Molybdenum-induced effects on nitrogen absorption and utilization under different nitrogen sources in Vitis vinifera

ABSTRACT
 Nitrogen (N) in different forms has been demonstrated to play significant roles in plants. However, little is known about molybdenum (Mo) effects on N absorption and utilization in grapevine seedlings grown under different N sources. The present study used a sand culture system to analyze the impact of Mo application (0 μM; 1 μM) on N absorption and utilization in grapevine (Vitislabrusca × V. vinifera ‘Shine Muscat’ (rootstock 3309 m)) young potted seedlings under different N sources (NO3 −, NH4NO3 and NH4 +). The different N forms and Mo application significantly influenced dry matter accumulation, and root architecture and activity. The effects of Mo on total N content followed the order of (NH4NO3 > NO3 − > NH4 +). Moreover, Mo and N induced VvMOT1 and VvNRT1.1 expression synergistically. Mo supply altered the utilization of NO3 −, NO2 −, and NH4 + in grapevines under different N sources. NH4NO3 showed the highest effect while NH4 + the least. Furthermore, the 15N-labeling experiment showed that the 15N content in shoot and root and the 15N-use efficiency were the highest after Mo application under NH4NO3 source, indicating the synergistic effects of Mo with the co-application of NO3 − and NH4 + sources. The study’s findings provide insights on Mo and N fertilizer utilization for cultivation and production practices in fruits.


Introduction
Nitrogen (N) is a primary macronutrient essential for plant function under natural ecosystems and production systems (Marschner 1995;Bell and Henschke 2005;Siddiqui et al. 2012;Rahman et al. 2021). Plants absorb two primary inorganic N forms, nitrate (NO 3 − ) and ammonium (NH 4 + ), from the soil (Li et al. 2013. NO 3 − needs to be converted to NH 4 + before it can be assimilated and used by plants . NH 4 + is assimilated into organic compounds via the glutamine synthetase (GS)/glutamate synthase (GOGAT) cycle. Studies have reported differences in effects on plant physiology among the various N forms. NH 4 + as the sole N source reduces N-use efficiency (NUE), root activity, nitrate reductase (NR) and sucrose phosphate synthase activities, overall photosynthesis, and N metabolism (Drath et al. 2008;Zhang et al. 2012;Li et al. 2013). Meanwhile, NO 3 − is not only an essential source of nitrogen for plants, but also functions as a signaling molecule. NO 3 − stimulates whole plant nutrient transport and assimilation in response to environmental changes (Liu, Niu, et al. 2017). Zhang et al. (2012) reported lower NUE and root activity in tobacco under a floating seeding system with NH 4 + than those with NO 3 − or urea ((NH 2 ) 2 CO-N)as the N source. Genomics and proteomics have shown that NO 3 − regulated the expression of genes encoding NR and a nitrate transporter (NRT1.1) and rapidly activated NO 3 − signaling (Wang et al. 2012;Alvarez et al. 2014;Bouguyon et al. 2016). Besides, research has demonstrated complementary effects and plant benefits with the application of a mixture of NO 3 − and NH 4 + than with either NO 3 − or NH 4 + source given alone (Britto and Kronzucker 2013;Zheng et al. 2015;Qin et al. 2017;Liu et al. 2019). A good N-absorption efficiency guarantees plant growth, development, and yield (Hirel et al. 2001;Kraiser et al. 2011;Xu et al. 2011). Several studies have positively correlated chlorophyll content, photosynthesis, and plant biomass with N availability . Still, less studies have reported the changes in these effects of different N sources in the presence of other important minerals (such as molybdenum) in plants. Molybdenum (Mo) is a trace element essential for plants (Arnon and Stout 1939;Mulder 1954;Siddiqui et al. 2021;Alamria et al. 2022). It is a key catalytic component of the NR molybdenum cofactor (Moco) with a crucial role in NO 3 − metabolism in higher plants (Mulder 1954;Kaiser et al. 2005;Mendel and Schwarz 2011;Tejada-Jiménez et al. 2013). However, excessive amounts of N and phosphate fertilizers with inadequate trace elements and organic fertilizers have resulted in soil acidification, which tends to bring about Mo deficiency problem (von Uexküll and Mutert 1995;Wang et al. 2002;Gao et al. 2016). Mo deficiency is common in plants grown in well-drained soils, acidic soils, and iron oxide-rich soils (Kaiser et al. 2005). Almost 70% of arable land in the world is acidic, while nearly 4 million ha of cultivable land in China is deficient in Mo (Wang et al. 2002). The traditional Mo application practices are the foliar application (0.02-0.05%) during the flowering period or period of plants showing symptoms of Mo deficiency(which is often confused with N deficiency). In China, the soil available molybdenum content of the Southwest producing area and the Vitis amurensis of Northeast producing area even below 0.1 mg kg −1 . The Arid and Semi-arid production area in the Loess Plateau, the Middle and Lower reaches of the Yellow River producing area and the Southern producing area were at the level of 0.10∼0.15 mg kg −1 . The threshold value of soil Mo deficiency is 0.15 mg kg −1 (Liu 2017). Therefore, there is an urgent need to correct Mo deficiency. Therefore, Mo deficiency is often associated with N stress (Dijkshoorn and Ismunadji 1972), increased NO3-concentration (Kaiser et al. 2005), and reduced plant biomass and yield (Kovács et al. 2015).
The deficiency of Mo mainly affects the biosynthesis of Moco, present in Mo enzymes, such as NR, sulfite oxidase (SO), aldehyde oxidase (AO), xanthine dehydrogenase (XDH), and the mitochondrial amidoxime-reducing component (mARC). In plants, the enzyme NR catalyzes the conversion of NO 3 − to nitrite (NO 2 − ); it regulates molybdate transporters (MOT1, molybdate transporter type 1) and NRTs at the transcript level (Sun et al. 2015;Liu et al. 2020) and plays a vital role in N fixation and assimilation (Mendel and Schwarz 2011). Studies have shown that the deficiency of Mo causes symptoms, such as leaf yellowing (Sun et al. 2009;Gao et al. 2016), small taproots and lateral roots (Gao et al. 2016), and irregular chloroplasts and unclear membrane structures (Liu et al. 2020), which are noticeably similar to N deficiency symptoms (Mulder et al. 1959). Studies have reported impaired MOT1 expression, molybdate (MoO 4 2− ) absorption, and NR activity in MOT1deficient mutants. The atmot1;2 Arabidopsis mutant had lower NO 3 − levels and NR activity than the wild-type plants (Gasber et al. 2011). These earlier findings suggested that Mo deficiency significantly affects N acquisition and assimilation. Meanwhile, a mutant deficient in NRT1.1 showed decreased NO 3 − absorption into guard cells (Wang et al. 2012), suggesting that regulating NRT1.1 may influence photosynthesis and N acquisition. However, no study has reported the effects of Mo application on plant Mo concentration and N absorption and its utilization in the presence of different N forms.
Understanding the physiological changes and the mechanisms improving photosynthesis through efficient N acquisition and utilization in grapevines with Mo application will contribute to less fertilizer use and better fruit quality. Therefore, the effects of Mo application with different N forms/sources on Mo absorption, chlorophyll content, root morphology and activity, and nitrate metabolism in grapevines were investigated. The study's findings will help understand the correlation between Mo and N and provide a scientific basis for rational N and Mo fertilization programs.

Measurement of growth parameters, root morphology, and activity
The whole seedling was separated into shoots and roots and dried first at 105°C for 30 min and then at 80°C for five days. The shoot and root dry weight and the total plant dry weight were recorded.
The roots were thoroughly washed, and the morphological features were determined using the WinRhizo image analysis system (V4.1 c; Regent Instruments, Quebec, Canada). Root activity was measured following the TTC (2,3,5-triphenyltetrazolium chloride) method (Liu et al. 2020).

Determination of Mo concentration, chlorophyll content of leaves, amino acid, and soluble protein content
Fresh grapevine samples (5-8 leaves) were separated into roots and shoots, dried to constant weight, and ground to a fine powder using a pulverizer. The powder was digested with 5 mL of 65% nitric acid (v/v) overnight, and the ash was dissolved in 2 mL of 30% hydrogen peroxide (H 2 O 2 , v/v). The Mo concentration in the sample was analyzed using a mass spectrometer (NexION™ 300 ICP-MS system; PerkinElmer, Waltham, MA, USA) following the method by Filipiak-Szok et al. (2014).
Chlorophyll was extracted from leaves with 50 mL of 80% aqueous acetone, and the absorbance values at 645 and 663 nm were measured using a spectrophotometer to determine the chlorophyll a and b concentrations (Liu, Xiao, et al. 2017).
The amino acid concentrations were examined using a Ninhydrin (triketohydrindene hydrate) Assay Kit (Comin, Suzhou, China) by absorption spectrometry. The sample solution (1 mL) was ground in 1 mL acetate buffer (2 mmol L −1 , pH 5.4) and 1 mL ninhydrin, which was incubated for 15 min in a boiling water bath and then cooled in water. After 5 min, the above mixture was diluted with 3 ml 60% ethanol. The absorbance of the mixture at 570 nm was read using a spectrometer.
The soluble protein contents were analyzed using a BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). The sample solution (0.1 ml) was ground in 2 mL BCA detection reagent and incubation for 30 min at 37°C. After cooling to room temperature, the sample absorbance was determined at 562 nm for the measurement of the soluble protein content. The total N content was measured using the Kjeldahl method (Ding et al. 2017).
The NO 3 − concentrations in the shoot and root samples were determined using the specific kits (Comin, Suzhou, China). Plant samples (1 g) were transferred to a 10 mL test tube and about 5 mL of water was added, which was then incubated for 30 min in a boiling water bath. The homogenates were centrifuged at 12,000g for 20 min after cooling and the supernatant was transferred to a tube to measure the NO 3 − content using the salicylic acid method. The NH 4 + concentrations in the shoot and root samples were measured by the specific kits following the manufacturer's guidelines (Comin, Suzhou, China). The reaction mixture contained 0.1 mL filtrate, 0.01 mL 10% K-Na tartrate, 2.4 mL redistilled water, and 0.1 mL Nessler reagent. A spectrophotometer was used to measure the absorbance at 425 nm after 5 min.

The 15 N labeling method and measurement of Nuse efficiency (NUE)
Grapevine seedlings were cultured with modified Hoagland solution with Ca( 15 NO 3 ) 2 (10.20 atom% 15 N, Shanghai Research Institute of Chemical Industry, SRICI), 15 NH 4 15 NO 3 (10.20 atom% 15 N, SRICI), and 15 NH 4 Cl (10.20 atom% 15 N, SRICI) for treatments with NO 3 − , NH 4 NO 3 , and NH 4 + as sources, respectively, for 50 days to determine N absorption and NUE. The samples (roots and shoots)were washed by branch water, detergent, branch water, and 1% hydrochloric acid in order, and then with deionized water for 3 times. The roots and shoots were separately placed in paper envelopes and dried first at 105°C for 30 min and then at 80°C for five days. The roots and shoots were homogenized using a mortar and pestle, filtered with a fine-mesh sieve (0.25 mm). The samples were analyzed using a stable-isotope ratio mass spectrometer (MAT-251, Thermo Finnigan, San Jose, CA, USA) to determine the total N content, and 15 NO 3 − -use efficiency at the Institute for Application of Atomic Energy (IAAE) of the Chinese Academy of Agricultural Sciences (CAAS) (Clarkson et al. 1996).
The calculation of 15 N was measured according to Ding et al. (2017) as follows: (2)
NiR activity assay was measured as follows: The enzyme extract (0.2 mL) was mixed with 1 mL 0.1 mol L −1 potassium phosphate buffer (pH 7.5), 0.05 mL, 10 mmol L −1 KNO 2 , 0.05 mL 15 mg mL −1 methylviologen, and then 50 mg mL −1 sodium dithionite (Na 2 S 2 O 4 ), dissolved in 100 mmol L −1 NaHCO 3 was added to initiate the reaction. The mixture was incubated for 30 min at 25°C and was terminated by vortexing until the methylviologen color had completely disappeared. The residual NO 2 − in the reaction mixture was measured by combining 0.2 mL of the mixture and 6.5 mL of water with 1.8 mL 10% (w/v) sulfanilamide prepared in HCl and 1.5 mL 1% (w/v) N (1-naphty1)-ethylenediamine dihydrochloride. The absorbance of the mixture was measured at 540 nm.
GS activity assay followed the protocol of our previous study (Liu, Xiao, et al. 2017). Fresh roots and shoots samples were ground in an extraction buffer containing 50 mmol L −1 Tris-HCl (pH 8.0), 2 mmol L −1 MgSO 4 ·7H 2 O, 2 mmol L −1 DTT, and 0.4 mol L −1 sucrose. The homogenate was centrifuged at 15,000g for 20 min at 4°C. Reaction mixture A comprised 0.1 mol L −1 Tris-HCl (pH 7.4), 80 mmol L −1 MgSO 4 ·7H 2 O, 20 mmol L −1 glutamic acid-Na, 20 mmol L −1 cysteine, and 2 mmol L −1 EGTA, and reaction mixture B contained mixture A and 80 mol L −1 hydroxylamine hydrochloride (pH 7.4). The final reaction mixture comprised of 1.6 mL of mixture B, 0.7 mL of the crude enzyme extract, and 0.7 mL of 40 mmol L −1 ATP, and was incubated for 30 min at 37°C. The reaction was ended with 1 mL of a color agent containing of 0.2 mol L −1 TCA, 0.37 mol L −1 FeCl 3 , and 0.6 mol L −1 HCl in 2% HCl. The absorbance of each supernatant was determined at 540 nm.

Total RNA extraction and quantitative RT-PCR
The roots and leaves were harvested and immediately frozen in liquid nitrogen. The total RNA was extracted from these samples using an RNA extraction kit (Tiangen Biotechnology, Beijing, China). The PrimeScript™ RT Reagent Kit with gDNA Eraser (Perfect Real Time) was used to generate the complementary deoxyribo nucleic acid (cDNA) for quantitative real-time polymerase chain reaction (qRT-PCR). The expression levels of Mo transporter genes, NO 3 − transporter genes, and Moco biosynthetic genes were measured using the SYBR Premix Ex Taq system (Takara Biotechnology, Dalian, China). The genes and primer sequences are listed in Supplementary  Table S2. The qRT-PCR was performed with an initial denaturation at 95°C for 10 min, followed by 40 cycles of amplification. The data were analyzed following the comparative threshold cycle (CT) (2 −ΔΔCT) method (Liu et al. 2020), using the Vvactin gene as the reference. Three independent biological replicates and three technical replicates were used for the analysis.

Statistical analysis
Data were analyzed using SPSS Statistics (Version 19.0; IBM Corp., Armonk, NY, USA). Duncan's multiple range test was used to measure the differences between pairs of means at P < 0.05. GraphPad Prism (Version 6.0) was used to plot the graphs and do an ANOVA to compare the treatments.

Mo application influences the plant parameters (growth, dry weight, root architecture, and activity) of grapevines seedlings under different N sources
Seedlings cultured with Mo grew better than those without Mo under different N supply, especially under NO 3 − or NH 4 NO 3 supply (Figure 1).
Among the different N forms applied, NH 4 NO 3 resulted in the highest plant dry weight while NH 4 + alone resulted in the lowest (Table 1). Mo application increased the shoot dry weight by 10.40%, 15.73%, and 9.94%, and the root dry weight by 16.43%, 34.84%, and 15.88% in the grapevine seedlings under NO 3 − , NH 4 NO 3 , and NH 4 + sources, respectively (Table 1). Mo application significantly increased the plant dry weight compared with the −Mo treatments (Table 1).
Among the various N sources, NH 4 NO 3 application led to the highest root architectural parameters and root activity, while NH 4 + alone resulted in the lowest values. Mo application significantly increased root length, volume, surface area, forks per root, tips per root, and root activity of grapevine seedlings (Table 2). Meanwhile, no considerable difference was observed in average root diameter between −Mo and +Mo treatments (  letters (a, b, c, d) indicate significant differences between the treatments (P < 0.05; Duncan-test).Significance levels are shown as:*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, and ns = non-significant. NH 4 NO 3 , and NH 4 + sources, respectively, compared with −Mo seedlings (Table 2).

Mo application influences Mo concentration, chlorophyll content, amino acids contents and the soluble protein under different nitrogen sources in grapevine seedlings
Mo application markedly increased Mo concentrations in shoot and root of grapevine seedlings under N supply (Table 3). Mo concentration was considerably higher in roots under NH 4 NO 3 than with NO 3 − or NH 4 + source alone (Table 3). However, no significant difference in Mo concentration in shoot and root was observed among the seedlings under different N sources grown without Mo (Table 3). The chlorophyll (a + b) content was maximum in grapevine seedlings cultured with NH 4 NO 3 in −Mo and +Mo treatments (Table 3). Compared with −Mo treatments, Mo application increased chlorophyll (a + b) content under NO 3 − and NH 4 NO 3 sources (Table 3). However, no differences were detected in chlorophyll b content and chlorophyll a/b ratio (data not shown).
Seedlings grown with NH 4 NO 3 as the N source exhibited the highest amino acid content among the treatments Table 2. Effects of molybdenum (Mo) application on the root length (A), root volume (B), tips per root (C), forks per root (D), surface area (E), average root diameter (F), and root activity (G) of grapevine seedlings under different nitrogen (N)  , and NH 4 NO 3 represent sole nitrate source, sole ammonium source, and co-application of ammonium and nitrate sources. Data are represented as mean ± SE (n = 3). Different lowercase letters (a, b, c, d) indicate significant differences between the treatments (P < 0.05; Duncan-test). Significance levels are shown as:*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, and ns = non-significant. , and NH 4 NO 3 represent sole nitrate source, sole ammonium source, and co-application of ammonium and nitrate sources. Data are represented as mean ± SE (n = 3). Different lowercase letters (a, b, c, d) indicate significant differences between the treatments (P < 0.05; Duncan-test). Significance levels are shown as:*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, and ns = non-significant.
( Table 3), consistent with the soluble protein concentrations in roots and shoots of grapevine seedlings (Table 3).

Effects of Mo application on the total N content, NO 3 − content and NH 4 + content in grapevine seedlings under different N sources
The N content in roots showed a significant difference when cultivated with different N supply treatments (Table 4). The shoots showed trends with a higher content in seedlings cultivated with NH 4 NO 3 than those with NO 3 − or NH 4 + . Mo increased the total N content in shoot and root of grapevine seedlings (Table 4). The total N content was the highest under NH 4 NO 3 treatment after Mo supply (Table 4). The NO 3 − concentration in roots and shoots was analyzed. Mo supply observably reduced the NO 3 − content in the presence of NO 3 − alone and with the co-application NH 4 NO 3 compared with NH 4 + alone (Table 4). Additionally, NH 4 + concentration was higher in the leaves and roots of grapevine seedlings with Mo than those without Mo in the presence of NO 3 − and NH 4 NO 3 sources (Table 4).

Mo application improves N absorption and NUE in grapevine seedlings under different N sources
The 15 N-labeling experiments showed that 15 N absorption in shoots and roots was the highest after Mo application under NH 4 NO 3 (  (Table 5).

Effects of Mo on the N metabolic enzyme activities and gene expression of VvMOT1 and VvNRT1.1
The NR activity was high in both roots and shoots of grapevine seedlings under NO 3 − and NH 4 NO 3 sources (Table 6); however, no difference was detected in seedlings with only NH 4 + as the N source between + Mo and −Mo treatments (Table 6). Meanwhile, NiR activity in the leaves and roots of grapevine seedlings cultured with Mo was higher than those without Mo, especially in the presence of NH 4 NO 3 (Table 6).
Mo application significantly increased GS activity in roots of grapevine seedlings under the NH 4 NO 3 source (Table 6). Meanwhile, GS activity in the shoots of grapevine seedlings with Mo was markedly higher than those without Mo under all sources (Table 6). A similar trend was observed in NADH-GOGAT activity in the grapevine seedlings (Table 6).
Mo application upregulated VvMOT1 gene expression in the shoots and roots in the presence of N sources (Figure 2  (A, B)), indicating the role of Mo in activating the VvMOT1 transcript. Under Mo application, the VvMOT1 expression level reached a maximum in both roots and shoots with N source NH 4 NO 3 (Figure 2(A, B)
Grapevine seedlings rely on their roots to perceive the changes in soil nutrients and ensure optimal absorption of nutrients. Therefore, root activity, size, and morphology are indices used to evaluate the nutrient absorption ability of grapevine seedlings (Gruber et al. 2013). In the present study, the results showed that different N forms significantly affected the root parameters, such as root length, root volume, tips per root, forks per root, surface area, and root activity, followed the same order of NH 4 NO 3 > NO 3 − > NH 4 + . In soybean, peanut, and strawberry, Mo application enhanced root activity (Liu and Yang 1999;Zhang et al. 2011;Liu et al. 2020). Nonetheless, the role of Mo in promoting nutrient absorption and subsequent plant growth by affecting root architecture remains unclear. In this study, Mo application increased root length, root volume, root surface area, tips per root, forks per root, and root activity of grapevine seedlings under different N sources, indicating the sensitivity of roots (morphology and activity) to Mo availability. Previous research has identified that longer roots show a larger root surface area, leading to more N absorption (Liu et al. 2020). Therefore, better root parameters indicate that Mo application might lead to efficient N absorption and assimilation. Interestingly, our results indicated that the interaction between Mo and N on root length, root volume, and root activity is synergetic, which might be beneficial to increase its absorption capacity for nutrients (N, Mo) of grapevine seedlings.
Our data showed that Mo application significantly increased Mo concentration of roots and shoots (Table 3). Similar reports were reported in winter wheat (Imran et al. 2019) and strawberry (Liu et al. 2020). However, the effect of the N treatments (different N forms) or the interaction between Mo and N on Mo concentration was not significant. The higher chlorophyll content has a positive correlation with plant growth and development . In terms of chlorophyll content seedlings responded with a significant difference in the N forms tested (Table 3). In addition, Mo induced significant increases in the chlorophyll content under NO 3 − and NH 4 NO 3 treatments, which was also reported by Imran et al. (2019). The chlorophyll content was maximum in grapevine seedlings cultured with NH 4 NO 3 and Mo, consistent with the highest N content and highest total biomass. Our results revealed that NH 4 NO 3 can obviously enhance amino acid and soluble protein of the grapevine seedlings and Mo supply exhibited induced effects on amino acid and soluble protein, indicating more nutrient contents in seedlings.
Plant cells absorb Mo, as MoO 4 2− , through molybdate transporters, especially those of the MOT1 family. In the present study, MoO 4 2− in the nutrient solution influenced VvMOT1 expression and its role in MoO 4 2− transport. Mo application markedly enhanced the expression of VvMOT1 in the shoots and roots under different N sources, consistent with the increased Mo concentration in the seedlings. Mo application increased vine Mo concentration under different N sources. These findings are consistent with Arabidopsis, strawberry, and rice (Tomatsu et al. 2007;Liu, Xiao, et al. 2017;Huang et al. 2018;Liu et al. 2020). Yet, the Table 6. Nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), and NADH-glutamate synthase (NADH-GOGAT) activities in roots and shoots of grapevine seedlings under different nitrogen (N)  , and NH 4 NO 3 represent sole nitrate source, sole ammonium source, and co-application of ammonium and nitrate sources. Data are represented as mean ± SE (n = 3). Different lowercase letters (a, b, c, d) indicate significant differences between the treatments (P < 0.05; Duncan-test).Significance levels are shown as: *P ≤ 0.05, **P ≤0.01, ***P ≤ 0.001, ****P ≤ 0.0001, and ns = non-significant.
characterization of VvMOT1 involved in MoO 4 2− absorption requires detailed analysis. In plants, the transporter NRT1.1 is responsible for NO 3 − absorption from soil or nutrient solution. The N treatments significantly influenced the expression of NRT1.1 by the ANOVA analysis ( Figure 2). Mo application enhanced the transcript levels of VvNRT1.1, which was consistent with Liu (2017) and Imran et al. (2019). The highest expression of NRT1.1 transporter gene was found in seddlings cultured with NH 4 NO 3 and Mo, indicating the combined effect of Mo and N on NRT1.1 expression is synergistic in grapevine young seedlings.
Our data showed that the N content in shoot showed no significant difference between NO 3 − and NH 4 + (but for NH 4-NO 3 )treatments. This finding was consistent with the findings of Lang et al. (2018) argue that none of the applied N forms (NO 3 −, NH 4 + , urea, arginine, and glutamine) influence the N content in leaves and wood in grapevine. However, the N content in root was significantly influenced by different N forms (Table 4). The N metabolic enzymes (NR, NiR, GS, NADH-GOGAT) activities were influenced by the N forms and Mo supply, but no significant difference was observed on the interaction between Mo and N ( Table  6). Mo application resulted in higher NR activity in both roots and shoots under different N sources, with a considerable effect in the presence of NH 4 NO 3 and NO 3 − sources. This observation is consistent with Imran et al. (2019) who indicates that the maximum NR activity in plants occurs when cultured with NH 4 NO 3 and Mo. As a result, Mo supply significantly decreased the NO 3 − content under NH 4 NO 3 and NO 3 − sources. In addition, grapevine seedlings grown with N in three different forms exhibited higher NiR, GS, and NADH-GOGAT activities, higher NH 4 + content, and total N content with Mo supply, suggesting Mo fertilizer's role in N absorption and assimilation. To verify the above results, this experiment with 15 N tracer technique revealed that N forms significantly influenced the 15 N absorption content and 15 NUE followed the order NH 4 NO 3-> NO 3 − > NH 4 + , which indicated that more 15 N was absorbed and utilized by seedlings grown with NH 4 NO 3 source. Obviously, with increasing 15 N absorption content, 15 NUE increased in seedlings supplied with Mo under all N sources. The data (Table 5) provided direct evidence that Mo and N promote 15 N absorption and 15 NUE in grapevine seedlings synergistically.

Conclusion
In grapevine seedlings, the N forms and Mo application significantly influenced dry weight and root architecture and activity. Mo application improved tissue Mo levels to ensure sufficient Mo for N assimilation. Additionally, the chlorophyll content, amino acid content, and the soluble protein were affected by the N forms and Mo application. Mo application induced VvMOT1 expression and NRT1.1 expression to ensure sufficient Mo and N absorption. The reduced tissue NO 3 − content and the enhanced NH 4 + content with higher N assimilation-related enzyme (NR, NiR, GS, and NADH-GOGAT) activities indicated improvement in N absorption and assimilation with NH 4 NO 3 and Mo supply. The 15 N absorption and 15 NUE improved after Mo application under different N sources, exhibiting a synergistic effect of Mo and N. Overall, Mo showed more complementary effects with nitrate-based sources than the sole NH 4 + source. In summary, Mo application together with nitrate-based nutrition may be used for grapevine in practice. Nonetheless, it is necessary to further explore the detailed interaction of Mo and N on N absorption and utilization in adult vines under field conditions. , and NH 4 NO 3 represent sole nitrate source, sole ammonium source, and co-application of ammonium and nitrate sources. Data are represented as mean ± SE (n = 3). Different lowercase letters (a, b, c, d) indicate significant differences between the treatments (P < 0.05; Duncan-test). Significance levels are shown as:*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, and ns = non-significant. Analysis of variance: A: N treatments ****, Mo treatments ****, N×Mo ****. B: N treatments ****, Mo treatments ****, N×Mo **. C: N treatments ****, Mo treatments ****, N×Mo ***. D: N treatments ****, Mo treatments ****, N×Mo *.

Notes on contributors
Li Liu, research assistant research interest: cultivation physiology and molecular biology of fruit tree, formation and regulation of fruit quality, mineral nutrition of fruit tree, nitrogen nutrition, molybdenum nutrition, calcium signaling.
Meng-meng An, research assistant research interest: cultivation physiology and molecular biology of fruit tree, mineral nutrition of Vitis vinifera.
Xiu-jie Li, associate professor, research interest:cultivation physiology of fruit tree, mineral nutrition of fruit tree, formation and regulation of fruit quality.
Zhen Han, research assistant, research interest: cultivation physiology and molecular biology of fruit tree, mineral nutrition of fruit tree, calcium nutrition.
Shao-xuan Li, associate professor, research interest: cultivation physiology and molecular biology of fruit tree, bioinformatics of fruit tree, photomorphogenesis.
Bo Li, professor, research interest: cultivation physiology and molecular biology of fruit tree, formation and regulation of fruit quality, mineral nutrition of fruit tree, nitrogen nutrition, calcium signaling.