Zinc and nitrogen mediate the regulation of growth, leading to the upregulation of antioxidant aptitude, physio-biochemical traits, and yield in wheat plants

An ample amount of water and soil nutrients is required for economic wheat production to meet the current food demands. Nitrogen (N) and zinc (Zn) fertigation in soils can produce a substantial wheat yield for a rapidly increasing population and bring a limelight to researchers. The present study was designed to ascertain N and Zn’s synergistic role in wheat growth, yield, and physio-biochemical traits. A pot experiment was laid out under a complete randomized design with four N levels (N1-0, N2-60, N3- 120, and N4-180 kg ha−1), Zn (T1-0, T2-5, T3-10, and T4-15 kg ha−1) with four replications. After the emergence of the plants, N and Zn fertigation was applied in the soil. The growth traits were considerably increased by combined applications as compared to the sole applications of the N and Zn. The photosynthetic pigments were found maximum due to combined applications of N and Zn, which were positively associated with biomass, growth, yield, and wheat grain quality. The combined application also substantially enhances the antioxidant enzyme activities to scavenge the ROS as H2O2 and reduce lipid peroxidation to protect the permeability of the biologic membranes. The combined higher applications of N and Zn were more responsive to ionic balance in a shoot by maintaining the Na+ for osmotic adjustments, accumulating more Ca2+ for cellular signaling; but, combined applications resulted in K+ reduction. Our present results suggest that appropriate sole or combined applications of N and Zn improve wheat's growth, yield, and antioxidant mechanisms. Previous studies lack sufficient information on N and Zn combined fertigation. We intend to investigate both the sole and combined roles of N and Zn to exploit their potential synergistic effects on wheat.

In the current era, different grain bio-fortification methods are used.In contrast, agronomic bio-fortification in cereal crops is considered a rapid and economical tool due to its low cost and sustainable strategy to improve wheat productivity, growth, and nutrient profiling.To meet wheat demands, N fertilizers are a critical tool for attaining the required productivity.The above-mentioned ideas suggest that wheat grown in soils with poor nutrient levels leads to malnutrition and seriously threatens humans and plants.Hence, it is a concern of the current era to enhance the nutritional contents of wheat and soils through numerous agronomic practices.A promising strategy that could help cereal producers improve nitrogen use efficiency is gene editing or molecular tools 38 .In previous findings, agricultural scientists primarily focused on the implications of single-element fertilizers on cereals, yet limited information is available on the combined fertigation of N and Zn.Present notations suggest we explore the combined and sole applications of the N and Zn on yield, growth, and physio-biochemical attributes.Moreover, we intend to determine the impact of N and Zn applications on shoot ions, cellular antioxidants, and organic osmolytes.We hypothesized that N and Zn fertigation might help improve wheat yield and grain quality through physiological and biochemical processes.

Experimental conditions, establishment, and layout
The seeds of one wheat genotype Akbar-19 seeds were procured from Ayub Agricultural Research Institute, Faisalabad-Pakistan. Pot experimentation was carried out in the research area of University of Agriculture community college, Postgraduate Agricultural Research Station (PARS) (coordinates: 31.3848o N, 72.996 o E, altitude 184.4 m a.s.1).Seeds of wheat were engrossed in 30% hydrogen peroxide (H 2 O 2 ) for 1 h for microbial disinfection, thoroughly washed with distilled water (H 2 O) for 2 h, dried and stowed until sowing.Ten uniformly selected seeds were sown into each plastic pot (630 cm 3 ) for growth and filled with soil (3 kg/pot).The soil in plastic pots has a sandy loam texture (Electrical conductivity/ECe 1.2 dS m −1 , pH 8.22, Na + 39.95 mg kg −1 , Cl − 9.98 mg kg −1 , Mg 2+ 250 mg kg −1 , organic matter-OM 0.87%, Zn 9.88 mg kg ─1 , N 599.3 mg kg −1 , P 7.5 mg kg −1 and K 123 mg kg −1 .After seedling emergence, plants were thinned to six for better growth to prevent nutrient deficiency until analysis and harvesting.The experimental units were arranged as completely randomized design with four replications for each treatment with a day/night temperature of 27 °C/21 °C and relative humidity of 55-65%.After sowing, half-strength Hoagland's nutrient solution (4.66  ) was uniformly added to all plastic pots for an interlude of 2 days until the development of seedlings took place.The field capacity was maintained throughout the work to prevent water deficit conditions.After each seven-day gap, the pots were reshuffled throughout the experimentation to avoid border and pseudo-replication effects.

Plant sampling
Plant sampling was done at the time of maturity until reaching growth stage 75 on the Zadok's scale (after 110 days of seed sowing) by following the method outlined by Zadoks et al. 39 .Fully matured flag leaves from each treated pot were taken and immediately stored at − 70 °C to determine the physiological and biochemical parameters, reactive oxygen species (ROS), and antioxidants assay.Harvesting was done after the maturity and growth and yield components were recorded.For this purpose, the floral parts, such as the leaf, shoot, and leaf, were washed with distilled water to remove the soil, and growth traits were recorded.

Measurement of growth traits
After harvesting, five plants from each treatment were used to determine the root and shoot fresh and dry biomass by using a digital weighing balance (± 0.01gm), while dry weight was taken after oven drying the samples for 72 h at by scale (± 0.001 gm) at 70 °C.After harvest, each plant's roots and shoot were separated from the plant and delicately washed by running tap water.A manual measuring scale recorded the root length and shoot length.Plant height was recorded from root to leaves with the help of a measuring scale.The leaves number was visually counted from each experimental pot under different treatments.The leaf area (LA) was measured using the formula L × W × 0.75.The leaf area ratio (LAR) was measured by ascertaining the ratio between the leaf area (LA) and shoot dry weight (SDW) of the plants by using the following equation;

Yield components
The number of fertile tillers (m -2 ) undertaken from each treatment.The length of spikelets was measured with measuring tape, and spike length (SL) was measured precisely.Manual calculations were used to find the number of spikes and spikelets per spike.After threshing, the grain's numbers were counted and averaged per plant.
(1) LAR = LA/ SDW www.nature.com/scientificreports/Leaf photosynthetic pigments Plant fresh leaf (0.25gm) were homogenized in an 80% acetone solution at 0-4o C to ascertain the photosynthetic pigments as chlorophyll a, b, and total chlorophyll (T.Chl) contents by following the Arnon, 1949 40 method and carotenoids were ascertained by Goodwin 41 .The extracted material was then filtered by using filter paper.The filtrate was centrifuged at 10,000 × at 4 °C for 5 min.Pigment absorption was noted at 480, 645, and 663 nm with the help of an ultraviolet-visible spectrophotometer (Hitachi-220, Japan).

Cellular antioxidants
To prepare enzyme extract, wheat leaves (0.5gm) were ground in 1% polyvinyl pyrrolidine, 1 mM ethylene diamine tetra acetic acid (EDTA), 0.05 M sodium phosphate buffer having a volume of 5 mL in a pestle mortar.The supernatant was collected after centrifugation at 20,000 rpm and 4 °C for 20 min to analyze antioxidant enzymes.The protein contents were estimated using method 42 , and bovine serum albumin was used to calculate the enzyme-specific activity.

Super oxide dismutase
The activity of superoxide dismutase (SOD) was recorded based on the inhibition level of nitro blue tetrazolium (NBT) reduction 43 .The 10 µL of extracted enzyme solution was added in 3 mL of reactants (200 mM methionine, 100 mM (Na 3 PO 4 ) phosphate buffer, 1.5 Na 2 CO 3 , 3 mM ethylene diamine tetra acetic acid (EDTA), 2.25 NBT, 0.06 mM concentration of riboflavin, and distilled water.The same set of test tubes was prepared as a control.Fluorescent bulbs irradiated both reactant and control test tubes for 15 min.After that, the test tubes were darkened with a black cloth for reaction termination purposes.A blank was prepared following the above method without irradiation.The levels of optical density (OD) were determined at 560 nm.One unit of SOD accounted for the quantity of enzyme inhibiting 50% of NBT photoreduction.

Peroxidase
The activity of peroxidase (POD) was determined in the presence of H 2 O 2 by following the method of 44 .A reactant solution was prepared in glass test tubes containing 3 mL of phosphate buffer, 10 mM H 2 O 2 , and 20 mL of guaiacol.The reaction was started after adding the enzyme extract, and specific absorption was recorded spectrophotometrically at 460 nm.

Hydrogen peroxide
The quantification of H 2 O 2 was based on ferrous ions oxidation induced by POD concentration.These ferrous ions react with xylenol in reactants 45 .The leaf material (0.5 gm) was grounded in sodium phosphate buffer, and homogenized was centrifuged at 10,000 × for 15 min.The supernatant was collected and reacted with the assay reagent.After 45 min, absorbance was recorded at 560 nm.

Lipid peroxidation/MDA
Lipids peroxidation (LPX) was measured using an amount of malondialdehyde (MDA) 46 .Thiobarbituric acid (TBA) and MDA were used to determine LPX concentration.The 250 mg leaf sample was homogenized in 5 mL of 0.1% (w/v) trichloroacetic acid (TCA) and centrifuged at 15,000 × g.The 1 mL aliquot was mixed with 2 mL of 0.5% TBA and 20% TCA.Reactants were placed in test tubes then incubated at 85 °C for 10 min, and the reaction was stopped in an icebox.The optical density was determined at 532 and 600 nm by spectrophotometer (Hitachi U2910, Tokyo, Japan).The optical density (532 -600 nm) was used to determine the final values.The 155 mM cm -1 as an extinction coefficient was used to count LPX.

Shoot ionic contents
Ionic contents in the shoot were recorded using the Wolf method 47 .The plant sample (0.25 g) dried and was added to digestion tubes with 5 mL of concentrated H 2 SO 4 and then incubated overnight.After incubation, 500 ml of 30% H 2 O 2 (V/V) was added.The test tubes were placed on a digestion plate, and the temperature was gradually raised till the formation of fumes at 350 °C.When the mixture cooled, then 30% H 2 O 2 was added.The process was repeated until a clear solution was formed.Deionized water was added to digested material to dilute it up to 50 ml.using a flame photometer, shoot K + Na + and Ca 2+ were determined (Portable flame photometer-PFP7, Jenway, and Staffordshire, United Kingdom).

Statistical analysis
The data visualization and statistical analysis were performed using the R statistical software (R Core Team, 2022) using the R integrated development environment R Studio (R Studio Team, 2022).Curated data were subjected to a two-way analysis of variance (ANOVA) to evaluate the interaction between N and Zn treatment at a significance level (p ≤ 0.05).The treatment means standard errors (SE) and level of significance (ANOVA) were compared by Tukey's honestly significant difference by using the R-integrated "Agricolae" package.The principle component analysis was done to evaluate the relationship between a variable of different traits and the possible impact of treatment with the help of R code "FactoMineR and ggplot2.The Clustered heatmap was constructed by R integrated code "pheatmap, while the correlation matrix was drawn by "GGally.

Ethics approval
The pot experiment and plant material collection were conducted per relevant institutional and national guidelines and legislation.

Growth attributes
The exogenous application of nitrogen (N) and zinc (T) considerably increases the growth traits of wheat.The sole applications of Zn notably increase wheat's shoot fresh weight (SFW); a more pronounced increase in FW occurred in response to the T4 level.The collective applications of Zn and N positively influence SFW, and the maximum mean value for this trait was evaluated for N4:T4.We observed a similar response for the shoot dry weight by applying Zn and N. The root fresh and dry weights were substantially increased as a result of both nutrient applications.The maximum mean values were found in response to T4 and N4 by showing a linear increase as application levels were increased.The root length (RL) and shoot length (SL) were considerably increased by an increase in the levels of Zn, the highest mean value drawn by T4.The combined application of Zn and N notably enhanced the RL and SL in response to higher regimes.The combined application of both nutrients causes a considerable increase in plant height (PH) and leaves per plant (NLPP).The maximum value for NLPP and plant height (PH) was secured by T4:N4 (Table 1).
The wheat genotype's leaf area increased as the nutrient treatment levels increased.The N4 with T4 responded to the maximum mean values, which showed a substantial increase.The leaf area ratio (LAR) has not substantially changed in response to T1:N1, T2:N2, and T3:N3.But a LAR notable increase in response to combined applications.The sole applications of Zn and N considerably improved the biologic yield of the wheat plants.The number of fertile tillers (NFT) per plant was maximum in response to higher N and Zn treatments.The number of spikelets showed the same increasing trend line pattern to improve the biologic yield of the wheat plant.No major change in mean values was evaluated for spike length (SPL) at low or higher treatments of the Zn and N. The yield parameters DTH (day to heading) and DTM (days to maturity) showed a substantial increase as the level of the treatment was increased (Table 2).

Photosynthetic pigments
The photosynthetic pigment Chl a was notably increased as the levels of N and Zn were increased.However, the most apparent change was evaluated due to N3:T3 and N4:T4.The Chl b contents were more under moderate supplementation of N1 and Zn (T2, T3), while no change occurred at N4:T4.The Chl b pointedly decreased as levels of Zn increased under N2 supplementations, while N3:T2 showed the least amount of the Chl b and N4 applications represented no substantial changes.The amount of chlorophyll a/b was maintained at N1 and various Zn fertigation, while other levels (N3, N4) were considerably increased as the level of Zn increased.The total chlorophyll had shown maximum mean values at N3 and N4 with a combined application of Zn.The carotenoid pigments were decreased in response to elevated levels of Zn and N, except in N1: Zn.

Shoot ionic contents
The Na + in the shoot increased in response to combined treatments; N4:T1, N4:T2, and N4:T4 maintain the Na + contents.The Ca 2+ was notably increased at the T3 level of Zn with combined fertigation of N. The N applications maintained the amount of K + in shoots; however, a substantial reduction occurred at elevated levels of N and Zn (Fig. 1).

Oxidative markers and antioxidants
The malondialdehyde contents in terms of lipid peroxidation were considerably reduced at higher applications of N and Zn to maintain the membrane integrity.The N3 and N1 applications with different levels of Zn substantially reduced reactive oxygen species (H 2 O 2 ) levels, while higher levels of N and Zn showed an increasing pattern of reactive oxygen species.
The cellular antioxidant 48 was increased due to N1-N3 with different Zn levels.The amount of peroxidase considerably enhanced N2 and N4 applications with varying levels of Zn.A substantial reduction was evaluated in response to N1 and N2 levels with various Zn regimes (Fig. 2).

Principal component analysis (PCA)
The effect of various N and Zn combined applications on growth, pigments, ions, cellular antioxidants and ROS were illustrated with the help of ellipsed PCAs.The closed and linear eigenvector demonstrated a noteworthy association between traits at discriminating levels of N and Zn.The explained variation for growth and photosynthetic pigments were PCA1 57.3% and PCA2 11.2 with an influential cumulative of 68.5%.The photosynthetic pigments Chl a, Caro, and Chl a/b were closely clustered toward the PCA1 by closely associating with SFW, SPL, RFW and RL growth traits.These pigments were lowered with negative eigenvalues by associating with SL, NOS, Chl a, and DTM.
The PCA biplot for ions, antioxidants ROS, and yield components of wheat represented explained variations of PC1 41.1% and PC2 19.9% (Total 61%).The K + and malondialdehyde (MDA) contents were closer by showing higher positive eigenvalues and excelled toward the PC2 side of the biplot.The traits POD, DTH, SPL, NG, and SOD were positively associated with each other and grouped toward the PC1 side of the biplot.The NOS, NFT, Ca 2+ and Na + were lowered, contributing to the negative side of PC1 with higher negative eigenvalues.The ROS was considerably associated with the various levels of the N by excelling toward the PC2 side (Fig. 3).

Discussion
The present study evaluates the combined effect of N and Zn on crop growth, yield, pigments, ions, ROS, and antioxidants in wheat plants.For years, the synergetic role of the available nutrients to plants has been well established 49 .The micro (Zn) and macronutrient (N) deficit profile soil is increasing enormously due to exhaustive crops and injudicious use of fertilizers, leading to malnutrition in humans 50 .As a critical nutrient, the N plays a key role in Zn sequestrations in the wheat's vegetative and reproductive parts of the wheat 51,52 .Nitrogen as a macronutrient enhances vegetative growth by regulating the morphologic, physiological, and biochemical mechanisms 53 .The Zn plays an indispensable role in development by regulating the levels of antioxidants and nucleic acid metabolism, a key part of carbonic anhydrase enzymes involved in auxin biosynthesis 54,55 .In the current work, the synergistic application of Zn and N substantially improves plant biomass as shoot and root fresh (FW) and dry (DW) weights and plant height (PH) in wheat.The increase in fresh and dry biomass and height might occur due to elevated pigment levels and antioxidants.The increase in plant biomass is directly www.nature.com/scientificreports/linked to the photosynthetic activity of the plants as N is a major component of the chlorophyll.The Zn essentially involves the biosynthesis of the proteins, the main part of ribosomes for their structural integrity and interaction with other elements involved in the growth of the plants.The N and Zn fertigation enhances wheat root length (RL) and shoot length.The increase in RL occurs to capture the immobilized Zn and N 56,57 .The SL increases the capacity to move nutrients toward aerial parts of the plant 58,59 .The increase in RL and SL mainly occurs due to the involvement of the Zn in cell division, cell proliferation and enhances the meristematic activities to facilitate the better translocation of nutrients across the root and shoot.Leaf area and numbers increase as the levels of Zn and N increase, this increase might occur to provide the maximum surface area to capture the solar light to enhance the photosynthesis.The exogenous supply of Zn and N increases the number of leaves and area in wheat 36 .The leaf area ratio (LAR) increased with increasing regimes of nutrients.The N and Zn applications in wheat markedly enhance the biomass by an increase in leaf area index, LAR, which ultimately helps the plant to increase the number of spikes and yield 60,61 .Plant yield is closely correlated with key yield components such as numbers of fertile tillers (NFT) and spikes (NOS) 36 .Various studies demonstrated the positive role of the sole application of N and Zn in increasing the NOS and NFT [62][63][64] .In the present work, the NFT and NOS spikes increased, contributing to an increase in yield performance and a positive effect on grain numbers and quality.An increase in NFT and NOS is an important facet in determining the final yield per plant 65,66 .The spike length (SPL) increases in wheat plants.This increase is related to the greater number of grains per spike and the possible role of Zn in cell division, enlargement, and elongation 67 .In current work, the numbers of grains per spike have increased due to combined applications.This increase is linked to more NOS, NFT, and an increase in LAR.Improved supply of N and Zn work parallel to an increase in spike numbers, eventually leading to more seed numbers 68 .The grain yield confers a positive relation with days to heading (DTH) and maturity (DTM) in wheat 69 .The Zn as a microelement has an essential constructive and catalytic role in various physiological and biochemical activities which ultimately results in a higher economic yield in wheat 69 .
Enhanced amounts of photosynthetic pigments play a pivotal role in photosynthetic performance by capturing more light photons and providing energy to thylakoid membranes 70 .The amount of Chl a, b, Chl a/b, and total chlorophyll enhanced due to wheat's combined fertigation of N and Zn.The increasing trend of photosynthetic pigments improves photosynthetic efficacy in cereal crops, increases photochemical activity, and improves parabolic relations with an increase in yield 71 .The carotenoid in the leaves of wheat increases as the levels of N and Zn increase.The carotenoids in higher plants correlated with the supply of N and Zn to the plants, ultimately linked to the yield 71 .Carotenoids are a vital pigment, light absorption, distribution, and transmission to the reaction centre and play an important role in growth and development 72 .Ion homeostasis is a central plant quench that regulates the normal metabolic process 73 .The amount of Na + in wheat under combined applications of N and Zn increased due to low supply while remaining constant at higher applications.The regulation of Na + is undertaken by various mechanisms such as more Zn sequestration, avoidance of absorbing Na + , compartmentalization in vacuoles, and exploitation of osmotic adjustment approaches.The Zn regulate the balance of K + /Na + by more accumulation of the K + in pant vegetative parts to maintain internal ion homeostasis.Also, Zn enhances the adenosine triphosphate by regulating the Na + /H + antiports for compartmentalization 74 .The N supplementation also plays a vital role in reducing the cellular Na + and improving the K + uptake in wheat 75 .The same pattern of increase in K + concentration was recorded in the current work, which might be related to the controlling mechanism of Na + due to N and Zn fertigation.As a divalent macromolecule, calcium plays an imperative role in membrane integrity, signaling, and sustaining the metabolism of crop plants 76 .The current illustration indicates a higher level of Ca 2+ , which is vital for normal metabolic processes and growth.
The Zn and N applications are dominant in scavenging reactive oxygen species (ROS) and free radicals and protecting membranes through lipid peroxidation by enhancing the amount of cellular antioxidants 77,78 .The antioxidants potentially reduce the production of the ROS cause a marked decrease in the amount of singlet oxygen and also elevate the organic osmolytes in cells.The activities of SOD enhanced as a result of N and Zn applications.This increase is interlinked with reducing LPX and H 2 O 2 contents in the leaves.The increasing signature of SOD is supported by ROS limitation, which helps the plant counteract oxidative damages under   Zn applications 79 .The rising capability of SOD is indispensable for the distribution of toxic radicles, protecting the membranes, photosynthetic apparatus, and nucleic acids, and providing functional stability as a result of N supplementation 75 .

Conclusion
Fertigation of nitrogen (N) and zinc (Zn) notably increases wheat growth by enhancing the plant's fresh and dry biomass.Both nutrients also improve vegetative growth traits such as RL, SL, PH, NLLP, and ultimately lead to an increase in yield components.The increase in growth traits and yield components was closely linked with higher levels of photosynthetic pigments, assisted by N and Zn combined applications.Additionally, N and Zn tempt changes in the level of antioxidants, which perform a decisive role in scavenging the ROS as H 2 O 2 and consequent decrease in lipid peroxidation to protect cellular membranes.The sole and combined applications of N and Zn efficiently maintain ionic statutes in shoots by balancing the Na + , Ca 2+ , and K + .The findings reported in this manuscript shed light on the intricate cross-talk between Zn and N, and unlock the pathways for future research to unravel molecular mechanisms.Moreover, further explorations by large-scale field experimentations could provide valuable insights into harnessing the role of N and Zn in agriculture. https://doi.org/10.1038/s41598-024-63423-ywww.nature.com/scientificreports/

Figure 1 .
Figure 1.Photosynthetic pigments and shoot ionic contents of wheat treated with different levels of Zn and N2.Means ± SE provided with error bars; different letter indicates significance (p ≤ 0.05) between N and Zn treatments.For an abbreviation description see the list at the start of the manuscript.

Figure 2 .
Figure 2. Oxidative stress markers (MDA, H 2 O 2 ) and cellular antioxidants of wheat treated with different levels of Zn and N2.Means ± SE provided with error bars; different letter indicates significance (p ≤ 0.05) between N and Zn treatments.For an abbreviation description see the list at the start of the manuscript.

Table 1 .
Growth traits of wheat treated with different levels of Zn and N. Means ± SE provided with error bars; different letter indicates significance (p ≤ 0.05) between N and Zn treatments.Means provided with error bars.The small letter indicates a significant (p ≤ 0.05) difference between Zn treatments and capital letters for N 2 .For an abbreviation description see the list at the start of the manuscript.