Potential role of multiwalled carbon nanotube priming in boosting nitrogen metabolism and nutritional value during the sprouting process

Potential role of multiwalled carbon nanotube priming in boosting nitrogen metabolism and nutritional value during the sprouting process | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Potential role of multiwalled carbon nanotube priming in boosting nitrogen metabolism and nutritional value during the sprouting process Yasmen Khaled, Momtaz M. Hegab, Mohammad K. Okla, Amal Mohamed AlGarawi, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4777965/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Oct, 2024 Read the published version in Journal of Nanoparticle Research → Version 1 posted 7 You are reading this latest preprint version Abstract Sprouts are well known for having a remarkable nutritional profile. Enhancing the tissue chemical composition and quality of sprouts is essential since these metabolites offer numerous health advantages. To this end, this study aimed to investigate the effects of priming with multiwalled carbon nanotubes (MWCNT) on the growth and nitrogen metabolism of four horticultural plants, namely T. foenum graecum , L. grandifloruum , L. sativum , and A. gravelones . The properties of our synthesized MWCNTs included three characteristic peaks 3434, 1539, and 1068 cm − 1 attributable to the stretching vibration of O–H, bending vibration, and C − O, respectively. MWCNT priming increased the sprouting process by inducing biomass and protein accumulation. MWCNT priming improved nitrogen metabolism, including amino acid and polyamine metabolism. At the amino acids level, there was an increase in amino acids levels (e.g., glycine, lysine, asparagine, and glutamic acid) as well as their metabolic enzyme activity including glutamine synthetase (GS), threonine synthase (TS), and glutamate synthase (GOGAT). Increased polyamine levels like spermine, putrescine, and spermidine were also associated with boosting their related biosynthetic enzyme activities i.e. , arginine decarboxylase (ADC), ornithine decarboxylase (ODC), spermidine synthase, and spermine synthase. This improvement of nitrogen metabolic pathways highlights the potential of MWCNT to boost the tissue chemical composition of horticultural plants. multiwalled carbon nanotubes sprouts N metabolism amino acids polyamine metabolism nutritive value Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Enhancing plant growth and productivity is an objective of agriculture farming, particularly under undesirable conditions. The application of nanotechnology has shown promise in enhancing the chemical and biological functions of plants. Nanotechnology has transformed several industries, including electronics, materials science, healthcare, and agriculture, by creating nanoparticles with specific properties. Carbon nanotubes (CNTs) nanomaterial is considered a unique substance due to its good properties in mechanical, electrical, and thermal applications. 1 Additionally, various industries depend on MWCNTs as a type of nanomaterials. With a diameter range from 1 to 3 nm, the Graphene sheets were used to form multi-walled carbon nanotubes. 2 Furthermore, the numerous layers enjoy higher stability in the thermal properties and mechanical strength than the single-walled carbon nanotubes (SWCNTs). Multiple investigations were carried out on MWCNTs due to their exceptional physicochemical characteristics such as electrical conductivity, chemical stability, and high surface area. 3 MWCNTs interact positively with plants in various manners, mainly they act on the improvement of the uptake and utilization of nutrients. Also, it was involved in enzyme activity alteration that was shared in nutrient assimilation like nitrogen which in turn significantly assisted in the plant metabolism enhancement. MWCNTs contributed to the absorption of nutrition and enhancement of the accumulation of soluble proteins in Malus hupehensis and free amino acids by alteration of (NR) activity that in turn improves plant growth. 4 There were a few investigations about the ability of MWCNTs the improve sprouts' nitrogen metabolism. The current study seeks to explore the impact of MWCNTs on the assimilation, absorption, and metabolic process of nitrogen in specific plant species. Many studies exhibited the importance of nitrogen molecules as a critical macronutrientional compound which was necessary in the creation of variable biomolecules, like nucleic acids, amino acids, and, chlorophyll. 5 This compound has a great role in the nitrogen metabolism yield, development, and plant growth through plant consumption of nitrogen. The environmental conditions impressed the nitrogen consumption which greatly affects the productivity of yields and plant development. Nitrogen metabolism is critical for sprout growth and development, which can be obtained from many sources such as ammonia (NH3) fertilizer. 6 Generally, nitrogen metabolism was considered the major influencer on the complete development of plant growth and nutrient uptake, utilization, and assimilation of nutrients in sprouts. 7 In plants, an accumulation of polyamines and amino acids influences various physiological and biochemical activities. They are required for the synthesis of proteins, growth, nitrogen metabolism, photosynthesis, and hormone regulation. They boost the nutritional quality of plants through their contributions to protein content, vitamins, secondary metabolites, and nutrient bioavailability. Moreover, the nitrogen shortage altered the sprout’s metabolism which in turn affected the concentration of vital metabolites and other organic acids that were required for plant enlargement and development. According to prior research, MWCNTs and nano-SiO2 raise the activities of the nitrogen metabolism-related enzymes GS, GOGAT, GAD, and GDH in maize leaves and roots. They can also boost pyruvate levels, which will aid in the synthesis of carbohydrates and the uptake of nitrogen, and eventually encourage plant growth. 8 Many investigators highlighted the role of silicon nanoparticles in the improvement of sugar metabolism, accumulation of osmoprotectants and antioxidants, synthesis of amino acids, and seedling growth of peas. 9 The current investigation aimed to explore the capacity of MWCNTs in the improvement of nitrogen metabolism for sprouts that belong to four medical commercial plant species T. foenum-graecum , L. sativum , A. graveolens , and L. grandiflorum . Due to the economic, medicinal, and substantial nutritional value of the targeted plants, they were chosen for the present study. The annual herb fenugreek is the common name for T. foenum-graecum and belongs to the Fabaceae family. Through phytochemical examination, it is composed of carbohydrates, amino acids, lipids, flavonoids, steroids, polyphenols, hydrocarbons, alkaloids, and saponins. 10 The Linaceae family includes L. grandiflorum with the common name flax. Their seeds were considered good provenance fibers of diet, omega-3 fatty acids, and lignans. Some studies were registered as anti-carcinic agents with anti-inflammatory properties and cardiovascular protection. 11 Concerned with the Brassicaceae family, it includes a fast-growing edible herb with the scientific name L. sativum . It was recorded that it was composed of important minerals like calcium and iron, vitamins A, C, and K, proteins, lipids, and carbs. 12 Finally, the Apiaceae family includes A. graveolens herb, whereas their seeds contain phenolic acids, steroids, flavonoids, and coumarins. 13 It was abundant in the medical field due to its antibacterial, anti-inflammatory, and digestive characteristics. Many contributions were added to the agriculture field through exploring innovative agricultural techniques, like MWCNTs in seed priming, which subsequently develop the activities of development and metabolism of examined plant tissue. This research attempts to explore the possibility of MWCNT-based seed priming to enhance N metabolism during the sprouting stage and thus raise the development and production of these significant plant species. 2. Materials And Methods 2.1. Carbon nanotubes preparation and characterization Multi-walled carbon nanotubes (MWCNTs) were acquired from the Centre of Nanomaterials (Beni Suef University, Beni Suef 62521, Egypt). Chemical vapor deposition (CVD) was used to prepare highly pure MWCNTs, as previously described in the literature. 14 An alumina tube furnace was filled with 0.5 g of the Fe/Co catalyst. A nitrogen gas flow rate of 70 mL min − 1 was used to heat the catalyst to 700°C. After heating the catalyst surface for 10 minutes at 700°C, acetylene gas was added to the alumina tube over the catalyst surface at a flow rate of 10 mL min − 1 for 30 minutes, and nitrogen gas was reduced to 30 mL min − 1 . Finally, the CNTs were collected once the tube furnace had cooled to room temperature. The surface form and size of MWCNTs were investigated using field emission scanning electron microscopy (FE-SEM) with a dispersive energy X-ray (EDX) analyzer (JSM-6700 F, JEOL Japan). To record the photographs, the operating voltage in the secondary emission mode was 15 kV. Fourier transform infrared spectroscopy (FTIR), using a Nicolet Nexus 670 FT-IR instrument, was used to determine the material composition. 2.2. Determination of total soluble protein The quantity of soluble proteins was measured using the Bradford technique. 15 Five milliliters of Coomassie blue reagent (100 milliliters of Coomassie brilliant blue G-250 dissolved in 50 milliliters of 95% ethanol, followed by 100 milliliters of 85% H 3 PO 4 and finally up to 1 liter of distilled water) with 0.1 milliliters of crude extract. Following 30 min, the absorbance was measured at 595 nm in comparison to a water-reagent blank. The bovine serum albumin reference curve was used to calculate the protein concentration (as mg protein g − 1 FWt). 2.3 Determination of nitrogen and nitrate reductase activity To determine the total nitrogen (N) content, 0.2 g of finely ground sprout samples were digested in H 2 SO 4 –H 2 O at 260 ◦C. A CN element analyzer (NC-2100, Carlo Erba Instruments, Milan, Italy) was used to measure the nitrogen levels. The nitrite-dependent NADH oxidation (A340) was used to evaluate the nitrate reductase (NR, EC 1.7.1.1) enzyme's activity. 16 2.4. Determination of amino acids and their metabolic enzymes A methanol extraction was performed on 100 mg fresh weight (FW) of sprout samples. 17 Using an HP-5 MS column, materials were separated and GC/MS (Hewlett Packard, Palo Alto, CA, USA) analysis was performed. To quantify the amino acids, 200 milligrams of FW sprout leaves were homogenized in 80% aqueous ethanol and centrifuged at 22,000× g for 25 minutes. Re-suspending the precipitates in chloroform required evaporating the supernatant. Once again dissolved in chloroform, the pellet was filtered using Millipore microfilters with a pore size of 0.2 µm. By using a Waters Acquity UPLC-tqd system with a 60/40 acetonitrile/water mobile phase ratio and a measuring wavelength of 254 nm, the amounts of amino acids were determined at 37 ◦ C and low pressure. The result was given as mg/g dry weight of the sample. The reduction of NADH at A340 was used to evaluate the activity of the enzyme glutamine synthetase (GS, EC 6.3.1.2), glutamate synthase (EC 1.4.7.1), and glutamine 2-oxoglutarate aminotransferase (GOGAT, EC 1.4.7.1). NADH oxidation reliant on 2-oxoglutarate is determined by GDH. By evaluating the production of γ-glutamyl hydroxamate at A340, GS activity was identified. By monitoring the glutamine-dependent NADH oxidation at A340, GOGAT activity was calculated. The nitrite-dependent NADH oxidation (A340) was used to evaluate the nitrate reductase (NR, EC 1.7.1.1) enzyme's activity. 15 2.5 Assessment of polyamines and their metabolic enzymes Samples were extracted in perchloric acid (Sigma) while being cooled to 4°C, according to Bregoli et al . 18 The extracts were then centrifuged at 15,000 g for 30 min. Sample supernatants and standard solutions were derivatized with dansyl chloride to find free polyamines. To create conjugated polyamines, samples were hydrolyzed for a whole night at 110°C in 6 N HCl. To ascertain the quantity of dedansylated polyamine derivatives, reverse phase HPLC (Shimadzu SIL10-ADvp; C18 column) was employed. The plant tissues were homogenized in 100 mM KPO4 (pH 7.5) and centrifuged at 25,000 RPM for 20 min to measure the activity of polyamine-related enzymes. The labeled CO2 that was released from L-[l-14C] Ar (55 mCi mmol − 1 ) and L-[l-14C] Orn (55 mCi mmol − 1 ) was used to evaluate the activity of the enzymes ornithine decarboxylase (ODC) and arginine decarboxylase (ADC). 19 Using a liquid scintillation counter, the trapped radioactivity was quantified. In a pH 8.0 Tris-HCl (0.1 M) reaction mixture, the activity of spermidine synthase (SpdS) was measured. 20 With the use of a reverse-phase HPLC fitted with a fluorescence detector, the amount of 50-deoxy-50-methylthioadenosyne produced was measured. The synthesis of methylthioadenosine served as a proxy for spermine synthase (SpmS) activity. 21 The approach of Gao et al . 22 and Sheteiwy et al . 23 was utilized to measure the enzyme responsible for polyamine-degradative enzymes, such as PAO. 2.6 Statistical analysis In the statistical analysis, the student's t-test was used. The statistical significance of the groups was shown by the p < 0.05 values. After being plotted using GraphPad Prism software version 8.4.2, the data are displayed as (means and standard error). Each experiment was conducted with three to five duplicates, with each copy consisting of a mixture of control and MWCNT-treated plants. 3. Results 3.1. Manufacture and characterization of MWCNTs 3.1.1 Field emission scanning electron microscopy (FE-SEM) FESEM images of commercially created MWCNTs are shown in (Fig. 1 ). These multilayered carbon nanotubes (MWCNTs) exhibit bending and entanglement and have lengths in the micrometer range and diameters between 40 and 100 nm. Once the Hummers procedure is applied, the MWCNTs undergo a notable morphological change. As can be observed from the low-magnification image (Fig. 1 a), when CNTs are successfully cut in the transverse direction, shorter particles with lengths of hundreds of nanometers are produced. 24 3.1.2. Fourier transform infrared spectroscopy (FTIR) The FTIR spectrum of MWCNTs exhibits prominent peaks at about 3434, 1539, and 1068 cm − 1 attributable to the stretching vibration of O–H, bending vibration, and C − O, respectively (Fig. 2 ). 25 While the peaks at 2375, and 2914 cm − 1 are related to CNT and C–H x respectively. 26 For MWCNTs to diffuse in water and integrate with the plant system, these covalent bonds are essential. integrate with the plant system. Water dispersion is crucial since water is how MWCNTs are transferred to plant seeds. 27 3.2. Growth The treatment of MWCNTs significantly increased the dry weight of four seedlings by 100%, 100%, 17 in T. foenum – graecum , L.grandiflorum , and L. sativum , respectively, compared with the control, however, A. graveolens did not exhibit a significant increase(Table 1 ). Table 1 Effect of Multiwalled carbon nanotubes priming on dry weight(g), total protein (mg/g Fwt), nitrogen content (mg/gDW), and amino acids content (µmol/ gFW) of four sprouts. Experiments were carried out in triplicate and the data are expressed as mean ± standard error (S.E); Level of significance, * p < 0.05; MWCNT, multiwalled carbon nanotubes. Trigonella foenum - graecum Linum grandiflorum Lepidium sativum Anethum graveolens Control MWCNT-treated Control MWCNT- treated Control MWCNT- treated Control MWCNT- treated Dry weight (g) 0.05 ± 0.01 0.1 ± 0.00* 0.02 ± 0.00 0.04 ± 0.00* 0.06 ± 0.00 0.07 ± 0.00* 0.05 ± 0.00 0.05 ± 0.00 Total protein (mg/g Fwt) 6.51 ± 0.35 13.33 ± 0.42* 20.33 ± 0.63 34.89 ± 1.49* 13.06 ± 0.36 17.33 ± 0.45* 8.64 ± 0.68 18.43 ± 1.40* Nitrogen mg/gDW 192.98 ± 11.62 229.28 ± 8.76 200.62 ± 11.93 240.74 ± 8.76 213.99 ± 10.11 252.21 ± 8.76* 208.26 ± 13.38 244.57 ± 10.64 Essential amino acids Lysine 4.84 ± 0.58 6.07 ± 0.45 5.54 ± 0.55 8.15 ± 1.83 5.16 ± 0.07 5.98 ± 0.13* 4.96 ± 0.27 6.43 ± 0.11* Histidine 1.78 ± 0.1 2.93 ± 0.28 * 2.62 ± 0.57 3.95 ± 0.99 0.95 ± 0.11 1.04 ± 0.14 1.73 ± 0.38 2.45 ± 0.37 Isoleucine 0.32 ± 0.08 0.33 ± 0.08 0.32 ± 0.11 0.24 ± 0.04 0.17 ± 0.04 0.53 ± 0.09* 1.25 ± 0.01 1.10 ± 0.05* Leucine 0.29 ± 0.07 0.39 ± 0.06 0.37 ± 0.09 0.26 ± 0.04 0.15 ± 0.04 0.23 ± 0.04 0.29 ± 0.07 0.38 ± 0.06 Methionine 0.17 ± 0.01 0.29 ± 0.05 0.40 ± 0.12 0.40 ± 0.07 0.19 ± 0.06 0.33 ± 0.09 0.45 ± 0.17 0.53 ± 0.11 Threonine 0.89 ± 0.49 0.86 ± 0.27 1.17 ± 0.53 1.38 ± 0.39 0.99 ± 0.50 1.24 ± 0.34 1.03 ± 0.39 1.33 ± 0.36 Valine 1.05 ± 0.05 0.90 ± 0.02 1.08 ± 0.00 1.23 ± 0.00* 0.98 ± 0.00 1.12 ± 0.02* 0.90 ± 0.05 1.13 ± 0.02* Phenylalanine 1.46 ± 0.55 1.83 ± 0.49 1.54 ± 0.71 1.98 ± 0.53 1.72 ± 0.68 1.63 ± 0.40 2.05 ± 0.77 1.25 ± 0.24 Arginine 0.71 ± 0.04 1.14 ± 0.16 1.53 ± 0.45 1.52 ± 0.19 0.62 ± 0.02 0.81 ± 0.14 1.19 ± 0.02 1.52 ± 0.17 Total EAAs 11.51 13.87 14.57 19.11 10.93 12.91 13.85 16.12 Non-essential amino acids Glycine 56.95 ± 1.85 95.12 ± 14.59 50.03 ± 0.92 58.47 ± 0.66* 50.07 ± 0.67 63.58 ± 1.01* 62.94 ± 1.9 74.30 ± 7.16 Alanine 11.71 ± 0.61 12.48 ± 0.24 8.89 ± 0.12 12.2 ± 0.26* 13.37 ± 0.77 10.29 ± 0.31* 10.36 ± 0.23 11.52 ± 0.07* Ornithine 0.52 ± 0.07 0.51 ± 0.09 0.48 ± 0.04 0.55 ± 0.04 0.43 ± 0.14 0.50 ± 0.06 1.51 ± 0.39 1.44 ± 0.18 Glutamine 0.56 ± 0.06 0.94 ± 0.12* 1.79 ± 0.41 1.53 ± 0.15 0.97 ± 0.30 1.47 ± 0.48 2.07 ± 0.68 2.44 ± 0.32 Asparagine 0.42 ± 0.08 0.70 ± 0.13 1.66 ± 0.04 1.48 ± 0.24 1.16 ± 0.03 2.28 ± 0.17* 3.53 ± 0.46 2.18 ± 0.47 Serine 0.65 ± 0.25 0.71 ± 0.16 0.39 ± 0.15 0.56 ± 0.16 0.59 ± 0.27 0.82 ± 0.24 0.81 ± 0.35 0.84 ± 0.20 Glutamic acid 1.15 ± 0.01 1.46 ± 0.01* 1.42 ± 0.00 1.67 ± 0.00* 1.41 ± 0.01 1.74 ± 0.01* 1.62 ± 0.02 1.83 ± 0.02* Aspartate 0.15 ± 0.01 0.13 ± 0.00 0.17 ± 0.00 0.21 ± 0.00* 0.18 ± 0.01 0.25 ± 0.01* 0.28 ± 0.03 0.30 ± 0.01 Cystine 0.71 ± 0.14 0.82 ± 0.07 0.61 ± 0.01 0.60 ± 0.03 0.30 ± 0.08 0.48 ± 0.02 0.67 ± 0.04 0.77 ± 0.02 Tyrosine 0.71 ± 0.05 0.77 ± 0.04 0.59 ± 0.04 0.41 ± 0.02* 0.29 ± 0.02 0.46 ± 0.03* 0.59 ± 0.04 0.68 ± 0.04 Total NEAAs 73.53 113.64 66.03 77.68 68.77 81.87 84.38 96.3 3.3. Protein, nitrogen, and nitrate reductase activity (NR) Functional proteins are found in soluble proteins, primarily in the form of enzymes. Their content increased by 104.76%, 71.62%, 32.70%, and 113.31%, in T. foenum – graecum , L. grandiflorum , L. sativum , and A. graveolens , respectively, as compared to the control (Table 1 ). MWCNT greatly impacted the N content in the four species as compared to control sprouts. N content increased by 19%,20%,18%, and 17% in T. foenum – graecum , L.grandiflorum , L. sativum , and A. graveolens , respectively (Table 1 ). Depending on the species, the activities of NR changed in response to MWCNT priming. In particular, NR activity rose relative to the control by 27%, 35%, and 10% in T. foenum-graecum , L. grandiflorum , and A. graveolens , respectively, but declined in L. sativum (Fig. 3 ). 3.4 Amino acids metabolism Amino acids are important for the growth and development of sprouts, serving as the building blocks for proteins, which are essential for various physiological processes. They are essential for enzyme activity, cellular metabolism, and the production of hormones and signaling chemicals. The nutritional value and stress tolerance of sprouts are influenced by both essential and non-essential amino acids, which also contribute to the overall health and productivity of the plant. Our results revealed that MWCNT treatment induced significant increases in the levels of the majority of the detected amino acids (including both essential and non-essential ones) (Table 1 ). The non-essential amino acids showed the highest concentrations 89%, 80%, 86%, and 86% in T. foenum - graecum , L. grandiflorum , L. sativum , and A. graveolens , respectively, compared to the control. Essential amino acids showed concentrations of 11%, 20%, 14%, and 14% in T. foenum - graecum , L. grandiflorum, L. sativum , and A. graveolens , respectively, as compared to the control. The amount of histidine, glutamine, and glutamic acid was increased significantly in T. foenum - graecum ; glycine, alanine, glutamic acid, valine, and lysine were enhanced in L. grandiflorum .; glycine, lysine, alanine, asparagine, isoleucine, valine, glutamic acid, aspartate, and tyrosine, were increased in L. sativum ; and lysine, alanine, isoleucine, valine and glutamic acid, glutamine, glycine, were elevated in A. graveolens . Variable trends were noticed among different sprouts. To understand the bases of increased levels of amino acids, we measured their related biosynthetic enzymes. This involved assessing the activity of key enzymes responsible for the biosynthesis of amino acids to provide insights into how MWCNT can boost the production of bioactive amino acids and overall plant metabolism. Key enzymes such as GS, protease, and GOGAT are necessary for plants to absorb and assimilate nitrogen. The sprouts of the four species responded differentially to the impacts of MWCNT on GDH, GOGAT, GS, arginase, and threonine synthase in terms of nitrogen metabolism (Fig. 3 ). Following treatment with MWCNTs, the activity of GDH in four sprouts rose in comparison to the control by 26%, 17%, 25%, and 11% in T. foenum-graecum , L. grandiflorum , L. sativum , and A. graveolens , respectively. Furthermore, MWCNT significantly increased arginase activity by 21% and 13% in L. grandiflorum and A. graveolens , respectively, but it reduced in T. foenum-graecum and L. sativum . GS activity decreased in L. grandiflorum and L. sativum , but increased by 13% and 14% in T. foenum-graecum and A. graveolens , respectively, compared to the control. Additionally, TS increased by 12%, 61%, and 16% in T. foenum-graecum , L. sativum , and A. graveolens , respectively, compared to the control, but decreased in L. grandiflorum . Following MWCNT treatment, GOGAT activity increased by 16%, 4%, 59%, and 14% in T. foenum-graecum , L. grandiflorum , L. sativum , and A. graveolens , respectively, compared to the control. 3.5 Polyamine metabolism 3.5.1. Polyamine levels Polyamines such as diamino propane, putrescine, cadaverine, spermine, spermidine, and agmatine were measured to study the impact of MWCNT priming on polyamine metabolism in sprouts of the four investigated species(Table 2 ). Our results showed that putrescine levels were generally elevated by 57%,50%,47%, and 28%, respectively in T. foenum-graecum , L. grandiflorum , L. sativum , and A. graveolens . Spermidine increased by 4%,77%,32%, and 39%, respectively in T. foenum-graecum , L. grandiflorum , L. sativum , and A. graveolens . In addition, Spermine enhanced by 116%,5%,61%, and 33%, respectively in T. foenum-graecum , L. grandiflorum , L. sativum , and A. graveolens . When compared to the control, diaminopropane levels in T. foenum-graecum , L. grandiflorum , L. sativum , and A. graveolens increased by 124%, 48%, 60%, and 46%, respectively. On the other hand, T. foenum-graecum , L. grandiflorum , L. sativum , and A. graveolens all showed notable increases in cadaverine after receiving MWCNT treatment: 98%, 54%, 38%, and 40%, respectively. Meanwhile, agmatine increased in L. grandiflorum , and L. sativum , while decreasing in T. foenum – graecum , and A. graveolens . Table 2 Effect of Multiwalled carbon nanotubes priming on polyamines(nmol/gFW) content of four sprouts. Experiments were carried out in triplicate and the data are expressed as mean ± standard error (S.E); Level of significance, * p < 0.05; MWCNT, multiwalled carbon nanotubes. Trigonella foenum - graecum Linum grandiflorum Lepidium sativum Anethum graveolens Control MWCNT-treated Control MWCNT- treated Control MWCNT- treated Control MWCNT- treated Di amino propane 3.53 ± 0.07 7.91 ± 0.50* 3.11 ± 0.03 4.61 ± 0.03* 3.12 ± 0.02 5.00 ± 0.04* 3.90 ± 0.07 5.69 ± 0.25* Putrescine 40.03 ± 2.69 62.90 ± 2.34* 45.59 ± 2.53 68.19 ± 4.41* 41.40 ± 0.32 60.95 ± 0.74* 40.25 ± 1.22 51.35 ± 0.37* Cadaverine 1.81 ± 0.06 3.59 ± 0.11* 2.78 ± 0.33 4.28 ± 0.44 0.99 ± 0.07 1.37 ± 0.10* 1.84 ± 0.22 2.57 ± 0.20 Spermidine 3.18 ± 2.49 3.31 ± 2.54 2.33 ± 1.77 4.13 ± 3.18 2.75 ± 2.12 3.65 ± 2.85 2.77 ± 2.14 3.85 ± 2.94 Spermine 19.41 ± 0.71 41.86 ± 3.32* 44.24 ± 2.14 46.36 ± 7.33 17.23 ± 0.34 27.68 ± 1.55* 33.21 ± 0.27 44.01 ± 2.78* Agmatine 0.54 ± 0.04 0.51 ± 0.04 0.49 ± 0.03 0.54 ± 0.02 0.49 ± 0.10 0.51 ± 0.03 1.62 ± 0.23 1.50 ± 0.08 3.5.2 Polyamine metabolism We measured the activity of plolyamines related enzymes such as arginine decarboxylase, ornithine decarboxylase, S-adenosyl-L-methionine decarboxylase, spermine synthase, spermidine synthase, arginine decarboxylase. We first focused on the polyamine-related enzymes including arginine decarboxylase and ornithine decarboxylase, which form putrescine from L-ornithine and can subsequently be converted into spermidine and spermine, to investigate the mechanism underlying the changes in polyamines (Fig. 4 ). S-adenosyl-L-methionine decarboxylase, which plays a role in the biosynthesis of spermidine and spermine from putrescine, as well as its involvement in the conversion of S-adenosyl methionine into S-adenosylmethioninamine, was also measured. This increase in putrescine levels was correlated with a significant increase in either ADC or ODC. Also, MWCNT treatment resulted in the accumulation of spermidine and spermine. This accumulation is consistent with the enhanced levels of spermidine synthase (SpdS), spermine synthase (SpmS), and S-adenosyl-L-methionine decarboxylase. The current results also demonstrated that both PAO and DAO were decreased in all species. 4. Discussion 4.1. Importance of N in improving sprouts' nutritive value Nitrogen (N) is necessary for many metabolic reactions that improve the chemical composition of sprout tissue. It improves the synthesis of both primary and secondary metabolites, enzyme activity, polyamines, chlorophyll synthesis, and overall nitrogen assimilation. Because of these aspects, sprouts have better more accurate growth, nutritional value, and health benefits; therefore, nitrogen is crucial for the growth and quality of sprouts. The nitrogen assimilation process, which has greatly influenced the tissue chemical composition of sprouts, needs nitrate reductase (NR). The process of transforming consumed nitrogen into forms that can be used is started when NR reduces nitrate to nitrite. During this process, the ammonium is formed and then subsequently incorporated into amino acids and proteins. Elevated (NR) activity stimulates growth, biomass accumulation, the formation of amino acids and proteins, and metabolic activity. MWCNTs can substantially boost NR activity, and affect vital enzymes that contribute to nitrogen assimilation. It may also greatly enhance the nutritional content and nitrogen metabolism of sprouts. The currently available outcomes demonstrate that there were observable rises in both N and NR in each sprout. Similarly, higher dosages of MWCNTs (such as 50, 100, and 200 µg/mL) show higher activity in the stimulation of nitrate absorption, assimilation, and nitrogen metabolism in sprouts. 4 Due to the higher biomass of sprouts that result from, nitrogen content, and a buildup of healthy bioactive substances such as proteins, amino acids, and antioxidants. 4.2. Increased essential amino acids and polyamines improved sprout tissue chemical composition It's well-known that amino acids have a crucial role in plant enlargement and development, whereas they stimulate nitrogen metabolism and nutrition absorption, and share in building blocks for proteins and precursors for certain metabolites. Particularly, the variation in nutritional and health-promoting characteristics greatly depends on the necessary amino acid constituents of those plants. The investigated sprout's construction was formed of both necessary and non-essential amino acids. The vital amino acids play a critical role in the enhancement of nutritional content such as vitamins, minerals, and bioactive substances during germination which in turn was important in plant growth and development. Comparatively, amino acids participate in the improvement of polyamine production and subsequently have a good impression on nitrogen metabolism, protein synthesis, enzyme activity, and the synthesis of bioactive metabolites. Additionally, polyamines participate in many physiological processes such as the availability of nutrients, malfunctioning phytic acid, increasing antioxidant stimulation, cell division, and adding to their nutritional value. The final result of this result was the refinement of the nutritional components of sprouts, making them a more useful and nutrient-dense food option. According to the species, MWCNTs can leave various impacts on polyamine yielding. Similar results were recorded in mung bean sprouts that have been impacted by MWCNT priming whereas they manage the breakdown of phytic acid through stimulation of polyamines, specifically spermidine and putrescine. 28 Nonetheless, it has been observed that MWCNT priming influences the polyamine metabolism of peach fruit in response to chilling damage (CI). The study concluded that MWCNT priming elevated the amounts of putrescine, spermine, and spermidine in peach fruit, which assisted in reducing the effects of CI. 29 4.3. Biochemical bases of increased amino acids and polyamines The main importance of amino acids is that they act as basic units for the construction of protein and play a crucial role in nutritional absorption and N metabolism, besides acting as a precursor for certain metabolites. Certainly, in the conversion of organic N from inorganic form, the amino acids act as intermediates, and the plant conveys the pathway of GS/ GOGAT to transform the inorganic N to glutamine and then glutamate. These amino acids were used widely by some proteins, nucleic acids, and other amino acids. The four sprouts in our investigation had higher total protein and amino acid contents after MWCNT treatment, which enhanced N metabolism. It has been demonstrated that MWCNTs stimulate the activity of important nitrogen metabolism-related enzymes, including GS and GOGAT. 30 As a result, nitrogen-containing substances like proteins and amino acids build up in plants. MWCNTs appear to have the ability to affect plant amino acid metabolism in general by stimulating the production and accumulation of amino acids, as evidenced by their regulation of enzymes such as GS and GOGAT. This is especially clear from a study where 100 mg/L MWCNTs boosted the activity of important enzymes involved in the metabolism of nitrogen, causing soluble proteins, carbohydrates (sugars and starch), and nitrogen to accumulate in maize plants. 31 Our results declared that the treatment with MWCNT caused a remarkable accumulation in the levels of Put as well as Spm and Spd in addition to their metabolic enzymes (ADC, ODC, and S-adenosylmethionine decarboxylase; SAMDC, Spd synthase, and Spm synthase). There were indirect impacts of MWCNTs on polyamine metabolism during the regulation of nitrogen metabolism due to the linear influence of the availability of nitrogen compounds on the homeostasis and synthesis of plant polyamines. For example, it has been demonstrated that MWCNT priming improves radish sprouts' polyamine metabolism, namely the accumulation of putrescine, spermine, and spermidine. 32 Our findings revealed that there was an elevation in the level of polyamines as well as the enzyme activities necessary for their synthesis, which include (ADC), (SpdS), and (ODC) during the application of MWCNT priming. Gondor et al . 33 stated a similar impact on the polyamine metabolism under different light conditions. The current outcomes demonstrated higher levels of putrescine and spermidine in both high and low-light conditions throughout the MWCNT treatment, on the other hand, there was no significant influence on spermine rates. With this intention, MWCNTs influence varies depending on the category of plant species in the polyamine synthesis. Amino acids operate as fundamental components in the synthesis of polyamines, which are vital for cell growth and differentiation. Polyamines are produced directly from the amino acids (ornithine and arginine), which are obtained via the nitrogen absorption pathway. Putrescine is generated when arginine undergoes a process known as ADC, which creates agmatine. Putrescine is produced when ODC decarboxylates ornithine, which is formed by transaminates glutamate. Key amino acids are used for the production of putrescine, the first polyamine. As a substrate, it serves to facilitate the formation of spermidine and spermine. The crucial amino acid called methionine is converted to (SAM), which is then decarboxylated by SAMDC to generate decarboxylated SAM. The correlation between amino acid metabolism and polyamine production is highlighted by the fact that decarboxylated SAM contributes aminopropyl groups for the formation of spermidine and spermine from putrescine. These pathways are major targets for improving the nutritional and biochemical qualities of sprouts since they are essential for plant growth, development, and stress responses. Conclusion Finally, the endpoint of the current study revealed that usage of four plant species A. graveolens, L. sativum , T. foenum-graecum , and L. grandiflorum that were treated with (MWCNTs) multi-walled carbon nanotubes showed well impact on the metabolism of nitrogen, amino acid, and polyamine. The data analysis exhibited a raised level of GS, NR, and GOGAT enzymes which stimulated the nitrogen composition and improved assimilation of nitrogen. It witnessed also an enhancement in protein synthesis by detecting higher levels of soluble proteins. Additionally, both essential and nonessential amino acids revealed a significant increase by treatment with MWCNTs. Therefore, the physiological activities of the plant like response to stress, enzyme activity, and cellular metabolism needed the improvement of the productivity of the plant besides the support of the general health that was achieved by increasing the level of amino acids. Not only amino acids but also, polyamines metabolism were enhanced by additional MWCNTs. The current analysis revealed the elevation of (ODC), the biosynthetic activities of enzymes including (ADC), and polyamines such as putrescine, spermidine, and spermine significantly. In conclusion, the MWCNT is considered a promising strategy for the enhancement of the metabolic process that is necessary for the adaptation and development of the plant. The agriculture nanotechnology opened the way for improving the crop yield and resistance which was proved through the current investigation. More studies were required to explore the promising mechanisms of MWCNT seeking the enhancement of the sustainable agriculture yield and resistance. Declarations Funding The authors extend their appreciation to the Researchers Supporting Project number (RSPD2024R931) King Saud University, Riyadh, Saud Arabia. Author Contribution A- Yasmen khaled (write the main manuscript-experimental setup-Resources-software-validation)B-Momtaz M. Hegaba(Data curation- formal analysis-project administration-supervision)C-Mohammad K. Okla(Data curation- funding-supervision- validation)D-Amal Mohamed AlGarawi(Data curation- funding-supervision- validation)E-Wael Z. Tawfik(Data curation- formal analysis-project administration-supervision)F-Gehad AbdElgayed(Data curation- supervision- validation)G-Mona Sayed (Data curation- formal analysis-project administration-supervision) Acknowledgement The authors extend their appreciation to the Researchers Supporting Project number (RSPD2024R931) King Saud University, Riyadh, Saud Arabia. References Slepičková Kasálková, N., Slepička, P., & Švorčík, V. (2021). Carbon nanostructures, nanolayers, and their composites. Nanomaterials, 11 (9), 2368. H. He, L.A. Pham-Huy, P. Dramou, D. Xiao, P. Zuo, C. Pham-Huy, Carbon nanotubes: applications in pharmacy and medicine, BioMed Res. Int. (2013). Oliveira, T. M., & Morais, S. (2018). New generation of electrochemical sensors based on multi-walled carbon nanotubes. Applied Sciences , 8 (10), 1925. Shi, J., Xun, M., Song, J., Li, J., Zhang, W., & Yang, H. (2023). 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A., & AbdElgawad, H. (2021). Silicon dioxide nanoparticles orchestrate carbon and nitrogen metabolism in pea seedlings to cope with broomrape infection. Environmental Science: Nano, 8 (7), 1960–1977. Nagulapalli Venkata, K. C., Swaroop, A., Bagchi, D., & Bishayee, A. (2017). A small plant with big benefits: Fenugreek (Trigonella foenum-graecum Linn.) for disease prevention and health promotion. Molecular nutrition & food research, 61(6), 1600950. Al-Madhagy, S., Ashmawy, N. S., Mamdouh, A., Eldahshan, O. A., & Farag, M. A. (2023). A comprehensive review of the health benefits of flaxseed oil in relation to its chemical composition and comparison with other omega-3-rich oils. European journal of medical research, 28(1), 240. Agarwal, N., & Sharma, S. (2013). Appraisal of garden cress (Lepidium sativum L.) and product development as an all pervasive and nutrition worthy food stuff. Annals of Food Science and Technology , 14 (1), 2013. Jana, S., & Shekhawat, G. S. (2010). 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Torrigiani, Peach (Prunus persica) fruit ripening: aminoethoxyvinylglycine (AVG) and exogenous polyamines affect ethylene emission and flesh firmness, Physiol. Plant 114 (2002) 472–481. H. Birecka, A.J. Bitonti, P.P. McCann, Assaying ornithine and arginine decarboxylases in some plant species, Plant Physiol. 79 (1985) 509–514, https://doi.org/10.1104/pp.79.2.509 . S.O. Yoon, Y.S. Lee, S.H. Lee, Y.D. Cho, Polyamine synthesis in plants: isolation and characterization of spermidine synthase from soybean (Glycine max) axes, Biochim. Biophys. Acta - Gen. Subj. 1475 (2000) 17–26, https://doi.org/10.1016/S0304-4165(00)00039-8 . A.L. Cason, Y. Ikeguchi, C. Skinner, T.C. Wood, K.R. Holden, H.A. Lubs, F. Martinez, R.J. Simensen, R.R. Stevenson, A.E. Pegg, C.E. Schwartz, X-linked spermine synthase gene (SMS) defect: the first polyamine deficiency syndrome, Eur. J. Hum. Genet. 11 (2003) 937–944. Gao, C., Sheteiwy, M.S., Lin, C., Guan, Y., Ulhassan, Z., Hu, J., 2021. Spermidine suppressed the inhibitory effects of polyamines inhibitors combination in maize (Zea mays L.) seedlings under chilling stress. Plants 10, 2421. Sheteiwy S, M, Shen, H, Xu, J, Guan, Y, Song, W, Hu, J, 2017. Seed polyamines metabolism induced by seed priming with spermidine and 5-aminolevulinic acid for chilling tolerance improvement in rice (Oryza sativa L.) seedlings. Environ. Exp. Bot. 137, 58–72. Wang, H., Wang, Y., Hu, Z., & Wang, X. (2012). Cutting and unzipping multiwalled carbon nanotubes into curved graphene nanosheets and their enhanced supercapacitor performance. ACS applied materials & interfaces, 4(12), 6827–6834. Misra, A., Tyagi, P. K., Rai, P., & Misra, D. S. (2007). FTIR spectroscopy of multiwalled carbon nanotubes: a simple approachto study the nitrogen doping. Journal of nanoscience and nanotechnology, 7(6), 1820–1823. Joshi, A., Kaur, S., Dharamvir, K., Nayyar, H., & Verma, G. (2018). Multi-walled carbon nanotubes applied through seed‐priming influence early germination, root hair, growth and yield of bread wheat (Triticum aestivum L.). Journal of the Science of Food and Agriculture, 98(8), 3148–3160. Szewczyk, A., Paździora, W., & Ekiert, H. (2023). The Influence of Exogenous Phenylalanine on the Accumulation of Secondary Metabolites in Agitated Shoot Cultures of Ruta graveolens L. Molecules, 28 (2), 727. Zhou, T., Wang, P., Yang, R., & Gu, Z. (2018). Polyamines regulating phytic acid degradation in mung bean sprouts. Journal of the Science of Food and Agriculture, 98(9), 3299–3308. Wuddineh, W., Minocha, R., & Minocha, S. C. (2018). Polyamines in the context of metabolic networks. Polyamines: methods and protocols, 1–23. Dai, J., Duan, L., & Dong, H. (2015). Comparative effect of nitrogen forms on nitrogen uptake and cotton growth under salinity stress. Journal of Plant Nutrition, 38(10), 1530–1543. Hu, Y., Zhang, P., Zhang, X., Liu, Y., Feng, S., Guo, D., … Dang, X. (2021). Multi-wall carbon nanotubes promote the growth of maize (Zea mays) by regulating carbon and nitrogen metabolism in leaves. Journal of Agricultural and Food Chemistry, 69(17), 4981–4991. Hasanuzzaman, M., Alhaithloul, H. A. S., Parvin, K., Bhuyan, M. B., Tanveer, M., Mohsin,S. M., … Fujita, M. (2019). Polyamine action under metal/metalloid stress: regulation of biosynthesis, metabolism, and molecular interactions. International Journal of Molecular Sciences, 20(13), 3215. Gondor, O. K., Tajti, J., Hamow, K. Á., Majláth, I., Szalai, G., Janda, T., & Pál, M. (2021). Polyamine metabolism under different light regimes in wheat. International Journal of Molecular Sciences, 22(21), 11717. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 21 Oct, 2024 Read the published version in Journal of Nanoparticle Research → Version 1 posted Editorial decision: Revision requested 31 Aug, 2024 Reviews received at journal 26 Aug, 2024 Reviewers agreed at journal 16 Aug, 2024 Reviewers invited by journal 15 Aug, 2024 Editor assigned by journal 26 Jul, 2024 Submission checks completed at journal 26 Jul, 2024 First submitted to journal 21 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4777965","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":343195509,"identity":"eb8aa376-cbe4-4025-a018-a86baf92ef72","order_by":0,"name":"Yasmen Khaled","email":"data:image/png;base64,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","orcid":"","institution":"Beni-suef University","correspondingAuthor":true,"prefix":"","firstName":"Yasmen","middleName":"","lastName":"Khaled","suffix":""},{"id":343195510,"identity":"9d88a977-222f-4dae-a681-4b43d6026bc3","order_by":1,"name":"Momtaz M. Hegab","email":"","orcid":"","institution":"Beni-suef University","correspondingAuthor":false,"prefix":"","firstName":"Momtaz","middleName":"M.","lastName":"Hegab","suffix":""},{"id":343195511,"identity":"e67e14ca-a1f2-49cf-9594-6950dcd29cd3","order_by":2,"name":"Mohammad K. Okla","email":"","orcid":"","institution":"King Saud University","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"K.","lastName":"Okla","suffix":""},{"id":343195512,"identity":"bbdf443e-0bb7-4c24-96ee-e1af35b3e3d7","order_by":3,"name":"Amal Mohamed AlGarawi","email":"","orcid":"","institution":"King Saud University","correspondingAuthor":false,"prefix":"","firstName":"Amal","middleName":"Mohamed","lastName":"AlGarawi","suffix":""},{"id":343195513,"identity":"32fb25aa-6c51-4aa4-9998-d86fc53f00ce","order_by":4,"name":"Wael Z. Tawfik","email":"","orcid":"","institution":"Beni-suef University","correspondingAuthor":false,"prefix":"","firstName":"Wael","middleName":"Z.","lastName":"Tawfik","suffix":""},{"id":343195514,"identity":"a7f6f4cf-38a0-46bc-8a80-54a6591b6c38","order_by":5,"name":"Gehad AbdElgayed","email":"","orcid":"","institution":"University of Antwerp","correspondingAuthor":false,"prefix":"","firstName":"Gehad","middleName":"","lastName":"AbdElgayed","suffix":""},{"id":343195515,"identity":"64a3a8d4-c730-4f92-8eb9-9b9b46455a40","order_by":6,"name":"Mona Sayed","email":"","orcid":"","institution":"Beni-suef University","correspondingAuthor":false,"prefix":"","firstName":"Mona","middleName":"","lastName":"Sayed","suffix":""}],"badges":[],"createdAt":"2024-07-21 19:08:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4777965/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4777965/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11051-024-06145-6","type":"published","date":"2024-10-21T15:57:50+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":63025566,"identity":"ca9b5574-c650-48fd-a0b2-258a6422858c","added_by":"auto","created_at":"2024-08-22 08:22:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":343608,"visible":true,"origin":"","legend":"\u003cp\u003eFESEM images of the MWCNTs (a,b)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4777965/v1/11226508fa72dbdcc5f5d57f.png"},{"id":63025565,"identity":"a7d370da-5aba-4db3-a43b-06a79653121e","added_by":"auto","created_at":"2024-08-22 08:22:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":106638,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra of MWCNTs.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4777965/v1/83a6ab3b7c9185d4f8cc4682.png"},{"id":63026304,"identity":"1df34de3-5cb1-432e-b531-3fd7df50fa71","added_by":"auto","created_at":"2024-08-22 08:30:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":248262,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Multiwalled carbon nanotubes priming on the activity of nitrogen metabolizing enzymes such as nitrate reductase (NR), glutamate dehydrogenase (GDH), glutamine synthetase (GS), glutamine oxoglutarate aminotransferase (GOGAT), threonine synthase (TS) and arginase (ARG) expressed as (µmol mg\u003csup\u003e−1\u003c/sup\u003e protein. min) in four sprouts. Experiments were carried out in triplicate and the data are expressed as mean ± standard error (S.E); Level of significance, * p \u0026lt; 0.05; MWCNT, multiwalled carbon nanotubes.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4777965/v1/53e3169f42b72fac8dc24998.png"},{"id":63025568,"identity":"ad34a040-9a73-482d-a435-f6feeca45bcd","added_by":"auto","created_at":"2024-08-22 08:22:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":160487,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different treatments on polyamine-related enzymes (µ mol mg\u003csup\u003e-1\u003c/sup\u003e Protein min) of four sprouts. Experiments were carried out in triplicate and the data are expressed as mean ± standard error (S.E); Level of significance, * p \u0026lt; 0.05; MWCNT, multiwalled carbon nanotubes\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4777965/v1/6d393a32f0378f4776ba1b48.png"},{"id":67681942,"identity":"d057625e-e5c4-4d13-9476-05785b950cea","added_by":"auto","created_at":"2024-10-28 16:11:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1859557,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4777965/v1/59a8a681-88f5-4221-b7ea-0d6ee02a9f00.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Potential role of multiwalled carbon nanotube priming in boosting nitrogen metabolism and nutritional value during the sprouting process","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eEnhancing plant growth and productivity is an objective of agriculture farming, particularly under undesirable conditions. The application of nanotechnology has shown promise in enhancing the chemical and biological functions of plants. Nanotechnology has transformed several industries, including electronics, materials science, healthcare, and agriculture, by creating nanoparticles with specific properties. Carbon nanotubes (CNTs) nanomaterial is considered a unique substance due to its good properties in mechanical, electrical, and thermal applications.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Additionally, various industries depend on MWCNTs as a type of nanomaterials. With a diameter range from 1 to 3 nm, the Graphene sheets were used to form multi-walled carbon nanotubes.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Furthermore, the numerous layers enjoy higher stability in the thermal properties and mechanical strength than the single-walled carbon nanotubes (SWCNTs). Multiple investigations were carried out on MWCNTs due to their exceptional physicochemical characteristics such as electrical conductivity, chemical stability, and high surface area.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e MWCNTs interact positively with plants in various manners, mainly they act on the improvement of the uptake and utilization of nutrients. Also, it was involved in enzyme activity alteration that was shared in nutrient assimilation like nitrogen which in turn significantly assisted in the plant metabolism enhancement. MWCNTs contributed to the absorption of nutrition and enhancement of the accumulation of soluble proteins in \u003cem\u003eMalus hupehensis\u003c/em\u003e and free amino acids by alteration of (NR) activity that in turn improves plant growth.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e There were a few investigations about the ability of MWCNTs the improve sprouts' nitrogen metabolism. The current study seeks to explore the impact of MWCNTs on the assimilation, absorption, and metabolic process of nitrogen in specific plant species.\u003c/p\u003e \u003cp\u003eMany studies exhibited the importance of nitrogen molecules as a critical macronutrientional compound which was necessary in the creation of variable biomolecules, like nucleic acids, amino acids, and, chlorophyll.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e This compound has a great role in the nitrogen metabolism yield, development, and plant growth through plant consumption of nitrogen. The environmental conditions impressed the nitrogen consumption which greatly affects the productivity of yields and plant development. Nitrogen metabolism is critical for sprout growth and development, which can be obtained from many sources such as ammonia (NH3) fertilizer.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e Generally, nitrogen metabolism was considered the major influencer on the complete development of plant growth and nutrient uptake, utilization, and assimilation of nutrients in sprouts.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e In plants, an accumulation of polyamines and amino acids influences various physiological and biochemical activities. They are required for the synthesis of proteins, growth, nitrogen metabolism, photosynthesis, and hormone regulation. They boost the nutritional quality of plants through their contributions to protein content, vitamins, secondary metabolites, and nutrient bioavailability. Moreover, the nitrogen shortage altered the sprout\u0026rsquo;s metabolism which in turn affected the concentration of vital metabolites and other organic acids that were required for plant enlargement and development. According to prior research, MWCNTs and nano-SiO2 raise the activities of the nitrogen metabolism-related enzymes GS, GOGAT, GAD, and GDH in maize leaves and roots. They can also boost pyruvate levels, which will aid in the synthesis of carbohydrates and the uptake of nitrogen, and eventually encourage plant growth.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e Many investigators highlighted the role of silicon nanoparticles in the improvement of sugar metabolism, accumulation of osmoprotectants and antioxidants, synthesis of amino acids, and seedling growth of peas.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe current investigation aimed to explore the capacity of MWCNTs in the improvement of nitrogen metabolism for sprouts that belong to four medical commercial plant species \u003cem\u003eT. foenum-graecum\u003c/em\u003e, \u003cem\u003eL. sativum\u003c/em\u003e, \u003cem\u003eA. graveolens\u003c/em\u003e, and \u003cem\u003eL. grandiflorum\u003c/em\u003e. Due to the economic, medicinal, and substantial nutritional value of the targeted plants, they were chosen for the present study. The annual herb fenugreek is the common name for \u003cem\u003eT. foenum-graecum\u003c/em\u003e and belongs to \u003cem\u003ethe Fabaceae\u003c/em\u003e family. Through phytochemical examination, it is composed of carbohydrates, amino acids, lipids, flavonoids, steroids, polyphenols, hydrocarbons, alkaloids, and saponins.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e The Linaceae family includes \u003cem\u003eL. grandiflorum\u003c/em\u003e with the common name flax. Their seeds were considered good provenance fibers of diet, omega-3 fatty acids, and lignans. Some studies were registered as anti-carcinic agents with anti-inflammatory properties and cardiovascular protection.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e Concerned with the \u003cem\u003eBrassicaceae\u003c/em\u003e family, it includes a fast-growing edible herb with the scientific name \u003cem\u003eL. sativum\u003c/em\u003e. It was recorded that it was composed of important minerals like calcium and iron, vitamins A, C, and K, proteins, lipids, and carbs.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Finally, the \u003cem\u003eApiaceae\u003c/em\u003e family includes \u003cem\u003eA. graveolens\u003c/em\u003e herb, whereas their seeds contain phenolic acids, steroids, flavonoids, and coumarins.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e It was abundant in the medical field due to its antibacterial, anti-inflammatory, and digestive characteristics. Many contributions were added to the agriculture field through exploring innovative agricultural techniques, like MWCNTs in seed priming, which subsequently develop the activities of development and metabolism of examined plant tissue. This research attempts to explore the possibility of MWCNT-based seed priming to enhance N metabolism during the sprouting stage and thus raise the development and production of these significant plant species.\u003c/p\u003e "},{"header":"2. Materials And Methods","content":"\n\u003ch3\u003e2.1. Carbon nanotubes preparation and characterization\u003c/h3\u003e\n\u003cp\u003eMulti-walled carbon nanotubes (MWCNTs) were acquired from the Centre of Nanomaterials (Beni Suef University, Beni Suef 62521, Egypt). Chemical vapor deposition (CVD) was used to prepare highly pure MWCNTs, as previously described in the literature.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e An alumina tube furnace was filled with 0.5 g of the Fe/Co catalyst. A nitrogen gas flow rate of 70 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was used to heat the catalyst to 700\u0026deg;C. After heating the catalyst surface for 10 minutes at 700\u0026deg;C, acetylene gas was added to the alumina tube over the catalyst surface at a flow rate of 10 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for 30 minutes, and nitrogen gas was reduced to 30 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Finally, the CNTs were collected once the tube furnace had cooled to room temperature.\u003c/p\u003e \u003cp\u003eThe surface form and size of MWCNTs were investigated using field emission scanning electron microscopy (FE-SEM) with a dispersive energy X-ray (EDX) analyzer (JSM-6700 F, JEOL Japan). To record the photographs, the operating voltage in the secondary emission mode was 15 kV. Fourier transform infrared spectroscopy (FTIR), using a Nicolet Nexus 670 FT-IR instrument, was used to determine the material composition.\u003c/p\u003e\n\u003ch3\u003e2.2. Determination of total soluble protein\u003c/h3\u003e\n\u003cp\u003eThe quantity of soluble proteins was measured using the Bradford technique.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Five milliliters of Coomassie blue reagent (100 milliliters of Coomassie brilliant blue G-250 dissolved in 50 milliliters of 95% ethanol, followed by 100 milliliters of 85% H\u003csub\u003e3\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e and finally up to 1 liter of distilled water) with 0.1 milliliters of crude extract. Following 30 min, the absorbance was measured at 595 nm in comparison to a water-reagent blank. The bovine serum albumin reference curve was used to calculate the protein concentration (as mg protein g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FWt).\u003c/p\u003e\n\u003ch3\u003e2.3 Determination of nitrogen and nitrate reductase activity\u003c/h3\u003e\n\u003cp\u003eTo determine the total nitrogen (N) content, 0.2 g of finely ground sprout samples were digested in H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e\u0026ndash;H\u003csub\u003e2\u003c/sub\u003eO at 260 ◦C. A CN element analyzer (NC-2100, Carlo Erba Instruments, Milan, Italy) was used to measure the nitrogen levels. The nitrite-dependent NADH oxidation (A340) was used to evaluate the nitrate reductase (NR, EC 1.7.1.1) enzyme's activity.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003ch3\u003e2.4. Determination of amino acids and their metabolic enzymes\u003c/h3\u003e\n\u003cp\u003eA methanol extraction was performed on 100 mg fresh weight (FW) of sprout samples.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Using an HP-5 MS column, materials were separated and GC/MS (Hewlett Packard, Palo Alto, CA, USA) analysis was performed. To quantify the amino acids, 200 milligrams of FW sprout leaves were homogenized in 80% aqueous ethanol and centrifuged at 22,000\u0026times; g for 25 minutes. Re-suspending the precipitates in chloroform required evaporating the supernatant. Once again dissolved in chloroform, the pellet was filtered using Millipore microfilters with a pore size of 0.2 \u0026micro;m. By using a Waters Acquity UPLC-tqd system with a 60/40 acetonitrile/water mobile phase ratio and a measuring wavelength of 254 nm, the amounts of amino acids were determined at 37 \u003csup\u003e◦\u003c/sup\u003eC and low pressure. The result was given as mg/g dry weight of the sample.\u003c/p\u003e \u003cp\u003eThe reduction of NADH at A340 was used to evaluate the activity of the enzyme glutamine synthetase (GS, EC 6.3.1.2), glutamate synthase (EC 1.4.7.1), and glutamine 2-oxoglutarate aminotransferase (GOGAT, EC 1.4.7.1). NADH oxidation reliant on 2-oxoglutarate is determined by GDH. By evaluating the production of γ-glutamyl hydroxamate at A340, GS activity was identified. By monitoring the glutamine-dependent NADH oxidation at A340, GOGAT activity was calculated. The nitrite-dependent NADH oxidation (A340) was used to evaluate the nitrate reductase (NR, EC 1.7.1.1) enzyme's activity. \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003ch3\u003e2.5 Assessment of polyamines and their metabolic enzymes\u003c/h3\u003e\n\u003cp\u003eSamples were extracted in perchloric acid (Sigma) while being cooled to 4\u0026deg;C, according to Bregoli \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e The extracts were then centrifuged at 15,000 g for 30 min. Sample supernatants and standard solutions were derivatized with dansyl chloride to find free polyamines. To create conjugated polyamines, samples were hydrolyzed for a whole night at 110\u0026deg;C in 6 N HCl. To ascertain the quantity of dedansylated polyamine derivatives, reverse phase HPLC (Shimadzu SIL10-ADvp; C18 column) was employed. The plant tissues were homogenized in 100 mM KPO4 (pH 7.5) and centrifuged at 25,000 RPM for 20 min to measure the activity of polyamine-related enzymes. The labeled CO2 that was released from L-[l-14C] Ar (55 mCi mmol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and L-[l-14C] Orn (55 mCi mmol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was used to evaluate the activity of the enzymes ornithine decarboxylase (ODC) and arginine decarboxylase (ADC).\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e Using a liquid scintillation counter, the trapped radioactivity was quantified. In a pH 8.0 Tris-HCl (0.1 M) reaction mixture, the activity of spermidine synthase (SpdS) was measured.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e With the use of a reverse-phase HPLC fitted with a fluorescence detector, the amount of 50-deoxy-50-methylthioadenosyne produced was measured. The synthesis of methylthioadenosine served as a proxy for spermine synthase (SpmS) activity.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e The approach of Gao \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e and Sheteiwy \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e was utilized to measure the enzyme responsible for polyamine-degradative enzymes, such as PAO.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical analysis\u003c/h2\u003e \u003cp\u003eIn the statistical analysis, the student's t-test was used. The statistical significance of the groups was shown by the p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 values. After being plotted using GraphPad Prism software version 8.4.2, the data are displayed as (means and standard error). Each experiment was conducted with three to five duplicates, with each copy consisting of a mixture of control and MWCNT-treated plants.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Manufacture and characterization of MWCNTs\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e3.1.1 Field emission scanning electron microscopy (FE-SEM)\u003c/h2\u003e \u003cp\u003eFESEM images of commercially created MWCNTs are shown in (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These multilayered carbon nanotubes (MWCNTs) exhibit bending and entanglement and have lengths in the micrometer range and diameters between 40 and 100 nm. Once the Hummers procedure is applied, the MWCNTs undergo a notable morphological change. As can be observed from the low-magnification image (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), when CNTs are successfully cut in the transverse direction, shorter particles with lengths of hundreds of nanometers are produced.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1.2. Fourier transform infrared spectroscopy (FTIR)\u003c/h2\u003e \u003cp\u003eThe FTIR spectrum of MWCNTs exhibits prominent peaks at about 3434, 1539, and 1068 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e attributable to the stretching vibration of O\u0026ndash;H, bending vibration, and C\u0026thinsp;\u0026minus;\u0026thinsp;O, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e While the peaks at 2375, and 2914 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are related to CNT and C\u0026ndash;H\u003csub\u003ex\u003c/sub\u003e respectively.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e For MWCNTs to diffuse in water and integrate with the plant system, these covalent bonds are essential. integrate with the plant system. Water dispersion is crucial since water is how MWCNTs are transferred to plant seeds.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Growth\u003c/h2\u003e \u003cp\u003eThe treatment of MWCNTs significantly increased the dry weight of four seedlings by 100%, 100%, 17 in \u003cem\u003eT. foenum \u0026ndash; graecum\u003c/em\u003e, \u003cem\u003eL.grandiflorum\u003c/em\u003e, and \u003cem\u003eL. sativum\u003c/em\u003e, respectively, compared with the control, however, \u003cem\u003eA. graveolens\u003c/em\u003e did not exhibit a significant increase(Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of Multiwalled carbon nanotubes priming on dry weight(g), total protein (mg/g Fwt), nitrogen content (mg/gDW), and amino acids content (\u0026micro;mol/ gFW) of four sprouts. Experiments were carried out in triplicate and the data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (S.E); Level of significance, * p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; MWCNT, multiwalled carbon nanotubes.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTrigonella foenum - graecum\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e\u003cem\u003eLinum grandiflorum\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u003cem\u003eLepidium sativum\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e\u003cem\u003eAnethum graveolens\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eMWCNT-treated\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eMWCNT- treated\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003eMWCNT- treated\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eMWCNT- treated\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDry weight (g)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal protein (mg/g Fwt)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e34.89\u0026thinsp;\u0026plusmn;\u0026thinsp;1.49*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e17.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e18.43\u0026thinsp;\u0026plusmn;\u0026thinsp;1.40*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNitrogen mg/gDW\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e192.98\u0026thinsp;\u0026plusmn;\u0026thinsp;11.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e229.28\u0026thinsp;\u0026plusmn;\u0026thinsp;8.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e200.62\u0026thinsp;\u0026plusmn;\u0026thinsp;11.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e240.74\u0026thinsp;\u0026plusmn;\u0026thinsp;8.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e213.99\u0026thinsp;\u0026plusmn;\u0026thinsp;10.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e252.21\u0026thinsp;\u0026plusmn;\u0026thinsp;8.76*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e208.26\u0026thinsp;\u0026plusmn;\u0026thinsp;13.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e244.57\u0026thinsp;\u0026plusmn;\u0026thinsp;10.64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEssential amino acids\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLysine\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.15\u0026thinsp;\u0026plusmn;\u0026thinsp;1.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e6.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHistidine\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIsoleucine\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLeucine\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMethionine\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eThreonine\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eValine\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePhenylalanine\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eArginine\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e 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\u003cp\u003e13.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e16.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNon-essential amino acids\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGlycine\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e56.95\u0026thinsp;\u0026plusmn;\u0026thinsp;1.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e95.12\u0026thinsp;\u0026plusmn;\u0026thinsp;14.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e58.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e50.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e63.58\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e62.94\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e74.30\u0026thinsp;\u0026plusmn;\u0026thinsp;7.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAlanine\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e11.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eOrnithine\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGlutamine\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAsparagine\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSerine\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGlutamic acid\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAspartate\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCystine\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTyrosine\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal NEAAs\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e73.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e113.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e66.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e77.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e68.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e81.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e84.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e96.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Protein, nitrogen, and nitrate reductase activity (NR)\u003c/h2\u003e \u003cp\u003eFunctional proteins are found in soluble proteins, primarily in the form of enzymes. Their content increased by 104.76%, 71.62%, 32.70%, and 113.31%, in \u003cem\u003eT. foenum \u0026ndash; graecum\u003c/em\u003e, \u003cem\u003eL. grandiflorum\u003c/em\u003e, \u003cem\u003eL. sativum\u003c/em\u003e, and \u003cem\u003eA. graveolens\u003c/em\u003e, respectively, as compared to the control (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). MWCNT greatly impacted the N content in the four species as compared to control sprouts. N content increased by 19%,20%,18%, and 17% in \u003cem\u003eT. foenum \u0026ndash; graecum\u003c/em\u003e, \u003cem\u003eL.grandiflorum\u003c/em\u003e, \u003cem\u003eL. sativum\u003c/em\u003e, and \u003cem\u003eA. graveolens\u003c/em\u003e, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Depending on the species, the activities of NR changed in response to MWCNT priming. In particular, NR activity rose relative to the control by 27%, 35%, and 10% in \u003cem\u003eT. foenum-graecum\u003c/em\u003e, \u003cem\u003eL. grandiflorum\u003c/em\u003e, and \u003cem\u003eA. graveolens\u003c/em\u003e, respectively, but declined in \u003cem\u003eL. sativum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Amino acids metabolism\u003c/h2\u003e \u003cp\u003eAmino acids are important for the growth and development of sprouts, serving as the building blocks for proteins, which are essential for various physiological processes. They are essential for enzyme activity, cellular metabolism, and the production of hormones and signaling chemicals. The nutritional value and stress tolerance of sprouts are influenced by both essential and non-essential amino acids, which also contribute to the overall health and productivity of the plant. Our results revealed that MWCNT treatment induced significant increases in the levels of the majority of the detected amino acids (including both essential and non-essential ones) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The non-essential amino acids showed the highest concentrations 89%, 80%, 86%, and 86% in \u003cem\u003eT. foenum - graecum\u003c/em\u003e, \u003cem\u003eL. grandiflorum\u003c/em\u003e, \u003cem\u003eL. sativum\u003c/em\u003e, and \u003cem\u003eA. graveolens\u003c/em\u003e, respectively, compared to the control. Essential amino acids showed concentrations of 11%, 20%, 14%, and 14% in \u003cem\u003eT. foenum - graecum\u003c/em\u003e, \u003cem\u003eL. grandiflorum, L. sativum\u003c/em\u003e, and \u003cem\u003eA. graveolens\u003c/em\u003e, respectively, as compared to the control. The amount of histidine, glutamine, and glutamic acid was increased significantly in \u003cem\u003eT. foenum - graecum\u003c/em\u003e; glycine, alanine, glutamic acid, valine, and lysine were enhanced in \u003cem\u003eL. grandiflorum\u003c/em\u003e.; glycine, lysine, alanine, asparagine, isoleucine, valine, glutamic acid, aspartate, and tyrosine, were increased in \u003cem\u003eL. sativum\u003c/em\u003e; and lysine, alanine, isoleucine, valine and glutamic acid, glutamine, glycine, were elevated in \u003cem\u003eA. graveolens\u003c/em\u003e. Variable trends were noticed among different sprouts.\u003c/p\u003e \u003cp\u003eTo understand the bases of increased levels of amino acids, we measured their related biosynthetic enzymes. This involved assessing the activity of key enzymes responsible for the biosynthesis of amino acids to provide insights into how MWCNT can boost the production of bioactive amino acids and overall plant metabolism. Key enzymes such as GS, protease, and GOGAT are necessary for plants to absorb and assimilate nitrogen. The sprouts of the four species responded differentially to the impacts of MWCNT on GDH, GOGAT, GS, arginase, and threonine synthase in terms of nitrogen metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFollowing treatment with MWCNTs, the activity of GDH in four sprouts rose in comparison to the control by 26%, 17%, 25%, and 11% in \u003cem\u003eT. foenum-graecum\u003c/em\u003e, \u003cem\u003eL. grandiflorum\u003c/em\u003e, \u003cem\u003eL. sativum\u003c/em\u003e, and \u003cem\u003eA. graveolens\u003c/em\u003e, respectively. Furthermore, MWCNT significantly increased arginase activity by 21% and 13% in \u003cem\u003eL. grandiflorum\u003c/em\u003e and \u003cem\u003eA. graveolens\u003c/em\u003e, respectively, but it reduced in \u003cem\u003eT. foenum-graecum\u003c/em\u003e and \u003cem\u003eL. sativum\u003c/em\u003e. GS activity decreased in \u003cem\u003eL. grandiflorum\u003c/em\u003e and \u003cem\u003eL. sativum\u003c/em\u003e, but increased by 13% and 14% in \u003cem\u003eT. foenum-graecum\u003c/em\u003e and \u003cem\u003eA. graveolens\u003c/em\u003e, respectively, compared to the control. Additionally, TS increased by 12%, 61%, and 16% in \u003cem\u003eT. foenum-graecum\u003c/em\u003e, \u003cem\u003eL. sativum\u003c/em\u003e, and \u003cem\u003eA. graveolens\u003c/em\u003e, respectively, compared to the control, but decreased in \u003cem\u003eL. grandiflorum\u003c/em\u003e. Following MWCNT treatment, GOGAT activity increased by 16%, 4%, 59%, and 14% in \u003cem\u003eT. foenum-graecum\u003c/em\u003e, \u003cem\u003eL. grandiflorum\u003c/em\u003e, \u003cem\u003eL. sativum\u003c/em\u003e, and \u003cem\u003eA. graveolens\u003c/em\u003e, respectively, compared to the control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Polyamine metabolism\u003c/h2\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e3.5.1. Polyamine levels\u003c/h2\u003e \u003cp\u003ePolyamines such as diamino propane, putrescine, cadaverine, spermine, spermidine, and agmatine were measured to study the impact of MWCNT priming on polyamine metabolism in sprouts of the four investigated species(Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Our results showed that putrescine levels were generally elevated by 57%,50%,47%, and 28%, respectively in \u003cem\u003eT. foenum-graecum\u003c/em\u003e, \u003cem\u003eL. grandiflorum\u003c/em\u003e, \u003cem\u003eL. sativum\u003c/em\u003e, and \u003cem\u003eA. graveolens\u003c/em\u003e. Spermidine increased by 4%,77%,32%, and 39%, respectively in \u003cem\u003eT. foenum-graecum\u003c/em\u003e, \u003cem\u003eL. grandiflorum\u003c/em\u003e, \u003cem\u003eL. sativum\u003c/em\u003e, and \u003cem\u003eA. graveolens\u003c/em\u003e. In addition, Spermine enhanced by 116%,5%,61%, and 33%, respectively in \u003cem\u003eT. foenum-graecum\u003c/em\u003e, \u003cem\u003eL. grandiflorum\u003c/em\u003e, \u003cem\u003eL. sativum\u003c/em\u003e, and \u003cem\u003eA. graveolens\u003c/em\u003e. When compared to the control, diaminopropane levels in \u003cem\u003eT. foenum-graecum\u003c/em\u003e, \u003cem\u003eL. grandiflorum\u003c/em\u003e, \u003cem\u003eL. sativum\u003c/em\u003e, and \u003cem\u003eA. graveolens\u003c/em\u003e increased by 124%, 48%, 60%, and 46%, respectively. On the other hand, \u003cem\u003eT. foenum-graecum\u003c/em\u003e, \u003cem\u003eL. grandiflorum\u003c/em\u003e, \u003cem\u003eL. sativum\u003c/em\u003e, and \u003cem\u003eA. graveolens\u003c/em\u003e all showed notable increases in cadaverine after receiving MWCNT treatment: 98%, 54%, 38%, and 40%, respectively. Meanwhile, agmatine increased in \u003cem\u003eL. grandiflorum\u003c/em\u003e, \u003cem\u003eand L. sativum\u003c/em\u003e, while decreasing in \u003cem\u003eT. foenum \u0026ndash; graecum\u003c/em\u003e, and \u003cem\u003eA. graveolens\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of Multiwalled carbon nanotubes priming on polyamines(nmol/gFW) content of four sprouts. Experiments were carried out in triplicate and the data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (S.E); Level of significance, * p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; MWCNT, multiwalled carbon nanotubes.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTrigonella foenum - graecum\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e\u003cem\u003eLinum grandiflorum\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u003cem\u003eLepidium sativum\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e\u003cem\u003eAnethum graveolens\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eMWCNT-treated\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eMWCNT- treated\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003eMWCNT- treated\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eMWCNT- treated\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDi amino propane\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e5.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePutrescine\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40.03\u0026thinsp;\u0026plusmn;\u0026thinsp;2.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e62.90\u0026thinsp;\u0026plusmn;\u0026thinsp;2.34*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e45.59\u0026thinsp;\u0026plusmn;\u0026thinsp;2.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e68.19\u0026thinsp;\u0026plusmn;\u0026thinsp;4.41*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e41.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e60.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e40.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e51.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCadaverine\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSpermidine\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.18\u0026thinsp;\u0026plusmn;\u0026thinsp;2.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.31\u0026thinsp;\u0026plusmn;\u0026thinsp;2.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.13\u0026thinsp;\u0026plusmn;\u0026thinsp;3.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.75\u0026thinsp;\u0026plusmn;\u0026thinsp;2.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.65\u0026thinsp;\u0026plusmn;\u0026thinsp;2.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.77\u0026thinsp;\u0026plusmn;\u0026thinsp;2.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.85\u0026thinsp;\u0026plusmn;\u0026thinsp;2.94\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSpermine\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41.86\u0026thinsp;\u0026plusmn;\u0026thinsp;3.32*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e44.24\u0026thinsp;\u0026plusmn;\u0026thinsp;2.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e46.36\u0026thinsp;\u0026plusmn;\u0026thinsp;7.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e17.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e27.68\u0026thinsp;\u0026plusmn;\u0026thinsp;1.55*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e33.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e44.01\u0026thinsp;\u0026plusmn;\u0026thinsp;2.78*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAgmatine\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.5.2 Polyamine metabolism\u003c/h2\u003e \u003cp\u003eWe measured the activity of plolyamines related enzymes such as arginine decarboxylase, ornithine decarboxylase, S-adenosyl-L-methionine decarboxylase, spermine synthase, spermidine synthase, arginine decarboxylase. We first focused on the polyamine-related enzymes including arginine decarboxylase and ornithine decarboxylase, which form putrescine from L-ornithine and can subsequently be converted into spermidine and spermine, to investigate the mechanism underlying the changes in polyamines (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). S-adenosyl-L-methionine decarboxylase, which plays a role in the biosynthesis of spermidine and spermine from putrescine, as well as its involvement in the conversion of S-adenosyl methionine into S-adenosylmethioninamine, was also measured. This increase in putrescine levels was correlated with a significant increase in either ADC or ODC. Also, MWCNT treatment resulted in the accumulation of spermidine and spermine. This accumulation is consistent with the enhanced levels of spermidine synthase (SpdS), spermine synthase (SpmS), and S-adenosyl-L-methionine decarboxylase. The current results also demonstrated that both PAO and DAO were decreased in all species.\u003c/p\u003e \u003c/div\u003e "},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e4.1. Importance of N in improving sprouts' nutritive value\u003c/h2\u003e \u003cp\u003eNitrogen (N) is necessary for many metabolic reactions that improve the chemical composition of sprout tissue. It improves the synthesis of both primary and secondary metabolites, enzyme activity, polyamines, chlorophyll synthesis, and overall nitrogen assimilation. Because of these aspects, sprouts have better more accurate growth, nutritional value, and health benefits; therefore, nitrogen is crucial for the growth and quality of sprouts. The nitrogen assimilation process, which has greatly influenced the tissue chemical composition of sprouts, needs nitrate reductase (NR). The process of transforming consumed nitrogen into forms that can be used is started when NR reduces nitrate to nitrite. During this process, the ammonium is formed and then subsequently incorporated into amino acids and proteins. Elevated (NR) activity stimulates growth, biomass accumulation, the formation of amino acids and proteins, and metabolic activity. MWCNTs can substantially boost NR activity, and affect vital enzymes that contribute to nitrogen assimilation. It may also greatly enhance the nutritional content and nitrogen metabolism of sprouts. The currently available outcomes demonstrate that there were observable rises in both N and NR in each sprout. Similarly, higher dosages of MWCNTs (such as 50, 100, and 200 \u0026micro;g/mL) show higher activity in the stimulation of nitrate absorption, assimilation, and nitrogen metabolism in sprouts.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e Due to the higher biomass of sprouts that result from, nitrogen content, and a buildup of healthy bioactive substances such as proteins, amino acids, and antioxidants.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e4.2. Increased essential amino acids and polyamines improved sprout tissue chemical composition\u003c/h2\u003e \u003cp\u003eIt's well-known that amino acids have a crucial role in plant enlargement and development, whereas they stimulate nitrogen metabolism and nutrition absorption, and share in building blocks for proteins and precursors for certain metabolites. Particularly, the variation in nutritional and health-promoting characteristics greatly depends on the necessary amino acid constituents of those plants. The investigated sprout's construction was formed of both necessary and non-essential amino acids. The vital amino acids play a critical role in the enhancement of nutritional content such as vitamins, minerals, and bioactive substances during germination which in turn was important in plant growth and development. Comparatively, amino acids participate in the improvement of polyamine production and subsequently have a good impression on nitrogen metabolism, protein synthesis, enzyme activity, and the synthesis of bioactive metabolites. Additionally, polyamines participate in many physiological processes such as the availability of nutrients, malfunctioning phytic acid, increasing antioxidant stimulation, cell division, and adding to their nutritional value. The final result of this result was the refinement of the nutritional components of sprouts, making them a more useful and nutrient-dense food option. According to the species, MWCNTs can leave various impacts on polyamine yielding. Similar results were recorded in mung bean sprouts that have been impacted by MWCNT priming whereas they manage the breakdown of phytic acid through stimulation of polyamines, specifically spermidine and putrescine.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e Nonetheless, it has been observed that MWCNT priming influences the polyamine metabolism of peach fruit in response to chilling damage (CI). The study concluded that MWCNT priming elevated the amounts of putrescine, spermine, and spermidine in peach fruit, which assisted in reducing the effects of CI.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e4.3. Biochemical bases of increased amino acids and polyamines\u003c/h2\u003e \u003cp\u003eThe main importance of amino acids is that they act as basic units for the construction of protein and play a crucial role in nutritional absorption and N metabolism, besides acting as a precursor for certain metabolites. Certainly, in the conversion of organic N from inorganic form, the amino acids act as intermediates, and the plant conveys the pathway of GS/ GOGAT to transform the inorganic N to glutamine and then glutamate. These amino acids were used widely by some proteins, nucleic acids, and other amino acids. The four sprouts in our investigation had higher total protein and amino acid contents after MWCNT treatment, which enhanced N metabolism. It has been demonstrated that MWCNTs stimulate the activity of important nitrogen metabolism-related enzymes, including GS and GOGAT.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e As a result, nitrogen-containing substances like proteins and amino acids build up in plants. MWCNTs appear to have the ability to affect plant amino acid metabolism in general by stimulating the production and accumulation of amino acids, as evidenced by their regulation of enzymes such as GS and GOGAT. This is especially clear from a study where 100 mg/L MWCNTs boosted the activity of important enzymes involved in the metabolism of nitrogen, causing soluble proteins, carbohydrates (sugars and starch), and nitrogen to accumulate in maize plants.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eOur results declared that the treatment with MWCNT caused a remarkable accumulation in the levels of Put as well as Spm and Spd in addition to their metabolic enzymes (ADC, ODC, and S-adenosylmethionine decarboxylase; SAMDC, Spd synthase, and Spm synthase). There were indirect impacts of MWCNTs on polyamine metabolism during the regulation of nitrogen metabolism due to the linear influence of the availability of nitrogen compounds on the homeostasis and synthesis of plant polyamines. For example, it has been demonstrated that MWCNT priming improves radish sprouts' polyamine metabolism, namely the accumulation of putrescine, spermine, and spermidine.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e Our findings revealed that there was an elevation in the level of polyamines as well as the enzyme activities necessary for their synthesis, which include (ADC), (SpdS), and (ODC) during the application of MWCNT priming. Gondor \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e stated a similar impact on the polyamine metabolism under different light conditions. The current outcomes demonstrated higher levels of putrescine and spermidine in both high and low-light conditions throughout the MWCNT treatment, on the other hand, there was no significant influence on spermine rates. With this intention, MWCNTs influence varies depending on the category of plant species in the polyamine synthesis.\u003c/p\u003e \u003cp\u003eAmino acids operate as fundamental components in the synthesis of polyamines, which are vital for cell growth and differentiation. Polyamines are produced directly from the amino acids (ornithine and arginine), which are obtained via the nitrogen absorption pathway. Putrescine is generated when arginine undergoes a process known as ADC, which creates agmatine. Putrescine is produced when ODC decarboxylates ornithine, which is formed by transaminates glutamate. Key amino acids are used for the production of putrescine, the first polyamine. As a substrate, it serves to facilitate the formation of spermidine and spermine. The crucial amino acid called methionine is converted to (SAM), which is then decarboxylated by SAMDC to generate decarboxylated SAM. The correlation between amino acid metabolism and polyamine production is highlighted by the fact that decarboxylated SAM contributes aminopropyl groups for the formation of spermidine and spermine from putrescine. These pathways are major targets for improving the nutritional and biochemical qualities of sprouts since they are essential for plant growth, development, and stress responses.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eFinally, the endpoint of the current study revealed that usage of four plant species \u003cem\u003eA. graveolens, L. sativum\u003c/em\u003e, \u003cem\u003eT. foenum-graecum\u003c/em\u003e, and \u003cem\u003eL. grandiflorum\u003c/em\u003e that were treated with (MWCNTs) multi-walled carbon nanotubes showed well impact on the metabolism of nitrogen, amino acid, and polyamine. The data analysis exhibited a raised level of GS, NR, and GOGAT enzymes which stimulated the nitrogen composition and improved assimilation of nitrogen. It witnessed also an enhancement in protein synthesis by detecting higher levels of soluble proteins. Additionally, both essential and nonessential amino acids revealed a significant increase by treatment with MWCNTs.\u003c/p\u003e \u003cp\u003eTherefore, the physiological activities of the plant like response to stress, enzyme activity, and cellular metabolism needed the improvement of the productivity of the plant besides the support of the general health that was achieved by increasing the level of amino acids. Not only amino acids but also, polyamines metabolism were enhanced by additional MWCNTs. The current analysis revealed the elevation of (ODC), the biosynthetic activities of enzymes including (ADC), and polyamines such as putrescine, spermidine, and spermine significantly. In conclusion, the MWCNT is considered a promising strategy for the enhancement of the metabolic process that is necessary for the adaptation and development of the plant. The agriculture nanotechnology opened the way for improving the crop yield and resistance which was proved through the current investigation. More studies were required to explore the promising mechanisms of MWCNT seeking the enhancement of the sustainable agriculture yield and resistance.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe authors extend their appreciation to the Researchers Supporting Project number (RSPD2024R931) King Saud University, Riyadh, Saud Arabia.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eA- Yasmen khaled (write the main manuscript-experimental setup-Resources-software-validation)B-Momtaz M. Hegaba(Data curation- formal analysis-project administration-supervision)C-Mohammad K. Okla(Data curation- funding-supervision- validation)D-Amal Mohamed AlGarawi(Data curation- funding-supervision- validation)E-Wael Z. Tawfik(Data curation- formal analysis-project administration-supervision)F-Gehad AbdElgayed(Data curation- supervision- validation)G-Mona Sayed (Data curation- formal analysis-project administration-supervision)\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e \u003cp\u003eThe authors extend their appreciation to the Researchers Supporting Project number (RSPD2024R931) King Saud University, Riyadh, Saud Arabia.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSlepičkov\u0026aacute; Kas\u0026aacute;lkov\u0026aacute;, N., Slepička, P., \u0026amp; Švorč\u0026iacute;k, V. (2021). Carbon nanostructures, nanolayers, and their composites. Nanomaterials, \u003cem\u003e11\u003c/em\u003e(9), 2368.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eH. He, L.A. Pham-Huy, P. Dramou, D. Xiao, P. Zuo, C. 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International Journal of Molecular Sciences, 22(21), 11717.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanoparticle-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nano","sideBox":"Learn more about [Journal of Nanoparticle Research](http://link.springer.com/journal/11051)","snPcode":"11051","submissionUrl":"https://submission.nature.com/new-submission/11051/3","title":"Journal of Nanoparticle Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"multiwalled carbon nanotubes, sprouts, N metabolism, amino acids, polyamine metabolism, nutritive value","lastPublishedDoi":"10.21203/rs.3.rs-4777965/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4777965/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSprouts are well known for having a remarkable nutritional profile. Enhancing the tissue chemical composition and quality of sprouts is essential since these metabolites offer numerous health advantages. To this end, this study aimed to investigate the effects of priming with multiwalled carbon nanotubes (MWCNT) on the growth and nitrogen metabolism of four horticultural plants, namely \u003cem\u003eT. foenum graecum\u003c/em\u003e, \u003cem\u003eL. grandifloruum\u003c/em\u003e, \u003cem\u003eL. sativum\u003c/em\u003e, and \u003cem\u003eA. gravelones\u003c/em\u003e. The properties of our synthesized MWCNTs included three characteristic peaks 3434, 1539, and 1068 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e attributable to the stretching vibration of O\u0026ndash;H, bending vibration, and C\u0026thinsp;\u0026minus;\u0026thinsp;O, respectively. MWCNT priming increased the sprouting process by inducing biomass and protein accumulation. MWCNT priming improved nitrogen metabolism, including amino acid and polyamine metabolism. At the amino acids level, there was an increase in amino acids levels (e.g., glycine, lysine, asparagine, and glutamic acid) as well as their metabolic enzyme activity including glutamine synthetase (GS), threonine synthase (TS), and glutamate synthase (GOGAT). Increased polyamine levels like spermine, putrescine, and spermidine were also associated with boosting their related biosynthetic enzyme activities \u003cem\u003ei.e.\u003c/em\u003e, arginine decarboxylase (ADC), ornithine decarboxylase (ODC), spermidine synthase, and spermine synthase. This improvement of nitrogen metabolic pathways highlights the potential of MWCNT to boost the tissue chemical composition of horticultural plants.\u003c/p\u003e","manuscriptTitle":"Potential role of multiwalled carbon nanotube priming in boosting nitrogen metabolism and nutritional value during the sprouting process","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-22 08:22:43","doi":"10.21203/rs.3.rs-4777965/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-31T13:47:36+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-26T14:25:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"326709762004227640775165295441860682801","date":"2024-08-16T12:21:07+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-15T21:41:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-26T20:49:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-26T04:23:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Nanoparticle Research","date":"2024-07-21T18:55:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanoparticle-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nano","sideBox":"Learn more about [Journal of Nanoparticle Research](http://link.springer.com/journal/11051)","snPcode":"11051","submissionUrl":"https://submission.nature.com/new-submission/11051/3","title":"Journal of Nanoparticle Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"741172b4-fd31-45b1-bc53-1f4b8ecc3f2d","owner":[],"postedDate":"August 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-28T16:03:30+00:00","versionOfRecord":{"articleIdentity":"rs-4777965","link":"https://doi.org/10.1007/s11051-024-06145-6","journal":{"identity":"journal-of-nanoparticle-research","isVorOnly":false,"title":"Journal of Nanoparticle Research"},"publishedOn":"2024-10-21 15:57:50","publishedOnDateReadable":"October 21st, 2024"},"versionCreatedAt":"2024-08-22 08:22:43","video":"","vorDoi":"10.1007/s11051-024-06145-6","vorDoiUrl":"https://doi.org/10.1007/s11051-024-06145-6","workflowStages":[]},"version":"v1","identity":"rs-4777965","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4777965","identity":"rs-4777965","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}