The pot experiment was carried out between April and July 2022 at the Agricultural Science Experimental Station of Weifang University (118°10’ E and 35°41’ N). The soil utilized in the experiment was loam, with 22.13 grams of organic matter per kilogram, 46.81 milligrams of N per kilogram, 33.25 milligrams of accessible phosphorus per kilogram, and 178.12 milligrams of potassium per kilogram. The test materials consisted of Malus domestica M9T337 dwarf rootstocks. Rootstocks were cultivated in a soil culture system within a growth chamber, exposed to natural light. The growth chamber maintained a temperature cycle of 28°C during the day and 18°C during the night, as well as a humidity level of 65%. On April 9th, when the seedlings reached 10 true leaves, they were moved into pots. The pots had a height of 17 cm and a diameter of 22 cm, and contained 2.5 kg of soil each and each treatment was repeated in 30 pots (replicates). Only one plant was placed in each pot. After a period of 14 days, rootstocks exhibiting consistent development were chosen for the purpose of conducting experiments (about 13 cm tall).

In the formal trial, Ca(NO₃)₂ was used as the only N source. The seedlings were treated for root application as follows: CK (Control, 10 mmol·L⁻¹ NO₃ ⁻-N), 100 μmol·L⁻¹ melatonin (MT, Kangwei Century Technology Co., Ltd.), the experimental concentration is referred to the study of Liang et al (Liang et al., 2018), 30 mmol·L⁻¹ NO₃ ⁻-N (HN), the experimental concentration is referred to the study of Liu et al (Liu et al., 2022), 30 mmol·L⁻¹ NO₃ ⁻-N + 100 μmol·L⁻¹ melatonin (HN+MT). For the ¹⁵N labeling of plants, we added 0.5 g (CK and MT) and 1.5 g (HN and HN+MT) Ca(¹⁵NO₃)₂ (10.22% abundance, Shanghai Yuan ye Biotechnology Co., Ltd.) to each pots, each pots was treated with 200ml, once every 2 days, for a total of three times. After 30 days of treatment, samples were obtained to determine different indicators.

The cleaned root systems that float on 5 mm of water in a 0.3 × 0.2 m Plexiglas tray. Roots be untangled and discrete with a brush to lessen root interlock. Greyscale root images got by tuning the parameters to ‘‘high’’ setting (resolution 300 by 300 dpi). Then WinRhizo software (WinRHIZO version 2012b, Regent Instruments Canada, Montreal, Canada) was using to analyze the whole root system (root length, Number of root tips, root surface area and root volume).

The root activity was determined by the 2,3,5-triphenyltetrazolium chloride (TTC) reduction method and expressed as the amount of TTC reduced by per gram of root (Gao et al., 2023).

The plant samples were washed and dried off with an absorbent paper. Then, the fresh weights of the aboveground and belowground parts were measured. The dry weights of the aboveground and below-ground parts were measured after drying the samples in an oven at 105°C for 30 min and at 80°C to constant weight (Sun et al., 2022).

The superoxide dismutase (SOD) activity was determined based on the ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT) (Meftahizadeh et al., 2023). The peroxidase (POD) activity was determined by a colorimetric method in a reaction mixture containing guaiacol as the substrate (Wu et al., 2023). The POD activity was determined based on the change in absorbance at 470 nm due to the oxidation of guaiacol to tetraguaiacol. The supernatant was mixed with and was allowed to run. The absorbance of the reaction mixture was read at 560 nm. The catalase (CAT) activity was determined by the ultraviolet spectrophotometric method (Ren et al., 2022) at 240 nm in a reaction mixture. The CAT activity was measured based on the decomposition of H₂O₂ with time. Ascorbate peroxidase (APX) activity was measured by a spectrophotometric method according to Sun et al (Sun et al., 2022).

The H₂O₂ was determined according to the methods described by Yan et al (Yan et al., 2023). Pre-cooled acetone was used to extract the H₂O₂ in the sample and detected by monitoring the absorbance of the titanium peroxide complex at 412 nm.

Sorbose (Sor), sucrose (Suc), glucose (Glu) and fructose (Fru) were performed as described by Aslam et al. (2019). The hexose phosphates glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P) were extracted and assayed as described by Chen et al. (2023).

After 27 days of treatment, ¹³C isotope labeling was performed. Ba¹³CO₃ (¹³C independence is 98%) was used as a selection marker, and the dosage was 0.2 g. The seedlings were placed together with the markers, fans, and reduced iron powder into a sealed marking room to make transparent film. The transmittance of sunlight in the labeling chamber was 95% of the natural light intensity. Labeling work started at 9:00 a.m., at which point the fan was turned on and the labeling chamber was sealed. One milliliter of hydrochloric acid (1 mol·L⁻¹) was injected into the beaker with a syringe every 0.5 h in order to maintain the concentration of CO₂, and the ¹³C labeling lasted for 4 h. In order to keep low temperature during the labeling process, an appropriate amount of ice was added to the bottom of the labeling chamber to limit the temperature within the range of 28–37°C. At the same time, three other plants were selected as the control (¹³C natural abundance), and destructive samples were taken on the third day after labeling and ¹³C was determined (Xu et al., 2020).

Biomass samples for ¹³C and ¹⁵N analyses were collected at the end of the labeling for ¹³C and ¹⁵N analyses. The samples were dried and then ground with an electric grinder and filtered with a 0.25 mm mesh screen. The abundance of ¹⁵N and ¹³C were measured with a ZHT-03 mass spectrometer from the Beijing Analytical Instrument Factory (Chinese Academy of Agricultural Sciences). Three replicates were conducted for each treatment. The formula is calculated according to Wang et al (Wang et al., 2020).

The NO₃ ⁻ flux at root surface were measured using a scanning non-invasive micro-test technique system (NMT 100 Series, United States) by Xuyue Sci. & Tech. Co., Ltd., Beijing, China. Net fluxes of NO₃ ⁻. Each selected root was tested for 10 min, with 5 replicates per group. After the test, we analyzed the data with MageFlux (imFluxes V2.0; YoungerUSA LLC, Amherst, MA 01002, USA), a data analysis software provided by Xuyue company (Zheng et al., 2023).

The ammonium-nitrogen (NH₄ ⁺-N) concentration was quantified using Nessler’s spectrophotometric technique (Molins-Legua et al., 2006). The leaf extract was combined with hypochlorite and phenol in a very alkaline environment to produce a water-soluble blue indophenol, with its absorbance measured at 625 nm. The activity of nitrate reductase (NR) in the leaves was assessed using sulfanilamide colorimetry (Zhao and Cang, 2015). The leaf extract was combined with sulfanilamide in hydrochloric acid and N-1-naphtyl-ethylenediamine to produce a red azo dye, with absorbance measured at 520 nm. Glutamine synthetase (GS) activity was assessed using the plant GS enzyme activity assay kit, and glutamic acid synthetase (GOGAT) activity was evaluated using the plant GOGAT enzyme activity assay kit (Jiangsu Enzyme Label Biotechnology Co., Ltd, Jiangsu, China), in accordance with the manufacturer’s instructions (Sun et al., 2022).

Genes involved in N assimilation such as nitrate reductase (MdNR), glutamine synthetase 1 (MdGS1), and nicotinamide adenine dinucleotide-glutamate synthase (MdNADH-GOGAT); nitrate transporters such as nitrate transporter 1.1/1.2/1.5/2.1 (MdNRT1.1/1.2/1.5/2.1); Sucrose synthase (MdSUSY1); sucrose transporter (MdSUT1); Phosphosucrose synthase (MdSPS1) and hexokinase (MdHK6) were selected for transcript analysis by quantitative real-time PCR (qRT-PCR).

Total RNA was extracted using an RNAprep Pure Plant Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. The RNA was reverse-transcribed into cDNA using a RevertAid First Strand cDNA Synthesis Kit (TransGen) in a 20 mL reaction. The quantitative real-time PCR (qRT-PCR) was performed in a 20 mL reaction mixture containing 10 mL of Green qPCR SuperMix, 1 mL of cDNA, 2 mL (1 mL of upstream and 1 mL of downstream primers) of primers and 7 mL of ddH₂O. The relative gene expression levels were calculated by the 2eDDCT method (Lv et al., 2012), and the MdActin gene was used as the internal control. These qRT-PCR experiments were performed with three technical replicates and three biological replicates. The primers used for qRT-PCR were listed in Supplementary Table S1 .

The data presented as means (± SD) of three replicates. The data were subjected to one-way ANOVA and the least significant difference test (Duncan’s new multiple range method, p ≤ 0.05) using SPSS 20.0 software (Statistics software, version 20.0, IBM, USA). Significant differences were determined at a probability level of P ≤ 0.05.

Compared to CK, HN significantly inhibited the root growth of seedlings ( Figure 1A ). It was found that the MT treatment greatly improved the root morphology of plants that were given CK, and the HN+MT treatment greatly improved the effects of high N on plant root growth. For example, the root length, surface area, volume, and number of root tips were 14.79%, 27.29%, 46.53%, and 39.16% higher in the HN+MT treatment than in the HN treatment ( Figures 1B–E ). The root activity also showed the same trend ( Figure 1F ). Figure 1A shows that different treatments have different effects on M9T337 rootstock growth. The HN treatment can greatly lower the plant’s biomass in its roots and leaves. On the other hand, the HN+MT treatment can greatly improve M9T337 rootstock growth, which was slowed down by the high N treatment. Compared with HN treatment, HN+MT treatment increased root and leaf growth by 11.38% and 28.01%, respectively ( Figures 1G, H ).

Compared to CK, MT treatment increases the net photosynthetic rate of M9T337 rootstocks, HN treatment reduces the net photosynthetic rate of M9T337, while high N treatment with the addition of melatonin can significantly reduce the decrease in the net photosynthesis rate caused by high N. The HN+MT treatment of M9T337 rootstocks increased the net photosynthetic rate by 19.65% compared to the HN treatment ( Figure 2A ). MT and HN treatment significantly increased the intercellular CO₂ concentration (Ci) of M9T337 rootstocks and reduced the stomatal conductance (Gs) and transpiration rate (Tr) compared to the control. Compared to HN treatment, HN+MT treatment significantly reduces the intercellular CO₂ concentration of M9T337 rootstocks and improves the stomatal conductance and transpiration rate. Intercellular CO₂ concentration decreased by 12.94% compared to HN treatment, and stomatal conductance and transpiration rates increased by 18.08% and 11.20%, respectively ( Figures 2B–D ).

MT treatment can significantly increase SOD activity, POD activity, and APX activity, contrary to HN treatment. Compared with CK treatment, SOD activity, POD activity, and APX activity of MT treatment were 12.56%, 19.45%, and 34.70% higher, respectively. However, compared with HN treatment, HN+MT treatment can significantly increase SOD activity, CAT activity, and APX activity under high N stress. Compared to HN treatment, HN+MT treatment had higher SOD activity, CAT activity, and APX activity (11.05%, 12.90%, and 5.92%, respectively) ( Figures 2E–I ).

The soluble sugar content in rootstock leaves and roots under different treatments were determined ( Figure 3 ). The content of sorbitol and sucrose in leaves under HN+MT treatment was lower than that under HN treatment and higher than that under MT treatment, while the content of sorbitol and sucrose in leaves under HN+MT treatment was higher than that under HN treatment and lower than that under MT treatment. Compared to the MT treatment, the contents of sorbitol and sucrose in leaves treated with HN+MT increased by 18.18% and 23.77%, respectively. In roots treated with HN+MT, the content of sorbitol and sucrose increased by 20.71% and 30.91%, respectively, compared to HN treatment ( Figures 3A, B, E, F ).

Leaves and roots treated with HN+MT had significantly higher fructose and glucose contents than those treated with HN. Compared with HN treatment, the fructose and glucose contents in leaves under HN+MT treatment were increased by 58.33% and 4.83%, respectively, and the fructose and glucose contents in roots were increased by 28.88% and 17.19%, respectively ( Figures 3C, D, G, H ).

The rhizosphere NO₃ ^– velocity of M9T337 rootstocks was significantly different under different treatments ( Figures 4A, B ). Negative values indicate NO₃ ^– influx, i.e., NO₃ ^– absorption, and positive values indicate efflux. Compared to CK, MT treatment promoted NO₃ ^– absorption, with the average value changing from -26.11 pmol·cm^–2·s^–1 to -35.73 pmol·cm^–2·s^–1. In HN treatment, NO₃ ^– is effluxed by roots, while in HN+MT treatment, NO₃ ^– flow in rhiza is influxed, with an average rate of around -10.21 pmol·cm^–2·s^–1. The results showed that high N treatment was not conducive for NO₃ ^– absorption, while high N treatment after adding melatonin is favorable to NO₃ ^– absorption.

N-metabolizing enzymes play important roles in the process of N assimilation. Under different treatments, the activity trend of NR, GS, and GOGAT in leaves and roots remained the same. Compared to CK, MT treatment increased the activities of NR, GS, and GOGAT, while HN treatment inhibited these three enzymes. MT+HN treatment could reduce the inhibition of NR, GS, and GOGAT activities in high N treatment. Compared to HN treatment, NR, GS, and GOGAT in leaves increased by 12.84%, 20.49%, and 43.92%, respectively, while NR, GS, and GOGAT in roots increased by 12.84%, 34.44%, and 61.54%, respectively ( Figures 4C–E ). According to Figures 4F–H , it can be found that, compared with CK, the nitrate N and amino acid contents of leaves and roots under HN treatment significantly increased, while the soluble protein contents decreased. Compared with HN treatment, MT+HN treatment increased soluble protein content.

The expression changes of the N transporter gene and the N channel protein gene in the root and leaf systems are shown in Figures 5A–N . The transcription levels of NRT1.1, NRT1.2, and NRT2.1 in the roots of M9T337 stock treated by HN were significantly lower than those treated by HN+MT. Under MT treatment, the expression levels of MdNR, MdGS1, MdGOGAT, MdSUSY1, MdSUT1, and MdHK6 in the root were the highest. These results indicated that high N significantly inhibited the expression of the N transporter gene and the N channel protein gene. Compared with HN treatment, the expression of the N transporter gene and the N channel protein gene in the root and leaf was significantly up-regulated by high N treatment with melatonin.

According to the heat map of relative expression analysis in Figure 5O , the expression of the N transporter gene and the N channel protein gene in roots and leaves was significantly down-regulated under HN treatment, while HN+MT treatment could significantly up-regulate the expression of the N transporter gene and the N channel protein gene in roots and leaves.

Under different treatments conditions, ¹³C accumulation was highest in all of the organs under the MT treatment, followed by the HN+MT and CK treatments. The least ¹³C accumulated in all of the organs and the entire plant under the HN treatment ( Figure 6A ). Compared with HN treatment, the root, stem, leave and total ¹³C accumulation of HN+MT treatment were 23.85%, 7.63%, 20.44% and 17.53% higher, respectively ( Figure 6A ). The distribution rate of ¹³C was mainly concentrated in the root of HN treatment, the stem of MT treatment and the leaf of HN+MT treatment ( Figure 6B ). The ¹⁵N accumulation of CK and MT treatment was significantly higher than that of HN and HN+MT treatment. Compared with HN treatment, root, stem, leaf and total N accumulation of HN+MT treatment were 11.45%, 3.52%, 14.98% and 11.17% lower, respectively ( Figure 6C ). It can be seen from Figure 6D that the C/N accumulation rate of HN+MT treatment and MT treatment is higher than that of CK and HN treatment. Compared with HN treatment, the C/N accumulation rate of HN+MT treatment was 32.55% higher.

In order to study the effects of different treatments on the rootstock of seedling M9T337, correlation analysis was carried out on its growth conditions, antioxidant protective enzymes (SOD, POD, CAT, APX) activity, soluble sugar content, carbon and N accumulation and distribution. As can be seen from Figure 7 , leaf sucrose content was significantly negatively correlated with root activity, leaf glucose content, root glucose content, volume of root, APX activity and POD activity. The was negatively correlated with SOD activity, root sorbitol content, root length and root activity. Leaf fructose content was positively correlated with ¹³C accumulation, root fructose content, CAT activity, number of root tips, APX activity and POD activity. Among them, ¹³C and ¹⁵N are negatively correlated.

The major way that crops take in water and nutrients from the soil is through their roots. Proper root shape and structure are critical for plant growth and development as well as for the uptake of nutrients from the soil (Mulaudzi et al., 2020). Plants frequently allocate biomass preferentially to the roots in order to increase their capacity for nutrient uptake in response to nutritional deficiencies. For instance, low P stress encourages lateral root growth and increases the number of root hairs, while low N stress induces root elongation (Hu et al., 2024). The findings indicated that melatonin may successfully reduce the negative effects of excessive N on root development and vitality. The use of melatonin may effectively ameliorate the reduction of root length, root spots, root surface area, and root volume caused by high N pressure. Properly increasing the melatonin supply helps optimize the form and structure of roots in high-N environments by maximizing the absorption of N, which in turn encourages root development ( Figures 1A–F ). We conducted further measurements of the apple rootstocks’ biological mass. Our findings indicate that the application of high levels of N hindered plant development. Moreover, the addition of melatonin successfully mitigated the inhibitory effects of high levels of nitrate on plant growth ( Figures 1G–I ). These findings are concordant to the results of Zhang et al. (2017).

We also identified the antioxidant system’s metabolites and associated enzyme activity in the rootstock roots. The ROS equilibrium is maintained by plants by means of their inherent antioxidant systems (Boaretto et al., 2020). Among them, APX, CAT, POD, and SOD are crucial for sustaining and scavenging ROS (Choudhury et al., 2017). Under normal development circumstances, antioxidant protection enzyme activity is in a balanced state, but under high N suppression conditions, SOD activity, CAT activity and APX activity falls, weakening the function of root antioxidants. It lowers the efficiency of H₂O₂ clearance, promoting membrane lipid peroxidation, finally leading to root oxidation damage. This explains why excessive nitrate on produces greater oxidative damage to the root ( Figures 2E–I ). However, melatonin can significantly alleviate the peroxide damage caused by high N, and is conducive to removing hydrogen peroxide and reducing lipid peroxidation.

Plants convert CO₂ dioxide into carbohydrates via photosynthesis, and these carbohydrates account for 90% of the biomass and agricultural productivity (Simkin et al., 2019). The carbohydrates essential for root development are transferred from the leaf, which shows that carbon partitioning is also a significant factor controlling root growth (Lambers et al., 2002). Some studies have shown that improving nitrogen (N) efficiency can lead to increased photosynthetic rates (Cechin and Valquilha, 2019). However, there is also evidence suggesting competition between nitrogen and carbon metabolism under high nitrogen conditions (Bloom, 2015). Our research indicates that under high nitrogen supply, the Pn, Gs, and Tr of apple rootstocks significantly decreased, while the addition of melatonin significantly enhanced photosynthetic capacity.

The effect of N in root growth promotion indicated via connections with carbohydrate content and metabolism (Li et al., 2018). In apples and certain other Rosaceae species, sorbitol and sucrose are the principal end-products of photosynthesis and are the main types of carbohydrates carried across large distances in the phloem (Sha et al., 2020). The findings revealed that high amounts of N compulsion and melatonin had a substantial influence on the accumulation and distribution of soluble sugar in M9T337 rootstock. Research has found that N fertilizer and melatonin have significant effects on plant carbon metabolism (Yan et al., 2023). In the absence of an increase in melatonin, high N coercion raised the level of peanut and mercury in the leaves and dramatically decreased the amounts of root-based peanut alcohol and mercury, showing that high nitrous coertion prevented the transfer of leaves from leaves to roots. This is comparable to the results of the research on apple (Malus hupehensis) (Zhao et al., 2020). Our results also show that high N stress limits the transport of photosynthetic products, as sorbitol content in leaves increases while sorbitol content and sucrose content in roots decreases. Increasing the supply of melatonin helps to alleviate the restriction of high N on the transport of photosynthetic products ( Figure 3 ). Sorbitol and sucrose, as photosynthetic products can be degraded to fructose and glucose by SDH and SuSy after unloading to the root via long-distance transport in the phloem (Zhao et al., 2020). High N coercion reduces the content of fructose and glucose in the root. High nitrous coertion may reduce the activity of the related enzymes, resulting in a decrease in the concentration of frucose and glycine in the roots, thereby affecting the cycle of triglyceride and starch synthesis, inhibiting root growth (Li et al., 2012). Further analysis shows that the addition of melatonin in high N conditions promotes the phosphoridation of the fructose and glucose, thus enhancing the absorption and utilization of M9T337 rootstocks.

To further investigate the accumulation and distribution of photosynthetic products, M9T337 rootstock was tagged with ¹³C isotope. We found that the accumulation of ¹³C in leaves, stems and roots was the highest with melatonin administration, whereas the accumulation of ¹³C in roots under high N treatment was the lowest. High N treatment hindered N buildup and lowered C/N accumulation rate. Therefore, high N treatment inhibited the transport of photosynthetic products to roots, while appropriately increasing the melatonin supply level could increase and promote the distribution of sorbitol and sucrose from leaves to roots and increase the inhibition, thus reducing the inhibition of high N treatment on root growth.

The application of N is frequently used in excess to achieve increased crop yields (Sun et al., 2020). Nevertheless, this study discovered that an overabundance of nitrate supply hindered the growth of the M9T337 apple rootstocks’ roots and decreased the ratio of roots to shoots, which was unfavorable for the absorption of nutrients and the growth of the rootstocks. After nitrate is absorbed and transported by the roots, it is reduced to nitrite, glutamine, and glutamate in sequence under the action of NR, GS, and GOGAT, and then further metabolized into amino acids and nitrogen-containing compounds. We quantified the absorption of NO₃ ⁻ with the use of non-invasive micro measuring techniques. The net fluxes of NO₃ ⁻ at the root surface was effluxed under circumstances of elevated nitrate levels, suggesting that high N concentrations hindered the absorption of NO₃ ⁻. This is due to when the intracellular N levels are too high, key enzymes in the metabolic pathways are inhibited to prevent further accumulation of N. N sensors, such as TARGET OF RAPAMYCIN (TOR) signaling pathway, are activated to reduce N uptake and utilization (Artins et al., 2024). This mechanism helps cells maintain N balance and avoid toxicity from excess N. It has been reported that genes encoding enzymes related to N assimilation are regulated by carbon and N interactions (Guti´errez et al., 2007). Our findings revealed that under circumstances of high N levels, the activities and expression of NR, GS, and GOGAT were dramatically suppressed, while melatonin can significantly relieve the symptoms, which may be related to the fact that melatonin changed the C/N ratio of seedlings.

The absorption of nutrients in plants is regulated by both internal signals inside the plant and external signals from the environment. When the intensity of nutrient supply surpasses the concentration needed for the plant to develop at its maximum rate, the uptake of nutrients is primarily controlled by the intensity of the nutrient pool. Nitrate uptake is mostly attributed to NRTs (Wang et al., 2012; Yuan et al., 2022). MdNRT1.1, MdNRT1.5 and MdNRT2.1 are significant NRTs that are predominantly engaged in NO₃ ⁻ absorption in the roots (Xuan et al., 2017). We observed that the expression levels of NRT1.1, NRT1.2 and NRT2.1 in the roots fell dramatically under high N conditions, which explained why high N stress hindered N uptake. However, it is not obvious if the reduced expression of these genes is regulated by internal or external N levels, and its precise mechanism requires additional exploration.

Excessive N application hampers the growth of M9T337 rootstocks by decreasing root-to-shoot ratios and nutrient absorption. High nitrate levels inhibit NO₃ ⁻ uptake, activate N sensors like the TOR pathway, suppress key N metabolism enzymes, and inhibit the synthesis of soluble proteins, while melatonin can alleviate these effects. Additionally, the expression of vital nitrate transporters decreases under high N conditions, highlighting the intricate relationship between nitrogen levels and nutrient absorption, with melatonin serving as a potential mitigator of high N stress.