The influence of combined carbon and nitrogen application on the growth and metabolism of Ilex rotunda

Daoliang Yan; Shuxian Zhao; Yuxi Chen; Chenyi You; Jiaxin Hu; Bingsong Zheng

doi:10.1186/s12870-026-08722-4

. 2026 Apr 11;26:874. doi: 10.1186/s12870-026-08722-4

The influence of combined carbon and nitrogen application on the growth and metabolism of Ilex rotunda

Daoliang Yan ^1,^✉, Shuxian Zhao ¹, Yuxi Chen ¹, Chenyi You ², Jiaxin Hu ¹, Bingsong Zheng ¹

PMCID: PMC13181908 PMID: 41965533

Abstract

When cultivating Ilex rotunda Thunb., the blind application of nitrogen fertilizer often resulted in poor growth and negatively impacted yield and quality. To explore the balancing effect of carbon fertilizer on excessive nitrogen fertilizer and improve the yield and quality of I. rotunda, two-year-old cuttings were sprayed with different concentrations of urea and α-ketoglutarate (α-KG) in 12 treatment groups. After 12 weeks of treatment, the growth, photosynthetic indexes, main physiological indexes and metabolome of I. rotunda were determined and analyzed. The results showed that applying appropriate concentrations of α-KG and urea to I. rotunda enhanced its photosynthetic performance, evidenced by increased net photosynthetic rate and stomatal conductance, reduced intercellular carbon dioxide concentration, and improved specific leaf area of I. rotunda. The activity of photosystem Ⅱ (PS Ⅱ) was enhanced. The activities of enzymes related to GS-GOGAT cycle were increased, and the co-accumulation of carbon and nitrogen metabolites was promoted; it effectively mitigated the adverse effects of excessive nitrogen on the plant. The application of 4 g·L⁻¹ urea + 40 mg·L⁻¹ α-KG (N2KG1) and 4 g·L⁻¹ urea + 80 mg·L⁻¹ α-KG (N2KG2) increased the levels of α-KG and citric acid. N2KG1 and N2KG2 had the most positive effects on the photosynthetic performance, physiological and biochemical indexes, and accumulation of carbon and nitrogen metabolites in I. rotunda leaves. Therefore, the exogenous application of appropriate concentrations of α-KG and urea can promote the growth of I. rotunda, enhance its adaptability to environmental conditions, and improve plant quality. This provides a theoretical basis for cultivating high-quality I. rotunda specimens.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-026-08722-4.

Keywords: I. rotunda, α-ketoglutarate, High nitrogen stress, Physiological and biochemical indexes, Metabolome

Introduction

Ilex rotunda Thunb., belonging to family Aquifoliaceae, is an evergreen tree. It is widely distributed in southern provinces of the Yangtze River Basin in China. It is resistant to barren soil, drought, and frost. It often grows in evergreen broadleaf forests, along forest edges, and near streams at altitudes between 400 and 1,100 meters. The leaves and stems of I. rotunda are rich in triterpenoids, flavonoids, saponins and other medicinal active ingredients [1]. Modern medicine believes that I. rotunda has various pharmacological effects and is effective in treating cardiovascular diseases, lowering lipids, protecting the liver, and combating tumors, inflammation, and bacteria [2–4].

Currently, nitrogen fertilizer is widely used in agriculture. However, the blind pursuit of higher crop yields has resulted in the excessive and unscientific application of nitrogen fertilizer. This imbalance in carbon and nitrogen metabolism seriously affects crop yield and quality. Meanwhile, the carbon element, which is also necessary for plant growth and development, has not received enough attention. Carbon deficiency in plants is the key factor that limits the full utilization of nitrogen and then affects plant yield and quality [5]. Plants mainly rely on carbon dioxide (CO₂) in the atmosphere to provide carbon sources in their natural growth. Currently, the main way to supplement carbon in vegetables grown artificially is to apply CO₂, a gaseous carbon fertilizer, in relatively closed environments, but it is not yet possible to replicate the use of CO₂ in the production of field crops, and increased application of CO₂ can seriously affect the quality of the crops [6].

Studies have shown that applying organic carbon fertilizer to plants can promote the accumulation of nitrogen in their bodies, and its effect is better than that of applying nitrogen fertilizer to plants. The reason is that organic carbon plays an important role in the nitrogen balance of plants [7, 8]. Excessive application of nitrogen to plants will lead to insufficient carbon skeleton in the body to synthesize various organic substances, which will affect the absorption and transformation of nitrogen. The supplement of organic carbon can promote the synthesis of amino acids, peptides, proteins and other organic matter, reduce the accumulation of inorganic nitrogen in plants, and promote the absorption and assimilation of nitrogen by crops.

In the process of daily planting and production, the commonly used carbon fertilizers can be divided into organic carbon fertilizers and inorganic carbon fertilizers. Among them, organic carbon fertilizer refers to organic carbon compounds that can be absorbed and utilized by plants, such as sugars, alcohols, acids, etc. In the process of supplementing carbon to plants, compared with inorganic carbon fertilizer, carbon fertilizer has higher water solubility, higher absorption efficiency, and more significant effect on promoting plant growth [9].

α-KG can be used as a new type of organic carbon fertilizer. It has been reported in the literature that spraying α-KG on wheat seedlings with low nitrogen treatment can increase the nitrogen assimilation of wheat seedlings [10]. Gong et al. reported that spraying appropriate concentrations of α-KG on soybean leaves under low nitrogen increased the total nitrogen fixation capacity of soybean nodules, promoted the accumulation of dry matter in soybeans, and improved the quality of soybeans [11]. And our previous research showed that applying a certain amount of organic carbon fertilizer α-KG to Dendrobium officinale can effectively alleviate the carbon supply shortage of plants, maintain the normal carbon and nitrogen metabolism balance of plants, and thus achieve the effect of improving crop quality and yield, which cannot be achieved by applying gas fertilizer CO₂ [12].

Therefore, to develop scientific and reasonable fertilization strategies, maintain the balance of carbon and nitrogen metabolism in I. rotunda, and improve its yield and quality, we plan to study the effects of different combinations of carbon and nitrogen treatments on its growth, main physiological indexes, and carbon and nitrogen metabolism. This will clarify the effects of α-KG and nitrogen on its main physiological indexes and growth, the balance effect of organic carbon fertilizer on nitrogen fertilizer and provide a new nutrient management scheme to improve its yield and quality.

Materials and methods

Test materials and treatment

The plant material used in this study are 2-year-old I. rotunda cutting seedling (approximately 25–30 cm tall), which was cultivated by the experimenters and sourced from the Ilex nursery of Zhejiang A&F University. It was identified by plant classification experts Yan Daoliang and Lou Luhuan. Test seedlings with consistent growth were selected and planted in plastic pots with a diameter of 30 cm and a height of 40 cm. The potting medium consists of a 3: 1 mixture of peat soil and river sand by volume. After a 14-day acclimatization period in the plastic pots, the test seedlings were randomly divided into 12 groups, with 30 seedlings in each group. From April to July 2024, the I. rotunda plants underwent carbon and nitrogen fertilization treatments in a greenhouse maintained at a temperature of (25 ± 3)℃. The treatment groups and methods are detailed in Table 1. The 12 treatment groups were divided into four groups: no nitrogen fertilizer treatment group (CK, KG1, KG2), low nitrogen concentration treatment group (CK, N1, N1KG1, N1KG2), medium nitrogen concentration treatment group (CK, N2, N2KG1, N2KG2) and high nitrogen concentration treatment group (CK, N3, N3KG1, N3KG2). During the treatment, carbon and nitrogen treatments were alternated, carbon and nitrogen treatments were performed once a week, for a total of 12 weeks, carbon and nitrogen combined application treatments for a total of 12 times. Spraying was applied to both the upper and lower surfaces of the leaves until small droplets naturally dripped off the leaf surface. Leaf samples were taken 12 weeks after the combined fertilization treatment. The collected leaves were quickly frozen in liquid nitrogen, and some were stored in a – 80℃ refrigerator for the measurement of carbon and nitrogen metabolism-related enzyme activities. Other leaves were washed with distilled water and dried in an 80℃ oven until they reached a constant weight. The dried samples were crushed using a high throughput tissue grinder (DHS LifeScience & Technology Co.,Ltd, China) for the measurement of various physiological indicators. Non targeted metabolomics analysis of I. rotunda was performed on five independent biological replicates, and all other experiments were performed on three independent biological replicates, each consisting of three seedlings, forming a pooled sample. The remaining plants were used as spare plants.

Table 1.

Carbon and Nitrogen Co-application Treatment for I. rotunda

Treatment	Spraying Methods
CK	Spraying tap water as control
KG1	Spraying 40 mg·L⁻¹ α-KG
KG2	Spraying 80 mg·L⁻¹ α-KG
N1	Spraying 2 g·L⁻¹ urea
N1KG1	Spraying 2 g·L⁻¹ urea + 40 mg·L⁻¹ α-KG
N1KG2	Spraying 2 g·L⁻¹ urea + 80 mg·L⁻¹ α-KG
N2	Spraying 4 g·L⁻¹ urea
N2KG1	Spraying 4 g·L⁻¹ urea + 40 mg·L⁻¹ α-KG
N2KG2	Spraying 4 g·L⁻¹ urea + 80 mg·L⁻¹ α-KG
N3	Spraying 8 g·L⁻¹ urea
N3KG1	Spraying 8 g·L⁻¹ urea + 40 mg·L⁻¹ α-KG
N3KG2	Spraying 8 g·L⁻¹ urea + 80 mg·L⁻¹ α-KG

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Determination of specific leaf area of blade

After sampling, the blade surface shall be wiped clean, and the fresh weight of the blade shall be measured immediately with a thousandth electronic balance (JA5003B, TECHCOMP, China). After that, the leaf surface area (cm²) was measured with a leaf area meter (YMJ-CH, TOP Cloud-agri, China). Then Put the blade in an oven, dry it to constant weight, and weigh its dry weight (g) with a electronic balance. Specific leaf area = blade surface area/blade dry weight.

Determination of photosynthetic parameters

The net photosynthetic rate, intercellular carbon dioxide concentration, stomatal conductance and transpiration rate of I. rotunda leaves were measured by Li-6800 portable photosynthesis instrument (LI-COR Biosciences, USA). The measurement time is 8:00–11:00 a.m. The parameters in the leaf chamber are set as follows: the CO₂ concentration is 400 µmol·mol⁻¹, the air flow rate is 500 µmol·s⁻¹, the pressure value is 0.2 kPa, the relative humidity is 50%, the temperature is 28℃, the ratio of red to blue light is 9:1, and the photosynthetic effective radiation intensity is 1,200 µmol·m⁻²·s⁻¹.

Determination of chlorophyll fluorescence parameters

YZQ-500 chlorophyll dynamic fluorescence instrument (Yi Zong Qi Technology [Beijing] Co., Ltd, China) was used to determine the chlorophyll fluorescence induction kinetics curve of I. rotunda leaves; the fluorescence kinetic parameters were calculated: the maximum photochemical quantum yield of PSⅡ ( Inline graphic ), the maximum quantum yield of non-photochemical quenching (), the quantum yield of electron transport (), and the maximum quantum yield of non-photochemical quenching ().

Formula:

Determination of soluble sugar, starch and soluble protein content

The content of soluble sugar and starch was determined by anthrone method [13]. Firstly, the standard curve of soluble sugar was drawn (the required reagents and their amounts are shown in Table S1). Then, 0.2 g dry leaf sample was weighed and placed in a test tube, 15 mL of Reverse osmosis water (RO water) was added and continuously heated in a boiling water bath for 30 minutes to extract the soluble sugar in the sample. The cooled sample extraction solution was filtered, and the filtered extraction solution was placed in a 100 mL volumetric flask. After that, the residue was washed several times with RO water and finally adjusted to a constant volume at the scale line. Finally, 1 mL sample extraction solution was added with 5 mL anthrone reagent, and the absorbance was measured at the wavelength of 620 nm, and the soluble sugar content was calculated according to the standard curve.

The starch in the leaves was hydrolyzed by acid and converted into soluble sugar to prepare the extraction solution: After soluble sugar was extracted, 0.1 g of dry sample residue of leaves was taken into a centrifuge tube, 1 mL of 52% perchloric acid was added, and the shaker was shaken for 30 minutes. After centrifugation at 8000 Revolutions Per Minute (rpm) for 15 minutes, the supernatant was transferred to a 100 mL volumetric flask. After that, the soluble sugar content was determined by the above anthrone method, and the starch content was calculated according to the soluble sugar content.

The content of soluble protein was determined by Coomassie brilliant blue method [14]. First, the standard curve of soluble protein was drawn (the required reagents and their amounts are shown in Table S2). RO water (0.9 mL) and sample extraction solution (0.1 mL) were added to the test tube, followed by 5 mL of Coomassie brilliant blue G-250 reagent, fully mixed, and let stand for 2 minutes. The absorbance was measured at 595 nm and recorded. The protein content was obtained by the standard curve of soluble protein.

Determination of nitrogen content

After drying and grinding the leaves into powder, weigh 0.1 g of the sample and determine the nitrogen content of the leaves by Kjeldahl method [15].

Determination of GS, ferredoxin-dependent glutamate synthase (Fd-GOGAT EC1.4.7.1) and NADH-glutamate synthase (NADH-GOGAT EC 1.4.1.14) activities

GS, Fd-GOGAT and NADH-GOGAT activity were determined with the corresponding kit (Suzhou Michy Biomedical Technology Co., Ltd, China).

Determination of antioxidant enzyme activity

During the determination, 0.2 g of I. rotunda leaves were placed in a pre-cooled mortar, and 2 mL of pre-cooled 50 mmol·L⁻¹ phosphate buffer with pH 7.8 was added to grind into a slurry, and buffer was added to rinse the mortar. The final volume was 8 mL, centrifuged at 10,000 rpm for 15 min at 4℃, and the supernatant was the enzyme extracting solution. The enzyme extracting solution was stored at 0℃ – 4℃ for determination of superoxide dismutase (SOD EC1.15.1.1) activity and peroxidase (POD EC1.11.1.7) activity.

The activity of POD was determined by guaiacol method [16]. Firstly, the POD reaction solution was prepared: 100 mL phosphate buffer solution (0.05 mol·L⁻¹, pH 6.0), 100 mL RO water, 100 μL guaiacol and 100 μL 30% H₂O₂ were placed in beakers in turn and then mixed and transferred to the reagent bottle. After that, 0.1 mL of enzyme solution was drawn into a colorimetric dish, 3 mL of POD reaction solution was added, and the reaction solution without enzyme extracting solution was used as a blank control. The absorbance value at the wavelength of 470 nm was read immediately, read once every 15 s, and the absorbance value in the first 3 minutes of the reaction was measured. Each treatment group was repeated three times, and the data obtained were averaged. The calculation method is as follows:

In the formula:

The activity of SOD was determined according to the method of Beauchamp and Fridovich [17]. The calculation method is as follows:

In the formula:

Metabonomic determination and analysis

After 12 weeks of treatment, the leaf samples of I. rotunda in CK, N2, N2KG1 and N2KG2 groups were collected and stored in the −80℃ ultra-low temperature refrigerator after quick freezing in liquid nitrogen. Take 100 mg of crushed leaf tissue sample, put it into EP tube, and add 500 μL of 80% methanol aqueous solution; vortex oscillation, ice bath standing for 5 min, set at 15,000 g, centrifugation at 4℃ for 20 min; take a certain amount of supernatant and dilute it with mass spectrometry grade water until the methanol content is 53%; After centrifugation at 15,000 g and 4℃ for 20 min, the supernatant was collected and analyzed with LC-MS.

CD 3.1 (Compound Discoverer 3.1) Soku software is used to process the offline data file. First, each metabolite is simply screened, including retention time, mass charge ratio and other parameters. Then, different samples were aligned by setting the retention time deviation of 0.2 minutes and the mass deviation of 5 ppm to improve the accuracy of identification. Then, the information such as mass deviation of 5 ppm, signal strength deviation of 30%, signal-to-noise ratio of 3, minimum signal strength and additive ions were used for peak extraction, and the peak area was quantified. Then, the target ions were integrated, and the molecular formula was predicted by molecular ion peaks and fragment ions, and compared with mzcloud, mzvault and masslist databases. The blank sample was used to remove the background ions, and the original quantitative results were standardized. Finally, the identification and relative quantitative results of metabolites were obtained. The data processing part is based on Linux operating system (CentOS version 6.6) and R, python software. The identified metabolites were annotated using KEGG database.

Data processing

One-way analysis of variance (ANOVA) and Duncan’s multiple range test were used to analyze the differences in the mean physiological data between treatment groups within each of the four groups. Excel, Origin 2022 and SPSS 2022 were used for data analysis and processing. Statistical significance is defined as P ≤ 0.05. The results were reported as mean ± standard error (SE) based on at least three biological replicates.

Results

Effects of combined application of carbon and nitrogen on the photosynthetic performance of I. rotunda

Effects of combined application of carbon and nitrogen on specific leaf area of I. rotunda

Under different nitrogen concentrations, 2 g·L⁻¹ and 4 g·L⁻¹ urea treatments could promote the increase of specific leaf area of I. rotunda, and the effect of 4 g·L⁻¹ urea treatment was better. Compared with CK treatment, N2KG1 and N2KG2 treatment groups significantly increased the specific leaf area of I. rotunda by 10.94% and 11.80%, respectively. The treatment of high nitrogen concentration (8 g·L⁻¹ urea) could not improve the specific leaf area of I. rotunda. Under different carbon concentrations, KG1 and KG2 treatments could promote the increase of specific leaf area of I. rotunda, and the effect of KG2 treatment was better, and its specific leaf area was significantly higher than that of CK (P<0.05), increased by 8.02%. In addition, the effect of combined applications of carbon and nitrogen was better than that of single application of carbon and nitrogen. N2KG2 was the best treatment among the 12 treatments (Fig. 1).

Fig. 1 — Changes in specific leaf area of *I. rotunda* under different carbon and nitrogen co-application conditions. Group1: No nitrogen. Group2: Low nitrogen. Group3: Medium nitrogen. Group4: High nitrogen. CK, KG1, KG2, N1, N2 and N3 refer to spraying tap water, 40 mg·L⁻¹ α-KG, 80 mg·L⁻¹ α-KG, 2 g·L⁻¹ urea, 4 g·L⁻¹ urea and 8 g·L⁻¹ urea. The CK treatment group underwent differential comparisons with other treatment groups within each of the four groups, hence the CK group appears four times in the figure. The data mean ± SE(n=3). The different lowercase letters indicate significant differences between the treatment groups within each of the four groups (P<0.05). Whiskers above the bars indicate the SE

Effects of combined application of carbon and nitrogen on photosynthetic parameters of I. rotunda

The photosynthetic parameters of I. rotunda were different with different treatments of carbon and nitrogen. Among them, the combination of carbon and nitrogen (especially N2KG1 treatment) has a more significant role in promoting the photosynthetic performance of I. rotunda, improving the net photosynthetic rate (Pn) and stomatal conductance (gs) of I. rotunda, and reducing the intercellular carbon dioxide concentration (Ci), which is more effective than applying organic carbon fertilizer or organic nitrogen fertilizer alone. The four photosynthetic parameters of N2KG1 treatment showed the best results, and its net photosynthetic rate was 41.48% higher than that of the control. In addition, high nitrogen treatment had a significant inhibitory effect on Photosynthesis of I. rotunda, which began to appear from the fourth week after treatment (Figure S1). Under the treatment of N3KG1 and N3KG2, the net photosynthetic rate and stomatal conductance of I. rotunda were significantly higher than those of N3 treatment, and the intercellular carbon dioxide concentration was significantly lower than that of N3 treatment (Fig. 2), indicating that exogenous carbon application alleviated the inhibition of nitrogen excess on Photosynthesis of I. rotunda to a certain extent.

Fig. 2 — Changes in photosynthetic parameters of *I. rotunda* under different carbon and nitrogen co-application conditions. A Changes in net photosynthetic Rate of *I. rotunda*. B Changes in transpiration rate of *I. rotunda*. C Changes in intercellular carbon dioxide concentration of *I. rotunda*. D Changes in stomatal conductance of *I. rotunda*. Group1: No nitrogen. Group2: Low nitrogen. Group3: Medium nitrogen. Group4: High nitrogen. CK, KG1, KG2, N1, N2 and N3 refer to spraying tap water, 40 mg·L⁻¹ α-KG, 80 mg·L⁻¹ α-KG, 2 g·L⁻¹ urea, 4 g·L⁻¹ urea and 8 g·L⁻¹ urea. The CK treatment group underwent differential comparisons with other treatment groups within each of the four groups, hence the CK group appears four times in each figure. The data mean ± SE (n=3). The different lowercase letters indicate significant differences between the treatment groups within each of the four groups (P<0.05). Whiskers above the bars indicate the SE

Effects of combined application of carbon and nitrogen on chlorophyll fluorescence parameters of I. rotunda

φPo represents the maximum photochemical efficiency of photosystem II, Ψo reflects the electron transfer efficiency caused by excitons captured by PS reaction center, φE_O represents the electron transfer efficiency of photosystem II, and φDo represents the efficiency of non-photochemical quenching. In the treatment group without nitrogen fertilizer, φPo, φE_O and ψo of KG1 and KG2 treatments were significantly higher than those of CK treatment, while φDo was significantly lower than that of CK treatment. In low nitrogen treatment group, φPo and Ψo of N1, N1KG1 and N1KG2 treatments were significantly higher than those of CK treatment, while φDo was significantly lower than that of CK treatment. The φE_O of N1, N1KG1 and N1KG2 treatments were significantly higher than that of CK treatment, and the φE_O of N1KG1 and N1KG2 treatments was significantly higher than that of N1 treatment. In the medium nitrogen treatment group, the φPo, φE_O and ψo of N2KG1 and N2KG2 treatments were significantly higher than those of CK treatment, and the φDo was significantly lower than that of CK treatment. Compared with CK treatment, φPo and ψo in N2 treatment were increased, but the difference was not significant. φE_O was significantly increased, and φDo was significantly decreased. In the high nitrogen treatment group, there was no significant difference in φPo and φDo among the treatments. The φE_O and Ψo of N3, N3KG1 and N3KG2 treatments were significantly lower than those of CK treatment (Fig. 3). The maximum photochemical quantum yield (φP_O) of PSⅡ in the optimal treatment N2KG1 was 22.39% higher than that in the CK treatment, indicating that the combined application of carbon and nitrogen could improve the activity of photosystem Ⅱ (PSⅡ).

Fig. 3 — Changes in chlorophyll fluorescence parameters of *I. rotunda* under different carbon and nitrogen co-application conditions. A Changes in φPo of *I. rotunda*. B Changes in φDo of *I. rotunda*. C Changes in φEo of *I. rotunda*. D Changes in ΨO of *I. rotunda*. Group1: No nitrogen. Group2: Low nitrogen. Group3: Medium nitrogen. Group4: High nitrogen. CK, KG1, KG2, N1, N2 and N3 refer to spraying tap water, 40 mg·L⁻¹ α-KG, 80 mg·L⁻¹ α-KG, 2 g·L⁻¹ urea, 4 g·L⁻¹ urea and 8 g·L⁻¹ urea. The CK treatment group underwent differential comparisons with other treatment groups within each of the four groups, hence the CK group appears four times in each figure. The data mean ± SE (n=3). The different lowercase letters indicate significant differences between the treatment groups within each of the four groups (P<0.05). Whiskers above the bars indicate the SE

Effects of combined application of carbon and nitrogen on important physiological indexes of I. rotunda leaves

Differences in soluble sugar, starch and soluble protein contents of I. rotunda leaves under different carbon and nitrogen fertilization conditions

It can be seen from Fig. 4A that the combined application of carbon and nitrogen has different degrees of influence on the content of soluble sugar in the leaves of I. rotunda. Under different nitrogen concentrations, N1 and N2 treatments could promote the accumulation of soluble sugar, and N2 treatment had better effect, while too high nitrogen concentration inhibited the accumulation of soluble sugar in I. rotunda. Under the treatment of different carbon concentrations, 40 and 80 mg·L⁻¹ α-KG could promote the accumulation of soluble sugar in I. rotunda, and the treatment of 80 mg·L⁻¹ α-KG had the best effect. In the combined application of carbon and nitrogen treatment, the soluble sugar content in the leaves of N2KG2 treatment was the highest, which was 62.98% higher than that of CK treatment. At the same time, compared with N2 treatment and KG2 treatment, the effect of N2KG2 treatment was also significantly better than the two treatments.

The starch content in the leaves of I. rotunda was correlated with different carbon and nitrogen treatments. Under the same nitrogen concentration treatment, 80 mg·L⁻¹ α-KG treatment had the most significant effect on the accumulation of I. rotunda starch. High nitrogen treatment had a certain inhibitory effect on the accumulation of starch content, and 80 mg·L⁻¹ α-KG had the most significant effect on the inhibition. N2KG2 was the best treatment, and its starch content was significantly higher than that of CK treatment (Fig. 4B).

The soluble protein content of each treatment group was significantly higher than that of CK treatment. In the single carbon treatment, the soluble protein content showed a certain upward trend with the increase of α-KG concentration. In the low nitrogen treatment group, the soluble protein contents of N1KG1 and N1KG2 treatments were significantly higher than that of CK and N1 treatments, and N1KG2 treatment performed best, with the soluble protein content of 11.02 mg·g⁻¹, 82.39% higher than that of CK treatment. In the medium nitrogen treatment group, the treatment effect of N2KG2 treatment was the best, and the soluble protein content reached 16.22 mg·g⁻¹, which was 168.45% higher than that of CK treatment. In the high nitrogen treatment group, the change trend of soluble protein content in I. rotunda was similar to that in the low nitrogen treatment. There was no significant difference in soluble protein content between N3KG1 and N3KG2 treatments, and they were significantly higher than those in CK and N3 treatments. The soluble protein content of N3KG2 treatment was the highest, reaching 16.10 mg·g⁻¹ (Fig. 4C).

Comprehensive analysis showed that N2KG1 and N2KG2 treatments significantly increased the contents of soluble sugar, soluble protein and starch in leaves of I. rotunda compared with CK treatment, and promoted the co-accumulation of carbon and nitrogen metabolites in plants.

Difference of total nitrogen content in leaves of I. rotunda under different combined application of carbon and nitrogen

It can be seen from Fig. 5 that in the treatments of applying nitrogen alone, the nitrogen content in the leaves of I. rotunda was significantly higher than that in the CK treatment. N1KG2, N2KG2 and N3KG2 in the combined application of carbon and nitrogen showed the best performance under all nitrogen concentrations, and their leaf nitrogen content showed an upward trend compared with N1, N2 and N3 treatments. Compared with N1 treatment, the nitrogen content of N1KG2 treatment increased by 34.11%, N2KG2 increased by 40.74% and N3KG2 increased by 48.95%. In the treatment group without nitrogen fertilizer, the leaf nitrogen content of KG1 and KG2 treatments were significantly higher than that of CK treatment, which increased by 29.23% and 31.42% respectively. The above results showed that the application of carbon fertilizer and nitrogen fertilizer could effectively improve the nitrogen content in the leaves of I. rotunda, and the effect of combined application of carbon and nitrogen was better than that of single application of carbon fertilizer and nitrogen fertilizer. In addition, with the increase of carbon fertilizer and nitrogen fertilizer concentration, the nitrogen content in the leaves of I. rotunda was also increased.

Difference of GS activity in leaves of I. rotunda under different combined application of carbon and nitrogen

It can be found from Fig. 6 that the GS activity in the leaves of I. rotunda was affected by the combined application of different carbon and nitrogen. In the treatment group without nitrogen application, the activities of GS in leaves treated with KG1 and KG2 were significantly higher than those treated with CK treatment. In the low nitrogen treatment group, the GS activity of 2 g·L⁻¹ urea combined with 40 mg·L⁻¹ and 80 mg·L⁻¹ α-KG treatment increased significantly compared with CK treatment, and the GS activity of N1KG2 treatment was the highest, reaching 14.07 μmol·min⁻¹·g⁻¹, which was 183.67% higher than CK treatment. In the medium nitrogen treatment group, the effect of 4 g·L⁻¹ urea combined with 80 mg·L⁻¹ α-KG treatment was the best, and the GS activity was 14.86 μmol·min⁻¹·g⁻¹, which was not significantly different from that of 40 mg·L⁻¹ α-KG treatment. In the high nitrogen treatment group, the change trend of GS activity was consistent with that in the medium nitrogen treatment group. The treatment with 8 g·L⁻¹ urea plus 80 mg·L⁻¹ α-KG had the best result, and the GS activity was 16.34 μmol·min⁻¹·g⁻¹, which was 229.44% higher than that in the CK treatment.

In conclusion, the change trend of GS activity in the groups without nitrogen, low nitrogen and medium nitrogen treatment was basically consistent with that of soluble sugar and protein content, but the change trend of GS activity in the high nitrogen treatment group was opposite to that of soluble sugar and protein content. Among the 12 treatments, N3KG2 treatment had the highest GS enzyme activity, but not N2KG1 and N2KG2 treatments.

Difference in activity of ferredoxin-dependent glutamate synthase (Fd-GOGAT) in leaves of I. rotunda under different combined application of carbon and nitrogen

It can be seen from Fig. 7 that both single carbon and combined application of carbon and nitrogen have an impact on the activity of Fd-GOGAT in the leaves of I. rotunda.

In the single carbon treatment group, the activity of Fd-GOGAT after KG1 and KG2 treatments was significantly higher than that of CK treatment, and the effect of KG2 treatment was better than that of KG1 treatment. The activity of Fd-GOGAT in the leaves of I. rotunda, and the activity of Fd-GOGAT in the combined application of carbon and nitrogen treatment group was also significantly higher than that in the single application of nitrogen treatment. Under the condition of low nitrogen treatment, the activity of Fd-GOGAT after N1KG2 treatment was the highest, which was 0.1557 μmol·min⁻¹·g⁻¹, significantly increased by 135.55% compared with CK. Under high nitrogen treatment, the activity of Fd-GOGAT in N3KG2 treatment was the highest, which was 0.2104 μmol·min⁻¹·g⁻¹, significantly increased by 218.31% compared with CK treatment. Under medium nitrogen treatment, the activity of Fd-GOGAT in N2KG1 treatment was the highest, reaching 0.1939 μmol·min⁻¹·g⁻¹. The activity of Fd-GOGAT in N2KG2 treatment was slightly lower than that in N2KG1 treatment, but slightly higher than that in N2 treatment, but the differences were not significant.

Difference of NADH-glutamate synthase (NADH-GOGAT) activity in leaves of I. rotunda under different combined application of carbon and nitrogen

It can be seen from Fig. 8 that the change trend of NADH-GOGAT activity in the leaves of I. rotunda is similar to that of GS. The application of 40 and 80 mg·L⁻¹ α-KG increased the NADH-GOGAT activity of I. rotunda. The effect of KG2 treatment was better than that of KG1 treatment. The NADH-GOGAT activity of KG2 treatment was 74.49% higher than that of CK treatment. In the low nitrogen treatment group, the NADH-GOGAT activity of N1KG2 treatment was 0.6436 μmol·min⁻¹·g⁻¹, which was 178.25% higher than that of CK treatment, but there was no significant difference. In addition, N1 treatment also made the NADH-GOGAT activity of I. rotunda plants significantly higher than that of CK treatment, which was 79.03% higher than that of CK treatment. The change trend of NADH-GOGAT activity under medium nitrogen treatment was consistent with that under low nitrogen treatment. At this time, the activity of NADH-GOGAT in N2 treatment was increased by 128.79% compared with CK treatment. The activity of NADH-GOGAT in N2KG2 combined application treatment was still the highest, reaching 0.7275 μmol·min⁻¹·g⁻¹, which was increased by 214.53% compared with CK treatment. In the high nitrogen treatment group, there was no significant difference in NADH-GOGAT activity among N3, N3KG1 and N3KG2 treatments, and the NADH-GOGAT activity of the three treatment groups was significantly higher than that of CK treatment. At this time, the enzyme activity under N3KG2 treatment was 202.25% higher than that of CK treatment.

Effects of combined application of different carbon and nitrogen on antioxidant enzyme activities of I. rotunda

It can be found from Fig. 9 that the activities of POD and SOD in leaves of I. rotunda in N3 treatment were significantly higher than those in CK treatment. There was no significant difference in the activities of POD and SOD between N1 and N2 treatment and CK treatment. The POD activity of N3KG1 treatment was still significantly higher than that of CK and N3 treatment, reaching 515.35 U·g⁻¹·min⁻¹, but the POD activity of N3KG1 treatment was significantly different from that of N3KG2 treatment at this time. The SOD activity of N3KG1 treatment was 479.23 U·g⁻¹·min⁻¹, and the SOD activity of N3KG2 treatment was significantly higher than that of N3KG1 treatment. The above results showed that the application of α-KG to I.rotunda plants under high nitrogen treatment could effectively improve the activities of POD and SOD in I. rotunda plants, and improve the ability of scavenging free radicals, so as to effectively alleviate the high nitrogen stress and enhance the stress resistance of plants. At the same time, with the extension of carbon and nitrogen treatment time, the activities of POD and SOD in each treatment group under high nitrogen treatment had an upward trend (Figure S2).

Non targeted metabolomics analysis of I. rotunda in different treatments of carbon and nitrogen fertilization

Total sample PCA analysis

Principal component analysis (PCA) is used to evaluate the overall distribution between all the tested samples and the reliability of the analysis process. It can be seen from Fig. 10 that there was a significant difference between the four treatment groups (P < 0.5), and the aggregation and repeatability of the samples in the group were good, indicating that the samples were reliable. The results of PCA analysis showed that the direction with the largest total data difference (PC1) explained 48.5% of the total data variability, the second direction of the total data difference (PC2) explained 16.6% of the total data variability, and the third direction of the total data difference (PC3) explained 12.4% of the total data variability. The above results indicate that there are significant differences in the enrichment of metabolites among the four groups of samples.

Fig. 10 — Principal component analysis (PCA) of *I. rotunda* samples

Cluster analysis of differential metabolites among different treatments of carbon and nitrogen fertilization

The cluster thermogram analysis of different metabolites showed that different treatments had a greater impact on the accumulation of carbon and nitrogen metabolites in I. rotunda, and it was obvious that there were differences in the accumulation of metabolites among different treatments. CK, N2, N2KG1 and N2KG2 were divided into four groups. N2KG1 and N2KG2 were far away from CK (Fig. 11), indicating that the metabolite difference between N1KG1, N2KG2 and CK was more significant than that between N2.

Fig. 11 — Hierarchical clustering heatmap of differential metabolites in I. rotunda

Differential metabolite analysis

The different metabolites are screened with the threshold values of VIP>1.0, FC>2.0 and FDR<0.05. After 12 weeks of treatment, the total number of metabolites with significant difference in the CK vs N2 comparison group is 321, of which the total number of up-regulated metabolites is 114 and the total number of down-regulated metabolites is 207. The total number of up-regulated and down-regulated metabolites in CK vs N2KG1 comparison group is 328, of which the total number of up-regulated metabolites is 100 and the total number of down-regulated metabolites is 228. The total number of differential metabolites in the CK vs N2KG2 comparison group is the largest, reaching 327. The total number of significantly increased metabolites is 69, and the total number of significantly decreased metabolites is 258. In the N2KG1 vs N2KG2 comparison group, a total of 208 significantly different metabolites are identified, with a total of 54 significantly up-regulated and 154 significantly down-regulated metabolites (Table 2).

Table 2.

Quantitative analysis of differential metabolites in I. rotunda under different treatments

Treatment	Total number of metabolite identifications	No. of metabolites with significant differences	No. of metabolites significantly up-regulated	No. of metabolites significantly down-regulated
CK vs N2	1,525	321	114	207
CK vs N2KG1	1,525	328	100	228
CK vs N2KG2	1,525	327	69	258
N2KG1vsN2KG2	1,525	208	54	154

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The identified differential metabolites were annotated to KEGG database. Among them, 221 different metabolites are distributed in metabolism, genetic information processing and environmental information processing (Fig. 12).

Fig. 12 — KEGG pathway annotation of differential metabolites in *I. rotunda*

By selecting the differential metabolites obtained from the differential comparison combination of CK vs N2KG1 and CK vs N2KG2 to draw a matchstick diagram, it can more clearly show the up and down regulation of metabolites and substances with large variation in differential multiples. The author transformed the fold change value of differential metabolites into logarithm based on 2 and took the top 20 metabolites of up and down for the display of matchstick diagram. Blue represents down regulation and red represents up regulation; the length of the rod represents the size of log2 (fold change); the size of the point represents the size of the VIP value. It can be seen from Fig. 13 that I. rotunda is affected by organic carbon and nitrogen fertilizer. A variety of glycosides and flavonoids with pharmacological activities are enriched in the leaves of I. rotunda and inhibit the synthesis of some terpenoids (dehydropachymic acid, pachymic acid, geranylgeraniol). Among the top 20 up-regulated metabolites in the CK vs N2KG1 comparison group, 13 metabolites have been proved to have pharmacological activities. These substances include flavonoids (linarin, robinin, tricin 5-o-hexoside, pelargonidin chloride), Glycosides (alpha hederin, ginsenoside RO, notoginsenoside R2, tubuloside A, saikosaponin E), Organic acids (salvianolic acid B and salvianolic acid D), Peptides (L-glutathione). 12 metabolites in the first 20 up-regulated metabolites of CK vs N2KG2 comparison group have also been proved to have pharmacological activities. These substances include flavonoids (C-pentosyl-apigenin O-p-coumaroylhexoside, tricin 5-o-hexoside, pelargonidin chloride, linarin), Glycosides (ginsenoside-Ro, rosamultin), Organic acids (testosterone undecanoate and salvianolic acid B). In addition, the contents of dulcitol, vincristine, miyabenol O and Glu-Gln (a dipeptide formed by the condensation of glutamic acid and glutamine) were increased. Previous studies have demonstrated that the application of exogenous flavonoids to plants subjected to abiotic stress can exert positive effects by inducing the expression of stress-responsive genes, thereby aiding plants in counteracting abiotic stress and maintaining normal growth and development. Among these interventions, the co-application of Robinin and chitosan triggers interactive mechanisms that enhance the plant’s drought resistance response, consequently improving plant performance under stress conditions. Additionally, certain organic acids participate in critical physiological processes such as mineral element loading in the xylem, maintenance of cellular pH and redox balance, enhancement of drought tolerance, and increased resistance to fungal pathogens.In addition, N2KG1 treatment resulted in a decrease in the content of malonic acid compared with the CK treatment. Malonic acid is a competitive inhibitor of succinate dehydrogenase and has a blocking effect on the TCA cycle, which indicates that the combined application of carbon and nitrogen promotes the TCA cycle to a certain extent. Since no more metabolites related to TCA cycle were found in the top20 differential metabolites among CK, N2KG1 and N2kKG2 treatments, we analyzed the original data and found two TCA cycle metabolic intermediates, α -KG and citric acid. According to the analysis in Fig. 14, the accumulation of α -KG and citric acid in I. rotunda plants under N2KG1 and N2KG2 treatments were significantly higher than those under CK treatment (P<0.001), and the content of citric acid in N2KG2 treatment was significantly higher than that under N2KG1 treatment (P<0.05), and the content of α-KG in N2KG2 treatment was significantly higher than that under N2KG1 treatment (P<0.01). The above results show that the combined application of organic carbon fertilizer and nitrogen fertilizer can affect the TCA cycle of plants, so as to promote the more efficient carbon and nitrogen metabolism of plants. It can also be seen from the analysis in Fig. 15 that the top 20 KEGG enrichment metabolic pathway of CK vs N2KG1 and CK vs N2KG2 includes the TCA cycle metabolic pathway and α-KG metabolic pathway, which on the other hand shows that the external application of organic carbon and nitrogen fertilizer has an impact on the carbon and nitrogen metabolism of I.rotunda.

Fig. 13 — Differential metabolite plot among different treatment groups of *I. rotunda.*A CK vs N2KG1. B CK vs N2KG2

Fig. 14 — Differences in intermediate metabolite levels of the tricarboxylic acid (TCA) cycle among different treatment groups in *I. rotunda*. A Citric acid. B α -KG. The black dot represents the mean value, the whisker line extends to the minimum and maximum values, and the black horizontal line represents the median (n=5).*, **and ***indicate significance at 0.05, 0.01 and 0.001 levels, respectively

Fig. 15 — KEGG enrichment factor plot of *I. rotunda* among different treatment groups. A CK vs N2KG1. B CK vs N2KG2

Analysis of pathways for enrichment of differential metabolites

The KEGG Top20 metabolic pathway of CKvsN2KG1 and CKvsN2KG2 differential metabolites was analyzed. It was found that the combined application of carbon and nitrogen affected the α-KG metabolic pathway of I. rotunda. Based on the above findings, we imported the original data into KEGG mapper website to draw the α-KG metabolic pathway map of I. rotunda in different treatment groups (CK vs N2KG1 and CK vs N2KG2). The circle represents metabolites, in which the red circle is marked as up-regulated differential metabolite, the green circle is marked as down-regulated differential metabolite, and the hollow circle is marked as no significant difference metabolite or unidentified metabolite. It can be seen from the analysis in Fig. 16 that the content of aspartic acid in I. rotunda leaves in N2KG1 treatment was lower than that in CK, while the content of citric acid, α-KG and glutamate in I. rotunda leaves in N2KG1 treatment was higher than that in the control treatment. Under N2KG2 treatment, the contents of aconitic acid and aspartic acid were decreased, while the contents of citric acid, α-KG and glutamate were increased.

Fig. 16 — Schematic diagram illustrating the enriched metabolic pathways of *I. rotunda* between different treatment groups. A CK vs N2KG1. B CK vs N2KG2

Discussion

Effects of combined application of different carbon and nitrogen on photosynthetic performance of I. rotunda

The change of plant specific leaf area is mainly affected by nutrients in the environment [18]. At the same time, the value of specific leaf area also reflects the light capture ability and competitive activity of plants [19, 20]. The results showed that the specific leaf area of I. rotunda treated with carbon and nitrogen fertilizer was significantly increased compared with the control, and N2KG2 treatment was the best. Bai et al. and Zhou et al. found that fertilization on Leymus chinensis significantly increased its specific leaf area, which is consistent with the results of this study [21, 22]. The research of Sun et al. showed that applying an appropriate concentration of organic carbon fertilizer to Paeonia suffruticosa can significantly improve its net photosynthetic rate, thus improving the photosynthetic efficiency and the quality of the used parts of the plant [23]. Yang et al. found that the net photosynthetic rate and other photosynthetic parameters of BlackBerry could be improved to some extent by applying nitrogen fertilizer of appropriate concentration, which indicated that the photosynthetic performance of Blackberry was indeed improved by applying nitrogen fertilizer of appropriate concentration [24]. The content of photosynthetic pigment and photosynthetic parameters of BlackBerry decreased significantly when the concentration of nitrogen fertilizer was too high. The above results are basically consistent with the results of this study. Chlorophyll fluorescence parameters not only reflect the activities of photosystem II and photosystem I, but also comprehensively reflect the photosynthetic state and performance of plants [18]. The results showed that N2KG1 treatment increased the maximum photochemical efficiency and the quantum yield for electron transfer in the PSⅡ reaction center after dark adaptation of I. rotunda, and thus increased the PSⅡ activity of I. rotunda. The results of Li et al. showed that the maximum photochemical efficiency and other fluorescence parameters of Gleditsia sinensis seedlings were improved when an appropriate concentration of nitrogen fertilizer was applied, indicating that the activity and light energy conversion efficiency of photosystem II were improved, but the application of excessive concentration of nitrogen reduced the activity of photosystem II of Gleditsia sinensis seedlings [25]. Zhang et al. found that the application of appropriate concentration of gaseous carbon fertilizer CO₂ to melon improved the electron transfer capacity of photosystem II, reduced NPQ and heat dissipation, thereby enhancing the photosynthetic performance of the plant [26]. The above results are consistent with this study.

Effects of different combined application of carbon and nitrogen on physiological indexes and carbon and nitrogen metabolism of I. rotunda

As an important metabolic intermediate in the tricarboxylic acid cycle (TCA cycle), α-KG is a key node connecting carbon and nitrogen metabolism in cells. α-KG is a precursor of glutamine, glutamic acid and other important amino acids. It is an organic acid necessary for plants to assimilate NO− 3and NH+ 4. After NO− 3 is absorbed into plants, it is converted into NH+ 4 through nitrate reductase (NR) and nitrite reductase (NiR), and NH+ 4 is further assimilated through the GS-GOGAT cycle [27]. Glutamine synthase (GS EC6.3.1.2) and glutamate synthase (GOAGT EC2.6.1.53) play a synergistic role in the GS-GOGAT cycle and are key enzymes in the nitrogen metabolism pathway of plants. GS catalyzes the synthesis of glutamine from glutamic acid and NH+ 4, which is the first step of nitrogen assimilation. Then GOGAT catalyzes the transfer of the amide group of glutamine to α-ketoglutaric acid to produce glutamate. Glutamate, as a key substrate for amino acid and protein synthesis, is a key compound for nitrogen assimilation [28]. The increase in the activity of key enzymes GS and GOAGT during nitrogen assimilation means that plants increase the efficiency of nitrogen assimilation. Leng et al. overexpressed GS in hybrid poplar under high NH+ 4 stress, and the genes related to amino acid biosynthesis, sucrose and starch degradation, galactose metabolism and antioxidant system in roots were significantly up-regulated, which indicated that GS was helpful to maintain the balance between carbon and nitrogen metabolism and alleviate the symptoms of ammonia poisoning [29].

In cell metabolism, the production and decomposition of α-KG involve various metabolic pathways. In the TCA cycle, isocitrate is oxidized and decarboxylated under the action of isocitrate dehydrogenase to produce α-KG, which can also be decarboxylated into α-KG, succinyl-CoA and carbon dioxide (CO₂) by α-KG dehydrogenase (a key enzyme in the TCA cycle). Glutamate can generate α-KG by glutamate dehydrogenase deamination or transamination [30]. In the metabolic process of the body, α-KG can directly participate in the conversion of TCA cycle into CO₂ without the preparation of glycolytic pathway and TCA cycle, providing energy for cells and improving the metabolic efficiency of the body. At the same time, it can also reduce the loss of glutamine and glutamic acid and indirectly increase the deposition of protein [31].

Soluble sugars play an important role in plant physiological processes, not only providing energy for plant metabolism, but also providing carbon skeleton of plant structural substances [32]. As the main energy storage material of plants, starch also plays an important role in the growth and development of plants [33]. In this study, the application of appropriate concentration of carbon fertilizer, nitrogen fertilizer and the combined application of carbon and nitrogen can significantly improve the soluble sugar content and starch content in the leaves of I. rotunda. At the 12th week of treatment, the content of soluble sugar and starch in leaves of I. rotunda in N2KG2 treatment was the highest, which may be due to the improvement of photosynthesis and the promotion of soluble sugar and starch synthesis of I. rotunda in the combination of carbon and nitrogen. Secondly, the application of urea and α-KG may improve the carbon metabolism of plants, thus directly increasing the content of soluble sugar and starch. Otálora et al. found in their research on Sonchus oleraceus that the soluble sugar content in S. oleraceus increased first and then decreased with the increase of nitrogen concentration sprayed on leaves [33]. Chung et al. showed that the application of high concentration urea to Lycium barbarum could reduce the soluble sugar content in L. barbarum leaves, but the application of carbon and nitrogen organic fertilizer could significantly improve the soluble sugar content in L. barbarum leaves [34]. The above results are consistent with the results of this study.

Soluble protein plays an important role in plant growth and development, nutrition supply, stress resistance and other aspects, and is one of the key factors to maintain the healthy growth of plants [35, 36]. In this study, the combined application of carbon and nitrogen fertilizer promoted the accumulation of soluble protein in I. rotunda, and the change trend of leaf nitrogen content was basically the same as that of soluble protein content in I. rotunda. This may be caused by the increase of nitrogen concentration in the growth environment of I. rotunda, but we found that the effect of nitrogen application alone on the accumulation of soluble protein and leaf nitrogen content of I. rotunda was worse than that of the combined application of carbon and nitrogen. Therefore, we speculate that the application of α-KG can improve the nitrogen use efficiency of I. rotunda, resulting in the increase of nitrogen and soluble protein content in plants. Cruz et al. showed that the increase of CO₂ concentration in the atmosphere improved the nitrogen fixation ability of papaya seedlings, which proved our previous scientific hypothesis [37]. Moreover, the enhancement of GS-GOGAT cycle further promotes the synthesis of amino acids such as glutamate in I. rotunda plants, which is consistent with the research results of Liu et al. [38].

In this study, the activities of metabolic enzymes related to carbon and nitrogen metabolism were affected by the application of α-KG, and the activities of GS and GOGAT enzymes were significantly increased. Zhang et al. have shown that the application of appropriate concentrations of organic acids to Capsicum annuum effectively improves the activities of GS enzyme and GOGAT enzyme in C. annuum [39]. Pang et al. also found that the activities of GS and GOGAT enzymes in Brassica campestris increased after applying organic carbon fertilizer of appropriate concentration [40]. The above results are consistent with the experimental results. As a key pathway of nitrogen assimilation, GS-GOGAT cycle mainly converts NH+ 4 into amino acids such as glutamate. The increase of GS and GOGAT enzyme activities indicated the enhancement of plant nitrogen use ability and amino acid synthesis. On the other hand, the urea sprayed on the leaves of I. rotunda in this study also increased the activities of GS and GOGAT enzymes, which may be because urea was directly absorbed by the leaves and decomposed into NH+ 4 by urease in the cytoplasm, which increased the content of NH+ 4 in the plant, thus improving the activities of GS and GOGAT enzymes [41, 42], which is consistent with the results of Chen et al. [43]. Therefore, the GS and GOGAT enzyme activities of I. rotunda leaves under the combined application of carbon and nitrogen were higher than those under the single nitrogen or single carbon treatment, which may be due to the synergistic effect of carbon fertilizer and nitrogen fertilizer.

High nitrogen stress will affect plant photosynthesis and inhibit plant growth [44, 45]. Xun et al. found that NH+ 4 as the only nitrogen source of tomato (Solanum lycopersicum) can inhibit plant growth, especially when the concentration of NH+ 4 is too high [46]. Vega-Mas et al. found that increasing carbon supply can effectively alleviate the ammonium stress on tomatoes and promote the synthesis and accumulation of amino acids [47]. In this study, excessive urea application on I. rotunda also affected the accumulation of plant carbon metabolites. This may be due to the high content of NH+ 4 in the plant after excessive urea is applied. In order to assimilate this part of ammonium, excessive carbon skeleton is consumed, resulting in carbon deficiency, which affects the normal carbon and nitrogen metabolism of the plant [48]. Otálora et al. found in the study of chicory that the application of high concentration of urea resulted in the increase of soluble protein content and the decrease of soluble total sugar content in chicory [33]. Hodges believes that the deficiency of α-KG in plants is the direct reason for the decline of ammonium assimilation efficiency [49]. In this study, after α-KG was applied to I. rotunda, the accumulation of carbon and nitrogen metabolites in the plant was significantly increased compared with the control, and the activities of enzymes related to carbon and nitrogen metabolism were also increased compared with the control, which effectively alleviated the effect of nitrogen stress and effectively confirmed Hodges’ conjecture. In addition, Wu et al. and Sun et al. also found that applying α-KG and other organic carbon fertilizers to plants can alleviate low temperature, weak light and salt stress [50, 51]. The analysis of antioxidant enzyme activities in the leaves of I. rotunda showed that high nitrogen treatment increased the activities of SOD and POD in the plant, and there was no significant change in the activities of SOD and POD under low nitrogen and medium nitrogen treatment compared with the control. It further shows that excessive application of nitrogen fertilizer has produced stress on I. rotunda, which is consistent with the research results of Xu et al. [52]. The increase of POD and SOD activity may be due to the damage of plant cell membrane caused by high nitrogen concentration applied to the plant, the destruction of cell structure and the increase of peroxide content [53]. SOD and POD are important antioxidant enzymes in plants. When plants are stressed, the improvement of their activities is conducive to improving the ability of plants to scavenge their own peroxides. In this study, the activities of SOD and POD in the combined application of carbon and nitrogen in the high nitrogen group were significantly higher than those in the N3 group, which indicated that the application of α-KG could further improve the ability of scavenging peroxide of I. rotunda, so as to enhance the resistance of plants. Zhang et al. found that increasing the CO₂ concentration in the tomato growth environment can improve the activities of POD and SOD of tomato and improve the plant resistance [54]. This is consistent with the experimental results.

Through non-targeted metabolomics analysis, it was found that among the top 20 up-regulated metabolites in the CK vs N2KG1 comparison group, 13 metabolites were substances that have been proved to have pharmacological activity. Among the top 20 up-regulated metabolites in the CK vs N2KG2 comparison group, 12 metabolites have been shown to have pharmacological activity. The differential metabolites that were up-regulated in the two comparison groups were salvianolic acid B, linarin, medicago sativa 5-o-hexoside, ginsenoside RO and pelargonidin. Regarding the reasons for the upregulation of flavonoids, we speculate that the combined application of carbon and nitrogen accelerates the TCA cycle, generating more glutamate and glutamine. These compounds then provide nitrogen sources via the shikimate pathway for phenylalanine, a key precursor in the synthesis of flavonoids. Among them, salvianolic acid B has been proved to have potential efficacy in the treatment of ischemic brain injury and myocardial infarction [55, 56]. Linarin, ginsenoside RO and pelargonidin have been proved to have the effect of inhibiting inflammation [57–59]. On the other hand, Al Jaouni et al. showed that the increase of CO₂ concentration in the environment led to the up regulation of glutamine, L-glutathione, anthocyanin and most flavonoids metabolites in two kinds of herbal medicines — Ocimum basilicum and Mentha piperita, which was consistent with the results of this study [60]. We also found that N2KG1 treatment reduced the content of malonic acid in I. rotunda plants compared with the CK group. Malonic acid is a competitive inhibitor of succinate dehydrogenase and has a blocking effect on the TCA cycle, which indicates that the combined application of carbon and nitrogen promotes the TCA cycle to a certain extent.

Both N2KG1 and N2KG2 treatments with combined application of carbon and nitrogen had an effect on the TCA cycle of I. rotunda. The contents of α-KG, citric acid and glutamic acid were increased and the content of aspartic acid was decreased by N2KG1 and N2KG2 treatment. We believe that the increase of α-KG and citric acid content indicates that the combined application of carbon and nitrogen regulates the TCA cycle of I. rotunda. Glutamic acid plays an important role in amino acid metabolism and protein synthesis in plants. Its accumulation in I. rotunda also shows that the quality of plants has been improved. Aspartic acid and glutamic acid are transformed into each other in plants, and the relationship is complex. The reason why the content of aspartic acid and glutamic acid is relatively down-regulated by the combined application of carbon and nitrogen remains to be studied. Therefore, in the next step, we will further study the effect of carbon fertilizer (α-KG) on the activities of sucrose synthase (SS), sucrose phosphate synthase (SPS), glutamate dehydrogenase (GDH), aspartate aminotransferase (TAS) and other enzymes of I. rotunda under the treatment of carbon and nitrogen combined application, and clarify the effect of carbon and nitrogen combined application on the carbon and nitrogen metabolism pathway of I. rotunda. The second is to explore the internal mechanism of α- KG combined with urea on promoting plant growth of I. rotunda from the perspective of gene pathway, in order to provide reference for directional breeding.

Additionally, the study by Sabir et al. on the chitinase gene family in Hordeum vulgare revealed that key genes exhibit distinct expression patterns in response to various stresses [61]. This provides a gene-level perspective for comprehending the plant stress response network. Subsequent studies can employ genomics and molecular biology methods to elucidate the molecular mechanism by which α-KG regulates the carbon and nitrogen metabolism of I. rotunda. This provides a foundation for the cultivation of high-quality germplasm. Irfan et al. found that exogenous substances can alleviate stress by regulating photosynthesis, providing ideas for future research [62, 63]. Musa et al. identified various bioactive compounds in Justicia adhatoda using gas chromatography-mass spectrometry [64]. Subsequent studies can learn from their analytical methods to further isolate and identify active components in I. rotunda.

Conclusion

This study examined the effects of α-KG and nitrogen on the growth and carbon and nitrogen metabolism of I. rotunda. Photosynthetic performance and physiological indexes were measured in 2-year-old I. rotunda cuttings after 12 weeks of treatment with different concentrations of α-KG and urea. At the same time, the leaves of each treatment group (CK, N2, N2KG1, and N2KG2) were analyzed by non-targeted metabolomics after 12 weeks of treatment at a 0.4% urea treatment level.

The results showed the application of appropriate concentration of α-KG and urea to I. rotunda increased the net photosynthetic rate and other photosynthetic indicators, enhanced the activity of photosystem II, improved the efficiency of light energy conversion, and promoted plant growth. At the same time, it also promoted the accumulation of soluble sugar, soluble protein and starch in I. rotunda, increased the nitrogen content in leaves, improved the nitrogen utilization ability of plants, and promoted the accumulation of nutrients. The activities of GS, FD-GOGAT and NADH-GOGAT enzymes in I. rotunda were increased, which regulated the GS-GOGAT cycle. The contents of α-KG and citric acid in I. rotunda were increased, and the TCA cycle was regulated. Under the combined application of carbon and nitrogen, the carbon and nitrogen metabolism efficiency of I. rotunda was effectively regulated. The N2KG1 and N2KG2 treatment groups showed the most promising results for each treatment. The application of α-KG to I. rotunda under high nitrogen stress increased the activities of POD and SOD, and increased the contents of soluble sugar and soluble protein in I. rotunda, which was beneficial to enhance the adaptation of I. rotunda to the environment. Metabolomic analysis of I. rotunda leaves from different treatment groups further demonstrated that the combined application of carbon and nitrogen sources enhanced both carbon and nitrogen metabolism in the plants, corroborating the physiological experimental findings. Therefore, the application of α-KG and urea can promote the growth of I. rotunda and improve plant resistance, which provides a theoretical basis for the scientific cultivation of I. rotunda.

Supplementary Information

Supplementary Material 1.^{(1.3MB, doc)}

Authors’ contributions

Writing—original draft preparation and editing, DY; writing—review, SZ and YC; DY, SZ and YC have the same contribution to the article and are the co-first author. Supervision—project administration and funding acquisition, CY. Putting forward the suggestions of the experiment and make some modifications to the content of the article, JH and BZ. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the open project of Key Laboratory of Quality Control and Resistance Breeding of Characteristic Horticultural Crops in Ningbo City, grant number NBYYL2023003.

Data availability

The metabolomics data have been deposited to MetaboLights repository with the study identifier MTBLS13301.Other data that support the findings of this study are openly available in FigShare and the supplementary material of this article.

Declarations

Ethics approval and consent to participate

This article does not involve research on humans, animals, or endangered species. All experimental studies and materials comply with relevant institutional, national, and international guidelines and laws. The plant (herbarium code 1747414) was identified by Hang Zhibiao.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

1.Yang B, Li H, Ruan Q, Xuan SX, Chen XJ, Cui H, et al. Effects of gut microbiota and ingredient-ingredient interaction on the pharmacokinetic properties of rotundic acid and pedunculoside. Planta Med. 2019;85:729–37. 10.1055/a-0902-5300. [DOI] [PubMed] [Google Scholar]
2.Khan F, Bamunuarachchi NI, Tabassum N, Kim YM. Caffeic acid and its derivatives:antimicrobial drugs toward microbial pathogens. J Agric Food Chem. 2021;69:2979–3004. 10.1021/acs.jafc.0c07579. [DOI] [PubMed] [Google Scholar]
3.Mirzaei S, Gholami MH, Zabolian A, Saleki H, Farahani MV, Hamzehlou S, et al. Caffeic acid and its derivatives as potential modulators of oncogenic molecular pathways: new hope in the fight against cancer. Pharmacol Res. 2021;171:105759. 10.1016/j.phrs.2021.105759. [DOI] [PubMed] [Google Scholar]
4.Silva H, Lopes NMF. Cardiovascular effects of caffeic acid and its derivatives: a comprehensive review. Front Physiol. 2021;11:595516. 10.3389/fphys.2020.595516. [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Wu XQ, Yao JY, Liao ZW, Liu YY, Chen XY. Study on the relationship between stress resistance and nitrogen application of microbial fertilizer for high yield under low light and temperature[J]. Meteorology and Disaster Reduction Research. 2024;47:221–6. 10.12013/qxyjzyj2024-027. [Google Scholar]
6.Yadav A, Jha A, Misra AK. Unraveling the effects of increasing human population on CO₂ level and crop yield: a study of deterministic and stochastic systems. Math Comput Simul. 2025;240:38–65. 10.1016/j.matcom.2025.07.007. [Google Scholar]
7.Zhang FF. Effects of controlled release nitrogenfertilizer and organic carbon fertilizer on Yieldand nitrogen use efficiency of direct-seedingrapeseed. Sichuan Agricultural University. 2021. 10.27345/d.cnki.gsnyu.2021.001271
8.Xiang QH. Effects of nitrogen reduction combinedwith organic carbon fertilizer on flue-cured tobacco. Hunan Agricultural University. 2021. 10.27136/d.cnki.ghunu.2021.000030
9.Liao ZW, Mao XY, Liu KX. Effect of organic carbon fertilizer on nutrient balance — analysis of carbon, a short board, in plant nutrition. Acta Pedol Sin. 2014;051:656–9. 10.11766/trxb201308300390. [Google Scholar]
10.Zhao LS, Ma HZ, Song QH. Effects of exogenous α-ketoglutarate on growth and nitrogen metabolism of wheat seedlings underadversity stress. Journal of Seed Industry Guide. 2025;04:67–72. 10.3969/j.issn.1003-4749.2025.04.014. [Google Scholar]
11.Gong Y, Gao J, Wang HM, Liang CZ, Li WT, Wen BW, et al. Effects of exogenous α⁃ketoglutaric acid on nitrogen assimilation and yield character of soybean under low nitrogen conditions. Soybean Sci. 2025;44:84–95. [Google Scholar]
12.Huang N, Li QX, Yan DL. Effects of foliar application of α-ketoglutaric acid on quality and nutrient content of dendrobium officinale . Journal of Zhejiang Agricultural Sciences. 2021;62. 10.16178/jissn0528901720211116 .
13.Zhao SJ, Liu HS, Dong XC. Experimental Guidance for Plant Physiology. Beijing: China Agricultural Science and Technology Press; 1998. p. 98–105. [Google Scholar]
14.Wang XP, Xing SL. Study on determination of protein content by Coomassie brilliant blue method. Tianjin Chem Ind. 2009;23:40–2. [Google Scholar]
15.Bremner JM. Determination of nitrogen in soil by the Kjeldahl method. J Agric Sci. 1960;55:11–33. 10.1017/S0021859600021572. [Google Scholar]
16.Chen JX, Wang XF. Experimental guidance of plant physiology. Guangzhou: South China University of Technology Press; 2015. p. 24–6. [Google Scholar]
17.Beauchamp C, Fridovich I. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem. 1971;44:276–87. 10.1016/0003-2697(71)90370-8. [DOI] [PubMed] [Google Scholar]
18.Gan TT, Zhao NJ, Yin GF, Chen M, Wang X, Liu JG, et al. Optimal chlorophyll fluorescence parameter selection for rapid and sensitive detection of lead toxicity to marine microalgae Nitzschia closterium based on chlorophyll fluorescence technology. J Photochem Photobiol B Biol. 2019;197:111551. 10.1016/j.jphotobiol.2019.111551. [DOI] [PubMed] [Google Scholar]
19.Garnier E, Shipley B, Roumet C, Laurent G. A standardized protocol for the determination of specific leaf area and leaf dry matter content. Functional Ecology. 2001;688–695. 10.1046/j.0269-8463.2001.00563.x.
20.Al Haj Khaled R, Duru M, Theau JP, Plantureux S, Cruz P. Variation in leaf traits through seasons and N availability levels and its consequences for ranking grassland species. Journal of Vegetation Science. 2005;16:391–398. 10.1111/j165411032005tb02378x .
21.Bai YT, Dai JZ, Xia JB, Yan RR, Yang HJ, Lv SJ, et al. Effects of fertilization on functional traits and stoichiometry of Leymus chinensis in cut meadow. Acta Agrestia Sin. 2021;29:2442–53. 10.11733/jissn10070435202111009.
22.Zhou YP, Zhang YG, Ma W, Liang XS, Ma XY, Wang ZW. Effects of nitrogen addition and water reduction on the traits of five plants in Hulunbeir Grassland. Ecol Environ Sci. 2020;29:41–8. 10.16258/j.cnki.1674-5906.2020.01.005. [Google Scholar]
23.Sun XH, Niu LX, Zhang MF, Zhang HW, Liu H, Zhao M, et al. Application of carbon-based nutrient fertilizer improved soil fertility and seed yield of Paeonia ostii ‘Feng Dan.’ Ind Crops Prod. 2024;212:118348. 10.1016/j.indcrop.2024.118348. [Google Scholar]
24.Yang Y, Huang ZJ, Wu YQ, Wu WL, Lyu LF, Li WL. Effects of nitrogen application level on the physiological characteristics, yield and fruit quality of blackberry. Sci Hortic. 2023;313:111915. 10.1016/j.scienta.2023.111915. [Google Scholar]
25.Li JQ, Tian JH, Yu LN, Ji YZ, Zheng JW, Li BH. Effects of fertilization on seedling growth and photosynthetic characteristics of gleditsia sinensis. Forestry and Ecological Sciences. 2024;39:28–33. 10.13320/j.cnki.hjfor.2024.0004. [Google Scholar]
26.Zhang ZP, Li T, Hou LP, Sun S, Xing GM. Effects of CO₂ enrichment on photosynthetic fluorescence of thin-skinned melon in greenhouse during vine elongation. Acta Agric Boreali-Sin. 2019;34:97–103. 10.7668/hbnxb.20190069. [Google Scholar]
27.Fortunato S, Nigro D, Lasorella C, Marcotuli I, Gadaleta A, de Pinto MC. The role of glutamine synthetase (GS) and glutamate synthase (GOGAT) in the improvement of nitrogen use efficiency in cereals. Biomolecules. 2023;13:1771. 10.3390/biom13121771. [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Stolarz M, Hanaka A. Glutamate and its role in the metabolism of plants and animals. Processes. 2025;13:2084. 10.3390/pr13072084. [Google Scholar]
29.Leng X, Wang H, Cao L, Chang RH, Zhang S, Xu CF, et al. Overexpressing GLUTAMINE SYNTHETASE 1;2 maintains carbon and nitrogen balance under high-ammonium conditions and results in increased tolerance to ammonium toxicity in hybrid poplar. J Exp Bot. 2024;75:4052–73. 10.1093/jxb/erae124. [DOI] [PubMed] [Google Scholar]
30.Voor SF, Cooper AJL, Pinto JT. Glutaminase I and Glutamine Transaminase-ω-Amidase Pathways in Colorectal Cancer: Metabolic Reprogramming and Emerging Therapeutic Strategies. Anal Biochem. 2025. 10.1016/j.ab.2025.115989. [DOI] [PubMed]
31.Xiao D, Zeng L, Yao K, Kong X, Wu G, Yin Y. The glutamine-alpha-ketoglutarate (AKG) metabolism and its nutritional implications. Amino Acids. 2016;48:2067–80. 10.1007/s00726-016-2254-8. [DOI] [PubMed] [Google Scholar]
32.Behera PK, Sarangi CS, Mishra D, Patra HK. Catalase, peroxidase and alkaline inorganic pyrophosphatase activities during petal development and senescence in Thevetia peruviana (Pers.) K. Schum. Isr J Plant Sci. 1987;36:15–23. 10.1080/0021213X.1987.10677062. [Google Scholar]
33.Otálora G, Piñero MC, López-Marín J, Varó P, del Amor FM. Effects of foliar nitrogen fertilization on the phenolic, mineral, and amino acid composition of escarole (Cichorium endivia L. var. latifolium). Sci Hortic. 2018;239:87–92. 10.1016/j.scienta.2018.05.031. [Google Scholar]
34.Chung RS, Chen CC, Ng LT. Nitrogen fertilization affects the growth performance, betaine and polysaccharide concentrations of Lycium barbarum. Ind Crops Prod. 2010;32:650–5. 10.1016/j.indcrop.2010.07.021. [Google Scholar]
35.Fisher FM, Zak JC, Cunningham GL, Whitford WG. Water and nitrogen effects on growth and allocation patterns of creosotebush in the northern Chihuahuan desert. J Range Manage. 1998;41:387–91. 10.2307/3899572. [Google Scholar]
36.Yang X, Tang ZY, Ji CG, Liu HY, Ma WH, Mohhamot A, et al. Scaling of nitrogen and phosphorus across plant organs in shrubland biomes across Northern China. Sci Rep. 2014;4:5448. 10.1038/srep05448. [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Cruz JL, Alves AAC, LeCain DR, Ellis DD, Morgan JA. Interactive effects between nitrogen fertilization and elevated CO₂ on growth and gas exchange of papaya seedlings. Sci Hortic. 2016;202:32–40. 10.1016/j.scienta.2016.02.010. [Google Scholar]
38.Liu ZX, Gao F, Li XD, Zhang JL. Source-sink coordinated peanut cultivar increases yield and kernel protein content through enhancing photosynthetic characteristics and regulating carbon and nitrogen metabolisms. Plant Physiol Biochem. 2024;206:108311. 10.1016/j.plaphy.2023.108311. [DOI] [PubMed] [Google Scholar]
39.Zhang XY, Gao Y, Liu CJ, Li J, Ai XZ, Bi HG. Effects of combining application of fulvic acid and chemical fertilizers on soil environment and nitrogen, phosphorus and potassium utilization efficiency of pepper in greenhouse. Journal of Shandong Agricultural University (Natural Science Edition). 2021;52:893–902. 10.3969/j.issn.1000-2324.2021.06.001. [Google Scholar]
40.Pang QQ, Chen RY, Liu HC, Song SW, Su W, Sun GW. Effects of fulvic acid on the growth and activities of nitrogen metabolism enzymes in pakchoi under NO₃^- stress. Acta Agric Zhejiangensis. 2015;27:2136–40. 10.3969/j.issn.1004-1524.2015.12.14. [Google Scholar]
41.Dong YH, He XY, Wang KX, Yang HY. Research progress in uptake and metabolism of foliar urea in plants. Plant Science Journal. 2023;41:552–62. 10.11913/PSJ.2095-0837.22270. [Google Scholar]
42.Witte CP, Tiller SA, Taylor MA, Davies HV. Leaf urea metabolism in potato. Urease activity profile and patterns of recovery and distribution of (15)N after foliar urea application in wild-type and urease-antisense transgenics. Plant Physiol. 2002;128:1129–36. 10.1104/pp.010506. [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Chen HJ, Huang XF, Shi WM, Kronzucker HJ, Hou LH, Yang HY, et al. Coordination of nitrogen uptake and assimilation favours the growth and competitiveness of moso bamboo over native tree species in high-NH+ 4 environments. J Plant Physiol. 2021;266:153508. 10.1016/j.jplph.2021.153508. [DOI] [PubMed] [Google Scholar]
44.Bittsánszky A, Pilinszky K, Gyulai G, Komives T. Overcoming ammonium toxicity. Plant Science. 2015;231:184–90. 10.1080/0021213X.1987.10677062. [DOI] [PubMed] [Google Scholar]
45.Esteban R, Ariz I, Cruz C, Moran JF. Mechanisms of ammonium toxicity and the quest for tolerance. Plant Sci. 2016;248:92–101. 10.1016/j.plantsci.2016.04.008. [DOI] [PubMed] [Google Scholar]
46.Xun ZL, Guo XF, Li YL, Wen XZ, Wang CQ, Wang Y. Quantitative proteomics analysis of tomato growth inhibition by ammonium nitrogen. Plant Physiol Biochem. 2020;154:129–41. 10.1016/j.plaphy.2020.05.036. [DOI] [PubMed] [Google Scholar]
47.Vega-Mas I, Pérez-Delgado CM, Marino D, Fuertes-Mendizábal T, González-Murua C, Márquez AJ, et al. Elevated CO₂ induces root defensive mechanisms in tomato plants when dealing with ammonium toxicity. Plant Cell Physiol. 2017;58:2112–25. 10.1093/pcp/pcx146. [DOI] [PubMed] [Google Scholar]
48.Britto DT, Kronzucker HJ. NH+ 4 toxicity in higher plants: a critical review. J Plant Physiol. 2002;159:567–84. 10.1078/0176-1617-0774. [Google Scholar]
49.Hodges M. Enzyme redundancy and the importance of 2‐oxoglutarate in plant ammonium assimilation. J Exp Bot. 2002;53:905–16. 10.1093/jexbot/53.370.905. [DOI] [PubMed] [Google Scholar]
50.Wu YL, Hu JX, Chen YX, Zheng BS, Yan DL. Effects of external application of α-ketoglutarate on growth, carbon, nitrogen and phosphorus accumulation and their stoichiometric relationships in Kosteletzkya virginica under salt stress. Journal of Agricultural Science and Technology. 2023;25:170–7. 10.13304/j.nykjdb.2022.0276. [Google Scholar]
51.Sun L, Suo QY, Zhao HH, Chen SH, Kang WQ. Effect of spraying organic carbon fertilizer on photosynthetic characteristics of wheat under weak light. Journal Of Northern Agriculture. 2021;49:65–70. 10.12190/j.issn.2096-1197.2021.01.10. [Google Scholar]
52.Xu SJ, Wang L, DeMu QG, Wang JB, Qi HX, Zhu GL. Responses of antioxidant enzyme activities,yield,and oil content to nitrogen fertilizer in dwarf castor hybrids in the Xiliaohe Plain. China Soil and Fertilizer. 2021;4:192–202. 10.11838/sfsc.1673-6257.20271. [Google Scholar]
53.Yang Q, Li Ym, Xiao K, Du YH. Effect of different amount of nitrogen on flag leaf senescence and yield components of wheat. J Agric Univ Hebei. 2002;25:5. 10.3969/j.issn.1000-1573.2002.04.005. [Google Scholar]
54.Zhang Y, Yao Q, Shi Y, Li XF, Hou LP, Xing GM, et al. Elevated CO₂ improves antioxidant capacity, ion homeostasis, and polyamine metabolism in tomato seedlings under Ca(NO₃)²⁻induced salt stress. Sci Hortic. 2020;273:109644. 10.1016/j.scienta.2020.109644. [Google Scholar]
55.Yan ML, Li ZM, Dai SJ, Li SY, Yu PP. The potential effect of salvianolic acid B against rat ischemic brain injury in combination with mesenchymal stem cells. J Chem Neuroanat. 2023;133:102338. 10.1016/j.jchemneu.2023.102338. [DOI] [PubMed] [Google Scholar]
56.Chen M, Li J, Peng J, Huang Y, Ouyang WJ, Liu XQ, et al. Linarin ameliorates innate inflammatory response in an experimental dry eye model via modulation of the NLRP3 inflammasome. Digit Chin Med. 2021;4:42–53. 10.1016/j.dcmed.2021.03.006. [Google Scholar]
57.Chen R, Zhu C, Xu L, Gu Y, Ren SJ, Bai H, et al. An injectable peptide hydrogel with excellent self-healing ability to continuously release salvianolic acid B for myocardial infarction. Biomaterials. 2021;274:120855. 10.1016/j.biomaterials.2021.120855. [DOI] [PubMed] [Google Scholar]
58.Xu HL, Chen GH, Wu YT, Xie LP, Tan ZB, Liu B, et al. Ginsenoside Ro, an oleanolic saponin of Panax ginseng, exerts anti-inflammatory effect by direct inhibiting toll like receptor 4 signaling pathway. J Ginseng Res. 2022;46:156–66. 10.1016/j.jgr.2021.05.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
59.Duarte LJ, Chaves VC, Nascimento MVPDS, Calvete E, Li MC, Ciraolo E, et al. Molecular mechanism of action of Pelargonidin-3-O-glucoside, the main anthocyanin responsible for the anti-inflammatory effect of strawberry fruits. Food Chem. 2018;247:56–65. 10.1016/j.foodchem.2017.12.015. [DOI] [PubMed] [Google Scholar]
60.Al Jaouni S, Saleh AM, Wadaan MAM, Hozzein WN, Selim S, AbdElgawad H. Elevated CO₂ induces a global metabolic change in basil (Ocimum basilicum L.) and peppermint (Mentha piperita L.) and improves their biological activity. J Plant Physiol. 2018;224:121–31. 10.1016/j.jplph.2018.03.01. [DOI] [PubMed] [Google Scholar]
61.Sabir IA, Nabi F, Manzoor MA, Ullah F, Saeed M, Hashem A, et al. Genome-wide identification of chitinase gene family in Hordeum vulgare: insights into stress response mechanisms and evolutionary dynamics. BMC Plant Biol. 2015;13:628. 10.1186/s12870-025-06475-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
62.Irfan M, Alam J, Ahmad I, Ali I, Gul H. Effects of exogenous and foliar applications of Brassinosteroid (BRs) and salt stress on the growth, yield and physiological parameters of Lycopersicon esculentum (Mill.). Plant Sci. 2017;4:88–101. 10.14719/pst.2017.4.3.218. [Google Scholar]
63.Irfan M, Jan G, Gul Jan FG, Murad W Influence of Ascorbic Acid against Salt stress on the Germination, Biochemicals and Micronutrients Concentrations of Solanum melongena (L.). Preprints. 2021;2021010278. 10.20944/preprints202101.0278.v1
64.Musa M, Jan G, Jan FG, Hamayun M, Irfan M, Rauf A, et al. Pharmacological activities and gas chromatography–mass spectrometry analysis for the identification of bioactive compounds from Justicia adhatoda L. Front Pharmacol. 2022;13:922388. 10.3389/fphar.2022.922388. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1.^{(1.3MB, doc)}

Data Availability Statement

[CR1] 1.Yang B, Li H, Ruan Q, Xuan SX, Chen XJ, Cui H, et al. Effects of gut microbiota and ingredient-ingredient interaction on the pharmacokinetic properties of rotundic acid and pedunculoside. Planta Med. 2019;85:729–37. 10.1055/a-0902-5300. [DOI] [PubMed] [Google Scholar]

[CR2] 2.Khan F, Bamunuarachchi NI, Tabassum N, Kim YM. Caffeic acid and its derivatives:antimicrobial drugs toward microbial pathogens. J Agric Food Chem. 2021;69:2979–3004. 10.1021/acs.jafc.0c07579. [DOI] [PubMed] [Google Scholar]

[CR3] 3.Mirzaei S, Gholami MH, Zabolian A, Saleki H, Farahani MV, Hamzehlou S, et al. Caffeic acid and its derivatives as potential modulators of oncogenic molecular pathways: new hope in the fight against cancer. Pharmacol Res. 2021;171:105759. 10.1016/j.phrs.2021.105759. [DOI] [PubMed] [Google Scholar]

[CR4] 4.Silva H, Lopes NMF. Cardiovascular effects of caffeic acid and its derivatives: a comprehensive review. Front Physiol. 2021;11:595516. 10.3389/fphys.2020.595516. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR5] 5.Wu XQ, Yao JY, Liao ZW, Liu YY, Chen XY. Study on the relationship between stress resistance and nitrogen application of microbial fertilizer for high yield under low light and temperature[J]. Meteorology and Disaster Reduction Research. 2024;47:221–6. 10.12013/qxyjzyj2024-027. [Google Scholar]

[CR6] 6.Yadav A, Jha A, Misra AK. Unraveling the effects of increasing human population on CO₂ level and crop yield: a study of deterministic and stochastic systems. Math Comput Simul. 2025;240:38–65. 10.1016/j.matcom.2025.07.007. [Google Scholar]

[CR7] 7.Zhang FF. Effects of controlled release nitrogenfertilizer and organic carbon fertilizer on Yieldand nitrogen use efficiency of direct-seedingrapeseed. Sichuan Agricultural University. 2021. 10.27345/d.cnki.gsnyu.2021.001271

[CR8] 8.Xiang QH. Effects of nitrogen reduction combinedwith organic carbon fertilizer on flue-cured tobacco. Hunan Agricultural University. 2021. 10.27136/d.cnki.ghunu.2021.000030

[CR9] 9.Liao ZW, Mao XY, Liu KX. Effect of organic carbon fertilizer on nutrient balance — analysis of carbon, a short board, in plant nutrition. Acta Pedol Sin. 2014;051:656–9. 10.11766/trxb201308300390. [Google Scholar]

[CR10] 10.Zhao LS, Ma HZ, Song QH. Effects of exogenous α-ketoglutarate on growth and nitrogen metabolism of wheat seedlings underadversity stress. Journal of Seed Industry Guide. 2025;04:67–72. 10.3969/j.issn.1003-4749.2025.04.014. [Google Scholar]

[CR11] 11.Gong Y, Gao J, Wang HM, Liang CZ, Li WT, Wen BW, et al. Effects of exogenous α⁃ketoglutaric acid on nitrogen assimilation and yield character of soybean under low nitrogen conditions. Soybean Sci. 2025;44:84–95. [Google Scholar]

[CR12] 12.Huang N, Li QX, Yan DL. Effects of foliar application of α-ketoglutaric acid on quality and nutrient content of dendrobium officinale . Journal of Zhejiang Agricultural Sciences. 2021;62. 10.16178/jissn0528901720211116 .

[CR13] 13.Zhao SJ, Liu HS, Dong XC. Experimental Guidance for Plant Physiology. Beijing: China Agricultural Science and Technology Press; 1998. p. 98–105. [Google Scholar]

[CR14] 14.Wang XP, Xing SL. Study on determination of protein content by Coomassie brilliant blue method. Tianjin Chem Ind. 2009;23:40–2. [Google Scholar]

[CR15] 15.Bremner JM. Determination of nitrogen in soil by the Kjeldahl method. J Agric Sci. 1960;55:11–33. 10.1017/S0021859600021572. [Google Scholar]

[CR16] 16.Chen JX, Wang XF. Experimental guidance of plant physiology. Guangzhou: South China University of Technology Press; 2015. p. 24–6. [Google Scholar]

[CR17] 17.Beauchamp C, Fridovich I. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem. 1971;44:276–87. 10.1016/0003-2697(71)90370-8. [DOI] [PubMed] [Google Scholar]

[CR18] 18.Gan TT, Zhao NJ, Yin GF, Chen M, Wang X, Liu JG, et al. Optimal chlorophyll fluorescence parameter selection for rapid and sensitive detection of lead toxicity to marine microalgae Nitzschia closterium based on chlorophyll fluorescence technology. J Photochem Photobiol B Biol. 2019;197:111551. 10.1016/j.jphotobiol.2019.111551. [DOI] [PubMed] [Google Scholar]

[CR19] 19.Garnier E, Shipley B, Roumet C, Laurent G. A standardized protocol for the determination of specific leaf area and leaf dry matter content. Functional Ecology. 2001;688–695. 10.1046/j.0269-8463.2001.00563.x.

[CR20] 20.Al Haj Khaled R, Duru M, Theau JP, Plantureux S, Cruz P. Variation in leaf traits through seasons and N availability levels and its consequences for ranking grassland species. Journal of Vegetation Science. 2005;16:391–398. 10.1111/j165411032005tb02378x .

[CR21] 21.Bai YT, Dai JZ, Xia JB, Yan RR, Yang HJ, Lv SJ, et al. Effects of fertilization on functional traits and stoichiometry of Leymus chinensis in cut meadow. Acta Agrestia Sin. 2021;29:2442–53. 10.11733/jissn10070435202111009.

[CR22] 22.Zhou YP, Zhang YG, Ma W, Liang XS, Ma XY, Wang ZW. Effects of nitrogen addition and water reduction on the traits of five plants in Hulunbeir Grassland. Ecol Environ Sci. 2020;29:41–8. 10.16258/j.cnki.1674-5906.2020.01.005. [Google Scholar]

[CR23] 23.Sun XH, Niu LX, Zhang MF, Zhang HW, Liu H, Zhao M, et al. Application of carbon-based nutrient fertilizer improved soil fertility and seed yield of Paeonia ostii ‘Feng Dan.’ Ind Crops Prod. 2024;212:118348. 10.1016/j.indcrop.2024.118348. [Google Scholar]

[CR24] 24.Yang Y, Huang ZJ, Wu YQ, Wu WL, Lyu LF, Li WL. Effects of nitrogen application level on the physiological characteristics, yield and fruit quality of blackberry. Sci Hortic. 2023;313:111915. 10.1016/j.scienta.2023.111915. [Google Scholar]

[CR25] 25.Li JQ, Tian JH, Yu LN, Ji YZ, Zheng JW, Li BH. Effects of fertilization on seedling growth and photosynthetic characteristics of gleditsia sinensis. Forestry and Ecological Sciences. 2024;39:28–33. 10.13320/j.cnki.hjfor.2024.0004. [Google Scholar]

[CR26] 26.Zhang ZP, Li T, Hou LP, Sun S, Xing GM. Effects of CO₂ enrichment on photosynthetic fluorescence of thin-skinned melon in greenhouse during vine elongation. Acta Agric Boreali-Sin. 2019;34:97–103. 10.7668/hbnxb.20190069. [Google Scholar]

[CR27] 27.Fortunato S, Nigro D, Lasorella C, Marcotuli I, Gadaleta A, de Pinto MC. The role of glutamine synthetase (GS) and glutamate synthase (GOGAT) in the improvement of nitrogen use efficiency in cereals. Biomolecules. 2023;13:1771. 10.3390/biom13121771. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR28] 28.Stolarz M, Hanaka A. Glutamate and its role in the metabolism of plants and animals. Processes. 2025;13:2084. 10.3390/pr13072084. [Google Scholar]

[CR29] 29.Leng X, Wang H, Cao L, Chang RH, Zhang S, Xu CF, et al. Overexpressing GLUTAMINE SYNTHETASE 1;2 maintains carbon and nitrogen balance under high-ammonium conditions and results in increased tolerance to ammonium toxicity in hybrid poplar. J Exp Bot. 2024;75:4052–73. 10.1093/jxb/erae124. [DOI] [PubMed] [Google Scholar]

[CR30] 30.Voor SF, Cooper AJL, Pinto JT. Glutaminase I and Glutamine Transaminase-ω-Amidase Pathways in Colorectal Cancer: Metabolic Reprogramming and Emerging Therapeutic Strategies. Anal Biochem. 2025. 10.1016/j.ab.2025.115989. [DOI] [PubMed]

[CR31] 31.Xiao D, Zeng L, Yao K, Kong X, Wu G, Yin Y. The glutamine-alpha-ketoglutarate (AKG) metabolism and its nutritional implications. Amino Acids. 2016;48:2067–80. 10.1007/s00726-016-2254-8. [DOI] [PubMed] [Google Scholar]

[CR32] 32.Behera PK, Sarangi CS, Mishra D, Patra HK. Catalase, peroxidase and alkaline inorganic pyrophosphatase activities during petal development and senescence in Thevetia peruviana (Pers.) K. Schum. Isr J Plant Sci. 1987;36:15–23. 10.1080/0021213X.1987.10677062. [Google Scholar]

[CR33] 33.Otálora G, Piñero MC, López-Marín J, Varó P, del Amor FM. Effects of foliar nitrogen fertilization on the phenolic, mineral, and amino acid composition of escarole (Cichorium endivia L. var. latifolium). Sci Hortic. 2018;239:87–92. 10.1016/j.scienta.2018.05.031. [Google Scholar]

[CR34] 34.Chung RS, Chen CC, Ng LT. Nitrogen fertilization affects the growth performance, betaine and polysaccharide concentrations of Lycium barbarum. Ind Crops Prod. 2010;32:650–5. 10.1016/j.indcrop.2010.07.021. [Google Scholar]

[CR35] 35.Fisher FM, Zak JC, Cunningham GL, Whitford WG. Water and nitrogen effects on growth and allocation patterns of creosotebush in the northern Chihuahuan desert. J Range Manage. 1998;41:387–91. 10.2307/3899572. [Google Scholar]

[CR36] 36.Yang X, Tang ZY, Ji CG, Liu HY, Ma WH, Mohhamot A, et al. Scaling of nitrogen and phosphorus across plant organs in shrubland biomes across Northern China. Sci Rep. 2014;4:5448. 10.1038/srep05448. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR37] 37.Cruz JL, Alves AAC, LeCain DR, Ellis DD, Morgan JA. Interactive effects between nitrogen fertilization and elevated CO₂ on growth and gas exchange of papaya seedlings. Sci Hortic. 2016;202:32–40. 10.1016/j.scienta.2016.02.010. [Google Scholar]

[CR38] 38.Liu ZX, Gao F, Li XD, Zhang JL. Source-sink coordinated peanut cultivar increases yield and kernel protein content through enhancing photosynthetic characteristics and regulating carbon and nitrogen metabolisms. Plant Physiol Biochem. 2024;206:108311. 10.1016/j.plaphy.2023.108311. [DOI] [PubMed] [Google Scholar]

[CR39] 39.Zhang XY, Gao Y, Liu CJ, Li J, Ai XZ, Bi HG. Effects of combining application of fulvic acid and chemical fertilizers on soil environment and nitrogen, phosphorus and potassium utilization efficiency of pepper in greenhouse. Journal of Shandong Agricultural University (Natural Science Edition). 2021;52:893–902. 10.3969/j.issn.1000-2324.2021.06.001. [Google Scholar]

[CR40] 40.Pang QQ, Chen RY, Liu HC, Song SW, Su W, Sun GW. Effects of fulvic acid on the growth and activities of nitrogen metabolism enzymes in pakchoi under NO₃^- stress. Acta Agric Zhejiangensis. 2015;27:2136–40. 10.3969/j.issn.1004-1524.2015.12.14. [Google Scholar]

[CR41] 41.Dong YH, He XY, Wang KX, Yang HY. Research progress in uptake and metabolism of foliar urea in plants. Plant Science Journal. 2023;41:552–62. 10.11913/PSJ.2095-0837.22270. [Google Scholar]

[CR42] 42.Witte CP, Tiller SA, Taylor MA, Davies HV. Leaf urea metabolism in potato. Urease activity profile and patterns of recovery and distribution of (15)N after foliar urea application in wild-type and urease-antisense transgenics. Plant Physiol. 2002;128:1129–36. 10.1104/pp.010506. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR43] 43.Chen HJ, Huang XF, Shi WM, Kronzucker HJ, Hou LH, Yang HY, et al. Coordination of nitrogen uptake and assimilation favours the growth and competitiveness of moso bamboo over native tree species in high-NH+ 4 environments. J Plant Physiol. 2021;266:153508. 10.1016/j.jplph.2021.153508. [DOI] [PubMed] [Google Scholar]

[CR44] 44.Bittsánszky A, Pilinszky K, Gyulai G, Komives T. Overcoming ammonium toxicity. Plant Science. 2015;231:184–90. 10.1080/0021213X.1987.10677062. [DOI] [PubMed] [Google Scholar]

[CR45] 45.Esteban R, Ariz I, Cruz C, Moran JF. Mechanisms of ammonium toxicity and the quest for tolerance. Plant Sci. 2016;248:92–101. 10.1016/j.plantsci.2016.04.008. [DOI] [PubMed] [Google Scholar]

[CR46] 46.Xun ZL, Guo XF, Li YL, Wen XZ, Wang CQ, Wang Y. Quantitative proteomics analysis of tomato growth inhibition by ammonium nitrogen. Plant Physiol Biochem. 2020;154:129–41. 10.1016/j.plaphy.2020.05.036. [DOI] [PubMed] [Google Scholar]

[CR47] 47.Vega-Mas I, Pérez-Delgado CM, Marino D, Fuertes-Mendizábal T, González-Murua C, Márquez AJ, et al. Elevated CO₂ induces root defensive mechanisms in tomato plants when dealing with ammonium toxicity. Plant Cell Physiol. 2017;58:2112–25. 10.1093/pcp/pcx146. [DOI] [PubMed] [Google Scholar]

[CR48] 48.Britto DT, Kronzucker HJ. NH+ 4 toxicity in higher plants: a critical review. J Plant Physiol. 2002;159:567–84. 10.1078/0176-1617-0774. [Google Scholar]

[CR49] 49.Hodges M. Enzyme redundancy and the importance of 2‐oxoglutarate in plant ammonium assimilation. J Exp Bot. 2002;53:905–16. 10.1093/jexbot/53.370.905. [DOI] [PubMed] [Google Scholar]

[CR50] 50.Wu YL, Hu JX, Chen YX, Zheng BS, Yan DL. Effects of external application of α-ketoglutarate on growth, carbon, nitrogen and phosphorus accumulation and their stoichiometric relationships in Kosteletzkya virginica under salt stress. Journal of Agricultural Science and Technology. 2023;25:170–7. 10.13304/j.nykjdb.2022.0276. [Google Scholar]

[CR51] 51.Sun L, Suo QY, Zhao HH, Chen SH, Kang WQ. Effect of spraying organic carbon fertilizer on photosynthetic characteristics of wheat under weak light. Journal Of Northern Agriculture. 2021;49:65–70. 10.12190/j.issn.2096-1197.2021.01.10. [Google Scholar]

[CR52] 52.Xu SJ, Wang L, DeMu QG, Wang JB, Qi HX, Zhu GL. Responses of antioxidant enzyme activities,yield,and oil content to nitrogen fertilizer in dwarf castor hybrids in the Xiliaohe Plain. China Soil and Fertilizer. 2021;4:192–202. 10.11838/sfsc.1673-6257.20271. [Google Scholar]

[CR53] 53.Yang Q, Li Ym, Xiao K, Du YH. Effect of different amount of nitrogen on flag leaf senescence and yield components of wheat. J Agric Univ Hebei. 2002;25:5. 10.3969/j.issn.1000-1573.2002.04.005. [Google Scholar]

[CR54] 54.Zhang Y, Yao Q, Shi Y, Li XF, Hou LP, Xing GM, et al. Elevated CO₂ improves antioxidant capacity, ion homeostasis, and polyamine metabolism in tomato seedlings under Ca(NO₃)²⁻induced salt stress. Sci Hortic. 2020;273:109644. 10.1016/j.scienta.2020.109644. [Google Scholar]

[CR55] 55.Yan ML, Li ZM, Dai SJ, Li SY, Yu PP. The potential effect of salvianolic acid B against rat ischemic brain injury in combination with mesenchymal stem cells. J Chem Neuroanat. 2023;133:102338. 10.1016/j.jchemneu.2023.102338. [DOI] [PubMed] [Google Scholar]

[CR56] 56.Chen M, Li J, Peng J, Huang Y, Ouyang WJ, Liu XQ, et al. Linarin ameliorates innate inflammatory response in an experimental dry eye model via modulation of the NLRP3 inflammasome. Digit Chin Med. 2021;4:42–53. 10.1016/j.dcmed.2021.03.006. [Google Scholar]

[CR57] 57.Chen R, Zhu C, Xu L, Gu Y, Ren SJ, Bai H, et al. An injectable peptide hydrogel with excellent self-healing ability to continuously release salvianolic acid B for myocardial infarction. Biomaterials. 2021;274:120855. 10.1016/j.biomaterials.2021.120855. [DOI] [PubMed] [Google Scholar]

[CR58] 58.Xu HL, Chen GH, Wu YT, Xie LP, Tan ZB, Liu B, et al. Ginsenoside Ro, an oleanolic saponin of Panax ginseng, exerts anti-inflammatory effect by direct inhibiting toll like receptor 4 signaling pathway. J Ginseng Res. 2022;46:156–66. 10.1016/j.jgr.2021.05.011. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR59] 59.Duarte LJ, Chaves VC, Nascimento MVPDS, Calvete E, Li MC, Ciraolo E, et al. Molecular mechanism of action of Pelargonidin-3-O-glucoside, the main anthocyanin responsible for the anti-inflammatory effect of strawberry fruits. Food Chem. 2018;247:56–65. 10.1016/j.foodchem.2017.12.015. [DOI] [PubMed] [Google Scholar]

[CR60] 60.Al Jaouni S, Saleh AM, Wadaan MAM, Hozzein WN, Selim S, AbdElgawad H. Elevated CO₂ induces a global metabolic change in basil (Ocimum basilicum L.) and peppermint (Mentha piperita L.) and improves their biological activity. J Plant Physiol. 2018;224:121–31. 10.1016/j.jplph.2018.03.01. [DOI] [PubMed] [Google Scholar]

[CR61] 61.Sabir IA, Nabi F, Manzoor MA, Ullah F, Saeed M, Hashem A, et al. Genome-wide identification of chitinase gene family in Hordeum vulgare: insights into stress response mechanisms and evolutionary dynamics. BMC Plant Biol. 2015;13:628. 10.1186/s12870-025-06475-0. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR62] 62.Irfan M, Alam J, Ahmad I, Ali I, Gul H. Effects of exogenous and foliar applications of Brassinosteroid (BRs) and salt stress on the growth, yield and physiological parameters of Lycopersicon esculentum (Mill.). Plant Sci. 2017;4:88–101. 10.14719/pst.2017.4.3.218. [Google Scholar]

[CR63] 63.Irfan M, Jan G, Gul Jan FG, Murad W Influence of Ascorbic Acid against Salt stress on the Germination, Biochemicals and Micronutrients Concentrations of Solanum melongena (L.). Preprints. 2021;2021010278. 10.20944/preprints202101.0278.v1

[CR64] 64.Musa M, Jan G, Jan FG, Hamayun M, Irfan M, Rauf A, et al. Pharmacological activities and gas chromatography–mass spectrometry analysis for the identification of bioactive compounds from Justicia adhatoda L. Front Pharmacol. 2022;13:922388. 10.3389/fphar.2022.922388. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

The influence of combined carbon and nitrogen application on the growth and metabolism of Ilex rotunda

Daoliang Yan

Shuxian Zhao

Yuxi Chen

Chenyi You

Jiaxin Hu

Bingsong Zheng

Abstract

Supplementary Information

Introduction

Materials and methods

Test materials and treatment

Table 1.

Determination of specific leaf area of blade

Determination of photosynthetic parameters

Determination of chlorophyll fluorescence parameters

Determination of soluble sugar, starch and soluble protein content

Determination of nitrogen content

Determination of GS, ferredoxin-dependent glutamate synthase (Fd-GOGAT EC1.4.7.1) and NADH-glutamate synthase (NADH-GOGAT EC 1.4.1.14) activities

Determination of antioxidant enzyme activity

Metabonomic determination and analysis

Data processing

Results

Effects of combined application of carbon and nitrogen on the photosynthetic performance of I. rotunda

Effects of combined application of carbon and nitrogen on specific leaf area of I. rotunda

Fig. 1.

Effects of combined application of carbon and nitrogen on photosynthetic parameters of I. rotunda

Fig. 2.

Effects of combined application of carbon and nitrogen on chlorophyll fluorescence parameters of I. rotunda

Fig. 3.

Effects of combined application of carbon and nitrogen on important physiological indexes of I. rotunda leaves

Differences in soluble sugar, starch and soluble protein contents of I. rotunda leaves under different carbon and nitrogen fertilization conditions

Fig. 4.

Difference of total nitrogen content in leaves of I. rotunda under different combined application of carbon and nitrogen

Fig. 5.

Difference of GS activity in leaves of I. rotunda under different combined application of carbon and nitrogen

Fig. 6.

Difference in activity of ferredoxin-dependent glutamate synthase (Fd-GOGAT) in leaves of I. rotunda under different combined application of carbon and nitrogen

Fig. 7.

Difference of NADH-glutamate synthase (NADH-GOGAT) activity in leaves of I. rotunda under different combined application of carbon and nitrogen

Fig. 8.

Effects of combined application of different carbon and nitrogen on antioxidant enzyme activities of I. rotunda

Fig. 9.

Non targeted metabolomics analysis of I. rotunda in different treatments of carbon and nitrogen fertilization

Total sample PCA analysis

Fig. 10.

Cluster analysis of differential metabolites among different treatments of carbon and nitrogen fertilization

Fig. 11.

Differential metabolite analysis

Table 2.

Fig. 12.

Fig. 13.

Fig. 14.

Fig. 15.

Analysis of pathways for enrichment of differential metabolites

Fig. 16.

Discussion

Effects of combined application of different carbon and nitrogen on photosynthetic performance of I. rotunda

Effects of different combined application of carbon and nitrogen on physiological indexes and carbon and nitrogen metabolism of I. rotunda

Conclusion

Supplementary Information

Authors’ contributions

Funding

Data availability

Declarations

Ethics approval and consent to participate

Consent for publication

Competing interests

Footnotes

References

Associated Data

Supplementary Materials

Data Availability Statement

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