Exogenous Cytokinins Regulate Nitrogen Metabolism in Soybean Under Low Phosphorus Stress
by
Yubo Yao
1, 
Yongguo Xue
1,
Jun Yan
2,
Xiaofei Tang
1,
Dan Cao
1,
Wenjin He
1,
Xiaoyan Luan
1,
Qi Liu
1,
Zifei Zhu
1 and
Xinlei Liu
1,* 1
Soybean Research Institute, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, China
2
Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Harbin 150081, China
*
Author to whom correspondence should be addressed.
Agronomy 2025, 15(6), 1459; https://doi.org/10.3390/agronomy15061459
Submission received: 12 May 2025
/
Revised: 2 June 2025
/
Accepted: 13 June 2025
/
Published: 16 June 2025
(This article belongs to the Special Issue Recent Developments in Biological Nitrogen Fixation in Plants)
Abstract
:Low phosphorus (P) stress impacts nitrogen (N) metabolism in soybeans. This study investigated the effects of exogenous cytokinin (Trans-Zeatin) on soybean N metabolism under low P stress by treating seeds with Trans-Zeatin and analyzing N accumulation, 15N abundance, nodule N fixation accumulation, nodule N fixation rate, nodule nitrogenase activity, soluble protein content, and free amino acid profiles. The results showed that exogenous cytokinin enhanced N accumulation in aboveground tissues, roots, and nodules, as well as nodule N fixation accumulation and fixation rate (from day 35 onward) under low P stress. Additionally, it promoted both acetylene reduction activity (ARA) and specific nitrogenase activity (SNA) in soybean nodules. By increasing the absorption of fertilizer-derived N, exogenous cytokinin alleviated the inhibitory effects of low P stress on the early growth and development of soybeans. Notably, under low P conditions, exogenous cytokinin significantly elevated the soluble protein content in nodules. However, the underlying mechanisms governing changes in free amino acid profiles require further investigation. This study provides a theoretical foundation for developing strategies to regulate soybean N metabolism under low P stress.
1. Introduction
Soybean (Glycine max [L.] Merr), as one of the world’s most extensively cultivated leguminous crops, plays a multifunctional role in global agriculture, serving as a primary source of food, oil, natural fertilizer, and high-quality livestock feed [1,2]. This multipurpose crop contributes substantially to the global food supply, accounting for approximately 30% of worldwide edible oil production and 69% of dietary protein intake [3].
Nitrogen represents a fundamental nutrient for plant physiology, serving as a core component of amino acids and nucleic acids in cellular structures [4]. Soybeans uniquely form symbiotic relationships with nitrogen-fixing rhizobia in soil, leading to root nodule formation. This symbiosis represents one of the most efficient forms of biological nitrogen fixation, converting atmospheric nitrogen (N2) into soluble, non-toxic ammonium (NH4+), which plant cells then use to synthesize diverse biomolecules [5,6]. Symbiotic nodulation is vital for leguminous crop growth [7], serving as a critical nitrogen source for soybean growth, development, and yield formation [8,9]. During key developmental stages, nitrogen fixed by nodules accounts for over 60% of the total nitrogen accumulated by soybeans [10].
Phosphorus is an important mineral nutrient required by plants, ranking as the second most growth-limiting macronutrient and playing a decisive role in plant physiological systems [11,12]. This element is integral to nearly all biological processes, including energy transfer, metabolic regulation, and protein activation [13,14,15]. The limited availability of P significantly restricts crop yields, making it a critical determinant of global crop growth and productivity [16]. The symbiotic nitrogen fixation (SNF) process in legume nodules is highly energy-intensive, requiring substantially more P for efficient energy transfer and optimal nodule function than non-nodulating plants [17,18]. P deficiency not only impairs nodule formation, development, and biomass in legumes [19,20,21] but also reduces nodule number [22] and hinders nitrogen and carbon fixation, photosynthesis, nitrogen uptake, and metabolism [4]. For example, Yao and Liu reported that low P stress caused significant reductions in nitrogen accumulation, nodule nitrogen fixation accumulation, and nodule nitrogen fixation rates [23].
Cytokinins (CKs), first identified during tobacco tissue culture [24], are a crucial class of plant hormones known for their diverse biological activities. These hormones regulate various aspects of plant growth and development [25] while also playing a central role in plants’ adaptation to biotic and abiotic stresses by modulating key physiological pathways [26,27,28,29,30]. In tobacco, CKs have been shown to influence chloroplast gene expression, specifically upregulating genes associated with Photosystem II (PSII) to enhance photosynthetic efficiency [31]. Notably, emerging evidence highlights the regulatory role of CKs in soybean nodule nitrogen fixation. As key signaling molecules, CKs are essential for mediating nodule formation [31]. Foliar application and seed treatment with CKs have been demonstrated to increase nodule number and enhance nodule nitrogen fixation, with seed treatments reportedly improving nitrogen fixation accumulation by 26% [31]. Moreover, CKs contribute to maintaining the stability of the nitrogen fixation process in nodules. Among CKs, trans-zeatin riboside exhibits potent cytokinin activity and elicits significant effects on plant growth when applied exogenously [32]. Our previous research revealed that exogenous trans-zeatin promotes carbohydrate accumulation in soybeans, leading to increased plant height, dry matter mass, and higher levels of fructose, sucrose, and starch in nodules under low P stress conditions [33]. These effects enhance the plant’s tolerance to low P stress. Building on these findings regarding the impact of exogenous trans-zeatin on carbon metabolism under low P stress, this study investigates its effects on nitrogen metabolism. By analyzing soybean nitrogen accumulation, nodulation, nitrogen fixation capacity, and changes in soluble protein and free amino acid levels in root nodules, we aim to elucidate the underlying mechanisms. This research provides a theoretical framework for understanding how exogenous trans-zeatin modulates nitrogen metabolism in soybeans under low P stress, offering insights for future agricultural applications.
2. Materials and Methods
2.1. Cytokinins Treatment
There were three experimental treatments in total, represented as P1 (P stress), P1 + CKs (P stress + cytokinin), and P31 (normal P, CK), respectively. Trans-zeatin (purchased from Beijing Soleibao Technology Co., Ltd., Beijing, China, with a purity of ≥99%) was used. Using the seed treatment method with a concentration of 1 × 10−9 mol·L−1. P1 and CK treatments used water instead of trans-zeatin. Soybean seeds were immersed in CKs solution or water for 4 h, air-dried, and stored at 4 °C until sowing [31].
2.2. Plant Materials and Sampling
This experiment was conducted at the experimental base of Heilongjiang Academy of Agricultural Sciences, Harbin, Heilongjiang Province, China (127°3′ E, 46°9′ N), in 2022 and 2023. Soybean plants (Suinong 14, SN14) were grown in pots filled with sand medium.
The nutrient solution composition and concentration (mg/L) and cultivation methods were the same as those described by Yao and Liu [23]. Samples (3 replicates for each treatment) were taken from 8:00 to 10:00 am every 7 days from the V3 (third trifoliate leaf) until the R5 stage. The plants were divided into three parts: the aboveground parts, roots, and nodules, and weighed after drying.
Nodules were collected three times (3 replicate samples for each treatment) after the 7th, 14th, 21st, 28th, 35th, 42nd, and 49th day of P stress treatment, frozen immediately in liquid nitrogen, stored at −80 °C, and then used for soluble protein content and free amino acid detection.
2.3. Measurement and Calculation Methods
Measurement and calculation methods were as described by Yao and Liu [23].
Statistical analyses were conducted using SPSS 19.0 (SPSS Inc., Chicago, IL, USA). T-tests were used to analyze the comparison between two sets of data, and ANOVA was used to compare three sets of data.
2.4. Nodule Nitrogenase Activity Detection
An acetylene reduction assay was used to measure the nitrogenase activity in nodules as described by Gremaud and Harper [34], Chen et al. [35]. Specific nitrogenase activity (SNA) was expressed as μmoles of ethylene formed per gram dry weight of nodules per hour. Acetylene reduction activity (ARA) was expressed as μmoles of ethylene formed per plant per hour.
2.5. Soluble Protein Content Detection
Measurement and calculation methods were as described by Yao and Liu [23].
2.6. Free Amino Acid Detection
The procedure followed the standard method [36] GB/T 8314-2013. Briefly, 0.1 g of the samples was ground into a fine powder using liquid nitrogen. Next, 1 mL of 10% acetic acid solution was added and mixed thoroughly. The homogenate was transferred to a 1.5 mL Eppendorf tube and incubated in a boiling water bath for 15 min. After cooling, the mixture was centrifuged at 10,000 rpm for 10 min at 4 °C. To 10 μL of the supernatant, 120 μL of acetic acid-sodium acetate buffer, 100 μL of 3% ninhydrin solution, and 10 μL of 0.3% ascorbic acid solution were added in sequence. The mixture was then heated in a boiling water bath for 15 min. Following incubation, the solution was vortexed and centrifuged at 8000 rpm for 15 min. Finally, absorbance was measured at 570 nm using a spectrophotometer.
Amino Acid Content (mg/g) = Detected Sample Concentration × Sample Volume ÷ Sample Weight × Dilution Factor
3. Results
3.1. The Effect of Exogenous Cytokinin on Nitrogen Accumulation in Soybean Under Low P Stress
Low P stress significantly inhibited nitrogen accumulation in soybean aboveground parts, roots, and nodules. Exogenous cytokinin was beneficial for increasing nitrogen accumulation in soybeans under low P stress, with increases of 1.15% (49 days)–35.18% (35 days), 1.11% (21 days)–49.86% (35 days), and 0.49% (14 days)–129.97% (35 days), respectively. The nitrogen accumulation of the aboveground parts showed a significant difference, except for 49 days. There was a significant difference in nitrogen accumulation in roots after 28 days. The nitrogen accumulation of nodules showed significant differences except for 14 days and 21 days (Table 1).
3.2. The Effect of Exogenous Cytokinin on 15N Abundance in Soybean Under Low P Stress
Under low P stress, the abundance of 15N in the aboveground parts, roots, and nodules of soybean was higher than that in the P31 treatment. Exogenous cytokinin promoted the absorption of fertilizer nitrogen by soybean aboveground parts, roots, and root nodules at 7 days, 14 days, 21 days, and 28 days under low P stress, but the abundance of 15N of P1 + CKs was lower than that in P1 treatment at 35 days (Table 2).
3.3. The Effect of Exogenous Cytokinin on Nodule Nitrogen Fixation Accumulation in Soybean Under Low P Stress
Low P stress significantly inhibited nitrogen fixation accumulation in soybean aboveground parts, roots, and nodules, while exogenous cytokinin was beneficial for increasing nitrogen fixation accumulation under low P stress. The nitrogen fixation accumulation of aboveground parts increased by 6.32% (28 days)–71.88% (35 days), showing a significant difference from the 35 days. The nitrogen fixation accumulation of roots increased by 6.67% (14 days)–69.72% (35 days), showing a significant difference from the 28th day. The nitrogen fixation accumulation of nodules increased by 6.45% (21 days)–159.30% (35 days), showing a significant difference except for 14 days and 21 days (Table 3).
The nitrogen fixation rate decreased in soybean aboveground parts, roots, and nodules under low P stress. The nitrogen fixation rate of P1 + CKs was lower than that of P1 at 7 days, 14 days, 21 days, and 28 days. The nitrogen fixation rate of the aboveground parts, roots, and nodules of P1 + CKs was higher than that of P1 at 35 days and reached a significant difference, except for the 49 days of nodules (Table 4).
3.4. The Effect of Exogenous Cytokinin on Nitrogenase Activity in Soybean Under Low P Stress
Low P stress significantly decreased soybean nodule number and weight, while the application of exogenous cytokinin increased the number and weight by 0.00% (14 days)–100.00% (35 days) and 16.49% (7 days)–202.48% (35 days), respectively. Except for the number of nodules at 14 days and 21 days, all others showed a significant difference (Table 5).
Low P stress inhibited the ARA of soybean, while exogenous cytokinin significantly increased the ARA at 28 days, 42 days, and 49 days compared with P1 (Figure 1).
Low P stress inhibited the SNA of soybean, while exogenous cytokinin significantly increased the SNA at 7 days, 21 days, 28 days, 42 days, and 49 days compared with P1 (Figure 2).
3.5. The Effect of Exogenous Cytokinin on Soluble Protein in Soybean Nodules Under Low P Stress
All sampling time results indicated that low P stress significantly inhibited the soluble protein content in soybean nodules. Exogenous cytokinin significantly increased the soluble protein content by 32.55% at 7 days, 15.08% at 14 days, 62.80% at 21 days, 42.30% at 42 days, and 25.85% at 49 days, respectively (Figure 3).
3.6. The Effect of Exogenous Cytokinin on Free Amino Acid in Soybean Nodules Under Low P Stress
The free amino acid content in P1 was higher than that in P31 of nodules. However, the effect of exogenous cytokinins on free amino acids in nodules was an irregular change compared with P1 (Figure 4).
4. Discussion
Cytokinins, a class of adenine-derived phytohormones renowned for stimulating cell proliferation [37], represent one of the most critical regulatory systems governing plant growth, development, and adaptability [38]. These hormones are indispensable in a wide array of plant physiological processes, including cell division, de novo organogenesis, shoot and root development, axillary bud growth, chlorophyll biosynthesis, and nutrient translocation [39,40]. Among them, trans-zeatin (tz), a well-characterized bioactive compound, serves as a central mediator of cytokinin-driven functions [41]. Building on prior research exploring the effects of low P stress on soybean growth, development, and nitrogen metabolism [22,42], the aim of this study is to elucidate the regulatory role of exogenous cytokinin in modulating nitrogen metabolism in soybeans under low P stress conditions.
Numerous studies have demonstrated the beneficial effects of cytokinin treatment on crop productivity [43,44]. Under adverse environmental conditions, cytokinin application has been shown to enhance both the yield and quality of agricultural products [45]. For example, seed pretreatment with zeatin in wheat improved stomatal conductance, photosynthetic efficiency, biomass, and yield under salt and drought stress [46]. Cytokinins are also critical for plant leaf development, exerting regulatory effects on the photosystem and photosynthetic processes [47,48,49,50]. In cytokinin (CK)-deficient Arabidopsis mutants, the apical meristems are smaller, and leaf cell production is reduced to only 3–4% of that in wild-type plants [51]. As leaves serve as the primary photosynthetic organs responsible for generating photosynthates, understanding the role of cytokinins in leaf development is fundamental to elucidating overall plant growth mechanisms. In this study, exogenous cytokinin treatment was found to increase the number and weight of nodules in soybeans. Given the close correlation between nitrogen accumulation and dry matter accumulation in soybeans, a key question arose: how does exogenous cytokinin affect nitrogen accumulation in soybeans? To address this, we investigated the impact of exogenous cytokinins on nitrogen accumulation in soybeans under low P stress. The results revealed that exogenous cytokinins promoted nitrogen accumulation in the aboveground parts, roots, and nodules of soybeans under low P stress, accompanied by an increase in nodule number and weight. These findings suggest that exogenous cytokinins enhance soybean tolerance to low P stress by modulating nitrogen metabolism and nodulation.
Bertell and Eliasson proposed that cytokinin-treated seeds result in reduced soil nitrogen uptake [52]. In contrast, our study revealed that under low P stress, exogenous cytokinin enhanced the uptake of fertilizer nitrogen by the aboveground parts, roots, and nodules of soybeans at 7, 14, 21, and 28 days after treatment. However, from the 35th day onwards, the abundance of 15N in the P1 + CKs treatment was lower than that in the P1 treatment, which partially diverges from the findings of Bertell and Eliasson. We hypothesize that under low P stress, exogenous cytokinin may mitigate the inhibitory effects of nodule nitrogen fixation on early soybean growth and development by enhancing the uptake of exogenous nitrogen (fertilizer nitrogen).
A well-established correlation exists between elevated cytokinin signaling and increased protein synthesis rates [53]. Previous investigations have consistently demonstrated that cytokinin treatments promote translation and enhance the protein synthesis rate [53,54,55,56,57]. It is hypothesized that the cytokinin signal directly influences the protein synthesis mechanism [53]. Recent proteomics studies have further expanded this understanding, revealing that cytokinin impacts on the protein synthesis mechanism are likely multifaceted. Alterations in cytokinin content have been shown to modify the abundance of proteins involved in ribosome biogenesis and translation [1,58]. In this study, we observed that exogenous cytokinin significantly increased the soluble protein content in nodules under low P stress. While this finding is novel, the underlying mechanisms by which cytokinin modulates protein synthesis remain incompletely understood. Potential contributing factors may include changes in ribosomal structures or transporter proteins associated with cytokinin trafficking and translocation [53]. As such, further research is needed to fully elucidate the intricate relationship between cytokinin and protein synthesis.
Amino acids serve as fundamental building blocks in the interconnected networks of plants’ primary metabolic pathways. Plants exude approximately 15% of assimilated nitrogen, with amino acids representing a major nitrogen-containing component in these exchanges [59]. Amino acid transport is essential for nutrient exchange between plants and their environment. When coupled with specific receptors, it may also mediate amino acid sensing and signaling during plant–microbe interactions [60]. Microbial chemoreceptors can recognize a wide spectrum of amino acids, guiding microbes toward the nutrient-rich rhizosphere around plant roots [61]. Under adverse stress conditions, plants actively regulate amino acid absorption, synthesis, and degradation to mitigate stress-induced damage [62,63]. Consequently, amino acid accumulation and metabolism represent critical adaptive responses of plants to abiotic stress [64]. Several studies have elucidated the dynamic role of amino acids under various experimental conditions. Saad et al. observed that in hydroponic culture, free amino acid levels and 17 specific amino acid types in root phloem increased as P supply decreased [65]. Medhi et al. investigated the combined effects of P and growth regulators on mung bean nodulation, demonstrating that their application significantly elevated the nitrogen, protein, sugar, and free amino acid contents in nodules [66]. In soybean, ethylene application caused a five-fold increase (from 250 to 1284 mg/100 g) in essential amino acid content and a six-fold increase (from 544 to 3478 mg/100 g) in non-essential amino acids in leaves [67]. In this study, the free amino acid content in the nodules of P1 was higher than that in P31. However, exogenous cytokinin treatment did not significantly affect the free amino acid levels in the nodules. Cytokinins are known to inhibit protein degradation in plants and facilitate the redistribution of essential amino acids, hormones, and other compounds to different tissues [32]. Notably, this experiment only monitored the free amino acid content in cytokinin-treated nodules. Future research should investigate whether amino acids are being redistributed to other soybean tissues, potentially explaining the inconsistent nodular amino acid level changes observed at different sampling times.
5. Conclusions
- (1)
- Exogenous cytokinin regulated soybean nitrogen metabolism by increasing nitrogen accumulation, nitrogen fixation accumulation, nitrogen fixation rate (at 35 days), and nitrogenase activity (ARA and SNA) in soybeans under low P stress.
- (2)
- Exogenous cytokinin enhanced the absorption of fertilizer nitrogen to alleviate the inhibition of low P stress on soybean early growth and development.
- (3)
- Exogenous cytokinin significantly increased the soluble protein content in nodules to regulate nitrogen metabolism under low P stress, while the changes in free amino acids in nodules were not obvious.
Author Contributions
Conceptualization, Y.Y.; formal analysis, Y.Y., Y.X. and J.Y.; investigation, X.T., X.L. (Xiaoyan Luan), W.H., Q.L. and Z.Z.; methodology, Y.Y. and D.C.; resources, D.C. and Y.X.; supervision, X.L. (Xinlei Liu) and X.L. (Xiaoyan Luan); validation, Q.L. and Z.Z.; visualization, J.Y. and W.H.; writing—original draft preparation, Y.Y. and X.T.; writing—review and editing, Y.Y. and X.L. (Xinlei Liu). All authors have read and agreed to the published version of the manuscript.
Funding
This work was supported by the Innovation Project of Agricultural Science and Technology Foundation under the Agricultural Science and Technology Innovation Leapfrog Project of Heilongjiang Province (CX25JC02) and Heilongjiang Province Postdoctoral Research Start-up Fund.
Data Availability Statement
The raw data used to assemble this article will be made available by the authors, without undue reservation.
Acknowledgments
We appreciate all colleagues who helped us in any way during the experiment but are not included in the author list.
Conflicts of Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
References
- Yuan, S.L.; Li, R.; Chen, H.F.; Zhang, C.J.; Chen, L.M.; Hao, Q.N.; Chen, S.L.; Shan, Z.H.; Yang, Z.L.; Zhang, X.J.; et al. RNA-Seq analysis of nodule development at five different developmental stages of soybean (Glycine max) inoculated with Bradyrhizobium japonicum strain 113-2. Sci. Rep. 2017, 7, 42248. [Google Scholar] [CrossRef]
- Guo, B.F.; Sun, L.P.; Jiang, S.Q.; Ren, H.L.; Sun, R.J.; Wei, Z.Y.; Hong, H.L.; Luan, X.Y.; Wang, J.; Wang, X.B.; et al. Correction to: Soybean genetic resources contributing to sustainable protein production. Theor. Appl. Genet. 2022, 135, 4123. [Google Scholar] [CrossRef]
- Zhao, M.L.; Guo, R.; Li, M.X.; Liu, Y.; Wang, X.X.; Fu, H.; Wang, S.Y.; Liu, X.Y.; Shi, L.X. Physiological characteristics and metabolomics reveal the tolerance mechanism to low nitrogen in Glycine soja leaves. Physiol. Plant. 2019, 168, 819–834. [Google Scholar] [CrossRef]
- Wang, Y.; Yang, Z.; Kong, Y.; Li, X.; Li, W.; Du, H.; Zhang, C. GmPAP12 Is Required for nodule development and nitrogen fixation under phosphorus starvation in soybean. Front. Plant Sci. 2020, 11, 450. [Google Scholar] [CrossRef]
- Soumare, A.; Diedhiou, A.G.; Thuita, M.; Hafidi, M.; Ouhdouch, Y.; Gopalakrishnan, S.; Kouisni, L. Exploiting biological nitrogen fixation: A route towards a sustainable agriculture. Plants 2020, 9, 1011. [Google Scholar] [CrossRef]
- Yang, Z.; Du, H.; Xing, X.; Li, W.; Kong, Y.; Li, X.; Zhang, C. A small heat shock protein, GmHSP17.9, from nodule confers symbiotic nitrogen fixation and seed yield in soybean. Plant Biotechnol. J. 2022, 20, 103–115. [Google Scholar] [CrossRef]
- Song, J.; Liu, Y.; Cai, W.; Zhou, S.; Fan, X.; Hu, H.; Ren, L.; Xue, Y. Unregulated GmAGL82 due to phosphorus deficiency positively regulates root nodule growth in soybean. Int. J. Mol. Sci. 2024, 25, 1802. [Google Scholar] [CrossRef]
- Saikia, S.P.; Jain, V. Biological nitrogen fixation with non-legumes: An achievable target or a dogma. Curr. Sci. 2007, 92, 317–322. [Google Scholar]
- Sur, S.; Bothra, A.K.; Sen, A. Symbiotic nitrogen fixation-A bioinformatics perspective. Biotechnology 2010, 9, 257–273. [Google Scholar] [CrossRef]
- Zhao, L.F.; Xu, Y.J.; Lai, X.H. Antagonistic endophytic bacteria associated with nodules of soybean (Glycine max L.) and plant growth-promoting properties. Braz. J. Microbiol. 2017, 308, 269–278. [Google Scholar] [CrossRef]
- Hawkesford, M.; Horst, W.; Kichey, T.; Lambers, H.; Schjoerring, J.; Møller, I.S.; White, P. Functions of Macronutrients. In Marschner’s Mineral Nutrition of Higher Plants, 3rd ed.; Marschner, P., Ed.; Academic Press: Amsterdam, The Netherlands, 2012; pp. 135–189. [Google Scholar] [CrossRef]
- Hou, E.; Luo, Y.; Kuang, Y.; Chen, C.; Lu, X.; Jiang, L.; Luo, X.; Wen, D. Global meta-analysis shows pervasive phosphorus limitation of aboveground plant production in natural terrestrial ecosystems. Nat. Commun. 2020, 11, 637. [Google Scholar] [CrossRef]
- Raghothama, K.G. Phosphate acquisition. Annu. Rev. Plant Phys. 1999, 50, 665–693. [Google Scholar] [CrossRef]
- Ham, B.K.; Chen, J.Y.; Yan, Y.; Lucas, W.J. Insights into plant phosphate sensing and signaling. Curr. Opin. Biotech. 2018, 49, 1–9. [Google Scholar] [CrossRef]
- Zhu, S.N.; Chen, Z.J.; Xie, B.X.; Guo, Q.; Chen, M.H.; Liang, C.Y.; Bai, Z.L.; Wang, X.R.; Wang, H.C.; Liao, H.; et al. A phosphate starvation responsive malate dehydrogenase, GmMDH12 mediates malate synthesis and nodule size in soybean (Glycine max). Environ. Exp. Bot. 2021, 189, 104560. [Google Scholar] [CrossRef]
- Liu, X.Q.; Cai, Y.P.; Yao, W.W.; Chen, L.; Hou, W.S. The soybean NUCLEAR FACTOR-Y C4 and α-EXPANSIN 7 module influences phosphorus uptake by regulating root morphology. Plant Physiol. 2025, 197, kiae478. [Google Scholar] [CrossRef]
- Makoudi, B.; Kabbadj, A.; Mouradi, M.; Amenc, L.; Domergue, O.; Blair, M.; Drevon, J.J.; Ghoulam, C. Phosphorus deficiency increases nodule phytase activity of faba bean-rhizobia symbiosis. Acta Physiol. Plant. 2018, 40, 63. [Google Scholar] [CrossRef]
- Ferguson, B.J.; Mens, C.; Hastwell, A.H.; Zhang, M.; Su, H.; Jones, C.H.; Chu, X.T.; Gresshoff, P.M. Legume nodulation: The host controls the party. Plant Cell Environ. 2019, 42, 41–51. [Google Scholar] [CrossRef]
- Taliman, N.; Dong, Q.; Echigo, K.; Raboy, V.; Saneoka, H. Effect of phosphorus fertilization on the growth, photosynthesis, nitrogen fixation, mineral accumulation, seed yield, and seed quality of a soybean low-phytate line. Plants 2019, 8, 119. [Google Scholar] [CrossRef]
- Wang, S.Q.; Han, X.Z.; Qiao, Y.F.; Yan, J.; Li, X.H. Nodule growth, nodulation and nitrogen fixation in soybean (Glycine max L.) as affected by P deficiency stress. Soybean Sci. 2009, 28, 1000–1003. [Google Scholar]
- Lu, M.Y.; Cheng, Z.Y.; Zhang, X.M.; Huang, P.H.; Fan, C.M.; Yu, G.L.; Chen, F.L.; Xu, K.; Chen, Q.S.; Miao, Y.C.; et al. Spatial divergence of PHR-PHT1 modules maintains phosphorus homeostasis in soybean nodules. Plant Physiol. 2020, 184, 01209–02019. [Google Scholar] [CrossRef]
- Li, H.; Wang, X.; Liang, Q.; Lyu, X.; Li, S.; Gong, Z.; Dong, S.; Yan, C.; Ma, C. Regulation of phosphorus supply on nodulation and nitrogen fixation in soybean plants with dual-root systems. Agronomy 2021, 11, 2354. [Google Scholar] [CrossRef]
- Yao, Y.B.; Liu, X.L. Responses of nitrogen metabolism pathways to low-phosphorus stress: Decrease in nitrogen accumulation and alterations in protein metabolism in soybeans. Agronomy 2025, 15, 836. [Google Scholar] [CrossRef]
- Miller, C.O.; Skoog, F.; Okomura, F.S. Isolation, structure and synthesis of kinetin, a substance promoting cell division. J. Am. Chem. Soc. 1956, 78, 1375–1380. [Google Scholar] [CrossRef]
- Wu, Y.F. Physiological Mechanism of Exogenous Cytokinin on Drought Stress Mitigation Effect of Meng Yuan Cerasus humilis; Inner Mongolia Agricultural University: Hohhot, China, 2022. [Google Scholar]
- Li, Y.; Zhao, J.H.; Li, J.R.; Qiao, B.C.; Liu, Z.X.; Gao, F.; Yang, D.Q.; Li, X.D. Effects of exogenous 6-ba on root growth and pod yield of flooded peanut at different growth stages. Sci. Agric. Sin. 2020, 53, 3665–3678. [Google Scholar] [CrossRef]
- Verslues, P.E. ABA and cytokinins: Challenge and opportunity for plant stress research. Plant Mol. Biol. 2016, 91, 629–640. [Google Scholar] [CrossRef]
- Lukatkin, A.S.; Gracheva, N.V.; Grishenkova, N.N.; Dukhovskis, P.V.; Brazaitite, A.A. Cytokinin-like growth regulators mitigate toxic action of zinc and nickel ions on maize seedling. Russ. J. Plant Physl. 2007, 54, 381–387. [Google Scholar] [CrossRef]
- Hong, J.P.; Kim, W.T. Isolation and functional characterization of the Ca-DREBLP1 gene encoding a dehydration-responsive element binding-factor-like protein 1 in hot pepper (Capsicum annuum L. cv. Pukang). Planta 2005, 220, 875–888. [Google Scholar] [CrossRef]
- Kulaeva, O.N.; Prokoptseva, O.S. Recent advances in the study of mechanisms of action of phytohormones. Biochemistry 2004, 69, 233–247. [Google Scholar] [CrossRef]
- Kempster, R.; Barat, M.; Bishop, L.; Rufino, M.; Borras, L.; Dodd, L.C. Genotype and cytokinin effects on soybean yield and biological nitrogen fixation across soil tempera. Ann. Appl. Biol. 2021, 178, 341–354. [Google Scholar] [CrossRef]
- Ullah, A.; Manghwar, H.; Shaban, M.; Khan, A.H.; Akbar, A.; Ali, U.; Ali, E.; Fahad, S. Phytohormones enhanced drought tolerance in plants: A coping strategy. Environ. Sci. Pollut. Res. Int. 2018, 25, 33103–33118. [Google Scholar] [CrossRef]
- Yao, Y.B.; Cheng, L.L.; Liu, X.L.; Xue, Y.G.; Tang, X.F.; Cao, D.; He, W.J.; Luan, X.Y. The Effect of Exogenous Cytokinin on Photosynthetic Products Accumulation and Enzyme Activity in Soybean Nodules under Phosphorus Stress. Soybean Sci. 2025, 44, 103–110. Available online: https://link.cnki.net/urlid/23.1227.S.20250423.1435.010 (accessed on 23 April 2025).
- Gremaud, M.F.; Harper, J.E. Selection and initial characterization of partially nitrate tolerant nodulation mutants of soybean. Plant Physiol. 1989, 89, 169–173. [Google Scholar] [CrossRef]
- Chen, H.; Di, W.; Yao, Y.B.; Gong, Z.P.; Ma, C.M. Study on nitrogenase activity and nitrogen fixation of different soybean varieties. J. Nucl. Agric. Sci. 2013, 3, 379–383. [Google Scholar]
- GB/T 8314-2013; Determination of Total Free Amino Acids in Tea. China Standard Press: Beijing, China, 2013.
- Yang, W.B.; Cortijo, S.; Korsbo, N.; Roszak, P.; Schiessl, K.; Gurzadyan, A.; Wightman, R.; Jönsson, H.; Meyerowitz, E. Molecular mechanism of cytokinin-activated cell division in Arabidopsis. Science 2021, 371, 1350–1355. [Google Scholar] [CrossRef] [PubMed]
- Hoyerová, K.; Hošek, P. New insights into the metabolism and role of cytokinin N-glucosides in plants. Front. Plant Sci. 2020, 11, 741. [Google Scholar] [CrossRef]
- Osugi, A.; Sakakibara, H. Q&A: How do plants respond to cytokinins and what is their importance? BMC Biol. 2015, 13, 102. [Google Scholar] [CrossRef]
- Kieber, J.J.; Schaller, G.E. Cytokinin signaling in plant development. Development 2018, 145, dev149344. [Google Scholar] [CrossRef]
- Schaller, G.E.; Bishopp, A.; Kieber, J.J. The yin-yang of hormones: Cytokinin and auxin interactions in plant development. Plant Cell 2015, 27, 44–63. [Google Scholar] [CrossRef]
- Yao, Y.B.; Wu, D.T.; Gong, Z.P.; Zhao, J.K.; Ma, C.M. Variation of nitrogen accumulation and yield in response to phosphorus nutrition of soybean (Glycine max L. Merr.). J. Plant Nutrition 2018, 41, 1138–1147. [Google Scholar] [CrossRef]
- Iqbal, M.; Ashraf, M.; Jamil, A. Seed enhancement with cytokinins: Changes in growth and grain yield in salt stressed wheat plants. Plant Growth Regul. 2006, 50, 29–39. [Google Scholar] [CrossRef]
- Kariali, E.; Mohapatra, P.K. Hormonal regulation of tiller dynamics in di erentially-tillering rice cultivars. Plant Growth Regul. 2007, 53, 215–223. [Google Scholar] [CrossRef]
- Zatloukal, M.; Plihalova, L.; Klaskova, J.; Spichal, L.; Koprna, R.; Dolezal, K. Substituted 6-anilino-9-heterocyclylpurine Derivatives for Inhibition of Plant Stress. U.S. Patent EP3233860A1, 25 October 2017. [Google Scholar]
- Alharby, H.; Alzahrani, Y.M.; Rady, M. Seeds pretreatment with zeatins or maize grain-derived organic biostimulant improved hormonal contents, polyamine gene expression, and salinity and drought tolerance of wheat. Int. J. Agric. Biol. 2020, 24, 714–724. [Google Scholar] [CrossRef]
- Yaronskaya, E.; Vershilovskaya, I.; Poers, Y.; Alawady, A.E.; Averina, N.; Grimm, B. Cytokinin effects on tetrapyrrole biosynthesis and photosynthetic activity in barley seedlings. Planta 2006, 224, 700–709. [Google Scholar] [CrossRef]
- Werner, T.; Holst, K.; Pörs, Y.; Guivar´ch, A.; Mustroph, A.; Chriqui, D.; Grimm, B.; Schm¨ulling, T. Cytokinin defiency causes distinct changes of sink and source parameters in tobacco shoots and roots. J. Exp. Bot. 2008, 59, 2659–2672. [Google Scholar] [CrossRef]
- Aswathi, K.P.R.; Kalaji, H.M.; Puthur, J.T. Seed priming of plants aiding in drought stress tolerance and faster recovery: A review. Plant Growth Regul. 2022, 97, 235–253. [Google Scholar] [CrossRef]
- Johnson, R.; Puthur, J.T. Biostimulant priming in oryza sativa: A novel approach to reprogram the functional biology under nutrient-deficient soil. Cereal Res. Commun. 2022, 50, 45–52. [Google Scholar] [CrossRef]
- Werner, T.; Motyka, V.; Strnad, M.; Schmulling, T. Regulation of plant growth by cytokinin. Proc. Natl. Acad. Sci. USA 2001, 98, 10487–10492. [Google Scholar] [CrossRef]
- Bertell, G.; Eliasson, L. Cytokinin effects on root growth and possible interactions with ethylene and indole-3-acetic acid. Physiol. Plantarum 1992, 84, 255–261. [Google Scholar] [CrossRef]
- Karunadasa, S.S.; Kurepa, J.; Shull, T.E.; Smalle, J.A. Cytokinin-induced protein synthesis suppresses growth and osmotic stress tolerance. New Phytol. 2020, 227, 50–64. [Google Scholar] [CrossRef]
- Klambt, D. Cytokinin effects on protein synthesis of in vitro systems of higher plants. Plant Cell Physiol. 1976, 17, 73–76. [Google Scholar] [CrossRef]
- Muren, R.C.; Fosket, D.E. Cytokinin-mediated translational control of protein synthesis in cultured cells of Glycine max. J. Exp. Bot. 1977, 28, 775–784. [Google Scholar] [CrossRef]
- Tepfer, D.A.; Fosket, D.E. Hormone-mediated translational control of protein synthesis in cultured cells of Glycine max. Dev. Biol. 1978, 62, 486–497. [Google Scholar] [CrossRef]
- Szweykowska, A.; Gwozdz, E.; Spychała, M. The cytokinin control of protein synthesis in plants. In Metabolism and Molecular Activities of Cytokinins; Guern, J., Peaud-Leno€ el, C., Eds.; Springer: Berlin, Germany, 1981; pp. 212–217. [Google Scholar]
- Černý, M.; Kuklová, A.; Hoehenwarter, W.; Fragner, L.; Novák, O.; Rotková, G.; Jedelsky, P.L.; Žáková, K.; Šmehilová, M.; Strnad, M.; et al. Proteome and metabolome profiling of cytokinin action in Arabidopsis identifying both distinct and similar responses to cytokinin down- and up-regulation. J. Exp. Bot. 2013, 64, 4193–4206. [Google Scholar] [CrossRef]
- Venturi, V.; Keel, C. Signaling in the rhizosphere. Trends Plant Sci. 2016, 21, 187–198. [Google Scholar] [CrossRef] [PubMed]
- Moormann, J.; Heinemann, B.; Hildebrandt, T.M. News about amino acid metabolism in plant-microbe interactions. Trends Biochem. Sci. 2022, 47, 10. [Google Scholar] [CrossRef] [PubMed]
- Yang, Y.L.; Pollard, A.M.; Höfler, C.; Poschet, G.; Wirtz, M.; Hell, R.; Sourjik, V. Relation between chemotaxis and consumption of amino acids in bacteria. Mol. Microbiol. 2015, 96, 1272–1282. [Google Scholar] [CrossRef]
- Hildebrandt, T.M. Synthesis versus degradation: Directions of amino acid metabolism during Arabidopsis abiotic stress response. Plant Mol. Biol. 2018, 98, 121–135. [Google Scholar] [CrossRef]
- Batista-Silva, W.; Heinemann, B.; Rugen, N.; Nunes-Nesi, A.; Araujo, W.L.; Braun, H.P.; Hildebrandt, T.M. The role of amino acid metabolism during abiotic stress release. Plant Cell Environ. 2019, 42, 1630–1644. [Google Scholar] [CrossRef]
- Planchet, E.; Limami, A.M. Amino Acid Synthesis under Abiotic Stress. In Amino Acids in Higher Plants; D’Mello, J.P.F., Ed.; CAB International: Wallingford, UK, 2015; pp. 262–276. [Google Scholar] [CrossRef]
- Saad, S.; Van, H.C.; Joachim, S.; Lam-Son Phan, T. Growth and nodulation of symbiotic Medicago truncatula at different levels of phosphorus availability. J. Exp. Bot. 2013, 64, 2701–2712. [Google Scholar] [CrossRef]
- Medhi, A.K.; Dhar, S.; Roy, A. Effect of different growth regulators and phosphorus levels on nodulation, yield and quality components in green gram. Indian J. Plant Physiol. 2014, 19, 74–78. [Google Scholar] [CrossRef]
- Ban, Y.J.; Song, Y.H.; Kim, J.Y.; Cha, J.Y.; Ali, I.; Baiseitova, A.; Shah, A.B.; Kim, W.Y.; Park, K.H. A Significant Change in Free Amino Acids of Soybean (Glycine max L. Merr) through Ethylene Application. Molecules 2021, 26, 1128. [Google Scholar] [CrossRef] [PubMed]
Figure 1.
Changes in ARA (C2H4 μmol·h−1·plant−1). The value is the data of year 2023. * denotes p < 0.05; ** denotes p < 0.01; *** denotes p < 0.001; ns denotes p > 0.05. The value is the data of year 2023.
Figure 1.
Changes in ARA (C2H4 μmol·h−1·plant−1). The value is the data of year 2023. * denotes p < 0.05; ** denotes p < 0.01; *** denotes p < 0.001; ns denotes p > 0.05. The value is the data of year 2023.
Figure 2.
Changes in SNA (C2H4 μmol·g−1 nodule dry mass·h−1). The value is the data of year 2023. * denotes p < 0.05; ** denotes p < 0.01; *** denotes p < 0.001; ns denotes p > 0.05. The value is the data of year 2023.
Figure 2.
Changes in SNA (C2H4 μmol·g−1 nodule dry mass·h−1). The value is the data of year 2023. * denotes p < 0.05; ** denotes p < 0.01; *** denotes p < 0.001; ns denotes p > 0.05. The value is the data of year 2023.
Figure 3.
Changes in soluble protein content (mg/g). *** denotes p < 0.005; **** denotes p < 0.001; ns denotes p > 0.05. The value is the data of year 2023.
Figure 3.
Changes in soluble protein content (mg/g). *** denotes p < 0.005; **** denotes p < 0.001; ns denotes p > 0.05. The value is the data of year 2023.
Figure 4.
Changes in free amino acid (mg/g). The value is the data of year 2023. * denotes p < 0.05; ** denotes p < 0.01; *** denotes p < 0.001; ns denotes p > 0.05. The value is the data of year 2023.
Figure 4.
Changes in free amino acid (mg/g). The value is the data of year 2023. * denotes p < 0.05; ** denotes p < 0.01; *** denotes p < 0.001; ns denotes p > 0.05. The value is the data of year 2023.
Table 1.
Changes in nitrogen accumulation (mg/plant).
| Treatments | Aboveground | Root | Nodule | |
|---|---|---|---|---|
| 7 days | P1 | 27.88 ± 0.72 c | 12.91 ± 0.13 b | 1.21 ± 0.05 c |
| P1 + CKs | 34.19 ± 0.26 b | 15.19 ± 0.18 a | 1.73 ± 0.01 b | |
| P31 | 53.20 ± 0.67 a | 11.31 ± 0.10 c | 7.10 ± 0.11 a | |
| 14 days | P1 | 38.73 ± 0.75 c | 13.33 ± 0.11 b | 2.04 ± 0.02 b |
| P1 + CKs | 45.28 ± 0.52 b | 14.83 ± 0.91 b | 2.05 ± 0.01 b | |
| P31 | 91.60 ± 0.36 a | 16.89 ± 0.03 a | 9.84 ± 0.20 a | |
| 21 days | P1 | 47.37 ± 0.93 c | 18.03 ± 0.59 b | 1.95 ± 0.13 b |
| P1 + CKs | 54.06 ± 0.42 b | 18.23 ± 0.04 b | 2.15 ± 0.06 b | |
| P31 | 184.36 ± 1.71 a | 41.45 ± 0.24 a | 25.73 ± 0.26 a | |
| 28 days | P1 | 69.09 ± 1.43 c | 22.70 ± 0.78 c | 2.45 ± 0.11 c |
| P1 + CKs | 86.85 ± 2.33 b | 31.67 ± 0.99 b | 4.07 ± 0.09 b | |
| P31 | 287.09 ± 3.39 a | 71.45 ± 0.56 a | 29.23 ± 0.07 a | |
| 35 days | P1 | 100.11 ± 1.68 c | 32.13 ± 0.66 c | 3.17 ± 0.05 c |
| P1 + CKs | 135.33 ± 0.43 b | 48.15 ± 0.79 b | 7.29 ± 0.07 b | |
| P31 | 439.45 ± 8.42 a | 91.36 ± 0.29 a | 36.62 ± 0.14 a | |
| 42 days | P1 | 181.05 ± 0.35 c | 55.86 ± 0.98 c | 4.81 ± 0.12 c |
| P1 + CKs | 206.69 ± 2.97 b | 68.45 ± 0.47 b | 9.59 ± 0.13 b | |
| P31 | 444.68 ± 1.43 a | 114.85 ± 3.59 a | 35.52 ± 0.51 a | |
| 49 days | P1 | 286.90 ± 2.34 b | 90.27 ± 1.17 b | 8.40 ± 0.07 c |
| P1 + CKs | 290.20 ± 6.87 b | 109.47 ± 0.93 a | 10.18 ± 0.11 b | |
| P31 | 565.46 ± 4.49 a | 112.84 ± 2.11 a | 36.37 ± 0.31 a | |
Vertical comparison. The data are represented as mean values ± standard error (with three replicates). The value is the average of the data in the years 2022 and 2023. Values with the same letters are not significantly different at the 5% level.
Table 2.
Changes in 15N abundance (%).
| Treatments | Aboveground | Root | Nodule | |
|---|---|---|---|---|
| 7 days | P1 | 1.90 ± 0.00 b | 2.07 ± 0.01 b | 0.96 ± 0.01 b |
| P1 + CKs | 2.03 ± 0.00 a | 2.17 ± 0.02 a | 1.18 ± 0.01 a | |
| P31 | 1.93 ± 0.02 b | 1.80 ± 0.03 c | 0.99 ± 0.00 b | |
| 14 days | P1 | 2.12 ± 0.00 a | 2.27 ± 0.01 a | 1.09 ± 0.00 b |
| P1 + CKs | 2.14 ± 0.04 a | 2.29 ± 0.04 a | 1.18 ± 0.02 a | |
| P31 | 1.92 ± 0.03 b | 2.13 ± 0.07 a | 0.70 ± 0.02 c | |
| 21 days | P1 | 2.30 ± 0.02 a | 2.26 ± 0.03 a | 1.11 ± 0.01 b |
| P1 + CKs | 2.29 ± 0.03 a | 2.29 ± 0.01 a | 1.20 ± 0.00 a | |
| P31 | 1.31 ± 0.01 b | 1.74 ± 0.00 b | 0.47 ± 0.01 c | |
| 28 days | P1 | 2.37 ± 0.01 b | 2.37 ± 0.01 b | 1.17 ± 0.02 a |
| P1 + CKs | 2.49 ± 0.01 a | 2.51 ± 0.00 a | 1.17 ± 0.02 a | |
| P31 | 1.36 ± 0.02 c | 1.80 ± 0.00 c | 0.52 ± 0.01 b | |
| 35 days | P1 | 2.44 ± 0.00 a | 2.44 ± 0.02 a | 1.20 ± 0.00 a |
| P1 + CKs | 2.24 ± 0.04 b | 2.33 ± 0.03 b | 1.02 ± 0.03 b | |
| P31 | 1.40 ± 0.00 c | 1.87 ± 0.01 c | 0.58 ± 0.03 c | |
| 42 days | P1 | 2.43 ± 0.05 a | 2.55 ± 0.02 a | 0.94 ± 0.03 a |
| P1 + CKs | 2.20 ± 0.00 b | 2.36 ± 0.00 b | 0.76 ± 0.02 b | |
| P31 | 1.48 ± 0.00 c | 1.87 ± 0.00 c | 0.54 ± 0.00 c | |
| 49 days | P1 | 2.29 ± 0.05 a | 2.45 ± 0.04 a | 0.83 ± 0.05 a |
| P1 + CKs | 2.07 ± 0.02 b | 2.28 ± 0.01 b | 0.72 ± 0.05 a | |
| P31 | 1.45 ± 0.02 c | 1.86 ± 0.02 c | 0.49 ± 0.02 b | |
Vertical comparison. The data are represented as mean values ± standard error (with three replicates). The value is the average of the data in the years 2022 and 2023. Values with the same letters are not significantly different at the 5% level.
Table 3.
Changes in nodule nitrogen fixation accumulation (mg/plant).
| Treatments | Aboveground | Root | Nodule | |
|---|---|---|---|---|
| 7 days | P1 | 11.00 ± 0.20 b | 4.52 ± 0.04 a | 0.84 ± 0.04 c |
| P1 + CKs | 11.91 ± 0.11 b | 4.83 ± 0.19 a | 1.10 ± 0.01 b | |
| P31 | 20.39 ± 0.17 a | 4.87 ± 0.06 a | 4.98 ± 0.10 a | |
| 14 days | P1 | 12.27 ± 0.41 b | 3.90 ± 0.01 b | 1.32 ± 0.00 b |
| P1 + CKs | 14.05 ± 0.11 b | 4.16 ± 0.17 b | 1.27 ± 0.02 b | |
| P31 | 35.84 ± 0.19 a | 5.62 ± 0.15 a | 7.71 ± 0.10 a | |
| 21 days | P1 | 13.02 ± 0.14 b | 5.17 ± 0.00 b | 1.24 ± 0.03 b |
| P1 + CKs | 14.75 ± 0.33 b | 5.05 ± 0.11 b | 1.32 ± 0.03 b | |
| P31 | 107.13 ± 1.03 a | 18.56 ± 0.57 a | 21.89 ± 0.31 a | |
| 28 days | P1 | 17.55 ± 0.02 b | 5.72 ± 0.16 c | 1.55 ± 0.08 c |
| P1 + CKs | 18.66 ± 0.09 b | 6.53 ± 0.32 b | 2.55 ± 0.01 b | |
| P31 | 159.88 ± 0.09 a | 30.13 ± 0.03 a | 24.52 ± 0.02 a | |
| 35 days | P1 | 23.08 ± 0.17 c | 7.63 ± 0.12 c | 1.99 ± 0.04 c |
| P1 + CKs | 39.67 ± 0.21 b | 12.95 ± 0.07 b | 5.16 ± 0.14 b | |
| P31 | 241.14 ± 0.41 a | 37.42 ± 0.09 a | 29.87 ± 0.24 a | |
| 42 days | P1 | 42.27 ± 0.31 c | 10.61 ± 0.16 c | 3.47 ± 0.04 c |
| P1 + CKs | 65.80 ± 0.33 b | 17.36 ± 0.15 b | 7.38 ± 0.16 b | |
| P31 | 233.83 ± 1.45 a | 44.40 ± 0.18 a | 29.49 ± 0.35 a | |
| 49 days | P1 | 80.04 ± 0.55 c | 20.41 ± 0.54 c | 6.29 ± 0.18 c |
| P1 + CKs | 100.74 ± 0.68 b | 31.20 ± 0.96 b | 8.00 ± 0.15 b | |
| P31 | 291.50 ± 4.58 a | 46.70 ± 0.04 a | 30.88 ± 0.44 a | |
Vertical comparison. The data are represented as mean values ± standard error (with three replicates). The value is the average data of years 2022 and 2023. Values with the same letters are not significantly different at the 5% level.
Table 4.
Changes in ratio of nodule nitrogen fixation (%).
| Treatments | Aboveground | Root | Nodule | |
|---|---|---|---|---|
| 7 days | P1 | 39.90 ± 0.23 a | 34.71 ± 0.46 b | 69.45 ± 0.36 a |
| P1 + CKs | 35.73 ± 0.11 b | 31.42 ± 0.82 c | 62.71 ± 0.50 b | |
| P31 | 39.13 ± 0.32 a | 42.98 ± 1.02 a | 68.85 ± 0.21 a | |
| 14 days | P1 | 32.64 ± 0.33 b | 28.10 ± 0.57 b | 65.57 ± 0.31 b |
| P1 + CKs | 32.25 ± 0.40 b | 27.41 ± 0.29 b | 62.48 ± 0.81 c | |
| P31 | 39.19 ± 1.00 a | 32.76 ± 0.28 a | 77.84 ± 0.68 a | |
| 21 days | P1 | 27.48 ± 0.88 b | 28.65 ± 0.98 b | 64.89 ± 0.56 b |
| P1 + CKs | 27.63 ± 0.96 b | 27.55 ± 0.55 b | 61.86 ± 0.16 c | |
| P31 | 58.49 ± 0.36 a | 45.08 ± 0.17 a | 85.10 ± 0.33 a | |
| 28 days | P1 | 25.27 ± 0.63 b | 24.97 ± 0.53 b | 63.12 ± 0.69 b |
| P1 + CKs | 21.42 ± 0.56 c | 20.59 ± 0.31 c | 63.05 ± 0.73 b | |
| P31 | 56.73 ± 0.91 a | 42.77 ± 0.18 a | 83.70 ± 0.37 a | |
| 35 days | P1 | 22.97 ± 0.14 c | 22.94 ± 0.79 c | 61.83 ± 0.18 c |
| P1 + CKs | 28.86 ± 0.25 b | 26.01 ± 0.15 b | 67.43 ± 0.99 b | |
| P31 | 55.54 ± 0.06 a | 40.85 ± 0.37 a | 81.59 ± 0.96 a | |
| 42 days | P1 | 23.10 ± 0.70 c | 19.33 ± 0.79 c | 70.08 ± 0.99 c |
| P1 + CKs | 30.24 ± 0.23 b | 25.08 ± 0.05 b | 75.85 ± 0.71 b | |
| P31 | 53.00 ± 0.24 a | 40.54 ± 0.14 a | 82.97 ± 0.17 a | |
| 49 days | P1 | 27.55 ± 0.79 c | 22.43 ± 0.27 c | 73.74 ± 0.82 b |
| P1 + CKs | 34.34 ± 0.88 b | 27.84 ± 0.61 b | 76.97 ± 0.69 b | |
| P31 | 53.78 ± 0.90 a | 41.08 ± 0.76 a | 84.38 ± 0.68 a | |
Vertical comparison. The data are represented as mean values ± standard error (with three replicates). The value is the average of the data in the years 2022 and 2023. Values with the same letters are not significantly different at the 5% level.
Table 5.
Changes in nodule number and weight (per plant and g/plant).
| Treatments | Number (per Plant) | Weight (g/Plant) | |
|---|---|---|---|
| 7 days | P1 | 0.03 ± 0.00 c | 30.33 ± 0.29 c |
| P1 + CKs | 0.04 ± 0.00 b | 35.33 ± 0.29 b | |
| P31 | 0.14 ± 0.00 a | 74.50 ± 0.67 a | |
| 14 days | P1 | 0.05 ± 0.00 b | 28.58 ± 0.06 c |
| P1 + CKs | 0.05 ± 0.00 b | 39.92 ± 0.24 b | |
| P31 | 0.20 ± 0.00 a | 64.92 ± 0.87 a | |
| 21 days | P1 | 0.05 ± 0.00 b | 28.50 ± 0.07 c |
| P1 + CKs | 0.06 ± 0.00 b | 42.42 ± 0.21 b | |
| P31 | 0.55 ± 0.01 a | 87.83 ± 0.46 a | |
| 28 days | P1 | 0.07 ± 0.00 c | 34.75 ± 0.34 c |
| P1 + CKs | 0.12 ± 0.00 b | 54.08 ± 0.78 b | |
| P31 | 0.67 ± 0.00 a | 97.00 ± 0.78 a | |
| 35 days | P1 | 0.09 ± 0.00 c | 30.25 ± 0.21 c |
| P1 + CKs | 0.18 ± 0.00 b | 91.50 ± 0.96 b | |
| P31 | 0.87 ± 0.00 a | 136.17 ± 1.54 a | |
| 42 days | P1 | 0.12 ± 0.00 c | 40.83 ± 0.29 c |
| P1 + CKs | 0.23 ± 0.00 b | 81.00 ± 0.09 b | |
| P31 | 0.81 ± 0.01 a | 225.08 ± 1.43 a | |
| 49 days | P1 | 0.22 ± 0.00 c | 71.75 ± 2.55 c |
| P1 + CKs | 0.36 ± 0.02 b | 109.92 ± 1.05 b | |
| P31 | 0.91 ± 0.01 a | 184.08 ± 1.78 a | |
Vertical comparison. The data are represented as mean values ± standard error (with three replicates). The value is the data of year 2023. Values with the same letters are not significantly different at the 5% level.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
MDPI and ACS Style
Yao, Y.; Xue, Y.; Yan, J.; Tang, X.; Cao, D.; He, W.; Luan, X.; Liu, Q.; Zhu, Z.; Liu, X. Exogenous Cytokinins Regulate Nitrogen Metabolism in Soybean Under Low Phosphorus Stress. Agronomy 2025, 15, 1459. https://doi.org/10.3390/agronomy15061459
AMA Style
Yao Y, Xue Y, Yan J, Tang X, Cao D, He W, Luan X, Liu Q, Zhu Z, Liu X. Exogenous Cytokinins Regulate Nitrogen Metabolism in Soybean Under Low Phosphorus Stress. Agronomy. 2025; 15(6):1459. https://doi.org/10.3390/agronomy15061459
Chicago/Turabian StyleYao, Yubo, Yongguo Xue, Jun Yan, Xiaofei Tang, Dan Cao, Wenjin He, Xiaoyan Luan, Qi Liu, Zifei Zhu, and Xinlei Liu. 2025. "Exogenous Cytokinins Regulate Nitrogen Metabolism in Soybean Under Low Phosphorus Stress" Agronomy 15, no. 6: 1459. https://doi.org/10.3390/agronomy15061459
APA StyleYao, Y., Xue, Y., Yan, J., Tang, X., Cao, D., He, W., Luan, X., Liu, Q., Zhu, Z., & Liu, X. (2025). Exogenous Cytokinins Regulate Nitrogen Metabolism in Soybean Under Low Phosphorus Stress. Agronomy, 15(6), 1459. https://doi.org/10.3390/agronomy15061459
Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.
