; Venkataramana, P.B.; Ndakidemi, P.A. Soybean-nodulating rhizobia: Ecology, characterization, diversity, and growth promoting functions. Front. Sustain. Food Syst. 2022, 6, 824444. [Google Scholar] [CrossRef]
  3. Oldroyd, G.E.D.; Downie, J.A. Calcium, kinases and nodulation signalling in legumes. Nat. Rev. Mol. Cell Biol. 2004, 5, 566–576. [Google Scholar] [CrossRef]
  4. Oke, V.; Long, S.R. Bacteroid formation in the Rhizobium-legume symbiosis. Curr. Opin. Microbiol. 1999, 2, 641–646. [Google Scholar] [CrossRef] [PubMed]
  5. Prell, J.; Poole, P. Metabolic changes of rhizobia in legume nodules. Trends Microbiol. 2006, 14, 161–168. [Google Scholar] [CrossRef]
  6. White, J.; Prell, J.; James, E.K.; Poole, P. Nutrient sharing between symbionts. Plant Physiol. 2007, 144, 604–614. [Google Scholar] [CrossRef] [PubMed]
  7. Salvagiotti, F.; Cassman, K.G.; Specht, J.E.; Walters, D.T.; Weiss, A.; Dobermann, A. Nitrogen uptake, fixation and response to fertilizer N in soybeans: A review. Field Crops Res. 2008, 108, 1–13. [Google Scholar] [CrossRef]
  8. Streeter, J.G. Carbohydrates in soybean nodules: II. Distribution of compounds in seedlings during the onset of nitrogen fixation. Plant Physiol. 1980, 66, 471–476. [Google Scholar] [CrossRef]
  9. Nishida, H.; Suzaki, T. Nitrate-mediated control of root nodule symbiosis. Curr. Opin. Plant Biol. 2018, 44, 129–136. [Google Scholar] [CrossRef]
  10. Voisin, A.S.; Salon, C.; Jeudy, C.; Warembourg, F.R. Root and nodule growth in Pisum sativum L. in relation to photosynthesis: Analysis using 13C-labelling. Ann. Bot. 2003, 92, 557–563. [Google Scholar] [CrossRef]
  11. Fujikake, H.; Yamazaki, A.; Ohtake, N.; Sueyoshi, K.; Matsuhashi, S.; Ito, T.; Mizuniwa, C.; Kume, T.; Hashimoto, S.; Ishioka, N.S.; et al. Quick and reversible inhibition of soybean root nodule growth by nitrate involves a decrease in sucrose supply to nodules. J. Exp. Bot. 2003, 54, 1379–1388. [Google Scholar] [CrossRef] [PubMed]
  12. Li, S.; Wu, C.; Liu, H.; Lyu, X.; Xiao, F.; Zhao, S.; Ma, C.; Yan, C.; Liu, Z.; Li, H.; et al. Systemic regulation of nodule structure and assimilated carbon distribution by nitrate in soybean. Front. Plant Sci. 2023, 14, 1101074. [Google Scholar] [CrossRef] [PubMed]
  13. Vernon, L.P.; Aronoff, S. Metabolism of soybean leaves. IV. Translocation from soybean leaves. Arch. Biochem. Biophys. 1952, 36, 383–398. [Google Scholar] [CrossRef]
  14. Xu, Q.; Wang, X.; Wang, N.; Li, S.; Yao, X.; Kuang, H.; Qiu, Z.; Ke, D.; Yang, W.; Guan, Y. Nitrogen inhibition of nitrogenase activity involves the modulation of cytosolic invertase in soybean nodule. J. Genet. Genom. 2024, 51, 1404–1412. [Google Scholar] [CrossRef]
  15. Streeter, J.G. Effect of nitrate in the rooting medium on carbohydrate composition of soybean nodules. Plant Physiol. 1981, 68, 840–844. [Google Scholar] [CrossRef] [PubMed]
  16. Gordon, A.J.; Minchin, F.R.; Skot, L.; James, C.L. Stress-induced declines in soybean N2 fixation are related to nodule sucrose synthase activity. Plant Physiol. 1997, 114, 937–946. [Google Scholar] [CrossRef]
  17. Gordon, A.J.; Skot, L.; James, C.L.; Minchin, F.R. Short-term metabolic responses of soybean root nodules to nitrate. J. Exp. Bot. 2002, 53, 423–428. [Google Scholar] [CrossRef]
  18. Udvardi, M.; Poole, P.S. Transport and metabolism in legume-rhizobia symbioses. Annu. Rev. Plant Biol. 2013, 64, 781–805. [Google Scholar] [CrossRef]
  19. Morell, M.; Copeland, L. Sucrose synthase of soybean nodules. Plant Physiol. 1985, 78, 149–154. [Google Scholar] [CrossRef]
  20. Baier, M.C.; Barsch, A.; Kuster, H.; Hohnjec, N. Antisense repression of the Medicago truncatula nodule-enhanced sucrose synthase leads to a handicapped nitrogen fixation mirrored by specific alterations in the symbiotic transcriptome and metabolome. Plant Physiol. 2007, 145, 1600–1618. [Google Scholar] [CrossRef]
  21. Gordon, A.J.; Minchin, F.R.; James, C.L.; Komina, O. Sucrose synthase in legume nodules is essential for nitrogen fixation. Plant Physiol. 1999, 120, 867–878. [Google Scholar] [CrossRef]
  22. Craig, J.; Barratt, P.; Tatge, H.; Déjardin, A.; Handley, L.; Gardner, C.D.; Barber, L.; Wang, T.; Hedley, C.; Martin, C.; et al. Mutations at the rug4 locus alter the carbon and nitrogen metabolism of pea plants through an effect on sucrose synthase. Plant J. 1999, 17, 353–362. [Google Scholar] [CrossRef]
  23. Lambert, I.; Pervent, M.; Le Queré, A.; Clément, G.; Tauzin, M.; Severac, D.; Benezech, C.; Tillard, P.; Martin-Magniette, M.L.; Colella, S.; et al. Responses of mature symbiotic nodules to the whole-plant systemic nitrogen signaling. J. Exp. Bot. 2020, 71, 5039–5052. [Google Scholar] [CrossRef] [PubMed]
  24. Sulieman, S.; Schulze, J.; Tran, L.S. N-feedback regulation is synchronized with nodule carbon alteration in Medicago truncatula under excessive nitrate or low phosphorus conditions. J. Plant Physiol. 2014, 171, 407–410. [Google Scholar] [CrossRef]
  25. Lyu, X.; Sun, C.; Zhang, J.; Wang, C.; Zhao, S.; Ma, C.; Li, S.; Li, H.; Gong, Z.; Yan, C. Integrated proteomics and metabolomics analysis of nitrogen system regulation on soybean plant nodulation and nitrogen fixation. Int. J. Mol. Sci. 2022, 23, 2545. [Google Scholar] [CrossRef]
  26. Heckmann, M.-O.; Drevon, J.-J.; Saglio, P.; Salsac, L. Effect of oxygen and malate on NO3− inhibition of nitrogenase in soybean nodules. Plant Physiol. 1989, 90, 224–229. [Google Scholar] [CrossRef] [PubMed]
  27. Vessey, J.K.; Walsh, K.B.; Layzell, D.B. Can a limitation in pholem supply to nodules account for the inhibitory effect of nitrate on nitrogenase activity in soybean? Physiol. Plant. 1988, 74, 137–146. [Google Scholar] [CrossRef]
  28. Lyu, X.; Li, M.; Li, X.; Li, S.; Yan, C.; Ma, C.; Gong, Z. Assessing the systematic effects of the concentration of nitrogen supplied to dual-root systems of soybean plants on nodulation and nitrogen fixation. Agronomy 2020, 10, 763. [Google Scholar] [CrossRef]
  29. Fehr, W.R.; Caviness, C.E.; Burmood, D.T.; Pennington, J.S. Stage of development descriptions for soybeans, Glycine max (L.) Merrill. Crop Sci. 1971, 11, 929–931. [Google Scholar] [CrossRef]
  30. Lyu, X.; Xia, X.; Wang, C.; Ma, C.; Dong, S.; Gong, Z. Effects of changes in applied nitrogen concentrations on nodulation, nitrogen fixation and nitrogen accumulation during the soybean growth period. Soil Sci. Plant Nutr. 2019, 65, 479–489. [Google Scholar] [CrossRef]
  31. Buysse, J.; Merckx, R. An improved colorimetric method to quantify sugar content of plant tissue. J. Exp. Bot. 1993, 44, 1627–1629. [Google Scholar] [CrossRef]
  32. Zrenner, R.; Salanoubat, M.; Willmitzer, L.; Sonnewald, U. Evidence of the crucial role of sucrose synthase for sink strength using transgenic potato plants (Solanum tuberosum L.). Plant J. 1995, 7, 97–107. [Google Scholar] [CrossRef]
  33. Scherer, R.; Rybka, A.C.P.; Ballus, C.A.; Meinhart, A.D.; Filho, J.T.; Godoy, H.T. Validation of a HPLC method for simultaneous determination of main organic acids in fruits and juices. Food Chem. 2012, 135, 150–154. [Google Scholar] [CrossRef]
  34. Qiang, B.; Chen, S.; Fan, Z.; Cao, L.; Li, X.; Fu, C.; Zhang, Y.; Jin, X. Effects of nitrogen application levels on soybean photosynthetic performance and yield: Insights from canopy nitrogen allocation studies. Field Crops Res. 2025, 326, 109871. [Google Scholar] [CrossRef]
  35. Zhou, H.; Yao, X.; Zhao, Q.; Zhang, W.; Zhang, B.; Xie, F. Rapid effect of nitrogen supply for soybean at the beginning fowering stage on biomass and sucrose metabolism. Sci. Rep. 2019, 9, 15530. [Google Scholar] [CrossRef] [PubMed]
  36. Fujikake, H.; Tamura, Y.; Ohtake, N.; Sueyoshi, K.; Ohyama, T. Photoassimilate partitioning in hypernodulation mutant of soybean (Glycine max (L.) Merr.) NOD1-3 and its parent williams in relation to nitrate inhibition of nodule growth. Soil Sci. Plant Nutr. 2003, 49, 583–590. [Google Scholar] [CrossRef]
  37. Qiang, B.; Zhou, W.; Zhong, X.; Fu, C.; Cao, L.; Zhang, Y.; Jin, X. Effect of nitrogen application levels on photosynthetic nitrogen distribution and use efficiency in soybean seedling leaves. J. Plant Physiol. 2023, 287, 154051. [Google Scholar] [CrossRef]
  38. Hansen, A.P.; Yoneyama, T.; Kouchi, H. Short-term nitrate effects on hydroponically-grown soybean cv. Bragg and its supernodulating mutant: I. carbon, nitrogen and mineral element distribution, respiration and the effect of nitrate on nitrogenase activity. J. Exp. Bot. 1992, 43, 1–7. [Google Scholar] [CrossRef]
  39. Oldroyd, G.E.D.; Leyser, O. A plant’s diet, surviving in a variable nutrient environment. Science 2020, 368, eaba0196. [Google Scholar] [CrossRef]
  40. Daimon, H.; Yoshioka, M. Responses of root nodule formation and nitrogen fixation activity to nitrate in a split-root system in peanut (Arachis hypogaea L.). J. Agron. Crop Sci. 2001, 187, 89–95. [Google Scholar] [CrossRef]
  41. Kouchi, H.; Akao, S.; Yoneyama, T. Respiratory utilization of 13C-labelled photosynthate in nodulated root systems of soybean plants. J. Exp. Bot. 1986, 37, 985–993. [Google Scholar] [CrossRef]
  42. Lyu, X.; Sun, C.; Lin, T.; Wang, X.; Li, S.; Zhao, S.; Gong, Z.; Wei, Z.; Yan, C.; Ma, C. Systemic regulation of soybean nodulation and nitrogen fixation by nitrogen via isoflavones. Front. Plant Sci. 2022, 13, 968496. [Google Scholar] [CrossRef]
  43. Mbah, G.C.; Dakora, F.D. Nitrate inhibition of N2 fixation and its effect on micronutrient accumulation in shoots of soybean (Glycine max L. Merr.), Bambara groundnut (Vigna subterranea L. Vedc) and Kersting’s groundnut (Macrotyloma geocarpum Harms.). Symbiosis 2018, 75, 205–216. [Google Scholar] [CrossRef] [PubMed]
  44. Streeter, J.G. Transport and metabolism of carbon and nitrogen in legume nodules. Adv. Bot. Res. 1991, 18, 129–187. [Google Scholar] [CrossRef]
  45. Hacin, J.I.; Bohlool, B.B.; Singleton, P.W. Partitioning of 14C-labelled photosynthate to developing nodules and roots of soybean (Glycine max). New Phytol. 1997, 137, 257–265. [Google Scholar] [CrossRef]
  46. Ke, X.; Xiao, H.; Peng, Y.; Xia, X.; Wang, X. Nitrogen deficiency modulates carbon allocation to promote nodule nitrogen fixation capacity in soybean. Exploration 2024, 4, 20230104. [Google Scholar] [CrossRef] [PubMed]
  47. Thummler, F.; Verma, D.P. Nodulin-100 of soybean is the subunit of sucrose synthase regulated by the availability of free heme in nodules. J. Biol. Chem. 1987, 262, 14730–14736. [Google Scholar] [CrossRef]
  48. Anthon, G.E.; Emerich, D.W. Developmental regulation of enzymes of sucrose and hexose metabolism in effective and ineffective soybean nodules. Plant Physiol. 1990, 92, 346–351. [Google Scholar] [CrossRef]
  49. Schulte, C.C.M.; Borah, K.; Wheatley, R.M.; Terpolilli, J.J.; Saalbach, G.; Crang, N.; de Groot, D.H.; Ratcliffe, R.G.; Kruger, N.J.; Papachristodoulou, A.; et al. Metabolic control of nitrogen fixation in rhizobium-legume symbioses. Sci. Adv. 2021, 7, eabh2433. [Google Scholar] [CrossRef]
  50. Bacanamwo, M.; Harper, J.E. The feedback mechanism of nitrate inhibition of nitrogenase activity in soybean may involve asparagine and/or products of its metabolism. Physiol. Plant. 1997, 100, 371–377. [Google Scholar] [CrossRef]
  51. Wang, X.; Zhang, Y.; Lian, Z.; Lyu, X.; Yan, C.; Yan, S.; Gong, Z.; Li, S.; Ma, C. Nitrate inhibits nodule nitrogen fixation by accumulating ureide in soybean plants. Plants 2024, 13, 2045. [Google Scholar] [CrossRef] [PubMed]
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Figure 1. Schematic illustration of the experimental treatments and the experimental time course. (A) Experimental treatments, (B) experimental time course.
Figure 1. Schematic illustration of the experimental treatments and the experimental time course. (A) Experimental treatments, (B) experimental time course.
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Figure 2. Dynamic changes in dry weight of soybean leaves (A), petioles (B), stems (C), non-nodulated roots (D), nodulated roots (E), nodulated root–nodule complexes (F), shoots (G), belowground parts (H), and the whole plant (I) in unilaterally nodulated dual-root soybeans following nitrate supply to the non-nodulated roots for 1, 3, 7, and 14 days. DAT refers to days after treatment. In the FN treatment, both root systems were supplied with a nitrogen-free nutrient solution. In the HN treatment, the non-nodulated roots received a nitrogen-containing solution (200 mg·L−1), while the nodulated roots continued to receive nitrogen-free solution. Data are presented as means ± SE (n = 3). Different lowercase letters indicate significant differences between the FN and HN treatments at the same treatment time points according to Student’s t-test (p < 0.05).
Figure 2. Dynamic changes in dry weight of soybean leaves (A), petioles (B), stems (C), non-nodulated roots (D), nodulated roots (E), nodulated root–nodule complexes (F), shoots (G), belowground parts (H), and the whole plant (I) in unilaterally nodulated dual-root soybeans following nitrate supply to the non-nodulated roots for 1, 3, 7, and 14 days. DAT refers to days after treatment. In the FN treatment, both root systems were supplied with a nitrogen-free nutrient solution. In the HN treatment, the non-nodulated roots received a nitrogen-containing solution (200 mg·L−1), while the nodulated roots continued to receive nitrogen-free solution. Data are presented as means ± SE (n = 3). Different lowercase letters indicate significant differences between the FN and HN treatments at the same treatment time points according to Student’s t-test (p < 0.05).
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Figure 3. Dynamic changes in dry weight proportion of soybean leaves (A), petioles (B), stems (C), non-nodulated roots (D), nodulated roots (E), nodulated root–nodule complexes (F), shoots (G), and belowground (H) in unilaterally nodulated dual-root soybeans following nitrate supply to the non-nodulated roots for 1, 3, 7, and 14 days. In the FN treatment, both root systems were supplied with a nitrogen-free nutrient solution. In the HN treatment, the non-nodulated roots received a nitrogen-containing solution (200 mg·L−1), while the nodulated roots continued to receive nitrogen-free solution. Data are presented as means ± SE (n = 3). Different lowercase letters indicate significant differences between the FN and HN treatments at the same treatment time points according to Student’s t-test (p < 0.05).
Figure 3. Dynamic changes in dry weight proportion of soybean leaves (A), petioles (B), stems (C), non-nodulated roots (D), nodulated roots (E), nodulated root–nodule complexes (F), shoots (G), and belowground (H) in unilaterally nodulated dual-root soybeans following nitrate supply to the non-nodulated roots for 1, 3, 7, and 14 days. In the FN treatment, both root systems were supplied with a nitrogen-free nutrient solution. In the HN treatment, the non-nodulated roots received a nitrogen-containing solution (200 mg·L−1), while the nodulated roots continued to receive nitrogen-free solution. Data are presented as means ± SE (n = 3). Different lowercase letters indicate significant differences between the FN and HN treatments at the same treatment time points according to Student’s t-test (p < 0.05).
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Figure 4. Dynamic changes in nodule dry weight (A), nodule dry weight proportion (B), nodule number (C), individual nodule dry weight (D), acetylene reduction assay (ARA) (E), and specific nitrogenase activity (SNA) (F) in the nodulated side roots of unilaterally nodulated dual-root soybeans following nitrate supply to the non-nodulated roots for 1, 3, 7, and 14 days. DAT refers to days after treatment. In the FN treatment, both root systems were supplied with a nitrogen-free nutrient solution. In the HN treatment, the non-nodulated roots received a nitrogen-containing solution (200 mg·L−1), while the nodulated roots continued to receive nitrogen-free solution. Data are presented as means ± SE (n = 3). Different lowercase letters indicate significant differences between the FN and HN treatments at the same treatment time points according to Student’s t-test (p < 0.05).
Figure 4. Dynamic changes in nodule dry weight (A), nodule dry weight proportion (B), nodule number (C), individual nodule dry weight (D), acetylene reduction assay (ARA) (E), and specific nitrogenase activity (SNA) (F) in the nodulated side roots of unilaterally nodulated dual-root soybeans following nitrate supply to the non-nodulated roots for 1, 3, 7, and 14 days. DAT refers to days after treatment. In the FN treatment, both root systems were supplied with a nitrogen-free nutrient solution. In the HN treatment, the non-nodulated roots received a nitrogen-containing solution (200 mg·L−1), while the nodulated roots continued to receive nitrogen-free solution. Data are presented as means ± SE (n = 3). Different lowercase letters indicate significant differences between the FN and HN treatments at the same treatment time points according to Student’s t-test (p < 0.05).
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Figure 5. Dynamic changes in sucrose content (A), Sucrose Synthase (SuSy) cleavage activity (B), and malate content (C) in the nodules of unilaterally nodulated dual-root soybeans following nitrate supply to the non-nodulated roots for 1, 3, 7, and 14 days. DAT refers to days after treatment. In the FN treatment, both root systems were supplied with a nitrogen-free nutrient solution. In the HN treatment, the non-nodulated roots received a nitrogen-containing solution (200 mg·L−1), while the nodulated roots continued to receive nitrogen-free solution. Data are presented as means ± SE (n = 3). Different lowercase letters indicate significant differences between the FN and HN treatments at the same treatment time points according to Student’s t-test (p < 0.05).
Figure 5. Dynamic changes in sucrose content (A), Sucrose Synthase (SuSy) cleavage activity (B), and malate content (C) in the nodules of unilaterally nodulated dual-root soybeans following nitrate supply to the non-nodulated roots for 1, 3, 7, and 14 days. DAT refers to days after treatment. In the FN treatment, both root systems were supplied with a nitrogen-free nutrient solution. In the HN treatment, the non-nodulated roots received a nitrogen-containing solution (200 mg·L−1), while the nodulated roots continued to receive nitrogen-free solution. Data are presented as means ± SE (n = 3). Different lowercase letters indicate significant differences between the FN and HN treatments at the same treatment time points according to Student’s t-test (p < 0.05).
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Wang, X.; Guo, T.; Zhang, Y.; Lyu, X.; Yan, S.; Yan, C.; Gong, Z.; Ma, C. Systemic Effects of Nitrate on Nitrogen Fixation and Sucrose Catabolism in Soybean (Glycine max (L.) Merr.) Nodules. Agronomy 2025, 15, 1032. https://doi.org/10.3390/agronomy15051032

AMA Style

Wang X, Guo T, Zhang Y, Lyu X, Yan S, Yan C, Gong Z, Ma C. Systemic Effects of Nitrate on Nitrogen Fixation and Sucrose Catabolism in Soybean (Glycine max (L.) Merr.) Nodules. Agronomy. 2025; 15(5):1032. https://doi.org/10.3390/agronomy15051032

Chicago/Turabian Style

Wang, Xuelai, Tong Guo, Yuchen Zhang, Xiaochen Lyu, Shuangshuang Yan, Chao Yan, Zhenping Gong, and Chunmei Ma. 2025. "Systemic Effects of Nitrate on Nitrogen Fixation and Sucrose Catabolism in Soybean (Glycine max (L.) Merr.) Nodules" Agronomy 15, no. 5: 1032. https://doi.org/10.3390/agronomy15051032

APA Style

Wang, X., Guo, T., Zhang, Y., Lyu, X., Yan, S., Yan, C., Gong, Z., & Ma, C. (2025). Systemic Effects of Nitrate on Nitrogen Fixation and Sucrose Catabolism in Soybean (Glycine max (L.) Merr.) Nodules. Agronomy, 15(5), 1032. https://doi.org/10.3390/agronomy15051032

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