Photosynthetic Performance and Urea Metabolism After Foliar Fertilization with Nickel and Urea in Cotton Plants

Abstract

The use of nickel (Ni) as a fertilizer remains a topic of debate, particularly in non-legume species, as Ni is required only in trace amounts for optimal plant function. Urea application in plants, whether foliar or root-based, relies on the urease enzyme to convert urea into NH_4⁺ and CO₂, with Ni serving as an essential cofactor. In this study, we conducted an experiment using a 2 × 2 factorial design, combining two urea concentrations [4% and 8% (w/v)] with the absence or presence of Ni (0.3 g L⁻¹ supplied as NiSO₄·6H₂O). Gas exchange parameters were measured two days after fertilization. We quantified urease enzyme activity, urea content, photosynthetic pigments, carbohydrates, and other nitrogenous metabolites. The presence of Ni during foliar urea fertilization significantly increased the photosynthetic rate and photosynthetic pigments, which we attributed to improved urea assimilation. The combination of urea and Ni enhanced urease activity, leading to higher levels of various nitrogenous metabolites. Ni positively influenced foliar urea assimilation, promoting its conversion into organic compounds, such as proteins, while mitigating the toxic effects associated with urea accumulation.

1. Introduction

Production costs and increased food demand are relevant for fertilizer use; this is inducing continuous pressure on the agricultural sector and industries to meet the growing food needs [1]. Nutrients such as N are of significant importance as their scarcity leads to rapid response and plants become more susceptible to abiotic and biotic stresses [2]; in this situation, a greater demand for inputs is common, requiring efficient use. Urea is considered an efficient source of N when it is applied to the leaves due to its high N content (46%), which is advantageous for cotton crops since cotton tolerates concentrated N solutions. Urea concentrations between 3 to 15% have been reported in cotton [3,4,5,6]. The application of urea on the leaves of cotton plants aims to provide N in a high yield, supplying N demand when the root extraction capacity is reduced during the flowering peak or late fruiting [7].

Dixon et al. [8] verified the presence of two Ni ions in the constitution of the metalloenzyme urease (urea amidohydrolase; EC 3.5.1.5.), which catalyzes the urea hydrolysis into ammonia (NH_4⁺) and carbon dioxide (CO₂). Since then, the scientific community has investigated the role of Ni in urea metabolism [9,10,11,12,13]. Studies have also deepened the knowledge of its role in other metabolic aspects, such as in the production of phytoalexins [14,15,16,17], in the hydrogenase enzyme [18], and in the glyoxalase I enzyme [19], which performs an important role in the degradation of a potent cytotoxic compound—methylglyoxal. This fact suggests that Ni may play an important role in the metabolism of antioxidants in plants, especially under stress [20].

Urease enzyme activity has been detected in several plants [21,22]. The absence of Ni compromises the biosynthesis of this enzyme, leading to urea accumulation and, consequently, necrotic lesions on leaf tips [10,14,23]. In situations where nitrogen (N) is supplied as urea in a hydroponic nutrient solution or via the leaves, the role of Ni becomes essential since it stimulates the primary metabolism and increases plants’ yield traits [24,25,26]. Ni and N application faces challenges in soils due to the low prevalence of soluble and readily absorbable urea [27].

The herbaceous cotton (Gossypium hirsutum L. r. latifolium Hutch.) is widely used for fiber production and the process to produce fibers demands high technology; therefore, there is a high monetary value associated with the fiber production chain due to the wide range of products produced [28,29]. Nitrogen is one of the most expensive inputs in the cotton production chain and needs 48 to 85 kg ha⁻¹ of N to produce 1 ton of seed cotton, depending on the variety used and edaphoclimatic conditions [30,31]. Nitrogen plays an important role in the plant [32] since it is a constituent or activator of enzymes, amino acids, nucleic acids, and chlorophyll [33], as well as stimulating the formation and development of flowering and fruiting buds [34].

Previous studies have reported that the application of Ni-associated N to the soil has no significant effect on the chlorophyll index (SPAD) [35] and on plant growth [36] in cotton plants. Other studies have found the beneficial effect of Ni in combination with the application of foliar urea in soybean plants, such as in decreasing the toxic effects of urea [37] and alleviating the harmful effects of glyphosate [38]. Nevertheless, few studies have investigated the physiological responses and N metabolism associated with Ni after foliar application in agronomic crops. This paper hypothesizes that, similar to leguminous plants, the presence of Ni during foliar urea fertilization will enhance its assimilation into organic nitrogen compounds, thereby improving the photosynthetic performance of the plant. Here, the behavior of gas exchange, carbohydrates, pigments, and N metabolism indicators were studied in cotton plants at the early adult reproductive stage newly fertilized with urea and Ni to demonstrate short-term changes in photosynthetic performance and metabolites related to urea metabolization.

2. Materials and Methods

2.1. Experimental Site

The experiment was carried out in an arch-type greenhouse, covered with a plastic film that diffuses transparent light, with a thickness of 1000 microns, in the municipality of Ilha Solteira, São Paulo State, Brazil. The experimental site belongs to the Agronomy Campus of the São Paulo State University Júlio de Mesquita Filho (UNESP) (20°25′06.0″ S and 51°20′29.7″ W).

2.2. Experimental Design and Treatments

The experimental design was completely randomized and arranged in a 2 × 2 factorial scheme comprising two N concentrations as CH₄N₂O [4% and 8% (w/v)] in the presence or absence of Ni as NiSO₄·6H₂O (0.3 g L⁻¹) applied on cotton leaves. The concentration applied was based on a dose screening performed by the author (U. Each plot consisted of a pot containing a cotton plant, with four plants per treatment.

2.3. Growing Conditions

The soil used was a Dystrophic Ultisol soil [39], collected in the experimental site of the Fazenda de Ensino e Pesquisa of UNESP in the municipality of Selvíria, Mato Grosso do Sul, Brazil (20°20′24.9″ S 51°24′19.7″ W) in areas adjacent to the plots for cultivation of agronomic crops. The particle size analysis of the composite sample of soil collected from the 0.00–0.40 cm layer contained the following fractions: 38% clay, 56% sand, and 6% silt. The chemical traits of the Ultisol used were as follows: pH = 5.2 (CaCl₂ 0.01M), organic matter = 15.0 g kg⁻¹ (colorimetric method), P = 2 mg kg⁻¹ (resin), K = 0.5 mmol_c kg⁻¹ (resin), Ca = 12 mmol_c kg⁻¹ (resin), Mg = 10 mmol_c kg⁻¹ (resin), potential acidity 18.0 mmol_c kg⁻¹ (SMP buffer), Al = 0.0 mmol_c kg⁻¹, Cu = 0.8 mg dm⁻³ (DTPA), Fe = 11 mg dm⁻³ (DTPA), Mn = 7.2 mg dm⁻³ (DTPA), Zn = 0.3 mg dm⁻³ (DTPA), and Ni < 2 mg dm⁻³ (ICP-OES, HNO₃ EPA. 3051A).

Before the experiment, the soil field capacity was determined [100% of the water mass (g) that the soil withstood] as described by [40]. During the experimental conduction, the replenishment of evapotranspired water for the plots (pots + plants) was achieved using suspended micro-sprinklers, which were activated twice a day (morning and afternoon), providing the water amount necessary for soil moisture to remain at 80% of field capacity.

The soil was placed in pots with a capacity of 8 dm⁻³ and we added CaCO₃ and MgCO₃ at a 3:1 ratio, increasing the base saturation to 70% [41]. The soil remained incubated for 50 days, keeping moisture at 80% of field capacity to allow for the reaction. Seven days before sowing, the soil was dried, chipped, sieved, and incorporated with 40 mg dm⁻³ N as urea, 200 mg dm⁻³ P as P₂O₅, 80 mg dm⁻³ K as KCl, 4 mg dm⁻³ Zn as ZnSO₄·7H₂O, and 0.8 mg dm⁻³ B as H₃BO₃. Eight cotton seeds cv. ‘TMG 81 WS’ were placed 2.5 cm below the soil surface of each pot. The plants were thinned to one representative plant per plot in the second week. The experiment was conducted during the 2019 year. Plants were grown until 42 days after planting; subsequently, treatments were applied as reported below.

2.4. Foliar Fertilization with Urea and Nickel

At 42 days after planting, automated irrigation was suspended to apply Ni (NiSO₄·6H₂O) and urea (CH₄N₂O) on the adaxial surfaces of the cotton leaves, according to the treatments, using an individual brush. At this time, cotton plants were at the growth stage 51 [42], with the first buds detectable (pin-head square), but still without the visible petals.

2.5. Gas Exchange Measurement

At 44 days after planting (2 days after fertilization), coinciding with the emission of the first flower buds detectable, the photosynthetic performance of the cotton plant was evaluated through gas exchange with the portable CIRAS-3 analyzer (Portable Photosynthesis System-PP Systems). Ambient light under photosynthetically active radiation (PAR) was used with an average of 1189 µmol m⁻² s⁻¹ with 390 µmol⁻¹ mol¹ of CO₂ as a reference to carry out the analyses.

The readings were taken from the apex on the first fully expanded leaves, between 9h30 and 12h00 in the morning, with the frequency of three analyses per plot on the same leaf, following the stability of measurements recorded in real time on the equipment screen for manual recording of each value. The equipment had been configured for automatic calibration. The photosynthetic rate values (A, µmol CO₂ m⁻² s⁻¹), stomatal conductance (gs, mol H₂O m⁻² s⁻¹), transpiration (E, mmol H₂O m⁻² s⁻¹), and internal carbon (Ci, μmol CO₂ m⁻² ar⁻¹) were measured. Subsequently, the instantaneous efficiency of carboxylation (EiC, mol ar⁻¹) was estimated (A/Ci).

2.6. Collection of Plant Material for Laboratory Analysis

Three days after fertilization with urea and Ni, the first and second leaves fully expanded were excised from the apex for analysis of N metabolites and soluble carbohydrates. The leaves were washed with deionized water and dried in paper towels. Then, a fraction of the fresh tissue was removed in vivo to evaluate the urease enzyme activity and content of urea and photosynthetic pigments. The remaining fresh tissue was placed in a −20 °C freezer.

2.7. Photosynthetic Pigments

Chlorophyll a (Chl a), chlorophyll b (Chl b), and carotenoid (CAR) contents were determined using the extracting agent dimethyl sulfoxide (DMSO). Leaf tissue (50 mg) was cut into 1 mm fragments and incubated in 7 mL of DMSO in the dark in a water bath at 65 °C for 30 min [43]. After readings in the spectrophotometer, the contents of the photosynthetic pigments were expressed in mg g⁻¹ FW.

2.8. Extraction and Measurement of Urease Enzyme Activity

The urease activity was determined through N-NH_4⁺ production [44]. The extract was prepared with adaptations of the method described by Hogan [21]. Fresh leaf samples (200 mg) were immersed in 8 mL of 0.1 M sodium phosphate buffer (pH 7.4) containing 0.21 M urea and N-propanol (pH 7.4). This extract was kept for 3 h in a water bath with manual shaking of the tube rack every 15 min. The reaction consisted of 500 µL of extract, 2.5 mL of reagent I—0.1 M phenol and 170 µM sodium nitroprusside (SNP)—and 2.5 mL of reagent II: 0.125 M sodium hydroxide, 0.15 M disodium phosphate, and 3% sodium hypochlorite (w/v). Then, the samples were incubated in a water bath at 37 °C for 35 min and the determination was carried out in a spectrophotometer at λ = 625 nm with the results expressed in µmol N-NH_4⁺ g⁻¹ FW h⁻¹.

2.9. Quantification of Urea Content

The urea content in cotton leaves was determined based on the reaction described by Kyllingsbæk [45] and adapted by Kojima et al. [46]. Fresh tissue (500 mg) was cut (1 mm) and then added to 2 mL of ice-cold formic acid. The material was centrifuged at 12,000 rpm at 4 °C for 15 min. A quantity of 60 µL of the supernatant was incubated in 2 mL of color-developing reagent [4.6 mM diacetylmonoxime, 1.28 mM thiosemicarbazide, 6.6% H₂SO₄ (v/v), 14.6 µM ferric chloride hexahydrate, and 0.006% orthophosphoric acid (v/v)] at 99 °C for 15 min followed by cooling in ice for 5 min. The determination was performed in a spectrophotometer at λ = 540 nm and the results were expressed in μmol g⁻¹ FW.

2.10. Extraction of Soluble Compounds

The soluble compounds were extracted according to Bieleski and Turner [47], using the MCW extractor solution (60% methanol, 25% chloroform, and 15% water) for water-soluble compounds and 0.1 M NaOH for protein extraction, as described by Lapaz et al. [48].

2.11. Quantification of Ammonia Content

The ammonia content was determined using the method described by McCullough [44]. The reaction consisted of 100 µL of water-soluble extract, 500 µL of reagent I (0.1 M phenol, 170 µM sodium nitroprusside (SNP)), and 500 µL of reagent II (0.125 M sodium hydroxide, 0.15 M disodium phosphate, and (3%) sodium hypochlorite (w/v)), followed by incubation in a water bath at 37 °C for 60 min. Ammonia content determination was performed in a spectrophotometer at λ = 630 nm with the results expressed in µmol g⁻¹ FW.

2.12. Quantification of Ureide Content

Total ureides were measured according to Van Der Drift et al. [49]. The test was divided into two phases. The first phase comprised the addition of 250 µL of the water-soluble extract in 500 µL distilled H₂O, one drop of 0.33% phenylhydrazine (w/v), and 250 µL of 0.5 M NaOH. Then, the samples were heated in a water bath (100 °C) for 8 min and cooled to room temperature. Quantities of 250 µL of 0.4 M phosphate buffer (pH 7) and 250 µL of 0.33% phenylhydrazine solution were added. After 5 min at room temperature, the assay was incubated in an ice bath for another 5 min, then 1.25 mL HCl p.a (−8 °C) and 250 µL of 1.65% P-ferrocyanide (w/v) were added. After 15 min at room temperature, the assay was removed from the ice bath and the determination was carried out in a spectrophotometer at λ = 535 nm. The results were expressed in μmol g⁻¹ FW. The ureide content was measured using the standard curve of allantoin solution.

2.13. Quantification of Amino Acid Content

Total amino acids were quantified with the acid ninhydrin method [50]. We used 100 µL of the water-soluble extract in 900 µL of H₂O, 500 µL of citrate buffer (pH 5.5), 200 µL of 5% ninhydrin (w/v) in methyl glycol, and 1 mL of 0.0002 M potassium cyanide solution. The samples were heated in a water bath at 100 °C for 20 min and placed to cool at room temperature for subsequent inclusion of 1 mL of 60% ethanol (v/v). The determination was carried out in a spectrophotometer at λ = 570 nm and the results were expressed in μmol g⁻¹ FW. Total amino acid content was measured using the standard curve of leucine solution.

2.14. Quantification of Protein Content

Total proteins were measured using the Coomassie blue reaction [51]. For the test, we used 50 µL of alkaline extract (sodium hydroxide fraction) in 2.5 mL of Bradford solution. After resting for 5 min, total protein content was determined in a spectrophotometer at λ = 595 nm. Protein content was measured using the standard curve of bovine serum albumin solution and the results were expressed in µmol g⁻¹ FW.

2.15. Quantification of Carbohydrate Content

Total carbohydrates were determined according to the anthrone method described by Yemm and Willis [52]. A quantity of 1 mL of the water-soluble extract was added to a test tube with 1 mL of anthrone 0.2% in sulfuric acid (w/v), followed by stirring and subsequent incubation in a water bath for 10 min and cooling at room temperature. Then, the determination was carried out in a spectrophotometer at λ = 620 nm. The concentration of total carbohydrates was measured using the standard curve of glucose solution and the results were expressed in µmol g⁻¹ FW.

2.16. Data Analysis

The data were first assessed for normality using the Shapiro–Wilk test (p < 0.05). Subsequently, analysis of variance (ANOVA) was conducted using the F test (p ≤ 0.05), with treatments treated as quantitative factors. Mean comparisons were performed using the Tukey test (p < 0.05). All statistical analyses were carried out using custom protocols implemented in the R software.

3. Results

The results were presented in two ways. For simple effects, the behavior of the variables was displayed in a table (

Table 1. Significance of the F-test and results of the Tukey test for variables with simple effect for substomatal concentration of carbon dioxide (Ci, μmol CO₂ m⁻² ar⁻¹), total soluble carbohydrates (TSC, µmol g⁻¹ FW), carotenoids (CAR, mg g⁻¹ FW), urease activity (µmol N–NH₄⁺ g⁻¹ FW h⁻¹), ammonia (µmol g⁻¹ FW), ureides (μmol g⁻¹ FW), and amino acids (μmol g⁻¹ FW) in cotton plants subjected to foliar fertilization with urea (4% and 8%) and Ni (0 and 0.3 g L⁻¹).

Factor	Ci	TSC	CAR	Urease	Ammonia	Ureides	Amino Acids
Urea	ns	**	ns	ns	**	*	***
Ni	**	ns	ns	***	ns	***	***
Urea × Ni	ns	ns	ns	ns	ns	ns	ns
Urea (%)
4	300.5	1.95 b	0.75	5.65	0.51 b	13.17 b	11.70 b
8	322.0	2.38 a	0.72	6.20	0.79 a	13.95 a	23.15 a
Ni (g L−1)
0	346,0 a	2.15	0.77	2.74 b	0.57	11.81 b	15.38 b
0.3	276.5 b	2.18	0.71	9.10 a	0.73	15.31 a	19.47 a

Significance by factorial analysis: ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001. Different letters in the same column indicate significant differences according to the Tukey test (p < 0.05).

), highlighting the differences between the isolated factors. When the interaction was significant, bar graphs were plotted (Figure 1, Figure 2 and Figure 3). We will begin by presenting the gas exchange data, followed by pigment analysis and concluding with N-metabolism data. The simple effects, along with any significant interactions, will be presented in three distinct moments within the same section. The corresponding table will be referenced to facilitate a clear and structured description of the results.

The traits TSC and ammonia showed an isolated effect only for the urea factor while Ci and urease showed an isolated effect only for the Ni factor on the ANOVA (Table 1). The concentrations of ureides and amino acids showed isolated effects for both factors on the ANOVA (Table 1). The average CAR concentration was 0.743 mg g⁻¹ FW and it was not significant for both factors on the ANOVA (Table 1). The remaining traits (A, gs, EiC, E, Chl a, Chl b, Chl a/b ratio, urea, and total protein) showed a significant effect of double interaction on the ANOVA (Figure 1, Figure 2 and Figure 3).

At 2 days after fertilization, A, gs, and EiC increased in the presence of Ni in both urea concentrations. This increase was more pronounced in plants that had received the 8% urea concentration, with increases of 94, 62, 45, 21, and 131% compared to plants fertilized with 8% urea in the absence of Ni (Figure 1I–III). The same behavior was also observed in E, although to a lesser extent compared to the previously mentioned variables (Figure 1IV). All these variables showed significance for the Ni × urea interaction. Our findings indicate that the combination of Ni and urea enhances gas exchange and that this effect is responsive to increasing concentrations of nitrogen fertilizer.

The variable Ci was not significant in the interaction between the factors. However, it was demonstrated that plants treated with Ni exhibited a 20% reduction in substomatal carbon compared to those without Ni application (Table 1). Soluble carbohydrates were also evaluated through simple effects based on the significance observed for the urea factor. The increase in urea concentration led to a 22% rise in this metabolite (Table 1).

In summary, we demonstrated that increasing the concentration of urea applied through foliar fertilization enhanced gas exchange. The presence of Ni reduced the internal CO₂ concentration, suggesting that carboxylation may have occurred more rapidly in plants treated with Ni. This hypothesis was supported by the higher instantaneous carboxylation efficiency observed in plants receiving Ni at both urea concentrations, with an additional increase of 8%. Furthermore, soluble carbohydrate data reinforced that higher urea levels also led to greater sugar availability.

In the presence of Ni in plants fertilized with 8% urea, Chl a and Chl b contents increased by 10.1% and 59.3%, respectively, compared to plants that did not receive Ni (Figure 2I,II). Thus, the Chl a/b ratio was also altered, decreasing from 5.8 to 4.1 when fertilized with 8% urea associated with Ni (Figure 2III). The CAR content was not significantly different for any fertilization condition (Table 1).

Chlorophyll a exhibited minimal variation across all treatments. However, a peak in chlorophyll b content was observed in the 8% urea treatment with Ni, which significantly altered the Chl a/b ratio. This suggests that, under these conditions, the plant may have enhanced its light-harvesting capacity by increasing the pool of chlorophyll b in the antenna complex, likely in response to the high nitrogen availability.

The protein content increased in the presence of Ni at both urea concentrations, and the highest values were observed in plants fertilized with 8% urea concentration, with increases of 141%. In plants fertilized with 8% urea in the absence of Ni, at 4%, following the same trend, the percentage increase was 63,7% (Figure 3I). This significant increase in the soluble protein pool suggests that a substantial portion of the N from the applied urea was assimilated into proteins, and Ni likely played a crucial role in facilitating this process.

The highest urea content was observed in plants fertilized with 8% urea in the absence of Ni. On the other hand, in the presence of Ni, the urea content decreased by 17 and 50.7% in plants fertilized with urea at 4 and 8%, respectively, compared to plants that had not received Ni (Figure 3II). The increase in foliar urea concentration, in contrast to the low protein content observed in treatments without Ni, reinforces the importance of Ni in the metabolization of the applied urea.

The activity of the urease enzyme was not significant for the urea factor or for the urea × Ni interaction. However, it showed high significance for the Ni factor. Plants treated with Ni exhibited urease activity more than three times higher than those without Ni application. The ammonia content showed significance only for the urea factor, with a 59.4% increase observed in plants treated with 8% urea (Table 1).

Significant variations were observed for both factors tested in relation to ureides and amino acids. For the urea factor, increases of 5.9% in ureides and 97.8% in amino acids were observed between the two concentrations tested. For the Ni factor, plants treated with Ni showed a 29.6% increase in ureide content and a 26.5% increase in amino acid content compared to those without Ni (Table 1). N metabolites responded to both N application and Ni alone, reinforcing our findings that Ni plays a key role in enhancing the conversion of urea into organic N compounds in this case.

Figure 4 showed that the Pearson correlation analysis highlighted significant relationships between physiological and metabolic variables in plants, providing a comprehensive way to uncover connections in the dataset that had not been explored in previous analyses. Transpiration showed a strong positive correlation with stomatal conductance and total protein content, indicating that higher stomatal opening and protein synthesis are associated with increased transpiration rates.

Urease exhibited a strong positive correlation with total ureide and total protein contents, reinforcing its role in converting urea into organic nitrogen compounds and revealing an intriguing, unexplained behavior: the accumulation of ureides in non-nodulated plants. Total ureide contents were also strongly correlated with total protein contents, raising the possibility of their importance in protein synthesis under high urea-N conditions.

The photosynthetic rate showed a strong negative correlation with Ci, indicating efficient CO₂ utilization, and a strong positive correlation with gs, highlighting the importance of stomatal opening for photosynthesis. Additionally, the positive correlation with total proteins suggests that higher photosynthetic rates enhance protein synthesis. Finally, the negative correlation with urea implies that plants with high photosynthetic activity may metabolize urea more rapidly.

Carotenoids, however, showed weak correlations, except for a moderate negative correlation with A, E and gs, indicating limited direct involvement in nitrogen metabolism. Finally, ammonia was moderately correlated with total amino acid and gas exchange traits, reinforcing its role as an intermediate in nitrogen assimilation. This analysis underscores the importance of urease and stomatal conductance in N metabolism and plant growth while revealing unexpected relationships that warrant further investigation.

4. Discussion

N fertilization with urea on cotton leaves favored gas exchange, increasing the A with split applications of urea in the field during the reproductive phase of cotton, where the need for photosynthates is high [53]. The premature photosynthetic decline of the cotton canopy as a consequence of the apparent remobilization of ribulose 1,5 biphosphate carboxylase-oxygenase (Rubisco) [54] suggests that extra and timely N fertilization could maintain the canopy photosynthetic capacity longer in the reproductive stage, which is likely to provide even greater yield increases [53]. The increase in A (Figure 1I) in response to urea and Ni fertilization was associated with increased CO₂ influx mediated by higher gs (Figure 1II), and also by higher EiC (Figure 1III), corroborated by their significant correlation (Figure 4).

In our study, Chl a and b increased with 8% urea and Ni fertilization (Figure 2I,II), changing the Chl a/b ratio (Figure 2III) due to a substantial increase in Chl b content, improving light absorption capacity in the antenna complex. Chl a is involved in the transformation of light energy into electrical energy and Chl b contributes to improving the light absorption capacity, acting in the absorption of blue–violet light [55]. However, Barcelos [35] did not observe significant effects of N and Ni fertilization in the soil on chlorophylls (SPAD) in cotton. Importantly, growth promotion is related to the improvement of urea-N assimilation and chlorophyll contents in urea-fed wheat plants when Ni is supplemented (0.01 and 0.05 mg Ni L⁻¹ in nutrient solution); the increase in chlorophyll content demonstrated the improvement in urea-N assimilation by the Ni supplement because chlorophyll is an N-containing compound [56].

Ni is a constituent of the urease enzyme responsible for breaking down urea into ammonia and carbon dioxide [8], participating in N recycling in the plant [20]. Therefore, some studies have been related to whether or not Ni is associated with urea applications to assess its Ni role, especially in the activity of the urease enzyme. Studies with cereals [11] pecan [15], and colza [57] reported increases in the urease enzyme activity in the presence of Ni and inefficiency in urea metabolism in Ni’s absence. Ni enrichment in soybean seeds and the application in nutrient solution reduced the toxic effects of urea accumulation after foliar fertilization, increasing the content of photosynthetic pigments and improving the nutritional statuses of plants. Ni-deficient plants took longer to show positive responses to foliar fertilization with 2% urea, indicating significantly quicker assimilation of urea N by Ni-fed plants [37].

Three days after urea and Ni application, the urease enzyme activity increased in cotton plants in the early adult reproductive stages (Table 1) and showed a strong correlation between urease activity, urea, ureides, and proteins (Figure 4). The foliar application of urea of 31 and 16 mg of N plant⁻¹, combined or not with the supply of 0.05 mg of Ni plant⁻¹, showed that Ni impaired N uptake in coffee seedlings because when Ni was present, the leaf always absorbed less N than when Ni was not present [33]. In our study, N absorption did not seem to be impaired in Ni’s presence since the urea content in the leaves decreased in both experimental urea concentrations (Figure 3II).

Once urea enters the plant cell or is generated as a metabolic by-product, its rapid metabolism or temporary accumulation is possible [58]. In senescent tissue, urea accumulation has already been verified as there is a need for the remobilization of reserves to sustain plant growth [59]. In non-senescent tissues, urea as the intact molecule is practically not accumulated [60]. Urea accumulation in cotton plants that have not received Ni can be a positive indicator of the Ni effect during urea metabolization after foliar fertilization, as demonstrated by the negative correlation of this metabolite with urease enzyme activity and protein content (Figure 4).

The ammonia content is expected to increase after urea fertilization as urease is dependent on Ni and urea for its activation [58]. In our study, the urease enzyme activity was unchanged at both experimental concentrations of urea (Table 1); however, the ammonia content was higher in plants that had received a greater amount of urea (Table 1). The increase in ammonia content was also observed after the application of 2% urea to potatoes; nevertheless, the urease enzyme activity did not appear to be induced by urea concentration [13]. Ammonia produced is assimilated mainly by the combined action of the enzymes glutamine synthetase (GS) and glutamate oxoglutarate aminotransferase (GOGAT) [61], which suggests the use of CO₂ resulting from the process of organic acid synthesis via tricarboxylic acid cycle [62]. This internal situation might be coordinated between C and N metabolism because glycolysis and the TCA cycle must produce energy and metabolic precursors, such as amino acids, for the storage of protein synthesis.

As expected, the amino acid contents also increased in the presence of urea and Ni (Table 1). The accumulation of amino acids in urea-fed plants is mainly related to the pool of the amino acids glutamine and asparagine used in the N translocation. High levels of these molecules decrease ammonia assimilation due to the feedback inhibition of assimilation products that are not efficiently translocated [57,61]. Ureides are found in several plants and their formation usually occurs after the oxidative degradation of nucleic acids and their purines [63,64]. Some plants contain high ureide contents in their tissues, such as legumes [32]. In this case, these ureides are substances associated with N storage and transport from biological fixation [65].

Ureides are economical molecules in situations of low A, such as in C3 plants in which the use of ureides for storage could be an alternative to save metabolic energy. The advantage of having ureides as N storage and transport molecules is their N: C ratio (1:1) while the amides glutamine and asparagine contain (1:2.5) and (1.2), respectively [66]. The positive correlation between urease enzyme activity, proteins, and ureides (Figure 4) indicates a possibility of using ureides as nitrogenous molecules for temporary storage in this situation, even under high A conditions (Figure 1I).

Many studies have addressed this topic, primarily in legumes, attributing productivity responses to Ni application [67,68,69,70]. However, in cotton, there is still limited information on the best practices for Ni application as a nutrient, with recent studies focusing mainly on root exposure [71,72]. We believe that the findings of this manuscript provide a comparative basis for more detailed studies on the use of foliar Ni, either combined or not with urea, in cotton.

5. Conclusions

The presence of Ni during foliar urea fertilization increased the photosynthetic rate and photosynthetic pigments, which we attributed to the enhanced urea assimilation process. Simultaneously, the combination of urea and Ni boosted urease enzyme activity, leading to higher levels of various N metabolites. The detection of ureides in cotton leaves following foliar fertilization with urea and Ni suggests their potential roles as storage forms of N. Furthermore, Ni can alleviate the stress induced by urea application, mitigating potential toxic effects and improving overall plant performance.

The increases in protein content and its positive correlation with gas exchange parameters suggest that short-term improvements in photosynthesis, coupled with elevated N metabolite levels in plants treated with urea and Ni, may have long-term benefits. These changes could support late-cycle cotton traits, such as flowering, by providing an abundant supply of N compounds to meet increased metabolic demands during reproductive stage.